AMPK Pathway Ameliorate

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1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function, and Exhibit Inhibition of Proliferation in Cancer Cells Sergio Valente, Paolo Mellini, Francesco Spallotta, Vincenzo Carafa, Angela Nebbioso, Lucia Polletta, Ilaria Carnevale, Serena Saladini, Daniela Trisciuoglio, Chiara Gabellini, Maria Tardugno, Clemens Zwergel, Chiara Cencioni, Sandra Atlante, Sébastien Moniot, Clemens Steegborn, Roberta Budriesi, Marco Tafani, Donatella Del Bufalo, Lucia Altucci, Carlo Gaetano, and Antonello Mai J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01117 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 24, 2015

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

1,4-Dihydropyridines

Active

on

the

SIRT1/AMPK

Pathway

Ameliorate Skin Repair and Mitochondrial Function, and Exhibit Inhibition of Proliferation in Cancer Cells

Sergio Valente,† Paolo Mellini,† Francesco Spallotta,° Vincenzo Carafa,# Angela Nebbioso,# Lucia Polletta,^ Ilaria Carnevale,^ Serena Saladini,^ Daniela Trisciuoglio,‡ Chiara Gabellini,‡ Maria Tardugno,† Clemens Zwergel,† Chiara Cencioni,° Sandra Atlante,° Sébastien Moniot,= Clemens Steegborn,= Roberta Budriesi,¶ Marco Tafani,^ Donatella Del Bufalo,‡ Lucia Altucci,#,% Carlo Gaetano,° Antonello Mai*,†,£

†

Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro

5, 00185 Rome, Italy °Division of Cardiovascular Epigenetics, Department of Cardiology, Goethe University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany #

Department of Biochemistry, Biophysics and General Pathology, Second University of Naples,

Vico L. De Crecchio 7, 80138 Naples, Italy ^Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy ‡

Regina Elena National Cancer Institute, Via Elio Chianesi, 53, 00144 Rome, Italy

=

Department of Biochemistry; University of Bayreuth; Bayreuth, Germany

1

ACS Paragon Plus Environment


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¶

Department of Pharmacy and Biotechnology, University of Bologna, Via Zamboni 33, 40126,

Bologna, Italy %

Institute of Genetics and Biophysics, IGB, Adriano Buzzati Traverso, Via P. Castellino 111,

80131, Naples, Italy £

Pasteur Institute, Cenci-Bolognetti Foundation, Sapienza University of Rome, P. le A. Moro 5,

00185 Rome, Italy

2


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ABSTRACT Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines bearing changes at the C2/C6, C3/C5, C4 or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by co-administration of compound C (9), a AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 µM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

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INTRODUCTION Histone deacetylases (HDACs) are among the most studied epigenetic enzymes for their involvement in control of gene expression and transcription as well as in many other cellular processes,

spanning

from

angiogenesis,

inflammation,

cell

growth,

migration,

and

differentiation, to metabolism homeostasis and neurodegeneration.1-3 Among the known four HDAC classes, the sirtuin family (class III HDACs) strongly differs from the others because its members (SIRT1-7) possess no sequence similarity with the “classical” deacetylases. They use a different mechanism for deacetylation, based on the NAD+ co-substrate that reacts with the acetyl-lysine furnishing nicotinamide, O-acetyl-ADP-ribose and the free amine, and they are insensitive to well-known class-I/II/IV HDAC inhibitors such as trichostatin A (TSA) and vorinostat.4-6 Due to their requirement of NAD+ for catalysis, a connection between energy metabolism and protein deacetylation became immediately evident.7 Several studies, based on the always growing number of both histone and non-histone protein targets identified for sirtuins, have firmly established that SIRTs can regulate many fundamental biological processes in response to a variety of cellular, environmental and/or nutritional stimuli.8,9 Dysregulation of sirtuin expression and/or activity is believed to be connected to a number of pathological

conditions,

such

as

cancer initiation

and

progression,10,11

neurodegeneration,12 inflammation,13 cardiovascular diseases,14 type 2 diabetes,15 stress susceptibility.16 Since the overexpression of most sirtuins mimics the beneficial effects of caloric restriction, they were predicted to prevent age-related diseases and to maintain metabolic homeostasis.17-19 Indeed, SIRT1 binds to and deacetylates a number of important transcription factors and proteins, including p53, E2F1, Ku70, Notch, PPARα, PPARγ, PGC-1α, LXR, FXR, FOXO1, FOXO3, NF-κB, and eNOS.1,20 In this way, SIRT1 exerts antiapoptotic and

4


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antidifferentiation properties, it modulates glucose as well as lipid metabolism, it protects microglia cells from degeneration and improved survival in mouse models of Huntington’s disease.21-25 The role of SIRTs in cancer is controversial, because many of them have been described as tumor suppressors as well as tumor promoters.11,26 Despite this, a number of SIRT1/2 inhibitors displayed cell death and potent antiproliferative activities in a panel of cancer cells including cancer stem cells.11,27,28 On the other hand, some SIRT1 activators proved to be beneficial not only for diseases related to metabolism, such as type 2 diabetes or obesity, but also for cardiovascular and neurodegenerative diseases.29-32 Compound 1 (resveratrol, RSV) (Figure 1) is the first molecule described as a SIRT1 activator, together with other natural polyphenols, and its administration in vivo extends lifespan in yeast,33 C. elegans,34 Drosophila,34 fish35 and bees.36 Mice treated with 1 showed improved mitochondrial functions and protection against fat diet-induced obesity.37 In obese mice, treatment with 1 led to increased healthspan and lifespan.38 Extensive high-throughput screening, sometime combined with molecular modelling techniques, led to identification of both natural and synthetic molecules with different chemical scaffolds able to activate SIRT1 in vitro (Figure 1).39-43 Among them, some SRT compounds (such as 2, SRT1720) have been also tested in vivo, and resembled most of the beneficial effects of 1.44 The most potent of them [for example, SRT210445 (Figure 1)] together with 1 entered clinical trials for the treatment of metabolic disorders, type 2 diabetes and/or sepsis,46 and the first positive data suggest that sirtuin activating compounds can be effectively used to treat metabolic disorders and inflammation in patients.

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N

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N N

O

N OH

O

S

HN

HO

HN

N S

O S O

N S

N

N

H2N N

N

N

OH

resveratrol (RSV) 1

HN

N

N

O

SRT1720, 2

pyrroloquinoxalines

SRT2104 H3CO

H3CO O

F

O

H N

O

OH Br

N

O

OCH3

terpenylated coumarins

OH

naphthohydrazones

Br

COOH O N H

O

H

HN

O

N O

H

COOH

O H

A2

HO

H

OH

dammarane triterpenes

Figure 1. Known natural and synthetic SIRT activators.

A strong debate was emerging around the effective SIRT1 activation by 1 and sirtuin activating compounds, because of the use in the enzyme assay of aminomethylcoumarin (AMC)or carboxytetramethylrhodamine (TAMRA)-labeled substrate, which allowed to see activation, whereas the tested natural substrates did not.47-49 Therefore, it was questioned if the action of 1 and sirtuin activating compounds on SIRT1 was a direct activation, or if rather it was the indirect result of modulation of other pathways, such as AMPK activation and/or PDE inhibition.50 In recent papers, direct SIRT1 activation was confirmed, and it was shown that there are sharp 6


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structural and positional requirements to see activation with natural substrates (such as FOXO3a and PGC-1α).51,52 Furthermore, E230K or E230A SIRT1 mutation abolishes enzyme activation as well as binding of sirtuin activating compounds, suggesting an assisted-allosteric activation mechanism in which the activators bind and stabilize the enzyme-substrate complex favoring deacetylation.51 Lastly, the crystal structure of a SRT compound bound to an engineered minimally functional human SIRT1 (mini-hSIRT1) has been reported, unambiguously confirming the direct allosteric activation of SIRT1 by small molecules.53 During our studies on sirtuin modulators,27,28,54-62 in 2009 we reported the design, synthesis and SIRT inhibiting/activating activity of a series of 1,4-dihydropyridines (DHPs).63 Particularly compounds 3a-c (Figure 2), in which the DHP scaffold carried a benzyl group at the N1 position, a phenyl ring at C4, and a double carbethoxy, carboxy, or carboxamido function at the C3/C5 positions, displayed SIRT activating properties when tested in a fluorescence assay (BioMol). In cellular contexts, 3a-c reduced the number of senescent cells of 30-40% in human mesenchymal stem cells (hMSCs),63 and 3a increased mitochondrial function and transcription factor A (mTFA) transcriptional activity in murine C2C12 myoblasts,63 and stimulated proliferation in human keratinocyte HaCaT cells via endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production, resembling in all cases the effects observed with 1.64 Interestingly, 3a accelerated tissue renewal in a mouse model of skin repair. In the same assay, 1 and the well-known HDACi TSA displayed the same behavior, suggesting the presence of a NOdependent crosstalk among class III and I HDACs in wound healing.64 Following our research on the DHPs as SIRT activators, we prepared novel analogues of 3ac, compounds 4-8, in which: i) two methyl groups have been inserted at the unsubstituted C2/C6 positions

of

3a-c

(compounds

ii)

4a-c);

7

two

asymmetric


carbethoxy/carboxy,


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

carbethoxy/carboxamido, or carboxy/carboxamido as well as symmetric cyano substitutions have been applied at the DHP C3/C5 positions (compounds 5a-d); iii) the C4-phenyl ring of 3a-c was replaced by heteroaromatic monocyclic or bicyclic rings (compounds 6a-d'); iv) a number of N1-aroyl analogues of 3a have been prepared (7a-p), the first retaining the phenyl ring at C4 and replacing the N1-benzyl with various aroyl/phenylsulfonyl portions (7a-g), and the others keeping fixed the benzoyl group at N1, and showing different (hetero)aryl rings at C4 (7h-p); v) a (4-methylpiperazin-1-yl)methyl group as a salifiable function has been inserted at the para position of the C4 phenyl ring in the 3a structure (compound 8), to improve its water solubility (Figure 2).

8


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Ar

R

C2H5OOC

R1

Ar Ar

COOC2H5 HOOC

N

N

H2NOC

CONH2

COOH N N

6a-j 5a 5b 5c 5d

6u-d'

R = COOC2H5, R1 = COOH

6k-t

R = COOC2H5, R1 = CONH2 R = COOH, R1 = CONH2 R = R1 = CN

Ar = 2-furyl, 2- and 3-thienyl, 2- and 5-thiazolyl, 4-biphenyl, 1- and 2-naphthyl, benzo[b]thiophen-2-yl, 3-quinolinyl

C2H5OOC R

R

R

COOC2H5

R N

H3C

N

CH3

N

O

Ar1

7a-f 4a R = COOC2H5 4b R = COOH 4c R = CONH2 .

H3C

Ar1 = phenyl, O-benzyl, 2-pyrazinyl, 1- and 2-naphthyl, 2-quinoxalyl

3a R = COOC2H5 3b R = COOH 3c R = CONH2

HCl

N

.

N

X

HCl

C2H5OOC C2H5OOC N O

O

O EtO

COOC2H5

COOC2H5

OEt

N O S O

7g 7h-p

N

X = 2-, 3- and 4-Me, 2-, 3- and 4-Cl, 2-, 3- and 4-OMe

8 Figure 2. The first DHP SIRT activators 3a-c and the novel 4-8 analogues.

9



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The new DHPs 4-8 were screened in vitro in an enzyme assay against SIRT1, to check their inhibiting or activating properties, and in HaCaT cells, to detect their capability to induce NO production. On selected compounds, in HaCaT cells we determined the SIRT1 activating activity, and studied the effect of NO release in the presence of 9 (compound C), a AMPK inhibitor,65 and 10 (EX-527),66 a SIRT1-selective inhibitor, to confirm the involvement of the AMPK/SIRT1 axis in the DHP-dependent NO production. Hence, the most efficient DHPs in NO production were in vivo assayed to test their ability to induce wound healing in a mouse model of skin repair. Selected DHP derivatives were also tested in C2C12 myoblasts to detect their effect on mitochondrial density, again in the absence and in the presence of 9 or 10. Afterwards, to ascertain the absence of any calcium channel antagonistic activity by the new DHPs, the prototypes 6c and 7a were tested on K+-depolarized guinea pig smooth muscles (aortic strips and ileum longitudinal smooth muscle) to assess their vascular- or non vascular-relaxant activity, respectively, using nifedipine as a reference drug. Finally, despite the marginal effects displayed by 3a-c in human leukemia U937 and breast cancer MCF7 cells,63 since SIRT1 could play a double sword edge activity as tumor promoter or tumor suppressor depending on the cellular context, environment and background, we tested selected novel DHPs in a panel of cancer cell lines to determine their effect on cell viability.

RESULTS AND DISCUSSION Chemistry. Compound 4a was synthesized according to the literature.67 After basic hydrolysis with 5M potassium hydroxide in ethanol at 80 °C, 4a gave the corresponding acid 4b, converted into the bis-amide 4c by using triethylamine, benzotriazole-1-yloxytris(dimethylamino)

10


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phosphonium hexafluorophosphate (BOP reagent), and 33% aqueous ammonia under N2 atmosphere and in anhydrous N,N-dimethylformamide (Scheme 1A). Using stoichiometric amount of 5M KOH in ethanol, 3a63 was converted into the monoester derivative 5a, which was then treated with triethylamine/BOP reagent and 33% ammonia to furnish the 3-carbethoxy-5-carboxamido analogue 5b. Further hydrolysis of 5a yielded the 3,5dicarboxylic acid 3b63 which was transformed into its monocarboxamido derivative 5c by the use of stoichiometric amounts of the same reagents (Scheme 1B). The 1-benzyl-4-phenyl-1,4-dihydropyridine-3,5-dicarbonitrile 5d was prepared through a dehydration reaction on 3c63 carried out with phosphorus pentoxide heated at 180 °C in dry N,Ndimethylformamide (Scheme 1C).

Scheme 1. Synthesis of Compounds 4a-c and 5a-da

11



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A O

O

EtO

O OEt

H3C

N

a

O

O

HO

CH3

b

OH

H3 C

4a

N

O

H2 N

NH2

H3 C

CH3

4b

N

CH3

4c

B O

O

EtO

O OEt

a

O

O

EtO

X

N

N

3a b

a

O

HO

X N

from 5a

5a X = OH 5b X = NH2

b

3b X = OH 5c X = NH2

C O

O

H2N

a

N NH2

N

c

N

N

3c

5d

Reagents and conditions: (a) 5M KOH, EtOH, 80 °C, overnight; (b) 1) Et3N, BOP reagent, dry

DMF, rt, 30 min, N2 atmosphere, 2) NH3 33%, rt, 30 min, N2 atmosphere; (c) P2O5, dry DMF, 180 °C, N2 atmosphere.

The

1-benzyl-3,5-dicarbethoxy-4-aryl-1,4-dihydropyridines

6a-j

were

prepared

by

multicomponent cyclocondensation between the opportune aldehydes, ethyl propiolate and benzylamine, heated at 80 °C in glacial acetic acid. Further alkaline hydrolysis of 6a-j at 80 °C

12


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in ethanol gave the corresponding 3,5-dicarboxy derivatives 6k-t, which were treated with triethylamine/BOP reagent and 33% ammonia to furnish the 3,5-dicarboxamide analogues 6u-d' (Scheme 2A). The N1-aroyl-3,5-carbethoxy-4-aryl DHPs 7a-f,h-p and the N1-phenylsulfonyl analogue 7g were prepared by using the following procedure: i) multicomponent cyclocondensation between (properly substituted) benzaldehyde, ethyl propiolate and ammonium acetate; ii) heating at 80 °C in glacial acetic acid to obtain the intermediate compounds 11a-j (see reff. 68-71 for 11a,68 11d,69 11e-g,70 11h,71 11i69 and 11j70); iii) N1-acylation with triethylamine and benzoyl chloride (for 7a and 7h-p) or the appropriate acyl chloride (for 7b-f) or phenylsulfonyl chloride (for 7g) to afford the desired compounds (Scheme 2B). Multicomponent reaction between ethyl propiolate, the commercially available 4-(4methylpiperazin-1-yl)methylbenzaldehyde and benzylamine, according to what reported for 6a-j, followed by treatment with 4M HCl in 1,4-dioxane afforded compound 8 as dihydrochloride salt (Scheme 2C).

Scheme 2. Synthesis of Compounds 6a-d', 7a-p and 8a

13



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A Ar

Ar

Ar = O

XOC

O

CHO

(a,k,u) (b,l,v) (c,m,w) (d,n,x) (e,o,y)

COX

OEt

N

NH2

(f,p,z)

6a-j X = OEt 6k-t X = OH c 6u-d' X = NH2

B X

N

(i,s,c')

b

(j,t,d')

X O

CHO

O a

OEt

O

O

EtO

OEt

AcONH4

from 11a

OEt N

N H O

X = H (a), 2- (b), 3- (c) and 4-Me (d) 2- (e), 3- (f) and 4-Cl (g) 2- (h), 3- (i) and 4-OMe (j)

X O

O EtO

OEt

from 11b-j from 11a

O

7g

7h-p X = 2- (h), 3- (i) and 4-Me (j) 2- (k), 3- (l) and 4-Cl (m) 2- (n), 3- (o) and 4-OMe (p)

C .

N

H3C

N

HCl

N

.

N

O CHO

HCl

a, g

O

O

O

OEt EtO

OEt

NH2 N

8

14

(a) OEt

N O S O


O

Ar1 =

O

EtO

e

f

Ar1

7a-f

O

N

EtO

O

EtO

d

11a-j

H3C

(h,r,b')

(g,q,a')

S

EtO

S

S

S

a

EtO

O

N

N S

O

(b)

N N

(c)

(d) N N

(e)

(f)

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a

Reagents and conditions: (a) CH3COOH, 80 °C; (b) 5M KOH, EtOH, 80 °C; (c) 1) Et3N, BOP

reagent, dry DMF, rt, 30 min, N2 atmosphere, 2) 33% NH3, rt, 30 min, N2 atmosphere; (d) Et3N, Ar1COCl, dry DCM, rt, overnight; (e) Et3N, PhSO2Cl, dry DCM, rt, overnight; (f) Et3N, PhCOCl, dry DCM, rt, overnight; (g) 4M HCl in 1,4-dioxane, tetrahydrofuran, 0 °C.

SIRT1 enzyme screening. The novel DHPs 4-8 were screened at 50 µM against SIRT1 using either the BioMol (Enzo) or the SIRTainty (EMD Millipore)51 assay. Moreover, we repeated the SIRTainty screen cloning full-length GST-tagged recombinant human SIRT1, after purified according to standard procedures, and using a p53-based untagged substrate. The SIRTainty assay coupled the SIRT1 deacetylase action with that of nicotinamidase PCN1, which catalyzes breakdown of the nicotinamide generated upon cleavage of NAD+ during the sirtuin-mediated deacetylation. The obtained ammonia is then quantized by reacting with ortho-phathalaldehyde (OPT) and dithiothreitol (DTT), with the formation of 1-alkylthioisoindoles detected at 460 nm.51 This method is particularly suitable to test SIRT modulators because non-tagged peptides are used as substrates avoiding the pitfalls of fluorescent substrates. We report in Table 1 the modulation of SIRT1 activity by 4-8 according to the SIRTainty (EMD Millipore) test (similar results were obtained with all the screen methods). Compound 2 and 3a were used as reference compounds. For some compounds, a high intrinsic fluorescence (IF) was detected.70 Given that the subtraction of the fluorescence may influence the in vitro screen results, we reported both the data without or with subtraction of IF for each compound. In some cases, the high IF did not allow to detect any modulation of SIRT1 activity (see compounds marked with ** in Table 1), even if in bio-based assay the regulation of SIRT1 was assessable (see below).

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Taken in account the data including IF (+ IF) reported in Table 1, we may appreciate that the insertion of methyl groups at the C2/C6 position of 3a-c (compounds 4a-c) led to moderate or no SIRT1

activation,

and

compounds

with

the

C3/C5

carbethoxy/carboxy

(5a),

carbethoxy/carboxamido (5b), carboxy/carboxamido (5c) or dicyano (5d) functions displayed similar or lower activation potency than 3a. About compounds 6, as a general trend those bearing the C3/C5 dicarbethoxy substitution (6a-j) were more efficient than the corresponding C3/C5 dicarboxy (6k-t) or dicarboxamido (6u-d') analogues, with the sole exception of the series bearing the 4-biphenyl residue at C4, in which the diester 6f and the dicarboxamido 6z displayed inhibition or no activity vs SIRT1, and the dicarboxy derivative 6p was instead an activator. Compounds bearing the C3/C5 dicarboxy and/or C3/C5 dicarboxamido substitution were either moderate activators, or displayed SIRT1 inhibition. The replacement of the N1-benzyl moiety of 3a with the benzoyl portion (7a) increased the SIRT1 activating property of the molecule, and also introduction at N1 of 2-pyrazinoyl (7c) or 2-naphthoyl (7e) group led to higher activation with respect to the prototype 3a. However, changes applied at the C4-phenyl ring of 7a by insertion of the methyl, chloro or methoxy group at ortho, meta or para position (compounds 7hp), despite their capability to still arouse SIRT1 activation, did not improve the SIRT1 activation ability of 7a. Compound 8, the water-soluble form of 3a, displayed slightly lower activation than the parent compound. For two representative compounds, 6c and 8, dose-response curves of their effets on SIRT1 activity have been reported in Figure S1 in Supporting Information. Also in this case the percentage values of SIRT1 activity in dependence of different concentrations of 6c or 8 have been drawn with and without intrinsic fluorescence.

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Table 1. Percentage of SIRT1 Activity in the Presence of DHP Derivatives 4-8 at 50 µM using the SIRTainty assay.a

cpd

R

R1

R2

R3

DMSO 2 3a 4a 4b 4c 5a 5b 5c 5d 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n

Ph Ph Ph Ph 2-furyl 2-thienyl 3-thienyl 2-thiazolyl 5-thiazolyl 4-biphenyl 1-naphthyl 2-naphthyl benzo[b]thiophen-2-yl 3-quinolinyl 2-furyl 2-thienyl 3-thienyl 2-thiazolyl

COOEt COOEt COOH CN COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOH COOH COOH COOH

COOH CONH2 CONH2 CN COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOH COOH COOH COOH

CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph

17


% SIRT1 activity + IFb - IFc 100 100 209 166 217 164 114 114 103 102 110 110 196 105 198 108 149 160 193 152 294 64* 246 117* 404 126* 300 282 221 133 61 61 187 ** 282 100* 139 39* 324 283 41 41 21 21 56 45 174 132


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6o 6p 6q 6r 6s 6t 6u 6v 6w 6x 6y 6z 6a' 6b' 6c' 6d' 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 8 a Values

COOH 5-thiazolyl 4-biphenyl COOH 1-naphthyl COOH 2-naphthyl COOH benzo[b]thiophen-2-yl COOH 3-quinolinyl COOH 2-furyl CONH2 2-thienyl CONH2 CONH2 3-thienyl CONH2 2-thiazolyl 5-thiazolyl CONH2 CONH2 4-biphenyl 1-naphthyl CONH2 2-naphthyl CONH2 benzo[b]thiophen-2-yl CONH2 3-quinolinyl CONH2 Ph COOEt Ph COOEt Ph COOEt Ph COOEt Ph COOEt Ph COOEt Ph COOEt 2-Me-Ph COOEt 3-Me-Ph COOEt 4-Me-Ph COOEt 2-Cl-Ph COOEt 3-Cl-Ph COOEt 4-Cl-Ph COOEt 2-OMe-Ph COOEt 3-OMe-Ph COOEt 4-OMe-Ph COOEt d PIP-CH2-Ph COOEt are reported as the average of

COOH COOH COOH COOH COOH COOH CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt at least

CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph COPh COOCH2Ph CO-2-pyrazinyl CO-1-naphthyl CO-2-naphthyl CO-2-quinoxalyl SO2Ph COPh COPh COPh COPh COPh COPh COPh COPh COPh CH2Ph three independent

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172 128 285 285 108 94 116 ** 38 7 209 167 77 62 55 49 218 97* 231 188 194 165 96 ** 145 ** 128 67* 127 37* 197 147 244 153* 206 158 242 171 179 ** 235 207 206 165 169 130 183 140 186 140 144 103 184 143 163 127 231 191 170 127 156 117 173 131 152 68* determinations. bValues

obtained without subtraction of intrinsic fluorescence (IF). cValues obtained with subtraction of IF. Note that for the compounds marked with the * a high IF was detected, and for those marked with the ** the value of IF was higher than that of the SIRT1 activity read by the instrument. d

PIP-CH2-Ph, 4-(4-Methylpiperazyn-1-yl)methylphenyl dihydrochloride.

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Due to the controversies arisen with 1, that was claimed to exert its beneficial effects on metabolism through phosphodiesterase 4 (PDE4) inhibition rather than through direct SIRT1 activation,50 selected DHP derivatives 3a, 6a, 6c, 6p, 7a and 7e were tested at 50 µM against PDE4, using rolipram (10 µM) as positive control. As it results from Figure 3, none among the tested compounds was able to inhibit PDE4.

Figure 3. PDE4 activity after treatment with DMSO (negative control), rolipram (10 µM), and the selected DHPs 3a, 6a, 6c, 6p, 7a and 7e (each at 50 µM). The PDE4 activity is reflected by the absorption at 620 nm. Error bars in the figure are standard deviations of three determinations.

NO release screening and SIRT1 activation in HaCaT cells. In endothelial cells (ECs), SIRT1 co-localize with and stimulate eNOS through deacetylation to increase NO release.72

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Inhibition or activation of SIRT1, by nicotinamide or 1 respectively, modulated endotheliumdependent vasodilation as well as NO bioavailability towards negative (by nicotinamide) or positive (by 1) values.72 Interestingly, AMPK phosphorylation of eNOS at Ser-633 has been shown to be required for its SIRT1-dependent deacetylation, highlighting the presence of the AMPK/SIRT1/eNOS axis in ECs.65 In the light of these findings, the new DHPs 5a-d, 6a-d', 7ag and 8 were tested in human keratinocyte HaCaT cells to determine their effect on NO production, monitored by 4,5-diaminofluorescein (DAF) (Figure 4A). Diethylenetriamine-nitric oxide (DETA/NO), a known NO donor, 1 and 3a were used as reference drugs. Nifedipine, a well known calcium channel antagonistic compound carrying the 1,4-DHP structure, was added for comparison. Cells were treated with the DHPs at different concentrations (1, 10, 25, and 50 µM) for 1, 3 and 5 h to select the best concentration for biological evaluations. In Figure 4A, the percentage values of DAF-positive cells with respect to the control (DMSO) recorded after 1 h of treatment with nifedipine, 1, 3a, 5a-d, 6a-d', 7a-g and 8 (all at 1 µM) or DETA/NO (at 500 µM) are shown. Compounds sharing the C3/C5 dicarbethoxy substitution and a 2-furyl (6a) and 3thienyl (6c) ring at the C4 position, as well as compound 7a, characterized by a phenyl at C4 and a benzoyl portion at N1, displayed the strongest NO release. Compounds 6d and 6j, characterized by a 2-thiazolyl or 3-quinolinyl ring at C4 and a C3/C5 dicarbethoxy functions, and compound 8, the water-soluble form of 3a, displayed similar NO induction as 3a (Figure 4A). For 6a, 6c and 7a the corresponding dose-response curves were also determined (Figure 4B). As we stated above, eNOS activation is driven by AMPK-dependent phophorylation and then SIRT1-dependent deacetylation.65 To ascertain if the extensive NO release observed with DHPs was effectively regulated by the AMPK/SIRT1 axis, the experiments were repeated by treating

20


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HaCaT cells with 6c, 7a or 8 alone and in combination with 9 (Figure 4D), a known AMPK inhibitor,65 or 10 (Figure 4D), a selective SIRT1 inhibitor,66 all tested at 10 µM. The results depicted in Figure 4C clearly showed that the combined uses of 6c, 7a or 8 with either 9 or 10 totally abated (with 9) or highly decreased (with 10) the NO release observed with the DHPs used as single agents. Our results demonstrated the selectivity for SIRT1 of compounds 6c, 7a and 8 on NO production, as well as the effect of 10, able to partially counteract the DHPs dependent outcome.

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Figure 4. A) Quantification of NO release by DAF staining in the human keratinocyte HaCat cells after 1 h of treatment with DMSO, DETA/NO (500 µM), 1, 3a, 5a-d, 6a-d', 7a-g and 8 (all at 1 µM). B) Dose-response curves for increased DAF staining for 6a, 6c and 7a. C) Quantification of NO release by DAF staining in HaCaT cells after 1 h of treatment with 6c, 7a or 8, used alone or in combination with the AMPK inhibitor 9 or the SIRT1-selective inhibitor 10, all tested at 10 µM. D) Chemical structures of 9 and 10.

To detect the DHP-driven SIRT1 activation directly in the cellular context, we measured the activity of SIRT1 in HaCaT nuclear extracts64 treated with 6a, 6c and 7a (1 µM). Sirtinol (25 µM) and 1 (1 µM) were used as negative and positive control, respectively. As shown in Figure 5A, all the tested DHPs induced a time-dependent SIRT1 activation when compared with DMSO, with 6a and 6c giving the highest activation. As expected, 1 was also able to increase SIRT1 activity while sirtinol behaved as an inhibitor. SIRT1 functional assay was also performed in HaCaT cells through Western blot analysis, monitoring changes in H4K16 acetylation levels after 1 h treatment with 6a, 6c and 7a (1 µM) (Figure 5B). The novel sirtuin activators induced H4K16 deacetylation in HaCaT cells when compared with DMSO, similarly to 1 and 3a, endorsing the assumption of SIRT1 activation mediated by the tested compounds.

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Figure 5. A) SIRT1 assay performed in HaCaT cells treated with 6a, 6c and 7a (1 µM). Sirtinol (25 µM) and RSV (1 µM) were used as negative and positive controls, respectively. B) Effects of 6a, 6c and 7a (1 h, 1 µM) on acetylation levels of H4K16Ac in HaCaT cells. 1 and 3a (1 µM) were used as positive controls. Relative densitometry is shown in the right panel.

Effect of 6a, 6c and 7a in wound healing. Treatment of a mouse model of skin repair with either HDAC inhibitors or SIRT activators such as 3a accelerated tissue regeneration and promoted wound healing.64 The mechanism of this effect involves an increase of cell proliferation, shown in keratinocyte HaCaT cells and depending from the high release of NO.

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We suggested the presence of a SIRT-NO-HDAC signaling cascade, in which SIRT activators were responsible of increased release of NO, and HDAC2 was inhibited through S-nitrosylation of key residues for its catalytic activity.64 Accordingly, we performed in vivo biopsy punch experiments with 6a, 6c and 7a (1 µM) to assess the capability of the novel DHPs to accelerate the wound healing process in mice. In coherence with our previous findings,64 6a, 6c and 7a decreased the epithelial gap and increased the percentage of wound closure respect to the solvent (DMSO) when topically administered each day for 14 days (Figure 6).

Figure 6. Time dependent evaluation of 6a, 6c and 7a (1 µM) in wound healing in CD1 mice. DMSO was used as solvent control. N (number of experiments) = 6.

Effects of 6c, 6k and 7a on mitochondrial function in murine C2C12 myoblasts. SIRT1 is known to promote mitochondrial biogenesis and activity through deacetylation and activation of PGC-1α.73 PGC-1α is also a target of AMPK,74 thus reinforcing the metabolic link established by the AMPK/SIRT1 axis. To determine the effect of selected DHPs on mitochondrial function,

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mouse C2C12 myoblasts were treated overnight with 6c, 6k (negative control) or 7a as single agents (each at 30 µM). In addition, 6c or 7a (30 µM) were also tested in combination with 9 or 10 (each at 5 µM), to detect their effects in the presence of an AMPK inhibitor or a SIRT1 inhibitor, respectively. Treatment of C2C12 cells with 6c or 7a alone highly enhanced the fluorescence intensity of the cells due to the mitochondrial specific probe Mitotracker Green, indicating increased mitochondrial density, whilst 6k displayed no effect, as expected. Coadministration of 6c or 7a with either 9 or 10 in part reverted this effect, showing that it is related to activation of the AMPK/SIRT1 axis (Figure 7A). The cellular effects of 6c, used alone or in combination with either 9 or 10, and of 6k as a negative control, were visualized by confocal microscopy (Figure 7B).

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B

Figure 7. Mitotracker Green assay performed on murine C2C12 myoblasts treated with 6c or 7a (30 µM), as single agent or in combination with 9 or 10, both at 5 µM. B) Effects on C2C12 myoblasts (Mitotracker Green assay) of 6c as single agent or in combination with 9 or 10, and of 6k used as a negative control. Confocal microscopy settings (laser intensity, pinhole, detector gain, amplifier offset and amplifier gain) were set on the intensity of Mitotracker fluorescence from wt untreated cells and maintained as such during the acquisition of treated cells. Afterward, images were only reduced in size without changing original settings.

Calcium channel antagonist activity. It is well known that compounds bearing the 1,4-DHP structure display high calcium channel antagonist activity. For this reason, we tested nifedipine in HaCaT cells to determine its effect on NO production (Figure 4A), and we found that it was

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unable to increase the NO level up to 50 µM (the maximum dose tested, data not shown). The SAR described for DHPs reported that normally the N1 substitution is detrimental for the calcium channel antagonsitic activity, but they are controversial in some cases.75,76 Thus, to ascertain that our 1,4-DHPs lack significant calcium-antagonist activity, we tested 6c and 7a on guinea-pig isolated vascular (aortic strips) and non-vascular [ileum longitudinal smooth muscle (GPILSM)] smooth muscle, to evaluate the percent inhibition of calcium-induced contraction on K+-depolarized (80 mM) aortic strips and GPILSM. Nifedipine was used as a refence drug. Data reported in Table S2 in Supporting Information clearly show that 6c and 7a were totally inactive to inhibit calcium-induced contraction in aortic strips (6c and 7a: 13% and 12% inhibition at 50 µM; nifedipine: IC50 = 9 nM), and displayed 266- and 720-fold lower inhibition activity than nifedipine in GPILSM, respectively. Antiproliferative effects of selected DHPs in a panel of cancer cells. Conflicting data reported in the literature support both activation and inhibition of SIRT1 as a strategy for cancer therapy. In fact, depending on the cellular context or specific signaling pathways or cancers, SIRT1 has been reported to be a tumor suppressor or a tumor promoter. In some cases, it deacetylates tumor-promoting transcription factors, such as NF-κB and HIF-1α, leading to their inactivation, and improves genomic stability and DNA repair. In other contexts, SIRT1 exerts antiapoptotic and antidifferentiation properties through deacetylation and inactivation of other transcription factors, such as p53 and members of the FOXO family, leading to cancer initiation and progression. In contrast to the great number of reports describing the anticancer activities of SIRT1 inhibitors, only few papers have been released on the beneficial effect of SIRT1 activators in cancer.77 Therefore, compound 7a and and the water-soluble derivative of 3a, compound 8, were

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tested in a panel of cancer cells (melanoma, lung, colon and ovary cancer cells) as representative members of our DHPs, to determine their effect on tumor cell viability. The IC50 (concentration able to inhibit 50% cell viability) values of 7a and 8 against the tested cancer cell lines after 72 h of treatment are reported in Table 3. The related dose-response curves are depicted in Figure 8 (for 8) and in Supporting Information (for 7a). As it is shown in Table 3, 8 was 5- to 20-fold more potent than 7a against the tested cancer cells, likely because of its increased water solubility. In particular, 8 displayed single-digit micromolar potency against the M14 melanoma cell line (IC50 = 8 µM), and lower effect (IC50s ranging between 10 to 35 µM) against all the other tested cell lines. Differently, 7a inhibited proliferation only in LOVO colon cancer cells (IC50 = 22 µM), and showed IC50s lower than 100 µM only in the other tested colon cancer cell lines. To confirm that the mechanism of action of 8 as anticancer agent involves increased histone deacetylation, western blot analyses have been performed treating M14 melanoma and H1299 lung cancer cells, as representative members of the most and less responsive cell lines, respectively, with 5, 10, and 20 µM doses of 8 for 48 h. After, the levels of acetyl-H4K16, a typical mark of SIRT1 deacetylating activity, were detected with specific antibodies. β–Actin was used for equal loading. Data depicted in Figure 9 clearly show that 8 was able to induce a dose-dependent decrease of acetyl-H4K16 levels, confirming its behaviour of SIRT1 activator in the tested cell lines.

Table 3. IC50 Values of 7a and 8 in a Panel of Human Cancer Cell Linesa cell line

cancer histotype

M14 SAN

melanoma melanoma

IC50 (µM, 72 h) 7a 8 153 ± 10 8±6 161 ± 7 16 ± 7

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A549 lung 169 ± 7 20 ± 12 H1299 lung 125 ± 10 24 ± 15 HT29 colon 80 ± 2 11 ± 3 LOVO colon 22 ± 3 15 ± 9 HCT116 colon 62 ± 1 NDb CAOV3 ovary >200 24 ± 10 HEY ovary 143 ± 18 15 ± 12 SKOV3 ovary ND 35 ± 20 A2780 ovary ND 10 ± 14 a Values represent the mean ± standard deviation (SD) of at least three separate experiments. b ND, not determined.

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Figure 8. Dose-response curves for antiproliferative activity of 8 in a panel of human cancer cell lines. The results are reported as “viability of drug-treated cells/viability of control cells” × 100 and represent the average ± SD of three independent experiments.

Figure 9. Dose-dependent increased deacetylation activity (acetyl-H4K16) by 8 in M14 melanoma and H1299 lung cancer cells. Relative densitometry is shown in the right panels.

Conclusion

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Sirtuins are NAD+-dependent Lys deacetylases that play a key role in cell survival, energy metabolism and ageing. The members of this family act in different cellular compartments, such as nucleus, cytoplasm or mitochondria, regulating fat and glucose metabolism, energy homeostasis and healthspan. Small molecule modulators of sirtuins are retained promising therapeutic targets for the treatment of age-related diseases such as cancer, type 2 diabetes, inflammatory disorders, and cardiovascular and neurodegenerative diseases. The 1-benzyl-3,5dicarbethoxy-4-phenyl-1,4-dihydropyridine 3a has been recently reported by us to improve the SIRT1 deacetylase activity and, from a biological point of view, to reduce the number of senescent cells in hMSCs, to improve mitochondrial function in murine C2C12 myoblasts, to stimulate proliferation in human HaCat cells via NO production and to accelerate the process of skin repair in an in vivo mouse model. To acquire further SAR information on the 3a structure, and to identify more potent and efficient analogues, we prepared a number of DHP derivatives 48 by introduction of methyl groups at the C2/C6 positions, by replacing the C3/C5-dicarbethoxy functions

with

asymmetric

carbethoxy/carboxy,

carbethoxy/carboxamido,

or

carboxy/carboxamido groups, by changing the C4-phenyl group with a mono- or bicyclic (hetero)aromatic ring, by replacing the N1-benzyl group with a N1-benzoyl/aroyl substituent, and by insertion of a (4-methylpiperazin-1-yl)methyl group in the 3a structure to improve its watersolubility (8). Among the studied modification, the substitution of the C4-phenyl ring of 3a with mono- or bicyclic heteroaromatic rings often led to increased potency in activating SIRT1 than 3a. A slight improvement of potency was also observed by changing the 3a N1-benzyl with the N1-benzoyl (7a) or -2-pyrazinoyl (7c) or 2-naphthoyl (7e) moiety. When tested in human keratinocyte HaCat cells to determine their ability to increase NO production respect to DMSO (control), the majority of the new compounds were able to augment the percentage of DAF-

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positive cells, used as a marker of NO production, with 6a, 6c, and 7a being more efficient than 3a. Dose-response curves for 6a, 6c and 7a were determined, and co-administration of 6c, 7a or 8 with either 9, a known AMPK inhibitor, or 10, a SIRT1-selective inhibitor, decreased the NO production observed with the treatment of the DHPs alone, confirming the involvement of the AMPK/SIRT1 axis in their effect. SIRT1 activation assay with 6a, 6c and 7a was repeated in the HaCat cells, where the increase of NO production was been observed, and functional test for SIRT1 activation was performed determining the level of acetyl-H4K16 in HaCat cells in the presence of the same DHPs. Importantly, when tested in a mouse model for wound healing, 6a, 6c and 7a were able to improve the percentage of closure of the wound during 14 days respect to DMSO used as solvent control. Furthermore, similarly to what previously observed with 3a, also 6c and 7a improved mitochondria function in murine C2C12 myoblasts, as detected by Mitotracker Green, and their effects were in part counteracted by the co-administration of either 9 or 10, reinforcing our conviction that the DHP-induced biological effects were due at least in part to activation of the AMPK/SIRT1 pathway also in this cell type. Despite their 1,4-DHP structure, 6c and 7a did not show any calcium channel antagonsitic activity, tested in guinea pig aortic strips as well as in ileum longitudinal smooth muscle. Due to the controversial role of SIRT1 in cancer, for which it has been described as either a tumor suppressor or a tumor promoter, we tested 7a and 8 against melanoma, lung, colon, and ovary cancer cells, to determine their potential antiproliferative effects. While 7a showed low micromolar potency only against LOVO colon cancer cells, 8 probably due to its increased water-solubility exerted high effect (IC50s in the 8-35 µM range) against all the tested cancer cells, and decreased the levels of acetyl-H4K16, a typical mark of SIRT1 deacetylating activity,

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in M14 melanoma and H1299 lung cancer cells, suggesting that SIRT activators could play a role also in cancer therapy.

Experimental Section Chemistry. Melting points were determined on a Buchi 530 melting point apparatus and are uncorrected. 1H NMR and

13

C NMR spectra were recorded at 400 MHz and 100 MHz,

respectively, on a Bruker AC 400 spectrometer; chemical shifts are reported in δ (ppm) units relative to the internal reference tetramethylsilane (Me4Si). EIMS spectra were recorded with a Fisons Trio 1000 spectrometer; only molecular ions (M+) and base peaks are given. All compounds were routinely checked by TLC and 1H NMR. TLC was performed on aluminumbacked silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots visualized by UV light. All solvents were reagent grade and, when necessary, were purified and dried by standard methods. Concentration of solutions after reactions and extractions involved the use of a rotary evaporator operating at reduced pressure of ca. 20 Torr. Organic solutions were dried over anhydrous sodium sulfate. Elemental analysis has been used to determine purity of the described compounds, that is >95%. Analytical results are within ± 0.40% of the theoretical values. All chemicals were purchased from Sigma Aldrich, Milan (Italy), or from Alfa Aesar, Karlsruhe (Germany), and were of the highest purity. Procedure for the Synthesis of 1-Benzyl-5-(ethoxycarbonyl)-4-phenyl-1,4-dihydropyridine3-carboxylic Acid (5a). A mixture of diethyl 1-benzyl-4-phenyl-1,4-dihydropyridine-3,5dicarboxylate 3a63 (2.55 mmol, 1 g) and 5M KOH (6.39 mmol, 0.36 g, 1.28 mL) in ethanol (10 mL) was stirred at 80 °C for 2.5 h. Afterward, the solvent was evaporated, the residue was eluted with water (30 mL) and the resulting solution acidified with 2M HCl. The obtained precipitate

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was filtered and dried to afford the pure compound 5a as a pale yellow solid that was recrystallized by acetonitrile/methanol. m.p.: 162-164 °C; yield: 68 %; 1H NMR (DMSO-d6) δ 1.26 (t, 3H, -OCH2CH3), 4.17 (q, 2H, -OCH2CH3), 4.79 (s, 2H, -NCH2Ph), 4.80 (s, 1H, ArCH), 6.93 (s, 1H, dihydropyridine proton), 7.0 (s, 1H, dihydropyridine proton), 7.28-7.38 (m, 10H, aromatic protons), 11.80 (bs, 1H, -COOH) ppm; 13C NMR (DMSO-d6) δ 14.2, 34.7, 57.9, 61.1, 105.5, 106.4, 127.2 (3C), 127.6, 128.5 (2C), 128.6 (2C), 128.7 (2C), 134.7, 136.1, 142.8, 144.0, 167.4, 168.3 ppm; MS (EI): m/z [M]+: 363.15. General Procedure for the Synthesis of Ethyl 1-Benzyl-5-carbamoyl-4-phenyl-1,4dihydropyridine-3-carboxylate

(5b)

and

1-Benzyl-5-carbamoyl-4-phenyl-1,4-

dihydropyridine-3-carboxylic Acid (5c). Example: Synthesis of 1-Benzyl-5-carbamoyl-4phenyl-1,4-dihydropyridine-3-carboxylic Acid (5c). Triethylamine (0.26 mL, 1.88 mmol) and BOP reagent (0.25 g, 0.565 mmol) were added under nitrogen atmosphere to a solution of compound 3b63 (0.47 mmol, 0.158 g) in dry N,N-dimethylformamide (5 mL), and the resulting mixture was stirred for 30 min at room temperature. After this time 33% aqueous ammonia (4.7 mmol, 0.27 mL) was added and the resulting mixture was stirring for further 30 min. The reaction was quenched by water (30 mL), the precipitate was filtered and washed with water (3 × 30 mL) and diethyl ether (3 × 30 mL) to provide 5c as a pale yellow solid that was recrystallized by methanol. m.p.: 190-192 °C; yield: 89 %; 1H NMR (DMSO-d6) δ 4.79 (s, 2H, -NCH2Ph), 4.80 (s, 1H, ArCH), 6.89-7.40 (m, 14H, -CONH2, dihydropyridine and aromatic protons), 11.80 (bs, 1H, -COOH) ppm;

13

C NMR (DMSO-d6) δ 32.9, 57.9, 105.5, 111.4, 127.2 (3C), 127.6, 128.5

(2C), 128.6 (2C), 128.7 (2C), 136.1, 141.5, 142.8, 144.0, 148.9, 168.2, 170.7 ppm; MS (EI): m/z [M]+: 334.13.

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Procedure for the Synthesis of 1-Benzyl-4-phenyl-1,4-dihydropyridine-3,5-dicarbonitrile (5d). A mixture of 1-benzyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxamide 3c63 (0.9 mmol, 0.3 g) and phosphorus pentoxide (2.27 mmol, 0.77 g) in dry N,N-dimethylformamide (3 mL) was stirred at 180 °C for 30 min. Then water (50 mL) was added to the reaction, and the suspension was extracted by ethyl acetate (3 × 30 mL), the collected organic phases were washed by saturated sodium chloride solution (3 × 30 mL), dried with sodium sulfate and concentrated to furnish a residue that was purified by silica gel chromatography eluting with ethyl acetate:nhexane 1:8 to give the pure compound 5d as a pale yellow solid that was recrystallized by benzene/acetonitrile. m.p.: 157-159 °C; yield: 64 %; 1H NMR (CDCl3) δ 4.62 (s, 2H, -NCH2Ph), 4.70 (s, 1H, ArCH), 6.90 (s, 2H, dihydropyridine protons), 7.28-7.38 (m, 10H, aromatic protons) ppm;

13

C NMR (CDCl3) δ 38.6, 57.9, 84.2 (2C), 116.7 (2C), 127.2 (3C), 127.2, 127.6, 128.6

(4C), 128.7 (2C), 136.1, 141.9 (2C), 142.1 ppm; MS (EI): m/z [M]+: 297.13. General Procedure for the Synthesis of Diethyl 4-Aryl-1-benzyl-1,4-dihydropyridine-3,5dicarboxylate 6a-j.

Example: Synthesis

of

Diethyl

1-Benzyl-4-(thiophen-3-yl)-1,4-

dihydropyridine-3,5-dicarboxylate (6c). Ethyl propiolate (18.84 mmol, 1.9 mL), thiophen-3carboxaldehyde (9.42 mmol, 0.82 mL) and benzylamine (9.42 mmol, 0.65 mL) in glacial acetic acid (0.5 mL) were heated at 80 °C for 30 min. After cooling, the mixture was poured into water (20 mL) and stirred for 1 h. The solid product was filtered and washed with diethyl ether (3 × 30 mL) to give the pure 6c as a pale yellow solid that was recrystallized by cyclohexane/toluene. m.p.: 110-112 °C; yield: 76 %; 1H NMR (CDCl3) δ 1.22 (t, 6H, -OCH2CH3), 4.13 (q, 4H, OCH2CH3), 4.59 (s, 2H, -NCH2Ph), 5.08 (s, 1H, ArCH), 6.99 (s, 2H, dihydropyridine protons), 7.14-7.15 (m, 1H, aromatic proton), 7.25-7.27 (m, 4H, aromatic protons), 7.36-7.41 (m, 3H, aromatic protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 32.1, 57.9, 61.1 (2C), 105.0 (2C), 120.5,

36


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124.8, 127.2 (2C), 127.6 (2C), 128.0, 128.6 (2C), 136.1, 137.6 (2C), 150.2, 166.6 (2C) ppm; MS (EI): m/z [M]+: 397.13. General

Procedure

for

the

Synthesis

of

1-Benzyl-2,6-dimethyl-4-phenyl-1,4-

dihydropyridine-3,5-dicarboxylic Acid (4b) and 4-Aryl-1-benzyl-1,4-dihydropyridine-3,5dicarboxylic

Acids

(6k-t).

Example:

Synthesis

of

1-benzyl-4-(thiazol-2-yl)-1,4-

dihydropyridine-3,5-dicarboxylic acid (6n). A mixture of 6d (2.55 mmol, 1.02 g) and 5M KOH (12.77 mmol, 0.7 g, 2.55 mL) in ethanol (10 mL) was stirred at 80 °C overnight. Afterward the solvent was evaporated, the residue was eluted with water (30 mL) and the resulting solution acidified with 2M HCl. The obtained precipitate was filtered and dried to afford the pure compound 6n as a pale yellow solid that was recrystallized by acetonitrile/methanol. m.p.: 218220 °C; yield: 67 %; 1H NMR (DMSO-d6) δ 4.81 (s, 2H, -NCH2Ph), 5.17 (s, 1H, ArCH), 7.297.33 (m, 3H, dihydropyridine protons and aromatic proton), 7.37-7.40 (m, 2H, aromatic protons), 7.45-7.46 (m, 2H, aromatic protons), 7.52 (d, 1H, aromatic proton), 7.62 (d, 1H, aromatic proton), 12.00 (bs, 2H, -COOH) ppm;

13

C NMR (DMSO-d6) δ 33.0, 63.6, 107.3 (2C), 118.7,

126.7, 126.9 (2C), 128.5 (2C), 136.5, 141.9, 148.9 (2C), 165.9, 171.3 (2C) ppm; MS (EI): m/z [M]+: 342.07. General

Procedure

for

the

Synthesis

of

1-Benzyl-2,6-dimethyl-4-phenyl-1,4-

dihydropyridine-3,5-dicarboxamide (4c) and 4-Aryl-1-benzyl-1,4-dihydropyridine-3,5dicarboxamides

(6u-d').

Example:

Synthesis

of

1-Benzyl-4-(naphthalen-2-yl)-1,4-

dihydropyridine-3,5-dicarboxamide (6b'). Triethylamine (0.52 mL, 3.76 mmol) and BOP reagent (0.5 g, 1.13 mmol) were added under nitrogen atmosphere to a solution of compound 6r (0.47 mmol, 0.181 g) in dry N,N-dimethylformamide (5 mL), and the resulting mixture was stirred for 30 min at room temperature. After this time 33% aqueous ammonia (4.7 mmol, 0.27

37



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mL) was added and the resulting mixture was stirring for further 30 min. The reaction was quenched by water (30 mL), the precipitate was filtered and washed with water (3 × 30 mL) and diethyl ether (3 × 30 mL) to provide 6b' as a pale yellow solid that was recrystallized by methanol. m.p.: >250 °C; yield: 78 %; 1H NMR (DMSO-d6) δ 4.66 (s, 2H, -NCH2Ph), 5.11 (s, 1H, ArCH), 6.78 (bs, 4H, -CONH2), 7.34-7.42 (m, 11H, dihydropyridine and aromatic protons), 7.64-7.66 (m, 1H, aromatic proton), 7.70-7.72 (m, 1H, aromatic proton), 7.80-7.82 (m, 1H, aromatic proton) ppm; 13C NMR (DMSO-d6) δ 45.4, 62.8, 110.2 (2C), 125.1, 126.0, 126.7, 126.9 (2C), 127.0, 127.3, 127.5, 127.6, 128.0, 128.5 (2C), 131.8, 133.7, 135.2, 136.5, 144.9 (2C), 171.0 (2C) ppm; MS (EI): m/z [M]+: 383.16. General

Procedure

for

the

Synthesis

of

Diethyl

4-(Substituted)phenyl-1,4-

dihydropyridine-3,5-dicarboxylate (11b,c). Example: Synthesis of diethyl 4-o-tolyl-1,4dihydropyridine-3,5-dicarboxylate (11b). Ethyl propiolate (17.84 mmol, 1.82 mL), 2tolualdehyde (8.92 mmol, 1.03 mL) and ammonium acetate (8.92 mmol, 0.7 g) were heated to 80 °C for 5 h in glacial acetic acid (5 mL). Back to room temperature, the reaction mixture was quenched with water (50 mL) and stirred for additional 10 min at room temperature and then extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with brine, dried with sodium sulfate and evaporated under vacuum. The crude product was purified via column chromatography on silica gel using ethyl acetate/n-hexane 1:2 as eluting system to afford compound 11b. m.p.: 93-96 °C; yield: 44 %; 1H NMR (CDCl3) δ 1.18-1.22 (t, 6H, -OCH2CH3); 2.68 (s, 3H, CH3), 4.02- 4.15 (m, 4H, -OCH2CH3); 5.09 (s, 1H, dihydropyridine proton); 6.24 (bs, 1H, -NH); 6.8-6.7 (m, 2H, thiophene protons); 7.03-7.36 (m, 6H, dihydropyridine protons and benzene protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 19.6, 41.6, 61.6 (2C), 108.2 (2C),

38


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122.7, 124.5, 125.6, 130.4, 135.7, 136.9, 138.5 (2C), 167.1 (2C) ppm; MS (EI): m/z [M]+: 315.15. General Procedure for the Synthesis of Diethyl 1-Aroyl- or 1-Phenylsulfonyl-4-Aryl-1,4dihydropyridine-3,5-dicarboxylate (7a-p). Example: Synthesis of Diethyl 1-Benzoyl-4-(3methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7o). Diethyl 4-(3-methoxyphenyl)1,4-dihydropyridine-3,5-dicarboxylate (11i) (0.17 mmol, 0.06 g), benzoyl chloride (0.26 mmol, 0.45 mL), and triethylamine (0.85 mmol, 0.12 mL) are stirred in dry dichloromethane (7 mL) at room temperature overnight. The reaction is then quenched with water (50 mL) and stirred for additional 10 min at room temperature and then extracted with ethyl acetate (4 × 30 mL). The organic layer is washed with brine, dried with sodium sulfate and evaporated at reduced pressure. The crude is purified via column chromatography on silica using ethyl acetate/n-hexane 1:5 as eluting system. m.p.: 68-71 °C; yield: 88 %; 1H NMR (CDCl3) δ 1.21-1.24 (m, 6H, -OCH2CH3), 3.82 (s, 3H, -OCH3), 4.10-4.17 (m, 4H, -OCH2CH3), 4.96 (s, 1H, ArCH-), 6.77-6.79 (m, 1H, aromatic proton), 6.93-6.97 (m, 2H, aromatic protons), 7.22-7.26 (m, 1H, aromatic proton), 7.547.58 (m, 2H, aromatic protons), 7.62-7.69 (m, 3H, aromatic protons), 8.12 (s, 2H, dihydropyridine protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 44.4, 55.8, 61.7 (2C), 108.0 (2C), 113.1, 113.3, 120.0, 127.5 (2C), 128.8 (2C), 129.6, 131.4, 132.1, 141.2 (2C), 143.2, 160.5, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 435.17. Procedure

for

the

Synthesis

of

Diethyl

1-Benzyl-4-(4-((4-methylpiperazin-1-

yl)methyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylate

dihydrochloride

(8).

Ethyl

propiolate (18.84 mmol, 1.9 mL), 4-((4-methylpiperazin-1-yl)methyl)benzaldehyde (9.42 mmol, 2.1 g) (from Acros Organics, Belgium) and benzylamine (9.42 mmol, 0.65 mL) in glacial acetic acid (5 mL) were heated at 80 °C for 1 h. After cooling, the mixture was poured into water (20

39



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mL) and extracted with dichloromethane (3 × 30 mL) to give a pale yellow residue that was purified by SiO2 column chromatography eluting with ethyl acetate. Afterwards the obtained pure compound was eluted in dry tetrahydrofuran (3 mL) followed by the addition of a cooled (0 °C) 4M HCl solution in 1,4-dioxane. After a period of 30 min the formed hydrochloric salt was filtered, washed with diethyl ether (3 × 5 mL), dried and recrystallized by ethanol to give pure 8. m.p.: 232-234 °C; yield: 83 %; 1H NMR (DMSO-d6) δ 1.11 (t, 6H, -OCH2CH3), 2.80 (s, 3H, NCH3), 3.35-3.45 (m, 10H, piperazine and PhCH2-piperazine protons), 4.00 (q, 4H, -OCH2CH3), 4.75 (s, 1H, PhCH), 4.84 (s, 2H, -NCH2Ph), 7.14 (d, 2H, benzene protons), 7.35-7.51 (m, 9H, benzene and dihydropyridine protons), 11.60 (bs, 2H, 2 x NH+ piperazine) ppm;

13

C NMR

(DMSO-d6) δ 14.1 (2C), 42.2, 44.3, 51.5 (2C), 54.5 (2C), 56.4, 61.6 (2C), 63.7, 108.1 (2C), 126.6, 126.9 (2C), 128.6 (2C), 128.8 (4C), 129.7, 136.5, 141.6, 146.2 (2C), 167.3 (2C) ppm; MS (EI): m/z [M]+: 503.28. Chemical and physical data, 1H NMR, 13C NMR and MS (EI) data for compounds 4-7,11. 1-Benzyl-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylic acid (4b). m.p.: 148-150 °C; recrystallization solvent: acetonitrile; yield: 87%; 1H NMR (DMSO-d6) δ 2.37 (s, 6H, -CH3), 4.90 (s, 2H, -NCH2Ph), 5.12 (s, 1H, ArCH-), 6.91-6.93 (m, 2H, benzene protons), 7.13-7.34 (m, 8H, benzene protons), 11.80 (bs, 2H –COOH) ppm; 13C NMR (DMSO-d6) δ 16.1 (2C), 37.3, 56.0, 103.0 (2C), 127.3 (3C), 127.6 (5C), 128.5 (2C), 137.5, 145.2, 147.8 (2C), 168.2 (2C) ppm; MS (EI): m/z [M]+: 363.15. 1-Benzyl-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxamide (4c). m.p.: 176-178 °C; recrystallization solvent: acetonitrile/methanol; yield: 76%; 1H NMR (DMSOd6) δ 2.12 (s, 6H, -CH3), 4.75 (s, 1H, ArCH-), 4.78 (s, 2H, -NCH2Ph), 6.81- (bs, 2H, -CONH2), 7.11-7.30 (m, 12H, benzene protons and -CONH2), 11.80 (bs, 2H –COOH) ppm;

40


13

C NMR

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(DMSO-d6) δ 16.2 (2C), 34.9, 50.6, 109.8 (2C), 127.2, 127.6 (5C), 127.8 (2C), 128.5 (2C), 137.5, 142.8, 146.3 (2C), 168.4 (2C) ppm; MS (EI): m/z [M]+: 361.18. Ethyl 1-Benzyl-5-carbamoyl-4-phenyl-1,4-dihydropyridine-3-carboxylate (5b). m.p.: 148-150 °C; recrystallization solvent: methanol; yield: 75%; 1H NMR (DMSO-d6) δ 1.26 (t, 3H, -OCH2CH3), 4.17 (q, 2H, -OCH2CH3), 4.79 (s, 2H, -NCH2Ph), 4.80 (s, 1H, ArCH), 6.897.40 (m, 14H, -CONH2, dihydropyridine and aromatic protons) ppm;

13

C NMR (DMSO-d6) δ

14.2, 31.8, 57.9, 61.1, 106.4, 111.4, 127.2 (3C), 127.6, 128.5 (2C), 128.6 (2C), 128.7 (2C), 134.7, 136.1, 141.5, 142.8, 167.4, 170.7 ppm; MS (EI): m/z [M]+: 362.16. Diethyl 1-Benzyl-4-(furan-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6a). m.p.: 82-85 °C; recrystallization solvent: cyclohexane; yield: 68%; 1H NMR (CDCl3) δ 1.24 (t, 6H, -OCH2CH3), 4.16 (q, 4H, -OCH2CH3), 4.79 (s, 2H, -NCH2Ph), 4.83 (s, 1H, ArCH), 5.885.89 (m, 1H, dihydropyridine proton), 6.28-6.29 (m, 1H, dihydropyridine proton), 7.30-7.42 (m, 8H, aromatic protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 38.3, 57.9, 61.1 (2C), 103.0, 110.4 (2C), 127.2 (2C), 127.6, 128.6 (2C), 136.1, 138.7 (2C), 142.3, 153.5, 166.5 (2C) ppm; MS (EI): m/z [M]+: 381.16. Diethyl 1-Benzyl-4-(thiophen-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6b). m.p.: 102-104 °C; recrystallization solvent: cyclohexane/toluene; yield: 67%; 1H NMR (CDCl3) δ 1.24 (t, 6H, -OCH2CH3), 4.16 (q, 4H, -OCH2CH3), 4.62 (s, 2H, -NCH2Ph), 5.27 (s, 1H, ArCH), 6.87-6.88 (m, 2H, dihydropyridine protons), 7.10-7.12 (m, 1H, aromatic protons), 7.27-7.29 (m, 4H, aromatic protons), 7.39-7.41 (m, 3H, aromatic protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 41.0, 57.9, 61.1 (2C), 108.3 (2C), 123.6, 126.2, 126.8, 127.2 (2C), 127.6, 128.6 (2C), 136.1, 137.4 (2C), 145.6, 166.5 (2C) ppm; MS (EI): m/z [M]+: 397.13. Diethyl 1-Benzyl-4-(thiazol-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6d).

41



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m.p.: 66-68 °C; recrystallization solvent: cyclohexane/toluene; yield: 74%; 1H NMR (CDCl3) δ 1.23 (t, 6H, -OCH2CH3), 4.16 (q, 4H, -OCH2CH3), 4.65 (s, 2H, -NCH2Ph), 5.47 (s, 1H, ArCH), 7.22-7.23 (m, 1H, dihydropyridine proton), 7.28-7.40 (m, 7H, dihydropyridine proton and aromatic protons), 7.69 (d, 1H, aromatic proton) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 31.0, 57.9, 61.1 (2C), 104.3 (2C), 120.2, 127.2 (2C), 127.6, 128.6 (2C), 136.1, 141.1, 141.3 (2C), 166.5 (2C), 176.3 ppm; MS (EI): m/z [M]+: 398.13. Diethyl 1-Benzyl-4-(thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6e). m.p.: 80-82 °C; recrystallization solvent: cyclohexane/toluene; yield: 74%; 11H NMR (CDCl3) δ 1.24 (t, 6H, -OCH2CH3), 4.16 (q, 4H, -OCH2CH3), 4.63 (s, 2H, -NCH2Ph), 5.32 (s, 1H, ArCH), 7.26-7.28 (m, 2H, dihydropyridine protons), 7.39-7.42 (m, 5H, aromatic protons), 7.62 (d, 1H, aromatic proton), 8.62 (d, 1H, aromatic proton) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 42.5, 57.9, 61.1 (2C), 108.3 (2C), 127.2 (2C), 127.6, 128.6 (2C), 136.1, 137.4 (2C), 152.9, 153.4, 154.8, 166.5 (2C) ppm; MS (EI): m/z [M]+: 398.13. Diethyl 4-([1,1'-Biphenyl]-4-yl)-1-benzyl-1,4-dihydropyridine-3,5-dicarboxylate (6f). m.p.: 111-113 °C; recrystallization solvent: cyclohexane; yield: 87%; 11H NMR (CDCl3) δ 1.21 (t, 6H, -OCH2CH3), 4.11 (q, 4H, -OCH2CH3), 4.62 (s, 2H, -NCH2Ph), 4.98 (s, 1H, ArCH), 7.287.48 (m, 14H, dihydropyridine protons and aromatic protons), 7.58 (d, 2H, aromatic protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 35.8, 57.9, 61.1 (2C), 106.4 (2C), 127.0 (2C), 127.2 (2C), 127.3 (2C), 127.6, 127.7, 128.1 (2C), 128.6 (2C), 128.9 (2C), 134.7 (2C), 136.1, 139.1, 140.1, 142.6, 167.4 (2C) ppm; MS (EI): m/z [M]+: 467.21. Diethyl 1-Benzyl-4-(naphthalen-1-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6g). m.p.: 165-167 °C; recrystallization solvent: cyclohexane; yield: 69%; 11H NMR (CDCl3) δ 0.91 (t, 6H, -OCH2CH3), 3.89 (q, 4H, -OCH2CH3), 4.65 (s, 2H, -NCH2Ph), 5.73 (s, 1H, ArCH), 7.35-

42


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7.47 (m, 11H, dihydropyridine protons and aromatic protons), 7.76 (d, 1H, aromatic proton), 7.87 (d, 1H, aromatic proton), 8.66 (d, 1H, aromatic proton) ppm;

13

C NMR (CDCl3) δ 14.2

(2C), 34.9, 57.9, 61.1 (2C), 105.0 (2C), 123.9, 125.2 (2C), 125.8, 126.4, 126.6, 127.2 (2C), 127.6, 128.4, 128.6 (2C), 132.1, 133.0 (2C), 133.5, 136.1, 137.3, 167.4 (2C) ppm; MS (EI): m/z [M]+: 441.19. Diethyl 1-Benzyl-4-(naphthalen-2-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6h). m.p.: 117-119 °C; recrystallization solvent: cyclohexane; yield: 56%; 11H NMR (CDCl3) δ 1.22 (t, 6H, -OCH2CH3), 4.13 (q, 4H, -OCH2CH3), 4.59 (s, 2H, -NCH2Ph), 5.08 (s, 1H, ArCH), 7.347.42 (m, 11H, dihydropyridine and aromatic protons), 7.64-7.66 (m, 1H, aromatic proton), 7.707.72 (m, 1H, aromatic proton), 7.80-7.82 (m, 1H, aromatic proton) ppm;

13

C NMR (CDCl3) δ

14.2 (2C), 37.2, 57.9, 61.1 (2C), 106.4 (2C), 125.5, 126.1, 127.2 (2C), 127.4, 127.5, 127.6, 127.8, 128.2, 128.6 (2C), 128.8, 132.4, 133.4, 134.7 (2C), 136.1, 142.5, 167.4 (2C) ppm; MS (EI): m/z [M]+: 441.19. Diethyl 4-(Benzo[b]thiophen-2-yl)-1-benzyl-1,4-dihydropyridine-3,5-dicarboxylate (6i). m.p.: 103-105 °C; recrystallization solvent: cyclohexane/toluene; yield: 77%; 11H NMR (CDCl3) δ 1.22 (t, 6H, -OCH2CH3), 4.13 (q, 4H, -OCH2CH3), 4.59 (s, 2H, -NCH2Ph), 5.08 (s, 1H, ArCH), 6.98-7.01 (s, 2H, dihydropyridine protons), 7.23-7.36 (m, 6H, aromatic protons), 7.49-7.53 (m, 2H, aromatic protons), 7.79 (d, 1H, aromatic proton), 7.97 (d, 1H, aromatic proton) ppm;

13

C

NMR (CDCl3) δ 14.2 (2C), 41.5, 57.9, 61.1 (2C), 108.3 (2C), 122.6, 125.0, 125.2, 127.2 (2C), 127.4, 127.6, 128.6 (2C), 132.6, 136.1, 137.4 (2C), 139.0, 139.4, 144.9, 166.5 (2C) ppm; MS (EI): m/z [M]+: 447.15. Diethyl 1-Benzyl-4-(quinolin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate (6j).

43



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Page 44 of 73

m.p.: 112-114 °C; recrystallization solvent: cyclohexane/toluene; yield: 73%; 11H NMR (CDCl3) δ 1.22 (t, 6H, -OCH2CH3), 4.13 (q, 4H, -OCH2CH3), 4.59 (s, 2H, -NCH2Ph), 5.08 (s, 1H, ArCH), 7.02 (s, 2H, dihydropyridine protons), 7.23-7.33 (m, 5H, aromatic protons), 7.59-8.03 (m, 5H, aromatic protons), 8.70 (s, 1H, aromatic proton) ppm; 13C NMR (CDCl3) δ 14.24, 34.28, 57.94, 61.07, 106.37, 126.93, 127.20, 127.64, 128.62, 128.69, 129.19, 129.57, 129.70, 131.02, 134.66, 136.12, 138.73, 146.15, 147.42, 167.39 ppm; MS (EI): m/z [M]+: 442.19. 1-Benzyl-4-(furan-2-yl)-1,4-dihydropyridine-3,5-dicarboxylic Acid (6k). m.p.: 190-193 °C; recrystallization solvent: acetonitrile/methanol; yield: 71%;

11

H NMR

(DMSO-d6) δ 4.79 (s, 2H, -NCH2Ph), 4.83 (s, 1H, ArCH), 5.88 (s, 1H, dihydropyridine proton), 6.28(s, 1H, dihydropyridine proton), 7.30-7.42 (m, 8H, aromatic protons), 11.88 (bs, 2H, COOH) ppm;

13

C NMR (DMSO-d6) δ 31.9, 63.6, 106.7, 107.3 (2C), 110.6, 126.7, 126.9 (2C),

128.5 (2C), 136.5, 142.1, 148.9 (2C), 152.5, 171.3 (2C) ppm; MS (EI): m/z [M]+: 325.09. 1-Benzyl-4-(thiophen-2-yl)-1,4-dihydropyridine-3,5-dicarboxylic Acid (6l). m.p.: 200-202 °C; recrystallization solvent: acetonitrile/methanol; yield: 82%;

11

H NMR

(DMSO-d6) δ 4.79 (s, 2H, -NCH2Ph), 4.99 (s, 1H, ArCH), 6.98 (m, 1H, dihydropyridine proton), 6.99 (m, 1H, dihydropyridine proton), 7.23-7.25 (m, 1H, aromatic proton), 7.30-7.41 (m, 7H, aromatic protons), 11.83 (bs, 2H, -COOH) ppm; 13C NMR (DMSO-d6) δ 34.8, 63.6, 107.3 (2C), 123.4, 125.5, 126.7, 126.9 (2C), 127.0, 128.5 (2C), 136.5, 139.7, 148.9 (2C), 171.3 (2C) ppm; MS (EI): m/z [M]+: 341.07. 1-Benzyl-4-(thiophen-3-yl)-1,4-dihydropyridine-3,5-dicarboxylic Acid (6m). m.p.: 206-208 °C; recrystallization solvent: acetonitrile/methanol; yield: 79%;

11

H NMR

(DMSO-d6) δ 4.76 (s, 2H, -NCH2Ph), 4.79 (s, 1H, ArCH), 6.85-6.89 (m, 2H, dihydropyridine protons), 7.30-7.41 (m, 8H, benzene protons), 11.83 (bs, 2H, -COOH) ppm; 13C NMR (DMSO-

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d6) δ 27.8, 57.9, 106.5 (2C), 120.5, 124.8, 127.2 (2C), 127.6, 128.0, 128.6 (2C), 136.1, 143.4 (2C), 150.2, 167.8 (2C) ppm; MS (EI): m/z [M]+: 341.07. 1-Benzyl-4-(thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxylic Acid (6o). m.p.: 198-200 °C; recrystallization solvent: acetonitrile/methanol; yield: 88%;

11

H NMR

(DMSO-d6) δ 4.60 (s, 2H, -NCH2Ph), 5.18 (s, 1H, ArCH), 7.09-7.11 (m, 2H, dihydropyridine protons), 7.27-7.35 (m, 5H, aromatic protons), 7.42 (d, 1H, aromatic proton), 8.66 (d, 1H, aromatic proton), 12.01 (bs, 2H, -COOH) ppm; 13C NMR (DMSO-d6) δ 34.3, 63.6, 107.3 (2C), 126.7, 126.9 (2C), 128.5 (2C), 133.3, 136.5, 141.5, 148.9 (2C), 153.9, 171.3 (2C) ppm; MS (EI): m/z [M]+: 342.07. 4-([1,1'-Biphenyl]-4-yl)-1-benzyl-1,4-dihydropyridine-3,5-dicarboxylic Acid (6p). m.p.: 211-213 °C; recrystallization solvent: methanol; yield: 72%; 11H NMR (DMSO-d6) 4.74 (s, 1H, ArCH), δ 4.78 (s, 2H, -NCH2Ph), 7.21 (d, 2H, aromatic protons), 7.35-7.48 (m, 12H, aromatic protons and dihydropyridine protons), 7.59-7.61 (m, 2H, aromatic protons), 11.83 (bs, 2H, -COOH) ppm;

13

C NMR (DMSO-d6) δ 43.8, 63.6, 107.3 (2C), 126.7, 126.9 (2C), 127.6,

127.8 (2C), 127.9 (2C), 128.5 (2C), 129.2 (2C), 129.5 (2C), 136.5, 137.8, 140.8, 143.3, 148.9 (2C), 171.3 (2C) ppm; MS (EI): m/z [M]+: 411.15. 1-Benzyl-4-(naphthalen-1-yl)-1,4-dihydropyridine-3,5-dicarboxylic Acid (6q). m.p.: 212-214 °C; recrystallization solvent: methanol; yield: 78%; 11H NMR (DMSO-d6) δ 4.84 (s, 2H, -NCH2Ph), 5.47 (s, 1H, ArCH), 7.23-7.24 (m, 1H, aromatic proton), 7.31-7.34 (m, 1H, aromatic proton), 7.38-7.49 (m, 9H, aromatic and dihydropyridine proton), 7.68-7.70 (m, 1H, aromatic proton), 7.80-7.82 (m, 1H, aromatic proton), 8.52-8.54 (m, 1H, aromatic proton), 11.83 (bs, 2H, -COOH) ppm;

13

C NMR (DMSO-d6) δ 42.1, 63.6, 107.3 (2C), 124.2, 125.5, 125.6,

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125.8, 126.5, 126.7, 126.9 (3C), 128.5 (2C), 128.6, 132.6, 133.5, 134.0, 136.5, 148.9 (2C), 171.3 (2C) ppm; MS (EI): m/z [M]+: 385.13. 1-Benzyl-4-(naphthalen-2-yl)-1,4-dihydropyridine-3,5-dicarboxylic Acid (6r). m.p.: 224-226 °C; recrystallization solvent: methanol; yield: 87%; 11H NMR (DMSO-d6) δ 4.66 (s, 2H, -NCH2Ph), 5.11 (s, 1H, ArCH), 7.34-7.42 (m, 11H, dihydropyridine and aromatic protons), 7.64-7.66 (m, 1H, aromatic proton), 7.70-7.72 (m, 1H, aromatic proton), 7.80-7.82 (m, 1H, aromatic proton), 11.83 (bs, 2H, -COOH) ppm;

13

C NMR (DMSO-d6) δ 44.2, 63.6, 107.3

(2C), 125.1, 126.0, 126.7, 126.9 (2C), 127.0, 127.3, 127.5, 127.6, 128.0, 128.5 (2C), 131.8, 133.7, 135.2, 136.5, 148.9 (2C), 171.3 (2C) ppm; MS (EI): m/z [M]+: 385.13. 4-(Benzo[b]thiophen-2-yl)-1-benzyl-1,4-dihydropyridine-3,5-dicarboxylic Acid (6s). m.p.: 66-68 °C; recrystallization solvent: acetonitrile/methanol; yield: 69%;

11

H NMR (DMSO-

d6) δ 4.59 (s, 2H, -NCH2Ph), 5.11 (s, 1H, ArCH), 6.98-7.01 (s, 2H, dihydropyridine protons), 7.23-7.36 (m, 6H, aromatic protons), 7.49-7.53 (m, 2H, aromatic protons), 7.79 (d, 1H, aromatic proton), 7.97 (d, 1H, aromatic proton), 11.83 (bs, 2H, -COOH) ppm;

13

C NMR (DMSO-d6) δ

35.6, 63.6, 107.3 (2C), 119.6, 122.8, 123.2, 124.3, 124.4, 126.7, 126.9 (2C), 128.5 (2C), 136.5, 139.7, 139.8, 140.2, 148.9 (2C), 171.3 (2C) ppm; MS (EI): m/z [M]+: 391.09. 1-Benzyl-4-(quinolin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylic Acid (6t). m.p.: 220-222 °C; recrystallization solvent: methanol; yield: 64%; 11H NMR (DMSO-d6) δ 4.59 (s, 2H, -NCH2Ph), 5.08 (s, 1H, ArCH), 7.00-7.03 (s, 2H, dihydropyridine protons), 7.23-7.33 (m, 5H, aromatic protons), 7.59-8.03 (m, 5H, aromatic protons), 8.70 (s, 1H, aromatic proton), 11.83 (bs, 2H, -COOH) ppm; 13C NMR (DMSO-d6) δ 44.2, 63.6, 107.3 (2C), 126.7, 126.8, 126.9 (2C), 127.7, 128.4, 128.5 (3C), 128.8, 130.1, 134.7, 136.5, 146.5, 148.9 (2C), 152.3, 171.3 (2C) ppm; MS (EI): m/z [M]+: 386.13.

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1-Benzyl-4-(furan-2-yl)-1,4-dihydropyridine-3,5-dicarboxamide (6u). m.p.: 251-254 °C; recrystallization solvent: methanol; yield: 65%; 11H NMR (DMSO-d6) δ 4.64 (s, 2H, -NCH2Ph), 5.08 (s, 1H, ArCH), 5.94-5.95 (m, 1H, dihydropyridine proton), 6.27-6.28 (m, 1H, dihydropyridine proton), 6.83 (bs, 4H, -CONH2), 7.26-7.42 (m, 8H, aromatic protons) ppm; 13

C NMR (DMSO-d6) δ 33.1, 62.8, 106.7, 110.2 (2C), 110.6, 126.7, 126.9 (2C), 128.5 (2C),

136.5, 142.1, 144.9 (2C), 152.5, 171.0 (2C) ppm; MS (EI): m/z [M]+: 323.13. 1-Benzyl-4-(thiophen-2-yl)-1,4-dihydropyridine-3,5-dicarboxamide (6v). m.p.: >250 °C; recrystallization solvent: methanol; yield: 78%; 11H NMR (DMSO-d6) δ 4.63 (s, 2H, -NCH2Ph), 5.26 (s, 1H, ArCH), 6.75-6.92 (m, 6H, dihydropyridine protons and -CONH2), 7.21-7.38 (m, 8H, aromatic protons) ppm; 13C NMR (DMSO-d6) δ 36.0, 62.8, 110.2 (2C), 123.4, 125.5, 126.7, 126.9 (2C), 127.0, 128.5 (2C), 136.5, 139.7, 144.9 (2C), 171.0 (2C) ppm; MS (EI): m/z [M]+: 339.10. 1-Benzyl-4-(thiophen-3-yl)-1,4-dihydropyridine-3,5-dicarboxamide (6w). m.p.: >250 °C; recrystallization solvent: methanol; yield: 73%; 11H NMR (DMSO-d6) δ 4.51 (s, 2H, -NCH2Ph), 4.96 (s, 1H, ArCH), 6.59-6.97 (m, 6H, dihydropyridine protons and -CONH2), 7.06-7.93 (m, 8H, aromatic protons) ppm; 13C NMR (DMSO-d6) δ 37.7, 57.9, 111.3 (2C), 120.5, 124.8, 127.2 (2C), 127.6, 128.0, 128.6 (2C), 136.1, 137.8 (2C), 150.2, 170.5 (2C) ppm; MS (EI): m/z [M]+: 339.10. 1-Benzyl-4-(thiazol-2-yl)-1,4-dihydropyridine-3,5-dicarboxamide (6x). m.p.: >250 °C; recrystallization solvent: acetonitrile/methanol; yield: 63%;

11

H NMR (DMSO-

d6) δ 4.67 (s, 2H, -NCH2Ph), 5.37 (s, 1H, ArCH), 6.91-7.03 (bs, 4H, -CONH2), 7.30-7.37 (m, 5H, aromatic protons and dihydropyridine protons), 7.44-7.45 (m, 2H, aromatic protons), 7.52 (d, 1H, aromatic proton), 7.62 (d, 1H, aromatic proton) ppm;

47

13

C NMR (DMSO-d6) δ 34.2, 62.8,



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110.2 (2C), 118.7, 126.7, 126.9 (2C), 128.5 (2C), 136.5, 141.9, 144.9 (2C), 165.9, 171.0 (2C) ppm; MS (EI): m/z [M]+: 340.10. 1-Benzyl-4-(thiazol-5-yl)-1,4-dihydropyridine-3,5-dicarboxamide (6y). m.p.: >250 °C; recrystallization solvent: acetonitrile/methanol; yield: 59%;

11

H NMR (DMSO-

d6) δ 4.63 (s, 2H, -NCH2Ph), 5.38 (s, 1H, ArCH), 6.91-7.03 (bs, 4H, -CONH2), 7.33-7.40 (m, 7H, dihydropyridine protons and aromatic protons), 7.48 (d, 1H, aromatic proton), 8.79 (d, 1H, aromatic proton) ppm; 13C NMR (DMSO-d6) δ 35.5, 62.8, 110.2 (2C), 126.7, 126.9 (2C), 128.5 (2C), 133.3, 136.5, 141.5, 144.9 (2C), 153.9, 171.0 (2C) ppm; MS (EI): m/z [M]+: 340.10. 4-([1,1'-Biphenyl]-4-yl)-1-benzyl-1,4-dihydropyridine-3,5-dicarboxamide (6z). m.p.: >250 °C; recrystallization solvent: methanol; yield: 69%; 11H NMR (DMSO-d6) δ 4.54 (s, 2H, -NCH2Ph), 5.05 (s, 1H, ArCH), 6.74 (bs, 4H, -CONH2), 7.31-8.42 (m, 16H, dihydropyridine protons and aromatic protons) ppm; 13C NMR (DMSO-d6) δ 45.0, 62.8, 110.2 (2C), 126.7, 126.9 (2C), 127.6, 127.8 (2C), 127.9 (2C), 128.5 (2C), 129.2 (2C), 129.5 (2C), 136.5, 137.8, 140.8, 143.3, 144.9 (2C), 171.0 (2C) ppm; MS (EI): m/z [M]+: 409.18. 1-Benzyl-4-(naphthalen-1-yl)-1,4-dihydropyridine-3,5-dicarboxamide (6a'). m.p.: 160-162 °C; recrystallization solvent: methanol; yield: 85%; 11H NMR (DMSO-d6) δ 4.66 (s, 2H, -NCH2Ph), 5.66 (s, 1H, ArCH), 6.63 (bs, 4H, -CONH2), 7.27-7.48 (m, 11H, dihydropyridine and aromatic protons), 7.67-7.69 (m, 1H, aromatic proton), 7.80-7.82 (m, 1H, aromatic proton), 8.63-8.64 (m, 1H, aromatic proton) ppm;

13

C NMR (DMSO-d6) δ 43.3, 62.8,

110.2 (2C), 124.2, 125.5, 125.6, 125.8, 126.5, 126.7, 126.9 (3C), 128.5 (2C), 128.6, 132.6, 133.5, 134.0, 136.5, 144.9 (2C), 171.0 (2C) ppm; MS (EI): m/z [M]+: 383.16. 4-(Benzo[b]thiophen-2-yl)-1-benzyl-1,4-dihydropyridine-3,5-dicarboxamide (6c').

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m.p.: >260 °C; recrystallization solvent: acetonitrile/methanol; yield: 81%;

11

H NMR (DMSO-

d6) δ 4.59 (s, 2H, -NCH2Ph), 5.11 (s, 1H, ArCH), 6.78-6.80 (bs, 4H, -CONH2), 6.98-7.01 (s, 2H, dihydropyridine protons), 7.23-7.36 (m, 6H, aromatic protons), 7.49-7.53 (m, 2H, aromatic protons), 7.79 (d, 1H, aromatic proton), 7.97 (d, 1H, aromatic proton), ppm; 13C NMR (DMSOd6) δ 36.8, 62.8, 110.2 (2C), 119.6, 122.8, 123.2, 124.3, 124.4, 126.7, 126.9 (2C), 128.5 (2C), 136.5, 139.7, 139.8, 140.2, 144.9 (2C), 171.0 (2C) ppm; MS (EI): m/z [M]+: 389.12. 1-Benzyl-4-(quinolin-3-yl)-1,4-dihydropyridine-3,5-dicarboxamide (6d'). m.p.: >260 °C; recrystallization solvent: methanol; yield: 61%; 11H NMR (DMSO-d6) δ 4.59 (s, 2H, -NCH2Ph), 5.08 (s, 1H, ArCH), 6.83 (bs, 4H, -CONH2), 7.01 (s, 2H, dihydropyridine protons), 7.23-7.33 (m, 5H, aromatic protons), 7.59-8.03 (m, 5H, aromatic protons), 8.70 (s, 1H, aromatic proton), ppm; 13C NMR (DMSO-d6) δ 45.4, 62.8, 110.2 (2C), 126.7, 126.8, 126.9 (2C), 127.7, 128.4, 128.5 (3C), 128.8, 130.1, 134.7, 136.5, 144.9 (2C), 146.5, 152.3, 171.0 (2C) ppm; MS (EI): m/z [M]+: 384.16. Diethyl 1-Benzoyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (7a). m.p.: 107-109 °C; recrystallization solvent: cyclohexane; yield: 81%; 11H NMR (CDCl3) δ 1.081.11 (m, 6H, -OCH2CH3), 4.01-4.07 (m, 4H, -OCH2CH3), 4.78 (s, 1H, ArCH-), 7.20-7.21 (m, 1H, aromatic protons), 7.29-7.35 (m, 4H, aromatic protons), 7.61-7.64 (m, 2H, aromatic protons), 7.68-7.73 (m, 4H, aromatic protons), 7.90 (s, 2H, dihydropyridine protons) ppm;

13

C

NMR (CDCl3) δ 14.2 (2C), 44.1, 61.7 (2C), 108.0 (2C), 125.7, 131.4, 132.1 127.5 (2C), 127.7 (2C), 128.6 (2C), 128.8 (2C), 141.2 (2C), 144.4, 167.2 (2C) ppm; MS (EI): m/z [M]+: 405.16. 1-Benzyl-3,5-diethyl-4-phenylpyridine-1,3,5(4H)-tricarboxylate (7b). m.p.: 85-87 °C; recrystallization solvent: cyclohexane; yield: 78%;

11

H NMR (CDCl3) δ 1.19-

1.26 (m, 6H, -OCH2CH3), 4.04-4.23 (m, 4H, -OCH2CH3), 4.87 (s, 1H, ArCH-), 5.40 (s, 2H, -

49



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CH2Ph), 7.18-7.22 (m, 1H, aromatic proton), 7.24-7.30 (m, 2H, aromatic protons), 7.42-7.48 (m, 5H, aromatic protons), 8.06 (s, 2H, dihydropyridine protons) ppm;

13

C NMR (CDCl3) δ 14.2

(2C), 44.1, 61.7 (2C), 108.0 (2C), 121.6 (2C), 125.5, 125.7, 127.7 (2C), 128.6 (2C), 129.1, 141.2 (2C), 144.4, 151.3, 151.8 167.2 (2C) ppm; MS (EI): m/z [M]+: 421.15. Diethyl 4-Phenyl-1-(pyrazine-2-carbonyl)-1,4-dihydropyridine-3,5-dicarboxylate (7c). m.p.: 127-129 °C; recrystallization solvent: cyclohexane; yield: 69%; 11H NMR (CDCl3) δ 1.101.14 (m, 6H, -OCH2CH3), 4.04-4.09 (m, 4H, -OCH2CH3), 4.80 (s, 1H, ArCH-), 7.18-7.22 (m, 1H, aromatic proton), 7.26-7.30 (m, 4H, aromatic protons), 8.19 (s, 2H, dihydropyridine protons), 8.85 (s, 1H, pyrazine proton), 8.86 (d, 1H, pyrazine proton) 9.20 (s, 1H, pyrazine proton) ppm;

13

C NMR (CDCl3) δ 14.2 (2C), 44.1, 61.7 (2C), 108.0 (2C), 125.7 (2C), 127.7

(2C), 128.6 (2C), 141.2 (2C), 144.4, 144.6, 144.7, 145.0, 146.0, 158.9, 167.2 (2C) ppm; MS (EI): m/z [M]+: 407.15. Diethyl 1-(1-Naphthoyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (7d). m.p.: 140-142 °C; recrystallization solvent: toluene; yield: 75%; 11H NMR (CDCl3) δ 1.06-1.16 (m, 6H, -OCH2CH3), 4.00-4.19 (m, 4H, -OCH2CH3), 4.95 (s, 1H, ArCH-), 7.22-7.41 (m, 6H, aromatic protons), 7.59-7.68 (m, 4H, dihydropyridine protons and aromatic protons), 6.28-6.29 (m, 1H, dihydropyridine proton), 7.90-8.10 (m, 4H, aromatic protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 41.6, 61.7 (2C), 108.0 (2C), 120.3, 122.6, 124.6, 125.6, 127.3, 128.5, 129.3, 130.1, 130.3, 131.9, 132.2, 133.6, 134.4, 134.5, 135.6, 136.8, 141.2 (2C), 161.2, 162.7 (2C) ppm; MS (EI): m/z [M]+: 455.17. Diethyl 1-(2-Naphthoyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (7e). m.p.: 123-125 °C; recrystallization solvent: cyclohexane; yield: 78%; 11H NMR (CDCl3) δ 1.171.21 (m, 6H, -OCH2CH3), 4.05-4.19 (m, 4H, -OCH2CH3), 4.99 (s, 1H, ArCH-), 7.24-7.25 (m,

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1H, aromatic proton), 7.33-7.35 (m, 2H, aromatic protons), 7.40-7.42 (m, 2H, aromatic protons), 7.65-7.72 (m, 3H, aromatic protons), 7.89-7.95 (m, 3H, aromatic protons), 8.23 (s, 1H, aromatic proton), 8.27 (s, 2H, dihydropyridine protons) ppm;

13

C NMR (CDCl3) δ 14.2 (2C), 61.7 (2C),

44.1, 108.0 (2C), 124.2, 125.7, 126.9, 127.7 (3C), 128.3, 128.5 128.6 (3C), 129.5, 132.5, 134.2, 135.6, 141.2 (2C), 144.4, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 455.17. Diethyl 4-Phenyl-1-(quinoxaline-2-carbonyl)-1,4-dihydropyridine-3,5-dicarboxylate (7f). m.p.: 142-144 °C; recrystallization solvent: toluene; yield: 65%; 11H NMR (CDCl3) δ 1.11-1.13 (m, 6H, -OCH2CH3), 4.04-4.10 (m, 4H, -OCH2CH3), 4.83 (s, 1H, ArCH-), 7.21-7.23 (m, 1H, aromatic proton), 7.30-7.34 (m, 4H, aromatic protons), 8.03-8.10 (m, 2H, quinoxaline protons), 8.18 (d, 1H, quinoxaline proton), 8.26 (d, 1H, quinoxaline proton), 8.45 (s, 2H, dihydropyridine protons), 9.41 (s, 1H, quinoxaline proton) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 44.1, 61.7 (2C), 108.0 (2C), 125.7, 127.7 (2C), 128.6 (2C), 128.9, 130.3, 130.7, 131.9, 140.3, 141.2 (2C), 142.9, 143.8, 144.0, 144.4, 158.9, 167.2 (2C) ppm; MS (EI): m/z [M]+: 457.16. Diethyl 4-Phenyl-1-(phenylsulfonyl)-1,4-dihydropyridine-3,5-dicarboxylate (7g). m.p.: 155-157 °C; recrystallization solvent: toluene; yield: 76%; 11H NMR (CDCl3) δ 1.14-1.23 (m, 6H, -OCH2CH3), 4.06-4.17 (m, 4H, -OCH2CH3), 4.79 (s, 1H, ArCH-), 6.84-6.92 (m, 2H, aromatic protons), 7.05-7.15 (m, 3H, aromatic protons), 7.66-7.72 (m, 2H, aromatic protons), 7.76-7.86 (m, 3H, aromatic proton and dihydropyridine protons), 7.91-8.00 (m, 2H, aromatic protons) ppm;

13

C NMR (CDCl3) δ 14.2 (2C), 44.1, 61.7 (2C), 108.0 (2C), 125.7, 127.3 (2C),

127.7 (2C), 128.6 (2C), 129.0 (2C), 131.9, 133.3 (2C), 136.6, 144.4, 167.2 (2C) ppm; MS (EI): m/z [M]+: 441.12. Diethyl 1-Benzoyl-4-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (7h).

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m.p.: 110-112 °C; recrystallization solvent: cyclohexane; yield: 82%; 11H NMR (CDCl3) δ 1.181.21 (m, 6H, -OCH2CH3), 2.71 (s, 3H, -CH3), 4.06-4.17 (m, 4H, -OCH2CH3), 5.11 (s, 1H, ArCH), 7.10-7.15 (m, 2H, aromatic protons), 7.17-7.18 (m, 2H, aromatic protons), 7.55-7.59 (m, 2H, aromatic protons), 7.63-7.66 (m, 1H, aromatic proton), 7.70-7.71 (m, 2H, aromatic protons), 8.13 (s, 2H, dihydropyridine protons) ppm;

13

C NMR (CDCl3) δ 14.2 (2C), 19.6, 41.6, 61.7 (2C),

108.0 (2C), 122.6, 124.6, 125.6, 127.5 (2C), 128.8 (2C), 130.3, 131.4, 132.1, 135.6, 136.8, 141.2 (2C), 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 419.17. Diethyl 1-Benzoyl-4-(m-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (7i). m.p.: 97-99 °C; recrystallization solvent: cyclohexane; yield: 83%;

11

H NMR (CDCl3) δ 1.20-

1.24 (m, 6H, -OCH2CH3), 2.36 (s, 3H, -CH3), 4.08-4.19 (m, 4H, -OCH2CH3), 4.93 (s, 1H, ArCH), 7.04 (d, 1H, aromatic proton), 7.14-7.23 (m, 3H, aromatic protons), 7.53-7.58 (m, 2H, aromatic protons), 7.62-7.69 (m, 3H, aromatic protons), 8.12 (s, 2H, dihydropyridine protons) ppm;

13

C

NMR (CDCl3) δ 14.2 (2C), 21.6, 44.4, 61.7 (2C), 108.0 (2C), 124.7, 126.0, 127.5 (2C), 128.5 128.8 (2C), 130.9, 131.4, 132.1 138.3, 141.2 (2C), 142.1, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 419.17. Diethyl 1-Benzoyl-4-(p-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (7j). m.p.: 119-121 °C; recrystallization solvent: cyclohexane; yield: 87%; 11H NMR (CDCl3) δ 1.201.25 (m, 6H, -OCH2CH3), 2.33 (s, 3H, -CH3), 4.06-4.18 (m, 4H, -OCH2CH3), 4.92 (s, 1H, ArCH), 7.13 (d, 2H, aromatic protons), 7.25 (d, 2H, aromatic protons), 7.53-7.57 (m, 2H, aromatic protons), 7.63 (d, 1H, aromatic proton), 7.68 (d, 2H, aromatic protons), 8.11 (s, 2H, dihydropyridine protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 21.3, 44.1, 61.7 (2C), 108.0 (2C), 127.5 (2C), 128.8 (2C), 128.9 (4C), 131.4, 132.1, 135.4, 141.2 (2C), 141.4, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 419.17.

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Diethyl 1-Benzoyl-4-(2-chlorophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7k). m.p.: 127-129 °C; recrystallization solvent: cyclohexane; yield: 80%; 11H NMR (CDCl3) δ 1.181.21 (m, 6H, -OCH2CH3), 4.06-4.19 (m, 4H, -OCH2CH3), 5.40 (s, 1H, ArCH-), 7.14-7.18 (m, 1H, aromatic proton), 7.23-7.26 (m, 1H, aromatic proton), 7.33-7.36 (m, 2H, aromatic protons), 7.54-7.58 (m, 2H, aromatic protons), 7.62-7.66 (m, 1H, aromatic proton), 7.68-7.71 (m, 2H, aromatic protons), 8.15 (s, 2H, dihydropyridine protons) ppm;

13

C NMR (CDCl3) δ 14.2 (2C),

39.0, 67.1 (2C), 108.0 (2C), 126.7, 127.1, 126.4, 127.5 (2C), 128.7, 128.8 (2C), 131.4 (2C), 132.1, 141.2 (2C), 143.7, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 439.12. Diethyl 1-Benzoyl-4-(3-chlorophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7l). m.p.: 120-122 °C; recrystallization solvent: cyclohexane; yield: 94%; 11H NMR (CDCl3) δ 1.211.24 (m, 6H, -OCH2CH3), 4.09-4.20 (m, 4H, -OCH2CH3), 4.95 (s, 1H, ArCH-), 7.21-7.24 (m, 1H, aromatic proton), 7.25-7.27 (m, 2H, aromatic protons), 7.33 (s, 1H, aromatic proton), 7.557.59 (m, 2H, aromatic protons), 7.65-7.69 (m, 3H, aromatic proton), 8.14 (s, 2H, dihydropyridine protons) ppm;

13

C NMR (CDCl3) δ 14.2 (2C), 43.6, 61.7 (2C), 108.0 (2C), 125.8 (2C), 127.5

(2C), 128.8 (3C), 130.0, 131.4, 132.1, 134.2, 141.2 (2C), 143.6, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 439.12. Diethyl 1-Benzoyl-4-(4-chlorophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7m). m.p.: 119-121°C; recrystallization solvent: cyclohexane; yield: 85%; 11H NMR (CDCl3) δ 1.201.23 (m, 6H, -OCH2CH3), 4.08-4.17 (m, 4H, -OCH2CH3), 4.95 (s, 1H, ArCH-), 7.30 (s, 4H, aromatic protons), 7.55-7.58 (m, 2H, aromatic protons), 7.63-7.68 (m, 3H, aromatic protons), 8.12 (s, 2H, dihydropyridine protons) ppm;

13

C NMR (CDCl3) δ 14.2 (2C), 44.1, 61.7 (2C),

108.0 (2C), 127.5 (2C) , 128.7 (2C), 128.8 (2C), 130.4 (2C), 131.3, 131.4, 132.1, 141.2 (2C), 142.5, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 439.12.

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Diethyl 1-Benzoyl-4-(2-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7n). m.p.: 137-140 °C; recrystallization solvent: cyclohexane/toluene; yield: 88%; 11H NMR (CDCl3) δ 1.18-1.21 (m, 6H, -OCH2CH3), 3.84 (s, 3H, -OCH3), 4.04-4.16 (m, 4H, -OCH2CH3), 5.14 (s, 1H, ArCH-), 6.88-6.95 (m, 2H, aromatic protons), 7.20-7.26 (m, 1H, aromatic proton), 7.33-7.36 (m, 1H, aromatic proton), 7.53-7.57 (m, 2H, aromatic protons), 7.61-7.65 (m, 1H, aromatic proton), 7.71-7.73 (m, 2H, aromatic protons), 8.10 (s, 2H, dihydropyridine protons) ppm;

13

C

NMR (CDCl3) δ 14.2 (2C), 38.2, 56.1, 61.7 (2C), 108.0 (2C), 112.2, 120.9, 121.0, 126.7, 128.8 (2C), 127.5 (2C), 130.0, 131.4, 132.1, 141.2 (2C), 158.6, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 435.17. Diethyl 1-Benzoyl-4-(4-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7p). m.p.: 185-187 °C; recrystallization solvent: toluene/acetonitrile; yield: 89%; 11H NMR (CDCl3) δ 1.20-1.24 (m, 6H, -OCH2CH3), 3.80 (s, 3H, -OCH3), 4.10-4.19 (m, 4H, -OCH2CH3), 4.91 (s, 1H, ArCH-), 6.85 (d, 2H, aromatic protons), 7.26 (s, 2H, aromatic protons), 7.54-7.58 (m, 2H, aromatic proton), 7.63 (d, 1H, aromatic proton), 7.67-7.69 (m, 2H, aromatic protons), 8.10 (s, 2H, dihydropyridine protons) ppm; 13C NMR (CDCl3) δ 14.2 (2C), 44.1, 55.8, 61.7 (2C), 108.0 (2C), 114.2 (2C), 127.5 (2C), 128.8 (2C); 130.0 (2C), 131.4, 132.1, 136.7, 141.2 (2C), 157.6, 161.2, 167.2 (2C) ppm; MS (EI): m/z [M]+: 435.17. Diethyl 4-(m-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (11c). m.p.: 130-133 °C; recrystallization solvent: cyclohexane; yield: 41%;

11

H NMR (CDCl3, 400

MHz) δ 1.22 (t, 6H, -OCH2CH3); 2.32 (s, 3H, CH3), 4.03- 4.18 (m, 4H, -OCH2CH3); 4.89 (s, 1H, dihydropyridine proton); 6.26 (bs, 1H, -NH); 6.99 (s, 1H, benzene proton), 7.13-7.16 (m, 3H, benzene protons), 7.34-7.36 (s, 2H, dihydropyridine protons) ppm; 13C NMR (CDCl3, 100 MHz)

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δ 14.2 (2C), 21.7, 44.4, 61.6 (2C), 108.2 (2C), 124.8, 126.2, 128.6, 130.8, 138.4 (3C), 142.2, 167.3 (2C) ppm; MS (EI): m/z [M]+: 315.15.

SIRT1 in Vitro Assays. The SIRT1 activity assay was performed using different methods. First we have used the BioMol Fluor de Lys assay (Enzo), following standard procedures. Secondly we have used the recombinant SIRT1 as described in the SIRTainty product sheet kit (EMD Millipore). The assay employs nicotinamidase to measure nicotinamide generated upon cleavage of NAD+ during sirtuin-mediated deacetylation of a substrate. SIRT1 enzyme, NAD+, acetylated peptide substrate, test compound, and nicotinamidase enzyme are combined and incubated for 30 min. The deacetylation of substrate produce nicotinamide. Then in a secondary reaction by nicotinamidase (NMase), nicotinamide is converted to nicotinic acid and free NH3 that reacts with a developer reagent to generate the signal that is read using a fluorescent plate reader (Ex 420 nm, Em 460 nm). The assays were carried out using the SIRTainty acetylated peptide substrate (H3K9) at 25 µM, 0.2 mM NAD+, and 5 Units of human recombinant SIRT1. Thirdly, we have also performed an ‘in house assay’ producing, purifying and using a GST-tag recombinant human full-length SIRT1 and His-tag NMase. All these assays were performed in triplicates. For all compounds tested, the intrinsic fluorescence (IF) was determined. In Table 1 data without subtraction of IF (+ IF) as well as data with subtraction of IF (- IF) have been reported. Compounds with an high intrinsic fluorescence have been indicated as * in Table 1. In some cases (marked as **), the IF was higher than the SIRT activity value read by the instrument. For these specific cases, the modulating activity given was mainly based and corroborated by the biological-based assays, since the IF may alter the in vitro standards.

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PDE4 Inhibition Assay. Activity of recombinant human PDE4D (SignalChem, Richmond, BC, Canada) was assayed with a modified Malachite Green assay as described by the manufacturer (Biomol Int., Plymouth Meeting, PA, USA). Assays contained 20 mU PDE4D in 10 mM TrisHcl pH 7.4 and 200 mM cAMP as the substrate. After 60 min incubation at 37 °C, 100 µL BIOMOL GREEN reagent was added and PDE activity determined by measuring phosphate release through absorption measurements at 620 nm (Biomol Int., Plymouth Meeting, PA, USA). Data shown are averages from three determinations. Compounds 3a, 6a, 6c, 6p, 7a and 7e were tested at 50 µM. Rolipram was used as a positive control and was tested at 10 µM. All compounds were dissolved in DMSO. Nitric oxide production. Cell culture and treatment. A cell line of transformed human keratinocytes (HaCaT) was cultured in DMEM (Lonza) supplemented with 10% FBS and antibiotics. Cells were treated with the DHPs at different concentrations (1, 10, 25, and 50 µM) for 1, 3 and 5 h to select the best concentration for biological evaluations. In all experiments, nifedipine, 1, 3a and DETA/NO were used as reference compounds and DMSO was used as solvent control. DAF-2D Assay. Nitric oxide production was evaluated by adding 5 µM 4,5-diaminofluorescein diacetate (DAF-2D) (Alexis) to the complete medium. At the end of treatment, cells were washed with PBS, trypsinized, centrifuged, and analyzed by flow cytometry (FACS) to detect intracellular NO production. Briefly, DAF-2D is a sensitive fluorescent indicator for the detection of intracellular NO. Upon entry into the cell, DAF-2D is transformed into the less cellpermeable DAF-2 by cellular esterases, thus preventing loss of signal due to diffusion of the molecule from the cell. In the presence of oxygen, DAF-2 reacts with NO to yield the highly fluorescent triazolofluorescein (DAF-2T). Fluorescence was monitored using excitation and

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emission wavelengths of 485 and 538 nm, respectively, and was read by FACS. All the DHPs were tested at 1 µM in HaCat cells for 1 h. Dose response curves were obtained for 6a, 6c and 7a by testing them at 5, 25 and 50 µM. SIRT1 activity in HaCat cells. SIRT1 activity in HaCat cells. A SIRT1 fluorimetric assay (CycLex) was performed according to the manufacturer instructions. Briefly, amino and carboxyl terminal of substrate peptide are, respectively, marked with a fluorophore and a quencher that can be activated only after a deacetylation reaction occurs. Thus, when SIRT1 performs deacetylation, quencher will separate from fluorophore, leading to fluorescence emission, that we measured by Berthold microplate readers system. To measure the effects of different DHPs on SIRT1 activity, 50 µg of total HaCaT cellular extract freshly lysated in RIPA buffer were incubated with 6a, 6c and 7a (1 µM). Sirtinol (25 µM) and 1 (1 µM) were used as negative and positive controls, respectively. The SIRT1 activity was followed during the first 4 hours of incubation reading the fluorescent signal by microplate readers system. Western Blotting. Cells were lysed in Laemmli buffer (WB) or radioimmune precipitation assay buffer (immunoprecipitation). WB was performed according to standard procedures. Antiacetylated histone H4 lysine 16 (WB, 1:1000, polyclonal, Abcam) was used as antibody. Histone H4 antibody (WB, 1:1000, polyclonal, Santa Cruz) was used for equal loading. All the tested drugs (1, 3a, 6a, 6c, and 7a) were tested at 1 µM. DMSO was used as solvent control. Data show representative results of at least three independent experiments. Animal Skin Wound model. Eight-week-old CD1 male mice were obtained from Charles River Laboratories, Inc. (Calco, Lecco, Italy) and were anesthetized with an intraperitoneal injection of 1 mg/kg medetomidine (Domitor, Vetem, Milan, Italy) and 75 mg/kg ketamine (Ketavet 100, Intervet Farmaceutici, Aprilia, Italy). The animal dorsum was clipped free of hair,

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and a full-thickness wound of 3.5-mm diameter was created using a biopsy punch. Drugs were applied daily at the indicated concentration in 20 µL of saline solution, directly in the wound area. Another group of mice was treated with diluting solvent in 20 µL of saline solution on the wound. The novel DHPs 6a, 6c and 7a were administered in the wound area each at 1 µM. DMSO was used as a solvent control. All experimental procedures complied with the Guidelines of the Italian National Institutes of Health and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee. Mitochondrial staining and confocal analysis in murine C2C12 myoblasts. Murine C2C12 myoblasts were plated on 13 mm cover slips in complete medium and incubated overnight at 37 °C under an atmosphere of 95% air and 5% CO2. The next day, the cover slips were washed with PBS and incubated in HBSS medium without serum at pH 7.4. Subsequently, the cells were either left untreated or treated for 16 h with the following compounds: 6c, 6k and 5a, both at 30 µM; 9 and 10, both at 5 µM; 6c (30 µM) plus 9 (5 µM); 6c (30 µM) plus 10 (5 µM); 7a (30 µM) plus 9 (5 µM); 7a (30 µM) plus 10 (5 µM). Thirty minutes before ending the treatment, the mitochondria dye Mitotracker Green (Life Technologies) was added at a final concentration of 200 nM. Treatments were ended by fixing the cells in 4% paraformaldehyde. The cover slips were then washed twice with PBS and permeabilized with 0.2% Triton X-100 for 10 min. Coverslips were mounted on antifade mountant (ProLongR Diamond Antifade Mountant, Life Technologies). Fluorescence intensity was visualized with a LSM 510 confocal microscope (Zeiss). Alternatively, cells (1000/well) were grown on a 96 well plate, treated and stained with Mitotracker green as described above. Finally, fluorescence from mitochondria was quantified using Glomax multi detection system (Promega).

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Statistical analysis. The results are expressed as means ± standard deviations (SD) and 95% confidence intervals (95% CI). Before using parametric tests, the assumption of normality was verified using the Shapiro-Wilk W-test. Student’s paired t-test was used to determine any significant differences before and after treatment. Significance was set at 0.05 (p ≤ 0.05). SPSS statistical software package (SPSS Inc., Version 13.0.1 for Windows Chicago, IL, USA) was used for all statistical calculations. Antiproliferative Activity in Cancer Cells. M14 and SAN (melanoma), A549 and H1299 (non-small cell lung carcinoma), HT29, LOVO and HCT116 (colon carcinoma), and CAOV3, HEY, SKOV3, and A2780 (ovary carcinoma) were cultured in RPMI (Invitrogen) with 10% fetal calf serum (Hyclone), 100 U/mL penicillin. Exponentially growing tumor cells were seeded in sextuplicates in 96-well (3 × 103 cells/well) and 24 h later cells were treated with 7a and 8 at concentrations ranging from 5 to 200 µM for 72 h. Cell viability was evaluated by 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

bromide

(MTT,

Sigma-Aldrich)

assay

as

previously described.78,79 The results are reported as “viability of drug-treated cells/viability of untreated cells” × 100 and represent the average ± SD of three independent experiments. The concentration of 7a and 8 that causes a 50% of cell viability inhibition (IC50) was calculated by CalcuSyn software (Biosoft). Western Blotting in M14 melanoma and H1299 lung cancer cells. Western blot analysis total protein extracts were performed as previously described.80 Immunodetection was performed using antibodies directed to: acetylated histone H4 (K16) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (Sigma-Aldrich), anti-mouse, anti-rabbit or anti-goat immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Cell Signaling; Amersham Biosciences, Freiburg, Germany). Antibody binding was visualized by enhanced chemiluminescence method

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(Amersham Biosciences) according to manufacturer’s specification and recorded on autoradiographic film (Amersham Biosciences). Densitometric evaluation was performed using Image J software and normalized with relative controls depending on the analysis.

ASSOCIATED CONTENT Supporting Information. Elemental Analysis for Compounds 4-8. Dose-response effects of 4c and 6 on SIRT1 activity. Activity of 6c and 7a on K+-depolarized Guinea Pig Smooth Muscle. Dose-response curves for antiproliferative activity of 7a in a panel of human cancer cell lines. AUTHOR INFORMATION Corresponding Author A.M.: phone, +39 06 49913392; fax, +39 06 49693268; E-mail, [email protected]. ACKNOWLEDGMENT This work was supported by FIRB grant n. RBFR10ZJQT, by RF-2010-2318330, by IITSapienza Project, by FP7 Projects BLUEPRINT/282510 and A-PARADDISE/602080, and AIRC grant IG14100. Daniela Trisciuoglio was supported by a Fondazione Umberto Veronesi Fellowship.

ABBREVIATIONS AMC, aminomethylcoumarin; AMPK, adenosine monophosphate-activated protein kinase; BOP reagent, benzotriazole-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate; CD1, cluster of differentiation 1; DAF, 4,5-diaminofluorescein; DAF-2D, 4,5-diaminofluorescein diacetate; DAF-2T fluorescent triazolofluorescein; DETA/NO, diethylenetriamine-nitric oxide;

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DHPs, 1,4-dihydropyridines; DMEM, Dulbecco modified Eagle's minimal essential medium; EC, endothelial cells; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FOXO, forkhead box O; FXR, farnesoid X receptor; GPILSM, guinea-pig ileum longitudinal smooth muscle; HIF-1α, hypoxia-inducible factor alpha; hMSCs, human mesenchymal stem cells; IF, intrinsic fluorescence; LXR, liver X receptor; mTFA, transcription factor A; OPT, ortho-phathalaldehyde; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; RSV, resveratrol; TAMRA, carboxytetramethylrhodamine; TSA, trichostatin A. REFERENCES (1)

Yang, X.-J.; Seto, E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and

yeast to mice and men. Nat. Rev. Mol. Cell Biol. 2008, 9, 206–218. (2)

Falkenberg, K. J.; Johnstone, R. W. Histone deacetylases and their inhibitors in cancer,

neurological diseases and immune disorders. Nat. Rev. Drug Discov. 2014, 13, 673-691. (3)

Zwergel, C.; Valente, S.; Jacob, C.; Mai, A. Emerging approaches for histone deacetylase

inhibitor drug discovery. Expert Opin. Drug Discov. 2015, 10, 599-613. (4)

Sebastián, C.; Satterstrom, F. K.; Haigis, M. C.; Mostoslavsky, R. From sirtuin biology to

human diseases: an update. J. Biol. Chem. 2012, 287, 42444–42452. (5)

Imai, S.; Guarente, L. NAD+ and sirtuins in aging and disease. Trends Cell Biol. 2014,

24, 464-471. (6)

Chen, B.; Zang, W.; Wang, J.; Huang, Y.; He, Y.; Yan, L.; Liu, J,.; Zheng, W. The

chemical biology of sirtuins. Chem. Soc. Rev. 2015, 44, 5246-5264. (7)

Guarente, L. Sirtuins as potential targets for metabolic syndrome. Nature 2006, 444, 868–

874.

61



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(8)

Page 62 of 73

Michishita, E.; Park, J. Y.; Burneskis, J. M.; Barrett, J. C.; Horikawa, I. Evolutionarily

conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol. Biol. Cell 2005, 16, 4623–4635. (9)

Baur, J. A.; Ungvari, Z.; Minor, R. K.; Le Couteur, D. G.; de Cabo, R. Are sirtuins viable

targets for improving healthspan and lifespan? Nat. Rev. Drug Discov. 2012, 11, 443–461. (10)

Yuan, H.; Su, L.; Chen, W. Y. The emerging and diverse roles of sirtuins in cancer: a

clinical perspective. OncoTargets Ther. 2013, 6, 1399–1416. (11)

Hu, J.; Jing, H.; Lin, H. Sirtuin inhibitors as anticancer agents. Future Med. Chem. 2014,

6, 945-966. (12)

Herskovits, A. Z.; Guarente, L. SIRT1 in neurodevelopment and brain senescence.

Neuron 2014, 81, 471-483. (13)

Vachharajani, V.; Liu, T.; McCall, C. E. Epigenetic coordination of acute systemic

inflammation: potential therapeutic targets. Expert Rev. Clin. Immunol. 2014, 10, 1141-1150. (14)

Cencioni, C.; Spallotta, F.; Mai, A.; Martelli, F.; Farsetti, A.; Zeiher, A. M.; Gaetano, C.

Sirtuin function in aging heart and vessels. J. Mol. Cell Cardiol. 2015, 83, 55-61. (15)

Kitada, M.; Koya, D. SIRT1 in type 2 diabetes: mechanisms and therapeutic potential.

Diabetes Metab. J. 2013, 37, 315-325. (16)

Wagner, G. R.; Hirschey, M. D. Nonenzymatic protein acylation as a carbon stress

regulated by sirtuin deacylases. Mol. Cell 2014, 54, 5-16. (17)

Wang, Y. Molecular links between caloric restriction and Sir2/SIRT1 activation.

Diabetes Metab. J. 2014, 38, 321-329. (18)

Michan, S. Calorie restriction and NAD⁺/sirtuin counteract the hallmarks of aging. Front.

Biosci. 2014, 19, 1300-1319.

62


Page 63 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(19)

Chang, H. C.; Guarente, L. SIRT1 and other sirtuins in metabolism. Trends Endocrinol.

Metab. 2014, 25, 138-145. (20)

Martínez-Redondo, P.; Vaquero, A. The diversity of histone versus nonhistone sirtuin

substrates. Genes Cancer 2013, 4, 148-163. (21)

Simmons, G. E. Jr, Pruitt, W. M.; Pruitt, K. Diverse roles of SIRT1 in cancer biology and

lipid metabolism. Int. J. Mol. Sci. 2015, 16, 950-965. (22)

Wang, F.; Chen, H. Z.; Lv, X.; Liu, D. P. SIRT1 as a novel potential treatment target for

vascular aging and age-related vascular diseases. Curr. Mol. Med. 2013, 13, 155-164. (23)

Xu, F.; Gao, Z.; Zhang, J.; Rivera, C. A.; Yin, J.; Weng, J.; Ye, J. Lack of SIRT1

(mammalian sirtuin 1) activity leads to liver steatosis in the SIRT1+/- mice: a role of lipid mobilization and inflammation. Endocrinology 2010, 151, 2504–2514. (24)

Chen, J.; Zhou, Y.; Mueller-Steiner, S.; Chen, L.-F.; Kwon, H.; Yi, S.; Mucke, L.; Gan,

L. SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-κB signaling. J. Biol. Chem. 2005, 280, 40364–40374. (25)

Jeong, H.; Cohen, D. E.; Cui, L.; Supinski, A.; Savas, J. N.; Mazzulli, J. R.; Yates, J. R.,

3rd; Bordone, L.; Guarente, L.; Krainc, D. Sirt1 mediates neuroprotection from mutant huntingtin by activation of the TORC1 and CREB transcriptional pathway. Nat. Med. 2012, 18, 159–165. (26)

Chen, Y.; Fu, L. L.; Wen, X.; Wang, X. Y.; Liu, J.; Cheng, Y.; Huang, J. Sirtuin-3

(SIRT3), a therapeutic target with oncogenic and tumor-suppressive function in cancer. Cell Death Dis. 2014, 5:e1047. (27)

Rotili, D.; Tarantino, D.; Nebbioso, A.; Paolini, C.; Huidobro, C.; Lara, E,.; Mellini, P.;

Lenoci, A.; Pezzi, R.; Botta, G.; Lahtela-Kakkonen, M.; Poso, A.; Steinkühler, C.; Gallinari, P.;

63



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 64 of 73

De Maria, R.; Fraga, M.; Esteller, M.; Altucci, L., Mai, A. Discovery of salermide-related sirtuin inhibitors: binding mode studies and antiproliferative effects in cancer cells including cancer stem cells. J. Med. Chem. 2012, 5, 10937-10947. (28)

Rotili, D.; Tarantino, D.; Carafa, V.; Paolini, C.; Schemies, J.; Jung, M.; Botta, G.; Di

Maro, S.; Novellino, E.; Steinkühler, C.; De Maria, R.; Gallinari, P.; Altucci, L.; Mai, A. Benzodeazaoxaflavins as sirtuin inhibitors with antiproliferative properties in cancer stem cells. J. Med. Chem. 2012, 55, 8193-8197. (29)

Hubbard, B. P.; Sinclair, D. A. Small molecule SIRT1 activators for the treatment of

aging and age-related diseases. Trends Pharmacol. Sci. 2014, 35, 146-154. (30)

D'Onofrio, N.; Vitiello, M.; Casale, R.; Servillo, L.; Giovane, A.; Balestrieri, M. L.

Sirtuins in vascular diseases: emerging roles and therapeutic potential. Biochim. Biophys. Acta 2015, 1852, 1311-1322. (31)

Scuderi, C.; Stecca, C.; Bronzuoli, M. R.; Rotili, D.; Valente, S.; Mai, A.; Steardo, L.

Sirtuin modulators control reactive gliosis in an in vitro model of Alzheimer's disease. Front. Pharmacol. 2014, 5:89. (32)

Mellini, P.; Valente, S; Mai, A. Sirtuin modulators: an updated patent review (2012 -

2014). Expert Opin. Ther. Pat. 2015, 25, 5-15. (33)

Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.; Lavu, S.; Wood, J. G.;

Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang, L.-L.; Scherer, B.; Sinclair, D. A. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003, 425, 191–196.

64


Page 65 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34)

Wood, J. G.; Rogina, B.; Lavu, S.; Howitz, K.; Helfand, S. L.; Tatar, M.; Sinclair, D.

Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 2004, 430, 686-689. (35)

Valenzano, D. R.; Terzibasi, E.; Genade, T.; Cattaneo, A.; Domenici, L.; Cellerino, A.

Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr. Biol. 2006, 16, 296–300. (36)

Rascon, B.; Hubbard, B. P.; Sinclair, D. A.; Amdam, G. V. The lifespan extension effects

of resveratrol are conserved in the honey bee and may be driven by a mechanism related to caloric restriction. Aging 2012, 4, 499–508. (37)

Milne, J. C.; Lambert, P. D.; Schenk, S.; Carney, D. P.; Smith, J. J.; Gagne, D. J.; Jin, L.;

Boss, O.; Perni, R. B.; Vu, C. B.; Bemis, J. E.; Xie, R.; Disch, J. S.; Ng, P. Y.; Nunes, J. J.; Lynch, A. V.; Yang, H.; Galonek, H.; Israelian, K.; Choy, W.; Iffland, A.; Lavu, S.; Medvedik, O.; Sinclair, D. A.; Olefsky, J. M.; Jirousek, M. R.; Elliott, P. J.; Westphal, C. H. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 2007, 450, 712– 716. (38)

Baur, J. A.; Pearson, K. J.; Price, N. L.; Jamieson, H. A.; Lerin, C.; Kalra, A.; Prabhu, V.

V.; Allard, J. S.; Lopez-Lluch, G.; Lewis, K.; Pistell, P. J.; Poosala, S.; Becker, K. G.; Boss, O.; Gwinn, D.; Wang, M.; Ramaswamy, S.; Fishbein, K. W.; Spencer, R. G.; Lakatta, E. G.; Le Couteur, D.; Shaw, R. J.; Navas, P.; Puigserver, P.; Ingram, D. K.; de Cabo, R.; Sinclair, D. A. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 2006, 444, 337342. (39)

Nayagam, V. M.; Wang, X.; Tan, Y. C.; Poulsen, A.; Goh, K. C.; Ng, T.; Wang, H.;

Song, H. Y.; Ni, B.; Entzeroth, M.; Stünkel, W. SIRT1 modulating compounds from high-

65



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 66 of 73

throughput screening as anti-inflammatory and insulin-sensitizing agents. J. Biomol. Screen. 2006, 11, 959-967. (40)

Dao, T. T.; Tran, T. L.; Kim, J.; Nguyen, P. H.; Lee, E. H.; Park, J.; Jang, I. S.; Oh, W. K.

Terpenylated coumarins as SIRT1 activators isolated from Ailanthus altissima. J. Nat. Prod. 2012, 75, 1332-1338. (41)

Wu, J.; Zhang, D.; Chen, L.; Li, J.; Wang, J.; Ning, C.; Yu, N.; Zhao, F.; Chen, D.; Chen,

X.; Chen, K.; Jiang, H.; Liu, H.; Liu, D. Discovery and mechanism study of SIRT1 activators that promote the deacetylation of fluorophore-labeled substrate. J. Med. Chem. 2013, 56, 761– 780. (42)

Pulla, V. K.; Alvala, M.; Sriram, D. S.; Viswanadha, S.; Sriram, D.; Yogeeswari. P.

Structure-based drug design of small molecule SIRT1 modulators to treat cancer and metabolic disorders. J. Mol. Graph. Model. 2014, 52, 46-56. (43)

Yang, J. L.; Ha, T. K.; Dhodary, B.; Kim, K. H.; Park, J.; Lee, C. H.; Kim, Y. C.; Oh, W.

K. Dammarane triterpenes as potential SIRT1 activators from the leaves of Panax ginseng. J. Nat. Prod. 2014, 77, 1615-1623. (44)

Sinclair, D. A.; Guarente, L. Small-molecule allosteric activators of sirtuins. Annu. Rev.

Pharmacol. Toxicol. 2014, 54, 363-380. (45)

Libri, V.; Brown, A. P.; Gambarota, G.; Haddad, J.; Shields, G. S.; Dawes, H.; Pinato, D.

J.; Hoffman, E.; Elliot, P. J.; Vlasuk, G. P.; Jacobson, E.; Wilkins, M. R.; Matthews, P. M. A pilot randomized, placebo controlled, double blind phase I trial of the novel SIRT1 activator SRT2104 in elderly volunteers. PLoS One 2012, 7, e51395. (46)

https://clinicaltrials.gov/ct2/results?term=sirt+activator&Search=Search

and

https://clinicaltrials.gov/ct2/results?term=SRT3025&Search=Search. Access date: Dec 17, 2015.

66


Page 67 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(47)

Borra, M. T.; Langer, M. R.; Slama, J. T.; Denu, J. M. Substrate specificity and kinetic

mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases. Biochemistry 2004, 43, 9877–9887. (48)

Kaeberlein, M.; McDonagh, T.; Heltweg, B.; Hixon, J.; Westman, E. A.; Caldwell, S. D.;

Napper, A.; Curtis, R.; Di Stefano, P. S.; Fields, S.; Bedalov, A.; Kennedy, B. K. Substratespecific activation of sirtuins by resveratrol. J. Biol. Chem. 2005, 280, 17038–17045. (49)

Pacholec, M.; Bleasdale, J. E.; Chrunyk, B.; Cunningham, D.; Flynn, D.; Garofalo, R. S.;

Griffith, D.; Griffor, M.; Loulakis, P.; Pabst, B.; Qiu, X.; Stockman, B.; Thanabal, V.; Varghese, A.; Ward, J.; Withka, J.; Ahn, K. SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J. Biol. Chem. 2010, 285, 8340–8351. (50)

Park, S. J.; Ahmad, F.; Philp, A.; Baar, K.; Williams, T.; Luo, H.; Ke, H.; Rehmann, H.;

Taussig, R.; Brown, A. L.; Kim, M. K.; Beaven, M. A.; Burgin, A. B.; Manganiello, V.; Chung, J. H. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell 2012, 148, 421–433. (51)

Hubbard, B. P., Gomes, A. P.; Dai, H.; Li, J.; Case, A. W.; Considine, T.; Riera, T. V.;

Lee, J. E.; E, S. Y.; Lamming, D. W.; Pentelute, B. L.; Schuman, E. R.; Stevens, L. A.; Ling, A. J.; Armour, S. M.; Michan, S.; Zhao, H.; Jiang, Y.; Sweitzer, S. M.; Blum, C. A.; Disch, J. S.; Ng, P. Y.; Howitz, K. T.; Rolo, A. P.; Hamuro, Y.; Moss, J.; Perni, R. B.; Ellis, J. L.; Vlasuk, G. P.; Sinclair, D. A. Evidence for a common mechanism of SIRT1 regulation by allosteric activators. Science 2013, 339, 1216-1219. (52)

Lakshminarasimhan, M.; Rauh, D.; Schutkowski, M.; Steegborn, C. Sirt1 activation by

resveratrol is substrate sequence-selective. Aging 2013, 5, 151-154.

67



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(53)

Page 68 of 73

Dai, H.; Case, A. W.; Riera, T. V.; Considine, T.; Lee, J. E.; Hamuro, Y.; Zhao, H.;

Jiang, Y.; Sweitzer, S. M.; Pietrak, B.; Schwartz, B.; Blum, C. A.; Disch, J. S.; Caldwell, R.; Szczepankiewicz, B.; Oalmann, C.; Yee, Ng. P.; White, B. H.; Casaubon, R.; Narayan, R.; Koppetsch, K.; Bourbonais, F.; Wu, B.; Wang, J.; Qian, D.; Jiang, F.; Mao, C.; Wang, M.; Hu, E.; Wu, J. C.; Perni, R. B.; Vlasuk, G. P.; Ellis, J. L. Crystallographic structure of a small molecule SIRT1 activator-enzyme complex. Nat. Commun. 2015, 6:7645. (54)

Mai, A.; Massa, S.; Lavu, S.; Pezzi, R.; Simeoni, S.; Ragno, R.; Mariotti, F. R.; Chiani,

F.; Camilloni, G.; Sinclair, D. A. Design, synthesis, and biological evaluation of sirtinol analogues as class III histone/protein deacetylase (sirtuin) inhibitors. J. Med. Chem. 2005, 48, 7789-7795. (55)

Mai, A.; Cheng, D.; Bedford, M. T.; Valente, S.; Nebbioso, A.; Perrone, A.; Brosch, G.;

Sbardella, G.; De Bellis, F.; Miceli, M.; Altucci, L. Epigenetic multiple ligands: mixed histone/protein methyltransferase, acetyltransferase, and class III deacetylase (sirtuin) inhibitors. J. Med. Chem. 2008, 51, 2279-2290. (56)

Lara, E.; Mai, A.; Calvanese, V.; Altucci, L.; Lopez-Nieva, P.; Martinez-Chantar, M. L.;

Varela-Rey, M.; Rotili, D.; Nebbioso, A.; Ropero, S.; Montoya, G.; Oyarzabal, J.; Velasco, S.; Serrano, M.; Witt, M.; Villar-Garea, A.; Imhof, A.; Mato, J. M.; Esteller, M.; Fraga, M. F. Salermide, a sirtuin inhibitor with a strong cancer-specific proapoptotic effect. Oncogene 2009, 28, 781-791. (57)

Rotili, D.; Tarantino, D.; Carafa, V.; Lara, E.; Meade, S.; Botta, G.; Nebbioso, A.;

Schemies, J.; Jung, M.; Kazantsev, A. G.; Esteller, M.; Fraga, M. F.; Altucci, L.; Mai, A. Identification of tri- and tetracyclic pyrimidinediones as sirtuin inhibitors. ChemMedChem 2010, 5, 674-677.

68


Page 69 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(58)

Pasco, M. Y.; Rotili, D.; Altucci, L.; Farina, F.; Rouleau, G. A.; Mai, A.; Néri, C.

Characterization of sirtuin inhibitors in nematodes expressing a muscular dystrophy protein reveals muscle cell and behavioral protection by specific sirtinol analogues. J. Med. Chem. 2010, 53, 1407-1411. (59)

Rotili, D.; Carafa, V.; Tarantino, D.; Botta, G.; Nebbioso, A.; Altucci, L.; Mai, A.

Simplification of the tetracyclic SIRT1-selective inhibitor MC2141: coumarin- and pyrimidinebased SIRT1/2 inhibitors with different selectivity profile. Bioorg. Med. Chem. 2011, 19, 36593668. (60)

Orecchia, A.; Scarponi, C.; Di Felice, F.; Cesarini, E.; Avitabile, S.; Mai, A.; Mauro, M.

L.; Sirri, V.; Zambruno, G.; Albanesi, C.; Camilloni, G.; Failla, C. M. Sirtinol treatment reduces inflammation in human dermal microvascular endothelial cells. PLoS ONE 2011, 6, e24307. (61)

Mellini, P.; Carafa, V.; Di Rienzo, B.; Rotili, D.; De Vita, D.; Cirilli, R.; Gallinella, B.;

Provvisiero, D. P.; Di Maro, S.; Novellino, E.; Altucci, L.; Mai, A. Carprofen analogues as sirtuin inhibitors: enzyme and cellular studies. ChemMedChem 2012, 7, 1905-1908. (62)

Mellini, P.; Kokkola, T.; Suuronen, T.; Salo, H. S.; Tolvanen, L.; Mai, A.; Lahtela-

Kakkonen, M.; Jarho, E. M. Screen of pseudopeptidic inhibitors of human sirtuins 1-3: two lead compounds with antiproliferative effects in cancer cells. J Med Chem 2013, 56, 6681-6695. (63)

Mai, A.; Valente, S.; Meade, S.; Carafa, V.; Tardugno, M.; Nebbioso, A.; Galmozzi, A.;

Mitro, N.; De Fabiani, E.; Altucci, L.; Kazantsev, A. Study of 1,4-dihydropyridine structural scaffold: discovery of novel sirtuin activators and inhibitors. J. Med. Chem. 2009, 52, 54965504. (64)

Spallotta, F.; Cencioni, C.; Straino, S.; Nanni, S.; Rosati, J.; Artuso, S.; Manni, I.,

Colussi, C.; Piaggio G.; Martelli, F.; Valente, S.; Mai, A.; Capogrossi, M. C.; Farsetti, A.;

69



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 70 of 73

Gaetano, C. A nitric oxide-dependent crosstalk between class I and III histone deacetylases accelerates skin repair. J. Biol. Chem. 2013, 288, 11004-11012. (65)

Chen, Z.; Peng, I.-C.; Cui, X.; Li, Y.-S.; Chien, S.; Shyy, J. Y.-J. Shear stress, SIRT1,

and vascular homeostasis. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 10268–10273. (66)

Napper, A. D.; Hixon, J.; McDonagh, T.; Keavey, K.; Pons, J. F.; Barker, J.; Yau, W. T.;

Amouzegh, P.; Flegg, A.; Hamelin, E.; Thomas, R. J.; Kates, M.; Jones, S.; Navia, M. A.; Saunders, J. O.; Di Stefano P. S.; Curtis, R. Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1. J. Med. Chem. 2005, 48, 8045-8054. (67)

Rimoli, M. G.; Avallone, L.; Zanarone, S.; Abignente, E.; Mangoni, A. Research on

heterocyclic compounds. XLIII. Synthetic studies on 1,4-dihydropyridine derivatives. J. Heterocyclic Chem. 2002, 39, 1117-1122. (68)

Funder, E. D.; Trads, J. B.; Gothelf, K.V. Oxidative activation of dihydropyridine amides

to reactive acyl donors. Org. Biomol. Chem. 2015, 13, 185-198. (69)

Molina, P.; Pastor, A.; Vilaplana, M. J. Unusual reactivity of (vinylimino)phosphoranes

and their utility in the preparation of pyridine and dihydropyridine derivatives J. Org. Chem. 1996, 61, 8094-8098. (70)

Sueki, S.; Takei, R.; Zaitsu, Y.; Abe, J.; Fukuda, A.; Seto, K.; Furukawa, Y.; Shimizu, I.

Synthesis of 1,4-dihydropyridines and their fluorescence properties. Eur. J. Org. Chem. 2014, 24, 5281-5301. (71)

Molina, P.; Pastor, A.; Vilaplana, M. J. Synthetic applications of vinyliminophosphoranes

based on the reactivity of the vinyl side chain. Preparation of dihydropyridines Tetrahedron Lett. 1995, 36, 8283-8286.

70


Page 71 of 73

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(72)

Mattagajasingh, I.; Kim, C.-S.; Naqvi, A.; Yamamori, T.; Hoffman, T. A.; Jung, S.-B.;

DeRicco, J.; Kasuno, K.; Irani, K. SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 14855– 14860. (73)

Gerhart-Hines, Z.; Rodgers, J. T.; Bare, O.; Lerin, C.; Kim, S. H.; Mostoslavsky, R.; Alt,

F. W.; Wu, Z.; Puigserver, P. Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1α. EMBO J. 2007, 26, 1913-1923. (74)

Jäger, S.; Handschin, C.; St-Pierre, J.; Spiegelman, B. M. AMP-activated protein kinase

(AMPK) action in skeletal muscle via direct phosphorylation of PGC-1α. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12017-12022. (75)

Ioan, P.; Carosati, E.; Micucci, M.; Cruciani, G.; Broccatelli, F.; Zhorov, B. S.; Chiarini,

A.; Budriesi, R. 1,4-Dihydropyridine Scaffold in Medicinal Chemistry, The Story so Far And Perspectives. (Part 1): Action in Ion Channels and GPCRs. Curr. Med. Chem. 2011, 18, 49014922. (76)

Carosati, E.; Ioan, P.; Micucci, M.; Broccatelli, F.; Cruciani, G.; Zhorov, B. S.; Chiarini,

A.; Budriesi, R. 1,4-Dihydropyridine Scaffold in Medicinal Chemistry, The Story so Far And Perspectives. (Part 2): Action in Other Targets and Antitargets. Curr. Med. Chem., 2012, 19, 4306-4323. (77)

Chauhan, D.; Bandi, M.; Singh, A.V.; Ray, A.; Raje, N.; Richardson, P.; Anderson, K.C.

Preclinical evaluation of a novel SIRT1 modulator SRT1720 in multiple myeloma cells. Br. J. Haematol. 2011, 155, 588–598. (78)

Trisciuoglio, D.; Ragazzoni, Y.; Pelosi, A.; Desideri, M.; Carradori, S.; Gabellini, C.;

Maresca, G.; Nescatelli, R.; Secci, D.; Bolasco, A.; Bizzarri, B.; Cavaliere, C.; D’Agnano, I.;

71



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 72 of 73

Filetici, P.; Ricci-Vitiani, L.; Rizzo, M. G.; Del Bufalo, D. CPTH6, a thiazole derivative, induces histone hypoacetylation and apoptosis in human leukemia cells. Clin. Cancer Res. 2012, 18, 475−486. (79)

Valente, S.; Trisciuoglio, D.; Tardugno, M.; Benedetti, R.; Labella, D.; Secci, D.;

Mercurio, C.; Boggio, R.; Tomassi, S.; Di Maro, S.; Novellino, E.; Altucci, L.; Del Bufalo, D.; Mai, A.; Cosconati, S. tert-Butylcarbamate-containing histone deacetylase inhibitors: apoptosis induction, cytodifferentiation, and antiproliferative activities in cancer cells. ChemMedChem 2013, 8, 800−811. (80)

Ragazzoni, Y.; Desideri, M.; Gabellini, C.; De Luca, T.; Carradori, S.; Secci, D.;

Nescatelli, R.; Candiloro, A.; Condello, M.; Meschini, S.; Del Bufalo, D.; Trisciuoglio, D. The thiazole derivative CPTH6 impairs autophagy. Cell Death Dis. 2013, 4, e524.

72


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AMPK Pathway Ameliorate

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