Synthesis and Potent Antimicrobial Activity of Some Novel N-(Alkyl)-2-Phenyl-1H-Benzimidazole-5-Carboxamidines

Abstract

A series of 22 novel 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidine derivatives were synthesized and evaluated for in vitro antibacterial activity against S. aureus and methicillin resistant S. aureus (MRSA), E. coli, E. faecalis and for antifungal activity against C. albicans. Compound 59 [1-(2,4-dichlorobenzyl)-N-(2-diethylaminoethyl)-1H-benzimidazole-5-carboxamidine], with a 3,4-dichlorophenyl group at the C-2 position, displayed the greatest activity (MIC = 3.12 μg/mL against both some bacteria and the fungus C. albicans).

Introduction

We have already reported the synthesis of a series of 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidine derivatives and their very potent antibacterial activities against S. aureus and methicillin resistant S. aureus [1]. The study revealed that compounds I–IV (Figure 1) exhibited the best activity, with MIC values of 0.78 - 0.39 μg/mL against these species. As part of a continuing program focused on development of new antimicrobial benzimidazole carboxamidines, we planned to modify the structure of compounds I–IV.

Figure 1.

Figure 1.

Results and Discussion

Chemistry

Syntheses of the target benzimidazoles (Table 1, Table 2) were achieved by two different methods, as shown in Scheme 1.

Scheme 1.

Scheme 1.

Method A involved nucleophilic displacement in DMF of the chloro group of 4-chloro-3-nitro-benzonitrile by reaction with several amines to give 1–8 (Table 3). The cyano group was then converted into the imidate ester, using a modified Pinner method [1], and the imidate esters were used directly to make the corresponding benzamidines 9–18 (Table 4). Their reduction with H₂, Pd/C produced 19–28 (Table 5). Condensation of these derivatives with the Na₂S₂O₅ adducts of several benzaldehydes afforded the corresponding benzimidazoles 45–52, 55 and 59–61 [2]. Method B involved cyclization of 29–32 with the Na₂S₂O₅ adducts of various benzaldehydes to afford 5-cyano-benzimidazoles 33–39 (Table 6), following this, the cyano groups were converted into the imidate esters, as in method A, and these were used to prepared the corresponding amidine compounds 40–44, 53, 54 and 56–58. For its practical advantages this method was used in particular for cyano-benzimidazoles, which have better solubility in EtOH, although the yields were low.

Table 1. Formulas and in vitro antibacterial and antifungal activities of 40 - 61.

No.	Substituents					Minimal inhibitory concentration, μg/mL
	R’	R1	R2	R3	R4	S .a.	MRSA	MRSA*	E. c.	E. f.	C. a.
40			Cl			>50	>50	>50	>50	>50	>50
41	CH(CH3)2		Cl		Cl	>50	>50	>50	>50	>50	>50
42	CH(CH3)2				F	>50	>50	>50	>50	>50	>50
43	(Et)2NCH2CH2				F	>50	>50	>50	>50	>50	>50
44	(Me)2NCH2CH2		Cl			>50	>50	>50	>50	>50	>50
45	(Me)2NCH2CH2			Cl	Cl	12.5	12.5	12.5	>50	50	12.5
46	CH(CH3)2	CH3			CN	>50	>50	>50	>50	>50	>50
47	CH(CH3)2	CH3		OCH3	OCH3	>50	>50	>50	>50	>50	>50
48	Cyclopropyl				COOCH3	>50	>50	>50	>50	>50	>50
49	PhCH2				COOH	>50	>50	>50	>50	>50	>50
50	PhCH2				COOCH3	12.5	25	3.12	>50	50	25
51	Cyclohexyl				COOH	>50	>50	>50	>50	>50	>50
52		n-butyl				>50	>50	>50	>50	>50	>50
53	(Me)2NCH2CH2	n-butyl			F	>50	>50	>50	>50	>50	>50
54	CH(CH3)2	CH(CH3)2			F	>50	>50	>50	>50	>50	>50
55	(Me)2NCH2CH2	Ph		Cl	Cl	12.5	3.12	12.5	>50	25	12.5
56	(Me)2NCH2CH2	PhCH2		Cl	Cl	12.5	12.5	12.5	>50	25	12.5
57	(Et)2NCH2CH2	PhCH2		Cl	Cl	12.5	12.5	6.25	50	12.5	12.5
58	(Me)2NCH2CH2	PhCH2	Cl		Cl	50	12.5	12.5	>50	50	25
59	(Et)2NCH2CH2	2,4-di-Cl- benzyl		Cl	Cl	3.12	3.12	3.12	12.5	6.25	3.12
60	(Et)2NCH2CH2	PhCH2CH2		OCH3	OCH3	>50	>50	>50	>50	>50	>50
61	Isobutyl	PhCH2CH2		Cl	Cl	6.25	6.25	6.25	>50	12.5	12.5
Ref			OH		t-butyl	0.78	0.78	0.78
Sult						0.39	25	25		1.56
Amp						0.78		50		0.78
Cip									0.39
Flu											1.56

S.a.: Staphylococcus aureus (ATCC 25923); MRSA (methicillin resistant Staphylococcus aureus, ATCC 43300); MRSA* (Methicillin resistant Staphylococcus aureus, clinical isolate); E. c.: Escherichia coli (ATCC 25922); E. f.: E. faecalis (ATCC 29212); C. a.: Candida albicans (ATCC 10231); Ref: this compound was found to be the most active compound against S. aureus by Weidner-Wells et al [3]; Sult: Sultamicillin; Amp: Ampicillin; Cip: Ciprofloxacin; Flu: Fluconazole

Table 1. Formulas and in vitro antibacterial and antifungal activities of 40 - 61.

No.	Substituents					Minimal inhibitory concentration, μg/mL
	R’	R1	R2	R3	R4	S .a.	MRSA	MRSA*	E. c.	E. f.	C. a.
40			Cl			>50	>50	>50	>50	>50	>50
41	CH(CH3)2		Cl		Cl	>50	>50	>50	>50	>50	>50
42	CH(CH3)2				F	>50	>50	>50	>50	>50	>50
43	(Et)2NCH2CH2				F	>50	>50	>50	>50	>50	>50
44	(Me)2NCH2CH2		Cl			>50	>50	>50	>50	>50	>50
45	(Me)2NCH2CH2			Cl	Cl	12.5	12.5	12.5	>50	50	12.5
46	CH(CH3)2	CH3			CN	>50	>50	>50	>50	>50	>50
47	CH(CH3)2	CH3		OCH3	OCH3	>50	>50	>50	>50	>50	>50
48	Cyclopropyl				COOCH3	>50	>50	>50	>50	>50	>50
49	PhCH2				COOH	>50	>50	>50	>50	>50	>50
50	PhCH2				COOCH3	12.5	25	3.12	>50	50	25
51	Cyclohexyl				COOH	>50	>50	>50	>50	>50	>50
52		n-butyl				>50	>50	>50	>50	>50	>50
53	(Me)2NCH2CH2	n-butyl			F	>50	>50	>50	>50	>50	>50
54	CH(CH3)2	CH(CH3)2			F	>50	>50	>50	>50	>50	>50
55	(Me)2NCH2CH2	Ph		Cl	Cl	12.5	3.12	12.5	>50	25	12.5
56	(Me)2NCH2CH2	PhCH2		Cl	Cl	12.5	12.5	12.5	>50	25	12.5
57	(Et)2NCH2CH2	PhCH2		Cl	Cl	12.5	12.5	6.25	50	12.5	12.5
58	(Me)2NCH2CH2	PhCH2	Cl		Cl	50	12.5	12.5	>50	50	25
59	(Et)2NCH2CH2	2,4-di-Cl- benzyl		Cl	Cl	3.12	3.12	3.12	12.5	6.25	3.12
60	(Et)2NCH2CH2	PhCH2CH2		OCH3	OCH3	>50	>50	>50	>50	>50	>50
61	Isobutyl	PhCH2CH2		Cl	Cl	6.25	6.25	6.25	>50	12.5	12.5
Ref			OH		t-butyl	0.78	0.78	0.78
Sult						0.39	25	25		1.56
Amp						0.78		50		0.78
Cip									0.39
Flu											1.56

S.a.: Staphylococcus aureus (ATCC 25923); MRSA (methicillin resistant Staphylococcus aureus, ATCC 43300); MRSA* (Methicillin resistant Staphylococcus aureus, clinical isolate); E. c.: Escherichia coli (ATCC 25922); E. f.: E. faecalis (ATCC 29212); C. a.: Candida albicans (ATCC 10231); Ref: this compound was found to be the most active compound against S. aureus by Weidner-Wells et al [3]; Sult: Sultamicillin; Amp: Ampicillin; Cip: Ciprofloxacin; Flu: Fluconazole

Antimicrobial Activity

The benzimidazoles 40–61 were tested by the macro-broth dilution [4] assay for in vitro antibacterial activity against Gram positive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA, a clinical isolate from a wound), Enterococcus faecalis and Gram negative Escherichia coli and for antifungal activity against Candida albicans. The MIC values are listed in Table 1. The synthesized compounds and reference drugs were dissolved in water or DMSO-water (40 %) at a concentration of 400 μg/mL. The concentration was adjusted to 100 μg/mL by four- fold dilution with media culture and bacteria solution at the first tube. Data was not taken for the initial solution because of the high DMSO concentration (10 %). We have already reported that 3,4-dichloro substitution on the 2-phenyl group of amidinobenzimidazoles plays an important role in their antibacterial activity [1]. Thus, the most active compound, 59, and less active compounds 45, and 55– 57 all have a 3,4-dichlorophenyl group at the C-2 position. Replacement of 3,4-dichloro substitution with other functions such as fluoro, cyano, methoxy, carboxyl or methyl ester caused a reduction in inhibitory activities, and only compound 50, having a methyl ester group, exhibited moderate activity against MRSA with a MIC of 3.12 μg/mL. More lipophilic substituents on the benzimidazole N-atom such as phenyl, benzyl and 2,4-dichlorobenzyl do lead to quite active compounds (55–59), however substitution with methyl, butyl and isopropyl (cf. 46, 47, 52–54) gave no significant activity. Introduction of N,N-diethylaminoethyl substitution on the cationic amidine 59 led to inhibitory activity against E. coli and C. albicans. This is a very important result, as it represents the first example to date of inhibitory activity against E. coli with these amidinobenzimidazoles. Except for compound 59, none of the compounds showed important inhibitory activity against E. faecalis and C. albicans.

Conclusions

Introduction of aromatic amidine groups into the benzimidazole system gives a good profile of Gram-positive antibacterial activity. In particular, 1-(2,4-dichlorobenzyl)-N-(2-diethylaminoethyl)-1H-benzimidazole-5-carboxamidine (59), having a 3,4-dichlorophenyl at the C-2 position, exhibited the greatest activity, with a MIC value of 3.12 μg/mL against S. aureus and MRSA. Detailed mechanistic studies are required to understand the potent activity of this compound.

Experimental

General

Uncorrected melting points were measured on an Electrothermal 9100 capillary melting point apparatus. ¹H-NMR spectra were recorded employing a Varian Mercury 400 MHz FT spectrometer, chemical shifts (δ) are in ppm relative to TMS, and coupling constants (J) are reported in Hertz. Mass spectra were taken on a Waters Micromass ZQ using the ESI(+) method. Microanalyses were performed by Leco CHNS-932. Some HCl salts of compounds 40–61 were prepared by using dry HCl gas in EtOH or isopropanol. All chemicals and solvents were purchased from Aldrich Chemical Co. or Fischer Scientific. Compounds 29–32 were synthesized as described in our previous study [2]

Table 2. Physical and spectral data for compounds 40 – 61.

No	Mp (OC)	Yield (%)		Formula Calculated Found	1H-NMR δ ppm (DMSO-d6) (if not stated otherwise)	MS (ESI+) m/z	Synthesis Method and Isolation Column Chromatography if not stated otherwise
40	>300	35		C14H11ClN4 . 2HCl . H2O C: 46.50 H: 4.18 N: 15.5 C: 46.24 H: 3.99 N: 15.3	7.59 - 7.97 (arom. 7H), 8.31 (s), 9.30 (s), 9.53 (s)	271 (100) 273 (33)	(B) Crys. Ethanolic HCl
41	>290	40		C17H16Cl2N4. 2HCl . 1.5H2O C: 45.66 H: 4.73 N: 12.5 C: 45.67 H: 4.45 N: 12.4	1.21 (d, 6H), 4.03 (m, 1H), 7.6 (m, 2H), 7.8(d, J=8.5, 1H), 7.83 (d, J=2, 1H), 7.92 (d, J=8.4, 1H), 8.05 (d, J=1.5, 1H), 9.06 (s), 9.42 (s), 9.54 (d)	347 (100) 349 (65) 351 (11)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 4)
42	>300	28		C17H17FN4. 2HCl . 1.5H2O C: 51.52 H: 5.59 N: 14.1 C: 51.90 H: 5.31 N: 14.1	1.29 (d, 6H), 4.08 (m, 1H), 7.51 (t, 2H), 7.67 (d, J=8.3, 1H), 7.87 (d, J=8.4, 1H), 8.08 (s, 1H), 8.44 (m, 2H), 9.08 (s), 9.48 (s), 9.62 (d)	297 (100)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
43	>300	41		C20H24FN5. 3HCl . 2H2O C: 48.15 H: 6.26 N: 14.1 C: 48.01 H: 6.02 N: 13.99	1.29 (t, 6H), 3.26 (4H), 3.46 (2H), 4.00 (2H), 7.56 (t, 2H), 7.84 (d, J=8.4, 1H), 7.92 (d, J=8.4, 1H), 8.28 (s, 1H), 8.52 (m, 2H), 9.67 (s), 9.93 (s), 10.21 (1H), 10.96 (s)	354 (100)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
44	100-110 bubb	37		* C18H20ClN5 . 4HCl . 1.5H2O. 0.5C2H6O C: 42.27 H: 5.97 N: 12.97 C: 42.54 H: 6.04 N: 13.01	(CD3OD): 3.05 (6H), 3.66 (t, 2H), 4.03 (t, 2H), 7.69 (m, 1H), 7.82 (m, 2H), 7.99 (d, 1H), 8.102 (s, 2H), 8.54 (s, 1H)	342 (100) 344 (33)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
45	95-100 bubb	18		C18H19Cl2N5. 1.5HCl 0.25H2O . 0.25C3H8O C: 49.99 H: 5.14 N: 15.54 C: 49.97 H: 5.14 N: 15.23	2.23 (s, 6H), 2.58 (t, 2H), 3.53 (t, 2H), 7.56 (dd, J=8.4, 1.6, 1H), 7.80 (d, J=8.8, 1H), 7.86 (d, J=8.4, 1H), 8.06 (s, 1H), 8.25 (dd, J= 8.4 1.8, 1H), 8.50 (d, J=1.8, 1H)	376 (100) 378 (69) 380 (11)	(A) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
46	200-210 bubb	30		C19H19N5 . 1.5H2O . 0.1C3H8O C: 66.15 H: 6.55 N: 19.98 C: 66.19 H: 5.97 N: 19.82	1.23 (d, 6H), 3.95 (m, 1H), 3.97 (s, 3H), 7.76 (m, 2H), 8.09 (m, 5H)	318 (100)	(A) CH2Cl2 : Isopropanol : Propylamine(100 : 50 : 4)
47	100-110 bubb	26		C20H24N4O2 . 1.8H2O C: 62.42 H: 7.22 N: 14.55 C: 62.62 H: 6.94 N: 14.21	(DMSO-d6 + 1 drop D2O): 1.17 (d, 6H), 3.85-3.90 (10H), 7.15 (d, J=8.4, 1H), 7.4 (s, 2H), 7.62 (m, 2H), 7.97 (s, 1H)	353 (100)	(A) CH2Cl2 : Isopropanol : Propylamine (100 : 50 : 3)
48	285-290 bubb	60		C19H18N4O2 . 3H2O . 0.3C3H8O C: 58.80 H: 6.54 N: 13.78 C: 58.72 H: 6.20 N: 13.36	0.77 (2H), 0.91 (d, 2H), 2.79 (1H), 3.89 (s, 3H), 7.53 (d, J=8, 1H), 7.67 (d, J=8.4, 1H), 8.04 (s, 1H), 8.09 (d, J=7.8, 2H), 8.38 (d, J=8, 2H)	335 (100)	(A) Cryst. Isopropanol
49	260-270 bubb	34	C22H18N4O2 . 2H2O C: 65.01 H: 5.45 N: 13.77 C: 64.95 H: 5.23 N: 13.78		4.73 (s, 2H), 7.35-8.30 (aromat. 12H)	371 (100)	(A) Cryst. EtOH
50	265-275 bubb	52	C23H20N4O2 . 0.25H2O C: 71.03 H: 5.31 N: 14.40 C: 71.07 H: 5.14 N: 14.32		3.88 (s, 3H), 4.53 (s, 2H), 7.27-7.56 (m, 7H), 8.03-8.06 (m, 3H), 8.37 (d, J=8.4, 2H),	385 (100)	(A) CH2Cl2 : Isopropanol : Propylamine (100 : 100 : 6)
51	>300	64	C21H22N4O2 . 2HCl . 1.5H2O C: 54.55 H: 5.88 N: 12.12 C: 54.26 H: 5.89 N: 12.62		1.21-1.42-1.63-1.77-1.98 (m, 10H), 3.78 (1H), 7.65 (d, J=8.4, 1H), 7.87 (d, J=8.4, 1H), 8.1 (s, 1H), 8.15 (d, J=8, 2H), 8.47 (d, J=7.6, 2H), 9.19 (s), 9.50 (s), 9.63 (d)	363 (100)	(A) Cryst. Ethanolic HCl
52	150-155 bubb	22	C18H20N4. 2HCl . 2.2H2O 0.5C3H8O C: 53.84 H: 7.04 N: 12.88 C: 53.98 H: *** N: 12.81		(Base) : 0.64 (t, 3H), 1.01 (m, 2H), 1.56 (m, 2H), 4.33 (t, 2H), 7.56 (m, 3H), 7.76 (m, 3H), 7.95 (d, J=8.6, 1H), 8.23 (d, J=1.4, 1H), 9.18 (s), 9.41 (s)	293 (100)	(A) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 4)
53	205-210 bubb	32	C22H28FN5 . 3HCl . 2H2O C: 50.14 H: 6.69 N: 13.29 C: 50.34 H: 6.52 N: 12.92		0.78 (t, 3H), 1.20 (m, 2H), 1.70 (m, 2H), 2.90 (s, 6H), 3.51 (t, 2H), 4.03 (t, 2H), 4.45 (t, 2H), 7.55 (m, 2H), 8.01 (m, 3H), 8.10 (d, J=8.6, 1H), 8.42 (d, J=1.3, 1H), 9.77 (s), 9.97 (s), 10.28 (s)	382 (100)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
54	115-120 bubb	20	C20H23FN4 . 3HCl . 1.25H2O C: 51.07 H: 6.11 N: 11.91 C: 51.05 H: 6.31 N: 11.55		1.34 (d, 6H), 1.66 (d, 6H), 4.2 (m, 1H), 4.8 (m, 1H), 7.54 (m, 2H), 7.74 (d, J=8.7, 1H), 7.86 (m, 2H), 8.22 (2H), 9.21 (s), 9.58 (s), 9.68 (d)	339 (100)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
55	295-300 bubb	30	C24H23Cl2N5 . 3HCl . 1.25H2O C: 49.34 H: 4.91 N: 11.98 C: 49.45 H: *** N: 11.87		2.85 (s, 6H), 3.44 (t, 2H), 3.92 (t, 2H), 7.37-7.80 (m, 10H), 8.41 (d, J=1.2, 1H), 9.52 (s), 9.83 (s), 10.07 (s), 10.81 (s)	452 (100) 454 (68) 456 (12)	(A) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 15)
56	130-135 bubb	49	C25H25Cl2N5 . 3HCl . 1.25H2O C: 50.18 H: 5.14 N: 11.7 C: 50.06 H: 5.22 N: 11.49		2.90 (6H), 3.50 (t, 2H), 4.03 (q, 2H), 5.74 (s, 2H), 7.04 (m, 2H),7.34 (m, 3H), 7.82 (m, 4H), 8.02 (d, J=1.9, 1H), 8.42 (d, J=1.4, 1H), 9.71 (s), 9.86 (s), 10.15 (d), 11.14 (s)	466 (100) 468 (68) 470 (11)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 5)
57	120-130 bubb	46	C27H29Cl2N5 . 3HCl . 2H2O C: 50.68 H: 5.67 N: 10.94 C: 50.66 H: 5.62 N: 10.92		1.27 (t, 6H), 3.24 (4H), 3.45 (2H), 3.99 (2H), 5.72 (s, 2H), 6.98 (m, 2H), 7.27 (m, 3H), 7.81 (m, 4H), 8.01 (d, J=2, 1H), 8.35 (s, 1H), 9.61 (s), 9.81 (s), 10.11 (s), 10.97 (s)	494 (100) 496 (68) 498 (12)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
58	**	33	** C25H25Cl2N5 . HCl		(CD3OD): 3.29 (6H), 3.63 (t, 2H), 3.99 (t, 2H), 5.57 (s, 2H), 7.03 (m, 2H), 7.26 (m, 3H), 7.61 (dd, J=8.4 , 2, 1H), 7.68 (d, J=8.1, 1H), 7.82 (d, J=2, 1H), 7.95 (m, 2H), 8.42 (s, 1H)	466 (100) 468 (71) 470 (13)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 0.5)
59	120-125 bubb	35	C27H27Cl4N5 . 3HCl . H2O C: 46.64 H: 4.71 N: 10.07 C: 46.80 H: 4.82 N: 9.86		1.25 (t, 6H), 3.23 (m, 4H), 3.41 (t, 2H), 3.84 (2H), 5.71 (s, 2H), 6.73 (d, J=8.8, 1H), 7.30 (dd, J=8.4 2, 1H), 7.62-7.81 (m, 5H), 7.93 (d, J=2, 1H), 8.35 (s, 1H), 9.64 (s), 9.83 (s), 10.13 (s), 10.96 (s)	562 (80) 564 (100) 566 (51) 568 (14)	(A) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 0.5)
60	127-130 bubb.	30	C30H37N5O2 . 3HCl . H2O C: 54.34 H: 6.99 N: 10.56 C: 54.40 H: 6.80 N: 10.45		1.29 (t, 6H), 3.08 (t, 2H), 3.25 (4H), 3.48 (d, 2H), 3.84 (s,3H), 3.89 (s,3H), 4.04 (q, 2H), 4.83 (t, 2H), 6.9 (m, 2H), 7.12-7.31 (m, 6H), 8.03 (d, J=8.8, 1H), 8.27 (d, J=8.8, 1H), 8.36 (s, 1H), 9.87 (s), 10.08 (s), 10.44 (s), 11.1 (s)	500 (100)	(A) CH2Cl2 : Isopropanol : iso-propylamine(90 : 30 : 2)
61	288-290	19	C26H26Cl2N4 . 1.5HCl C: 60.04 H: 5.33 N: 10.77 C: 60.05 H: 5.50 N: 10.66		0.96 (d,6H), 2.06 (m, 1H), 2.93 (t, 2H), 3.16 (t, 2H), 4.63 (t, 2H), 6.67 (d, J=7.2, 2H), 7.03 (t, J=7.2, 2H), 7.11 (m, 1H), 7.45 (2H), 7.70 (m, 2H), 8.01 (d, J=8.8, 1H), 8.12 (s, 1H), 9.02 (s), 9.44 (s), 9.75 (s)	465 (100) 467 (67) 469 (11)	(A) CH2Cl2 : Isopropanol : ethylamine(100 : 50 : 0.5)

* Hygroscopic; ** Very hygroscopic, not measurable; *** No satisfactory result.

Table 2. Physical and spectral data for compounds 40 – 61.

No	Mp (OC)	Yield (%)		Formula Calculated Found	1H-NMR δ ppm (DMSO-d6) (if not stated otherwise)	MS (ESI+) m/z	Synthesis Method and Isolation Column Chromatography if not stated otherwise
40	>300	35		C14H11ClN4 . 2HCl . H2O C: 46.50 H: 4.18 N: 15.5 C: 46.24 H: 3.99 N: 15.3	7.59 - 7.97 (arom. 7H), 8.31 (s), 9.30 (s), 9.53 (s)	271 (100) 273 (33)	(B) Crys. Ethanolic HCl
41	>290	40		C17H16Cl2N4. 2HCl . 1.5H2O C: 45.66 H: 4.73 N: 12.5 C: 45.67 H: 4.45 N: 12.4	1.21 (d, 6H), 4.03 (m, 1H), 7.6 (m, 2H), 7.8(d, J=8.5, 1H), 7.83 (d, J=2, 1H), 7.92 (d, J=8.4, 1H), 8.05 (d, J=1.5, 1H), 9.06 (s), 9.42 (s), 9.54 (d)	347 (100) 349 (65) 351 (11)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 4)
42	>300	28		C17H17FN4. 2HCl . 1.5H2O C: 51.52 H: 5.59 N: 14.1 C: 51.90 H: 5.31 N: 14.1	1.29 (d, 6H), 4.08 (m, 1H), 7.51 (t, 2H), 7.67 (d, J=8.3, 1H), 7.87 (d, J=8.4, 1H), 8.08 (s, 1H), 8.44 (m, 2H), 9.08 (s), 9.48 (s), 9.62 (d)	297 (100)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
43	>300	41		C20H24FN5. 3HCl . 2H2O C: 48.15 H: 6.26 N: 14.1 C: 48.01 H: 6.02 N: 13.99	1.29 (t, 6H), 3.26 (4H), 3.46 (2H), 4.00 (2H), 7.56 (t, 2H), 7.84 (d, J=8.4, 1H), 7.92 (d, J=8.4, 1H), 8.28 (s, 1H), 8.52 (m, 2H), 9.67 (s), 9.93 (s), 10.21 (1H), 10.96 (s)	354 (100)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
44	100-110 bubb	37		* C18H20ClN5 . 4HCl . 1.5H2O. 0.5C2H6O C: 42.27 H: 5.97 N: 12.97 C: 42.54 H: 6.04 N: 13.01	(CD3OD): 3.05 (6H), 3.66 (t, 2H), 4.03 (t, 2H), 7.69 (m, 1H), 7.82 (m, 2H), 7.99 (d, 1H), 8.102 (s, 2H), 8.54 (s, 1H)	342 (100) 344 (33)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 60 : 3)
45	95-100 bubb	18		C18H19Cl2N5. 1.5HCl 0.25H2O . 0.25C3H8O C: 49.99 H: 5.14 N: 15.54 C: 49.97 H: 5.14 N: 15.23	2.23 (s, 6H), 2.58 (t, 2H), 3.53 (t, 2H), 7.56 (dd, J=8.4, 1.6, 1H), 7.80 (d, J=8.8, 1H), 7.86 (d, J=8.4, 1H), 8.06 (s, 1H), 8.25 (dd, J= 8.4 1.8, 1H), 8.50 (d, J=1.8, 1H)	376 (100) 378 (69) 380 (11)	(A) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
46	200-210 bubb	30		C19H19N5 . 1.5H2O . 0.1C3H8O C: 66.15 H: 6.55 N: 19.98 C: 66.19 H: 5.97 N: 19.82	1.23 (d, 6H), 3.95 (m, 1H), 3.97 (s, 3H), 7.76 (m, 2H), 8.09 (m, 5H)	318 (100)	(A) CH2Cl2 : Isopropanol : Propylamine(100 : 50 : 4)
47	100-110 bubb	26		C20H24N4O2 . 1.8H2O C: 62.42 H: 7.22 N: 14.55 C: 62.62 H: 6.94 N: 14.21	(DMSO-d6 + 1 drop D2O): 1.17 (d, 6H), 3.85-3.90 (10H), 7.15 (d, J=8.4, 1H), 7.4 (s, 2H), 7.62 (m, 2H), 7.97 (s, 1H)	353 (100)	(A) CH2Cl2 : Isopropanol : Propylamine (100 : 50 : 3)
48	285-290 bubb	60		C19H18N4O2 . 3H2O . 0.3C3H8O C: 58.80 H: 6.54 N: 13.78 C: 58.72 H: 6.20 N: 13.36	0.77 (2H), 0.91 (d, 2H), 2.79 (1H), 3.89 (s, 3H), 7.53 (d, J=8, 1H), 7.67 (d, J=8.4, 1H), 8.04 (s, 1H), 8.09 (d, J=7.8, 2H), 8.38 (d, J=8, 2H)	335 (100)	(A) Cryst. Isopropanol
49	260-270 bubb	34	C22H18N4O2 . 2H2O C: 65.01 H: 5.45 N: 13.77 C: 64.95 H: 5.23 N: 13.78		4.73 (s, 2H), 7.35-8.30 (aromat. 12H)	371 (100)	(A) Cryst. EtOH
50	265-275 bubb	52	C23H20N4O2 . 0.25H2O C: 71.03 H: 5.31 N: 14.40 C: 71.07 H: 5.14 N: 14.32		3.88 (s, 3H), 4.53 (s, 2H), 7.27-7.56 (m, 7H), 8.03-8.06 (m, 3H), 8.37 (d, J=8.4, 2H),	385 (100)	(A) CH2Cl2 : Isopropanol : Propylamine (100 : 100 : 6)
51	>300	64	C21H22N4O2 . 2HCl . 1.5H2O C: 54.55 H: 5.88 N: 12.12 C: 54.26 H: 5.89 N: 12.62		1.21-1.42-1.63-1.77-1.98 (m, 10H), 3.78 (1H), 7.65 (d, J=8.4, 1H), 7.87 (d, J=8.4, 1H), 8.1 (s, 1H), 8.15 (d, J=8, 2H), 8.47 (d, J=7.6, 2H), 9.19 (s), 9.50 (s), 9.63 (d)	363 (100)	(A) Cryst. Ethanolic HCl
52	150-155 bubb	22	C18H20N4. 2HCl . 2.2H2O 0.5C3H8O C: 53.84 H: 7.04 N: 12.88 C: 53.98 H: *** N: 12.81		(Base) : 0.64 (t, 3H), 1.01 (m, 2H), 1.56 (m, 2H), 4.33 (t, 2H), 7.56 (m, 3H), 7.76 (m, 3H), 7.95 (d, J=8.6, 1H), 8.23 (d, J=1.4, 1H), 9.18 (s), 9.41 (s)	293 (100)	(A) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 4)
53	205-210 bubb	32	C22H28FN5 . 3HCl . 2H2O C: 50.14 H: 6.69 N: 13.29 C: 50.34 H: 6.52 N: 12.92		0.78 (t, 3H), 1.20 (m, 2H), 1.70 (m, 2H), 2.90 (s, 6H), 3.51 (t, 2H), 4.03 (t, 2H), 4.45 (t, 2H), 7.55 (m, 2H), 8.01 (m, 3H), 8.10 (d, J=8.6, 1H), 8.42 (d, J=1.3, 1H), 9.77 (s), 9.97 (s), 10.28 (s)	382 (100)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
54	115-120 bubb	20	C20H23FN4 . 3HCl . 1.25H2O C: 51.07 H: 6.11 N: 11.91 C: 51.05 H: 6.31 N: 11.55		1.34 (d, 6H), 1.66 (d, 6H), 4.2 (m, 1H), 4.8 (m, 1H), 7.54 (m, 2H), 7.74 (d, J=8.7, 1H), 7.86 (m, 2H), 8.22 (2H), 9.21 (s), 9.58 (s), 9.68 (d)	339 (100)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
55	295-300 bubb	30	C24H23Cl2N5 . 3HCl . 1.25H2O C: 49.34 H: 4.91 N: 11.98 C: 49.45 H: *** N: 11.87		2.85 (s, 6H), 3.44 (t, 2H), 3.92 (t, 2H), 7.37-7.80 (m, 10H), 8.41 (d, J=1.2, 1H), 9.52 (s), 9.83 (s), 10.07 (s), 10.81 (s)	452 (100) 454 (68) 456 (12)	(A) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 15)
56	130-135 bubb	49	C25H25Cl2N5 . 3HCl . 1.25H2O C: 50.18 H: 5.14 N: 11.7 C: 50.06 H: 5.22 N: 11.49		2.90 (6H), 3.50 (t, 2H), 4.03 (q, 2H), 5.74 (s, 2H), 7.04 (m, 2H),7.34 (m, 3H), 7.82 (m, 4H), 8.02 (d, J=1.9, 1H), 8.42 (d, J=1.4, 1H), 9.71 (s), 9.86 (s), 10.15 (d), 11.14 (s)	466 (100) 468 (68) 470 (11)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 5)
57	120-130 bubb	46	C27H29Cl2N5 . 3HCl . 2H2O C: 50.68 H: 5.67 N: 10.94 C: 50.66 H: 5.62 N: 10.92		1.27 (t, 6H), 3.24 (4H), 3.45 (2H), 3.99 (2H), 5.72 (s, 2H), 6.98 (m, 2H), 7.27 (m, 3H), 7.81 (m, 4H), 8.01 (d, J=2, 1H), 8.35 (s, 1H), 9.61 (s), 9.81 (s), 10.11 (s), 10.97 (s)	494 (100) 496 (68) 498 (12)	(B) CH2Cl2 : Isopropanol : Ethylamine (100 : 50 : 3)
58	**	33	** C25H25Cl2N5 . HCl		(CD3OD): 3.29 (6H), 3.63 (t, 2H), 3.99 (t, 2H), 5.57 (s, 2H), 7.03 (m, 2H), 7.26 (m, 3H), 7.61 (dd, J=8.4 , 2, 1H), 7.68 (d, J=8.1, 1H), 7.82 (d, J=2, 1H), 7.95 (m, 2H), 8.42 (s, 1H)	466 (100) 468 (71) 470 (13)	(B) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 0.5)
59	120-125 bubb	35	C27H27Cl4N5 . 3HCl . H2O C: 46.64 H: 4.71 N: 10.07 C: 46.80 H: 4.82 N: 9.86		1.25 (t, 6H), 3.23 (m, 4H), 3.41 (t, 2H), 3.84 (2H), 5.71 (s, 2H), 6.73 (d, J=8.8, 1H), 7.30 (dd, J=8.4 2, 1H), 7.62-7.81 (m, 5H), 7.93 (d, J=2, 1H), 8.35 (s, 1H), 9.64 (s), 9.83 (s), 10.13 (s), 10.96 (s)	562 (80) 564 (100) 566 (51) 568 (14)	(A) CH2Cl2 : Isopropanol : NH3 (100 : 50 : 0.5)
60	127-130 bubb.	30	C30H37N5O2 . 3HCl . H2O C: 54.34 H: 6.99 N: 10.56 C: 54.40 H: 6.80 N: 10.45		1.29 (t, 6H), 3.08 (t, 2H), 3.25 (4H), 3.48 (d, 2H), 3.84 (s,3H), 3.89 (s,3H), 4.04 (q, 2H), 4.83 (t, 2H), 6.9 (m, 2H), 7.12-7.31 (m, 6H), 8.03 (d, J=8.8, 1H), 8.27 (d, J=8.8, 1H), 8.36 (s, 1H), 9.87 (s), 10.08 (s), 10.44 (s), 11.1 (s)	500 (100)	(A) CH2Cl2 : Isopropanol : iso-propylamine(90 : 30 : 2)
61	288-290	19	C26H26Cl2N4 . 1.5HCl C: 60.04 H: 5.33 N: 10.77 C: 60.05 H: 5.50 N: 10.66		0.96 (d,6H), 2.06 (m, 1H), 2.93 (t, 2H), 3.16 (t, 2H), 4.63 (t, 2H), 6.67 (d, J=7.2, 2H), 7.03 (t, J=7.2, 2H), 7.11 (m, 1H), 7.45 (2H), 7.70 (m, 2H), 8.01 (d, J=8.8, 1H), 8.12 (s, 1H), 9.02 (s), 9.44 (s), 9.75 (s)	465 (100) 467 (67) 469 (11)	(A) CH2Cl2 : Isopropanol : ethylamine(100 : 50 : 0.5)

* Hygroscopic; ** Very hygroscopic, not measurable; *** No satisfactory result.

4-(2,4-Dichlorobenzyl)amino-3-nitrobenzonitrile (7)

A mixture of 2,4-dichlorobenzylamine (3.52 g, 20 mmol) and 4-chloro-3-nitrobenzonitrile (2 g, 10.95 mmol) in DMF (3 mL) was heated under reflux for 4h at 120°C. The mixture was allowed to cool and EtOH was added. The resultant yellow precipitate was filtered, washed with water and crystallised from EtOAc-n-hexane; yield 57 %; mp: 192^oC; ¹H-NMR (CDCl₃): 4.66 (d, J=5.6, 2H), 6.8 (d, J=9.2, 1H), 7.24 (m, 2H), 7.47 (d, J=2, 1H), 7.58 (dd, J_o=9.2, J_m=2, 1H), 8.55 (d, J=2, 1H), 8.78 (br.t, 1H).

4-(2-Phenylethyl)amino-3-nitrobenzonitrile (8)

2-Phenylethylamine (1.3 g, 10.8 mmol) and 4-chloro-3-nitrobenzonitrile (1g, 5.4 mmol) were allowed to react for 2 h, at 100^oC and the product wasisolated as described for 7; yield 79 %; mp: 111^oC; ¹H-NMR (CDCl₃): 3.04 (t, J=7.1, 2H), 3.61 (q, J=6.8, 2H), 6.89 (d, J=8.8, 1H), 7.25-7.37 (m, 4H), 7.58 (dd, J_o=8.8, J_m=2, 1H), 8.44 (br.t, 1H), 8.47 (d, J=2, 1H).

Table 3. Formulas and melting points of 1–8.

Comp	R1	Formula	Ref.
1	H	C7H5N3O2	Commercial
2	methyl	C8H7N3O2	Lit [1]
3	iso-propyl	C10H11N3O2	Lit [2]
4	n-butyl	C11H13N3O2	Lit [2]
5	phenyl	C13H9N3O2	lit [5] 126oC, 126 oC
6	benzyl	C14H11N3O2	Lit [2]
7	2,4-dichlorobenzyl	C14H9Cl2N3O2	---
8	PhCH2CH2	C15H13N3O2	---

Table 3. Formulas and melting points of 1–8.

Comp	R1	Formula	Ref.
1	H	C7H5N3O2	Commercial
2	methyl	C8H7N3O2	Lit [1]
3	iso-propyl	C10H11N3O2	Lit [2]
4	n-butyl	C11H13N3O2	Lit [2]
5	phenyl	C13H9N3O2	lit [5] 126oC, 126 oC
6	benzyl	C14H11N3O2	Lit [2]
7	2,4-dichlorobenzyl	C14H9Cl2N3O2	---
8	PhCH2CH2	C15H13N3O2	---

General Procedure for Synthesis of 9–18, 40–44, 53, 54, 56–58

1–8 (4.5 mmol) and 33–39 (Table 6, 1 mmol) were suspended in absolute EtOH, cooled in a ice-salt bath, and dry HCl gas was passed through the solution for 40 min. The solution was stirred in a stoppered flask at room temperature for 3 days and then diluted with dry ether. The imidate esters precipitated as yellow solids, which were washed with ether then dried under vacuum at room temperature. All imidate esters were used directly without characterisation. A suspension of imidate ester HCl in absolute EtOH was stirred with corresponding the amines (1.5 - 2 fold excess) overnight at 25-30^oC. The reaction mixture was evaporated and diluted with ether, the precipitate was filtered, washed with ether, then dried. Compounds 9–18 (Table 4) were used without purification as HCl salts for the next steps since they were prepared completely pure. In contrast, crude products 40–44, 53, 54, 56–58 were treated with dilute Na₂CO₃ solution, then water. Further purification methods are given in Table 2.

Table 4. Formulas, spectroscopic data, m.p. and yields of 9 - 18.

Comp	R’	R1	Formula	NMR δ ppm (DMSO-d6)	Mass (ESI+)	mp (oC)	Yield (%)
9		butyl	C11H16N4O2	0.895 (t, 3H), 1.34 (m, 2H), 1.56 (m, 2H), 3.41 (q, 2H), 7.22 (d, J=9.2, 1H), 7.95 (dd, J=9.2, 2.4, 1H), 8.6 (t, 1H), 8.67 (d, J=2.4, 1H), 9.2 (br.s)	237 (100)	200-4	62
10	iso-propyl	methyl	C11H16N4O2			Lit [1]
11	(CH3)2N(CH2)2		C11H17N5O2	2.24 (s, 6H), 2.58 (t, 2H), 3.5 (t, 2H), 7.14 (d, J=8.8,1H), 7.5 (dd, J=8.8, 2, 1H), 8.09 (br.s, 2H), 8.48 (d, J=2, 1H).	252 (100)	220-230 (bubb) Lit [6]
12	cyclo-propyl		C10H12N4O2			Lit [1]
13	cyclo-hexyl		C13H18N4O2			Lit [1]
14	Benzyl		C14H14N4O2			Lit [1]
15	(CH3)2N(CH2)2	phenyl	C17H21N5O2	2.32 (s, 6H), 2.69 (2H), 3.4 (br.s), 3.56 (t, 2H), 7.12 (d, J=9..3, 1H), 7.28-7.49 (m, 5H), 7.85 (dd, J=9.2, 2.3, 1H), 8.61 (d, J=2.3, 1H), 9.85 (s, 1H)	328 (100)	Hygros. 110 (bubb)	87
16	iso-butyl	PhCH2CH2	C19H24N4O2	(CD3OD): 1.05 (d, 6H), 2.07 (m,1H), 3.05 (t, 2H), 3.25 (d, 2H), 3.72 (m, 2H), 7.21 (m, 2H), 7.3 (m, 4H), 7.79 (dd, J=9.2 2.4, 1H), 8.53 (t, 1H), 8.59 (d, J=2.4, 1H)	341 (100)	247-9	75
17	(C2H5)2N(CH2)2	PhCH2CH2	C21H29N5O2	(CD3OD): 1.11 (t, 6H), 2.68 (q, 4H), 2.78 (t, 2H), 3.03 (t, 2H), 3.57 (t, 2H), 4.7 (s, 2H), 7.21 (m, 2H), 7.3 (m, 4H), 7.81 (dd, J=8.8 ,2.4, 1H), 8.62 (d, J=2.4, 1H).	384 (100)	236-9	85
18	(C2H5)2N(CH2)2	2,4-dichloro benzyl	C20H25Cl2N5O2	(CD3OD): 1.11 (t, 6H), 2.68 (q, 4H), 2.79 (t, 2H), 3.57 (t, 2H), 4.78 (t, 2H), 6.98 (d, J=9.2, 1H), 7.30 (dd, J=8.6 2.4, 1H), 7.37 (d, J=8.8, 1H), 7.53 (d, J=2, 1H), 7.78 (dd, J=9.2, 2.4, 1H), 8.68 (d, J=2.4, 1H)	438(100) 440 (65) 442 (12)	203-5	89

Table 4. Formulas, spectroscopic data, m.p. and yields of 9 - 18.

Comp	R’	R1	Formula	NMR δ ppm (DMSO-d6)	Mass (ESI+)	mp (oC)	Yield (%)
9		butyl	C11H16N4O2	0.895 (t, 3H), 1.34 (m, 2H), 1.56 (m, 2H), 3.41 (q, 2H), 7.22 (d, J=9.2, 1H), 7.95 (dd, J=9.2, 2.4, 1H), 8.6 (t, 1H), 8.67 (d, J=2.4, 1H), 9.2 (br.s)	237 (100)	200-4	62
10	iso-propyl	methyl	C11H16N4O2			Lit [1]
11	(CH3)2N(CH2)2		C11H17N5O2	2.24 (s, 6H), 2.58 (t, 2H), 3.5 (t, 2H), 7.14 (d, J=8.8,1H), 7.5 (dd, J=8.8, 2, 1H), 8.09 (br.s, 2H), 8.48 (d, J=2, 1H).	252 (100)	220-230 (bubb) Lit [6]
12	cyclo-propyl		C10H12N4O2			Lit [1]
13	cyclo-hexyl		C13H18N4O2			Lit [1]
14	Benzyl		C14H14N4O2			Lit [1]
15	(CH3)2N(CH2)2	phenyl	C17H21N5O2	2.32 (s, 6H), 2.69 (2H), 3.4 (br.s), 3.56 (t, 2H), 7.12 (d, J=9..3, 1H), 7.28-7.49 (m, 5H), 7.85 (dd, J=9.2, 2.3, 1H), 8.61 (d, J=2.3, 1H), 9.85 (s, 1H)	328 (100)	Hygros. 110 (bubb)	87
16	iso-butyl	PhCH2CH2	C19H24N4O2	(CD3OD): 1.05 (d, 6H), 2.07 (m,1H), 3.05 (t, 2H), 3.25 (d, 2H), 3.72 (m, 2H), 7.21 (m, 2H), 7.3 (m, 4H), 7.79 (dd, J=9.2 2.4, 1H), 8.53 (t, 1H), 8.59 (d, J=2.4, 1H)	341 (100)	247-9	75
17	(C2H5)2N(CH2)2	PhCH2CH2	C21H29N5O2	(CD3OD): 1.11 (t, 6H), 2.68 (q, 4H), 2.78 (t, 2H), 3.03 (t, 2H), 3.57 (t, 2H), 4.7 (s, 2H), 7.21 (m, 2H), 7.3 (m, 4H), 7.81 (dd, J=8.8 ,2.4, 1H), 8.62 (d, J=2.4, 1H).	384 (100)	236-9	85
18	(C2H5)2N(CH2)2	2,4-dichloro benzyl	C20H25Cl2N5O2	(CD3OD): 1.11 (t, 6H), 2.68 (q, 4H), 2.79 (t, 2H), 3.57 (t, 2H), 4.78 (t, 2H), 6.98 (d, J=9.2, 1H), 7.30 (dd, J=8.6 2.4, 1H), 7.37 (d, J=8.8, 1H), 7.53 (d, J=2, 1H), 7.78 (dd, J=9.2, 2.4, 1H), 8.68 (d, J=2.4, 1H)	438(100) 440 (65) 442 (12)	203-5	89

Table 5. Formulas, spectroscopic data, mp and yields of 19 - 28.

Comp	R’	R1	Formula	NMR δ ppm (DMSO-d6)	MS (ESI+)	mp (oC)	Yield (%)
19		butyl	C11H18N4	0.88 (t, 3H), 1.36 (m, 2H), 1.56 (m, 2H), 3.1 (t, 2H), 5.01 (br.s, 2H), 5.57 (br.s, 2H), 6.46 (d, J=8, 1H), 6.9 (s, 1H), 7.07 (d, J=8.4, 1H), 8.58 (s, 2H), 8.74 (s, 2H).	207(100)	285	94
20	iso-propyl	methyl	C11H18N4			Lit [1]
21	(CH3)2N(CH2)2		C11H19N5	(+ one drop D2O): 2.2 (s, 6H), 2.54 (t, 2H), 3.4 (t, 2H), 6.57 (d, J=8, 1H), 6.85 (m, 2H)	222(100)	Lit [6]
22	cyclo-propyl		C10H14N4			Lit [1]
23	cyclo-hexyl		C13H20N4			Lit [1]
24	benzyl		C14H16N4			Lit [1]
25	(CH3)2N(CH2)2	phenyl	C17H23N5	(+ one drop D2O) : 2.27 (s, 6H), 2.63 (t, 2H), 3.5 (t, 2H), 6.85-7.26 (8H)	298(100)	*	90
26	iso-butyl	-CH2CH2 Ph	C19H26N4	0.92 (d, 6H), 1.96 (m, 1H), 2.9 (t, 2H), 3.17 (t, 2H), 3.31(2H), 4.39 (t, 1H), 5.03 (2H), 5.66 (t,1H), 6.57 (d, J=8.4, 1H), 6.87 (d, J=1.6, 1H), 6.98 (dd, J=8.4 1.6, 1H), 7.2-7.3 (m, 5H), 8.57 (s, 1H), 8.9 (s, 1H)	311(100)	98-100	94
27	(C2H5)2N(CH2)2	-CH2CH2 Ph	C21H31N5	(CD3OD): 1.09 (t, 6H), 2.65 (q, 4H), 2.77 (t, 2H), 2.96 (t, 2H), 3.47 (t, 2H), 3.52 (t, 2H), 6.68 (d, J=8, 1H), 7.01 (d, J=2, 1H), 7.14-7.3 (m, 6H)	354(100)	*	95
28	(C2H5)2N(CH2)2	2,4-dichloro-benzyl	C20H27Cl2N5	(CD3OD): 1.14 (t, 6H), 2.76 (4H), 2.89 (2H), 3.57 (t, 2H), 4.54 (s, 2H), 6.37 (d, J=8, 1H), 7.06 (d, J=2, 2H), 7.25 (dd, J=8.2 2, 1H), 7.31 (d, J=8.4, 1H), 7.49 (d, J=2, 1H)	408(100) 410(65) 412(11)	*	92

* No sharp melting point.

Table 5. Formulas, spectroscopic data, mp and yields of 19 - 28.

Comp	R’	R1	Formula	NMR δ ppm (DMSO-d6)	MS (ESI+)	mp (oC)	Yield (%)
19		butyl	C11H18N4	0.88 (t, 3H), 1.36 (m, 2H), 1.56 (m, 2H), 3.1 (t, 2H), 5.01 (br.s, 2H), 5.57 (br.s, 2H), 6.46 (d, J=8, 1H), 6.9 (s, 1H), 7.07 (d, J=8.4, 1H), 8.58 (s, 2H), 8.74 (s, 2H).	207(100)	285	94
20	iso-propyl	methyl	C11H18N4			Lit [1]
21	(CH3)2N(CH2)2		C11H19N5	(+ one drop D2O): 2.2 (s, 6H), 2.54 (t, 2H), 3.4 (t, 2H), 6.57 (d, J=8, 1H), 6.85 (m, 2H)	222(100)	Lit [6]
22	cyclo-propyl		C10H14N4			Lit [1]
23	cyclo-hexyl		C13H20N4			Lit [1]
24	benzyl		C14H16N4			Lit [1]
25	(CH3)2N(CH2)2	phenyl	C17H23N5	(+ one drop D2O) : 2.27 (s, 6H), 2.63 (t, 2H), 3.5 (t, 2H), 6.85-7.26 (8H)	298(100)	*	90
26	iso-butyl	-CH2CH2 Ph	C19H26N4	0.92 (d, 6H), 1.96 (m, 1H), 2.9 (t, 2H), 3.17 (t, 2H), 3.31(2H), 4.39 (t, 1H), 5.03 (2H), 5.66 (t,1H), 6.57 (d, J=8.4, 1H), 6.87 (d, J=1.6, 1H), 6.98 (dd, J=8.4 1.6, 1H), 7.2-7.3 (m, 5H), 8.57 (s, 1H), 8.9 (s, 1H)	311(100)	98-100	94
27	(C2H5)2N(CH2)2	-CH2CH2 Ph	C21H31N5	(CD3OD): 1.09 (t, 6H), 2.65 (q, 4H), 2.77 (t, 2H), 2.96 (t, 2H), 3.47 (t, 2H), 3.52 (t, 2H), 6.68 (d, J=8, 1H), 7.01 (d, J=2, 1H), 7.14-7.3 (m, 6H)	354(100)	*	95
28	(C2H5)2N(CH2)2	2,4-dichloro-benzyl	C20H27Cl2N5	(CD3OD): 1.14 (t, 6H), 2.76 (4H), 2.89 (2H), 3.57 (t, 2H), 4.54 (s, 2H), 6.37 (d, J=8, 1H), 7.06 (d, J=2, 2H), 7.25 (dd, J=8.2 2, 1H), 7.31 (d, J=8.4, 1H), 7.49 (d, J=2, 1H)	408(100) 410(65) 412(11)	*	92

* No sharp melting point.

General Procedure for Synthesis of 19 – 28

Compound 9–18 (3.5 mmol) in EtOH (75 mL) was subjected to hydrogenation using 40 psi of H₂ and 10 % Pd-C (40mg) until uptake of H₂ ceased. The catalyst was filtered on a bed of Celite, washed with EtOH, and the filtrate was concentrated in vacuo. The crude o-phenylenediamines (grey–purple– black in colour) were used for the subsequent steps without crystallisation (Table 5). In order to prevent halogen reduction of compound 28, 15 psi of H₂ pressure was employed.

Table 6. Formulas, spectroscopic data, mp and yields of 33 - 39.

Comp	R1	R2	R3	R4	Formula	NMR δ ppm (CDCl3)	Mass (ESI+)	mp (oC)	Yield (%)	İsolation
33		Cl			C14H8ClN3			Lit [2]
34		Cl		Cl	C14H7Cl2N3			Lit [2]
35				F	C14H8FN3			Lit [2]
36	iso-propyl			F	C17H14FN3			Lit [2]
37	n-butyl			F	C18H16FN3			Lit [2]
38	benzyl		Cl	Cl	C21H13Cl2N3	5.47 (s, 2H), 7.05 (m, 2H), 7.3-7.55 (m,7H),7.82 (d, J=2, 1H), 8.16 (d, J=1.5, 1H)	91(100) 378(50) 380(33) 382(6)	192	75	Cryst. EtOH-water
39	benzyl	Cl		Cl	C21H13Cl2N3	5.27 (s, 2H), 6.91(m, 2H), 7.26-7.57 (m, 8H), 8.16 (1H)	91(100) 378(54) 380(31) 382(5)	195-6	73	Cryst. EtOH-water

Table 6. Formulas, spectroscopic data, mp and yields of 33 - 39.

Comp	R1	R2	R3	R4	Formula	NMR δ ppm (CDCl3)	Mass (ESI+)	mp (oC)	Yield (%)	İsolation
33		Cl			C14H8ClN3			Lit [2]
34		Cl		Cl	C14H7Cl2N3			Lit [2]
35				F	C14H8FN3			Lit [2]
36	iso-propyl			F	C17H14FN3			Lit [2]
37	n-butyl			F	C18H16FN3			Lit [2]
38	benzyl		Cl	Cl	C21H13Cl2N3	5.47 (s, 2H), 7.05 (m, 2H), 7.3-7.55 (m,7H),7.82 (d, J=2, 1H), 8.16 (d, J=1.5, 1H)	91(100) 378(50) 380(33) 382(6)	192	75	Cryst. EtOH-water
39	benzyl	Cl		Cl	C21H13Cl2N3	5.27 (s, 2H), 6.91(m, 2H), 7.26-7.57 (m, 8H), 8.16 (1H)	91(100) 378(54) 380(31) 382(5)	195-6	73	Cryst. EtOH-water

General Procedure for Synthesis of 33–39, 45–52, 55, 59–61

The corresponding benzaldehydes (15 mmol) were dissolved in EtOH (50 mL) and sodium metabisulfite (1.6 g) in H₂O (10 mL) was added in portions. The reaction mixture was stirred vigorously and more EtOH was added. The mixture was kept in a refrigerator for a several hours. The precipitate was filtered and dried (yields over 93 %). The mixture of these salts (1 mmol) and 19–28 and 29–32 (1 mmol) in DMF (1-2 mL) was heated at 120^oC for 4h. The reaction mixture was cooled, poured into H₂O, and the solid was filtered. Purification methods are given in Table 2 and Table 6.