Synthesis and Antiprotozoal Activity of Cationic 1,4-Diphenyl-1H-1,2,3-Triazoles

Abstract

Novel dicationic triazoles 1–60 were synthesized by the Pinner method from the corresponding dinitriles, prepared via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The type and the placement of cationic moieties as well as the nature of aromatic substituents influenced in vitro antiprotozoal activities of compounds 1–60 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani and their cytotoxicity for mammalian cells. Eight congeners displayed antitrypanosomal IC₅₀ values below 10 nM. Thirty-nine dications were more potent against P. falciparum than pentamidine (IC₅₀ = 58 nM) and eight analogues were more active than artemisinin (IC₅₀ = 6 nM). Diimidazoline 60 exhibited antiplasmodial IC₅₀ value of 0.6 nM. Seven congeners administered at 4 × 5 mg/kg by the intraperitoneal route cured at least three out of four animals in the acute mouse model of African trypanosomiasis. At 4 × 1 mg/kg, diamidine 46 displayed better antitrypanosomal efficacy than melarsoprol, curing all infected mice.

Introduction

In recent years, the number of treatment failures associated with the development of drug resistant parasites for many infectious diseases, such as malaria,^1-4 human African trypanosomiasis^5-7 (HAT^a or sleeping sickness), and leishmaniasis^8-11 has increased with an alarming rate. The majority of people suffering from these infections live in the poorest regions. The need to replace inexpensive commonly used medications that are loosing efficacy due to the development of parasite resistance with pricier alternatives, or to rely on drug combinations, increases the economic burden on the affected nations. In the case of malaria, the structural and mechanistic resemblances of the existing treatments escalate the risk of cross-resistance and potential failures for newly introduced drug candidates. Current therapies approved for use against HAT (which is fatal without treatment) and leishmaniasis require a long course of parenteral administration and suffer from unacceptable toxicity and prolonged dosing.^{6, 7, 12} While new medications for visceral leishmaniasis have recently become available,^{13, 14} the high cost of treatment limits their broad application in developing countries. Therefore, there is an urgent need for safer and more affordable therapies that are effective against these re-emerging infections.

Although aromatic diamidines have been long known to possess a wide range of antimicrobial activities,^15-32 only 1,5-bis(4-amidinophenoxy)pentane (pentamidine) (Figure 1) has been widely used to treat humans. The drug has found practical applications against early stage Trypanosoma brucei gambiense HAT,^{6, 7, 33} antimony-resistant leishmaniasis,^{12, 34} and Pneumocystis jiroveci (formerly P. carinii) pneumonia.^35-37 Additionally, pentamidine demonstrates some antimalarial potency,^{23, 38, 39} although it was never approved to treat the disease. Because both cationic moieties in pentamidine are ionized at physiological pH, the drug has low oral activity due to its inability to pass through cellular membranes. This reduces the practicality of pentamidine treatments in remote regions where oral administration of medications would present the most sensible option.

Figure 1.

Structures of pentamidine, pafuramidine, and furamidine.

Recently, an orally active prodrug 2,5-bis(4-methoxyamidinophenyl)furan⁴⁰ (pafuramidine) (Figure 1) of 2,5-bis(4-amidinophenyl)furan²⁰ (furamidine) reached Phase III clinical trials against HAT and P. jiroveci, although newly recognized possibilities of hepatic and renal toxicity of furamidine in humans are likely to preclude its further development. In a search for novel molecules with improved antiprotozoal activity and reduced toxicity a large number of furamidine-related analogues possessing various aromatic linkers have been synthesized.^{19, 21, 29, 41-49} Previously, we described excellent antitrypanosomal and antiplasmodial activities of select cationic diphenyl isoxazoles⁵⁰ and 2-phenyl benzofurans.⁵¹ Lately, the 1,2,3-triazole fragment has attracted our attention as a suitable isosteric replacement for the central 5-membered ring because many compounds possessing 1,2,3-triazole fragment exhibit useful biological properties^52-63 and because 1,4-diphenyl 1,2,3-triazoles would retain strong geometrical resemblance to furamidine.

The chemistry of 1,2,3-triazoles has been known for more than a century (for a recent review see Ref.⁶⁴). Among several possible ways to construct the 5-membered triazole ring, the most suitable for us was the Huisgen 1,3-dipolar cycloaddition⁶⁵ of organic azides to terminal alkynes. This method, which normally required prolonged usage of elevated temperatures and usually afforded mixtures of 1,4- and 1,5-disubstituted 1,2,3-triazoles,^{66, 67} has been recently revitalized by the discovery that Cu(I) salts facilitate the addition of azides to alkynes at ambient temperature affording exclusively 1,4-disubstituted isomers.^{68, 69} Because of its high regioselectivity, mild reaction conditions, and excellent yields of desired products, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has found multiple applications in material science, bioconjugate chemistry, and drug discovery.^70-76

In our search for novel safer and more potent antiprotozoal drug candidates, we herein report the synthesis of novel cationic 1,4-diphenyl-1,2,3-triazoles 1–60 utilizing the CuAAC methodology. Compounds 1–60 were evaluated in vitro for antiprotozoal potency versus T. brucei rhodesiense (STIB900), chloroquine resistant Plasmodium falciparum (K1), axenic amastigotes of Leishmania donovani (MHOM/SD/62/1S–CL2_D), and for cytotoxicity against rat myoblast cells (L6). Dications exhibiting high antitrypanosomal activities in vitro were screened in vivo in the STIB900 acute mouse model of African trypanosomiasis.

Chemistry

3-Azidobenzonitrile⁷⁷ (61) and 4-azidobenzonitrile^{77, 78} (62) were obtained by diazotization/azidation of the corresponding commercially available aminobenzonitriles following the published procedure.^{79, 80} Synthesis of novel phenyl azides 63–65 is depicted in Scheme 1. 4-Methoxy-3-nitrobenzonitrile (67), prepared by O-methylation of commercially available 4-hydroxy-3-nitrobenzonitrile (66) with MeI and K₂CO₃ in DMF, underwent catalytic hydrogenation to give 3-amino-4-methoxybenzonitrile (68). Compound 68 was diazotized with sodium nitrite in diluted hydrochloric acid at 0–5 °C followed by azidation of the resulting diazonium chloride with sodium azide to afford 3-azido-4-methoxybenzonitrile (63) in 64% yield over the three steps. 4-Bromo-o-anisidine (70) was synthesized in 50% yield by bromination of o-anisidine (69) with bromine in glacial acetic acid as previously described.⁸¹ Compound 70 was reacted with CuCN in DMF–pyridine (5:1) mixture to give 4-amino-3-methoxybenzonitrile⁸² (71) in 54% yield. Treatment of 71 with sodium nitrite in hydrochloric acid at 0–5 °C followed by reaction with sodium azide afforded 4-azido-3-methoxybenzonitrile (64) in 94% yield. 3-Azido-4-hydroxybenzonitrile (65) was obtained in 55% overall yield from 66 by catalytic hydrogenation over 10% Pd/C followed by the diazotization/azidation of the resulting 3-amino-4-hydroxybenzonitrile (72). Ethynyl benzonitriles 73–76 were synthesized as reported earlier.⁵⁰

Scheme 1a.

^a Reagents and conditions: (i) H₂, 50 psi, 10% Pd/C, MeOH; (ii) MeI, K₂CO₃, DMF, ambient temp; (iii) NaNO₂, 10% aq HCl, 0–5 °C, 1 h, then NaN₃, H₂O, 0–5 °C, 1 h; (iv) Br₂, AcOH; (v) CuCN, DMF–Py (5:1), reflux.

1,4-Dicyanophenyl-1H-1,2,3-triazoles 77–89 and 93 were prepared by the copper(I)-catalyzed 1,3-dipolar cycloaddition^{70, 74} of 3- or 4-azidobenzonitriles 61–65 to ethynyl benzonitriles 73–76 in aqueous t-BuOH or DMSO following the published protocol (Scheme 2).⁶⁹ Hydroxy substituted dinitriles 90, 92, 94, and 96 were synthesized from the corresponding methoxy substituted congeners 82, 84, 86, and 88 in 89–99% yields by treatment with BBr₃ in CH₂Cl₂. Attempts to employ this method for demethylation of methoxy derivatives 83 and 87 were unsuccessful. Consequently, compounds 91 and 95 were obtained in 89% yields by treatment of the dinitriles 83 and 87 with melted pyridine hydrochloride at 150–160 °C following the previously reported procedure.⁵¹ Dicationic 1,4-diphenyl-1H-1,2,3-triazoles 1–60 (Tables 1-4) were synthesized using the modified Pinner method⁸³ (Scheme 3). Thus, dinitriles 77–96 were transformed to imidate esters, which reacted with ethanolic solutions of ammonia, isopropylamine, or ethylenediamine at ambient temperature followed by treatment with aqueous HCl to afford congeners 1–60 as dihydrochloride salts.

Scheme 2a.

^a Reagents and conditions: (i) Sodium ascorbate, CuSO₄·5H₂O, DMF–H₂O (9:1) or t-BuOH–H₂O (1:1), 24–48 h; (ii) BBr₃, CH₂Cl₂, 0 °C–ambient temp; (iii) pyridine hydrochloride, 150–160 °C, 3 h.

Table 1.

Cytotoxicity and in Vitro Antiprotozoal Activity of Dications 1–15.

compd	R	R1	R2	Cytotoxicity	T. brucei rhodesienseg		P. falciparumi		L. donovanik
				IC50 (μM)	IC50 (μM)	SITh	IC50 (μM)	SIPj	IC50 (μM)	SILl
1	Am	H	H	219	0.083	2639	0.021	10429	48	5
2	i-PrAm	H	H	>184	14.8	>12	0.118	>1559	>100	NDm
3	Im	H	H	>197	14.1	>14	0.365	>540	>100	ND
4	Am	OMe	H	>224	0.047	>4766	0.028	>8000	>100	ND
5	i-PrAm	OMe	H	131	10.1	13	0.125	1048	>100	ND
6	Im	OMe	H	>172	7.83	>22	0.030	>5733	>100	ND
7	Am	H	OMe	>200	1.65	>121	0.024	>8333	>100	ND
8	i-PrAm	H	OMe	>176	1.25	>141	0.106	>1660	>100	ND
9	Im	H	OMe	>176	0.006	>29333	0.014	>12571	4.9	>36
10	Am	OH	H	>209	49.6	>4	8.93	>23	>100	ND
11	i-PrAm	OH	H	>167	13.9	>12	0.739	>226	>100	ND
12	Im	OH	H	>174	102	>2	0.325	>535	>100	ND
13	Am	H	OH	>136	0.174	>782	0.036	>3778	20	>7
14	i-PrAm	H	OH	>170	10.9	>16	0.229	>742	>100	ND
15	Im	H	OH	>183	11.7	>16	0.052	>3519	>100	ND
PMDa				46.6	0.003	15533	0.058	803	1.8	25
MLSPb				7.78	0.004	1945
CQc				117			0.124	944
ATMSd				450			0.006	75000
PPTe				0.020

^aPMD, Pentamidine.

^bMLSP, melarsoprol

^cCQ, chloroquine.

^dATMS, artemisinin.

^ePPT, podophyllotoxin.

^fCytotoxicity (L6 rat myoblast cells). Average of duplicate determinations. Maximum test concentration was 90 μg/mL. Based on the molecular weight of tested compounds, different > μM values were obtained from the conversion of 90 μg/mL to the μM scale.

^gTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.

^hSelectivity index for T. brucei rhodesiense (SI_T), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (T. brucei rhodesiense)].

ⁱPlasmodium falciparum (K1, resistant to chloroquine). Average of duplicate determinations.

^jSelectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (P. falciparum)].

^kLeishmania donovani (MHOM/SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.

^lSelectivity index for L. donovani (SI_L), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (L. donovani)].

^mND, not determined.

Table 4.

Cytotoxicity and in Vitro Antiprotozoal Activity of Dications 46–60.

compd	R	R1	R2	Cytotoxicityf	T. brucei rhodesienseg		P. falciparumi		L. donovanik
				IC50 (μM)	IC50 (μM)	SITh	IC50 (μM)	SIPj	IC50 (μM)	SILl
46	Am	H	H	8.10	0.004	2025	0.002	4050	9.1	<1
47	i-PrAm	H	H	>183	0.201	>910	0.005	>36600	25	>7
48	Im	H	H	101	0.053	1906	0.002	50500	>100	NDm
49	Am	OMe	H	>183	0.038	>4816	0.009	>20333	>40	ND
50	i-PrAm	OMe	H	>164	0.328	>500	0.019	>8632	>100	ND
51	Im	OMe	H	>176	0.675	>261	0.017	>10353	>100	ND
52	Am	H	OMe	59.4	0.031	1916	0.002	29700	13	5
53	i-PrAm	H	OMe	>166	0.201	>826	0.013	>12769	>100	ND
54	Im	H	OMe	142	0.116	1224	0.011	12909	>100	ND
55	Am	OH	H	>216	0.501	>431	0.170	>1271	>100	ND
56	i-PrAm	OH	H	>178	1.38	>129	0.800	>223	>100	ND
57	Im	OH	H	>198	0.998	>198	0.041	>4829	>100	ND
58	Am	H	OH	49.3	0.016	3081	0.002	24650	4.3	11
59	i-PrAm	H	OH	>180	0.037	>4865	0.007	>25714	>50	ND
60	Im	H	OH	>190	0.038	>5000	0.0006	>316667	24	>8
PMDa				46.6	0.003	15533	0.058	803	1.8	25
MLSPb				7.78	0.004	1945
CQc				117			0.124	944
ATMSd				450			0.006	75000
PPTe				0.020

^aPMD, Pentamidine.

^bMLSP, melarsoprol.

^cCQ, chloroquine.

^dATMS, artemisinin.

^ePPT, podophyllotoxin.

^fCytotoxicity (L6 rat myoblast cells). Average of duplicate determinations. Maximum test concentration was 90 μg/mL. Based on the molecular weight of tested compounds, different > μM values were obtained from the conversion of 90 μg/mL to the μM scale.

^gTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.

^hSelectivity index for T. brucei rhodesiense (SI_T), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (T. brucei rhodesiense)].

ⁱPlasmodium falciparum (K1, resistant to chloroquine). Average of duplicate determinations.

^jSelectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (P. falciparum)].

^kLeishmania donovani (MHOM/SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.

^lSelectivity index for L. donovani (SI_L), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (L. donovani)].

^mND, not determined.

Scheme 3a.

^a Reagents and conditions: (i) HCl gas, 1,4-Dioxane–EtOH (3:1); (ii) appropriate amine, EtOH, ambient temp; (iii) 3M HCl, EtOH.

Results and Discussion

In our recent study of 1,4-diphenyl isoxazoles, we found that several methoxy substituted dications were more active against T. brucei rhodesiense and P. falciparum compared to their chloro and nitro substituted analogues.⁵⁰ Similarly, the introduction of methoxy and hydroxy substituents improved antitrypanosomal and antiplasmodial activities and reduced cytotoxicities of select 2-phenyl benzofurans.⁵¹ Here, we investigate how the substitution on amidine groups, varying the position of the cationic moieties, and the attachment of methoxy and hydroxy groups to the aromatic rings influences antiprotozoal properties of novel cationic diphenyl triazoles 1–60. The results of the in vitro testing of the compounds 1–60 against the bloodstream form of T. brucei rhodesiense trypomastigotes (STIB900), chloroquine resistant P. falciparum (K1), axenic amastigotes of L. donovani (MHOM/SD/62/1S-CL2_D), and the assessment of their cytotoxicity against rat myoblast cells (L6) are summarized in Tables 1-4. For comparison, we included the activities of pentamidine, melarsoprol (T. brucei rhodesiense), chloroquine and artemisinin (P. falciparum), and podophyllotoxin (L6). To differentiate between cytotoxicity for parasite and mammalian cells, three in vitro selectivity indeces⁸⁴ were calculated as follows: antitrypanosomal selectivity index SI_T, expressed as the ratio [IC₅₀ (L6) / IC₅₀ (T. brucei rhodesiense)], antiplasmodial selectivity index SI_P, expressed as the ratio [IC₅₀ (L6) / IC₅₀ (P. falciparum)], and antileishmanial selectivity index SI_L, expressed as the ratio [IC₅₀ (L6) / IC₅₀ (L. donovani)]. The results of the in vivo screening of select dications in the STIB900 acute mouse model of trypanosomiasis are presented in Table 5.

Table 5.

Efficacy of Select Cationic Triazoles in the T. brucei rhodesiense STIB900 Mouse Model

compound	in vitro IC50 (nM)a	in vivob
		dose (mg/kg)c	curesd	MRD f
melarsoprol	4	4 × 8	4/4	–
		4 × 2	4/4	–
		4 × 1	2/4	20
pentamidine	3	4 × 20	2/4	26
		4 × 5	2/4	20
1	83	4 × 5	0/4	10
4	47	4 × 5	0/4	17
9	6	4 × 5	4/4	–
		4 × 1	0/4	12
		1 × 10	3/4	14
13	174	4 × 5	0/4	14.75
16	8	4 × 5	1/4	17.33
19	11	4 × 5	0/4	13
22	8	4 × 5	2/4	15.5
23	77	4 × 5	toxice	–
24	21	4 × 5	0/4	26
25	175	4 × 5	0/4	14
28	6	4 × 5	4/4	–
29	116	4 ×5	0/4	28.75
30	43	4 × 5	0/4	10.75
31	6	4 × 5	1/4	18
34	16	4 × 5	1/4	23
37	5	4 × 5	4/4	–
		4 × 1	0/4	9.5
		1 × 10	0/4	23.5
43	8	4 × 5	2/4	39
44	77	4 × 5	1/4	18
46	4	4 × 5	4/4	–
		4 × 1	4/4	–
		1 × 10	3/4	27
		4 × 0.5	0/4	25.5
47	201	4 × 5	0/2e	8
48	53	4 × 5	0/4	8
49	38	4 × 5	0/4	34
52	31	4 × 5	3/4	60
58	16	4 × 5	4/4	–
59	37	4 × 5	4/4	–
60	38	4 × 5	0/4	10

^aAverage of duplicate determinations.

^bSTIB900 acute mouse model.

^cIntraperitoneal administration.

^dNumber of mice that survive and are parasite free for 60 days.

^eMice died due to toxicity.

^fMean relapse day, untreated control mice have a high parasitamia load on day 7 and would expire between day 7 and 10 postinfection.

Structure–Activity Relationship

Cytotoxicity Study

Except for diamidines 1, 16, 31, 37, 46, 52, and 58, bis(N-isopropyl)amidines 5 and 32 and diimidazolines 18, 33, 38, and 54, all tested triazoles exhibited no cytotoxicity for L6 mammalian cells at the maximum dose of 90 μg/mL. Out of thirteen congeners active in the L6 assay, four compounds (1, 5, 16, and 18) possessed one cationic moiety in the 5′-position and nine were 4′,5 - (31–33, 37) and 4,4′-disubstituted analogues (46, 48, 52, 54, 58). Bis(N-isopropyl)amidines and diimidazolines without methoxy and hydroxy substituents on aromatic rings were less cytotoxic for mammalian cells than the corresponding diamidines. Placement of one cationic group in the 4-position increased cytotoxicities of 4,5′- and 4,4′-disubstituted diamidines 16 and 46 compared to the 5,5′-disubstituted analogue 1 nearly 25-fold. Although 4′,5-disubstituted congener 31 also displayed increased cytotoxicity with respect to diamidine 1, it was less cytotoxic than 4,5′- and 4′,4-disubstituted isomers 16 and 46. Apart from 4-substituted diamidines 16 and 46, all tested congeners 1–60 exhibited lower cytotoxicities than pentamidine.

In Vitro Antitrypanosomal Activity

All compounds 1–60 were active in vitro against T. brucei rhodesiense showing antitrypanosomal IC₅₀ values ranging from 4 nM to 102 μM. Eight diamidines (16, 19, 22, 28, 31, 37, 43, and 46) and diimidazoline 9 displayed antitrypanosomal IC₅₀ values comparable to that of melarsoprol (IC₅₀ = 4 nM) and pentamidine (IC₅₀ = 3 nM). Diamidine 46 bearing cationic groups in the 4,4′-positions was the most potent compounds in the series with antitrypanosomal IC₅₀ value of 4 nM.

The N-substitution on amidine groups has been shown to reduce antitrypanosomal properties of aromatic diamidines. In this study, unsubstituted diamidines, except congeners 7 and 10, also exhibited higher in vitro activities against T. brucei rhodesiense than the corresponding bis(N-isopropyl)amidines and diimidazolines, corroborating previously published results.^{23, 50, 85-89} For instance, 5,5′-disubstituted bis(N-isopropyl)amidine 2 and diimidazoline 3 were nearly 170-fold less active against T. brucei rhodesiense compared to diamidine 1 (Table 1). This loss of antitrypanosomal activity was further enhanced by the introduction of the methoxy and hydroxy groups on aromatic rings. For example, antitrypanosomal potencies of 4,5′-disubstituted bis(N-isopropyl)amidine 17 and diimidazoline 18 decreased 67- and 25-fold compared to that of diamidine 16. However, their 2-methoxy substituted analogues 20 and 21 were 178- and 170-fold less active against T. brucei rhodesiense than the corresponding congener 19 (Table 2). Similarly, in the series of the 4′,5-disubstituted triazoles (Table 3), activities of bis(N-isopropyl)amidine 32 and diimidazoline 33 for the pathogen decreased 77-fold compared to diamidine 31. At the same time, dications 38 and 39 bearing the methoxy group in the 2′-position were 141- and 804-fold less active against T. brucei rhodesiense than diamidine 37. This pattern continued among the 4,4′-disubstituted congeners (Table 4). Overall, antitrypanosomal potency of dications without methoxy and hydroxy groups on aromatic rings decreased in the order Am > Im > i-PrAm, whereas compounds possessing these substituents displayed no apparent correlation between the nature of cationic moieties and activities versus T. brucei rhodesiense.

Table 2.

Cytotoxicity and in Vitro Antiprotozoal Activity of Dications 16–30.

compd	R	R1	R2	Cytotoxicity	T. brucei rhodesienseg		P. falciparumi		L. donovanik
				IC50 (μM)	IC50 (μM)	SITh	IC50 (μM)	SIPj	IC50 (μM)	SILl
16	Am	H	H	8.60	0.008	1075	0.003	2867	12	<1
17	i-PrAm	H	H	>190	0.539	>353	0.038	>5000	>100	NDm
18	Im	H	H	127	0.200	635	0.084	1512	>100	ND
19	Am	OMe	H	>192	0.011	>17455	0.025	>7680	33	>6
20	i-PrAm	OMe	H	>159	1.96	>81	0.055	>2891	>100	ND
21	Im	OMe	H	>173	1.87	>93	0.058	>2983	>100	ND
22	Am	H	OMe	>198	0.008	>24750	0.007	>28286	4.3	>46
23	i-PrAm	H	OMe	>177	0.077	>2299	0.026	>6808	>100	ND
24	Im	H	OMe	>188	0.021	>8952	0.010	>18800	>100	ND
25	Am	OH	H	>209	0.175	>1194	0.175	>1194	>100	ND
26	i-PrAm	OH	H	>187	18.7	>10	1.19	>157	>100	ND
27	Im	OH	H	>187	11.1	>17	0.159	>1176	>100	ND
28	Am	H	OH	>219	0.006	>36500	0.022	>9955	>50	ND
29	i-PrAm	H	OH	>179	0.116	>1543	0.028	>6393	>100	ND
30	Im	H	OH	>190	0.043	>4419	0.009	>21111	43	>4
PMDa				46.6	0.003	15533	0.058	803	1.8	25
MLSPb				7.78	0.004	1945
CQc				117			0.124	944
ATMSd				450			0.006	75000
PPTe				0.020

^aPMD, Pentamidine.

^bMLSP, melarsoprol.

^cCQ, chloroquine.

^dATMS, artemisinin.

^ePPT, podophyllotoxin.

^fCytotoxicity (L6 rat myoblast cells). Average of duplicate determinations. Maximum test concentration was 90 μg/mL. Based on the molecular weight of tested compounds, different > μM values were obtained from the conversion of 90 μg/mL to the μM scale.

^gTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.

^hSelectivity index for T. brucei rhodesiense (SI_T), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (T. brucei rhodesiense)].

ⁱPlasmodium falciparum (K1, resistant to chloroquine). Average of duplicate determinations.

^jSelectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (P. falciparum)].

^kLeishmania donovani (MHOM/SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.

^lSelectivity index for L. donovani (SI_L), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (L. donovani)].

^mND, not determined.

Table 3.

Cytotoxicity and in Vitro Antiprotozoal Activity of Dications 31–45.

compd	R	R1	R2	Cytotoxicity	T. brucei rhodesienseg		P. falciparumi		L. donovanik
				IC50 (μM)	IC50 (μM)	SITh	IC50 (μM)	SIPj	IC50 (μM)	SILl
31	Am	H	H	86.0	0.006	14333	0.004	21500	17	5
32	i-PrAm	H	H	193	0.462	418	0.043	4488	58	3
33	Im	H	H	133	0.461	289	0.095	1400	>100	NDm
34	Am	OMe	H	>201	0.016	>12563	0.017	>11824	31	>6
35	i-PrAm	OMe	H	>173	0.447	>387	0.195	>887	>100	ND
36	Im	OMe	H	>170	0.162	>1049	0.022	>7727	>100	ND
37	Am	H	OMe	164	0.005	32800	0.007	23429	11	15
38	i-PrAm	H	OMe	>170	0.703	>242	0.034	>5000	32	>5
39	Im	H	OMe	>172	4.02	>43	0.064	>2688	>100	ND
40	Am	OH	H	>203	6.64	>31	1.23	>165	>100	ND
41	i-PrAm	OH	H	>170	15.1	>11	5.27	>32	>100	ND
42	Im	OH	H	>175	19.6	>9	0.885	>198	>100	ND
43	Am	H	OH	>210	0.008	>26250	0.007	>30000	8.0	>26
44	i-PrAm	H	OH	>175	0.077	>2273	0.049	>3571	>100	ND
45	Im	H	OH	>183	0.606	>302	0.016	>11438	>100	ND
PMDa				46.6	0.003	15533	0.058	803	1.8	25
MLSPb				7.78	0.004	1945
CQc				117			0.124	944
ATMSd				450			0.006	75000
PPTe				0.020

^aPMD, Pentamidine.

^bMLSP, melarsoprol.

^cCQ, chloroquine.

^dATMS, artemisinin.

^ePPT, podophyllotoxin.

^fCytotoxicity (L6 rat myoblast cells). Average of duplicate determinations. Maximum test concentration was 90 μg/mL. Based on the molecular weight of tested compounds, different > μM values were obtained from the conversion of 90 μg/mL to the μM scale.

^gTrypanosoma brucei rhodesiense (STIB900). Average of duplicate determinations.

^hSelectivity index for T. brucei rhodesiense (SI_T), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (T. brucei rhodesiense)].

ⁱPlasmodium falciparum (K1, resistant to chloroquine). Average of duplicate determinations.

^jSelectivity index for P. falciparum (SI_P), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (P. falciparum)].

^kLeishmania donovani (MHOM/SD/62/1S-CL2_D) axenic amastigotes. Average of duplicate determinations.

^lSelectivity index for L. donovani (SI_L), expressed as the ratio [IC₅₀ (L6)/IC₅₀ (L. donovani)].

^mND, not determined.

Placement of cationic groups on aromatic rings affected the antitrypanosomal properties of triazoles 1–60. Among the dications lacking methoxy and hydroxy substituents, 4,4′-disubstituted analogues 46–48 were more active against T. brucei rhodesiense compared to isomers possessing cationic moieties in the 5- or 5′-positions. Alternatively, 4,4′-disubstituted diamidines 49, 52, and 58 bearing methoxy or hydroxy groups exhibited lower antitrypanosomal activities than the corresponding 4,5′- and 4′,5-disubstituted analogues 19, 22, and 28 and 34, 37, and 43, respectively. In the case of methoxy and hydroxy substituted dications, all 5,5′-disubstituted congeners, except 2′-methoxy diimidazoline 9 and 2-hydroxy bis(N-isopropyl)amidine 11, were less active against T. brucei rhodesiense than their counterparts possessing at least one of the cationic moieties in the 4- or 4′-positions. Thus, among the methoxy and hydroxy substituted analogues, 4,5′-disubtituted diamidines as well as 4,4′-substituted bis(N-isopropyl)amidines and diimidazolines were the most potent against T. brucei rhodesiense.

The effect of the methoxy and hydroxy substitution on antitrypanosomal activities of dications 1–60 varied based on the type and the position of cationic moieties and the nature of aromatic substituents. For example, in the series of the 5,5′-disubstituted analogues 1–15 (Table 1), the attachment of the methoxy group to the 2-position of diamidine 1 and diimidazoline 3 improved activities of the resulting 2-methoxy analogues 4 and 6 against T. brucei rhodesiense with respect to the parent compounds. At the same time, 2-hydroxy substituted congeners 10 and 12 displayed antitrypanosomal potencies 600- and 7-fold lower relative to dications 1 and 3. 2-Methoxy substituted diamidine 4 and diimidazoline 6 were 1000- and 13-fold as active against T. brucei rhodesiense as their 2-O-demethylated analogues 10 and 12, while both bis(N-isopropyl)amidines 5 and isomer 11, bearing methoxy and hydroxy substituents in the 2-position, exhibited antitrypanosomal potencies comparable to that of congener 2. Similarly, activities of 2′-substituted methoxy and hydroxy diamidines 7 and 13 against T. brucei rhodesiense decreased 20- and 2-fold relative to the unsubstituted congener 1, while 2′-methoxy and 2′-hydroxy bis(N-isopropyl)amidines 8 and 14 were more potent than analogue 2. In the case of diimidazolines 9 and 15, the attachment of the 2′-hydroxy group only marginally improved antitrypanosomal activity of congener 15, while 2′-methoxy substituted diimidazoline 9 was nearly 2350-times as active against the pathogen as unsubstituted analogue 3.

In the series of the 4,5′-disubstituted congeners 16–30 (Table 2), diamidines 19, 22, and 28 possessing 2- and 2′-methoxy as well as 2′-hydroxy groups displayed antitrypanosomal potencies comparable to that of the unsubstituted dication 16. At the same time, the introduction of the 2-hydroxy substituent reduced the activity of the resulting congener 25 against the pathogen nearly 22-fold relative to diamidine 16. 2-Substituted bis(N-isopropyl)amidines and diimidazolines 20, 21, 26, and 27 were less active versus T. brucei rhodesiense than dications 17 and 18, while the attachment of the methoxy or hydroxy groups to the 2′-position increased antitrypanosomal activities of congeners 23, 24, 29, and 30.

Antitrypanosomal properties of the 4′,5-disubstituted dications 31–45 (Table 3) varied depending on the nature of cationic moieties and aromatic substituents. For example, while the placement of the methoxy and hydroxy groups in the 2-position reduced activities of diamidines 34 and 40 against T. brucei rhodesiense relative to congener 31, 2′-substituted diamidines 37 and 43 displayed potencies comparable to that of the unsubstituted analogue 31. Bis(N-isopropyl)amidines and diimidazolines, however, demonstrated different trends. For instance, antitrypanosomal activities of 2-methoxy substituted bis(N-isopropyl)amidine 35 and diimidazoline 36 improved compared to congeners 32 and 33, while the placement of the hydroxy group in the same position reduced the activity of bis(N-isopropyl)amidine 41 and diimidazoline 42 against the pathogen 33- and 43-fold, respectively. On the other hand, antitrypanosomal activities of 2′-methoxy substituted bis(N-isopropyl)amidine 38 and diimidazoline 39 decreased relative to dications 32 and 33. At the same time, 2′-hydroxy substituted congener 44 was more potent against T. brucei rhodesiense than bis(N-isopropyl)amidine 32, while dication 45 was less active than diimidazoline 33.

Except for 2′-hydroxy substituted bis(N-isopropyl)amidine 59 and diimidazoline 60, the introduction of the methoxy and hydroxy groups on aromatic rings did not improve activities of 4,4′-disubstituted congeners 49–60 against T. brucei rhodesiense relative to analogues 46–48 (Table 4). For example, placement of the hydroxy group in the 2-position reduced the antitrypanosomal activity of diamidine 55 125-fold compared to dication 46. Bis(N-isopropyl)amidine 59 and diimidazoline 60 were more potent than the corresponding unsubstituted analogues 47, 48 and dications 53, 54, possessing the methoxy group in the 2′-position.

Dications 1–60 displayed in vitro antitrypanosomal selectivity indices SI_T, varied from 2 to 36500. All diamidines except congeners 7, 10, and 58 possessed higher selectivity indices than bis(N-isopropyl)amidines and diimidazolines, which correlate with our earlier findings for 2-phenyl benzofurans.⁵¹ Six dications (9, 19, 22, 28, 37, and 43) exhibited antitrypanosomal selectivity indices greater than that of pentamidine (SI_T = 15533), while 4,5′-disubstituted diamidine 28 had the highest selectivity index for T. brucei rhodesiense in the series (SI_T = 36500).

In Vitro Antiplasmodial Activity

All tested dications 1–60 exhibited in vitro antiplasmodial activities with IC₅₀ values ranging from 0.6 nM to 8.93 μM. Thirty-nine dications, including sixteen diamidines (1, 4, 7, 13, 16, 19, 22, 28, 31, 34, 37 43, 46, 49, 52, and 58), eleven bis(N-isopropyl)amidines (17, 20, 23, 29, 32, 38, 44, 47, 50, 53, and 59), and twelve diimidazolines (6, 9, 15, 24, 30, 36, 45, 48, 51, 54, 57, and 60), were more potent in vitro against P. falciparum than pentamidine (IC₅₀ = 58 nM). Eight analogues (16, 31, 46–48, 52, 58, and 60) displayed better antiplasmodial activities in vitro than artemisinin (IC₅₀ = 6 nM). 4,4′-Disubstituted diimidazoline 60 showing antiplasmodial IC₅₀ value of 0.6 nM was the most potent compounds in the series against P. falciparum.

In this study, the influence of the N-alkylation of cationic moieties on antiplasmodial properties of tested dications 1–60 was not conclusive. Thus, in the series of congeners lacking methoxy and hydroxy substituents on aromatic rings, diamidines were generally more active against P. falciparum than bis(N-isopropyl)amidines and diimidazolines. At the same time, among the methoxy and hydroxy substituted analogues, we found no apparent correlation between the nature of cationic moieties and antiplasmodial activities.

Placement of cationic moieties in the 4- or 4′-positions increased in vitro antiplasmodial activities of tested congeners. For example, all 4,5′-disubstituted congeners 16–30, except for 2-methoxy substituted diimidazoline 21 and 2-hydroxy substituted bis(N-isopropyl)amidine 26, displayed higher antiplasmodial activities compared to isomers 1–15 bearing cationic moieties in the 5,5′-positions. Similarly, congeners 31–45 were more active against P. falciparum than 5,5′-disubstituted isomers 1–15 except for bis(N-isopropyl)amidines 35 and 41, possessing the methoxy and hydroxy groups in the 2-position, and 2′-methoxy substituted diimidazoline 39 and 2-hydroxy substituted isomer 42. 4,4′-Disubstituted dications 46–60 were more active against P. falciparum than dications 1–45 bearing at least one cationic moiety in the 5- or 5′-positions, except for 2′-methoxy substituted diimidazoline 54 and 2-hydroxy substituted bis(N-isopropyl)amidine 56 that were less potent than the corresponding 5,5′-disubstituted analogue 11 and 4,5′-disubstituted isomer 24, respectively. All 4,4′-disubstituted congeners, excluding analogues 55–57 possessing the hydroxy group in the 2-position, exhibited antiplasmodial IC₅₀ values below 20 nM.

The effect of the methoxy and hydroxy substitution on antiplasmodial activities of dications 1–60 varied depending on the nature and the position of cationic moieties. For example, in the series of 5,5′-disubstituted analogues (Table 1), 2- and 2′-methoxy substituted diamidines 4 and 7 as well as the corresponding bis(N-isopropyl)amidines 5 and 8 exhibited in vitro activities versus P. falciparum comparable to those of unsubstituted dications 1 and 2. At the same time, activities of 2′-hydroxy substituted diamidine 13 and bis(N-isopropyl)amidine 14 against the parasite decreased slightly compared to analogues 1 and 2, while placement of the hydroxy group in the 2-position significantly affected antiplasmodial activities of dications 10–12. Thus, 2-hydroxy substituted diamidine 10 was 425- and 320-fold less potent against P. falciparum compared to unsubstituted dication 1 and its 2-methoxy substituted analogue 4. Similarly, bis(N-isopropyl)amidine 11 exhibited 7- and 6-fold lower antiplasmodial activity relative to congeners 2 and 5. On the other hand, 2- and 2′-methoxy substituted diimidazolines 6 and 9 and 2′-hydroxy substituted analogue 15 were 12-, 26-, and 7-fold more active against P. falciparum than unsubstituted diimidazoline 3, while dication 12 possessing hydroxy group in the 2-position displayed antiplasmodial activity comparable to that of 3.

Among the 4,5′-disubstituted analogues 16–30 (Table 2), methoxy and hydroxy substituted diamidines 19, 22, 25, and 28 were less active in vitro against P. falciparum than unsubstituted analogue 16. In the case of bis(N-isopropyl)amidines, placement of the methoxy or hydroxy group in the 2-position reduced antiplasmodial activities of dications 20 and 26 compared to unsubstituted isomer 17, while 2′-methoxy- and hydroxy substituted congeners 23 and 29 exhibited higher activities against the parasite. At the same time, all 2- and 2′-substituted diimidazolines except for congener 27, possessing the hydroxy group in the 2-position, were more active against P. falciparum than unsubstituted dication 18.

In the series of 4′,5-disubstituted congeners 31–45 (Table 3), the effect of the methoxy or hydroxy substitution on aromatic rings on antiplasmodial potencies depended on the type of the cationic moieties and the position of aromatic substituents. For example, 2′-methoxy and 2′-hydroxy substituted diamidines 37 and 43 were only marginally less active against P. falciparum compared to the unsubstituted analogue 31, while the antiplasmodial activity of dication 34, possessing the methoxy group in the 2-position, decreased 4-fold. At the same time, 2-hydroxy substituted diamidine 40 was 308-fold less active versus the parasite than congener 31. Also, bis(N-isopropyl)amidines 38 and 44, possessing the methoxy and hydroxy groups in the 2′-position, displayed antiplasmodial activities comparable to that of dication 32. On the other hand, 2-methoxy substituted analogue 35 and 2-hydroxy substituted isomer 41 were 5- and 123-fold less active against P. falciparum than unsubstituted bis(N-isopropyl)amidine 32. All methoxy and hydroxy substituted diimidazolines except congener 42, possessing the hydroxy group in the 2-position, displayed improved antiplasmodial activities compared to the unsubstituted diimidazoline 33.

Similarly, the antiplasmodial properties of analogues 46–60 (Table 4) also depended on the placement of aromatic substituents. Thus, regardless of the nature of the cationic moieties, the attachment of methoxy or hydroxy groups in the 2-position reduced antiplasmodial activities of congeners 49–51 and 55–57, respectively, compared to the unsubstituted analogues 46–48. At the same time, 2′-methoxy and 2′-hydroxy substituted diamidines 52 and 58 and bis(N-isopropyl)amidines 53 and 59 displayed comparable activities against P. falciparum relative to unsubstituted congeners 46 and 47. The antiplasmodial potency of diimidazoline 54, possessing the methoxy group in the 2′-position, decreased relative to that of unsubstituted congener 48, while 2′-hydroxy substituted analogue 60 was 3 times as active against the parasite as diimidazoline 48 and 10 times as potent as artemisinin. Diimidazoline 60 (IC₅₀ = 0.6 nM) was the most active compound among all tested congeners 1–60 against P. falciparum. Compound 60 was tested in vivo in the P. berghei mouse model. When administered subcutaneously at 30 mg/kg daily for four days, diimidazoline 60 did not reduce the blood parasite count (data not shown). This result, although discouraging, came at no surprise, because aromatic diamidines do not usually display good activity in this model. For example, orally active pafuramidine dosed at 100 mg twice a day for 5 days has demonstrated 90% efficacy in uncomplicated P. falciparum malaria and 80% in P. vivax malaria in humans,⁹⁰ even though furamidine was inactive against trophozoite-induced P. berghei infection in mice.^{20, 85}

Compounds 1–60 displayed in vitro antiplasmodial selectivity indices SI_P, varied from 23 to >316667. Among dications with identical substitution pattern on aromatic rings (unsubstituted, methoxy- or hydroxy substituted), the selectivity indices of 5,5′- and 4′,5-disubstituted congeners 1–15 and 31–45 for P. falciparum correlated with their activities against the parasite. Thus, congeners 1, 4, 9, and 13, showing superior activities among dications possessing the same aromatic substituents, also displayed maximum selectivity indices for P. falciparum. The selectivity indices of congeners 16–30 varied depending on the nature of cationic moieties and the substitution on the aromatic rings. For example, in the case of unsubstituted or methoxy substituted dications 16–24, diamidines 16, 19, and 22 demonstrated maximum antiplasmodial selectivity indices, while among the hydroxy substituted analogues 25–30, selectivity indices of diimidazolines 27 and 30 were the highest. Antiplasmodial selectivity indices of congeners 22–24 and 28–30 bearing methoxy or hydroxy groups in the 2′-position increased compared to both dications 16–18 lacking aromatic substituents and to the corresponding 5,5′-disubstituted analogues 7–9 and 13–15. Except for 2-hydroxy substituted congeners 10–12 and 40–42 and bis(N-isopropyl)amidines 26 and 56 also possessing the hydroxy group in the 2-position, dications 1–60 exhibited superior antiplasmodial selectivity indices for P. falciparum compared to pentamidine. 4,4′-Disubstituted diimidazoline 60 exhibited the highest antiplasmodial selectivity index in the series (SI_P = >316667), which was nearly 400 times greater than that of pentamidine.

In Vitro Antileishmanial Activity

Among the 5,5′-disubstituted triazoles 1–15 (Table 1), only diamidine 1 and its 2′-hydroxy substituted isomer 13 as well as diimidazoline 9, possessing the methoxy group in the 2′-position, were active against L. donovani. The substitution on the amidine moieties had a distinct effect on the antileishmanial properties of 4,5′-disubstituted congeners 16–30 (Table 2). Only diamidine 16 and the corresponding 2- and 2′-substituted analogues 19 and 22, as well as dications 28 and 30 bearing the 2′-hydroxy group were active against L. donovani. Attachment of the 2′-methoxy substituent improved the antileishmanial potency of the diamidine 22 compared to congener 16. Antileishmanial potency of congeners 31–45 (Table 3) diminished with the substitution on the amidine moieties. Except for diamidines 31, 34, 37, and 43 and bis(N-isopropyl)amidines 32 and 38, the 4′,5-substituted congeners were inactive against L. donovani. Antileishmanial activity of the dications 31–45 improved compared to their 5,5′-substituted counterparts 1–15. 2′-Hydroxy substituted diamidine 43 was more active than both the unsubstituted dication 31 and its 2′-methoxy analogue 37. The substitution on amidine groups or on aromatic rings in most cases reduced the antileishmanial potency of congeners 46–60 (Table 4). Except for 2′-hydroxy substituted diamidine 58 and diimidazoline 60, all 4,4′- disubstituted congeners 49–60 bearing methoxy or hydroxy groups on aromatic rings were less active against L. donovani than unsubstituted dications 46–48. Similar to 4′,5-disubstituted isomers, 2′-hydroxy substituted diamidine 58 was the most active compound among dications 46–60, demonstrating improved antileishmanial potency compared to both unsubstituted dication 46 and 2′-methoxy substituted congener 52.

In Vivo Antitrypanosomal Activity

Selected 1,4-diphenyl-1H-1,2,3-triazoles exhibiting promising in vitro activities against T. brucei rhodesiense were evaluated in the STIB900 mouse model of African trypanosomiasis (Table 5). The screening was conducted using intraperitoneal dosing at 5 mg/kg daily for 4 days. Diamidines 22, 28, 37, 43, 46, 52, and 58, bis(N-isopropyl)amidine 59, and diimidazoline 9 displayed excellent in vivo efficacies curing at least two out of four animals.

With the exception of 2′-hydroxy substituted bis(N-isopropyl)amidine 59, N-substitution on the cationic fragments reduced antitrypanosomal efficacies of diphenyl triazoles. For example, all tested diimidazolines except 5,5′-disubstituted dication 9, possessing the methoxy group in the 2′-position, displayed no curative activity at this dosage. Besides, 4,5′-disubstituted bis(N-isopropyl)amidine 23 and 4,4′-disubstituted analogue 47 were found to be toxic in the in vivo model despite showing no in vitro toxicity. However, this lack of correlation between the in vitro and the in vivo data is not surprising and quite common. The purpose of the in vitro cytotoxicity assay is to identify and discharge compounds exhibiting antiprotozoal activity due to general cellular toxicity.⁸⁴ Because only toxic effects at a single cellular level can be observed in vitro, it is impossible to detect the organ-specific toxicity of the compound in question until in vivo animal tests are conducted.

The position of attachment of cationic moieties influenced the efficacy of tested dications in the acute mouse model of African trypanosomiasis (compare 1, 16, 31, and 46). For example, 5,5′-disubstituted congener 1 displayed no activity, while diamidines 16 and 31 bearing one cationic group in the 4- or 4′-positions revealed improved antitrypanosomal efficacies, curing one out of four mice. At the same time, 4,4′-disubstituted diamidine 46 provided cures to all infected animals.

The placement of methoxy and hydroxy groups affected antitrypanosomal efficacies of tested analogues depending on the placement of cationic moieties. For example, antitrypanosomal potency of 4,5′- and 4′,5-substituted diamidines 19 and 34 bearing the methoxy group in the 2-position did not improve compared to their unsubstituted counterparts 16 and 31. On the other hand, 2′-methoxy substituted congeners 22 and 37 and 2′-hydroxy substituted analogues 28 and 43 were more active than diamidines 16 and 31, lacking aromatic substitution. At the same time, the introduction of the aromatic substituents reduced antitrypanosomal efficacy of 4,4′-disubstituted congeners with respect to diamidine 46.

Dications 9, 37, and 46, which cured all mice at the 5 mg/kg dosing, were subjected to a dose-response evaluation. In a single-dose regimen at 10 mg/kg, diimidazoline 9 and diamidine 46 cured three out of four animals. When administered at 1 mg/kg daily for 4 days, dications 9 and 37 provided no cures, while congener 46 retained its in vivo efficacy curing all mice. Further reduction of the administered dosage of diamidine 46 to 0.5 mg/kg daily for 4 days afforded no cures but substantially postponed relapse time for infected animals compared to untreated controls. Overall, dication 46 exhibited excellent potency in the STIB900 acute mouse model of African trypanosomiasis. To our knowledge, this is the first aromatic diamidine showing in vivo efficacy superior to that of melarsoprol.

Summary

A series of cationic 1,4-diphenyl-1H-1,2,3-triazoles 1–60 was synthesized by the Pinner method from the corresponding dinitriles, prepared via the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). Dications 1–60 were tested in vitro against T. brucei rhodesiense, P. falciparum, and L. donovani as well as for cytotoxicity against mammalian cells. The cytotoxicities of triazoles 1–60 were lower compared to that of pentamidine and were not significantly affected by the alkylation on the amidine groups or the substitution on the aromatic rings. However, the placement of the cationic moiety in the 4-position increased the cytotoxicities of diamidines 16 and 46 with respect to the drug. Except for the congeners 7 and 10, unsubstituted diamidines exhibited higher in vitro activities against T. brucei rhodesiensethan bis(N-isopropyl)amidines and diimidazolines, corroborating previously published results.^{50, 85-87, 89} The majority of diamidines also exhibited higher antiplasmodial and antileishmanial activities compared to N-substituted analogues. The position of attachment of cationic groups influenced antiprotozoal properties of triazoles 1–60. For example, placement of the cationic fragments in the 5,5′-position of the aromatic rings afforded congeners that were in most cases less potent against T. brucei rhodesiense, P. falciparum and L. donovani than isomers bearing at least one cationic moiety in the 4- or 4′-positions. The introduction of the hydroxy group in the 2-position of the phenyl ring significantly reduced activities of tested congeners against T. brucei rhodesiense, P. falciparum and L. donovani, perhaps due to the formation of the intramolecular hydrogen bond between the hydrogen atom of the 2-hydroxy group and the 2-nitrogen of the triazole ring. Diamidines 28, 37, 46, and 58, bis(N-isopropyl)amidine 59, and diimidazoline 9 exhibited excellent in vivo efficacies in the acute mouse model of trypanosomiasis curing all infected animals when administered intraperitoneally at 5 mg/kg daily for 4 days. Even at a lower dose of 1 mg/kg diamidine 46 retained its curative potency exhibiting in vivo efficacy superior to that of melarsoprol. Promising antiprotozoal activity of cationic 1,4-diphenyl-1H-1,2,3-triazoles 1–60 in vitro and excellent efficacy of select congeners in the acute mouse model of African trypanosomiasis warrant further investigation of this class of compounds as potential antitrypanosomal drug candidates.

Experimental Section

Biology

Preparation of Compounds

Compounds were dissolved in 100% dimethylsulfoxide (DMSO) and finally diluted in culture medium prior to the assay. The DMSO concentration never exceeded 1% in the in vitro assays. For in vivo experiments, the compounds were dissolved in DMSO and further diluted with distilled H₂O to a final DMSO concentration of 10% prior to injection into the animals.

In Vitro Cytotoxicity Assay (L6 Rat Myoblast Cells)

IC₅₀ values were determined using the Alamar blue assay⁹¹ and were carried out twice independently and in duplicate. Briefly, 4000 L6 cells were seeded in RPMI 1640 medium supplemented with L-glutamine 2 mM, HEPES 5.95 g/L, NaHCO₃ 2 g/L, and 10% fetal bovine serum in 96-well microtiter plates. The serial drug dilutions were incubated for 70 h at 37 °C under a humidified 5% CO₂ atmosphere. The viability marker Alamar blue (12.5 mg resazurin (Sigma) dissolved in 100 ml phosphate buffered saline) (10 μL) was then added to each well and the plate was incubated for additional 2–3 h. The plates were read in a Spectramax Gemini XS microplate fluorescence scanner (Molecular Devices) using an excitation wavelength 536 nm and an emission wavelength 588 nm. The IC₅₀ values were calculated from the sigmoidal inhibition curves using the SoftmaxPro software.

In Vitro Growth Inhibition Assay of T. brucei rhodesiense (STIB900)

IC₅₀ values were determined using the Alamar blue assay and were carried out twice independently and in duplicate. Briefly, the compounds were tested in Minimum Essential Medium with Earle’s salts, supplemented as previously described⁹² with the following modifications: 2-mercaptoethanol 0.2 mM, sodium pyruvate 1 mM, hypoxanthine 0.5 mM, and 15% heat-inactivated horse serum. Serial drug dilutions were prepared in 96-well microtiter plates and each well inoculated with 2000 bloodstream forms and incubated for 70 h at 37 °C under a humidified 5% CO₂ atmosphere. The viability marker Alamar blue (12.5 mg resazurin (Sigma) dissolved in 100 ml phosphate buffered saline) (10 μL) was then added to each well and the plate was incubated for additional 2–6 h. The plates were read in a Spectramax Gemini XS microplate fluorescence scanner (Molecular Devices) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC₅₀ values were calculated from the sigmoidal inhibition curves using the SoftmaxPro software.

In Vitro Growth Inhibition Assay of P. falciparum(K1)

The determination of IC₅₀ values against erythrocytic stages of P. falciparumwas carried out twice independently and in duplicate using the [³H]-hypoxanthine incorporation assay.^{93, 94} Briefly, the compounds were tested in RPMI 1640 medium 10.44 g/L, supplemented with Hepes 5.94 g/L, Albumax II 5 g/L, sodium bicarbonate 2.1 g/L, and neomycin 100 mg/L in 96-well microtiter plates. Infected human red blood cells in medium (hematocrit 1.25%, parasitemia 0.3%) were incubated with the drug dilutions in an atmosphere of 93% N₂, 4% CO₂, 3% O₂ at 37 °C. After 48 h, [³H]-hypoxanthine (0.5 μCi/well) was added and the plates were incubated for additional 24 h under the same conditions. The wells were harvested with a Betaplate cell harvester and transferred on a glass fiber filter. Viability was assessed by measuring the incorporation of [³H]-hypoxanthine by a Betaplate liquid scintillation counter (Wallac, Zurich, Switzerland). The IC₅₀ values were calculated from the sigmoidal inhibition curves using MS Excel.

Antileishmanial Assay

Axenic amastigotes of L. donovani (WHO designation MHOM/SD/62/1S-CL2_D) were adapted from promastigotes and grown in the amastigote medium described previously⁹⁵ at 37 ^oC. In a final volume of 60 μL, 6 × 10⁴ parasites were added to each well of a 96-well plate except for negative control wells. Standard and test compounds were added as appropriate using 2-fold dilutions to allow a range of concentrations to be tested. Plates were then incubated at 37 ^°C for 72 h in a humidified environment containing 5% CO₂. The tetrazolium dye-based CellTiter reagent (Promega, Madison, WI) was used to assess parasite growth.⁹⁶ Several hours after adding 12 μL of the CellTiter reagent to each well of the plate, absorbance readings were taken at 490 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA). SoftMax Pro software (Amersham Biosciences, Piscataway, NJ) was used to calculate IC₅₀ values by employing the dose–response equation y = [(a – d)/(1 + (x/c)^b)] + d, where x = compound concentration, y = absorbance at 490 nm, a = upper asymptote, b = slope, c = IC₅₀ value, and d = lower asymptote.

STIB900 Acute Mouse Model of Trypanosomiasis

Experiments were performed as previously reported⁹⁷ with minor modifications. Briefly, female NMRI mice were infected intraperitoneally (ip) with 2 × 10⁴ STIB900 bloodstream forms. Experimental groups of four mice were treated ip with tested dications on 4 consecutive days from day 3 to day 6 postinfection. A control group was infected but remained untreated. After drug treatment, parasitamia of all animals was checked by tail blood examination on day 7, day 10, then twice a week until day 30 followed by once a week until 60 days postinfection. After detection of parasitamia, the day of parasitamia relapse was recorded to calculate the MRD and mice were euthanized. Surviving and aparasitemic mice at day 60 were considered cured and then euthanized.

Chemistry

General Experimental Information

All chemicals and solvents were purchased from Aldrich Chemical Co., Fisher Scientific, or Acros Organics and were used without further purification. The purity of all novel compounds was confirmed to exceed 95% by elemental analysis and HPLC. Uncorrected melting points were measured on a Thomas–Hoover capillary melting point apparatus. ¹H NMR spectra were recorded on a Varian Gemini 2000 spectrometer operating at 300 MHz. Chemical shifts are reported in ppm relative to tetramethylsilane. Anhydrous ethanol was distilled over Mg/I₂ immediately prior to use. Reaction mixtures were monitored by TLC using Whatman silica gel 250 μm UV₂₅₄ plates or by reversed phase HPLC. Organic layers of extraction mixtures were washed with saturated NaCl solution and dried over Na₂SO₄ or MgSO₄ before being evaporated under reduced pressure. Flash column chromatography was performed using Davisil grade 633, type 60A silica gel (200–425 mesh). Analytical HPLC chromatograms were recorded on an Agilent 1200 chromatograph using an Agilent Zorbax SB C8 column (4.6 mm × 75 mm, 3.5 μm) and UV photodiode array detection at 230, 254, 265, 290, and 320 nm. The column temperature was maintained at 40 °C. Mobile phases consisted of mixtures of methanol (0–95%) and water, both solvents containing formic acid (80 mM), ammonium formate (20 mM), and triethylamine (15 mM). Flow rates were maintained at 1.5 mL/min. In method A, the concentration of methanol was increased linearly from 0 to 28.5% over 6 min, from 28.5 to 71.25% over 4 min, from 71.25 to 95% over 0.5 min, and maintained at 95% for 2 min before re-equilibration. In method B, the concentration of methanol was increased linearly from 28.5 to 95% over 10 min and then maintained at 95% for 2 min before re-equilibration.

Preparative Reversed Phase HPLC

Preparative reversed phase HPLC was performed on a Varian ProStar Chromatography Workstation configured with two PS-215 pumps fitted with 50 mL pump heads, a Dynamax Microsorb C18 (60 Å) column (41.4 mm × 250 mm, 8 μm), PS-320 variable wavelength UV–vis detector, and a PS-701 fraction collector. Mobile phases consisted of mixtures of acetonitrile (0–75%) in water containing formic acid (40 mM) and ammonium formate (10 mM). Flow rates were maintained at 40 mL/min. Detector wavelengths and mobile phase gradients were optimized for the individual compounds. Select fractions were analyzed for purity as described above for analytical HPLC. Residues of evaporated pooled purified fractions were reconstituted in water and lyophilized on a VirTis BenchTop 6K lyophilizer. The lyophilized compounds were dissolved in ethanol and converted into HCl salts with aqueous HCl.

Flash Chromatography of Amidines on C₁₈ Reversed Phase Silica Gel

The chromatographic column was half-filled with acetonitrile and packed with a slurry of C₁₈ Silica Gel (70 g) in acetonitrile (70–100 mL). The excess acetonitrile was drained out, and the top of the column was covered with a 2 cm pad of sand. The column was equilibrated with 150 mL of initial mobile phase consisting of water containing formic acid (40 mM) and ammonium formate (10 mM). A concentrated reaction mixture was dissolved in the initial mobile phase. In case of low solubility, heating of the mixture and/or addition of a small amount of methanol as a cosolvent were performed. After the reaction mixture was applied to the column, the elution began with initial mobile phase (150 mL) to remove the excess amine and then with a mobile phase consisting of a mixture of acetonitrile (0–75%) in water containing formic acid (40 mM) and ammonium formate (10 mM). Acetonitrile concentrations varied for each individual compound and contained 50–70% of calculated amount of acetonitrile at the point of the retention time of the compound in analytical method A. After the purification was completed, the column was washed with acetonitrile (3 × 100 mL), ethanol (100 mL), deionized water (2 × 100 mL), and kept in acetonitrile or acetonitrile–water mixture. Select fractions were analyzed for purity as described above for analytical HPLC. Residues of evaporated pooled purified fractions were reconstituted in water and lyophilized on a VirTis BenchTop 6K lyophilizer. The lyophilized compounds were dissolved in ethanol and converted into HCl salts with aqueous HCl.

Low resolution ESI mass spectra were recorded on an Agilent Technologies 1100 series LC/MSD trap spectrometer. Elemental analyses were performed by Atlantic Microlab, Norcross, GA, and were within ±0.4% of calculated values.

General Procedure for Synthesis of Dications (1–60). 1,4-Bis(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (1)

A mixture of dry 1,4-dioxane (60 mL) and dry EtOH (15 mL) in a three-neck 250 mL flask equipped with a gas inlet tube, a thermometer, and a drying tube was saturated with gaseous HCl at 0 °C. 1,4-Bis(3-cyanophenyl)-1H-1,2,3-triazole (77) (1.51 g, 5.57 mmol) was added in one portion, the flask was sealed, and the mixture was stirred at ambient temperature until the starting material was no longer detectable by HPLC. The reaction mixture was diluted with dry ether (100 ml). A formed precipitate was filtered off under argon, washed with diethyl ether, and dried under high vacuum over KOH to give diimidate ester (2.27 g, 94%), which was split in three portions and reacted immediately with the appropriate amines.

To a suspension of the diimidate ester (0.75 g, 1.72 mmol) in dry EtOH (20 ml) was added saturated ethanolic ammonia (10 mL). The reaction mixture was sealed and stirred at ambient temperature. The progress of the reaction was monitored by HPLC. After four days, the mixture was diluted with dry diethyl ether and cooled in a freezer. A resulting precipitate was filtered off, washed with ether and dried under vacuum to give 0.49 g of crude material, which was purified by reverse phase column chromatography and recrystallized from 1.5 N HCl to give 1, as a white solid (0.21 g, 32%); mp > 350 °C (dec). ¹H NMR (DMSO-d₆) δ 9.82 (s, 1H), 9.74 (br s, 2H), 9.60 (br s, 2H), 9.47 (br s, 2H), 9.36 (br s, 2H), 8.58 (s, 1H), 8.50 (s, 1H), 8.34 (d, J = 7.7 Hz, 1H), 8.26 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.91 (dd, J = 7.7 and 7.7 Hz, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.78 (dd, J = 7.7 and 7.7 Hz, 1H). HPLC (method A) t_R 5.12 min (100 area %). M/Z 306.8 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇·2HCl·1.3H₂O) C, H, N, Cl.

1,4-Bis(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (2)

White solid (0.36 g, 45%); mp 265–267 °C (dec). ¹H NMR (DMSO-d₆) δ 9.90 (s, 1H), 9.86 (br s, 6H), 8.48 (s, 1H), 8.37 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.25 (m, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.88 (dd, J = 7.7 and 7.7 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.75 (dd, J = 7.7 and 7.7 Hz, 1H), 4.18 (m, 2H), 1.33 (d, J = 6.7 Hz, 6H), 1.31 (d, J = 6.7 Hz, 6H). HPLC (method A) t_R 7.41 min (100 area %). M/Z 390.8 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇·2HCl·1.5H₂O) C, H, N, Cl.

1,4-Bis[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (3)

White solid (0.35 g, 45%); mp 353–355 °C (dec). ¹H NMR (DMSO-d₆) δ 11.1 (br s, 4H), 9.82 (s, 1H), 8.94 (s, 1H), 8.81 (s, 1H), 8.34 (d, J = 8.2 Hz, 1H), 8.25 (d, J = 7.7 Hz, 1H), 8.24 (d, J = 7.7 Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.94 (dd, J = 8.2 and 8.2 Hz, 1H), 7.81 (dd, J = 7.7 and 7.7 Hz, 1H), 4.06 (s, 8H). HPLC (method A) t_R 6.79 min (100 area %). M/Z 358.8 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇·2HCl·1.5H₂O) C, H, N, Cl.

1-(5-Amidino-2-methoxyphenyl)-4-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (4)

White solid (0.63 g, 60%); mp > 243 °C (dec). ¹H NMR (DMSO-d₆) δ 9.60 (br s, 2H), 9.51 (br s, 2H), 9.30 (br s, 2H), 9.24 (s, 1H), 9.23 (br s, 2H), 8.50 (s, 1H), 8.32 (d, J = 1.8 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H), 8.14 (dd, J = 8.8 and 1.8 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.76 (dd, J = 7.7 and 7.7 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 4.03 (s, 3H). HPLC (method A) t_R 5.97 min (100 area %). M/Z 336.2 (MH⁺ of free base). Anal. (C₁₇H₁₇N₇O·2HCl·3H₂O) C, H, N, Cl.

1-(5-N-Isopropylamidino-2-methoxyphenyl)-4-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (5)

White solid (0.61 g, 48%); mp > 248 °C (dec). ¹H NMR (DMSO-d₆) δ 9.83 (d, J = 6.0 Hz, 1H), 9.75 (d, J = 6.8 Hz, 1H), 9.67 (br s, 1H), 9.62 (br s, 1H), 9.31 (br s, 1H), 9.29 (s, 1H), 9.22 (br s, 1H), 8.39 (s, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 2.2 Hz, 1H), 8.02 (dd, J = 8.8 and 2.2 Hz, 1H), 7.76 (m, 2H), 7.59 (d, J = 8.8 Hz, 1H), 4.13 (m, 2H), 4.01 (s, 3H), 1.30 (s, 12H). HPLC (method A) t_R 7.84 min (100 area %). M/Z 420.6 (MH⁺ of free base). Anal. (C₂₃H₂₉N₇O·2.4HCl·2.6H₂O) C, H, N, Cl.

1-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-4-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (6)

White solid (0.78 g, 65%); mp > 250 °C (dec). ¹H NMR (DMSO-d₆) δ 11.15 (br s, 2H), 10.93 (br s, 2H), 9.34 (s, 1H), 8.96 (s, 1H), 8.57 (s, 1H), 8.40 (d, J = 8.2 Hz, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.79 (dd, J = 8.0 and 7.7 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 4.06 (s, 4H), 4.04 (s, 4H), 4.01 (s, 3H). HPLC (method A) t_R 7.43 min (100 area %). M/Z 388.3 (MH⁺ of free base). Anal. (C₂₁H₂₁N₇O·2.5HCl·2.5H₂O) C, H, N, Cl.

4-(5-Amidino-2-methoxyphenyl)-1-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (7)

White solid (0.58 g, 70%); mp > 246 °C (dec). ¹H NMR (DMSO-d₆) δ 9.82 (br s, 2H), 9.48 (br s, 4H), 9.46 (s, 1H), 9.17 (br s, 2H), 8.72 (d, J = 2.3 Hz, 1H), 8.62 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.93 (dd, J = 8.8 and 2.3 Hz, 1H), 7.88 (dd, J = 8.0 and 8.0 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 4.14 (s, 3H). HPLC (method A) t_R 6.44 min (100 area %). M/Z 336.5 (MH⁺ of free base). Anal. (C₁₇H₁₇N₇O·2HCl·2.3H₂O) C, H, N, Cl.

4-(5-N-Isopropylamidino-2-methoxyphenyl)-1-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (8)

White solid (0.66 g, 66%); mp > 275 °C (dec). ¹H NMR (DMSO-d₆) δ 10.14 (br s, 1H), 9.93 (br s, 1H), 9.63 (d, J = 6 Hz, 1H), 9.54 (s, 1H), 9.50 (br s, 1H), 9.42 (br s, 1H), 9.13 (br s, 1H), 8.59 (d, J = 2.3 Hz, 1H), 8.52 (s, 1H), 8.35 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.86 (dd, J = 7.8 and 7.8 Hz, 1H), 7.82 (dd, J = 8.8 and 2.3 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 4.14 (m, 2H), 4.13 (s, 3H), 1.33 (d, J = 6.5 Hz, 6H), 1.31 (d, J = 6.5 Hz, 6H). HPLC (method A) t_R 8.24 min (100 area %). M/Z 420.6 (MH⁺ of free base). Anal. (C₂₃H₂₉N₇O·2HCl·1.1H₂O) C, H, N, Cl.

4-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-1-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (9)

White solid (0.64 g, 69%); mp > 235 °C (dec). ¹H NMR (DMSO-d₆) δ 11.38 (br s, 2H), 10.74 (br s, 2H), 9.53 (s, 1H), 9.08 (s, 1H), 8.87 (d, J = 2.3 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.16 (dd, J = 8.8 and 2.3 Hz, 1H), 7.92 (dd, J = 8.0 and 8.0 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 4.18 (s, 3H), 4.06 (s, 4H), 4.01 (s, 4H). HPLC (method A) t_R 7.83 min (100 area %). M/Z 388.8 (MH⁺ of free base). Anal. (C₂₁H₂₁N₇O·2HCl·2.9H₂O) C, H, N, Cl.

1-(5-Amidino-2-hydroxyphenyl)-4-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (10)

Brown solid (0.30 g, 41%); mp 260 °C (dec). ¹H NMR (DMSO-d₆) δ 12.29 (s, 1H), 9.57 (s, 2H), 9.39 (s, 2H), 9.29 (s, 2H), 9.22 (s, 1H), 9.12 (s, 2H), 8.48 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.28 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.75 (dd, J = 7.7 and 7.7 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H). HPLC (method A) t_R 5.09 min (100 area %). M/Z 322.2 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2HCl·2H₂O) C, H, N, Cl.

1-(5-N-Isopropylamidino-2-hydroxyphenyl)-4-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (11)

Brown solid (0.26 g, 29%); mp > 245 °C (dec). ¹H NMR (DMSO-d₆) δ 12.24 (s, 1H), 9.79 (d, J = 7.7 Hz, 1H), 9.62 (br s, 1H), 9.60 (d, J = 7.0 Hz, 1H), 9.48 (br s, 1H), 9.28 (br s, 1H), 9.25 (s, 1H), 9.10 (br s, 1H), 8.36 (s, 1H), 8.30 (d, J = 1.8 Hz, 1H), 8.12 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.74 (br s, 2H), 7.52 (d, J = 8.8 Hz, 1H), 4.11 (m, 2H), 1.31 (d, J = 6.4 Hz, 6H), 1.28 (d, J = 6.4 Hz, 6H). HPLC (method A) t_R 7.57 min (100 area %). M/Z 406.2 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2.3HCl·2.8H₂O) C, H, N, Cl.

1-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-4-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (12)

Brown solid (0.41 g, 49%); mp 247–250 °C (dec). ¹H NMR (DMSO-d₆) δ 12.26 (s, 1H), 11.07 (s, 2H), 10.76 (s, 2H), 9.26 (s, 1H), 8.85 (s, 1H), 8.49 (s, 1H), 8.36 (d, J = 8.2 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.78 (dd, J = 7.7 and 7.7 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 4.04 (s, 4H), 3.98 (s, 4H). HPLC (method A) t_R 7.00 min (100 area %). M/Z 374.2 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2.5HCl·3H₂O) C, H, N, Cl.

4-(5-Amidino-2-hydroxyphenyl)-1-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (13)

Purple solid (0.23 g, 41%); mp > 278 °C (dec). ¹H NMR (DMSO-d₆) δ 11.87 (s, 1H), 9.73 (br s, 2H), 9.45 (br s, 2H), 9.35 (br s, 2H), 9.27 (s, 1H), 9.05 (br s, 2H), 8.68 (d, J = 2.4 Hz, 1H), 8.51 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 8.7 and 2.4 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H). HPLC (method A) t_R 5.64 min (100 area %). M/Z 322.2 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2HCl·1.5H₂O) C, H, N, Cl.

4-(5-N-Isopropylamidino-2-hydroxyphenyl)-1-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (14)

Purple solid (0.26 g, 37%); mp > 245 °C (dec). ¹H NMR (DMSO-d₆) δ 11.80 (s, 1H), 9.93 (d, J = 8.8 Hz, 1H), 9.76 (br s, 1H), 9.51 (d, J = 8.9 Hz, 1H), 9.38 (br s, 2H), 9.27 (s, 1H), 9.00 (br s, 1H), 8.52 (d, J = 2.2 Hz, 1H), 8.40 (s, 1H), 8.35 (d, J = 8.7 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.85 (m, 1H), 7.63 (dd, J = 8.8 and 2.2Hz, 1H), 7.39 (d, J = 8.8Hz, 1H), 4.12 (m, 2H), 1.33 (d, J = 6.4 Hz, 6H), 1.31 (d, J = 6.4 Hz, 6H). HPLC (method A) t_R 7.97 min (100 area %). M/Z 406.2 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2.4HCl·2H₂O) C, H, N, Cl.

4-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-1-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (15)

White solid (0.19 g, 29%); mp > 307 °C (dec). ¹H NMR (DMSO-d₆) δ 11.33 (s, 1H), 10.64 (br s, 4H), 9.29 (s, 1H), 8.85 (s, 1H), 8.80 (d, J = 2.4 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.05 (dd, J = 8.7 and 2.4 Hz, 1H), 7.91 (dd, J = 8.0 and 8.0 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 4.06 (s, 4H), 3.99 (s, 4H). HPLC (method A) t_R 7.51 min (100 area %). M/Z 374.2 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2HCl·1.7H₂O·0.3EtOH) C, H, N, Cl.

1-(4-Amidinophenyl)-4-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (16)

White solid (0.33 g, 57%); mp 274–277 °C (dec). ¹H NMR (DMSO-d₆) δ 9.80 (s, 1H), 9.62 (br s, 4H), 9.39 (br s, 4H), 8.55 (s, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.25 (d, J = 8.8 Hz, 2H), 8.15 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.2 Hz, 1H), 7.81 (dd, J = 8.2 and 8.2 Hz, 1H). HPLC (method A) t_R 5.25 min (100 area %). Anal. (C₁₆H₁₅N₇·2HCl·1.2H₂O) C, H, N, Cl.

1-(4-N-Isopropylamidinophenyl)-4-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (17)

White solid (0.33 g, 45%); mp 335–336 °C (dec). ¹H NMR (DMSO-d₆) δ 9.84 (s, 1H), 9.70 (m, 6H), 8.40 (s, 1H), 8.28 (m, 1H), 8.24 (d, J = 8.8 Hz, 2H), 8.04 (d, J = 8.8 Hz, 2H), 7.76 (m, 2H), 4.14 (m, 2H), 1.32 (d, J = 6.6 Hz, 6H), 1.30 (d, J = 6.6 Hz, 6H). HPLC (method A) t_R 7.65 min (100 area %). Anal. (C₂₂H₂₇N₇·2HCl·0.6H₂O) C, H, N, Cl.

1-[4-(4,5-Dihydro-1H-imidazol-2-yl)phenyl]-4-(3-(4,5-dihydro-1H-imidazol-2-yl)phenyl)-1H-1,2,3-triazole Dihydrochloride (18)

Off-white solid (0.34 g, 51%); mp > 272 °C (dec). ¹H NMR (DMSO-d₆) δ 11.1 (br s, 4H), 9.82 (s, 1H), 8.90 (s, 1H), 8.38 (d, J = 8.2 Hz, 2H), 8.29 (d, J = 8.2 Hz, 1H), 8.26 (d, J = 8.2 Hz, 2H), 8.10 (d, J = 7.7 Hz, 1H), 7.82 (dd, J = 8.2 and 7.7 Hz, 1H), 4.05 (s, 8H). HPLC (method A) t_R 6.94 min (100 area %). Anal. (C₂₀H₁₉N₇·2.2HCl·1.6H₂O) C, H, N, Cl.

1-(4-Amidino-2-methoxyphenyl)-4-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (19)

White solid (0.55 g, 52%); mp 127–130 °C. ¹H NMR (DMSO-d₆) δ 9.73 (br s, 2H), 9.62 (br s, 2H), 9.44 (br s, 2H), 9.34 (br s, 2H), 9.30 (s, 1H), 8.53 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.88 (d, J = 1.9 Hz, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.76 (dd, J = 7.7 and 7.7 Hz, 1H), 7.66 (dd, J = 8.3 and 1.9 Hz, 1H), 4.06 (s, 3H). HPLC (method A) t_R 5.89 min (100 area %). M/Z 336.2 (MH⁺ of free base). Anal. (C₁₇H₁₇N₇O·2.2HCl·3H₂O) C, H, N, Cl.

1-(4-N-Isopropylamidino-2-methoxyphenyl)-4-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (20)

Off-white solid (0.60 g, 47%); mp 80–83 °C (dec). ¹H NMR (DMSO-d₆) δ 9.95 (d, J = 7.2 Hz, 1H), 9.81 (d, J = 7.0 Hz, 1H), 9.80 (br s, 1H), 9.64 (br s, 1H), 9.40 (br s, 1H), 9.30 (s, 1H), 9.29 (br s, 1H), 8.40 (s, 1H), 8.29 (m, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.78 (br s, 1H), 7.75 (m, 1H), 7.73 (dd, J = 7.7 and 7.7 Hz, 1H), 7.55 (dd, J = 8.3 and 1.6 Hz, 1H), 4.14 (m, 2H), 4.06 (s, 3H), 1.33 (d, J = 6.2 Hz, 6H), 1.31 (d, J = 6.2 Hz, 6H). HPLC (method A) t_R 7.96 min (100 area %). M/Z 420.2 (MH⁺ of free base). Anal. (C₂₃H₂₉N₇O·2.5HCl·3H₂O) C, H, N, Cl.

1-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-4-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (21)

Off-white solid (0.82 g, 69%); mp > 135 °C (dec). ¹H NMR (DMSO-d₆) δ 11.24 (s, 2H), 11.08 (s, 2H), 9.38 (s, 1H), 8.91 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.23 (d, J = 1.6 Hz, 1H), 8.10 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.88 (dd, J = 8.3 and 1.6 Hz, 1H), 7.79 (dd, J = 7.7 and 7.7 Hz, 1H), 4.08 (s, 3H), 4.06 (s, 4H), 4.05 (s, 4H). HPLC (method A) t_R 7.48 min (100 area %). M/Z 388.3 (MH⁺ of free base). Anal. (C₂₁H₂₁N₇O·2.2HCl·3H₂O) C, H, N, Cl.

4-(5-Amidino-2-methoxyphenyl)-1-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (22)

White solid (0.53 g, 65%); mp 270–272 °C (dec). ¹H NMR (DMSO-d₆) δ 9.62 (br s, 2H), 9.45 (br s, 4H), 9.30 (s, 1H), 9.20 (br s, 2H), 8.72 (d, J = 2.2 Hz, 1H), 8.35 (d, J = 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.96 (dd, J = 8.8 and 2.2 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 4.12 (s, 3H). HPLC (method A) t_R 6.38 min (100 area %). Anal. (C₁₇H₁₇N₇O·2HCl·2.1H₂O·0.2EtOH) C, H, N, Cl.

4-(5-N-Isopropylamidino-2-methoxyphenyl)-1-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (23)

White solid (0.18 g, 19%); mp 283–284 °C (dec). ¹H NMR (DMSO-d₆) δ 9.62 (br s, 4H), 9.30 (s, 1H), 9.15 (br s, 2H), 8.58 (d, J = 2.2 Hz, 1H), 8.33 (d, J = 8.8 Hz, 2H), 8.03 (d, J = 8.8 Hz, 2H), 7.83 (dd, J = 8.8 and 2.2 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 4.17 (m, 2H), 4.11 (s, 3H), 1.32 (d, J = 6.6 Hz, 6H), 1.30 (d, J = 6.6 Hz, 6H). HPLC (method A) t_R 8.31 min (100 area %). Anal. (C₂₃H₂₉N₇O·2HCl·0.9H₂O) C, H, N, Cl.

4-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-1-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (24)

White solid (0.50 g, 55%); mp 315–317 °C (dec). ¹H NMR (DMSO-d₆) δ 10.9 (br s, 2H), 10.6 (br s, 2H), 9.31 (s, 1H), 8.87 (br s, 1H), 8.41 (d, J = 8.8 Hz, 2H), 8.29 (d, J = 8.8 Hz, 2H), 8.10 (dd, J = 8.2 and 8.2 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 4.13 (s, 3H), 4.06 (s, 4H), 4.01 (s, 4H). HPLC (method A) t_R 7.88 min (100 area %). Anal. (C₂₁H₂₁N₇O·2HCl·1H₂O) C, H, N, Cl.

1-(4-Amidino-2-hydroxyphenyl)-4-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (25)

Light-brown solid (0.91 g, 70%); mp > 230 °C (dec). ¹H NMR (DMSO-d₆) δ 11.83 (br s, 1H), 9.57 (br s, 4H), 9.33 (br s, 4H), 9.26 (s, 1H), 8.50 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.75 (dd, J = 7.8 and 7.8 Hz, 1H), 7.66 (s, 1H), 7.42 (d, J = 8.2 Hz, 1H). HPLC (method A) t_R 5.39 min (100 area %). M/Z 322.2 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2HCl·2H₂O) C, H, N, Cl.

1-(4-N-Isopropylamidino-2-hydroxyphenyl)-4-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (26)

Grey solid (0.55 g, 35%); mp > 235 °C (dec). ¹H NMR (DMSO-d₆) δ 11.84 (s, 1H), 9.78 (br s, 2H), 9.60 (br s, 2H), 9.25 (br s, 3H), 8.36 (s, 1H), 8.32 (m, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.73 (m, 2H), 7.61 (s, 1H), 7.34 (d, J = 8.2 Hz, 1H), 4.11 (m, 2H), 1.31 (d, J = 6.2 Hz, 6H), 1.29 (d, J = 6.2 Hz, 6H). HPLC (method A) t_R 7.71 min (100 area %). M/Z 406.3 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2HCl·2H₂O) C, H, N, Cl.

1-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-4-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (27)

Yellow solid (0.75 g, 51%); mp > 250 °C (dec). ¹H NMR (DMSO-d₆) δ 10.96 (br s, 4H), 9.31 (s, 1H), 8.85 (s, 1H), 8.37 (d, J = 7.7 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.74 (m, 1H), 7.62 (d, J = 8.3 Hz, 1H), 4.04 (s, 8H). HPLC (method A) t_R 7.17 min (100 area %). M/Z 374.2 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2HCl·2H₂O) C, H, N, Cl.

4-(5-Amidino-2-hydroxyphenyl)-1-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (28)

Purple solid (0.54 g, 57%); mp > 251 °C (dec). ¹H NMR (DMSO-d₆) δ 11.80 (s, 1H), 9.61 (br s, 2H), 9.36 (br s, 2H), 9.33 (br s, 2H), 9.22 (s, 1H), 9.03 (br s, 2H), 8.65 (d, J = 2.5 Hz, 1H), 8.31 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 8.8 Hz, 2H), 7.76 (dd, J = 8.6 and 2.5 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H). HPLC (method A) t_R 5.74 min (100 area %). M/Z 322.7 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2HCl·0.9H₂O) C, H, N, Cl.

4-(5-N-Isopropylamidino-2-hydroxyphenyl)-1-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (29)

White solid (0.66 g, 57%); mp > 255 °C (dec). ¹H NMR (DMSO-d₆) δ 11.77 (s, 1H), 9.83 (d, J = 8.8 Hz, 1H), 9.67 (br s, 1H), 9.53 (d, J = 7.6 Hz, 1H), 9.39 (br s, 1H), 9.33 (br s, 1H), 9.22 (s, 1H), 9.01 (br s, 1H), 8.51 (d, J = 2.3 Hz, 1H), 8.30 (d, J = 8.6 Hz, 2H), 8.00 (d, J = 8.6 Hz, 2H), 7.65 (dd, J = 8.7 and 2.3 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 4.11 (m, 2H), 1.31 (d, J = 6.2 Hz, 6H), 1.29 (d, J = 6.2 Hz, 6H). HPLC (method A) t_R 8.10 min (100 area %). M/Z 406.7 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2HCl·1.4H₂O) C, H, N, Cl.

4-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-1-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (30)

White solid (0.62 g, 58%); mp > 307 °C (dec). ¹H NMR (DMSO-d₆) δ 12.08 (br s, 1H), 11.09 (br s, 2H), 10.67 (br s, 2H), 9.24 (s, 1H), 8.81 (s, 1H), 8.36 (s, 4H), 7.99 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 4.04 (s, 4H), 3.99 (s, 4H). HPLC (method A) t_R 7.62 min (100 area %). M/Z 374.7 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2HCl·1.5H₂O) C, H, N, Cl.

1-(3-Amidinophenyl)-4-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (31)

White solid (0.09 g, 16%); mp 342–345 °C (dec). ¹H NMR (DMSO-d₆) δ 9.87 (s, 1H), 9.75 (br s, 2H), 9.50 (br s, 2H), 9.46 (br s, 2H), 9.28 (br s, 2H), 8.61 (s, 1H), 8.35 (d, J = 8.2 Hz, 1H), 8.17 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 8.01 (d, J = 7.7 Hz, 1H), 7.91 (dd, J = 8.2 and 7.7 Hz, 1H). HPLC (method A) t_R 5.19 min (100 area %). Anal. (C₁₆H₁₅N₇·2HCl·2H₂O) C, H, N, Cl.

1-(3-N-Isopropylamidinophenyl)-4-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (32)

White solid (0.11 g, 15%); mp 324–325 °C (dec). ¹H NMR (DMSO-d₆) δ 9.99 (d, J = 8.2 Hz, 1H), 9.92 (s, 1H), 9.81 (br s, 1H), 9.72 (d, J = 7.7 Hz, 1H), 9.58 (br s, 1H), 9.44 (br s, 1H), 9.25 (br s, 1H), 8.49 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.16 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.90 (d, J = 7.7 Hz, 1H), 7.87 (dd, J = 7.7 and 7.7 Hz, 1H), 4.15 (m, 2H), 1.32 (d, J = 6.6 Hz, 6H), 1.30 (d, J = 6.6 Hz, 6H). HPLC (method A) t_R 7.63 min (100 area %). Anal. (C₂₂H₂₇N₇·2HCl·1.1H₂O) C, H, N, Cl.

1-[3-(4,5-Dihydro-1H-imidazol-2-yl)phenyl]-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (33)

Off-white solid (0.10 g, 18%); mp > 275 °C (dec). ¹H NMR (DMSO-d₆) δ 11.2 (br s, 1H), 10.9 (br s, 2H), 9.92 (s, 1H), 9.01 (s, 1H), 8.38 (d, J = 8.2 Hz, 1H), 8.30 – 8.15 (m, 6H), 7.94 (dd, J = 8.2 and 8.2 Hz, 1H), 4.07 (s, 4H), 4.03 (s, 4H). HPLC (method A) t_R 6.86 min (100 area %). Anal. (C₂₀H₁₉N₇·2HCl·1.9H₂O) C, H, N, Cl.

1-(5-Amidino-2-methoxyphenyl)-4-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (34)

White solid (0.65 g, 57%); mp > 247 °C (dec). ¹H NMR (DMSO-d₆) δ 9.51 (br s, 4H), 9.30 (s, 1H), 9.26 (br s, 4H), 8.31 (d, J = 1.8 Hz, 1H), 8.20 (d, J = 8.2 Hz, 2H), 8.15 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.2 Hz, 1H), 4.03 (s, 3H). HPLC (method A) t_R 6.03 min (100 area %). M/Z 336.2 (MH⁺ of free base). Anal. (C₁₇H₁₇N₇O·2HCl·2.2H₂O) C, H, N, Cl.

1-(5-N-Isopropylamidino-2-methoxyphenyl)-4-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (35)

White solid (0.63 g, 46%); mp > 250 °C (dec). ¹H NMR (DMSO-d₆) δ 9.65 (d, J = 6.0 Hz, 2H), 9.51 (br s, 2H), 9.29 (s, 1H), 9.12 (br s, 2H), 8.20 (d, J = 8.8 Hz, 2H), 8.17 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.87 (d, J = 7.8 Hz, 2H), 7.60 (d, J = 8.8 Hz, 1H), 4.08 (m, 2H), 4.01 (s, 3H), 1.30 (s, 12H). HPLC (method A) t_R 8.05 min (100 area %). M/Z 420.3 (MH⁺ of free base). Anal. (C₂₃H₂₉N₇O·2HCl·1.5H₂O) C, H, N, Cl.

1-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (36)

White solid (0.76 g, 59%); mp 250–258 °C. ¹H NMR (DMSO-d₆/CD₃OD) δ 9.19 (s, 1H), 8.47 (d, J = 2.4 Hz, 1H), 8.26 (d, J = 8.2 Hz, 2H), 8.19 (d, J = 8.2 Hz, 1H), 8.08 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.2 Hz, 1H), 4.08 (s, 8H), 4.05 (s, 3H). HPLC (method A) t_R 7.42 min (100 area %). M/Z 388.2 (MH⁺ of free base). Anal. (C₂₁H₂₁N₇O·2.4HCl·3.1H₂O) C, H, N, Cl.

4-(4-Amidino-2-methoxyphenyl)-1-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (37)

White solid (0.35 g, 44%); mp > 238 °C (dec). ¹H NMR (DMSO-d₆) δ 9.78 (br s, 2H), 9.56 (br s, 2H), 9.44 (s, 1H), 9.40 (br s, 2H), 9.26 (br s, 2H), 8.59 (m, 1H), 8.42 (d, J = 8.2 Hz, 1H), 8.40 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.90 (dd, J = 7.9 and 7.9 Hz, 1H), 7.69 (d, J = 1.4 Hz, 1H), 7.62 (dd, J = 8.2 and 1.4 Hz, 1H), 4.15 (s, 3H). HPLC (method A) t_R 6.41 min (100 area %). M/Z 336.7 (MH⁺ of free base). Anal. (C₁₇H₁₇N₇O·2.2HCl·1.4H₂O) C, H, N, Cl.

4-(4-N-Isopropylamidino-2-methoxyphenyl)-1-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (38)

White solid (0.64 g, 68%); mp > 240 °C (dec). ¹H NMR (DMSO-d₆) δ 10.04 (d, J = 7.2 Hz, 1H), 9.85 (br s, 1H), 9.74 (d, J = 7.4 Hz, 1H), 9.62 (br s, 1H), 9.48 (s, 1H), 9.36 (br s, 1H), 9.23 (br s, 1H), 8.48 (s, 1H), 8.41 (d, J = 8.1 Hz, 1H), 8.36 (d, J = 7.4 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.86 (dd, J = 7.6 and 7.4 Hz, 1H), 7.57 (s, 1H), 7.49 (d, J = 8.1 Hz, 1H), 4.15 (s, 3H), 4.13 (m, 2H), 1.33 (d, J = 6.2Hz, 6H), 1.31 (d, J = 6.2 Hz, 6H). HPLC (method A) t_R 8.26 min (100 area %). M/Z 420.7 (MH⁺ of free base). Anal. (C₂₃H₂₉N₇O·2HCl·2H₂O) C, H, N, Cl.

4-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-1-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (39)

White solid (0.82 g, 94%); mp > 268 °C (dec). ¹H NMR (DMSO-d₆) δ 11.31 (br s, 2H), 10.97 (br s, 2H), 9.52 (s, 1H), 9.01 (s, 1H), 8.47 (dd, J = 8.2 and 1.6 Hz, 1H), 8.45 (d, J = 8.2 Hz, 1H), 8.18 (d, J = 8.2 Hz, 1H), 7.98 (br s, 1H), 7.93 (dd, J = 8.2 and 8.2 Hz, 1H), 7.80 (dd, J = 8.2 and 1.6 Hz, 1H), 4.16 (s, 3H), 4.07 (s, 4H), 4.04 (s, 4H). HPLC (method A) t_R 7.88 min (100 area %). M/Z 388.6 (MH⁺ of free base). Anal. (C₂₁H₂₁N₇O·2HCl·3.5H₂O) C, H, N, Cl.

1-(5-Amidino-2-hydroxyphenyl)-4-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (40)

Dark-red solid (0.22 g, 50%); mp 240 °C (dec). ¹H NMR (DMSO-d₆) δ 12.24 (s, 1H), 9.48 (br s, 2H), 9.38 (br s, 2H), 9.28 (s, 1H), 9.22 (br s, 2H), 9.12 (br s, 2H), 8.25 (s, 1H), 8.22 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.2 Hz, 2H), 7.96 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H). HPLC (method A) t_R 5.13 min (100 area %). M/Z 322.7 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2.2HCl·2.3H₂O) C, H, N, Cl.

1-(5-N-Isopropylamidino-2-hydroxyphenyl)-4-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (41)

Dark-red solid (0.25 g, 47%); mp > 235 °C (dec). ¹H NMR (DMSO-d₆) δ 12.14 (s, 1H), 9.66 (d, J = 7.5 Hz, 1H), 9.56 (d, J = 7.6 Hz, 1H), 9.52 (br s, 1H), 9.45 (br s, 1H), 9.28 (s, 1H), 9.17 (br s, 1H), 9.06 (br s, 1H), 8.20 (d, J = 8.1 Hz, 2H), 8.10 (s, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 4.08 (m, 2H), 1.31 (d, J = 6.4 Hz, 6H), 1.29 (d, J = 6.4 Hz, 6H). HPLC (method A) t_R 7.74 min (100 area %). M/Z 406.9 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2.1HCl·2.6H₂O) C, H, N, Cl.

1-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (42)

Dark-red solid (0.16 g, 32%); mp 245 °C (dec). ¹H NMR (DMSO-d₆) δ 12.51 (s, 1H), 10.87 (s, 2H), 10.72 (s, 2H), 9.31 (s, 1H), 8.47 (s, 1H), 8.26 (d, J = 8.3 Hz, 2H), 8.20 (d, J = 8.3 Hz, 2H), 8.16 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 4.03 (s, 4H), 3.99 (s, 4H). HPLC (method A) t_R 6.98 min (100 area %). M/Z 374.7 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2.2HCl·3.3H₂O) C, H, N, Cl.

4-(4-Amidino-2-hydroxyphenyl)-1-(3-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (43)

Off-white solid (0.38 g, 46%); mp > 240 °C (dec). ¹H NMR (DMSO-d₆) δ 11.36 (s, 1H), 9.72 (br s, 2H), 9.46 (br s, 2H), 9.43 (br s, 2H), 9.29 (s, 1H), 9.22 (s, 2H), 8.49 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.86 (dd, J = 8.0 and 8.0 Hz, 1H), 7.52 (d, J = 1.6 Hz, 1H), 7.36 (dd, J = 8.2 and 1.6 Hz, 1H). HPLC (method A) t_R 5.81 min (100 area %). M/Z 322.6 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2.5HCl·0.9H₂O) C, H, N, Cl.

4-(4-N-Isopropylamidino-2-hydroxyphenyl)-1-(3-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (44)

Off-white solid (0.42 g, 42%); mp > 245 °C (dec). ¹H NMR (DMSO-d₆) δ 11.38 (s, 1H), 9.92 (d, J = 7.7 Hz, 1H), 9.74 (br s, 1H), 9.66 (d, J = 7.7 Hz, 1H), 9.49 (br s, 1H), 9.39 (br s, 1H), 9.28 (s, 1H), 9.16 (br s, 1H), 8.40 (s, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H,), 7.82 (dd, J = 7.8 and 7.8 Hz, 1H), 7.48 (s, 1H), 7.30 (d, J = 8.2 Hz, 1H), 4.11 (m, 2H), 1.32 (d, J = 6.3 Hz, 6H), 1.28 (d, J = 6.3 Hz, 6H). HPLC (method A) t_R 8.04 min (100 area %). M/Z 406.6 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2HCl·1H₂O·0.4EtOH) C, H, N, Cl.

4-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-1-[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (45)

Off-white solid (0.56 g, 60%); mp > 260 °C (dec). ¹H NMR (DMSO-d₆) δ 11.46 (br s, 1H), 10.95 (br s, 4H), 9.35 (s, 1H), 8.84 (s, 1H), 8.44 (dd, J = 8.2 and 1.2 Hz, 1H), 8.35 (d, J = 8.2 Hz, 1H), 8.20 (d, J = 8.2 Hz, 1H), 7.91 (dd, J = 8.2 and 8.2 Hz, 1H), 7.62 (d, J = 1.2 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 4.06 (s, 4H), 4.01 (s, 4H). HPLC (method A) t_R 7.51 min (97.1 area %). M/Z 374.9 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2HCl·2.5H₂O) C, H, N, Cl.

1,4-Bis(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (46)

Off-white solid (0.22 g, 48%); mp 357–359 °C. ¹H NMR (DMSO-d₆) δ 9.80 (s, 1H), 9.46 (br s, 6H), 8.26 (d, J = 8.8 Hz, 2H), 8.19 (d, J = 8.2 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.2 Hz, 2H). HPLC (method A) t_R 5.46 min (100 area %). Anal. (C₁₆H₁₅N₇·2HCl·0.8H₂O) C, H, N, Cl.

1,4-Bis(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (47)

White solid (0.09 g, 16%); mp 315–318 °C (dec). ¹H NMR (DMSO-d₆) δ 9.82 (d, J = 8.1 Hz, 1H), 9.81 (s, 1H), 9.70 (d, J = 7.7 Hz, 1H), 9.68 (br s, 1H), 9.56 (br s, 1H), 9.32 (br s, 1H), 9.23 (br s, 1H), 8.24 (d, J = 8.8 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 4.12 (m, 2H), 1.31 (d, J = 6.7 Hz, 12H). HPLC (method A) t_R 7.82 min (100 area %). Anal. (C₂₂H₂₇N₇·2HCl·1.6H₂O) C, H, N, Cl.

1,4-Bis(4-(4,5-dihydro-1H-imidazol-2-yl)phenyl)-1H-1,2,3-triazole Dihydrochloride (48)

Off-white solid (0.32 g, 66%); mp > 360 °C. ¹H NMR (DMSO-d₆) δ 10.6 (br s, 4H), 9.73 (s, 1H), 8.27 (m, 4H), 8.22 (d, J = 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 1H), 4.06 (s, 8H). HPLC (method A) t_R 7.07 min (100 area %). Anal. (C₂₀H₁₉N₇·2HCl·0.6H₂O) C, H, N, Cl.

1-(4-Amidino-2-methoxyphenyl)-4-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (49)

Yellow solid (0.81 g, 66%); mp 315–317 °C (dec). ¹H NMR (DMSO-d₆) δ 9.76 (br s, 2H), 9.54 (br s, 2H), 9.49 (br s, 2H), 9.33 (s, 1H), 9.30 (br s, 2H), 8.22 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 3H), 7.89 (d, J = 1.8 Hz, 1H), 7.67 (dd, J = 8.4 and 1.7 Hz, 1H), 4.06 (s, 3H). HPLC (method A) t_R 5.91 min (100 area %). M/Z 336.1 (MH⁺ of free base). Anal. (C₁₇H₁₇N₇O·2.2HCl·4.3H₂O) C, H, N, Cl.

1-(4-N-Isopropylamidino-2-methoxyphenyl)-4-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (50)

Yellow solid (0.90 g, 61%); mp 245–247 °C (dec). ¹H NMR (DMSO-d₆) δ 9.91 (d, J = 7.6 Hz, 1H), 9.78 (br s, 1H), 9.69 (d, J = 7.4 Hz, 1H), 9.55 (br s, 1H), 9.38 (br s, 1H), 9.31 (s, 1H), 9.20 (br s, 1H), 8.20 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 1.8 Hz, 1H), 7.55 (dd, J = 8.2 and 1.8 Hz, 1H), 4.13 (m, 2H), 4.06 (s, 3H), 1.32 (d, J = 6.7 Hz, 6H), 1.30 (d, J = 6.7 Hz, 6H). HPLC (method A) t_R 8.07 min (100 area %). M/Z 420.2 (MH⁺ of free base). Anal. (C₂₃H₂₉N₇O·2.5HCl·2.2H₂O) C, H, N, Cl.

1-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (51)

Yellow solid (1.00 g, 73%); mp > 320 °C (dec). ¹H NMR (DMSO-d₆) δ 11.11 (br s, 4H), 9.36 (s, 1H), 8.28 (d, J = 1.6 Hz, 1H), 8.24 (s, 4H), 8.06 (d, J = 8.3 Hz, 1H), 7.90 (dd, J = 8.3 and 1.6 Hz, 1H), 4.06 (s, 4H), 4.05 (s, 4H), 4.02 (s, 3H). HPLC (method A) t_R 7.47 min (100 area %). M/Z 388.2 (MH⁺ of free base). Anal. (C₂₁H₂₁N₇O·2.2HCl·2.4H₂O) C, H, N, Cl.

4-(4-Amidino-2-methoxyphenyl)-1-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (52)

White solid (0.34 g, 59%); mp 301–302 °C (dec). ¹H NMR (DMSO-d₆) δ 9.50 (br s, 8H), 9.33 (s, 1H), 8.41 (d, J = 8.2 Hz, 1H), 8.36 (d, J = 8.8 Hz, 2H), 8.13 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 1.6 Hz, 1H), 7.62 (dd, J = 8.2 and 1.6 Hz, 1H), 4.13 (s, 3H). HPLC (method A) t_R 6.43 min (100 area %). Anal. (C₁₇H₁₇N₇O·2HCl·1.8H₂O) C, H, N, Cl.

4-(4-N-Isopropylamidino-2-methoxyphenyl)-1-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (53)

Off-white solid (0.38 g, 54%); mp 267–270 °C (dec). ¹H NMR (DMSO-d₆) δ 9.83 (d, J = 7.7 Hz, 1H), 9.77 (d, J = 8.1 Hz, 1H), 9.67 (br s, 1H), 9.65 (br s, 1H), 9.31 (s, 1H), 9.30 (br s, 1H), 9.25 (br s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 8.33 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H), 7.59 (br s, 1H), 7.50 (d, J = 8.2 Hz, 1H), 4.14 (s, 3H), 4.13 (m, 2H), 1.32 (d, J = 6.6 Hz, 12H). HPLC (method A) t_R 8.31 min (100 area %). Anal. (C₂₃H₂₉N₇O·2HCl·2.7H₂O) C, H, N, Cl.

4-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-methoxyphenyl]-1-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (54)

Off-white solid (0.39 g, 59%); mp 320–321 °C (dec). ¹H NMR (DMSO-d₆) δ 11.0 (br s, 4H), 9.35 (s, 1H), 8.44 (d, J = 8.2 Hz, 1H), 8.39 (d, J = 8.8 Hz, 2H), 8.35 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 1.1 Hz, 1H), 7.80 (dd, J = 8.2 and 1.1 Hz, 1H), 4.13 (s, 3H), 4.05 (s, 4H), 4.04 (s, 4H). HPLC (method A) t_R 7.93 min (100 area %). Anal. (C₂₁H₂₃N₇O·2HCl·1.8H₂O) C, H, N, Cl.

1-(4-Amidino-2-hydroxyphenyl)-4-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (55)

Off-white solid (0.95 g, 84%); mp > 315 °C (dec). ¹H NMR (DMSO-d₆) δ 11.78 (s, 1H), 9.56 (br s, 2H), 9.50 (br s, 2H), 9.32 (br s, 2H), 9.30 (s, 1H), 9.27 (br s, 2H), 8.23 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.3 Hz, 1H), 7.65 (s, 1H), 7.42 (d, J = 8.3 Hz, 1H). HPLC (method A) t_R 5.43 min (100 area %). M/Z 322.2 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2HCl·1.2H₂O) C, H, N, Cl.

1-(4-N-Isopropylamidino-2-hydroxyphenyl)-4-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (56)

White solid (0.53 g, 38%); mp > 258 °C (dec). ¹H NMR (DMSO-d₆) δ 11.84 (s, 1H), 9.79 (d, J = 7.7 Hz, 1H), 9.69 (d, J = 6.7 Hz, 1H), 9.61 (br s, 1H), 9.54 (br s, 1H), 9.29 (br s, 2H), 9.21 (br s, 1H), 8.21 (d, J = 7.9 Hz, 2H), 7.91 (d, J = 8.2 Hz, 2H), 7.88 (d, J = 7.9 Hz, 1H), 7.61 (s, 1H), 7.33 (d, J = 8.2 Hz, 1H), 4.11 (m, 2H), 1.30 (d, J = 6.2 Hz, 6H), 1.29 (d, J = 6.2 Hz, 6H). HPLC (method A) t_R 7.87 min (100 area %). M/Z 406.3 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2HCl·1.5H₂O) C, H, N, Cl.

1-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (57)

White solid (0.40 g, 31%); mp > 360 °C. ¹H NMR (DMSO-d₆) δ 10.72 (br s, 4H), 9.33 (s, 1H), 8.27 (d, J = 8.3 Hz, 2H), 8.14 (d, J = 8.3 Hz, 2H), 8.00 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.55 (d, J = 8.3 Hz, 1H), 4.03 (s, 8H). HPLC (method A) t_R 7.21 min (100 area %). M/Z 374.3 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2HCl·0.5H₂O) C, H, N, Cl.

4-(4-Amidino-2-hydroxyphenyl)-1-(4-amidinophenyl)-1H-1,2,3-triazole Dihydrochloride (58)

White solid (0.34 g, 42%); mp > 312 °C (dec). ¹H NMR (DMSO-d₆) δ 11.33 (s, 1H), 9.62 (br s, 2H), 9.44 (br s, 2H), 9.38 (br s, 2H), 9.28 (s, 1H), 9.22 (br s, 2H), 8.34 (d, J = 8.8 Hz, 2H), 8.32 (d, J = 8.2 Hz, 1H), 8.11 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 1.7 Hz, 1H), 7.38 (dd, J = 8.2 and 1.7 Hz, 1H). HPLC (method A) t_R 5.95 min (100 area %). M/Z 322.5 (MH⁺ of free base). Anal. (C₁₆H₁₅N₇O·2HCl·1.2H₂O) C, H, N, Cl.

4-(4-N-Isopropylamidino-2-hydroxyphenyl)-1-(4-N-isopropylamidinophenyl)-1H-1,2,3-triazole Dihydrochloride (59)

White solid (0.54 g, 55%); mp > 286 °C (dec). ¹H NMR (DMSO-d₆) δ 11.36 (s, 1H), 9.80 (d, J = 8.0 Hz, 1H), 9.66 (d, J = 8.0 Hz, 1H), 9.65 (br s, 1H), 9.49 (br s, 1H), 9.31 (br s, 1H), 9.24 (s, 1H), 9.17 (br s, 1H), 8.31 (d, J = 7.7 Hz, 1H), 8.30 (d, J = 8.8 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 1.6 Hz, 1H), 7.29 (dd, J = 8.0 and 1.6 Hz, 1H), 4.12 (m, 2H), 1.31 (d, J = 6.7 Hz, 6H), 1.29 (d, J = 6.7 Hz, 6H). HPLC (method A) t_R 8.18 min (100 area %). M/Z 406.7 (MH⁺ of free base). Anal. (C₂₂H₂₇N₇O·2HCl·1.2H₂O) C, H, N, Cl.

4-[4-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxyphenyl]-1-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1H-1,2,3-triazole Dihydrochloride (60)

Light-yellow solid (0.52 g, 57%); mp > 345 °C (dec). ¹H NMR (DMSO-d₆) δ 11.34 (s, 1H), 10.62 (br s, 4H), 9.27 (s, 1H), 8.36 (d, J = 8.2 Hz, 2H), 8.24 (d, J = 8.2 Hz, 2H), 8.09 (d, J = 1.7 Hz, 1H), 7.54 (dd, J = 8.2 and 1.7 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 4.03 (s, 4H), 4.01 (s, 4H). HPLC (method A) t_R 7.60 min (100 area %). M/Z 374.5 (MH⁺ of free base). Anal. (C₂₀H₁₉N₇O·2HCl·1.5H₂O) C, H, N, Cl.

3-Azido-4-methoxybenzonitrile (63)

A mixture of 4-hydroxy-3-nitrobenzonitrile (66) (14.7 g, 89.4 mmol), K₂CO₃ (14.2 g, 103 mmol), and MeI (14.2 g, 100 mmol) in dry DMF (150 mL) was stirred at ambient temperature. The progress of the reaction was monitored by HPLC. Upon completion, the mixture was poured into iced water and a formed precipitated was collected by filtration, washed with water, and dried to afford 4-methoxy-3-nitrobenzonitrile (67) (11.5 g, 73%). ¹H NMR (DMSO-d₆) δ 8.49 (d, J = 1.8 Hz, 1H), 8.16 (dd, J = 8.8 and 1.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 4.02 (s, 3H). HPLC (method B) t_R 4.26 min (100 area %).

A suspension of 67 (11.0 g, 61.8 mmol) and 10% Pd/C (1.00 g) in MeOH (250 mL) was hydrogenated in a Parr apparatus at 60 psi overnight. The reaction mixture was filtered through a pad of Celite, which was rinsed with MeOH. The filtrate was concentrated and dried under vacuum to yield 3-amino-4-methoxybenzonitrile (68) (9.00 g, 98%). ¹H NMR (DMSO-d₆) δ 6.98 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.90 (s, 1H), 5.22 (br s, 2H), 3.83 (s, 3H). HPLC (method B) t_R 3.28 min (98.6 area %).

A solution of NaNO₂ (4.20 g, 60.0 mmol) in water (30 mL) was added to a solution of 68 (8.50 g, 57.4 mmol) in 10% aqueous HCl (120 mL) at 0–5 °C. The reaction mixture was stirred at this temperature for 30 min and then a solution of NaN₃ (4.55 g, 70.0 mmol) in water (60 mL) was added dropwise. The mixture was stirred for 1 h and a precipitate was filtered off, washed with water, and dried to give 63 as a white solid (9.00 g, 91%); mp 63–65 °C. ¹H NMR (DMSO-d₆) δ 7.66 (dd, J = 8.6 and 1.8 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 3.93 (s, 3H). HPLC (method B) t_R 5.78 min (96.2 area %). Anal. (C₈H₆N₄O) C, H, N.

4-Azido-3-methoxybenzonitrile (64)

A solution of NaNO₂ (3.50 g, 50.0 mmol) in water (30 mL) was added to a solution of 4-amino-3-methoxybenzonitrile (71) (7.00 g, 47.2 mmol) in 10% aqueous HCl (120 mL) at 0–5 °C. The reaction mixture was stirred at this temperature for 30 min and then a solution of NaN₃ (3.90 g, 60.0 mmol) in water (50 mL) was added dropwise. The mixture was stirred for 1 h and a precipitate was filtered off, washed with water, and dried to give 64 as a white solid (7.75 g, 94%); mp 97–98 °C. ¹H NMR (DMSO-d₆) δ 7.56 (d, J = 1.6 Hz, 1H), 7.41 (dd, J = 8.2 and 1.6 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 3.88 (s, 3H). HPLC (method B) t_R 5.77 min (100 area %). Anal. (C₈H₆N₄O) C, H, N.

3-Azido-4-hydroxybenzonitrile (65)

A solution of NaNO₂ (1.54 g, 22.0 mmol) in water (10 mL) was added to a solution of 3-amino-4-hydroxybenzonitrile (72) (2.68 g, 20.0 mmol) in 10% aqueous HCl (40 mL) at 0–5 °C. The reaction mixture was stirred at this temperature for 30 min and then a solution of NaN₃ (1.56 g, 24.0 mmol) in water (20 mL) was added dropwise. The mixture was stirred for 1 h and a precipitate was filtered off, washed with water, and dried to give 65 as a white solid (2.00 g, 63%); mp 108 °C (dec). ¹H NMR (DMSO-d₆) δ 11.40 (s, 1H), 7.47 (dd, J = 8.1 and 2.0 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H). HPLC (method B) t_R 4.65 min (100 area %). Anal. (C₇H₄N₄O) C, H, N.

4-Amino-3-methoxybenzonitrile⁸² (71)

A mixture of 4-bromo-o-anisidine⁸¹ (70) (18.9 g, 93.5 mmol) and CuCN (10.7 g, 119 mmol) in dry DMF (50 mL) and pyridine (10 mL) was refluxed overnight. The mixture was cooled, diluted with CHCl₃, and filtered through a pad of Celite. A filtrate was concentrated and dried under vacuum to give crude, which was purified by column chromatography eluting with CHCl₃ to afford 71 (7.50 g, 54%); mp 46–48 °C (No lit.⁸² mp). ¹H NMR (DMSO-d₆) δ 7.13 (d, J = 1.8 Hz, 1H), 7.12 (dd, J = 8.4 and 1.8 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 5.80 (br s, 2H), 3.81 (s, 3H). HPLC (method B) t_R 3.53 min (100 area %). Anal. (C₈H₈N₂O) C, H, N.

3-Amino-4-hydroxybenzonitrile (72)

A suspension of 3-nitro-4-hydroxybenzonitrile 66 (10.0 g, 60.9 mmol) and 10% Pd/C (1.00 g) in MeOH (250 mL) was hydrogenated in a Parr apparatus at 60 psi for 2 h. The reaction mixture was filtered through a pad of Celite, which was rinsed with MeOH. The filtrate was concentrated and dried under vacuum to yield 72 (7.15 g, 88%); mp 153–154 °C. ¹H NMR (DMSO-d₆) δ 10.21 (br s, 1H), 8.86 (d, J = 2.0 Hz, 1H), 8.83 (dd, J = 7.8 and 2.0 Hz, 1H), 6.75 (d, J = 7.8 Hz, 1H), 4.99 (br s, 2H). HPLC (method A) t_R 4.95 min (100 area %). Anal. (C₇H₆N₂O) C, H, N.

General Procedure for Synthesis of Dinitriles 77–89, 93. 1,4-Bis(3-cyanophenyl)-1H-1,2,3-triazole (77)

To a suspension of 3-azidobenzonitrile (61) (1.44 g, 10.0 mmol) and 3-ethynylbenzonitrile (73) (1.27 g, 10.0 mmol) in a 1:1 mixture of water and t-butyl alcohol (40 ml) was added a solution of sodium ascorbate (0.20 g, 1 mmol) in water (5 ml) followed by copper(II) sulfate pentahydrate (0.025 g, 0.1 mmol) in water (1 ml). The mixture was stirred at ambient temperature overnight, at which point HPLC analysis indicated presence of starting materials in the mixture. A solution of sodium ascorbate (0.20 g, 1 mmol) in water (5 ml) was added and the mixture was stirred for 24 hrs at 70 °C to complete the reaction. The reaction mixture was diluted with water, a precipitate was collected by filtration, washed with water (3 × 50 mL), and dried under vacuum to give crude (2.60 g, 96%), which was recrystallized to afford 77 as a light-yellow solid; mp 218–219 °C (EtOAc/EtOH). ¹H NMR (DMSO-d₆) δ 9.56 (s, 1H), 8.45 (dd, J = 1.6 and 1.6 Hz, 1H), 8.32 (dd, J = 1.1 and 1.1 Hz, 1H), 8.29 (dd, J = 8.2 and 1.6 Hz, 1H), 8.26 (dd, J = 8.2 and 1.6 Hz, 1H), 8.02 (dd, J = 7.7 and 1.1 Hz, 1H), 7.89 (dd, J = 7.7 and 1.1 Hz, 1H), 7.87 (dd, J = 7.7 and 7.7 Hz, 1H), 7.75 (dd, J = 8.2 and 8.2 Hz, 1H). HPLC (method B) t_R 6.56 min (100 area %). Anal. (C₁₆H₉N₅) C, H, N.

1-(4-Cyanophenyl)-4-(3-cyanophenyl)-1H-1,2,3-triazole (78)

Following the procedure described above for 77, 78 was prepared from 4-azidobenzonitrile (62) (1.44 g, 10.0 mmol) and 73 (1.27 g, 10.0 mmol). Off-white solid (2.47 g, 91%); mp 217–219 °C (EtOAc). ¹H NMR (DMSO-d₆) δ 9.61 (s, 1H), 8.34 (dd, J = 1.6 and 1.6 Hz, 1H), 8.27 (dd, J = 8.2 and 1.6 Hz, 1H), 8.17 (s, 4H), 7.88 (dd, J = 8.2 and 1.6 Hz, 1H), 7.75 (dd, J = 8.2 and 8.2 Hz, 1H). HPLC (method B) t_R 6.59 min (100 area %). Anal. (C₁₆H₉N₅) C, H, N.

1-(3-Cyanophenyl)-4-(4-cyanophenyl)-1H-1,2,3-triazole (79)

Following the procedure described above for 77, 79 was prepared from 61 (1.44 g, 10.0 mmol) and 4-ethynylbenzonitrile (74) (1.27 g, 10.0 mmol). Off-white solid (2.47 g, 91%); mp 223–224 °C (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 9.61 (s, 1H), 8.47 (dd, J = 1.1 and 1.1 Hz, 1H), 8.34 (dd, J = 8.2 and 1.1 Hz, 1H), 8.11 (d, J = 8.2 Hz, 2H), 8.03 (dd, J = 8.2 and 1.1 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.87 (dd, J = 8.2 and 8.2 Hz, 1H). HPLC (method B) t_R 6.57 min (100 area %). Anal. (C₁₆H₉N₅) C, H, N.

1,4-Bis(4-cyanophenyl)-1H-1,2,3-triazole (80)

Following the procedure described above for 77, 80 was prepared from 62 (1.44 g, 10.0 mmol) and 74 (1.27 g, 10.0 mmol). Orange solid (2.38 g, 88%); mp 261–263 °C (DMF). ¹H NMR (DMSO-d₆) δ 9.66 (s, 1H), 8.18 (s, 4H), 8.12 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H). HPLC (method B) t_R 6.60 min (100 area %). Anal. (C₁₆H₉N₅) C, H, N.

1-(5-Cyano-2-methoxyphenyl)-4-(3-cyanophenyl)-1H-1,2,3-triazole (81)

Following the procedure described above for 77, 81 was prepared from 63 (1.74 g, 10.0 mmol) and 73 (1.27 g, 10.0 mmol) in a mixture of DMSO (45 mL) and water (15 mL). White solid (2.81 g, 93%); mp 228–230 °C (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 9.18 (s, 1H), 8.40 (s, 1H), 8.31 (d, J = 7.7 Hz, 1H), 8.27 (d, J = 1.8 Hz, 1H), 8.10 (dd, J = 8.8 and 1.8 Hz, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.73 (dd, J = 7.7 and 7.7 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 4.00 (s, 3H). HPLC (method B) t_R 6.52 min (100 area %). Anal. (C₁₇H₁₁N₅O) C, H, N.

4-(5-Cyano-2-methoxyphenyl)-1-(3-cyanophenyl)-1H-1,2,3-triazole (82)

Following the procedure described above for 77, 82 was prepared from 61 (2.88 g, 20.0 mmol) and 3-ethynyl-4-methoxybenzonitrile (75) (3.14 g, 20.0 mmol) in a mixture of DMSO (90 mL) and water (20 mL). White solid (4.90 g, 81%); mp 280–281 °C (Py). ¹H NMR (DMSO-d₆) δ 9.23 (s, 1H), 8.59 (s, 1H), 8.52 (d, J = 1.5 Hz, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.90 (dd, J = 8.8 and 1.5 Hz, 1H), 7.88 (dd, J = 7.8 and 7.8 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 4.09 (s, 3H). HPLC (method B) t_R 7.04 min (100 area %). Anal. (C₁₇H₁₁N₅O) C, H, N.

1-(4-Cyano-2-methoxyphenyl)-4-(3-cyanophenyl)-1H-1,2,3-triazole (83)

Following the procedure described above for 77, 83 was prepared from 64 (3.70 g, 21.0 mmol) and 73 (2.70 g, 21.0 mmol) in a mixture of DMSO (70 mL) and water (30 mL). Dark-yellow solid (6.30 g, 99%); mp 228–230 °C (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 9.25 (s, 1H), 8.42 (s, 1H), 8.33 (dd, J = 8.1 and 1.3 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 1.3 Hz, 1H), 7.86 (dd, J = 7.8 and 1.3 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.69 (dd, J = 8.1 and 1.3 Hz, 1H), 4.00 (s, 3H). HPLC (method B) t_R 6.74 min (100 area %). Anal. (C₁₇H₁₁N₅O·0.2EtOH·0.1H₂O) C, H, N.

4-(5-Cyano-2-methoxyphenyl)-1-(4-cyanophenyl)-1H-1,2,3-triazole (84)

Following the procedure described above for 77, 84 was prepared from 62 (1.44 g, 10.0 mmol) and 75 (1.60 g, 11.0 mmol). White solid (2.39 g, 79%); mp 288–289 °C (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 9.24 (s, 1H), 8.51 (d, J = 1.6 Hz, 1H), 8.29 (d, J = 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.90 (dd, J = 8.8 and 1.6 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 4.08 (s, 3H). HPLC (method B) t_R 6.98 min (100 area %). Anal. (C₁₇H₁₁N₅O) C, H, N.

1-(5-Cyano-2-methoxyphenyl)-4-(4-cyanophenyl)-1H-1,2,3-triazole (85)

Following the procedure described above for 77, 85 was prepared from 63 (1.74 g, 10.0 mmol) and 74 (1.27 g, 10.0 mmol) in a mixture of DMSO (45 mL) and water (15 mL). Off-white solid (2.95 g, 98%); mp 235–237 °C (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 9.22 (s, 1H), 8.27 (d, J = 1.8 Hz, 1H), 8.14 (d, J = 8.3 Hz, 2H), 8.10 (dd, J = 8.7 and 1.8 Hz, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.7 Hz, 1H), 3.99 (s, 3H). HPLC (method B) t_R 6.51 min (100 area %). Anal. (C₁₇H₁₁N₅O) C, H, N.

4-(4-Cyano-2-methoxyphenyl)-1-(3-cyanophenyl)-1H-1,2,3-triazole (86)

Following the procedure described above for 77, 86 was prepared from 61 (2.88 g, 20.0 mmol) and 4-ethynyl-3-methoxybenzonitrile (76) (3.14 g, 20.0 mmol) in a mixture of DMSO (90 mL) and water (20 mL). Off-white solid (5.87 g, 97%); mp 236–237 °C (Py). ¹H NMR (DMSO-d₆) δ 9.27 (s, 1H), 8.59 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.85 (dd, J = 8.0 and 8.0 Hz, 1H), 7.69 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 4.07 (s, 3H). HPLC (method B) t_R 7.24 min (100 area %). Anal. (C₁₇H₁₁N₅O) C, H, N.

1-(4-Cyano-2-methoxyphenyl)-4-(4-cyanophenyl)-1H-1,2,3-triazole (87)

Following the procedure described above for 77, 87 was prepared from 64 (3.70 g, 21.0 mmol) and 74 (2.70 g, 21.0 mmol) in a mixture of DMSO (70 mL) and water (30 mL). Yellow solid (6.15 g, 97%); mp 240–242 °C (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 9.28 (s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.2 Hz, 3H), 7.93 (d, J = 1.4 Hz, 1H), 7.69 (dd, J = 8.2 and 1.4 Hz, 1H), 3.99 (s, 3H). HPLC (method B) t_R 6.75 min (100 area %). Anal. (C₁₇H₁₁N₅O·0.3H₂O) C, H, N.

4-(4-Cyano-2-methoxyphenyl)-1-(4-cyanophenyl)-1H-1,2,3-triazole (88)

Following the procedure described above for 77, 88 was prepared from 62 (3.50 g, 24.3 mmol) and 76 (4.40 g, 28.0 mmol). White solid (7.03 g, 96%); mp 286–287 °C (DMF). ¹H NMR (DMSO-d₆) δ 9.29 (s, 1H), 8.37 (d, J = 8.2 Hz, 1H), 8.29 (d, J = 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 1.6 Hz, 1H), 7.77 (dd, J = 8.2 and 1.6 Hz, 1H), 4.06 (s, 3H). HPLC (method B) t_R 7.20 min (100 area %). Anal. (C₁₇H₁₁N₅O) C, H, N.

1-(5-Cyano-2-hydroxyphenyl)-4-(3-cyanophenyl)-1H-1,2,3-triazole (89)

Following the procedure described above for 77, 89 was prepared from 65 (1.60 g, 10.0 mmol) and 73 (1.27 g, 10.0 mmol) in a mixture of DMSO (45 mL) and water (15 mL). Purple solid (2.06 g, 72%); mp 285–287 °C (EtOH). ¹H NMR (DMSO-d₆) δ 11.98 (s, 1H), 9.17 (s, 1H), 8.41 (s, 1H), 8.31 (d, J = 7.7 Hz, 1H), 8.20 (d, J = 1.8 Hz, 1H), 7.92–7.80 (m, 2H), 7.72 (dd, J = 7.7 and 7.7 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H). HPLC (method B) t_R 6.33 min (100 area %). Anal. (C₁₆H₉N₅O·0.2EtOH) C, H, N.

General Procedure for Synthesis of Dinitriles 90, 92, 94, 96. 4-(5-Cyano-2-hydroxyphenyl)-1-(3-cyanophenyl)-1H-1,2,3-triazole (90)

To a suspension of 82 (2.55 g, 8.46 mmol) in dry CH₂Cl₂ (120 mL) at 0 °C was added 1M solution of BBr₃ in CH₂Cl₂ (12 mL, 12 mmol). The mixture was stirred at 0 °C for 1h and then at ambient temperature. The progress of the reaction was monitored by HPLC. Upon completion, the reaction mixture was cooled in the ice bath and EtOH (30 mL) was added dropwise. A precipitate was separated, washed with EtOH, and dried under vacuum to give crude (2.40 g, 98%), which was recrystallized to afford 90. Off-white solid; mp 291 °C (dec) (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 11.56 (s, 1H), 9.17 (s, 1H), 8.57 (s, 1H), 8.43 (d, J = 2.1 Hz, 1H), 8.38 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.82 (dd, J = 7.7 and 7.7 Hz, 1H), 7.69 (dd, J = 8.5 and 2.1 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H). HPLC (method B) t_R 6.63 min (100 area %). Anal. (C₁₆H₉N₅O) C, H, N.

General Procedure for Synthesis of Dinitriles 91, 95

1-(4-Cyano-2-hydroxyphenyl)-4-(3-cyanophenyl)-1H-1,2,3-triazole (91)

To molten pyridine hydrochloride (24 g) was added 83 (4.00 g, 13.3 mmol), and the reaction mixture was kept at 160–180 °C for 3 h, cooled to 80–90 °C and diluted with water (100 mL). A formed precipitate, was separated, washed with water, and dried to give 91 as a grey solid (3.40 g, 89%); mp 284–285 °C (EtOH). ¹H NMR (DMSO-d₆) δ 11.62 (s, 1H), 9.24 (s, 1H), 8.43 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.86 (dd, J = 7.7 and 1.6 Hz, 1H), 7.72 (dd, J = 7.7 and 7.7 Hz, 1H), 7.52 (dd, J = 8.8 and 1.6 Hz, 1H), 7.51 (s, 1H). HPLC (method B) t_R 6.68 min (100 area %). Anal. (C₁₆H₉N₅O₂·0.3EtOH·0.3H₂O) C, H, N.

4-(5-Cyano-2-hydroxyphenyl)-1-(4-cyanophenyl)-1H-1,2,3-triazole (92)

Following the procedure described above for 90, 92 was prepared from 84 (3.50 g, 11.6 mmol) and 1M BBr₃ in CH₂Cl₂ (15 mL). Off-white solid (2.98 g, 89%); mp 282 °C (dec) (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 11.59 (s, 1H), 9.17 (s, 1H), 8.43 (d, J = 2.2 Hz, 1H), 8.26 (d, J = 8.9 Hz, 2H), 8.12 (d, J = 8.9 Hz, 2H), 7.70 (dd, J = 8.5 and 2.2 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H). HPLC (method B) t_R 6.65 min (100 area %). Anal. (C₁₆H₉N₅O·0.3H₂O) C, H, N.

1-(5-Cyano-2-hydroxyphenyl)-4-(4-cyanophenyl)-1H-1,2,3-triazole (93)

Following the procedure described above for 77, 93 was prepared from 65 (1.60 g, 10.0 mmol) and 74 (1.27 g, 10.0 mmol) in a mixture of DMSO (45 mL) and water (15 mL). Brown solid (1.30 g, 45%); mp 285–287 °C (EtOH). ¹H NMR (DMSO-d₆) δ 11.98 (s, 1H), 9.21 (s, 1H), 8.20 (s, 1H), 8.15 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 7.7 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H). HPLC (method B) t_R 6.34 min (100 area %). Anal. (C₁₆H₉N₅O·0.2DMF·0.3H₂O) C, H, N.

4-(4-Cyano-2-hydroxyphenyl)-1-(3-cyanophenyl)-1H-1,2,3-triazole (94)

Following the procedure described above for 90, 94 was prepared from 86 (2.38 g, 7.90 mmol) and 1M BBr₃ in CH₂Cl₂ (11 mL). White solid (2.20 g, 97%); mp 263 °C (dec) (MeCN). ¹H NMR (DMSO-d₆) δ 11.17 (s, 1H), 9.24 (s, 1H), 8.59 (s, 1H), 8.39 (dd, J = 7.8 and 2.2 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.83 (dd, J = 7.8 and 7.8 Hz, 1H), 7.42 (dd, J = 8.0 and 1.5 Hz, 1H), 7.35 (d, J = 1.5 Hz, 1H). HPLC (method B) t_R 6.90 min (100 area %). Anal. (C₁₆H₉N₅O) C, H, N.

1-(4-Cyano-2-hydroxyphenyl)-4-(4-cyanophenyl)-1H-1,2,3-triazole (95)

Following the procedure described above for 91, 95 was prepared from 87 (2.00 g, 6.50 mmol) as an off-white solid (1.70, 89%); mp 276–278 °C (EtOH/DMF). ¹H NMR (DMSO-d₆) δ 11.64 (s, 1H), 9.28 (s, 1H), 8.17 (d, J = 8.1 Hz, 2H), 7.98 (d, J = 8.1 Hz, 2H), 7.93 (d, J = 8.2 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.50 (s, 1H). HPLC (method B) t_R 6.68 min (100 area %). Anal. (C₁₆H₉N₅O₂·0.2EtOH·0.4H₂O) C, H, N.

4-(4-Cyano-2-hydroxyphenyl)-1-(4-cyanophenyl)-1H-1,2,3-triazole (96)

Following the procedure described above for 90, 96 was prepared from 88 (5.18 g, 17.2 mmol) and 1M BBr₃ in CH₂Cl₂ (24 mL). White solid (4.90 g, 99%); mp 293 °C (dec) (DMF). ¹H NMR (DMSO-d₆) δ 11.19 (s, 1H), 9.23 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 8.8 Hz, 2H), 7.41 (dd, J = 8.0 and 1.5 Hz, 1H), 7.35 (d, J = 1.5 Hz, 1H). HPLC (method B) t_R 6.92 min (100 area %). Anal. (C₁₆H₉N₅O) C, H, N.