Synthesis and selective inhibition of human monoamine oxidases of a large scaffold of (4,5-substituted-thiazol-2-yl)hydrazones

Franco Chimenti; Daniela Secci; Adriana Bolasco; Paola Chimenti; Arianna Granese; Simone Carradori; Melissa D'Ascenzio; Matilde Yáñez; Francisco Orallo

doi:10.1039/C0MD00014K

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DOI: 10.1039/C0MD00014K (Concise Article) Med. Chem. Commun., 2010, 1, 61-72

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Synthesis and selective inhibition of human monoamine oxidases of a large scaffold of (4,5-substituted-thiazol-2-yl)hydrazones†

Franco Chimenti ^a, Daniela Secci *^a, Adriana Bolasco ^a, Paola Chimenti ^a, Arianna Granese ^a, Simone Carradori ^a, Melissa D'Ascenzio ^a, Matilde Yáñez ^b and Francisco Orallo ^b
^aDipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy. E-mail: daniela.secci@uniroma1.it; Fax: +39 06 49913772; Tel: +39 06 49913149
^bDepartamento de Farmacología and Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur, E-15782 Santiago de Compostela, La Coruña, Spain

Received 18th February 2010 , Accepted 21st March 2010

First published on 28th April 2010

Abstract

A large class of (4,5-substituted-thiazol-2-yl)hydrazone derivatives (1–66) was synthesized in good yield (82–99%) and characterized by elemental analysis and ¹H NMR studies. The compounds were assayed for their in vitro human monoamine oxidase inhibitory activity and selectivity. Most of them showed IC₅₀ values in the micromolar range. Our aim was to evaluate the importance of a bulky group at C2, C4, and C5 positions of the thiazole nucleus in modulating the biological activity of this scaffold.

1 Introduction

Monoamine oxidases (MAOs; EC 1.4.3.4) are flavoenzymes which exist in two isoforms, MAO-A and MAO-B, sharing about 70% amino acid identity. They are identified by separated encoding genes and can be distinguished on the basis of tissue distribution, inhibitor sensitivity, and substrate selectivity. They catalyze the oxidative deamination of endogenous and exogenous monoamines, playing a significant role in the control of intracellular concentration of monoaminergic neurotransmitters or neuromodulators such as 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NA), and dietary amines. In particular, hMAO-A is present in catecholaminergic neurons, placenta, and fibroblasts; it preferentially deaminates 5-HT and NA and is irreversibly inhibited by low concentrations of clorgyline. Instead, hMAO-B is concentrated in hystaminergic and serotoninergic neurons, glial cells, and lymphocytes; it prefers substrates like phenylethylamines and is selectively inhibited by R-(−)-deprenyl. Dopamine and tyramine are common substrates of both isoforms.^1–3 By inhibiting MAO, the degradation of these monoamines is blocked, leading to an increase in their availability for physiological functions. Indeed, the rapid degradation of these molecules ensures the proper functioning of synaptic neurotransmission and is critically important for the regulation of emotion and other brain functions. The monoaminergic hypothesis of depression, in fact, postulates that the lack of adequate concentrations of monoamines (NA, 5-HT, and DA) in the synaptic cleft could be the cause of the abnormal behaviors that characterize this pathology. The enzymatic isoform which is principally involved in the metabolism of such neurotransmitters is hMAO-A. On the basis of this assumption, the medical treatment of depression makes use of a large number of hMAO-A inhibitors (MAO-I). These molecules, in fact, are not only able to increase the concentration of NA and 5-HT in the synaptic cleft, but they even reduce the oxidative stress caused by the metabolism of such neurotransmitters and, in chronic administration, they can determine an increase of expression of neurotrophic factors (BDNF) whose action is essential to guarantee neuronal plasticity and survival.

Furthermore, the observation that hMAO-B levels in humans increase 4–5-fold on aging presents a rationale for the involvement of hMAO-B in age-related neurological disorders such as Parkinson's and Alzheimer's disease. Increased hMAO-B activity would be expected to diminish dopamine concentration and to release catalytic reaction products (H₂O₂). Dopamine has been involved in R-synuclein aggregation implicated in the etiology of Parkinson's disease, while high levels of H₂O₂ in the cell promote apoptotic signaling events and the development of Parkinson's disease. Inhibition of hMAO-B allows a sort of neuroprotection against this bioactivation and treatment of pre-Parkinson's patients with selective hMAO-B inhibitors has been shown to be effective in reducing the development of this neurodegeneration.⁴ As hMAO-A inhibitors are used for the treatment of mental disorders, particularly depression, and hMAO-B inhibitors are used as coadjuvants for the treatment of Parkinson's and Alzheimer's disease, the aim of researchers is to synthesize selective inhibitors that would permit control over this enzymatic activity.^5–7

In previous studies conducted by our research group,^8–10 numerous substituted thiazolylhydrazones have been investigated as hMAO inhibitors. In this paper, our interest is to point out the influence of steric hindrance on the inhibitory activity and selectivity of a wide series of new 2-thiazolylhydrazones on which the portion interacting with the active site of the enzymes (substituents at C4 and C5 of the thiazole nucleus) was opportunely modified on the basis of the information we extrapolated from the interaction pattern among these isoforms and their inhibitors.^11,12 From molecular modelling studies previously reported by us,^8,9 the corresponding residues Ile335 (hMAO-A) and Tyr326 (hMAO-B) appeared relevant to explain the molecular recognition of this scaffold, because they oriented the substituent in C4 of the inhibitor in the catalytic site. In particular, in the B isoform Tyr326, due to its consistent steric effects, induced the inhibitor to interact with the FAD. Conversely, the hMAO-A Ile335, characterized by a lower hindrance with respect to the corresponding hMAO-B residue, allowed the ligand to assume multiple poses into the binding cleft with higher enthalpic and entropic penalties. In addition, the presence of a methyl group at C5 of the thiazole has not been evaluated before. Finally, the choice of different aliphatic, cycloaliphatic, heterocyclic, and bicyclic moieties on the C [double bond, length as m-dash] N group (C2 of the thiazole) allowed us to better comprehend the steric and electronic influence on the enzyme-inhibitor interaction and to modulate the chemical structure of these derivatives.

2 Results and discussion

4,5-Substituted-2-thiazolylhydrazone derivatives (1–66) were synthesized in high yields (82–99%) as reported in our previous communications.^8–10 Different carbonyl compounds reacted directly with thiosemicarbazide in 2-propanol with catalytic amounts of acetic acid, and the obtained thiosemicarbazones were subsequently converted into 4,5-substituted-2-thiazolylhydrazones by reaction with α-bromo acetylnaphthalene or α-bromo propiophenone in the same solvent (Hantzsch reaction) (Scheme 1).


	Scheme 1 Reagents and conditions: (i) CH₃COOH (cat.), 2-propanol, rt; (ii) α-bromo acetylnaphthalene or α-bromo propiophenone, 2-propanol, rt.

All synthesized products were purified by washing them with petroleum ether and diethyl ether and, if required, by chromatography (SiO₂, ethyl acetate–hexane) before characterization by ¹H NMR and elemental analysis. Moreover, the presence of a C [double bond, length as m-dash] N double bond can give rise to isomeric geometry E/Z. The ¹H NMR (in CDCl₃) spectra analysis revealed that the E isomer was more favoured and stable than the Z-configuration. The amounts of both conformers were measured by area integration of the signal relative to the CH₃ protons (area ratio of proton signals E [thin space (1/6-em)] :Z was generally 6:1). The low-field signal was assigned to the E isomer, since it is widely accepted in thiosemicarbazone derivatives.¹³ Our choice, as reaction medium, of a polar alcoholic solvent appeared to be preferred to obtain the E-configuration and limit the interconversion according to the results of our previous theoretical and chromatographic study for similar compounds.¹⁴

The biological activity of the test drugs (assayed as E/Z mixture) on hMAOs was investigated by measuring their effects on the production of hydrogen peroxide (H₂O₂) from p-tyramine (a common substrate for both hMAO-A and hMAO-B), using the Amplex Red MAO assay kit and microsomal MAO isoforms prepared from insect cells (BTI-TN-5B1-4) infected with recombinant baculovirus containing cDNA inserts for hMAO-A or hMAO-B. The production of H₂O₂ catalyzed by MAO isoforms can be detected using 10-acetyl-3,7-dihydroxyphenoxazine, a non-fluorescent and highly sensitive probe, that reacts with H₂O₂ in the presence of horseradish peroxidase to produce a fluorescent product, resorufin. In this study hMAO activity was evaluated using the above method following the general procedure previously described by us.¹⁵ The test drugs (new compounds and reference inhibitors) themselves were unable to react directly with the Amplex Red reagent, which indicates that these drugs do not interfere with the measurements. On the other hand, in our experiments and under our experimental conditions, the control activity of hMAO-A and hMAO-B, using p-tyramine, was 165 ± 2 pmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min (n = 20). Most tested drugs concentration-dependently inhibited this enzymatic control activity.

Structure–activity relationships (SARs) were inferred from data of enzymatic experiments reported in Table 1, 2, and 3. All tested compounds inhibited hMAOs at micromolar concentrations with ratio values ranging from 0.043 (55) to >16.67 (45). All compounds bearing a naphthalene moiety on C4 of the thiazole ring (1–9, 19–27, and 37–50), to which for practical reasons we will refer as “first series”, possessed a greater inhibitory activity on hMAO-A than on hMAO-B. This evidence could be justified on the basis of the detailed informations that PDB provides about the active site of both isoforms: the active site of hMAO-A is characterized by a single large hydrophobic cavity which adapts itself to the naphthalene moiety better than the two small hydrophobic cavities of hMAO-B.^11,12 In addition, compounds bearing a less bulky substituent (benzene) on C4 of the thiazole ring and a CH₃ group on C5 (10–18, 28–36, 51–66), the “second series”, were characterized by a decrease of selectivity towards MAO-A. Moreover, these compounds were not as active as the former ones, probably because of the effect of steric hindrance of a methyl group on C5 of the thiazole. Molecular modelling studies reported for similar compounds,³ in fact, proved that this region of the thiazole moiety was involved in the interaction between inhibitor and FAD cofactor which was placed inside the active site of the enzyme and was responsible of its oxidative activity. Therefore small changes of substituent dimension in this position were scarcely tolerated.

Table 1 Structures and biological activity of aliphatic derivatives 1–18^a


Comp	R	R₁	R₂	R₃	IC₅₀ hMAO-A	IC₅₀ hMAO-B	Ratio
a C = Clorgyline, D = R-(−)-deprenyl, I = Iproniazid, M = Moclobemide, Is = Isatin. Ratio: hMAO-A selectivity index = IC_50 (_hMAO-B₎/IC_50 (_hMAO-A₎. Each IC₅₀ value is the mean ± S.E.M. from five experiments. b Level of statistical significance: P < 0.01 versus the corresponding IC₅₀ values obtained against hMAO-B, as determined by ANOVA/Dunnett's. c Level of statistical significance: P < 0.05 versus the corresponding IC₅₀ values obtained against hMAO-B, as determined by ANOVA/Dunnett's. d 100 μM inhibits the corresponding hMAO activity by approximately 40–45%. At higher concentration the compounds precipitate. e Values obtained under the assumption that the corresponding IC₅₀ against hMAO-B is the highest concentration tested (100 μM).
1	CH₃	CH₃	naphthalen-2-yl	H	1.56 ± 0.07 μM^b	3.55 ± 0.29 μM	2.27
2	CH₃	CH₂CH₃	naphthalen-2-yl	H	1.74 ± 0.08 μM^c	2.65 ± 0.19 μM	1.52
3	CH₃	(CH₂)₂CH₃	naphthalen-2-yl	H	1.81 ± 0.07 μM^c	3.11 ± 0.16 μM	1.72
4	CH₂CH₃	CH₂CH₃	naphthalen-2-yl	H	1.86 ± 0.06 μM	2.32 ± 0.03 μM	1.25
5	CH₃	CH₂CH(CH₃)₂	naphthalen-2-yl	H	2.31 ± 0.16 μM^c	3.56 ± 0.06 μM	1.54
6	CH₃	CH₂CH₂CHCH₂	naphthalen-2-yl	H	1.37 ± 0.08 μM^b	3.94 ± 0.25 μM	2.86
7	CH₃	(CH₂)₄CH₃	naphthalen-2-yl	H	2.45 ± 0.12 μM^b	15.96 ± 0.45 μM	6.67
8	CH₂CH₃	(CH₂)₃CH₃	naphthalen-2-yl	H	2.93 ± 0.12 μM	3.76 ± 0.13 μM	1.28
9	CH₃	(CH₂)₅CH₃	naphthalen-2-yl	H	15.84 ± 0.99 μM^b		>6.25^e
10	CH₃	CH₃	phenyl	CH₃	2.55 ± 0.17 μM^b	5.28 ± 0.36 μM	2.08
11	CH₃	CH₂CH₃	phenyl	CH₃	1.55 ± 0.07 μM^c	1.53 ± 0.21 μM	1.0
12	CH₃	(CH₂)₂CH₃	phenyl	CH₃	2.52 ± 0.13 μM^c	2.31 ± 0.08 μM	0.90
13	CH₂CH₃	CH₂CH₃	phenyl	CH₃	1.65 ± 0.09 μM	2.45 ± 0.14 μM	1.49
14	CH₃	CH₂CH(CH₃)₂	phenyl	CH₃	2.40 ± 0.13 μM^c	2.78 ± 0.12 μM	1.16
15	CH₃	CH₂CH₂CHCH₂	phenyl	CH₃	6.97 ± 0.43 μM^c	8.85 ± 0.45 μM	1.27
16	CH₃	(CH₂)₄CH₃	phenyl	CH₃	3.69 ± 0.11 μM^b	6.00 ± 0.21 μM	1.64
17	CH₂CH₃	(CH₂)₃CH₃	phenyl	CH₃	4.13 ± 0.22 μM	4.78 ± 0.17 μM	1.16
18	CH₃	(CH₂)₅CH₃	phenyl	CH₃	3.91 ± 0.19 μM^b	3.75 ± 0.12 μM	1.04

Table 2 Structures and biological activity of cycloaliphatic derivatives 19–36^a


Comp	n	R₂	R₃	R₄	IC₅₀ hMAO-A	IC₅₀ hMAO-B	Ratio
a C = Clorgyline, D = R-(−)-deprenyl, I = Iproniazid, M = Moclobemide, Is = Isatin. Ratio: hMAO-A selectivity index = IC_50 (_hMAO-B₎/IC_50 (_hMAO-A₎. Each IC₅₀ value is the mean ± S.E.M. from five experiments. b Level of statistical significance: P < 0.01 versus the corresponding IC₅₀ values obtained against hMAO-B, as determined by ANOVA/Dunnett's. c Level of statistical significance: P < 0.05 versus the corresponding IC₅₀ values obtained against hMAO-B, as determined by ANOVA/Dunnett's.
19	1	naphthalen-2-yl	H	H	1.21 ± 0.06 μM^c	2.19 ± 0.07 μM	1.82
20	1	naphthalen-2-yl	H	2-CH₃	2.60 ± 0.08 μM	2.88 ± 0.06 μM	1.11
21	1	naphthalen-2-yl	H	3-CH₃	1.91 ± 0.09 μM^c	3.49 ± 0.05 μM	1.82
22	2	naphthalen-2-yl	H	H	1.98 ± 0.11 μM	3.19 ± 0.07 μM	1.61
23	2	naphthalen-2-yl	H	2-CH₃	2.01 ± 0.08 μM^c	4.53 ± 0.18 μM	2.27
24	2	naphthalen-2-yl	H	3-CH₃	1.94 ± 0.07 μM^b	10.84 ± 1.08 μM	5.56
25	2	naphthalen-2-yl	H	4-CH₃	1.95 ± 0.09 μM^b	11.53 ± 0.51 μM	5.88
26	3	naphthalen-2-yl	H	H	2.33 ± 0.15 μM^b	12.61 ± 0.87 μM	5.56
27	4	naphthalen-2-yl	H	H	2.91 ± 0.19 μM^b	7.55 ± 0.36 μM	2.63
28	1	phenyl	CH₃	H	0.55 ± 0.02 μM^c	1.41 ± 0.08 μM	2.56
29	1	phenyl	CH₃	2-CH₃	2.44 ± 0.14 μM	1.33 ± 0.02 μM	0.56
30	1	phenyl	CH₃	3-CH₃	2.29 ± 0.16 μM^c	2.31 ± 0.04 μM	1.01
31	2	phenyl	CH₃	H	1.78 ± 0.07 μM	1.79 ± 0.02 μM	1.01
32	2	phenyl	CH₃	2-CH₃	3.23 ± 0.21 μM^c	1.11 ± 0.03 μM	0.34
33	2	phenyl	CH₃	3-CH₃	3.63 ± 0.26 μM^b	2.88 ± 0.12 μM	0.76
34	2	phenyl	CH₃	4-CH₃	3.90 ± 0.24 μM^b	2.52 ± 0.06 μM	0.66
35	3	phenyl	CH₃	H	2.11 ± 0.17 μM^b	1.96 ± 0.12 μM	0.90
36	4	phenyl	CH₃	H	7.07 ± 0.49 μM	2.31 ± 0.06 μM	0.32

Table 3 Structures and biological activity of heterocyclic derivatives 37–66 and reference drugs^a


Comp	Cy	R₁	R₂	R₃	IC₅₀ hMAO-A	IC₅₀ hMAO-B	Ratio
a C = Clorgyline, D = R-(−)-deprenyl, I = Iproniazid, M = Moclobemide, Is = Isatin. Ratio: hMAO-A selectivity index = IC_50 (_hMAO-B₎/IC_50 (_hMAO-A₎. Each IC₅₀ value is the mean ± S.E.M. from five experiments. b Level of statistical significance: P < 0.01 versus the corresponding IC₅₀ values obtained against hMAO-B, as determined by ANOVA/Dunnett's. c Level of statistical significance: P < 0.05 versus the corresponding IC₅₀ values obtained against hMAO-B, as determined by ANOVA/Dunnett's. d Inactive at 1 mM (highest concentration tested). e 100 μM inhibits the corresponding hMAO activity by approximately 40–45%. At higher concentration the compounds precipitate. f Values obtained under the assumption that the corresponding IC₅₀ against hMAO-B is the highest concentration tested (100 μM). g Value obtained under the assumption that the corresponding IC₅₀ against hMAO-B is the highest concentration tested (1 mM). h Value obtained under the assumption that the corresponding IC₅₀ against hMAO-A is the highest concentration tested (100 μM). i Ref. 16. j Ref. 17.
37	fur-2-yl	H	naphthalen-2-yl	H	2.04 ± 0.07 μM	2.76 ± 0.09 μM	1.35
38	fur-2-yl	CH₃	naphthalen-2-yl	H	1.77 ± 0.06 μM	2.29 ± 0.13 μM	1.30
39	thiophen-2-yl	H	naphthalen-2-yl	H	2.71 ± 0.13 μM^c	4.63 ± 0.19 μM	1.72
40	thiophen-2-yl	CH₃	naphthalen-2-yl	H	3.73 ± 0.21 μM^b	14.53 ± 1.08 μM	3.85
41	cyclohexyl	CH₃	naphthalen-2-yl	H	6.95 ± 0.29 μM		>14.29^f
42	phenyl	CH₃	naphthalen-2-yl	H	2.00 ± 0.13 μM^b	6.23 ± 0.17 μM	3.13
43	pyridin-3-yl	H	naphthalen-2-yl	H	2.57 ± 0.16 μM^c	4.76 ± 0.29 μM	1.85
44	pyridin-4-yl	H	naphthalen-2-yl	H	8.67 ± 0.71 μM	7.48 ± 0.46 μM	0.83
45	benzodioxol-5-yl	H	naphthalen-2-yl	H	5.92 ± 0.13 μM		>16.67^f
46	indol-3-yl	H	naphthalen-2-yl	H	2.34 ± 0.16 μM^c	5.99 ± 0.38 μM	2.56
47	naphthalen-1-yl	H	naphthalen-2-yl	H	16.38 ± 1.09 μM	22.90 ± 1.12 μM	1.41
48	naphthalen-2-yl	CH₃	naphthalen-2-yl	H
49	coumarin-3-yl	CH₃	naphthalen-2-yl	H	6.47 ± 0.21 μM^c	10.84 ± 0.76 μM	1.67
50	ferrocen-2-yl	CH₃	naphthalen-2-yl	H	6.63 ± 0.58 μM		>14.28^f
51	fur-2-yl	H	phenyl	CH₃	1.79 ± 0.04 μM	3.06 ± 0.14 μM	1.72
52	fur-2-yl	CH₃	phenyl	CH₃	1.68 ± 0.06 μM	2.10 ± 0.08 μM	1.25
53	thiophen-2-yl	H	phenyl	CH₃	3.38 ± 0.17 μM^c	3.05 ± 0.09 μM	0.90
54	thiophen-2-yl	CH₃	phenyl	CH₃	2.67 ± 0.11 μM^b	2.34 ± 0.06 μM	0.90
55	thiazol-2-yl	CH₃	phenyl	CH₃	6.52 ± 0.21 μM^b	0.28 ± 0.01 μM^b	0.043
56	cyclohexyl	CH₃	phenyl	CH₃	7.65 ± 0.18 μM	6.14 ± 0.12 μM	0.83
57	phenyl	CH₃	phenyl	CH₃	2.24 ± 0.15 μM^b	2.23 ± 0.10 μM	1.0
58	pyridin-2-yl	CH₃	phenyl	CH₃	2.39 ± 0.16 μM^b	0.88 ± 0.05 μM	0.37
59	pyridin-3-yl	H	phenyl	CH₃	1.83 ± 0.03 μM^c	2.22 ± 0.04 μM	1.22
60	pyridin-4-yl	H	phenyl	CH₃	8.29 ± 0.37 μM^b	1.37 ± 0.10 μM	0.16
61	benzodioxol-5-yl	H	phenyl	CH₃	8.63 ± 0.38 μM	6.57 ± 0.23 μM	0.76
62	indol-3-yl	H	phenyl	CH₃	3.12 ± 0.21 μM^c	1.40 ± 0.05 μM	0.45
63	naphthalen-1-yl	H	phenyl	CH₃
64	naphthalen-2-yl	CH₃	phenyl	CH₃
65	coumarin-3-yl	CH₃	phenyl	CH₃	9.18 ± 0.41 μM^c	3.02 ± 0.07 μM	0.33
66	ferrocen-2-yl	CH₃	phenyl	CH₃	9.09 ± 0.86 μM	8.14 ± 0.47 μM	0.90
C					4.46 ± 0.22 nM^b	61.35 ± 1.13 μM	13698
D					67.25 ± 1.02 μM	19.60 ± 0.86 nM	0.00029
I					6.56 ± 0.26 μM	7.54 ± 0.36 μM	1.14
M					361.38 ± 19.37 μM		>2.77^g
Is						18.75 ± 1.24 μM	<0.18^h

More interesting information about activity and selectivity of thiazole derivatives on hMAOs could be gathered from the analysis of the influence that different substituents on the hydrazonic nitrogen (C2 of the thiazole nucleus) had on biological data of both series. Aliphatic moieties (1–18) showed that hMAO inhibitory activity and selectivity were largely influenced by the chain length, in particular when even the C4 position was substituted with bulky functional groups (1–9). Elongation of the alkyl chain produced a slight reduction of hMAO-A inhibition, but an increase of selectivity towards hMAO-A (derivatives 7 and 9 with ratio 6.67 and >6.25 respectively) could be determined, while the presence of a double bond at the end of the aliphatic chain (6) endowed the compound with discrete hMAO-A selectivity (IC₅₀ hMAO-A = 2.45 ± 0.12 μM and ratio 2.86). Instead, when the C4 position was substituted with benzene (10–18), the elongation of the chain led to only slight differences in activity (with the only exception of compound 15) or selectivity.

On the other hand, carbocyclic derivatives (19–36) showed a surprising coherent behavior in both series: as well as the aliphatic ones, they lost activity when the ring dimension increased: the most active compounds, in fact, were the smallest ones (cyclopentane derivatives 19 and 28 with IC₅₀ hMAO-A = 1.21 ± 0.06 μM and IC₅₀ hMAO-A = 0.55 ± 0.02 μM, respectively). The introduction of a methyl group on the aliphatic ring (20–21, 23–25, 29–30, 32–34) caused a reduction of activity in comparison to the corresponding non-substituted compounds (compare 19 to 20, 22 to 23, 28 to 29, and 31 to 32). However, it is easily noticeable that the presence of a methyl group on C2′ of the aliphatic ring, especially in the first series, influenced activity more than the same substitution on C3′ or C4′ (compare 20 to 21 and 23 to 24–25), while, in the second series, the decrease of activity on hMAO-A due to steric effects on the hydrazonic carbon caused an inversion of selectivity in compounds 29 and 32–36, which, on the contrary, maintained a good inhibitory activity on hMAO-B because of the less bulky substituent on C4 of the thiazole.

The anti-hMAO activity and selectivity associated with the presence of a heterocyclic moiety (37–66) on the hydrazonic nitrogen was also influenced by steric hindrance. The bulkiest compounds of the naphthalene derivatives (45–50), in fact, displayed both low inhibitory activity or the best selectivity toward hMAO-A (compound 45, IC₅₀ hMAO-A = 5.92 ± 0.13 μM and ratio >16.67). Conversely, compounds bearing a smaller heterocycle in that position (37–43) were quite potent but less selective hMAO-A inhibitors.

Regarding compounds 51–66, on which the naphthalene moiety was substituted with a phenyl ring and a methyl group was introduced at C5 of the thiazole nucleus, a surprising inversion of selectivity towards hMAO-B (compound 55, IC₅₀ hMAO-A = 6.52 ± 0.21 μM, IC₅₀ hMAO-B = 0.28 ± 0.01 μM, and ratio 0.043) was observed, confirming the hypothesis that the decrease of steric hindrance also on the C2 (hydrazone substituent) of the thiazole ring determines a reduction of selectivity toward hMAO-A which, in the case of heterocyclic derivatives at the hydrazonic nitrogen, resulted in a dramatic change of activity.

3 Experimental

Starting materials and reagents were obtained from commercial suppliers and were used without purification. Melting points (mp) were determined by the capillary method on an FP62 apparatus (Mettler-Toledo) and are uncorrected. ¹H NMR spectra were recorded at 400 MHz on a Bruker spectrometer using DMSO-d₆ or CDCl₃ as solvent. Chemical shifts are expressed as δ units (ppm) relative to TMS. Coupling constants J are expressed in hertz (Hz). Elemental analyses for C, H, and N were determined with a Perkin-Elmer 240 B microanalyzer and the analytical results were ≥ 95% purity for all compounds. All reactions were monitored by TLC performed on 0.2 mm thick silica gel plates (60 F₂₅₄ Merck). Preparative flash column chromatography was carried out on silica gel (230–400 mesh, G60 Merck). Organic solutions were dried over anhydrous sodium sulfate. Concentration and evaporation of the solvent after reaction or extraction was carried out on a rotary evaporator (Büchi Rotavapor) operating at reduced pressure.

3.1 General procedure for the synthesis of derivatives 1–66

The appropriate carbonyl compound, aldehyde or ketone, (1 g, 50 mmol) was dissolved in 100 mL of 2-propanol and stirred with an equimolar quantity of thiosemicarbazide for 24 h at room temperature with catalytic amounts of acetic acid. Within 30 min the starting reaction mixture turned into a suspension and the precipitated thiosemicarbazone was filtered, washed with a suitable solvent (petroleum ether, hexane, diethyl ether), and dried. The structures of the synthesized intermediates were confirmed by ¹H NMR. Equimolar amounts of the prepared thiosemicarbazone (25 mmol), α-bromo-acetylnaphthalene (1–9, 19–27, and 37–50) or α-bromopropiophenone (10–18, 28–36, and 51–66) both suspended in 2-propanol, were reacted at room temperature under magnetic stirring for 2 h. The obtained powder was filtered on Gooch filter, washed with adequate solvents, and dried under vacuum to give compounds 1–66 in high yield. All the yields are on an isolated basis.

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(propan-2-ylidene)hydrazine (1). (1.05 g, 99%); ivory solid; mp 222–223 °C; (Found: C, 68.32; H, 5.38; N, 14.94. C₁₆H₁₅N₃S requires C, 68.30; H, 5.37; N, 14.93%); δ_H(400 MHz, CDCl₃, Me₄Si) 2.14 (3 H, s, CH₃), 2.25 (3 H, s, CH₃), 6.81 (1 H, s, C(5)H-thiazole), 7.56 (2 H, m, naphthalene), 7.69 (1 H, s, naphthalene), 7.86 (3 H, m, naphthalene), 8.46 (1 H, s, naphthalene), 12.46 (1 H, br s, NH, D₂O exch).

1-(Butan-2-ylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (2). (0.99 g, 99%); ivory solid; mp 180–181 °C; (Found: C, 69.10; H, 5.81; N, 14.23. C₁₇H₁₇N₃S requires C, 69.12; H, 5.80; N, 14.22%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.16 (3 H, m, CH₃), 1.25 (3 H, s, CH₃), 2.56 (2 H, m, CH₂), 6.69 (1 H, s, C(5)H-thiazole), 7.53 (2 H, m, naphthalene), 7.72 (1 H, m, naphthalene), 7.90 (3 H, m, naphthalene), 8.53 (1 H, s, naphthalene), 12.35 (1 H, br s, NH, D₂O exch.).

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(pentan-2-ylidene)hydrazine (3). (0.99 g, 99%); cream solid; mp 181–184 °C; (Found: C, 69.89; H, 6.18; N, 13.59. C₁₈H₁₉N₃S requires C, 69.87; H, 6.19; N, 13.58%); δ_H(400 MHz, DMSO-d₆) 0.98 (3 H, m, CH₃), 1.66 (2 H, m, CH₂), 2.15 (3 H, s, CH₃), 2.37 (2 H, m, CH₂), 6.70 (1 H, s, C(5)H-thiazole), 7.47 (2 H, m, naphthalene), 7.68 (1 H, m, naphthalene), 7.84 (3 H, m, naphthalene), 8.40 (1 H, s, naphthalene), 12.29 (1 H, br s, NH, D₂O exch.).

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(pentan-3-ylidene)hydrazine (4). (0.90 g, 97%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); cream solid; mp 196–198 °C; (Found: C, 69.88; H, 6.18; N, 13.59. C₁₈H₁₉N₃S requires C, 69.87; H, 6.19; N, 13.58%); δ_H(400 MHz, DMSO-d₆) 1.16 (3 H, m, CH₃), 1.25 (3 H, m, CH₃), 2.42 (2 H, m, CH₂), 2.56 (2 H, m, CH₂), 6.69 (1 H, s, C(5)H-thiazole), 7.52 (2 H, m, naphthalene), 7.72 (1 H, m, naphthalene), 7.90 (3 H, m, naphthalene), 8.53 (1 H, s, naphthalene), 12.35 (1 H, br s, NH, D₂O exch.).

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(4-methylpentan-2-ylidene)hydrazine (5). (0.96 g, 99%); yellow solid; mp 119–120 °C; (Found: C, 70.54; H, 6.55; N, 12.98. C₁₉H₂₁N₃S requires C, 70.55; H, 6.54; N, 12.99%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.95 (6 H, d, J 6.6 Hz, 2 × CH₃), 2.05 (1 H, m, J 6.6 Hz, J 7.2 Hz, CH(CH₃)₂), 2.15 (3 H, s, CH₃C [double bond, length as m-dash]

), 2.26 (2 H, d, J 7.2 Hz, CH₂), 6.81 (1 H, s, C(5)H-thiazole), 7.55 (2 H, m, naphthalene), 7.57 (1 H, m, naphthalene), 7.78 (3 H, m, naphthalene), 8.27 (1 H, s, naphthalene), 12.51 (1 H, br s, NH, D₂O exch.).

1-(Hex-5-en-2-ylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (6). (0.83 g, 88%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); light yellow solid; mp 195–197 °C; (Found: C, 70.97; H, 5.97; N, 13.06. C₁₉H₁₉N₃S requires C, 70.99; H, 5.96; N, 13.07%); δ_H(400 MHz, CDCl₃, Me₄Si) 2.20 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.38 (2 H, m, CH₂CH [double bond, length as m-dash]

), 2.50 (2 H, m, CH₂C [double bond, length as m-dash]

N), 5.07 (2 H, m, CH₂ [double bond, length as m-dash]

), 5.83 (1 H, m, CH [double bond, length as m-dash]

), 6.81 (1 H, s, C(5)H-thiazole), 7.55 (2 H, m, naphthalene), 7.71 (1 H, m, naphthalene), 7.92 (3 H, m, naphthalene), 8.27 (1 H, s, naphthalene), 12.43 (1 H, br s, NH, D₂O exch.).

1-(Heptan-2-ylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (7). (0.87 g, 99%) green–yellow solid; mp 133–135 °C; (Found: C, 71.20; H, 6.86; N, 12.44. C₂₀H₂₃N₃S requires C, 71.18; H, 6.87; N, 12.45%); δ_H(400 MHz, DMSO-d₆) 0.91 (3 H, m, CH₃CH₂), 1.34 (2 H, m, CH₂), 1.57 (2 H, m, CH₂), 1.62 (2 H, m, CH₂), 2.19 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.37 (2 H, m, CH₂C [double bond, length as m-dash]

N), 6.69 (1 H, s, C(5)H-thiazole), 7.54 (2 H, m, naphthalene), 7.76 (1 H, m, naphthalene), 7.97 (3 H, m, naphthalene), 8.67 (1 H, s, naphthalene), 12.30 (1 H, br s, NH, D₂O exch.).

1-(Heptan-3-ylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (8). (0.82 g, 93%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); camel solid; mp 170–172 °C; (Found: C, 71.17; H, 6.88; N, 12.46. C₂₀H₂₃N₃S requires C, 71.18; H, 6.87; N, 12.45%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.98 (3 H, m, CH₃CH₂), 1.15 (3 H, m, CH₃CH₂C [double bond, length as m-dash]

N), 1.56 (2 H, m, CH₃CH₂C [double bond, length as m-dash]

N), 1–61 (2 H, m, CH₂C [double bond, length as m-dash]

N), 2.41 (2 H, m, CH₂), 2.54 (2 H, m, CH₂), 6.80 (1 H, s, C(5)H-thiazole), 7.54 (2 H, m, naphthalene), 7.71 (1 H, m, naphthalene), 7.83 (3 H, m, naphthalene), 8.28 (1 H, s, naphthalene), 12.52 (1 H, br s, NH, D₂O exch.).

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(octan-2-ylidene)hydrazine (9). (0.73 g, 84%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); camel solid; mp 145–146 °C; (Found: C, 71.74; H, 7.18; N, 11.96. C₂₁H₂₅N₃S requires C, 71.75; H, 7.17; N, 11.95%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.91 (3 H, m, CH₃CH₂), 1.32 (2 H, m, CH₂), 1.36 (2 H, m, CH₂), 1.39 (2 H, m, CH₂), 1.59 (2H, m, CH₂), 2.20 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.37 (2 H, m, CH₂C [double bond, length as m-dash]

N), 6.81 (1 H, s, C(5)H-thiazole), 7.55 (2 H, m, naphthalene), 7.74 (1 H, m, naphthalene), 7.93 (3 H, m, naphthalene), 8.29 (1 H, s, naphthalene), 12.63 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(propan-2-ylidene)hydrazine (10). (0.91 g, 94%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); white solid; mp 235–237 °C; (Found: C, 63.65; H, 6.15; N, 17.14. C₁₃H₁₅N₃S requires C, 63.64; H, 6.16; N, 17.13%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.97 (3 H, s, CH₃), 1.99 (3 H, s, CH₃), 2.33 (3 H, s, CH₃-thiazole), 7.38 (1 H, m, phenyl), 7.54 (2 H, m, phenyl), 7.59 (2 H, m, phenyl), 12.46 (1 H, br s, NH, D₂O exch).

1-(Butan-2-ylidene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (11). (0.87 g, 99%); sand solid; mp 180–183 °C; (Found: C, 64.84; H, 6.60; N, 16.21. C₁₄H₁₇N₃S requires C, 64.83; H, 6.61; N, 16.20%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.15 (3 H, m, CH₃), 1.25 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.56 (2 H, m, CH₂), 6.69 (3 H, s, CH₃-thiazole), 7.39 (1 H, m, phenyl), 7.53 (2 H, m, phenyl), 7.62 (2 H, m, phenyl), 12.35 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(pentan-2-ylidene)hydrazine (12). (0.81 g, 99%); orange solid; mp 163–165 °C; (Found: C, 65.88; H, 7.01; N, 15.38. C₁₅H₁₉N₃S requires C, 65.90; H, 7.00; N, 15.37%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.98 (3 H, m, CH₃), 1.66 (2 H, m, CH₂), 2.15 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.37 (2 H, m, CH₂), 6.70 (3 H, s, CH₃-thiazole), 7.83 (1 H, m, phenyl), 7.86 (2 H, m, phenyl), 7.87 (2 H, m, phenyl), 12.29 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(pentan-3-ylidene)hydrazine (13). (0.67 g, 82%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); sand solid; mp 135–137 °C; (Found: C, 65.89; H, 6.99; N, 15.37. C₁₅H₁₉N₃S requires C, 65.90; H, 7.00; N, 15.37%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.17 (3 H, m, CH₃), 1.24 (3 H, m, CH₃), 2.42 (2 H, m, CH₂), 2.57 (2 H, m, CH₂), 6.69 (3 H, s, CH₃-thiazole), 7.41 (1 H, m, phenyl), 7.51 (2 H, m, phenyl), 7.56 (2 H, m, phenyl), 12.35 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(4-methylpentan-2-ylidene)hydrazine (14). (0.76 g, 99%); sand solid; mp 150–155 °C; (Found: C, 66.87; H, 7.35; N, 14.63. C₁₆H₂₁N₃S requires C, 66.86; H, 7.36; N, 14.62%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.95 (6 H, m, 2 × CH₃), 1.92 (1 H, m, CH(CH₃)₂), 2.15 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.26 (2 H, m, CH₂), 6.81 (3 H, s, CH₃-thiazole), 7.28 (1 H, m, phenyl), 7.47 (2 H, m, phenyl), 7.54 (2 H, m, phenyl), 12.51 (1 H, br s, NH, D₂O exch.).

1-(Hex-5-en-2-ylidene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (15). (0.82 g, 99%); camel solid; mp 132–140 °C; (Found: C, 67.32; H, 6.72; N, 14.71. C₁₆H₁₉N₃S requires C, 67.33; H, 6.71; N, 14.72%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.96 (3 H, s, CH₃C [double bond, length as m-dash]

), 2.32 (2 H, m, CH₂CH [double bond, length as m-dash]

), 2.49 (2 H, m, CH₂C [double bond, length as m-dash]

N), 5.09 (2 H, m, CH₂ [double bond, length as m-dash]

), 5.83 (1 H, m, CH [double bond, length as m-dash]

), 6.81 (3 H, s, CH₃-thiazole), 7.39 (5 H, m, Ar), 12.43 (1 H, br s, NH, D₂O exch.).

1-(Heptan-2-ylidene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (16). (0.79 g, 99%); ivory solid; mp 143–145 °C; (Found: C, 67.75; H, 7.68; N, 13.95. C₁₇H₂₃N₃S requires C, 67.73; H, 7.69; N, 13.94%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.92 (3 H, m, CH₃CH₂), 1.35 (2 H, m, CH₂), 1.57 (2 H, m, CH₂), 1.62 (2 H, m, CH₂), 2.19 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.37 (2 H, m, CH₂C [double bond, length as m-dash]

N), 6.69 (3 H, s, CH₃-thiazole), 7.42 (1 H, m, phenyl), 7.47 (2 H, m, phenyl), 7.57 (2 H, m, phenyl), 12.30 (1 H, br s, NH, D₂O exch.).

1-(Heptan-3-ylidene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (17). (0.66 g, 84%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); light pink solid; mp 75–77 °C; (Found: C, 67.74; H, 7.69; N, 13.95. C₁₇H₂₃N₃S requires C, 67.73; H, 7.69; N, 13.94%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.99 (3 H, m, CH₃CH₂), 1.15 (3 H, m, CH₃CH₂C [double bond, length as m-dash]

N), 1.56 (2 H, m, CH₂), 1.61 (2 H, m, CH₂), 2.41 (2 H, m, CH₃CH₂C [double bond, length as m-dash]

N), 2.54 (2 H, m, CH₂C [double bond, length as m-dash]

N), 6.80 (3 H, s, CH₃-thiazole), 7.43 (1 H, m, phenyl), 7.48 (2 H, m, phenyl), 7.55 (2 H, m, phenyl), 12.52 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(octan-2-ylidene)hydrazine (18). (0.93 g, 98%); light brown solid; mp 117–119 °C; (Found: C, 68.51; H, 8.00; N, 13.33. C₁₇H₂₃N₃S requires C, 68.53; H, 7.99; N, 13.32%); δ_H(400 MHz, CDCl₃, Me₄Si) 0.92 (3 H, m, CH₃CH₂), 1.34 (2 H, m, CH₂), 1.40 (2 H, m, CH₂), 1.45 (2 H, m, CH₂), 1.59 (2 H, m, CH₂), 2.20 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.37 (2 H, m, CH₂C [double bond, length as m-dash]

N), 6.81 (3 H, s, CH₃-thiazole), 7.55 (1 H, m, phenyl), 7.60 (2 H, m, phenyl), 7.64 (2 H, m, phenyl), 12.63 (1 H, br s, NH, D₂O exch.).

1-Cyclopentylidene-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (19). (0.97 g, 99%); pink solid; mp 212–213 °C; (Found: C, 70.34; H, 5.58; N, 13.66. C₁₈H₁₇N₃S requires C, 70.33; H, 5.57; N, 13.67%); δ_H(400 MHz, DMSO-d₆) 1.86 (2 H, m, CH₂-cyclopentyl), 1.95 (2 H, m, CH₂-cyclopentyl), 2.54 (2 H, m, CH₂-cyclopentyl), 2.66 (2 H, m, CH₂-cyclopentyl), 6.67 (1 H, s, C(5)H-thiazole), 7.45 (2 H, m, naphthalene), 7.67 (1 H, m, naphthalene), 7.85 (3 H, m, naphthalene), 8.44 (1 H, s, naphthalene), 12.28 (1 H, br s, NH, D₂O exch.).

1-(2-Methylcyclopentylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (20). (0.89 g, 99%); yellow solid; mp 170–171 °C; (Found: C, 70.97; H, 5.97; N, 13.06. C₁₉H₁₉N₃S requires C, 70.99; H, 5.96; N, 13.07%); δ_H(400 MHz, DMSO-d₆) 1.20 (3 H, s, CH₃), 1.22 (1 H, m, CH₃CH-cyclopentyl), 1.61 (2 H, m, CH₂-cyclopentyl), 1.90 (2 H, m, CH₂-cyclopentyl), 2.77 (2 H, m, CH₂-cyclopentyl), 6.68 (1 H, s, C(5)H-thiazole), 7.54, (2 H, m, naphthalene), 7.74 (1 H, m, naphthalene), 7.95 (3 H, m, naphthalene), 8.49 (1 H, s, naphthalene), 12.28 (1 H, br s, NH, D₂O exch.).

1-(3-Methylcyclopentylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (21). (0.86 g, 93%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); yellow solid; mp 205–207 °C; (Found: C, 70.98; H, 5.95; N, 13.08. C₁₉H₁₉N₃S requires C, 70.99; H, 5.96; N, 13.07%); δ_H(400 MHz, DMSO-d₆) 1.02 (3 H, m, CH₃), 1.13 (1 H, m, CH₃CH-cyclopentyl), 1.90 (2 H, m, CH₂-cyclopentyl), 2.38 (2 H, m, CH₂-cyclopentyl), 2.66 (2 H, m, CH₂-cyclopentyl), 6.68 (1 H, s, C(5)H-thiazole), 7.39 (2 H, m, naphthalene), 7.55 (1 H, m, naphthalene), 7.76 (3 H, m, naphthalene), 8.33 (1 H, s, naphthalene), 10.70 (1 H, br s, NH, D₂O exch.).

1-Cyclohexyliden-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (22). (0.92 g, 99%); yellow solid; mp 195–196 °C; (Found: C, 70.97; H, 5.95; N, 13.06. C₁₉H₁₉N₃S requires C, 70.99; H, 5.96; N, 13.07%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.59 (2 H, m, CH₂-cyclohexyl), 1.64 (2 H, m, CH₂-cyclohexyl), 1.69 (2 H, m, CH₂-cyclohexyl), 1.77 (2 H, m, CH₂-cyclohexyl), 2.59 (2 H, m, CH₂-cyclohexyl), 6.68 (1 H, s, C(5)H-thiazole), 7.72 (2 H, m, naphthalene), 7.88 (1 H, m, naphthalene), 7.95 (3 H, m, naphthalene), 8.50 (1 H, s, naphthalene), 12.28 (1 H, br s, NH, D₂O exch.).

1-(2-Methylcyclohexylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (23). (0.76 g, 87%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); sand solid; mp 161–162 °C; (Found: C, 71.63; H, 6.32; N, 12.54. C₂₀H₂₁N₃S requires C, 71.61; H, 6.31; N, 12.53%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.16 (3 H, m, CH₃CH-cyclohexyl), 1.32 (1 H, m, CH₃CH-cyclohexyl), 1.60 (2 H, m, CH₂-cyclohexyl), 1.83 (2 H, m, CH₂-cyclohexyl), 2.00 (2 H, m, CH₂-cyclohexyl), 2.19 (2 H, m, CH₂-cyclohexyl), 6.80 (1 H, s, C(5)H-thiazole), 7.55 (2 H, m, naphthalene), 7.73 (1 H, m, naphthalene), 7.85 (3 H, m, naphthalene), 8.27 (1 H, s, naphthalene), 12.53 (1 H, br s, NH, D₂O exch.).

1-(3-Methylcyclohexylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (24). (0.75 g, 86%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); sand solid; mp 185–186 °C; (Found: C, 71.60; H, 6.30; N, 12.54. C₂₀H₂₁N₃S requires C, 71.61; H, 6.31; N, 12.53%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.02 (3 H, m, CH₃CH-cyclohexyl), 1.10 (2 H, m, CH₂-cyclohexyl), 1.60 (1 H, m, CH₃CH-cyclohexyl), 1.68 (2 H, m, CH₂-cyclohexyl), 2.36 (2 H, m, CH₂-cyclohexyl), 3.19 (2 H, m, CH₂-cyclohexyl), 6.67 (1 H, s, C(5)H-thiazole), 7.27 (2 H, m, naphthalene), 7.43 (1 H, naphthalene), 7.64 (3 H, naphthalene), 8.15 (1 H, s, naphthalene), 12.28 (1 H, br s, NH, D₂O exch.).

1-(4-Methylcyclohexylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (25). (0.89 g, 99%); sand solid; mp 186–187 °C; (Found: C, 71.62; H, 6.30; N, 12.52. C₂₀H₂₁N₃S requires C, 71.61; H, 6.31; N, 12.53%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.23 (3 H, m, CH₃CH-cyclohexyl), 1.87 (1 H, m, CH₃CH-cyclohexyl), 1.97 (2 H, m, CH₂-cyclohexyl), 2.42 (2 H, m, CH₂-cyclohexyl), 2.61 (2 H, m, CH₂-cyclohexyl), 3.09 (2 H, m, CH₂-cyclohexyl), 6.80 (1 H, s, C(5)H-thiazole), 7.54 (2 H, m, naphthalene), 7.73 (1 H, m, naphthalene), 7.96 (3 H, m, naphthalene), 8.25 (1 H, s, naphthalene), 12.10 (1 H, br s, NH, D₂O exch.).

1-Cycloheptylidene-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (26). (0.90 g, 99%); sand solid; mp 198–199 °C; (Found: C, 71.63; H, 6.30; N, 12.54. C₂₀H₂₁N₃S requires C, 71.61; H, 6.31; N, 12.53%); δ_H(400 MHz, DMSO-d₆) 1.59 (2 H, m, CH₂-cycloheptyl), 1.63 (2 H, m, CH₂-cycloheptyl), 1.66 (2 H, m, CH₂-cycloheptyl), 1.72 (2 H, m, CH₂-cycloheptyl), 2.84 (2 H, m, CH₂-cycloheptyl), 2.89 (2 H, m, CH₂-cycloheptyl), 7.26 (1 H, s, C(5)H-thiazole), 7.65 (2 H, m, naphthalene), 7.74 (1 H, m, naphthalene), 7.85 (3 H, m, naphthalene), 8.41 (1 H, s, naphthalene), 12.30 (1 H, br s, NH, D₂O exch.).

1-Cyclooctylidene-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (27). (0.75 g, 85%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); sand solid; mp 169–170 °C; (Found: C, 72.15; H, 6.64; N, 12.01. C₂₁H₂₃N₃S requires C, 72.17; H, 6.63; N, 12.02%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.27 (2 H, m, CH₂-cyclooctyl), 1.59 (2 H, m, CH₂-cyclooctyl), 1.72 (2 H, m, CH₂-cyclooctyl), 1.78 (2 H, m, CH₂-cyclooctyl), 2.09 (2 H, m, CH₂-cyclooctyl), 2.44 (2 H, m, CH₂-cyclooctyl), 2.58 (2 H, m, CH₂-cyclooctyl), 6.81 (1 H, s, C(5)H-thiazole), 7.54 (2 H, m, naphthalene), 7.80 (1 H, m, naphthalene), 8.08 (3 H, m, naphthalene), 8.39 (1 H, s, naphthalene), 12.33 (1 H, br s, NH, D₂O exch.).

1-Cyclopentylidene-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (28). (0.95 g, 99%); white solid; mp 203–205 °C; (Found: C, 66.38; H, 6.30; N, 15.49. C₁₅H₁₇N₃S requires C, 66.39; H, 6.31; N, 15.48%); δ_H(400 MHz, DMSO-d₆) 1.80 (2 H, m, CH₂-cyclopentyl), 1.91 (2 H, m, CH₂-cyclopentyl), 2.39 (2 H, m, CH₂-cyclopentyl), 2.57 (2 H, m, CH₂-cyclopentyl), 6.67 (3 H, s, CH₃-thiazole), 7.32 (1 H, m, phenyl), 7.47 (2 H, m, phenyl), 7.57 (2 H, m, phenyl), 12.28 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(2-methylcyclopentylidene)hydrazine (29). (0.82 g, 99%); sand solid; mp 163–165 °C; (Found: C, 67.35; H, 6.72; N, 14.72. C₁₆H₁₉N₃S requires C, 67.33; H, 6.71; N, 14.72%); δ_H(400 MHz, DMSO-d₆) 1.22 (3 H, m, CH₃CH-cyclopentyl), 1.26 (1 H, m, CH₃CH-cyclopentyl), 1.56 (2 H, m, CH₂-cyclopentyl), 2.39 (2 H, m, CH₂-cyclopentyl), 2.78 (2 H, m, CH₂-cyclopentyl), 6.65 (3 H, s, CH₃-thiazole), 7.38 (1 H, m, phenyl), 7.42 (2 H, m, phenyl), 7.50 (2 H, m, phenyl), 12.28 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(3-methylcyclopentylidene)hydrazine (30). (0.82 g, 99%); yellow ochre solid; mp 181–183 °C; (Found: C, 67.34; H, 6.71; N, 14.73. C₁₆H₁₉N₃S requires C, 67.33; H, 6.71; N, 14.72%); δ_H(400 MHz, DMSO-d₆) 1.16 (3 H, m, CH₃CH-cyclopentyl), 1.22 (1 H, m, CH₃CH-cyclopentyl), 1.94 (2 H, m, CH₂-cyclopentyl), 2.33 (2 H, m, CH₂-cyclopentyl), 2.66 (2 H, m, CH₂-cyclopentyl), 6.71 (3 H, s, CH₃-thiazole), 7.41 (1 H, m, phenyl), 7.46 (2 H, m, phenyl), 7.59 (2 H, m, phenyl), 10.70 (1 H, br s, NH, D₂O exch.).

1-Cyclohexylidene-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (31). (0.82 g, 99%); yellow ochre solid; mp 195–196 °C; (Found: C, 67.34; H, 6.72; N, 14.71. C₁₆H₁₉N₃S requires C, 67.33; H, 6.71; N, 14.72%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.52 (2 H, m, CH₂-cyclohexyl), 1.67 (2 H, m, CH₂-cyclohexyl), 1.75 (2 H, m, CH₂-cyclohexyl), 1.83 (2 H, m, CH₂-cyclohexyl), 2.58 (2 H, m, CH₂-cyclohexyl), 6.78 (3 H, s, CH₃-thiazole), 7.28 (1 H, m, phenyl), 7.35 (2 H, m, phenyl), 7.49 (2 H, m, phenyl), 12.28 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(2-methylcyclohexylidene)hydrazine (32). (0.68 g, 84%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); ivory solid; mp 155–157 °C; (Found: C, 68.20; H, 7.08; N, 14.02. C₁₇H₂₁N₃S requires C, 68.19; H, 7.07; N, 14.03%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.20 (3 H, m, CH₃CH-cyclohexyl), 1.36 (1 H, m, CH₃CH-cyclohexyl), 1.68 (2 H, m, CH₂-cyclohexyl), 1.76 (2 H, m, CH₂-cyclohexyl), 1.92 (2 H, m, CH₂-cyclohexyl), 2.56 (2 H, m, CH₂-cyclohexyl), 6.77 (3 H, s, CH₃-thiazole), 7.32 (1 H, m, phenyl), 7.44 (2 H, m, phenyl), 7.52 (2 H, m, phenyl), 12.53 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazine (33). (0.79 g, 99%); light brown solid; mp 159–162 °C; (Found: C, 68.21; H, 7.07; N, 14.02. C₁₇H₂₁N₃S requires C, 68.19; H, 7.07; N, 14.03%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.08 (3 H, m, CH₃CH-cyclohexyl), 1.24 (2 H, m, CH₂-cyclohexyl), 1.32 (1 H, m, CH₃CH-cyclohexyl), 1.66 (2 H, m, CH₂-cyclohexyl), 1.72 (2 H, m, CH₂-cyclohexyl), 2.39 (2 H, m, CH₂-cyclohexyl), 3.02 (2 H, m, CH₂-cyclohexyl), 6.78 (3 H, s, CH₃-thiazole), 7.29 (1 H, m, phenyl), 7.39 (2 H, m, phenyl), 7.54 (2 H, m, phenyl), 12.28 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(4-methylcyclohexylidene)hydrazine (34). (0.80 g, 99%); light brown solid; mp 158–160 °C; (Found: C, 68.21; H, 7.08; N, 14.04. C₁₇H₂₁N₃S requires C, 68.19; H, 7.07; N, 14.03%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.19 (3 H, m, CH₃CH-cyclohexyl), 1.74 (1 H, m, CH₃CH-cyclohexyl), 1.90 (2 H, m, CH₂-cyclohexyl), 2.28 (2 H, m, CH₂-cyclohexyl), 2.64 (2 H, m, CH₂-cyclohexyl), 3.00 (2 H, m, CH₂-cyclohexyl), 6.81 (3 H, s, CH₃-thiazole), 7.32 (1 H, m, phenyl), 7.48 (2 H, m, phenyl), 7.53 (2 H, m, phenyl), 12.10 (1 H, br s, NH, D₂O exch.).

1-Cycloheptylidene-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (35). (0.77 g, 99%); ivory solid; mp 175–177 °C; (Found: C, 68.20; H, 7.06; N, 14.03. C₁₇H₂₁N₃S requires C, 68.19; H, 7.07; N, 14.03%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.50 (2 H, m, CH₂-cycloheptyl), 1.61 (2 H, m, CH₂-cycloheptyl), 1.70 (2 H, m, CH₂-cycloheptyl), 1.76 (2 H, m, CH₂-cycloheptyl), 2.84 (2 H, m, CH₂-cycloheptyl), 2.90 (2 H, m, CH₂-cycloheptyl), 6.83 (3 H, s, CH₃-thiazole), 12.30 (1 H, br s, NH, D₂O exch.).

1-Cyclooctylidene-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (36). (0.64 g, 82%, purified by chromatography: SiO₂, ethyl acetate/hexane 1/1); white solid; mp 135–137 °C; (Found: C, 68.98; H, 7.41; N, 13.42. C₁₈H₂₃N₃S requires C, 68.97; H, 7.40; N, 13.41%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.49 (2 H, m, CH₂-cyclooctyl), 1.56 (2 H, m, CH₂-cyclooctyl), 1.68 (2 H, m, CH₂-cyclooctyl), 1.81 (2 H, m, CH₂-cyclooctyl), 1.95 (2 H, m, CH₂-cyclooctyl), 2.28 (2 H, m, CH₂-cyclooctyl), 2.49 (2 H, m, CH₂-cyclooctyl), 6.81 (3 H, s, CH₃-thiazole), 7.35 (1 H, m, phenyl), 7.44 (2 H, m, phenyl) 7.52 (2 H, m, phenyl), 12.33 (1 H, br s, NH, D₂O exch.).

1-(Furan-2-ylmethylene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (37). (0.89 g, 99%); camel solid; mp 249–250 °C; (Found: C, 67.70; H, 4.11; N, 13.15. C₁₈H₁₃N₃OS requires C, 67.69; H, 4.10; N, 13.16%); δ_H(400 MHz, CDCl₃, Me₄Si) 7.73 (1 H, m, C(3)H-furan), 7.78 (1 H, m, C(4)H-furan), 7.90 (1 H, C(5)H-thiazole), 7.46 (2 H, m, naphthalene), 7.65 (1 H, m, naphthalene), 7.79 (1 H, m, C(5)H-furan), 7.81 (3 H, m, naphthalene), 7.92 (1 H, s, CH = N), 8.46 (1 H, s, naphthalene), 11.95 (1 H, br s, NH, D₂O exch.).

1-(1-(Furan-2-yl)ethylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (38). (0.77 g, 85%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); camel solid; mp 245–247 °C; (Found: C, 68.46; H, 4.54; N, 12.61. C₁₉H₁₅N₃OS requires C, 68.45; H, 4.53; N, 12.60%); δ_H(400 MHz, CDCl₃, Me₄Si) 2.23 (3 H, s, CH₃C [double bond, length as m-dash]

N), 6.61 (1 H, m, C(3)H-furan), 6.76 (1 H, m, C(4)H-furan), 7.31 (1 H, s, C(5)H-thiazole), 7.52 (2 H, m, naphthalene), 7.59 (1 H, m, C(5)H-furan), 7.68 (1 H, m, naphthalene), 7.76 (3 H, m, naphthalene), 8.46 (1 H, s, naphthalene), 11.28 (1 H, br s, NH, D₂O exch.).

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(thiophen-2-ylmethylene)hydrazine (39). (0.87 g, 99%); yellow ochre solid; mp 244–245 °C; (Found: C, 64.43; H, 3.92; N, 12.54. C₁₈H₁₃N₃S₂ requires C, 64.45; H, 3.91; N, 12.53%); δ_H(400 MHz, DMSO-d₆) 6.80 (1 H, s, C(5)H-thiazole), 7.09 (1 H, m, C(3)H-thiophene), 7.31 (1 H, m, C(5)H-thiophene), 7.57 (1 H, m, C(4)H-thiophene), 7.64 (2 H, m, naphthalene), 7.72 (1 H, m, naphthalene), 7.89 (3 H, m, naphthalene), 8.22 (1 H, s, CH = N), 8.64 (1 H, s, naphthalene), 12.22 (1 H, br s, NH, D₂O exch.).

1-(1-Cyclohexylethylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (41). (0.86 g, 86%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); ivory solid; mp 208–210 °C; (Found: C, 72.19; H, 6.64; N, 12.03. C₂₁H₂₃N₃S requires C, 72.17; H, 6.63; N, 12.02%); δ_H(400 MHz, CDCl₃, Me₄Si) 1.60 (2 H, m, CH₂-cyclohexyl), 1.64 (2 H, m, CH₂-cyclohexyl), 1.69 (2 H, m, CH₂-cyclohexyl), 2.34 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.60 (2 H, m, CH₂-cyclohexyl), 2.83 (2 H, m, CH₂-cyclohexyl), 6.68 (1 H, s, C(5)H-thiazole), 7.74 (2 H, m, naphthalene), 7.88 (1 H, m, naphthalene), 7.96 (3 H, m, naphthalene), 8.70 (1 H, s, naphthalene), 12.31 (1 H, br s, NH, D₂O exch.).

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(pyridin-3-ylmethylene)hydrazine (43). (0.85 g, 99%); chrome yellow solid; mp 210–211 °C; (Found: C, 69.09; H, 4.28; N, 16.97. C₁₉H₁₄N₄S requires C, 69.07; H, 4.27; N, 16.96%); δ_H(400 MHz, DMSO-d₆) 7.54 (2 H, m, naphthalene), 7.58 (1 H, m, C(2)H-pyridine), 7.86 (1 H, m, naphthalene), 8.05 (3 H, m, naphthalene), 8.15 (1 H, s, CH = N), 8.38 (1 H, s, C(5)H-thiazole) 8.49 (1 H, m, C(6)H-pyridine), 8.55 (1 H, s, naphthalene), 8.76 (1 H, m, C(4)H-pyridine), 8.99 (1 H, m, C(5)H-pyridine), 11.36 (1 H, br s, NH, D₂O exch.).

1-(4-(Naphthalen-2-yl)thiazol-2-yl)-2-(pyridin-4-ylmethylene)hydrazine (44). (0.85 g, 99%); orange solid; mp 250–252 °C; (Found: C, 69.09; H, 4.28; N, 16.97. C₁₉H₁₄N₄S requires C, 69.07; H, 4.27; N, 16.96%); δ_H(400 MHz, DMSO-d₆) 7.40 (1 H, s, C(5)H-thiazole), 7.45 (1 H, m, C(3)H-pyridine), 7.58 (1 H, m, C(5)H-pyridine), 7.73 (2 H, m, naphthalene), 7.82 (1 H, m, naphthalene), 8.12 (3 H, m, naphthalene), 8.15 (1 H, s, CH [double bond, length as m-dash]

N), 8.26 (1 H, s, naphthalene), 8.39 (1 H, m, C(2)H-pyridine), 8.82 (1 H, m, C(6)H-pyridine), 13.10 (1 H, br s, NH, D₂O exch.).

1-(Benzodioxol-5-ylmethylene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (45). (0.85 g, 99%); salmon solid; mp 244–245 °C; (Found: C, 67.56; H, 4.06; N, 11.24. C₂₁H₁₅N₃O₂S requires C, 67.54; H, 4.05; N, 11.25%); δ_H(400 MHz, DMSO-d₆) 6.06 (2 H, s, OCH₂O), 6.90 (1 H, s, C(5)H-thiazole), 7.16 (1 H, m, benzodioxole), 7.27 (2 H, m, benzodioxole), 7.46 (2 H, m, naphthalene), 7.52 (1 H, m, naphthalene), 7.66 (3 H, m, naphthalene), 7.98 (1 H, s, CH [double bond, length as m-dash]

N), 8.55 (1 H, s, naphthalene), 12.19 (1 H, br s, NH, D₂O exch.).

1-((1H-Indol-3-yl)methylene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (46). (0.78 g, 85%, purified by chromatography: SiO₂, ethyl acetate/hexane 5/1); light pink solid; mp 258–259 °C; (Found: C, 71.70; H, 4.37; N, 15.20. C₂₂H₁₆N₄S requires C, 71.71; H, 4.38; N, 15.21%); δ_H(400 MHz, DMSO-d₆) 7.10 (1 H, s, C(5)H-thiazole), 7.32 (1 H, m, C(6)H-indole), 7.43 (1 H, m, C(7)H-indole), 7.55 (2 H, m, naphthalene), 7.64 (1 H, m, naphthalene), 7.77 (1 H, s, C(2)H-indole), 7.83 (3 H, m, naphthalene), 8.23 (1 H, s, C(4)H-indole), 8.28 (1 H, s, CH [double bond, length as m-dash]

N), 8.55 (1 H, s, naphthalene), 11.55 (1 H, br s, NH, D₂O exch.), 12.15 (1 H, br s, NH, D₂O exch.).

1-(Naphthalen-1-ylmethylene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (47). (0.71 g, 86%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); orange solid; mp 198–199 °C; (Found: C, 75.95; H, 4.51; N, 11.06. C₂₄H₁₇N₃S requires C, 75.96; H, 4.52; N, 11.07%); δ_H(400 MHz, DMSO-d₆) 6.98 (1 H, s, C(5)H-thiazole), 7.34 (4 H, m, naphthalene), 7.57 (2 H, m, naphthalene), 7.97 (6 H, m, naphthalene), 8.33 (1 H, s, naphthalene), 8.36 (1 H, s, naphthalene), 8.91 (1 H, s, CH [double bond, length as m-dash]

N), 11.45 (1 H, br s, NH, D₂O exch.).

1-(1-(2H-2-Oxo-chromen-3-yl)ethylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (49). (0.77 g, 99%); yellow solid; mp 210–211 °C; (Found: C, 70.03; H, 4.17; N, 10.20. C₂₄H₁₇N₃O₂S requires C, 70.05; H, 4.16; N, 10.21%); δ_H(400 MHz, DMSO-d₆) 2.27 (3 H, s, CH₃C [double bond, length as m-dash]

N), 7.05 (1 H, s, C(5)H-thiazole), 7.34 (2 H, m, naphthalene), 7.44 (1 H, m, C(6)H-chromene), 7.48 (1 H, m, C(8)H-chromene) 7.56 (1 H, m, C(7)H-chromene), 7.66 (1 H, m, naphthalene), 7.76 (1 H, m, C(5)H-chromene), 7.94 (3 H, m, naphthalene), 8.16 (1 H, s, naphthalene), 8.64 (1 H, s, CH [double bond, length as m-dash]

N), 11.40 (1 H, br s, NH, D₂O exch.).

1-(1-(Ferrocen-1-yl)ethylidene)-2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazine (50). (0.70 g, 94%, purified by chromatography: SiO₂, ethyl acetate/hexane 5/1); brown solid; mp 185–186 °C; (Found: C, 66.21; H, 5.12; N, 9.28. C₂₅H₂₃FeN₃S requires C, 66.23; H, 5.11; N, 9.27%); δ_H(400 MHz, DMSO-d₆) 2.48 (3 H, s, CH₃C [double bond, length as m-dash]

N), 4.20 (5 H, s, ferrocene), 4.48 (2 H, m, ferrocene), 4.70 (2 H, m, ferrocene), 6.82 (1 H, s, C(5)H-thiazole), 7.56 (2 H, m, naphthalene), 7.73 (1 H, m, naphthalene), 7.86 (3 H, m, naphthalene), 8.29 (1 H, s, naphthalene), 12.40 (1 H, br s, NH, D₂O exch.).

1-(1-(Furan-2-yl)ethylidene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (52). (0.75 g, 85%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); ivory solid; mp 225–227 °C; (Found: C, 64.63; H, 5.09; N, 14.14. C₁₆H₁₅N₃OS requires C, 64.62; H, 5.08; N, 14.13%); δ_H(400 MHz, DMSO-d₆) 2.17 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.33 (3 H, s, CH₃-thiazole), 6.72 (1 H, m, C(3)H-furan), 6.98 (1 H, m, C(4)H-furan), 7.22 (1 H, m, phenyl), 7.38 (2 H, m, phenyl), 7.56 (1 H, m, C(5)H-furan), 7.63 (2 H, m, phenyl), 7.65 (1 H, m, C(5)H-furan), 11.28 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(1-(thiophen-2-yl)ethylidene)hydrazine (54). (0.76 g, 97%); light orange solid; mp 210–212 °C; (Found: C, 61.30; H, 4.81; N, 13.42. C₁₆H₁₅N₃S₂ requires C, 61.31; H, 4.82; N, 13.41%); δ_H(400 MHz, DMSO-d₆) 2.33 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.37 (3 H, s, CH₃-thiazole), 7.12 (1 H, m, C(3)H-thiophene), 7.31 (1 H, m, C(5)H-thiophene), 7.37 (1 H, m, phenyl), 7.40 (1 H, m, C(4)H-thiophene), 7.58 (2 H, m, phenyl), 7.87 (2 H, m, phenyl), 11.40 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(1-(thiazol-2-yl)ethylidene)hydrazine (55). (0.78 g, 99%); salmon solid; mp 250–251 °C; (Found: C, 57.31; H, 4.50; N, 17.83. C₁₅H₁₄N₄S₂ requires C, 57.30; H, 4.49; N, 17.82%); δ_H(400 MHz, DMSO-d₆) 2.28 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.39 (3 H, s, CH₃-thiazole), 7.42 (1 H, m, phenyl), 7.59 (2 H, m, phenyl), 7.64 (2 H, m, phenyl), 7.74 (1 H, m, C(5)H-thiazole), 8.51 (1 H, m, C(4)H-thiazole), 12.31 (1 H, br s, NH, D₂O exch.).

1-(1-Cyclohexylethylidene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (56). (0.78 g, 99%); light yellow solid; mp 175–177 °C; (Found: C, 68.95; H, 7.41; N, 13.42. C₁₈H₂₃N₃S requires C, 68.97; H, 7.40; N, 13.41%); δ_H(400 MHz, DMSO-d₆) 1.97 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.87 (2 H, m, CH₂-cyclohexyl), 2.96 (2 H, m, CH₂-cyclohexyl), 3.16 (2 H, m, CH₂-cyclohexyl), 3.66 (2 H, m, CH₂-cyclohexyl), 7.58 (1 H, m, phenyl), 7.79 (2 H, m, phenyl), 7.97 (2 H, m, phenyl), 12.10 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(1-phenylethylidene)hydrazine (57). (0.79 g, 99%); green–yellow solid; mp 144–146 °C; (Found: C, 70.34; H, 5.58; N, 13.68. C₁₈H₁₇N₃S requires C, 70.33; H, 5.57; N, 13.67%); δ_H(400 MHz, CDCl₃, Me₄Si) 2.21 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.39 (3 H, s, CH₃-thiazole), 7.33 (2 H, m, phenyl), 7.45 (2 H, m, phenyl), 7.52 (1 H, m, phenyl), 7.68 (1 H, m, phenyl), 7.76 (1 H, m, phenyl), 7.93 (2 H, m, phenyl), 8.02 (2 H, m, phenyl), 11.39 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)hydrazine (58). (0.65 g, 82%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); orange solid; mp 215–220 °C; (Found: C, 66.23; H, 5.22; N, 18.16. C₁₇H₁₆N₄S requires C, 66.21; H, 5.23; N, 18.17%); δ_H(400 MHz, DMSO-d₆) 2.27 (3 H, s, CH₃-thiazole), 7.30 (1 H, m, phenyl), 7.38 (2 H, m, phenyl), 7.52 (2 H, m, phenyl), 7.61 (2 H, m, C(5)H-pyridine), 7.67 (2 H, m, C(4)H-pyridine), 8.17 (1 H, s, CH [double bond, length as m-dash]

N), 8.40 (1 H, m, C(3)H-pyridine), 8.46 (1 H, m, C(6)H-pyridine), 11.36 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(pyridin-4-ylmethylene)hydrazine (60). (0.64 g, 82%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); red solid; mp 220–223 °C; (Found: C, 65.29; H, 4.79; N, 19.02. C₁₆H₁₄N₄S requires C, 65.28; H, 4.79; N, 19.03%); δ_H(400 MHz, DMSO-d₆) 2.19 (3 H, s, CH₃-thiazole), 7.16 (1 H, m, phenyl), 7.28 (2 H, m, phenyl), 7.36 (2 H, m, phenyl), 7.54 (1 H, m, C(3)H-pyridine), 7.61 (1 H, m, C(5)H-pyridine), 7.98 (1 H, s, CH [double bond, length as m-dash]

N), 8.30 (1 H, m, C(2)H-pyridine), 8.35 (1 H, m, C(6)H-pyridine), 12.19 (1 H, br s, NH, D₂O exch.).

1-(Benzodioxol-5-ylmethylene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (61). (0.66 g, 99%); ivory solid; mp 240–242 °C; (Found: C, 64.10; H, 4.47; N, 12.46. C₁₈H₁₅N₃O₂S requires C, 64.08; H, 4.48; N, 12.45%); δ_H(400 MHz, DMSO-d₆) 2.25 (3 H, s, CH₃-thiazole), 6.31 (2 H, s, OCH₂O), 7.22 (1 H, m, benzodioxole), 7.29 (1 H, m, phenyl), 7.39 (2 H, m, benzodioxole), 7.56 (2 H, m, phenyl), 7.96 (2 H, m, phenyl), 7.98 (1 H, s, CH [double bond, length as m-dash]

N), 12.50 (1 H, br s, NH, D₂O exch.).

1-((1H-Indol-3-yl)methylene)-2-(5-methyl-4-phenylthiazol-2-yl)hydrazine (62). (0.65 g, 86%, purified by chromatography: SiO₂, ethyl acetate/hexane 5/1); sand solid; mp 198–200 °C; (Found: C, 68.66; H, 4.86; N, 16.85. C₁₉H₁₆N₄S requires C, 68.65; H, 4.85; N, 16.85%); δ_H(400 MHz, DMSO-d₆) 2.22 (3 H, s, CH₃-thiazole), 7.29 (1 H, m, phenyl), 7.36 (1 H, m, C(6)H-indole), 7.47 (1 H, m, C(7)H-indole), 7.52 (2 H, m, phenyl), 7.59 (2 H, m, phenyl), 7.83 (1 H, m, C(2)H-indole), 7.98 (1 H, s, C(2)H-indole), 8.23 (1 H, s, CH [double bond, length as m-dash]

N), 11.55 (1 H, br s, NH, D₂O exch.), 12.80 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(naphthalen-1-ylmethylene)hydrazine (63). (0.71 g, 99%); ivory solid; mp 192–194 °C; (Found: C, 73.45; H, 4.98; N, 12.22. C₂₁H₁₇N₃S requires C, 73.44; H, 4.99; N, 12.23%); δ_H(400 MHz, DMSO-d₆) 2.16 (3 H, s, CH₃-thiazole), 7.43 (1 H, m, phenyl), 7.58 (2 H, m, naphthalene), 7.60 (2 H, m, phenyl), 7.64 (2 H, m, phenyl), 7.72 (1 H, m, naphthalene), 7.98 (3 H, m, naphthalene), 8.21 (1 H, s, CH [double bond, length as m-dash]

N), 11.45 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(1-(naphthalen-2-yl)ethylidene)hydrazine (64). (0.62 g, 88%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); ivory solid; mp 200–201 °C; (Found: C, 73.91; H, 5.35; N, 11.76. C₂₂H₁₉N₃S requires C, 73.92; H, 5.36; N, 11.75%); δ_H(400 MHz, DMSO-d₆) 2.27 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.42 (3 H, s, CH₃-thiazole), 7.28 (1 H, m, phenyl), 7.36 (2 H, m, naphthalene), 7.42 (2 H, m, phenyl), 7.49 (2 H, m, phenyl), 7.71 (1 H, m, naphthalene), 7.88 (3 H, m, naphthalene), 11.30 (1 H, br s, NH, D₂O exch.).

1-(5-Methyl-4-phenylthiazol-2-yl)-2-(1-(2H-2-oxo-chromen-3-yl)ethylidene)hydrazine (65). (0.68 g, 90%, purified by chromatography: SiO₂, ethyl acetate/hexane 3/1); light yellow solid; mp 215–218 °C; (Found: C, 67.20; H, 4.56; N, 11.20. C₂₁H₁₇N₃O₂S requires C, 67.18; H, 4.56; N, 11.19%); δ_H(400 MHz, DMSO-d₆) 2.27 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.31 (3 H, s, CH₃-thiazole), 7.33 (1 H, m, phenyl), 7.39 (2 H, m, phenyl), 7.45 (2 H, m, phenyl), 7.52 (1 H, m, C(6)H-chromene), 7.56 (1 H, m, C(8)H-chromene), 7.67 (1 H, m, C(7)H-chromene), 7.94 (1 H, m, C(5)H-chromene), 8.31 (1 H, s, CH [double bond, length as m-dash]

N), 11.40 (1 H, br s, NH, D₂O exch.).

1-(Ferrocen-1-yl)-2-(5-methyl-4-phenylthiazol-2-yl)ethylidene)hydrazine (66). (0.62 g, 90%, purified by chromatography: SiO₂, ethyl acetate/hexane 5/1); brown solid; mp 175–176 °C; (Found: C, 63.63; H, 5.09; N, 10.11. C₂₂H₂₁FeN₃S requires C, 63.62; H, 5.10; N, 10.12%); δ_H(400 MHz, DMSO-d₆) 2.21 (3 H, s, CH₃C [double bond, length as m-dash]

N), 2.48 (3 H, s, CH₃-thiazole), 4.20 (5 H, s, ferrocene), 4.39 (2 H, m, ferrocene), 4.52 (2 H, m, ferrocene), 7.37 (1 H, m, phenyl), 7.45 (2 H, m, phenyl), 7.51 (2 H, m, phenyl), 12.40 (1 H, br s, NH, D₂O exch.).

3.2 Determination of hMAO isoform activity.

The effects of the test compounds on the enzymatic activity of hMAO isoform were evaluated by a fluorimetric method. Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) containing different concentrations of the test drugs (new compounds or reference inhibitors) and adequate amounts of recombinant hMAO-A or hMAO-B required and adjusted to obtain in our experimental conditions the same reaction velocity, i.e., to oxidize (in the control group) 165 pmol of p-tyramine/min (hMAO-A: 1.1 μg protein; specific activity: 150 nmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 μg protein; specific activity: 22 nmol of p-tyramine transformed/min/mg protein) were incubated for 15 min at 37 °C in a flat-black-bottom 96-well microtest plate (BD Biosciences, Franklin Lakes, NJ, USA) placed in the dark multimode microplate reader chamber. After this incubation period, the reaction was started by adding (final concentrations) 200 μM Amplex Red reagent, 1 U/mL horseradish peroxidase and 1 mM p-tyramine (final saturating concentration). The production of H₂O₂ and, consequently, of resorufin was quantified at 37 °C in a multidetection microplate fluorescence reader (Fluostar Optima, BMG Labtech GmbH, Offenburg, Germany) based on the fluorescence generated (excitation, 545 nm, emission, 590 nm) over a 15 min period, in which the fluorescence increased linearly. Control experiments were carried out simultaneously by replacing the test drugs (new compounds and reference inhibitors) with appropriate dilutions of the vehicles. In addition, the possible capacity of the above test drugs to modify the fluorescence generated in the reaction mixture due to non-enzymatic inhibition (e.g., for directly reacting with Amplex Red reagent) was determined by adding these drugs to solutions containing only the Amplex Red reagent in a sodium phosphate buffer. The specific fluorescence emission (used to obtain the final results) was calculated after subtraction of the background activity, which was determined from vials containing all components except the hMAO isoforms, which were replaced by a sodium phosphate buffer solution. In our experimental conditions, this background activity was practically negligible.

4 Conclusion

These findings increase our confidence in the 2-thiazolylhydrazone model and stimulate us to continue our investigations in designing more potent and selective analogues. The results obtained in this study indicate that some of the compounds tested may have interesting therapeutic potential as original chemical models (templates) for the design and subsequent development of new drugs useful for improving the pharmacological treatment of major depressive disorders. Finally, in Scheme 2, we have collected all SAR studies which could be drawn about this scaffold.^8–10


	Scheme 2 SAR studies of MAO inhibition by 2-thiazolylhydrazone scaffold.

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Footnote

† Electronic supplementary information (ESI) available: Pharmacological studies. See DOI: 10.1039/c0md00014k

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