Synthesis and activity of azaterphenyl diamidines against Trypanosoma brucei rhodesiense and Plasmodium falciparum

Abstract

A series of azaterphenyl diamidines has been synthesized and evaluated for in vitro antiprotozoal activity against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) and in vivo efficacy in the STIB900 acute mouse model for T. b. r. Six of the 13 compounds showed IC₅₀ values less than 7 nM against T. b. r. Twelve of those exhibited IC₅₀ values less than 6 nM against P. f. and six of those showed IC₅₀ values ≤0.6 nM, which are more than 25-fold as potent as furamidine. Moreover, two of them showed more than 40-fold selectivity for P. f. versus T. b. r. Three compounds 15b, 19d and 19e exhibited in vivo efficacy against T. b. r. much superior to furamidine, and equivalent to or better than azafuramidine. The antiparasitic activity of these diamidines depends on the ring nitrogen atom(s) location relative to the amidine groups and generally correlates with DNA binding affinity.

1. Introduction

Human African trypanosomiasis (HAT) and malaria, which are caused by the protozoan parasites Trypanosoma brucei and Plasmodium sp., infect millions of people in large parts of the world each year.¹ Numerous aromatic or heterocyclic diamidines exhibit potent antiprotozoal activity against HAT and malaria.² However, pentamidine 1 (Fig. 1) is the only one of this class which has seen significant human clinical use and it has been used to treat 1st stage HAT for over half a century.² Furamidine 2a, a diphenyl furan diamidine analogue, has been shown to be more potent and less toxic than pentamidine in murine models of trypanosomiasis.³ The oral prodrug of furamidine 2b (pafuramidine) showed promising results in Phase I and II clinical trials against both HAT and malaria. ^1–3 Unfortunately, in an additional safety study of pafuramidine parallelling the Phase III trials, liver and kidney toxicities in some volunteers were found and the development of pafuramidine was suspended.³ Introduction of an N-atom into one of phenyl ring of furamidine resulted in an aza-analogue of furamidine 3a, which exhibited more potent in vivo antitrypanosomal activity than pentamidine and furamidine, although all three have similar in vitro activities.⁴ The methoxyamidine prodrug of azafuramidine 3b was found to be quite effective against 2nd stage HAT.⁵ Although diamidines have been used therapeutically since the 1950s, the antiparasitic mode of action of such diamidines is not well understood. A long-hypothesized mechanism of action arose from their binding to the minor groove of DNA at AT rich sites in the nucleus or kinetoplast,⁶ which has been suggested to interfere with DNA-associated enzymes, such as topoisomerase II, and possibly direct inhibition of transcription.⁷ Recent investigations have shown that both furamidine 2a and azafuramidine 3a accumulated within trypanosomes at millimolar concentration, with intracellular concentrations over 15,000-fold higher than mouse plasma concentrations.⁸ Although the results of this study showed that the extent of accumulation is not directly correlated with killing of the trypanosomes, the selective concentration of diamidines in parasite mitochondria (kinetoplast) appears to represent a pivotal step in their antiparasitic activity.⁸

Figure 1.

Aromatic or heterocyclic diamidine antiprotozoal agents.

The previous design of DNA minor groove binding diamidine analogues has focused on crescent shaped molecules which closely fit the curvature of the groove, such as pentamidine, furamidine and their analogues.^2,9 Recently, linear dicationic diamidines CGP40215A 4 and a benzimidazole diamidine 5 have shown strong DNA minor groove binding and potent antiparasitic activity.¹⁰ Both compounds were found to simulate the curved structure of DNA minor groove by incorporation of a water molecule into the recognition complex with DNA.¹⁰ Based on the discovery of this new binding mode, we recently prepared a series of linear terphenyl diamidines which showed significant DNA minor groove binding affinity and low nanomolar antiprotozoal activity against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.).¹¹ The parent terphenyl compound 6a is more effective than furamidine in an acute mouse model for T. b. r. STIB 900. Due to the promising results found in the furan diamidine system, we first synthesized three aza-analogues 6b–d of terphenyl diamidine 6a by introduction of a nitrogen atom into the central phenyl ring or both of the terminal phenyl rings.¹¹ Both aza-diamidine compounds 6b–c in which a nitrogen atom has been placed in the central phenyl ring or meta to both of the amidine groups in the terminal phenyl rings are more effective than furamidine in an acute mouse model for T. b. r. STIB 900. The diaza-analogue 6d is of particular interest because it exhibits very strong activity (IC₅₀ = 0.5 nM) against P. f. and at the same time shows a 32-fold selectivity for P. f. compared to antitrypanosomal activity.¹¹ More interestingly, 6d binds specifically to a GC-rich sequence more strongly than to the usual AT recognition, which is the first non-polyamide, synthetic compound to specifically recognize a DNA sequence with a majority of GC base pairs.¹² Therefore, we prepared additional analogues of this series of azaterphenyl diamidines in order to investigate structure–activity relationships (SAR) and their DNA binding profiles. More recently, we have reported that this series of terphenyl and azaterphenyl diamidines exhibit potent in vitro antileishmanial activity.¹³ The results indicate that the antileishmanial activity of these dications strongly depends on the ring N-atom position relative to the amidine groups and correlates with the DNA minor groove binding affinity. Here, we describe their synthesis, in vitro activities against T. b. r. and P. f. and in vivo efficacy in the T. b. r. STIB 900 acute mouse model.

2. Results and discussion

2.1. Chemistry

The syntheses of the azaterphenyl diamidines begins with Suzuki coupling of the appropriate aryl halides with the corresponding aryl boronic acids or esters to yield the azaterphenyl bisnitriles (Schemes 1–3).¹¹ The bis-nitriles were converted to the diamidines by the action of lithium trimethylsilylamide [LiN(TMS)₂] in THF. As illustrated in Scheme 1, azaterphenyl diamidine analogues 10a–c with nitrogen atom(s) in both of the terminal aryl rings (pyridyl, pyrimidinyl and pyrazinyl ring) were prepared from 5-bromo-2-pyridinecarbonitrile 7a, 5-bromo-2-pyrimidine-carbonitrile 7b and 5-chloro-2-pyrazinecarbonitrile 7c¹⁴ by coupling with 1,4-phenylenebisboronic acid 8 in two steps. Scheme 2 shows the syntheses of the diamidines 10a–c with the two nitrogen atoms in the central aryl ring (pyrazinyl, pyridazinyl and pyrimidnyl ring) as well as pyridinyl rings as both the terminal units. Scheme 3 outlines the approach used to prepare the compounds 19a–g with phenyl or pyridyl ring in the central ring and nitrogen atom(s) in one terminal aryl ring. The bis-nitriles 18a–g were obtained from 4-bromophenylboronic acid 16a or 2-chloropyridine-5-boronic acid 16b in two step Suzuki coupling reactions.

Scheme 1.

Reagents and conditions: (i) Pd(PPh₃)₄, Na₂CO₃, toluene, 80 °C; (ii) (a) LiN(TMS)₂, THF; (b) HCl (gas), EtOH.

Scheme 3.

Reagents and conditions: (i) Pd(pph₃)₄, NaCO₃, toluene. 80 °C; (ii) 4-cyanophenylboronic acid, Pd(PPh₃)₄, NaCO₃, toluene, 80 °C; (iii) (a) LiN(TMS)₂, THF; (b) HCl (gas), EtOH.

Scheme 2.

Reagents and conditions: (i) Pd(PPh₃)₄, Na₂CO₃, toluene, 80 °C; (ii) (a) LiN(TMS)₂, THF; (b) HCl (gas), EtOH.

2.2. Biology

The results for the evaluation of the 13 azaterphenyl diamidine analogues against T. b. r. STIB900 and P. f. K1¹⁶ and their DNA binding affinities¹³ are shown in Table 1. For comparative and SAR purposes, the analogous data for pentamidine 1, furamidine 2a, azafuramidine 3a and the terphenyl analogues 6a–d are also included in Table 1. Six of the 13 compounds showed antitrypanosomal IC₅₀ values ≤7 nM, comparable to that of pentamidine and furamidine. Another six compounds exhibited IC₅₀ values between 7 and 32 nM, and only one compound 10c gave poor antitrypanosomal activity (IC₅₀ > 7 μM). More interestingly, these azaterphenyl dications exhibited very potent activities against P. f. in vitro. Six of the 13 compounds showed IC₅₀ values ≤0.6 nM, which are over 25-fold more potent than furamidine (IC₅₀ = 15.5 nM). Another six of the compounds exhibited IC₅₀ values between 1.1 and 5.7 nM, and only one compound 10c gave moderate antimalaria activity (IC₅₀ = 65 nM). Two of the most active compounds 15a and 15c showed more than 40-fold selectivity for P. f. compared to T. b. r. Compound 15c was 106 times more active against P. f. (IC₅₀ = 0.3 nM) than against T. b. r. (IC₅₀ = 32 nM). Compound 15c is one of the most potent compounds against P. f. K1 in the STI in vitro screen.

Table 1.

DNA affinities and antiprotozoan activity for azaterphenyl diamidines

Code	Aryl type	A	B	D	M	G	Q	ΔTma (°C)	T. b. r.b IC50 (nM)	P. f.b IC50 (nM)	Selectivity c	Cytotoxicityd IC50 (nM)
1	/	/	/	/	/	/	/	12.6	2.2	46.4	0.05	2100
2a	/	/	/	/	/	/	/	25	4.5	15.5	0.3	6400
3a	/	/	/	/	/	/	/	19.3	6.5	6.5	1.0	77,900
6a	I	CH	CH	CH	CH	CH	CH	17.1	5	1.4	3.6	22,100
6b	I	CH	CH	CH	N	CH	CH	9.2	47	10.1	4.7	25,600
6c	I	N	CH	CH	CH	CH	CH	18.7	2	10	0.2	49,900
6d	I	N	CH	N	CH	CH	CH	8.1	16	0.9	17.8	40,900
10a	I	CH	CH	CH	CH	N	CH	17	1	0.4	2.5	1200
10b	I	CH	CH	CH	CH	N	N	12.8	6	0.6	10.0	2200
10c	I	CH	CH	CH	N	CH	N	3.9	7131	65.4	109.0	46,500
15a	I	CH	N	N	CH	CH	CH	8.0	18	0.4	45.0	42,500
15b	I	N	N	CH	CH	CH	CH	16.9	14	1.3	10.8	31,300
15c	I	N	CH	N	CH	N	CH	6.8	32	0.3	106.7	5300
19a	II	CH	/	/	CH	N	CH	19.5	3	0.3	10	2800
19b	II	CH	/	/	N	CH	CH	11.7	11	1.3	8.5	26,700
19c	II	CH	/	/	CH	N	N	16.0	4	0.6	6.7	4700
19d	II	N	/	/	CH	N	CH	15.2	6	1.1	5.5	19,900
19e	II	N	/	/	N	CH	CH	13.8	7	2.5	2.8	28,300
19f	II	N	/	/	CH	N	N	14.9	12	1.5	8.0	6400
19g	II	N	/	/	N	CH	N	12.1	14	5.7	2.5	93,400

^aIncrease in thermal melting of poly(dA–dT)₂; see Refs. 11 and 13.

^bThe T. b. r. (Trypanosoma brucei rhodesiense) strain was STIB900, and the P. f. (Plasmodium falciparum) strain was K1. The Values were average of duplicate determinations; see Ref. 16.

^cSelectivity was the ratio [IC₅₀ (T. b. r.)/IC₅₀ (P. f.)].

^dCytotoxicity was evaluated using cultured L6 rat myoblast cells; see Refs. 13 and 17.

Our previous study has shown that the number and location of nitrogen atoms has a significant impact on the antileishmanial activity for these linear rigid-rod systems.¹³ The results from the current study of these azaterphenyl compounds against T. b. r. and P. f. are generally consistent with that observed in the antileishmanial study. Compounds 10a and 19a, in which a nitrogen atom has been replaced ortho to one or both of the amidine groups, showed a five- and two-fold increase in potency compared to the parent terphenyl diamidine 6a against T. b. r. and a three- and four-fold increased potency against P. f. The two isomeric compounds 6b and 19b, in which the nitrogen atom is meta to the amidine, exhibited a significant loss in potency against both T. b. r. and P. f., compared to 10a and 19a. Introduction of two nitrogen atoms ortho to one amidine, 19c, resulted in a modest reduction of activity against both T. b. r. and P. f. compared to 19a. Interestingly, if two nitrogen atoms are introduced ortho to both of the amidine groups, 10b, a six-fold reduction in activity against T. b. r. was observed, conversely a slight enhancement in activity against P. f. was seen. For compound 10c, in which one of the nitrogen atoms is meta and another is ortho to both amidine units, a significant reduction in activity against both T. b. r. and P. f. was seen. Introduction of one nitrogen atom in the central ring (6c) led to a two-fold increase in activity against T. b. r., however, a seven-fold reduction in activity against P. f., compared to the parent terphenyl compound 6a.¹¹ In contrast, introduction of two nitrogen atoms in the central ring (6d, 15a–c) resulted in a more than two-fold reduction in activity against T. b. r., however, a three-fold increase in activity against P. f., except 15b which showed comparable activity, compared to the parent terphenyl compound 6a. Introduction of one nitrogen atom in the central ring (6d) in compound 19a with a nitrogen atom in one terminal aryl ring, led to moderate reduction in activity against T. b. r. and P. f. However, it is noteworthy that introduction of two nitrogen atoms in the central ring (6e and 6f) in the compounds 19b and 19c showed no apparent effect on either antitrypanosomal or antiplasmodial activity.

The ΔTm values of these linear azaterphenyl diamidines, which range from high values of 17–19 °C to low ones of 4–6 °C, are lower than that of crescent shape molecules e.g. furamidine (ΔTm=25 °C).¹³ Our previous study found that the compounds which exhibit the higher ΔTm values showed the higher antileishmanial activity and the weaker binding compounds show low activity.¹³ A similar trend is found for T. b. r., but not for P. f. The potent antitrypanosomal compounds, which showed IC₅₀ values less than 7 nM, exhibited higher ΔTm values (12 °C or greater). On the other hand, the less potent antitrypanosomal compounds, which showed IC₅₀ values of more than 18 nM, exhibited lower ΔTm values (less than 10 °C). These results are generally consistent with the observed relationship between DNA binding affinities and antileishmanial activity of this series azaterphenyl diamidines. However, the correlation between ΔTm values and antiplasmodial activity is complex. For example, the potent compounds 6d, 15a and 15c which IC₅₀ values of ≤0.9 nM showed ΔTm values of 6.8–8.1 °C. In contrast, the potent compounds 10a, 10b, 19a and 19c with IC₅₀ values of ≤0.6 nM showed significantly higher ΔTm values of 12.8–19.5 °C. Clearly, other factors such as transport, induced changes in DNA topology or protein–DNA complex inhibition, are important in determining the antiplasmodial activity of these linear-rod molecules. It is perhaps also significant that diamidine compounds localize only in the nuclear DNA of plasmodia¹⁵ but they are in both the mitochondrial kinetoplast DNA and in the nucleus in trypanosomes.^8c

Given the promising in vitro T. b. r. activity of these analogues, we have evaluated them in the stringent STIB900 acute mouse model for T. b. r.¹⁶ The results are shown in Table 2 and for comparative purposes the in vivo data for pentamidine 1, furamidine 2a, azafuramidine 3a and the terphenyl analogues 6a–d in the same model are also presented. On intraperitoneal dosing all of the dications showed a significant increase in survival time for the treated animals compared to untreated controls. Three of the thirteen compounds, 15b, 19d and 19e, gave superior and four compounds, 10b, 19b, 19c and 19g, identical results to that for furamidine in this model at the dose of 5 mg/kg. Five compounds were not able to cure any mice although they extended the survival significantly. Two compounds 15b and 19d were as effective as the azafuramdine 3a giving 3/4 cures at 5 mg/kg. The best result obtained was for compound 19e, which showed 4/4 cures at a dose of 5 mg/kg. It is noteworthy that although the compounds 6b and 19b, in which the nitrogen atom is meta to the amidine, exhibited a significant loss of in vitro potency against T. b. r. compared to their isomeric compounds 10a and 19a, in which the nitrogen atom is ortho to the amidine, the in vivo efficacy of 6b and 19b (2/4 and 1/4 cure, respectively) was superior to the ortho isomers 10a and 19a (0/4 cure). The pyridinyl analogue 19e was also more effective than the ortho isomer 19d in this mouse model, even though these two isomers exhibited almost similar in vitro antitrypanosomal activity. These results are consistent with those observed for the azafuramidine system.⁴ Since previous study of the bis-amidoximes and bis-O-methylamidoximes prodrugs of the terphenyl diamidine analogues showed poor bioconversion and were not effective on oral administration,¹¹ we did not prepare their bis-amidoximes and bis-O-methylamidoximes prodrugs during this study. However, further studies to develop novel orally effective prodrugs of this highly active series of azaterphenyl diamidines are underway.

Table 2.

In vitro and in vivo anti-trypanosomal activity of azaterphenyl diamidines in the STIB900 mouse model^a

Code	T. b. r. IC50 (nM)	Dosageb (ip, mg/kg)	Cures c	Survival (days) d
1	2.2	20	2/4	>57.5
		5	1/4	>38
2a	4.5	20	3/4	>57.75
		5	1/4	>46
3a	6.5	20	4/4	>60
		5	3/4	>54.5
6a	5	20	2/4	>47.75
6b	47	5	2/4	>60
6c	2	5	2/4	>42
6d	16	5	0/4	23.5
10a	1	5	0/4	>51.5
10b	6	5	1/4	>43.25
10c	7131	NA
15a	18	5	0/4	36.5
15b	14	5	3/4	>49.25
15c	32	5	0/4	34
19a	3	5	0/4	>48.75
19b	11	5	1/4	>56.0
19c	4	5	1/4	>46.25
19d	6	5	3/4	>60.0
19e	7	5	4/4	>60.0
19f	12	5	0/4	>43.25
19g	14	5	1/4	>31.75

^aSee Ref. 16 for details of STIB900 mouse model.

^bDosage was for four days; ip, intraperitoneal.

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

^dAverage days of survive; untreated control expires between day 7 and 9 post-infection.

In summary, we have prepared a series of azaterphenyl diamidine analogues that exhibit potent in vitro activity against both T. b. r. and P. f., and showed promising activity on intraperitoneal administration in the T. b. r. STIB900 acute mouse model. Six of the 13 compounds showed IC₅₀ values ≤0.6 nM against P. f., and two of them showed more than 40-fold selectivity for P. f. versus T. b. r. Three compounds 15b, 19d and 19e exhibited in vivo efficacy (3/4 or 4/4 cure at 5 mg/kg dosage, ip) in the stringent STIB900 model much superior to that of furamidine, and comparable to or better than azafuramidine. SAR information shows that the number and location of nitrogen atoms has a significant effect on antiparasitic activity. This series of azaterphenyl diamidines merit further investigation as novel potent antiparasitic agents.

3. Experimental

3.1. Biology

3.1.1. Efficacy evaluation

In vitro assays with T. b. r. STIB900 and P. f. K1 strain as well as the efficacy study in an acute mouse model for T. b. r. STIB900 were carried out as previously reported.¹⁶

3.2. Chemistry

Melting points were determined on a Mel-Temp 3.0 melting point apparatus, and are uncorrected. TLC analysis was carried out on Silica Gel 60 F254 precoated aluminum sheets using UV light for detection. ¹H and ¹³C NMR spectra were recorded on a Varian Unity Plus 300 MHz or Bruker 400 MHz spectrometer using indicated solvents. Mass spectra was obtained from the Georgia State University Mass Spectrometry Laboratory, Atlanta, GA. Elemental analysis were performed by Atlantic Microlab Inc., Norcross, GA, and are within ±0.4 of the theoretical values. The compounds reported as salts frequently analyzed correctly for fractional moles of water and/or ethanol of solvation. In each case, proton NMR showed the presence of the indicated solvents. All chemicals and solvents were purchase from Aldrich Chemical Co., VWR International or Frontier Scientific.

3.2.1. 1,4-Bis-(2′-cyanopyridin-5′-yl)phenylene (9a)

To a stirred solution of 5-bromo-2-pyridinecarbonitrile 7a (6.60 g, 36.0 mmol), and tetrakis(triphenylphosphine) palladium (1.80 g, 1.56 mmol) in toluene (100 mL) under a nitrogen atmosphere was added 50 mL of a 2 M aqueous solution of Na₂CO₃ followed by 1,4-phenylenebisboronic acid 8 (3.90 g, 24.0 mmol) in 50 mL of methanol. The vigorously stirred mixture was heat at 80 °C overnight. After cooling, the solution was filtered, and the precipitate was washed with toluene, water and ether, to afford the title compound as a white solid 9a (4.70 g, 92% yield); mp > 300 °C. ¹H NMR (DMSO-d₆): δ 7.99 (s, 4H), 8.08 (d, J = 8.0 Hz, 2H), 8.38 (dd, J = 2.0, 8.0 Hz, 2H), 9.14 (d, J = 2.0 Hz, 2H). Anal. Calcd for C₁₈H₁₀N₄–0.3H₂O: C, 75.14; H, 3.71; N, 19.47. Found: C, 75.37; H, 3.51; N, 19.15.

3.2.2. 1,4-Bis-(2′-amidinopyridin-5′-yl)-phenylene hydrochloride salt (10a)

The dinitrile 9a (570 mg, 2.0 mmol), suspended in freshly distilled THF (30 mL), was treated with lithium trimethylsilylamide (1 M solution in THF, 15 mL, 15.0 mmol), and the reaction was allowed to stir overnight at room temperature. The reaction mixture was then cooled to 0 °C and HCl saturated ethanol (15 mL) was added, whereupon a precipitate started forming. The mixture was left to stir overnight, after which it was diluted with ether, and the resultant solid was collected by filtration. The diamidine was purified by neutralization with 1 N NaOH followed by filtration of the resultant solid and washing with water (3×). Finally, the dried free base was stirred with ethanolic HCl overnight and diluted with ether, and the solid which formed was filtered and dried to give diamidine salt 10a in 68% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.11 (s, 4H), 8.47 (d, J = 8.4 Hz, 2H), 8.60 (dd, J = 2.0, 8.4 Hz, 2H), 9.24 (d, J = 2.0 Hz, 2H), 9.41 (s, 4H), 9.66 (s, 4H). ¹³C NMR (DMSO-d₆): δ 161.6, 147.4, 142.5, 138.5, 135.8, 135.4, 127.8, 123.1. HRMS: m/z 317.1517 (M+1) (calculated for C₁₈H₁₇N₆, 317.1515). Anal. Calcd for C₁₈H₁₆N₆–2.0HCl–0.3H₂O: C, 54.78; H, 4.75; N, 21.29. Found: C, 55.03; H, 4.60; N, 21.01.

3.2.3. 1,4-Bis-(2′-amidinopyrimidin-5′-yl)-phenylene hydrochloride salt (10b)

The same procedure described for 1,4-bis-(2′-cyanopyridin-5′-yl)phenylene 9a was used by employing 5-bromo-2-pyrimidine-carbonitrile 7b and 1,4-phenylenebisboronic acid 8 to furnish 1,4-bis-(2′-cyanopyrimidin-5′-yl)-phenylene 9b in 69% yield; mp >300 °C.¹H NMR (DMSO-d₆): δ 8.16 (s, 4H), 9.50 (s, 4H). The compound was used directly in the next step.

The same procedure described for the preparation of 10a was used starting with the dinitrile 9b; 68% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.24 (s, 4H), 9.57 (s, 4H), 9.60 (s, 4H), 9.76 (s, 4H). HRMS: m/z 319.1418 (M+1) (calculated for C₁₆H₁₅N₈, 319.1420). Anal. Calcd for C₁₆H₁₄N₈–2.0HCl–2.5H₂O: C, 44.05; H, 4.83; N, 25.68. Found: C, 44.39; H, 4.55; N, 25.28.

3.2.4. 1,4-Bis-(2′-cyanopyrazin-5′-yl)phenylene (9c)

The same procedure described for 1,4-bis-(2′-cyanopyridin-5′-yl)phenylene 9a was used by employing 5-chloro-2-pyrazinecarbonitrile 7c¹⁴ and 1,4-phenylenebisboronic acid 8 to furnish the title compound 9c in 74% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.39 (s, 4H), 9.22 (s, 2H), 9.69 (s, 2H). ¹³C NMR (DMSO-d₆): δ 151.1, 147.2, 145.9, 136.2, 129.1, 128.0, 116.1. HRMS: m/z 285.0886 (M+1) (calculated for C₁₆H₉N₆, 285.0889).

3.2.5. 1,4-Bis-(2′-amidinopyrazin-5′-yl)-phenylene hydrochloride salt (10c)

The same procedure described for the preparation of 10a was used starting with the dinitrile 9c; 55% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.64 (s, 4H), 9.47 (s, 2H), 9.71 (s, 4H), 9.78 (s, 2H), 9.92 (s, 4H). HRMS: m/z 319.1419 (M+1) (calculated for C₁₆H₁₅N₈, 319.1420). Anal. Calcd for C₁₆H₁₄N₈–2.0HCl–0.65H₂O: C, 47.69; H, 4.33; N, 27.81. Found: C, 48.04; H, 4.18; N, 27.52.

3.2.6. 2,5-Bis-(4′-cyanophenyl)-pyrazine (14a)

The same procedure described for 1,4-bis-(2′-cyanopyridin-5′-yl)phenylene 9a was used by employing 2,5-dibromopyrazine 11a¹⁸ and 4-cyanophenylboronic acid 12 to furnish the title compound 14a in 52% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.99 (d, J = 8.4 Hz, 4H), 8.39 (d, J = 8.4 Hz, 4H), 9.42 (s, 2H). Anal. Calcd for C₁₈H₁₀N₄–0.3H₂O: C, 75.14; H, 3.71; N, 19.47. Found: C, 75.26; H, 3.47; N, 19.70.

3.2.7. 2,5-Bis-(4′-amidinophenyl)-pyrazine hydrochloride salt (15a)

The same procedure described for the preparation of 10a was used starting with the dinitrile 14a; 76% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.05 (d, J = 8.4 Hz, 4H), 8.45 (d, J = 8.4 Hz, 4H), 9.37 (s, 4H), 9.53 (s, 2H), 9.58 (s, 4H). ¹³C NMR (DMSO-d₆): δ 165.1, 149.0, 142.1, 140.2, 129.2, 129.0, 127.0. Anal. Calcd for C₁₈H₁₆N₆–2.0HCl–0.55H₂O: C, 54.16; H, 4.82; N, 21.05. Found: C, 54.41; H, 4.75; N, 20.71.

3.2.8. 2,5-Bis-(4′-cyanophenyl)-pyridazine (14b)

The same procedure described for 1,4-bis-(2′-cyanopyridin-5′-yl)phenylene 9a was used employing 3,6-dichloropyridazine 11b and 4-cyanophenylboronic acid 12 to furnish the title compound 14b in 80% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.05 (d, J = 8.4 Hz, 4H), 8.43 (d, J = 8.4 Hz, 4H), 8.51 (s, 2H). MS: m/z 282 (M+1). Anal. Calcd for C₁₈H₁₀N₄–0.25H₂O: C, 75.38; H, 3.69; N, 19.54. Found: C, 75.11; H, 3.56; N, 19.75.

3.2.9. 2,5-Bis-(4′-amidinophenyl)-pyridazine hydrochloride salt (15b)

The same procedure described for the preparation of 10a was used starting with the dinitrile 14b; 72% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.61 (d, J = 8.4 Hz, 4H), 8.06 (d, J = 8.4 Hz, 4H), 8.13 (s, 2H), 8.83 (s, 4H), 9.10 (s, 4H). Anal. Calcd for C₁₈H₁₆N₆-2.5HCl-0.5EtOH: C, 53.00; H, 5.03; N, 19.52. Found: C, 53.27; H, 4.85; N, 19.38.

3.2.10. 2,5-Bis-(2′-cyanopyridin-5′-yl)-pyrimidine (14c)

The same procedure described for 1,4-bis-(2′-cyanopyridin-5′-yl)phenylene 9a was used employing 5-bromo-2-chloropyrimidine 11c and 2-cyanopyridine-5-boronic acid pinacol ester 13 to furnish the title compound 14c in 78% yield; mp >300 °C. ¹H NMR (DMSO-d₆):): δ 8.24 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.61 (dd, J = 2.0, 8.4 Hz, 1H), 8.93 (dd, J = 2.0, 8.4 Hz, 1H), 9.32 (d, J = 2.0 Hz, 1H), 9.50 (s, 2H), 9.68 (d, J = 2.0 Hz, 1H). HRMS: m/z 285.0884 (M+1) (calculated for C₁₆H₉N₆, 285.0875).

3.2.11. 2,5-Bis-(2′-amidinopyridin-5′-yl)-pyrimidine hydrochloride salt (15c)

The same procedure described for the preparation of 10a was used starting with the dinitrile 14c; 67% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.51 (d, J = 8.4 Hz, 1H), 8.53 (d, J = 8.4 Hz, 1H), 8.77 (dd, J = 2.0, 8.4 Hz, 1H), 9.08 (dd, J = 2.0, 8.4 Hz, 1H), 9.40 (d, J = 2.0 Hz, 1H), 9.47 (s, 4H), 9.60 (s, 2H), 9.72 (s, 2H), 9.75 (s, 2H), 9.76 (d, J = 2.0 Hz, 1H). HRMS: m/z 319.1425 (M+1) (calculated for C₁₆H₁₅N₈, 319.1420). Anal. Calcd for C₁₆H₁₄N₈–2.0HCl–0.8H₂O: C, 47.37; H, 4.37; N, 27.62. Found: C, 47.47; H, 4.14; N, 27.32.

3.2.12. 5-(4′-Bromophenyl)-2-pyridinecarbonitrile (17a)

5-Bromo-2-cyanopyridine 7a and 4-bromophenylboronic acid 16a were reacted under the above-mentioned Suzuki coupling conditions to give the target nitrile 17a, which was purified by column chromatography (EtOAc/hexane, 80:20); yield 82%; mp 138–140 °C. ¹H NMR (DMSO-d₆): δ 7.73 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 8.10 (d, J = 8.0 Hz, 1H), 8.34 (dd, J = 2.4, 8.0 Hz, 1H), 9.08 (d, J = 2.4 Hz, 1H). ¹³C NMR (DMSO-d₆): δ 149.2, 137.8, 135.4, 134.5, 132.2, 131.4, 129.5, 129.1, 123.2, 117.6. HRMS: m/z 258.9866 (M+1) (calculated for C₁₂H₈N₂Br, 258.9871).

3.2.13. Phenyl[1,1′]phenyl[4′,5″]pyridinyl-4,2″-bis-carbonitrile (18a)

The nitrile 17a and 4-cyanophenylboronic acid were reacted under the above-mentioned Suzuki coupling conditions to give the target dinitrile 18a; yield 84%; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.90–7.97 (m, 8H), 8.08 (d, J = 8.4 Hz, 1H). 8.38 (dd, J = 2.0, 8.4 Hz, 1H), 9.13 (d, J = 2.0 Hz, 1H). Anal. Calcd for C₁₉H₁₁N₃–0.3H₂O: C, 79.59; H, 4.08; N, 14.66. Found: C, 79.65; H, 3.91; N, 14.43.

3.2.14. Phenyl[1,1′]phenyl[4′,5″]pyridinyl-4,2″-bis-amidine hydrochloride salt (19a)

The same procedure described for the preparation of 10a was used starting with the dinitrile 18a; 65% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.97 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.44 (d, J = 8.4 Hz, 1H), 8.58 (dd, J = 2.0, 8.4 Hz, 1H), 9.16 (s, 2H), 9.22 (d, J = 2.0 Hz, 1H), 9.40 (s, 2H), 9.44 (s, 2H), 9.64 (s, 2H). HRMS: m/z 316.1551 (M+1) (calculated for C₁₉H₁₈N₅, 316.1562). Anal. Calcd for C₁₉H₁₇N₅–2.0HCl–0.6H₂O: C, 57.18; H, 5.10; N, 17.55. Found: C, 57.46; H, 5.08; N, 17.25.

3.2.15. 2-(4′-Bromophenyl)-5-pyridinecarbonitrile (17b)

The same procedure described for 5-(4′-bromophenyl)-2-pyridinecarbonitrile 17a was used by employing 2-bromo-5-cyanopyridine 7d and 4-bromophenylboronic acid 16a to furnish the title compound 17b in 78% yield; mp 157–159 °C. ¹H NMR (DMSO-d₆): δ 7.74 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 8.8 Hz, 2H), 8.22 (d, J = 8.4 Hz, 1H), 8.40 (dd, J = 2.4, 8.4 Hz, 1H), 9.09 (d, J = 2.4 Hz, 1H). ¹³C NMR (DMSO-d₆): δ 157.9, 152.6, 141.1, 136.1, 132.0, 129.2, 124.4, 120.2, 117.2, 107.7. HRMS: m/z 258.9871 (M+1) (calculated for C₁₂H₈N₂Br, 258.9871).

3.2.16. Phenyl[1,1′]phenyl[4′,2″]pyridinyl-4,5″-bis-carbonitrile (18b)

The nitrile 17b and 4-cyanophenylboronic acid were reacted under the above-mentioned Suzuki coupling conditions to give the target dinitrile 18b; yield 94%; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.68 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.0 Hz, 1H). 8.42 (dd, J = 2.0, 8.0 Hz, 1H), 9.17 (d, J = 2.0 Hz, 1H). HRMS: m/z 282.1038 (M+1) (calculated for C₁₉H₁₂N₃, 282.1031).

3.2.17. Phenyl[1,1′]phenyl[4′,2″]pyridinyl-4,5″-bis-amidine hydrochloride salt (19b)

The same procedure described for the preparation of 10a was used starting with the dinitrile 18b; 75% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.98 (d, J = 8.4 Hz, 4H), 8.04 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.33–8.37 (m, 4H), 9.11 (s, 1H), 9.26 (s, 2H), 9.38 (s, 2H), 9.49 (s, 2H), 9.66 (s, 2H). ¹³C NMR (DMSO-d₆): δ 165.2, 163.8, 159.3, 149.0, 144.2, 140.0, 137.5, 137.1, 128.9, 127.9, 127.7, 127.6, 127.1, 123.0, 119.9. Anal. Calcd for C₁₉H₁₇N₅–2.0HCl–0.9H₂O: C, 56.42; H, 5.18; N, 17.30. Found: C, 56.74; H, 4.94; N, 16.90.

3.2.18. Phenyl[1,1′]phenyl[4′,5″]pyrimidinyl-4,2″-biscarbonitrile (18c)

The same procedure described for 5-(4′-bromophenyl)-2-pyridinecarbonitrile 17a was used by employing 5-bromo-2-cyanopyrimidine 7b and 4-bromophenylboronic acid 16a to furnish 5-(4′-Bromophenyl)-2-pyrimidinecarbonitrile 17c in 58% yield; mp 202–204 °C. ¹H NMR (DMSO-d₆): δ 7.79 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 9.39 (s, 2H). The compound was used directly in the next step. The above nitrile 17c and 4-cyanophenylboronic acid were reacted under the above-mentioned Suzuki coupling conditions to give the target dinitrile 18c; yield 74%; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.95–8.01 (m, 6H), 8.08 (d, J = 8.4 Hz, 2H), 9.47 (s, 2H). HRMS: m/z 283.0983 (M+1) (calculated for C₁₈H₁₁N₄, 283.0984).

3.2.19. Phenyl[1,1′]phenyl[4′,5″]pyrimidinyl-4,2″-bis-amidine hydrochloride salt (19c)

The same procedure described for the preparation of 10a was used starting with the dinitrile 18c; 75% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.97 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.15 (d, J = 8.4 Hz, 2H), 9.13 (s, 2H), 9.44 (s, 2H), 9.54 (s, 2H), 9.57 (s, 2H), 9.76 (s, 2H). ¹³C NMR (DMSO-d₆): δ 165.1, 159.5, 155.6, 151.6, 144.0, 139.7, 135.0, 132.5, 128.9, 128.4, 128.0, 127.3, 127.2. Anal. Calcd for C₁₈H₁₆N₆–2.0HCl–0.6H₂O: C, 54.04; H, 4.84; N, 21.00. Found: C, 54.29; H, 4.68; N, 20.86.

3.2.20. 5-(2′-Chloropyridin-5′-yl)-2-pyridinecarbonitrile (17d)

The same procedure described for 5-(4′-bromophenyl)-2-pyridinecarbonitrile 17a was used by employing 5-bromo-2-cyanopyridine 7a and 2-chloropyridine-5-boronic acid 16b to furnish the title compound 17d in 98% yield; mp 220–224 °C. ¹H NMR (DMSO-d₆): δ 8.22 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 8.4 Hz, 1H), 8.51 (dd, J = 2.0, 8.4 Hz, 1H), 8.75 (dd, J = 2.0, 8.0 Hz, 1H), 9.19 (d, J = 2.0 Hz, 1H), 9.48 (d, J = 2.0 Hz, 1H). ¹³C NMR (DMSO-d₆): δ 151.1, 149.4, 148.6, 135.9, 135.0, 132.0, 130.6, 129.1, 124.6, 117.5. HRMS: m/z 216.0334 (M+1) (calculated for C₁₁H₇N₃Cl, 216.0329).

3.2.21. Phenyl[1,2′]pyridinyl[5′,5″]pyridinyl-4,2″-bis-carbonitrile (18d)

The nitrile 17d and 4-cyanophenylboronic acid were reacted under the above-mentioned Suzuki coupling conditions to give the target dinitrile 18d; yield 80%; mp 270–272 °C. ¹H NM R¹H NMR (DMSO-d₆): δ 7.99 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.4 Hz, 2H), 8.17 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.39 (dd, J = 2.0, 8.4 Hz, 1H), 8.75 (dd, J = 2.0, 8.4 Hz, 1H), 9.18 (d, J = 1.6 Hz, 1H), 9.52 (d, J = 2.0 Hz, 1H). ¹³C NMR (DMSO-d₆): δ 151.6, 148.9, 148.0, 140.5, 136.3, 135.5, 134.9, 133.7, 132.5, 132.3, 128.6, 127.4, 121.4, 118.1, 117.0, 110.9. HRMS: m/z 283.0974 (M+1) (calculated for C₁₈H₁₁N₄, 283.0984).

3.2.22. Phenyl[1,2′]pyridinyl[5′,5″]pyridinyl-4,2″-bis-amidine hydrochloride salt (19d)

The same procedure described for the preparation of 10a was used starting with the dinitrile 18d; 71% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.00 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 8.4 Hz, 1H), 8.43 (d, J = 8.4 Hz, 2H), 8.47–8.51 (m, 2H), 8.66 (dd, J = 2.0, 8.4 Hz, 1H), 9.18 (s, 2H), 9.26 (d, J = 2.0 Hz, 1H), 9.30 (d, J = 2.0 Hz, 1H), 9.42 (s, 2H), 9.46 (s, 2H), 9.66 (s, 2H). HRMS: m/z 317.1502 (M+1) (calculated for C₁₈H₁₇N₆, 317.1515). Anal. Calcd for C₁₈H₁₆N₆–2.0HCl–1.0H₂O: C, 53.08; H, 4.95; N, 20.63. Found: C, 53.28; H, 4.59; N, 20.34.

3.2.23. 2-(2′-Chloropyridin-5′-yl)-5-pyridinecarbonitrile (17e)

The same procedure described for 5-(4′-bromophenyl)-2-pyridinecarbonitrile 17a was used by employing 2-bromo-5-cyanopyridine 7d and 2-chloropyridine-5-boronic acid 16b to furnish the title compound 17e in 83% yield; mp 198–200 °C. ¹H NMR (DMSO-d₆): δ 7.68 (d, J = 8.4 Hz, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.45 (dd, J = 2.0, 8.4 Hz, 1H), 8.55 (dd, J = 2.4, 8.4 Hz, 1H), 9.12 (d, J = 2.0 Hz, 1H), 9.15 (d, J = 2.4 Hz, 1H). ¹³C NMR (DMSO-d₆): δ 155.8, 152.7, 152.0, 148.7, 141.3, 138.1, 132.0, 124.6, 120.8, 117.0, 108.4. HRMS: m/z 216.0323 (M+1) (calculated for C₁₁H₇N₃Cl, 216.0329).

3.2.24. Phenyl[1,2′]pyridinyl[5′,2″]pyridinyl-4,5″-bis-carbonitrile (18e)

The nitrile 17e and 4-cyanophenylboronic acid were reacted under the above-mentioned Suzuki coupling conditions to give the target dinitrile 18e; yield 76%; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.00 (d, J = 8.4 Hz, 2H), 8.29 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 8.4 Hz, 3H), 8.48 (dd, J = 2.0, 8.4 Hz, 1H), 8.68 (dd, J = 2.0, 8.4 Hz, 1H), 9.16 (d, J = 2.0 Hz, 1H), 9.48 (d, J = 2.0 Hz, 1H). HRMS: m/z 283.0976 (M+1) (calculated for C₁₈H₁₁N₄, 283.0984).

3.2.25. Phenyl[1,2′]pyridinyl[5′,2″]pyridinyl-4,5″-bis-amidine hydrochloride salt (19e)

The same procedure described for the preparation of 10a was used starting with the dinitrile 18e; 57% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.00 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 8.4 Hz, 1H), 8.42–8.44 (m, 4H), 8.71 (dd, J = 2.0, 8.4 Hz, 1H), 9.14 (d, J = 2.0 Hz, 1H), 9.21 (s, 2H), 9.35 (s, 2H), 9.48 (s, 2H), 9.52 (d, J = 2.0 Hz, 1H), 9.65 (s, 2H). ¹³C NMR (DMSO-d₆): δ 165.2, 163.7, 157.4, 155.4, 149.2, 148.5, 142.6, 137.7, 136.0, 132.3, 128.8, 128.7, 127.1, 123.7, 121.3, 120.4. Anal. Calcd for C₁₈H₁₆N₆–2.5HCl–1.5H₂O: C, 49.75; H, 4.99; N, 19.34. Found: C, 50.03; H, 4.73; N, 19.25.

3.2.26. 5-(2′-Chloropyridin-5′-yl)-2-pyrimidinecarbonitrile (17f)

The same procedure described for 5-(4′-bromophenyl)-2-pyridinecarbonitrile 17a was used by employing 5-bromo-2-cyanopyrimidine 7b and 2-chloropyridine-5-boronic acid 16b to furnish the title compound 17f in 27% yield; mp 158–160 °C. ¹H NMR (DMSO-d₆): δ 7.77 (d, J = 8.4 Hz, 2H), 8.41 (dd, J = 2.4, 8.4 Hz, 1H), 8.97 (d, J = 2.4 Hz, 1H), 9.46 (s, 2H). ¹³C NMR (DMSO-d₆): δ 156.5, 151.7, 148.7, 143.1, 138.6, 132.0, 127.8, 124.8, 116.1. HRMS: m/z 217.0288 (M+1) (calculated for C₁₀H₆N₄Cl, 217.0281).

3.2.27. Phenyl[1,2′]pyridinyl[5′,5″]pyrimidinyl-4,2″-bis-carbonitrile (18f)

The nitrile 17f and 4-cyanophenylboronic acid were reacted under the above-mentioned Suzuki coupling conditions to give the target dinitrile 18f; yield 77%; mp 272–274 °C. ¹H NMR (DMSO-d₆): δ 8.01 (d, J = 8.4 Hz, 2H), 8.34 (d, J = 8.4 Hz, 1H), 8.40 (d, J = 8.4 Hz, 2H), 8.53 (dd, J = 2.0, 8.4 Hz, 1H), 9.28 (d, J = 2.0 Hz, 1H), 9.55 (s, 2H). ¹³C NMR (DMSO-d₆): δ 156.4, 154.9, 148.5, 142.9, 141.8, 136.3, 132.9, 132.5, 127.9, 127.5, 121.3, 118.7, 116.2, 112.0. HRMS: m/z 284.0949 (M+1) (calculated for C₁₇H₁₀N₅, 284.0936).

3.2.28. Phenyl[1,2′]pyridinyl[5′,5″] pyrimidinyl-4,2″-bisamidine hydrochloride salt (19f)

The same procedure described for the preparation of 10a was used starting with the dinitrile 18f; 51% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.99 (d, J = 8.4 Hz, 2H), 8.38 (d, J = 8.4 Hz, 1H), 8.44 (d, J = 8.4 Hz, 2H), 8.59 (dd, J = 2.0, 8.4 Hz, 1H), 9.16 (s, 2H), 9.34 (d, J = 2.0 Hz, 1H), 9.47 (s, 2H), 9.60 (s, 2H), 9.62 (s, 2H), 9.79 (s, 2H). ¹³C NMR (DMSO-d₆): δ 165.2, 159.5, 155.9, 155.1, 152.0, 148.7, 142.4, 136.5, 132.8, 128.8, 128.8, 128.1, 127.0, 121.2. HRMS: m/z 318.1464 (M+1) (calculated for C₁₇H₁₆N₇, 318.1467). Anal. Calcd for C₁₇H₁₅N₇–2.0HCl–0.6H₂O: C, 50.91; H, 4.57; N, 24.45. Found: C, 50.68; H, 4.49; N, 24.13.

3.2.29. 5-(2′-Chloropyridin-5′-yl)-2-pyrazinecarbonitrile (17g)

The same procedure described for 5-(4′-bromophenyl)-2-pyridinecarbonitrile 17a was used employing 2-chloro-5-cyanopyrazine 7c and 2-chloropyridine-5-boronic acid 16b to furnish the title compound 17g in 47% yield; mp 118–120 °C. ¹H NMR (DMSO-d₆): δ 7.75 (d, J = 8.4 Hz, 1H), 8.58 (dd, J = 2.4, 8.4 Hz, 1H), 9.18 (d, J = 2.4 Hz, 1H), 9.25 (s, 1H), 9.65 (s, 1H). ¹³C NMR (DMSO-d₆): δ 152.4, 149.1, 148.7, 147.6, 146.0, 138.2, 129.6, 129.1, 124.8, 115.9. HRMS: m/z 217.0273 (M+1) (calculated for C₁₀H₆N₄Cl, 217.0281).

3.2.30. Phenyl[1,2′]pyridinyl[5′,5″]pyrazinyl-4,2″-bis-carbonitrile (18g)

The nitrile 17g and 4-cyanophenylboronic acid were reacted under the above-mentioned Suzuki coupling conditions to give the target dinitrile 18g; yield 70%; mp >300 °C. ¹H NMR (DMSO-d₆): δ 8.01 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 8.4 Hz, 2H), 8.68 (dd, J = 2.0, 8.4 Hz, 1H), 9.25 (s, 1H), 9.48 (s, 1H), 9.72 (s, 1H). HRMS: m/z 284.0948 (M+1) (calculated for C₁₇H₁₀N₅, 284.0936).

3.2.31. Phenyl[1,2′]pyridinyl[5′,5″]pyrizinyl-4,2″-bis-amidine hydrochloride salt (19g)

The same procedure described for the preparation of 10a was used starting with the dinitrile 18g; 75% yield; mp >300 °C. ¹H NMR (DMSO-d₆): δ 7.80 (d, J = 8.4 Hz, 2H), 8.38 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 8.4 Hz, 2H), 8.97 (dd, J = 2.4, 8.4 Hz, 1H), 9.20 (s, 2H), 9.49 (s, 3H), 9.67 (s, 2H), 9.74 (d, J = 2.0 Hz, 1H), 9.79 (s, 1H), 9.91 (s, 2H). ¹³C NMR (DMSO-d₆): δ 165.2, 160.7, 155.7, 148.9, 148.4, 146.5, 142.9, 142.4, 139.4, 136.4, 129.7, 128.8, 128.7, 127.1, 121.1. Anal. Calcd for C₁₇H₁₅N₇–2.7HCl–2.0H₂O: C, 45.19; H, 4.84; N, 21.70. Found: C, 45.52; H, 4.71; N, 21.34.