Design, synthesis and biological evaluation of stereo- and regioisomers of amino aryl esters as multidrug resistance (MDR) reversers

European Journal of Medicinal Chemistry 182 (2019) 1 11655

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis and biological evaluation of stereo- and

regioisomers of amino aryl esters as multidrug resistance (MDR)

reversers

,

Elisabetta Teodori ^{a *}, Marialessandra Contino ^b, Chiara Riganti ^c, Gianluca Bartolucci ^a,

Laura Braconi ^a, Dina Manetti ^a, Maria Novella Romanelli ^a, Alfonso Trezza ^d,

Asimidis Athanasios ^d, Ottavia Spiga ^d, Maria Grazia Perrone ^b, Roberta Giampietro ^b,

Elena Gazzano ^c, Milena Salerno ^e, Nicola Antonio Colabufo ^b, Silvia Dei ^a

^aDepartment 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

^bDepartment of Pharmacy-Drug Sciences, University of Bari “A. Moro”, via Orabona 4, 70125, Bari, Italy

^cDepartment of Oncology, University of Turin, Via Santena 5/bis, 10126, Torino, Italy

^dDepartment of Biotechnology, Chemistry and Pharmacy, University of Siena, via Aldo Moro 2, 53100, Siena, Italy

e

ꢀ

Universite Paris 13, Sorbonne Paris Cite, Laboratoire CSPBAT, CNRS (UMR 7244), UFR-SMBH, 74 rue Marcel Cachin, 93017, Bobigny, France

a r t i c l e i n f o

a b s t r a c t

Article history:

Stereo- and regioisomers of a series of N,N-bis(alkanol)amine aryl ester derivatives have been prepared

and studied as multidrug resistance (MDR) modulators. The new compounds contain a 2-(methyl)propyl

chain combined with a 3-, 5- or 7-methylenes long chain and carry different aromatic ester portions.

Thus, these compounds have a methyl group on the 3-methylenes chain and represent branched ho-

mologues of previously studied derivatives. The introduction of the methyl group gives origin to a

stereogenic center and consequently to (R) and (S) enantiomers. In the pirarubicin uptake assay on K562/

DOX cell line these compounds showed good activity and efﬁcacy and in many cases enantioselectivity

was observed. Docking studies conﬁrmed the inﬂuence of the stereocenter on the interaction in the P-gp

pocket. The P-gp interaction mechanism and selectivity towards MRP1 and BCRP were also evaluated on

MDCK transfected cells overexpressing the three transporters. Almost all these compounds inhibited

both P-gp and BCRP, but only derivatives with speciﬁc structural characteristics showed MRP1 activity.

Moreover, two compounds, (S)-3 and (R)-7, showed the ability to induce collateral sensitivity (CS) against

MDR cells. Therefore, these two CS-promoting agents could be considered interesting leads for the

development of selective cytotoxic agents for drug-resistant cells.

Received 27 May 2019

Received in revised form

26 August 2019

Accepted 27 August 2019

Available online 30 August 2019

Keywords:

MDR reversers

P-gp modulators

Enantiomers

Molecular docking

K562 cells

MDCK cells

Collateral sensitivity

Human plasma stability

1. Introduction

The anticancer chemotherapy is often impaired by the resis-

tance that cancer cells develop to cytotoxic drugs after an exposure

Abbreviations: P-gp, P-glycoprotein; MRP1, multidrug-resistance-associated

protein-1; BCRP, breast cancer resistance protein; ROS, reactive oxygen species; CS,

collateral sensitivity; K562/DOX, doxorubicin-resistant erythroleukemia K562 cells;

DOX, Doxorubicin; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimmide hy-

drochloride; DMAP, 4-dimethylaminopyridine; PDB, Protein Data Bank; MDCK,

Madin-Darby Canine Kidney; P_app, apparent permeability; BA, basolateral to apical;

AB, apical to basolateral; Calcein-AM, calcein acetoxy methyl ester; SR, selectivity

ratio; NAC, N-acetyl-cysteine; RLU, relative luminescence units; PBS, phosphate

buffer solution; KEE, ketoprofene ethylester; TR, retention time.

period. This resistance is called multidrug resistance (MDR) when it

is towards a multiplicity of structurally unrelated chemothera-

peutic drugs [1]. This cross-resistance is often due to an enhanced

energy dependent drug efﬂux, caused by the overexpression of

some transmembrane ATP-Binding Cassette (ABC) transporter

proteins. They are able to transport substrates through the mem-

brane against the concentration gradient [2,3] and are constitu-

tively expressed in many tissues playing important protective roles

through the regulation of the permeability of substances through

* Corresponding author.

E-mail address: elisabetta.teodori@uni ﬁ .it (E. Teodori).

https://doi.org/10.1016/j.ejmech.2019.111655

2

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

biological membranes [4]. The overexpression of these proteins in

cancer cells causes the reduction of the intracellular concentration

of antineoplastic agents. The transport proteins P-glycoprotein (P-

gp, ABCB1), Multidrug-Resistance-associated Protein-1 (MRP1,

ABCC1), and Breast Cancer Resistance Protein (BCRP, ABCG2) have

mainly been associated with MDR as they were found overex-

pressed in malignant tissues [5e7]. P-gp was the ﬁrst ABC trans-

porter to be discovered playing a role in cancer cells drug resistance

[8,9]. In fact, it is overexpressed in several tumor cells as a result of

anticancer treatment and is responsible of an acquired resistance to

a number of structurally and mechanistically unrelated chemo-

therapeutic drugs [10,11]. MRP1 is overexpressed in cancer cells

that are not sensitive to several chemotherapeutic drugs such as

doxorubicin, methotrexate, cisplatin, etoposide and vincristine

[12]. The most recently discovered ABC efﬂux protein involved in

multidrug-resistance is BCRP. Its overexpression in many solid tu-

mors and leukemia, together with P-gp, impairs the efﬁcacy of

many chemotherapeutic agents [13]. BCRP was also found

expressed in cancer stem cells causing their insensitivity to anti-

cancer drugs and consequently the long-lasting ineffectiveness of

many chemotherapy treatments [14,15]. P-gp and BCRP are the two

prevalent ABC efﬂux proteins placed at the blood-brain barrier

(BBB) and are the cause of the reduced BBB penetration of many

drugs, including antineoplastic agents, that are actively extruded

being substrate of these two efﬂux pumps [16,17].

A number of approaches to reverse MDR have been extensively

studied and the identiﬁcation of ABC transporter protein inhibitors

has been considered an appropriate strategy. For this reason, many

modulators of these proteins have been synthesized over the past

few decades. These compounds are deﬁned chemosensitizers, in

fact, when co-administered with anticancer agents which are

substrates of ABC transporters, they are able to restore their

effectiveness in resistant tumor cells [18,19].

In the last years several ABC transporter modulators have been

discovered; they are classiﬁed as ﬁrst-, second- or third-generation,

according to their chronology and characteristics [20e23]. Several

of them have reached clinical trials [24e27], however no substan-

tial beneﬁts have been established. The observed problems are

mainly due to their low potency and toxicity and also to cyto-

chrome isoform inhibitory effects [28]; even though the more

recently discovered MDR reversing agents show lower negative

properties.

paradoxical hypersensitivity, known as “collateral sensitivity” (CS),

as well as the development of rationally-designed new sensitizer

compounds are still under intensive investigation [35,36].

In previous papers we described the design and synthesis, and

in vitro studies of many series of MDR modulators characterized by

the presence of a basic nitrogen atom connected to two different

aromatic ester portions by two linkers with different length and

ﬂexibility, such as polymethylenic chains or cyclohexane rings

[37,38]. In particular, several N,N-bis(alkanol)amine aryl esters

were synthesized; these compounds were characterized by

different combinations of aromatic ester residues connected to the

N-methylated basic portion by two polymethylenic chains of vari-

able length. Most of the synthesized compounds showed to be

potent and efﬁcacious P-gp-dependent MDR reversers [37e42].

Some combinations of aromatic moieties and spacers seem to be

very positive; in fact, the (E)-3-(3,4,5-trimethoxyphenyl)vinyl

moiety with the 3,4,5-trimethoxyphenyl or the anthracene resi-

dues, and spacers of a total length of 6, 8 or 10 methylenes, gave

interesting results (compounds I-X, structure A, Chart 1).

In the present study we explored the consequence of the

modiﬁcation of a three methylenes chain by the introduction of a

methyl group on the position 2. The steric hindrance introduced in

the chain could inﬂuence the binding mode of these derivatives and

also prevent the enzymatic hydrolysis of the corresponding ester

function. As a matter of fact, in a previous study [42] it was

observed that in a series of derivatives with two chains of different

length, the hydrolysis occurs, in particular, when the ester group

was carried by a 3-methylenes N-alkyl chain. Moreover, this

modiﬁcation gives origin to a stereogenic center and the two

possible enantiomers (R) and (S) could show different biological

activity. For this purpose, we synthesized a new series of com-

pounds with a 2-(methyl)propyl chain combined with a 3-, 5- or 7-

methylenes long chain, in order to connect the two aromatic

moieties with spacers of 6, 8 or 10 methylenes total length. All the

possible stereo- and regioisomers were obtained (compounds

1e12, structure B, Chart 1).

The new derivatives were studied on the doxorubicin-resistant

erythroleukemia K562 cell line (K562/DOX) to evaluate their

reversal activity by the pirarubicin uptake assay. Molecular docking

simulation studies were performed in order to identify the binding

mode of these compounds in the P-gp binding pocket.

The compounds were also studied to evaluate their P-gp inter-

action proﬁle and selectivity towards two other ABC transporters,

MRP1 and BCRP. For these studies Madin-Darby Canine Kidney

(MDCK) transfected cells were used (MDCK-MDR1, MDCK-MRP1

and MDCK-BCRP cells overexpressing P-gp, MRP1 and BCRP,

respectively). Among the compounds displaying the best P-gp ac-

tivity and selectivity proﬁle in these tests, the enantiomers (R)-3

and (R)-7 were selected for the co-administration study in the

presence of doxorubicin in MDCK-MDR1 cells. This study has been

performed in order to conﬁrm the ability of these compounds to

reverse MDR, restoring the access of the chemotherapeutic drug

Consequently, the search for more potent and efﬁcacious MDR

reversers is still ongoing with the aim of overcoming some of the

obstacles concerning the use of chemosensitizers.

Compounds able to interact with ABC transporters present

different chemical structures; however, some general features for

the binding with these proteins have been identiﬁed, for instance

the presence of aromatic moieties and one or more protonable

nitrogen atoms, the ability of establishing hydrogen bond in-

teractions and high lipophilicity [29]. These features are in agree-

ment with the information collected on the structure of ABC

transporters suggesting that they are characterized, in particular for

P-gp, by large and polymorphous recognition sites where different

compounds can be accommodated in a plurality of binding modes

establishing hydrophobic interactions, hydrogen and

bonds [30,31].

pꢀp, p-ion

In the last years, high-throughput screening of pharmacological

libraries progressively identiﬁed also speciﬁc compounds which

were unexpectedly more effective as cytotoxic agents in drug-

resistant than in drug-sensitive cells [32e34]. Compounds

increasing reactive oxygen species (ROS), interfering with the en-

ergy metabolism and the synthesis of ATP and altering the mem-

brane ﬂuidity exert selective toxicity in P-gp- and MRP1-

overexpressing cells. However, the molecular bases of this

Chart 1. General structure of lead and newly designed compounds.

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

3

into the cells. Moreover, collateral sensitivity studies were per-

formed for all compounds 1e12 on MDCK-MDR1, MDCK-MRP1 cells

and on the parental MDCK cells, in order to evaluate the selective

cytotoxicity of the synthesized compounds towards cells over-

expressing the two pumps P-gp and MRP1. Two compounds were

able to selectively kill MDR cells without cytotoxic effects towards

the parental cells. They can be considered as CS-promoting agents

and were further studied for their effect on the intracellular ROS

level.

Finally, the stability of this series of molecules towards hydro-

lysis was evaluated. In fact, the presence of the ester groups, in

particular, can compromise the stability of these compounds to-

wards plasma enzymes. For this purpose, a series of experiments in

phosphate buffer solution (PBS) and human plasma were per-

formed and the degradation proﬁles were evaluated.

(see Experimental section for details). The (S) bromoalkyl esters

13e15 were transformed in the corresponding (S) derivatives

16e18 with NaI in acetone, to obtain higher yields in the next re-

action. The (S) enantiomers 16e18 were then transformed into the

(R) secondary amines 19e27 by reaction with the commercially

available amino alcohols, 3-aminopropan-1-ol and 5-

aminopentan-1-ol, or the already reported 7-aminoheptan-1-ol

[43], using standard procedures. The absolute conﬁguration

assigned to stereocenters changes by passing from iodo to amino

derivatives due to the different priority of the groups linked to the

stereogenic center. The (R) secondary amines were then alkylated

by reductive methylation with HCOOH/HCHO to give the corre-

sponding tertiary amines 28e36. Title compounds (R) 1e12 were

eventually obtained by reaction of (R) 28e36 with the appropriate

carboxylic acid in the presence of EDCI and DMAP in anhydrous

CH₂Cl₂, or by the corresponding acyl chloride obtained as described

before. The proper acids used are the same as described above: (E)-

3-(3,4,5-trimethoxyphenyl)acrylic acid, 3,4,5-trimethoxybenzoic

acid and anthracene-9-carboxylic acid. The (S) enantiomers of

compounds 1e12 were obtained starting from (R)-3-bromo-2-

(methyl)propan-1-ol and following the same procedures described

for the (R) enantiomers. For biological tests, all compounds were

transformed into HCl salts.

2. Chemistry

The reaction pathways used to synthesize the (R) enantiomers of

compounds 1e12 are reported in Scheme 1. The (S) bromoesters

13e15 were obtained by esteriﬁcation of the commercially avail-

able chiral synthon (S)-3-bromo-2-(methyl)propan-1-ol with (E)-

3-(3,4,5-trimethoxyphenyl)acrylic acid, 3,4,5-trimethoxybenzoic

acid or anthracene-9-carboxylic acid. To obtain the desired ester,

the mixture of carboxylic acid and bromoalkyl alcohol was treated

with the activating agent EDCI, in the presence of DMAP in anhy-

drous CH₂Cl₂, or the suitable acid was transformed in the corre-

sponding acyl chloride by reaction with SOCl₂in ethanol-free CHCl₃

The enantiomeric excess (ee) of (R) and (S) enantiomers of

compounds 1e12 was assessed by enantioselective liquid chro-

matography coupled with diode array detector (LC-DAD) analysis.

To perform this analysis, different elution conditions were

employed (see Experimental section). All enantiomers showed

Scheme 1. Reagents and conditions: (I) SOCl₂, ethanol-free CHCl₃or EDCI, DMAP, an. CH₂Cl₂; for details, see the Experimental section; (II) NaI, acetone; (III) H₂N(CH₂)_nOH

(n ¼ 3,5,7), K₂CO₃, CH₃CN; (IV) HCOOH/HCHO; (V) Ar¹COCl, ethanol-free CHCl₃or Ar¹COOH, EDCI, DMAP, an. CH₂Cl₂. The (S) enantiomers of compounds 1e12 were obtained starting

from (R)-3-bromo-2-(methyl)propan-1-ol and following the same procedures (see the Experimental section).

4

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

ee ꢁ 95%, that is the maximal evaluable value with the used

method, except compounds 4, 7 and 12 whose enantiomer pairs

(R)/(S) did not reach a sufﬁcient resolution to assess their ee values;

anyway, since the synthetic pathway and the used enantiomeric

reagents were common for all products, it was reasonable that also

these compounds maintained the same enantiomeric excess

(ꢁ95%).

propyl chain were more potent than those of the corresponding

regioisomers with this group on the 5-methylenes chain (compare

(S)-5 vs (S)-7 and (S)-6 vs (S)-8). The regioisomers of the set with

n ¼ 7 showed instead a different behavior since the more potent

compounds have the (E)-3-(3,4,5-trimethoxyphenyl)vinyl moiety

on the 7-methylenes chain (compare (S)-9 vs (S)-11 and (S)-10 vs

(S)-12). In particular, the eutomer (S)-12 was 11 times more potent

than (S)-10 ([I]_0.5¼ 0.08

mM and 0.89 mM. Moreover, in this set, the

two enantiomers were almost equally potent except for compound

12 (ER ¼ 2.5).

3. Results

3.1. Modulation of pirarubicin uptake on K562/DOX cells

As regards the efﬁcacy, these compounds showed a_maxvalues

between 0.65 and 0.99 indicating that were able to reverse P-gp-

dependent pirarubicin extrusion with rather high amount and in

some cases the resistance was even completely reversed (a_maxclose

to 1).

Compounds (R) and (S) 1e12 were studied for the P-gp modu-

lating activity by the pirarubicin uptake assay on K562/DOX

doxorubicin resistant cells, that overexpress only the membrane

glycoprotein P-gp [44e48], following protocols reported in previ-

ous papers [49,50].

Altogether, these results suggest that a methyl group at the

stereogenic center of the 3-methylenes linker differently inﬂuences

the activity of these compounds depending on the total length of

the molecule. In fact, the presence of this group exerts a favourable

effect, in comparison with the corresponding achiral derivatives, in

compounds with n ¼ 3 (compare 1e4 vs I and II); on the contrary,

this feature appears to have lower inﬂuence on the activity for

compounds with a 5-methylenes chain (compare 5e8 vs III-VI),

The results obtained are reported in Table 1 together with those

of the corresponding previously synthetized compounds (I-X) with

an achiral three methylenes chain, and verapamil, used as reference

compound. The data reported in Table 1 indicate that all the new

compounds inhibited the P-gp activity with high potency since

their [I]_0.5values fell in the nanomolar range; in addition, in many

cases these compounds completely reversed P-gp-dependent

pirarubicin extrusion (a_maxclose to 1). Their potency and afﬁnity

values were higher than those of the reference compound verap-

amil. A thorough analysis of the results indicated that the activity

([I]_0.5) is inﬂuenced by the length of the linear chain (n value) and

only in part by the combination of the aromatic residues. In most

cases the two (R) and (S) enantiomers showed different potency

values although there is no regular trend in the behavior of the two

enantiomers. In fact, the (S) enantiomer was often the most potent,

but not in the case of compounds 2 and 3, that showed an inverted

enantioselectivity, and compounds 7, 9, 10 and 11 that showed

almost equally active (R) and (S) enantiomers. In any case, the in-

ﬂuence of the stereogenic center on the activity of these com-

pounds was more or less pronounced depending on the total length

of the molecule. In fact, in the set with n ¼ 3 (1e4), the two en-

antiomers were in general more potent than the corresponding

achiral compound with the same length and residues, particularly

and is deﬁnitely not productive for compounds with

a 7-

methylenes chain (compare 9e12 vs VII-X). On the other hand, it

is important to note that, as reported above, the achiral compounds

with a total spacer of 10 methylenes (compounds VII-X) showed a

very good proﬁle both in terms of potency and efﬁcacy with all

combinations of aromatic groups; molecular modeling studies

indicated that these molecules are able to interact in a productive

manner with the P-gp interaction site [42]. In these new molecules

the methyl group on the stereogenic center apparently prevents the

best interaction of this set of compounds with the protein binding

region.

3.2. Molecular modeling studies

Docking studies were performed to investigate the interaction

mode of the new compounds in the P-gp binding pocket and to

elucidate the inﬂuence of the stereocenter on their P-gp-inhibitory

activity. For this purpose, MolSoft was used to perform a ﬂexible

docking calculation in order to predict the correct binding geom-

etry, the potential binding pocket of the protein, scoring functions

and the bond types of the enantiomers with the surrounding amino

acid binding pocket [51]. Molecular docking studies were per-

formed into 3D crystal of human P-gp (PDB code 6C0V) [52] and the

binding region of our compounds was identiﬁed based on 3D

crystal structure of Mus musculus P-gp in complex with the P-gp

inhibitor BDE-100 (PDB code 4XWK) [53]. The binding mode within

the P-gp binding region of compounds 1e12 was compared with

that of the corresponding achiral analogues I-X (Table 1), studied

with the same method. The docking energy scores of the tested

compounds are listed in Table 1. Fig. 1 describes the correlation

in the case of the two enantiomers of 1 (compare I [I]_0.5¼ 0.60

mM

with (R)-1 and (S)-1 [I]_0.5¼ 0.14 and 0.09

m

M, respectively). In the

set with n ¼ 5 (5e8), the eutomer (the most active enantiomer) (S)-

5 showed a [I]_0.5value lower than that of the corresponding achiral

analogue III ([I]_0.5¼ 0.08

mM and 0.27 mM, respectively). In the

other cases the activity of the achiral analogues was equally or

slightly higher than that of the eutomers (compare IV vs (S)-7, V vs

(S)-6, VI vs (S)-8). Regarding the set with n ¼ 7 (9e12), all the

achiral derivatives, VII-X, were always more potent than the two

enantiomers of the corresponding chiral compounds (VII vs (R) and

(S) 9, VIII vs (R) and (S) 11, IX vs (R) and (S) 10, X vs (R) and (S) 12).

For what concerns the ester groups, the activity is not much

affected by the nature of the aromatic groups; in fact, there is no

regular trend for the inﬂuence of the type and the relative position

of the aromatic moieties. Anyway, within each set the combination

of the aromatic residues inﬂuences the enantioselectivity. In fact, in

the set with n ¼ 3, the two eutomers of the regioisomers 1 and 3

showed an opposite conﬁguration, (S)-1 and (R)-3, and different

enantioselectivity, since the eudismic ratios (ER, here deﬁned for

sake of clarity as the ratio between the [I]_0.5of the (R) enantiomer

and the [I]_0.5of the (S) one) are 1.5 and 0.2, respectively. Com-

pounds 2 and 4 showed similar behavior even if, also in this case,

the enantioselectivity is inverted and the two eutomers are (R)-2

and (S)-4. In the set with n ¼ 5 the eutomers of compounds with

the (E)-3-(3,4,5-trimethoxyphenyl)vinyl moiety on the 2-(methyl)

between the calculated binding free energies (DG) and the activ-

ities of the compounds on the pirarubicin uptake test, expressed as

p[I]_0.5. The correlation coefﬁcient (R²) value in Fig. 1 suggests that

computational analysis results strictly correlate with the biological

activities on the pirarubicin uptake test.

MolSoft docking results revealed that all achiral and chiral

compounds showed moderate to high binding afﬁnity towards P-

gp with binding energy ranging from ꢀ3.80 to ꢀ25.06 kcal/mol.

They identiﬁed a binding region with two pockets: one (site 1) is

located in the inner central chamber surrounded by hydrophobic

residues, which is the common binding site of ligands in complex

crystal structures, while site 2 is a deeper binding site where the

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

5

Table 1

MDR-reversing activity of enantiomers of the target compounds 1e12 evaluated on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) by the pirarubicin uptake

assay.

Ar¹

[I]_0.5

m

M^a

ER^b

1.5

a_max

^dDG (Kcal/mol)

c

Compd

Struct.

Conf.

n

Ar

(R)-1

(S)-1

(R)-3

(S)-3

I^e

(R)-2

(S)-2

(R)-4

(S)-4

II^e

(R)-5

(S)-5

III^f

(R)-7

(S)-7

IV^f

(R)-6

(S)-6

V^f

(R)-8

(S)-8

VI^f

(R)-9

(S)-9

VII^g

(R)-11

(S)-11

VIII^g

(R)-10

(S)-10

IX^g

B

A

B

A

B

A

B

A

B

A

B

A

B

A

B

A

B

A

B

A

R

S

R

S

3

5

7

a

b

a

c

b

a

b

c

a

c

b

a

c

a

b

a

c

0.14 0.03

0.09 0.07

0.11 0.02

0.54 0.15

0.60 0.15

0.08 0.03

0.18 0.02

0.17 0.04

0.04 0.01

0.18 0.05

0.40 0.01

0.08 0.02

0.27 0.05

0.35 0.10

0.24 0.07

0.12 0.02

0.08 0.03

0.03 0.007

0.04 0.02

0.23 0.08

0.08 0.02

0.04 0.01

0.59 0.17

0.38 0.11

0.08 0.03

0.12 0.04

0.13 0.05

0.06 0.02

0.97 0.28

0.89 0.21

0.04 0.01

0.20 0.06

0.08 0.02

0.01 0.003

1.60 0.30

0.99 0.01

0.75 0.04

0.99 0.01

0.89 0.10

0.90 0.05

0.99 0.10

0.79 0.09

0.80 0.06

0.99 0.01

0.78 0.03

0.99 0.30

0.99 0.01

0.97 0.02

0.72 0.07

0.75 0.06

0.99 0.01

0.88 0.03

0.79 0.01

0.94 0.03

0.89 0.06

0.75 0.05

0.98 0.02

0.81 0.05

0.74 0.05

0.97 0.03

0.83 0.05

0.70 0.04

0.99 0.01

0.65 0.07

0.80 0.05

0.88 0.08

0.93 0.05

0.99 0.01

0.93 0.04

0.70 0.07

ꢀ15.94

ꢀ16.66

ꢀ16.12

ꢀ5.90

0.2

ꢀ5.50

R

S

R

S

0.4

4.2

ꢀ16.80

ꢀ15.86

ꢀ15.90

ꢀ20.30

ꢀ15.30

ꢀ7.10

c

a

b

a

c

R

S

5.0

1.4

2.6

2.9

1.5

1

ꢀ16.90

ꢀ9.10

R

S

ꢀ9.90

ꢀ10.20

ꢀ16.80

ꢀ17.10

ꢀ22.70

ꢀ20.00

ꢀ9.56

R

S

R

S

c

ꢀ17.50

ꢀ19.80

ꢀ5.70

R

S

a

b

a

c

ꢀ8.70

ꢀ18.00

ꢀ16.30

ꢀ16.50

ꢀ19.30

ꢀ3.80

R

S

R

S

1.1

2.5

ꢀ4.00

ꢀ20.50

ꢀ10.10

ꢀ20.74

ꢀ25.06

ꢀ1.80

(R)-12

(S)-12

X^g

R

S

a

c

Ver.

a

Potency of the MDR-modulator: concentration of the compound that causes a 50% increase in the nuclear concentration of pirarubicin (

a

¼ 0.5).

b

c

ER ¼ eudismic ratio ([I]_0.5(R) enantiomer/[I]_0.5(S) enantiomer).

Efﬁcacy of the MDR-modulator: maximum increase that can be obtained in the nuclear concentration of pirarubicin in resistant cells;

a value 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). Results are expressed as the mean SE of three inde-

pendent experiments done at least three times.

d

Predicted binding afﬁnity.

See ref. 37.

See ref. 40.

See ref. 42.

e

f

g

residues from transmembrane helixes 4, 5, 8 and 9 are able to

establish interactions with both hydrophobic and polar residues

[54], as showed in Fig. 2. The achiral compound X showed the

on one of the two linkers. Moreover, the presence of the stereo-

center inﬂuences the pose of the enantiomer couples. As an

example, in Fig. 3 B we observed how the two enantiomers (R)-6

and (S)-6 achieved similar poses showing comparable binding free

energy values (Table 1). Differently, the two enantiomers (R)-5 and

(S)-5 showed different poses and binding energies (Fig. 3C and

Table 1), in accordance with the results on the pirarubicin uptake

test where the two enantiomers showed an ER ¼ 5.0 (Table 1).

In conclusion, we can therefore state that chiral molecules

interact at the P-gp binding pocket in different ways with respect to

achiral analogues, in particular, with a 7-methylene chain; the

latter derivatives, in fact, can reach easily the end of the pocket

forming important interactions with Leu 225, Phe 193, 200 and 204,

and Val 338. Anyway, many chiral molecules are able to form strong

interactions with binding protein residues that are crucial for the

lowest binding energy computational value (

mol). It interacts at both the P-gp binding sites forming three

D

G ¼ ꢀ25.06 kcal/

p-

stacking bonds with the three aromatic rings of the anthracene

moiety and phenylalanine residues (site 1), and hydrophobic in-

teractions with isoleucine, leucine, phenylalanine and valine resi-

dues (sites 1 and 2) (Fig. 3 A).

On the contrary, molecular docking of chiral compounds

showed that they occupy only a part of the binding region estab-

lishing interactions with different binding pocket residues than the

achiral compounds. In particular, we observed that all chiral com-

pounds do not reach the same binding poses of the best achiral

derivative (Fig. 3), probably due to the presence of the methyl group

6

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

that represents passive diffusion, and AB, from the apical to baso-

lateral compartments, that represents active transport; 2) ATP cell

depletion in cells overexpressing the transporter MDCK-MDR1 [57]

which measures the ATP consumption due to transport mediated

by the pump; generally, a substrate, being transported by the

pump, induces ATP cell depletion (unambiguous substrate, cate-

gory I), while a P-gp inhibitor does not induce ATP consumption.

(BA/AB) < 2 indicates that the compound can be considered an in-

hibitor, also taking into account the results of the ATPase assay and

Calcein-AM modulation, (BA/AB) > 2 indicates that the compound

is classiﬁed as a substrate [58,59]. However, another substrate

category (known as category IIB3) displaying a P_appvalue > 2 but

not inducing an ATP cell depletion is also reported [60].

Finally, the compounds were also studied on cells over-

expressing MRP1 (MDCK-MRP1 cells) or BCRP (MDCK-BCRP cells)

to evaluate their inhibitory activity toward two other ABC proteins,

MRP1 and BCRP, by the inhibition of the transport of Calcein-AM

(MRP1 substrate) or the ﬂuorescent probe Hoechst 33342 (BCRP

substrate), respectively.

Fig. 1. Correlation between the calculated MolSoft tool binding free energies (DG) and

the experimental p[I]_0.5values, expressed as

ported in Table 1.

mM concentration, for P-gp that are re-

P-gp, MRP1 and BCRP expression levels were periodically ana-

lysed by immunoblotting analysis in MDCK-MDR1, MDCK-MRP1

and MDCK-BCRP cells, respectively, using tubulin as control of

equal protein loading. Details of the method and a representative

Western blot analysis are reported in the Supplementary data.

The results of the assays described above are reported in Table 2

together with those on verapamil, used as reference compound.

As shown in Table 2, all compounds were able to inhibit the P-gp

Calcein-AM transport with EC₅₀values ranging from 0.073 to

mechanism of action and inhibitory effect proposed by us in this

study.

3.3. Characterization of P-gp interacting proﬁle and ABC

transporters selectivity on transfected MDCK cells

The new compounds were further studied on MDCK-MDR1

(Madin-Darby Canine Kidney-MDR1) cells overexpressing P-gp,

by inhibition of the transport of Calcein-AM, a proﬂuorescent probe

that is a P-gp substrate.

1.40 mM, indicating, in this assay, high or moderate activities on P-

gp. The EC₅₀values are, in some cases ((R)-3, (R)-7, (S)-7 and (S)-9)

The P-gp interaction mechanism of the compounds was also

investigated and, besides the P-gp potency assay on MDCK-MDR1

cells, other two combined assays were employed, as previously

reported [55]: 1) apparent permeability (P_app) determination (BA/

AB) in Caco-2 cell monolayer [56] which measures the ratio be-

tween two ﬂuxes, BA, from the basolateral to apical compartments,

lower than that of the reference compound verapamil

(EC₅₀¼ 0.50

mM).

Stereochemistry does not seem to inﬂuence the P-gp inhibition

on MDCK-MDR1 cells of these compounds. In fact, in many cases

the two enantiomers showed similar EC₅₀values, or with a small

Fig. 2. 3D representation of human P-gp in the lipid bilayer (green). MolSoft program identiﬁed two sites inside the protein and predicted binding-pockets are shown in orange

surface surrounded by binding region residues represented with blue ball and sticks. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the

Web version of this article.)

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

7

Fig. 3. 3D representation of human P-gp binding region (orange) inside the lipid bilayer (green). (A) Blue balls and sticks represent residues of P-gp binding pocket with the best

active achiral compound X in orange. (B-C) Blue balls and sticks represent residues of P-gp binding pocket with enantiomers (R)-6 (magenta) and (S)-6 (green), (panel B) and (R)-5

(red) and (S)-5 (yellow), (panel C). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the Web version of this article.)

difference ranging from 1.5 to 2.8 times, except the two enantio-

mers (R)-3 and (S)-3 that showed a difference of 7.5 times

verapamil, with the exception of compounds (S)-1, (R)-3, (S)-3 and

(S)-5. Also in this case the stereochemistry has little inﬂuence on

the activity, but not in the case of 1, 5, 9 and 3, where only enan-

(EC₅₀¼ 0.073

mM and 0.55 mM, respectively). Anyway, also in this

test, there is a non-homogeneous S/R preference since the eutomers

of compounds 1 and 3 showed a R absolute conﬁguration while

those of compounds 7, 9 and 10 showed a S absolute conﬁguration.

The total length of the molecule does not seem to inﬂuence this

kind of activity while the nature of the aromatic residues partly

affects the activity; in fact, compounds with EC₅₀values lower than,

tiomer (R)-3 was able to inhibit MRP1 (EC₅₀¼ 7.3

mM) and (S)-3 was

completely inactive.

The apparent permeability values (P_app) indicated that all com-

pounds had a BA/AB ratio > 2 and were not able to induce ATP

consumption, with the exception of compound (S)-1; therefore, (S)-

1 could be considered a P-gp unambiguous substrate (category I)

and all the other compounds were not transported substrates

(category IIB3) [60].

or roughly equal to 0.5 mM ((R)-3, (S)-3, (R)-7, (S)-7, (S)-9 and (S)-11)

have the combination of the (E)-3-(3,4,5-trimethoxyphenyl)vinyl

moiety with the 3,4,5-trimethoxyphenyl one, while the presence of

the anthracene residue gave higher EC₅₀values ranging from 0.76

3.4. Co-administration assay

to 1.40 mM.

In this test, these compounds showed a lightly different proﬁle

than that in the pirarubicin uptake test, but it must be considered

that the cell lines used for the two assays are different.

Compounds that displayed the best P-gp activity in the test on P-

gp transfected cells (MDCK-MDR1) ((R)-3, EC₅₀¼ 0.073

endowed with a good P-gp activity and selectivity proﬁle towards

the other two sister proteins ((R)-7, EC₅₀¼ 0.31 M, 5.8 M, 41

vs P-gp, MRP1 and BCRP, respectively), have been tested in co-

administration with the antineoplastic drug doxorubicin at 10

mM) or

All compounds, except (R)-5, were able to inhibit BCRP but with

m

mM

activities lower than that of verapamil (EC₅₀¼ 0.9

mM). Also in this

case stereochemistry has low inﬂuence on the activity with some

exception (1, 2, 5, 6, 7). In particular, enantiomer (S)-5 was able to

mM

in MDCK cells overexpressing MDR1. Doxorubicin alone did not

exert cytotoxicity effects since it is a P-gp substrate and was unable

to overcome MDR1; when doxorubicin was added in the presence

of (R)-3 and (R)-7, its activity was restored because of the inhibition

of the pump carried out by the two compounds. (R)-3 and (R)-7

were tested at two different doses in a range near their EC₅₀values

inhibit BCRP (EC₅₀¼ 4.2

mM) while (R)-5 was completely inactive

up to a 100 M.

m

The inhibitory activity on MRP1 is inﬂuenced by the nature of

the aromatic residues since all anthracene derivatives were inactive

on this transporter; the other compounds were able to inhibit

MRP1 Calcein-AM transport with potencies higher than that of

(100 nM and 1 mM for (R)-3, and 500 nM and 1 mM for (R)-7)

8

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

Table 2

Biological results of enantiomers 1e12: inhibition activity on MDCK-MDR1, MDCK-MRP1 and MDCK-BCRP cells overexpressing each transporter P-gp, MRP1 and BCRP, ATP cell

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

Ar¹

EC₅₀

P-gp

m

M^a

ATP cell depletion

P_app

b

Conf

n

Ar

Compd

MRP1

BCRP

(R)-1

(S)-1

(R)-3

(S)-3

(R)-2

(S)-2

(R)-4

(S)-4

(R)-5

(S)-5

(R)-7

(S)-7

(R)-6

(S)-6

(R)-8

(S)-8

(R)-9

(S)-9

(R)-11

(S)-11

(R)-10

(S)-10

(R)-12

(S)-12

Ver.

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

3

5

7

a

b

a

c

b

a

c

0.71 0.10

1.38 0.20

0.073 0.01

0.55 0.10

0.95 0.20

0.91 0.20

0.96 0.10

0.91 0.18

1.00 0.16

1.10 0.20

0.31 0.05

0.11 0.02

0.72 0.10

0.69 0.14

0.81 0.16

0.76 0.10

0.82 0.15

0.32 0.05

0.65 0.13

0.55 0.10

1.40 0.20

0.93 0.18

0.99 0.19

1.04 0.20

0.50 0.1

5.6 1.12

12.7 2.5

7.3 1.4

NA

33.5 5.8

3.7 0.70

9.5 1.90

6.4 1.30

5.9 1.20

2.3 0.40

6.8 1.36

11.0 2.0

NA

4.2 0.80

41.0 8.2

8.1 1.62

2.3 0.46

4.7 0.90

4.6 0.92

4.6 0.90

4.7 0.84

2.3 0.40

4.3 0.80

2.5 0.50

6.4 1.20

5.6 1.12

6.2 1.24

7.2 1.40

0.9 0.20

no

yes^c

no

yes^d

7.6

5

6.9

7.5

13

30

7.5

15

c

a

b

a

c

NA

c

a

b

a

c

3.9 0.77

10.1 2.0

5.8 1.20

4.4 0.88

NA

3.7 0.72

1.9 0.32

3.5 0.70

2.8 0.60

NA

6.8 3.0

5

8

6.5

6.6

>20

18

15

16

17

c

a

b

a

c

a

c

a

b

a

c

10.3

14.4

20

23

6.1

9.2

18

c

a

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

b

c

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

Percentage of the effect at a concentration of 10 M (20%).

M (20%). NA ¼ not active.

m

d

Percentage of the effect at a concentration of 1

m

showing a reduction of viability about 30% at 1

respectively (Fig. 4).

m

M and 500 nM,

strategy to overcome MDR by using compounds able to selectively

kill MDR cells without cytotoxic effects towards the parental cells

[35,36,61e63]. CS can be quantitatively assessed by measuring the

selectivity ratio (SR) between the cytotoxicity of the compound vs

parental cells and its cytotoxicity vs MDR-derived cells (EC₅₀

parental/EC₅₀MDR). SR < 1 indicates that MDR cells are resistant to

the ligand that is probably transported by the MDR pump; SR > 1

3.5. Collateral sensitivity study and effect on ROS production

All compounds have been tested for their ability to induce

collateral sensitivity (CS) against MDR1 or MRP1. CS is a new

Fig. 4. In vitro cell growth experiments performed on MDCK-MDR1 cells in the presence of 10 mM doxorubicin (Doxo) alone (white bar), compounds (R)-3 and (R)-7 at different

doses alone and in co-administration with Doxo. Each bar represents the mean SEM of two experiments performed in triplicate. One-way ANOVA analysis: **p < 0.005.

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

9

indicates that the cytotoxicity of the ligand vs MDR cells is higher

than that vs parental cells. A compound displaying a SR ꢁ 2 is

generally deﬁned as CS-promoting agent.

Among the tested series, only compounds (S)-3 and (R)-7 dis-

played a SR > 2 towards MDR pumps.

increased production of ROS in MDR1-and MRP1-overexpressing

cells, respectively.

MDCK-MDR1 cells (panels A and B) were grown for 24 h in fresh

medium (ctrl) or with 500 nM (S)-3 alone or in the presence of the

ROS scavenger N-acetyl-cysteine (NAC, 10 mM). MDCK-MRP1 cells

(panels C and D) were grown for 24 h in fresh medium (ctrl) or with

500 nM (R)-7 alone or in the presence of the ROS scavenger N-

acetyl-cysteine (NAC, 10 mM). Panels A and C: ROS were measured

ﬂuorimetrically, in duplicates (n ¼ 3). Data are presented as

Indeed, (S)-3 has a SR ¼ 2.5 vs MDCK-MDR1 cells (EC₅₀¼ 100

on MDCK-wt cells and EC₅₀¼ 40 M on MDCK-MDR1 cells) and (R)-

7 has a SR ¼ 8.5 vs MDCK-MRP1 cells (EC₅₀¼ 100 M on MDCK-wt

M on MDCK-MRP1 cells). Therefore, (S)-3 and

mM

m

cells and EC₅₀¼ 11.7

m

(R)-7 can be considered as CS-promoting agents on P-gp and MRP1,

respectively.

means

SD. ****p < 0.0001 vs ctrl; ^ꢂꢂꢂꢂp < 0.0001: NAC-treated

cells vs (S)-3 and (R)-7, respectively. Panels B and D: cell viability

was measured with a chemiluminescence-based assay, in quadru-

plicates (n ¼ 3). Data are presented as means SD. ****p < 0.0001:

vs ctrl; ^ꢂꢂp < 0.001: NAC-treated cells vs (S)-3 and (R)-7, respec-

tively [63].

One of the mechanisms at the basis of CS is the increased pro-

duction of ROS in cells overexpressing ABC transporters [35]. To

investigate whether this mechanism was involved in the sensitizing

effects elicited by (S)-3 and (R)-7, the intracellular ROS in MDCK-

MDR1, for compound (S)-3, and MDCK-MRP1 cells for compound

(R)-7, was measured. (S)-3 and (R)-7 signiﬁcantly increased intra-

cellular ROS in MDCK-MDR1 (Fig. 5A) and MDCK-MRP1 (Fig. 5C)

cells, respectively. To verify whether the increased ROS mediated

the reduction in cell viability elicited by the compounds, the cells

were co-incubated with (S)-3 or (R)-7 plus the ROS scavenger N-

acetyl-cysteine (NAC), at a concentration that abrogated the in-

crease in ROS induced by the compounds (Fig. 5 A and C). While (S)-

3 or (R)-7 alone signiﬁcantly decreased cell viability, the co-

incubation with NAC partially but signiﬁcantly rescued cell

viability (Fig. 5 B and D).

3.6. Chemical stability test

The chemical stability of compounds 1e12 was evaluated in

phosphate buffer solution (PBS) and in human plasma samples to

verify if spontaneous or enzymatic hydrolysis occurs. The analyses

were carried out by LC-MS/MS method operating in Multiple Re-

action Monitoring (MRM) mode [64].

The LC-MS/MS system and experimental conditions used are

reported in the Supplementary data.

The solution stability of each compound was veriﬁed by moni-

toring the variation of analyte concentration at different incubation

times in PBS and human plasma samples. The evaluation of the

These results suggest that at least part of the mechanism at the

basis of the collateral sensitivity of (S)-3 or (R)-7 relies on the

Fig. 5. Effects of (S)-3 and (R)-7 on MDCK-MDR1 and MDCK-MRP1 ROS levels and viability.

10

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

Table 3

Half-life (t_1/2) values of reference and studied compounds in PBS e human plasma samples.

Compd

PBS t_1/2error (min)

Human plasma t_1/2error (min)

Compd

PBS t_1/2error (min)

Human plasma t_1/2error (min)

KEE

n.d.

107 16

117 10

(R)-1

(S)-1

(R)-2

(S)-2

(R)-3

(S)-3

(R)-4

(S)-4

(R)-5

(S)-5

(R)-6

(S)-6

ꢁ240

(R)-7

(S)-7

(R)-8

(S)-8

(R)-9

(S)-9

(R)-10

(S)-10

(R)-11

(S)-11

(R)-12

(S)-12

ꢁ240

74 27

29

7

ꢁ240

54 12

ꢁ240

30

9

ꢁ240

29 15

18

6

ꢁ240

n.d.: not determined.

employed LC-MS/MS methods and the processed raw data are re-

ported in the Supplementary data. The obtained results demon-

strated that all the compounds were stable in PBS and most of them

also in human plasma. In fact, only the degradation plots, in human

plasma, of (R)-1, (S)-1, (R)-3, (R)-5, (S)-5, (R)-9 and (S)-9 showed a

derivatives. Since 240 min is the maximum time of observation in

our experimental protocol, it is possible to assume that their half-

life values could be equal or greater than 240 min.

The degradation products of compounds (R)-1, (S)-1, (R)-3, (R)-5,

(S)-5, (R)-9 and (S)-9 were also examined to determine the possible

mechanism of the enzymatic hydrolysis. The results proved that

these compounds were hydrolyzed at the ester group linked to the

(E)-3-(3,4,5-trimethoxyphenyl)vinyl moiety (a, see Chart 1), with

formation of the corresponding N-alkyl alcohol and of the (E)-3-

(3,4,5-trimethoxyphenyl)acrylic acid (Supplementary data,

Fig. S26). Noteworthy is that the hydrolysis takes place only when

the (E)-3-(3,4,5-trimethoxyphenyl)vinyl ester is linked to the three

methylenes 2-(methyl)propyl chain and combined with the 3,4,5-

trimethoxybenzoic moiety (b, see Chart 1) (e.g. (R)-1 and (S)-1).

As a matter of fact, the enzyme activity is prevented when the (E)-

signiﬁcant decay rate (k value ꢃ ꢀ2 10^ꢀ3ln

mM/min), and their

calculated half-life values varied between 18 and 117 min (Table 3).

As an example, the human plasma degradation plots of the enan-

tiomer couples (R)-1 and (S)-1 are reported in Fig. 6. The plots of all

compounds are reported in the Supplementary data (Figs. S2eS25).

The half-life value of the reference compound ketoprofene ethyl-

ester (KEE) indicated that the human batch employed was enzy-

matically active (half-life < 2 h) [65].

The k values of the stable compounds were close to 0; therefore,

extremely high half-life values can be calculated for these

Fig. 6. Degradation plots of the enantiomers couples (R)-1 (top), (S)-1 (bottom) in PBS (square blue) and human plasma (red triangle) samples. (For interpretation of the references

to colour in this ﬁgure legend, the reader is referred to the Web version of this article.)

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

11

Fig. 7. Degradation plots of the enantiomers couples (R)-3 (top), (S)-3 (bottom) in PBS (square blue) and human plasma (red triangle) samples. (For interpretation of the references

to colour in this ﬁgure legend, the reader is referred to the Web version of this article.)

3-(3,4,5-trimethoxyphenyl)vinyl moiety is combined with the

anthracene residue (c, see Chart 1) (i.e. (R)-2 and (S)-2). This

behavior was already highlighted in the previously studied achiral

series with a 3-methylenes chain [42] (see the achiral derivative I)

and it is therefore conﬁrmed also in this chiral series carrying a

branched 3-methylenes chain (2-(methyl)propyl chain). However,

this feature depends on the conﬁguration of the stereogenic center,

in fact, the (S) enantiomers always showed a degradation rate

higher than the corresponding (R) ones (see (S)-1, (S)-5 and (S)-9)

(Table 3). On the contrary, (R)-3 and (S)-3, having the (E)-3-(3,4,5-

trimethoxyphenyl)vinyl moiety linked to the linear three methy-

lenes alkyl chain, showed a different behavior. As a matter of fact,

pirarubicin extrusion (a_maxclose to 1). In most cases the (R) and (S)

isomers showed different potency values although there is no

regular trend in the behavior of the two enantiomers. In compari-

son with the corresponding achiral derivatives, the presence of the

methyl group on the stereogenic center appears to exert a clear

positive effect only in the set with n ¼ 3, since these compounds

were in general more potent than the corresponding achiral de-

rivatives showing the same length and residues.

Docking simulations were employed to elucidate the inﬂuence

of the stereocenter on the binding mode of these compounds in the

P-gp pocket site. The chiral compounds appeared to have a binding

mode partly different from that of achiral ones, forming in-

teractions with different binding pocket residues. In particular, the

presence of the methyl group at the stereocenter prevents de-

rivatives with n ¼ 7 from reaching the same binding poses of the

corresponding most active achiral derivatives; however, the added

methyl group is probably able to establish favourable interactions

for compounds with n ¼ 3.

the (R) enantiomer suffers

a remarkable degradation (t_1/

¼ 54 12 min) while the (S) one did not show a signiﬁcant

2

enzymatic hydrolysis (Table 3 and Fig. 7).

4. Discussion

The P-gp interaction proﬁle and selectivity towards MRP1 and

BCRP of the new compounds were investigated on MDCK trans-

fected cells overexpressing the transporters P-gp, MRP1 and BCRP.

In this test, all compounds were able to inhibit P-gp efﬂux ac-

tivity and small differences in activity and enantioselectivity were

observed throughout the series. These results are lightly different

from those found in the pirarubicin uptake test, but it must be kept

in mind that the cell lines used in these two tests are different

(Madin-Darby Canine Kidney (MDCK) transfected cells and

doxorubicin-resistant erythroleukemia K562 cells, respectively).

As for the P-gp-interacting proﬁle, only compound (S)-1 can be

considered a P-gp unambiguous substrate (category I) whereas all

the other compounds were not transported substrates (category

IIB3).

In this work we reported a new series of N,N-bis(alkanol)amine

aryl ester derivatives studied as MDR modulators. The new com-

pounds contain a 2-(methyl)propyl chain combined with a 3-, 5- or

7-methylenes long chain and bearing different aromatic ester

portions. These compounds represent branched homologues of

previously studied derivatives (I-X). The introduction of a methyl

group in these molecules gives origin to a stereogenic center and

consequently to (R) and (S) enantiomers.

The biological behavior of these compounds was studied by

various tests that allowed us to highlight different characteristics.

The MDR reversal activity was evaluated by the pirarubicin

uptake assay on doxorubicin-resistant erythroleukemia K562 cell

line (K562/DOX). In this test these compounds showed good

inhibitory activity with potency values ([I]_0.5) in the nanomolar

range and, in general, completely reversed P-gp-dependent

The co-administration assay on the enantiomers (R)-3 and (R)-7

12

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

proved that they were able in reversing resistance to doxorubicin

since the activity of the anticancer drug in MDCK-MDR1 cells was

restored.

6. Experimental section

6.1. Chemistry

All compounds, except (R)-5, were able to inhibit BCRP, while

only molecules bearing the combination of the two aromatic

All melting points were taken on a Büchi apparatus and are

uncorrected. NMR spectra were recorded on a Bruker Avance 400

spectrometer (400 MHz for ¹H NMR, 100 MHz for ¹³C). ¹H and ¹³C

NMR spectra were measured at room temperature (25 ^ꢂC) in an

appropriate solvent. ¹H and ¹³C chemical shifts are expressed in

moieties

(E)-3-(3,4,5-trimethoxyphenyl)vinyl

and

3,4,5-

trimethoxyphenyl showed MRP1 inhibition activity. These results

conﬁrmed the same outcome already found in our previous paper

[42], namely that the anthracene residue is not positive for the

MRP1 inhibition.

ppm (d) referred to TMS. Spectral data are reported using the

Of great interest was the ability to induce collateral sensitivity

showed by (S)-3 and (R)-7. Both compounds indeed can be

considered as CS-promoting agents towards MDR pumps, as they

proved to be able to selectively kill MDR cells (MDCK-MDR1 and

MDCK-MRP1 cells, respectively) without cytotoxic effects towards

the parental cells, displaying a SR > 2. The mechanism at the basis

of the collateral sensitivity of these two compounds relies at least in

part on the increased production of ROS in MDR1-and MRP1-

overexpressing cells, respectively.

Finally, most compounds of this new series of MDR inhibitors

were stable in PBS and human plasma, despite the presence of the

two ester groups. Only (R)-3 and the two enantiomers of com-

pounds 1, 5 and 9 showed a signiﬁcant decay rate in the human

plasma; the hydrolysis occurs at the ester group linked to the (E)-3-

(3,4,5-trimethoxyphenyl)vinyl moiety and the degradation rate

depends on the conﬁguration of the streocenter of the molecules.

following abbreviations: s ¼ singlet, bs ¼ broad singlet, d ¼ doublet,

dd ¼ doublet of doublets, t ¼ triplet, m ¼ multiplet, and coupling

constants are reported in Hz, followed by integration. Chromato-

graphic separations were performed on a silica gel column by

gravity chromatography (Kieselgel 40, 0.063e0.200 mm; Merck) or

ﬂash chromatography (Kieselgel 40, 0.040e0.063 mm; Merck).

Yields are given after puriﬁcation, unless otherwise stated.

The enantiomeric excess (ee) and purity of ﬁnal compounds

were determined by Agilent 1200 liquid chromatography system

composed by autosampler, binary pumps, column oven and diode-

array detector (LC-DAD) operating in UV range (210e400 nm). The

operating conditions were reported in Supplementary data.

High resolution mass spectrometry (HR-MS) analyses of ﬁnal

compounds were performed with a Thermo Finnigan LTQ Orbitrap

mass spectrometer equipped with an electrospray ionization

source (ESI). The analyses were carried out introducing, via syringe

pump at 10 m mg/mL in mQ

L min^ꢀ1, the sample solution (1.0

water:acetonitrile 50:50), in positive ion mode. These experimental

conditions allow the monitoring of protonated molecules of the

studied compounds ([MþH]^þspecies), that they were measured

with a proper dwell time to achieve 60,000 units of resolution at

Full Width at Half Maximum (FWHM). Elemental composition of

compounds was calculated on the basis of their measured accurate

masses, accepting only results with an attribution error less than

5 ppm and a not integer RDB (double bond/ring equivalents) value,

in order to consider only the protonated species [66].

When reactions were performed in anhydrous conditions, the

mixtures were maintained under nitrogen. Free bases (R) and (S)

1e12 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. Compounds

were named following IUPAC rules as applied by ChemBioDraw

Ultra 14.0 software.

5. Conclusions

In this study we designed and synthesized a new series of N,N-

bis(alkanol)amine aryl esters bearing a chiral alkyl chain allowing

the identiﬁcation of interesting MDR reversers. The presence of a

methyl group at the stereocenter causes in many cases enantiose-

lectivity, a characteristic that is not common among MDR trans-

porter protein ligands. Docking studies conﬁrmed the inﬂuence of

the stereocenter on the interaction of these compounds in the P-gp

pocket.

Interestingly, nearly all these compounds were able to inhibit

both P-gp and BCRP; since these two transporter proteins were

often found co-expressed in several tumors, this behaviour could

be an interesting feature of this series of derivatives. The inhibitory

activity on MRP1 is inﬂuenced by the nature of the aromatic resi-

dues since all anthracene derivatives are inactive allowing us to

identify the molecular determinants for this activity.

6.1.1. (S) (E)-3-Bromo-2-methylpropyl 3-(3,4,5-trimethoxyphenyl)

Also noteworthy is the identiﬁcation of two compounds, (S)-3

and (R)-7, able to induce collateral sensitivity (CS) against MDR1-

and MRP1-overexpressing cells. Therefore, these two compounds

can be considered as CS-promoting agents and could represent

interesting leads for the development of selective cytotoxic agents

for drug-resistant cells. In a biological perspective, these results are

remarkable at least for two reasons. First, it was possible to identify

two compounds, (S)-3 and (R)-7, that exhibited anti-cancer activity

in the low micromolar/nanomolar range, i.e. a range that are

comparable to most common chemotherapeutic drugs. Second,

while several sensitizers for MDR1-expressing cells have been

designed and tested at preclinical level, the number of sensitizers

effective against MRP1-expressing cells is signiﬁcantly lower. Our

study provides a comprehensive view of synthesis, physical-

chemical properties and biological efﬁcacy of two new potent and

MDR1/MRP1-expressing cells sensitizer compounds.

acrylate (S)-13

To

a solution of 467.0 mg (1.96 mmol) of ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid in 6 mL of CHCl₃(free of ethanol),

0.25 mL (3.9 mmol) of SOCl₂were added and the solution was

stirred for 4 h at 60 ^ꢂC. 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 (S)-3-bromo-2-(methyl)propan-1-ol

(0.20 mL, 1.96 mmol) was added. The mixture was maintained at

room temperature for 18 h 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

crude product was then puriﬁed by ﬂash chromatography using

cyclohexane/ethyl acetate 70:30 as eluent. The desired compound

was obtained as an oil. Yield: 98.4%.

Taking altogether these results into account, this series of MDR

modulators seems very promising and deserves further investiga-

tion. Work is underway to expand structure activity relationships

and to improve potency and efﬁcacy in this series of molecules.

¹H NMR (400 MHz, CDCl₃)

6.70 (s, 2H, CH arom.); 6.30 (d, J ¼ 15.6 Hz, 1H, CH]CH); 4.17e4.12

(m, 1H, OCHH); 4.10e4.05 (m, 1H, OCHH); 3.82 (s, 6H, OCH₃); 3.81

(s, 3H, OCH₃); 3.46e3.38 (m, 2H, CH₂Br); 2.22e2.17 (m, 1H, CH);

d: 7.54 (d, J ¼ 15.6 Hz, 1H, CH]CH);

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

13

1.05 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹³C APT NMR (CDCl₃)

: 166.61 (C]O); 153.40 (C); 145.04 (CH]

Yield: 99%.

d

¹H NMR (400 MHz, CDCl₃)

d: 8.53 (s, 1H, CH. arom.); 8.07e8.02

CH); 140.19 (C); 129.74 (C); 116.89 (CH]CH); 105.28 (CH arom.);

66.99 (CH₂); 60.88 (OCH₃); 56.12 (OCH₃); 36.92 (CH₂); 34.77 (CH);

15.75 (CH₃) ppm.

(m, 4H, CH arom.); 7.58e7.48 (m, 4H, CH arom.); 4.65e4.56 (m, 2H,

OCH₂); 3.55e3.46 (m, 2H, CH₂Br); 2.44e2.36 (m, 1H, CH); 1.18 (d,

J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.2. (R) (E)-3-Bromo-2-methylpropyl 3-(3,4,5-trimethoxyphenyl)

acrylate (R)-13

6.1.7. (S) (E)-3-Iodo-2-methylpropyl 3-(3,4,5-trimethoxyphenyl)

acrylate (S)-16

Following the same procedure described for compound (S)-13,

starting from (R)-3-bromo-2-(methyl)propan-1-ol, compound (R)-

13 was obtained as an oil. Yield: 95.9%.

To a solution of 719.4 mg (1.93 mmol)) of compound (S)-13 in

8 ml of acetone, NaI (1.16 g, 7.70 mmol) was added and the resulting

mixture was maintained at reﬂux in the dark for 18 h. The reaction

mixture was cooled to room temperature, and the solvent was

removed under reduced pressure; the residue was dissolved in

CH₂Cl₂and washed three times with water. The organic layer was

dried with Na₂SO₄, and the solvent was removed under reduced

pressure yielding 721.4 mg (89%) of the desired compound (S)-16 as

a solid which was used as such for the next reaction. Mp: 81e84 ^ꢂC.

¹H NMR (400 MHz, CDCl₃)

d: 7.60 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.75 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.24e4.19

(m, 1H, OCHH); 4.16e4.011 (m, 1H, OCHH); 3.89 (s, 6H, OCH₃); 3.88

(s, 3H, OCH₃); 3.55e3.42 (m, 2H, CH₂Br); 2.35e2.23 (m, 1H, CH);

1.11 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹³C APT NMR (CDCl₃)

d: 166.72 (C]O); 153.46 (C); 145.12 (CH]

CH); 129.78 (C); 116.94 (CH]CH); 105.31 (CH arom.); 66.60 (CH₂);

60.98 (OCH₃); 56.19 (OCH₃); 36.90 (CH₂); 34.82 (CH); 15.80 (CH₃)

ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.59 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.75 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.19e4.14

(m, 1H, OCHH); 4.10e4.02 (m, 1H, OCHH); 3.88 (s, 6H, OCH₃); 3.87

(s, 3H, OCH₃); 3.33e3.22 (m, 2H, CH₂I); 1.99e1.88 (m, 1H, CH); 1.07

(d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

6.1.3. (S)-3-Bromo-2-methylpropyl 3,4,5-trimethoxybenzoate (S)-

14

A solution of 416 mg (1.96 mmol) of 3,4,5-trimethoxybenzoic

acid in 15 mL of anhydrous CH₂Cl₂was cooled at 0 ^ꢂC and 0.14 mL

of (S)-3-bromo-2-(methyl)propan-1-ol (1.31 mmol), 128 mg

(1.05 mmol) of 4-dimethylaminopiridine (DMAP) and 452 mg

(2.36 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (EDCI) were added in this order. The reaction

mixture was stirred at 0 ^ꢂC for 1 h and at room temperature for 18 h

and treated with CH₂Cl₂. The organic layer was washed three times

with water and 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 chroma-

tography using cyclohexane/ethyl acetate 80:20 as eluent, yielding

426.4 mg (94%) of the desired compound as an oil.

6.1.8. (R) (E)-3-Iodo-2-methylpropyl 3-(3,4,5-trimethoxyphenyl)

acrylate (R)-16

Following the same procedure described for compound (S)-16,

starting from 625.6 mg (1.60 mmol) of (R)-13, compound (R)-16

was obtained as an oil. Yield: 93%.

¹H NMR (400 MHz, CDCl₃)

d

: 7.55 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.71 (s, 2H, CH arom.); 6.30 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.16e4.07

(m, 1H, OCHH); 4.05e3.97 (m, 1H, OCHH); 3.84 (s, 6H, OCH₃); 3.83

(s, 3H, OCH₃); 3.31e3.18 (m, 2H, CH₂I); 1.95e1.85 (m, 1H, CH); 1.03

(d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.9. (S)-3-Iodo-2-methylpropyl 3,4,5-trimethoxybenzoate (S)-17

Following the same procedure described for compound (S)-16,

starting from 214.1 mg (0.62 mmol) of (S)-14, compound (S)-17 was

obtained as an oil. Yield: 99%.

¹H NMR (400 MHz, CDCl₃)

d: 7.28 (s, 2H, CH arom.); 4.33e4.23

(m, 2H, OCH₂); 3.89 (s, 9H, OCH₃); 3.40 (d, J ¼ 6.4 Hz, 2H, CH₂Br);

2.38e2.32 (m, 1H, CH); 1.03 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.27 (s, 2H, CH arom.); 4.25 (dd,

J ¼ 11.2 Hz, J ¼ 5.6 Hz,1H, OCHH); 4.16 (dd, J ¼ 11.2 Hz, J ¼ 7.2 Hz,1H,

OCHH); 3.88 (s, 9H, OCH₃); 3.31e3.23 (m, 2H, CH₂I); 2.05e1.98 (m,

1H, CH); 1.08 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.4. (R)-3-Bromo-2-methylpropyl 3,4,5-trimethoxybenzoate (R)-

14

Following the same procedure described for compound (S)-14,

starting from (R)-3-bromo-2-(methyl)propan-1-ol, compound (R)-

14 was obtained as an oil. Yield: 98%.

6.1.10. (R)-3-Iodo-2-methylpropyl 3,4,5-trimethoxybenzoate (R)-17

Following the same procedure described for compound (S)-16,

starting from 112.2 mg (0.32 mmol) of (R)-14, compound (R)-17

was obtained as an oil. Yield: 98%.

¹H NMR (400 MHz, CDCl₃)

d: 7.17 (s, 2H, CH arom.); 4.20e4.11

(m, 2H, OCH₂); 3.78 (s, 9H, OCH₃); 3.37 (d, J ¼ 5.6 Hz, 2H, CH₂Br);

2.26e2.18 (m, 1H, CH); 1.02 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.16 (s, 2H, CH arom.); 4.14 (dd,

J ¼ 11.2 Hz, J ¼ 5.6 Hz, 1H, OCHH); 4.05 (dd, J ¼ 11.2 Hz, J ¼ 5.6 Hz,

1H, OCHH); 3.78 (s, 9H, OCH₃); 3.20e3.12 (m, 2H, CH₂I); 1.96e1.86

(m, 1H, CH); 0.97 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.5. (S)-3-Bromo-2-methylpropyl anthracene-9-carboxylate (S)-

15

Following the same procedure described for compound (S)-13,

starting from anthracene-9-carboxylic acid and (S)-3-bromo-2-

(methyl)propan-1-ol, compound (S)-15 was obtained as an oil.

Yield: 97%.

6.1.11. (S)-3-Iodo-2-methylpropyl anthracene-9-carboxylate (S)-18

Following the same procedure described for compound (S)-16,

starting from 208.6 mg (0.54 mmol) of (S)-15, compound (S)-18 was

obtained as an oil. Yield: 99%.

¹H NMR (CDCl₃)

d: 8.51 (s, 1H, CH arom.); 8.07e8.01 (m, 4H, CH

arom.); 7.58e7.48 (m, 4H, CH arom.); 4.65e4.56 (m, 2H, OCH₂);

3.55e3.46 (m, 2H, CH₂Br); 2.42e2.38 (m, 1H, CH); 1.18 (d, J ¼ 6.8 Hz,

3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 8.52 (s, 1H, CH arom.); 8.09e8.01

(m, 4H, CH arom.); 7.59e7.48 (m, 4H, CH arom.); 4.61e4.59 (m, 1H,

OCHH); 4.47e4.46 (m, 1H, OCHH); 3.32e3.29 (m, 2H, CH₂I);

2.07e1.99 (m, 1H, CH); 1.14 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.6. (R)-3-Bromo-2-methylpropyl anthracene-9-carboxylate (R)-

15

Following the same procedure described for compound (S)-13,

starting from anthracene-9-carboxylic acid and (R)-3-bromo-2-

(methyl)propan-1-ol, compound (R)-15 was obtained as an oil.

6.1.12. (R)-3-Iodo-2-methylpropyl anthracene-9-carboxylate (R)-18

Following the same procedure described for compound (S)-16,

starting from 112.7 mg (0.32 mmol) of (R)-15, compound (R)-18 was

obtained as an oil. Yield: 96%.

14

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

¹H NMR (400 MHz, CDCl₃)

d

: 8.54 (s, 1H, CH arom.); 8.07e8.02

CH₂); 0.96 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.13.5. (R)-3-((3-hydroxypropyl)amino)-2-methylpropyl

(m, 4H, CH arom.); 7.62e7.48 (m, 4H, CH arom.); 4.58 (dd,

J ¼ 11.2 Hz, J ¼ 5.2 Hz, 1H, OCHH); 4.48 (dd, J ¼ 11.2 Hz, J ¼ 7.2 Hz,

1H, OCHH); 3.34e3.23 (m, 2H, CH₂I); 2.08e2.00 (m,1H, CH); 1.16 (d,

J ¼ 7.2 Hz, 3H, CH₃) ppm.

anthra-

cene-9-carboxylate (R)-21. Starting from (S)-18 and 3-

aminopropan-1-ol, compound (R)-21 was obtained as an oil.

Yield: 77%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.1.

6.1.13. General procedure for the synthesis of the (S) and (R)

enantiomers of the hydroxyaminoesters 19e27

¹H NMR (400 MHz, CDCl₃)

d: 8.50 (s, 1H, CH arom.); 8.04e7.98

The appropriate haloester ((S) and (R)-16, (S) and (R)-17, (S) and

(R)-18) (1 mmol), K₂CO₃(1 mmol) and the suitable amino-

alkylalcohol (3-aminopropan-1-ol, 5-aminopentan-1-ol or 7-

aminoheptan-1-ol [43]) (2 mmol) were dissolved in 1 mL of anhy-

drous CH₃CN. The mixture was heated at 80 ^ꢂC for 5e10 h. The re-

action 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.

(m, 4H, CH arom.); 7.56e7.45 (m, 4H, CH arom.); 4.52 (d, J ¼ 6.0 Hz,

2H, OCH₂); 3.57 (t, J ¼ 5.2 Hz, 2H, OCH₂); 3.05 (s, 2H, NH and OH);

2.81 (t, J ¼ 5.6 Hz, 2H, NCH₂); 2.72 (dd, J ¼ 11.6 Hz, J ¼ 6.0 Hz, 1H,

NCHH); 2.56 (dd, J ¼ 11.6 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.22e2.14 (m,

1H, CH); 1.64e1.60 (m, 2H, CH₂); 1.07 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.13.6. (S)-3-((3-hydroxypropyl)amino)-2-methylpropyl

anthra-

cene-9-carboxylate (S)-21. Starting from (R)-18 and 3-

aminopropan-1-ol, compound (S)-21 was obtained as an oil.

Yield: 29%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.1.

6.1.13.1. (R) (E)-3-((3-hydroxypropyl)amino)-2-methylpropyl 3-

(3,4,5-trimethoxyphenyl)acrylate (R)-19. Starting from (S)-16 and 3-

aminopropan-1-ol, compound (R)-19 was obtained as an oil. Yield:

50%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.1.

¹H NMR (400 MHz, CDCl₃)

d: 8.48 (s, 1H, CH arom.); 8.03e7.97

(m, 4H, CH arom.); 7.53e7.44 (m, 4H, CH arom); 4.50 (d, J ¼ 6.0 Hz,

2H, OCH₂); 3.75 (t, J ¼ 5.6 Hz, 2H, OCH₂); 2.92 (s, 2H, NH and OH);

2.80 (t, J ¼ 5.6 Hz, 2H, NCH₂); 2.71 (dd, J ¼ 11.6 Hz, J ¼ 6.4 Hz, 1H,

NCHH); 2.56 (dd, J ¼ 11.6 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.22e2.14 (m,

1H, CH); 1.64e1.61 (m, 2H, CH₂); 1.06 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.54 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.72 (s, 2H, CH arom.); 6.30 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.12e4.03

(m, 2H, OCH₂); 3.84 (s, 6H, OCH₃); 3.82 (s, 3H, OCH₃); 3.75 (t,

J ¼ 5.6 Hz, 2H, OCH₂); 3.30e3.11 (bs, 2H, OH and NH); 2.83 (t,

J ¼ 6.0 Hz, 2H, NCH₂); 2.68e2.59 (m, 1H, NCHH); 2.53e2.45 (m, 1H,

NCHH); 2.05e1.97 (m, 1H, CH); 1.71e1.60 (m, 2H, CH₂); 0.97 (d,

J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.13.7. (R) (E)-3-((5-hydroxypentyl)amino)-2-methylpropyl 3-

(3,4,5-trimethoxyphenyl)acrylate (R)-22. Starting from (S)-16 and 5-

aminopentan-1-ol, compound (R)-22 was obtained as an oil. Yield:

47%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:6:0.8.

¹H NMR (400 MHz, CDCl₃)

d: 7.56 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.1.13.2. (S) (E)-3-((3-hydroxypropyl)amino)-2-methylpropyl 3-

(3,4,5-trimethoxyphenyl)acrylate (S)-19. Starting from (R)-16 and 3-

aminopropan-1-ol, compound (S)-19 was obtained as an oil. Yield:

28%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.1.

ESI-MS: 368.3[MþH]^þ.

6.73 (s, 2H, CH arom.); 6.32 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.11 (d,

J ¼ 5.6 Hz, 2H, OCH₂); 3.86 (s, 6H, OCH₃); 3.85 (s, 3H, OCH₃); 3.59 (t,

J ¼ 6.8 Hz, 2H, OCH₂); 2.68e2.51 (m, 3H, NCH₂and NCHH);

2.51e2.42 (m, 1H, NCHH); 2.11e2.01 (m, 1H, CH); 2.00e1.88 (bs, 2H,

OH and NH); 1.60e1.45 (m, 4H, CH₂); 1.43e1.32 (m, 2H, CH₂); 0.99

(d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d

: 7.59 (d, 1H, J ¼ 16.0 Hz, CH]CH);

6.76 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.18e4.05

(m, 2H, OCH₂); 3.93e3.85 (m, 9H, OCH₃); 3.81 (t, J ¼ 5.2 Hz, 2H,

OCH₂); 3.08 (bs, 2H, OH and NH); 2.90 (t, J ¼ 5.6 Hz, 2H, NCH₂);

2.72e2.67 (m, 1H, NCHH); 2.58e2.53 (m, 1H, NCHH); 2.15e2.06 (m,

1H, CH); 1.77e1.68 (m, 2H, CH₂); 1.02 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.13.8. (S) (E)-3-((5-hydroxypentyl)amino)-2-methylpropyl 3-

(3,4,5-trimethoxyphenyl)acrylate (S)-22. Starting from (R)-16 and 5-

aminopentan-1-ol, compound (S)-22 was obtained as an oil. Yield:

46%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:6:0.8.

¹H NMR (400 MHz, CDCl₃)

d

: 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.1.13.3. (R)-3-((3-hydroxypropyl)amino)-2-methylpropyl 3,4,5-

trimethoxybenzoate (R)-20. Starting from (S)-17 and 3-

aminopropan-1-ol, compound (R)-20 was obtained as an oil.

Yield: 53%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.1.

6.75 (s, 2H, CH arom.); 6.33 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.14 (d,

J ¼ 6.0 Hz, 2H, OCH₂); 3.88 (s, 6H, OCH₃); 3.87 (s, 3H, OCH₃); 3.63 (t,

J ¼ 6.4 Hz, 2H, OCH₂); 2.67e2.53 (m, 3H, NCH₂and NCHH);

2.52e2.44 (m, 1H, NCHH); 2.41e2.30 (bs, 2H, OH and NH);

2.09e2.00 (m, 1H, CH); 1.59e1.42 (m, 4H, CH₂); 1.41e1.32 (m, 2H,

CH₂); 0.98 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.28 (s, 2H, CH arom.); 4.22 (d,

J ¼ 6.0 Hz, 2H, OCH₂); 3.89 (s, 9H, OCH₃); 3.79 (t, J ¼ 5.2 Hz, 2H,

OCH₂); 2.88e2.85 (m, 2H, NCH₂); 2.68 (dd, J ¼ 11.8 Hz, J ¼ 6.4 Hz,

1H, NCHH); 2.56 (dd, J ¼ 11.8 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.15e2.10

(m, 1H, CH); 1.65e1.62 (m, 2H, CH₂); 1.04 (d, J ¼ 6.8 Hz, 3H, CH₃)

ppm.

¹³C APT NMR (100 MHz, CDCl₃)

d: 167.08 (C]O); 153.40 (C);

144.87 (CH]CH); 140.13 (C); 129.84 (C); 117.17 (CH]CH); 105.28

(CH arom.); 67.47 (CH₂); 62.28 (CH₂); 60.93 (OCH₃); 56.16 (OCH₃);

52.92 (CH₂); 49.80 (CH₂); 33.10 (CH); 32.35 (CH₂); 29.40 (CH₂);

23.39 (CH₂); 15.50 (CH₃) ppm.

6.1.13.4. (S)-3-((3-hydroxypropyl)amino)-2-methylpropyl 3,4,5-

trimethoxybenzoate (S)-20. Starting from (R)-17 and 3-

aminopropan-1-ol, compound (S)-20 was obtained as an oil.

Yield: 68%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.1.

6.1.13.9. (R)-3-((5-hydroxypentyl)amino)-2-methylpropyl 3,4,5-

trimethoxybenzoate (R)-23. Starting from (S)-17 and 5-

aminopentan-1-ol, compound (R)-23 was obtained as an oil.

Yield: 63%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.5.

¹H NMR (400 MHz, CDCl₃)

d

: 7.20 (s, 2H, CH arom.); 4.15 (d,

¹H NMR (400 MHz, CDCl₃)

d: 7.28 (s, 2H, CH arom.); 4.29e4.20

J ¼ 6.0 Hz, 2H, OCH₂); 3.81 (s, 9H, OCH₃); 3.70 (t, J ¼ 5.6 Hz, 2H,

OCH₂); 3.01 (s, 2H, NH and OH); 2.78 (t, J ¼ 5.6 Hz, 2H, NCH₂); 2.61

(dd, J ¼ 12.0 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.48 (dd, J ¼ 12.0 Hz,

J ¼ 6.8 Hz, 1H, NCHH); 2.09e2.01 (m, 1H, CH); 1.64e1.58 (m, 2H,

(m, 2H, OCH₂); 3.89 (s, 9H, OCH₃); 3.61 (t, J ¼ 6.4 Hz, 2H, OCH₂);

2.67 (dd, J ¼ 12.0 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.62 (t, J ¼ 7.2 Hz, 2H,

CH₂); 2.55 (dd, J ¼ 12.0 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.17e2.12 (m, 1H,

CH); 1.84 (s, 2H, NH and OH); 1.59e1.48 (m, 4H, CH₂); 1.43e1.39 (m,

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

15

2H, CH₂); 1.04 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

aminoheptan-1-ol [43], compound (R)-26 was obtained as an oil.

Yield: 41%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.5.

6.1.13.10. (S)-3-((5-hydroxypentyl)amino)-2-methylpropyl 3,4,5-

trimethoxybenzoate (S)-23. Starting from (R)-17 and 5-

aminopentan-1-ol, compound (S)-23 was obtained as an oil.

Yield: 57%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.5.

¹H NMR (400 MHz, CDCl₃)

d: 7.21 (s, 2H, CH arom.); 4.19 (d,

J ¼ 6.0 Hz, 2H, OCH₂); 3.82 (s, 9H, OCH₃); 3.50 (t, J ¼ 6.4 Hz, 2H,

OCH₂); 3.44 (s, 2H, NH and OH); 2.70 (dd, J ¼ 12.4 Hz, J ¼ 6.8 Hz, 1H,

NCHH); 2.64e2.55 (m, 3H, NCH₂and NCHH); 2.26e2.18 (m, 1H,

CH); 1.51e1.42 (m, 4H, CH₂); 1.32e1.18 (m, 6H, CH₂); 1.02 (d,

J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.19 (s, 2H, CH arom.); 4.15 (dd,

J ¼ 6.0 Hz, J ¼ 2.0 Hz, 2H, OCH₂); 3.80 (s, 9H, OCH₃); 3.49 (t,

J ¼ 6.4 Hz, 2H, OCH₂); 2.58 (dd, J ¼ 12.0 Hz, J ¼ 6.8 Hz 1H, NCHH);

2.51 (t, J ¼ 7.2 Hz, 2H, NCH₂); 2.45 (dd, J ¼ 12.0 Hz, J ¼ 6.8 Hz, 1H,

NCHH); 2.24 (s, 2H, NH and OH); 2.10e2.02 (m, 1H, CH); 1.49e1.38

(m, 4H, CH₂); 1.33e1.27 (m, 2H, CH₂); 0.95 (d, J ¼ 6.8 Hz, 3H, CH₃)

ppm.

6.1.13.16. (S)-3-((7-hydroxyheptyl)amino)-2-methylpropyl 3,4,5-

trimethoxybenzoate (S)-26. Starting from (R)-17 and 7-

aminoheptan-1-ol [43], compound (S)-26 was obtained as an oil.

Yield: 50%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

90:10:1.

6.1.13.11. (R)-3-((5-hydroxypentyl)amino)-2-methylpropyl anthra-

cene-9-carboxylate (R)-24. Starting from (S)-18 and 5-

aminopentan-1-ol, compound (R)-24 was obtained as an oil.

Yield: 50%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:6:0.8.

¹H NMR (400 MHz, CDCl₃)

d: 7.22 (s, 2H, CH arom.); 4.18 (d,

J ¼ 6.0 Hz, 2H, OCH₂); 3.83 (s, 9H, OCH₃); 3.53 (t, J ¼ 6.4 Hz, 2H,

OCH₂); 2.62 (dd, J ¼ 12.0 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.54e2.46 (m,

3H, NCH₂and NCHH); 2.12e2.09 (m, 3H, CH, NH and OH);

1.48e1.40 (m, 4H, CH₂); 1.34e1.20 (m, 6H, CH₂); 0.98 (d, J ¼ 6.8 Hz,

3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 8.50 (s, 1H, CH arom.); 8.04e7.99

(m, 4H, CH arom.); 7.55e7.45 (m, 4H, CH arom.); 4.55 (d, J ¼ 6.4 Hz,

2H, OCH₂); 3.54 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.73e2.55 (m, 4H, NCH₂);

2.45e2.17 (m, 1H, CH); 1.85 (s, 2H, NH and OH); 1.52e1.39 (m, 4H,

CH₂); 1.34e1.27 (m, 2H, CH₂); 1.07 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.13.17. (R)-3-((7-hydroxyheptyl)amino)-2-methylpropyl anthra-

cene-9-carboxylate (R)-27. Starting from (S)-18 and 7-

aminoheptan-1-ol [43], compound (R)-27 was obtained as an oil.

Yield: 45%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.5.

6.1.13.12. (S)-3-((5-hydroxypentyl)amino)-2-methylpropyl anthra-

cene-9-carboxylate (S)-24. Starting from (R)-18 and 5-

aminopentan-1-ol, compound (S)-24 was obtained as an oil.

Yield: 44%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:6:0.8.

¹H NMR (400 MHz, CDCl₃)

d: 8.47 (s, 1H, CH arom.); 8.01e7.96

(m, 4H, CH arom.); 7.51e7.42 (m, 4H, CH arom.); 4.52 (d, J ¼ 5.6 Hz,

2H, OCH₂); 3.52 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.68 (dd, J ¼ 12.0 Hz,

6.8 Hz, 1H, NCHH); 2.57e2.51 (m, 3H, NCH₂and NCHH); 2.23e2.17

(m,1H, CH); 1.46e1.38 (m, 4H, CH₂); 1.28e1.15 (m, 6H, CH₂); 1.04 (d,

J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 8.52 (s, 1H, CH arom.); 8.05e8.00

(m, 4H, CH arom.); 7.55e7.46 (m, 4H, CH arom.); 4.56 (d, J ¼ 6.0 Hz,

2H, OCH₂); 3.57 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.75e2.57 (m, 4H, NCH₂);

2.26e2.19 (m, 1H, CH); 1.79 (s, 2H, NH and OH); 1.55e1.42 (m, 4H,

CH₂); 1.37e1.30 (m, 2H, CH₂); 1.08 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.13.18. (S)-3-((7-hydroxyheptyl)amino)-2-methylpropyl anthra-

cene-9-carboxylate (S)-27. Starting from (R)-18 and 7-

aminoheptan-1-ol [43], compound (S)-27 was obtained as an oil.

Yield: 56%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.5.

6.1.13.13. (R) (E)-3-((7-hydroxyheptyl)amino)-2-methylpropyl 3-

(3,4,5-trimethoxyphenyl)acrylate (R)-25. Starting from (S)-16 and 7-

aminoheptan-1-ol [43], compound (R)-25 was obtained as an oil.

Yield: 50%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.5.

¹H NMR (400 MHz, CDCl₃)

d: 8.46 (s, 1H, CH arom.); 8.02e7.95

(m, 4H, CH arom.); 7.51e7.42 (m, 4H, CH arom.); 4.52 (d, J ¼ 6.0 Hz,

2H, OCH₂); 3.53 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.68 (dd, J ¼ 11.6 Hz,

J ¼ 6.8 Hz, 1H, NCHH); 2.56e2.50 (m, 3H, NCH₂and NCHH);

2.22e2.15 (m, 1H, CH); 1.97 (s, 2H, NH and OH); 1.48e1.36 (m, 4H,

CH₂); 1.27e1.15 (m, 6H, CH₂); 1.04 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d

: 7.58 (d, J ¼ 15.6 Hz, 1H, CH]CH);

6.75 (s, 2H, CH arom.); 6.34 (d, J ¼ 15.6 Hz, 1H, CH]CH); 4.14 (d,

J ¼ 6.0 Hz, 2H, OCH₂); 3.90 (s, 6H, OCH₃); 3.88 (s, 3H, OCH₃); 3.61 (t,

J ¼ 6.6 Hz, 2H, OCH₂); 2.68e2.50 (m, 4H, NCH₂); 2.10e2.06 (m, 1H,

CH); 1.76 (s, 2H, NH and OH); 1.54e1.50 (m, 4H, CH₂); 1.45e1.26 (m,

6H, CH₂); 1.01 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.14. General procedure for the synthesis of the (S) and (R)

enantiomers of the (hydroxyalkyl) methylaminoesters 28e36

A 1 mmol portion of the appropriate hydroxyaminoester ((R)

and (S) 19e27) 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 ^ꢂC for 4e5 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 and the

residue puriﬁed by ﬂash chromatography, yielding a yellow oil.

6.1.13.14. (S) (E)-3-((7-hydroxyheptyl)amino)-2-methylpropyl 3-

(3,4,5-trimethoxyphenyl)acrylate (S)-25. Starting from (R)-16 and

7-aminoheptan-1-ol [43], compound (S)-25 was obtained as an oil.

Yield: 25%. Chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

95:5:0.5.

¹H NMR (400 MHz, CDCl₃)

d: 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.74 (s, 2H, CH arom.); 6.33 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.14 (dd,

J ¼ 6.0 Hz, J ¼ 2.4 Hz, 2H, OCH₂); 3.86 (s, 6H, OCH₃); 3.85 (s, 3H,

OCH₃); 3.60 (t, J ¼ 6.6 Hz, 2H, OCH₂); 2.65 (dd, J ¼ 12.0 Hz,

J ¼ 6.8 Hz, 1H, NCHH); 2.58 (t, J ¼ 7.2 Hz, 2H, NCH₂); 2.51 (dd,

J ¼ 12.0 Hz, J ¼ 6.8 Hz, 1H, NCHH); 2.11e2.03 (m, 1H, CH); 1.68 (s,

2H, NH and OH); 1.55e1.46 (m, 4H, CH₂); 1.44e1.27 (m, 6H, CH₂);

1.01 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.14.1. (R)-3-((3-hydroxypropyl)(methyl)amino)-2-methylpropyl

(E)-3-(3,4,5-trimethoxyphenyl) acrylate (R)-28. From (R)-19. Chro-

matographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.5. Yield: 64%.

¹H NMR (400 MHz, CDCl₃)

d

: 7.57 (d, J ¼ 15.6 Hz, 1H, CH]CH);

6.73 (s, 2H, CH arom.); 6.33 (d, J ¼ 15.6 Hz, 1H, CH]CH); 4.20e4.13

(m, 1H, OCHH); 4.08e3.98 (m, 1H, OCHH); 3.86 (s, 6H, OCH₃); 3.85

(s, 3H, OCH₃); 3.77 (t, J ¼ 5.2 Hz, 2H, OCH₂); 2.61 (t, J ¼ 5.6 Hz, 2H,

NCH₂); 2.44e2.35 (m, 1H, NCHH); 2.34e2.25 (m, 1H, NCHH); 2.29

6.1.13.15. (R)-3-((7-hydroxyheptyl)amino)-2-methylpropyl 3,4,5-

trimethoxybenzoate (R)-26. Starting from (S)-17 and 7-

16

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

(s, 3H, NCH₃); 2.18e2.06 (m, 1H, CH); 1.76e1.63 (m, 2H, CH₂); 1.02

(d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.14.7. (R)-3-((5-hydroxypentyl)(methyl)amino)-2-methylpropyl

(E)-3-(3,4,5-trimethoxyphenyl) acrylate (R)-31. From (R)-22. Chro-

matographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 93%.

¹³C APT NMR (CDCl₃)

d: 166.94 (C]O); 153.43 (C); 144.87 (CH]

CH); 140.19 (C); 129.87 (C); 117.18 (CH]CH); 105.33 (CH arom.);

67.80 (CH₂); 63.88 (CH₂); 62.05 (CH₂); 60.92 (OCH₃); 58.54 (CH₂);

56.17 (OCH₃); 42.08 (NCH₃); 30.97 (CH); 27.85 (CH₂); 15.65 (CH₃)

ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.57 (d, J ¼ 15.6 Hz 1H, CH]CH);

6.74 (s, H, 2CH arom.); 6.33 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.28e4.21

(m, 1H, OCHH); 4.06e3.98 (m, 1H, OCHH); 3.87 (s, 6H, OCH₃); 3.86

(s, 3H, OCH₃); 3.61 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.36e2.26 (m, 3H, NCH₂

and NCHH); 2.18 (s, 3H, NCH₃); 2.17e2.10 (m, 1H, NCHH); 2.09e2.01

(m,1H, CH); 1.98 (bs,1H, OH); 1.60e1.52 (m, 2H, CH₂); 1.51e1.41 (m,

2H, CH₂); 1.41e1.32 (m, 2H, CH₂); 0.98 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.14.2. (S)-3-((3-hydroxypropyl)(methyl)amino)-2-methylpropyl

(E)-3-(3,4,5-trimethoxyphenyl) acrylate (S)-28. From (S)-19. Chro-

matographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.5. Yield: 79%.

¹³C APT NMR (CDCl₃)

d: 167.16 (C]O); 153.43 (C); 144.62 (CH]

¹H NMR (400 MHz, CDCl₃)

d: 7.60 (d, J ¼ 16.0 Hz, 1H, CH]CH);

CH); 139.81 (C); 129.94 (C); 117.44 (CH]CH); 105.27 (CH arom.);

68.18 (CH₂); 62.83 (CH₂); 61.32 (CH₂); 60.95 (OCH₃); 58.10 (CH₂);

56.18 (OCH₃); 42.74 (NCH₃); 32.55 (CH₂); 31.27 (CH); 26.99 (CH₂);

23.47 (CH₂); 15.91 (CH₃) ppm.

6.76 (s, 2H, CH arom.); 6.35 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.22e4.16

(m, 1H, OCHH); 4.10e4.02 (m, 1H, OCHH); 3.89 (s, 6H, OCH₃); 3.87

(s, 3H, OCH₃); 3.81 (t, J ¼ 5.2 Hz 2H, OCH₂); 2.73e2.65 (m, 2H,

NCH₂); 2.51e2.44 (m,1H, NCHH); 2.43e2.36 (m,1H, NCHH); 2.35 (s,

3H, NCH₃); 2.26e2.14 (m, 1H, CH); 1.81e1.70 (m, 2H, CH₂); 1.06 (d,

J ¼ 6.4 Hz, 3H, CH₃) ppm.

6.1.14.8. (S)-3-((5-hydroxypentyl)(methyl)amino)-2-methylpropyl

(E)-3-(3,4,5-trimethoxyphenyl) acrylate (S)-31. From (S)-22. Chro-

matographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 94%.

¹³C APT NMR (CDCl₃)

d: 166.95 (C]O); 153.43 (C); 144.87 (CH]

¹H NMR (400 MHz, CDCl₃)

d: 7.58 (d, J ¼ 15.6 Hz, 1H, CH]CH);

CH); 140.16 (C); 129.88 (C); 117.19 (CH]CH); 105.30 (CH arom.);

67.84 (CH₂); 64.03 (CH₂); 62.08 (CH₂); 60.95 (OCH₃); 58.70 (CH₂);

56.17 (OCH₃); 42.12 (NCH₃); 30.99 (CH); 27.84 (CH₂); 15.70 (CH₃)

ppm.

6.74 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.28e4.19

(m, 1H, OCHH); 4.07e3.98 (m, 1H, OCHH); 3.87 (s, 6H, OCH₃); 3.86

(s, 3H, OCH₃); 3.61 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.37e2.25 (m, 3H, NCH₂

and NCHH); 2.19 (s, 3H, NCH₃); 2.18e2.11 (m, 1H, NCHH); 2.10e2.01

(m, 1H, CH); 1.60e1.50 (m, 2H, CH₂); 1.50e1.42 (m, 2H, CH₂);

1.41e1.32 (m, 2H, CH₂); 0.99 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.14.3. (R)-3-((3-hydroxypropyl)(methyl)amino)-2-methylpropyl

3,4,5-trimethoxybenzoate (R)-29. From (R)-20. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.5. Yield: 82%.

6.1.14.9. (R)-3-((5-hydroxypentyl)(methyl)amino)-2-methylpropyl

3,4,5-trimethoxybenzoate (R)-32. From (R)-23. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 84%.

¹H NMR (400 MHz, CDCl₃)

d: 7.28 (s, 2H, CH arom.); 4.28 (dd,

J ¼ 10.8 Hz, J ¼ 5.2 Hz, 1H, OCHH); 4.13 (dd, J ¼ 10.8 Hz, J ¼ 6.0 Hz

1H, OCHH); 3.88 (s, 9H, OCH₃); 3.77 (t, J ¼ 5.2 Hz, 2H, OCH₂); 2.57 (t,

J ¼ 5.2 Hz, 2H, NCH₂); 2.39e2.35 (m, 1H, NCHH); 2.28e2.16 (m, 2H,

NCHH and CH); 2.24 (s, 3H, NCH₃); 1.71e1.67 (m, 2H, CH₂); 1.02 (d,

J ¼ 6.4 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.28 (s, 2H, CH arom.); 4.33 (dd,

J ¼ 10.8 Hz, J ¼ 4.4 Hz, 1H, OCHH); 4.11 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz,

1H, OCHH); 3.88 (s, 9H, OCH₃); 3.59 (t, J ¼ 6.4 Hz, 2H, OCH₂);

2.32e2.22 (m, 3H, NCH₂and NCHH); 2.18 (s, 3H, NCH₃); 2.16e2.11

(m, 2H, NCHH and CH); 1.54e1.41 (m, 4H, CH₂); 1.38e1.34 (m, 2H,

CH₂); 1.00 (d, J ¼ 6.0 Hz, 3H, CH₃) ppm.

6.1.14.4. (S)-3-((3-hydroxypropyl)(methyl)amino)-2-methylpropyl

3,4,5-trimethoxybenzoate (S)-29. From (S)-20. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.5. Yield: 84%.

6.1.14.10. (S)-3-((5-hydroxypentyl)(methyl)amino)-2-methylpropyl

3,4,5-trimethoxybenzoate (S)-32. From (S)-23. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 88%.

¹H NMR (400 MHz, CDCl₃)

d: 7.23 (s, 2H, CH arom.); 4.23 (dd,

J ¼ 10.8 Hz, J ¼ 5.2 Hz, 1H, OCHH); 4.07 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz,

1H, OCHH); 3.83 (s, 9H, OCH₃); 3.71 (t, J ¼ 5.2 Hz, 2H, CH₂); 2.52 (t,

J ¼ 5.6 Hz, 2H, CH₂); 2.32 (dd, J ¼ 12.0 Hz, J ¼ 6.4 Hz, 1H, NCHH);

2.23e2.11 (m, 2H, NCHH and CH); 2.19 (s, 3H, NCH₃); 1.65e1.62 (m,

2H, CH₂); 0.97 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.23 (s, 2H, CH arom.); 4.28 (dd,

J ¼ 10.8 Hz, J ¼ 4.8 Hz, 1H, OCHH); 4.06 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz,

1H, OCHH); 3.83 (s, 9H, OCH₃); 3.53 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.46 (s,

1H, OH); 2.31e2.25 (m, 3H, NCH₂and NCHH); 2.14 (s, 3H, NCH₃);

2.13e2.05 (m, 2H, NCHH and CH); 1.51e1.37 (m, 4H, CH₂); 1.33e1.27

(m, 2H, CH₂); 0.96 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

6.1.14.5. (R)-3-((3-hydroxypropyl)(methyl)amino)-2-methylpropyl

anthracene-9-carboxylate (R)-30. From (R)-21. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 98%.

6.1.14.11. (R)-3-((5-hydroxypentyl)(methyl)amino)-2-methylpropyl

anthracene-9-carboxylate (R)-33. From (R)-24. Chromatographic

eluent: CH₂Cl₂/CH₃OH 90:10. Yield: 99%.

¹H NMR (400 MHz, CDCl₃)

d: 8.52 (s, 1H, CH arom.); 8.06e8.01

(m, 4H, CH arom.); 7.56e7.47 (m, 4H, CH arom.); 4.61 (dd,

J ¼ 10.8 Hz, J ¼ 6.8 Hz, 1H, OCHH); 4.48 (dd, J ¼ 10.8 Hz, J ¼ 5.2 Hz,

1H, OCHH); 3.80 (t, J ¼ 5.2 Hz, 2H, OCH₂); 2.58 (t, J ¼ 5.2 Hz, 2H,

NCH₂); 2.46e2.42 (m, 1H, CH); 2.35e2.26 (m, 2H, NCH₂); 2.27 (s,

3H, NCH₃); 1.72e1.68 (m, 2H, CH₂); 1.10 (d, J ¼ 5.6 Hz 3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 8.51 (s, 1H, CH arom.); 8.07e8.00

(m, 4H, CH arom.); 7.55e7.46 (m, 4H, CH arom.); 4.66 (dd,

J ¼ 10.8 Hz, J ¼ 4.4 Hz, 1H, OCHH), 4.48 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz,

1H, OCHH); 3.59 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.41e2.30 (m, 3H, NCH₂

and NCHH); 2.23 (s, 3H, NCH₃); 2.20e2.16 (m, 2H, NCHH and CH);

1.56e1.43 (m, 4H, CH₂); 1.39e1.33 (m, 2H, CH₂); 1.06 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

6.1.14.6. (S)-3-((3-hydroxypropyl)(methyl)amino)-2-methylpropyl

anthracene-9-carboxylate (S)-30. From (S)-21. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 92%.

¹H NMR (400 MHz, CDCl₃)

d: 8.48 (s, 1H, CH arom.); 8.00 (m, 4H,

6.1.14.12. (S)-3-((5-hydroxypentyl)(methyl)amino)-2-methylpropyl

anthracene-9-carboxylate (S)-33. From (S)-24. Chromatographic

eluent: CH₂Cl₂/CH₃OH 90:10. Yield: 95%.

CH arom.); 7.52e7.43 (m, 4H, CH arom.); 4.58 (dd, J ¼ 10.8 Hz,

J ¼ 4.8 Hz 1H, OCHH); 4.45 (dd, J ¼ 10.8 Hz, J ¼ 5.6 Hz, 1H, OCHH);

3.77 (t, J ¼ 5.2 Hz, 2H, OCH₂); 2.53 (t, J ¼ 6.0 Hz, 2H, NCH₂); 2.32 (dd,

J ¼ 15.2 Hz, J ¼ 10.0 Hz, 1H, NCHH); 2.26e2.23 (m, 2H, NCHH and

CH); 2.23 (s, 3H, NCH₃); 1.69e1.63 (m, 2H, CH₂); 1.07 (d, J ¼ 6.0 Hz,

3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 8.51 (s, 1H, CH arom.); 8.07e8.00

(m, 4H, CH arom.); 7.55e7.46 (m, 4H, CH arom.); 4.66 (dd,

J ¼ 10.8 Hz, J ¼ 4.4 Hz, 1H, OCHH), 4.48 (dd, J ¼ 10.8 Hz, J ¼ 6.0 Hz,

1H, OCHH); 3.59 (t, J ¼ 6.4 Hz, 2H, OCH₂); 2.42e2.25 (m, 3H, NCH₂

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

17

and NCHH); 2.22 (s, 3H, NCH₃); 2.20e2.16 (m, 2H, NCHH and CH);

1.57e1.44 (m, 4H, CH₂); 1.39e1.33 (m, 2H, CH₂); 1.06 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

(s, 1H, OH); 1.50e1.41 (m, 4H, CH₂); 1.32e1.18 (m, 6H, CH₂); 1.04 (d,

J ¼ 6.0 Hz, 3H, CH₃) ppm.

6.1.15. General procedures for the synthesis of the (S) and (R)

enantiomers of the diesters 1e12

Diester compounds were synthesized using two different gen-

eral procedures:

6.1.14.13. (R)-3-((7-hydroxyheptyl)(methyl)amino)-2-methylpropyl

(E)-3-(3,4,5-trimethoxyphenyl) acrylate (R)-34. From (R)-25. Chro-

matographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 89%.

¹H NMR (400 MHz, CDCl₃)

d: 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.73 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.12 (m,

2H, OCH₂); 3.88 (s, 6H, OCH₃); 3.86 (s, 3H, OCH₃); 3.61 (t, J ¼ 6.6 Hz,

2H, OCH₂); 2.32e2.27 (m, 3H, NCH₂and NCHH); 2.18 (s, 3H, NCH₃);

2.19e2.00 (m, 2H, NCHH and CH); 1.53 (s, 2H, NH and OH);

1.45e1.36 (m, 4H, CH₂); 1.34e1.18 (m, 6H, CH₂); 0.98 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

6.1.15.1. General procedure A. A solution of the suitable (hydrox-

yalkyl)methylaminoester (0.250 mmol) in 5 mL of an. CH₂Cl₂was

cooled at 0 ^ꢂC and 0.360 mmol of the appropriate carboxylic acid

((E)-3-(3,4,5-trimethoxyphenyl)acrylic

acid

or

3,4,5-

trimethoxybenzoic 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 ^ꢂC 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

solution was concentrated to dryness and the crude product was

puriﬁed by ﬂash chromatography with the eluting systems

described below. The pure compounds were transformed into the

corresponding white solid hydrochloride salts that were crystal-

lized from abs. ethanol/petroleum ether.

6.1.14.14. (S)-3-((7-hydroxyheptyl)(methyl)amino)-2-methylpropyl

(E)-3-(3,4,5-trimethoxyphenyl) acrylate (S)-34. From (S)-25. Chro-

matographic eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 97%.

¹H NMR (400 MHz, CDCl₃)

d

: 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH);

6.73 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.12 (m,

2H, OCH₂), 3.87 (s, 6H, OCH₃); 3.85 (s, 3H, OCH₃); 3.61 (t, J ¼ 6.6 Hz,

2H, OCH₂); 2.32e2.27 (m, 3H, NCH₂and NCHH); 2.18 (s, 3H, NCH₃);

2.19e2.00 (m, 2H, NCHH and CH); 1.55e1.51 (m, 2H, CH₂);

1.50e1.40 (m, 2H, CH₂); 1.28e1.15 (m, 6H, CH₂); 0.98 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

6.1.15.2. General procedure B. A 0.250 mmol portion of the appro-

priate carboxylic acid ((E)-3-(3,4,5-trimethoxyphenyl)acrylic 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 ^ꢂC for 4e5 h.

After cooling to room temperature, the solution was concen-

trated to dryness; the mixture was washed twice with cyclohexane

and then organic layer was removed by a rotary evaporation. The

acyl chloride obtained was dissolved in CHCl₃(free of ethanol), and

the appropriate (hydroxyalkyl)methylaminoester (1 eq) was added.

The mixture was heated to 60 ^ꢂC for 4 h, cooled to room tempera-

ture and then CH₂Cl₂was added. The organic phase was washed

with 10% NaOH solution, dried with Na₂SO₄and then concentrated

to dryness. The crude products obtained were puriﬁed by ﬂash

chromatography with the eluting systems described below, and the

desired compounds were obtained as oil.

6.1.14.15. (R)-3-((7-hydroxyheptyl)(methyl)amino)-2-methylpropyl

3,4,5-trimethoxybenzoate (R)-35. From (R)-26. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 91%.

¹H NMR (400 MHz, CDCl₃)

d: 7.21 (s, 2H, CH arom.); 4.25 (dd,

J ¼ 10.4 Hz, J ¼ 4.4 Hz, 1H, OCHH); 4.04 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz,

1H, OCHH); 3.81 (s, 9H, OCH₃); 3.50 (t, J ¼ 6.8 Hz, 2H, OCH₂);

2.29e2.19 (m, 4H, NCHH, CH₂and OH); 2.14e2.03 (m, 2H, NCHH

and CH); 2.11 (s, 3H, NCH₃); 1.45e1.34 (m, 4H, CH₂); 1.26e1.12 (m,

6H, CH₂); 0.93 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

6.1.14.16. (S)-3-((7-hydroxyheptyl)(methyl)amino)-2-methylpropyl

3,4,5-trimethoxybenzoate (S)-35. From (S)-26. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 97%.

¹H NMR (400 MHz, CDCl₃)

d: 7.24 (s, 2H, CH arom.); 4.29 (dd,

J ¼ 10.8 Hz, 4.4 Hz, 1H, OCHH); 4.07 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H,

OCHH); 3.84 (s, 9H, OCH₃); 3.55 (t, J ¼ 6.8 Hz, 2H, OCH₂); 2.32e2.23

(m, 3H, NCHH and CH₂); 2.17e2.06 (m, 2H, NCHH and CH); 2.15 (s,

3H, NCH₃); 1.50e1.38 (m, 4H, CH₂); 1.28e1.15 (m, 6H, CH₂); 0.97 (d,

J ¼ 6.4 Hz, 3H, CH₃) ppm.

All the compounds were transformed into the corresponding

hydrochloride as white solid. The salts were crystallized from abs.

ethanol/petroleum ether.

6.1.15.3. (R) (E)-3-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl 3,4,5-trimethoxybenzoate (R)-1.

Procedure A, starting from (R)-28 and 3,4,5-trimethoxybenzoic

acid.

6.1.14.17. (R)-3-((7-hydroxyheptyl)(methyl)amino)-2-methylpropyl

anthracene-9-carboxylate (R)-36. From (R)-27. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 93:7:0.3. Yield: 90%.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 74%.

¹H NMR (400 MHz, CDCl₃)

d: 8.48 (s, 1H, CH arom.); 8.04e7.97

(m, 4H, CH arom.); 7.52e7.43 (m, 4H, CH arom.); 4.61 (dd,

J ¼ 10.4 Hz, J ¼ 4.0 Hz, 1H, OCHH); 4.46 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz,

1H, OCHH); 3.57 (t, J ¼ 6.8 Hz, 2H, OCH₂); 2.38e2.27 (m, 3H, NCHH

and CH₂); 2.19 (s, 3H, NCH₃); 2.19e2.13 (m, 2H, NCHH and CH);

1.50e1.42 (m, 4H, CH₂); 1.34e1.20 (m, 6H, CH₂); 1.04 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.56 (d, J ¼ 16.0 Hz, 1H, CH]CH);

7.26 (s, 2H, CH arom.); 6.72 (s, 2H, CH arom.); 6.31 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.36 (t, J ¼ 6.4 Hz, 2H, OCH₂); 4.22 (dd, J ¼ 10.8 Hz,

J ¼ 5.2 Hz, 1H, OCHH); 4.03 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H, OCHH);

3.86 (s, 9H, OCH₃); 3.85 (s, 6H, OCH₃); 3.84 (s, 3H, OCH₃); 2.63e2.50

(m, 2H, NCH₂); 2.49e2.38 (m, 1H, NCHH); 2.30 (s, 3H, NCH₃);

2.29e2.20 (m, 1H, NCHH); 2.18e2.03 (m, 1H, CH); 2.02e1.90 (m, 2H,

CH₂); 1.01 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

6.1.14.18. (S)-3-((7-hydroxyheptyl)(methyl)amino)-2-methylpropyl

anthracene-9-carboxylate (S)-36. From (S)-27. Chromatographic

eluent: CH₂Cl₂/CH₃OH/NH₄OH 90:10:1. Yield: 92%.

¹³C APT NMR (CDCl₃)

d: 167.00 (C]O); 166.16 (C]O); 153.41 (C);

152.89 (C); 144.61 (CH]CH); 142.15 (C); 140.09 (C); 129.90 (C);

125.42(C); 117.34 (CH]CH); 106.78 (CH arom.); 105.24 (CH arom.);

67.96 (CH₂); 63.47 (CH₂); 61.34 (CH₂); 60.92 (OCH₃); 60.85 (OCH₃);

56.19 (OCH₃); 56.14 (OCH₃); 54.59 (CH₂); 42.64 (NCH₃); 31.34 (CH);

26.76 (CH₂); 15.79 (CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 8.47 (s, 1H, CH arom.); 8.05e7.96

(m, 4H, CH arom.); 7.52e7.43 (m, 4H, CH arom.); 4.63 (dd,

J ¼ 10.8 Hz, J ¼ 4.4 Hz, 1H, OCHH); 4.46 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz,

1H, OCHH); 3.56 (t, J ¼ 6.8 Hz, 2H, OCH₂); 2.37e2.27 (m, 3H, NCHH

and CH₂); 2.19 (s, 3H, NCH₃); 2.19e2.14 (m, 2H, NCHH and CH); 1.95

ESI-HRMS (m/z) calculated for [MþH]^þion species

18

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

C₃₀H₄₂NO₁₀¼ 576.2803, found 576.2797.

Hydrochloride: mp 50e55 ^ꢂC.

NCHH); 2.12e2.02 (m, 3H, NCH₂and CH); 1.02 (d, J ¼ 6.8 Hz, 3H,

CH₃) ppm.

¹³C APT NMR (CDCl₃)

d

: 169.59 (C]O); 167.01 (C]O); 153.38

6.1.15.4. (S) (E)-3-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl 3,4,5-trimethoxybenzoate (S)-1.

Procedure A, starting from (S)-28 and 3,4,5-trimethoxybenzoic

acid.

(C); 144.88 (CH]CH); 142.55 (C); 130.96 (C); 129.84 (C); 129.33 (CH

arom.); 128.64 (CH arom.); 128.38 (C); 128.25 (C); 127.00 (CH

arom.); 125.49 (CH arom.); 124.93 (CH arom.); 117.15 (CH]CH);

105.24 (CH arom.); 67.61 (CH₂); 63.86 (CH₂); 60.96 (OCH₃); 60.92

(CH₂); 56.12 (OCH₃); 54.72 (CH₂); 42.30 (NCH₃); 30.99 (CH); 26.22

(CH₂); 15.94 (CH₃) ppm.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 61%.

¹H NMR (400 MHz, CDCl₃)

d: 7.55 (d, J ¼ 16.0 Hz, 1H, CH]CH);

ESI-HRMS (m/z) calculated for [MþH]^þion species

7.25 (s, 2H, CH arom.); 6.72 (s, 2H, CH arom.); 6.31 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.34 (t, J ¼ 6.8 Hz, 2H, OCH₂); 4.22 (dd, J ¼ 10.8 Hz,

J ¼ 4.8 Hz, 1H, OCHH); 4.00 (dd, J ¼ 10.8 Hz, J ¼ 6.8 Hz, 1H, OCHH);

3.86 (s, 9H, OCH₃); 3.85 (s, 6H, OCH₃); 3.84 (s, 3H, OCH₃); 2.50e2.44

(m, 2H, NCH₂); 2.33 (dd, J ¼ 12.0 Hz, J ¼ 7.6 Hz, 1H, NCHH); 2.22 (s,

3H, NCH₃); 2.19e2.14 (m, 1H, NCHH); 2.10e2.01 (m, 1H, CH);

1.93e1.86 (m, 2H, CH₂); 0.97 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

C

₃₅H₄₀NO₇¼ 586.2799, found 586.2792.

Hydrochloride: mp 80e82 ^ꢂC.

6.1.15.7. (R) (E)-2-methyl-3-(methyl(3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl 3,4,5-trimethoxybenzoate (R)-3.

Procedure A, starting from (R)-29 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

¹³C APT NMR (CDCl₃)

d

: 166.96 (C]O); 166.17 (C]O); 153.42

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 96:4. Yield:

86%.

(C); 152.91 (C); 144.79 (CH]CH); 142.22 (C); 140.14 (C); 129.85 (C);

125.28 (C); 117.18 (CH]CH); 106.81 (CH arom.); 105.26 (CH arom.);

67.79 (CH₂); 63.25 (CH₂); 61.10 (CH₂); 60.93 (OCH₃); 60.87 (OCH₃);

56.22 (OCH₃); 56.15 (OCH₃); 54.55 (CH₂); 42.44 (NCH₃); 31.12 (CH);

26.39 (CH₂); 15.89 (CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.57 (d, J ¼ 16.0 Hz, 1H, CH]CH);

7.28 (s, 2H, CH arom.); 6.74 (s, 2H, CH arom.); 6.32 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.36 (dd, J ¼ 10.4 Hz, J ¼ 4.8 Hz, 1H, OCHH); 4.25 (t,

J ¼ 6.4 Hz, 2H, OCH₂); 4.14 (dd, J ¼ 10.4 Hz, J ¼ 6.4 Hz, 1H, OCHH);

3.88 (s, 9H, OCH₃); 3.87 (s, 9H, OCH₃); 2.58e2.35 (m, 3H, NCHH and

NCH₂); 2.25 (s, 3H, NCH₃); 2.24e2.10 (m, 2H, NCHH and CH);

1.90e1.80 (m, 2H, CH₂); 1.03 (d, J ¼ 6.0 Hz, 3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₀H₄₂NO₁₀¼ 576.2803, found 576.2804.

Hydrochloride: mp 46e50 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 166.96 (C]O); 166.22 (C]O); 153.43

6.1.15.5. (R) (E)-3-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl anthracene-9-carboxylate (R)-2.

Procedure B, starting from (R)-28 and anthracene-9-carboxylic

acid.

(C); 152.93 (C); 144.70 (CH]CH); 129.90 (C); 125.47 (C); 117.32

(CH]CH); 106.78 (CH arom.); 105.23 (CH arom.); 68.44 (CH₂);

62.87 (CH₂); 61.32 (CH₂); 60.97 (OCH₃); 60.91 (OCH₃); 56.21

(OCH₃); 56.16 (OCH₃); 54.74 (CH₂); 42.66 (NCH₃); 31.41 (CH); 26.73

(CH₂); 15.90 (CH₃) ppm.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 37%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

¹H NMR (400 MHz, CDCl₃)

d

: 8.50 (s, 1H, CH arom.); 8.04e7.99

C

₃₀H₄₂NO₁₀¼ 576.2803, found 576.2802.

(m, 4H, CH arom.); 7.56 (d, J ¼ 16.0 Hz, 1H, CH]CH); 7.53e7.45 (m,

4H, CH arom.); 6.69 (s, 2H, CH arom.); 6.32 (d, J ¼ 16.0 Hz, 1H, CH]

CH); 4.69 (t, J ¼ 6.4 Hz, 2H, OCH₂); 4.25 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz,

1H, OCHH); 4.08 (dd, J ¼ 10.8 Hz, J ¼ 6.0 Hz, 1H, OCHH); 3.86 (s, 3H,

OCH₃); 3.82 (s, 6H, OCH₃); 2.72e2.58 (m, 2H, NCH₂); 2.50e2.40 (m,

1H, NCHH); 2.31 (s, 3H, NCH₃); 2.30e2.22 (m, 1H, NCHH);

2.20e2.03 (m, 3H, CH₂and CH); 1.04 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

Hydrochloride: mp 49e53 ^ꢂC.

6.1.15.8. (S) (E)-2-methyl-3-(methyl(3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl 3,4,5-trimethoxybenzoate (S)-3.

Procedure A, starting from (S)-29 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 96:4. Yield:

81%.

¹³C APT NMR (CDCl₃)

d: 169.69 (C]O); 167.10 (C]O); 153.37 (C);

144.65 (CH]CH); 142.65 (C); 130.98 (C); 129.92 (C); 129.23 (CH

arom.); 128.61 (CH arom.); 128.38 (C); 128.08 (C); 126.93 (CH

arom.); 125.46 (CH arom.); 125.00 (CH arom.); 117.38 (CH]CH);

105.21 (CH arom.); 67.86 (CH₂); 64.16 (CH₂); 61.25 (CH₂); 60.95

(OCH₃); 56.11 (OCH₃); 54.78 (CH₂); 42.57 (NCH₃); 31.30 (CH); 26.75

(CH₂); 15.83 (CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.54 (d, J ¼ 16.0 Hz, 1H, CH]CH);

7.25 (s, 2H, CH arom.); 6.71 (s, 2H, CH arom.); 6.28 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.32 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz, 1H, OCHH); 4.22 (t,

J ¼ 6.8 Hz, 2H, OCH₂); 4.11 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H, OCHH);

3.85 (s, 9H, OCH₃); 3.84 (s, 6H, OCH₃); 3.83 (s, 3H, OCH₃); 2.44 (t,

J ¼ 6.8 Hz, 2H, NCH₂); 2.35 (dd, J ¼ 11.6 Hz, J ¼ 7.2 Hz, 1H, NCHH);

2.21 (s, 3H, NCH₃); 2.19e2.10 (m, 2H, NCHH and CH); 1.86e1.79 (m,

2H, CH₂); 0.99 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₅H₄₀NO₇¼ 586.2799, found 586.2791.

Hydrochloride: mp 87e92 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 166.91 (C]O); 166.19 (C]O); 153.45

(C); 152.94 (C); 144.65 (CH]CH); 140.22 (C); 129.91 (C); 125.49 (C);

117.35 (CH]CH); 106.88 (CH arom.); 105.34 (CH arom.); 68.43

(CH₂); 62.87 (CH₂); 61.37 (CH₂); 60.92 (OCH₃); 60.86 (OCH₃); 56.21

(OCH₃); 56.17 (OCH₃); 54.76 (CH₂); 42.66 (NCH₃); 31.47 (CH); 26.78

(CH₂); 15.86 (CH₃) ppm.

6.1.15.6. (S) (E)-3-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl anthracene-9-carboxylate (S)-2.

Procedure B, starting from (S)-28 and anthracene-9-carboxylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 32%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

¹H NMR (400 MHz, CDCl₃)

d: 8.50 (s, 1H, CH arom.); 8.03 (d,

C

₃₀H₄₂NO₁₀¼ 576.2803, found 576.2805.

J ¼ 8.8 Hz, 2H, CH arom.); 8.00 (d, J ¼ 8.8 Hz, 2H, CH arom.); 7.56 (d,

J ¼ 16.0 Hz, 1H, CH]CH); 7.53e7.45 (m, 4H, CH arom.); 6.69 (s, 2H,

CH arom.); 6.33 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.69 (t, J ¼ 6.4 Hz, 2H,

OCH₂); 4.26 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz, 1H, OCHH); 4.08 (dd,

J ¼ 10.8 Hz, J ¼ 6.0 Hz, 1H, OCHH); 3.86 (s, 3H, OCH₃); 3.82 (s, 6H,

OCH₃); 2.56 (t, J ¼ 6.8 Hz, 2H, NCH₂); 2.41 (dd, J ¼ 12.4 Hz, J ¼ 7.6 Hz,

1H, NCHH); 2.27 (s, 3H, NCH₃); 2.22 (dd, J ¼ 12.4 Hz, J ¼ 7.2 Hz, 1H,

Hydrochloride: mp 50e55 ^ꢂC.

6.1.15.9. (R) (E)-2-methyl-3-(methyl(3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl anthracene-9-carboxylate (R)-4.

Procedure A, starting from (R)-30 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 98:2. Yield:

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

19

67%.

¹H NMR (400 MHz, CDCl₃)

58.12 (CH₂); 56.25 (OCH₃); 56.18 (OCH₃); 42.69 (NCH₃); 31.28 (CH);

28.72 (CH₂); 26.96 (CH₂); 23.84 (CH₂); 15.88 (CH₃) ppm.

d: 8.49 (s, 1H, CH arom.); 8.04 (d,

J ¼ 8.8 Hz, 2H, CH arom.); 8.00 (d, J ¼ 8.8 Hz, 2H, CH arom.); 7.56 (d,

J ¼ 16.0 Hz, 1H, CH]CH); 7.52e7.44 (m, 4H, CH arom.); 6.68 (s, 2H,

CH arom.); 6.31 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.66 (dd, J ¼ 10.8 Hz,

J ¼ 4.0 Hz, 1H, OCHH); 4.50 (dd, J ¼ 10.8 Hz, J ¼ 5.6 Hz, 1H, OCHH);

4.27 (t, J ¼ 6.0 Hz, 2H, OCH₂); 3.86 (s, 3H, OCH₃); 3.81 (s, 6H, OCH₃);

2.60e2.39 (m, 3H, NCHH and NCH₂); 2.28 (s, 3H, NCH₃); 2.27e2.17

(m, 2H, NCHH and CH); 1.98e1.82 (m, 2H, CH₂); 1.09 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₂H₄₆NO₁₀¼ 604.3116, found 604.3115.

Hydrochloride: mp 95e98 ^ꢂC.

6.1.15.12. (S) (E)-5-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) pentyl 3,4,5-trimethoxybenzoate (S)-5.

Procedure A, starting from (S)-31 and 3,4,5-trimethoxybenzoic

acid.

¹³C APT NMR (CDCl₃)

d

: 169.75 (C]O); 167.01 (C]O); 153.37

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 28.5%.

(C); 144.74 (CH]CH); 140.32 (C); 130.99 (C); 129.81 (C); 129.20

(CH]CH); 128.60 (CH arom); 128.41 (C); 126.90 (CH arom); 125.46

(CH arom); 125.06 (CH arom); 117.29 (CH]CH); 105.26 (CH arom.);

69.12 (CH₂); 62.64 (CH₂), 61.18 (CH₂); 60.93 (OCH₃); 56.10 (OCH₃);

54.49 (CH₂); 42.60 (NCH₃); 31.21 (CH); 26.65 (CH₂); 16.04 (CH₃)

ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.57 (d, J ¼ 16.0 Hz, 1H, CH]CH);

7.27 (s, 2H, CH arom.); 6.74 (s, 2H, CH arom.); 6.33 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.29 (t, J ¼ 6.4 Hz, 2H, OCH₂); 4.23 (dd, J ¼ 10.8 Hz,

J ¼ 4.8 Hz, 1H, OCHH); 4.00 (dd, J ¼ 10.8 Hz, J ¼ 6.8 Hz, 1H, OCHH);

3.88 (s, 9H, OCH₃); 3.87 (s, 6H, OCH₃); 3.86 (s, 3H, OCH₃); 2.38e2.27

(m, 3H, NCHH and NCH₂); 2.19 (s, 3H, NCH₃); 2.18e2.14 (m, 1H,

NCHH); 2.10e2.00 (m, 1H, CH); 1.80e1.72 (m, 2H, CH₂); 1.54e1.40

(m, 4H, CH₂); 0.98 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₅H₄₀NO₇¼ 586.2799, found 586.2793.

Hydrochloride: mp 96e99 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 167.05 (C]O); 166.24 (C]O); 153.43

6.1.15.10. (S) (E)-2-methyl-3-(methyl(3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) propyl anthracene-9-carboxylate (S)-4.

Procedure A, starting from (R)-30 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

(C); 152.91 (C); 144.58 (CH]CH); 142.17 (C); 140.12 (C); 129.93 (C);

125.50 (C); 117.43 (CH]CH); 106.82 (CH arom.); 105.25 (CH arom.);

68.12 (CH₂); 65.18 (CH₂); 61.34 (CH₂); 60.94 (OCH₃); 60.89 (OCH₃);

58.14 (CH₂); 56.23 (OCH₃); 56.16 (OCH₃); 42.73 (NCH₃); 31.30 (CH);

28.72 (CH₂); 27.05 (CH₂); 23.84 (CH₂); 15.86 (CH₃) ppm.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 98:2. Yield:

70%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

¹H NMR (400 MHz, CDCl₃)

d: 8.46 (s, 1H, CH arom.); 8.02 (d,

C

₃₂H₄₆NO₁₀¼ 604.3116, found 604.3125.

J ¼ 8.8 Hz, 2H, CH arom.); 7.97 (d, J ¼ 8.8 Hz, 2H, CH arom.); 7.53 (d,

J ¼ 16.0 Hz, 1H, CH]CH); 7.49e7.42 (m, 4H, CH arom.); 6.65 (s, 2H,

CH arom.); 6.29 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.63 (dd, J ¼ 10.8 Hz,

J ¼ 4.0 Hz, 1H, OCHH); 4.46 (dd, J ¼ 10.8 Hz, J ¼ 6.0 Hz, 1H, OCHH);

4.25 (t, J ¼ 6.4 Hz, 2H, OCH₂); 3.83 (s, 3H, OCH₃); 3.78 (s, 6H, OCH₃);

2.51e2.40 (m, 3H, NCHH and NCH₂); 2.23 (s, 3H, NCH₃); 2.23e2.18

(m, 2H, NCHH and CH); 1.88e1.81 (m, 2H, CH₂); 1.04 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

Hydrochloride: mp 90e95 ^ꢂC.

6.1.15.13. (R) (E)-5-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) pentyl anthracene-9-carboxylate (R)-6.

Procedure B, starting from (R)-31 and anthracene-9-carboxylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 54%.

¹H NMR (400 MHz, CDCl₃)

d: 8.51 (s, 1H, CH arom.); 8.04e8.00

¹³C APT NMR (CDCl₃)

d

: 169.87 (C]O); 167.01 (C]O); 153.34

(m, 4H, CH arom.); 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH); 7.55e7.45 (m,

4H, CH arom.); 6.73 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]

CH); 4.61 (t, J ¼ 6.4 Hz, 2H, OCH₂); 4.23 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz,

1H, OCHH); 4.03 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H, OCHH); 3.87 (s, 3H,

OCH₃); 3.85 (s, 6H, OCH₃); 2.41e2.29 (m, 3H, NCHH and NCH₂); 2.21

(s, 3H, NCH₃); 2.20e2.11 (m, 1H, NCHH); 2.10e2.04 (m, 1H, CH);

1.94e1.87 (m, 2H, CH₂); 1.59e1.46 (m, 4H, CH₂); 0.99 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

(C); 144.69 (CH]CH); 141.85 (C); 130.97 (C); 129.89 (C); 129.18 (CH

arom.); 128.60 (CH arom.); 128.38 (C); 126.89 (CH arom.); 125.46

(CH arom.); 125.08 (CH arom.); 117.36 (CH]CH); 105.14 (CH

arom.); 69.27 (CH₂); 62.78 (CH₂); 61.31 (CH₂); 60.96 (OCH₃); 56.16

(OCH₃); 56.07 (OCH₃); 54.59 (CH₂); 42.62 (NCH₃); 31.28 (CH); 26.68

(CH₂); 16.00 (CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C₃₅H₄₀NO₇¼ 586.2799, found 586.2792.

¹³C APT NMR (CDCl₃)

d: 169.73 (C]O); 167.06 (C]O); 153.42

Hydrochloride: mp 88e92 ^ꢂC.

(C); 144.64 (CH]CH); 131.00 (C); 129.93 (C); 129.22 (CH arom.);

128.61 (CH arom.); 128.38 (C); 126.92 (CH arom.); 125.46 (CH

arom.); 125.00 (CH arom.); 117.41 (CH]CH); 105.26 (CH arom.);

68.05 (CH₂); 65.84 (CH₂); 61.22 (CH₂); 60.96 (OCH₃); 58.03 (CH₂);

56.16 (OCH₃); 42.65 (NCH₃); 31.25 (CH); 28.70 (CH₂); 26.79 (CH₂);

23.90 (CH₂); 15.89 (CH₃) ppm.

6.1.15.11. (R) (E)-5-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) pentyl 3,4,5-trimethoxybenzoate (R)-5.

Procedure A, starting from (R)-31 and 3,4,5-trimethoxybenzoic

acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 70.5%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₇H₄₄NO₇¼ 614.3112, found 614.3102.

¹H NMR (400 MHz, CDCl₃)

d: 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH);

Hydrochloride: mp 79e83 ^ꢂC.

7.28 (s, 2H, CH arom.); 6.74 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.30 (t, J ¼ 6.8 Hz, 2H, OCH₂); 4.24 (dd, J ¼ 10.8 Hz,

J ¼ 4.8 Hz, 1H, OCHH); 4.02 (dd, J ¼ 10.8 Hz, J ¼ 6.8 Hz, 1H, OCHH);

3.89 (s, 9H, OCH₃); 3.88 (s, 6H, OCH₃); 3.87 (s, 3H, OCH₃); 2.37e2.30

(m, 3H, NCHH and NCH₂); 2.21 (s, 3H, NCH₃); 2.20e2.12 (m, 1H,

NCHH); 2.11e2.04 (m, 1H, CH); 1.82e1.72 (m, 2H, CH₂); 1.59e1.40

(m, 4H, CH₂); 0.99 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

6.1.15.14. (S) (E)-5-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) pentyl anthracene-9-carboxylate (S)-6.

Procedure B, starting from (S)-31 and anthracene-9-carboxylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH/NH₄OH

98:2:0.2. Yield: 71%.

ESI-MS: 614.2 [MþH]^þ.

¹³C APT NMR (CDCl₃)

d: 167.04 (C]O); 166.25 (C]O); 153.45

¹H NMR (400 MHz, CDCl₃)

d: 8.50 (s, 1H, CH arom.); 8.04e7.99

(C); 152.93 (C); 144.62 (CH]CH); 142.23 (C); 140.17 (C); 129.93 (C);

125.49 (C); 117.40 (CH]CH); 106.86 (CH arom.); 105.30 (CH arom.);

68.09 (CH₂); 65.16 (CH₂); 61.30 (CH₂); 60.95 (OCH₃); 60.89 (OCH₃);

(m, 4H, CH arom.); 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH); 7.55e7.45 (m,

4H, CH arom.); 6.73 (s, 2H, CH arom.); 6.34 (d, J ¼ 16.0 Hz, 1H, CH]

CH); 4.62 (t, J ¼ 6.8 Hz, 2H, OCH₂); 4.24 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz,

20

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

1H, OCHH); 4.03 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H, OCHH); 3.87 (s, 3H,

OCH₃); 3.85 (s, 6H, OCH₃); 2.37e2.29 (m, 3H, NCHH and NCH₂);

2.20 (s, 3H, NCH₃); 2.18e2.13 (m, 1H, NCHH); 2.11e2.04 (m, 1H, CH);

1.94e1.86 (m, 2H, CH₂); 1.59e1.46 (m, 4H, CH₂); 0.98 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

Procedure A, starting from (R)-33 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 97:3. Yield:

78%.

¹H NMR (400 MHz, CDCl₃)

d: 8.51 (s, 1H, CH arom.); 8.05 (d,

¹³C APT NMR (CDCl₃)

d: 169.73 (C]O); 167.07 (C]O); 153.42

J ¼ 8.8 Hz, 2H, CH arom.); 8.01 (d, J ¼ 8.8 Hz, 2H, CH arom.); 7.58 (d,

J ¼ 16.0 Hz, 1H, CH]CH); 7.54e7.45 (m, 4H, CH arom.); 6.72 (s, 2H,

CH arom.); 6.35 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.65 (dd, J ¼ 10.4 Hz,

J ¼ 4.0 Hz, 1H, OCHH); 4.49 (dd, J ¼ 10.4 Hz, J ¼ 6.0 Hz, 1H, OCHH);

4.19 (t, J ¼ 6.4 Hz, 2H, OCH₂); 3.86 (s, 3H, OCH₃); 3.84 (s, 6H, OCH₃);

2.43e2.30 (m, 3H, NCHH and NCH₂); 2.23 (s, 3H, NCH₃); 2.22e2.17

(m, 2H, NCHH and CH); 1.72e1.65 (m, 2H, CH₂); 1.53e1.42 (m, 4H,

CH₂); 1.06 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

(C); 144.59 (CH]CH); 131.00 (C); 129.95 (C); 129.21 (CH arom.);

128.61 (CH arom.); 128.38 (C); 128.16 (C); 126.91 (CH arom.);

125.45 (CH arom.); 125.01 (CH arom.); 117.46 (CH]CH); 105.24 (CH

arom.); 68.11 (CH₂); 65.86 (CH₂); 61.31 (CH₂); 60.96 (OCH₃); 58.09

(CH₂); 56.15 (OCH₃); 42.73 (NCH₃); 31.32 (CH); 28.72 (CH₂); 26.95

(CH₂); 23.91 (CH₂); 15.87 (CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₇H₄₄NO₇¼ 614.3112, found 614.3104.

Hydrochloride: mp 76e80 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 169.83 (C]O); 167.10 (C]O); 153.37 (C);

144.63 (CH]CH); 139.95 (C); 131.00 (C); 129.92 (C); 129.20 (CH

arom); 128.63 (CH arom); 128.38 (C); 126.86 (CH arom); 125.42 (CH

arom); 125.15 (CH arom); 117.47 (CH]CH); 105.13 (CH arom); 69.33

(CH₂); 64.61 (CH₂); 61.28 (CH₂); 60.98 (OCH₃); 58.13 (CH₂); 56.13

(OCH₃); 42.72 (NCH₃); 31.22 (CH); 28.73 (CH₂); 27.00 (CH₂); 23.83

(CH₂); 16.10 (CH₃) ppm.

6.1.15.15. (R) (E)-2-methyl-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)pentyl)amino) propyl 3,4,5-trimethoxybenzoate (R)-7.

Procedure B, starting from (R)-32 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 96:4. Yield:

78%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₇H₄₄NO₇¼ 614.3112, found 614.3115.

¹H NMR (400 MHz, CDCl₃)

d: 7.57 (d, J ¼ 16.0 Hz, 1H, CH]CH);

Hydrochloride: mp 129e132 ^ꢂC.

7.28 (s, 2H, CH arom.); 6.74 (s, 2H, CH arom.); 6.33 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.34 (dd, J ¼ 10.8 Hz, J ¼ 4.4 Hz, 1H, OCHH); 4.17 (t,

J ¼ 6.4 Hz, 2H, OCH₂); 4.13 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H, OCHH);

3.88 (s, 9H, OCH₃); 3.87 (s, 9H, OCH₃); 2.36e2.31 (m, 3H, NCHH and

NCH₂); 2.22 (s, 3H, NCH₃); 2.21e2.10 (m, 2H, NCHH and CH);

1.71e1.65 (m, 2H, CH₂); 1.56e1.35 (m, 4H, CH₂); 1.02 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

6.1.15.18. (S) (E)-2-methyl-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)pentyl)amino) propyl anthracene-9-carboxylate (S)-8.

Procedure A, starting from (S)-33 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 97:3. Yield:

72%.

¹³C APT NMR (CDCl₃)

d: 167.04 (C]O); 166.27 (C]O); 153.89

¹H NMR (400 MHz, CDCl₃)

d: 8.50 (s, 1H, CH arom.); 8.05 (d,

(C); 152.94 (C); 144.61 (CH]CH); 142.19 (C); 129.93 (C); 125.48 (C);

117.42 (CH]CH); 106.81 (CH arom.); 105.24 (CH arom.); 68.49

(CH₂); 64.55 (CH₂); 60.96 (CH₂); 60.90 (OCH₃); 58.14 (CH₂); 56.25

(OCH₃); 56.23 (OCH₃); 42.77 (NCH₃); 31.31 (CH); 28.68 (CH₂); 27.00

(CH₂); 23.80 (CH₂); 15.99 (CH₃) ppm.

J ¼ 8.8 Hz, 2H, CH arom.); 8.00 (d, J ¼ 8.8 Hz, 2H, CH arom.); 7.58 (d,

J ¼ 16.0 Hz, 1H, CH]CH); 7.54e7.45 (m, 4H, CH arom.); 6.72 (s, 2H,

CH arom.); 6.35 (d, 1H, J ¼ 16.0 Hz, CH]CH); 4.65 (dd, J ¼ 10.8 Hz,

J ¼ 4.4 Hz, 1H, OCHH); 4.49 (dd, J ¼ 10.8 Hz, J ¼ 6.0 Hz, 1H, OCHH);

4.19 (t, J ¼ 6.4 Hz, 2H, OCH₂); 3.86 (s, 3H, OCH₃); 3.83 (s, 6H, OCH₃);

2.42e2.30 (m, 3H, NCHH and NCH₂); 2.22 (s, 3H, NCH₃); 2.25e2.15

(m, 2H, NCHH and CH); 1.72e1.65 (m, 2H, CH₂); 1.53e1.42 (m, 4H,

CH₂); 1.06 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₂H₄₆NO₁₀¼ 604.3116, found 604.3110.

Hydrochloride: mp 53e57 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 169.86 (C]O); 167.08 (C]O); 153.38

6.1.15.16. (S) (E)-2-methyl-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)pentyl)amino) propyl 3,4,5-trimethoxybenzoate (S)-7.

Procedure B, starting from (S)-32 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

(C); 144.62 (CH]CH); 139.97 (C); 130.98 (C); 129.95 (C); 129.17 (CH

arom); 128.61 (CH arom); 128.39 (C); 126.88 (CH arom); 125.47 (CH

arom); 125.11 (CH arom); 117.47 (CH]CH); 105.15 (CH arom); 69.32

(CH₂); 64.60 (CH₂); 61.25 (CH₂); 60.97 (OCH₃); 58.13 (CH₂); 56.10

(OCH₃); 42.76 (NCH₃); 31.23 (CH); 28.70 (CH₂); 26.99 (CH₂); 23.85

(CH₂); 16.08 (CH₃) ppm.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 96:4. Yield:

67%.

¹H NMR (400 MHz, CDCl₃)

d: 7.51 (d, J ¼ 16.0 Hz, 1H, CH]CH);

ESI-HRMS (m/z) calculated for [MþH]^þion species

7.22 (s, 2H, CH arom.); 6.68 (s, 2H, CH arom.); 6.27 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.28 (dd, J ¼ 10.8 Hz, J ¼ 4.4 Hz, 1H, OCHH); 4.12e4.05

(m, 3H, OCHH and OCH₂); 3.82 (s, 9H, OCH₃); 3.80 (s, 6H, OCH₃);

3.79 (s, 3H, OCH₃); 2.29e2.20 (m, 3H, NCHH and NCH₂); 2.14 (s, 3H,

NCH₃); 2.13e2.06 (m, 2H, NCHH and CH); 1.66e1.55 (m, 2H, CH₂);

1.46e1.30 (m, 4H, CH₂); 0.96 (d, J ¼ 6.0 Hz, 3H, CH₃) ppm.

C

₃₇H₄₄NO₇¼ 614.3112, found 614.3117.

Hydrochloride: mp 88e99 ^ꢂC.

6.1.15.19. (R) (E)-7-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) heptyl 3,4,5-trimethoxybenzoate (R)-9.

Procedure B, starting from (R)-34 and 3,4,5-trimethoxybenzoic

acid.

¹³C APT NMR (CDCl₃)

d: 166.98 (C]O); 166.18 (C]O); 153.39

(C); 152.89 (C); 144.56 (CH]CH); 142.13 (C); 140.03 (C); 129.93 (C);

125.50 (C); 117.43 (CH]CH); 106.74 (CH arom.); 105.19 (CH arom.);

68.50 (CH₂); 64.55 (CH₂); 61.32 (CH₂); 60.91 (OCH₃); 60.86 (OCH₃);

58.13 (CH₂); 56.17 (OCH₃); 56.11 (OCH₃); 42.78 (NCH₃); 31.33 (CH);

28.66 (CH₂); 27.01 (CH₂); 23.78 (CH₂); 15.93 (CH₃) ppm.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 97:3. Yield:

21%.

¹H NMR (400 MHz, CDCl₃)

d: 7.58 (d, J ¼ 15.6 Hz, 1H, CH]CH);

7.28 (s, 2H, CH arom.); 6.74 (s, 2H, CH arom); 6.33 (d, J ¼ 15.6 Hz,1H,

CH]CH); 4.28 (t, J ¼ 6.8 Hz, 2H, OCH₂); 4.23 (dd, J ¼ 10.8 Hz,

J ¼ 5.2 Hz, 1H, OCHH); 4.05 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H, OCHH);

3.89 (s, 9H, OCH₃); 3.88 (s, 6H, OCH₃); 3.87 (s, 3H, OCH₃); 2.47e2.43

(m, 3H, NCHH and NCH₂); 2.31 (s, 3H, NCH₃); 2.30e2.26 (m, 1H,

NCHH); 2.19e2.09 (m, 1H, CH); 1.70e1.59 (m, 2H, CH₂); 1.48e1.29

(m, 8H, CH₂); 1.03 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₂H₄₆NO₁₀¼ 604.3116, found 604.3106.

Hydrochloride: mp 59e62 ^ꢂC.

6.1.15.17. (R) (E)-2-methyl-3-(methyl(5-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)pentyl)amino) propyl anthracene-9-carboxylate (R)-8.

¹³C APT NMR (CDCl₃)

d: 167.00 (C]O); 166.27 (C]O); 153.45

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

21

(C); 152.92 (C); 144.77 (CH]CH); 142.78 (C); 129.88 (C); 125.51 (C);

¹H NMR (400 MHz, CDCl₃)

d

: 8.51 (s, 1H, CH arom.); 8.05e8.00

117.26 (CH]CH); 106.83 (CH arom.); 105.29 (CH arom.); 67.92

(CH₂); 65.18 (CH₂); 60.96 (CH₂); 60.67 (OCH₃); 57.76 (CH₂); 56.10

(OCH₃); 42.19 (NCH₃); 30.87 (CH); 29.16 (CH₂) 28.69 (CH₂) 27.26

(CH₂) 25.97 (CH₂) 16.03 (CH₃) ppm.

(m, 4H, CH arom); 7.59 (d, J ¼ 16.0 Hz, 1H, CH]CH); 7.55e7.46 (m,

4H, CH arom.); 6.74 (s, 2H, CH arom); 6.35 (d, J ¼ 16.0 Hz, 1H, CH]

CH); 4.61 (t, J ¼ 6.8 Hz, 2H, OCH₂); 4.25 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz,

1H, OCHH); 4.03 (dd, J ¼ 10.8 Hz, J ¼ 6.8 Hz, 1H, OCHH); 3.87 (s, 3H,

OCH₃); 3.86 (s, 6H, OCH₃); 2.35e2.28 (m, 3H, NCHH and NCH₂); 2.19

(s, 3H, NCH₃); 2.17e2.02 (m, 2H, NCHH and CH); 1.91e1.84 (m, 2H,

CH₂); 1.53e1.30 (m, 8H, CH₂); 0.99 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₄H₅₀NO₁₀¼ 632.3429, found 632.3434.

Hydrochloride: mp 67e70 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 167.07 (C]O); 153.44 (C); 144.55 (CH]

6 .1.15 . 2 0 . ( S ) ( E ) - 7 - ( m e t h yl ( 2 - m e t hyl - 3 - ( ( 3 - ( 3 , 4 , 5 -

trimethoxyphenyl)acryloyl)oxy)propyl)amino) heptyl 3,4,5-

trimethoxybenzoate (S)-9. Procedure A, starting from (S)-34 and

3,4,5-trimethoxybenzoic acid.

CH); 131.01 (C); 129.96 (C); 129.19 (CH arom.); 128.61 (CH arom.);

128.39 (C); 126.90 (CH arom.); 125.45 (CH arom.); 125.03 (CH

arom.); 117.49 (CH]CH); 105.25 (CH arom.); 68.20 (CH₂); 65.92

(CH₂); 61.30 (CH₂); 60.97 (OCH₃); 58.28 (CH₂); 56.17 (OCH₃); 42.78

(NCH₃); 31.30 (CH); 29.21 (CH₂); 28.75 (CH₂); 27.32 (CH₂); 26.10

(CH₂); 15.91 (CH₃) ppm.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 95:5. Yield:

77%.

¹H NMR (400 MHz, CDCl₃)

d: 7.58 (d, J ¼ 16.0 Hz, 1H, CH]CH);

ESI-HRMS (m/z) calculated for [MþH]^þion species

7.28 (s, 2H, CH arom.); 6.75 (s, 2H, CH arom); 6.34 (d, J ¼ 16.0 Hz,1H,

CH]CH); 4.29 (t, J ¼ 6.8 Hz, 2H, OCH₂); 4.24 (dd, J ¼ 10.8 Hz,

J ¼ 4.4 Hz, 1H, OCHH); 4.02 (dd, J ¼ 10.8 Hz, J ¼ 6.8 Hz, 1H, OCHH);

3.90 (s, 9H, OCH₃); 3.88 (s, 6H, OCH₃); 3.87 (s, 3H, OCH₃); 2.40e2.25

(m, 3H, NCHH and NCH₂); 2.20 (s, 3H, NCH₃); 2.19e2.03 (m, 2H,

NCHH and CH); 1.77e1.71 (m, 2H, CH₂); 1.50e1.24 (m, 8H, CH₂);

1.00 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

C

₃₉H₄₈NO₇¼ 642.3425, found 642.3437.

Hydrochloride: mp 125e127 ^ꢂC.

6 .1.15 . 2 3 . ( R ) ( E ) - 2 - m e t hyl - 3 - ( m e t hyl ( 7 - ( ( 3 - ( 3 , 4 , 5 -

trimethoxyphenyl)acryloyl)oxy)heptyl)amino)p ropyl 3,4,5-

trimethoxybenzoate (R)-11. Procedure B, starting from (R)-35 and

((E)-3-(3,4,5-trimethoxyphenyl)acrylic acid.

¹³C APT NMR (CDCl₃)

d: 167.04 (C]O); 166.27 (C]O); 153.45

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 98:2. Yield:

41%.

(C); 152.92 (C); 144.61 (CH]CH); 142.80 (C); 140.30 (C); 129.93 (C);

125.54 (C); 117.42 (CH]CH); 106.83 (CH arom.); 105.27 (CH arom.);

68.11 (CH₂); 65.23 (CH₂); 61.23 (CH₂); 60.96 (OCH₃); 60.90 (OCH₃);

58.25 (CH₂); 56.25 (OCH₃); 56.18 (OCH₃); 42.71 (NCH₃); 31.22 (CH);

29.23 (CH₂); 28.72 (CH₂); 27.30 (CH₂); 26.00 (CH₂); 15.93 (CH₃)

ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.54 (d, J ¼ 15.6 Hz, 1H, CH]CH);

7.25 (s, 2H, CH arom.); 6.71 (s, 2H, CH arom.); 6.30 (d, J ¼ 15.6 Hz,

1H, CH]CH); 4.30 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz, 1H, OCHH); 4.15e4.07

(m, 3H, OCH₂and OCHH); 3.85 (s, 9H, OCH₃); 3.83 (s, 6H, OCH₃);

3.82 (s, 3H, OCH₃); 2.33e2.27 (m, 3H, NCH₂and NCHH); 2.18 (s, 3H,

NCH₃); 2.17e2.11 (m, 2H, NCHH and CH); 1.65e1.59 (m, 2H, CH₂);

1.42e1.20 (m, 8H, CH₂); 0.98 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₄H₅₀NO₁₀¼ 632.3429, found 632.3430.

Hydrochloride: mp 71e75 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 167.02 (C]O); 166.21 (C]O); 153.42

(C); 152.92 (C); 144.55 (CH]CH); 129.95 (C); 125.51 (C); 117.49

(CH]CH); 106.79 (CH arom.); 105.22 (CH arom.); 68.53 (CH₂);

64.63 (CH₂); 61.28 (CH₂); 60.94 (OCH₃); 60.89 (OCH₃); 58.30 (CH₂);

56.21 (OCH₃); 56.15 (OCH₃); 42.78 (NCH₃); 31.30 (CH); 29.21 (CH₂);

28.70 (CH₂); 27.29 (CH₂); 27.18 (CH₂); 25.95 (CH₂); 15.98 (CH₃)

ppm.

6.1.15.21. (R) (E)-7-(methyl(2-methyl-3-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)propyl)amino) heptyl anthracene-9-carboxylate (R)-10.

Procedure B, starting from (R)-34 and anthracene-9-carboxylic

acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 97:3. Yield:

58%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

¹H NMR (400 MHz, CDCl₃)

d

: 8.51 (s, 1H, CH arom.); 8.05e8.00

C

₃₄H₅₀NO₁₀¼ 632.3429, found 632.3420.

(m, 4H, CH arom.); 7.59 (d, J ¼ 16.0 Hz, 1H, CH]CH); 7.55e7.46 (m,

4H, CH arom.); 6.74 (s, 2H, CH arom); 6.35 (d, J ¼ 16.0 Hz, 1H, CH]

CH); 4.60 (t, J ¼ 6.4 Hz, 2H, OCH₂); 4.24 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz,

1H, OCHH); 4.03 (dd, J ¼ 10.8 Hz, J ¼ 6.4 Hz, 1H, OCHH); 3.87 (s, 3H,

OCH₃); 3.86 (s, 6H, OCH₃); 2.33e2.28 (m, 3H, NCH₂and NCHH); 2.18

(s, 3H, NCH₃); 2.17e2.06 (m, 2H, NCHH and CH); 1.91e1.83 (m, 2H,

CH₂); 1.53e1.30 (m, 8H, CH₂); 1.00 (d, J ¼ 6.8 Hz, 3H, CH₃) ppm.

Hydrochloride: mp 81e85 ^ꢂC.

6.1.15.24. (S) (E)-2-methyl-3-(methyl(7-((3-(3,4,5-trimethoxyphenyl)

acryloyl)oxy)heptyl)amino) propyl 3,4,5-trimethoxybenzoate (S)-11.

Procedure B, starting from (S)-35 and ((E)-3-(3,4,5-

trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 98:2. Yield:

27%.

¹³C APT NMR (CDCl₃)

d: 167.08 (C]O); 153.41 (C); 144.52 (CH]

CH); 131.00 (C); 129.95 (C); 129.17 (CH arom.); 128.63 (CH arom.);

128.42 (C); 126.88 (CH arom.); 125.43 (CH arom.); 125.04 (CH

arom.); 117.51 (CH]CH); 105.22 (CH arom.); 68.25 (CH₂); 65.90

(CH₂); 61.32 (CH₂); 60.99 (OCH₃); 58.25 (CH₂); 56.21 (OCH₃); 42.72

(NCH₃); 31.28 (CH); 29.25 (CH₂); 28.75 (CH₂); 27.35 (CH₂); 26.12

(CH₂); 15.89 (CH₃) ppm.

¹H NMR (400 MHz, CDCl₃)

d: 7.55 (d, J ¼ 16.0 Hz, 1H, CH]CH);

7.26 (s, 2H, CH arom.); 6.72 (s, 2H, CH arom.); 6.31 (d, J ¼ 16.0 Hz,

1H, CH]CH); 4.31 (dd, J ¼ 10.8 Hz, J ¼ 4.8 Hz, 1H, OCHH); 4.16e4.09

(m, 3H, OCH₂and OCHH); 3.86 (s, 9H, OCH₃); 3.85 (s, 6H, OCH₃);

3.84 (s, 3H, OCH₃); 2.40e2.30 (m, 3H, NCH₂and NCHH); 2.22 (s, 3H,

NCH₃); 2.21e2.10 (m, 2H, NCHH and CH); 1.66e1.61 (m, 2H, CH₂);

1.50e1.40 (m, 2H, CH₂); 1.39e1.20 (m, 6H, CH₂); 1.01 (d, J ¼ 6.4 Hz,

3H, CH₃) ppm.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₉H₄₈NO₇¼ 642.3425, found 642.3425.

Hydrochloride: mp 100e103 ^ꢂC.

¹³C APT NMR (CDCl₃)

d: 167.05 (C]O); 166.22 (C]O); 153.43

(C); 152.94 (C); 144.57 (CH]CH); 129.96 (C); 125.48 (C); 117.49

(CH]CH); 106.82 (CH arom.); 105.23 (CH arom.); 68.50 (CH₂);

64.64 (CH₂); 61.18 (CH₂); 60.96 (OCH₃); 60.91 (OCH₃); 58.22 (CH₂);

56.23 (OCH₃); 56.16 (OCH₃); 42.69 (NCH₃); 31.24 (CH); 29.21 (CH₂);

28.71 (CH₂); 27.29 (CH₂); 25.95 (CH₂); 16.03 (CH₃) ppm.

6 .1.15 . 2 2 . ( S ) ( E ) - 7 - ( m e t h yl ( 2 - m e t hyl - 3 - ( ( 3 - ( 3 , 4 , 5 -

trimethoxyphenyl)acryloyl)oxy)propyl)amino) heptyl anthracene-9-

carboxylate (S)-10. Procedure B, starting from (S)-34 and anthra-

cene-9-carboxylic acid.

Free base: chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH

93:7:0.3. Yield: 46%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C₃₄H₅₀NO₁₀¼ 632.3429, found 632.3429.

22

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

Hydrochloride: mp 86e93 ^ꢂC.

CH₂); 1.05 (d, J ¼ 6.0 Hz, 3H, CH₃) ppm.

¹³C APT NMR (CDCl₃)

: 169.79 (C]O); 167.04 (C]O); 153.42

d

(C); 144.55 (CH]CH); 131.00 (C); 129.97 (C); 129.18 (CH arom.);

128.60 (CH arom.); 128.41 (C); 128.28 (C); 126.87 (CH arom.);

125.46 (CH arom.); 125.11 (CH arom.); 117.52 (CH]CH); 105.24 (CH

arom.); 69.27 (CH₂); 64.65 (CH₂); 61.13 (CH₂); 60.95 (OCH₃); 58.27

(CH₂); 56.14 (OCH₃); 42.69 (NCH₃); 31.17 (CH); 29.19 (CH₂); 28.73

(CH₂); 27.28 (CH₂); 25.96 (CH₂); 16.12 (CH₃) ppm.

6 .1.15 . 2 5 . ( R ) ( E ) - 2 - m e t h yl - 3 - ( m e t h yl ( 7 - ( ( 3 - ( 3 , 4 , 5 -

trimethoxyphenyl)acryloyl)oxy)heptyl)amino) propyl anthracene-9-

carboxylate (R)-12. Procedure B, starting from (R)-36 and ((E)-3-

(3,4,5-trimethoxyphenyl)acrylic acid.

Free base: chromatographic eluent: CH₂Cl₂/MeOH/NH₄OH

93:7:0.3. Yield: 77%.

ESI-HRMS (m/z) calculated for [MþH]^þion species

¹H NMR (400 MHz, CDCl₃)

d: 8.48 (s, 1H, CH arom.); 8.02 (d,

C

₃₉H₄₈NO₇¼ 642.3425, found 642.3415.

J ¼ 8.4 Hz, 2H, CH arom.); 7.98 (d, J ¼ 8.4 Hz, 2H, CH arom.); 7.56 (d,

J ¼ 16.0 Hz, 1H, CH]CH); 7.51e7.43 (m, 4H, CH arom.); 6.71 (s, 2H,

CH arom.); 6.31 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.60 (dd, J ¼ 10.8 Hz,

J ¼ 4.4 Hz, 1H, OCHH); 4.47 (dd, J ¼ 10.8 Hz, J ¼ 5.6 Hz, 1H, OCHH);

4.15 (t, J ¼ 6.8 Hz, 2H, OCH₂); 3.84 (s, 3H, OCH₃); 3.83 (s, 6H, OCH₃);

2.45e2.35 (m, 3H, NCH₂and NCHH); 2.24 (s, 3H, NCH₃); 2.24e2.22

(m, 2H, NCHH and CH); 1.66e1.61 (m, 2H, CH₂); 1.48e1.37 (m, 2H,

CH₂); 1.36e1.20 (m, 6H, CH₂); 1.06 (d, J ¼ 6.4 Hz, 3H, CH₃) ppm.

Hydrochloride: mp 84e89 ^ꢂC.

6.1.16. Enantiomeric excess (ee) of (R) and (S) enantiomers

evaluation

The separation of racemic mixture of the studied compounds

was carried out by Agilent 1200 liquid chromatography system

composed by autosampler, binary pumps, column oven and diode-

array detector (LC-DAD) operating in UV range (210e400 nm). The

analyses were performed by using a Phenomenex Lux Cellulose-3

¹³C APT NMR (CDCl₃)

d: 169.80 (C]O); 167.06 (C]O); 153.42

(C); 144.57 (CH]CH); 131.01 (C); 129.96 (C); 129.20 (CH arom.);

128.61 (CH arom.); 128.42 (C); 126.89 (CH arom.); 125.47 (CH

arom.); 125.11 (CH arom.); 117.51 (CH]CH); 105.23 (CH arom.);

69.26 (CH₂); 64.66 (CH₂); 60.95 (CH₂); 58.12 (CH₂); 56.15 (OCH₃);

42.56 (NCH₃); 31.09 (CH); 29.18 (CH₂); 28.72 (CH₂); 27.29 (CH₂);

26.80 (CH₂); 25.95 (CH₂); 16.15 (CH₃) ppm.

column 250 mm length, 4.6 mm internal diameter and 5

ticle size, in isocratic elution. The sample injection volume was

20 L. The elution conditions employed to carry out the resolution

mm par-

m

of racemic mixtures of compounds 1, 2, 3, 5, 6, 8, 9, 10 and 11 are

reported in Table 4 and the Retention Time (TR), ee values and

resolution (R) between the enantiomers of the same compounds

are reported in Table 5.

ESI-HRMS (m/z) calculated for [MþH]^þion species

C

₃₉H₄₈NO₇¼ 642.3425, found 642.3425.

Hydrochloride: mp 67e73 ^ꢂC.

6.2. Biological assay

6 .1.15 . 2 6 . ( S ) ( E ) - 2 - m e t h yl - 3 - ( m e t hyl ( 7 - ( ( 3 - ( 3 , 4 , 5 -

trimethoxyphenyl)acryloyl)oxy)heptyl)amino) propyl anthracene-9-

carboxylate (S)-12. Procedure B, starting from (R)-36 and ((E)-3-

(3,4,5-trimethoxyphenyl)acrylic acid.

6.2.1. Cell lines and cultures

The K562 is an undifferentiated erythroleukemia cell line orig-

inally derived from a patient with chronic myelogenous leukemia

[48]. The K562 leukemia cells and the P-gp over-expressing K562/

DOX cells were obtained from Prof. J.P. Marie (Hopital Hotel-Dieu,

Paris, France). The cells were cultured following a previously re-

ported protocol [67].

MDCK-MDR1, MDCK-MRP1 and MDCK-BCRP cells are a gift of

Prof. P. Borst, NKI-AVL Institute, Amsterdam, Nederland. MDCK cells

were grown in DMEM high glucose supplemented with 10% fetal

Free base: chromatographic eluent: CH₂Cl₂/CH₃OH 98:2. Yield:

29%.

¹H NMR (400 MHz, CDCl₃)

d: 8.47 (s, 1H, CH arom.); 8.01 (d,

J ¼ 8.4 Hz, 2H, CH arom.); 7.97 (d, J ¼ 8.4 Hz, 2H, CH arom.); 7.56 (d,

J ¼ 16.0 Hz, 1H, CH]CH); 7.51e7.42 (m, 4H, CH arom.); 6.70 (s, 2H,

CH arom.); 6.31 (d, J ¼ 16.0 Hz, 1H, CH]CH); 4.60 (dd, J ¼ 10.8 Hz,

J ¼ 4.4 Hz, 1H, OCHH); 4.46 (dd, J ¼ 10.8 Hz, J ¼ 6.0 Hz, 1H, OCHH);

4.15 (t, J ¼ 6.8 Hz, 2H, OCH₂); 3.84 (s, 3H, OCH₃); 3.82 (s, 6H, OCH₃);

2.45e2.30 (m, 3H, NCH₂and NCHH); 2.22 (s, 3H, NCH₃); 2.22e2.20

(m, 2H, NCHH and CH); 1.67e1.61 (m, 2H, CH₂); 1.48e1.20 (m, 8H,

bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL

streptomycin, in a humidiﬁed incubator at 37 ^ꢂC with a 5% CO₂

atmosphere. Caco-2 cells were a gift of Dr. Aldo Cavallini and Dr.

Table 4

Elution conditions employed to carry out the resolution of racemic mixtures of compounds 1, 2, 3, 5, 6, 8, 9, 10 and 11.

Racemic mixture

Mobile phase^a

Flow (mL min^ꢀ1

)

T (^ꢂC)

UV (nm)

(R)-1/(S)-1, (R)-3/(S)-3, (R)-5/(S)-5, (R)-9/(S)-9, (R)-11/(S)-11

(R)-2/(S)-2

(R)-6/(S)-6, (R)-10/(S)-10

(R)-8/(S)-8

A

B

C

D

0.5

0.8

0.5

20

10

20

300

250

a

methanol:isopropanol 60:40 (v/v), with 0.1% of DEA; B: methanol:acetonitrile 95:5 (v/v), with 0.1% of DEA; C: methanol:acetonitrile 90:10 (v/v), with 0.1% of DEA; D:

methanol:acetonitrile 98:2 (v/v), with 0.1% of DEA.

Table 5

Retention time (TR), ee values and resolution (R) between the enantiomers of compounds 1, 2, 3, 5, 6, 8, 9, 10 and 11.

Compd

TR (min)

ee %

R value

Compd

TR (min)

ee %

R value

(R)-1

(S)-1

(R)-2

(S)-2

(R)-3

(S)-3

(R)-5

(S)-5

22.28 0.02

19.36 0.03

16.47 0.05

14.73 0.04

19.70 0.20

21.70 0.20

22.70 0.60

19.10 0.50

ꢁ95

2.18 0.04

(R)-6

(S)-6

(R)-8

(S)-8

(R)-9

(S)-9

(R)-10

(S)-10

12.02 0.01

9.27 0.01

23.22 0.06

24.80 0.07

22.72 0.02

20.13 0.02

11.01 0.01

9.11 0.01

ꢁ95

3.88 0.01

1.22 0.04

1.43 0.03

2.78 0.05

1.18 0.01

1.89 0.05

3.12 0.01

E. Teodori et al. / European Journal of Medicinal Chemistry 182 (2019) 111655

23

Caterina Messa from the Laboratory of Biochemistry, National

Institute for Digestive Diseases, “S. de Bellis”, Bari (Italy).

calculation, MolSoft was used [51], then for each compound, the

molecular docking study was carried out by the software. The grid

box was set to 20 Å ꢄ 20 Å ꢄ 28 Å with a grid space value of 1 Å. The

binding box was centred at the previously reported helix TM4

(W232), TM5 (R296, I299, I306), TM6 (Y310, F336), TM7 (F928),

TM8 (F303, Y307, F770) and TM12 (M986, Q990, F994), involved in

the substrate-binding region [73e76]. MolSoft program was used

setting default parameters and a maximum of 50 poses per ligand

were collected. The results were analysed using PyMOL v2.0 [77]. To

ﬁnd network of interaction we used Ligplot þ v.1.4.5 [78] and

Protein-Ligand Interaction Proﬁler [79] all the sets were by default.

6.2.2. Drugs and materials

Puriﬁed verapamil and pirarubicin were purchased by Sigma-

Aldrich (Milan

- Italy). Concentrations were determined by

diluting stock solutions to approximately 10^ꢀ5M and using

ε₄₈₀¼ 11500 M^ꢀ1cm^ꢀ1. Stock solutions were prepared just before

use. Buffer solutions were HEPES buffer containing 5 mM HEPES,

132 mM NaCl, 3.5 mM CaCl₂, 5 mM glucose, at pH 7.3.

Cell culture reagents were purchased from Celbio s.r.l. (Milano,

Italy). CulturePlate 96/wells plates were purchased from Perki-

nElmer Life Science; Calcein-AM, bisBenzimide H 33342 trihydro-

chloride were obtained from Sigma-Aldrich (Milan, Italy).

6.4. Stability test

6.4.1. Chemicals

6.2.3. Modulation of pirarubicin uptake on K562/DOX cells

The uptake of pirarubicin in cells was followed by monitoring

the decrease in the ﬂuorescence signal at 590 nm (l_ex¼ 480 nm)

according to the previously described method [68,69].

Acetonitrile (Chromasolv), formic acid and ammonium formate

(MS grade), NaCl, KCl, Na₂HPO₄2H₂O, KH₂PO₄(Reagent grade) and

verapamil hydrochloride (analytical standard, used as internal

standard), ketoprofen and enalapril (analytical standard) were

purchased by Sigma-Aldrich (Milan, Italy). Ketoprofen Ethyl Ester

(KEE) were obtained by Fisher's reaction from ketoprofen and

ethanol.

6.2.4. Characterization of P-gp interacting proﬁle and ABC

transporters selectivity on transfected MDCK cells

Calcein-AM and Hoechst 33342 experiments, ATPlite assay and

Permeability experiments were carried out as previously described

[42]. Details are reported in the Supplementary data.

MilliQ water 18 MU

cm^ꢀ1was obtained from Millipore's

Simplicity system (Milan - Italy).

Phosphate buffer solution (PBS) was prepared by adding

8.01 g L^ꢀ1of NaCl, 0.2 g L^ꢀ1of KCl, 1.78 g L^ꢀ1of Na₂HPO₄2H₂O and

0.27 g L^ꢀ1of KH₂PO₄. Human plasma was collected from healthy

male volunteer and kept at ꢀ80 ^ꢂC until use.

6.2.5. Antiproliferative assay

Determination of cell growth was performed using the MTT

assay at 48 h and 72 h [70]. Data were analysed by one-way ANOVA

for repeated measures. Results are expressed as mean SD of at

2e3 independent experiments in triplicates. Statistical signiﬁcance

was accepted at a level of P < 0.05. Details are reported in the

Supplementary data.

6.4.2. Preparation of samples

Each sample was prepared adding 10

mL of working solution

1e100 L of tested matrix (PBS or rat plasma or human plasma) in

m

microcentrifuge tubes. The obtained solutions correspond to 1

of analyte.

Each set of samples was incubated in triplicate at four different

times, 0, 30, 60 and 120 min at 37 ^ꢂC. Therefore, the degradation

proﬁle of each analyte was represented by a batch of 12 samples (4

incubation times x 3 replicates). After the incubation, the samples

mM

6.2.6. Co-administration assay

The co-administration assay with doxorubicin was performed in

MDCK-MDR1 cells at 48 h as reported with minor modiﬁcation [71].

Details are reported in the Supplementary data.

were added with 300 mL of ISTD solution and centrifuged (room

6.2.7. Collateral sensitivity (CS) study

temperature for 5 min at 10000 rpm). The supernatants were

transferred in autosampler vials and dried under a gentle stream of

nitrogen.

The dried samples were dissolved in 1.0 mL of 10 mM of formic

acid in mQ water:acetonitrile 80:20 solution. The obtained sample

solutions were analysed by LC-MS/MS method described in the

Supplementary data.

All the compounds have been tested for their cytotoxic activity

at 24 h on MDCK-MDR1 and MDCK-MRP1 cells and on the parental

MDCK cells by MTT assay.

To evaluate the mechanisms involved in CS, reactive oxygen

species (ROS) were measured using the ﬂuorescent probe 5-(and-

6)-chloromethyl-2⁰,7⁰-dichlorodihydro-ﬂuorescein

diacetate-

acetoxymethyl ester (DCFDA-AM), as previously reported [72].

The results were expressed as nmoles/mg cellular proteins. Cell

viability with the compounds and ROS scavenger was measured by

the ATPlite Luminescence Assay System (PerkinElmer, Waltham,

MA), as per manufacturer's instructions, using a Synergy HT Multi-

Detection Microplate Reader (BioTek Instruments, Winooski, VT).

The relative luminescence units (RLU) of untreated cells were

considered as 100% viability; results were expressed as a percent-

age of viable cells versus untreated cells.

Acknowledgements

This work was supported by grants from the University of

Florence (Fondo Ricerca Ateneo RICATEN16 and RICATEN17) and by

Italian Association for Cancer Research (AIRC; IG21408 to CR).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2019.1 1 1655.

6.3. Molecular docking studies

The crystal structure of human P-gp with in the ATP-bound,

outward-facing conformation (PDB ID: 6C0V) [52], was down-

loaded from PDB database.

The molecular structures of synthesized compounds were

designed with Chemdraw program, and then optimized prior to

docking procedure, by using the Dock Prep application.

In order to prepare the ligands and receptor for docking

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Article Doi

Design, synthesis and biological evaluation of stereo- and regioisomers of amino aryl esters as multidrug resistance (MDR) reversers

DOI: 10.1016/j.ejmech.2019.111655

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