Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to the discovery of a series of highly potent P-glycoprotein-based multidrug resistance (MDR) modulators

Accepted Manuscript

Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to

the discovery of a series of highly potent P-glycoprotein-based multidrug-resistance

(MDR) modulators

Silvia Dei, Laura Braconi, Alfonso Trezza, Marta Menicatti, Marialessandra Contino,

Marcella Coronnello, Niccolò Chiaramonte, Dina Manetti, Maria Grazia Perrone,

Maria Novella Romanelli, Chatchanok Udomtanakunchai, Nicola Antonio Colabufo,

Gianluca Bartolucci, Ottavia Spiga, Milena Salerno, Elisabetta Teodori

PII:

S0223-5234(19)30282-X

DOI:

https://doi.org/10.1016/j.ejmech.2019.03.054

EJMECH 11224

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 26 November 2018

Revised Date: 21 March 2019

Accepted Date: 22 March 2019

Please cite this article as: S. Dei, L. Braconi, A. Trezza, M. Menicatti, M. Contino, M. Coronnello,

Niccolò. Chiaramonte, D. Manetti, M.G. Perrone, M.N. Romanelli, C. Udomtanakunchai, N.A. Colabufo,

G. Bartolucci, O. Spiga, M. Salerno, E. Teodori, Modulation of the spacer in N,N-bis(alkanol)amine

aryl ester heterodimers led to the discovery of a series of highly potent P-glycoprotein-based

multidrug-resistance (MDR) modulators, European Journal of Medicinal Chemistry (2019), doi: https://

doi.org/10.1016/j.ejmech.2019.03.054.

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ACCEPTED MANUSCRIPT

Graphical abstract

m + n = 10

CH₃

O

N

O

Ar₁

Ar

potent P-gp-dependent

MDR reversers

(CH₂)_m

(CH₂)_n

OCH₃

a

O

Ar, Ar₁=

OCH₃

H₃CO

b

c

docking model of a representative

compound (in purple) and P-gp

:

ACCEPTED MANUSCRIPT

Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to the

discovery of a series of highly potent P-glycoprotein-based multidrug-resistance (MDR)

modulators

Silvia Dei,^a* Laura Braconi,^aAlfonso Trezza,^bMarta Menicatti,^aMarialessandra Contino,^cMarcella

Coronnello,^dNiccolò Chiaramonte,^aDina Manetti,^aMaria Grazia Perrone,^cMaria Novella

Romanelli,^aChatchanok Udomtanakunchai,^eNicola Antonio Colabufo,^cGianluca Bartolucci,^a

Ottavia Spiga,^bMilena Salerno,^fElisabetta Teodori^a

a

Department of Neuroscience, Psychology, Drug Research and Child’s Health - Section of

Pharmaceutical and Nutraceutical Sciences, University of Florence, via Ugo Schiff 6, 50019 Sesto

Fiorentino (FI), Italy.

b

Department of Biotechnology, Chemistry and Pharmacy, (Department of Excellence 2018-2022),

University of Siena, via Aldo Moro 2, 53100 Siena, Italy

^cDepartment of Pharmacy – Drug Sciences, University of Bari Aldo Moro, via Orabona 4, 70125,

Bari, Italy.

d

Department of Health Sciences - Section of Clinical Pharmacology and Oncology, University of

Florence, Viale Pieraccini 6, 50139 Firenze, Italy.

e

Department of Radiologic Technology, Faculty of Associated Medical Sciences, Chang Mai

University, 50200, Thailand.

f

University of Paris 13, Sorbonne Paris Cité, Laboratoire CSPBAT, CNRS (UMR 7244), UFR-

SMBH, 74 rue Marcel Cachin, 93017 Bobigny, France

* corresponding author:

Silvia Dei, Department of Neuroscience, Psychology, Drug Research and Child’s Health - Section

of Pharmaceutical and Nutraceutical Sciences, University of Florence, via Ugo Schiff 6, 50019

Sesto Fiorentino (FI), Italy. Tel +39 055 4573689. Email: silvia.dei@unifi.it

Abstract

In this study, a new series of N,N-bis(alkanol)amine aryl ester heterodimers was synthesized and

studied. The new compounds were designed based on the structures of our previous arylamine ester

derivatives endowed with high P-gp-dependent multidrug resistance reversing activity on a

multidrug-resistant leukemia cell line. All new compounds were active in the pirarubicin uptake

assay on the doxorubicin–resistant erythroleukemia K562 cells (K562/DOX). Compounds bearing a

linker made up of 10 methylenes showed unprecedented high reversal activities regardless of the

combination of aromatic moieties. Docking results obtained by an in silico study supported the data

obtained by the biological tests and a study devoted to establish the chemical stability in phosphate

buffer solution (PBS) and human plasma showed that only a few compounds exhibited a significant

degradation in the human plasma matrix. Ten selected non-hydrolysable derivatives were able to

inhibit the P-gp-mediated rhodamine-123 efflux on K562/DOX cells, and the evaluation of their

apparent permeability and ATP consumption on other cell lines suggested that the compounds can

behave as unambiguous or not transported substrates. The activity of these the compounds on the

transport proteins breast cancer resistance protein (BCRP) and multidrug resistance associated

protein 1 (MRP1) was also analyzed. All tested derivatives displayed a moderate potency on the

BCRP overexpressing cells; while only four molecules showed to be effective on MRP1

overexpressing cells, highlighting a clear structural requirement for selectivity. In conclusion, we

have identified a new very powerful series of compounds which represent interesting leads for the

development of new potent and efficacious P-gp-dependent MDR modulators.

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Keywords

P-gp modulators; BCRP modulators; MRP1 modulators; MDR reversers; pirarubicin uptake;

molecular docking; human plasma stability; rhodamine-123 efflux; ATPase activity; apparent

permeability.

List of Abbreviations

P-gp,

P-glycoprotein;

DOX,

Doxorubicin;

EDCI,

1-(3-dimethylaminopropyl)-3-

ethylcarbodiimmide hydrochloride; DMAP, 4-dimethylaminopyridine; TPSA, topological polar

surface area; PDB, Protein Data Bank; PBS, phosphate buffer solution; KEE, ketoprofene

ethylester; Rhd123, rhodamine-123; MDCK, Madin-Darby Canin Kidney; BCRP, Breast Cancer

Resistance Protein; MRP1, Multidrug Resistance associated Protein 1; Caco-2 cells, colorectal

adenocarcinoma cells.

1. Introduction

The resistance of cancer cells to cytotoxic drugs is a significant limitation to successful

chemotherapy. Multidrug resistance (MDR) is a type of acquired drug resistance to multiple classes

of structurally and mechanistically unrelated anticancer drugs [1].

MDR is a complex phenomenon which can derive from different biochemical mechanisms. In the

case of the so called classical MDR, cells accumulate a lower intracellular concentration of drug as

a result of an accelerated efflux of the antitumor agents mediated by an ATP-dependent process.

The main mechanism responsible of this transport is the overexpression of integral membrane

transporters such as P-glycoprotein (P-gp, ABCB1) [2], Breast Cancer Resistance Protein (BCRP,

ABCG2) [3] and Multidrug Resistance associated protein 1 (MRP1, ABCC1) [4], belonging to the

ABC (ATP Binding Cassette) protein family.

P-gp is a membrane glycoprotein present, beside cancer cells, in several important tissues and

blood-tissue barriers, where it regulates some important physiological processes such as the

secretion of lipophilic molecules and the extrusion of exogenous agents [5]. Unfortunately, this

efflux protein is overexpressed in cancer cells as a result of the upregulation of the human MDR1

gene expression and it extrudes the chemotherapeutic drug from the cells, lowering its concentration

below that necessary for anticancer action [6].

Since its discovery and the elucidation of the mechanism of action, P-gp has been considered a

suitable target for circumventing transporter-dependent MDR. MDR reversers (chemosensitizers)

are P-gp modulators that administered in combination with cytotoxic agents, which are substrates of

the efflux pump, could restore their efficacy in resistant cancer cells [7,8].

Verapamil was the first compound showing P-gp modulating activity and, together with many other

molecules, belongs to the first generation of P-gp modulators. However, the toxicity of this first

series of compounds prevented their clinical use and, at present, verapamil is only used as gold

standard in biological assays. Since then, many P-gp modulators, belonging to three generations of

compounds have been identified [9,10]. Several of them have reached pre-clinical or clinical trials

[11,12], but none of these compounds has been approved for therapy, because of their low potency,

toxicity and inhibitory effect on isoforms of cytochrome [13], although some of the latest MDR

reversing compounds show a safer profile.

The failure of pre-clinical and clinical investigational studies led to considerable pessimism

regarding the validity of such therapeutic approach to overcome MDR [14]. Nevertheless, the

search for new, safer, more potent and efficacious multidrug transporter modulators is still of

interest. In fact, MDR occurs also in brain disorders such as epilepsy, depression, and

schizophrenia: in these diseases, about 20-40 % of the patients develop resistance to current

therapeutic drugs [15]. Another field of investigation regards Parkinson’s and Alzheimer’s diseases:

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recent evidences suggest that a decrease in P-gp expression and function at blood brain barrier

occur in these diseases [16]. Consequently, there are several reasons for medicinal chemists to

continue the search for molecules that could be used to modulate P-gp.

Information on the interaction site of P-gp suggest that it is a large, flexible drug binding domain

where molecules can accommodate in a plurality of binding modes, establishing π−π, ion-π,

hydrogen bonds and hydrophobic interactions [17]. So, in the search for efficient P-gp modulating

MDR reversers, in the last years we have designed and studied several families of basic molecules

bearing suitably positioned aromatic rings, assuming that the presence of aromatic moieties and of

one or more protonable nitrogen atoms is an important property for the P-gp interaction. In our drug

design strategy, these molecular features have been connected by linkers of different length and

flexibility [18,19]. In particular, a high structural flexibility would allow the molecules to choose

the most productive binding mode within the P-gp recognition site. This approach, labeled as

“polyvalency”, had been already used successfully by other researchers, who synthesized several

homodimers as MDR inhibitors [20-22].

Based on the polyvalency approach, we synthesized several N,N-bis(alkanol)amine aryl esters

characterized by the presence of a basic nitrogen atom linked to two different aromatic ester

portions by two polymethylenic chains of variable length as spacers. This approach provided good

results since most of the synthesized compounds showed to be very potent MDR reversers in a

human leukemia cell line [18,23,24].

The first series of compounds were characterized by different combinations of aromatic residues

connected to the N-methylated basic portion by two identical polymethylenic chains of variable

length as spacers [18,23,24]; best results were obtained by the combination of two chains

constituted by 4 or 5 methylenes, with a spacer total length corresponding to 8 or 10 methylenes and

one nitrogen atom. Then, we explored the consequences of varying the relative position of the

nitrogen in the chain, abandoning the symmetry of the linkers. These derivatives conserved two

aromatic ester portions, and a chain of nine components, eight methylenes and one nitrogen atom,

but the position of the nitrogen atom was changed according to the length of the two linkers (3- and

5-methylenes long, structure A, Chart 1) [24]. In this way the nitrogen atom was at different

distances from the two aromatic moieties, according to the length of the two different spacers. The

new asymmetric compounds showed an outstanding potency if compared to the symmetric isomers;

in particular the combination of the (E)-3-(3,4,5-trimethoxyphenyl)vinyl (also named trans-3,4,5-

trimethoxycinnamyl) moiety with the 3,4,5-trimethoxyphenyl and anthracene residues (compounds

I-IV, structure A, Chart 1) that were already present in the most potent of the previously

synthesized compounds [18,23,24] gave rise to the best results of the series. On the bases of these

unexpected results, in the present study we decided to widen the series of asymmetrical derivatives

by synthesizing all the possible isomers obtained by the combination of different spacers of 2-8

units, for a total length of 8, 9 and 10 methylenes, bearing the aromatic moieties described above

(compounds 1-28, structure B, Chart 1).

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Chart 1. Structure of the reference compounds I-IV and of the derivatives 1-28 synthesized in this

paper.

The reversal activity of the new compounds 1-28 was evaluated by the pirarubicin uptake assay in

doxorubicin-resistant erythroleukemia K562 cells (K562/DOX). Moreover, a molecular docking

simulation was performed to identify the potential binding poses of compounds and their

mechanism of interaction within the P-gp binding pocket. Finally, a study devoted to establishing

the chemical stability of these compounds was planned. In fact, it is well known that the ester

group, present in the chemical structure of studied compounds, can be susceptible to hydrolysis by

the plasma enzymes. Therefore, a series of experiments concerning the stability of these compounds

were performed.

On the bases of the obtained results, the derivatives displaying the best activity on pirarubicin assay

and a good chemical stability were tested to define their interacting mechanism vs P-gp by

evaluating their effect on P-gp mediated rhodamine-123 efflux on K562/DOX cell line. Moreover,

the activity of the same compounds was measured on another cell line overexpressing P-gp, the

transfected Madin-Darby Canine Kidney-MDR1 (MDCK-MDR1) one, and on two cell lines

overexpressing the sister pumps MRP1 and BCRP, MDCK-MRP1 and MDCK-BCRP respectively,

to evaluate their selectivity. Finally, their apparent permeability on Caco-2 cells and their effect on

ATP cell depletion on MDCK-MDR1 cells were also analyzed.

2. Chemistry

The reaction pathways used to synthesize the designed derivatives (1-28) are reported in Schemes 1

and 2. The haloesters 29-40 were synthesized by esterification of the suitable haloalkyl alcohol (2-

bromoethan-1-ol, 3-bromopropan-1-ol, 4-chlorobutan-1-ol, 5-chloropentan-1-ol, 6-chlorohexan-1-

ol, 7-bromoheptan-1-ol and 8-bromooctan-1-ol) with the commercially available (E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid, 3,4,5-trimethoxybenzoic acid or anthracene-9-carboxylic acid

(Scheme 1). These esters were obtained by transformation of the carboxylic acid in the

corresponding acyl chloride by reaction with SOCl₂in ethanol-free CHCl₃(for details, see the

Experimental section). Derivatives 29, 30, 32, 33, 35 had already been described by our group [18,

24].

The chloroalkyl esters 31- 34 were transformed in the corresponding iodo derivatives 41-44 with

NaI in acetone, to achieve higher yields in the following reaction. The bromo esters 29-30, 35-40

and the iodo esters 41-44 were then transformed into the secondary amines 45-54 by reaction with

the corresponding aminoalcohol using standard procedures (Scheme 2). These compounds were

alkylated by reductive methylation with HCOOH/HCHO to give the corresponding tertiary amines

55-64. In order to obtain the (2-hydroxyethyl)methylaminoesters 65-72, the appropriate haloester

4

ACCEPTED MANUSCRIPT

was reacted with the commercially available 2-methylaminoethan-1-ol, yielding directly the desired

tertiary amines 65-72.

Final compounds 1-28 were eventually obtained by reaction of 55-72 with the proper carboxylic

acid using EDCI and DMAP in anhydrous CH₂Cl₂, or by transformation of the carboxylic acid in

the corresponding acyl chloride as described before. The suitable acids are the same described

previously: (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid, 3,4,5-trimethoxybenzoic acid and

anthracene-9-carboxylic acid.

Scheme 1. Reagents and conditions: (I) SOCl₂, CHCl₃; (II) CHCl₃; (III) NaI, acetone.

5

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Scheme 2. Reagents and conditions: (I) H₂N(CH₂)_mOH (m= 3, 4, 5, 6), an. CH₃CN; (II)

CH₃NH(CH₂)₂OH, an. CH₃CN; (III) HCOOH/CH₂O, EtOH; (IV) Ar₁COCl, CHCl₃or EDCI,

DMAP, an. CH₂Cl₂.

6

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3. Results and discussion

3.1. Modulation of pirarubicin uptake on K562 cells

The P-gp modulating ability of compounds 1-28 was evaluated on K562/DOX doxorubicin resistant

cells. K562 is a human leukemia cell line established from a patient with chronic myelogenous

leukemia in blast transformation [25]. K562/DOX cells overexpress almost exclusively the

membrane glycoprotein P-gp [26-29]. P-gp expression in both sensitive K562 and K562/DOX cells

was checked by cytometric analysis with a P-gp-specific antibody (JSB-1 monoclonal antibody);

the obtained results showed an increase in the fluorescence ratio of about 3 times in the resistant

line. Moreover, application of a second methodology, the Real-time PCR analysis, indicated a

substantial increase in mRNA expression level of the ATP binding cassette transporter ABCB1

(175 folds) in the resistant cell line compared with the parental one, confirming an increased

presence of P-gp in the K562/DOX resistant cell line. Details are reported in the Experimental

Section and in the Supplementary Data (Figures S34 and S35).

The uptake of THP-adriamycin (pirarubicin) was measured by a continuous spectrofluorometric

signal of anthracycline at 590 nm (λ_ex= 480 nm) after cell incubation, following the protocols

reported in previous papers [30,31]. The P-gp modulating activity of the studied compounds on the

pirarubicin uptake test is expressed by: i) [I]_0.5, which measures the potency of the modulator and

represents the concentration that causes a half-maximal increase (α = 0.5) in the nuclear

concentration of pirarubicin, and ii) α_max, which represents the efficacy of the modulator and is the

maximum increase in the nuclear concentration of pirarubicin in resistant cells that can be obtained

with a given compound. The value of α varies between 0 (in the absence of the modulator) and 1

(when the amount of pirarubicin in resistant cells is the same as in sensitive cells).

The results obtained are reported in Table 1 together with those of verapamil, the gold standard of

P-gp activity inhibition, used as reference compound. 3,4,5-Trimethoxyphenyl derivatives I and II

and their anthracene analogs III (GDE6) and IV (FRA77) [24] have been added for comparison,

since they are the previously obtained asymmetric derivatives.

All the newly synthesized compounds were able to inhibit the activity of P-gp; their potencies and

efficacies were higher than those of verapamil. In fact, all molecules showed potency values ([I]_0.5)

in the submicromolar or nanomolar range and in many cases were able to completely reverse P-gp-

dependent pirarubicin extrusion (α_maxclose to 1).

A thorough evaluation of the potency values indicated that both the chain length and the

combination of the aromatic residues have some influence on the activity of our compounds. In fact,

in the case of derivatives 1-4, which carried a total spacer of 8-methylenes due to the combination

of two chains of 2 and 6 methylenes, the anthracene derivative 3 showed the best result, with an

[I]_0.5value of 0.04 µM, as it happened for the two anthracene regioisomers III and IV which were

used as lead compounds. The other compounds of the set, 1, 2 and 4, had lower potencies.

In the case of a total length of 9 methylenes, which can be obtained by three different combinations

of spacers, the presence of the anthracene residue conferred excellent properties to the molecules:

all the isomers, except for compound 16, showed a nanomolar activity (compounds 7, 8, 11, 12 and

15 with [I]_0.5values between 0.02 and 0.04 µM). The corresponding derivatives carrying the

combination of trans-3,4,5-trimethoxycinnamyl moiety with the 3,4,5-trimethoxyphenyl residue

showed a similar nanomolar activity only in the case of the two regioisomers 13 and 14.

The best results were obtained with the isomers with a total spacer of 10 methylenes; remarkably,

all the compounds (the three series 17-20, 21-24, 25-28) showed outstanding potencies in the

nanomolar range, regardless of the combination of aromatic moieties, with unprecedented results in

these series of derivatives.

As regards the efficacy values, almost all the compounds were able to completely reverse P-gp-

dependent pirarubicin extrusion (α_max= 0.90-0.99). In a little number of derivatives, some lower

efficacies can be highlighted (compounds 1, 4, and 14 with α_maxvalues of 0.77 or 0.79).

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Altogether, these data seem to confirm our previous results [23,24] that the MDR modulating

activity of this family of molecules can be related on the one hand to the nature of the aromatic

moieties and on the other to two characteristics of the linker, both the position of the nitrogen and

the total length of the tether. More interestingly, however, from these results it also comes to light

that the compounds which present a total spacer of 10 methylenes (17-28) are a new series of very

potent and efficacious compounds, regardless of the combination of aromatic moieties and of chain

lengths. In these series, only the total distance of the aromatic esters seems to be the crucial factor

for the activity. Therefore, we compared the potencies of these asymmetric compounds 17-28 with

the results obtained by the corresponding symmetric isomers V and VI already described by us [18].

As shown in Table 1 these two analogs, particularly the 3,4,5-trimethoxyphenyl derivative V,

showed a lower potency and efficacy. This comparison confirms the result already found in our

previous paper [24]. In fact, although the nitrogen position has not a clear influence on the activity,

if this atom is at the center of the spacer the activity of the compounds decreases.

Table 1

MDR-reversing activity and predictor parameters for binding free energy, drug transport properties

and lipophilicity of compounds 1-28.

e

total number of

methylenes in

the spacer

∆G

(Kcal/

mol)^c

-7

TPSA logP_o/w

b

compd

n

m

Ar

Ar₁

[I]_0.5μM^a

α_max

(Å²)^d

I^f

II ^f

5

6

5

6

7

6

3

2

4

3

2

4

a

b

a

c

b

a

c

0.12±0.02

0.27±0.05

0.04±0.01

0.04±0.02

0.19+0.07

0.46±0.07

0.04+0.01

0.20±0.08

0.10 + 0.04

0.27 + 0.12

0.03 + 0.01

0.04 + 0.01

0.24 + 0.08

0.39 + 0.12

0.03 + 0.001

0.04 + 0.01

0.07+0.02

0.02 + 0.01

0.17±0.08

0.01+0.001

0.07 + 0.002

0.99 ±0.01

0.97±0.02

0.98 ±0.02

0.94±0.03

0.77+0.04

0.91±0.03

0.94+0.03

0.79±0.06

0.94+ 0.05

0.99 + 0.01

0.94 + 0.02

0.90 + 0.04

0.99 + 0.01

0.92 + 0.03

0.99 + 0.01

0.79+0.04

0.98 + 0.02

0.88±0.08

0.99+0.01

0.99 + 0.01

111.22

83.53

6.30

6.26

6.42

6.38

6.31

6.38

6.30

5.86

6.26

6.60

6.11

6.21

6.53

6.56

6.35

6.22

6.23

6.58

6.31

6.11

6.91

6.89

-6.2

-7.4

-8.3

-6.4

-6.3

-7.9

-6.9

-7

8

9

III GDE6^f

IV FRA77^f

a

b

a

c

83.53

1

2

a

b

a

c

111.22

83.53

3

4

a

b

a

c

83.53

5

a

b

a

c

111.22

83.53

6

-6.9

-7

7

8

a

b

a

c

-7.8

-6.8

-6.5

-8

83.53

9

a

b

a

c

111.22

83.53

10

11

12

13

14

15

16

17

18

a

b

a

c

-8.4

-7

83.53

a

b

a

c

111.22

83.53

-7.5

-7

9

a

b

a

83.53

a

b

-8

111.22

10

-7.4

8

ACCEPTED MANUSCRIPT

19

6

7

8

5

4

3

2

5

a

c

a

b

a

c

0.02+0.005

0.01 + 0.002

0.06 + 0.02

0.08 + 0.03

0.01 + 0.003

0.04 ± 0.01

0.02 ± 0.01

0.04 ± 0.01

0.05 ± 0.02

0.04 ± 0.01

0.80 ± 0.20

0.10 ± 0.02

1.60 ± 0.30

0.94+0.05

0.95 + 0.05

0.99 + 0.01

0.97±0.03

0.93 ± 0.04

0.88 ± 0.08

0.93 ± 0.03

0.91 ± 0.02

0.99 ± 0.01

0.96 ± 0.02

0.84 ± 0.09

0.80 ± 0.07

0.70 ± 0.07

-8.7

-7.7

-7

83.53

111.22

83.53

111.22

83.53

111.22

83.53

63.95

6.66

6.74

6.90

6.95

6.67

6.71

6.91

6.89

6.81

6.80

6.90

4.50

20

21

a

b

a

c

22

-7.4

-8

10

23

24

a

b

a

c

-8.3

-7.3

-7.5

-8.1

-7.2

-6.7

-6.9

-7.8

25

a

b

a

c

26

10

27

28

V^g

VI^g

a

b

c

a

Verapamil^h

b

^aConcentration of the inhibitor that causes a 50% increase in nuclear concentration of pirarubicin (α = 0.5). Efficacy

of MDR-modulator and maximum increase that can be obtained in the nuclear concentration of pirarubicin in resistant

cells. Results are expressed as the mean ± SE of three independent experiments done at least three times. ^cPredicted

binding affinity. ^dPredicted topological polar surface area (TPSA). ^eThe n-octanol/water partition coefficient (log P_o/w

)

was obtained by using implicit log P method (iLOGP) ^fSee within ref. [24]: compounds I, II, III and IV are labelled as

13, 12, 16, and 15 respectively. ^gSee within ref. [18]: compounds V and VI are labelled as 11 and 12 respectively. ^hsee

ref. [24].

3.2. Molecular modeling studies

In order to elucidate the binding mode of the compounds in the P-gp interaction site, and possibly to

explain their remarkable inhibitory activity, an in silico study was performed. Therefore, since the

computational analysis provides a reliable structural basis to undertake structural investigations of

drug binding, we chose to study the capacity of the compounds to interact with P-gp using in silico

docking techniques. To validate and strengthen the computational modelling approach [32,33], we

first performed docking calculations with all our compounds on the basis of their in vitro activity.

Briefly, using Autodock Vina XB scoring function [34], we found, for all the compounds, the

binding energy for the best docking poses, which were all located within the inner chamber of the

protein. The good correlation between in vitro and in silico calculations (Figure S33,

Supplementary Data) provided confidence for using our modelling strategy to investigate putative

interactions with P-gp. The crystal structure of human P-gp (PDB code 6C0V) was used as

Molecular docking target [35], while, to identify the potential binding pocket of our compounds, we

referred to the 3D structure of Mus musculus P-gp in complex with BDE-100 (PDB code 4XWK)

[36,37]. As a matter of fact, this compound showed to be a P-gp inhibitor with a IC₅₀of 23.2±2.9

µM [37]. Compounds were clustered into sets “1” and “2”, based on the presence (set 1) or absence

(set 2) of the anthracene group. The ligand binding mode was visually analyzed: all compounds

docked with similar poses (Figure 1), showing interactions that appeared similar to those

established by verapamil with the binding site residues. The computation of docking energies shows

that all molecules can interact with the binding site, displaying a good thermodynamic affinity.

Nevertheless, some differences can be highlighted, depending on the aromatic residues and the total

length of the spacer, which modify the binding profiles. The docking results showed that the more

active compounds, which show a calculated binding free energy (∆G value) smaller than -7.8, are

decorated in most cases by the anthracene ester group; in addition, the compounds characterized by

a linker of 10 methylenes never exhibit ∆G higher than -7.

Also, the analyses of predicted lipophilicity (logP) and of the topological polar surface area (TPSA),

by using SwissADME [38] were performed. The parameter TPSA is defined as the sum of surfaces

of polar atoms in the molecule and is useful to identify the druggability of ligands, since it can be a

measure of the ability of the compounds to permeate cells. Molecules with a TPSA value greater

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ACCEPTED MANUSCRIPT

than 140 Å² tend to be poor in permeation of cell membranes and are considered poor drug-like

compounds [39].

These parameters may explain the differences between [I]_0.5and efficacy values against ∆G, in

particular as regard the standard verapamil. In fact, comparing the new synthesized compounds with

verapamil, it can be observed how, despite a similar ∆G value, verapamil displays lower potency

and efficacy with respect to the derivatives described in the paper. This behavior could be related to

its lower lipophilicity and permeability.

Fig. 1. Overview of the best poses of compounds 1-28 within the P-gp binding pocket. A) The P-gp 3D

structure is shown in grey cartoon and surface representation. The bilayer is reported in yellow surface.

BDE100 (red), Verapamil (black) and the studied ligands (coloured) are shown inside the P-gp binding

pocket with stick models. B) and C) show a closer view of the binding mode of set “1” and set “2”,

respectively.

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To gain more information on the remarkable inhibitory activity of the new compounds, we also

carried out a ligand-binding-site interaction network analyses on derivatives 17 and 23, which are

two very potent compounds representative of the two clusters. In fact, it could be interesting to

evaluate which type of interactions could be established between the protein and these active

compounds, identifying the involved residues, and making a comparison with those formed by other

well-known inhibitors.

Crystal structure of the mouse P-gp complexed with BDE-100 compound (PDB code 4XWK), was

used as reference point to study protein binding interactions [34]. BDE-100 forms hydrophobic

interactions with different aromatic residues but only one π-stacking bond with Tyr-303; this is

enough for its inhibitory capacity. Additionally, in a recent work a P-gp modulator was showed to

bind two sites of internal protein cavity also identified in co-crystal structures in both the inward-

open and the inward-closed transporter [40], showing the same binding region of our docking

results.

Figure 2 shows the 3D molecular docking models of the interaction between P-gp and the standard

verapamil (pointed up in panel B), and the very active compounds 23 and 17, which are

representative of cluster “1” and “2”, respectively (panel C and D). Verapamil docked into the

human P-gp (PDB code 6C0V) [35], is surrounded by several lipophilic or aromatic residues, but as

well as BDE-100, is able to form only one π-stacking interaction. On the contrary, compounds 17

and 23 can form a higher number of interactions with different residues of the binding site: the

presence of long polymethylenic chains allows numerous hydrophobic interactions within the

binding pocket, in addition to the capability to form stronger aromatic interactions compared to

BDE-100 and verapamil. For instance, for 23 the presence of the anthracene group favors the

formation of three π-stacking bonds between the three rings of the aromatic moiety and Phe-335,

Phe-728 and Phe-759 residues of the P-gp binding site, while both BDE-100 and verapamil can

form only one π-stacking interaction. As regard compound 17, the higher number of hydrophobic

interactions of the spacers, compared to verapamil, stabilizes its binding into the active pocket

despite the formation of only one π-stacking bond.

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Fig. 2: 3D Molecular docking models of the interaction between compounds and P-gp. A) The P-gp 3D

structure is shown in grey cartoon and the bilayer is reported as green surface. B) Verapamil in yellow, C)

Compound 23 in purple, D) Compound 17 in blue. For clarity only interacting residues are displayed, with

hydrophobic residues in green and π-stacking in orange respectively.

In summary, the docking results supported the data obtained by the biological tests: all molecules

are able to interact with the binding site with a good thermodynamic affinity: the differences in the

activity can be related to the ∆G values. The good activity of the compounds bearing an anthracene

ester group can be partially explained by the higher number of π-stacking bonds that these

molecules are able to establish with the protein.

3.3. Chemical stability tests

The chemical stability tests on these compounds were carried out both in phosphate buffer solution

(PBS) and in human plasma, to distinguish between spontaneous or enzymatic hydrolysis

respectively.

The stability analyses were performed by LC-MS/MS methods operating in product ion scan mode,

in the appropriate m/z range to ensure the fragment ions detection. The LC-MS/MS system and the

parameters used in this study were reported in the Supplementary Data section.

The stability of each compound, in both reported matrices, was evaluated by monitoring the

variation of its concentration at different incubation times. By plotting these data (analyte

concentrations vs the incubation time) their respective degradation profiles were obtained.

Generally, when the substrate concentration is smaller than its Michaelis–Menten constant (K_M), the

enzymatic degradation rate is described by a first-order kinetic.

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Therefore, plotting the natural logarithm of the quantitative data versus the incubation time, a linear

function can be used, and its slope represents the degradation rate constant (k). Therefore, the half-

life (t_1/2) of each tested compound can be calculated as follows:

ln(0.50µM )

t_{1/ 2}

=

k

The plots of the natural logarithm of the quantitative data versus the incubation time of all the

studied compounds were analysed. The obtained results demonstrated that all the compounds were

stable in PBS and most of them also in human plasma. In fact, only the degradation plots of II, 10,

18 and 22 in human plasma showed a significant decay rate (k value), and their calculated half-life

values (t_1/2values between 39 min. and 123 min), were reported in Table 2. Furthermore, the half-

life value of ketoprofene ethylester (KEE), used as reference compound, demonstrated that the

employed human batch was enzymatically active (half-life < 2 h) [30]. At the contrary, the k values

of the other studied compounds were close to 0; consequently, for these derivatives, extremely high

t

_1/2values can be calculated. Since under the proposed experimental conditions a half-life over 240

min is not measurable, it is reasonable to consider that their half-life values could be equal or

greater than 240 min. The human plasma degradation profiles of compound 22 and of its stable

isomer 21 are reported as an example (Figure 3); the plots of the other compounds are reported in

the Supplementary Data section.

Table 2

Half-life values of reference and studied compounds.

PBS

t _1/2± error

(min)

Human plasma

t _1/2± error

(min)

>240

123 ± 53

>240

45 ± 13

>240

41 ± 19

>240

15

16

17

18

19

20

21

22

23

24

25

26

27

28

I

Comp.

KEE

1

n.d.

107 ± 16

>240

39 ± 6

>240

2

3

4

5

6

7

8

9

10

11

12

13

14

II

V

n.d.: not determined

13

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Initial Concentration

PBS

H-pl

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

-3.5

-4

-5

0

5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125

Time (min.)

1

0.5

0

-0.5

-1

-1.5

-2

-2.5

-3

-3.5

-4

-5

0

5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125

Time (min.)

Fig. 3: Degradation profiles in PBS (blue) and human plasma (red) of compound 21 (top) and compound 22

(bottom).

The degradation products of II, 10, 18 and 22 compounds were also investigated to establish the

possible enzyme hydrolytic mechanism. The results confirmed that these compounds were degraded

for hydrolysis of the ester group linked to the trans-3,4,5-trimethoxycinnamyl moiety, with

formation of the corresponding N-alkyl alcohol and of the free trimethoxycinnamic acid. As an

example, the chromatograms of compound 22 in human plasma at two incubation times (0 and 120

min respectively) is reported in Figure 4.

14

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Chromatogram Plots

kCounts

T=0 min.

1.50

1.25

1.00

0.75

0.50

0.25

Parent compound

0.00

kCounts

1.50

T=120 min.

1.25

1.00

0.75

0.50

0.25

0.00

N-Alkyl alcohol

Parent compound

minutes

2.0

2.5

3.0

3.5

4.0

Fig. 4: LC-MS/MS chromatographic profiles of compound 22 in human plasma at the initial (up) and final

(bottom) incubation time.

Interestingly, the hydrolysis occurs only when the trans-3,4,5-trimethoxycinnamic ester is

combined with the 3,4,5-trimethoxybenzoic moiety, while the combination with the anthracene

group prevents the enzyme activity (i.e. anthracene analogues 24 and/or 12). Furthermore,

hydrolysis occurs only when the N-alkyl chain length of the trans-3,4,5-trimethoxycinnamyl portion

is of three methylenes: different values prevent the hydrolysis (i.e. 26 and/or V). Only 18, bearing a

N-alkyl chain length of four methylenes in the cinnamyl portion, shows an appraisable degradation,

but its k value is three times lower with respect to the analogues carrying a chain with three

methylene units. Compound

6, characterized by the same structural feature of 18, is instead

completely stable in human plasma, indicating that the chain composed by 4 methylenes confers a

higher stability.

3.4 Inhibition of P-gp-mediated rhodamine-123 (Rhd 123) efflux on K562/DOX cells

Based on the interesting results obtained, the effect of the most active compounds was further

evaluated on the P-gp function. For this test, all the compounds which presented a total spacer of 10

methylenes, apart from the hydrolysable derivatives 18 and 22, were selected. The ability of

compounds 17, 19-21, and 23-28 to inhibit the transport activity of the pump was measured

evaluating the P-gp specific substrate rhodamine-123 intracellular accumulation on the resistant

K562/DOX cell lines as described in the Experimental Section. Cells were loaded with rhodamine-

123 for 30 min; after this accumulation period, the efflux of the fluorochrome was evaluated after

10 min more, both in the presence and in the absence of modulators tested at 1.0 µM concentration.

The intracellular accumulation of Rhd 123 was also evaluated on the parental line K562

(Experimental Section). Because of the low level of P-gp present in this cell line, the rhodamine-

123 accumulated in the uptake phase was completely retained by the cell after 15 and 30 min of

efflux time (Figure S36 Supplementary Data).Therefore, the inhibiting activity of the selected

compounds was not evaluated on the parental cell line.

The inhibition of P-gp-mediated Rhd 123 efflux of compounds 17

cells is reported in Figure 5. In Figure 5A, the effect of Rhd 123 alone and of the compounds added

at 1.0 M concentrations is reported, expressed as fluorescence ratio value, that is the ratio between

, 19-21, and 23-28 on K562/DOX

µ

median fluorescence intensity value acquired at the end of the efflux phase, and that acquired at the

15

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end of the uptake phase. The values were expressed as percentage. The calculated ratios are

reported in the Supplementary Data (Table S4). Verapamil, tested at 3.0 µM concentration, was

used as reference compound. In Figure 5B, some typical fluorescence curves are reported, obtained

in the absence (panel a) or in the presence of verapamil (panel b) or of two representative

compounds, the potent isomers 19 and 20 (panels c-d). The fluorescence curves of the other tested

compounds are reported in the Supplementary Data (Figure S37).

A

100

80

60

40

20

0

Rhd

Cpd

+ + +

+ + + + + + + + +

-

panel a

RHD

panel b

B

120

90

60

30

0

120

90

60

30

0

RHD + Ver

Cellular autofluorescence

RHD Uptake

RHD Efflux

10¹10²10³10⁴10⁵10⁶10¹10²10³10⁴

10⁶

10⁵

Fluorescence channels

panel c

panel d

RHD + comp. 20

RHD + comp. 19

120

90

60

30

0

120

90

60

30

0

10⁵

Fluorescence channels

10⁴

10⁶

10¹10²10³

10¹10²

10⁴

10⁵10⁶

10³

Fluorescence channels

Fig. 5. Inhibition of P-gp-mediated Rhd 123 efflux of compounds 17, 19-21 and 23-28 on K562/DOX cells.

(A) Fluorescence ratio values in K562/DOX cells incubated with Rhd 123 in the absence (control) and in the

presence of compounds 17, 19-21 and 23-28 tested at 1.0 µM concentration. The efflux of the fluorochrome

was evaluated after 10 min as reported in Experimental Section. The data were expressed as the mean ± SE

of three independent experiments done at least three times. The figure also shows the reference compound,

verapamil tested at 3.0 µM concentration. (B) The fluorescence curves were calculated after 30 min Rhd 123

uptake and 10 min of efflux in Rhd 123-free medium. Each peak is identified by a fluorescence channel

number and designates the amount of intracellular Rhd 123 in each sample in the absence and in presence of

the tested compounds. Autofluorescence histogram (black line): cells incubated in media alone. Uptake

16

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histogram (red line): cells incubated 30 minutes in media with 5 µM rhodamine-123. Efflux histogram (blue

line): cells incubated 30 minutes in media with 5 µM rhodamine-123 in the absence (panel a) or in the

presence of verapamil (panel b), compound 19 (panel c), or 20 (panel d), then washed and incubated in

rhodamine-free media in the absence (panel a) and in presence of compounds (panels b-d) for 10 minutes.

The fluorescence ratio values showed that all the compounds were able to inhibit the efflux of Rhd

123, although to different extent. The most potent compounds were in order 19

, 24, 20, 21, 26, 25;

derivatives 23 27 17 and 28 were slightly more or equally active with respect to verapamil,

,

although tested at lower concentration than the reference (1 and 3 µM, respectively).

In particular, the anthracene derivatives 19 and 24 showed an outstanding activity, and were able to

maintain a high Rhd 123 intracellular fluorescence, showing an efflux of the fluorochrome of just

7.2% and 15.3% respectively; 20 the regioisomer of 19, and the 3,4,5-trimethoxybenzoic derivative

21 were able to maintain 64.6% and 58.4% of the intracellular fluorescence after 10 minutes of

efflux, still showing a considerable ability in inhibiting the efflux of Rhd123.

The same result is highlighted by analyzing the fluorescence curves. The treatment with the

regioisomers 19 and 20 caused a Rhd 123 intracellular retention higher than the untreated sample

(panel a), as evidenced by the blue curves overlaid on the red ones of the uptake. These derivatives

were able to retain almost all the Rhd 123 in the cells showing a P-gp inhibiting activity much

higher than verapamil.

3.5 Transfected MDCK cells: characterization of P-gp interacting mechanism and selectivity vs the

other MDR transporters

The effect of the most active compounds on K562/DOX cells, already selected for the inhibition of

the P-gp-mediated rhodamine-123 efflux test, was further evaluated. The potency of compounds 17

,

19-21, and 23-28 was tested on a different cell line overexpressing P-gp, the Madin-Darby Canine

Kidney-MDR1 (MDCK-MDR1) cells, measuring their inhibiting activity of the transport of a

profluorescent probe, Calcein-AM, that is a P-gp substrate (Table 3).

The P-gp interacting profile was also investigated combining other two biological assays: the

apparent permeability (P_app) determination in Caco-2 cell monolayer, and the ATP cell depletion in

MDCK-MDR1 cells [41]. The first assay measures the ratio between two fluxes: 1) BA,

representative of passive diffusion from the basolateral to apical compartments; 2) AB,

representative of active transport, from the apical to basolateral compartments. If the ratio BA/AB is

< 2, the compound is classified as an inhibitor (also considering the results of the ATPase activity).

If the ratio is > 2, the compound is classified as a substrate.

The detection of the ATP consumption in cells overexpressing P-gp measures the transport of the

tested compound. Only a P-gp unambiguous substrate, as transported, induces a depletion of ATP

while a P-gp inhibitor, as not transported, does not induce ATP consumption. Moreover, another

substrate category, known as category IIB₃, displaying a P_appvalue > 2 but not inducing an ATP cell

depletion is also reported [42]

.

At last, the activity of the compounds vs two other sister proteins, MRP1 and BCRP, was tested

(Table 3) by evaluating the ability of the ligands to inhibit the transport of Calcein-AM (MRP1

substrate) in cells overexpressing MRP1 (MDCK-MRP1) and of the fluorescent probe Hoechst

33342 (BCRP substrate) in cells overexpressing BCRP (MDCK-BCRP cells).

Expression levels of P-gp, MRP1 and BCRP were periodically analyzed by immunoblotting

analysis in MDCK-MDR1, MDCK-MRP1 and MDCK-BCRP cells respectively, as described in the

Experimental Section. Tubulin was used as control of equal protein loading. A representative

western blot analysis is reported in the Supplementary Data (Figure S38).

17

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Table 3

Biological evaluation of derivatives 17

,

19-21,23-28: inhibition activity on MDCK-MDR1, MDCK-

MRP1 and MDCK-BCRP cells overexpressing each transporter, ATP cell depletion in MDCK-

MDR1 and apparent permeability (P_app) determination (BA/AB) in Caco-2 cell monolayer.

EC₅₀µM^a

ATP cell

depletion

b

Compd

17

19

20

21

23

24

25

P-gp

MRP1

BCRP

P_app

0.20±0.03 5.08±1.01 16.0±2.9

0.52±0.10

0.85±0.17

0.21±0.11 3.0±0.60

0.23±0.05

0.26±0.05

0.30±0.10 4.5±0.89

0.22±0.04 2.2±0.42

0.34±0.07

0.92±0.20

0.50±0.10

Y

N

Y

N

Y^c

7.5

11

NA

3.8±0.72

3.3±0.55

8.1±1.59

3.9±0.78

3.0±0.49

3.8±0.70

4.5±0.87

5.4±1.06

5.1±1.02

NT

27

5.2

11

NA

25

7.1

6.8

14.4

4.6

18

26

27

28

NA

6.8±3.0

verapamil

a

b

Values are the mean ±SEM of two independent experiments, with samples in triplicate. Apparent

c

permeability estimation: values are from two independent experiments, with samples in duplicate.

Percentage of the effect at a concentration of 1 µM (20%). NA = not active. NT = not tested.

As shown in Table 3, on MDCK-MDR1 cells the compounds displayed good activities vs P-gp

(EC₅₀ranging from 0.20 µM to 0.92 µM). More in details, compounds 17, 23, 24 and 26 showed the

higher potency (EC₅₀=0.20 µM, 0.23 µM, 0.26 µM and 0.22 µM respectively). The trend in potency

is slightly different from that shown in the pirarubicin uptake test, but it is not surprising since the

cell lines are different. However, it is interesting to note that derivatives 17 and 23 were among the

most active compounds also in the pirarubicin uptake assay. Moreover, all the compounds showed a

moderate activity vs BCRP (EC₅₀ranging from 3.0 µM to 16.0 µM); however, in this test they were

at least 10-fold less active with respect to P-gp assay. Finally, only derivatives 17, 21, 25 and 26

showed some activity vs MRP1, while the other ligands were completely inactive. These four

compounds share the same combination of aromatic moieties, namely the (E)-3-(3,4,5-

trimethoxyphenyl)vinyl residue and the 3,4,5-trimethoxyphenyl one; it can be concluded that the

presence of the anthracene moiety is not favorable for the activity on this pump. So, a structural

requirement for the selectivity vs MRP1 could be highlighted.

The apparent permeability determination P_app(BA/AB) in Caco-2 cell monolayer indicated that all

compounds had a BA/AB ratio > 2; thus, considering also the results of the ATP consumption test,

compounds 17 and 20 can be defined unambiguous substrates while all the other compounds were

classified as substrate belonging to category IIB₃, as above reported.

4. Conclusions

In the present study, we designed and synthesized several new N,N-bis(alkanol)amine aryl ester

heterodimers by modulating the combination of aromatic moieties and the length of the methylenic

chain. We achieved all the possible isomers obtained by the combination of different spacers of 2-8

units, for a total length of 8, 9 and 10 methylenes, bearing the aromatic moieties described above.

These derivatives were as first evaluated for their P-gp inhibitory activity on doxorubicin-resistant

erythroleukemia K562 cell line (K562/DOX). The new compounds showed good P-gp modulating

ability in the pirarubicin assay: all molecules showed potency values ([I]_0.5) in the submicromolar or

nanomolar range and in many cases were able to completely reverse P-gp-dependent pirarubicin

extrusion (α_maxclose to 1).

18

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The best results were obtained with the isomers showing a total spacer of 10 methylenes; in fact, all

the compounds showed outstanding potencies in the nanomolar range, regardless of the

combination of aromatic moieties, with unprecedented results in these series of derivatives. In this

group of compounds, the nitrogen position and therefore the distance of this atom from the

combined aromatic residues have not a clear influence on the activity. So, the total distance of the

aromatic esters seems to be the crucial factor for the activity. However, comparing the potencies of

the compounds 17-28 with the activities obtained by two already described symmetric isomers

V

and VI [18] (Table 1), the same result already found in our previous paper [24] was confirmed: the

nitrogen position has not a clear influence on the activity, but if this atom is at the center of the

spacer the activity of the compounds decreases.

To explain the remarkable activity of the new compounds, we carried out a ligand-binding-site

interaction network analyses on all the derivatives. Using a computational approach, we highlighted

that all the molecules can interact with the protein binding site, displaying a good thermodynamic

affinity. Some compounds however showed better binding outlines, characterized by smallest

binding energies (

activity of the compounds bearing an anthracene ester group could be partially explained by the

higher number of -stacking bond that these molecules are able to establish with the protein.

∆G) that showed a certain correlation with the experimental activities. The good

π

Interesting information was obtained from the stability experiments performed on these new series

of MDR inhibitors both in phosphate buffer solution (PBS) and in human plasma. The compounds,

despite the presence of two ester groups, were stable in both the tested matrices. Only four

compounds (II, 10, 18 and 22) exhibited a significant degradation in the human plasma matrix,

showing the hydrolysis of the ester group present on the 3,4,5-trimethoxycinnamyl moiety of the

molecule. Interestingly, these compounds presented a common characteristic in their chemical

structures which reasonably favors the enzymatic activity. In fact, it was observed that the

hydrolysis occurs only in the presence of the 3,4,5-trimethoxyphenyl moiety and when the N-alkyl

chain linked with trans-3,4,5-trimethoxycinnamyl portion has a length of three methylenes.

Therefore, the substitution of aromatic moiety (e.g. in the case of anthracene analogues) or the

modification of the length of the N-alkyl chain, prevents the enzymatic activity.

The compounds which present a total spacer of 10 methylenes, apart from the hydrolysable

derivatives 18 and 22, were selected to further characterize the biological profile of the series. The

chosen molecules were tested on another resistant cell line which overexpresses P-gp, the

transfected MDCK-MDR1 cell line. The obtained results confirmed that our derivatives showed a

good P-gp inhibiting activity also in these cells. To deepen the evaluation of their effect on P-gp

function, three different tests were performed. The ability of the compounds of inhibiting the efflux

of rhodamine-123 was tested at 1 µM concentration on K562/DOX cells; all the derivatives were

able to inhibit the P-gp-mediated efflux with a noteworthy efficacy, which was at least the same of

verapamil tested at 3 µM concentration. Evaluation of apparent permeability on Caco-2 cells and

ATP consumption on MDCK-MDR1 cell line suggested that compounds 17 and 20 behave as

unambiguous substrates, while the other derivatives can be classified as IIB₃substrates.

The selectivity profile of the compounds vs two sister proteins was also analyzed, evaluating their

effect on cells overexpressing MRP1 and BCRP (MDCK-MRP1 and MDCK-BCRP cell lines

respectively). Our derivatives displayed a moderate potency on the BCRP overexpressing cells;

since this efflux transporter BCRP is often co-expressed with MDR1, the presence of some activity

also on this pump could be an interesting property of these series of derivatives. On the contrary,

only four molecules showed to be effective on MDCK-MRP1 cell line, and a clear structural

requirement for the selectivity vs MRP1 could be highlighted.

In conclusion, we have identified a new very powerful series of P-gp-dependent MDR inhibitors

showing a N,N-bis(alkanol)amine aryl ester scaffold. Interestingly, all the compounds carrying a

linker composed by 10 methylenes in different combination showed an excellent pharmacological

profile. Docking studies confirmed that these molecules can interact in a fruitful manner with the P-

gp binding pocket, and evaluation of their chemical stability in PBS and human plasma confirmed

19

ACCEPTED MANUSCRIPT

that these compounds are in most cases stable in both media, allowing us to predict their in vivo

bioavailability. In this series, derivatives bearing the 3,4,5-trimethoxyphenyl moiety appear very

active in inhibiting P-gp both in K562/DOX and in MDCK-MDR1 cell lines, but maintain a

moderate activity on BCRP and MRP1 overexpressing cells; derivatives bearing the anthracene

residue instead show a good profile of activity on P-gp overexpressing cells and on MDCK- BCRP

cell line but do not present any activity on MDCK-MRP1, highlighting interesting differences in

their selectivity profile.

5. Experimental

5.1. Chemistry

All melting points were taken on a Büchi apparatus and are uncorrected. NMR spectra were

1

13

recorded on a Bruker Avance 400 spectrometer (400 MHz for H-NMR, 100 MHz for C-NMR).

Chromatographic separations were performed on a silica gel column by gravity chromatography

(Kieselgel 40, 0.063-0.200 mm; Merck) or flash chromatography (Kieselgel 40, 0.040-0.063 mm;

Merck). Yields are given after purification, unless otherwise stated.

ESI-MS spectra were obtained using a Varian 1200L triple quadrupole system (Palo Alto, CA,

USA) equipped by Elettrospray Source (ESI) operating in both positive and negative ions.

The data were acquired in scan mode between the range 150-800 m/z by introducing the sample

solution, via syringe pump at 10 µL min⁻¹. The sample solution of each analyte was freshly

prepared by diluting its stock solution (1 mg mL^-1in acetonitrile) up to a concentration of 1.0

µg

mL^-1in mixture of mQ water: acetonitrile 50:50 (v/v).

In the used instrumental conditions, the most abundant signal for the analytes showed to be the

protonated ([M+H]⁺) or deprotonated ([M-H]^-) molecule ion species.

Compounds 1-28 were obtained in a purity

≥ 95%. Their combustion analyses are indicated by

symbols, and the analytical results are within ±0.4% of the theoretical values. Compounds were

named following IUPAC rules as applied by ChemBioDraw Ultra 14.0 software. When reactions

were performed in anhydrous conditions, the mixtures were maintained under nitrogen. Free bases

1-28 were transformed into the hydrochloride by treatment with a solution of acetyl chloride (1.1

eq) in anhydrous CH₃OH. The salts were crystallized from abs. ethanol/petroleum ether.

5.1.1. General procedure for the synthesis of haloesters 29-40

A 1 mmol portion of the appropriate carboxylic acid (trans-3-(3,4,5-trimethoxyphenyl)acrylic acid,

3,4,5-trimethoxybenzoic acid or anthracene-9-carboxylic acid) was transformed into the acyl

chloride by reaction with SOCl₂(2 mmol) in 5 mL of CHCl₃(free of ethanol) at 60 °C for 4-5 h.

The reaction mixture was cooled to rt, and the solvent was removed under reduced pressure; the

mixture was then treated twice with cyclohexane and the solvent removed under reduce pressure.

The acyl chloride obtained was dissolved in CHCl₃(free of ethanol), and the suitable alcohol (2-

bromoethan-1-ol, 3-bromopropan-1-ol, 4-chlorobutan-1-ol, 5-chloropentan-1-ol, 6-bromoexan-1-ol,

7-bromeptan-1-ol or 8-bromooctan-1-ol) (0.9 eq) was added. The mixture was heated to 60 °C.

After 4 h, the reaction mixture was cooled to rt, and treated with CH₂Cl₂. The resulting organic

layer was washed with 10% NaOH solution, dried with Na₂SO₄, and the solvent was removed under

reduced pressure. The substances obtained were purified by flash chromatography in the case of 31

,

38 39 and 40, otherwise were used as such for the next reaction. Compounds 29 30 32 33 and 35

,

were already synthesized by our group with the same procedure [18,24].

5.1.1.1. (E)-4-chlorobutyl 3-(3,4,5-trimethoxyphenyl)acrylate 31

1

Pale yellow oil. Chromatographic eluent: cyclohexane/ethyl acetate70:30. Yield: 68.0%. H-NMR

(CDCl₃) : 7.60 (d, J=16.0 Hz, 1H, CH=CH); 6.75 (s, 2H, CH arom.); 6.33 (d, J=16.0 Hz, 1H,

δ

CH=CH); 4.24 (t, J=6.0 Hz, 2H, CH₂O); 3.89 (s, 6H, OCH₃); 3.88 (s, 3H, OCH₃); 3.60 (t, J=6.0 Hz,

2H, CH₂Cl); 1.98-1.82 (m, 4H, CH₂) ppm. ¹³C APT NMR (CDCl₃) δ: 153.46 (C=O); 144.90

20

ACCEPTED MANUSCRIPT

(CH=CH); 129.85 (C); 117.17 (CH=CH); 105.29 (CH arom.); 63.67 (CH₂); 60.97 (OCH₃); 56.18

(OCH₃); 44.50 (CH₂); 29.23 (CH₂); 26.21 (CH₂) ppm.

5.1.1.2. 6-chlorohexyl anthracene-9-carboxylate 34

1

Yellow oil. Yield: 87.1 %. H-NMR (CDCl₃)

δ: 8.52 (s, 1H, CH arom.); 8.15-7.97 (m, 4H, CH

arom.); 7.62-7.42 (m, 4H, CH arom.); 4.63 (t, J=6.8 Hz, 2H, CH₂O); 3.54 (t, J=4.8 Hz, 2H, CH₂Cl);

1.96-1.85 (m, 2H, CH₂); 1.85-1.76 (m 2H, CH₂); 1.63-1.46 (m, 4H, CH₂) ppm.

5.1.1.3. 7-bromoheptyl 3,4,5-trimethoxybenzoate 36

1

Pale yellow oil. Yield: 37.8%. H-NMR (CDCl₃)

δ: 7.28 (s, 2H, CH arom.); 4.29 (t, J=6.8 Hz, 2H,

CH₂O); 3.90 (s, 6H, OCH₃); 3.89 (s, 3H, OCH₃); 3.39 (t, J=6.8 Hz, 2H, CH₂Br); 1.90-1.81 (m, 2H,

CH₂); 1.80-1.70 (m, 2H, CH₂); 1.50-1.32 (m, 6H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.22

(C=O); 152.91 (C); 142.16 (C); 125.46 (C); 106.79 (CH arom.); 62.21 (CH₂); 60.88 (OCH₃); 56.24

(OCH₃); 33.80 (CH₂); 32.65 (CH₂); 28.63 (CH₂); 28.38 (CH₂); 28.02 (CH₂); 25.82 (CH₂) ppm.

5.1.1.4. 7-bromoheptyl anthracene-9-carboxylate 37

1

Orange oil. Yield: 41.2%. H-NMR (CDCl₃)

δ: 8.49 (s, 1H, CH arom.); 8.08 (d, J=8.4 Hz, 2H, CH

arom.); 8.05 (d, J=8.4 Hz, 2H, CH arom.); 7.60-7.40 (m, 4H, CH arom.); 4.62 (t, J=6.4 Hz, 2H,

13

CH₂O); 3.38 (t, J=6.8 Hz, 2H, CH₂Br); 1.92-1.78 (m, 4H, CH₂); 1.60-1.30 (m, 6H, CH₂) ppm. C

APT NMR (CDCl₃) δ: 169.74 (C=O); 131.02 (C); 129.26 (CH arom.); 128.66 (CH arom.); 128.41

(C); 129.96 (CH arom.); 125.48 (CH arom.); 125.02 (CH arom.); 65.81 (CH₂); 33.88 (CH₂); 32.66

(CH₂); 29.13 (CH₂); 28.77 (CH₂); 28.70 (CH₂); 26.06 (CH₂) ppm.

5.1.1.5. (E)-8-bromooctyl 3-(3,4,5-trimethoxyphenyl)acrylate 38

Yellow oil. Chromatographic eluent: cyclohexane/ethyl acetate70:30. Yield: 40.2%. H-NMR

1

(CDCl₃) δ: 7.55 (d, J=16.0 Hz, 1H, CH=CH); 6.72 (s, 2H, CH arom.); 6.31 (d, J=16.0 Hz, 1H,

CH=CH); 4.16 (t, J=6.8 Hz, 2H, CH₂O); 3.85 (s, 6H, OCH₃); 3.84 (s, 3H, OCH₃); 3.37 (t, J=6.8 Hz,

2H, CH₂Br); 1.90-1.81 (m, 2H, CH₂); 1.76-1.65 (m, 2H, CH₂); 1.44-1.24 (m, 8H, CH₂) ppm. ¹³C

APT NMR (CDCl₃) δ: 167.07 (C=O); 153.43 (C); 144.61 (CH=CH); 129.96 (C); 117.48 (CH=CH);

105.20 (CH arom.); 64.64 (CH₂); 60.98 (OCH₃); 56.16 (OCH₃); 33.98 (CH₂); 32.74 (CH₂); 29.07

(CH₂); 28.70 (CH₂); 28.63 (CH₂); 28.05 (CH₂); 25.87 (CH₂) ppm. ESI-MS m/z (%): 429 (100)

[M+H]⁺.

5.1.1.6. 8-bromooctyl 3,4,5-trimethoxybenzoate 39

Pale yellow oil. Chromatographic eluent: cyclohexane/ethyl acetate 80:20. Yield: 37.3%. H-NMR

1

(CDCl₃) δ: 7.27 (s, 2H, CH arom.); 4.28 (t, J=6.8 Hz, 2H, CH₂O,); 3.88 (s, 6H, OCH₃); 3.87 (s, 3H,

OCH₃); 3.37 (t, J=6.4 Hz, 2H, CH₂Br); 1.86-1.80 (m, 2H, CH₂); 1.78-1.69 (m, 2H, CH₂); 1.43-1.22

(m, 8H, CH₂) ppm.

5.1.1.7. 8-bromooctyl anthracene-9-carboxylate 40

Yellow oil. Chromatographic eluent: cyclohexane/ethyl acetate 80:20. Yield:79.6%. H-NMR

1

(CDCl₃) δ: 8.50 (s, 1H, CH arom.); 8.07 (d, J=8.8 Hz, 2H, CH arom.); 8.00 (d, J=8.4 Hz, 2H, CH

arom.,); 7.57-7.48 (m, 4H, CH arom.); 4.63 (t, J=6.8 Hz, 2H, CH₂O); 3.38 (t, J=4.8 Hz, 2H,

13

CH₂Br); 1.95-1.77 (m, 4H, CH₂); 1.47-1.38 (m, 8H, CH₂) ppm. C APT NMR (CDCl₃) δ: 169.78

(C=O); 131.03 (C); 129.25 (CH arom.); 128.66 (CH arom.); 128.41 (C); 126.95 (CH arom.); 125.49

(CH arom.); 125.04 (CH arom.); 70.92 (CH₂); 65.91 (CH₂); 33.99 (CH₂); 32.76 (CH₂); 29.16 (CH₂);

28.66 (CH₂); 28.07 (CH₂); 26.02 (CH₂) ppm.

5.1.2. General procedure for the synthesis of iodoesters 41-44

A 1 mmol portion of the suitable chloroester (31-34) was dissolved in acetone. To this solution NaI

(4 eq) was added, and the resulting mixture was maintained 24 h at reflux in the dark. The reaction

mixture was cooled to room temperature, and the solvent was removed under reduced pressure; the

residue was dissolved in CH₂Cl₂and washed with water. The organic layer was dried with Na₂SO₄,

and the solvent was removed under reduced pressure yielding a yellow oil which was used as such

for the next reaction. Compound 42 was already synthesized by our group with the same procedure

[24].

21

ACCEPTED MANUSCRIPT

5.1.2.1. (E)-4-iodobutyl 3-(3,4,5-trimethoxyphenyl)acrylate 41

Yield: 94.7%. ¹H-NMR (CDCl₃)

δ: 7.57 (d, J=16.0 Hz, 1H, CH=CH); 6.73 (s, 2H, CH arom.); 6.31

(d, J=16.0 Hz, 1H, CH=CH); 4.20 (t, J=6.0 Hz, 2H, CH₂O); 3.86 (s, 6H, OCH₃); 3.85 (s, 3H,

OCH₃); 3.21 (t, J=6.8 Hz, 2H, CH₂I); 1.98-1.87 (m, 2H, CH₂); 1.84-1.74 (m, 2H, CH₂) ppm.

5.1.2.2. (E)-6-iodohexyl 3-(3,4,5-trimethoxyphenyl)acrylate 43

Yield: 95.9%. ¹H-NMR (CDCl₃)

δ: 7.58 (d, J=16.0 Hz, 1H, CH=CH); 6.74 (s, 2H, CH arom.); 6.33

(d, J=15.6 Hz, 1H, CH=CH); 4.19 (t, J=6.8 Hz, 2H, CH₂O); 3.88 (s, 6H, OCH₃); 3.87 (s, 3H,

OCH₃); 3.19 (t, J=6.8 Hz, 2H, CH₂I); 1.91-1.79 (m, 2H, CH₂); 1.86-1.68 (m, 2H, CH₂); 1.50-1.38

(m, 4H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.98 (C=O); 153.44 (C); 144.66 (CH=CH); 140.16

(C); 129.92 (C); 117.40 (CH=CH); 105.28 (CH arom.); 64.61 (CH₂); 60.95 (OCH₃); 56.18 (OCH₃);

33.13 (CH₂); 29.83 (CH₂); 26.54 (CH₂); 25.10 (CH₂); 6.79 (CH₂) ppm.

5.1.2.3. 6-iodohexyl anthracene-9-carboxylate 44

Yield: 90.4%. ¹H-NMR (CDCl₃)

δ: 8.52 (s, 1H, CH arom.); 8.15-7.97 (m, 4H, CH arom.); 7.65-7.42

(m, 4H, CH arom.); 4.63 (t, J=6.4 Hz, 2H, CH₂O); 3.17 (t, J=6.8 Hz, 2H, CH₂I); 1.99-1.73 (m, 4H,

CH₂); 1.60-1.37 (m, 4H, CH₂) ppm.

5.1.3. General procedure for the synthesis of hydroxyaminoesters 45-54

The appropriate haloester (29, 30, 35-37, 41-43) (1 mmol) and the suitable aminoalkylalcohol (3-

aminopropan-1-ol, 4-aminobutan-1-ol, 5-aminopentan-1-ol or 6-aminohexan-1-ol) (2 mmol) were

dissolved in 1 mL of anhydrous CH₃CN. The mixture was heated at 80 °C for 5-10 h. The reaction

mixture was cooled to room temperature, treated with CH₂Cl₂and the organic layer was washed

with 10% NaOH solution. After drying with Na₂SO₄, the solvent was removed under reduced

pressure and the residue purified by flash chromatography, yielding a pale-yellow oil. 52 was used

as such in the next reaction.

5.1.3.1. (E)-2-((6-Hydroxyhexyl)amino)ethyl 3-(3,4,5- trimethoxyphenyl)acrylate 45

1

Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 90:10:1. Yield: 26.3%. H-NMR (CDCl₃)

δ: 7.56

(d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.33 (d, J=16.0 Hz, 1H, CH=CH); 4.27 (t,

J=4.8 Hz, 2H, CH₂OH); 3.85 (s, 6H, OCH₃); 3.83 (s, 3H, OCH₃); 3.61-3.53 (m, 2H, CH₂O); 2.89 (t,

J=4.8 Hz, 2H, NCH₂,); 2.61 (t, J=6.4 Hz, 2H, NCH₂); 1.59-1.41 (m, 4H, CH₂); 1.40-1.23 (m, 4H,

CH₂) ppm. ESI-MS m/z (%): 382 (100) [M+H]⁺.

5.1.3.2. (E)-4-((5-Hydroxypentil)amino)butyl 3-(3,4,5- trimethoxyphenyl)acrylate 46

1

Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 90:10:1. Yield: 35.6%. H-NMR (CDCl₃)

δ: 7.53

(d, J=15.6 Hz, 1H, CH=CH); 6.70 (s, 2H, CH arom.); 6.29 (d, J=15.6 Hz, 1H, CH=CH); 4.14 (t,

J=6.8 Hz, 2H, CH₂O); 3.83 (s, 6H, OCH₃); 3.82 (s, 3H, OCH₃); 3.56 (t, J=6.4 Hz, 2H, CH₂OH);

3.21 (bs, 1H, OH); 2.64-2.57 (m, 4H, NCH₂); 1.78-1.68 (m, 2H, CH₂); 1.68-1.42 (m, 6H, CH₂);

1.42-1.32 (m, 2H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.95 (C=O); 153.38 (C); 144.63

(CH=CH); 140.05 (C); 129.87 (C); 117.37 (CH=CH); 105.19 (CH arom.); 64.26 (CH₂); 62.19

(CH₂); 60.91 (OCH₃); 56.12 (OCH₃); 49.52 (CH₂); 49.26 (CH₂); 29.53 (CH₂); 29.06 (CH₂); 28.57

(CH₂); 28.24 (CH₂); 26.55 (CH₂) ppm.

5.1.3.3. (E)-5-((4-Hydroxybutyl)amino)pentyl 3-(3,4,5- trimethoxyphenyl)acrylate 47

Chromatographic eluent: abs. ethanol/ CH₂Cl₂/petroleum ether/NH₄OH 65:340:60:8. Yield: 72.5%.

¹H-NMR (CDCl₃)

δ: 7.53 (d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.29 (d, J=16.0 Hz,

1H, CH=CH); 4.13 (t, J=6.8 Hz, 2H, CH₂O); 3.83 (s, 6H, OCH₃); 3.82 (s, 3H, OCH₃); 3.51 (t, J=5.0

Hz, 2H, CH₂OH); 3.44 (bs, 1H, OH); 2.61-2.56 (m, 4H, NCH₂); 1.72-1.48 (m, 8H, CH₂); 1.49-1.32

(m, 2H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.98 (C=O); 153.38 (C); 144.61 (CH=CH); 140.04

(C); 129.91 (C); 117.39 (CH=CH); 105.21 (CH arom.); 64.36 (CH₂); 62.44 (CH₂); 60.90 (OCH₃);

56.12 (OCH₃); 49.64 (CH₂); 49.32 (CH₂); 32.39 (CH₂); 29.31 (CH₂); 26.59 (CH₂); 26.44 (CH₂);

23.73 (CH₂) ppm.

5.1.3.4. (E)-3-((6-Hydroxyhexyll)amino)propyl 3-(3,4,5-trimethoxyphenyl)acrylate 48

22

ACCEPTED MANUSCRIPT

Chromatographic eluent: abs. ethanol/ CH₂Cl₂/petroleum ether/NH₄OH 65:340:60:8. Yield: 51.3%.

¹H-NMR (CDCl₃)

δ: 7.60 (d, J=16.0 Hz, 1H, CH=CH); 6.74 (s, 2H, CH arom.); 6.32 (d, J=16.0 Hz,

1H, CH=CH); 4.26 (t, J=6.0 Hz, 2H, CH₂O); 3.87 (s, 6H, OCH₃); 3.86 (s, 3H, OCH₃); 3.61 (t, J=6.8

Hz, 2H, CH₂OH); 2.72 (t, J=7.2 Hz, 2H, NCH₂); 2.61 (t, J=7.2 Hz, 2H, NCH₂); 1.96-1.85 (m, 2H,

CH₂); 1.83 (bs, 1H, NH); 1.61-1.58 (m, 4H, CH₂); 1.40-1.30 (m, 4H, CH₂) ppm. ¹³C APT NMR

(CDCl₃) δ: 166.99 (C=O); 153.43 (C); 144.81 (CH=CH); 140.16 (C); 129.87 (C); 117.25 (CH=CH);

105.26 (CH arom.); 62.80 (CH₂); 62.68 (CH₂); 60.96 (OCH₃); 56.17 (OCH₃); 49.81 (CH₂); 46.61

(CH₂); 32.65 (CH₂); 29.88 (CH₂); 29.22 (CH₂); 27.04 (CH₂); 25.62 (CH₂) ppm.

5.1.3.5. (E)-6-((3-Hydroxypropyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate 49

Chromatographic eluent: abs. ethanol/ CH₂Cl₂/petroleum ether/NH₄OH 65:340:60:8. Yield: 80.3%.

¹H-NMR (CDCl₃)

δ: 7.50 (d, J=15.6 Hz, 1H, CH=CH); 6.78 (s, 2H, CH arom.); 6.27 (d, J=15.6 Hz

1H, CH=CH); 4.11 (t, J=6.8 Hz, 2H, CH₂O); 3.80 (s, 6H, OCH₃); 3.79 (s, 3H, OCH₃); 3.69 (t, J=5.6

Hz, 2H, CH₂OH); 3.51 (bs, 2H, NH+OH); 2.79 (t, J=6.0 Hz, 2H, NCH₂); 2.56 (t, J=6.8 Hz, 2H,

13

NCH₂); 1.69-1.57 (m, 4H, CH₂); 1.52-1.41 (m, 2H, CH₂); 1.40-1.22 (m, 4H, CH₂) ppm. C APT

NMR (CDCl₃) δ: 166.98 (C=O); 153.35 (C); 144.57 (CH=CH); 140.01 (C); 129.89 (C); 117.37

(CH=CH); 105.20 (CH arom.); 64.43 (CH₂); 63.48 (CH₂); 60.86 (OCH₃); 56.10 (OCH₃); 49.49

(CH₂); 49.37 (CH₂); 30.48 (CH₂); 29.42 (CH₂); 28.61 (CH₂); 28.81 (CH₂); 25.78 (CH₂) ppm.

5.1.3.6. (E)-4-((6-Hydroxyhexyl)amino)butyl 3-(3,4,5-trimethoxyphenyl)acrylate 50

1

Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 90:10:1. Yield: 57.4%. H-NMR (CDCl₃)

δ: 7.50

(d, J=16.0 Hz, 1H, CH=CH); 6.67 (s, 2H, CH arom.); 6.25 (d, J=16.0 Hz, 1H, CH=CH); 4.13 (t,

J=6.8 Hz, 2H, CH₂O); 3.80 (s, 6H, OCH₃); 3.78 (s, 3H, OCH₃); 3.50 (t, J=6.4 Hz, 2H, CH₂OH);

2.57 (t, J=6.8 Hz, 2H, NCH₂); 2.53 (t, J=7.2 Hz, 2H, NCH₂); 2.45 (bs, 2H, OH+NH); 1.71-1.60 (m,

2H, CH₂); 1.59-1.49 (m, 2H, CH₂); 1.58-1.45 (m, 4H, CH₂); 1.33-1.20 (m, 4H, CH₂) ppm. ¹³C APT

NMR (CDCl₃) δ: 166.92 (C=O); 153.35 (C); 144.63 (CH=CH); 140.04 (C); 129.85 (C); 117.29

(CH=CH); 105.20 (CH arom.); 64.28 (CH₂); 62.24 (CH₂); 60.86 (OCH₃); 56.09 (OCH₃); 49.74

(CH₂); 49.40 (CH₂); 32.64 (CH₂); 29.75 (CH₂); 27.05 (CH₂); 26.55 (CH₂); 26.31 (CH₂); 25.67

(CH₂) ppm.

5.1.3.7. (E)-6-((4-Hydroxybutyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate 51

Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 95:5:0.5. Yield: 70.3%. ¹H-NMR (CDCl₃)

δ: 7.58

(d, J=15.6 Hz, 1H, CH=CH); 6.75 (s, 2H, CH arom.); 6.34 (d, J=15.6 Hz, 1H, CH=CH); 4.19 (t,

J=6.4 Hz, 2H, CH₂O); 3.89 (s, 6H, OCH₃); 3.87 (s, 3H, OCH₃); 3.57 (t, J=6.4 Hz, 2H, CH₂OH);

2.68-2.58 (m, 4H, NCH₂); 1.72-1.58 (m, 6H, CH₂); 1.60-1.51 (m, 2H, CH₂); 1.47-1.32 (m, 4H,

CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 167.27 (C=O); 153.44 (C); 144.61 (CH=CH); 129.96 (C);

117.48 (CH=CH); 105.27 (CH arom.); 64.52 (CH₂); 62.58 (CH₂); 60.97 (OCH₃); 56.18 (OCH₃);

49.66 (CH₂); 49.39 (CH₂); 32.60 (CH₂); 29.53 (CH₂); 28.83 (CH₂); 28.66 (CH₂); 26.93 (CH₂);

25.85 (CH₂) ppm.

5.1.3.8. (E)-6-((3-Hydroxypropyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate 52

Yield: 72.3%. ¹H-NMR (CDCl₃)

δ: 7.53 (d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.29

(d, J=16.0 Hz, 1H, CH=CH); 4.14 (t, J=6.8 Hz, 2H, CH₂O); 3.83 (s, 6H, OCH₃); 3.82 (s, 3H,

OCH₃); 3.73 (t, J=5.6 Hz, 2H, CH₂OH); 2.91 (bs, 2H, NH+OH); 2.80 (t, J=6.0 Hz, 2H, NCH₂); 2.55

(t, J=7.2 Hz, 2H, NCH₂); 1.72-1.62 (m, 4H, CH₂); 1.50-1.38 (m, 2H, CH₂); 1.38-1.25 (m, 6H, CH₂)

13

ppm. C APT NMR (CDCl₃)

δ: 167.05 (C=O); 153.42 (C); 144.58 (CH=CH); 140.10 (C); 129.95

(C); 117.48 (CH=CH); 105.27 (CH arom.); 64.61 (CH₂); 64.19 (CH₂); 60.93 (OCH₃); 56.16

(OCH₃); 49.87 (CH₂); 49.71 (CH₂); 30.56 (CH₂); 29.74 (CH₂); 29.12 (CH₂); 28.68 (CH₂); 27.12

(CH₂); 25.88 (CH₂) ppm.

5.1.3.9. 6-((3-Hydroxypropyl)amino)hexyl 3,4,5-trimethoxybenzoate 53

Chromatographic eluent: abs. ethanol/ CH₂Cl₂/petroleum ether/NH₄OH 65:340:60:8. Yield: 71.4%.

¹H-NMR (CDCl₃)

δ: 7.24 (s, 2H, CH arom.); 4.24 (t, J=6.8 Hz, 2H, CH₂O); 3.85 (s, 6H, OCH₃);

3.84 (s, 3H, OCH₃); 3.73 (t, J=5.6 Hz, 2H, CH₂OH); 3.21 (bs, 1H, OH); 2.80 (t, J=6.0 Hz, 2H,

NCH₂); 2.55 (t, J=6.8 Hz, 2H, NCH₂); 1.76-1.67 (m, 2H, CH₂); 1.67-61 (m, 2H, CH₂); 1.48-1.23

13

(m, 8H, CH₂) ppm. C APT NMR (CDCl₃)

δ: 166.21 (C=O); 152.87 (C); 142.12 (C); 125.48 (C);

23

ACCEPTED MANUSCRIPT

106.78 (CH arom.); 65.15 (CH₂); 64.00 (CH₂); 60.84 (OCH₃); 56.20 (OCH₃); 49.76 (CH₂); 49.70

(CH₂); 30.64 (CH₂); 29.74 (CH₂); 29.30 (CH₂); 28.65 (CH₂); 27.11 (CH₂); 25.89 (CH₂) ppm.

5.1.3.10. 6-((3- Hydroxypropyl)amino)hexyl anthracene-9-carboxylate 54

Chromatographic eluent: abs. ethanol/ CH₂Cl₂/petroleum ether/NH₄OH 65:340:60:8. Yield: 48.4%.

¹H-NMR (CDCl₃)

δ: 8.50 (s, 1H, CH arom.); 8.03 (d, J=8.8 Hz, 2H, CH arom.); 8.00 (d, J=8.4 Hz,

2H, CH arom.); 7.55-7.40 (m, 4H, CH arom.); 4.60 (t, J=6.8 Hz, 2H, CH₂O); 3.78 (t, J=5.2 Hz, 2H,

CH₂OH); 3.21 (bs, 1H, OH); 2.82 (t, J=5.6 Hz, 2H, NCH₂); 2.56 (t, J=7.2 Hz, 2H, NCH₂); 1.90-1.81

(m, 2H, CH₂); 1.72-1.61 (m, 2H, CH₂); 1.50-1.20 (m, 8H, CH₂) ppm.

5.1.4. General procedure for the synthesis of (hydroxyalkyl)methylaminoesters 55-64

A 1 mmol portion of the appropriate hydroxyaminoester (45-54) was dissolved in 5 mL of

anhydrous ethanol and HCOOH (17 mmol) and 37% HCHO solution (5 mmol) were added. The

mixture was heated to 80 °C for 4-5 h and concentrated in vacuo. The residue was then dissolved in

CH₂Cl₂and the organic layer was washed with 10% NaOH solution. After drying with Na₂SO₄, the

solvent was removed under reduced pressure, giving a pure compound as yellow oil which was used

as such for the next reaction.

5.1.4.1. (E)-2-((6-Hydroxyhexyl)(methyl)amino)ethyl 3-(3,4,5-trimethoxyphenyl)acrylate 55

Yield: 80.7%. ¹H-NMR (CDCl₃)

δ: 7.61 (d, J=16.0 Hz, 1H, CH=CH); 6.75 (s, 2H, CH arom.); 6.37

(d, J=16.0 Hz, 1H, CH=CH); 4.33 (t, J=6.0 Hz, 2H, CH₂O); 3.88 (s, 6H, OCH₃); 3.87 (s, 3H,

OCH₃); 3.62 (t, J=6.4 Hz, 2H, CH₂O); 2.75 (t, J=6.0 Hz, 2H, NCH₂); 2.47 (t, J=7.2 Hz, 2H, NCH₂);

2.36 (s, 3H, NCH₃); 1.63 -1.48 (m, 4H, CH₂); 1.46-1.28 (m, 4H, CH₂) ppm.

5.1.4.2. (E)-4-((5-Hydroxypentyl)(methyl)amino)butyl 3-(3,4,5-trimethoxyphenyl)acrylate 56

Yield: 92.0%. ¹H-NMR (CDCl₃)

δ: 7.54 (d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.30

(d, J=16.0 Hz, 1H, CH=CH); 4.17 (t, J=6.8 Hz, 2H, CH₂O); 3.84 (s, 6H, OCH₃); 3.83 (s, 3H,

OCH₃); 3.58 (t, J=6.8 Hz, 2H, CH₂O); 2.40-2.25 (m, 4H, NCH₂); 2.16 (s, 3H, NCH₃); 1.74-1.50 (m,

6H, CH₂); 1.50-1.40 (m, 2H, CH₂); 1.40-1.32 (m, 2H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.99

(C=O); 153.39 (C); 144.62 (CH=CH); 140.05 (C); 129.91 (C); 117.40 (CH=CH); 105.20 (CH

arom.); 64.43 (CH₂); 62.62 (CH₂); 60.92 (OCH₃); 57.68 (CH₂); 57.35 (CH₂); 56.13 (OCH₃); 41.11

(NCH₃); 32.48 (CH₂); 28.88 (CH₂); 26.76 (CH₂); 24.14 (CH₂); 23.91 (CH₂) ppm.

5.1.4.3. (E)-5-((4-Hydroxybutyl)(methyl)amino)pentyl 3-(3,4,5-trimethoxyphenyl)acrylate 57

Yield: 85.0%. ¹H-NMR (CDCl₃)

δ: 7.54 (d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.30

(d, J=16.0 Hz, 1H, CH=CH); 4.15 (t, J=6.8 Hz, 2H, CH₂O); 3.84 (s, 6H, OCH₃); 3.83 (s, 3H,

OCH₃); 3.51 (m, J=5.6 Hz, 2H, CH₂O); 2.41-2.30 (m, 4H, NCH₂); 2.19 (s, 3H, NCH₃); 1.74-1.51

(m, 6H, CH₂); 1.51-1.50 (m, 2H, CH₂); 1.48-1.32 (m, 2H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ:

166.96 (C=O); 153.39 (C); 144.57 (CH=CH); 140.06 (C); 129.93 (C); 117.42 (CH=CH); 105.22

(CH arom.); 64.39 (CH₂); 62.60 (CH₂); 60.90 (OCH₃); 58.17 (CH₂); 57.74 (CH₂); 56.13 (OCH₃);

41.21 (NCH₃); 32.54 (CH₂); 28.62 (CH₂); 26.45 (CH₂); 26.14 (CH₂); 23.89 (CH₂) ppm.

5.1.4.4. (E)-3-((6-Hydroxyhexyl)(methyl)amino)propyl 3-(3,4,5-trimethoxyphenyl)acrylate 58

Yield: 91.1%. ¹H-NMR (CDCl₃)

δ: 7.54 (d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.29

(d, J=16.0 Hz, 1H, CH=CH); 4.19 (t, J=6.8 Hz, 2H, CH₂O); 3.84 (s, 6H, OCH₃); 3.82 (s, 3H,

OCH₃); 3.56 (t, J=6.8 Hz, 2H, CH₂O); 2.42 (t, J=7.2 Hz, 2H, NCH₂); 2.30 (t, J=7.6 Hz, 2H, NCH₂);

2.18 (s, 3H, NCH₃); 1.89-1.77 (m, 2H, CH₂); 1.54-1.49 (m, 2H, CH₂); 1.49-1.40 (m, 2H, CH₂);

1.40-1.20 (m, 4H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.95 (C=O); 153.40 (C); 144.68

(CH=CH); 140.11 (C); 129.88 (C); 117.31 (CH=CH); 105.25 (CH arom.); 63.03 (CH₂); 62.56

(CH₂); 60.91 (OCH₃); 57.58 (CH₂); 56.14 (OCH₃); 54.18 (CH₂); 42.09 (NCH₃); 32.67 (CH₂); 27.16

(CH₂); 27.08 (CH₂); 26.54 (CH₂); 25.64 (CH₂) ppm.

5.1.4.5. (E)-6-((3-Hydroxypropyl)(methyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate 59

Yield: 96.3%. ¹H-NMR (CDCl₃)

δ: 7.49 (d, J=15.6 Hz, 1H, CH=CH); 6.67 (s, 2H, CH arom.); 6.26

(d, J=15.6 Hz, 1H, CH=CH); 4.10 (t, J=6.4 Hz, 2H, CH₂O); 3.79 (s, 6H, OCH₃); 3.78 (s, 3H,

OCH₃); 3.69 (t, J=5.6 Hz, 2H, CH₂O); 2.48 (t, J=6.0 Hz, 2H, NCH₂); 2.27 (t, J=7.2 Hz, 2H, NCH₂);

24

ACCEPTED MANUSCRIPT

2.14 (s, 3H, NCH₃); 1.68-1.58 (m, 4H, CH₂); 1.48-1.20 (m, 6H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.91 (C=O); 153.34 (C); 144.50 (CH=CH); 140.01 (C); 129.89 (C); 117.41 (CH=CH); 105.19

(CH arom.); 64.45 (CH₂); 64.42 (CH₂); 60.83 (OCH₃); 58.30 (CH₂); 57.97 (CH₂); 56.07 (OCH₃);

41.88 (NCH₃); 28.62 (CH₂); 27.78 (CH₂); 27.09 (CH₂); 26.91 (CH₂); 22.66 (CH₂) ppm.

5.1.4.6. (E)-4-(6-Hydroxyhexyl(methyl)amino)butyl 3-(3,4,5-trimethoxyphenyl)acrylate 60

Yield: 96.9%. ¹H-NMR (CDCl₃)

δ: 7.50 (d, J=16.0 Hz, 1H, CH=CH); 6.68 (s, 2H, CH arom.); 6.26

(d, J=16.0 Hz, 1H, CH=CH); 4.13 (t, J=6.4 Hz, 2H, CH₂O); 3.80 (s, 6H, OCH₃); 3.79 (s, 3H,

OCH₃); 3.51 (t, J=6.8 Hz, 2H, CH₂O); 2.86 (bs, 1H, OH); 2.30-2.22 (m, 4H, NCH₂); 2.12 (s, 3H,

NCH₃); 1.65-1.59 (m, 2H, CH₂); 1.54-1.44 (m, 4H, CH₂); 1.43-1.33 (m, 2H, CH₂); 1.32-1.18 (m,

4H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.97 (C=O); 153.34 (C); 144.61 (CH=CH); 139.99

(C); 129.87 (C); 118.00 (CH=CH); 105.17 (CH arom.); 64.39 (CH₂); 62.40 (CH₂); 60.87 (OCH₃);

57.66 (CH₂); 57.25 (CH₂); 56.08 (OCH₃); 42.10 (NCH₃); 32.66 (CH₂); 27.25 (CH₂); 27.06 (CH₂);

26.73 (CH₂); 25.67 (CH₂); 23.63 (CH₂) ppm.

5.1.4.7. (E)-6-(4-Hydroxybutyl(methyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate 61

Yield: 93.1%. ¹H-NMR (CDCl₃)

δ: 7.53 (d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.30

(d, J=16.0 Hz, 1H, CH=CH); 4.14 (t, J=6.4 Hz, 2H, CH₂O); 3.84 (s, 6H, OCH₃); 3.83 (s, 3H,

OCH₃); 3.52-3.47 (m, 2H, CH₂O); 2.36-2.24 (m, 4H, NCH₂); 2.17 (s, 3H, NCH₃); 1.70-1.55 (m,

6H, CH₂); 1.54-1.44 (m, 2H, CH₂); 1.53-1.23 (m, 4H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.96

(C=O); 153.40 (C); 144.51 (CH=CH); 140.07 (C); 129.94 (C); 117.47 (CH=CH); 105.23 (CH

arom.); 64.49 (CH₂); 62.63 (CH₂); 60.88 (OCH₃); 58.23 (CH₂); 57.87 (CH₂); 56.12 (OCH₃); 41.23

(NCH₃); 32.66 (CH₂); 28.64 (CH₂); 27.09 (CH₂); 26.74 (CH₂); 26.30 (CH₂); 25.88 (CH₂) ppm.

5.1.4.8. (E)-6-((3-Hydroxypropyl(methyl)amino)hexyl 3-(3,4,5-trimethoxyphenyl)acrylate 62

Yield: 97.6%. ¹H-NMR (CDCl₃)

δ: 7.56 (d, J=15.6 Hz, 1H, CH=CH); 6.73 (s, 2H, CH arom.); 6.31

(d, J=15.6 Hz, 1H, CH=CH); 4.16 (t, J=6.4 Hz, 2H, CH₂O); 3.86 (s, 6H, OCH₃); 3.85 (s, 3H,

OCH₃); 3.76 (t, J=5.2 Hz, 2H, CH₂O); 2.56 (t, J=5.6 Hz, 2H, NCH₂); 2.33 (t, J=7.2 Hz, 2H, NCH₂);

2.21 (s, 3H, NCH₃); 1.72-1.63 (m, 4H, CH₂); 1.50-1.42 (m, 2H, CH₂); 1.42-1.25 (m, 6H, CH₂) ppm.

5.1.4.9. 6-((3-Hydroxypropyl)(methyl)amino)hexyl 3,4,5-trimethoxybenzoate 63

1

Yield: 79.8%. H-NMR (CDCl₃)

δ: 7.26 (s, 2H, CH arom.); 4.27 (t, J=6.8 Hz, 2H, CH₂O); 3.87 (s,

6H, OCH₃); 3.86 (s, 3H, OCH₃); 3.75 (t, J=5.4 Hz, 2H, CH₂O); 2.47 (t, J=5.6 Hz, 2H, NCH₂); 2.32

(t, J=7.6 Hz, 2H, NCH₂); 2.20 (s, 3H, NCH₃); 1.80-1.69 (m, 2H, CH₂); 1.68-1.62 (m, 2H, CH₂);

1.50-1.23 (m, 8H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.24 (C=O); 152.88 (C); 142.10 (C);

125.50 (C); 106.77 (CH arom.); 65.18 (CH₂); 64.59 (CH₂); 60.86 (OCH₃); 58.50 (CH₂); 58.06

(CH₂); 56.21 (OCH₃); 41.85 (NCH₃); 29.16 (CH₂); 28.67 (CH₂); 27.65 (CH₂); 27.17 (CH₂); 27.10

(CH₂); 25.92 (CH₂) ppm.

5.1.4.10. 6-((3-Hydroxypropyl)(methyl)amino)hexyl anthracene-9-carboxylate 64

Yield: 87.2%. ¹H-NMR (CDCl₃)

δ: 8.50 (s, 1H, CH arom.); 8.03 (d, J=8.8 Hz, 2H, CH arom.); 8.00

(d, J=8.4 Hz, 2H, CH arom.); 7.55-7.42 (m, 4H, CH arom.); 4.61 (t, J=6.8 Hz, 2H, CH₂O); 3.78 (t,

J=5.2 Hz, 2H, CH₂O); 2.56 (t, J=5.6 Hz, 2H, NCH₂); 2.34 (t, J=7.2 Hz, 2H, NCH₂); 2.21 (s, 3H,

13

NCH₃); 1.93-1.86 (m, 2H, CH₂); 1.70-1.64 (m, 2H, CH₂); 1.50-1.20 (m, 8H, CH₂) ppm. C APT

NMR (CDCl₃) δ: 169.77 (C=O); 131.01 (C); 129.20 (CH arom.); 128.61 (CH arom.); 128.39 (C);

129.92 (CH arom.); 125.46 (CH arom.); 125.02 (CH arom.); 65.91 (CH₂); 64.61 (CH₂); 58.45

(CH₂); 58.07 (CH₂); 41.85 (NCH₃); 29.15 (CH₂); 28.72 (CH₂); 27.65 (CH₂); 27.18 (CH₂); 27.03

(CH₂); 26.03 (CH₂) ppm.

5.1.5. General procedure for the synthesis of (2-hydroxyethyl)methylaminoesters 65-72

The appropriate haloester (35-40, 43, 4) (1 mmol) and 2-methylaminoethan-1-ol (2 mmol) were

dissolved in 3 mL of anhydrous CH₃CN. The mixture was maintained at rt for 48 h. The reaction

mixture was treated with CH₂Cl₂and the organic layer was washed with 10% NaOH solution. After

drying with Na₂SO₄, the solvent was removed under reduced pressure.

The substances obtained were used as such for the next reaction (65

flash chromatography in the case of 68 69 70 71 and 72

,

66

,

67), or were purified by

,

.

25

ACCEPTED MANUSCRIPT

5.1.5.1. (E)-6-((2- Hydroxyethyl) methylamino)hexyl 3-(3,4,5- trimethoxyphenyl)acrylate 65

1

Yellow oil. Yield: 91.0%. H-NMR (CDCl₃)

δ: 7.56 (d, J=16.0 Hz, 1H, CH=CH); 6.73 (s, 2H, CH

arom.); 6.32 (d, J=16.0 Hz, 1H, CH=CH); 4.17 (t, J=6.8 Hz, 2H, CH₂O); 3.86 (s, 6H, OCH₃); 3.85

(s, 3H, OCH₃); 3.55 (t, J=5.6 Hz, 2H, CH₂OH); 2.99 (bs, 1H, OH); 2.49 (t, J=5.6 Hz, 2H, NCH₂);

2.37 (t, J=7.2 Hz, 2H, NCH₂);2.21 (s, 3H, NCH₃); 1.77-1.60 (m, 2H, CH₂); 1.53-1.42 (m, 2H, CH₂);

1.41-1.26 (m, 4H, CH₂) ppm.

5.1.5.2. 6-((2-Hydroxyethyl)methylamino)hexyl anthracene-9-carboxylate 66

1

Yellow oil. Yield: 94.9%. H-NMR (CDCl₃)

δ: 8.48 (s, 1H, CH arom.); 8.05-7.87 (m, 4H, CH

arom.); 7.56-7.40 (m, 4H, CH arom.); 4.62 (t, J=6.8 Hz, 2H, CH₂O); 3.57 (t, J=5.6 Hz, 2H,

CH₂OH); 2.50 (t, J=5.6 Hz, 2H, NCH₂); 2.39 (t, J=6.8 Hz, 2H, NCH₂); 2.23 (s, 3H, NCH₃); 1.95-

1.86 (m, 2H, CH₂); 1.57-1.43 (m, 4H, CH₂); 1.42-1.30 (m, 2H, CH₂) ppm.

5.1.5.3. (E)-7-((2- Hydroxyethyl)methylamino)heptyl 3-(3,4,5- trimethoxyphenyl)acrylate 67

Yellow-orange oil. Yield 93.2%.¹H-NMR (CDCl₃)

δ: 7.52 (d, J=16.0 Hz, 1H, CH=CH); 6.69 (s,

2H, CH arom.); 6.28 (d, J=16.0 Hz, 1H, CH=CH); 4.13 (t, J=6.4 Hz, 2H, CH₂O); 3.82 (s, 6H,

OCH₃); 3.81 (s, 3H, OCH₃); 3.51 (t, J=5.6 Hz, 2H, CH₂OH); 2.99 (bs, 1H, OH); 2.44 (t, J=5.6 Hz,

2H, NCH₂); 2.32 (t, J=7.2 Hz, 2H, NCH₂); 2.16 (s, 3H, NCH₃); 1.69-1.59 (m, 2H, CH₂); 1.48-1.20

(m, 8H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.98 (C=O); 153.37 (C); 144.52 (CH=CH); 140.05

(C); 129.92 (C); 117.44 (CH=CH); 105.23 (CH arom.); 64.56 (CH₂); 60.86 (OCH₃); 58.86 (CH₂);

58.41 (CH₂); 57.72 (CH₂); 56.10 (OCH₃); 41.63 (NCH₃); 29.13 (CH₂); 28.66 (CH₂); 27.14 (CH₂);

25.89 (CH₂) ppm.

5.1.5.4. 7-((2-Hydroxyethyl)methylamino)heptyl 3,4,5-trimethoxybenzoate 68

Oil. Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 95:5:0.5. Yield: 77.3%. H-NMR (CDCl₃)

1

δ:

7.27 (s, 2H, CH arom.); 4.28 (t, J=6.8 Hz, 2H, CH₂O); 3.89 (s, 6H, OCH₃); 3.88 (s, 3H, OCH₃);

3.55 (t, J=5.6 Hz, 2H, CH₂OH); 2.92 (bs, 1H, OH); 2.50 (t, J=5.6 Hz, 2H, NCH₂); 2.38 (t, J=7.6 Hz,

2H, NCH₂); 2.22 (s, 3H, NCH₃); 1.81-1.70 (m, 2H, CH₂); 1.50-1.28 (m, 8H, CH₂) ppm.

5.1.5.5. 7-((2-Hydroxyethyl)methylamino)heptyl anthracene-9-carboxylate 69

1

Pale yellow oil. Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 96:4:0.4. Yield: 66%. H-NMR

(CDCl₃) δ: 8.51 (s, 1H, CH arom.); 8.04 (d, J=8.8 Hz, 2H, CH arom.); 8.05 (d, J=8.4 Hz, 2H, CH

arom.); 7.58-7.40 (m, 4H, CH arom.); 4.61 (t, J=6.8 Hz, 2H, CH₂O); 3.57 (t, J=5.6 Hz, 2H,

CH₂OH); 2.92 (bs, 1H, OH); 2.51 (t, J=5.6 Hz, 2H, NCH₂); 2.39 (t, J=7.6 Hz, 2H, NCH₂; 2.25 (s,

3H, NCH₃); 1.94-1.83 (m 2H, CH₂); 1.50-1.20 (m, 8H, CH₂) ppm.

5.1.5.6. (E)-8-(2- Hydroxyethyl)methylamino)octyl 3-(3,4,5-trimethoxyphenyl)acrylate 70

1

Pale yellow oil. Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 90:10:1. Yield: 56.1%. H-NMR

(CDCl₃) δ: 7.54 (d, J=16.0 Hz, 1H, CH=CH); 6.71 (s, 2H, CH arom.); 6.30 (d, J=16.0 Hz, 1H,

CH=CH); 4.15 (t, J=6.8 Hz, 2H, CH₂O); 3.84 (s, 6H, OCH₃); 3.83 (s, 3H, OCH₃); 3.54 (t, J=5.6 Hz,

2H, CH₂OH); 2.96 (bs, 1H, OH); 2.47 (t, J=5.6 Hz, 2H, NCH₂); 2.35 (t, J=7.2 Hz, 2H, NCH₂); 2.19

(s, 3H, NCH₃); 1.70-1.59 (m, 2H, CH₂); 1.47-1.38 (m, 2H, CH₂); 1.37-1.19 (m, 8H, CH₂) ppm. ¹³C

APT NMR (CDCl₃) δ: 167.02 (C=O); 153.39 (C); 144.54 (CH=CH); 140.01 (C); 129.94 (C);

117.47 (CH=CH); 105.18 (CH arom.); 64.64 (CH₂); 60.91 (OCH₃); 58.81 (CH₂); 58.33 (CH₂);

57.74 (CH₂); 56.11 (OCH₃); 41.60 (NCH₃); 29.40 (CH₂); 29.22 (CH₂); 28.71 (CH₂); 27.21 (CH₂);

27.18 (CH₂); 25.91 (CH₂) ppm.

5.1.5.7. 8-(2- Hydroxyethyl)methylamino)octyl 3-(3,4,5- trimethoxybenzoate 71

Pale yellow oil. Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 95:5:0.5. Yield: 75.6%. ¹H-NMR

(CDCl₃) δ: 7.28 (s, 2H, CH arom.); 4.29 (t, J=6.8 Hz, 2H, CH₂O); 3.89 (s, 6H, OCH₃); 3.88 (s, 3H,

OCH₃); 3.56 (t, J=5.2 Hz, 2H, CH₂OH); 2.50 (t, J=5.2 Hz, 2H, CH₂N); 2.37 (t, J=7.2 Hz, 2H,

CH₂N); 2.22 (s, 3H, NCH₃); 1.81-1.70 (m, 2H, CH₂); 1.50-1.22 (m, 10H, CH₂) ppm.

5.1.5.8. 8-(2- Hydroxyethyl)methylamino)octyl anthracene-9-carboxylate 72

Yellow oil. Chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 90:10:0.1. Yield: 32.4%.

¹H-NMR (CDCl₃)

δ: 8.50 (s, 1H, CH arom.); 8.05-8.00 (m, 4H, CH arom.); 7.61-7.47 (m, 4H, CH

arom.); 4.61 (t, J=6.8 Hz, 2H, CH₂O); 3.57 (t, J=5.2 Hz, 2H, CH₂OH); 2.50 (t, J=5.2 Hz, 2H,

26

ACCEPTED MANUSCRIPT

NCH₂); 2.37 (t, J=7.2 Hz, 2H, NCH₂); 2.20 (s, 3H, NCH₃); 1.93-1.82 (m, 2H, CH₂); 1.53-1.42 (m,

2H, CH₂); 1.51-1.20 (m, 8H, CH₂) ppm.

5.1.6. General procedures for the synthesis of diester compounds 1-28

Diester compounds were synthesized using two different general procedures.

General procedure A.

A solution of the suitable (hydroxyalkyl)methylaminoester (0.250 mmol) in 5 mL of an. CH₂Cl₂

was cooled at 0 °C and 0.360 mmol of the appropriate carboxylic acid, 0.083 mmol of 4-

dimethylaminopiridine (DMAP) and 0.500 mmol of 1-(3-dimethylaminopropyl)-3-

ethylcarbodiimide hydrochloride (EDCl) were added. The reaction mixture was stirred for 1 h at

0 °C and 48 h at room temperature. Then CH₂Cl₂was added and the organic layer was washed

twice with a saturated solution of NaHCO₃. After drying with Na₂SO₄, the solvent was removed

under reduced pressure. The crude product was then purified by flash chromatography using the

appropriate eluting system, yielding the desired compound as an oil. All the compounds were

transformed into the corresponding hydrochloride as white solid. The salts were crystallized from

abs. ethanol/petroleum ether.

General procedure B.

A 0.250 mmol portion of the appropriate carboxylic acid ((E)-3-(3,4,5-trimethoxyphenyl)acrylic

acid, 3,4,5-trimethoxybenzoic acid or anthracene-9-carboxylic acid) was transformed into the acyl

chloride by reaction with SOCl₂(2 eq) in 3 mL of CHCl₃(free of ethanol) at 60 °C for 4-5 h.

The reaction mixture was cooled to rt, and the solvent was removed under reduced pressure; the

mixture was then treated twice with cyclohexane and the solvent removed under reduce pressure.

The acyl chloride obtained was dissolved in CHCl₃(free of ethanol), and the suitable

(hydroxyalkyl)methylaminoester (1 eq) was added. The mixture was heated to 60 °C. After 4 h, the

reaction mixture was cooled to rt, and treated with CH₂Cl₂. The resulting organic layer was washed

with 10% NaOH solution, dried with Na₂SO₄, and the solvent was removed under reduced pressure.

The substances obtained were purified by flash chromatography using the appropriate eluting

system, yielding the desired compound as an oil. All the compounds were transformed into the

corresponding hydrochloride as white solid. The salts were crystallized from abs. ethanol/petroleum

ether.

5.1.6.1. (E)-2-(Methyl-(6-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy))hexyl)amino)ethyl 3,4,5-

trimethoxybenzoate 1

Procedure A, starting from 65 and 3,4,5-trimethoxybenzoic acid.

Free base: chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 98:2:0.2. Yield: 69.1.%.

¹H-NMR (CDCl₃)

δ: 7.52 (d, J=16.0 Hz, 1H, CH=CH); 7.23 (s, 2H, CH arom.); 6.69 (s, 2H, CH

arom.); 6.28 (d, J=16.0 Hz, 1H, CH=CH); 4.35 (t, J=6.0 Hz, 2H, CH₂O); 4.11 (t, J=6.8 Hz, 2H,

CH₂O); 3.82 (s, 9H, OCH₃); 3.81 (s, 6H, OCH₃); 3.80 (s, 3H, OCH₃); 2.73 (t, J=6.0 Hz, 2H, NCH₂);

2.41 (t, J=7.6 Hz, 2H, NCH₂); 2.29 (s, 3H, NCH₃); 1.65-1.58 (m, 2H, CH₂); 1.50-1.40 (m, 2H,

CH₂); 1.40-1.23 (m, 4H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 166.90 (C=O); 166.05 (C=O);

153.36 (C); 152.86 (C); 144.53 (CH=CH); 142.20 (C); 140.02 (C); 129.88 (C); 125.16 (C); 117.39

(CH=CH); 106.84 (CH arom.); 105.18 (CH arom.); 64.44 (CH₂); 62.92 (CH₂); 60.85 (OCH₃); 60.81

(OCH₃); 57.83 (NCH₂); 56.16 (OCH₃); 56.08 (OCH₃); 55.57 (NCH₂); 42.67 (NCH₃); 28.67 (CH₂);

27.19 (CH₂); 26.96 (CH₂); 25.86 (CH₂) ppm.

Hydrochloride: mp 90-92 °C. Anal: C₃₁H₄₄ClNO₁₀(C, H, N).

5.1.6.2. (E)-6-(Methyl-(2-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)ethyl)amino)hexyl

3,4,5-trimethoxybenzoate 2

Procedure A, starting from 55 and 3,4,5-trimethoxybenzoic acid.

Free base: chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 98:2:0.2. Yield: 68.8.%.

¹H-NMR (CDCl₃)

δ: 7.58 (d, J=16.0 Hz, 1H, CH=CH); 7.26 (s, 2H, CH arom.); 6.72 (s, 2H, CH

arom.); 6.35 (d, J=16.0 Hz, 1H, CH=CH); 4.32-4.25 (m, 4H, CH₂O); 3.87 (s, 6H, OCH₃); 3.86 (s,

27

ACCEPTED MANUSCRIPT

3H, OCH₃); 3.85 (s, 6H, OCH₃); 3.84 (s, 3H, OCH₃); 2.72 (t, J=6.0 Hz, 2H, NCH₂); 2.45 (t, J=7.6

Hz, 2H, NCH₂); 2.33 (s, 3H, NCH₃); 1.78-1.71 (m, 2H, CH₂); 1.58-1.30 (m, 6H, CH₂) ppm. ¹³C

APT NMR (CDCl₃) δ: 166.85 (C=O); 166.21 (C=O); 153.42 (C); 152.91 (C); 144.97 (CH=CH);

142.16 (C); 140.17(C); 129.83 (C); 125.46 (C); 117.15 (CH=CH); 106.84 (CH arom.); 105.31 (CH

arom.); 65.07 (CH₂); 61.88 (CH₂); 60.92 (OCH₃); 60.87 (OCH₃); 57.80 (NCH₂); 56.23 (OCH₃);

56.14 (OCH₃); 55.60 (NCH₂); 42.53 (NCH₃); 28.72 (CH₂); 27.03 (CH₂); 26.87 (CH₂); 25.92 (CH₂)

ppm.

Hydrochloride: mp 97-98 °C. Anal: C₃₁H₄₄ClNO₁₀(C, H, N).

5.1.6.3.

(E)-2-(Methyl-(6-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)hexyl)amino)ethyl

anthracene-9- carboxylate 3

Procedure B, starting from 65 and anthracene-9-carboxylic acid.

Free base: chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 98:2:0.2. Yield: 32.8.%.

¹H-NMR (CDCl₃)

δ: 8.49 (s, 1H, CH arom.); 8.14 (d, J=8.8 Hz, 2H, CH arom.); 7.99 (d, J=8.4 Hz,

2H, CH arom.); 7.58 (d, J=16.0 Hz, 1H, CH=CH); 7.54-7.44 (m, 4H, CH arom.); 6.73 (s, 2H, CH

arom.); 6.33 (d, J=16.0 Hz, 1H, CH=CH); 4.75 (t, J=5.6 Hz, 2H, CH₂O); 4.14 (t, J=6.4 Hz, 2H,

CH₂O); 3.87 (s, 3H, OCH₃); 3.85 (s, 6H, OCH₃); 2.90 (t, J=5.6 Hz, 2H, NCH₂); 2.51 (t, J=7.6 Hz,

2H, NCH₂); 2.39 (s, 3H, NCH₃); 1.69-1.51 (m, 4H, CH₂); 1.43-1.21 (m, 4H, CH₂) ppm. ¹³C APT

NMR (CDCl₃) δ: 169.45 (C=O); 167.02 (C=O); 153.42 (C); 144.59 (CH=CH); 140.07 (C); 130.97

(C); 129.95 (C); 129.37 (CH arom.); 128.58 (CH arom.); 128.47 (C); 127.86 (C); 126.91 (CH

arom.); 125.46 (CH arom.); 125.23 (CH arom.); 117.48 (CH=CH); 105.22 (CH arom.); 64.54

(CH₂); 62.98 (CH₂); 60.97 (OCH₃); 57.79 (CH₂); 56.14 (OCH₃); 55.91 (CH₂); 42.37 (NCH₃); 28.68

(CH₂); 27.03 (CH₂); 25.90 (CH₂) ppm.

Hydrochloride: mp 84-86 °C. Anal: C₃₆H₄₂ClNO₇(C, H, N).

5.1.6.4.

(E)-6-(Methyl-(2-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)ethyl)amino)hexyl

anthracene-9- carboxylate 4

Procedure A, starting from 66 and (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 99:1:0.1. Yield: 39.7.%.

¹H-NMR (CDCl₃) δ: 8.47 (s, 1H, CH arom.); 8.02 (d, J=8.8 Hz, 2H, CH arom.); 7.97 (d, J=8.4 Hz,

2H, CH arom.); 7.60 (d, J=16.0 Hz, 1H, CH=CH); 7.55-7.43 (m, 4H, CH arom.); 6.72 (s, 2H, CH

arom.); 6.38 (d, J=16.0 Hz, 1H, CH=CH); 4.59 (t, J=6.8 Hz, 2H, CH₂O); 4.29 (t, J=6.0 Hz, 2H,

CH₂O); 3.85 (s, 3H, OCH₃); 3.82 (s, 6H, OCH₃); 2.68 (t, J=5.6 Hz, 2H, NCH₂); 2.40 (t, J=7.2 Hz,

2H, NCH₂); 2.29 (s, 3H, NCH₃); 1.90-1.83 (m, 2H, CH₂); 1.54-1.41 (m, 4H, CH₂); 1.41-1.33 (m,

2H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ: 169.69 (C=O); 166.93 (C=O); 153.41 (C); 144.86

(CH=CH); 140.11 (C); 130.97 (C); 129.89 (C); 129.22 (CH arom.); 128.62 (CH arom.); 128.36 (C);

128.15 (C); 126.92 (CH arom.); 125.45 (CH arom.); 124.98 (CH arom.); 117.30 (CH=CH); 105.24

(CH arom.); 65.80 (CH₂); 62.15 (CH₂); 60.94 (OCH₃); 57.86 (CH₂); 56.11 (OCH₃); 55.72 (CH₂);

42.66 (NCH₃); 28.75 (CH₂); 27.04 (CH₂); 26.05 (CH₂) ppm.

Hydrochloride: mp 61-63 °C. Anal: C₃₆H₄₂ClNO₇(C, H, N).

5.1.6.5. (E)-4-(Methyl-(5-((3-(3,4,5-trimethoxyphenyl)acryloyl)oxy)pentyl)amino)butyl 3,4,5-

trimethoxybenzoate 5

Procedure B, starting from 57 and 3,4,5-trimethoxybenzoic acid.

Free base: chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH 95:5:0.5. Yield: 83.1.%.

¹H-NMR (CDCl₃)

δ: 7.58 (d, J=16.0 Hz, 1H, CH=CH); 7.28 (s, 2H, CH arom.); 6.74 (s, 2H, CH

arom.); 6.33 (d, J=16.0 Hz, 1H, CH=CH); 4.31 (t, J=6.8 Hz, 2H, CH₂O); 4.19 (t, J=6.4 Hz, 2H,

CH₂O); 3.89 (s, 3H, OCH₃); 3.88 (s, 6H, OCH₃); 3.87 (s, 6H, OCH₃); 3.86 (s, 3H, OCH₃); 2.41-2.33

(m, 4H, NCH₂); 2.21 (s, 3H, NCH₃); 1.82-1.75 (m, 2H, CH₂); 1.75-1.67 (m, 2H, CH₂); 1.64-1.56

(m, 2H, CH₂); 1.55-1.48 (m, 2H, CH₂); 1.44-1.36 (m, 2H, CH₂) ppm. ¹³C APT NMR (CDCl₃)

δ:

166.96 (C=O); 166.20 (C=O); 153.41 (C); 152.90 (C); 144.58 (CH=CH); 142.19 (C); 140.10 (C);

129.91 (C); 125.41 (C); 117.42 (CH=CH); 106.82 (CH arom.); 105.23 (CH arom.); 65.03 (CH₂);

64.50 (CH₂); 60.91 (OCH₃); 60.86 (OCH₃); 57.68 (CH₂); 57.30 (CH₂); 56.22 (OCH₃); 56.13

28

Article Doi

Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to the discovery of a series of highly potent P-glycoprotein-based multidrug resistance (MDR) modulators

DOI: 10.1016/j.ejmech.2019.03.054

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Article abstract of DOI:10.1016/j.ejmech.2019.03.054

Full text of DOI:10.1016/j.ejmech.2019.03.054

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