Synthesis, SAR and selectivity of 2-acyl- and 2-cyano-1-hetarylalkyl-guanidines at the four histamine receptor subtypes: a bioisosteric approach

Abstract

In the search for potential bioisosteres of the 4-imidazolyl ring in acylguanidines (e.g. UR-AK24), known to possess affinity to several histamine receptor subtypes (H_xR, x = 1–4), and cyanoguanidine-type H₄R agonists (e.g. UR-PI376), the contribution of various heterocycles to agonism, antagonism and HR subtype selectivity was studied (recombinant human H_1,2,3,4Rs, isolated guinea pig organs (H₁R, H₂R)). While minor structural modifications of UR-PI376 analogues were not tolerated regarding H₄R agonism, in the case of the acylguanidines, a 1,2,4-triazole ring shifted the selectivity toward the H₂R.

---

Introduction

The physiological and pathophysiological effects of the biogenic amine histamine are mediated through four receptor subtypes, referred to as H₁, H₂, H₃ and H₄ receptors (H₁R, H₂R, H₃R, and H₄R), all belonging to class A of G protein coupled receptors.^1–4 H₁R and H₂R antagonists have been used for decades in the treatment of allergic conditions and as antiulcer drugs, respectively. The H₃R is mainly expressed in the brain and is considered a promising drug target, for instance, for the treatment of attention-deficit hyperactivity disorder, Alzheimer's disease, Parkinson's disease, sleep disorders or obesity.⁵ The H₄R was discovered by several groups based on its high sequence homology with the H₃R.^6–12 The expression on hematopoietic cells such as mast cells, basophils, eosinophils, T-cells and dendritic cells suggests a role of the H₄R in the regulation of immune responses and inflammation.^13–15 Therefore, the H₄R is considered a potential drug target for the treatment of diseases like allergic rhinitis, rheumatoid arthritis, bronchial asthma and pruritus.^16–18 Recent reports on β-arrestin-mediated signalling^19–21 of H₄R ligands and partial agonistic effects of the standard H₄R antagonist JNJ-7777120 (ref. 22) at certain H₄R species orthologs suggest that the interpretation of ligand effects in vivo in terms of agonism or antagonism should be interpreted with caution.^19,23 Hence, both selective antagonists and agonists are required as pharmacological tools to further explore the role of the H₄R.^15,24

Guanidine-type compounds like arpromidine and analogues represent highly potent H₂R agonists.^25,26 Drawbacks due to the strongly basic guanidine, e.g. very low oral bioavailability and lack of penetration across the blood brain barrier, were eliminated by replacing the guanidine group with a considerably less basic acylguanidine moiety (Fig. 1, UR-AK24 (1)).^27–29 However, these N^G-acylated imidazolylpropylguanidines developed as H₂R agonists lacked selectivity toward the hH₃R and hH₄R.³⁰ By analogy with the H₂R agonist amthamine (2),³¹ the replacement of the imidazole ring in acylguanidine-type ligands by a 2-amino-thiazole ring, resulted in selective H₂R agonists (3 and 4).^27,32,33 In contrast potent selective agonists for the hH₄R were obtained by replacing the basic acylguanidine group with a cyanoguanidine moiety as in UR-PI376 (5).³⁴ The highest potency resided in imidazolylalkylcyanoguanidines with a tetramethylene linker, connecting the imidazole and the cyanoguanidine moiety. Unlike hH₄R agonists such as 5-methylhistamine,³⁵ VUF-8430 (ref. 36) or OUP-16 (ref. 37) compound 5 is devoid of agonistic activities at hHR subtypes other than the hH₄R.³⁴

Fig. 1 Structures of the selective H₄R agonists 5-methylhistamine, VUF8430 and OUP-16, the N^G-acylated imidazolylpropylguanidine UR-AK24 (1), which is active at the H₂R, H₃R and H₄R, the H₂R agonist amthamine (2), related potent and selective acylguanidine-type H₂R agonists 3 and 4 and the potent and selective H₄R agonist UR-PI376 (5).

The previous results suggest that the bioisosteric approach harbours the potential of further increasing the preference or the selectivity of hetarylalkylguanidines for a certain HR subtype. In the present work various heterocycles were introduced to replace the 1H-imidazol-4-yl ring (Fig. 2). Special attention was paid to substructures known from early studies on hetaryl analogues of histamine.^38,39 These structural modifications were combined with an acylguanidine or a cyanoguanidine moiety as less basic or non-basic guanidine replacements.

Fig. 2 Imidazole replacement in acylguanidine-type non-selective H₂R agonists and in cyanoguanidine-type H₄R agonists. Overview of the introduced aromatic rings.

Results and discussion

Chemistry

Synthesis of the acylguanidines. The amines and alcohols required for the preparation of the arylpropylguanidines 44–52 were synthesized as depicted in Scheme 1. Reduction of 6 with LiAlH₄ followed by aminomethylation in a Mannich reaction⁴⁰ gave the furan analog 8. The imidazolylpropanol 10 was prepared by deprotonation of the methyl group in 9 with n-BuLi in THF and treatment with oxirane as an electrophile.⁴¹ Introduction of a three-membered carbon chain to the pyrazole core was performed by C–C coupling of the trityl-protected iodopyrazole 11 with propargyl alcohol under Sonogashira conditions⁴² using Pd(PPh₃)₂Cl₂ and CuI as catalysts. Hydrogenation over Pd/C (10%) provided the pyrazolylpropanol 14. The triazolylpropanol 20 was obtained in five steps starting from 1H-1,2,4-triazole (15). After trityl-protection of 15,^43,4416 was treated with n-BuLi and DMF in THF to afford the aldehyde 17.⁴⁵ Elongation of the side chain by two carbon atoms was carried out via the Horner–Wadsworth–Emmons reaction employing triethyl phosphonoacetate.⁴⁶ Subsequent hydrogenation of the C=C double bond and reduction of the ethyl ester yielded 20. Conversion of the pyridylpropanols 21–23 to the corresponding phthalimides 24–26 under Mitsunobu conditions⁴⁷ followed by hydrazinolysis gave the pyridylpropylamines 27–29.⁴⁸

Scheme 1 Synthesis of the arylpropyl alcohols 8, 10, 14 and 20, and the pyridylpropylamines 27–29. Reagents and conditions: (i) LiAlH₄ (2 eq.), Et₂O, overnight, 0 °C → rt; (ii) NH(CH₃)₂·HCl (1.6 eq.), (CH₂O)_n (1.6 eq.), EtOH, overnight, reflux; (iii) oxirane (5 eq.), n-BuLi (1.1 eq.), THF, overnight, −78 °C → rt; (iv) TrCl (1 eq.), NEt₃ (1.2 eq.), DCM, 12 h, 0 °C → rt; (v) Pd(PPh₃)₂Cl₂ (0.03 eq.), CuI (0.05 eq.), DIPA (4.5 eq.), propargyl alcohol (1.1 eq.), DMF, 48 h, −15 °C → rt; (vi) H₂, Pd/C (10%) (cat.), MeOH, overnight, rt; (vii) TrCl (1 eq.), NEt₃ (1 eq.), DCM, overnight, rt; (viii) TMEDA (1 eq.), n-BuLi (1.1 eq.), DMF (0.9 eq.), THF, 12 h, −78 °C; (ix) triethyl phosphonoacetate (1.2 eq.), NaH (60% dispersion in mineral oil) (1.2 eq.), THF, overnight, rt; (x) H₂, Pd/C (10%) (cat.), EtOH/THF, overnight, rt; (xi) LiAlH₄ (2 eq.), THF, 2 h, 0 °C → reflux; (xii) phthalimide (1.1 eq.), PPh₃(1.1 eq.), DIAD (1.1 eq.), THF, overnight, 0 °C → rt. (xiii) N₂H₄·H₂O (6 eq.), EtOH, overnight, rt.

The arylpropylguanidines 44–52 were synthesized starting from the corresponding arylpropyl alcohols 8, 10, 14 and 20 or arylpropylamines 27–31 (Scheme 2). Conversion of the alcohol to the di-Cbz-protected guanidines 35–38 was accomplished under Mitsunobu conditions⁴⁷ by analogy with the procedure described by Feichtinger et al.⁴⁹ The arylpropylamines 27–31 were treated with the triflyl-di-Cbz-protected guanidine 34 (ref. 49) to give the di-Cbz protected arylpropylguanidines 39–43. Finally, the arylpropylguanidines 44–52 were obtained by hydrogenolytic cleavage of the Cbz groups. The N^G-acylated arylpropylguanidines were prepared as outlined in Scheme 2. Coupling of the CDI-activated carboxylic acids^50,5153–56 to the arylpropylguanidines 44–52 gave the acylguanidines 57–78.²⁷ Tritylated heterocycles were deprotected under acidic conditions yielding 79–86.

Scheme 2 Synthesis of the N^G-acylated arylpropylguanidines 57–86. Reagents and conditions: (i) benzyl chloroformate (3 eq.), NaOH (5 eq.), H₂O/DCM, 20 h, 0 °C; (ii) Tf₂O (1 eq.), NaH (60% dispersion in mineral oil) (2 eq.), chlorobenzene, overnight, −45 °C → rt; (iii) PPh₃ (1.5 eq.), DIAD (1.5 eq.), THF, overnight, 0 °C → rt; (iv) NEt₃ (1 eq.), DCM, overnight, rt; (v) H₂, Pd/C (10%) (cat.), MeOH, 3 h, rt; (vi) (a) CDI (1.2 eq.), NaH (60% dispersion in mineral oil) (2 eq.), THF, 5 h, rt; (b) for trityl-protected intermediates 71–78: TFA (20%), DCM, 5 h, rt.

Synthesis of the cyanoguanidines. The amines 92–106 required for the preparation of the cyanoguanidines 107–133 were synthesized as recently reported.⁵² The synthesis of 107–133 was accomplished by analogy with a previously described procedure (Scheme 3).³⁴ Diphenyl cyanocarbonimidate (87)⁵³ and the primary amines 88–89 gave the isourea intermediates 90–91, which were treated with 92–106 in acetonitrile in a microwave oven to yield 107–133.⁵⁴

Scheme 3 Synthesis of the cyanoguanidines 107–133. Reagents and conditions: (i) 2-propanol, 1 h, rt; (ii) MeCN, microwave 150 °C, 15 min.

Pharmacological results and discussion. The acylguanidines 57–86 were investigated for histamine receptor agonism or antagonism in steady-state GTPase assays using [³²P] or [³³P] radiolabeled GTP. These experiments were performed using membrane preparations of Sf9 insect cells expressing the following proteins: hH₁R plus regulator of G protein signalling 4 (RGS4), hH₂R-Gsα_S fusion protein, hH₃R plus Gα_i2 plus Gβ₁γ₂ plus RGS4 or hH₄R-RGS19 fusion protein plus Gα_i2 plus Gβ₁γ₂ (Table 1).^27,55,56 Selected compounds were additionally investigated for H₁R and H₂R activity at the guinea pig (gp) ileum and the spontaneously beating guinea pig right atrium, respectively (Table 2). The cyanoguanidines 107–133 were investigated at the hH₁R as described above and at the other three HR subtypes in [³⁵S]GTPγS binding assays using membrane preparations of Sf9 cell expressing the hH₂R-Gsα_S fusion protein, the hH₃R plus Gα_i2 plus Gβ₁γ₂ or the hH₄R plus Gα_i2 plus Gβ₁γ₂ (Table 3).^57,58 In the following agonistic potencies are expressed as pEC₅₀ (−log EC₅₀) values. Intrinsic activities (α) refer to the maximal response induced by the standard agonist histamine. Compounds identified to be inactive as agonists (α < 0.1 or negative values, respectively, determined in the agonist mode) were investigated in the antagonist mode. The pK_B values of neutral antagonists and inverse agonists were determined from the concentration-dependent inhibition of the histamine-induced increase in [³⁵S]GTPγS binding or [γ³²P]GTP ([γ³³P]GTP) hydrolysis, respectively.

Table 1 Potencies and efficacies of the prepared acylguanidines 57–86 at hH₁R, hH₂R, hH₃R and hH₄R in the steady-state GTPase assay^a,b

Compound	hH1R		hH2R		hH3R		hH4R
	pEC50 or (pKB)	N	pEC50 or (pKB)	N	pEC50 or (pKB)	N	pEC50 or (pKB)	N
a Steady-state GTPase activity in Sf9 insect cell membranes expressing the hH1R + RGS4, hH2R-GsαS fusion protein, hH3R + Gαi2 + Gβ1γ2 + RGS4 or hH4R-RGS19 fusion protein + Gαi2 + Gβ1γ2 was determined as described in the ESI. N gives the number of independent experiments performed in duplicate. b n.d.: not determined.
Histamine	6.72 ± 0.02 (ref. 28)		5.92 ± 0.11 (ref. 28)		7.60 ± 0.05	3	7.92 ± 0.11	8
	α: 1.00		a: 1.00		a: 1.00		a: 1.00
UR-AK24 (1)	(<5)^27		7.17 (ref. 29)		8.60 ± 0.11	2	7.82 ± 0.01	2
	—		α: 0.87		a: 0.24 ± 0.02		a: 0.84 ± 0.06
Thioperamide	n.d.		n.d.		(7.01 ± 0.08)	5	(6.96 ± 0.06)	6
					a: −0.71 ± 0.6		a: −0.95 ± 0.07
57	(5.05 ± 0.06)	2	(5.89 ± 0.04)	2	(<5)	2	(<5)	2
	a: −0.02 ± 0.05		a: −0.10 ± 0.00		a: −0.19 ± 0.03		n.d.
58	(5.26 ± 0.03)	2	(5.89 ± 0.08)	2	(5.10 ± 0.0)	2	(<5)	2
	a: −0.01 ± 0.01		a: −0.12 ± 0.01		a: −0.24 ± 0.01		n.d.
59	(5.34 ± 0.01)	2	6.08 ± 0.11	3	(<5)	2	(<5)	2
	a: 0.21 ± 0.03		a: 0.30 ± 0.01		a: −0.17 ± 0.01		n.d.
60	(5.41 ± 0.02)	2	(5.68 ± 0.01)	2	(5.35 ± 0.04)	2	(<5)	2
	a: 0.10 ± 0.02		a: −0.00 ± 0.03		a: −0.44 ± 0.01		n.d.
61	(<5)	2	(5.27 ± 0.0)	2	(<5)	2	(<5)	2
	n.d.		a: −0.11 ± 0.05		a: −0.01 ± 0.01		n.d.
62	(5.04 ± 0.04)	2	(6.14 ± 0.08)	2	(<5)	2	(<5)	2
	a: 0.06 ± 0.04		a: −0.16 ± 0.05		a: −0.14 ± 0.01		n.d.
63	(<5)	2	(5.54 ± 0.01)	2	(<5)	2	(<5)	2
	a: 0.12 ± 0.06		a: 0.00 ± 0.09		a: −0.07 ± 0.06		n.d.
64	(<5)	2	(6.14 ± 0.01)	2	(5.06 ± 0.01)	2	(<5)	2
	a: 0.08 ± 0.07		a: −0.12 ± 0.06		a: −0.08 ± 0.07		n.d.
65	(<5)	2	(6.52 ± 0.03)	3	(5.92 ± 0.01)	2	(<5)	2
	a: 0.04 ± 0.01		a: −0.12 ± 0.02		a: −0.64 ± 0.00		n.d.
66	n.d.		(6.51 ± 0.12)	2	(5.77 ± 0.04)	2	(<5)	2
			a: −0.12 ± 0.02		a: −0.73 ± 0.01		a: −0.14 ± 0.05
67	(5.11 ± 0.01)	2	(7.03 ± 0.13	2	(5.89 ± 0.07)	2	(<5)	2
	a: −0.02 ± 0.00		a: −0.16 ± 0.02		a: −0.65 ± 0.01		a: −0.16 ± 0.09
68	n.d.		(6.13 ± 0.12)	2	(5.51 ± 0.03)	2	(<5)	2
			a: −0.13 ± 0.02		a: −0.62 ± 0.04		n.d.
69	(<5)	2	(<5)	2	(<5)	2	6.00 ± 0.13	2
	n.d.		n.d.		a: −0.05 ± 0.03		a: 0.77 ± 0.01
70	(<5)	2	(5.82 ± 0.03)	2	(<5)	2	(<5)	2
	a: 0.01 ± 0.01		a: −0.07 ± 0.02		a: −0.07 ± 0.02		n.d.
79	(<5)	2	(5.30 ± 0.02)	2	(5.41 ± 0.11)	2	5.54 ± 0.19	2
	a: 0.09 ± 0.06		a: 0.03 ± 0.03		a: −0.32 ± 0.02		a: 0.35 ± 0.01
80	n.d.		(5.26 ± 0.14)	2	(5.35 ± 0.14)	2	6.11 ± 0.12	3
			a: 0.04 ± 0.00		a: −0.46 ± 0.02		a: 0.50 ± 0.04
81	(5.07 ± 0.09)	2	(6.13 ± 0.12)	2	(5.47 ± 0.04)	2	(5.85 ± 0.15)	2
	a: 0.04 ± 0.01		a: −0.04 ± 0.06		a: −0.33 ± 0.00		a: −0.08 ± 0.19
82	n.d.		(5.19 ± 0.11)	2	(5.28 ± 0.04)	2	(<5)	3
			a: −0.00 ± 0.00		a: −0.46 ± 0.01		n.d.
83	(<5)	4	6.62 ± 0.11	2	(<5)	2	(<5)	2
	n.d.		a: 0.44 ± 0.01		a: 0.04 ± 0.01		n.d.
84	(5.06 ± 0.03)	2	(5.42 ± 0.09)	2	(<5)	2	(<5)	2
	a: 0.06 ± 0.02		a: 0.08 ± 0.04		a: −0.02 ± 0.01		n.d.
85	(<5)	2	6.13 ± 0.03	3	(<5)	2	Inactive	2
	n.d.		a: 0.66 ± 0.02		a: −0.03 ± 0.00
86	(<5)	2	6.39 ± 0.01	2	(<5)	2	Inactive	3
	a: 0.06 ± 0.02		a: 0.42 ± 0.01		a: −0.03 ± 0.02

---

Table 2 Pharmacological activities of selected compounds at the guinea pig ileum (gpH₁R) and the guinea pig right atrium (gpH₂R)

Compound	gpH1R		gpH2R
	pA2	N	pEC50^b/(pA2)/[pD^'2]^c/α^d	N
a Number of experiments. b pEC50 was calculated from the mean shift ΔpEC50 of the agonist curve relative to the histamine reference curve by equation: pEC50 = 6.00 + 0.13 + ΔpEC50. Summand 0.13 represents the mean desensitization observed for control organs when two successive curves for histamine were performed (0.13 ± 0.02, N = 16). The SEM given for pEC50 is the SEM calculated for ΔpEC50. c pD^'2 values given in brackets for compounds producing a significant, concentration-dependent reduction of the maximal response of histamine. d Efficacy, maximal response, relative to the maximal increase in heart rate induced by the reference compound histamine. e E max (histamine) at 10 μM: 0.68 ± 0.01. f E max (histamine) at 30 μM: 0.69 ± 0.03. g E max (histamine) at 30 μM: 0.54 ± 0.09. h pA2 of cimetidine (10 μM, N = 2): 6.32 ± 0.06. For experimental details, cf. ESI.
Histamine	—	—	6.00 ± 0.10	>50
			a: 1.00 ± 0.02
UR-AK24 (1)^27	5.87 ± 0.14	4	7.80 ± 0.07	4
			a: 0.99 ± 0.02
UR-PG276 (3)	n.d.		(<4.5)	2
			[< 4.5]
59	n.d.		6.71 ± 0.04	3
			a: 0.26 ± 0.03
61	n.d.		(<4.5)	2
			[4.22 ± 0.04]
63	n.d.		(4.72 ± 0.34)	2
			[4.54 ± 0.03]
65	5.52 ± 0.06	18	(6.28 ± 0.13)	2
			a: 0^e
69	5.95 ± 0.05	18	(<4.5)	2
			[4.16 ± 0.05]
			a: 0^f
79	5.63 ± 0.04	18	(4.90 ± 0.16)	2
			[4.44 ± 0.15]
			a: <10^g
83	5.42 ± 0.10	15	6.33 ± 0.07	3
			a: 0.54 ± 0.03
84	5.59 ± 0.09	17	6.44 ± 0.11	3
			a: 0.41 ± 0.05
85	5.83 ± 0.07	16	7.00 ± 0.07^h	3
			a: 1.00 ± 0.02
86	5.79 ± 0.04	16	6.69 ± 0.02	3
			a: 0.83 ± 0.06

---

Table 3 Potencies and efficacies of the cyanoguanidines 107–133 at the hHR subtypes in the [³⁵S]GTPγS assay^a or the steady-state [³²P]GTPase assay^b,c

Compound	hH1R		hH2R		hH3R		hH4R
	pEC50 or (pKB)	N	pEC50 or (pKB)	N	EC50 or (pKB)	N	EC50 or (pKB)	N
a Functional [^35S]GTPγS binding assay with membrane preparations of Sf9 cells expressing the hH3R + Gαi2 + Gβ1γ2 or the hH4R + Gαi2 + Gβ1γ2 or the hH2R-Gsαs fusion protein were performed as described in the ESI. b Steady-state GTPase activity in Sf9 cell membranes expressing the hH1R + RGS4 was determined as described under Pharmacological methods. c Reaction mixtures contained ligands at a concentration range from 1 nM to 1 mM as appropriate to generate saturated concentration/response curves. N gives the number of independent experiments performed in duplicate. The intrinsic activity (α) of histamine was set to 1.00 and α values of other compounds were referred to this value. The α values of neutral antagonists and inverse agonists were determined at a concentration of 10 μM. The pKB values of neutral antagonists and inverse agonists were determined in the antagonist mode versus histamine as the agonist.
Histamine	6.72 ± 0.02 (ref. 28)		5.92 ± 0.11 (ref. 28)		7.89 ± 0.07	3	7.96 ± 0.12	5
	α: 1.00		a: 1.00		a: 1.00		a: 1.00
UR-PI376 (5)	(<5)^34		(<5)^34		(6.14 ± 0.02)	2	7.43 ± 0.04	3
	α: 0.07		α: 0.08		a: −0.52 ± 0.05		a: 0.88 ± 0.08
Cimetidine	n.d.		(5.77 ± 0.11)^62		n.d.		(<5)^35
			a: −0.08 ± 0.01
107	(<5)	2	(<5)	2	(<5)	2	(<5)	2
	α: 0.01 ± 0.03		a: 0.04 ± 0.02		a: −0.13 ± 0.08		a: −0.23 ± 0.03
108	(<5.30)	2	4.79 ± 0.01	2	(<5)	2	(6.21 ± 0.04)	2
	a: −0.01 ± 0.03		a: −0.05 ± 0.02		a: −0.47 ± 0.04		a: −0.24 ± 0.11
109	Inactive	2	(<5)	2	(<5)	2	6.26 ± 0.02	2
			a: −0.02 ± 0.01		a: −0.52 ± 0.04		a: 0.74 ± 0.02
110	(<5.30)	2	(<5)	2	(<5)	2	6.33 ± 0.03	2
	a: 0.03 ± 0.05		a: −0.05 ± 0.02		a: −1.11 ± 0.03		a: 0.40 ± 0.07
111	(<5)	2	(<5)	2	5.97 ± 0.04	2	5.44 ± 0.04	2
	a: −0.03 ± 0.02		a: 0.04 ± 0.01		a: 0.23 ± 0.03		a: 0.60 ± 0.17
112	(<5.30)	2	(5.36 ± 0.04)	2	(5.50 ± 0.02)	2	6.61 ± 0.0	2
	a: −0.03 ± 0.01		a: −0.06 ± 0.02		a: −0.65 ± 0.02		a: 0.37 ± 0.1
113	Inactive	2	Inactive	2	Inactive	2	Inactive	2
114	(<5)	2	(<5)	2	Inactive	2	Inactive	2
	a: −0.02 ± 0.01		a: −0.04 ± 0.0
115	(<5)	2	(<5)	2	(5.54 ± 0.01)	2	(5.43 ± 0.09)	2
	a: −0.02 ± 0.04		a: −0.07 ± 0.01		a: −1.03 ± 0.01		a: −0.83 ± 0.06
116	(<5)	2	(<5)	2	(<5)	2	Inactive	2
	a: −0.01 ± 0.03		a: −0.07 ± 0.01		a: −0.26 ± 0.03		a: 0.06 ± 0.03
117	(<5)	2	(<5)	2	(<5)	2	(<5)	2
	a: 0.03 ± 0.01		a: −0.08 ± 0.00		a: −0.42 ± 0.06		a: −0.05 ± 0.04
118	Inactive	2	<5	2	Inactive	2	Inactive	2
			a: 0.31 ± 0.02
119	(<5)	2	(<5)	2	Inactive	2	Inactive	2
	a: 0.01 ± 0.04		a: 0.03 ± 0.02
120	Inactive	2	(<5)	2	Inactive	2	Inactive	2
			a: −0.03 ± 0.0
121	Inactive	2	(<5)	2	Inactive	2	Inactive	2
			a: −0.01 ± 0.04
122	Inactive	2	Inactive	2	Inactive	2	Inactive	2
123	Inactive	2	(<5)	2	Inactive	2	Inactive	2
			a: −0.09 ± 0.01
124	Inactive	2	(<5)	2	Inactive	2	Inactive	2
			a: −0.02 ± 0.0
125	Inactive	2	(<5)	2	Inactive	2	Inactive	2
			a: −0.05 ± 0.00
126	Inactive	2	Inactive	2	(<5)	2	(<5)	2
					a: −0.03 ± 0.04		a: −0.13 ± 0.05
127	(<5)	2	(<5)	2	(<5)	2	(<5)	2
	a: 0.02 ± 0.03		a: −0.12 ± 0.02		a: −0.21 ± 0.02		a: −0.10 ± 0.01
128	(<5)	2	Inactive	2	(<5)	2	(<5)	2
	a: 0.03 ± 0.03				a: −0.10 ± 0.03		a: −0.11 ± 0.03
129	(<5)	2	(<5)	2	(<5)	2	(<5)	2
	a: −0.01 ± 0.01		a: −0.13 ± 0.01		a: −0.36 ± 0.04		a: −0.34 ± 0.01
130	Inactive	2	Inactive	2	(<5)	2	(<5)	2
					a: −0.16 ± 0.02		a: −0.11 ± 0.03
131	(<5)	2	(<5)	2	(<5)	2	Inactive	2
	a: 0.01 ± 0.03		a: −0.08 ± 0.02		a: −1.43 ± 0.14
132	(<5)	2	Inactive	2	(<5)	2	Inactive	2
	a: 0.03 ± 0.02				a: −0.24 ± 0.05
133	(<5)	2	(<5)	2	(5.14 ± 0.01)	2	(<5)	2
	a: 0.01 ± 0.02		a: −0.11 ± 0.01		a: −0.71 ± 0.03		a: −0.09 ± 0.05

---

Acylguanidines 57–86 (Tables 1 and 2). When the imidazole ring in acylguanidines such as 1 was replaced by a phenyl ring (57 and 58), the agonistic potencies at the hH_2,3,4Rs dramatically decreased. However, in terms of antagonism at the hH₂R, these compounds (pK_B = 5.89) were comparable to the H₂R antagonists cimetidine or ranitidine.⁵⁹

Replacing the imidazole ring in 1 with a 2-pyridyl ring resulted in an hH₂R partial agonist (59) with a potency comparable to that of the endogenous ligand histamine (pEC₅₀ = 6.08, E_max = 0.30). At the hH₁R, this compound also behaved as a weak partial agonist. This is in agreement with data for the 2-pyridyl analogue of histamine, betahistine, which is a weak agonist at the hH₁R and hH₂R.^56,59 The bulky diphenylpropanoyl residue in 60 was not tolerated in terms of agonistic potency. At the hH₃R and hH₄R, 59 and 60 were almost inactive. The 3- and 4-pyridyl analogues 61–64 displayed moderate antagonism at the hH₂R and negligible activities at the other HR subtypes.

Replacement of the imidazole ring by the 5-[(dimethylamino)methyl]furan-2-yl group, reminiscent of the H₂R antagonist ranitidine, afforded hH₂R antagonists (65–68): all prepared compounds turned out to be superior to ranitidine (pK_B ∼ 6.10)⁵⁹ at the hH₂R, with the highest antagonist activity exhibited by the diphenylpropanoylguanidine 67 (pK_B = 7.03). 65–68 were weak inverse agonists at the hH₃R and almost inactive at the hH₁R and hH₄R.

Apart from other heterocycles, isomers of the 1H-imidazol-4-yl ring were investigated. The 1H-imidazol-1-yl isomer (69) was comparable with UR-AK24 (1) regarding intrinsic activity (E_max: 0.77 vs. 0.84) at the hH₄R, but the potency was 65-fold lower (pEC₅₀: 6 vs. 7.82). The activities (69) at the other HRs were negligible (pK_B < 5). However, the results for 69 suggest that, in contrast to other HR subtypes, an imidazole–NH group is obviously dispensible, though it is crucial to obtain highly potent hH₄R agonists. As obvious from the diphenylpropanoyl analogue 70, which is almost inactive at the hH₄R, the hH₄R agonism strongly depends on the constitution of the acyl residue.

For the 1H-imidazol-2-yl isomers 79–82 similar pharmacological activities at the hH₄R were observed as for the 1H-imidazol-1-yl isomers 69 and 70. Both 3-arylbutanoylguanidines (79 and 80) exhibited moderate partial agonistic potencies and low intrinsic activities. Similar to the isomer 70, diarylpropanoyl residues (81 and 82) abolished agonism at the hH₄R. At the other HR subtypes, 79–82 were very weak antagonists or inverse agonists.

The results for the imidazole isomers suggest that, regarding agonism and compared to the other HR subtypes, the hH₄R tolerates some modifications in the arrangement of side chains and nitrogen atoms in the heterocycle.

Exchange of the imidazole ring in UR-AK24 by a 1H-pyrazol-4-yl ring (83) resulted in a moderate decrease in potency and efficacy at the hH₂R (pEC₅₀ = 6.62, E_max = 0.44). Interestingly, this compound was virtually inactive at all other HRs, suggesting the pyrazole ring to be an appropriate bioisostere of the imidazole ring to shift the receptor subtype selectivity toward the hH₂R. However, the bulky diphenylpropanoyl residue in 84 was deleterious for agonistic activity at the hH₂R.

Compared to the pyrazole 83, the 1H-1,2,4-triazol-3-yl analogue 85 was more efficacious, but slightly less potent at the hH₂R (pEC₅₀ = 6.13, E_max = 0.66). In contrast to the pyrazole 84, the triazole analog 86 with a diphenylpropanoyl moiety was an hH₂R partial agonist with slightly higher potency than 85 (pEC₅₀ = 6.39, E_max = 0.42). This suggests that the acylguanidines containing a triazole or a pyrazole ring can adopt different binding modes at the hH₂R. Like the pyrazoles 83 and 84, the triazoles 85 and 86 were almost inactive at the other HRs.

The basicity of pyrazole (pK_a ≈ 3)⁶⁰ and triazole (pK_a ≈ 3)⁶⁰ is considerably lower than that of imidazole (pK_a ≈ 7).⁶⁰ This may be interpreted as a hint that the presence of a heterocycle, which is positively charged at physiological pH value, is not required for hH₂R activation. Moreover, the modification of the acyl residue in triazolylalkylguanidines obviously harbours the potential of increasing H₂R selectivity.

The results from isolated guinea pig organs were essentially in line with the data gained from recombinant human H₁ and H₂ receptors, but, in general, the guinea pig receptors proved to be more sensitive than the human orthologs. At the guinea pig ileum the investigated N^G-acylated arylpropylguanidines (Table 2) were moderate H₁R antagonists. Like at the hH₂R, introduction of a phenyl (58), 3-pyridyl (61), 4-pyridyl (63), 5-[(dimethylamino)-methyl]furan-2-yl (65), 1H-imidazol-1-yl (69) and 1H-imidazol-2-yl moiety (79) resulted in a loss of agonistic efficacy at the gpH₂R relative to compound 1 and yielded weak gpH₂R antagonists. Remarkably, the H₂R antagonist activity of compound 65 was comparable to that of cimetidine at the guinea pig right atrium.²⁷

In accordance with the results at the hH₂R, the exchange of the 1H-imidazol-4-yl ring in UR-AK24 (1) by a 2-pyridyl ring (59) resulted in a gpH₂R partial agonist with lower potency and efficacy than 1 (E_max = 0.26). This tendency is reminiscent of the close histamine analogues 2-(2-pyridyl)ethanamine and betahistine, which likewise display weak partial agonism at the guinea pig right atrium.⁶¹ Replacing the 1H-imidazol-4-yl by a 1H-pyrazol-4-yl ring (83 and 84) resulted in compounds with retained gpH₂R partial agonistic activity. However, relative to UR-AK24 (1), the potency decreased by about one order of magnitude. In contrast to 83 and 84, the analogues bearing a 1H-1,2,4-triazol-3-yl ring (85, 86) and UR-AK24 were equiefficacious, though 6-fold less potent at the guinea pig right atrium. These findings support the hypothesis that the 1H-1,2,4-triazol-3-yl ring is a potential bioisostere of the 1H-imidazol-4-yl moiety with respect to gpH₂R affinity.

Cyanoguanidines 107–133 (Table 3). The investigation of the synthesized cyanoguanidines for functional activity at the hH₄R revealed a high sensitivity even towards minor structural modifications and corroborated, in this respect, previous results.³⁴ All cyanoguanidines bearing heterocycles other than imidazole revealed only negligible partial agonism or antagonism, or were even inactive at all four histamine receptor subtypes. The phenylthioethyl substituted aminopyrimidine derivative 115 was the only compound with inverse agonistic activity in the lower micromolar range (pK_B ∼ 5.5) at both, the hH₄R and the hH₃R.

As expected, the compounds 107–112, bearing a methyl substituted imidazole ring, showed some activity at the H₄R. The 2-methylimidazole derivatives 107 and 108 with a tetramethylene chain connecting imidazole and cyanoguanidine were weak inverse agonists at the H₄R, devoid of noteworthy activity at the other HR subtypes. As reported previously, the phenylthioethyl residue confers higher potency at the H₄R compared to a methyl substitution. Reducing the spacer chain length to three carbon atoms provides the hH₄R partial agonists 109 and 110 with pEC₅₀ values around 6.3 and no agonistic activity at the other three histamine receptors. This is in agreement with the results for the amines 92 and 93.⁵² Compound 111, the carba analogue of cimetidine,⁶¹ was a weak partial agonist at the hH₄R (pEC₅₀ = 5.44) and the hH₃R (pEC₅₀ = 5.97) and showed only very weak antagonistic properties at the hH₁R and at the hH₂R. This is in accordance with data reported for H₂R antagonism at guinea pig right atrium and the rat uterus, where 111 was inferior to cimetidine by a factor of 6 to 10.⁶¹ The phenylthioethyl cyanoguanidine 112 was 15 times more potent as an hH₄R agonist than the methyl cyanoguanidine 111. With a pEC₅₀ value of 6.61 at the H₄R, the 5-methyl analogue of UR-PI376, compound 112, showed a more than 10-fold selectivity for the H₄R over the H₃R and the other HR subtypes. Nevertheless, none of the investigated hetarylalkylcyanoguanidines was superior to UR-PI376 in terms of H₄R agonistic potency or receptor subtype selectivity.

Conclusions

In summary, for most of the investigated acylguanidines the replacement of the 1H-imidazol-4-yl ring with isomers or other heterocycles resulted in considerably reduced potency and efficacy at the hH₂R, hH₃R and hH₄R. This underlines the substantial contribution of an appropriate arrangement of the nitrogen atoms in the heterocycle for binding to the H₂R, H₃R and H₄R and for stabilizing an active conformation of the H₂R and H₄R. Strikingly, the imidazol-1-yl-propylguanidine derivative 69 displayed a comparable maximal response as its isomer, reference compound UR-AK24 (1), at the H₄R subtype, although, the potency was about 50-fold lower. As these acylguanidines were poorly active at the other HR subtypes and the hH₄R activity largely depended on the type of acyl residue, further modifications in this moiety appear promising with respect to the development of more potent and selective hH₄R agonists.

Introduction of a 2-pyridyl (59 and 60), a 1H-pyrazol-4-yl (83 and 84) or a 1H-1,2,4-triazol-3-yl (85 and 86) ring provided compounds exhibiting partial to full agonist activity at the hH₂R and gpH₂R. Except for the 2-pyridyl analogues, these compounds had negligible activities at the other hHR subtypes. In particular, the triazole ring was identified as a promising bioisostere for the imidazole ring with respect to H₂R activity. At the gpH₂R, the N^G-acylated triazolylpropylguanidines (85 and 86) were equiefficacious to UR-AK24, but devoid of activities at the other hHR subtypes, suggesting the 1H-1,2,4-triazol-3-yl ring as a potential bioisostere for the design of H₂R selective ligands. 2-Aminothiazole analogues of acylguanidine-type H₂R ligands are described^33,63 as highly selective agonists with higher potencies compared to the triazole analogs. However, compared to the aminothiazoles, the triazole ring is considered as relatively stable against enzymatic oxidation.⁶⁴ This may offer an alternative to improve the drug-like properties.

The cyanoguanidines derived from OUP-16 and UR-PI376 revealed high sensitivity against both, replacement of the heterocycle and minor structural modifications such as methyl-substitution of the imidazole ring. None of the analogues showed improved potency and/or H₄R selectivity compared to UR-PI376. Except for the heterocycle, the cyanoguanidines are devoid of basic groups. Since previous studies revealed higher potency of H₄R agonists with retained basicity in the central structural motif, e.g. acylguanidines, a combination of bioisosteric replacement of both, imidazole ring and cyanoguanidine moiety, should be considered in future ligand design.

In conclusion, the presented data suggest alternative bioisosteric approaches, including the synthesis and pharmacological evaluation of additional heterocyclic analogues of known histamine receptor ligands, with respect to retained/increased potency, improved receptor subtype selectivity and drug-like properties.

Acknowledgements

The authors are grateful to Maria Beer-Kroen, Kerstin Fisch, Karin Schadendorf, Gertraud Wilberg and Astrid Seefeld for expert technical assistance. This work was supported by the Graduate Training Program (Graduiertenkolleg) GRK 760 of the Deutsche Forschungsgemeinschaft and the COST program BM0806 (H4R network) of the European Union.

Notes and references

1. S. J. Hill, C. R. Ganellin, H. Timmerman, J.-C. Schwartz, N. P. Shankley, J. M. Young, W. Schunack, R. Levi and H. L. Haas, Pharmacol. Rev., 1997, 49, 253–278.
2. L. B. Hough, Mol. Pharmacol., 2001, 59, 415–419.
3. R. Seifert, A. Strasser, E. H. Schneider, D. Neumann, S. Dove and A. Buschauer, Trends Pharmacol. Sci., 2013, 34, 33–58.
4. A. Strasser, H.-J. Wittmann, A. Buschauer, E. H. Schneider and R. Seifert, Trends Pharmacol. Sci., 2013, 34, 13–32.
5. K. Sander, T. Kottke and H. Stark, Biol. Pharm. Bull., 2008, 31, 2163–2181.
6. T. Oda, N. Morikawa, Y. Saito, Y. Masuho and S. Matsumoto, J. Biol. Chem., 2000, 275, 36781–36786.
7. T. Nakamura, H. Itadani, Y. Hidaka, M. Ohta and K. Tanaka, Biochem. Biophys. Res. Commun., 2000, 279, 615–620.
8. Y. Zhu, D. Michalovich, H.-L. Wu, K. B. Tan, G. M. Dytko, I. J. Mannan, R. Boyce, J. Alston, L. A. Tierney, X. Li, N. C. Herrity, L. Vawter, H. M. Sarau, R. S. Ames, C. M. Davenport, J. P. Hieble, S. Wilson, D. J. Bergsma and L. R. Fitzgerald, Mol. Pharmacol., 2001, 59, 434–441.
9. K. L. Morse, J. Behan, T. M. Laz, R. E. West, Jr, S. A. Greenfeder, J. C. Anthes, S. Umland, Y. Wan, R. W. Hipkin, W. Gonsiorek, N. Shin, E. L. Gustafson, X. Qiao, S. Wang, J. A. Hedrick, J. Greene, M. Bayne and F. J. Monsma, Jr, J. Pharmacol. Exp. Ther., 2001, 296, 1058–1066.
10. F. Coge, S.-P. Guenin, H. Rique, J. A. Boutin and J.-P. Galizzi, Biochem. Biophys. Res. Commun., 2001, 284, 301–309.
11. C. Liu, X.-J. Ma, X. Jiang, S. J. Wilson, C. L. Hofstra, J. Blevitt, J. Pyati, X. Li, W. Chai, N. Carruthers and T. W. Lovenberg, Mol. Pharmacol., 2001, 59, 420–426.
12. T. Nguyen, D. A. Shapiro, S. R. George, V. Setola, D. K. Lee, R. Cheng, L. Rauser, S. P. Lee, K. R. Lynch, B. L. Roth and B. F. O'Dowd, Mol. Pharmacol., 2001, 59, 427–433.
13. H. Engelhardt, R. A. Smits, R. Leurs, E. Haaksma and I. J. de Esch, Curr. Opin. Drug Discovery Dev., 2009, 12, 628–643.
14. E. Tiligada, E. Zampeli, K. Sander and H. Stark, Expert Opin. Invest. Drugs, 2009, 18, 1519–1531.
15. R. Kiss and G. M. Keseru, Expert Opin. Ther. Pat., 2009, 19, 119–135.
16. R. A. Smits, R. Leurs and I. J. P. de Esch, Drug Discovery Today, 2009, 14, 745–753.
17. J. M. Cowden, J. P. Riley, J. Y. Ma, R. L. Thurmond and P. J. Dunford, Respir. Res., 2010, 11, 86.
18. J. M. Cowden, M. Zhang, P. J. Dunford and R. L. Thurmond, J. Invest. Dermatol., 2010, 130, 1023–1033.
19. R. Seifert, E. H. Schneider, S. Dove, I. Brunskole, D. Neumann, A. Strasser and A. Buschauer, Mol. Pharmacol., 2011, 79, 631–638.
20. S. Nijmeijer, H. F. Vischer, E. M. Rosethorne, S. J. Charlton and R. Leurs, Mol. Pharmacol., 2012, 82, 1174–1182.
21. E. M. Rosethorne and S. J. Charlton, Mol. Pharmacol., 2011, 79, 749–757.
22. J. A. Jablonowski, C. A. Grice, W. Chai, C. A. Dvorak, J. D. Venable, A. K. Kwok, K. S. Ly, J. Wei, S. M. Baker, P. J. Desai, W. Jiang, S. J. Wilson, R. L. Thurmond, L. Karlsson, J. P. Edwards, T. W. Lovenberg and N. I. Carruthers, J. Med. Chem., 2003, 46, 3957–3960.
23. D. Neumann, S. Beermann and R. Seifert, Pharmacology, 2010, 85, 217–223.
24. P. Igel, S. Dove and A. Buschauer, Bioorg. Med. Chem. Lett., 2010, 20, 7191–7199.
25. A. Buschauer, J. Med. Chem., 1989, 32, 1963–1970.
26. A. Buschauer, A. Friese-Kimmel, G. Baumann and W. Schunack, Eur. J. Med. Chem., 1992, 27, 321–330.
27. P. Ghorai, A. Kraus, M. Keller, C. Goette, P. Igel, E. Schneider, D. Schnell, G. Bernhardt, S. Dove, M. Zabel, S. Elz, R. Seifert and A. Buschauer, J. Med. Chem., 2008, 51, 7193–7204.
28. S.-X. Xie, P. Ghorai, Q.-Z. Ye, A. Buschauer and R. Seifert, J. Pharmacol. Exp. Ther., 2006, 317, 139–146.
29. S.-X. Xie, A. Kraus, P. Ghorai, Q.-Z. Ye, S. Elz, A. Buschauer and R. Seifert, J. Pharmacol. Exp. Ther., 2006, 317, 1262–1268.
30. P. Igel, E. Schneider, D. Schnell, S. Elz, R. Seifert and A. Buschauer, J. Med. Chem., 2009, 52, 2623–2627.
31. G. Coruzzi, H. Timmerman, M. Adami and G. Bertaccini, Naunyn-Schmiedeberg's Arch. Pharmacol., 1993, 348, 77–81.
32. P. Ghorai, A. Kraus, T. Birnkammer, R. Geyer, G. Bernhardt, S. Dove, R. Seifert, S. Elz and A. Buschauer, Bioorg. Med. Chem. Lett., 2010, 20, 3173–3176.
33. A. Kraus, P. Ghorai, T. Birnkammer, D. Schnell, S. Elz, R. Seifert, S. Dove, G. Bernhardt and A. Buschauer, ChemMedChem, 2009, 4, 232–240.
34. P. Igel, R. Geyer, A. Strasser, S. Dove, R. Seifert and A. Buschauer, J. Med. Chem., 2009, 52, 6297–6313.
35. H. D. Lim, R. M. van Rijn, P. Ling, R. A. Bakker, R. L. Thurmond and R. Leurs, J. Pharmacol. Exp. Ther., 2005, 314, 1310–1321.
36. H. D. Lim, R. A. Smits, R. A. Bakker, C. M. E. vanDam, I. J. P. deEsch and R. Leurs, J. Med. Chem., 2006, 49, 6650–6651.
37. T. Hashimoto, S. Harusawa, L. Araki, O. P. Zuiderveld, M. J. Smit, T. Imazu, S. Takashima, Y. Yamamoto, Y. Sakamoto, T. Kurihara, R. Leurs, R. A. Bakker and A. Yamatodani, J. Med. Chem., 2003, 46, 3162–3165.
38. Chemistry and Structure–Activity Relationships of Drugs Acting at Histamine Receptors, ed. C. R. Ganellin, Wright PSG, Bristol, London, Boston, 1982.
39. C. R. Ganellin, Pharmacochemistry of H1 and H₂ Receptors, Wiley-Liss, Inc., 1992.
40. J. Navarro, Pharmazie, 1995, 50, 12–15.
41. B. Striegl, Doctoral thesis, University of Regensburg, Germany, 2006.
42. K. Sonogashira, Y. Tohda and N. Hagihara, Tetrahedron Lett., 1975, 16, 4467–4470.
43. P. G. Bulger, I. F. Cottrell, C. J. Cowden, A. J. Davies and U.-H. Dolling, Tetrahedron Lett., 2000, 41, 1297–1301.
44. M. Kunze, Doctoral thesis, University of Regensburg, Germany, 2006.
45. Application: J. M. Cox, P. Bellini, R. Barrett, R. M. Ellis and T. R. Hawkes (Zeneca Ltd.), WO Pat., WO 9315610, 1993, Chem. Abstr., 1993, 120, 134813.
46. W. S. Wadsworth and W. D. Emmons, J. Am. Chem. Soc., 1961, 83, 1733–1738.
47. O. Mitsunobu, M. Yamada and T. Mukaiyama, Bull. Chem. Soc. Jpn., 1967, 40, 935–939.
48. Application: J. R. Allen, S. A. Hitchcock, B. Liu and W. W. Turner, Jr. (Eli Lilly Co.), WO Pat., WO 2005009941, 2005, Chem. Abstr., 2005, 142, 197700.
49. K. Feichtinger, H. L. Sings, T. J. Baker, K. Matthews and M. Goodman, J. Org. Chem., 1998, 63, 8432–8439.
50. R. Paul and G. W. Anderson, J. Am. Chem. Soc., 1960, 82, 4596–4600.
51. R. Paul and G. W. Anderson, J. Org. Chem., 1962, 27, 2094–2099.
52. R. Geyer, M. Kaske, P. Baumeister and A. Buschauer, Arch. Pharm., 2014, 347 DOI:10.1002/ardp.201300316 , in press.
53. R. L. Webb and C. S. Labaw, J. Heterocycl. Chem., 1982, 19, 1205–1206.
54. R. Geyer and A. Buschauer, Arch. Pharm., 2011, 344, 775–785.
55. M. T. Kelley, T. Bürckstümmer, K. Wenzel-Seifert, S. Dove, A. Buschauer and R. Seifert, Mol. Pharmacol., 2001, 60, 1210–1225.
56. R. Seifert, K. Wenzel-Seifert, T. Bürckstümmer, H. H. Pertz, W. Schunack, S. Dove, A. Buschauer and S. Elz, J. Pharmacol. Exp. Ther., 2003, 305, 1104–1115.
57. T. Asano, S. E. Pedersen, C. W. Scott and E. Ross, Biochemistry, 1984, 23, 5460–5467.
58. G. Hilf, P. Gierschik and K. H. Jakobs, Eur. J. Biochem., 1989, 186, 725–731.
59. H. Preuss, P. Ghorai, A. Kraus, S. Dove, A. Buschauer and R. Seifert, J. Pharmacol. Exp. Ther., 2007, 321, 983–995.
60. pK_a-value was calculated with ACD Labs 9.0, Advanced Chemistry Development, Toronto (Canada).
61. G. J. Durant, J. C. Emmett, C. R. Ganellin, P. D. Miles, M. E. Parsons, H. D. Prain and G. R. White, J. Med. Chem., 1977, 20, 901–906.
62. H. Preuss, Doctoral thesis, University of Regensburg, 2007.
63. T. Birnkammer, A. Spickenreither, I. Brunskole, M. Lopuch, N. Kagermeier, G. Bernhardt, S. Dove, R. Seifert, S. Elz and A. Buschauer, J. Med. Chem., 2012, 55, 1147–1160.
64. D. K. Dalvie, A. S. Kalgutkar, S. C. Khojasteh-Bakht, R. S. Obach and J. P. O'Donnell, Chem. Res. Toxicol., 2002, 15, 269–299.

---

Footnotes

† Electronic supplementary information (ESI) available: Synthesis procedures, analytical data and pharmacological methods. See DOI: 10.1039/c3md00245d

‡ These authors contributed equally to this work.

---