Piperazines as nootropic agents: New derivatives of the potent cognition-enhancer DM235 carrying hydrophilic substituents

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

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Piperazines as nootropic agents: New derivatives of the potent cognition-enhancer DM235 carrying hydrophilic substituents Maria Vittoria Martino a, Luca Guandalini a, Lorenzo Di Cesare Mannelli b, Marta Menicatti a, Gianluca Bartolucci a, Silvia Dei a, Dina Manetti a, Elisabetta Teodori a, Carla Ghelardini b, Maria Novella Romanelli a,⇑ a University of Florence, Department of Neuroscience, Psychology, Drug Research and Child’s Health, Section of Pharmaceutical and Nutraceutical Sciences, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy b University of Florence, Department of Neuroscience, Psychology, Drug Research and Child’s Health, Section of Pharmacology and Toxicology, Viale Gaetano Pieraccini 6, 50100 Florence, Italy

a r t i c l e

i n f o

Article history: Received 19 December 2016 Revised 23 January 2017 Accepted 24 January 2017 Available online xxxx Keywords: Sunifiram Cognition-enhancers Piracetam-like compounds Mouse passive-avoidance test Piperazines

a b s t r a c t The piperazine ring of the potent nootropic drug DM235 has been decorated with H-bond donor and acceptor groups (CH2OH, CH2OMe, CH2OCOMe, COOEt); the aim was to insert new functional groups, suitable for further chemical manipulation. The influence of these modifications on nootropic activity was assessed by means of the mouse passive avoidance test; some of the newly synthesized molecules (alcohol 7b, acetate 8b and ester 10d) showed interesting in vivo potency. This makes it possible to use these functional groups for adding other residues, in order to increase molecular diversity, or for anchoring a biotin group, to obtain compounds useful to capture the biological target. Moreover, the new compounds will improve our knowledge of structure activity relationships of this family of drugs. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Cognitive enhancers are compounds able to improve the physiological processes related to cognition, such as attention, concentration, memory and learning; they are used as a pharmacological treatment of cognitive deficits.1 Memory enhancement is particularly important, since a loss of memory is one of the consequences of ageing and characterizes disorders such as Alzheimer’s disease (AD) and other forms of dementia, or conditions such as Mild Cognitive Impairment. Several strategies to improve cognition are presently under investigation, and new ones are being searched for, since very often the promising results found in animal models had not translated into therapeutics.2,3 Some years ago we discovered a new class of nootropics, exemplified by DM2324 (Unifiram) and DM235 (Sunifiram)5 (respectively, 1 and 2, Chart 1), which were able to revert scopolamine induced amnesia in rodents. DM232 contains the 2-oxopirrolidine ring, and for this reason can be formally related to the well-known nootropic piracetam; however, differently from piracetam-like compounds, this ring is not essential for activity, since the ⇑ Corresponding author. E-mail address: [email protected] (M.N. Romanelli).

seco-derivative DM235 is equipotent with 1. DM232 and DM235 were the most active among a large series of compounds synthesized within this research, with several other structural analogues showing very close potency, as for instance 3 or 4 (Chart 1)5,6; high potency was associated to the acyl and sulfonyl groups that decorate compounds 1–4. There is evidence that the nootropic activity of 1 and 2 involves cholinergic and glutamatergic transmission7,8; however, both compounds, as well as piracetam, did not show affinity for these receptors, nor for the most common neurotransmitter receptors, transporters and ion channels. This has made difficult to discover their mechanism of action: as a matter of fact, despite the interesting biological profile of these molecules, their development has been hampered because their biological target has not been elucidated. A recent paper has reviewed the medicinal chemistry of this class of compounds.9 The necessity to screen the compounds in vivo, owing to the lack of knowledge of the biological target, has made difficult to establish robust structure-activity relationships, thus complicating the optimization of the lead compounds. Nevertheless, many related compounds were found to show nootropic activity at doses below 1 mg kg
1, demonstrating that this class of compounds is endowed with fairly good potency. The initial work, mainly focused on the discovery of the appropriate acyl

http://dx.doi.org/10.1016/j.bmc.2017.02.019 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx O

O

O

Et N

N

O

N

N O2 S

N NH 2

O

1 DM232 MED 0.001 mg/kg ip

F

O

HN

Me

O2 S

12a

O

Et N R1

N

N

O2 S O

b

Me H N

R1

9a R=Me

COPh N

c

a

d

OR

e

N COEt

N COEt

8b R=Ac 9b R=Me

7b (R = H)

10b

COOEt

8a R=Ac

e

COPh N COOEt

12b

N

OR

7a (R = H)

COOEt

N COEt

d

N COEt

a

F

F

F

N O2 S

Ph F

F

H N

3 MED 0.01 mg/kg ip

COPh N

c

COOEt N COEt 10a

COOEt

F

Piracetam MED 30 mg/kg ip O

4 MED 0.01 mg/kg ip

N H

N

N

b

O

2 DM235 MED 0.001 mg/kg ip

Me

COPh

COPh N

N

N H 11

R 1 = Me (5a-d ), Ph (6a-d),CH 2OH (7a-d), CH2 OCOCH 3 (8a-d ), CH2 OCH 3(9a-d), COOEt (10a-d ),

O 2S N

f

N H 12c

d

O 2S N

c

N

COOEt

H N

COOEt

O 2S N

N COMe 12d

OR

N

COOEt

COMe

10c

7c (R = H)

e

8c R=Ac 9c R=Me

F O 2S N

COOEt

f

d

COMe

F

Chart 1.

and sulfonyl groups on the amidic N atoms, was followed by structural changes of the piperazine ring, which was varied in size and in the position of the amide linkages; another modification was the attachment of lipophilic substituents, such as a methyl or phenyl group, close to the amide moiety (compounds 5 and 6, Chart 1). As a continuation of this research, we have prepared and tested a series of 2- or 3-subsituted piperazines carrying a polar oxygencontaining substituent (compounds 7a-d–10a-d, Chart 1); this modification was based on the hypothesis that a group able to establish, within the binding site, a strong interaction such as H bond, could possibly bring an increase of affinity when compared to the methyl or phenyl group of compounds 5 and 6. At the same time, hydroxymethyl and carboxylic groups, if well tolerated, could be a possible anchor point for the introduction of other residues, to increase molecular diversity, or for the attachment of fragments like biotin, to obtain compounds useful to capture the biological target. Following this reasoning, both H-bond donor (CH2OH) and acceptor (CH2OMe, CH2OCOMe, COOEt) groups were inserted. Since the acetoxy and carbethoxy groups could be vulnerable to hydrolysis, their stability was checked in rat plasma; anyway, we thought that both ester functions, even if not completely stable, could improve diffusion into the CNS.

O 2S N

d

c N COMe 10d

d OR

N COMe 7d (R = H)

e

8d R=Ac 9d R=Me

Reagents: a) PhCOCl, Et 3N; b) C 2H 5COCl, Et3 N; c) NaBH 4, MeOH, THF; d) CH3 COCl, Et3 N; e) CH3 I, NaH; f) 4-F-C 6 H4 SO2 Cl, Et3 N.

Scheme 1.

were obtained starting from D and L serine methyl esters, which were transformed into (S) and (R)(4-benzylpiperazin-2-yl)methanol 13 according to Naylor.11 To prepare enantiopure 7b (Scheme 2), (S)-1311 was treated with benzoyl chloride to give (S)-14; catalytic hydrogenation gave (S)-15 which was reacted with propionyl chloride to give (S)-7b. Treatment of (S)-13 with 4-fluorobenzenesolfonyl chloride gave (S)-16; removal of the protecting group by catalytic hydrogenation and reaction with acetyl chloride gave (S)-8d. Starting from (R)-13, (R)-7b and (R)-8d were obtained in the same way.

2.2. Passive-avoidance test 2. Results and discussion 2.1. Chemistry The synthesis of compounds 7a-d–10a-d is reported in Scheme 1. At first racemic mixtures were prepared, limiting the synthesis of enantiomers to the most interesting derivatives. Therefore, ethyl piperazine-2-carboxylate 11 was chosen as starting point and prepared according to Rudolf10; then, it was sequentially treated with the suitable acyl or sulfonyl chlorides to give 10a-d through intermediate compounds 12a-d. In the first step, the reaction was performed with less than 1 equivalent of the suitable acyl or sulfonyl chloride, to minimize the double addition product, so the yields of 12a-d were between 44 and 68%. Reduction of the ester function of derivatives 10a-d with sodium borohydride gave alcohols 7a-d, which were transformed into ester 8a-d by reaction with acetyl chloride and into ethers 9a-d by reaction with sodium hydride and methyl iodide. Since some of the new compounds displayed interesting activity (7b and 8d, Table 1), their enantiomers were also prepared in order to study enantioselectivity. The enantiomers of 7b and 8d

The compounds were tested in the mouse passive-avoidance test of Jarvik and Kopp,12 using a procedure slightly modified by us.13 The ability of the synthesized compounds to revert scopolamine-induced amnesia is reported in Table 1. The results are expressed as the Minimal Effective Dose (MED, mg kg
1); compounds 5a-d and 6a-d are shown for comparison. The compounds were tested in 1:10 dilutions, up to the dose of 10 mg kg
1; details of the experiments and statistical analysis are shown in Table S1 (Supplementary Data). With the exception of 8b, 8c and 9c, all compounds are able to revert amnesia induced by the muscarinic antagonist scopolamine, with MED 3-300 times lower with respect to piracetam. The data reported in Table 1 show that a hydroxymethyl group is better tolerated when it is close to the aromatic amide moiety (7b and 7d, MED 0.1 and 1.0 mg kg
1, respectively) than when it is close to the acetyl or propionyl groups (7a and 7c, MED 10 mg kg
1). This is similar to what happened for the methyl derivatives 5a-d, but different with respect to 6a-d, for which the best position of the phenyl ring was close to the propionyl moiety. The presence of a OH group still allows brain penetration, since alcohols 7a-d displayed activity in the passive-avoidance test; this modification

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Table 1 Minimal Effective Dose (MED, mg kg
1),a Clog P values and plasma half life of compounds 7a-d–10a-d in the mouse passive-avoidance test. Compounds 5a-d and 6a-d are reported for comparison.

COEt N

X

N

Y

X Y MED Clog Pd t1/2 (min)

5ab

6ac

7a

8a

9a

10a

5bb

6bc

7b

8b

9b

10b

CH3 H >10 2.53 n.d.

Ph H 0.1 3.56 >240f

CH2OH H 10 1.58 >240e

CH2OAc H 1.0 2.06 154e

CH2OMe H 10 2.03 >240e

COOEt H 1.0 2.06 12e

H CH3 0.1 2.53 n.d.

H Ph 1.0 3.56 >240f

H CH2OH 0.1 1.58 >240e

H CH2OAc >10 2.06 100e

H CH2OMe 1.0 2.03 >240e

H COOEt 1.0 2.06 26e

5cb

6cc

7c

8c

9c

10c

5db

6dc

7d

8d

9d

10d

CH3 H 1.0 1.16 n.d.

Ph H >10 2.19 >240f

CH2OH H 10 0.20 >240e

CH2OAc H >10 0.69 30e

CH2OMe H >10 0.66 >240e

COOEt H 1.0 0.69 37e

H CH3 0.1 1.16 n.d.

H Ph 10 2.19 >240f

H CH2OH 1.0 0.20 >240e

H CH2OAc 0.1 0.69 29e

H CH2OMe 1.0 0.66 >240e

H COOEt 0.1 0.69 21e

O

COMe

X Y MED Clog Pd t1/2 (min)

X

N

N Y SO2 F

n.d.: not determined. a All compounds were dissolved in saline and injected s.c. 20 min before the training session; each value represents the mean of 12–14 mice. Scopolamine (S, 1.5 mg kg
1 i.p.) was injected immediately after punishment. b From Ref. 5. c From Ref. 14. d Calculated at www.rdchemicals.com/drug-relevant-properties.html. e Determined in rat plasma by extrapolation of the concentration-time curves. f Stability measured in human plasma; see Ref. 14.

Ph

OH

H N

O N

a N

Ph

OH

N O

(S)-15

(S)-14

Ph OH

c

N H

Ph

(S)- 13

O

OH

N

N

b

N Ph

O

Et

(S)-7b

d F

O O S N

OH

N Ph (S)-16

b

O O S N

F

OH

e

N H

O O

Me

N Me

(S)-17

F

O O S N

O

(S)-8d

Reagents: a) PhCOCl, Et 3N; b) H 2/Pd/C; c) EtCOCl, Et 3N; d) 4F-C 6H 4SO 2Cl, Et 3 N; e) CH 3COCl, NEt 3. The same sequence has been used to prepare (R)-7b and (R)-8d, starting from (R)-13.

If the results reported in Table 1 are viewed according to the position of the substituent (a-d series), it is possible to see that a small group on the piperazine ring (compounds 5a-d and 7a-d – 9a-d) is better tolerated when closed to the sulfonamide or benzamide functions (d and b series, respectively); as a matter of fact, when it is closed to the aliphatic amide function (a and c series), MED values are often higher than 1 mg kg
1. Exception to this trend is acetate 8b, whose MED is higher than 10 mg kg
1. For the phenyl derivatives 6a-d, which show a different order of activity, it was suggested that the binding mode could be affected by conformational factors driven by the position of the phenyl ring.14 Due to the good potency of 7b and 8d, the R and S forms were prepared and tested, but in both cases no difference in potency was observed for the enantiomers (Table S1, Supplementary Data). This is not surprising since in this class of compounds a low enantioselectivity was detected in some instances,14,15 while in others it was absent.13,16 We did not further investigate the possible reason (pharmacokinetic, pharmacodynamic or both) for this finding.

Scheme 2.

2.3. Physicochemical and pharmacokinetic properties improves activity in the a series (7a, MED 10 mg kg
1, compared to 5a, MED > 10 mg kg
1), while in the other series (b-d) the activity is decreased or left unchanged. Methylation of the alcoholic group does not improve activity, since ethers 9a-d are equally or less potent than alcohols 7a-d. It seems therefore that the H-bond donor or acceptor properties in this part of the molecule are not important for the interaction with the biological target; another possible explanation may be that the possible increase of potency is balanced by an increase of polarity and, as a consequence, by a reduction of brain concentration. Acetylation of the alcoholic group is productive for 8a and 8d, which are ten times more active than their alcohol analogues 7a and 7d, but not for 8b and 8c, which are inactive (MED > 10 mg kg
1). On the contrary, a carbethoxy function seems well tolerated on all the four series: the most active ethyl ester is 10d (MED 0.1 mg kg
1), but 10a-c show MED values only ten times higher.

To take into account the possible influence of physicochemical properties, CLogP values were calculated using a web facility (Table 1), and the variation of lipophilicity among the a-d series was compared. As expected, sulfonamides (c and d series) are more hydrophilic than acyl amides (a and b series), but no correlation can be found between ClogP and MED. However, although compounds 7–10 are less lipophilic with respect to 5–6, an activity 1 mg kg
1 is an indirect indication that the compounds enter the SNC in sufficient amount. Since the compounds have been parenterally administered, thus avoiding a first-pass metabolism, we thought that plasma stability could be an important process determining the amount of compound available for CNS penetration. Since some of the substances carry a labile ester function, their stability in rat plasma and in phosphate buffer has been measured using a previously published protocol.14 Compounds were monitored over a period of 2 h, taking samples at 0, 30, 60 and 120 min. Enalapril was used

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

Fig. 1. Chromatograms showing the degradation of acetates 8a (left) and 8b (right) at t = 0 (red) and t = 120 min (blue). ISTD = Internal Standard.

as reference, to check the hydrolytic activity of the employed rat plasma batch: under these experimental conditions, its half-life was <2 h. All compounds, as well as the reference substances, were stable in phosphate buffer; hydroxymethyl and methoxy derivatives (7a-d and 9a-d, respectively) showed no appreciable degradation also in rat plasma (Table 1). These experiments confirm the stability of the piperazine amidic moiety in plasma, already found for compounds 6a-d.14 On the contrary, esters 8a-d and 10a-d underwent extensive hydrolysis. Carbethoxy derivatives 10a-d are rapidly converted into the corresponding acids (Table 1): after 60 min their concentration dropped well below 50%. Acetates 8a-d are transformed into the hydroxymethyl derivative 7a-d, at a rate which is different depending on the kind of amide moiety: benzamides 8a and 8b seems more resistant to hydrolysis than sulfonamides 8c and 8d, since the amount of recovered ester after 2 h is very low for 8c and 8d (10%), but higher for 8a and 8b (60% and 50%, respectively; Fig. 1). Although plasma stability is only one of the many processes affecting the amount of compound crossing the blood-brain barrier, these preliminary data suggest that the inactivity of 8b and 8c may not be due only to plasma degradation, since their products of hydrolysis (7b and 7c) shows activity in the passive-avoidance test. On the other hand, since 8d, one of the most active among the newly synthesized compounds, undergoes extensive plasma degradation, its interaction with the biological target could even be stronger than what is apparent from its MED. A similar reasoning can be done for the carbethoxy derivatives, since a COOH group largely limits passive diffusion into the CNS. Obviously, other mechanisms, such as active transport, might be operative. However, the good in vivo potency of 10a-d suggests that these substances could be interesting lead compounds for further structural variations.

3. Conclusions In conclusion, we have synthesized a new series of piperazines, some of which show interesting nootropic potency (MED  1 mg kg
1) in the mouse passive avoidance test. The new compounds allow to derive further structure-activity relationships: in particular it was found that a hydroxymethyl moiety (as in compound 7a) and a carbethoxy group (compounds 10a-d) are well tolerated. Therefore, both groups could represent a new site for further functionalization, to increase molecular diversity or to add a suitably linked biotin residue, which could help to isolate the biological target, and to clarify the mechanism of action of this class of nootropics. This work is underway, and it will reported in due time.

4. Experimental 4.1. Chemistry All melting points were taken on a Büchi apparatus and are uncorrected. NMR spectra were recorded on a Brucker Avance 400 spectrometer (400 MHz for 1H NMR, 100 MHz for 13C). Chromatographic separations were performed on a silica gel column by gravity chromatography (Kieselgel 40, 0.063–0.200 mm; Merck) or flash chromatography (Kieselgel 40, 0.040–0.063 mm; Merck). Yields are given after purification, unless differently stated. When reactions were performed under anhydrous conditions, the mixtures were maintained under nitrogen. High resolution MS analysis were determined with a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with an electrospray ionization source (ESI). Analysis were carried out in positive ion mode observing protonated molecules [M+H]+ using a proper dwell time acquisition to achieve 30,000 units of resolution at Full Width at Half Maximum (FWHM). Elemental composition of compounds were calculated on the basis of their measured accurate masses, accepting only results with an attribution error less than 5 ppm and a not integer RDB (double bond/ring equivalents) value, in order to consider only the protonated species (Table S5, Supplementary Material).17 Compounds were named following IUPAC rules as applied by Reaxys (version 1.0.9619) software. 4.1.1. General procedure for the synthesis of acyl and sulfonyl amides To a solution of the suitable amine (0.3 g, 1 eq) and Et3N (1.1 eq) in anhydrous CH2Cl2 (5 mL), at 0 °C and under N2 atmosphere, the suitable acyl or sulfonyl chloride (0.7 eq for the synthesis of 12a-d, 1 eq for the synthesis of the other derivatives) in anhydrous CH2Cl2 (7 mL) was slowly added dropwise. The reaction was left stirring at RT for 2 h; for the synthesis of 12a-d the reaction was stopped when from TLC there was evidence for the formation of the product of double addition. The mixture was then washed twice with sat. aq. NaHCO3; after drying (Na2SO4), the solvent was removed under vacuum, and the residue was purified by flash chromatography. By this method the following compounds were obtained: 4.1.1.1. Ethyl 4-benzoyl-1-propionylpiperazine-2-carboxylate 10a. From 12a and propionyl chloride. Eluent: CH2Cl2/MeOH 97:3. Oil. Yield: 27%. 1H NMR (CDCl3, unresolved spectrum) d: 1.04–1.30 (m, 6H, CH3); 2.09–2.42 (m, 2H, COCH2); 2.82–3.06 (m, 1H); 3.07–3.28 (m, 1H); 3.41–3.55 (m, 1H); 3.58–3.63 (m, 1H); 3.86– 4.61 (m, 4H); 5.08 (bs, 1H, H-2); 7.25–7.38 (m, 5H, Ar) ppm. 13C NMR (CDCl3) d: 9.09 (COCH2CH3); 13.92 (OCH2CH3); 26.13 (CH2); 26.50 (CH2); 38.81 (CH2); 42.42 (CH2); 52.04 (CH); 55.68 (CH); 61.64 (OCH2); 62.09 (OCH2); 126.22 (CH); 128.47 (CH); 130.10

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

(CH); 134.84 (C); 135.02 (C); 169.38 (CO); 170.69 (CO); 173.66 (CO); 174.14 (CO) ppm. ESI-LC/MS: m/z 319.1 [M+H]+. ESI-HRMS calcd for C17H23N2O4 319.1652 (319.1658), found 319.1654. 4.1.1.2. Ethyl 1-benzoyl-4-propionylpiperazine-2-carboxylate 10b. From 12b and benzoyl chloride. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Oil. Yield: 96%. 1H NMR (CDCl3, mixture of conformers, unresolved spectrum) d: 1.08–1.15 (m, 3H, COCH2CH3), 1.23–1.35 (m, 3H, OCH2CH3); 2.23–2.54 (m, 2H, COCH2); 2.55–2.72 (m, 0.5H); 2.80–2.96 (m, 0.25H); 3.00–3.39 (m, 2H); 3.61–3.74 (m, 1.5H); 4.21–4.24 (m, 2H, OCH2); 4.31–4.68 (m, 2H); 4.92–5.18 (m, 0.25H); 5.45 (bs, 0.5H); 7.35–7.48 (m, 5H, Ar) ppm. 13C NMR (APT, CDCl3, mixture of conformers) d: 9.11 (COCH2CH3); 14.20 (OCH2CH3); 25.83, 39.62, 40.58 (NCH2); 41.12 (COCH2); 42.24, 45.19, 45.63 (NCH2); 52.54 (CH); 58.32 (CH); 62.17 (OCH2); 126.70, 126.99, 128.67, 130.22 (CH); 134.74, 135.02 (C); 168.96, 171.42, 172.72 (CO) ppm. ESI-LC/MS: m/z 319.1 [M+H]+. ESI-HRMS calcd for C17H23N2O4 318.1580, found 319.1654. 4.1.1.3. Ehyl 1-acetyl-4-(4-fluorophenylsulfonyl)piperazine-2-carboxylate 10c. From 12c and acetyl chloride. Eluent: CH2Cl2/ MeOH/NH3 98:2:0.2. Oil. Yield: 54%. 1H NMR (CDCl3, mixture of conformers A/B 3:7) d: 1.27 (t, J = 7.2 Hz, 3H, CH3); 1.99 (s, 0.9H, COCH3A); 2.08 (s, 2.1H, COCH3B); 2.28–2.51 (m, 2H); 2.93–3.00 (m, 0.3H, HB); 3.56–3.75 (m, 2.7H); 4.10–4.55 (m, 3H, CH2O + 1H); 4.40–4.50 (m, 0.3H, H-2A); 5.27 (s, 0.7H, H-2B); 7.21 (t, J = 8.4 Hz, 2H, H-30 + H-50 ); 7.75 (dd, J = 8.4 Hz, 4.8 Hz, 2H, H-20 + H-60 ) ppm. 13C NMR (CDCl3, APT, mixture of conformers) d: 14.15 (CH3); 21.05 (CH3COB); 21.26 (CH3COA); 38.39 (CH2N); 43.11 (CH2N); 45.36 (CH2N); 45.51 (CH2N); 47.06 (CH2N); 51.31 (C-2A); 56.37 (C-2B); 62.01 (OCH2A); 62.50 (OCH2B); 115.77 (d, JC-F = 23.0 Hz, C-30 + C-50 ); 130.48 (d, JC-F = 9.0 Hz, C-20 + C-60 ); 131.52 (C-10 ); 165.46 (d, JC-F = 255.0 Hz, C-40 ); 168.27 (CO); 170.55 (CO) ppm. ESI-LC/MS: m/z 359.0 [M+H]+. ESI-HRMS calcd for C15H20FN2O5S 359.1071, found 359.1076. 4.1.1.4. Ethyl 4-acetyl-1-(4-fluorophenylsulfonyl)piperazine-2-carboxylate 10d. From 12d and 4-fluorobenzenesolfonyl chloride. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Oil. Yield: 94%. 1H NMR (CDCl3, mixture of conformers A/B 4:6) d: 1.12 (t, J = 7.0 Hz, 1.8H, CH2-

5

3.41 (m, 3.5 H); 3.52–3.81 (m, 0.5 H); 3.98 (bs, 2H, CH2O); 4.25– 4.43 (m, 0.5 H); 7.14–7.38 (m, 5H, Ar) ppm. [13C] NMR (CDCl3, CPD) d: 14.01 (CH3); 42.33 (CH2); 44.20 (CH2); 47.99 (CH2); 49.42 (CH2); 56.69 (CH); 61.16 (CH2O); 126.95 (CH); 128.36 (CH); 129.72 (CH); 135.39 (C); 170.45 (CO); 170.69 (CO) ppm. 4.1.1.6. Ethyl 4-propionylpiperazine-2-carboxylate 12b. From 1110 and propionyl chloride. Eluent: CH2Cl2/MeOH/NH3 97:3:0.3. Oil. Yield: 44%. 1H NMR (CDCl3, mixture of conformers A + B  1:1) d: 1.13 (t, J = 7.2 Hz, 3H, propionyl CH3); 1.24 (t, J = 7.2 Hz, 1.5 H, CH2CH3A); 1.28 (t, J = 7.2 Hz, 1.5 H, CH2CH3B); 2.12 (s, 1H, NH); 2.32 (q, J = 7.2, 1H, COCH2A); 2.39 (q, J = 7.2, 1H, COCH2B); 2.71–2.77 (m, 1H, H-3A + H-6A); 2.92–2.97 (m,0.5 H, H-6B); 3.05–3.10 (m, 1H, H-6A + H-6B); 3.17–3.22 (m, 0.5 H, H-5A); 3.38–3.45 (m, 1.5 H, H2B + H-2A + H-3B); 3.55–3.60 (m, 1H, H-5A + H-5B); 3.73–3.77 (m, 1H, H-3B + H-5B); 4.15–4.23 (m, 2H, OCH2); 4.48 (dd, 0.5 H, J = 2.8 Hz, J = 13.2 Hz, H-3A) ppm. 13C NMR (APT, CDCl3, mixture of conformers A + B  1:1) d: 9.31 (propionyl CH3B); 9.44 (propionyl CH3A); 14.20 (ethyl CH3); 26.19 (COCH2B); 26.55 (COCH2A); 41.73(C-3B); 43.73 (C-3A); 43.78 (C-6B); 44.70 (C-6A); 45.86 (C5A); 47.01 (C-5B); 56.80 (C-2A); 57.02 (C-2B); 61.25 (OCH2A); 61.49 (OCH2B); 170.97 (CO); 172.50 (CO) ppm. 4.1.1.7. Ethyl 4-(4-fluorophenylsulfonyl)piperazine-2-carboxylate 12c. From 1110 and 4-fluorobenzenesolfonyl chloride. Eluent: CH2Cl2/ MeOH/NH3 98:2:0.2. Oil. Yield: 52%. 1H NMR (CDCl3) d: 1.17 (t, J = 7.2 Hz, 3H, CH3); 2.19 (s, 1H, NH); 2.45–2.53 (m, 1H, H-5); 2.55–2.65 (m, 1H, H-3); 2.72–2.80 (m, 1H, H-6); 2.98 (dt, J = 12.0 Hz, 4.0 Hz, 1H, H-6); 3.18–3.25 (m, 1H, H-5); 3.45 (dd, J = 8.4 Hz, 3.2 Hz, 1H, H-2); 3.52 (dd, J = 11.2 Hz, 2.4 Hz, 1H, H-3); 4.07 (q, J = 7.2 Hz, 2H, OCH2); 7.11 (t, J = 8.4 Hz, 2H, H-30 + H-50 ); 7.66 (dd, J = 8.6 Hz, 5.0 Hz, 2H, H-20 + H-60 ) ppm. 13C NMR (APT, CDCl3) d: 14.04 (CH3); 44.63 (C-6); 45.28 (C-5); 47.40 (C-3); 56.34 (C-2); 60.78 (OCH2); 116.35 (d, JC-F = 23 Hz, C-30 + C-50 ); 130.37 (d, JC-F = 10 Hz, C-20 + C-60 ); 131.12 (C-10 ); 165.24 (d, JC-F = 254 Hz, C-40 ); 170.21 (CO) ppm. 4.1.1.8. Ethyl 4-acetylpiperazine-2-carboxylate 12d. From 1110 and acetyl chloride. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Oil. Yield: 49%. 1H NMR (CDCl3, mixture of conformers A + B  1:1 d: 1.14

CH3B); 1.15 (t, J = 7.0 Hz, 1.2H, CH2CH3A); 2.01 (s, 1.2H, COCH3A); 2.06 (s, 1.8H, COCH3B); 2.68 (dt, J = 13.1 Hz, 3.6 Hz, 0.6H, H-5B); 2.96 (dd, J = 12.8 Hz, 4.0 Hz, 0.4H, H-3A); 3.11 (dt, J = 12.4 Hz, 3.2 Hz, 0.6H, H-6B); 3.28 (td, J = 12.8 Hz, 3.6 Hz, 0.4H, H-5A); 3.38 (dd, J = 13.6 Hz, 4.0 Hz, 0.6H, H-3B); 3.51 (td, J = 12.0 Hz, 3.2 Hz, 0.4H, H-6A); 3.70–3.79 (m, 1.4H, H-6A + H-6B + H-5A); 3.92–3.98

(t, J = 7.2 Hz, 1.5 H, CH2CH3A); 1.17 (t, J = 7.2 Hz, 1.5 H, CH2CH3B); 1.96 (s, 1.5 H, CH3COA); 2.00 (s, 1.5 H, CH3COB); 2.25 (bs, 1H, NHA+B); 2.62–2.68 (m, 1H, H3A + H6A); 2.79–2.84 (m, 0.5 H, H6B); 2.90–2.99 (m, 1H, H6A + H6B); 3.07–3.13 (m, 0.5 H, H5A); 3.28 (dd, J = 9.2 Hz, 3.6 Hz, 0.5 H, H2A); 3.31–3.39 (m, 1H, H2B + H5B); 3.45–3.63 (m,

(m, 1.4H, OCH2A + OCHHB); 4.04–4.12 (m, 0.6H, OCHHB); 4.35 (d, J = 13.2 Hz, 0.6H, H-3B); 4.55–4.63 (m, 1H, H-2A + H-5B); 4.72 (s, 0.6H, H-2B); 4.99 (d, J = 13.6 Hz, 0.4H, H-3A); 7.14–7.19 (m, 4H, H-30 A+B + H-50 A+B); 7.83–7.75 (m, 4H, H-20 A+B + H-60 A+B) ppm. 13C NMR (APT, CDCl3, mixture of conformers A/B 4:6) d: 14.03 (CH2-

J = 7.2 Hz, 1H, CH2CH3B); 4.34 (dd, J = 13.3 Hz, 2.8 Hz, 0.5H, H3A) ppm. 13C NMR (APT, CDCl3, mixture of conformers) d: 14.14

CH3); 20.90 (COCH3B); 21.04 (COCH3A); 40.78 (C-5B); 41.89 (C6A); 42.11 (C-6B); 43.71 (C-3A); 45.83 (C-5A); 48.20 (C-3B); 54.61 (C-2A); 55.54 (C-2B); 61.74 (OCH2A); 62.14 (OCH2B); 116.24 (d, JC-F = 22 Hz, ppm, C-30 + C-50 ); 130.03 (d, JC-F = 10 Hz, C-20 + C-60 ); 134.88 (C-10 A); 135.71 (C-10 B); 165.24 (d, JC-F = 255 Hz, C-40 ); 168.49 (COB); 168.70 (COA); 169.01 (COA); 169.20 (COB) ppm. ESI-LC/MS: m/z 359.0 [M+H]+. ESI-HRMS calcd for C15H20FN2O5S 359.1071, found 359.1074. 4.1.1.5. Ethyl 4-benzoylpiperazine-2-carboxylate 12a. From 1110 and benzoyl chloride. Eluent: CH2Cl2/MeOH/NH3 97:3:0.3. Oil. Yield: 68%. [1H] NMR (CDCl3, unresolved spectrum) d: 1.04 (bs, 3H, CH3); 2.46 (s, 1H, NH); 2.46–2.68 (m, 1.5 H); 2.85 (bs, 1H); 2.95–

2H, 2H3B + H5A + H5B); 4.06 (q, J = 7.2 Hz, 1H, CH2CH3A); 4.12 (q,

(CH2CH3(A+B)); 21.14 (COCH3B); 21.33 (COCH3A); 41.48 (C3B); 43.49 (C3A + C6B); 44.53 (C6A); 46.64 (C5A); 47.74 (C5B); 56.61 (C2A); 56.80 (C2B); 61.20 (OCH2A); 61.44 (OCH2B); 169.19 (CO);170.92 (CO) ppm. 4.1.1.9. (S)-[4-Benzyl-2-(hydroxymethyl)piperazin-1-yl](phenyl) methanone (S)-14. From (S)-1311 and benzoyl chloride. Brownish gummy solid. Yields: 60%. 1H NMR (CDCl3 mixture of conformers, unresolved spectrum) d: 1.92–2.40 (m, 2H); 2.60–2.92 (m, 1.5H); 2.95–3.06 (m, 0.5H); 3.10–3.30 (m, 0.5H); 3.35–3.68 (m, 4H); 3.70–3.80 (m, 0.5H); 3.83–4.08 (m, 2H); 4.35–4.60 (m, 0.5H); 4.65–4.85 (m, 0.5H); 7.10–7.50 (m, 10H, Ar) ppm. 13C NMR (CDCl3, APT) d: 45.21 (CH2N); 50.42 (C-2); 52.84 (CH2N); 54.26 (ArCH2); 62.76 (CH2O); 64.56 (CH2N); 127.00 (CH); 127.46 (CH); 128.48 (CH); 128.91 (CH); 129.67 (CH); 135.88 (C); 137.25 (C); 171.61 (CO) ppm.

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

In the same way, starting from (R)-13, (R)-14 was obtained in 61% yields. NMR spectra are the same as its enantiomer. 4.1.1.10. (S) and (R)-1-(4-Benzoyl-3-(hydroxymethyl)piperazin-1-yl) propan-1-one (S) and (R)-7b. From (S)-15 or (R)-15 and propionyl chloride. Yield: 71% and 58%, respectively. Chemical and physical characteristics are the same of the racemate. 4.1.1.11. (S)-[4-Benzyl-1-(4-fluorophenylsulfonyl)piperazin-2-yl] methanol (S)-16. From (S)-1311 and 4-fluorobenzenesulfonyl chloride. Oil. Yields: 51%. 1H NMR CDCl3 d: 2.03 (dt, J = 12.0 Hz, 3.6 Hz, 1H, H-5); 2.20 (dd, J = 11.6 Hz, 3.2 Hz, 1H, H-3); 2.74 (d, J = 12.0 Hz, 1H, H-5); 2.91 (d, J = 11.6 Hz, 1H, H-3); 3.40 (s, 2H, CH2Ar); 3.55 (td, J = 12.4 Hz, 2.8 Hz, 1H, H-6); 3.64 (bs, 1H, OH); 3.70–3.75 (m, 2H, H-6 + CHHO); 3.88–3.95 (m, 2H, CHHO + H-2); 7.13–7.30 (m, 7H, aromatics); 7.70–7.85 (m, 2H, H-20 + H-60 ) ppm. 13C NMR (CDCl3, APT) d: 42.73 (C-6); 51.98 (C-5); 53.92 (C-2); 54.61 (C-3); 62.76 (PhCH2); 64.57 (CH2O); 116.44 (d, JC-F = 22.0 Hz, C-30 + C-50 ); 127.67 (CH); 128.56 (CH); 128.95 (CH); 129.72 (d, JC-F = 9.0 Hz, C20 + C-60 ); 136.60 (C); 136.90 (C-10 ); 165.04 (d, JC-F = 253.0 Hz, C40 ) ppm. In the same way, starting from (R)-13, (R)-16 was obtained in 27% yields. NMR spectra are the same as its enantiomer. 4.1.1.12. (S) and (R)-(4-Acetyl-1-(4-fluorophenylsulfonyl)piperazin-2yl)methyl acetate (S) and (R)-8d. From (S) or (R)-17 and acetyl chloride (2 eq). Yield: 55% and 40%, respectively. Chemical and physical characteristics are the same of the racemate. 4.1.2. General procedure for the synthesis of compounds 7a-d Compounds 10a-d (0.25–0.30 g) were dissolved in 15 mL of a mixture of THF and EtOH (10:1), cooled at 0 °C, then NaBH4 (1 eq) was added in portions. The mixture was allowed to warm to RT and left stirring under nitrogen for 24 h. When the reaction was not complete, the mixture was heated at 60 °C for 300 . After removal of the solvent under vacuum, the residue was treated with ice, then partitioned between H2O and CH2Cl2. Drying (Na2SO4) and removal of the solvent gave a residue which was purified by flash chromatography. By this method the following compounds were obtained: 4.1.2.1. 1-(4-Benzoyl-2-(hydroxymethyl)piperazin-1-yl)propan-1-one 7a. Oil. Eluent: CH2Cl2/MeOH/NH3 90:10:0.5. Yield: 40%. 1H NMR (CDCl3, unresolved spectrum) d: 1.01–1.20 (m, 3H, CH3); 2.22– 2.48 (m, 2H, CH2CO); 2.62–2.80 (m, 1H); 2.91–3.30 (m, 2H); 3.30–3.92 (m, 4H, CH2OH + H); 4.01–4.12 (m, 1H, H-2); 4.26–4.87 (m, 2H); 7.40 (bs, 5H, phenyl) ppm. 13C NMR (APT, CDCl3, mixture of conformers) d: 9.27 (CH3); 9.46 (CH3); 26.29 (CH2CO); 26.84 (CH2CO); 37.56 (CH2); 42.51 (CH2); 47.79 (CH2); 54.39 (CH); 58.75 (CH2O); 59.71 (CH2O); 127.22 (CH); 128.70 (CH); 130.31 (CH); 134.24 (C); 134.71 (C); 171.72 (CO); 173.78 (CO) ppm. ESILC/MS: m/z 277.1 [M+H]+. ESI-HRMS calcd for C15H21N2O3 277.1547, found 277.1547. 4.1.2.2. 1-(4-Benzoyl-3-(hydroxymethyl)piperazin-1-yl)propan-1-one 7b. Oil. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Yield: 78%. 1H NMR (CDCl3, unresolved spectrum) d: 1.07–1.14 (m, 3H, CH3); 2.22– 2.48 (m, 2H, CH2CO); 2.67 (t, J = 12.4 Hz, 1H); 2.70–3.21 (m, 2H); 3.35–4.12 (m, 5H); 4.38–4.72 (m, 2H); 7.37–7.39 (m, 5H, phenyl) ppm. 13C NMR (CDCl3, mixture of conformers) d: 9.24, 9.44 (CH3); 26.07, 26.33 (COCH2); 38.11, 41.47, 44.13, 45.51 (CH2); 51.20 (CH); 58.53(CH2O); 126.90, 128.67, 130.01 (CH); 135.36 (C); 171.77, 173.42 (C) ppm. ESI-LC/MS: m/z 277.1 [M+H]+. ESIHRMS calcd for C15H21N2O3 277.1547, found 277.1548.

4.1.2.3. 1-(4-(4-Fluorophenylsulfonyl)-2-(hydroxymethyl)piperazin1-yl)ethanone 7c. Oil. Eluent: CH2Cl2/MeOH/NH3 90:10:1. Yield: 67%. 1H NMR (CDCl3, mixture of conformers A/B  4:6) d: 2.03 (s, 1.2H, CH3A); 2.08 (s, 1.8H, CH3B); 2.18–2.24 (dt, J = 11.8, 4.8 Hz, 0.6H, HB); 2.28–2.43 (m, 1.4H, 2HA + HB); 2.87–3.03 (m, 1H, HB + OHA); 3.46 (t, J = 11.2 Hz, 0.4H, HA); 3.61–4.01 (m, 5.6H, CH2OHA+B + H-2B + 3HA + 2HB + OHB); 4.49 (d, J = 14.0 Hz, 0.6H, HB); 4.69– 4.77 (m, 0.4H, H-2A); 7.20–7.24 (m, 2H, H-30 + H-50 ); 7.72–7.74 (m, 2H, H-20 + H-60 ) ppm. 13C NMR (APT, CDCl3, mixture of conformers) d: 21.42, 21.61 (CH3A + CH3B); 36.28, 42.02, 45.45, 45.74, 45.97 (C piperazine); 49.39 (C2A); 54.77 (C2B); 59.31, 59.43 (CH2OA + CH2OB); 116.63 (d, JC-F = 22 Hz, ppm, C-30 + C-50 ); 130.39 (d, JC-F = 9 Hz, C-20 + C-60 ); 131.26, 131.37 (C10 A + C10 B); 165.44 (d, JC-F = 255 Hz, C40 (A+B)); 170.46 (CO); 170.60 (CO) ppm. ESI-LC/MS: m/z 317.0 [M+H]+. ESI-HRMS calcd for C13H18FN2O4S 317.0966, found 317.0967. 4.1.2.4. 1-(4-(4-Fluorophenylsulfonyl)-3-(hydroxymethyl)piperazin1-yl)ethanone 7d Oil. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Yield: 62%. 1H NMR (CDCl3, mixture of conformers A + B  1:1) d: 2.05 (s, 3H, CH3); 2.58 (dt, J = 13.0 Hz, 3.6 Hz, 0.5H); 2.73 (dd, J = 14.0 Hz, J = 3.6 Hz, 0.5H); 3.02–3.25 (m, 2H + OH); 3.44 (d, J = 7.2 Hz, 1H, OCHH); 3.58 (d, J = 7.2 Hz, 1H, OCHH); 3.59–3.79 (m, 1.5H); 3.96–4.03 (m, 1.5H, H-3A + H-3B + H); 4.36 (d, J = 13.2 Hz, 0.5H); 4.50 (d, J = 14 Hz, 0.5H); 7.15–7.19 (m, 2H, H30 + H-50 ); 7.81–7.82 (m, 2H, H-20 + H-60 ) ppm. 13C NMR (APT, CDCl3, mixture of conformers A + B  1:1) d: 21.06, 20.95 (CH3); 40.55, 41.13, 41.34, 44.97, 46.05 (NCH2); 54.03, 54.26 (C-3A + C-3B); 58.40, 58.46 (CH2OHA + CH2OHB); 116.69 (d, JC-F = 23 Hz, ppm, C-30 + C-50 ); 129.75 (d, JC-F = 9 Hz, C-20 + C-60 ); 136.29 (C-10 ); 165.21 (d, JC-F = 254 Hz, C-40 ); 170.20, 170.61 (COA + COB) ppm. ESI-LC/MS: 317.0 [M+H]+ m/z. ESI-HRMS calcd for C13H18FN2O4S 317.0966, found 317.0967. 4.1.3. General procedure for the synthesis of compounds 8a-d Compounds 7a-d (0.03–0.05 g) were dissolved in 5 mL of anhydrous CH2Cl2, NEt3 (1.2 eq) was added, and acetyl chloride (1.2 eq, solubilised into 2 mL CH2Cl2) was added dropwise at 0 °C. The mixture was left stirring at RT for 3 h, then it was treated with sat. NaHCO3 and extracted with CH2Cl2. Drying (Na2SO4) and removal of the solvent gave a residue which was purified by flash chromatography. By this method the following compounds were obtained: 4.1.3.1. (4-Benzoyl-1-propionylpiperazin-2-yl)methyl acetate 8a. Oil. Eluent: CH2Cl2/MeOH 97:3. Yield: 56%. 1H NMR (CDCl3, unresolved spectrum) d: 1.05–1.22 (m, 3H, CH3CH2CO); 1.72–2.18 (m, 3H, CH3CO); 2.23–2.42 (m, 2H, CH2CO); 2.76–3.30 (m, 2H); 3.30–3.42 (m, 0.5H); 3.52–4.90 (m, 6.5H); 7.29–7.43 (m, 5H, Ar) ppm. 13C NMR (CDCl3, mixture of conformers) d: 9.27, 9.44 (CH3CH2); 20.61 (CH3CO); 26.14, 26.79 (CH2CO); 36.88, 41.22, 42.28 (CH2N); 47.15 (C-2); 60.31 (CH2O); 127.07, 128.64, 130.14 (CH); 134.92 (C); 170.56, 171.15, 173.16 (CO) ppm. ESI-LC/MS: m/z 319.1 [M+H]+. ESI-HRMS calcd for C17H23N2O4 319.1652, found 319.1654. 4.1.3.2. (1-Benzoyl-4-propionylpiperazin-2-yl)methyl acetate 8b. Oil. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Yield: 81%. 1H NMR (CDCl3, unresolved spectrum) d: 1.14 (t, 3H, J = 7.2 Hz, CH2CH3); 2.03 (s, 3H, CH3CO); 2.25–2.42 (m, 2H, CH2CO); 2.62–2.74 (m, 0.7H); 2.82–2.94 (m, 0.7H), 3.09–3.43 (m, 2H), 3.58–4.15 (m, 5H); 4.90– 5.20 (m, 0.6H), 7.38–7.43 (m, 5H, Ar) ppm. 13C NMR (CDCl3) d: 9.32 (CH2CH3); 20.81 (CH3CO); 26.12, 26.42 (CH2CO); 41.51 (CH2); 45.25 (CH2); 47.16 (C-2); 60.46 (CH2O); 126.93, 128.72,

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

130.17 (CH); 135.28 (C); 172.89 (CO) ppm. ESI-LC/MS: m/z 319.1 [M+H]+. ESI-HRMS calcd for C17H23N2O4 319.1652, found 319.1652. 4.1.3.3. (1-Acetyl-4-(4-fluorophenylsulfonyl)piperazin-2-yl)methyl acetate 8c. Oil. Yield: 95%. 1H NMR (CDCl3, mixture of conformers A/B  4:6) d: 2.02 (s, 3H, CH3); 2.06 (s, 3H, CH3); 2.18–2.46 (m, 2H); 2.94 (t, J = 13.0 Hz, 0.6H, HB); 3.54 (t, J = 13.0 Hz, 0.4H, HA); 3.62–3.88 (m, 2H); 4.09–4.18 (m, 0.4H, H-2A); 4.19–4.32 (m, 2.2H, H-2B + OCH2B + OCHHA); 4.47–4.56 (m, 1H, HB + OCHHA); 4.93–5.02 (m, 0.4H, HA); 7.22 (t, J = 8.6 Hz, 2H, H-30 + H-50 ); 7.72– 7.77 (m, 2H, H-20 + H-60 ) ppm. 13C NMR (APT, CDCl3) d: 20.71, 21.08, 21.54, (CH3); 36.09, 41.74, 45.69, 45.79, 45.98, 46.27, (NCH2); 51.76 (C-2); 60.17, 60.68 (CH2O); 116.62 (d, JC-F = 23 Hz, C-30 + C-50 ); 130.45 (d, JC-F = 9 Hz, C-20 + C-60 ); 131.24 (C-10 ); 165.49 (d, JC-F = 255 Hz, C-40 ); 169.64, 170.47 (CO) ppm. ESI-LC/ MS: m/z 359.1 [M+H]+. ESI-HRMS calcd for C15H20FN2O5S 359.1071, found 359.1073. 4.1.3.4. (4-Acetyl-1-(4-fluorophenylsulfonyl)piperazin-2-yl)methyl acetate 8d. Oil. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Yield: 77%. 1 H NMR (CDCl3, mixture of conformers A/B  1:1) d: 1.98 (s, 1.5H), 2.03 (s, 3H), 2.03 (s, 1.5H) (CH3): 2.58 (dt, J = 12.8 Hz, 3.6 Hz, 0.5H, H-5A); 2.72 (dd, J = 13.6 Hz, 4.0 Hz, 0.5H, H-3B); 3.03–3.14 (m, 1H); 3.18–3.27 (m, 1H); 3.63–3.82 (m, 2H); 3.94– 4.06 (m, 1H, CHHOA + CHHOB); 4.09–4.15 (m, 1H, CHHOA + CHHOB); 4.21–4.27 (m, 1H, H-2A + H-2B); 4.48–4.52 (m, 1H, H3B + H-5A); 7.18–7.26 (m, 2H, H-30 + H-50 ); 7.84–7.87 (m, 2H, H20 + H-60 ) ppm. 13C NMR (APT, CDCl3, mixture of conformers) d: 20.71 (CH3); 20.94 (CH3); 21.13 (CH3); 40.66, 40.84, 40.96, 41.06 (C-3, C-5); 45.48, 46.19 (C-6); 51.10, 51.87 (C-2); 59.81, 60.03 (CH2O); 116.68 (d, JC-F = 22 Hz, ppm, C-30 + C-50 ); 129.75 (d, JC-F = 9 Hz, C-20 + C-60 ); 136.35, 136.57 (C-10 A + C-10 B); 165.23 (d, JC-F = 255 Hz, C-40 ); 169.48, 170.19, 170.51 (CO) ppm. ESI-LC/MS: m/z 359.1 [M+H]+. ESI-HRMS calcd for C15H20FN2O5S 359.1071, found 359.1073. 4.1.4. General procedure for the synthesis of compounds 9a-d Compounds 7a-d (0.05 g) were dissolved in anhydrous THF (5 mL), then NaH (50% oil dispersion, 2 eq) was added at 0 °C and under nitrogen atmosphere. The white suspension was left stirring for 0.5 h, then CH3I (2 eq), dissolved in anTHF (2 mL), was added dropwise. The mixture was left stirring at RT overnight, then ice was added and the solvent was removed under vacuum. The residue was partitioned between H2O and CH2Cl2; the organic phase was collected dried over Na2SO4, then the solvent was removed. The residue was purified by flash chromatography. 4.1.4.1. 1-(4-Benzoyl-2-(methoxymethyl)piperazin-1-yl)propan-1-one 9a. Oil. Eluent: CH2Cl2/MeOH 97:3. Yield: 90%. 1H NMR (CDCl3, unresolved spectrum) d: 1.13 (t, J = 7.4 Hz, 3H, CH2CH3); 2.25– 2.52 (m, 2H, COCH2); 2.72–4.35 (m, 10H); 4.38–4.81 (m, 2H, CHH + H-2); 7.31–7.58 (m, 5H, Ar) ppm.

13

C NMR (CDCl3, APT

+ CPD, mixture of conformers) d: 9.43 (CCH3); 26.28, 26.81 (COCH2); 37.01, 41.32, 42.20, 46.75, 47.39 (NCH2); 47.85 (CH); 58.83, 59.30 (OCH3); 68.98, 70.03 (OCH2); 127.10, 128.59, 130.10 (CH); 135.08 (C); 171.29, 172.98 (CO) ppm. ESI-HRMS calcd for C16H23N2O3 291.1703, found 291.1705. 4.1.4.2. 1-(4-Benzoyl-3-(methoxymethyl)piperazin-1-yl)propan-1-one 9b. Oil. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Yield: 72%. 1H NMR (CDCl3, unresolved spectrum) d: 1.12 (t, J = 7.2 Hz, 3H, CH2CH3); 2.27–2.42 (m, 2H, COCH2); 2.64 (t, J = 12.8 Hz, 1H); 2.75–3.62 (m, 9H); 3.70–4.11 (m, 1H); 4.40–4.96 (m, 1H); 7.35–7.38 (m, 5H, Ar) ppm. 13C NMR (CDCl3, APT, mixture of conformers) d: 9.26, 9.38

7

(CH2CH3); 26.41 (COCH2); 41.39, 45.20 (CH2); 58.97 (OCH3); 59.17 (CH); 68.20, 69.35 (OCH2); 126.80, 127.13, 128.52, 128.66, 129.79, 129.96 (CH); 135.42, 135.71 (C); 172.62, 173.18 (CO) ppm. ESI-LC/MS: m/z 291.1 [M+H]+. ESI-HRMS calcd for C16H23N2O3 291.1703, found 291.1706. 4.1.4.3. 1-(4-(4-Fluorophenylsulfonyl)-2-(methoxymethyl)piperazin1-yl)ethanone 9c. Oil. Eluent: CH2Cl2/MeOH/NH3 97:3:0.3. Yield: 60%. 1H NMR (CDCl3, mixture of conformers A:B 2:1) d: 2.04–2.10 (m, 3H, COCH3); 2.15–2.41 (m, 2HA+B); 2.90 (t, J = 12.4 Hz, 0.66H, HA); 3.36–3.47 (m, 3.34H, OCH3 + HB); 3.48–3.69 (m, 2.34H, OCH2 + HB); 3.72–3.85 (m, 2H); 3.95–4.05 (m, 0.66H, H-2A); 4.53 (d, J = 13.2 Hz, 0.66H, HA); 4.85–4.90 (m, 0.34H, H-2B); 7.20–7.26 (m, 2H, H-30 + H-50 ); 7.73–7.77 (m, 2H, H-20 + H-60 ) ppm. 13C NMR (CDCl3, APT, mixture of conformers) d: 21.33, 21.59 (COCH3); 36.34, 42.03, 45.62, 45.75, 46.36 (NCH2); 46.67 (C-2B); 53.08 (C-2A); 59.11, 59.42 (OCH3); 68.26, 69.52 (OCH2); 116.58 (d, JC-F = 23 Hz, ppm, C-30 + C-50 ); 130.38 (d, JC-F = 9 Hz, C-20 + C-60 ); 131.44 (C-10 ); 165.42 (d, JC-F = 254 Hz, C-40 ); 170.12 (CO) ppm. ESI-LC/MS: m/z 331.1 [M+H]+. ESI-HRMS calcd for C14H20FN2O4S 331.1122, found 331.1125. 4.1.4.4. 1-(4-(4-Fluorophenylsulfonyl)-3-(methoxymethyl)piperazin1-yl)ethanone 9d. Oil. Eluent: CH2Cl2/MeOH/NH3 95:5:0.5. Yield: 50%. 1H NMR (CDCl3, (mixture of conformers A/B 75:25) d: 2.15 (s, 3H, COCH3); 2.58 (dt, J = 12.8 Hz, 3.9 Hz, 0.75H, HA); 2.82 (dd, J = 13.6 Hz, 4 Hz, 0.25H, HB); 3.02–3.12 (m, 1.5H, 2HA); 3.12–3.24 (m, 1.25H, 1HA + 2HB); 3.26–3.35 (m, 3.5H, OCH3 + 2HB); 3.41 (7, J = 9.6 Hz, 0.75H, HA); 3.61–3.68 (m, 1H, HA + HB); 3.68–3.72 (m, .025H, HB); 3.94 (d, J = 14.0 Hz, 0.75H, HA); 4.05–4.12 (m, 0.75H, H-2A); 4.13–4.20 (m, 0.25H, H-2B); 4.48 (d, J = 13.6 Hz, 1H, HA + HB); 7.18–7.26 (m, 2H, H-30 + H-50 ); 7.83–7.86 (m, 2H, H-20 + H-60 ) ppm. 13C NMR (CDCl3, mixture of conformers) d: 20.88 (COCH3); 40.63, 41.15, 41.62, 45.54, 45.76 (NCH2); 51.80 (C-2A); 52.72 (C-2B); 58.90 (OCH3); 68.01 (OCH2A); 69.87(OCH2B); 116.57 (d, JC-F = 22 Hz, ppm, C-30 + C-50 ); 129.74 (d, JC-F = 9 Hz, C-20 + C-60 ); 136.57, 136.35 (C-10 ); 165.20 (d, JC-F = 255 Hz, C-40 ); 169.85 (CO) ppm. ESI-LC/MS: m/z 331.2 [M+H]+. ESI-HRMS calcd for C14H20FN2O4S 331.1122, found 331.1125. 4.1.5. (S) (2-(Hydroxymethyl)piperazin-1-yl)(phenyl)methanone (S)15 (S)-14 (0.2 g) in abs EtOH (20 mL) was hydrogenated over Pd/C (50 mg) for 1 night at 72 psi. Filtration and removal of solvent gave a residue which was purified by flash chromatography (CH2Cl2/ MeOH/NH3 97:3:0.3) obtaining the title compound as a thick oil. Yield: 60%. 1H NMR CDCl3 (unresolved spectrum) d: 2.50–3.30 (m, 7H); 3.30–3.60 (m, 1H); 3.60–4.10 (m, 2H); 4.39–4.45 (m, 0.5H); 4.60–4.80 (m, 0.5H); 7.30–7.40 (m, 5H, Ar) ppm. 13C NMR CDCl3 d: 45.92 (CH2N); 46.56 (CH2O); 126.87 (CH); 128.57 (CH); 129.70 (CH); 135.89 (C); 171.88 (CO) ppm. In the same way, (R)15 was ontained from (R)-14 in 82% yields. NMR spectra are the same as its enantiomer. 4.1.6. (S)-(1-(4-Fluorophenylsulfonyl)piperazin-2-yl)methanol (S)-17 Following the procedure reported for (S)-15, starting from (S)16, (S)-17 was obtained in 16% yields. 1H NMR (CDCl3) d: 2.60– 2.72 (m, 3H, H-5 + NH + OH); 2.80 (dd, J = 12.0, 3.6 Hz, 1H, H-6); 2.94 (d, J = 12.0 Hz, 1H, H-5); 3.15 (d, J = 12.0 Hz, 1H, H-6); 3.45 (td, J = 12.4, 3.2 Hz, 1H, H-3); 3.66 (d, J = 12.4 Hz, 1H, H-3); 3.75– 4.10 (m, 3H, H-2 + CH2OH); 7.17 (t, J = 9.0 Hz, 2H, H-30 + H-50 ); 7.82–7.86 (m, 2H, H-20 + H-60 ) ppm. 13C NMR (CDCl3, APT) d: 42.81 (C-6); 45.19 (C-5); 47.53 (C-3); 53.36 (C-2); 63.77 (CH2O); 116.47 (d, JC-F = 22.0 Hz, C-30 + C-50 ); 129.70 (d, JC-F = 9.0 Hz, C-20 + C-60 ); 137.07 (C-10 ); 165.05 (d, JC-F = 254.0 Hz, C-40 ) ppm. In the

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

same way, (R)-17 was ontained from (R)-16 in 22% yields. NMR spectra are the same as its enantiomer. 4.2. Pharmacology 4.2.1. Animals Male CD-1 albino mice (Harlan, Varese, Italy) weighing approximately 22–25 g at the beginning of the experimental procedure, were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least one week after their arrival. Twelve mice were housed per cage (size 26  41 cm); animals were fed a standard laboratory diet and tap water ad libitum, and kept at 23 ± 1 °C with a 12 h light/ dark cycle, light at 7 a.m. All animal manipulations were carried out according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive of 24 November 1986 (86/609/EEC). The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence. Experiments involving animals have been reported according to ARRIVE guidelines. All efforts were made to minimize animal suffering and to reduce the number of animals used. 4.2.2. Passive-avoidance test The test12 was performed according to a previously reported protocol.13,18 All investigated drugs were dissolved in saline containing 10% DMSO and injected s.c. in a 1:10 dilution sequence, 20 min before the training session; the amnesic drug scopolamine was injected immediately after termination of the training session. Saline treated mice received an additional injection of saline immediately after the training session as control of scopolamine injection. Compounds 5a-d and 6a-d were used as the reference drugs. Details on the time spent in the two sessions and statistical analysis are shown in Table S1 (Supplementary Data). All compounds elicited their effect without changing either gross behavior or motor coordination, as revealed by the rota-rod test (performed as reported in Ref. 18, data not shown). None of the drugs, at the active doses, increased the number of falls from the rotating rod in comparison with saline-treated mice. The number of falls in the rota-rod test progressively decreased since mice learned how to balance on the rotating rod. The spontaneous motility and inspection activity of mice was unmodified by the administration of the studied compounds as revealed by the hole-board test in comparison with saline-treated mice (performed as reported in Ref. 18, data not shown). 4.2.3. Statistical analysis All experimental results are given as the mean ± S.E.M. Analysis of variance (ANOVA), followed by Fisher’s Protected Least Significant Difference (PLSD) procedure for post hoc comparison, was used to verify significance between two means. Data were analyzed with the StatView software for the Macintosh (1992). P values of less than 0.05 were considered significant. 4.3. Plasma stability assay 4.3.1. Chemicals Acetonitrile, ethanol (Chromasolv), formic acid and ammonium formate (MS grade), NaCl, KCl, Na2HPO42H2O, KH2PO4 (Reagent grade), Verapamil, Enalapril (analytical standards) were purchased by Sigma-Aldrich (Milan, Italy). Verapamil was used as internal standard (ISTD) for quantitative analysis of the studied com-

pounds. MilliQ water 18 MX was obtained from Millipore’s Simplicity system (Milan - Italy). Phosphate buffer solution (PBS) was prepared by adding 8.01 g L
1 of NaCl, 0.2 g L
1 of KCl, 1.78 g L
1 of Na2HPO42H2O and 0.27 g L
1 of KH2PO4. The rat plasma batch was collected from Sprague Dawley male rats and was kept at
80 °C until use. 4.3.2. Instrumental The LC-MS/MS analysis was carried out using a Varian 1200L triple quadrupole system (Palo Alto, CA, USA) equipped by two Prostar 210 pumps, a Prostar 410 autosampler and an Electrospray Source (ESI) operating in positive ions. The ion sources and ion optics parameters were optimized during the calibration of the instrument introducing, via syringe pump at 10 lL min
1, a 1 lg mL
1 tuning solution. Raw-data were collected an processed by Varian Workstation Vers. 6.8 software. G-Therm 015 thermostatic oven was used to maintained the samples at 37 °C during the test of degradation. 4.3.3. Standard solutions and calibration curves Stock solutions of analytes and internal standard (ISTD) were prepared in acetonitrile at 1.0 mM and stored at 4 °C. Working solutions of each analyte were freshly prepared by diluting stock solutions up to a concentration of 10 lM and 1 lM (Working solution 1 and 2 respectively) in mixture of mQ water:acetonitrile 80:20 (v/v). The ISTD working solution was prepared in acetonitrile at 50 ng mL
1 (ISTD solution). A six levels calibration curve was prepared by adding proper volumes of working solution of each analyte to a constant volume of ISTD solution (300 lL corresponding to 15 ng of ISTD). The obtained solutions were dried under a gentle nitrogen stream and dissolved in 1.5 mL of mQ water:acetonitrile 50:50 (v/v) added with 10 mM of formic acid. Final concentrations of calibration levels were: 0; 0.10; 0.20; 0.50; 0.75 and 1.00 lM. All calibration levels were analyzed six times by LC-MS/MS method. 4.3.4. LC-MS/MS method The chromatographic parameters employed to analyze the samples were tuned to minimize the run time and were reported as follows: – column, Pursuit XRs C18 50 mm length, 2 mm internal diameter and 3 lm particle size purchased from Agilent Technologies (Palo Alto, CA, USA), – acidic mobile phase, composed by 10 mM of formic acid solution (solvent A) and 10 mM of formic acid in acetonitrile (solvent B), – flow rate and the injection volume were 0.25 mL min
1 and 5 lL respectively. The elution gradient is shown in Table S2 (Supplementary Material). The analyses were acquired in Multiple Reaction Monitoring (MRM), parameters are reported in Table S3 (Supplementary Material), using 100 ms of dwell time and argon at 2.0 mTorr as collision gas. 4.3.5. Sample preparation Each sample was prepared adding 10 lL of Working solution 1– 100 lL of PBS or rat plasma. The obtained solutions correspond to 1 lM of analyte. Each set of samples was incubated in triplicate at four different times, 0, 30, 60 and 120 min. at 37 °C. Therefore the degradation profile of each analyte was represented by a batch of 12 samples (4 incubation times  3 replicates). After the incubation, the samples were added with 300 lL of ISTD solution and

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M.V. Martino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx

centrifuged. The supernatants were transferred in autosampler vials and dried under a gentle stream of nitrogen. The dried samples were dissolved in 1.5 mL of mQ water:acetonitrile 80:20 added with 10 mM of formic acid. The obtained sample solutions were analyzed by LC-MS/MS method described above. 4.3.6. Linearity and LOD Calibration curves of analytes were obtained by plotting the peak area ratios (PAR), between quantitation ions of analyte and ISTD, versus the nominal concentration of the calibration solution. A linear regression analysis was applied to obtain the best fitting function between the calibration points. The precision was evaluated through the relative standard deviation (RSD%) of the quantitative data of the replicate analysis of highest level of calibration curves. In order to obtain reliable LOD values, the standard deviation of response and slope approach was employed.19 The estimated standard deviations of responses were obtained by the standard deviation of y-intercepts (SDY-I) of regression lines. The obtained linear regressions, the linearity coefficients, precision and the estimated LOD values for each analyte are reported in Table S4 (Supplementary Material). 4.3.7. Degradation profiles The degradation profiles were obtained by monitoring the variation of analyte concentration at different incubation times. Through the comparison between the results obtained in PBS and rat plasma samples it is possible distinguish between the chemical stability of each analyte. Enalapril degradation experiments were carried out to check the hydrolytic activity of rat plasma batch employed. In order to summarize all experimental data, the halflife (t1/2) of studied compounds under all conditions were reported in Table S5 (Supplementary Material). Acknowledgments This work was supported by grants from MIUR (Italy) (National Interest Research Project PRIN 2009, 2009ESXPT2_002) and from the University of Florence (ex 60%). A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.019.

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