Synthesis and pharmacological evaluation of benzannulated derivatives as potent and selective sigma-1 protein ligands

Accepted Manuscript Synthesis And Pharmacological Evaluation Of Benzannulated Derivatives As Potent And Selective Sigma-1 Protein Ligands Marion Donnier-Maréchal, Pascal Carato, Delphine Le Broc, Christophe Furman, Patricia Melnyk PII:

S0223-5234(14)00044-0

DOI:

10.1016/j.ejmech.2014.01.013

Reference:

EJMECH 6666

To appear in:

European Journal of Medicinal Chemistry

Received Date: 7 June 2013 Revised Date:

8 January 2014

Accepted Date: 9 January 2014

Please cite this article as: M. Donnier-Maréchal, P. Carato, D. Le Broc, C. Furman, P. Melnyk, Synthesis And Pharmacological Evaluation Of Benzannulated Derivatives As Potent And Selective Sigma-1 Protein Ligands, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.01.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Synthesis and pharmacological evaluation of benzannulated derivatives as potent and selective sigma-1 protein ligands

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Marion Donnier-Maréchala,b, Pascal Caratoa,b, Delphine Le Broca,c, Christophe Furmana,c and Patricia Melnyka,b,*

a

Univ Lille Nord de France, F-59000 Lille, France UDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France c UDSL, EA 4483, UFR Pharmacie, F-59000 Lille, France

O Y

n

N

N

N

X

O N

Linker

X = H, Br, Cl

N

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N

R1 N R2

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X n = 0 or 1 Y = O or S

N

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b

N

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A series of benzannulated derivatives were synthesised as sigma 1 ligands. Some of them showed excellent affinity for sigma 1 receptor and good selectivity for sigma 2 receptor. The cytotoxic effects were also evaluated.

ACCEPTED MANUSCRIPT SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF BENZANNULATED DERIVATIVES AS POTENT AND SELECTIVE SIGMA-1 PROTEIN LIGANDS

Marion Donnier-Maréchala,b, Pascal Caratoa,b,*, Delphine Le Broca,c, Christophe Furmana,c and

Univ Lille Nord de France, F-59000 Lille, France

c

UDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France

UDSL, EA 4483, UFR Pharmacie, F-59000 Lille, France

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b

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a

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Patricia Melnyka,b

Mail adress : UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille List of e-mail adresses :

[email protected], [email protected], delphine.lebroc@univ-

Corresponding author :

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lille2.fr, [email protected], [email protected]

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Pascal Carato, EA4481, UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille tel : 33 (0)3 20 96 49 66 – fax : 33 (0)3 20 96 49 13

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mail : [email protected]

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ACCEPTED MANUSCRIPT Abstract The σ1 proteins are considered to be a new class of target structures for several central nervous system disorders, including depression, anxiety, psychosis, and Parkinson's and Alzheimer's diseases. Recently, the involvement of these receptors in neuropathic pain and cancer has also been observed. So far, only a few ligands are in clinical trials. In a continuation of our previous studies on the development of σ1 ligands, a new series of

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benzannulated heterocycles was designed and synthesised. In vitro competition binding assays showed that many of them possessed high σ1 receptor affinity (Ki = 0.6 to 10.3 nM), and good σ2/σ1 subtype selectivity, without cytotoxic effects on SY5Y cells (human neuroblastoma cell

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line).

DCM,

Dichloromethane;

DMEM,

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Abbreviations: Dulbecco’s

Modified

Eagle

Medium;

DMSO,

Dimethylsulphoxide; PHPLC, Purity determined by HPLC; TLC, thin layer chromatography;

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TFA, Trifluoroacetic acid; tR, HPLC retention time

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ACCEPTED MANUSCRIPT 1. Introduction Originally proposed as a subtype of opioid receptors [1], σ receptors are now recognised as a unique protein family. They have a characteristic distribution in the central nervous system, but they are also widely present in the peripheral organs and tissues such as lung, liver, kidney

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and heart [2-4]. Based on ligand selectivity assays, two subtypes have been identified and designated σ1 and σ2 [5-6]. These subtypes differ in size, anatomical distribution and ligand selectivity. While the human σ1 receptor has been cloned from various tissues, and is well characterised at functional and structural level, the σ2 receptor has not been yet cloned from

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any species, and is less well known [7].

The σ1 receptors are integral membrane proteins consisting of 223 amino acids with two transmembrane domains [8]. They primarily reside in the specialised endoplasmic reticulum

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(ER) membrane directly apposing mitochondria, the so-called MAM (mitochondrialassociated endoplasmic reticulum membrane), and modulate Ca2+ efflux from ER by acting as molecular chaperones of inositol (1,4,5)-triphosphate receptors [9]. However, the localisation of σ1 receptors is dynamic in nature. Indeed, they are also able to translocate from the MAM to the plasma membrane [10], where they regulate a variety of functional proteins, including

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ion channels, receptors and kinases. It has been shown that the σ1 receptors are involved in the regulation of numerous neurotransmitter systems such as the cholinergic, dopaminergic and glutamatergic neurotransmission [11-12]. Although the signal transduction pathway after activation of σ1 receptors is not completely understood, there is more and more evidence to

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suggest that they represent a potential therapeutic target in many diseases. Indeed, since their discovery, the σ1 receptors have been implicated in various pathologies, including

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neurological and psychiatric disorders. Thus, ligands that bind to these receptors have been proposed to exhibit effects in several therapeutic areas such as mnesic disorders (Alzheimer’s disease and amnesia) [13], drug addiction [14], depression, anxiety [15], epilepsy, multiple sclerosis [16], Parkinson’s disease [17], stroke and pain. Recently, σ1 receptors have been described as inter-organelle signalling modulators, thus being potentially involved in misfolded protein diseases [18]. Furthermore, the σ1 receptors are overexpressed in tumour cells, making them a possible target for cancer treatment [19]. Several compounds have undergone clinical trials, but no selective σ1 receptor ligands have so far been marketed [20]. A phase 2 study to evaluate Anavex 2-73 in patients with Alzheimer’s disease is ongoing [21]. Moreover, the effects of Igmesine on depressive patients have been studied in phase 3 clinical trials [22]. 3

ACCEPTED MANUSCRIPT Biological and pharmacological investigations have pointed out several classes of structurally different compounds with high affinity for the σ1 receptors. Based on optimisation studies, the pharmacophoric model of these ligands has been reported to consist of an amine binding site flanked on either side by hydrophobic pockets that display bulk tolerance [23]. A large diversity of hydrophobic moieties have already been described, including essentially

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heterocycles such as benzomorphanes, benzofurans [24], benzothiazolinones [25] and benzoxazolinones [26].

Work in our laboratory has focused on development of selective σ1 receptor ligands with diverse therapeutic applications. In previous papers we presented the affinity of more

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constrained series containing tetrahydroisoquinoline-hydantoin (tic-hydantoin) structure [2730]. Compounds 1 and 2 (Figure 1) were identified as efficient σ1 ligands [27-29] with

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nanomolar affinity (1(S): Ki = 4.5 nM, 1(R): Ki = 7.1 nM, 2: Ki = 5.3 nM), low affinity for σ2 receptor ligands (1(S): Ki = 496 nM, 1(R): Ki = 1000 nM, 2: Ki = 545 nM), good σ2/σ1 selectivity (1(S): σ2/σ1 = 110, 1(R): σ2/σ1 = 141, 2: σ2/σ1 = 103) and very low cytotoxicity (CC50 > 100 µM), providing a high selectivity index (CC50/IC50 > 14,000).

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Figure 1

These σ1 ligands were evaluated in different pharmacological models. Compound 1(R) was identified as a potent anti-cocaine agent which is able to increase cocaine-induced locomotor stimulation and sensitisation [31]. The S-enantiomer of compound 1 brought about a 57%

neuroprotective

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decrease of infarct volume in an ischaemia model [32]. Finally, when evaluated as agents,

these

compounds

showed

strong

anti-inflammatory

and

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neuroprotective effects in an experimental autoimmune encephalomyelitis model [submitted for publication]. Although high potency and efficacy in in vivo experiments is a prerequisite for a candidate drug, the ADME profile is also essential in the perspective of drug development. Tic-hydantoin derivative 1 showed all properties compatible with development except metabolic stability. Indeed, despite chemical stability in neutral and acidic media, they demonstrated a low metabolic stability [31]. The major part of metabolites resulted from tichydantoin instability. However, among all the metabolites of compound 1, demethylated and debenzylated compounds have been identified (respectively 5% and 9% of all metabolites, unpublished data). An isoindoline moiety was thus introduced to avoid this metabolic instability. 4

ACCEPTED MANUSCRIPT In this paper, we report our efforts to replace the tic-hydantoin core with different heterocycles. Many benzannulated moieties have already proved their interest in the design of novel σ1 ligands. Based on the results obtained with benzoxazolinone derivatives [26], we decided to focus our work on the evaluation of related heterocycles. In the light of previous results with tic-hydantoin series, both side chains of lead compounds 1 and 2 (i.e.

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propylbenzylmethylamine and propylisoindoline) were selected for the study. Moreover, the presence of chlorine in many known σ1 ligands, such as haloperidol, guided our work on the introduction of halogens on the heterocyclic moieties.

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2. Chemistry

A series of benzannulated derivatives were synthesised and investigated for their affinity

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towards σ1 receptors. The heterocycles used were either commercially available, such as 2methyl-1H-benzimidazole and 1-methyl-1,3-dihydro-2H-benzimidazol-2-one, or synthesised. 2-benzoxazolinone and 5-bromo-2-benzoxazolinone were synthesised from the corresponding 2-aminophenol derivatives according to the method previously reported by Nachman et al. [33]. 2-Benzothiazolinone and 5-bromo-2-benzothiazolinone were prepared from the corresponding 2-nitrobenzenethiol derivatives and triphosgene [34]. An aromatic bromination

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reaction of 2-benzoxazolinone or 2-benzothiazolinone was performed in chloroform with bromine to obtain 6-bromo derivatives [35,36]. Ring closure of 4-bromo-2-aminophenol with ethyl bromoacetate in the presence of potassium ethoxide gave rise to 6-bromo-1,4benzoxazinone [37]. Finally, 6-bromo-1,4-benzothiazinone was synthesised from 1,4-

al. [38].

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dibromo-2-nitrobenzene according to a multi-step reaction previously described by Badger et

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According to the general procedure shown in scheme 1, a library of 18 novel benzannulated derivatives was synthesised. Synthesis started from 3-bromo-1-chloropropane, and reaction with the appropriate amines yielded amino side chains 4-5. The preparation of compounds 6-7 involved direct nucleophilic substitution using various heterocycles and potassium carbonate in DMF to obtain our desired derivatives. Heterocycles a-l were all introduced on N-benzyl-3-chloro-N-methylpropan-1-amine 4. For derivatives 7, only heterocycles a, c, e, g, i and k were used. The structures of all final compounds were determined by 1H NMR,

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C NMR and LC-MS analyses. Purity was evaluated under HPLC

conditions and exceeded 96%. Results are presented in the experimental section. For the

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ACCEPTED MANUSCRIPT pharmacological evaluation, all final products were converted into their water soluble hydrochloride salts.

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Schema 1

3. Results and Discussion 3.1 Biological evaluations and SAR

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Receptor affinities were investigated in competition experiments with radioligands according to the methods of Ganapathy et al. [39]. In the σ1 assay, the selective ligand [3H]

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(+)-pentazocine was employed as the radioligand. Since a σ2 selective radioligand was not commercially available, the non selective ligand [3H]-DTG was employed for σ2 assay, in the presence of an excess of non-tritiated (+)-pentazocine, which selectively occupies σ1 receptors. In both assays, Jurkat cell membranes were used as a source of receptors. The Ki values for σ1 receptors were determined from the corresponding IC50 values for each compound 6-7. For compounds showing high σ1 affinity, the Ki values for σ2 receptors

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and the σ2/σ1 selectivity ratios were also calculated (Table 1).

The tic-hydantoin moiety was replaced with various benzannulated heterocycles such as benzoxazolinone, benzothiazolinone, benzoxazinone, benzothiazinone, [40] 2-methyl-1Hbenzimidazole or 1-methyl-1,3-dihydro-2H-benzimidazol-2-one, substituted with halogen

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atoms (Cl, Br) or unsubstituted. From the results obtained, it appears that the replacement of the tic-hydantoin core with different heterocycles can strongly affect the σ1 affinity. Indeed,

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although a variety of modulations leads to a dramatic loss of affinity compared to the lead compounds 1 and 2, high affinity for σ1 receptors is maintained, or significantly improved for some derivatives. The whole structure-activity relationship was described and analysed afterwards.

With respect to the nature of the heterocycle, the presence of oxygen, nitrogen or sulphur atoms appeared to influence σ1 affinity, except for the halogenated derivatives. The benzothiazolinone derivative 6e (Ki = 10.3 nM) displayed an affinity 10-fold higher than the benzoxazolinone derivative 6a (Ki > 100 nM), the 1-methyl-1,3-dihydro-2H-benzimidazol-2one derivative 6k (Ki > 100 nM) or even the 2-methyl-1H-benzimidazole derivative 6l (Ki > 100 nM). Moreover, if the size of the heterocycle was increased from 5 to 6 atoms, the 6

ACCEPTED MANUSCRIPT presence of an oxygen atom conferred a complete loss of affinity (6i, Ki > 100 nM), whereas the sulphur analogue 6j maintained good affinity (Ki = 2.2 nM). Since many known σ1 ligands contain one or two halogens, the introduction of this kind of atom on our structures should be of interest. For this study, bromine and chlorine were inserted on the heterocyclic moiety. For the majority of halogenated compounds, a significant

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increase in affinity was noticed compared to the non-halogenated analogues. In the case of halogenated benzothiazolinonic compounds 6e and 6g, the affinity towards σ1 receptors was increased from 2 to 10-fold. For benzoxazolinone derivatives 6a and 6c, the improvement was even greater. For these two families of derivatives, whatever the nature of the heterocycle, the

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halogenated compounds (6b-d and 6f-g) showed useful σ1 affinity, with a Ki value less than 7 nM. The nature of the halogen appeared to modulate the affinity for σ1 receptors. The brominated compounds 6b and 6f showed σ1 affinity at least 2-fold better than their

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chlorinated analogues 6d and 6h. Conversely, the position of the halogen atom seemed to have no influence on affinity. Indeed, the 5-bromo derivatives 6b, 6f and the 6-bromo derivatives 6c, 6g all showed a Ki value around 1-3 nM.

Modification of the amino side chain on the tic-hydantoin family had a beneficial effect on metabolic stability without any significant decrease in the affinity of the ligands. However, for

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the new benzannulated derivatives, replacement of the methylbenzylamine moiety with the isoindoline moiety caused a dramatic loss of affinity. Indeed, compared to the methylbenzylamine derivative 6c, which showed a Ki value of 0.9 nM, the isoindoline analogue 7c displayed a poor affinity for σ1 receptors (Ki > 100 nM). Except for compounds

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7e and 7g, which showed good affinity (Ki = 30.7 nM and 5.7 nM, respectively), all derivatives 7 showed a Ki value above 100 nM. For these two compounds, the presence of the bromo substituent should be noted.

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In addition to the σ1 affinity, the selectivity of this new class of potent σ1 ligands for σ2 receptors was also investigated. The results clearly demonstrate a lower σ2 affinity of the benzannulated derivatives 6-7, and thus indicate some selectivity for the σ1 over the σ2 subtype. From the results obtained, it seems that the benzothiazoline derivatives 6f, 6g and 6h show better selectivity, with σ2/σ1 ratios between 35 and 48, than the oxygenated analogues 6b, 6c and 6d. In the case of sulphur heterocycles, the modification of the ring size seems to have no influence on σ2/σ1 selectivity (compare 6b and 6c with 6j).

Table 1

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ACCEPTED MANUSCRIPT 3.2 Evaluation of cytotoxic effects To determine the potential cytotoxic effects of our synthetic derivatives, the human neuroblastoma cell line SY5Y was treated with the whole compounds at different concentrations up to 100 µM. The cell viability was calculated using MTT colorimetry. As shown in Table 1, all benzannulated derivatives exhibited moderate to low cytotoxicity. In

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general, benzothiazole derivatives showed higher cytotoxicity than benzoxazole and 2methyl-1H-benzimidazole derivatives. Moreover, the isoindoline side chain conferred lower cytotoxicity than the methylbenzylamine moiety. Seven compounds (6a, 6d, 7a, 7c, 7e, 7i and 7k) resulted in an IC50 value above 100 µM. The other compounds displayed more moderate,

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though reasonable, cytotoxicity, with IC50 values between 10 µM and 50 µM. Among these 18 novel benzannulated σ1 ligands, compounds 6b, 6c, 6d and 6g (IC50 = 14.2 µM, 58.8 µM, 100 µM and 13.2 µM, respectively) showed interesting results, with selectivity indices (σ1)

ratio) above 10,000. The best compounds, 6c and 6g, respectively showed

selectivity indices of 63,291 and 23,250.

4. Conclusion

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(IC50(SY5Y)/Ki

Sigma-1 receptors are associated with various neurological and psychiatric disorders, and

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the identification of potent ligands for them is of great interest. We synthesised a series of halogenated and non-halogenated benzannulated derivatives, and evaluated their binding affinity for σ receptors and their cytotoxic effect on neuronal cells. Several high affinity σ1 ligands with significant selectivity for σ1 versus σ2, and with low toxicity were identified. The

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presence of bromine or chlorine in the benzannulated part of our derivatives significantly improved the affinity for σ1 receptors in most cases. Taking into account that the presence of an halogen is high importance in this structure, the best result was obtained with 3-[3-(N-

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benzyl-N-methylamino)propyl]-6-bromobenzothiazolin-2-one (6g), which displayed a high affinity for σ1 receptor (Ki = 0.6 nM), a σ2/σ1 ratio of 48, and an SY5Y/σ1 ratio of 23,250. This work underlines the importance of halogen atoms on the heterocyclic part of the ligands, which is in agreement with the literature. Further work is in progress to characterise this compound in several in vivo models of pathologies related to σ1 receptors.

5. Experimental section 5.1 Chemistry 5.1.1 General 8

ACCEPTED MANUSCRIPT Chemicals and solvents were obtained from commercial sources, and used without further purification unless otherwise noted. Reactions were monitored by TLC performed on Macherey-Nagel Alugram® Sil 60/UV254 sheets (thickness 0.2mm). Purification of products was carried out by either column chromatography or thick layer chromatography. Column chromatography was carried out on using Macherey-Nagel silica gel (230-400 mesh). Thick

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layer chromatography was performed on glass plates coated with Macherey-Nagel Sil/UV254 (thickness 2 mm), from which the pure compounds were extracted with the following solvent system: DCM/MeOH(NH3), 90:10. NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at 300 MHz for 1Hand 75 MHz for

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C). Chemical shifts are

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expressed in ppm relative to either tetramethylsilane (TMS) or to residual proton signal in deuterated solvents. Chemical shifts are reported as position (δ in ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd = double doublet, br = broad and m

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= multiplet), coupling constant (J in Hz), relative integral and assignment. The attributions of protons and carbons were achieved by analysis of 2D experiments (COSY, HSQC and HMBC). Mass spectra were recorded on a Varian triple quadrupole 1200W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using electrospray ionisation and a UV detector (diode array). The purity of final compounds was

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verified by two types of high pressure liquid chromatography (HPLC) columns: C18 Interchrom UPTISPHERE and C4 Interchrom UPTISPHERE. Analytical HPLC was performed on a Shimadzu LC-2010AHT system equipped with a UV detector set at 254 nm and 215 nm. Compounds were dissolved in 50 µL methanol and 950 µL buffer A, and

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injected into the system. The following eluent systems were used: buffer A (H2O/TFA, 100:0.1) and buffer B (CH3CN/H2O/TFA, 80:20:0.1). HPLC retention times (HPLC tR) were

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obtained at a flow rate of 0.2 mL/min for 35 min using the following conditions: a gradient run from 100% of buffer A over1 min, then to 100% of buffer B over the next 30 min.

5.1.5 N-Benzyl-3-chloro-N-methylpropan-1-amine 4 [41] A 2.0 g (16.9 mmol) amount of N-methylbenzylamine was dissolved in 50 mL DMF. After addition of potassium carbonate (4.6 g, 33.0 mmol), the resulting mixture was heated at 70°C. After 30 minutes, the solution was allowed to cool to room temperature. A 4.9 mL (49.5 mmol) amount of 1-bromo-3-chloropropane was added, and the reaction mixture stirred at room temperature for 24 hours. Then the excess potassium carbonate was filtered off and the solvent removed under reduced pressure. Purification by column chromatography (DCM) was performed and enabled collection of the product as a colourless oil (3.2 g, 95%). TLC: Rf 0.4 9

ACCEPTED MANUSCRIPT (DCM:MeOH(NH3), 99:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.40-7.22 (m, 5H, Haro); 3.65 (t, 3J = 7Hz, 2H, CH2); 3.52 (s, 2H, CH2); 2.55 (t, 3J = 7Hz, 2H, CH2); 2.21 (s, 3H, CH3); 1.98 (p, 3J = 7 Hz, 2H, CH2). LCMS (ESI+): Calc. for [M+H]: 198.09; 200.09. Found: 198.01; 199.96. HPLC (C4, 35 min): tR 8.01 min, PHPLC 96%; HPLC (C18, 35 min): tR 14.69 min,

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PHPLC 96%. 5.1.6 N-(3-Chloropropyl)isoindoline 5

A 1.0 g (8.4 mmol) amount of isoindoline was dissolved in 25 mL of acetonitrile. After addition of potassium carbonate (2.3 g, 16.8 mmol), the resulting mixture was heated at 70°C.

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After 30 minutes, the solution was allowed to cool to room temperature. A 4.2 mL (47.2 mmol) amount of 1-bromo-3-chloropropane was added and the reaction mixture stirred at room temperature for 24 hours. The solvent was removed under reduced pressure and 60 mL

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water added to the residue. The product was extracted with 3 x 30 mL dichloromethane. The combined organic fractions were washed with water and dried over sodium sulphate. The solvent was evaporated under reduced pressure. Purification by column chromatography (DCM:MeOH(NH3), 99:1 (v/v)) was performed and enabled collection of the product as a brown liquid (1.27 g, 77%). TLC: Rf 0.7 (DCM:MeOH(NH3), 9:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.21 (s, 4H, Haro); 3.95 (s, 4H, 2 CH2); 3.69 (t, 3J = 7Hz, 2H, CH2); 2.90 (t, 3J =

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7Hz, 2H, CH2); 2.07 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 140.0 (2 Caro); 126.7 (2 Caro); 122.3 (2 Caro); 59.1 (2 CH2); 53.0 (CH2); 43.1 (CH2); 31.9 (CH2). LCMS

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(ESI+): Calc. for [M+H]: 196.05; 198.07. Found: 195.95; 197.91.

5.1.7 General procedure for final compounds

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One equivalent of appropriate heterocyclic derivative was dissolved in DMF. Three equivalents of potassium carbonate and 1.2 equivalent of the appropriate 3-chloropropan-1amine derivative were added. The resulting mixture was heated at 70°C until disappearance of the starting material. The reaction was monitored by TLC. After 24 to 96 hours, the solvent was removed under reduced pressure, and water added to the residue. The crude product was extracted with dichloromethane. The combined organic fractions were washed with water and dried over magnesium sulphate. Purification by thick layer chromatography or column chromatography was performed.

5.1.7.1 3-[3-(N-Benzyl-N-methylamino)propyl]benzoxazolin-2-one 6a 10

ACCEPTED MANUSCRIPT The compound was purified by column chromatography (CycloHex:EtOAc, 3:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a yellow oil (346 mg, 79%). TLC: Rf 0.4 (CycloHex:EtOAc, 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.40-7.25 (m, 5H, Haro); 7.23-6.98 (m, 4H, Haro); 3.93 (t, 3J = 7 Hz, 2H, CH2); 3.51 (s, 2H, CH2); 2.50 (t, 3J = 7 Hz, 2H, CH2); 2.22 (s, 3H, CH3); 2.00 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 154.6 (CO); 142.7 (Caro);

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138.9 (Caro); 131.4 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 123.8 (Caro); 122.2 (Caro); 110.0 (Caro); 108.4 (Caro); 62.3 (CH2); 54.2 (CH2); 42.1 (CH3); 40.3 (CH2); 25.6 (CH2). LCMS (ESI+): Calc. for [M+H]: 297.15; Found: 297.00. HPLC (C4, 35 min): tR 9.57 min, PHPLC 99%;

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HPLC (C18, 35 min): tR 17.06 min, PHPLC 99%.

5.1.7.2 3-[3-(N-Benzyl-N-methylamino)propyl]-5-bromobenzoxazolin-2-one 6b The compound was purified by thick layer chromatography (CycloHex:EtOAc, 1:1 (v/v) +

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0.1% MeOH(NH3)), and obtained as a brown oil (150 mg, 57%). TLC: Rf 0.4 (CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.35-7.19 (m, 6H, H4, Haro); 7.16 (dd, 3J6-7= 9 Hz, 4J6-4 = 2 Hz, 1H, H6); 6.76 (d, 3J7-6 = 9 Hz, 1H, H7); 4.08 (t, 3J = 7 Hz, 2H, CH2); 3.55 (s, 2H, CH2); 2.57 (t, 3J = 7 Hz, 2H, CH2); 2.29 (s, 3H, CH3); 1.96 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 153.9 (CO); 146.3 (Caro); 138.9 (Caro); 131.7 (Caro);

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129.2 (2 Caro); 128.2 (2 Caro); 127.4 (Caro); 126.7 (Caro); 114.2 (Caro); 113.6 (Caro); 112.2 (Caro); 62.4 (CH2); 53.6 (CH2); 42.4 (CH3); 42.0 (CH2); 26.8 (CH2). LCMS (ESI+): Calc. for [M+H]: 375.04; 377.06. Found: 375.20; 377.20. HPLC (C4, 35 min): tR 13.93 min, PHPLC 96%; HPLC

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(C18, 35 min): tR 17.48 min, PHPLC 98%.

5.1.7.3 3-[3-(N-Benzyl-N-methylamino)propyl]-6-bromobenzoxazolin-2-one 6c

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The compound was purified by column chromatography (CycloHex:EtOAc, 1:1 (v/v) + 0.1% MeOH(NH3)), and obtained as a yellow oil (500 mg, 95%). TLC: Rf 0.5 (CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.37-7.17 (m, 7H, H5, H7, Haro); 6.88 (d, 3J = 8 Hz, 1H, H4); 3.89 (t, 3J = 7 Hz, 2H, CH2); 3.48 (s, 2H, CH2); 2.45 (t, 3J = 7 Hz, 2H, CH2); 2.19 (s, 3H, CH3); 1.96 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 154.0 (CO); 143.1 (Caro); 138.9 (Caro); 130.6 (Caro); 128.9 (Caro); 128.4 (2 Caro); 127.1 (2 Caro); 126.7 (Caro); 114.4 (Caro); 113.5 (Caro); 109.4 (Caro); 62.4 (CH2); 54.1 (CH2); 42.1 (CH3); 40.5 (CH2); 25.6 (CH2). LCMS (ESI+): Calc. for [M+H]: 375.06; 377.06. Found: 375.10; 377.00. HPLC (C4, 35 min): tR 20.55 min, PHPLC 96%; HPLC (C18, 35 min): tR 20.21 min, PHPLC 96%.

11

ACCEPTED MANUSCRIPT 5.1.7.4 3-[3-(N-Benzyl-N-methylamino)propyl]-5-chlorobenzoxazolin-2-one 6d The compound was purified by column chromatography (PE:EtOAc, 7:3 (v/v) + 0.1% MeOH(NH3)), and obtained as a colourless oil (130 mg, 23%). TLC: Rf 0.3 (PE:EtOAc 7:3, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.37-7.20 (m, 5H, Haro); 7.17-6.98 (m, 3H, Haro); 3.88 (t, 3

J = 7 Hz, 2H, CH2); 3.50 (s, 2H, CH2); 2.47 (t, 3J = 7 Hz, 2H, CH2); 2.20 (s, 3H, CH3); 1.97

RI PT

(p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 154.4 (CO); 141.1 (Caro); 138.7 (Caro); 132.4 (Caro); 129.3 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 122.1 (Caro); 110.8 (Caro); 109.0 (Caro); 62.4 (CH2); 54.0 (CH2); 42.0 (CH3); 40.5 (CH2); 25.5 (CH2).LCMS (ESI+): Calc. for [M+H]: 331.11; 333.11. Found: 330.94; 331.97. HPLC (C4, 35 min): tR 20.97 min, PHPLC

SC

99%; HPLC (C18, 35 min): tR 19.50 min, PHPLC 99%.

5.1.7.5 3-[3-(N-Benzyl-N-methylamino)propyl]benzothiazolin-2-one 6e

M AN U

The compound was purified by column chromatography (CycloHex:EtOAc, 3:2, (v/v) + 0.1% MeOH(NH3)), and obtained as a pale yellow oil (223 mg, 54%). TLC: Rf 0.7 (CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.46-7.10 (m, 9H, H4, H5, H6, H7, Haro); 4.03 (t, 3J = 7 Hz, 2H, CH2); 3.51 (s, 2H, CH2); 2.49 (t, 3J = 7 Hz, 2H, CH2); 2.32 (s, 3H, CH3); 1.95 (p, 3J = 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3) δ: 169.9 (CO); 139.0

TE D

(Caro); 137.3 (Caro); 128.9 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 126.3 (Caro); 123.0 (Caro); 122.8 (Caro); 122.6 (Caro); 110.7 (Caro); 62.4 (CH2); 54.4 (CH2); 42.1 (CH3); 41.0 (CH2); 25.6 (CH2). LCMS (ESI+): Calc. for [M+H]: 313.13. Found: 313.03. HPLC (C4, 35 min): tR 10.27 min,

EP

PHPLC 98%; HPLC (C18, 35 min): tR 18.30 min, PHPLC 99%. 5.1.7.6 3-[3-(N-Benzyl-N-methylamino)propyl]-5-bromobenzothiazolin-2-one 6f

AC C

The pure compound was purified by column chromatography (PE:EtOAc, 8:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a yellowish oil (450 mg, 88%). TLC: Rf 0.6 (CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.41-7.20 (m, 8H, H4, H6, H7, Haro); 4.00 (t, 3J = 7 Hz, 2H, CH2); 3.53 (s, 2H, CH2); 2.48 (t, 3J = 7 Hz, 2H, CH2); 2.22 (s, 3H, CH3); 1.94 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 169.5 (CO); 138.8 (Caro); 138.5 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 125.9 (Caro); 123.7 (Caro); 121.6 (Caro); 119.8 (Caro); 113.9 (Caro); 62.3 (CH2); 54.1 (CH2); 42.1 (CH3); 41.2 (CH2); 25.5 (CH2). LCMS (ESI+): Calc. for [M+H]: 391.04; 393.04. Found: 391.10; 393.10. HPLC (C4, 35 min): tR 17.59 min, PHPLC 98%; HPLC (C18, 35 min): tR 20.67 min, PHPLC 98%.

12

ACCEPTED MANUSCRIPT 5.1.7.7 3-[3-(N-Benzyl-N-methylamino)propyl]-6-bromobenzothiazolin-2-one 6g The compound was purified by column chromatography (PE:EtOAc, 8:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a yellow oil (440 mg, 86%). TLC: Rf 0.7 (CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.54 (d, 4J = 2Hz, 1H, H7); 7.39 (dd, 3J = 8 Hz, 4J = 2Hz, 1H, H5); 7.37-7.24 (m, 5H, Haro); 7.00 (d, 3J = 8 Hz, 1H, H4); 4.00 (t, 3J = 7 Hz, 2H,

RI PT

CH2); 3.50 (s, 2H, CH2); 2.47 (t, 3J = 7 Hz, 2H, CH2); 2.21 (s, 3H, CH3); 1.94 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 169.1 (CO); 139.0 (Caro); 136.3 (Caro); 129.3 (Caro); 128.9 (2 Caro); 128.3 (2 Caro); 127.0 (Caro); 125.1 (C7); 124.6 (Caro); 115.3 (Caro); 111.9 (C4); 62.4 (CH2); 54.3 (CH2); 42.1 (CH3); 41.1 (CH2); 25.5 (CH2). LCMS (ESI+): Calc. for [M+H]:

SC

391.04; 393.04. Found: 391.20; 393.20. HPLC (C4, 35 min): tR 21.56 min, PHPLC 97%; HPLC (C18, 35 min): tR 20.99 min, PHPLC 97%.

M AN U

5.1.7.8 3-[3-(N-Benzyl-N-methylamino)propyl]-5-chlorobenzothiazolin-2-one 6h The compound was purified by thick layer chromatography (CycloHex:EtOAc, 1:1 (v/v) + 0.1% MeOH(NH3)), and obtained as a yellow oil (510 mg, 91%). TLC: Rf 0.6 (CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.38-7.22 (m, 6H, H7 and Haro); 7.19 (d, 4J = 2 Hz, 1H, H4); 7.12 (dd, 3J = 8 Hz, 4J = 2Hz, 1H, H5); 3.99 (t, 3J = 7 Hz, 2H,

TE D

CH2); 3.52 (s, 2H, CH2); 2.48 (t, 3J = 7 Hz, 2H, CH2); 2.22 (s, 3H, CH3); 1.93 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 169.8 (CO); 138.7 (Caro); 138.3 (Caro); 132.4 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 123.4 (Caro); 123.1 (Caro); 121.0 (Caro); 111.2 (Caro); 62.2 (CH2); 54.1 (CH2); 42.1 (CH3); 41.2 (CH2); 25.5 (CH2). LCMS (ESI+): Calc. for [M+H]:

EP

347.09; 349.09. Found: 347.20; 349.20. HPLC (C4, 35 min): tR 20.99 min, PHPLC 96%; HPLC

5.1.7.9

AC C

(C18, 35 min): tR 20.71 min, PHPLC 97%. 4-[3-(N-Benzyl-N-methylamino)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-

benzoxazine 6i

The compound was purified by thick layer chromatography (CycloHex:EtOAc, 1:1 (v/v) + 0.1% MeOH(NH3)), and obtained as a colourless oil (65 mg, 62%). TLC: Rf 0.5 (CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.39-7.23 (m, 6H, H5 and Haro); 7.13 (dd, 3J7-8 = 8 Hz, 3J7-5 = 2 Hz, 1H, H7); 6.86 (d, 3J8-7= 8 Hz, 1H, H8); 4.58 (s, 2H, H2); 3.95(t, 3J= 7 Hz, 2H, CH2); 3.59 (s, 2H, CH2); 2.52 (t, 3J= 7 Hz, 2H, CH2); 2.28 (s, 3H, CH3); 1.89 (p, 3J= 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3) δ: 163.9 (CO); 144.4 (Caro); 137.9 (Caro); 130.0 (Caro); 129.2 (2 Caro); 128.4 (2 Caro); 127.3 (Caro); 126.4 (C7); 118.5 (Caro); 118.0 13

ACCEPTED MANUSCRIPT (C8); 115.1 (Caro); 67.5 (CH2); 62.1 (CH2); 54.1 (CH2); 42.0 (CH3); 39.6 (CH2); 24.6 (CH2). LCMS (ESI+): Calc. for [M+H]: 389.08; 391.08. Found: 388.80; 390.80. HPLC (C4, 35 min): tR 11.30 min, PHPLC 98%; HPLC (C18, 35 min): tR 20.16 min, PHPLC 97%. 5.1.7.10

4-[3-(N-Benzyl-N-methylamino)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-

RI PT

benzothiazine 6j The compound was purified by column chromatography (PE:EtOAc, 8:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a yellow oil (220 mg, 47%). TLC: Rf 0.5 (PE:EtOAc 7:3, v/v). 1

H NMR (300 MHz, CDCl3) δ: 7.44 (d, 4J5-7 = 2 Hz, 1H, H5); 7.36-7.10 (m, 7H, H7, H8 and

Haro); 4.03 (t, 3J = 7Hz, 2H, CH2); 3.50 (s, 2H, CH2); 3.36 (s, 2H, H2); 2.44 (t, 3J = 7 Hz, 2H,

SC

CH2); 2.21 (s, 3H, CH2); 1.86 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 164.7 (CO); 140.7 (Caro); 139.0 (Caro); 129.5 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.0 (Caro); 126.2

M AN U

(Caro); 122.9 (Caro); 121.0 (Caro); 120.6 (Caro); 62.3 (CH2); 54.2 (CH2); 43.4 (CH3); 42.2 (CH2); 31.4 (CH2); 25.2 (CH2). LCMS (ESI+): Calc. for [M+H]: 405.05; 407.05. Found: 404.92; 406.92. HPLC (C4, 35 min): tR 21.96 min, PHPLC 98%; HPLC (C18, 35 min): tR 12.75 min, PHPLC 98%.

3-[3-(N-Benzyl-N-methylamino)propyl]-1-methyl-1,3-dihydro-2H-benzimidazol-2-

TE D

5.1.7.11 one 6k

The compound was purified by thick layer chromatography (DCM: MeOH(NH3), 98:2 (v/v)), and obtained as a pale green oil (54 mg, 22%). TLC: Rf 0.2 DCM:MeOH(NH3), 98:2, v/v). 1H

EP

NMR (300 MHz, CDCl3) δ: 7.36-6.95 (m, 9H, H4, H5, H6, H7, Haro); 3.97 (t, 3J= 7 Hz, 2H, CH2); 3.43 (s, 2H, CH2); 3.53 (s, 3H, CH3); 2.51 (t, 3J= 7 Hz, 2H, CH2); 2.22 (s, 3H, CH3);

AC C

1.99 (p, 3J= 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 154.4 (CO); 130.1 (Caro); 129.5 (Caro); 129.1 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 121.2 (Caro); 121.0 (2 Caro); 107.6 (Caro); 107.4 (Caro); 62.2 (CH2); 54.5 (CH2); 42.0 (CH3); 39.2 (CH2); 27.1 (CH3); 26.2 (CH2). LCMS (ESI+): Calc. for [M+H]: 310.19. Found: 310.09. HPLC (C4, 35 min): tR 9.74 min, PHPLC 99%; HPLC (C18, 35 min): tR 16.79 min, PHPLC 99%. 5.1.7.12 1-[3-(N-Benzyl-N-methylamino)propyl]-2-methyl-1H-benzimidazole 6l The compound was purified by column chromatography (DCM:MeOH(NH3), 97:3 (v/v)), and obtained as a colourless oil (283 mg, 64%). TLC: Rf 0.5 (DCM:MeOH(NH3), 9:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.70 (m, 1H, Haro); 7.38-7.18 (m, 8H, Haro); 4.19 (t, 3J = 7 Hz, 14

ACCEPTED MANUSCRIPT 2H, CH2); 3.51 (s, 2H, CH2); 2.62 (s, 3H, CH3); 2.42 (t, 3J = 7 Hz, 2H, CH2); 2.21 (s, 3H, CH3); 1.98 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 151.6 (C2); 142.7 (Caro); 138.8 (Caro); 135.1 (Caro); 128.9 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 121.9 (Caro); 121.7 (Caro); 119.0 (Caro); 109.2 (Caro); 62.3 (CH2); 54.0 (CH2); 42.1 (CH3); 41.5 (CH2); 27.4 (CH2); 13.9 (CH3). LCMS (ESI+): Calc. for [M+H]: 294.19. Found: 294.10. HPLC (C4, 35 min): tR 15.35

RI PT

min, PHPLC 99%; HPLC (C18, 35 min): tR 12.34 min, PHPLC 98%. 5.1.7.13 3-[3-(Isoindolin-2-yl)propyl]benzoxazolin-2-one 7a

The compound was purified by column chromatography (CycloHex:EtOAc, 3:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a beige solid (152 mg, 70%). TLC: Rf 0.5 (CycloHex:EtOAc,

SC

1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.19 (s, 4H, Haro); 7.18-7.08 (m, 4H, Haro); 4.00 (t, 3J = 7 Hz, 2H, CH2); 3.96 (s, 4H, 2CH2); 2.82 (t, 3J = 7 Hz, 2H, CH2); 2.09 (p, 3J = 7 Hz, 2H,

M AN U

CH2).13C NMR (75 MHz, CDCl3) δ: 154.6 (CO); 142.7 (Caro); 139.5 (2 Caro); 131.5 (Caro); 126.9 (2 Caro); 123.8 (Caro); 122.3 (3 Caro); 110.0 (Caro); 108.5 (Caro); 58.9 (2 CH2); 52.5 (CH2); 40.0 (CH2); 27.0 (CH2). LCMS (ESI+): Calc. for [M+H]: 295.14. Found: 295.20. HPLC (C4, 35 min): tR 9.57 min, PHPLC 99%; HPLC (C18, 35 min): tR 16.47 min, PHPLC 99%.

TE D

5.1.7.14 3-[3-(Isoindolin-2-yl)propyl]-6-bromobenzoxazolin-2-one 7c The compound was purified by thick layer chromatography (CycloHex:EtOAc, 3:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a brown oil (340 mg, 65%). TLC: Rf 0.4 (CycloHex:EtOAc, 3:2, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.36-7.26 (m, 2H, H5 and H7);

EP

7.22 (s, 4H, Haro); 7.00 (d, 3J = 9 Hz, 1H, H4); 3.98 (t, 3J = 7 Hz, 2H, CH2); 3.91 (s, 4H, 2CH2); 2.77 (t, 3J = 7 Hz, 2H, CH2); 2.05 (p, 3J = 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3)

AC C

δ: 154.1 (CO); 143.0 (Caro); 139.8 (2 Caro); 130.8 (Caro); 126.9 (2 Caro); 126.7 (Caro); 123.7 (Caro); 122.3 (2 Caro); 114.5 (Caro); 113.5 (Caro); 109.6 (Caro); 58.8 (2 CH2); 52.2 (CH2); 40.1 (CH2); 26.9 (CH2). LCMS (ESI+): Calc. for [M+H]: 373.04; 375.04. Found: 372.90; 374.90. HPLC (C4, 35 min): tR 12.24 min, PHPLC 96%; HPLC (C18, 35 min): tR 19.40 min, PHPLC 96%. 5.1.7.15 3-[3-(Isoindolin-2-yl)propyl]benzothiazolin-2-one 7e The pure compound was purified by thick layer chromatography (CycloHex:EtOAc, 3:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a brown oil (162 mg, 52%). TLC: Rf 0.3 (DCM:MeOH(NH3), 95:5, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.36-7.14 (m, 8H, H4, H5, H6, H7, Haro); 4.13 (t, 3J = 7 Hz, 2H, CH2); 3.97 (s, 4H, 2CH2); 2.82 (t, 3J = 7 Hz, 2H, CH2); 2.05 15

ACCEPTED MANUSCRIPT (p, 3J = 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3) δ: 170.0 (CO); 139.9 (2 Caro); 137.4 (Caro); 126.8 (2 Caro); 126.3 (Caro); 123.0 (Caro); 122.8 (Caro); 122.6 (Caro); 122.3 (2 Caro); 110.8 (Caro); 59.0 (2 CH2); 52.9 (CH2); 40.8 (CH2); 27.1 (CH2). LCMS (ESI+): Calc. for [M+H]: 311.12. Found: 311.10. HPLC (C4, 35 min): tR 10.50 min, PHPLC 97%; HPLC (C18, 35 min): tR

RI PT

17.98 min, PHPLC 97%. 5.1.7.16 3-[3-(Isoindolin-2-yl)propyl]-6-bromobenzothiazolin-2-one 7g

The compound was purified by thick layer chromatography (CycloHex:EtOAc, 3:2 (v/v) + 0.1% MeOH(NH3)), and obtained as a colourless oil (324 mg, 64%). TLC: Rf 0.5

SC

(CycloHex:EtOAc 3:2, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.57 (d, 4J7-5= 2 Hz, 1H, H7); 7.43 (dd, 3J5-4= 8 Hz, 4J5-7 = 2 Hz, 1H, H5); 7.24 (s, 4H, Haro); 7.14 (d, 3J = 8 Hz, 1H, H4); 4.10 (t, 3J = 7 Hz, 2H, CH2); 3.97 (s, 4H, 2CH2); 2.80 (t, 3J = 7 Hz, 2H, CH2); 2.04 (p, 3J = 7

M AN U

Hz, CH2).13C NMR (75 MHz, CDCl3) δ: 169.4 (CO); 139.6 (2 Caro); 136.5 (Caro); 129.3 (C5); 126.9 (2 Caro); 125.1 (C7); 124.5 (Caro); 122.3 (2 Caro); 115.4 (Caro); 112.1 (C4); 58.9 (2 CH2); 52.5 (CH2); 40.9 (CH2); 26.9 (CH2). LCMS (ESI+): Calc. for [M+H]: 389.03; 391.03. Found: 389.00; 391.02. HPLC (C4, 35 min): tR 13.36 min, PHPLC 99%; HPLC (C18, 35 min): tR 20.49

TE D

min, PHPLC 99%.

5.1.7.17 3-[3-(Isoindolin-2-yl)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-benzoxazine 7i The compound was purified by column chromatography (CycloHex:EtOAc, 9:1 (v/v) + 0.1% MeOH(NH3)), and obtained as a yellowish oil (55 mg, 66%). TLC: Rf 0.5 (CycloHex:EtOAc

EP

3:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.39 (d, 3J 5-7 = 2 Hz, 1H, H5); 7.22 (s, 4H, Haro); 7.10 (dd, 3J7-8 = 8 Hz, 3J7-5 = 2 Hz, 1H, H7); 6.86 (d, 3J8-7= 8 Hz, 1H, H8); 4.60 (s, 2H, H2);

AC C

4.06 (t, 3J= 7 Hz, 2H, CH2); 3.97 (s, 4H, 2CH2); 2.81 (t, 3J= 7 Hz, 2H, CH2); 1.96 (p, 3J= 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 163.9 (CO); 144.3 (Caro); 139.7 (2 Caro); 130.2 (Caro); 126.8 (2 Caro); 126.4 (C7); 122.3 (2 Caro); 118.4 (C8); 118.2 (C5); 115.1 (Caro); 65.5 (CH2); 59.0 (2 CH2); 52.8 (CH2); 39.5 (CH2); 26.3 (CH2). LCMS (ESI+): Calc. for [M+H]: 387.07; 389.07. Found: 386.90; 388.90. HPLC (C4, 35 min): tR 10.99 min, PHPLC 97%; HPLC (C18, 35 min): tR 19.76 min, PHPLC 97%. 5.1.7.18 3-[3-(Isoindolin-2-yl)propyl]-1-methyl-1,3-dihydro-2H-benzimidazol-2-one 7k The compound was purified by column chromatography (DCM:MeOH(NH3), 97:3 (v/v)), and obtained as a brown oil (145 mg, 62%). TLC: Rf 0.4 DCM:MeOH(NH3), 95:5, v/v). 1H NMR 16

ACCEPTED MANUSCRIPT (300 MHz, CDCl3) δ: 7.14-6.96 (m, 8H, H4, H5, H6, H7, Haro); 4.05 (t, 3J= 7 Hz, 2H, CH2); 3.96 (s, 4H, 2CH2); 3.43 (s, 3H, CH3); 2.81 (t, 3J= 7 Hz, 2H, CH2); 2.06 (p, 3J= 7 Hz, 2H, CH2).

13

C NMR (75 MHz, CDCl3) δ: 154.5 (CO); 139.8 (2 Caro); 130.0 (Caro); 129.6 (Caro);

126.8 (2 Caro); 122.3 (2 Caro); 121.2 (Caro); 121.0 (Caro); 107.7 (Caro); 107.4 (Caro); 59.0 (2 CH2); 52.9 (CH2); 39.0 (CH2); 27.7 (CH2); 27.1 (CH3). LCMS (ESI+): Calc. for [M+H]:

RI PT

308.17. Found: 308.20. HPLC (C4, 35 min): tR 10.03 min, PHPLC 97%; HPLC (C18, 35 min): tR 16.47 min, PHPLC 97%.

SC

5.2 In vitro testing 5.2.1 Assay for binding to σ receptors

M AN U

The σ binding assays were performed by CEREP (Poitiers, France), according to Ganapathy et al.[36]. The σ1 binding assay was carried out by incubating Jurkat cell membranes (10-20 mg protein per tube) with [3H](+)-pentazocine (15 nM) and a range of concentrations of test compounds, at 37°C for 2 hours, in 5 mM Tris/HCl buffer (pH = 7.4). The σ2 binding assay was performed by incubating Jurkat cell membranes (10-20 mg protein per tube) with [3H]-DTG (25 nM) in the presence of (+)-pentazocine (1 µM) to saturate σ1

TE D

receptors, and a range of concentrations of test compounds, at room temperature for 1 hour in 5 mM TrisHCl buffer (pH = 7.4). The final assay volume was 0.5 mL. Binding was terminated by rapid filtration through Whatman GF/B filters, which were then washed with 5 x 1 mL ice-cold NaCl solution and allowed to dry before bound radioactivity was measured

EP

using liquid scintillation counting. Nonspecific binding was determined, in both assays, under similar conditions, but in the presence of 10 µM unlabelled haloperidol. Inhibition constants

(1973).

AC C

(Ki) were calculated from the IC50 values according to the method of Cheng and Prusoff

5.2.2 Cell culture and cytotoxicity assay The human neuroblastoma cell line (SY5Y) was cultured in DMEM (Dulbecco’s Modified Eagle Medium) (Gibco) supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 IU/mL penicillin, 1 mM non-essential amino acids and 10% (v/v) heat-inactivated foetal bovine serum (Sigma Aldrich), and grown at 37°C in a humidified incubator with 5% CO2. Cells were seeded at 2,000 cells per well onto 96-well plates in DMEM medium. Cells were starved for 24 hours to obtain synchronous cultures, and were then incubated in culture 17

ACCEPTED MANUSCRIPT medium that contained various concentrations of test compounds, each dissolved in less than 0.1% DMSO. After 72 hours of incubation, cell growth was estimated by the colorimetric MTT (thiazolyl blue tetrazolium bromide) assay.

RI PT

Acknowledgements This work was supported by Lille 2 University, FRI “J’innove” PRES Univ Lille Nord de France Grants. MDM is the recipient of a fellowship from Lille 2 University. The 300 MHz NMR facilities were funded by the Région Nord-Pas de Calais (France), the Ministère de la

SC

Jeunesse, de l’Education Nationale et de la Recherche (MJENR) and the Fonds Européens de Développement Régional (FEDER). We express our thanks to Jiaqui He for her contribution

M AN U

in organic synthesis.

References

[4] [5]

[6] [7]

[8] [9] [10] [11]

[12]

TE D

[3]

EP

[2]

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ACCEPTED MANUSCRIPT Table 1: σ1 and σ2 affinity and cytotoxicity evaluation of target compounds 6 and 7 Compound

Ki (nM)

Ki (σ2)/

IC50 (µM)

IC50/

σ2

Ki (σ1)

SY5Y

Ki (σ1)

Haloperidol

11.9

175

15

-

-

1(S)

4.5

496*

110

> 100

> 20,000

1(R)

7.1

764*

108

19.5

2

5.3

416*

78

-

6a

> 100

nd

-

> 100

-

6b

1.2

15.9

13

14.2

11,463

6c

0.9

34.9

37

6d

6.4

100.5

16

6e

10.3

6f

3.1

110.5

6g

0.6

27.3

6h

4.7

165.4

6i

> 100

nd

6j

2.2

47.7

6k

> 100

6l

> 100

7a

>100

7c

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σ1

2,746

63,291

100.0

15,500

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58.8

973

35

26.5

8,417

48

13.2

23,250

35

9.9

2,124

-

13.4

-

22

18.7

8,631

nd

-

73.8

-

nd

-

50.2

-

nd

-

> 100

-

>100

nd

-

> 100

-

7e

30.3

nd

-

> 100

> 3,000

7g

5.7

nd

31.4

5,471

7i

> 100

nd

-

> 100

-

7k

> 100

nd

-

> 100

-

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10.0

Mean IC50 values for 2-3 independent experiments are shown with less than 10% deviation. * Rat cerebral cortex membranes were used as a source of σ2 receptors. nd: Not determined

ACCEPTED MANUSCRIPT Cl

iii

N

N

N

i 4 Cl

X

Br

6

X = H, Br, Cl

3

iii X

5

Heterocycles: 6-7

a b c d

X=H X = 5-Br X = 6-Br X = 5-Cl

S

N

e f g h

O N

Y

i Y=O j Y=S Br

X

X

X=H X = 5-Br X = 6-Br X = 5-Cl

7

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N

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O

O

O

O

N

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Cl

ii

N

N

k

N

N l

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Scheme 1: Reagents and conditions: i: N-methylbenzylamine (0.34 eq), K2CO3 (0.66 eq), DMF, 70°C to rt, 24h (95%); ii: isoindoline (0.18 eq), K2CO3 (0.36 eq), CH3CN, 70°C to rt, 24 h (77%); iii: 3chloropropan-1-amine derivative 4 or 5 (1.2 eq), various heterocycles (1 eq), K2CO3 (3 eq), DMF, 70°C (23-95%).

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O

N

N

N

N

O

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N

O 2

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> A series of sigma-1 ligands was synthesised. > The affinity for sigma-1 and sigma-2 receptors was determined. > Most ligands showed nanomolar affinity for sigma-1 receptor. > A good selectivity towards sigma-2 was obtained.

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> A very low cytotoxicity was measured on SY5Y cells.

ACCEPTED MANUSCRIPT SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF BENZANNULATED DERIVATIVES AS POTENT AND SELECTIVE SIGMA-1 PROTEIN LIGANDS

a

Univ Lille Nord de France, F-59000 Lille, France UDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France c UDSL, EA 4483, UFR Pharmacie, F-59000 Lille, France b

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Mail adress : UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille

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Marion Donnier-Maréchala,b, Pascal Caratoa,b,*, Delphine Le Broca,c, Christophe Furmana,c and Patricia Melnyka,b

List of e-mail adresses :

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[email protected], [email protected], [email protected], [email protected], [email protected]

Corresponding author :

Pascal Carato, EA4481, UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille

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tel : 33 (0)3 20 96 49 66 – fax : 33 (0)3 20 96 49 13 Mail : [email protected]

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N-Benzyl-3-chloro-N-methylpropan-1-amine 4

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N-(3-Chloropropyl)isoindoline 5

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3-[3-(N-Benzyl-N-methylamino)propyl]-5-bromobenzoxazolin-2-one 6b

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3-[3-(N-Benzyl-N-methylamino)propyl]-6-bromobenzoxazolin-2-one 6c

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3-[3-(N-Benzyl-N-methylamino)propyl]-5-chlorobenzoxazolin-2-one 6d

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3-[3-(N-Benzyl-N-methylamino)propyl]benzothiazolin-2-one 6e

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3-[3-(N-Benzyl-N-methylamino)propyl]-5-bromobenzothiazolin-2-one 6f

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3-[3-(N-Benzyl-N-methylamino)propyl]-6-bromobenzothiazolin-2-one 6g

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4-[3-(N-Benzyl-N-methylamino)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-benzoxazine 6i

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4-[3-(N-Benzyl-N-methylamino)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-benzothiazine 6j

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3-[3-(N-Benzyl-N-methylamino)propyl]-1-methyl-1,3-dihydro-2H-benzimidazol-2-one 6k

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1-[3-(N-Benzyl-N-methylamino)propyl]-2-methyl-1H-benzimidazole 6l

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3-[3-(Isoindolin-2-yl)propyl]benzoxazolin-2-one 7a

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3-[3-(Isoindolin-2-yl)propyl]benzothiazolin-2-one 7e

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3-[3-(Isoindolin-2-yl)propyl]-1-methyl-1,3-dihydro-2H-benzimidazol-2-one 7k