Synthesis, DNA binding and antileishmanial activity of low molecular weight bis-arylimidamides

Abstract

The effects of reducing the molecular weight of the antileishmanial compound DB766 on DNA binding affinity, antileishmanial activity and cytotoxicity are reported. The bis-arylimidamides were prepared by the coupling of aryl S-(2-naphthylmethyl)thioimidates with the corresponding amines. Specifically, we have prepared new series of bis-arylimidamides which include 3a, 3b, 6, 9a, 9b, 9c, 13, and 18. Three compounds 9a, 9c, and 18 bind to DNA with similar or moderately lower affinity to that of DB766, the rest of these compounds either show quite weak binding or no binding at all to DNA. Compounds 9a, 9c, and 13 were the most active against L. amazonensis showing IC₅₀ values of less than 1 µM, so they were screened against intracellular L. donovani showing outstanding activity with IC₅₀.values of 25–79 nM. Despite exhibiting little in vitro cytotoxicity these three compounds were quite toxic to mice.

1. Introduction

In rural developing countries protozoan parasitic diseases, which have plagued mankind for centuries, continue to cause significant public health problems. Leishmaniasis, a potentially fatal protozoal tropical disease, is caused by parasites of the genus Leishmania which include as many as 20 species that are pathogenic for humans [1]. The three main clinical manifestations of leishmaniasis are cutaneous, mucocutaneous, and visceral leishmaniasis, with symptoms ranging from skin and mucosal ulceration to a systemic infection that is fatal if left untreated [2]. An estimated 12 million people are currently infected with Leishmania and up to 350 million people in 88 countries are at risk of infection [3]. Approximately 2 million new cases of leishmaniasis are believed to occur annually, 0.5 million involving visceral leishmaniasis and 1.5 million with cutaneous leishmaniasis. A number of drugs have been used to treat leishmaniasis including pentostam, glucantime, amphotericin B, miltefosine, and pentamidine, but all of these have one or more limitations, including the development of resistance, cost, parenteral administration, long treatment regimens, and various toxicities [4–8]. From this brief survey of the current status of chemotherapy for this disease, it is clear that an urgent need exists for development of new effective and safe therapies for treating leishmaniasis.

In our efforts to find promising antileishmanial agents from aromatic diamidines and their analogues, we identified a new class of molecules, arylimidamides ( I , Figure 1, AIAs; previously referred to as “reversed” amidines [9, 10], that exhibited submicromolar 50% inhibitory concentrations (IC₅₀s) in the axenic Leishmania donovani amastigote assay and the L. donovani infected macrophage assay [9]. Sub 100-nanomolar IC₅₀ values were found in a few cases against L. amazonensis intracellular parasites [11] and nanomolar IC₅₀.values were observed against promastigotes of L. infantum, L. major and L. tropica [12,13]. DB766 (Figure 1), a relatively high molecular weight compound, is among the most potent antileishmanial AIAs, with an IC₅₀.value < 0.1 µM. This in vitro activity is similar to that of amphotericin B and substantially greater than miltefosine and paromomycin [14]. The AIAs, including DB766, also have significant activity against T. cruzi 15]. Recently, anti-inflammatory and anticancer activity of this class of compounds has been described [16].

Fig. 1.

Lead bis-arylimidamides.

AIAs significantly differ from classical diamidines (pentamidine/furamidine analogues) as they are much less basic (pKs in the 7–8 range as opposed to 10–12 for amidines) and this property is thought to give rise to their enhanced oral bioavailability [14]. Little is known about the mode of action of AIAs, despite the fact that they were originally conceived as DNA minor groove binders [10]. Their DNA affinity has been found to be highly variable with structure, ranging from quite weak to moderately strong [10,14,17]. It has been shown that the T. cruzi activity of AIAs is not correlated with their binding to T. cruzi kinetoplast DNA [17]. Given the potent antiprotozoal activity and promising pharmacokinetic properties of AIAs, despite their relatively high molecular weights (e.g. DB766), we decided to embark upon SAR studies of this class of compounds by exploring low molecular weight AIAs. In this study, we have prepared four different types of low molecular weight AIAs including (i) replacement of the diphenylfuran moiety of DB766 with only a phenyl group (3a, 3b, 6), (ii) removing the furan ring of DB766 (9a, 13), (iii) replacement of the furan ring of DB766 with a single atom (9b, 9c), (iv) replacement of the furan ring of DB766 with a 3-atom linker which can approximate the geometry of the furan ring of DB766 (18).

2. Results and Discussion

2. 1. Chemistry

Scheme 1 outlines our approach to the synthesis of the target compounds 3a,b. Compounds 3a,b were prepared by stirring the diamine 1 with 2.2 equivalents of S-(2-naphthylmethyl)thioimidate hydrobromide 2a,b [9,10] in anhydrous ethanol/acetonitrile mixture at room temperature to give the hydrobromide salt. Purification was achieved by conversion to the free base followed by making the dihydrochloride salts of 3a,b.

Scheme 1.

Reagents and conditions (a) i-EtOH, CH₃CN, NaOH; ii-EtOH/HCl.

Scheme 2 outlines the synthesis of target compound 6. 2-Aminopyridine 5 was allowed to react with dicyanobenzene 4 in the presence of sodium bis(trimethylsilyl)amide, a strong non-nucleophilic base, in tetrahydrofuran at −80°C [18] followed by addition of ethanolic hydrochloride to form the hydrochloride salt of compound 6.

Scheme 2.

Reagents and conditions (a) i) NaN(TMS)₂, THF, −80 °C, ii) EtOH/HCl.

Scheme 3 outlines the synthesis of target compounds 9a–c. The commercial dinitro compounds 7a–c were reduced through reaction with a mixture of hydrazine and Raney nickel in methanol [19] to give the corresponding diamines 8a–c in very good yield (87–95%). These diamines were allowed to react with S-(2-naphthylmethyl)-2-pyridylthioimidate hydrobromide 2a to give the corresponding arylimidamides 9a–c as discussed before.

Scheme 3.

Reagent and conditions (a) Raney Ni, NH₂NH₂, MeOH; (b) i) S-(2-naphthylmethyl)-2-pyridylthioimidate HBr, EtOH, CH₃CN, NaOH; ii) EtOH/HCl.

Scheme 4 outlines the synthesis of the target compound 13. The dinitro biaryl compound 11 was achieved by a one-pot Suzuki-Miyaura homocoupling process [20] using the bromo compound 10, bis(pinacolato)diboron, PdCl₂(dppf)₂, and potassium acetate in dimethylformamide as solvent. The dinitro compound 11 was reduced to the diamine 12 using hydrazine and Raney nickel as discussed before. The diamine was conveniently converted to the arylimidamide 13 through reaction with S-(2-naphthylmethyl)-2-pyridylthioimidate hydrobromide 2a.

Scheme 4.

Reagents and conditions (a) Bis(pinacolato)diboron, PdCl₂(dppf), KOAc, DMF; (b) Raney Ni, NH₂NH₂, MeOH; (c) i) S-(2-naphthylmethyl)-2-pyridylthioimidate HBr, EtOH, CH₃CN, NaOH; ii) EtOH/HCl.

Scheme 5 outlines the synthesis of the target compound 18. The dinitro compound 16 was prepared through alkylation of 4-nitrobenzyl alcohol 14 with p-nitrobenzyl bromide 15 using sodium hydride as the base and a catalytic amount of sodium iodide in dimethylformamide as solvent. The diamine 17 was obtained in good yield through reduction of the dinitro compound 16 using hydrazine and Raney nickel as discussed before. The arylimidamide 18 was conveniently prepared through reaction of the diamine 17 with S-(2-naphthylmethyl)-2-pyridylthioimidate hydrobromide 2a.

Scheme 5.

Reagents and conditions (a) NaH, NaI, DMF; (b) Raney Ni, NH₂NH₂, MeOH; (c) i) S-(2-naphthylmethyl)-2-pyridylthioimidate HBr, EtOH, CH₃CN, NaOH; ii) EtOH/HCl.

2. 2. Biology

Table 1 contains the DNA binding affinities for the new lower molecular weight AIAs as well as the in vitro activity for these compounds againstLeishmania spp. For comparative purposes, similar data for DB766 are included. The thermal melting increase ΔTm (Tm of complex – Tm of free DNA) is a rapid and reliable method for ranking binding affinities for large numbers of aryldiamidines and arylimidamides [21]. The ΔTm values for the complexes between poly (dA-dT) and the new analogues range from 0 to 6.0°C. The three smallest AIAs (3a, 3b, 6) essentially do not bind to DNA. The parent biphenyl analogue 9a binds with the same affinity as DB766,however, the bis-i-propoxybiphenyl analogue 13 essentially does not bind to DNA, likely due to the quite twisted biphenyl unit. The two analogues with one atom linkers between the two phenyl groups 9b and 9c give ΔTm values of 2.1 and 5.2 °C, respectively. The ΔTm differences are also likely to be due to differences in the twist of the two molecules. The molecule with a three atom linker 18 exhibits modest DNA binding with a ΔTm value of 4.0°C.

Table 1.

DNA binding, in vitro antileishmanial and cytotoxicity data molecular weight arylimidamides.

code	IC50a L. a. (µM)	IC50b L. d. (µM)	IC50c Cytotox (µM)	Δ Tmd (°C)
DB766	0.087e	0.036e	3.0	6.0
3a	>10	NDf	>212	0.0
3b	ND	ND	>207	0.0
6	5.1	ND	>202	0.5
9a	0.76	0.079	46.9	6.0
9b	>10	ND	93.5	2.1
9c	0.67	0.028	5.0	5.2
13	0.14	0.025	22.9	0.1
18	1.1	ND	21.6	4.0

^aIC₅₀ values obtained against intracellular L. a. [14].

^bIC₅₀ values obtained against intracellular L. d. [14].

^cCytotoxicity (IC₅₀) was evaluated using cultured L6 rat myoblast cells [24,25].

^dIncrease in thermal melting of poly(dA-dT)n in °C [21].

^eValues taken from [14].

^fND, not determined.

Low MW arylimidamides as analogues of DB 766 were prepared

Arylimidamides are synthesized from aryl thioimidates and corresponding diamines

New arylimidamides showed L. d. IC₅₀ values of 25–79 nM and acceptable cytotoxicity

DNA binding affinities and in vivo toxicity reported

Also in Table 1, the activity of the new AIAs against L. amazonensis in vitro and their cytotoxicity to L6 rat myoblast cells is presented. The lowest molecular weight analogues in this study 3a, 6, and 3b are, in general, the least active against L. a., giving IC₅₀.values ranging from 5 to > 10 µM. The biphenyl analogues 9a and 13 show promising activity with IC₅₀.values of less than 1 µM. The two analogues 9c and 9b, which have the two AIA separated by a single atom linker, show unexpectedly divergent IC₅₀.values of 0.67 and >10 µM. The compound 18, with a 3-atom linker which can approximate the geometry of the furan ring of DB766, gives an IC₅₀.value of 1.1 µM. The selectivity indices (SI = IC₅₀cytotox / IC₅₀L. d .) of the three most active compounds 9a, 9c, and 13 are 594, 178 and 916, respectively. To further evaluate the antileishmanial potential of low molecular weight AIAs, the three most active compounds (9a, 13, 9c) with submicromolar IC₅₀.values against L. amazonensis were screened against intracellular L. donovani (see Table 1). These compounds apparently enter macrophages rather well as the IC₅₀.values for (9a, 13, 9c) are 79, 25 and 28 nM, respectively. Given this activity and selectivity, these compounds (9a, 13, 9c) were advanced to animal studies. Unfortunately, in preliminary toxicity evaluations all three of these compounds caused severe tremors in uninfected animals 10 minutes after administered intraperitoneally at a dose of 30 mg/kg. Considering the potency of these compounds against L. donovani in vitro we tested one of these compounds (9a) for its toxicity at a dose of 10 mg/kg, again by the intraperitoneal route. However, similar adverse reactions were also observed 40 minutes after administration of this lower dose of 9a. Due to their high in vivo toxicity, these compounds could not be evaluated in the murine visceral leishmaniasis model.

3. Conclusion

Four small series of low molecular weight AIAs derived from DB766 have been synthesized and three compounds from two of the series were found to have excellent activity versus intracellular L. donovani and acceptable selectivity indices. Our studies show that relatively low molecular weight AIAs can retain high activity against Leishmania sp. However, these molecules were highly toxic to mice and additional efforts are required to determine if structural modifications can be found which retain antileishmanial activity and eliminate toxicity.

4. Experimental

4.1. Biology

4.1.1. In vitro Efficacy Studies

The antileishmanial efficacy of the compounds against intracellular Leishmania amazonensis parasites was measured as described by Delfin et al 22]. Efficacy of the compounds against intracellular Leishmania donovani was assessed by the method of Zhu et al 23].

4.1.2. Cytotoxicity Measurements

The in vitro cytotoxicity of the compounds was determined as described by Bakunov et al 24].

4.1.3. Tm Measurements

Thermal melting experiments were conducted with a Cary 300 spectrophotometer. Cuvettes for the experiment were mounted in a thermal block and the solution temperatures monitored by a thermistor in the reference cuvette. Temperatures were maintained under computer control and increased at 0.5 °C/min. The experiments were conducted in 1 cm path length quartz curvettes in CAC 10 buffer (cacodylic acid 10 mM, EDTA 1 mM, NaCl 100 mM with NaOH added to give pH = 7.0). The concentrations of DNA were determined by measuring its absorbance at 260 nm. A ratio of 0.3 moles compound per mole of DNA was used for the complex and DNA alone was used as a control [21]. ΔTm values were determined by the peak in first derivative curves (dA/dT).

4.2 Chemistry

All commercial solvents and reagents were used without purification. 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 Bruker 400 MHz spectrometer using the indicated solvents. Mass spectra were obtained from the Georgia State University Mass Spectrometry Laboratory, Atlanta, GA. The compounds reported as salts frequently analyzed correctly for fractional moles of water and/or other solvents; in each case ¹H NMR spectra were consistent with the presence of water or organic solvent(s).Elemental analyses were performed by Atlantic Microlab Inc., Norcross, GA.

General procedure for the synthesis of the arylimidamides dihydrochloride: 3a,b, 6, 9a-c, 13,18

S-(2-Naphthylmethyl)-2-pyridyl or pyrimidylthioimidate hydrobromide 2a,b (2.2 mmol) was added to a cooled solution of the diamine (1 mmol) in a mixture of dry ethanol (30 mL) and dry acetonitrile (10 mL) in an ice bath. The reaction mixture was allowed to come to room temperature and was stirred overnight. After the disappearance of the starting material, the organic solvent was evaporated under reduced pressure to yield a crude oil product. Dry ether (50 mL) was added to the crude material and the mixture was stirred at room temperature for 4 h. The red precipitate was filtered and washed with dry ether. The solid was dissolved in ethanol (5 ml); the solution was cooled to 0 °C in an ice bath and 10% NaOH was added until the pH reached approximately 10. The free base was extracted with ethyl acetate (3 ×100 mL). The organic layer was washed with distilled water, dried over dry K₂CO₃, filtered and concentrated under reduced pressure. The resulting suspension was crystallized by adding dry ether and then filtered. The free base was suspended in dry ethanol (10 mL) and cooled to 0 °C in an ice bath. Freshly prepared anhydrous HCl-ethanol (2 mL) was added to the suspension and the mixture was stirred at room temperature overnight. The resulting red solution was concentrated under reduced pressure. The red crude solid was crystallized from a dry ethanol / ether mixture and filtered.

N,N'-(1,4-phenylene)dipicolinimidamide hydrochloride (3a)

White solid, yield: 68%; mp 200–202 °C; ¹H NMR (DMSO-d₆), δ (ppm): 7.65 (brs, 4H), 7.89 (dd, J = 4.0, 8.0 Hz, 2H), 8.23-8.26 (m, 2H), 8.53 (d, J = 8.0 Hz, 2H), 8.93 (d, J = 4.0 Hz, 2H), 9.41 (s, 2H), 10.24 (s, 2H), 12.01 (s, 2H); ¹³C NMR (DMSO-d₆), δ (ppm): 124.7, 129.1, 129.2, 135.4, 139.2, 144.6, 150.3, 160.8; ESI-MS: m/z calc. for C₁₈H₁₆N₆: 316.36, found: 316.20 (base M₊); Anal. Calc. for C₁₈H₁₆N₆. 2 HCl. 1.55 H₂O. 0.1 Et₂O: C, 52.04; H, 5.24; N, 19.79. Found: C, 51.68; H, 4.98; N, 19.44.

N,N'-(1,4-phenylene)dipyrimidine-2-carboximidamide hydrochloride (3b)

White solid, yield: 59%; mp 220–222 °C; ¹H NMR (DMSO-d₆), δ (ppm): 7.64–7.65 (m, 4H), 8.00-8.06 (m, 2H), 9.19–9.24 (m, 4H), 9.53 (s, 2H), 10.25 (s, 2H), 12.20 (s, 2H); ¹³C NMR (DMSO-d₆), δ (ppm): 125.5, 129.0, 135.4, 153.5, 158.5, 158.9; ESI-MS: m/z calc. for C₁₆H₁₄N₈: 318.34, found: 319.20 (base M⁺+ H₊); Anal. Calc. for C₁₆H₁₄N₈. 2 HCl. 1.75 H₂O. 0.25 EtOH: C, 45.63; H, 4.87; N, 25.80. Found: C, 45.54; H, 4.66; N, 25.47.

N ¹,N ⁴-di(pyridin-2-yl)terephthalimidamide hydrochloride (6)

Sodium bis(trimethylsilyl)amide 1M solution in tetrahydrofuran (1.58 g, 9.48 ml, 8.59 mmol) was added dropwise to a solution of 2-aminopyridine (0.8 g, 8.59 mmol) in tetrahydrofuran (10 mL) at −80 °C. Stirring was continued for 1 h, then 1,4-dicyanobenzene (0.5 g, 3.9 mmol) in tetrahydrofuran was added dropwise at −80 °C. The reaction mixture was allowed to come to room temperature and was stirred overnight. The reaction mixture was then cooled to 0 °C and HCl saturated ethanol (3 ml) was added. The mixture was stirred for 5 h, diluted with ether and the resultant solid was collected by filtration. Purification was via free base formation by neutralization with 1N sodium hydroxide solution followed by filtration of the resultant solid that was washed with water. Finally, the dry free base was stirred with saturated ethanolic HCl for 8 h, diluted with ether, and the solid which formed was filtered and dried to give the target compound as hydrochloride salt.

White solid, yield: 42%; mp 190–192 °C; ¹H NMR (DMSO-d₆), δ (ppm): 7.43-7.46 (m, 2H), 7.83 (d, J = 8.0 Hz, 2H), 8.08 (d, J = 8.0 Hz, 2H), 8.25 (br s, 4H), 8.55-8.56 (br s, 2H), 11.30 (s, 2H), 11.95 (s, 2H), 12.85 (s, 2H); ¹³C NMR (DMSO-d₆), δ (ppm): 124.7, 127.6, 129.3, 135.4, 139.2, 144.6, 149.7, 163.1; ESI-MS: m/z calc. for C₁₈H₁₆N₆: 316.36, found: 316.20 (base M₊); Anal. Calc. for C₁₈H₁₆N₆. 3 HCl. 1 H₂O: C, 48.71; H, 4.76; N, 18.93. Found: C, 48.71; H, 4.63; N, 18.58.

General procedure for the reduction of the dinitro compounds: 8a–c, 12, 17

The commercially available or synthesized dinitro compounds (1 mmol) were dissolved in methanol and 4 equivalents of hydrazine were added followed by a pinch of Raney nickel and the mixture was heated to 50 °C. A vigorous evolution of hydrogen gas ensued and the solution slowly turned colorless in 2–3 h time. It was cooled and passed through a pad of celite and washed with ethyl acetate, dried and concentrated to obtain the diamine which was subsequently washed with hexane to obtain sufficiently pure compounds to use directly in the next step as judged from ¹H NMR.

Benzidine (8a)

Yield: 95%; mp 128–130 °C; ¹H NMR (CDCl₃), δ (ppm): 3.56 (br s, 4H), 6.68 (d, J = 8.2 Hz, 4H), 7.21 (d, J = 8.2 Hz, 4H); ¹³C NMR (CDCl₃), δ (ppm): 115.2, 122.4, 132.2, 143.1; ESI-MS: Calc. for C₁₂H₁₂N₂: 184.1, found 185.2 (M+H⁺).

4,4'-Methylenedianiline (8b)

Yield: 93%; mp 97–99 °C; ¹H NMR (DMSO-d₆), δ (ppm): 3.54 (br s, 4H), 3.79 (s, 2H), 6.61 (d, J = 8.2 Hz, 4H), 6.99 (d, J = 8.2 Hz, 4H); ¹³C NMR (DMSO-d₆), δ (ppm): 40.0, 115.1, 129.4, 131.7, 144.1; ESI-MS: Calc. for C₁₃H₁₄N₂: 198.1, found 199.2 (M+H⁺).

4,4'-Oxydianiline (8c)

Yield: 87%, mp 190–191 °C; ¹H NMR (CDCl₃), δ (ppm): 6.86 (d, J = 8.8 Hz, 4H), 6.97 (br s, 4H), 7.41 (d, J = 8.8 Hz, 4H) ¹³C NMR (CDCl₃), δ (ppm): 119.9, 120.7, 152.4, 154.1; ESI-MS: Calc. for C₁₂H₁₂N₂O: 200.1, found 201.2 (M+H⁺).

N,N'-([1,1'-biphenyl]-4,4'-diyl)dipicolinimidamide hydrochloride (9a)

Yield: 97%; mp 210–212 °C; ¹H NMR (DMSO-d₆), δ (ppm): 6.89 (d, J = 8.2 Hz, 4H), 7.84–7.87 (m, 2H), 7.89 (d, J = 8.2 Hz, 4H), 8.22–8.25 (m, 2H), 8.68 (br s, 2H), 8.89 (d, J = 4.0 Hz, 2H), 10.21 (br s, 2H), 11.41 (br s, 2H), 12.05 (br s, 2H); ¹³C NMR (DMSO-d₆), δ (ppm): 114.8, 123.1, 132.8, 138.3, 142.8, 144.5, 148.1, 149.9, 153.4, 159.2; ESI-MS: Calc. for C₂₄H₂₀N₆: 392.2, found 393.3 (base M+H⁺); Anal. Calc. for C₂₄H₂₀N₆. 3.5 HCl. 1 H₂O: C, 53.57; H, 4.77; N, 15.61. Found: C, 53.20; H, 4.66; N, 15.61.

N,N'-(methylenebis(4,1-phenylene))dipicolinimidamide hydrochloride (9b)

Yield: 98%; mp 220–223 °C; ¹H NMR (DMSO-d₆), δ (ppm): 3.74 (s, 2H), 6.78 (d, J = 8.0 Hz, 4H), 7.70 (d, J = 8.0 Hz, 4H), 7.84 (br s, 2H), 8.05–8.06 (m, 2H), 8.42 (d, J = 7.6 Hz, 2H), 8.91 (d, J = 4.0 Hz, 2H), 9.37 (br s, 2H), 10.07 (br s, 2H), 11.73 (br s, 2H); ¹³C NMR (DMSO-d₆), δ (ppm): 39.6, 114.3, 128.5, 132.9, 137.8, 138.3, 144.2, 145.3, 149.3, 155.0, 158.8; ESI-MS: Calc. for C₂₅H₂₂N₆: 406.2, found 407.3 (base M+H⁺); Anal. Calc. for C₂₅H₂₂N₆. 4 HCl. 0.5 H₂O: C, 53.49; H, 4.84; N, 14.97. Found: C, 53.15; H, 4.90; N, 14.58.

N,N'-(oxybis(4,1-phenylene))dipicolinimidamide hydrochloride (9c)

Yield: 87%; mp 280 °C (dec.); ¹H NMR (DMSO-d₆), δ (ppm): 7.01 (d, J = 8.1 Hz, 4H), 7.56 (d, J = 8.1 Hz, 4H), 7.86 (br s, 2H), 8.21–8.23 (m, 2H), 8.43 (d, J = 7.6 Hz, 2H), 8.90 (d, J = 4.0 Hz, 2H), 9.39 (br s, 2H), 10.27 (br s, 2H), 11.71 (br s, 2H); ¹³C NMR (DMSO-d₆), δ (ppm): 119.4, 123.1, 149.7, 150.3, 153.4, 153.9, 155.0, 156.8, 159.4; ESI-MS (+): Calc. for C₂₄H₂₀N₆O: 408.2, found 409.3 (base M+H⁺); Anal. Calc. for C₂₄H₂₀N₆O. 3.5 HCl. 1.4 H₂O: C, 51.35; H, 4.72; N, 14.97. Found: C, 51.60; H, 4.67; N, 14.47.

2,2'-Di-isopropoxy-4,4'-dinitro-1,1'-biphenyl (11)

1-Bromo-2-isopropoxy-4-nitrobenzene (2 g, 7.69 mmol) was dissolved in 20 mL of dry DMF and potassium acetate (1.5 g, 15.38 mmol), bis(pinacolato)diboron (1.95 g, 7.69 mmol) and PdCl₂(dppf)₂ (5 mol %) was added and heated for 8 h at 90 °C. The black solution was cooled, passed through celite and washed with ethyl acetate. The ethyl acetate filtrate was washed with ice cold water, concentrated and the desired biphenyl compound was purified from it as a light yellow solid by column chromatography with hexane-ethyl acetate as the eluent.

Yield: 46%; mp 156–158 °C; ¹H NMR (CDCl₃), δ (ppm): 1.34 (d, J = 6.4 Hz, 12H), 4.63 (septet, J = 6.4 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 2.0 Hz, 2H), 7.80 (dd, J = 8.1 Hz, 2.0 Hz, 2H); ¹³C NMR (CDCl₃), δ (ppm): 22.0, 71.7, 109.1, 116.1, 129.2, 130.9, 147.6, 155.2. ESI-MS: Calc. for C₁₈H₂₀N₂O₆: 360.1, found 361.2 (M+H⁺). This compound was used in the next step without further characterization.

2,2'-Di-isopropoxy-[1,1'-biphenyl]-4,4'-diamine (12)

Yield: 91%; mp 123–125 °C; ¹H NMR (CDCl₃), δ (ppm): 1.23 (d, J = 6 Hz, 12H), 3.63 (br s, 4H), 4.34 (septet, J = 6.0 Hz, 2H), 6.31 (d, J = 2.2 Hz, 2H), 6.35 (dd, J = 8.0 Hz, 2.2 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H); ¹³C NMR (CDCl₃), δ (ppm): 22.3, 71.0, 102.8, 108.2, 113.4, 122.6, 146.7, 148.1, 150.8, 151.7, 152.9, 155.9, 156.1, 159.2; ESI-MS: Calc. for C₁₈H₂₄N₂O₂: 300.2, found 301.3 (M+H⁺). This compound was used in the next step without further characterization.

N,N'-(2,2'-di-isopropoxy-[1,1'-biphenyl]-4,4'-diyl)dipicolinimidamide hydrochloride (13)

Yield: 95%; mp 261–264 °C; ¹H NMR (DMSO-d₆), δ (ppm): 1.28 (d, J = 6.0 Hz, 12H), 4.41 (septet, J = 6.0 Hz, 2H), 6.68 (d, J = 2.2 Hz, 2H), 6.71 (dd, J = 8.0 Hz, 2.2 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.78 (m, 2H), 8.24 (m, 2H), 8.66 (d, J = 7.8 Hz, 2H), 8.91 (d, J = 4.0 Hz, 2H), 10.32 (br s, 2H), 11.36 (br s, 2H), 12.01 (br s, 2H); ¹³C NMR (DMSO-d₆), δ (ppm): 23.1, 72.1, 105.9, 111.8, 116.5, 124.2, 137.2, 138.8, 145.2, 147.2, 150.3, 156.2, 157.1, 159.3; ESI-MS: Calc. for C₃₀H₃₂N₆O₂ (base): 508.3, found 509.4 (M+H⁺); Anal. Calc. for C₃₀H₃₂N₆O₂. 2 HCl. 1.4 H₂O: C, 59.45; H, 6.12; N, 13.87. Found: C, 59.55; H, 6.05; N, 13.64.

4,4'-(Oxybis(methylene))bis(nitrobenzene) (16)

4-Nitrobenzyl alcohol (1.2 g, 7.84 mmol) was dissolved in 5 mL of dry DMF, cooled in an ice bath and NaH (60 % dispersion in mineral oil) (0.41 g, 10.2 mmol) was added. After 10–15 min, a solution of 4-nitrobenzyl bromide (1.54 g, 7.13 mmol) in 2 mL of DMF was added followed by a catalytic amount of NaI. After allowing the reaction mixture to warm to room temperature over a period of 4 h, it was quenched by the addition of water to obtain a yellow solid which was washed with ether and recrystallized from ethanol.

Yield: 52%; mp 231–233 °C; ¹H NMR (DMSO-d₆), δ (ppm): 4.51 (s, 4H), 7.84 (d, J = 8.0 Hz, 2H), 8.10 (d, J = 8.0 Hz, 2H); ¹³C NMR (DMSO-d₆), δ (ppm), 74.1, 119.4, 126.1, 143.1, 144.5; ESI-MS: Calc. for C₁₄H₁₂N₂O₅: 288.1, found 289.2 (M+H⁺). This compound was used in the next step without further characterization.

4,4'-(Oxybis(methylene))dianiline (17)

Yield: 92%; mp 178–180 °C; ¹H NMR (DMSO-d₆), δ (ppm): 3.45 (br s, 4H), 4.59 (s, 4H), 6.93 (d, J = 8.1 Hz), 7.14 (d, J = 8.1 Hz); ¹³C NMR (DMSO-d₆), δ (ppm): 75.2, 116.3, 126.9, 128.9, 146.1; ESI-MS: Calc. for C₁₄H₁₆N₂O: 228.1, found 229.2 (M+H⁺); This compound was used in the next step without further characterization.

N,N'-((Oxybis(methylene))bis(4,1-phenylene))dipicolinimidamide hydrochloride (18)

Yield: 73%; mp 280–282 °C (dec); ¹H NMR (DMSO-d₆), δ (ppm): 4.51 (s, 4H), 7.01 (d, J = 8.2 Hz, 4H), 7.42 (d, J = 8.2 Hz, 4H), 7.81 (m, 2H), 8.24 (dd, J = 7.8, 6.8 Hz, 2H), 8.39 (d, J = 7.8 Hz, 2H), 8.89 (d, J = 4.0 Hz, 2H), 10.26 (br s, 2H), 11.38 (br s, 2H), 12.02 (br s, 2H). ¹³C NMR (DMSO-d₆), δ (ppm): 74.4, 118.2, 126.2, 132.4, 144.1, 147.6, 150.7, 152.3, 152.8, 158.2, and 159.5; ESI-MS: Calc. for C₂₆H₂₄N₆O (base): 436.2, found 437.3 (free base M+H⁺); Anal. Calc. for C₂₆H₂₄N₆O. 3.5 HCl. 0.5 H₂O: C, 54.48; H, 5.01; N, 14.66. Found: C, 54.35; H, 5.04; N, 14.44.