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

Accepted Manuscript

Synthesis and pharmacological evaluation of benzamide derivatives as potent and

selective sigma-1 protein ligands

Marion Donnier-Marechal, Pascal Carato, Paul-Emmanuel Larchanché, Séverine

Ravez, Rajaa Boulahjar, Amélie Barczyk, Bénédicte Oxombre, Patrick Vermersch,

Patricia Melnyk

PII:

S0223-5234(17)30536-6

10.1016/j.ejmech.2017.07.014

EJMECH 9577

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 31 March 2017

Revised Date: 22 May 2017

Accepted Date: 10 July 2017

Please cite this article as: M. Donnier-Marechal, P. Carato, P.-E. Larchanché, Sé. Ravez, R. Boulahjar,

Amé. Barczyk, Béé. Oxombre, P. Vermersch, P. Melnyk, Synthesis and pharmacological evaluation of

benzamide derivatives as potent and selective sigma-1 protein ligands, European Journal of Medicinal

Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.014.

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SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF BENZAMIDE

ACCEPTED MANUSCRIPT

DERIVATIVES AS POTENT AND SELECTIVE SIGMA-1 PROTEIN LIGANDS

Marion Donnier-Marechal^a, Pascal Carato^a,*,#, Paul-Emmanuel Larchanché^a, Séverine Ravez^a, Rajaa

Boulahjar^a, Amélie Barczyk^b, Bénédicte Oxombre^b, Patrick Vermersch^band Patricia Melnyk^a,*

^aUniv. Lille, Inserm, CHU Lille, UMR-S 1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT

Neurosciences et Cancer, F-59000 Lille, France

^bUniv. Lille, Inserm, CHU Lille, U995 - LIRIC - Lille Inflammation Research International Center, F-

59000 Lille, France

Mail adress : Faculté de Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille, France

List of e-mail adresses :

marion.donnier-marechal@univ-lyon1.fr, pascal.carato@univ-poitiers.fr, paul-

emmanuel.larchanche@univ-lille2.fr, amelie.barczyk@univ-lille2.fr, benedicte.vanteghem@univ-

lille2.fr, patrick.vermersch@univ-lille2.fr, patricia.melnyk@univ-lille2.fr

* Corresponding authors

# Present address : Univ Poitiers, CIC INSERM 1402, F-86073 Poitiers, France.

1

Graphical Abstract

ACCEPTED MANUSCRIPT

A series of benzamide-derived compounds was synthesized, evaluated in vitro toward their S1R, S2R

affinities and cytotoxic effects on SY5Y cells and selectivities on a panel of 40 receptors.

2

ABSTRACT

ACCEPTED MANUSCRIPT

A series of novel benzamide-derived compounds was designed, synthesized and pharmacologically

evaluated. Among all 37 synthesized compounds, two series were developed with the modulation of the

nature, the position of atoms or groups on the benzamide scaffold, but also the nature of the amine group

separated from the benzamide with 2, 3 or 4 methylene groups. In vitro competition binding assays

against sigma proteins (sigma-1 S1R and sigma-2 S2R) revealed that most of them conferred S2R/S1R

selectivity toward without cytotoxic effects on SY5Y cells, especially with the first series with

compounds 7a-z. Some selected compounds were also evaluated for their agonist and antagonist activities

on a panel of 40 receptors. Results showed the importance of the nature and the position with halogeno

atom on the benzamide scaffold, the length chain but also the contribution of the hydrophobic part on the

amine group. Among them, compounds 7i, w, y with Cl, CN or NO₂groups at the 4-position of the

benzamide scaffold showed excellent affinity for S1R (Ki = 1.2 to 3.6 nM), selectivity for S2R (Ki up to

1400 nM) and high selectivity index (IC_50(SY5Y)/Ki

ratio from 28 000 to 83 000). Futhermore, these

(S1R)

compounds presented an excellent safety profile over 40 other receptors. These derivatives will be

selected for further biological investigations.

Key words: benzamide, sigma protein, CNS

3

1. Introduction

ACCEPTED MANUSCRIPT

More than 40 years after their discovery and due to their involvement in various CNS pathologies, the

sigma proteins continue to be widely studied [1-3]. These proteins have been first described as opioid

receptors [4] and then classified in the literature as distinct sigma-1 and sigma-2 receptors (S1R and S2R)

differing from their pharmacologically and ligand binding profile, anatomical distribution, function and

molecular size [5-8]. The protein S2R has not been cloned from any species, while the S1R protein was

cloned from various mouse [9], rat [10] and human [11] tissues.

The S1R have been identified as a small-ligand regulated chaperone protein. They are located at the

interface between endoplasmic reticulum (ER) and mitochondria called mitochondria associated

membrane (MAM) [12,13]. They modulate the Ca²⁺homeostasis under conditions of cellular stress

[13,14], but also the K⁺and Cl^-channels [15,16]. Additionally, S1R have been shown to regulate the

cholinergic [17,18], adrenergic [19,20], dopaminergic [21,22], glutamatergic [21] and serotoninergic [23]

neurotransmissions.

S1R are involved in the regulation of numerous neurotransmitter systems, the signal transduction pathway

is not completely understood but S1R pharmacological modulators have been shown to modulate several

CNS diseases such as pain [24,25], depression [26] or schizophrenia [27,28], but also neurodegenerative

disorders like Alzheimer’s and Parkinson’s diseases [29-31]. Thus, the synthesis of such S1R ligands

represents a promising strategy to develop novel treaments for various CNS diseases or even cancers [32-

33]. Many drugs described in the literature show affinity for S1R, and some of them have undergone

clinical trials, but no selective S1R ligands have so far been marketed [34]. A phase 2 study is ongoing to

evaluate Anavex 2-73 – a mixed muscarinic and S1R agonist - in patients with Alzheimer’s disease

[35,36].

Numerous studies described the synthesis, as well as the biological and pharmacological activities of S1R

ligands that exhibit Ki values in the nanomolar range, Among them, previous work of our laboratory

underlines a first series of tetrahydroisoquinoline-hydantoin (tic-hydantoin) compounds (Figure 1) as

efficient S1R ligands (Ki = 4.5 to 7.1 nM), with low affinity for S2R (Ki = 500 to 1000 nM) and therefore

high S2R/S1R selectivity (100 to 140). These S1R ligands were evaluated in different pharmacological

models. One derivative increased cocaine-induced locomotor stimulation and sensitisation in anti-cocaine

evaluation [37], an other one brought about a 57% decrease of infarct volume in an ischaemia model [38].

These compounds were described as neuroprotective agents by their strong anti-inflammatory and

neuroprotective effects in an experimental model of multiple sclerosis [39]. Despite chemical stability of

tic-hydantoin derivatives in neutral and acidic media, they demonstrated a low metabolic stability,

preventing from the perspective of drug development.

4

N

ACCEPTED MANUSCRIPT

N

X

O

Y

O

N

X

X: NH, O

N

Y

N

Y

O

X

N

Y: O, S

R

N

H

X: O, S

Y: H, Cl, Br

R: H, Ph

Tic-hydantoin series

Benzannulated series

Tricyclic heterocycle series

Figure 1: Tic-hydantoin, benzannulated and tricyclic series with propyl-N-methyl-N-benzylamine or isoindoline chains

Continuing our efforts in the synthesis of S1R ligands to solve this problem of metabolic stability, other

works (Figure 1) were dedicated to replacement of the tic-hydantoin moieties with different tricyclic

heterocycles [40] or benzannulated scaffold [41]. Though the S1R affinity was maintained for most of the

compounds (Ki = 0.6 to 30 nM), but the S2R/S1R selectivity remained low (< 30).

Thus we report here our efforts to simplify the structure and replace the different heterocycle moieties by

benzamide scaffold with a length chain of 2, 3 or 4 methylene functions separated from the amine part

with benzylmethylamine to afford compounds 7a-z (Figure 2) [42]. The design of these compounds could

be compared to 4-IBP [43], which was recently cocrystallized with S1R [44]. In a first series, numerous

substitutions on the benzamide scaffold were adopted by varying the nature and the position of halogeno

atoms (Cl, Br) and groups (alkyl, CF₃, OCH₃, NO₂, N(CH₃)₂, CN). Considering the good results with

compounds 7a-z, we developed a second series of derivatives 8a-h where we kept the 4-chlorobenzamide

moieties and modulated the nature of the amine part with N-phenylethyl-N-methylamine, isoindoline,

pyrrolidine, morpholine and methylpiperazine (Figure 2). In this second series, compounds 8a-h were

synthesized with length chain of 2 or 3 methylene groups between the benzamide and the functionalized

amine. In this series, we also evaluated the importance of the hydrophobic part with the phenyl ring and

the hydrophilic part with morpholine and piperazine moieties. In a third series, analogues of the

promising compound 7i were prepared while modifying the linker. On the benzamide scaffold (7i), the

amide function was changed by sulfamide function to give compound 9, the benzamide was reduced in

benzylamine to furnish derivative 10 and the retroamide was also synthesized with compound 13 (Figure

2).

O

N

R2

X

N

H

N

m

R ₃

H

R1

Cl

7a-z

8a-h

9, 10, 13

X: SO₂NH, CH₂NH, NHCO

R₃: alkyl, halogen, CF₃, OCH₃, NO₂, N(CH₃)_2,CN

m= 2,3,4

R ₂

N

R ₁

N

O

N

5

^{Figure 2:}A^StrC^ucC^tuE^reP^oT^{f f}EⁱⁿD^{al c}M^omA^poN^unU^dsS⁷C^a-R^z,I⁸P^a-T^{h, 9, 10, 13}

2. Chemistry

To synthesize final compounds 7a-z, a first attempt was realized in two steps starting from commercially

avalaible 4-chlorophenylacyl chloride (Scheme 1). Reaction with 3-bromopropylamine in

dichloromethane with 4-chlorophenylacyl chloride gave the intermediate 1. Nucleophilic substitution in

DMF with potassium carbonate and isoindoline as reagent, as the second step, did not afford the

corresponding isoindoline compound but derivative 2 obtained by cyclization of bromo compound 1.

O

b

a

Cl

N

Br

N

H

Cl

2

1

Scheme 1: attempt to synthesized final compounds

Reagents and conditions: (a): 3-bromopropylamine, CH₂Cl₂, 12h, 0°C to rt, 65%; (b): isoindoline, potassium carbonate,

DMF, 12h, 70°C, 70%.

In a first time, diamino compounds 5a-f were prepared in two or three steps and final compounds 7a-z

were synthesized (Scheme 2). From the first route, diamines 5a-c were synthesized in three steps. 3-

Bromopropylamine was protected with Boc group in CH₂Cl₂with triethylamine and di-tert-

butylcarbonate to afford derivative 3, which reacted with the corresponding substituted amine (HNR₁R₂)

in CH₃CN or DMF with potassium carbonate to give compounds 4a-c. These derivatives 4a-c were

deprotected in 3M HCl in dry dioxane to give compounds 5a-c. From the second route, the ethyl and

butyl analogues of benzylmethylpropane-1,3-diamine (5a) were synthesized in two steps (Scheme 2).

Starting from bromoalkylphtalimides, substitution reaction in DMF with potassium carbonate and

benzylmethylamine afforded compounds 6a-b, which were deprotected in ethanol with hydrazine to give

intermediates 5d-e. In the series with m= 2 we also synthetize the superior analogue of compound 5d with

derivative 5f which contained methylethylbenzylamine group as NR₁R₂.

R ₂

N

R ₁

R ₂

N

R ₂

N

a

b

c

Cpd

BocHN

Br

H₂N

Br

R ₁

3

4a-c

5a-c

N

5a

5b

O

R ₂

N

m

e

d

R ₂

H₂N

N

Br

R ₁

m

O

5d-f

O

5c

N

6a-c

Cpd

m

m= 2, 4

6a, 5d 6b, 5e

6c, 5f

2

4

2

R ₂

N

R ₁

N

Scheme 2: Preparation of diamine compounds 5a-f

Reagents and conditions: (a): di-tert-butylcarbonate, triethylamine, CH₂Cl₂, 18h, rt, 97%; (b): HNR₁R₂, potassium carbonate,

DMF or CH₃CN, 24h, 70°C, 65-81%; (c): 3M HCl in dry dioxane, 18h, rt, 83-98%; (d): benzylmethylamine, potassium

carbonate, DMF, 16h, 90°C, 52-64%; (e): H₂NNH₂, EtOH 96°, 16h, reflux, 55-73%.

6

These diamine intermediates 5a,d,e afforded final compounds 7a-z (Scheme 3). Compounds 7a-z were

ACCEPTED MANUSCRIPT

synthesized according two routes, starting from commercially substituted benzoyl acids, in

dichloromethane with hydroxybenzotriazole and O-benzotriazole-N,N,N’,N’-tetramethyluronium

hexafluorophosphate at room temperature or with commercially substituted benzoyl chlorides, in

dichloromethane with triethylamine The SAR study of compounds 7a-z showed that the substitution of

compounds 7h-j with a chlorine atom in para position induced good affinities for S1R, high S2R/S1R

selectivities and low cytotoxicities (Table 1). We thus decided to synthesize a second series with 4-

chlorobenzamide compounds 8a-h using the same experimental conditions. We synthesized compounds 9

and 10 respectively as the sulfonyl and reduced analogues of the amide compound 7i (Scheme 3).

Derivative 9 was synthesized starting from the 4-chlorophenyl sulfonyl chloride in dichloromethane with

trimethylamine and the diamine intermediate 5a. Derivative 10 was obtained by a reductive amination of

diamine intermediate 5a in MeOH with 4-chlorobenzaldehyde, followed by the addition of sodium

borohydride. We also decided to synthesize the retroamide analogue 13 of compound 7i. Starting from 4-

chloroaniline, reaction in dichloromethane with 4-chlorobutyryl chloride gave compound 11. This

derivative 11 never afforded derivative 13 in DMF with potassium carbonate or in CH₃CN with

triethylamine and benzylmethylamine, but gave compound 12 after cyclization. The desired derivative 13

was obtained from compound 11 in benzylmethylamine which reacted both as base and solvent.

O

a or b

N

Y

m

H₂N

N

m

R ₃

H

+

m= 2-4

Y = Cl or OH

5a,d,e

7a-x

O

R ₂

R2

a or b

N

Y

H₂N

N

H

+

R1

R ₁

m

m= 2-3

Cl

Y = Cl or OH

5b-c

8a-h

O

S

a

N

H

Cl

+

H₂N

N

Cl

9

5a

O

c

N

H

N

H

+

H₂N

N

10

5a

H

N

H

NH₂

f

d

N

Cl

O

Cl

11

e

13

N

O

Cl

12

Scheme 3: Synthesis of final compounds 7a-x, 8a-h, 9, 10 and 13

Reagents and conditions: (a): triethylamine, CH₂Cl₂, 12h, 0°C to rt, 7-69%; (b): HOBt, HBtu, DIEA, CH₂Cl₂, 12h, rt, 31-

75%; (c): i: MeOH, 18h, rt; ii: NaBH₄, 2h, rt, 35%; (d): 4-chlorobutyryl chloride, CH₂Cl₂, 12h, 0°C to rt, 87%; (e):

7

benzylmethylamine, potassium carbonate, DMF, 12h, 70°C or Et₃N, CH₃CN, 12h, 50°C; 67% (f): benzylmethylamine, 18h, rt,

ACCEPTED MANUSCRIPT

37-80%.

3. Results and Discussion

3.1 Biological evaluations and SAR

S1R and S2R affinities were investigated in competition experiments with radioligands according to the

methods described by Ganapathy [45]. In the S1R assay, the selective ligand [³H] (+)-pentazocine was

employed as the radioligand. Since a selective S2R radioligand was not commercially available, the non-

selective ligand [³H]-DTG was employed in the S2R assay, in the presence of an excess of non-tritiated

(+)-pentazocine, which selectively occupies S1R. In both assays, Jurkat cell membranes were used as a

source of receptors.

The K_ivalues for S1R and S2R were determined from the corresponding IC₅₀values for each compounds

7a-z, 8a-h, 9, 10, and 13. The S2R/S1R selectivity ratios were also calculated (Table 1).

In the first series, as all compounds 7b-x exhibited good affinities for S1R (Ki = 1.1 to 47 nM) except for

derivative 7a (Ki > 100 nM), we could conclude that substitution on the benzamide scaffold was

necessary. The substitution with alkyl group, as propyl, butyl and tert-butyl for compounds 7b-d induced

excellent affinities for S1R with Ki = 1.6 to 7.3 nM and good selectivity toward S2R with a S2R/S1R

ratio of 126 expecially for derivative 7d. The modulation of the tert-butyl group of compound 7d by

trifluoromethyl group with 7e, allowed to keep excellent S1R affinity but led to selectivity decrease with

a S2R/S1R ratio of 70.

The substitution with one halogen atom, such as Cl or Br, gave high affinities for S1R (Ki = 1.7 to 47

nM). On the contrary, the substitution with a fluorine decreased S1R affinity dramatically (compound

7n). In this series, the best affinities were obtained when benzamide moiety was substituted at the 4

position with a chlorine or bromine atom (7h, 7m). Substitution in meta position (for 7g Ki = 10 nM, 7l

Ki = 3.9 nM) or ortho position (for 7f Ki = 47 nM, 7k Ki = 42 nM) induced a decrease in S1R affinity, but

also a meaningful decrease for S2R affinity. Therefore, S2R/S1R selectivities remained modest in the

case of substitutions in meta or ortho position compared to the para-substituted compound 7h with

excellent affinity for S1R (Ki = 3.6 nM) and selectivity S2R/S1R of 236. According to these results, we

decided to introduce two halogens, with two chlorines (compounds 7o-r) or one fluorine and one bromine

(compound 7s). In this set of compounds 7n-s, S1R and S2R affinities, and thus selectivities were in the

same range. For the last compounds described in table 1 (compounds 7t-x), the nature and the position of

the substitutents were modulated with methoxy, dimethylamine, cyano and nitro groups. Substitution with

methoxy group (compounds 7t-u) gave moderate affinities for S1R (Ki = 37-40 nM) with a S2R/S1R

ratio higher than 27. Compounds 7v-z with dimethylamine, cyano and nitro groups allowed good

affinities for S1R (Ki = 1.2-24 nM) and especially derivative 7y with the best S2R/S1R selectivity ratio of

389. Finally, we also noticed the influence of the length chain between the benzamide scaffold and the

8

benzylmethylamine. Indeed the decrease in the chain length (from butyl to ethyl) induced a higher

ACCEPTED MANUSCRIPT

selectivity (compare compounds 7h-j, S2R/S1R = 12 to 236). Moreover, as a comparison, in the case of

cyano substitution, a slight modification in the length’s chain drastically modified the affinities and

selectivity (see compounds 7w and 7x).

Table 1 : Evaluation of S1R, S2R affinities and cytotoxicity of target compounds 7a-z

Ki (nM)^a

IC₅₀

(µM)

IC₅₀/Ki

(σ₁)

Compounds

R₃

m

Ki (σ₂)/

Ki (σ₁)

σ₁

σ₂

SY5Y ^b

> 100

90.2

> 100

60.2

7a

7b

7c

7d

7e

7f

7g

7h

7i

7j

7k

7l

7m

7n

7o

7p

7q

7r

H

4-Pr

4-Bu

4-tBu

4-CF₃

2-Cl

3-Cl

4-Cl

3

2

3

4

3

2

3

2

3

2

3

> 100

3.7

7.3

1.6

1.4

47

>> 400 ^a

160

49

200

98

> 1200

340

850

190

20

> 1200

350

160

470

310

110

59

48

-

43

7

126

70

> 26

34

236

60

12

> 29

90

76

24

31

17

54

2

69

-

> 27 027

12 356

> 62 500

> 71 429

> 2 128

> 10 000

> 27 778

> 31 250

> 58 824

> 2 381

> 25 641

28 667

10

3.6

3.2

1.7

42

3.9

2.1

20

2-Br

3-Br

4-Br

4-F

> 100

56.6

> 5 000

> 10 000

> 15 385

51 455

2,3-Cl

3,4-Cl

3,5-Cl

2-F-4-Br

3-OCH₃

4-OCH₃

3-N(CH₃)₂

4-CN

4-NO₂

10

6.5

1.1

23

2.9

40

60.0

2 609

7s

7t

200

> 100

100.0

> 100

> 34 483

> 2 500

2 702

> 52 632

> 83 334

> 4 167

> 27 778

> 23 256

> 1200

321

> 30

> 27

159

142

> 50

389

84

7u

7v

7w

7x

7y

7z

37

1.9

1.2

24

3.6

4.3

170

> 1200

1400

360

^aMean K_ivalues for 2-3 independent experiments are shown with less than 10% deviation.

^bCompound concentration causing 50% of cell death after 24 h treatment.

We then modulated the nature of the amine (HNR₁R₂) and the length’s chain with m = 2, 3 between this

amine group and the 4-chlorobenzamide. We preserved the chlorine atom at the 4 position, considering

the good and maintained affinities and selectivities obtained with compounds 7h-i. Physicochemical

properties of compound 7i were evaluated and shown the potency of this compound with high LLe,

solubility and metabolic stability (Figure 3).

O

MWt = 316.8

logD = 2.51 [46]

LLE = 6.0 [47]

N

H

Solubility (pH7.4) = 173µM [48]

Metabolic stability (hLM) = 77% [49]

Cl

7i

Figure 3: Physicochemical properties of compound 7i

9

The modulation of the nature of the amine group, benzylmethylamine (7i) to 2-(phenylethyl)methylamine

ACCEPTED MANUSCRIPT

(8a), led to good affinity S1R (Ki = 4.3 nM), but low selectivity toward S2R (Ki = 200 nM) with a

S2R/S1R ratio of 47. The restricted conformation of the benzylmethylamine group with isoindoline

moiety for compound 8b afforded acceptable affinity for S1R (Ki = 19 nM) but low selectivity toward

S2R (Ki = 350 nM) with a S2R/S1R ratio of 16. For compounds 8c-h, various substituted amine, such as

pyrrolidine, morpholine and piperazine, were introduced and we modulated the length’s chain with two or

three methylene. As expected, the elimination of the phenyl ring with these derivatives 8c-d, identified as

an essential hydrophobic part, and the replacement by morpholine or piperazine for derivatives 8e-h, as

hydrophilic moieties, induced a dramatic lost of affinity for S1R (Ki ≥100 nM).

Table 2 : Evaluation of S1R, S2R affinities and cytotoxicity of target compounds 8a-h

Ki (nM)^a

Ki (σ₂)/

Ki (σ₁)

IC₅₀

(µM)

SY5Y ^b

IC₅₀/Ki (σ₁)

Compounds

m

-NR₁R₂

σ₁

σ₂

8a

8b

3

4.3

200

47

> 100

> 23 256

> 5 264

N

19

350

16

> 100

8c

8d

8e

2

3

2

> 100

> 200

-

> 100

-

N

O

8f

3

> 100

> 200

-

> 100

-

8g

8h

2

3

> 100

> 200

-

> 100

-

N

^a-dsee footnotes of Table 1

Starting from 7i, we decided to replace the amide linker with a sulfonylamide function (9), to reduce the

amide to amine function (10) and to synthesize the retroamide derivative (13) (Table 3). These three

compounds 9, 10 and 13 displayed good affinities for S1R (Ki = 2.9-6.5 nM) but low selectivities toward

S2R with a S2R/S1R ratio of 26-35.

Table 3 : Evaluation of S1R, S2R affinities and cytotoxicity of target compounds 9, 10, 13

Ki (nM)^a

IC₅₀(µM) IC₅₀/Ki (σ₁)

SY5Y ^b

Compounds

X

Ki (σ₂)/

Ki (σ₁)

σ₁

σ₂

SO₂NH

CH₂NH

NHCO

5.8

2.9

6.5

200

95

170

35

33

26

> 100

> 17 242

> 34 483

> 15 385

9

10

13

^a-dsee footnotes of Table 1

10

ACCEPTED MANUSCRIPT

3.2 Docking study of benzamide 7i

To investigate the potential binding mode of benzamide derivatives, compound 7i was docked into

the binding site of S1R [44]. As illustrated in Figure 3, the representative docking solutions highlighted

common interactions of S1R ligands. The benzyl moiety interact with hydrophobic regions of the receptor

and the p-chlorobenzamide scaffold is stabilized by an aromatic stacking interaction with the Tyr103. In

addition, the basic amine of 7i is engaged in a persistent salt bridge with the carboxylate group of the

highly conserved Glu172.

Figure 3: Docking of compound 7i in the binding site of S1R (PDB Entry: 5HK2)

3.3 Evaluation of cytotoxic effects

To determine the potential cytotoxic effects of our compounds on CNS tissues, a human

neuroblastoma cell line was treated with the whole compounds at different concentrations up to 100 µM.

Cell viability was calculated using a colorimetric MTT assay.

All target compounds exhibited very low cytotoxicity (Table 1, 2, 3) with selectivity index between 2 128

and 76 923 (IC_50(SY5Y)/Ki _(S1R)ratio). In particular, derivatives 7i and 7p with high affinities for S1R (Ki =

1.3-3.6 nM) and S2R/S1R selectivities (236-238) showed excellent selectivity index of 31 250 and 15 385

respectively. Compound 7y with the best selectivity for S2R (S2R/S1R = 389) also showed excellent

selectivity index of 27 778.

3.4 Selectivity profile over a panel of 40 recombinant human receptors

Among these compounds, eleven were selected regarding their affinity, selectivity, ligand efficiency and

chemical diversity. Compounds (7b, 7e, 7h, 7i, 7l, 7m, 7t, 7u, 7w, 7y, 7z, 8a) were tested on their agonist

and antagonist activities on 40 receptors relevant for CNS diseases like serotonine, adenosine, adrenergic,

cannabinoide, dopamine, histamine, muscarinic and others receptors. They were tested at 10µM, in

11

duplicate, on the Aequorin [50], cAMP HTRF^TM[51] and GTPγ³⁵S [52] binding assays as described in

ACCEPTED MANUSCRIPT

Table 4.

None of the studied compounds provided significant agonist activity at 10 µM, except compound 8a which

showed an agonist activity on serotonine receptors (5-HT_2B, 5-HT_2Cand 5-HT₇).

Significant antagonist activity was observed at 10 µM for serotonine (5-HT_2B, 5-HT_2Cand 5-HT₃),

adrenergic (all subtypes), histamine H1, dopamine D4 and muscarinic M1 receptors for compounds 7b, 7e,

7i, 7l, 7m, 8a with propyl, trifluoromethyl groups or chlorine, bromine atoms. In these cases, the position of

the substitution (meta or para) or the length’s chain played a significant part in the antagonist activities of

the compounds. For exemple, 4-bromo benzamide 7m was more selective than the 3-bromo derivative 7l,

which showed significant antagonist activity on 5-HT_2B, 5-HT_2C, α_2Aand H1 receptors. For 4-

chlorobenzamide derivatives, propyl chain and benzylmethylamine seemed optimal (compound 7i) as the

decrease from propyl to ethyl (compound 7h) induced activity on 5-HT_2A,5-HT_2Cand D2 receptors. In a

similar manner, the stretching from benzylamine (compound 7i) to phenethylamine (compound 8a) led to

an increased activity on 5-HT_1B,5-HT_2B,5-HT_2C,5-HT_3,α_2A, α_2B, H1 and M2 receptors.

12

ACCEPTED MANUSCRIPT

5-HT_1A

5-HT_1B

5-HT_2A

5-HT_2B

5-HT_2C

5-HT₃

5-HT₄

5-HT_5a

5-HT₆

5-HT₇

A₁

A_2A

A₃

Beta₁

Beta₂

Alpha_1a

Alpha_1b

Alpha_2a

Alpha_2b

AT1

[a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b]

7b

7e

7h

7i

55

75 63 94

90

75

51

96

79

96

92

97

93

82

52

56

62

95

72

70

96

73

97

81

56

54

66

52

66 62 92

58

7l

78

54

85

79

57

7m

7t

7u

7w

7y

7z

8a

75

62

89

84

63

98

80

65

92 75 95

130

105

83

CB1

CCK1

D1

D2

Eta

H1

H2

M1

M2

M3

M4

NK1

NK2

NMDA

[b]

NPY1

Delta

Kappa

Mu

SST4

V1_a

VPAC1

[a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b]

[a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b] [a] [b]

7b

7e

7h

7i

98

88

90

69

89

96

89

61

7l

74

57

58

7m

7t

ND

7u

7w

7y

7z

8a

55

66

60

85

86

90

54

Table 4 : [a]: Agonist activity at 10 µM in duplicate; [b] : Antagonist activity at 10 µM in duplicate. For the agonist activity, data are expressed as the percentage of the activity of the

reference agonist. For the antagonist activity, data are expressed as the percentage of inhibition of the reference agonist activity In green, 0-50%, in white 51-80%, in red 81-100%.

Numerical value are indicated over 51%. ND, not determined.

13

ACCEPTED MANUSCRIPT

Compounds 7t, 7u, 7w, 7y could be considered as selective compounds because they show excellent profil

toward all receptors and hight selectivities for S1R. In particular, methoxy derivatives 7t and 7u showed no

activity upon near all tested receptors at a concentration of 10 µM. Compounds 7w, 7y with cyano and nitro

group showed low affinities for H₁, 5-HT₃and µ opioid receptors. Once again, the length’s chain between

the benzylmethylamine and the benzamide scaffold was important. When we compared the nitro

compounds 7y and 7z the elongation of the ethyl (7y) to propyl chain (7z) induced higher affinities for

antagonist activities for α_1aand M₁. It is important to emphasize that compound 7y showed an excellent

profil S1R toward S2R (S1R : Ki = 3.6 nM, S2R : Ki > 1400 nM, S2R/S1R= 389) and an excellent profil for

agonist and antagonist activities against all the tested receptors.

4. Conclusion

Starting from previous work in our lab and in the literature, we used benzamide moiety as tic-hydantoin

surrogate and various modulations were realized. In the first series, we modulated the nature and the

position of atoms or groups on the benzamide moiety and kept the benzylmethylamine separated with

alkyl chain with 2 to 4 methylene group from the heterocycle, to give compounds 7a-z. In a second series,

4-chlorobenzamide moiety was kept and the nature of the amine group was modulated (HNR₁R₂) to

afford derivatives 8a-h. Then, three compounds were prepared as the sulfonyl (9), reduced (10) and

retroamide (13) analogues of derivative 7i. The K_ivalues for S1R and S2R were determined from the

corresponding IC₅₀values for each compound. The modulation with chlorine or bromine atoms gave

good affinities for S1R and selectivities toward S2R. The length’s chain also played an important part

from these 2 parameters. As expected, the introduction of hydrophilic moieties in compounds 8c-h

induced a dramatic lost of affinities for S1R, underlining the major importance of both hydrophobic

moieties highlighted in the Glennon’s pharmacophore. All together this study underlined the interest of

compounds 7i, w, y with Cl, CN or NO₂groups at the 4-position of the benzamide scaffold showed

excellent affinity for S1R (Ki = 1.2 to 3.6 nM), selectivity for S2R (Ki up to 1400 nM) and high

selectivity index (from 28 000 to 83 000). Physicochemical properties of compound 7i are very

compatible with drug development. Futhermore, these compounds showed a highly selective profile over

40 other receptors. These derivatives will be considered for further biological investigations.

5. Experimental

5.1 Chemistry

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

14

60/UV₂₅₄sheets (thickness 0.2mm). Purification of products was carried out by either column

ACCEPTED MANUSCRIPT

chromatography or thick layer chromatography. Column chromatography was carried out on using

Macherey-Nagel silica gel (230-400 mesh). Thick 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. IR spectra were performed on a Bruker

Alpha FT-IR Spectrometer. NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at

1

300 MHz for H and 75 MHz for ¹³C). Chemical shifts are 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 = 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). HRMS experiments were performed on Q

Exactive Benchtop LC-MS/MS (Thermo Scientific). The purity of final compounds was 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 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 t_R) were

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.

Preparation of N-(3-bromopropyl)-4-chlorobenzamide (1)

To a solution of 3-bromopropylamine hydrobromide (2.86 mmol, 625 mg) in 10 mL of DCM at 0°C was

added slowly a solution of 4-chlorobenzoyl chloride (2.86 mmol, 363 µL) in 5 mL of DCM. The resulting

mixture was stirred at room temperature for 12 hours. Then the solution was diluted with DCM and

washed successively with HCl (2M) solution, NaHCO₃saturated solution and NaCl solution. The organic

layer was dried over magnesium sulfate and evaporated. The crude product 2.1 was purified by column

chromatography (DCM: cyclohexane, 5:5 (v/v)). Yield: 65%. Mp: 84°C. ¹H NMR (300 MHz, CDCl₃), δ:

7.72 (d, J = 9.1 Hz, 2H, H₂, H₆); 7.38 (d, J = 9.2 Hz, 2H, H₃,H₅); 6.69 (br s, 1H, NH); 3.60 (q, J = 7.1 Hz,

2H, H_a); 3.48 (t, J = 7.3 Hz, 2H, H_c); 2.19 (p, J = 7.1 Hz, 2H, H_b). ¹³C NMR (75 MHz, CDCl₃) δ: 166.8

(CO); 137.8 (C_aro); 132.7 (C_aro); 128.8 (2 C_aro); 128.4 (2 C_aro); 38.8 (CH₂); 32.0 (CH₂); 31.0 (CH₂). LCMS

m/z calc for [M+H⁺]: 275.9, 277.9; found: 276.0; 278.0.

2-(4-Chlorophenyl)-5,6-dihydro-4H-1,3-oxazine (2)

15

To a solution of isoindoline (20.4 µL, 0.18 mmol) in 1 mL of DMF was added a 24 mg (0.18 mmol)

ACCEPTED MANUSCRIPT

amount of potassium carbonate. The resulting mixture was stirred at 70°C. After 30 minutes, the 4-

chloro- N-(3-bromopropyl)-4-chlorobenzamide (50 mg, 0.18 mmol) was added and the reacting mixture

was stirred at 70°C overnight. The solvent was removed under reduced pressure and 15 mL of water

added to the residue. The crude product was extracted with 3x15 mL of DCM. The combined organic

fractions were washed with water and dried over magnesium sulphate. The compound was purified by

column chromatography (DCM:MeOH(NH₃), 99:1 (v/v)) and obtained as a colourless oil. Yield: 70%.

Mp= 84°C. ¹H NMR (300 MHz, CDCl₃) δ: 7.83 (d, J = 9.2 Hz, 2H, H₂, H₆); 7.34 (d, J = 9.1 Hz, 2H, H₃,

H₅); 4.36 (t, J = 6.3 Hz, 2H, CH₂); 3.61 (t, J = 6.1 Hz, 2H, CH₂); 1.99 (p, J = 6.3 Hz, 2H, CH₂). ¹³C NMR

(75 MHz, CDCl₃) δ: 154.7 (OCN); 136.4 (C_aro); 132.6 (C_aro); 128.3 (2 C_aro); 128.2 (2 C_aro); 65.2 (CH₂);

42.6 (CH₂); 21.9 (CH₂). LCMS (ESI⁺): LCMS m/z calc for [M+H⁺]: 196.0, 198.0; found: 196.0; 198.0.

2. Preparation of diamine intermediates (5a-e)

tert-Butyl-3-bromopropylcarbamate (3)

3-Bromopropylamine (13.70 mmol, 3.0 g) was dissolved in 80 mL of DCM. After addition of

triethylamine (16.4 mmol, 2.32 mL) and di-tert-butylcarbonate (13.70 mmol, 3.0 g), the resulting mixture

was stirred at room temperature overnight. Then, it was washed with 2 x 60 mL of a citric acid solution

(5%) and 50 mL of saturated brine solution. The organic fraction was dried over magnesium sulphate and

concentrated to give the desired product as yellow oil of sufficient purity for use without purification in

1

the next step. Yield: 97%. H NMR (300 MHz, CDCl₃), δ: 4.67 (br s, 1H, NH); 3.45 (t, J = 7.4 Hz, 2H,

CH₂); 3.28 (q, J = 7.1 Hz, 2H, CH₂); 2.06 (p, ³J = 7 Hz, 2H, CH₂); 1.46 (s, 9H, C(CH₃)₃).

General procedure for the synthesis of substituted compounds 4a-c

To a mixture of tert-butyl-3-bromopropylcarbamate 1.2 (5.6 mmol, 1.3 eq, 1.3 g) and N,N-disubstituted

amine (4.3 mmol, 1 eq) in 30 mL of DMF was added potassium carbonate (13.0 mmol, 3 eq, 1.8 g). The

resulting mixture was heated at 70°C for 24 hours. Then, the solvent was removed under reduced pressure

and water (80 mL) added to the residue. The crude product was extracted with 3 x 60 mL of DCM. The

combined organic fractions were washed with 60 mL of water and dried over magnesium sulphate. The

solvent was evaporated and the crude product collected as yellow oil. Purification by column

chromatography (DCM: MeOH(NH₃), 9.7:0.3 (v/v)) was performed.

tert-Butyl-3-[benzyl(methyl)amino]propyl carbamate (4a)

1

Yield: 74%. H NMR (300 MHz, CDCl₃), δ: 7.36-7.19 (m, 5H, H_aro); 5.61 (br s, 1H, NH); 3.48 (s, 2H,

H_e); 3.20 (q, J = 6.4 Hz, 2H, CH₂); 2.45 (t, J= 6.3 Hz, 2H, CH₂); 2.17 (s, 3H, CH₃); 1.68 (p, J = 6.2 Hz,

2H, CH₂); 1.46 (s, 9H, C(CH₃)₃). ¹³C NMR (75 MHz, CDCl₃) δ: 156.1 (CO); 139.0 (C_aro); 129.0 (2 C_aro);

16

128.3 (2 C_aro); 127.0 (C_aro); 78.7 (C(CH₃)₃); 62.7 (CH ); 56.5 (CH₂); 42.1 (CH₃); 40.1 (CH₂); 28.5

ACCEPTED MAN²USCRIPT

(C(CH₃)₃); 26.6 (CH₂). LCMS m/z calc for [M+H⁺]: 279.2; found: 279.1.

tert-Butyl-3-[N-methyl-N-(2-phenylethyl)amino] propyl carbamate (4b)

Yield: 81%. ¹H NMR (300 MHz, CDCl₃), δ: 7.30 (m, 2H, H_aro); 7.20 (m, 2H, H_aro); 5.66 (br s, 1H, NH);

3.16 (m, 2H, CH₂); 2.79 (m, 2H, CH₂); ); 2.61 (m, 2H, CH₂); 2.45 (t, J = 6.8 Hz, 2H, CH₂); 2.28 (s, 3H,

13

CH₃); 1.65 (q, J = 6.6 Hz, 2H, CH₂); 1.47 (s, 9H, C(CH₃)₃). C NMR (75 MHz, CDCl₃) δ: 156.1 (CO);

140.4 (C_aro); 128.7 (2 C_aro); 128.4 (2 C_aro); 126.0 (C_aro); 78.8 (C(CH₃)₃); 59.6 (CH₂); 55.9 (CH₂); 42.1

(CH₃); 39.7 (CH₂); 33.9 (CH₂); 28.5 (C(CH₃)₃); 27.0 (CH₂). LCMS m/z calc for [M+H- tertBut]⁺: 237.1;

found: 237.0.

tert-Butyl-3-(isoindolin-2-yl)propylcarbamate (4c)

Yield: 65%. ¹H NMR (300 MHz, CDCl₃), δ: 7.20 (m, 4H, H_aro); 5.12 (br s, 1H, NH); 3.96 (s, 4H, 2 CH₂);

3.24 (m, 2H, CH₂); 2.79 (t, J = 6.0 Hz, 2H, CH₂); 1.75 (q, J = 6.7 Hz, 2H, CH₂); 1.44 (s, 9H, C(CH₃)₃).

¹³C NMR (75 MHz, CDCl₃) δ: 156.1 (CO); 139.7 (2 C_aro); 126.8 (2 C_aro); 122.3 (2 C_aro); 79.1 (C(CH₃)₃);

59.1 (2 CH₂); 54.0 (CH₂); 39.5 (CH₂); 28.7 (CH₂); 28.5 (C(CH₃)₃). LCMS m/z calc for [M+H- tertBut]⁺:

221.2; found: 221.0.

General procedure for the synthesis of substituted compounds 6a-c

To a mixture of N-(bromoethyl or bromobutyl)phtalimide (5.9 mmol) and benzylmethylamine (7.1 mmol,

0.8 mL) or 2-phenylethylamine (7.1 mmol, 0.9 mL) in 30 mL of DMF was added potassium carbonate

(11.80 mmol, 1.6 g). The resulting mixture was heated at 90°C for 16 hours. Then, inorganics were

eliminated by filtration and the solvent was removed under reduced pressure. Purification by column

chromatography (DCM: MeOH(NH₃), 9.9:0.1 (v/v)) was performed.

2-[2-(Benzylmethylamino)ethyl]-2,3-dihydro-1H-isoindole-1,3-dione (6a)

1

Yield 52%. H NMR (300 MHz, CDCl₃), δ: 7.88 (m, 2H, H_aro); 7.72 (m, 2H, H_aro); 7.09-7.11 (m, 5H,

H_aro); 3.82 (t, J = 6.3 Hz, 2H, CH₂); 3.55 (s, 2H, CH₂); 2.70 (t, J = 6.3 Hz, 2H, CH₂); 2.31 (s, 3H, CH₃).

¹³C NMR (75 MHz, CDCl₃) δ: 168.3 (2 CO); 138.8 (C_aro); 133.8 (2 C_aro); 132.3 (2 C_aro); 128.9 (2 C_aro);

128.1 (2 C_aro); 126.9 (C_aro); 123.1 (2 C_aro); 62.1 (CH₂); 54.5 (CH₂); 42.2 (CH₃); 35.9 (CH₂). LCMS m/z

calc for [M+H⁺]: 295.1; found: .295.0.

2-[4-(Benzylmethylamino)butyl]-2,3-dihydro-1H-isoindole-1,3-dione (6b)

1

Yield 53%. H NMR (300 MHz, CDCl₃), δ: 7.82 (m, 2H, H_aro); 7.69 (m, 2H, H_aro); 7.15-7.32 (m, 5H,

H_aro); 3.71 (t, J = 7.2 Hz, 2H, CH₂); 3.48 (s, 2H, CH₂); 2.41 (t, J = 7.2 Hz, 2H, CH₂); 2.18 (s, 3H, CH₃);

1.74 (m, 2H, CH₂); 1.55 (m, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 168.4 (2 CO); 138.9 (C_aro); 133.9

17

(2 C_aro); 132.1 (C_aro); 129.0 (2 C_aro); 128.2 (2 C_aro); 126.9 (2 C_aro); 123.1 (2 C_aro); 62.4 (CH₂); 56.8 (CH₂);

ACCEPTED MANUSCRIPT

42.1 (CH₃); 37.9 (CH₂); 26.5 (CH₂); 24.6 (CH₂). LCMS m/z calc for [M+H⁺]: 323.1; found: 323.0.

2-[2-(N-methyl-N-(2-phenylethyl)amino)ethyl]-2,3-dihydro-1H-isoindole-1,3-dione (6c)

1

Yield: 64%. H NMR (300 MHz, CDCl₃), δ: 7.85 (m, 2H, H_aro); 7.70 (m, 2H, H_aro); 7.05-7.20 (m, 5H,

13

H_aro); 3.80 (t, J = 6.6 Hz, 2H, CH₂); 2.62-2.75 (m, 6H, 3CH₂); 2.38 (s, 3H, CH₃). C NMR (75 MHz,

CDCl₃) δ: 168.3 (CO); 140.3 (C_aro); 133.8 (C_aro); 132.2 (C_aro); 128.7 (C_aro); 128.2 (C_aro); 125.8 (C_aro);

123.1 (C_aro); 59.4 (CH₂); 54.7 (CH₂); 42.2 (CH₃); 35.8 (CH₂); 33.7 (CH₂). LCMS m/z calc for [M+H⁺]:

309.1; found: 309.0.

General procedure for the synthesis of substitutedcompounds 5a-c

The compound 1.3 (1 eq) was dissolved in 72 mL of 3M HCl in dry 1,4-dioxane. The resulting mixture

was stirred at room temperature overnight. Then, the solvent was removed under reduced pressure to give

the desired product of sufficient purity for use without purification in the next step.

N¹-Benzyl-N¹-methylpropane-1,3-diamine (5a)

1

Yield 98%. Mp: 77°C. H NMR (300 MHz, CDCl₃), δ: 11.27 (br s, 1H, NH); 8.37 (br s, 3H, NH); 7.66-

7.41 (m, 5H, H_aro); 4.27 (m, 2H, CH₂), 3.27-2.28 (m, 4H, 2 CH₂); 2.60 (s, 3H, CH₃); 2.13 (p, J = 7.4 Hz,

13

2H, CH₂). C NMR (75 MHz, CDCl₃) δ: 123.0 (C_aro); 130.3 (C_aro); 129.9 (2 C_aro); 129.2 (2 C_aro); 58.6

(CH₂); 52.1 (CH₂); 39.0 (CH₃); 36.7 (CH₂); 22.0 (CH₂). LCMS m/z calc for [M+H⁺]: 179.1; found: .179.0

N¹-Benzyl-N¹-(2-phenylethyl)propane-1,3-diamine (5b)

Yield: 88%. ¹H NMR (300 MHz, CDCl₃), δ: 7.38-7.22 (m, 5H, H_aro); 6.20 (br s, 2H, NH₂); 3.37-3.24 (m,

13

4H, CH₂); 3.19-3.01 (m, 4H, 2 CH₂); 2.83 (s, 3H, CH₃); 2.15 (q, J = 7.4 Hz, 2H, CH₂). C NMR (75

MHz, CDCl₃) δ: 137.0 (C_aro); 128.7 (2 C_aro); 128.6 (2 C_aro); 126.7 (C_aro); 57.6 (CH₂) 39.6 (CH₃); 37.3

(CH₂); 30.6 (CH₂); 22.4 (CH₂). LCMS m/z calc for [M+H⁺]: 193.1; found: 193.0.

3-(Isoindolin-2-yl)propan-1-amine (5c)

Yield: 83%. ¹H NMR (300 MHz, CDCl₃), δ: 8.04 (m, 4H, H_aro); 5.58 (br s, 2H, NH₂); 4.12 (t, J = 7.9 Hz,

2H, CH₂); 3.91 (s, 4H, CH₂), 3.74 (t, J = 7.6 Hz, 2H, CH₂); 2.71 (t, J = 7.8 Hz, 2H, CH₂). ¹³C NMR (75

MHz, CDCl₃) δ: 133.9 (2 C_aro); 128.6 (2 C_aro); 122.6 (2 C_aro); 58.5 (2 CH₂); 51.7 (CH₂); 36.6 (CH₂); 23.7

(CH₂). LCMS m/z calc for [M+H⁺]: 177.1; found: 177.0.

General procedure for the synthesis of substituted compounds 5d-f

In 50 mL of ethanol 96°, was added a mixture of 2-[N,N-disubstitutedaminoalkyl]-2,3-dihydro-1H-

isoindole-1,3-dione (6.2 mmol) and hydrazine hydrate (62.0 mmol, 3.1 mL). The resulting mixture was

18

heated at reflux for 16 hours. The solution was removed under reduced pressure. The mineral was

ACCEPTED MANUSCRIPT

eliminated by filtration and washed with ethyl acetate. The filtrate was evaporated under reduced pressure

to give an oily residue. Purification by column chromatography (DCM: MeOH(NH₃), 9:1 (v/v)) was

performed.

N¹-Benzyl-N¹-methylethane-1,2-diamine (5d)

1

Yield 55%. H NMR (300 MHz, CDCl₃), δ: 7.35-7.30 (m, 5H, H_aro); 4.90 (br s, 2H, NH₂); 3.58 (s, 2H,

CH₂); 2.91 (t, J = 6.3 Hz, 2H, CH₂); 2.60 (t, J = 6.2 Hz, 2H, CH₂); 2.22 (s, 3H, CH₃). ¹³C NMR (75 MHz,

CDCl₃) δ:138.0 (C_aro); 129.0 (2 C_aro); 128.0 (2 C_aro); 127.0 (C_aro); 62.1 (CH₂); 55.9 (CH₂); 40.7 (CH₃);

37.4 (CH₂). LCMS m/z calc for [M+H⁺]: 165.1; found: 165.0.

N¹-Benzyl-N¹-methylbutane-1,4-diamine (5e)

1

Yield 73%. H NMR (300 MHz, CDCl₃), δ: 7.35-7.25 (m, 5H, H_aro); 3.50 (s, 2H, CH₂); 2.72 (t, J = 6.8

Hz, 2H, CH₂); 2.39 (t, J = 6.9 Hz, 2H, CH₂); 2.22 (s, 5H, NH₂, CH₃); 1.61-1.45 (m, 4H, 2 CH₂). ¹³C NMR

(75 MHz, CDCl₃) δ: 139.1 (C_aro); 129.10 (2 C_aro); 128.2 (2 C_aro); 126.9 (C_aro); 62.3 (CH₂); 57.2 (CH₂);

42.1 (CH₃); 42.0 (CH₂); 31.4 (CH₂); 24.7 (CH₂). LCMS m/z calc for [M+H⁺]: 193.1; found: 193.0.

N¹-(2-phenylethyl)-N¹-methylethane-1,2-diamine dihydrochloride (5f)

1

Yield: 64%. H NMR (300 MHz, CDCl₃), δ: 7.30-7.22 (m, 2H, H_aro); 7.20-7.10 (m, 3H, H_aro); 2.80-2.70

(m, 4H, 2CH₂); 2.62 (m, 2H, CH₂); 2.47 (t, J = 6.0 Hz, 2H, CH₂); 2.26 (s, 3H, CH₃). ¹³C NMR (75 MHz,

CDCl₃) δ: 140.5 (C_aro); 128.7 (C_aro); 128.3 (C_aro); 125.9 (C_aro); 59.7 (CH₂); 59.6 (CH₂); 42.1 (CH₃); 39.2

(CH₂); 33.8 (CH₂). LCMS m/z calc for [M+H⁺]: 179.1; found: 179.1.

General procedure E1

A solution of N,N-disubstitutedalkyldiamine 5a-f or commercially available amine (1 eq), and

triethylamine (3 eq) in 10 mL of DCM was cooled at 0°C. A solution of substituted benzoylchloride (1

eq) in 5 mL of DCM was added slowly. The resulting mixture was stirred at room temperature overnight.

The solution was evaporated under reduced pressure. An aqueous solution of 3% sodium hydroxide (20

mL) was added and the mixture was stirred for 1 hour. The solution was extracted with DCM. The

organic layer was dried over magnesium sulphate and concentrated to give an oily product. The residue

was purified by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) or column chromatography

(DCM:MeOH(NH₃), 9:1 (v/v)).

General procedure E2

Reaction was carried out under nitrogen atmosphere. In 10 mL of DCM, benzoic acid (1 eq), HOBt (1.2

eq) and HBtu (1.2 eq) were added and stirred at room temperature for 10 minutes. A solution of N,N-

19

disubstitutedalkyldiamine 5a-f or commercially available amine (1 eq) and DIEA (15 eq) in DCM was

ACCEPTED MANUSCRIPT

added to the reacting mixture. After stirring at room temperature for 24 hours, the solvent was removed

under reduced pressure and DCM was added to the residue. The solution was washed with a solution of

NaHCO₃(5%) then saturated NaCl solution. The organic layer was dried over magnesium sulfate and

evaporated under reduced pressure. The crude product was purified by thick layer chromatography

(DCM:MeOH(NH₃), 9:1 (v/v)) or column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)).

N-[3-(N-benzyl-N-methylamino)propyl]benzamide (7a)

1

Procedure E2. Purification by thick layer chromatography was performed. Yield: 75%. H NMR (300

MHz, CDCl₃), δ: 8.05 (br s, 1H, NH); 7.70 (m, 2H, H₂, H₆); 7.49-7.30 (m, 3H, H₃, H₄, H₅); 7.29-7.22 (m,

5H, H_aro); 3.61-3.52 (m, 4H, 2 CH₂); 2.65 (t, J = 6.7 Hz, 2H, CH₂); 2.29 (s, 3H, CH₃); 1.84 (p, J = 6.8 Hz,

2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 167.3 (CO); 137.8 (C_aro); 134.7 (C_aro); 131.1 (2 C_aro); 129.4 (2

C_aro); 128.4 (C_aro); 128.4 (2 C_aro); 127.4 (C_aro); 126.9 (2 C_aro); 63.1 (CH₂); 56.9 (CH₂); 41.6 (CH₃); 40.1

(CH₂); 25.3 (CH₂). LCMS m/z calc for [M+H⁺]: 283.2; found: 283.1. HPLC (C₄, 35 min): t_R17.5 min,

P_HPLC97%; HPLC (C₁₈, 35 min): t_R15.2 min, P_HPLC99%.

N-[3-(benzylmethylamino)propyl]-4-propylbenzamide (7b)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 61%. ¹H NMR (300 MHz, CDCl₃), δ: 7.90 (br s, 1H, NH) ; 7.60 (d, J = 8.2 Hz, 2H, H₂, H₆); 7.29-

7.20 (m, 5H, H_aro); 7.13 (d, J = 8.1 Hz, 2H, H₃, H₅); 3.55 (t, J = 6.0 Hz, 2H, CH₂); 3.50 (s, 2H, CH₂); 2.60

(m, 4H, 2 CH₂); 2.23 (s, 3H, CH₃); 1.80 (p, J = 6.0 Hz, 2H, CH₂); 1.62 (s, J = 7.5 Hz, 2H, CH₂); 0.95 (t, J

13

= 7.3 Hz, 2H, CH₃). C NMR (75 MHz, CDCl₃) δ: 167.2 (CO); 146.1 (C_aro); 138.4 (C_aro); 132.2 (C_aro);

129.3 (C_aro); 128.5 (C_aro); 128.4 (C_aro); 127.2 (C_aro); 126.9 (C_aro); 63.3 (CH₂); 57.1 (CH₂); 42.1 (CH₃);

40.2 (CH₂); 37.9 (CH₂); 25.5 (CH₂); 24.3 (CH₂); 14.2 (CH₃). LCMS m/z calc for [M+H⁺]: 325.2; found:

325.0. HRMS: calcd for (C₂₁H₂₈ON₂)H⁺: 325.22744; found: 325.22617. HPLC (C₄, 35 min): t_R16.5 min,

P_HPLC96%; HPLC (C₁₈, 35 min): t_R22.5 min, P_HPLC97%.

N-[3-(benzylmethylamino)propyl]-4-butylbenzamide (7c)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 30%. ¹H NMR (300 MHz, CDCl₃), δ: 7.97 (br s, 1H, NH) ; 7.60 (d, J = 8.2 Hz, 2H, H₂, H₆); 7.29-

7.23 (m, 5H, H_aro); 7.13 (d, J = 8.2 Hz, 2H, H₃, H₅); 3.56 (m, 4H, 2CH₂); 2.64 (m, 4H, 2CH₂); 2.28 (s, 3H,

CH₃); 1.83 (p, J = 6.1 Hz, 2H, CH₂); 1.62 (m, 2H, CH₂); 1.35 (m, 2H, CH₂); 0.97 (t, J = 7.1 Hz, 2H, CH₃).

¹³C NMR (75 MHz, CDCl₃) δ: 167.7 (CO); 146.7 (C_aro); 138.0 (C_aro); 132.5 (C_aro); 129.8 (2 C_aro); 128.8

(4 C_aro); 127.9 (C_aro); 127.3 (2 C_aro); 63.5 (CH₂); 57.1 (CH₂); 41.9 (CH₃); 40.3 (CH₂); 35.9 (CH₂); 33.7

(CH₂); 25.7 (CH₂); 22.6 (CH₂); 14.3 (CH₃). LCMS m/z calc for [M+H⁺]: 339.2; found: 339.0. HPLC (C₄,

35 min): t_R16.0 min, P_HPLC96%; HPLC (C₁₈, 35 min): t_R21.9 min, P_HPLC97%.

20

ACCEPTED MANUSCRIPT

N-[3-(benzylmethylamino)propyl]-4-tert-butylbenzamide (7d)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 27%. H NMR (300 MHz, CDCl₃), δ: 8.03 (br s, 1H, NH); 7.63 (d, J = 8.5 Hz, 2H, H₂, H₆); 7.37

(d, J = 8.5 Hz, 2H, H₃, H₅); 7.29-7.24 (m, 5H, H_aro); 3.57 (m, 4H, 2CH₂); 2.63 (t, J = 6.0 Hz, 2H, CH₂);

13

2.28 (s, 3H, NCH₃); 1.84 (p, J = 5.9 Hz, 2H, CH₂); 1.32 (s, 9H, 3CH₃). C NMR (75 MHz, CDCl₃) δ:

167.1 (CO); 154.4 (C_aro); 137.8 (C_aro); 131.8 (C_aro); 129.5 (2 C_aro); 128.4 (2 C_aro); 127.4 (C_aro); 126.8 (2

C_aro); 125.3 (2 C_aro); 63.1 (CH₂); 56.9 (CH₂); 41.5 (CH₃); 40.1 (CH₂); 34.8 (C); 31.2 (CH₃); 25.3 (CH₂).

LCMS m/z calc for [M+H⁺]: 339.2; found: 339.0. HPLC (C₄, 35 min): t_R17.2 min, P_HPLC97%; HPLC

(C₁₈, 35 min): t_R23.5 min, P_HPLC98%.

N-[3-(benzylmethylamino)propyl]-4-trifluoromethylbenzamide (7e)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 57%. ¹H NMR (300 MHz, CDCl₃), δ: 8.50 (br s, 1H, NH) ; 7.78 (d, J = 8.2 Hz, 2H, H₂, H₆); 7.59

(d, J = 8.2 Hz, 2H, H₃, H₅); 7.28-7.21 (m, 5H, H_aro); 3.63-3.55 (m, 4H, 2 CH₂); 2.68 (t, J = 5.8 Hz, 2H,

CH₂); 2.30 (s, 3H, CH₃); 1.85 (p, J = 5.7 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 165.8 (CO); 138.0

(C_aro); 129.5 (2 C_aro); 128.5 (2 C_aro); 127.7 (C_aro); 127.4 (2 C_aro); 125.5 (2 C_aro); 125.4 (2 C_aro); 62.9 (CH₂);

57.1 (CH₂); 41.3 (CH₃); 40.5 (CH₂); 24.8 (CH₂). LCMS m/z calc for [M+H⁺]: 351.1; found: 351.0.

HRMS: calcd for (C₁₉H₂₁ON₂F₃)H⁺: 351.16787; found: 351.16663. HPLC (C₄, 35 min): t_R18.5 min,

P_HPLC96%; HPLC (C₁₈, 35 min): t_R25.2 min, P_HPLC97%.

N-[3-(benzylmethylamino)propyl]-2-chlorobenzamide (7f)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 46%. ¹H NMR (300 MHz, CDCl₃), δ: 7.52 (dd, J = 7.0 Hz, J = 1.9 Hz, 1H, H₆); 7.39-7.25 (m, 4H,

NH, H₃, H₄, H₅); 7.17-7.09 (m, 5H, H_aro); 3.54 (q, J = 6.2 Hz, 2H, CH₂); 3.49 (s, 2H, CH₂); 2.58 (t, J = 6.3

13

Hz, 2H, CH₂); 2.21 (s, 3H, NCH₃); 1.83 (p, J = 6.2 Hz, 2H, CH₂). C NMR (75 MHz, CDCl₃) δ: 166.7

(CO); 137.9 (C_aro); 136.0 (C_aro); 130.8 (C_aro); 130.7 (C_aro); 130.1 (C_aro); 129.5 (C_aro); 128.9 (2 C_aro); 128.3

(2 C_aro); 127.2 (C_aro); 126.9 (C_aro); 62.6 (CH₂); 56.0 (CH₂); 41.8 (CH₃); 39.7 (CH₂); 25.3 (CH₂). LCMS

m/z calc for [M+H⁺]: 317.1, 319.1; found: 317.0, 319.0. HPLC (C₄, 35 min): t_R12.6 min, P_HPLC95%;

HPLC (C₁₈, 35 min): t_R17.1 min, P_HPLC96%.

N-[3-(benzylmethylamino)propyl]-3-chlorobenzamide (7g)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 8%. ¹H NMR (300 MHz, CDCl₃), δ: 8.22 (br s, 1H, NH); 7.78 (dd, J = 1.5 Hz, J = 2.0 Hz, 1H, H₂);

7.68 (d, J = 8.0 Hz, 1H, H₆); 7.52 (d, J = 7.8 Hz, 1H, H₄); 7.52 (t, J = 7.3 Hz, 1H, H₅); 7.32-7.22 (m, 5H,

H_aro); 3.58-3.51 (m, 4H, 2CH₂); 2.63 (t, J = 7.5 Hz, 2H, CH₂); 2.30 (s, 3H, CH₃); 1.84 (p, J = 6.4 Hz, 2H,

21

13

CH₂). C NMR (75 MHz, CDCl₃) δ: 165.8 (CO); 136.5 (C ); 134.6 (C_aro); 131.2 (C_aro); 129.7 (C_aro);

ACCEPTED MANUS^aC^roRIPT

129.4 (2 C_aro); 128.5 (2 C_aro); 127.7 (2 C_aro); 127.5 (C_aro); 125.0 (C_aro); 63.1 (CH₂); 56.7 (CH₂); 41.6

(CH₃); 40.4 (CH₂); 24.8 (CH₂). LCMS m/z calc for [M+H⁺]: 317.1, 319.1; found: 317.0, 319.0. HPLC

(C₄, 35 min): t_R14.8 min, P_HPLC97%; HPLC (C₁₈, 35 min): t_R20.2 min, P_HPLC97%.

N-[3-(2-(N-methylbenzyl)amino)ethyl]-4-chlorobenzamide (7h)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 20%. ¹H NMR (300 MHz, CDCl₃), δ: 7.67 (d, J = 8.6 Hz, 2H, H₂, H₆); 7.41 (d, J = 8.6 Hz, 2H, H₃,

H₅); 7.23-7,33 (m, 5H, H_aro); 6.78 (br s, 1H, NH); 3.56 (s, 2H, CH₂); 3.51 (q, J = 5.2 Hz, 2H, CH₂); 2.62

13

(t, J = 5.9 Hz, 2H, CH₂); 2.31 (s, 3H, NCH₃). C NMR (75 MHz, CDCl₃) δ: 166.4 (CO); 138.6 (C_aro);

137.5 (C_aro); 133.1 (C_aro); 129.4 (2 C_aro); 129.1 (2 C_aro); 128.9 (2 C_aro); 128.7 (2 C_aro); 127.4 (C_aro); 62.3

(CH₂); 54.9 (CH₂); 42.1 (CH₃); 37.1 (CH₂). LCMS m/z calc for [M+H⁺]: 303.1, 305.1; found: 303.0,

305.0. HRMS: calcd for (C₁₇H₁₉ON₂Cl)H⁺: 303.12531; found: 303.12587. HPLC (C₄, 35 min): t_R14.2

min, P_HPLC97%; HPLC (C₁₈, 35 min): t_R19.3 min, P_HPLC97%.

N-[3-(benzylmethylamino)propyl]-4-chlorobenzamide (7i)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 55%. IR (cm^-1): 1625 (C=O), 3012 (NH). H NMR (300 MHz, CDCl₃), δ: 8.25 (br s, 1H, NH);

7.60 (d, J = 9 Hz, 2H, H₂, H₆); 7.33-7,21 (m, 7H, H₃, H₅, H_aro); 3.62-3.51 (m, 4H, 2CH₂); 2.65 (t, J = 5.4

Hz, 2H, CH₂); 2.27 (s, 3H, NCH₃); 1.83 (p, J = 5.5 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 166.0

(CO); 137.8 (C_aro); 137.3 (C_aro); 133.1 (C_aro); 129.4 (2 C_aro); 128.6 (2 C_aro); 128.5 (2 C_aro); 128.4 (2 C_aro);

127.5 (C_aro); 63.2 (CH₂); 57.2 (CH₂); 41.5 (CH₃); 40.5 (CH₂); 25.0 (CH₂). LCMS m/z calc for [M+H⁺]:

317.1, 319.1; found: 316.9, 318.9. HRMS: calcd for (C₁₈H₂₁ON₂Cl)H⁺: 317.14152; found: 317.14083.

HPLC (C₄, 35 min): t_R7.7 min, P_HPLC98%; HPLC (C₁₈, 35 min): t_R18.0 min, P_HPLC98%.

N-[4-(benzylmethylamino)butyl]-4-chlorobenzamide (7j)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 25%. ¹H NMR (300 MHz, CDCl₃), δ: 7.60 (d, J = 9.2 Hz, 2H, H₂, H₆); 7.41 (br s, 1H, NH); 7.33-

7,26 (m, 7H, H₃, H₅, H_aro); 3.53 (s, 2H, CH₂); 3.45 (q, J = 6.2 Hz, 2H, CH₂); 2.45 (t, J = 6.4 Hz, 2H, CH₂);

13

2.17 (s, 3H, NCH₃); 1.73-1.62 (m, 4H, 2CH₂). C NMR (75 MHz, CDCl₃) δ: 166.6 (CO); 137.7 (C_aro);

137.3 (C_aro); 133.3 (C_aro); 129.3 (2 C_aro); 128.6 (2 C_aro); 128.5 (2 C_aro); 128.4 (2 C_aro); 127.4 (C_aro); 61.8

(CH₂); 56.7 (CH₂); 42.1 (CH₃); 39.9 (CH₂); 27.1 (CH₂); 24.8 (CH₂). LCMS m/z calc for [M+H⁺]: 331.1,

333.1; found: 331.0, 333.0. HPLC (C₄, 35 min): t_R14.8 min, P_HPLC96%; HPLC (C₁₈, 35 min): t_R20.2

min, P_HPLC97%.

N-[3-(benzylmethylamino)propyl]-2-bromobenzamide (7k)

22

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

ACCEPTED MANUSCRIPT

1

Yield: 52%. H NMR (300 MHz, CDCl₃), δ: 7.56 (dd, J = 7.8 Hz, J = 1.3 Hz, 1H, H₆); 7.47 (br s, 1H,

NH); 7.37 (dd, J = 7.4 Hz, J = 2.0 Hz, 1H, H₃); 7.31 (td, J = 7.3 Hz, J = 1.2 Hz, 1H, H_aro); 7.24 (td, J = 7.4

Hz, J = 1.9 Hz, 1H, H_aro); 7.16-7.07 (m, 5H, H_aro); 3.52 (q, J = 6.2 Hz, 2H, CH₂); 3.48 (s, 2H, CH₂); 2.57

(t, J = 6.1 Hz, 2H, CH₂); 2.19 (s, 3H, NCH₃); 1.80 (p, J = 6.2 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃)

δ: 167.8 (CO); 138.6 (C_aro); 138.0 (C_aro); 133.2 (C_aro); 130.8 (C_aro); 129.0 (2 C_aro); 128.9 (2 C_aro); 128.3

(C_aro); 127.4 (C_aro); 127.1 (C_aro); 119.5 (C_aro); 62.6 (CH₂); 56.1 (CH₂); 41.8 (CH₃); 39.6 (CH₂); 25.3

(CH₂). LCMS m/z calc for [M+H⁺]: 361.1; 363.1; found: 360.9; 362.9. HPLC (C₄, 35 min): t_R13.0 min,

P_HPLC96%; HPLC (C₁₈, 35 min): t_R17.8 min, P_HPLC96%.

N-[3-(benzylmethylamino)propyl]-3-bromobenzamide (7l)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 53%. H NMR (300 MHz, CDCl₃), δ: 8.27 (br s, 1H, NH); 7.82 (t, J = 1.8 Hz, 2H, H₂); 7.55 (m,

1H, H₅); 7.28-7.17 (m, 7H, H₄, H₆, H_aro); 3.55 (m, 4H, 2CH₂); 2.62 (t, J = 6.0 Hz, 2H, CH₂); 2.30 (s, 3H,

13

NCH₃); 1.83 (p, J = 5.9 Hz, 2H, CH₂). C NMR (75 MHz, CDCl₃) δ: 165.7 (CO); 137.6 (C_aro); 136.7

(C_aro); 134.0 (C_aro); 130.3 (C_aro); 129.9 (C_aro); 129.4 (2 C_aro); 128.5 (2 C_aro); 127.6 (C_aro); 125.4 (C_aro);

122.6 (C_aro); 63.1 (CH₂); 56.8 (CH₂); 41.8 (CH₃); 40.4 (CH₂); 24.9 (CH₂). LCMS m/z calc for [M+H⁺]:

361.1; 363.1; found: 360.9; 362.9. HRMS: calcd for (C₁₈H₂₁ON₂Br)H⁺: 361.09100; found: 361.08984.

HPLC (C₄, 35 min): t_R14.8 min, P_HPLC98%; HPLC (C₁₈, 35 min): t_R20.2 min, P_HPLC98%.

N-[3-(benzylmethylamino)propyl]-4-bromobenzamide (7m)

Procedure E2. Purification by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 67%. ¹H NMR (300 MHz, CDCl₃), δ : 8.29 (br s, 1H, NH); 7.54 (d, J = 9.1 Hz, 2H, H₂, H₆); 7.46

(d, J = 9.2 Hz, 2H, H₃, H₅); 7.33-7.20 (m, 5H, H_aro); 3.65-3.47 (m, 4H, 2 CH₂); 2.66 (t, J = 5.9 Hz, 2H,

CH₂); 2.29 (s, 3H, CH₃); 1.84 (p, J = 5.8 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 166.2 (CO); 137.5

(C_aro); 133.5 (C_aro); 131.6 (2 C_aro); 129.5 (2 C_aro); 128.6 (2 C_aro); 128.5 (2 C_aro); 127.7 (C_aro); 125.7 (C_aro);

63.0 (CH₂); 57.0 (CH₂); 41.4 (CH₃); 40.3 (CH₂); 24.9 (CH₂). LCMS m/z calc for [M+H⁺]: 361.1; 363.1;

found: 360.9; 362.9. HRMS: calcd for (C₁₈H₂₁ON₂Br)H⁺: 361.09100; found: 361.08948. HPLC (C₄, 35

min): t_R8.8 min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R18.4 min, P_HPLC>99%.

N-[3-(benzylmethylamino)propyl]-4-fluorobenzamide (7n)

Procedure E2. Purification by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 58%. ¹H NMR (300 MHz, CDCl₃), δ : 8.20 (br s, 1H, NH); 7.66 (m, 2H, H₂and H₆); 7.28-7.24 (m,

5H, H_aro); 7.00 (t, ³J_3-F= 9 Hz, 2H, H₃and H₅); 3.60-3.52 (m, 4H, H_aet H_e); 2.65 (t, ³J = 6 Hz, 2H, H_c);

2.28 (s, 3H, H_d); 1.83 (p, ³J = 6 Hz, 2H, H_b). ¹³C NMR (75 MHz, CDCl₃) δ: 166.1 (CO); 164.5 (d, ¹J_4-F

=

4

3

250 Hz, C₄); 137.6 (C_aro); 130.8 (d, J_1-F= 3 Hz, C₁); 129.5 (2 C_aro); 129.2 (d, J_2-F= 9 Hz, C₂and C₆);

23

128.5 (2 C_aro); 127.6 (C_aro); 115.3 (d, ²J_3-F= 20 Hz, C and C₅); 63.1 (CH₂); 57.1 (CH₂); 41.4 (CH₃); 40.3

ACCEPTED M³ANUSCRIPT

(CH₂); 25.0 (CH₂). LCMS m/z calc for [M+H⁺]: 301.2; found: 301.1. HPLC (C₄, 35 min): t_R8.4 min,

P_HPLC>99%; HPLC (C₁₈, 35 min): t_R16.3 min, P_HPLC>99%.

N-[3-(benzylmethylamino)propyl]-2,3-dichlorobenzamide (7o)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 66%. H NMR (300 MHz, CDCl₃), δ: 7.58 (br s, 1H, NH); 7.51 (dd, J = 7.9 Hz, J = 1.6 Hz, 1H,

H₆); 7.32 (dd, J = 7.7 Hz, J = 1.6 Hz, 1H, H₄); 7.25 (m, 1H, H₅); 7.17-7.07 (m, 5H, H_aro); 3.55 (q, J = 6.7

Hz, 2H, CH₂); 3.48 (s, 2H, CH₂); 2.58 (t, J = 5.9 Hz, 2H, CH₂); 2.21 (s, 3H, NCH₃); 1.82 (p, J = 6.0 Hz,

2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 166.2 (CO); 138.6 (C_aro); 137.8 (C_aro); 133.7 (C_aro); 131.3 (C_aro);

128.9 (C_aro); 128.3 (2 C_aro); 127.6 (2 C_aro); 127.5 (C_aro); 127.3 (C_aro); 127.0 (C_aro); 62.7 (CH₂); 56.0 (CH₂);

41.8 (CH₃); 39.7 (CH₂); 25.1 (CH₂). LCMS m/z calc for [M+H⁺]: 351.1, 353.1, 355.1; found: 351.0,

353.0, 355.0. HPLC (C₄, 35 min): t_R14.7 min, P_HPLC98%; HPLC (C₁₈, 35 min): t_R20.0 min, P_HPLC98%.

N-[2-(benzylmethylamino)ethyl]-2,3-dichlorobenzamide (7p)

Procedure E1. Purification by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 18%. ¹H NMR (300 MHz, CDCl₃), δ: 7.60 (d, J = 8.3 Hz, 1H, H_aro); 7.44 (d, J = 2.0 Hz, 1H, H_aro);

7.32 (dd, J = 8.4 Hz J = 2.0 Hz, 1H, H_aro); 7.30-7.25 (m, 1H, H_aro); 6.90 (br s, 1H, NH); 3.59-3.52 (m, 4H,

13

2 CH₂); 2.63 (d, J = 5.6 Hz, 2H, CH₂); 2.26 (s, 3H, NCH₃). C NMR (75 MHz, CDCl₃) δ: 165.3 (CO);

138.4 (C_aro); 136.6 (C_aro); 133.5 (C_aro); 131.6 (C_aro); 131.4 (C_aro); 130.0 (C_aro); 129.0 (2 C_aro); 128.4 (2

C_aro); 127.4 (C_aro); 127.3 (C_aro); 62.4 (CH₂); 55.0 (CH₂); 41.6 (CH₃); 37.3 (CH₂). LCMS m/z calc for

[M+H⁺]: 337.1; 341.1 ; found: 337.0; 341.0. HPLC (C₄, 35 min): t_R15.0 min, P_HPLC97%; HPLC (C₁₈, 35

min): t_R20.5 min, P_HPLC97%.

N-[3-(benzylmethylamino)propyl]-3,4-dichlorobenzamide (7q)

Procedure E2. Purification by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 31%. ¹H NMR (300 MHz, CDCl₃), δ: 8.44 (br s, 1H, NH) ; 7.82 (d, J = 2.0 Hz, 1H, H₂); 7.47 (dd, J

= 8.2 Hz, J = 2.0 Hz, 1H, H₆); 7.38 (d, J = 8.3 Hz, 1H, H₅); 7.31-7.22 (m, 5H, H_aro); 3.60 (s, 2H, CH₂);

3.56 (q, J = 6.1 Hz, 2H, CH₂); 2.68 (t, J = 6.1 Hz, 2H, CH₂); 2.33 (s, 3H, CH₃); 1.86 (p, J = 6.2 Hz, 2H,

13

CH₂). C NMR (75 MHz, CDCl₃) δ: 164.9 (CO); 135.4 (C_aro); 134.5 (C_aro); 132.9 (C_aro); 130.4 (2 C_aro);

129.5 (2 C_aro); 129.3 (C_aro); 128.6 (2 C_aro); 127.8 (C_aro); 126.1 (C_aro); 62.8 (CH₂); 56.7 (CH₂); 41.4 (CH₃);

40.3 (CH₂); 24.7 (CH₂). LCMS m/z calc for [M+H⁺]: 351.1, 353.1, 355.1; found: 350.9, 352.9, 354.9.

HPLC (C₄, 35 min): t_R15.6 min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R19.8 min, P_HPLC>99%.

N-[3-(benzylmethylamino)propyl]-3,5-dichlorobenzamide (7r)

24

Procedure E2. Purification by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

ACCEPTED MANUSCRIPT

Yield: 34%. ¹H NMR (300 MHz, CDCl₃), δ: 8.42 (br s, 1H, NH) ; 7.55 (m, 2H, H₂, H₆); 7.44 (m, 1H, H₄);

7.28-7.20 (m, 5H, H_aro); 3.57-3.51 (m, 4H, 2 CH₂); 2.63 (t, J = 6.1 Hz, 2H, CH₂); 2.32 (s, 3H, CH₃); 1.82

13

(p, J = 6.0 Hz, 2H, CH₂). C NMR (75 MHz, CDCl₃) δ: 164.6 (CO); 137.7 (C_aro); 137.5 (C_aro); 135.3

(C_aro); 130.9 (2 C_aro); 129.3 (2 C_aro); 128.5 (2 C_aro); 127.6 (C_aro); 125.7 (2 C_aro); 63.0 (CH₂); 56.7 (CH₂);

41.8 (CH₃); 40.7 (CH₂); 24.8 (CH₂). LCMS m/z calc for [M+H⁺]: 351.1, 353.1, 355.1; found: 350.9,

352.9, 354.9. HPLC (C₄, 35 min): t_R21.9 min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R20.3 min, P_HPLC

>99%.

N-[3-(benzylmethylamino)propyl]-4-bromo-2-fluorobenzamide (7s)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 22%. H NMR (300 MHz, MeOD), δ: 7.60 (d, J = 8.2 Hz, 1H, H₆); 7.50 (m, 1H, H_aro); 7.44 (m,

1H, H_aro); 7.22-7.34 (m, 5H, H_aro); 3.59 (s, 2H, CH₂); 3.43 (t, J = 6.7 Hz, 2H, CH₂); 2.56 (t, J = 7.1 Hz,

2H, CH₂); 2.28 (s, 3H, CH₃); 1.85 (p, J = 7.0 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 162.5 (CO);

132.9 (C_aro); 129.5 (C_aro); 128.7 (C_aro); 128.4 (2 C_aro); 128.1 (2 C_aro); 125.1 (C_aro); 119.8 (C_aro); 119.4

(C_aro); 62.4 (CH₂); 55.3 (CH₂); 41.4 (CH₃); 29.7 (CH₂); 25.4 (CH₂). LCMS m/z calc for [M+H⁺]: 379.1;

381.1 found: 379.0; 381.0 HPLC (C₄, 35 min): t_R15.1 min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R20.6

min, P_HPLC>99%.

N-[3-(benzylmethylamino)propyl]-3-methoxybenzamide (7t)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 35%. ¹H NMR (300 MHz, CDCl₃), δ: 8.10 (br s, 1H, NH); 7.35 (m, 1H, H₂); 7.28-7.14 (m, 7H, H₅,

H₆, H_aro); 7.11 (m, 1H, H₄); 3.78 (s, 3H, OCH₃); 3.55 (m, 4H, 2CH₂); 2.63 (t, J = 6.2 Hz, 2H, CH₂); 2.28

13

(s, 3H, NCH₃); 1.83 (p, J = 6.4 Hz, 2H, CH₂). C NMR (75 MHz, CDCl₃) δ: 165.3 (CO); 159.7 (C_aro);

137.6 (C_aro); 136.2 (C_aro); 129.4 (3 C_aro); 128.4 (2 C_aro); 127.4 (C_aro); 118.8 (C_aro); 117.6 (C_aro); 112.1

(C_aro); 63.0 (CH₂); 56.8 (CH₂); 55.3 (OCH₃); 41.5 (NCH₃); 40.2 (CH₂); 25.2 (CH₂). LCMS m/z calc for

[M+H⁺]: 313.2; found: 313.0. HRMS: calcd for (C₁₉H₂₄O₂N₂)H⁺: 313.19105; found: 313.18948. HPLC

(C₄, 35 min): t_R13.8 min, P_HPLC98%; HPLC (C₁₈, 35 min): t_R18.8 min, P_HPLC98%.

N-[3-(benzylmethylamino)propyl]-4-methoxybenzamide (7u)

Procedure E2. Purification by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 73%. H NMR (300 MHz, CDCl₃), δ: 7.97 (br s, 1H, NH) ; 7.67 (m, 2H, H₂, H₆); 7.32-7.24 (m,

5H, H_aro); 6.84 (m, 2H, H₃, H₅); 3.84 (s, 3H, OCH₃); 3.60-3.52 (m, 4H, 2 CH₂); 2.65 (t, J = 6.0 Hz, 2H,

CH₂); 2.29 (s, 3H, CH₃); 1.85 (p, J = 6.1 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 166.9 (CO); 162.0

(C_aro); 137.2 (C_aro); 129.6 (2 C_aro); 128.8 (2 C_aro); 128.5 (2 C_aro); 127.7 (C_aro); 125.0 (C_aro); 113.6 (2 C_aro);

62.9 (CH₂); 56.7 (CH₂); 55.4 (OCH₃); 41.4 (CH₃); 39.9 (CH₂); 25.3 (CH₂). LCMS m/z calc for [M+H⁺]:

25

313.2; found: 313.0. HRMS: calcd for (C₁₉H O₂N₂)H⁺: 313.19105; found: 313.18976. HPLC (C₄, 35

ACCEPT²E⁴D MANUSCRIPT

min): t_R18.1 min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R16.2 min, P_HPLC98%.

N-[3-(benzylmethylamino)propyl]-3-dimethylaminobenzamide (7v)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 11%. H NMR (300 MHz, CDCl₃), δ: 7.90 (br s, 1H, NH); 7.28-7.24 (m, 5H, H_aro); 7.22-7.19 (m,

2H, H₂, H₅); 6.92 (d, J = 7.5 Hz, 1H, H₆); 6.82 (dd, J = 8.1 Hz, J = 2.2 Hz, 1H, H₄); 3.62-3.51 (m, 4H, 2

CH₂); 2.95 (s, 6H, 2 CH₃); 2.64 (t, J = 6.0 Hz, 2H, CH₂); 2.31 (s, 3H, CH₃); 1.85 (p, J = 5.8 Hz, 2H, CH₂).

¹³C NMR (75 MHz, CDCl₃) δ: 162.4 (CO); 138.5 (C_aro); 133.1 (2 C_aro); 129.0 (2 C_aro); 128.2 (2 C_aro);

128.0 (2 C_aro); 127.1 (C_aro); 119.7 (C_aro); 119.4 (C_aro); 63.0 (CH₂); 55.9 (CH₂); 50.9 (CH₂); 41.9 (3 CH₃);

39.9 (CH₂); 25.6 (CH₂). LCMS m/z calc for [M+H⁺]: 326.2; found: 326.0. HPLC (C₄, 35 min): t_R14.5

min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R19.8 min, P_HPLC>99%.

N-[2-(benzylmethylamino)ethyl]-4-cyanobenzamide (7w)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 33%. H NMR (300 MHz, CDCl₃), δ: 8.00 (d, 2H, J = 7.3 Hz, 2H, H_aro); 7.57 (d, 2H, J = 7.4 Hz,

2H, H_aro); 7.30-7.24 (m, 5 H, H_aro); 6.71 (br s, 1H, NH); 3.53 (s, 2H, CH₂); 3.00 (t, 2H, J = 5.5 Hz, CH₂);

2.65 (t, 2H, J = 5.7 Hz, CH₂); 2.18 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 171.4 (CO); 137.5 (C_aro);

131.7 (2 C_aro); 129.8 (2 C_aro); 129.0 (2 C_aro); 128.5 (2 C_aro); 127.5 (C_aro); 118.7 (C_aro); 114.0 (C_aro); 62.2

(CH₂); 54.7 (CH₂); 41.6 (CH₃); 37.0 (CH₂). LCMS m/z calc for [M+H⁺]: 294.1; found: 294.0. HRMS:

calcd for (C₁₈H₁₉ON₃)H⁺: 294.16009; found: 294.15906. HPLC (C₄, 35 min): t_R12.7 min, P_HPLC97%;

HPLC (C₁₈, 35 min): t_R17.3 min, P_HPLC97%.

N-[3-(benzylmethylamino)propyl]-4-cyanobenzamide (7x)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 7%. ¹H NMR (300 MHz, CDCl₃), δ: 8.61 (br s, 1H, NH); 7.71 (d, J = 8.2 Hz, 2H, H₃, H₅); 7.61 (d,

J = 8.3 Hz, 2H, H₂, H₆); 7.30-7.20 (m, 5H, H_aro); 3.62 (q, J = 6.4 Hz, 2H, CH₂); 3.55 (s, 2H, CH₂); 2.69 (t,

13

J = 5.8 Hz, 2H, CH₂); 2.27 (s, 3H, CH₃); 1.84 (p, J = 6.0 Hz, 2H, CH₂). C NMR (75 MHz, CDCl₃) δ:

165.3 (CO); 138.4 (C_aro); 136.8 (C_aro); 132.2 (2 C_aro); 129.6 (2 C_aro); 128.6 (2 C_aro); 127.7 (2 C_aro); 118.2

(C_aro); 114.6 (C_aro); 63.3 (CH₂); 57.6 (CH₂); 41.5 (CH₃); 40.9 (CH₂); 25.1 (CH₂). LCMS m/z calc for

[M+H⁺]: 308.1; found: 308.0. HPLC (C₄, 35 min): t_R13.2 min, P_HPLC98%; HPLC (C₁₈, 35 min): t_R18.0

min, P_HPLC>99%.

N-[2-(benzylmethylamino)ethyl]-4-nitrobenzamide (7y)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 52%. ¹H NMR (300 MHz, CDCl₃), δ: 8.30 (d, J = 8.8 Hz, 2H, H_aro); 7.85 (d, J = 8.7 Hz, 2H, H_aro);

26

7.34-7.28 (m, 5H, H_aro); 6.85 (br s, 1H, NH); 3.58 (s, 2H, CH ); 3.53 (q J = 4.9 Hz, 2H, CH₂); 2.64 (t, J =

ACCEPTED MANUS²CRIPT

5.6 Hz, 2H, CH₂); 2.34 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 165.2 (CO); 149.5 (C_aro); 140.3

(C_aro); 138.6 (C_aro); 129.1 (2 C_aro); 128.8 (2 C_aro); 128.5 (2 C_aro); 127.5 (C_aro); 123.8 (2 C_aro); 62.3 (CH₂);

54.6 (CH₂); 42.2 (CH₃); 37.3 (CH₂). LCMS (ESI⁺): calc for [M+H⁺]: 314.1; found: 314.1. HRMS: calcd

for (C₁₇H₁₉O₃N₃)H⁺: 314.14992; found: 314.14905. HPLC (C₄, 35 min): t_R15.1 min, P_HPLC>99%; HPLC

(C₁₈, 35 min): t_R20.6 min, P_HPLC>99%.

N-[3-(benzylmethylamino)propyl]-4-nitrobenzamide (7z)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 50%. H NMR (300 MHz, CDCl₃), δ: 8.68 (br s, 1H, NH); 8.17 (d, J = 8.8 Hz, 2H, H₃, H₅); 7.79

(d, J = 8.8 Hz, 2H, H₂, H₆); 7.32-7.22 (m, 5H, H_aro); 3.65-3.56 (m, 4H, 2CH₂); 2.72 (t, J = 5.6 Hz, 2H,

13

CH₂); 2.32 (s, 3H, NCH₃); 1.88 (p, J = 5.7 Hz, 2H, CH₂). C NMR (75 MHz, CDCl₃) δ: 166.7 (CO);

149.3 (C_aro); 140.1 (C_aro); 137.1 (C_aro); 129.6 (2 C_aro); 128.6 (2 C_aro); 128.1 (2 C_aro); 127.8 (C_aro); 123.6 (2

C_aro); 63.5 (CH₂); 57.6 (CH₂); 41.7 (CH₃); 41.1 (CH₂); 25.0 (CH₂). LCMS m/z calc for [M+H⁺]: 328.1;

found: 328.0. HRMS: calcd for (C₁₈H₂₁O₃N₃)H⁺: 328.16557; found: 328.16388. HPLC (C₄, 35 min): t_R

13.6 min, P_HPLC96%; HPLC (C₁₈, 35 min): t_R18.6 min, P_HPLC96%.

N-[3-(N-methyl-2-phenylethylamino)propyl]-4-chlorobenzamide (8a)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 69%. H NMR (300 MHz, CDCl₃), δ: 8.25 (br s, 1H, NH); 7.58 (d, J = 8.3 Hz, 2H, H₂, H₆); 7.34

(d, J = 8.4 Hz, 2H, H₃, H₅); 7.26-7.12 (m, 5H, H_aro); 3.53 (q, J = 6.2 Hz, 2H, CH₂); 2.82 (m, 2H, CH₂);

2.75 (m, 2H, CH₂); 2.66 (t, J = 6.0 Hz, 2H, CH₂); 2.35 (s, 3H, NCH₃); 1.80 (p, J = 5.9 Hz, 2H, CH₂). ¹³C

NMR (75 MHz, CDCl₃) δ: 165.9 (CO); 139.5 (C_aro); 137.2 (C_aro); 133.1 (C_aro); 128.6 (4 C_aro); 128.5 (2

C_aro); 128.3 (2 C_aro); 126.3 (C_aro); 59.6 (CH₂); 57.1 (CH₂); 41.7 (CH₃); 40.4 (CH₂); 33.3 (CH₂); 24.3

(CH₂). LCMS m/z calc for [M+H⁺]: 331.1, 333.1; found: 331.0, 333.0. HRMS: calcd for

(C₁₉H₂₃ON₂Cl)H⁺: 331.15717; found: 331.15601. HPLC (C₄, 35 min): t_R15.5 min, P_HPLC>99%; HPLC

(C₁₈, 35 min): t_R21.2 min, P_HPLC>99%.

N-[3-(isoindolin-2-yl)methylamino)propyl]-4-chlorobenzamide (8b)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 36%. ¹H NMR (300 MHz, CDCl₃), δ: 9.00 (br s, 1H, NH); 7.48 (d, J = 8.6 Hz, 2H, H₂, H₆); 7.31-

7.21 (m, 4H, H_aro); 7.03 (d, J = 8.5 Hz, 2H, H₃, H₅); 4.02 (s, 4H, 2CH₂); 3.63 (q, J = 5.0 Hz, 2H, CH₂);

3.05 (t, J = 5.8 Hz, 2H, CH₂); 1.89 (p, J = 5.8 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 165.6 (CO);

139.2 (2 C_aro); 137.1 (C_aro); 132.6 (C_aro); 128.4 (2 C_aro); 128.2 (2 C_aro); 127.2 (2 C_aro); 122.4 (2 C_aro); 58.6

(2 CH₂); 55.3 (CH₂); 40.8 (CH₂); 25.9 (CH₂). LCMS m/z calc for [M+H⁺]: 315.1, 317.1; found: 315.0,

317.0. HPLC (C₄, 35 min): t_R14.6 min, P_HPLC97%; HPLC (C₁₈, 35 min): t_R20.0 min, P_HPLC98%.

27

ACCEPTED MANUSCRIPT

N-[2-pyrrolidinylethyl]-4-chlorobenzamide (8c)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 22%. H NMR (300 MHz, CDCl₃), δ: 7.82 (d, J = 8.5 Hz, 2H, H₂, H₆); 7.51 (br s, 1H, NH); 7.48

(d, J = 8.5 Hz, 2H, H₃, H₅); 3.63 (q, J = 6.1 Hz, 2H, CH₂); 2.91 (t, J = 6.0 Hz, 2H, CH₂); 2.78 (m, 4H,

2CH₂); 1.90 (m, 4H, 2CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 165.6 (CO); 137.6 (C_aro); 132.6 (C_aro); 128.7

(4 C_aro); 54.9 (CH₂); 54.0 (2 CH₂); 37.9 (CH₂); 23.4 (2 CH₂). LCMS m/z calc for [M+H⁺]: 253.1, 255.1;

found: 253.0, 255.0. HPLC (C₄, 35 min): t_R12.1 min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R16.5 min,

P_HPLC>99%.

N-[3-pyrrolidinylpropyl]-4-chlorobenzamide (8d)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 7%. ¹H NMR (300 MHz, CDCl₃), δ: 8.48 (br s, 1H, NH); 7.96 (d, J = 8.8 Hz, 2H, H₂, H₆); 7.39 (d,

J = 8.8 Hz, 2H, H₃, H₅); 3.77 (m, 2H, CH₂); 3.63 (m, 2H, CH₂); 3.18 (q, J = 6.1 Hz, 2H, CH₂); 2.84 (m,

2H, CH₂); 2.37-1.88 (m, 6H, 3 CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 166.7 (CO); 137.8 (C_aro); 131.9

(C_aro); 129.3 (2 C_aro); 129.1 (2 C_aro); 54.1 (2 CH₂); 53.0 (CH₂); 36.7 (CH₂); 25.6 (CH₂); 23.6 (2 CH₂).

LCMS m/z calc for [M+H⁺]: 267.1, 269.1; found: 267.0, 269.0. HPLC (C₄, 35 min): t_R12.7 min, P_HPLC

98%; HPLC (C₁₈, 35 min): t_R17.3 min, P_HPLC98%.

N-[2-morpholinethyl]-4-chlorobenzamide (8e)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 35%. ¹H NMR (300 MHz, CDCl₃), δ: 7.66 (d, J = 8.6 Hz, 2H, H₂, H₆); 7.32 (d, J = 8.6 Hz, 2H, H₃,

H₅); 6.99 (br s, 1H, NH); 3.65 (m, 4H, 2CH₂);3.45 (q, J = 6.0 Hz, 2H, CH₂); 2.53 (t, J = 6.2 Hz, 2H, CH₂);

2.42 (m, 4H, 2CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 166.4 (CO); 137.5 (C_aro);132.9 (C_aro); 128.7 (2 C_aro);

128.4 (2 C_aro); 66.9 (2 CH₂); 56.9 (CH₂); 53.3 (2 CH₂); 36.2 (CH₂). LCMS m/z calc for [M+H⁺]: 269.1,

271.1; found: 269.0, 271.0. HPLC (C₄, 35 min): t_R12.2 min, P_HPLC98%; HPLC (C₁₈, 35 min): t_R16.6

min, P_HPLC98%.

N-[3-morpholinpropyl]-4-chlorobenzamide (8f)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 42%. H NMR (300 MHz, CDCl₃), δ: 8.09 (br s, 1H, NH); 7.73 (d, J = 8.6 Hz, 2H, H₂, H₆); 7.48

(d, J = 8.6 Hz, 2H, H₃, H₅); 3.67 (m, 4H, 2CH₂); 3.52 (q, J = 6.1 Hz, 2H, CH₂); 2.58-2.43 (m, 6H, 3CH₂);

1.76 (p, J = 6.1 Hz, 2H, CH₂). ¹³C NMR (75 MHz, CDCl₃) δ: 166.8 (CO); 137.8 (C_aro); 133.5 (C_aro); 129.1

(2 C_aro); 128.8 (2 C_aro); 67.2 (2 CH₂); 58.7 (CH₂); 54.4 (2 CH₂); 40.8 (CH₂); 24.5 (CH₂). LCMS m/z calc

for [M+H⁺]: 283.1, 285.1; found: 283.0, 285.0. HPLC (C₄, 35 min): t_R11.5 min, P_HPLC98%; HPLC (C₁₈,

35 min): t_R15.7 min, P_HPLC99%.

28

ACCEPTED MANUSCRIPT

N-[2-piperazinylethyl]-4-chlorobenzamide (8g)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

Yield: 42%. ¹H NMR (300 MHz, CDCl₃), δ: 7.72 (d, J = 7.9 Hz, 2H, H₂, H₆); 7.37 (d, J = 7.9 Hz, 2H, H₃,

H₅); 6.94 (br s, 1H, NH); 3.54 (q, J = 5.5 Hz, 2H, CH₂); 2.67-2.48 (m, 10H, 5CH₂); 2.32 (s, 3H, NCH₃).

¹³C NMR (75 MHz, CDCl₃) δ: 166.3 (CO); 137.6 (C_aro); 132.9 (C_aro); 128.7 (2 C_aro); 128.4 (2 C_aro); 56.2

(CH₂); 54.8 (2 CH₂); 52.5 (2 CH₂); 45.8 (CH₃); 36.4 (CH₂). LCMS m/z calc for [M+H⁺]: 282.1, 284.1;

found: 282.0, 284.0. HPLC (C₄, 35 min): t_R13.0 min, P_HPLC96%; HPLC (C₁₈, 35 min): t_R17.8 min, P_HPLC

96%.

N-[3-piperazinylpropyl]-4-chlorobenzamide (8h)

Procedure E1. Purification by column chromatography (DCM:MeOH(NH₃), 9:1 (v/v)) was performed.

1

Yield: 43%. H NMR (300 MHz, CDCl₃), δ: 8.28 (br s, 1H, NH); 7.78 (d, J = 8.5 Hz, 2H, H₂, H₆); 7.39

(d, J = 8.5 Hz, 2H, H₃, H₅); 3.56 (q, J = 5.5 Hz, 2H, CH₂); 2.62 (t, J = 5.7 Hz, 2H, CH₂); 2.58 -2.42 (m,

13

8H, 4CH₂); 2.32 (s, 3H, NCH₃); 1.81 (p, J = 5.8 Hz, 2H, CH₂). C NMR (75 MHz, CDCl₃) δ: 166.3

(CO); 137.4 (C_aro); 133.1 (C_aro); 128.6 (4 C_aro); 57.9 (CH₂); 54.7 (2 CH₂); 52.9 (2 CH₂); 45.9 (CH₃); 40.5

(CH₂); 24.1 (CH₂). LCMS m/z calc for [M+H⁺]: 296.1, 298.1; found: 296.0, 298.0. HPLC (C₄, 35 min): t_R

11.6 min, P_HPLC95%; HPLC (C₁₈, 35 min): t_R15.9 min, P_HPLC95%.

N-[3-(benzylmethylamino)propyl]-4-chlorobenzensulfonamide (9)

Procedure E1. Purification by column chromatography (petroleum ether:DCM:MeOH(NH₃), 5:4:1 (v/v))

was performed. Yield: 60%. IR (cm^-1): 1323 and 1156 (SO₂). ¹H NMR (300 MHz, CDCl₃), δ: 7.76 (d, J =

9.1 Hz, 2H, H₂, H₆); 7.47(d, J = 9.2 Hz, 2H, H₃, H₅); 7.36-7.22 (m, 5H, H_aro); 3.46 (s, 2H, CH₂); 3.05 (t, J

= 7.2 Hz, 2H, CH₂); 2.45 (t, J = 7.1 Hz, 2H, CH₂); 2.18 (s, 3H, CH₃); 1.67 (p, J = 7.0 Hz, 2H, CH₂). ¹³C

NMR (75 MHz, CDCl₃) δ: 138.8 (C_aro); 138.7 (C_aro); 137.9 (C_aro); 129.3 (2 C_aro); 129.0 (2 C_aro); 128.6 (2

C_aro); 128.5 (2 C_aro); 127.4 (C_aro); 62.8 (CH₂); 56.8 (CH₂); 44.0 (CH₃); 41.9 (CH₂); 24.7 (CH₂). LCMS m/z

calc for [M+H⁺]: 353.1, 355.1; found: 353.0, 355.0. HRMS: calcd for (C₁₇H₂₁O₂N₂ClS)H⁺: 353.10850;

found: 353.10782. HPLC (C₄, 35 min): t_R13.8 min, P_HPLC>99%; HPLC (C₁₈, 35 min): t_R18.6 min, P_HPLC

>99%.

N-(4-chlorobenzyl)-3-(benzylmethylamino)propanamine (10)

A solution of 4-chlorobenzaldehyde (0.55 mmol, 77 mg) and N¹-benzyl-N¹-methylpropane-1,3-diamine

5a (0.55 mmol, 100 mg) in MeOH (10 mL) was stirred at room temperature for 16 hours. Then, sodium

borohydride (0.82 mmol, 32 mg) was added and the reaction media was stirred for 3 hours. The mixture

was quenched with water (5 mL) and evaporated under reduce pressure. The crude product was purified

1

by thick layer chromatography (DCM:MeOH(NH₃), 9:1 (v/v)). Yield: 35%. IR (cm^-1): 2950 (NH). H

29

Article Doi

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

DOI: 10.1016/j.ejmech.2017.07.014

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Article abstract of DOI:10.1016/j.ejmech.2017.07.014

Full text of DOI:10.1016/j.ejmech.2017.07.014

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