FIELD OF THE INVENTION

The present invention relates to chemical heterocyclic compounds and synthesis thereof, said compounds having biological activity as Transient Receptor Potential cation channel, subfamily Vanilloid type 1 (TRPV1 ) ligands are potentially useful for use as medicaments, in particular for use in the treatment of pain, inflammation, cancer and vascular disorders.

STATE OF THE ART

The Transient Receptor Potential (TRP) channels are a large family of plasma membrane non-selective cation channels, classified into several subfamilies on the basis of their primary structure and involved in the transduction of a remarkable range of stimuli such as temperature, mechanical and osmotic stimuli, electrical charge, light, olfactive and taste stimuli, xenobiotic substances, endogenous lipids, etc.. ¹ Among these, the TRPV1 (transient receptor potential cation channel, subfamily Vanilloid type 1 ) has been the first TRP to be cloned by Caterina and coworkers in 1997, ² and the best characterized founding member of the thermo-TRP subfamily of sensory transducers.

The TRPV1 shows a high Ca ²⁺ permeability, abundantly expressed in sensory neurons ³ and also in higher brain structures ⁴ which are involved in pain processing and neurogenic inflammatory response, overlapping only in part with those of endocannabinoid system. In fact, TRPV1 is a polymodal nociceptor, up-regulated during chronic pain conditions, and exhibits a dynamic threshold of activation that could be significantly lowered by inflammatory agents such as mild acidification, temperature higher than 43 °C and various, mostly noxious, natural products. ¹ Among these, the natural pungent principle capsaicin was the first TRPV1 agonist described and its potential analgesic effects were known long before to the identification of its receptor. ⁵ Capsaicin (irans-8-methyl-/V-vanillyl-6-nonenamide) is a crystalline, lipophilic, colorless and odorless alkaloid found primarily in the Capsicum fruit, ⁶ responsible of its spicy flavour. Its action is unique in that the initial stimulation of the receptor is followed by a lasting refractory state, traditionally termed desensitization, silencing the whole nerve terminal. ⁷ However, capsaicin's pungency limits its use in clinical trials, so that the characterization and extraction/synthesis of non-pungent analogues is still in progress.

Available experimental evidence indicates that vanilloid agonists, exerting their antinociceptive actions through TRPV1 receptor-mediated selective neurotoxic/neurodegenerative effects directed against somatic and visceral C-fibre nociceptive primary afferent fibres, might be part of analgesic drugs which do not cause addiction and tachyphylaxis. ⁸

An ultrapotent capsaicin natural analogue is resiniferatoxin (RTX), while an early structure-activity relationship (SAR) study around capsaicin identified the first generation TRPV1 antagonist, capsazepine ⁹ (see structures in figure 1 ). The clinical use of the ultrapotent capsaicin analogue RTX is hampered by a number of problems, as RTX is a complex, highly hydrophobic molecule, expensive to manifacture and difficult to keep in solution. ¹⁰

Both agonists and antagonists of this non-selective cation channel are being evaluated as potential analgesics. However, TRPV1 agonists and antagonists are not equivalent therapeutic approaches and their clinical use is not mutually exclusive. Capsaicin-sensitive nerves express a myriad of receptors that are relevant to pain and inflammation of which TRPV1 is just one; moreover, TRPV1 agonists silence the whole nerve terminal, whereas antagonists selectively impair TRPV1 receptors. As a result, agonist compounds are expected to be more powerful analgesic drugs than antagonists as they simultaneously block all receptors on capsaicin-sensitive nerves.

Hence, TRPV1 channels have become a promising pain relief drug target, representing the key mediator in pain processing and neurogenic inflammatory response, and have opened the door to development of new type of analgesics. The structure of capsaicin and related compounds, called capsaicinoids, provided the basis for developing structurally different TRPV1 modulators, both agonists and antagonists. Three structural features have been identified as critical for the receptor interaction (Figure 2), namely, the lipophilic moiety (nonenyl chain for capsaicin, replaceable by a long, insaturated alkyl chain possibly bringing a terminal aromatic nucleus), a polar head (3-methoxy-4-hydroxybenzyl portion) and the connecting functionality (an amide for capsaicin, an ester for resiniferatoxin). ¹¹ Both H-bond donor/acceptor properties of the aromatic substituents on the vanillyl moiety are important for potent agonist activity and, in particular, the 4-phenolic hydroxyl group represents a crucial pharmacophoric element. ¹² On the other hand, lipophilicity is relevant for the bioactivity, since TRPV1 agonists need to cross the cell membrane to reach their binding site.

The aim of the authors was to design and synthesize a novel class of small-molecule TRPV1 ligands, endowed with agonist or antagonist effect, chemically stable, easily prepared through simple synthetic pathways and characterized by a positive biological profile.

SUMMARY OF THE INVENTION

Subject-matter of the present invention is a compound of formula (I)

Ar-(CH ₂ )m-Y-(CH2)n-An

(I)

wherein

m is an integer number varying from 0 to 6;

n is an integer number varying from 0 to 2;

Y is C =O)NH, NHC(=O)NH, NHC(=O)O, C(=O)O or

An is an aromatic ring selected in the group consisting of

Ri is H, I, (CH ₂ )4-CH ₃ , C≡CH-(CH ₂ )2-CH ₃ , Ph,

Rs is H, OH, OMe, O ⁱ Pr or CI;

R ₄ is H or OMe;

Rs is OMe, COOEt, CN, F, SO2CH3 or CH2OH;

X ₂ is O, S or NMe.

Excluding a compound wherein Y is CONH and n is 2 when R ₂ = R3 = OMe and R ₄ = H, when R ₃ = R ₄ = OMe and R2 = H, when R ₃ = R ₄ = H and R2 = OMe and when An = Benzo[c |[1 ,3]dioxol-5-ylmethyl-.

The compounds of general formula (I) according to the invention are chemically stable and were tested for their ability to bind the transient receptor potential vanilloid-type 1 channel, TRPV1 , showing potential activity in illnesses related to pain, inflammatory, cancer, and vascular disorders.

Subject-matter of the present invention is therefore also a compound of formula (I) as above described for use as a medicament, in particular for use in the treatment of pain, inflammation, cancer and vascular disorders. In particular compounds of formula (I) wherein Y is NHC(=0)NH or NHC(=0)0 are able to bind also Fatty Acid Amide Hydrolase (FAAH) thus being novel dual FAAH/TRPV1 ligands that might prove useful for the treatment of neuropathic and inflammatory pain as well as of other conditions in which TRP channels and/or cannabinoid receptors have already been shown to be targeted with success in preclinical and clinical studies (e.g. metabolic and hepatic dysfunctions, neurological or neuropsychiatric disorders, cancer, bone and gastrointestinal disorders, autoimmune diseases and allergies). The compounds of the invention, appropriately radiolabeled, can also be advantageously used for their ability to bind the endovanilloid receptor channel, TRPV1 , as pharmacological and/or diagnostic tools, in particular as radiotracers for mapping TRPV1 receptors in (patho)physiological conditions.

The present invention relates also to a process for preparing a compound of formula (I) according to the invention, said process using a compound of formula (II)

Ar-(CH ₂ )m-COOH

(II)

and a compound of formula (III)

(HI)

wherein X3 is NH2 or OH or N3.

wherein Ar, m, n and An are as above described.

BRIEF DESCRIPTION OF THE FIGURES

FIG.1 - shows the structures of Capsaicin, Resiniferatoxin (RTX) and Capsazepine. FIG.2 - shows critical chemical features of Capsaicin.

FIG. 3 - shows the synthetic scheme 1 for some embodiment compounds according to the invention; Method A: amine, CMC, HOBt, dry DCM, rt, overnight; Method B: amine hydrochloride and DMAP in dry DCM, CMC, HOBt, rt, overnight; Method C: amine, HBTU, HOBt, DIPEA, dry DMF; Method D: i) 4-methoxybenzylamine, CMC, HOBt, dry DCM, rt; ii) dry toluene, BBr ₃ , 100 °C.

FIG. 4 - shows the synthetic scheme 2 for some embodiment compounds according to the invention; Method E: a) dry toluene, DPPA, EtsN, 80 °C, overnight; b) appropriate amine, Et3N, 80 °C 1 h then room temperature overnight. Method F: a) dry toluene, DPPA, EtsN, 80 °C, overnight; b) 4-OMe-benzylamine, EtsN, 80 °C 1 h then room temperature overnight; c) dry toluene, BBr3, 100 °C 1 h.

FIG. 5 - shows the suitable synthetic scheme 3 for some embodiment compounds according to the invention.

FIG. 6 - shows the suitable synthetic scheme 4 for some embodiment compounds according to the invention; a) dry toluene, DPPA, E.3N , 80-90 °C, overnight; b) appropriate alcohol, DMAP, 80-90 °C 4-24 h then room temperature overnight. FIG. 7 - shows a possible synthetic scheme 5 for some embodiment compounds according to the invention.

FIG. 8 - shows levels of HNE protein-adducts induced by GO treatment in HaCaT cells, measured by Western blot. Data are representative of three experiments. Cells were pre-treated with MSP-3 for 24 h and then treated with GO for 1 h. Quantifications of HNE protein-adduct bands is shown as ratio of ΗΝΕ/β-actin (bottom panel). Data are expressed as arbitrary units (average of three different experiments, ^* vs control, # vs GO).

FIG. 9 - shows the cytotoxicity of compound MSP3, MSP18 and MSP20 in HaCaT cell line, concentrations ranging from 0.1 to 10 μΜ, after 24 and 48 h. A) LDH release assay and B) Alamar Blu assay. Data are expressing as percentage of control (average of five different experiments).

FIG. 10 - shows the results of formalin test of peripheral acute and inflammatory pain in rats; T = MSP18 treated rats; CTR = control (vehicle treated rats). Effect of MSP18 (1 mg/Kg, i.p.) on formalin induced pain in rats. Data are reported as total duration of behaviour in 1 h time observation (mean ± SEM). ^* P < 0.05; ^** P < 0.001 . DETAILED DESCRIPTION OF THE INVENTION

According to the invention are preferred compounds of formula (I) wherein

Ar is

Are also preferred compounds of formula (I) wherein

m is an integer number varying from 0 to 3;

n is an integer number varying from 0 to 2.

Are either preferred compounds of formula (I) wherein

Y is C(=0)NH or NHC(=0)NH.

Preferably An is an aromatic ring selected in the group consisting of

R ₂ is OH, OMe or CI ;

R is H.

Preferred compounds according to the invention are those wherein Ar is

m is an integer number varying from 0 to 3;

n is an integer number varying from 0 to 2;

Y is C(=0)NH or NHC(=0)NH;

An is an aromatic ring selected in the group consisting of

R ₂ is OH, OMe or CI;

R ₃ is H, OH, OMe or CI;

R is H.

More preferred are those wherein

Ar is

Xi is NH, or S; m is an integer number varying from 0 to 3;

n is an integer number varying from 1 to 2;

Y is C(=0)NH or is NHC(=0)NH ;

An is an aromatic ring selected in the group consisting of

R ₂ is OH ;

Rs is OH or OMe;

R is H.

Particularly preferred compounds of the invention are:

/V-(4-hydroxyphenyl)-4-(thiophen-2-yl)butanamide, /V-(4-hydroxy-3- methoxybenzyl)-4-(thiophen-2-yl)butanamide, /V-(3,4-dihydroxyphenethyl)-4- (thiophen-2-yl)butanamide, /V-(pyridin-4-ylmethyl)-4-(thiophen-2-yl)butanamide, Λ/- (benzo[d|[1 ,3]dioxol-5-ylmethyl)-4-(thiophen-2-yl)butanamide, Λ/-(3,4- dimethoxybenzyl)-4-(thiophen-2-yl)butanamide, 4-(thiophen-2-yl)-/V-(thiophen-2- ylmethyl)butanamide, /V-(3,4-dichlorobenzyl)-4-(thiophen-2-yl)butanamide, N-(4- methoxybenzyl)-4-(thiophen-2-yl)butanamide, /V-(4-hydroxybenzyl)-4-(thiophen-2- yl)butanamide, /V-(4-hydroxy-3-methoxybenzyl)-4-(5-iodothiophen-2- yl)butanamide, /V-(3,4-dihydroxyphenethyl)-4-(5-iodothiophen-2-yl)butanamid e, 3- (benzo[fc]thiophen-2-yl)-/V-(4-hydroxy-3-methoxybenzyl)propa namide, 3- (benzo[fc]thiophen-2-yl)-/V-(3,4-dichlorobenzyl)propanamide, 3-(benzo[fc]thiophen- 2-yl)-/V-(4-methoxybenzyl)propanamide, 3-(benzo[fc]thiophen-2-yl)-/V-(4- hydroxybenzyl)propanamide, 2-(5-hydroxy-1 /-/-indol-3-yl)-/V-(4-hydroxy-3- methoxybenzyl)acetamide, /V-(4-hydroxy-3-methoxybenzyl)-1 H-indol-2- carboxamide, and /V-(3,4-dichlorobenzyl)-1 H-indol-2-carboxamide, 3- (benzo[fc]thiophen-2-yl)-/V-(3,4-dihydroxyphenethyl)propanam ide, Λ/-(3,4- dihydroxyphenethyl)-1 /-/-indole-2-carboxamide, N-(3-chloro-4-methoxyphenethyl)- 4-(thiophen-2-yl)butanamide, N-(3,4-dichlorobenzyl)-4-(5-iodothiophen-2- yl)butanamide, N-(3-chloro-4-methoxyphenethyl)-4-(5-iodothiophen-2- yl)butanamide, 3-(benzo[k]thiophen-2-yl)-/V-(3-chloro-4- methoxyphenethyl)propanamide, 1 -(3,4-dichlorobenzyl)-3-(1 /-/-indol-2-yl)urea, 1 -(2- (benzo[fc]thiophen-2-yl)ethyl)-3-(4-hydroxy-3-methoxybenzyl) urea, 1 -(2- (benzo[k]thiophen-2-yl)ethyl)-3-(4-methoxybenzyl)urea, 1 -(2-(benzo[£>]thiophen-2- yl)ethyl)-3-(4-hydroxybenzyl)urea, 1 -(4-hydroxy-3-methoxybenzyl)-3-(1 H-indol-2- yl)urea, 1 -(3,4-dichlorobenzyl)-3-(3-(thiophen-2-yl)propyl)urea, and 1 -(4-hydroxy-3- methoxybenzyl)-3-(3-(thiophen-2-yl)propyl)urea, 1 -(2-(benzo[£>]thiophen-2-yl)ethyl)- 3-(3,4-dichlorobenzyl)urea, 1 -(2-(benzo[fc]thiophen-2-yl)ethyl)-3-(3,4- dihydroxyphenethyl)urea, 1 -(4-hydroxy-3-methoxybenzyl)-3-(3-(5-iodothiophen-2- yl)propyl)urea, 1 -(3,4-dichlorobenzyl)-3-(3-(5-iodothiophen-2-yl)propyl)urea, Λ/-(4- hydroxy-3-methoxybenzyl)-4-(5-phenylthiophen-2-yl)butanamide , 1 -(3,4- dihydroxyphenethyl)-3-(3-(5-iodothiophen-2-yl)propyl)urea, 1 -(3,4- dihydroxyphenethyl)-3-(3-(thiophen-2-yl)propyl)urea, /V-(3,4-dichlorobenzyl)-4-(5- phenylthiophen-2-yl)butanamide, /V-(4-hydroxy-3-methoxybenzyl)-4-(5-(pyridin-3- yl)thiophen-2-yl)butanamide, /V-(3,4-dichlorobenzyl)-4-(5-(pyridin-3-yl)thiophen-2- yl)butanamide, /V-(4-hydroxyphenyl)-4-(5-(pyridin-3-yl)thiophen-2-yl)butana mide.

A compound of formula (I) according to the invention wherein Y is C(=0)NH can be obtained contacting a compound of formula (II) as above described

Ar-(CH ₂ )m-COOH

(II)

with a compound of formula (III) as above described

(III).

Such a coupling reaction can be promoted with coupling activating agents well known in the art preferably 1 -cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) and 2-ethoxy-1 -ethoxycarbonyl-1 ,2-dihydroquinoline (EEDQ). ¹³ A compound of formula (I) according to the invention wherein Y is C(=0)NH were prepared using as starting material a series of selected aromatic amines which were condensed, according to the synthetic procedures depicted in Scheme 1 . 4- (thiophen-2-yl)butanoic acid, or similar compounds, such as 3-(benzo[£>]thiophen-2- yl)propanoic acid, 2-(5-hydroxy-1 /-/-indol-3-yl)acetic acid, 1 /-/-indole-2-carboxylic acid, 4-(5-iodothien-2-yl)butanoic acid, 4-(5-phenylthiophen-2-yl)butanoic acid, or 4- [5-(pyridin-3-yl)thiophen-2-yl]butanoic acid can be obtained from commercial sources or by simple organic chemistry reactions. Besides the 3-methoxy-4- hydroxybenzylamides (i.e., vanillamides), several aromatic amides were obtained from different aromatic amines, characterized by polar substituents (i.e. 4- hydroxyaniline, 4-methoxybenzylamine, 3-hydroxytyramine, 3,4- dichlorobenzylamine, 3,4-dimethoxybenzylamine, 3-chloro-4- methoxyphenethylamine, benzo[c |[1 ,3]dioxol-5-ylmethanamine), or heteroaromatic amines (i.e. pyridin-4-ylmethanamine and thiophen-2-ylmethanamine).

A compound of formula (I) according to the invention wherein Y is NHC(=0)NH can be prepared contacting a compound of formula (II)

Ar-(CH ₂ )m-COOH

(II)

with diphenylphosphoryl azide (DPPA) ¹⁴ and subsequently contacting the resulted compound with a compound of formula (111) as above described and particularly with an amine of formula (Ilia)

(Ilia)

Among a wide range of chemical structures characterizing the vanilloid ligands, literature data reported that their potency and functional activity (agonist versus antagonist) can be modulated by the manipulation of the amide functionality ¹ ' ⁹ ' ¹⁵ and/or the introduction of the appropriate substituents. ¹⁶ ' ¹⁷ Moreover, the chemical similarity between endocannabinoid and N-acyl-vanillylamide agonists of TRPV1 ¹ is well documented; in particular, several "hybrid" Fatty Acid Amide Hydrolase FAAH/TRPV1 blocker molecules contain in their structure an urea or a carbamate group. Aimed at investigating how the nature of the connecting functionality between the amide head and the lipophilic moiety can affect TRPV1 and endocannabinoid enzyme interactions, for an aspect the present invention is related to a series of urea derivatives of formula (I) (see table 2). For this purpose, 4-(thiophen-2- yl)butanoic acid, or similar compounds, such as 3-(benzo[£>]thiophen-2-yl)propanoic acid, 1 /-/-indole-2-carboxylic acid, 4-(5-iodothien-2-yl)butanoic acid, 4-(5- phenylthiophen-2-yl)butanoic acid or 4-[5-(pyridin-3-yl)thiophen-2-yl]butanoic acid, which can be obtained from commercial sources or by simple organic chemistry reactions, were converted in their corresponding isocyanates via the Curtius rearrangement using diphenylphosphoryl azide (DPPA) which reacted further with the appropriate amines (i.e. 3-methoxy-4-hydroxybenzylamine, 4- methoxybenzylamine, 3-hydroxytyramine, 3,4-dichlorobenzylamine) in one pot, as depicted in Scheme 2.

A compound of formula (I) according to the invention wherein Y is NHC(=0)0 can be obtained contacting a compound of formula (II) as above described

Ar-(CH ₂ )m-COOH

(II)

with diphenylphosphoryl azide (DPPA) ¹⁴ ' ¹⁸ and subsequently contacting the resulted compound with a compound of formula (III) as above described and particularly with an alcohol of formula (1Mb)

(1 Mb).

For this purpose, 4-(thiophen-2-yl)butanoic acid, or similar compounds, such as 3- (benzo[£>]thiophen-2-yl)propanoic acid, 1 /-/-indole-2-carboxylic acid, 4-(5-iodothien- 2-yl)butanoic acid, 4-(5-phenylthiophen-2-yl)butanoic acid, or 4-[5-(pyridin-3- yl)thiophen-2-yl]butanoic acid, which can be obtained from commercial sources or by simple organic chemistry reactions, were converted in their corresponding isocyanates via the Curtius rearrangement using diphenylphosphoryl azide (DPPA) which reacted further with the appropriate alcohols (i.e. vanillyl alcohol, 3- hydroxytyrosol, 3,4-dichlorobenzyl alcohol, 4-chlorobenzyl alcohol, 3,5-dimethoxy- 4-hydroxybenzyl alcohol, 3,4-difluorobenzyl alcohol, piperonyl alcohol, 3-(2- hydroxyethyl)indole) in one pot, as depicted in Scheme 4.

In previous studies, ¹⁹ ani/-(1 ,4) triazole capsaicinoid analogues were synthesized and evaluated for their activity at TRPV1 , CBi and CB2 receptors. In fact, although apparently metabolically inert, the 1 ,2,3-triazole ring is a combination of H-bond donor and acceptor sites capable of mimicking the hydrogen bonding acidity and basicity of an amide bond (peptide bond), but not its capability of being hydrolysed.

A compound of formula (I) according to the invention wherein Y is can be obtained contacting a compound of formula (II) as above described

Ar-(CH ₂ )m-COOH

(II)

with a compound of formula (III) ¹⁹ as above described and particularly with an azide of formula (III )

(Nlc)

For this purpose, 4-(thiophen-2-yl)butanoic acid, or similar compounds, such as 3- (benzo[£>]thiophen-2-yl)propanoic acid, 1 /-/-indole-2-carboxylic acid, 4-(5-iodothien- 2-yl)butanoic acid, 4-(5-phenylthiophen-2-yl)butanoic acid, or 4-[5-(pyridin-3- yl)thiophen-2-yl]butanoic acid, which can be obtained from commercial sources or by simple organic chemistry reactions, were converted to their corresponding aldehydes, ²⁰ which in turn were trasformed without the need to isolate the alkyne intermediates into the 1 ,4-disubstituted 1 ,2,3-triazoles in good yields by a one-pot Cu-catalyzed azide-alkyne click reaction ²¹ (Scheme 5).

Despite the wide chemical diversity of agonist and antagonist compounds, one of the aspect of the present invention was the identification of new TRPV1 ligands, chemically characterized by a 4-(thiophen-2-yl)butanoic acid amide moiety and endowed with agonist activity. Considering how the chemical decoration of the thiophene ring and/or the electronic/steric properties of amide can affect the interaction with the vanilloid receptor and modulate the TRPV1 activity, it is describe here a whole set of derivatives, characterized by modifications both on the acidic moiety and the aromatic amidic head, which are chemically stable and easily prepared through a simple synthetic pathway.

Compounds of the invention, in particular the synthesized analogues MSP2-MSP3, MSP6 and MSP11 -MSP35, MSP44, and MSP52-MSP55 were evaluated for their binding and their agonist/antagonist properties ²² using a functional Ca ²⁺ uptake assay in HEK-293 cells stably over-expressing recombinant human TRPV1 receptors. The results of these assays are summarized in Table 1 and Table 2. The efficacy of the agonists was determined by comparing their effect with the analogous effect observed with 4 μΜ of ionomycin, while the potency (ECso values) of compounds was determined as the concentration of test substances required to produce half-maximal increases in [Ca ²⁺ ]i. Antagonism/desensitizing behaviour was evaluated against capsaicin (0.1 μΜ) and data were expressed as the concentration exerting a half-maximal inhibition of agonist-induced [Ca ²⁺ ]i elevation (ICso). Many of the newly synthesized compounds were found to activate TRPV1 channel, confirming our hypothesis that the 4-(thiophen-2-yl)butanoic acid amide moiety is a valuable scaffold for the design of a novel class of TRPV1 ligands. In particular, all the active compounds showed an agonist effect, potencies ranging from submicromolar to low nanomolar values. Compound MSP3, our first identified TRPV1 ligand, has about the 80% of efficacy compared to ionomycin (4 μΜ) and an ECso value of 0.87 μΜ. Comparable behaviour is exhibited by compound MSP25 with about 67% of efficacy and an ECso of 0.56 μΜ, compound MSP27 with about 78% of efficacy and an ECso of 0.53 μΜ, compound MSP30 with about 66% of efficacy and an ECso of 0.1 1 μΜ, and MSP52 with about 48% of efficacy and an ECso of 0.15 μΜ. Compounds MSP18, MSP20, and MSP44 are the most potent derivatives with efficacy of approximately 80%, for the first two compounds, and 70% for the third, and ECso of 7.45, 46 and 1 .6 nM, respectively. For the latter three compounds ECso (for activation) and ICso (for desensitization) values are of the same order of magnitude and thus activation and desensitization potencies are comparable (see Table 1 ). Also derivative MSP34 showed a potency value in the nanomolar range (efficacy about 71 %, ECso = 29.3 nM), furthermore behaving as the first example in this series of FAAH inhibitor (ICso = 8.01 μΜ, see also Table 2). Regarding structure-activity relationships (SAR), previous studies on both agonists and antagonists, characterized by wide chemical diversity, indicated some common features: a lipophilic moiety (i.e. a long, unsaturated alkyl chain in some instances bringing a terminal aromatic group) and a polar head (i.e. 3-methoxy-4- hydroxybenzyl functionality). In agreement with aforementioned SAR, all active derivatives have an aromatic amide bringing polar substituents. Specifically, most potent/desensitizing compounds maintain the vanillamine functionality (see MSP3, MSP18, MSP20, MSP25, MSP34, and MSP44), but also 3,4-dihydroxyphenylethyl- (MSP9 and MSP27) and 3,4-dichlorobenzyl- (MSP15, MSP26, MSP30, and MSP52) groups are well tolerated in the amide head. Moreover, the preliminary goal of replacing the unsaturated fatty acid characterizing the natural capsaicin with a stable and readily available lipophilic moiety could be reached by introducing heteroaromatic carboxylic acids. In fact, the 4-(thien-2-yl)butanoic acid fragment seems to well mimic the unsaturated acyl portion present in the capsaicin structure (compound MSP3); further introduction on the thiophene ring of a iodine atom (MSP18) or its condensation with an additional aromatic ring (MSP20 and MSP34) or the replacement of the iodine atom with a phenyl ring (MSP44 and MSP52) gave the most potent compounds, suggesting a significant role for liphophilicity or the need of a more extensive π-π hydrophobic interaction. Consistently with the observed results, indole scaffold furnished an active derivative (MSP25) while the introduction in the aromatic moiety of a polar substituent (MSP24) seems to be detrimental for TRPV1 binding. Additional study aimed at investigating as the connecting functionality between the amide head and the lipophilic moiety can affect TRPV1 and endocannabinoid enzymatic interactions, has led to identify the first "hybrid" FAAH inhibitor/TRPV1 agonist; in fact, thirteen urea analogues were synthesized and some of these evaluated in TRPV1 assay and FAAH inhibition. The few data currently available indicate that chemical modification of the amide functionality into the urea moiety was well tolerated and, when the amide head was the vanillyl group, the potent vanilloid ligand behaved also as a FAAH inhibitor. Some of the most interesting compounds (i.e. MSP15, MSP18, MSP19, MSP20, MSP25 and MSP26, see Table 1 ) have been also evaluated for their ability to interact with both cannabinoid receptors, exploring the selectivity of these new derivatives. In fact, is well documented the overlap between the ligand recognition properties of some TRP channels and the cannabinoid receptors and/or the enzymes catalyzing anandamide cellular re-uptake and hydrolysis. All compounds tested in the preliminary screening on HEK cell membranes transfected with the human CBi or CB2 receptor showed Ki values greater than 10 μΜ and hence proved to be devoid of any affinity for both cannabinoid receptor subtypes and showed complete selectivity towards TRPV1 receptors.

Furthermore, because capsaicinoid antioxidant properties also contributes to their beneficial effects, ²³ especially on cardiovascular system, and capsaicin possesses some structural requirements for free radical-scavenging activity, ²⁴ the antioxidant properties of MSP3 and the biological profile, i.e. cytotoxicity and viability behaviour, of compounds MSP3, MSP18 and MSP20 have been investigated. In fact, the 4- methoxy-3-hydroxy moiety in the aromatic ring of the amide (vanillyl residue) is one of the structural arrangements imparting high antioxidant activity.

Thus, to verify the protective role of compound MSP3 against oxidative stress (OS), HaCaT cells ²⁵ were treated with glucose oxidase (GO) to generate hydrogen peroxide (H2O2). As a consequence of the induction of OS, the level of lipid peroxidation was measured based on the presence of the alpha-beta unsaturated aldehyde protein adducts. In particular the protein adducts with 4-hydroxy-2- nonenale (HNE), a very reactive and toxic aldehyde, was analysed in this study. Therefore, the formation of HNE protein-adducts in keratinocytes pre-treated with or without compound MSP3 was evaluated. ²⁶ As shown in figure 7, the GO treatment induced a significant increase of HNE protein-adduct levels, effect prevented in a dose-dependent fashion by pre-treatment with compound MSP3.

Citotoxicity of the compounds MSP3, MSP18 and MSP20 was evaluated in HaCaT cells by means of LDH release. ²⁷ As shown in figure 8A, the treatment with the tested compounds (concentrations ranging from 0.1 to 10 μΜ) did not cause any cytotoxic effect in HaCaT cells at 24 while the only derivative maintaining this profile at 48 h was MSP20. These results were confirmed also by Alamar Blu assay, figure 8B, with no differences in cell viability between control and cell treated with all compounds after 24 h. In this assay, MSP3 did not inhibit cellular viability also at 48 h for lower concentrations (0.1 and 1 μΜ).

These results indicate that the compounds according to the invention are a novel class of TRPV1 ligands, demonstrating that compounds active at TRPV1 receptors can be obtained replacing the unsaturated acid of naturally occurring capsaicin by heteroaromatic carboxylic acids. Furthermore it has been also partly explored the role of the connecting functionality, identifying the urea moiety as the key chemical feature able to modulate the receptor versus the endocannabinoid-hydrolyzing enzyme interaction, thus achieving desired cannabimimetic effects, and underlying that further studies are advisable to elucidate the structure-activity relationships within this new class of compounds.

In addition the biological effect of the compounds MSP3, MSP18 and MSP20 has been analysed in cell culture by detecting their potential cytotoxicity. Moreover, compound MSP3 has been studied in preventing lipid peroxidation and biological results showed this derivative to be able to counteract OS in HaCaT cells without exerting any cytotoxic effect.

This strategy could be exploited to develop new potential pharmacological tools and/or anti-inflammatory/analgesic agents, characterized by a positive biological profile.

Moreover, the chemical structure of the compounds according to the invention provides the opportunity of introducing a radiolabeled atom (i.e. iodine-125 in compound MSP18, and analogous derivatives, or carbon-1 1 in the methoxy substituent of all the vanillamides), to develop new radiotracers for Positron Emission Tomography (PET) imaging. These radiotracers might be of great relevance for mapping TRPV1 receptors in (patho)physiological conditions. EXPERIMENTAL SECTION

Chemistry

Chemicals, Materials, and Methods. All starting materials, reagents and solvents were purchased from common commercial suppliers and were used as received unless otherwise indicated. Organic solutions were dried over anhydrous sodium sulphate and concentrated with a Buchi rotary evaporator R-1 10 equipped with KNF N 820 FT 18 vacuum pump. Melting points were determined on a Kofler hot stage apparatus (K) or using a Mettler FPI apparatus (2 °C/min, M) or a Gallenkamp melting point apparatus (G) and are uncorrected. ¹ H NMR and ¹³ C NMR spectra were recorded in the indicated solvent at 25 °C on a Bruker AC200F or on a Bruker Advance DPX400 or on a Bruker Avance300MHz spectrometer and chemical shifts were expressed as δ (ppm). The chromatography-mass spectrometry (LC-MS) system consisted of a Triple QuadTM System API 3200 or of an Agilent 1 100 series liquid cromatograph system including a 1 100 MSD model VL benchtop mass spectrometer with API-ES interface, a binary high-pressure gradient pump (0.4 mL/min low flow rate, employing a binary solvent system of 95/5 methanol/water), and a solvent degassing unit. Nitrogen (purity 99.995%) was used as nebulizer and drying gas. UV detection was monitored at 254 nm. Mass spectra were acquired in positive or negative mode scanning over the mass range m/z of 150-1500. IR spectra were recorded on a Perkin-Elmer FT-IR Spectrum TWO (Nujol dispersion or CHC solution). The structures of final compounds were unambiguously assessed by ¹ H NMR, ¹³ C NMR, MS and/or IR. All compounds were checked for purity by TLC on Merck 60 F254 silica plates. For column chromatography, Merck 60 silica gel, 230-400 mesh, was used. Final products were purified by a Biotage flash chromatography system with columns 12.25 mm, packed with KP-Sil, 60A, 32-63 μΜ. Compound purity was assessed by elemental analysis on a Perkin-Elmer elemental apparatus model 240 for C, H, N, and the data are within ±0.4% of the theoretical values. All the tested compounds possessed a purity >95.0%.

Method A. A solution of the appropriate acid (1 .0 eq.), the appropriate amine (1 .5 eq.) and /V-hydroxybenzotriazole (HOBt) (1 .2 eq.) in dry DCM (2.5 ml_) was cooled at 0 °C and stirred under N2 atmosphere for 15'. Thereafter a solution of 1 - cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) (1 .5 eq.) in dry DCM (3.5 ml_) was added dropwise. The mixture was stirred at rt for 24 hours. After this time, the solvent was distilled off under vacuum and the residue was washed twice with a 5% NaHCO3 solution and extracted with chloroform. The collected organic layers, dried on anhydrous Na2SO ₄ , were concentrated and the crude compound purified by flash chromatograpy on silica gel with the appropriate eluent mixtures.

Method B. The amine hydrochloride (1 .5 eq.) was suspended in dry DCM (5.0 ml_), DMAP added and the mixture allowed to stir at room temperature for 30' under nitrogen atmosphere. Afterward, a solution of the appropriate acid (1 .0 eq.) in dry DCM (2.0 ml_), HOBt (1 .2 eq.) solid and a solution of CMC (1 .5 eq.) in dry DCM (3.0 ml_) were added respectively. The mixture was stirred at rt for 24 hours. After this time, the solvent was distilled off under vacuum and the residue was washed twice with a 5% NaHCO3 solution and extracted with chloroform. The collected organic layers, dried on anhydrous Na2S0 ₄ , were concentrated and the crude compound purified by flash chromatograpy on silica gel with the appropriate eluent mixtures. Method C. The 4-(5-(pyridin-3-yl)thiophen-2-yl)butanoic acid (1 .0 eq.) was dissolved in dry DMF (5-7 ml_) and HOBt (1 .0 eq.) HBTU (2.0 eq.), DIPEA (3.0 eq.) and the appropriate amine (1 .4 eq.) were added to the solution. After stirring at room temperature for 30 min, more DIPEA (3.0 eq.) was added and the reaction mixture was stirred at room temperature overnight. Then the reaction mixture was diluted with saturated NH ₄ CI solution and extracted with ethyl acetate (4 times). The collected organic layers were washed twice with brine, dried over anhydrous Na2S0 ₄ , filtered and evaporated to dryness. The crude residue was purified by flash chromatography on silica gel with the appropriate eluent mixtures.

Method D. A solution of the appropriate acid (1 .0 eq.), the appropriate amine (1 .5 eq., 4-methoxybenzylamine) and /V-hydroxybenzotriazole (HOBt) (1 .2 eq.) in dry DCM (2.5 ml_) was cooled at 0 °C and stirred under N2 atmosphere for 15'. Thereafter, a solution of 1 -cyclohexyl-3-(2-morpholiinoethyl)carbodiimide (CMC) (1 .5 eq.) in dry DCM (3.5 ml_) was added dropwise. The mixture was stirred at rt for 24 hours, then the solvent was distilled off under vacuum and the residue was washed twice with a 5% NaHCO3 solution, then with a saturated NH ₄ CI solution and finally extracted with chloroform. The collected organic layers, dried on anhydrous Na2SO ₄ , were concentrated and the crude compound (crude MSP16 or MSP22) used in the next reaction without further purification. To a solution of crude compound (1 .0 eq.) in dry toluene, heated at 50 °C for 15 minutes, was added BBr3 (4.0 eq.) and the mixture was stirred at 100 °C for 1 hour. Afterward, the reaction mixture was allowed to reach room temperature, recooled to 0 °C and cautiously treated dropwise with H2O. The organic layer was separated and washed with NaCI saturated solution and 2 N NaOH (3-4 times). The alkaline aqueous phase was treated with concentrated HCI to pH 2 and extracted with E.2O. The dried and filtered organic layer afforded the pure compound after evaporation of solvent.

Method E. The appropriate carboxylic acid (1 .0 eq.) was dissolved in dry toluene (15 ml_), and diphenylphosphoryl azide (1 .17 eq.) was added. Triethylamine (1 .2 eq.) was added dropwise and the reaction mixture was stirred first at room temperature for 30'and then heated to 80 °C overnight. A solution of the desired amine (0.9 eq.) in dry toluene together with triethylamine (1 .2 eq.) was added and the heating continued for 1 h at 80 °C. After this time, the reaction mixture was allowed to reach room temperature and stirred overnight. The solvent was evaporated to dryness under reduced pressure, and the residue was diluted with ethyl acetate (50 ml_). The organic layer was washed with NH ₄ CI saturated solution (two times) and once with brine. The solvent was then dried on Na2S0 ₄ , filtered and evaporated furnishing a crude material which was purified by flash chromatography on silica gel with the appropriate eluent mixtures.

Method F. The appropriate carboxylic acid (1 .0 eq.) was dissolved in dry toluene (15 ml_), and diphenylphosphoryl azide (1 .17 eq.) was added. Triethylamine (1 .2 eq.) was added dropwise and the reaction mixture was stirred first at room temperature for 30'and then heated to 80 °C overnight. A solution of the 4-methoxybenzylamine (0.9 eq.) in dry toluene together with triethylamine (1 .2 eq.) was added and the heating continued for 1 h at 80 °C. After this time, the reaction mixture was allowed to reach room temperature and stirred overnight. The solvent was evaporated to dryness under reduced pressure, and the residue was diluted with ethyl acetate (50 ml_). The organic layer was washed with NH ₄ CI saturated solution (two times) and once with brine. The solvent was then dryied on Na2S0 ₄ , filtered and evaporated furnishing a crude material (MSP35) used in the next reaction without further purification. To a solution of crude compound (1 .0 eq.) in dry toluene, heated at 50 °C for 15 minutes, was added BBr3 (4.0 eq.) and the mixture was stirred at 100 °C for 1 hour. Afterward, the reaction mixture was allowed to reach room temperature, recooled to 0 °C and cautiously treated dropwise with H2O. The organic layer was separated and washed with NaCI saturated solution and 2 N NaOH (3-4 times). The alkaline aqueous phase was treated with concentrated HCI to pH 2 and extracted with Et20. The dried and filtered organic layer afforded the pure compound after evaporation of solvent.

General procedure for the synthesis of 4-(5-iodothiophen-2-yl)butanoic acid. To a solution of the 4-(thiophen-2-yl)butanoic acid (1 .0 eq.) in methanol (10 ml_) 4-5 drops of concentrated H2S0 ₄ were added and the solution refluxed for 3 h until starting acid disappear; the solution was cooled, the solvent concentrated and the residue diluted with chloroform. The organic layer was washed twice with a 5% NaHC03 solution, dried on anhydrous Na2S0 ₄ , filtered and concentrated. To a solution of the obtained crude methyl ester in dry acetonitrile, under stirring and nitrogen atmosphere, /V-iodosuccinimide was added and the reaction mixture refluxed for 3 h. After cooling, the solvent was concentrated and the residue taken up into chloroform. The pale pink organic layer was washed with a sodium thiosulfate solution (two times), then with a NaCI saturated solution and dried. The filtered organic solution was concentrated to afford a crude material, which was purified by flash chromatograpy on silica gel, eluent chloroform. The collected methyl 4-(5- iodothiophen-2-yl)butanoate was dissolved in a mixture of methanol and sodium hydroxide solution (2:1 ) and refluxed for about 2 h, until starting ester could not be detected by TLC. The aqueous phase was acidified first with concentrated HCI and then with 2 N HCI and extracted more times (3-4) with chloroform. The collected organic layer were dried on anhydrous Na2S0 ₄ , filtered and concentrated to dryness under reduced pressure. The crude 4-(5-iodothiophen-2-yl)butanoic acid was was purified by flash chromatograpy on silica gel, eluent chloroform.

General procedure for the synthesis of 4-(5-phenylthiophen-2-yl)butanoic acid and 4-[5-(pyridin-3-yl)thiophen-2-yl]butanoic acid. To a mixture of PdCl2(PPh3)2 (0.05 eq.), potassium fluoride (2.5 eq.), and iodobenzene or 3-iodopyridine (1 .0 eq.) in 3- 4 ml_ of dry DMSO was added methyl 4-(thiophen-2-yl)butanoate (1 .2 eq.) under an nitrogen atmosphere. AgN03 was then added in portions (0.25 eq. x 5) for 1 h x 5, heating the suspension at 100 °C for 5 h. After cooling to room temperature, the mixture was passed through a Celite pad, which was washed with chloroform repeatedly. The filtrate was washed with brine twice, dried on anhydrous Na2S0 ₄ , filtered and concentrated to dryness under reduced pressure. The crude material was purified by chromatography on silica gel, eluent chloroform.

At room temperature most of the synthesized compounds are in the solid state and stable for a long time.

Examples

/V-(4-Hydroxyphenyl)-4-(thiophen-2-yl)butanamide (MSP2). This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 4- hydroxyaniline, following Method A. Pure compound was isolated as yellow solid (yield 72.6%, eluent: CHCIs/MeOH 48/2).

Mp 96.7 °C (G). IR (KBr): 3280, 1644 cm ^"1 . ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.3 (d, 2H, J = 8.7 Hz), 7.18-7.13 (m, 1 H), 6.92-6.86 (m, 1 H), 6.84-6.78 (m, 1 H), 6.72 (d, 2H, J = 8.7 Hz), 2.93- 2.82 (m, 2H), 2.4-2.2 (m, 2H), 2.1 -1 .8 (m, 2H). ¹³ C-NMR (300 MHz) CHsOD: 171 .29, 152.9, 143.0, 129.2, 125.3, 121 .8, 121 .7 120.9 (C x2), 1 13.7 (C x2), 34.4, 27.6, 25.9. MS (E.I.) m/z: 261 [M] ⁺ (33), 151 (100), 109 (97), 97 (58). Anal, calcd. for Ci ₄ Hi ₅ N0 ₂ S (MW 261 .34): C, 64.34%; H, 5.79%; N, 5.36%. Found: C, 64.28%; H, 5.81 %; N, 5.34%.

/V-(4-Hydroxy-3-methoxybenzyl)-4-(thiophen-2-yl)butanamide (MSP3).

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 4-hydroxy-3- methoxybenzylamine hydrochloride, following Method B. The pure compound was isolated as brown oil (yield 20%, eluent: CHCIs/MeOH 48/2).

IR (Nujol): 3287, 1645 cm ^"1 . Ή-NMR (300 MHz) CDCIs δ (ppm): 7.04 (dd, 1 H, J =

4.0 Hz, J = 1 .0 Hz.), 6.9-6.6 (m, 5H), 4.26 (d, 2H, J = 5.6 Hz), 3.78 (s, 3H), 2.80 (t, 2H, J = 7.5 Hz.), 2.19 (t, 2H, J = 7.0 Hz), 2.01 -1 .91 (m, 2H). ³ C-NMR (300 MHz) CDCIs: 172.5, 146.7, 145.1 , 144.1 , 130.0, 126.7, 124.5, 123.2, 120.7, 1 14.4, 1 10.7, 55.9, 43.6, 35.5, 29.0, 27.4. MS (E.I.) m/z: 305 [M] ⁺ (22), 137 (100). Anal, calcd. for C16H19NO3S (MW 305.39): C, 62.93%; H, 6.27%; N, 4.59%. Found: C, 62.91 %; H, 6.28%; N, 4.58%.

/V-(3,4-Dihydroxyphenethyl)-4-(thiophen-2-yl)butanamide (MSP6).

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 3- hydroxytyramine hydrochloride, following Method B. Pure compound was isolated as dark yellow oil (yield 30%, eluent: CHCIs/MeOH 48/2).

IR (Nujol): 3450, 1672 cm ^"1 . ¹ H-NMR (300 MHz) CDCIs δ (ppm): 6.98 (dd, 1 H, J =

4.1 Hz, J = 0.9 Hz), 6.79 (dd, 1 H, J = 5.0 Hz, J = 3.4 Hz), 6.78 (d, 1 H, J = 7.9 Hz), 6.66-6.56 (m, 2H), 6.42 (d, 1 H, J = 7.9 Hz), 3.31 (d, 2H, J = 6.0 Hz), 2.67 (t, 2H, J =

7.2 Hz), 2.53 (t, 2H, J= 6.8Hz), 2.06 (t, 2H, J= 7.4 Hz), 1 .89-1 .80 (m, 2H). ¹³ C-NMR (300 MHz) CDCIs: 173.9, 144.3, 143.9, 143.1 , 130.4, 126.8, 124.6, 123.3, 120.5,

1 15.5, 1 15.3, 41 .0, 35.5, 34.7, 28.9, 27.3. MS (E.I.) m/z: 305 [M] ⁺ (9), 170 (21 ), 153 (36), 136 (100), 123 (8), 97 (21 ). Anal, calcd. for C16H19NO3S (MW 305.39): C, 62.93%; H, 6.27%; N, 4.59%. Found: C, 62.89%; H, 6.29%; N, 4.60%.

/V-(Pyridin-4-ylmethyl)-4-(thiophen-2-yl)butanamide (MSP11 ).

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and pyridin-4- yimethanamine, following Method A. Pure compound was isolated as white solid (yield 82%, eluent: CHCIs/MeOH 50/2).

Mp 96-98 °C (K). ¹ H-NMR (200 MHz) CDCIs δ (ppm): 8.51 (d, 2H, J = 5.3 Hz), 7.19 (d, 2H, J = 5.5 Hz), 7.1 1 (dd, 1 H, J = 1 .5 Hz, J = 5.1 Hz), 6.90 (dd, 1 H, J = 3.6 Hz, J = 5.1 Hz), 6.77 (dd, 1 H, J = 4.0 Hz, J = 1 .9 Hz), 6.13 (br s, 1 H, disappears on treatment with D2O), 4.43 (d, 2H, J = 6.1 Hz), 2.88 (t, 2H, J = 7.1 Hz), 2.30 (t, 2H, J = 7.2 Hz), 2.12-1 .96 (m, 2H). ¹³ C-NMR (200 MHz) CDCIs: 172.7, 149.6 (x2), 147.9, 143.9, 126.8, 124.5, 123.2 (x2), 122.3, 42.2, 35.2, 29.1 , 27.3. MS (ESI) m/z: 261 [M+H] ⁺ (25), 283 [M+Na] ⁺ (100). Anal, calcd. for Ci ₄ Hi ₆ N ₂ OS (MW 260.36): C, 64.58%; H, 6.19%; N, 10.76%. Found: C, 64.49%; H, 6.21 %; N, 10.73%.

/V-(Benzo[d][1 ,3]dioxol-5-ylmethyl)-4-(thiophen-2-yl)butanamide (MSP12).

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and benzo[c |[1 ,3]dioxol-5-ylmethanamine, following Method A. Pure compound was isolated as white solid (yield 86%, eluent: CHCIs/MeOH 50/1 ).

Mp 90.5-91 .5 °C (K). ¹ H-NMR (200 MHz) CDCIs δ (ppm): 7.10 (d, 2H, J = 5.0 Hz), 6.89 (dd, 1 H, J= 3.4 Hz, J= 5.1 Hz), 6.75-6.73 (m, 3H), 5.93 (s, 2H), 5.64 (br s, 1 H, disappears on treatment with D2O), 4.32 (d, 2H, J= 5.5 Hz), 2.87 (t, 2H, J= 7.2 Hz), 2.23 (t, 2H, J= 7.2 Hz), 2.10-1 .99 (m, 2H). ¹³ C-NMR (200 MHz) CDCIs: 172.1 , 147.9, 146.9, 144.1 , 132.1 , 126.7, 124.5, 123.2, 121 .0, 108.4, 108.2, 101 .0, 43.4, 35.4, 29.1 , 27.4. MS (ESI) m/z: 304 [M+H] ⁺ (15), 326 [M+Na] ⁺ (100). Anal, calcd. for C16H17NO3S (MW 303.38): C, 63.34%; H, 5.65%; N, 4.62%. Found: C, 64.49%; H, 6.21 %; N, 10.73%.

/V-(3 ₁ 4-Dimethoxybenzyl)-4-(thiophen-2-yl)butanamide (MSP13).

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 3,4- dimethoxybenzylamine, following Method A. The pure compound was isolated as pale yellow solid (yield 80%, eluent: CHCIs/MeOH 50/1 ).

Mp 87-89 °C (K). ¹ H-NMR (200 MHz) CDCIs δ (ppm): 7.08 (d, 2H, J = 5.1 Hz), 6.88 (dd, 1 H, J = 3.3 Hz, J = 5.1 Hz), 6.78-6.73 (m, 3H), 5.83 (br s, 1 H, disappears on treatment with D ₂ 0), 4.33 (d, 2H, J = 5.7 Hz), 3.83 (s, 6H), 2.85 (t, 2H, J = 7.2 Hz), 2.22 (t, 2H, J= 7.0 Hz), 2.08-1 .98 (m, 2H). ¹³ C-NMR (200 MHz) CDCIs: 172.1 , 149.1 , 148.4, 144.1 , 130.9, 126.7, 124.5, 123.2, 120.1 , 1 1 1 .2, 1 1 1 .1 , 55.9, 55.8, 43.4, 35.5, 29.1 , 27.4. MS (ESI) m/z: 304 [M+H] ⁺ (15), 326 [M+Na] ⁺ (100). Anal, calcd. for C17H21 NO3S (MW 319.42): C, 63.92%; H, 6.63%; N, 4.39%. Found: C, 63.80%; H, 6.65%; N, 4.38%.

4-(Thiophen-2-yl)-/V-(thiophen-2-ylmethyl)butanamide (MSP14).

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and thiophen-2- ylmethanamine, following Method A. The pure compound was isolated as cream solid (yield 84%, eluent: CHCIs).

Mp 63-64 °C (K). ¹ H-NMR (200 MHz) CDCIs δ (ppm): 7.22 (dd, 1 H, J = 2.2 Hz, J = 4.4 Hz), 7.1 1 (dd, 1 H, J = 1 .2 Hz, J = 5.1 Hz), 6.95-6.88 (m, 3H), 6.77-6.75 (m, 1 H), 5.77 (br s, 1 H, disappears on treatment with D2O), 4.60 (d, 2H, J = 5.7 Hz), 2.87 (t, 2H, J = 7.0 Hz), 2.23 (t, 2H, J = 6.9 Hz), 2.1 1 -1 .96 (m, 2H). ³ C-NMR (200 MHz) CDCIs: 172.1 , 144.2, 141 .1 , 126.9, 126.8, 126.0, 125.2, 124.6, 123.2, 38.3, 35.4, 29.1 , 27.4. MS (ESI) m/z: 304 [M+H] ⁺ (15), 326 [M+Na] ⁺ (100). Anal, calcd. for C13H15NOS2 (MW 265.39): C, 58.83%; H, 5.70%; N, 5.28%. Found: C, 58.71 %; H, 5.72%; N, 5.27%.

/V-(3,4-Dichlorobenzyl)-4-(thiophen-2-yl)butanamide (MSP15).

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 3,4- dichlorobenzylamine following Method A. Pure compound was isolated as yellow solid (yield 25%, eluent: CHCIs/MeOH 48/2).

Mp 73.6 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.36-7.30 (m, 2H), 7.10-7.00 (m, 1 H), 6.93- 6.86 (m, 1 H), 6.75 (d, 1 H, J = 3.3 Hz), 6.20 (s, 1 H), 4.30 (d, 2H, J = 6.0 Hz), 2.90 (t, 2H, J = 7.1 Hz), 2.25 (t, 2H, J = 7.1 Hz), 2.05-1 .15 (m, 2H). ³ C- NMR (300 MHz) CDCIs: 172.6, 144.0, 138.8, 132.6, 131 .4, 130.6, 129.5, 127.0, 126.8, 124.6, 123.3, 42.3, 35.3, 29.1 , 27.3. MS (E.I.) m/z: 326 [M] ⁺ (100). Anal, calcd. for C15H15CI2NOS (MW 328.26): C, 54.88%; H, 4.61 %; N, 4.27%. Found: C, 54.75%; H, 4.63%; N, 4.28%.

/V-(4-Methoxybenzyl)-4-(thiophen-2-yl)butanamide (MSP16). This compound was prepared from 4-(thiophen-2-yl)butanoic and 4- methoxybenzylamine following Method A. Pure compound was isolated as white solid (yield 19%, eluent: CHCIs/MeOH 48/2).

Mp 77.4 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.13 (d, 2H, J = 8.2 Hz), 7.04 (d, 1 H, J = 5.2 Hz), 6.83 (dd, 1 H, J = 3.3 Hz, J = 5.2 Hz), 6.80 (d, 2H, J = 8.5 Hz), 6.70 (d, 1 H, J= 2.4 Hz), 5.54 (br s, 1 H), 4.30 (d, 2H, J= 5.3 Hz), 3.73 (s, 3H), 2.81 (t, 2H, J = 7.4 Hz), 2.16 (t, 2H, J = 7.4 Hz), 2.01 -1 .94 (m, 2H). ¹³ C-NMR (300 MHz) CDCIs: 172.1 , 159.2, 144.2, 130.4, 129.2 (x2), 126.8, 124.5, 123.2, 1 14.1 (x2), 55.3, 43.1 , 35.5, 29.1 , 27.4. MS (E.I.) m/z: 289 [M] ⁺ (100). Anal, calcd. for C16H19NO2S (MW 289.39): C, 66.41 %; H, 6.62%; N, 4.84%. Found: C, 66.32%; H, 6.64%; N, 4.83%.

/V-(4-Hydroxybenzyl)-4-(thiophen-2-yl)butanamide (MSP17).

This compound was prepared from 4-(thiophen-2-yl)butanoic and 4- methoxybenzylamine following Method D. Pure compound was isolated as brown solid (yield 7%).

Mp 105.5 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 6.99 (m, 3H), 6.8-6.5 (m, 4H), 6.10 (br s, 1 H), 4.20 (d, 2H, J = 5.4 Hz), 2.75 (t, 2H, J = 7.0 Hz), 2.15 (t, 2H, J = 7,7 Hz), 1 .90 (m, 2H). ¹³ C-NMR (300 MHz) CDCIs: 173.2, 156.1 , 144.0, 129.2 (x2), 129.1 , 126.8, 124.6, 123.2, 1 15.8 (x2), 43.3, 35.5, 29.1 , 27.5. MS (E.I.) m/z: 275 [M] ⁺ (100). Anal, calcd. for C15H17NO2S (MW 275.37): C, 65.43%; H, 6.22%; N, 5.09%. Found: C, 65.33%; H, 6.24%; N, 5.10%.

A/-(4-Hydroxy-3-methoxybenzyl)-4-(5-iodothiophen-2-yl)butana mide (MSP18).

This compound was prepared from 4-(5-iodothiophen-2-yl)butanoic acid and 4- hydroxy-3-methoxybenzylamine hydrochloride, following Method B. The pure compound was isolated as yellow oil (yield 70%, eluent: CHCh/MeOH 50/2).

¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.05 (d, 1 H, J= 3.6 Hz), 6.84 (d, 1 H, J= 8.0 Hz), 6.77 (d, 1 H, J = 1 .2 Hz), 6.72 (dd, 1 H, J = 8.2 Hz, J = 1 .3 Hz), 6.43 (d, 1 H, J = 3.5 Hz), 5.67 (br s, 1 H), 4.31 (d, 2H, J = 5.6 Hz), 3.85 (s, 3H), 2.83 (t, 2H, J = 7.3 Hz.), 2.20 (t, 2H, J= 7.3 Hz), 2.04-1 .94 (m, 2H). ¹³ C-NMR (400 MHz) CDCIs: 171 .9, 150.5, 146.7, 145.2, 136.7, 130.2, 126.4, 120.9, 1 14.5, 1 1 0.8, 69.9, 56.0, 43.6, 35.3, 29.4, 27.1 . MS (ESI) m/z: 454 [M+Na] ⁺ (75), 470 [M+K] ⁺ (100). Anal, calcd. for CieHislNOsS (MW 431 .29): C, 44.56%; H, 4.21 %; N, 3.25%. Found: C, 44.50%; H, 4.22%; N, 3.24%.

A/-(3,4-Dihydroxyphenethyl)-4-(5-iodothiophen-2-yl)butanamid e (MSP19).

This compound was prepared from 4-(5-iodothiophen-2-yl)butanoic and 3- hydroxytyramine hydrochloride, following Method B. Pure compound was isolated as pasty yellow solid (yield 60%, eluent: CHCIs/MeOH 47/3).

Mp < 40 °C (K). ¹ H-NMR (200 MHz) CDCIs δ (ppm): 6.98 (d, 1 H, J = 3.4 Hz), 6.78 (d, 1 H, J = 8.0 Hz), 6.71 (d, 1 H, J = 1 .9 Hz), 6.52 (dd, 1 H, J = 1 .6 Hz, J = 8.0 Hz), 6.38 (d, 1 H, J = 3.6 Hz), 5.70 (t, 1 H, J = 5.6 Hz), 3.48-3.38 (m, 2H), 2.73 (t, 2H, J = 7.4 Hz), 2.64 (t, 2H, J = 7.0 Hz), 2.09 (t, 2H, J = 7.7 Hz), 1 .96-1 .85 (m, 2H). MS (ESI) m/z: 454 [M+Na] ⁺ (100). Anal, calcd. for CieHislNOsS (MW 431 .29): C, 44.56%; H, 4.21 %; N, 3.25%. Found: C, 44.49%; H, 4.22%; N, 3.26%.

3-(Benzo[ ?]thiophen-2-yl)-/\/-(4-hydroxy-3-methoxybenzyl)propanamide (MSP20).

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 4- hydroxy-3-methoxybenzylamine hydrochloride, following Method B. Pure compound was isolated as white solid (yield 10%, eluent: CHC /MeOH 48/2). Mp = 129.8 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.72 (dd, 2H, J = 8.4-29.8 Hz), 7.27 (m, 2H), 7.00 (d, 1 H, J = 0.66 Hz), 6.69 (m, 2H), 6.57 (dd, 1 H, J = 1 .9 Hz, J = 8.0 Hz), 5.85 (s, 1 H), 5.70 (s, 1 H), 4.30 (d, 2H, J = 5.6 Hz), 3.70 (s, 3H), 3.26 (t, 2H, J = 7.3 Hz), 2.59 (t, 2H, J = 7.2 Hz). ³ C-NMR (300 MHz) CDCIs: 171 .1 , 146.6, 145.0, 144.2, 140.0, 139.3, 129.9, 124.2, 123.7, 122.9, 122.1 , 121 .5, 120.7, 1 14.3, 1 10.6, 55.8, 43.6, 37.9, 26.7. MS (E.I.) m/z: 341 [M] ⁺ (100). Anal, calcd. for C19H19NO3S (MW 341 .42): C, 66.84%; H, 5.61 %; N, 4.10%. Found: C, 66.75%; H, 5.63%; N, 4.05%.

3-(Benzo[fo]thiophen-2-yl)-/V-(3,4-dichlorobenzyl)propanamid e (MSP21).

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 3,4-dichlorobenzylamine, following Method A. Pure compound was isolated as white solid (yield 76%, eluent: CHCIs/MeOH 48/2).

Mp = 126.1 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.70 (dd, 2H, J = 8.0 Hz, J = 2.4 Hz), 7.4-7.2 (m, 3H), 7.10 (d, 1 H, J = 8.2 Hz), 7.01 (s, 1 H), 6.81 (dd, 1 H, J = 8.2 Hz, J = 2.0 Hz), 6.30 (s, 1 H), 4.30 (d, 2H, J = 5.9 Hz), 3.30 (t, 2H, J = 6.7 Hz), 2.70 (t, 2H, J = 7.1 Hz). ¹³ C-NMR (300 MHz) CDCIs: 171 .6, 144.0, 139.9, 139.3, 138.4, 132.4, 131 .2, 130.4, 129.3, 126.74, 124.33 123.9, 123.0, 122.2, 121 .6, 42.3, 37.8, 26.7. MS (E. I.) m/z: 363 [M] ⁺ (100). ). Anal, calcd. for C18CI2H15NOS (MW 364.29): C, 59.35%; H, 4.15%; N, 3.84%. Found: C, 59.26%; H, 4.16%; N, 3.85%. 3-(Benzo[ ?]thiophen-2-yl)-/\/-(4-methoxybenzyl)propanamide (MSP22).

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 4- methoxybenzylamine, following Method A. Pure compound was isolated as yellow solid (yield 95%, eluent: CHCIs/MeOH 48/2).

Mp = 165.8 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.70 (dd, 2H, J = 7.4 Hz, J = 6.0 Hz), 7.30 (m, 2H), 7.0 (m, 3H), 6.65 (m, 2H), 5.80 (s, 1 H), 4.32 (d, 2H, J = 5.5 Hz), 3.74 (s, 3H), 3.28 (t, 2H, J = 7.2 Hz), 2.60 (t, 2H, J = 7.2 Hz). ¹³ C-NMR (300 MHz) CDCIs: 171 .1 , 158.9, 144.2, 140.0, 139.4, 129.9, 128.9 (x2), 124.2, 123.7, 123.0, 122.2, 121 .6, 1 13.5 (x2), 55.2, 43.1 , 38.1 , 26.8. MS (E. I.) m/z: 325 [M] ⁺ (100). Anal, calcd. for C19H19NO2S (MW 325.42): C, 70.12%; H, 5.88%; N, 4.30%. Found: C, 69.97%; H, 5.90%; N, 4.31 %.

3-(Benzo[ ?]thiophen-2-yl)-/\/-(4-hydroxybenzyl)propanamide (MSP23).

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 4- methoxybenzylamine, following Method D. Pure compound was isolated as white solid (yield 17%).

Mp = 182.3 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.65 (dd, 2H, J = 7.6, J = 6.4 Hz), 7.20 (m, 2H), 6.95 (s, 1 H), 6.90 (d, 2H, J = 8.2 Hz), 6.60 (d, 2H, J = 7.8 Hz), 5.80 (br s, 1 H), 4.25 (d, 2H, J= 5.8 Hz), 3.20 (t, 2H, J= 7.5 Hz), 2.55 (t, 2H, J = 7.3 Hz). ¹³ C-NMR (300 MHz) CDCIs: 171 .1 , 156.0, 144.3, 139.3, 138.3, 134.4, 129.1 (x2), 124.2, 123.7, 122.9, 122.1 , 121 .5, 1 15.5 (x2), 43.2, 38.0, 26.7. MS (E. I.) m/z: 31 1 [M] ⁺ (100). Anal, calcd. for C18H17NO2S (MW 31 1 .40): C, 69.43%; H, 5.50%; N, 4.50%. Found: C, 69.27%; H, 5.62%; N, 4.40%.

2-(5-Hydroxy-1 W-indol-3-yl)-/V-(4-hydroxy-3-methoxybenzyl)acetamide

(MSP24).

This compound was prepared from 2-(5-hydroxy-1 /-/-indol-3-yl)acetic acid and 4- hydroxy-3-methoxybenzylamine hydrochloride, following Method B. Pure compound was isolated as pale pink solid (yield 35%, eluent: CHCIs/MeOH 47/3). Mp 143-145 °C (K). ¹ H-NMR (200 MHz) Acetone-c/e δ (ppm): 9.90 (s,1 H), 7.80 (br s, 1 H), 7.47 (br s, 1 H), 7.20-7.18 (m, 2H), 7.03 (s, 1 H), 6.72-6.66 (m, 4H), 4.27 (d, 2H, J = 5.7 Hz), 3.6 (s, 5H). ¹³ C-NMR (400 MHz) Acetone-c/e: 171 .3, 150.9, 147.4, 145.4, 131 .5, 130.8, 128.3, 124.7, 124.5, 120.0, 1 14.6, 1 1 1 .7, 1 10.8, 108.2, 102.8, 55.2, 42.4, 33.3. MS (ESI) m/z: 349 [M+Na] ⁺ (90), 365 [M+K] ⁺ (40), 675 [2M+Na] ⁺ (100). Anal, calcd. for 0ι ₈ Ηι ₈ Ν ₂ Ο ₄ (MW 326.35): C, 66.25%; H, 5.56%; N, 8.58%. Found: C, 66.12%; H, 5.58%; N, 8.57%.

/V-(4-Hydroxy-3-methoxybenzyl)-1 H-indole-2-carboxamide (MSP25).

This compound was prepared from 1 /-/-indole-2-carboxylic acid and 4-hydroxy-3- methoxybenzylamine hydrochloride, following Method B. Pure compound was isolated as pink solid (yield 89%, eluent: CHCIs/MeOH 48/2).

Mp = 178.8 °C (G). ¹ H-NMR (300 MHz) MeOD-c/ ₄ δ (ppm): 7.15 (d, 1 H, J = 7.7 Hz),

7.00 (d, 1 H, J = 8.4 Hz), 6.65 (m, 4H), 6.55 (s, 1 H), 6.35 (m, 1 H), 4.50 (s, 2H), 4.00

(s, 3H). ¹³ C-NMR (300 MHz) MeOD-c/ ₄ : 160.1 , 145.2, 144.0, 143.0, 134.0, 133.0, 127.0, 125.0, 122.0, 121 .0, 1 10.9, 1 17.5, 1 17.3, 109.2, 108.6, 52.5, 40.1 . MS (E.I.) m/z: 296 [M] ⁺ (100). Anal, calcd. for C17H16N2O3 (MW 296.32): C, 68.91 %; H,

5.44%; N, 9.45%. Found: C, 68.75%; H, 5.46%; N, 9.47%.

/V-(3,4-Dichlorobenzyl)-1 rV-indole-2-carboxamide (MSP26).

This compound was prepared from 1 /-/-indole-2-carboxylic acid and 3,4- dichlorobenzylamine, following Method A. Pure compound was isolated as violet solid (yield 61 %, eluent: CHCIs/MeOH 48/2).

Mp = 174.1 °C (G). ¹ H-NMR (300 MHz) MeOD-c/ ₄ δ (ppm): 10.1 (br s, 1 H), 7.65 (br s, 1 H), 7.30-7.25 (m, 1 H), 7.20-7.1 1 (m, 2H), 7.05 (s, 1 H), 7.01 -6.90 (m, 2H), 6.89- 6.80 (m, 2H), 6.82-6.70 (m, 2H). ¹³ C-NMR (300 MHz) MeOD-c/ ₄ : 161 .3, 138.3, 135.4, 132.0, 131 .37, 131 .0, 130.4, 129.3, 128.9, 128.6, 1 26.9, 122.2, 1 19.8, 1 18.3, 1 10.1 , 40.2. MS (E.I.) m/z: 318 [M] ⁺ (100). Anal, calcd. for C16H12CI2N2O (MW 319.19): C, 60.21 %; H, 3.79%; N, 8.78%. Found: C, 60.15%; H, 3.80%; N, 8.76%.

3-(Benzo[ ?]thiophen-2-yl)-/\/-(3,4-dihydroxyphenethyl)propanamide (MSP27) This compound was prepared from 3-(benzo[fc]thiophen-2-yl)propanoic acid and 3- hydroxytyramine hydrochloride, following Method B. Pure compound was isolated as brown solid (yield 31 %, eluent: CHCIs/MeOH 4/1 ). Mp = 133.9 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.75 (d, 1 H, J = 7.4 Hz), 7.65 (d, 1 H, J = 7.0 Hz), 7.30 (m, 2H), 7.00 (s, 1 H), 6.75 (d, 1 H, J = 7.9 Hz), 6.60 (s, 1 H), 6.45 (d, 1 H, J= 8.1 Hz), 5.60 (br s, 2H), 3.45 (t, 2H, J= 6.0 Hz), 3.25 (t, 2H, J = 7.4 Hz), 2.55 (m, 4H). ¹³ C-NMR (300 MHz) CDCIs: 162.0, 145.3, 144.9, 140.3, 139.7, 139.3, 138.5, 131 .1 , 124.1 , 123.6, 123.2, 122.0, 1 19.9, 1 19.1 , 1 14.8, 40.9,

37.0, 36.1 , 27.3. MS (E.I.) m/z: 341 [M] ⁺ (100). Anal, calcd. for C19H19NO3S (MW 341 .42): C, 62.84%; H, 5.61 %; N, 4.10%. Found: C, 62.65%; H, 5.63%; N, 4.1 1 %. /V-(3,4-Dihydroxyphenethyl)-1 rV-indole-2-carboxamide (MSP28)

This compound was prepared from 1 /-/-indole-2-carboxylic acid and 3- hydroxytyramine hydrochloride, following Method B. Pure compound was isolated as light brown solid (yield 10 %, eluent: CHCIs/MeOH 48/2).

Mp = 221 .5 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 9.20 (br s, 1 H), 7.65 (d, 1 H, J= 8.1 Hz), 7.45 (m, 2H), 7.30 (m, 2H), 6.40 (s, 1 H), 6.25 (s, 1 H), 6.00 (s, 1 H), 3.70 (d, 2H, J = 6.4 Hz), 2.80 (m, 2H). ¹³ C-NMR (300 MHz) CDCIs: 145.0, 143.5, 136.7, 131 .1 , 127.8, 125.2, 123.5, 121 .5, 120.5, 1 19.9, 1 19.9, 1 15.8, 1 15.2, 1 12.4, 1 12.1 ,

40.1 , 35.1 . MS (E.I.) m/z: 296 [M] ⁺ (100). Anal, calcd. for C17H16N2O3 (MW 296.32): C, 68.91 %; H, 5.44%; N, 9.45%. Found: C, 68.73%; H, 5.46%; N, 9.44%.

/V-(3-Chloro-4-methoxyphenethyl)-4-(thiophen-2-yl)butanam ide (MSP29)

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 3-chloro-4- methoxyphenethylamine, following Method A. Pure compound was isolated as white solid (yield 70.0 %, eluent: CHCIs/MeOH 50/1 ).

Mp 68-70 °C (K). ¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.13 (d, 1 H, J= 1 .5 Hz), 7.04 (d, 1 H, J = 5.2 Hz), 6.97 (dd, 1 H, J = 8.4 Hz, J = 1 .5 Hz), 6.84 (t, 1 H, J = 4.2 Hz), 6.79 (d, 1 H, J = 8.4 Hz), 6.68 (d, 1 H, J = 3.0 Hz), 5.36 (br s, 1 H), 3.80 (s, 3H), 3.40 (q, 2H, J = 6.5 Hz), 2.77 (t, 2H, J = 7.2 Hz), 2.67 (t, 2H, J = 6.9 Hz), 2.09 (t, 2H, J = 7.4 Hz), 1 .96-1 .88 (m, 2H). ¹³ C-NMR (400 MHz) CDCIs: 172.5, 153.6, 144.2, 132.1 , 130.4, 128.0, 126.8, 124.5, 123.2, 122.3, 1 12.3, 56.2, 40.6, 35.5, 34.5, 29.2, 27.5. MS (ESI) m/z: 338 [M+H] ⁺ (100), 360 [M+Na] ⁺ (20). Anal, calcd. for C17H20CINO2S (MW 337.86): C, 60.43%; H, 5.97%; N, 4.15%. Found: C, 60.32%; H, 5.99%; N, 4.14%.

/V-(3,4-Dichlorobenzyl)-4-(5-iodothiophen-2-yl)butanamide (MSP30) This compound was prepared from 4-(5-iodothiophen-2-yl)butanoic acid and 3,4- dichlorobenzylamine, following Method A. Pure compound was isolated as white solid (yield 65.0 %, eluent: CHCIs).

Mp 80-82 °C (K). ¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.38 (d, 1 H, J = 8.2 Hz), 7.32 (d, 1 H, J = 1 .7 Hz), 7.09 (dd, 1 H, J = 1 .7 Hz, J = 8.2 Hz), 7.02 (d, 1 H, J = 3.5 Hz), 6.46 (d, 1 H, J = 3.4 Hz), 5.71 (br s, 1 H), 4.36 (d, 2H, J = 5.8 Hz), 2.85 (t, 2H, J = 7.3 Hz), 2.23, (t, 2H, J = 7.3 Hz), 2.03-1 .93 (m, 2H). ¹³ C-NMR (400 MHz) CDCIs: 172.2, 150.4, 138.7, 136.8, 132.7, 131 .6, 130.7, 129.6, 126.9, 126.5, 70.0, 42.4, 35.1 , 29.3, 27.0. MS (ESI) m/z: 455 [M+H] ⁺ (100). Anal, calcd. for CisHuCWNOS (MW 454.15): C, 39.67%; H, 3.1 1 %; N, 3.08%. Found: C, 39.65%; H, 3.12%; N, 3.09%.

A/-(3-Chloro-4-methoxyphenethyl)-4-(5-iodothiophen-2-yl)b utanamide

(MSP31)

This compound was prepared from 4-(5-iodothiophen-2-yl)butanoic acid and 3- chloro-4-methoxyphenethylamine, following Method A. Pure compound was isolated as transparent oil (yield 68.0 %, eluent: CHCI3).

¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.17 (d, 1 H, J= 1 .9 Hz), 7.03-6.99 (m, 2H), 6.82 (d, 1 H, J = 9.0 Hz), 6.43 (d, 1 H, J = 3.7 Hz), 5.37 (br s, 1 H), 3.86 (s, 3H), 3.48-3.43 (m, 2H), 2.79 (t, 2H, J = 7.2 Hz), 2.71 (t, 2H, J = 6.9 Hz), 2.13 (t, 2H, J = 7.3 Hz), 1 .96-1 .89 (m, 2H). MS (ESI) m/z: 464 [M+H] ⁺ (23), 486 [M+Na] ⁺ (100). Anal, calcd. for C17H19CIINO2S (MW 463.76): C, 44.03%; H, 4.13%; N, 3.02%. Found: C, 44.00%; H, 4.14%; N, 3.01 %.

3-(Benzo[ ?]thiophen-2-yl)-/\/-(3-chloro-4-methoxyphenethyl)propanamid e (MSP32)

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 3- chloro-4-methoxyphenethylamine, following Method A. Pure compound was isolated as white solid (yield 75.0 %, eluent: CHCIs/MeOH 50/1 ).

Mp = 148.8 °C (M). ¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.73 (d, 1 H, J = 7.9 Hz), 7.64 (d, 1 H, J = 7.7 Hz), 7.34-7.22 (m, 2H), 7.09 (d, 1 H, J = 2.0 Hz), 7.01 (s, 1 H), 6.84 (dd, 1 H, J = 2.0 Hz, J = 8.3 Hz), 6.68 (d, 1 H, J = 8.3 Hz), 5.38 (br s, 1 H), 3.83 (s, 3H), 3.45-3.40 (m, 2H), 3.23 (t, 2H, J = 7.3 Hz), 2.64 (t, 2H, J = 6.9 Hz), 2.53 (t, 2H, J = 7.3 Hz). ¹³ C-NMR (400 MHz) CDCIs: 171 .3, 153.7, 144.4, 140.1 , 139.4, 131 .9, 130.3, 127.9, 124.3, 123.7, 123.0, 122.3, 122.2, 121 .5, 1 12.2, 56.2, 40.6, 38.0, 34.4, 26.7. MS (ESI) m/z: 374 [M+H] ⁺ (35), 396 [M+Na] ⁺ (100). Anal, calcd. for C20H20CINO2S (MW 373.90): C, 64.25%; H, 5.39%; N, 3.75%. Found: C, 64.18%; H, 5.41 %; N, 3.74%.

1-(3,4-Dichlorobenzyl)-3-(1 H-indol-2-yl)urea (MSP33)

This compound was prepared from 1 /-/-indole-2-carboxylic acid and 3,4- dichlorobenzylamine, following Method E. Pure compound was isolated as orange solid (yield 27 %, eluent: n.Hex/EtOAc 2/1 .).

Mp = 61 .0 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 9.95 (br s, 1 H), 8.30 (s, 2H),

7.90 (d, 2H, J= 1 .0 Hz), 7.73 (dd, 1 H, J= 1 .8 Hz, J = 8.2 Hz), 7.60 (m, 2H), 7.51 (d, 2H, J = 8.3 Hz), 7.43 (m, 2H), 7.18 (dd, 1 H, J = 1 .9 Hz, J = 8.2 Hz). ¹³ C-NMR (300

MHz) CDCIs: 160.0, 139.1 , 135.7, 135.7, 133.2, 132.54, 131 .3, 131 .2, 131 .1 , 130.7,

130.5, 129.8, 128.4, 127.4, 127.2, 63.5. MS (E.I.) m/z: 333 [M] ⁺ (100). Anal, calcd. for C16C12H13N3O (MW 334.20): C, 57.50%; H, 3.92%; N, 12.57%. Found: C,

57.41 %; H, 3.93%; N, 12.55%.

1 -(2-(Benzo[6]thiophen-2-yl)ethyl)-3-(4-hydroxy-3-methoxybenz yl)urea

(MSP34)

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 4- hydroxy-3-methoxybenzylamine hydrochloride, following Method E. Pure compound was isolated as pasty light brown oil (yield 46,0 %, eluent: CH2Cl2/n.Hex 3/1 ).

¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.65 (m, 2H), 7.30 (m, 3H), 6.95 (m, 1 H), 6.70 (m, 2H), 5.10 (br s, 1 H), 4.15 (d, 2H, J= 18.8 Hz), 3.75 (s, 3H), 3.44 (dd, 2H, J= 6.5 Hz, J = 12.6 Hz), 2.99 (t, 2H, J = 6.5 Hz). ¹³ C-NMR (300 MHz) CDCIs: 158.2, 154.1 , 150.5, 146.7, 144.9, 142.7, 140.0, 139.4, 129.8, 124.2, 123.7, 122.9, 120.2, 1 14.3, 1 10.2, 56.7, 44.4, 41 .1 , 31 .5. MS (E.I.) m/z: 356 [M] ⁺ (100). Anal, calcd. for C19H20N2O3S (MW 356.44): C, 64.02%; H, 5.66%; N, 7.86%. Found: C, 63.86%; H, 5.68%; N, 7.85%.

1-(2-(Benzo[6]thiophen-2-yl)ethyl)-3-(4-methoxybenzyl)urea (MSP35)

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 4- methoxybenzylamine, following Method E. Pure compound was isolated as yellow solid (yield 30.0%, eluent: n.Hex/EtOAc 1 /2). Mp = 142.8 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.72 (d, 1H, J = 7.7 Hz), 7.74 (d, 1 H, J= 7.1 Hz), 7.30 (m, 2H), 7.09 (d, 2H, J= 8.4 Hz), 6.95 (s, 1 H), 6.75 (d, 2H, J= 8.6 Hz), 4.94 (br s, 1H), 4.82 (br s, 1H), 4.14 (d, 2H, J= 5.6 Hz), 3.75 (s, 3H), 3.42 (dd, 2H, J= 6.3 Hz, J= 12.5 Hz), 2.98 (t, 2H, J= 6.4 Hz). ³ C-NMR (300 MHz) CDCIs: 158.8, 158.0, 142.8, 140.0, 139.5, 128.7 (x2), 127.9, 126.7, 124.2, 122.9, 122.1, 121.9, 113.9 (x2), 55.2, 47.0, 41.1, 31.5. MS (E.I.) m/z: 340 [M] ⁺ (100). Anal, calcd. for C19H20N2O2S (MW 340.44): C, 67.03%; H, 5.92%; N, 8.23%. Found: C, 66.94%; H, 5.94%; N, 8.24%.

1-(2-(Benzo[6]thiophen-2-yl)ethyl)-3-(4-hydroxybenzyl)urea (MSP36)

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 4- methoxybenzylamine, following Method F. Pure compound was isolated as brown oil (yield 31.0%).

¹ H-NMR (300 MHz) CDCIs δ (ppm): 7.75 (s, 1H), 7.50 (s, 1H), 7.20 (m, 2H), 6.90 (m, 2H), 6.62 (d, 3H, J= 8.3 Hz), 4.04 (s, 2H), 3.36 (t, 2H, J= 7.0 Hz), 3.05 (m, 2H). MS (E.I.) m/z: 326 [M] ⁺ (100). Anal, calcd. for C18H18N2O2S (MW 326,42): C, 66.23%; H, 5.56%; N, 8.58%. Found: C, 66.12%; H, 5.57%; N, 8.56%.

1-(4-Hydroxy-3-methoxybenzyl)-3-(1 H-indol-2-yl)urea (MSP37)

This compound was prepared from 1 /-/-indole-2-carboxylic acid and 4-hydroxy-3- methoxybenzylamine hydrochloride, following Method E. Pure compound was isolated as yellow solid (yield 8.0%, recrystallized from CHCIs/petroleum ether).

Mp = 178.1 °C (G). ¹ H-NMR (300 MHz) CDCIs δ (ppm): 9.65 (br s, 1H), 7.75 (d, 1H, J= 6.8 Hz), 7.60 (d, 1 H, J= 6.8 Hz), 7.40-6.90 (m, 6H), 6.80 (s, 1 H), 6.55 (br s, 2H), 4.60 (s, 2H), 3.80 (s, 3H). MS (E.I.) m/z: 311 [M] ⁺ (100). Anal, calcd. for C17H17N3O3 (MW 311.34): C, 65.58%; H, 5.50%; N, 13.50%. Found: C, 65.44%; H, 5.52%; N, 13.48%.

1 -(3,4-Dichlorobenzyl)-3-(3-(thiophen-2-yl)propyl)urea (MSP38)

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 3,4- dichlorobenzylamine, following Method E. Pure compound was isolated as white needles (yield 62.0 %, eluent: CHC /MeOH 48/2, recrystallization from n.Hexane). Mp 86-88 °C (K). ¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.37-7.35 (m, 2H), 7.11 (dd, 1H, J=2.0 Hz, J=6.1 Hz), 7.09 (s, 1H), 6.89 (dd, 1H, J=3.5 Hz, J=5.1 Hz), 6.76 (d, 1H, J= 3.3 Hz), 4.29 (s, 2H), 3.23 (t, 2H, J= 6.9 Hz), 2.85 (t, 2H, J= 7.4 Hz), 1 .91 -1 .84 (m, 2H). MS (ESI) m/z: 366 [M+Na] ⁺ (100). Anal, calcd. for C15H16CI2N2OS (MW 343.27): C, 52.48%; H, 4.70%; N, 8.16%. Found: C, 52.35%; H, 4.72%; N, 8.14%.

1-(4-Hydroxy-3-methoxybenzyl)-3-(3-(thiophen-2-yl)propyl)ure a (MSP39)

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 4-hydroxy-3- methoxybenzylamine hydrochloride, following Method E. Pure compound was isolated as white solid (yield 58.0 %, eluent: CHCIs/MeOH 47/3).

Mp 1 15-1 17 °C (K). ¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.08 (d, 1 H, J = 5.2 Hz), 6.88 (dd, 1 H, J = 3.5 Hz, J = 8.5 Hz), 6.86-6.80 (m, 2H), 6.74-6.73 (m, 2H), 5.60 (br s, 1 H), 4.22 (s, 2H), 3.84 (s, 3H), 3.20 (t, 2H, J = 6.9 Hz), 2.82 (t, 2H, J = 7.5 Hz), 1 .94-1 .80 (m, 2H). ¹³ C-NMR (400 MHz) CDCIs: 158.1 , 147.1 , 145.1 , 144.2, 130.9, 126.8, 124.4, 123.2, 120.4, 1 14.4, 1 10.3, 55.9, 44.6, 39.9, 32.1 , 27.1 . MS (ESI) m/z: 343 [M+Na] ⁺ (100). Anal, calcd. for C16H20N2O3S (MW 320.41 ): C, 59.98%; H, 6.29%; N, 8.74%. Found: C, 59.83%; H, 6.31 %; N, 8.1 2%.

1 -(2-(benzo[6]thiophen-2-yl)ethyl)-3-(3,4-dichlorobenzyl)urea (MSP40)

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 3,4-dichlorobenzylamine, following Method E. Pure compound was isolated as white solid (yield 60.0%, eluent: CHCIs/MeOH 50/0.5).

Mp 151 -153 °C (K). ¹ H-NMR (400 MHz) CDCIs δ (ppm): 7.74 (d, 1 H, J = 7.7 Hz), 7.66 (d, 1 H, J = 7.6 Hz), 7.33-7.28 (m, 4H), 7.05 (d, 1 H, J = 8.3 Hz), 7.02 (s, 1 H), 4.53 (br s, 1 H), 4.42 (br s, 1 H), 4.28 (d, 2H, J = 5.4 Hz), 3.58-3.53 (m, 2H), 3.10 (t, 2H, J = 6.3 Hz). ³ C-NMR (400 MHz, CD3COCD3) δ (ppm): 157.9, 143.5, 142.6, 140.4, 139.5, 130.3, 129.7, 127.2, 124.1 , 123.6, 123.2, 122.9, 122.0, 121 .7, 120.2, 42.2, 40.9, 31 .4. MS (ESI) m/z: 380 [M+H] ⁺ (20), 402 [M+Na] ⁺ (100), 418 [M+K] ⁺ (18). Anal, calcd. for C18H16CI2N2OS (MW 379.30): C, 57.00%; H, 4.25%; N, 7.39%. Found: C, 56.84%; H, 4.33%; N, 7.33%.

1-(2-(benzo[6]thiophen-2-yl)ethyl)-3-(3,4-dihydroxyphenethyl )urea (MSP41)

This compound was prepared from 3-(benzo[£>]thiophen-2-yl)propanoic acid and 3,4-dihydroxyphenethylamine, following Method E. Pure compound was isolated as pale pink solid (yield 20.0%, eluent: CHCIs/MeOH 48/2).

Mp 99-101 °C (K). ¹ H-NMR (400 MHz, CDsOD) δ (ppm): 7.73 (d, 1 H, J = 7.8 Hz), 7.65 (d, 1 H, J = 7.6 Hz), 7.27-7.19 (m, 2H), 7.05 (s, 1 H), 6.65 (d, 1 H, J = 8.0 Hz), 6.62 (d, 1 H, J = 1 .9 Hz), 6.48 (dd, 1 H, J = 1 .9 Hz, J = 8.0 Hz), 3.42 (t, 2H, J = 6.8 Hz), 3.29-3.24 (m, 2H), 3.02 (t, 2H, J = 6.7 Hz), 2.57 (t, 2H, J = 7.1 Hz). ¹³ C-NMR (400 MHz, CDsOD) δ (ppm): 159.6, 149.4, 144.8, 143.3, 142.8, 140.2, 130.9, 123.8, 123.3, 122.5, 121 .5 (x2), 1 19.7, 1 15.5, 1 15.0, 41 .3, 40.7, 35.4, 31 .0. MS (ESI) m/z: 357 [M+H] ⁺ (25), 379 [M+Na] ⁺ (100), 395 [M+K] ⁺ (25). Anal, calcd. for C19H20N2O3S (MW 356.44): C, 64.02%; H, 5.66%; N, 7.86%. Found: C, 63.88%; H, 5.74%; N, 7.78%.

1-(4-hydroxy-3-methoxybenzyl)-3-(3-(5-iodothiophen-2-yl)prop yl)urea

(MSP42)

This compound was prepared from 4-(5-iodothiophen-2-yl)butanoic acid and 4- hydroxy-3-methoxybenzylamine, following Method E. Pure compound was isolated as white solid (yield 25.0 %, eluent: CHCIs/MeOH 50/2).

Mp 134-136 °C (K). ¹ H-NMR (400 MHz, CDsOD) δ (ppm): 7.25-7.12 (m, 2H), 7.04- 7.01 (m, 1 H), 6.84 (s, 1 H), 6.51 (d, 1 H, J = 3.1 Hz), 4.18 (s, 2H), 3.80 (s, 3H), 3.14 (t, 2H, J = 6.7 Hz), 2.80 (t, 2H, J = 7.6 Hz), 1 .83-1 .74 (m, 2H). ¹³ C-NMR (400 MHz, CDsOD) δ (ppm): 159.8, 136.6, 131 .4, 128.8, 126.1 , 123.1 , 120.0, 1 19.6, 1 14.7, 1 10.8, 69.3, 55.0, 43.3, 38.8, 31 .9, 26.6. MS (ESI) m/z: 447 [M+H] ⁺ (10), 469 [M+Na] ⁺ (100), 485 [M+K] ⁺ (5). Anal, calcd. for C16H19IN2O3S (MW 446.30): C, 43.06%; H, 4.29%; N, 6.28%. Found: C, 42.92%; H, 4.35%; N, 6.16%.

1-(3,4-dichlorobenzyl)-3-(3-(5-iodothiophen-2-yl)propyl)u rea (MSP43)

This compound was prepared from 4-(5-iodothiophen-2-yl)butanoic acid and 3,4- dichlorobenzylamine, following Method E. Pure compound was isolated as white solid (yield 45.0 %, eluent: CHCIs/MeOH 50/0.5).

Mp 131 -133 °C (K). ¹ H-NMR (400 MHz, CDCIs) δ (ppm): 7.38-7.35 (m, 2H), 7.10 (d, 1 H, J = 8.2 Hz), 7.02 (d, 1 H, J = 3.6 Hz), 6.49 (d, 1 H, J = 3.4 Hz), 4.29 (s, 2H), 3.21 (t, 2H, J =6.8 Hz), 2.82 (t, 2H, J = 7.2 Hz), 1 .87-1 .79 (m, 2H). ³ C-NMR (400 MHz, CDsOD) δ (ppm): 158.1 , 149.4, 139.8, 135.1 , 128.6, 127.3, 125.1 , 124.6, 123.3, 120.1 , 67.9, 40.7, 37.4, 30.3, 25.3. MS (ESI) m/z: 504 [M+CI]- (100). Anal, calcd. for C15H15CI2IN2OS (MW 469.17): C, 38.40%; H, 3.22%; N, 5.97%. Found: C, 38.24%; H, 3.26%; N, 5.93%.

/V-(4-hydroxy-3-methoxybenzyl)-4-(5-phenylthiophen-2-yl)buta namide

(MSP44) This compound was prepared from 4-(5-phenylthiophen-2-yl)butanoic acid and 4- hydroxy-3-methoxybenzylamine, following Method B. Pure compound was isolated as pasty yellow solid (yield 60.0 %, eluent: CHCIs/MeOH 50/2).

Mp < 30 °C (K). ¹ H-NMR (400 MHz, CDCIs) δ (ppm): 7.33 (t, 2H, J = 5.6 Hz), 7.31 - 7.30 (m, 2H), 7.23-7.20 (m, 1 H), 7.08 (t, 1 H, J= 3.4 Hz), 6.84 (dd, 1 H, J = 2.9 Hz, J = 8.0 Hz), 6.78 (s, 1 H), 6.74-6.70 (m, 2H), 5.71 (br s, 1 H), 4.33 (d, 2H, J = 2.7 Hz), 3.84 (s, 3H), 2.86 (t, 2H, J= 5.2 Hz), 2.25 (t, 2H, J= 7.3 Hz), 2.08-2.01 (m, 2H). ¹³ C- NMR (400 MHz, CDCIs) δ (ppm): 172.2, 146.7, 145.2, 143.9, 142.2, 134.5, 130.2, 128.8 (x2), 127.2, 125.6, 125.5 (x2), 122.8, 120.9, 1 14.4, 1 10.8, 56.0, 43.7, 35.5, 29.4, 27.3. MS (ESI) m/z: 382 [M+H] ⁺ (30), 404 [M+Na] ⁺ (100), 420 [M+K] ⁺ (20). Anal, calcd. for C22H23NO3S (MW 381 .49): C, 69.26%; H, 6.08%; N, 3.67%. Found: C, 69.07%; H, 6.16%; N, 3.65%.

1-(3,4-dihydroxyphenethyl)-3-(3-(5-iodothiophen-2-yl)propyl) urea (MSP49)

This compound was prepared from 4-(5-iodothiophen-2-yl)butanoic acid and 3,4- dihydroxyphenethylamine, following Method E. Pure compound was isolated as brown oil (yield 20.0 %, eluent: CHCIs/MeOH 48/2).

¹ H-NMR (400 MHz, CDsOD) δ (ppm): 7.02 (d, 1 H, J = 3.5 Hz), 6.67-6.62 (m, 2H), 6.51 (s, 1 H), 6.49 (d, 1 H, J = 3.5 Hz), 3.26 (t, 2H, J = 7.3 Hz), 3.10 (t, 2H, J = 6.8 Hz), 2.80 (t, 2H, J = 7.5 Hz), 2.58 (t, 2H, J = 7.2 Hz), 1 .79-1 .72 (m, 2H). ¹³ C-NMR (400 MHz, CDsOD) δ (ppm): 159.8, 150.9, 144.9, 143.3, 136.6, 130.9, 126.1 , 1 19.7, 1 15.5, 1 15.0, 69.3, 41 .4, 38.7, 35.4, 31 .8, 26.8. MS (ESI) m/z: 447 [M+H] ⁺ (40), 469 [M+Na] ⁺ (100), 485 [M+K] ⁺ (15). Anal, calcd. for C16H19IN2O3S (MW 446.30): C, 43.06%; H, 4.29%; N, 6.28%. Found: C, 42.98%; H, 4.33%; N, 6.22%.

1-(3,4-dihydroxyphenethyl)-3-(3-(thiophen-2-yl)propyl)ure a (MSP51)

This compound was prepared from 4-(thiophen-2-yl)butanoic acid and 3,4- dihydroxyphenethylamine, following Method E. Pure compound was isolated as brown oil (yield 25.0 %, eluent: CHCIs/MeOH 50/2).

¹ H-NMR (400 MHz, CDsOD) δ (ppm): 7.12 (d, 1 H, J = 5.0 Hz), 6.86 (t, 1 H, J = 4.1 Hz), 6.78 (s, 1 H), 6.66 (d, 1 H, J = 8.0 Hz), 6.62 (s, 1 H), 6.50 (d, 1 H, J = 7.8 Hz), 3.26 (t, 2H, J= 7.4 Hz), 3.1 1 (t, 2H, J= 6.8 Hz), 2.81 (t, 2H, J = 7.5 Hz), 2.58 (t, 2H, J = 7.0 Hz), 1 .82-1 .74 (m, 2H). ¹³ C-NMR (400 MHz, CDsOD) δ (ppm): 159.8, 144.9, 144.3, 143.3, 130.9, 126.3, 124.0, 122.6, 1 19.7, 1 1 5.5, 1 15.5, 41 .4, 38.9, 35.5, 32.1 , 26.6. MS (ESI) m/z: 321 [M+H] ⁺ (40), 343 [M+Na] ⁺ (50), 359 [M+K] ⁺ (100). Anal, calcd. for C16H20N2O3S (MW 320.41 ): C, 59.98%; H, 6.29%; N, 8.74%. Found: C, 59.72%; H, 6.37%; N, 8.69%.

A/-(3,4-dichlorobenzyl)-4-(5-phenylthiophen-2-yl)butanamide (MSP52)

This compound was prepared from 4-(5-phenylthiophen-2-yl)butanoic acid and 3,4- dichlorobenzylamine, following Method A. Pure compound was isolated as pasty white solid (yield 40.0 %, eluent: CHCIs/MeOH 50/1 ).

Mp 104-106 °C (K). ¹ H-NMR (400 MHz, CDCIs) δ (ppm): 7.51 -7.49 (m, 2H), 7.31 (t, 4H, J = 7.1 Hz), 7.19 (t, 1 H, J = 6.6 Hz), 7.08 (d, 1 H, J = 3.5 Hz), 7.04 (d, 1 H, J = 8.2 Hz), 6.70 (d, 1 H, J = 3.0 Hz), 6.1 1 (br s, 1 H), 4.30 (d, 2H, J = 5.9 Hz), 2.83 (d, 2H, J = 7.2 Hz), 2.24 (t, 2H, J = 7.2 Hz), 2.05-1 .97 (m, 2H). ¹³ C-NMR (400 MHz, CDCIs) δ (ppm): 172.5, 143.8, 142.2, 138.8, 134.5, 132.6, 1 31 .4, 130.6, 129.5, 128.9 (x2), 127.2, 127.0, 125.7, 125.5 (x2), 122.8, 42.4, 35.2, 29.4, 27.1 . MS (ESI) m/z: 427 [M+Na] ⁺ (100). Anal, calcd. for C21 H19CI2NOS (MW 404.35): C, 62.38%; H, 4.74%; N, 3.46%. Found: C, 62.26%; H, 4.80%; N, 3.44%.

^(4-hydroxy-3-methoxybenzyl)-4-(5-(pyridin-3-yl)thiophen-2-y l)butanamide (MSP53)

This compound was prepared from 4-[5-(pyridine-3-yl)thiophen-2-yl]butanoic acid and 4-hydroxy-3-methoxybenzylamine, following Method C. Pure compound was isolated as pasty yellow solid (yield 75.0 %, eluent: CHCIs/MeOH 50/2).

Mp < 30 °C. ¹ H-NMR (400 MHz) CDCIs δ (ppm): 8.77 (s, 1 H), 8.45 (s, 1 H), 7.77 (d, 1 H, J= 7.9 Hz), 7.28-7.25 (m, 1 H), 7.14 (d, 1 H, J= 3.5 Hz), 6.84 (d, 1 H, J= 8.0 Hz), 6.78 (s, 1 H), 6.75-6.73 (m, 2H), 5.76 (br s, 1 H), 4.33 (d, 2H, J = 5.6 Hz), 3.84 (s, 3H), 2.87 (t, 2H, J = 7.3 Hz), 2.25 (t, 2H, J = 7.3 Hz), 2.09-2.02 (m, 2H). ¹³ C-NMR (400 MHz) CDCIs: 172.0, 147.8, 146.8, 146.3, 145.6, 145.3, 138.0, 132.7, 130.5, 130.2, 125.9, 124.1 , 123.7, 120.9, 1 14.5, 1 10.8, 56.0, 43.6, 35.4, 29.5, 27.2. MS (ESI) m/z: 383 [M+H] ⁺ (100), 405 [M+Na] ⁺ (35). Anal, calcd. for C21 H22N2O3S (MW 382.48): C, 65.95%; H, 5.80%; N, 7.32%. Found: C, 65.87%; H, 5.92%; N, 7.24%. /V-(3,4-dichlorobenzyl)-4-(5-(pyridin-3-yl)thiophen-2-yl)but anamide (MSP54) This compound was prepared from 4-[5-(pyridine-3-yl)thiophen-2-yl]butanoic acid and 3,4-dichlorobenzylamine, following Method C. Pure compound was isolated as white solid (yield 90.0 %, eluent: CHCIs/MeOH 50/1 ). Mp 94-96 °C (K). ¹ H-NMR (400 MHz) CDCIs δ (ppm): 8.69 (s, 1 H), 8.38 (d, 1 H, J = 4.2 Hz), 7.72 (d, 1 H, J = 7.9 Hz), 7.33-7.30 (m, 2H), 7.22 (dd, 1 H, J = 4.8 Hz, J = 8.0 Hz), 7.10 (d, 1 H, J = 3.6 Hz), 7.06 (dd, 1 H, J = 1 .8 Hz, J = 8.2 Hz), 6.72 (d, 1 H, J = 3.5 Hz), 6.45 (br s, 1 H), 4.33 (d, 2H, J = 5.9 Hz), 2.84 (t, 2H, J = 7.3 Hz), 2.27 (t, 2H, J = 7.3 Hz), 2.06-1 .99 (m, 2H). ³ C-NMR (400 MHz) Acetone-c/e δ (ppm): 171 .8, 148.0, 146.1 , 145.9, 141 .2, 138.0, 132.1 , 131 .6, 130.4, 130.1 , 129.5, 129.4, 127.6, 126.0, 124.4, 123.7, 41 .5, 34.6, 28.9, 27.3. MS (ESI) m/z: 406 [M+H] ⁺ (100), 427 [M+Na] ⁺ (35). Anal, calcd. for C20H18CI2N2OS (MW 405.34): C, 59.26%; H, 4.48%; N, 6.91 %. Found: C, 59.14%; H, 4.54%; N, 6.87%.

^(4-hydroxyphenyl)-4-(5-(pyridin-3-yl)thiophen-2-yl)butan amide (MSP55)

This compound was prepared from 4-[5-(pyridine-3-yl)thiophen-2-yl]butanoic acid and 4-hydroxyaniline, following Method C. Pure compound was isolated as white solid (yield 45.0 %, eluent: CHCIs/MeOH 50/2).

Mp 166-168 °C (K). ¹ H-NMR (400 MHz) MeOD δ (ppm): 8.73 (s, 1 H), 8.38 (d, 1 H, J = 4.7 Hz), 7.98 (d, 1 H, J = 8.1 Hz), 7.40 (dd, 1 H, J = 4.9 Hz, J = 8.0 Hz), 7.31 (d, 1 H, J = 3.6 Hz), 7.27 (d, 2H, J = 8.8 Hz), 6.88 (d, 1 H, J = 3.6 Hz), 6.69 (d, 2H, J = 8.8 Hz), 2.92 (t, 2H, J = 7.4 Hz), 2.40 (t, 2H, J = 7.4 Hz), 2.10-2.02 (m, 2H). ¹³ C- NMR (400 MHz) MeOD: 172.1 , 152.8, 146.9, 146.1 , 145.2, 137.9, 133.1 , 131 .0, 130.3, 126.0, 124.5, 124.1 , 122.0 (x2), 1 14.8 (x2), 35.3, 29.1 , 27.3. MS (ESI) m/z: 339 [M+H] ⁺ (100), 361 [M+Na] ⁺ (40). Anal, calcd. for C19H18N2O2S (MW 338.42): C, 67.43%; H, 5.36%; N, 8.28%. Found: C, 67.35%; H, 5.38%; N, 8.24%.

Biological methods

Procedure for TRPV1 channel assay. HEK293 (human embryonic kidney) cells stably over-expressing recombinant human TRPV1 were grown on 100 mm diameter Petri dishes as mono-layers in minimum essential medium (EMEM) supplemented with nonessential amino acids, 10% foetal bovine serum, and 2 mM glutamine, and maintained at 5% CO2 at 37 °C. Stable expression of channels was checked by quantitative PCR (data not shown). The effect of the substances on intracellular Ca ²⁺ concentration ([Ca ^2+] i) was determined by using Fluo-4, a selective intracellular fluorescent probe for Ca ²⁺ . On the day of the experiment, cells were loaded for 1 h at room temperature with the methyl ester Fluo-4-AM (4 μΜ; Invitrogen) in EMEM without foetal bovine serum, then were washed twice in tyroide's buffer (145 mM NaCI, 2.5 mM KCI, 1 .5 mM CaCI ₂ , 1 .2 mM MgCI ₂ , 10 mM D-glucose, and 10 mM HEPES, pH 7.4), resuspended in the same buffer, and transferred to the quartz cuvette of the spectrofluorimeter (Perkin-Elmer LS50B; Perkin-Elmer Life and Analitycal Sciences, Waltham, MA, USA) under continuous stirring. [Ca ²⁺ ]i was determined before and after the addition of various concentrations of test compounds by measuring cell fluorescence (λεχ = 488 nm, λΕΜ = 516 nm) at 25 °C. Curve fitting (sigmoidal dose-response variable slope) and parameter estimation were performed with GraphPad Prism® (GraphPad Software Inc., San Diego, CA). Potency was expressed as the concentration of test substance exerting half-maximal agonist effect (i.e., half-maximal increases in [Ca ²⁺ ]i) (ECso). The efficacy of the agonists was first determined by normalizing their effect to the maximum Ca ²⁺ influx effect on [Ca ²⁺ ]i observed with application of 4 μΜ ionomycin (Sigma). The values of the effect on [Ca ²⁺ ]i in wild type HEK293 (i.e., not transfected with any construct) cells were taken as baseline and subtracted from the values obtained from transfected cells. Antagonist/desensitizing behaviour was evaluated against capsaicin (0.1 μΜ) for TRPV1 , by adding the test compounds in the quartz cuvette 5 min before stimulation of cells with agonists. Data are expressed as the concentration exerting a half-maximal inhibition of agonist-induced [Ca ²⁺ ]i elevation (IC50), which was calculated again using GraphPad Prism® software. The effect on [Ca ²⁺ ]i exerted by agonist alone was taken as 100%. Dose response curves were fitted by sigmoidal regression with variable slope. Statistical analysis of the data was performed by analysis of variance at each point using ANOVA followed by the Bonferroni's test.

CHO cells. Chinese hamster ovary cells (CHO) stably transfected with cDNA encoding human cannabinoid CBi receptors were maintained at 37 °C and 5% CO2 Dulbecco's modified Eagle's medium nutrient mixture F-12 HAM supplemented with 1 mM L-glutamine, 10% foetal bovine serum and 0.6% penicillin-streptomycin together with G418 (600 μ mL ¹ ). These cells were passaged twice a week using non-enzymatic cell dissociation solution.

CBi and CB2 Binding Assay. The preliminary screening was carried out using three concentrations (5, 10 and 25 μΜ) of each compound, membranes from HEK- 293 cells stably transfected with either the human CBi or CB2 receptors and [ ³ H]-(- )-c/ ^' s-3-[2-hydroxy-4-(1 , 1 -dimethylheptyl)-phenyl]-frans-4-(3-hydroxypropyl)- cyclohexanol ([ ³ H]CP-55,940) (Ka = 0.18 nM for CBi and 0.31 nM for CB ₂ receptors) as the high affinity ligand as described by the manufacturer (Perkin Elmer, Italy). ²⁸ Compounds that displaced [ ³ H]CP-55,940 by more than 50% at 10 μΜ were further analyzed by carrying out a complete dose-response curve (using 1 , 10, 50, 100, 500, 1000 nM concentrations). Displacement curves were generated by incubating drugs with [ ³ H]CP-55,940 (0.14 nM for CBi and 0.084 μΜ for CB ₂ binding assay). In all cases, Ki values were calculated by applying the Cheng-Prusoff equation ²⁹ to the IC50 values (obtained by GraphPad) for the displacement of the bound radioligand by increasing concentrations of the test compounds. The concentrations used to carry out the dose-responses allow us to calculate IC50, and hence Ki, with at least a 10 nM resolution. Data are reported as mean value ± SEM of three separate experiments performed in duplicate.

Fatty acid amide hydrolase assay. The effect of increasing concentrations of the test compounds on the enzymatic hydrolysis of [ ¹⁴ C] anandamide was studied as described previously, by using membranes prepared from rat brain (which express high amounts of FAAH). In brief, the whole rat brain was homogenized at 4 °C in 50 mM Tris-HCI buffer, pH 7.0, by using an ultraturrax and a douce homogenizer. Homogenates were first centrifuged at 800 g for 15 min to get rid of the debris and the supernatant was centrifuged at 10 000 g for 30 min. The pellet from this latter centrifugation was used for the assay. Membranes (100 μ9) were incubated in 50 mM Tris-HCI, pH 9, for 30 min at 37 °C with increasing concentrations (up to 25 μΜ) of the test compounds and with synthetic N-arachidonoyl-[ ¹⁴ C]-ethanolamine (55 mCi/mmol, ARC St. Louis, MO, USA) properly diluted with AEA (Tocris Bioscience, Avonmouth, Bristol, UK) to the final concentration of 4 μΜ. After incubation, the amount of [ ¹⁴ C]-ethanolamine produced was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with 2 volumes of CHCI3/CH3OH 1 /1 (by vol.). Water-soluble [ ¹⁴ C]-ethanolamine produced from [ ¹⁴ C]- anandamide was used as a measure of anandamide hydrolysis.

Monoacylglycerol lipase assay. The hydrolysis of 2-AG was measured by incubating the 10,000 g cytosolic fraction (100 μg/sample) of COS-7 cells (which express high amounts of MAGL), homogenized as described above for rat brain (but without using the ultraturrax in this case), in Tris-HCI 50 mM, at pH 7 at 37 °C for 20 min, with synthetic 2-arachidonoyl-[ ³ H]-glycerol (40 Ci/mmol, ARC St. Louis, MO, USA) properly diluted with 2-AG (Cayman Chemicals, Ann Arbor, Ml, USA) to the final concentration of 20 μΜ. After incubation, the amount of [ ³ H]-glycerol produced was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with 2 volumes of CHCI3/CH3OH 11λ (by vol.). Water-soluble [ ³ H]- glycerol produced from [ ³ H]-2-AG was used as a measure of 2-AG hydrolysis. Cell Culture and Treatment. HaCaT cells were grown in Dulbecco's modified Eagle's medium High Glucose (Lonza, Milan, Italy), supplemented with 1 0% FBS, 100 U/ml penicillin, 1 00 μg/mL streptomycin and 2 mM L-glutamine as previously described. ²⁵ Cells were incubated at 37 °C for 24 h in 95% air/5% CO2 until 80% confluency. HaCaT cells were pre-treated with compound 1 for 24 h. The compound was dissolved in DMSO at a concentration of 1 0 μΜ as a stock solution. The stock was diluted to the required concentrations directly in the medium. The final concentration of DMSO in culture medium during compounds treatment did not exceed 0.1 % (v/v). After treatment, cells were collected or treated with glucose oxidase (1 U/mL, 1 h) and processed as described below.

Cellular Viability. Viability studies were performed at 24 and 48 h after treatments by Alamar Blue (Resazurin sodium salt from Sigma) and LDH (EuroClone Milan, Italy) release assay. LDH release was measured enzymatically as previously described. ²⁷ Prior to each assay, the cells were lysed with 0.1 % (V/V) Triton X-1 00 in culture media for 30 min at 37 °C to obtain a representative maximal LDH release as the positive control (1 00% toxicity). Alamar blue stock solution in phosphate buffered saline (pH 7.4, 1 mg/mL) was prepared to obtain final working concentration of 0.1 mg/mL in cell culture medium. Colour change of the redox indicator resazurin was measured at 585 nm (excitation) and 590 nm (emission) wavelengths to evaluate oxidized versus reduced forms of the reagent respectively. Western blot analysis. Total cell lysates were extracted in solubilization buffer containing 50 mM Tris (pH 7.5), 1 50 mM NaCI, 1 0% glycerol, 1 % Nonidet P-40, 1 mM EGTA, 0.1 % SDS, 5 mM N-ethylmaleamide (Sigma-Aldrich Corp.), protease and phosphatase inhibitor cocktails (Sigma-Aldrich Corp.) as previously described. ²⁶ Samples of 60 μg protein in 3X loading buffer (65 mM Tris base, pH 7.4, 20% glycerol, 2% sodium dodecyl sulfate, 5% β-mercaptoethanol and 1 % bromophenol blue) were boiled for 5 min, loaded onto 10% sodium dodecyl sulphate-polyacrylamide electrophoresis gels and separated by molecular size. The gels were then electro-blotted onto nitrocellulose membranes which were then blocked for 1 h in Tris-buffered saline, pH 7.5, containing 0.5% Tween 20 and 5% milk. Membranes were incubated overnight at 4 °C with the primary antibody, goat anti-HNE (Millipore Corporation, Billerica, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (anti-goat) for 1 h, and the bound antibodies were detected using chemiluminescence (BioRad, Milan, Italy). Images of the bands were digitized and the densitometry of the bands were performed using Image-J software.

DCFH-DA assay. After pre-treatment with MSP3 at different doses (1 , 10, 30, and

100 μΜ, respectively) for 24 h, HaCaT cells (5 x 10 ⁴ cell/mL) were incubated with 20 μΜ DCFH-DA in the loading medium in 95% air/5% CO2 at 37 °C for 30 minutes. After this time, cells were treated with GO for different times (15, 30, 45 and 60 minutes) and the fluorescence of the cells from each well was measured at 485 nm (excitation filter) and 530 nm (emission filter) by using a plate reader (TECAN-infinite M200). Preliminary study on the antinociceptive activity of compound MSP18 in rats

The experimental procedure used here was in accordance with the Italian law on the "Protection of animals used for experimental and other scientific reasons". Formalin Test. Animal care and all animal procedures were in compliance with Italian (D.L. 1 16/92) and EEC (O.J. of EC L358/1 18/12/1986) regulations on the protection of laboratory animals. Guidelines of the International Association for the Study of Pain were also followed. Rats were divided into SHAM or FORM groups (N = 7-8 for group). FORM animals received dilute formalin (10%, 50 μΙ) s.c. in the right dorsal hind paw. Sham animals were merely pricked with the syringe needle, without injection of any substances. To evaluate the effect of MSP18, the latter (or the vehicle) was i.p. injected (1 mg/Kg) 15 min before formalin injection. The rats were then placed in the Open Field apparatus (a square transparent Plexiglas cage, 50 x 50 x 30 cm) and its behaviour was recorded for 60 min. To determine pain intensity and to verify the behavioural effects of treatment, pain-related and spontaneous behaviours were considered:

a) pain-related behaviours (formalin-induced responses): licking duration (time spent licking the injected foot); flexing duration (time spent with the leg held off the floor, flexed close to the body); paw-jerk frequency (number of phasic flexions of the leg).

b) spontaneous behaviours: rearing frequency (number of times the animal stood on its fore limbs); activity duration (time spent sniffing and looking around the environment, even the time spent washing or scratching the face or body).

Results. Based on the data obtained in in vitro assay, authors decided to investigate whether compound MSP18 displayed any analgesic activity in vivo. To this end, the formalin test of peripheral acute and inflammatory pain in rats was employed. This test is a well-established model of persistent pain in which rats exhibit a transient, biphasic pattern of pain behaviour. The early, short lasting first phase of the nociceptive response is characterized by the activation of C and Αδ fibres and is followed first by a quiescent period and then by a second, prolonged phase of tonic pain, involving an inflammatory reaction in peripheral tissue, ³⁰ an activation of primary afferent nerve fibres ³¹ and the development of central nervous system (CNS) sensitization. ^32,33 From the results obtained with this pain model we found that MSP18 treated rats have shown to decrease significantly the licking, the flexing and the paw jerk duration as compared to control rats. Moreover MSP18 treated rats did not modified the rearing and spontaneous behaviour (locomotion, grooming, crouching) as compared to control rats (figure 9).

Table 1. In vitro TRPV1 efficacy, potency (ECso) and desensitization (ICso) of synthesized compounds MSP1 -MSP26 in comparison with capsaicin and RTX. ^a CBi and CB ₂ Receptor Affinity (Ki Values) of compounds MSP15,

55,212-2. ^b

separate experiments performed in duplicate and are expressed as Ki (μΜ), for CBi and CB2 binding assays. Reference compounds were tested under the same conditions in this study, n.a. = IC50 > 10 in the preliminary screening carried out with rat brain and spleen membranes. ^c As percent of the effect of ionomycin (4 μΜ). ^d Determined against the effect of capsaicin after a 5 min pre-incubation with each compound (0.1 μΜ). n.a. = not active, n.d. = not determined. Reference [34]. Table 2. In vitro TRPV1 efficacy, potency (ECso) and desensitization (ICso) of the synthesized ureas MSP33-39 in comparison with capsaicin and RTX ^a and inhibitory activity on FAAH and MAGL (ICso values) ^b

^a Data are means ± SEM of N = 3 determinations. Data are mean values of at least three separate experiments. Standard errors of means (SEM) are not shown for the sake of clarity and were never higher than 5% of the mean. ^c As percent of the effect of ionomycin (4 μΜ). ^d Determined against the effect of capsaicin after a 5 min pre-incubation with each compound (0.1 μΜ). n.a. = not active, n.d. = not yet determined.

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