Design, synthesis and in vitro biological evaluation of a novel class of anti-adenovirus agents based on 3-amino-1,2-propanediol

Bioorganic Chemistry 114 (2021) 105095

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Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Design, synthesis and in vitro biological evaluation of a novel class of

anti-adenovirus agents based on 3-amino-1,2-propanediol

,

b

,

c,

,

,*

Sarah Mazzotta^a

¹, Judith Berastegui-Cabrera^{d 1}, Margarita Vega-Holm^a

,

María del Rosario García-Lozano^{a e}, Marta Carretero-Ledesma^d, Francesca Aiello^b,

a

d,*

d

,*

Jose Manuel Vega-Perez , Jeronimo Pachon ^,f, Fernando Iglesias-Guerra^a

,

´

Javier Sanchez-Cespedes

^aDepartment of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville, E-41071 Seville, Spain

^bDepartment of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende (CS), Italy

^cDepartment of Pharmaceutical Sciences, University of Milan, 20133 Milan, Italy

^dUnit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of

Seville, E41013 Seville, Spain

^eInstitute of Biomedicine of Seville (IBiS), SeLiver Group, University Hospital Virgen del Rocío/CSIC/University of Seville, E41013 Seville, Spain

^fDepartment of Medicine, University of Seville, E-41009 Seville, Spain

A R T I C L E I N F O

A B S T R A C T

Keywords:

Adenovirus

Antiviral drugs

Aminoalcohol

Ester

Nowadays there is not an effective drug for the treatment of infections caused by human adenovirus (HAdV)

which supposes a clinical challenge, especially for paediatric and immunosuppressed patients. Here, we describe

the design, synthesis and biological evaluation as anti-adenovirus agents of a new library (57 compounds) of

diester, monoester and triazole derivatives based on 3-amino-1,2-propanediol skeleton.

Seven compounds (17, 20, 26, 34, 44, 60 and 66) were selected based on their high anti-HAdV activity at low

micromolar concentration (IC₅₀from 2.47 to 5.75 µM) and low cytotoxicity (CC₅₀from 28.70 to >200 µM). In

addition, our mechanistic assays revealed that compounds 20 and 44 might be targeting speciﬁcally the HAdV

DNA replication process, and compound 66 would be targeting HAdV E1A mRNA transcription. For compounds

17, 20, 34 and 60, the mechanism of action seems to be associated with later steps after HAdV DNA replication.

Carbamate

Triazole

Urea

1. Introduction

respiratory (1–3, 5 and 7) [6,7], conjunctival (8, 19 and 37) [8] and

gastrointestinal infections (40 and 41) [9], presenting signiﬁcant

Adenovirus (HAdV) represents a signiﬁcant viral pathogen, respon-

sible for a wide range of global clinical diseases, mostly in immuno-

compromised patients [1,2]. It’s a non-enveloped virus consisting of an

icosahedral capsid containing a linear double-stranded DNA (dsDNA)

genome of 34–36 kb. HAdVs belong to the family Adenoviridae that in-

cludes more than 100 serotypes classiﬁed into 7 species (HAdV A-G)

[3–5]. HAdV serotypes are traditionally associated with self-limited

morbidity and mortality in immunosuppressed patients and also in

healthy individuals, being a well-known causative agent of community-

acquired pneumonia [1,10].

At present, there are no drugs speciﬁcally approved to treat HAdV

infections. Usually, drugs approved for the treatment of other viral in-

fections are used off-label to manage serious HAdV-induced diseases,

such as ribavirin (1), ganciclovir (GCV, 2), cidofovir (CDV, 3) and

Abbreviations: BCV, brincidofovir; CC₅₀, cytotoxic concentration 50%; CDV, cidofovir; COSY, correlation spectroscopy; CuAAC, copper(I)-catalysed alkyne-azide

1,3-dipolar cycloaddition; DCM, dichloromethane; DEPT, distortionless enhancement by polarization transfer; DMAP, 4-(dimethylamino)-pyridine; DMF, dime-

thylformamide; EMEM, minimum essential medium Eagle; FBS, fetal bovine serum; GCV, ganciclovir; HAdV, human adenovirus; HMBC, heteronuclear multiple bond

correlation; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HSQC, heteronuclear single quantum correlation; IC₅₀, half maximal inhibitory concen-

tration; mcpba, meta-chloroperbenzoyc acid; NOESY, nuclear overhauser effect spectroscopy; SAR, structure-activity relationship; SI, selectivity index; THF, tetra-

hydrofuran; TLC, thin layer chromatography; TMS, tetramethylsilane.

* Corresponding authors.

´

E-mail addresses: mvegaholm@us.es (M. Vega-Holm), iglesias@us.es (F. Iglesias-Guerra), jsanchez-ibis@us.es (J. Sanchez-Cespedes).

1

S.M., J.B.C joint as co-ﬁrst authors.

https://doi.org/10.1016/j.bioorg.2021.105095

Received 7 March 2021; Received in revised form 9 May 2021; Accepted 10 June 2021

Available online 13 June 2021

Published by Elsevier Inc.

This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

brincidofovir (BCV, 4) (Fig. 1), which display no suitable clinical results

in terms of efﬁcacy or safety [11–13]. CDV, an acyclic nucleoside

analogue of cytosine, is a broad spectrum antiviral drug approved for the

treatment of cytomegalovirus (CMV) retinitis and represents, in most

cases, the drug of choice in the clinical setting [14,15]. Unfortunately,

its low oral bioavailability, and its nephrotoxicity and myelosuppression

effects, are undesirable factors that limit the use of CDV [16].

derived benzoic acid esters (11a and 11b, Fig. 1) searching for new

effective HAdV agents [22–25].

As part of our continuous efforts to develop new anti-HAdV com-

pounds with suitable active/safety proﬁles and different mode of action

[22–25], in this work we describe the design, synthesis and biological

evaluation of a library (57 compounds) of 3-amino-1,2-propanediol

derivatives.

The unsatisfactory results of current antiviral drugs highlight the

need to ﬁnd new effective anti-HAdV agents. Drug repurposing of

existing drugs approved for other pharmacological indication is gaining

interest as strategy for the development of new antiviral drugs. In this

sense, the salicylanilide anthelmintic drug niclosamide (5, Fig. 1) was

reported to display signiﬁcant anti-HAdV activity, targeting the micro-

tubular transport of the viral particles to the nucleus [17]. Among nat-

ural products, polyphenolic compounds have been found to have

antiviral activity, such as epigallocatechin gallate (6, Fig. 1), that

showed inhibitory activity against several viruses including HAdV [18].

During the years, interesting novel structural diversiﬁed molecular

entities have been described as new potential antiviral candidates

against HAdV infections. Many of these compounds include common

structural features such as amide, urea, esters functions and aromatic

portions [19–25].

2. Results and discussion

2.1. Chemistry

2.1.1. Design

We aimed to develop a set of new anti-HAdV molecules keeping in

mind the structure of CDV (that represents one of the current therapeutic

options for severe HAdV infections). Our approach involves the analysis

of cidofovir molecule in which three regions can be identiﬁed as points

for structural modiﬁcations to generate chemical diversity (Fig. 2).

1) The central skeleton that can be considered a three carbon ami-

noglycerol chain. Related to this, the ﬁrst step was the election of the

central core as starting point for the following chemical construction

process: 3-amino-1,2-propanediol.

A series of new 5-aminouracil derivatives were synthesized by

Nikitenko et al. with antiviral activity against HAdV5 (7, Fig. 1) [19]. A

set of optimized salicylamide derivatives with potent anti-HAdV activity

at low micromolar concentrations was recently described (8, Fig. 1)

[20], as well as a library of benzoic acid esters (9, Fig. 1) [21].

Our group synthesized a new set of ureas/thioureas derivatives based

on piperazine scaffold (10, Fig. 1) as well as a collection of serinol

2) The nitrogen pyrimidine base. In our model we replaced it with an

acyclic aryl urea moiety introduced at the nitrogen of the skeleton. Aryl

urea derivatives are an important class of compounds possessing a wide

spectrum of biological activities, such as anticancer, antibacterial,

antiviral, etc [22–26].

3) The phosphonate group at secondary position. Carbon 1 and 2

positions become important points of structural variability of our

Fig. 1. Current drugs employed for severe HAdV infections and novel compounds with in vitro anti-HAdV activity.

2

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Fig. 2. Analysis of the CDV structure and following design strategy.

general backbone. Our main purposes were: mono or diacylation of the

hydroxyls (benzoic acid esters) and their replacement by 1,2,3-triazole

rings.

1,2,3-Triazole ring is a well-known scaffold that has widespread

presence in different compounds characterized by several biological

activities; antiviral, antibacterial, antitumoral, among others [33–35].

Application of bioisosteric substitution is a common technique in the

structural modiﬁcation of active compounds to design analogues. In

addition, triazoles are among the most common amide bond isosteres

and have been widely employed in the peptidomimetics ﬁeld as well as

in the design of new analogues of bioactive compounds [36–39].

Related to our work, we focused on their use as ester isosteres, in an

attempt to obtain analogues with signiﬁcant anti-HAdV activity. In case

they generated promising derivatives, these would be expected to have

reduced in vivo susceptibility to enzymatic degradation, compared to the

ester prototypes [36].

Based on the relevance of aromatic esters as privileged functional

groups for anti-HAdV activity [18,21] and on the promising results

demonstrated by some serinol-derived aromatic diesters in the inhibi-

tion of HAdV infection [25], ﬁrstly a set of diester derivatives from the

isomeric 3-amino-1,2-propanediol (core present in antiviral compounds

[27,28]) was designed, in order to explore the effect of this different

skeleton on the anti-HAdV activity. Our attention was also focused on di-

and trimethoxy substitutions, frequently present in anticancer and

antiviral compounds [29–31]. In this sense and aiming to further explore

the presence of methoxy groups, a trimethoxycynnamic moiety was also

inserted, as it is a privileged structural scaffold broadly distributed in

natural products (esters and amides derivatives) with diverse and

important biological functions, included antiviral activities [32].

To evaluate the impact of the presence of a free hydroxyl group, as in

CDV, in providing active compounds, a small collection of monoester

derivatives of 3-amino-1,2-propanediol, in position 1 and 2, was

designed.

2.1.2. Synthesis

Based on this design, the ﬁrst structural modiﬁcation was the intro-

duction of the aryl urea moiety at the nitrogen with different substituents

on the phenyl ring. The synthesis of these compounds was carried out by

reaction between 3-amino-1,2-propanediol (12), commercially available,

and the appropriate phenyl isocyanate, using three different substituted

Fig. 3. Three different approaches for the introduction of chemical diversity.

3

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

phenyl isocyanates (p-CF₃, p-Cl and p-CH₃as models of electron with-

drawing and electron-releasing groups) (Scheme 1).

Once these urea derivatives (13–17) were prepared, three different

approaches were carried out to generate ﬁnal compounds (Fig. 3).

Route A, the preparation of a set of diester derivatives.

Route B, the selective monoacylation of hydroxyl groups of the

aminoalcohol chain.

monocarbamate 65 the same synthetic methodology was employed,

except for the acylation of secondary alcohol that was performed using

the appropriate phenyl isocyanate in the presence of 4-dimethylamino-

pyridine (DMAP) (Scheme 2, Table 2).

At this point it should be noticed that during the preparation of the

trimethoxycinnamic diester analogue 45, the monester derivative at

primary position was isolated, compound 66 (Table 2).

Route C, the introduction of 1,2,3-triazole rings, located at position 1

or 2 of the aminoalcohol chain.

2.1.2.3. Route C. Synthetic route for the preparation of N-phenyl-

aminocarbonyl 1,2,3-triazole derivatives. We generated a set of 4-

substituted-1,2,3-triazole derivatives following two different method-

ologies for the introduction of the triazole moiety at both positions of the

carbon chain.

2.1.2.1. Route A. Synthesis of N-phenylaminocarbonyl diester derivatives.

Diester derivatives (18–46) were synthesized by an acylation reaction of

both hydroxyl groups of the urea intermediate (13–17), with the cor-

responding acyl chloride or carboxylic acid to give the ﬁnal product in a

good yield (Scheme 1, Table 1).

For the preparation of compounds 71–76 (Table 3), in which the

heterocyclic nucleus is located at primary position, we employed allyl-

amine as the starting material for the synthetic procedure (67). Once the

p-Cl substituted urea derivative 68 was prepared, an oxidation reaction

of oleﬁn, using meta-chloroperbenzoyc acid (mCPBA) in dry tetrahy-

drofuran (THF), furnished the corresponding oxirane 69. In the next

step, the acid-catalysed ring opening reaction using NH₄Cl and sodium

azide under ethanol reﬂux, gave the azide derivative 70. Finally, the

synthesis of 1,2,3-triazole ring (1,4 adduct) was performed through a

click chemistry approach, the copper(I)-catalysed alkyne-azide 1,3-

dipolar cycloaddition (CuAAC) reaction (Scheme 3). The reaction pro-

ceeded using the appropriate terminal alkyne, CuSO₄as a source of pre-

catalyst Cu^IIand sodium ascorbate as reducing agent, in t-BuOH-H₂O at

room temperature. This reaction represents the most used “click” reac-

tion to obtain 1,2,3-triazole, due to its reliability, speciﬁcity, and

biocompatibility [40,41].

At this step, the structural variability on benzoyl moiety was exam-

ined as follows: methyl and methoxy groups as representative electron

releasing substituents, and CN, NO₂and CF₃as electron-withdrawing

groups (to explore the effects of the electronic properties on the anti-

viral activity) as well as di-and trimethoxy substitutions. The trime-

thoxycinnamoyl derivatives were prepared.

2.1.2.2. Route B. Synthesis of N-phenylaminocarbonyl monoester and

carbamate derivatives. Two different synthetic approaches were

employed for the generation of monoester derivatives from ureas 13 and

14, depending on the position of the acyl function on the aminoalcohol

skeleton. For the preparation of acylated derivatives at primary position

(47–49, Table 2), a selective O-acylation reaction of primary alcohol

group of 13 was performed. The reaction was carried out in dichloro-

methane (DCM) at –40 ^◦C by controlling the stoichiometric conditions,

using the appropriate acylating agent (p- or o-CH_3,p-CF₃benzoyl chlo-

rides) and pyridine (Scheme 2).

For the introduction of the triazole ring at secondary position more

steps were required and are depicted in Scheme 4. From the urea de-

rivative (p-Cl, 13) the ﬁrst step involved the selective protection of the

primary alcohol with the benzoyl group (77). The acylated product 77

was mesyled in THF (78) for the subsequent nucleophilic substitution

with sodium azide (in dimethylformamide, DMF, at 85 ^◦C), furnishing

the introduction of the azide group in position 2 (79). The click chem-

istry reaction between deprotected azide derivative 80 and the appro-

priate substituted alkyne, in the same conditions described above,

afforded ﬁnal 1,2,3-triazole derivatives 81–86 (Table 3).

The introduction of the ester function on the secondary alcohol

required an acyl protection and deprotection strategy. In the ﬁrst step,

the monoacylation of primary hydroxyl of the urea derivative (13, 14)

proceeded following the same reaction condition described above, but

using chloroacetyl ester as protecting group (50 and 51). During the

subsequent step, the intermediates 52–57 were generated by the intro-

duction of the desired benzoyl ester function at secondary position.

Finally, the deprotection of position 1 through the reaction with thio-

urea in CH₃CN-H₂O at 60 ^◦C removed the chloroacetyl ester, giving the

monoacylated compounds 59–64 in good yields. For the synthesis of

Compounds 71–76 and 81–86 were fully characterized by NMR

spectroscopy (see Experimental Section).

To establish the regiochemistry of the click reaction heteronuclear

Scheme 1. Synthesis of N-phenylaminocarbonyl diester derivatives 18–46.

4

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Percentage of plaque formation inhibition (%)^a

Table 1

Benzoic acid and cynnamic acid diesters 18–46 from route A.

Compound

CC₅₀

(μ

M)^b

R¹

R²

R³

R⁴

R⁵

R⁶

18 (A)

19 (A)

20 (A)

21 (A)

22 (A)

23 (A)

24 (A)

25 (A)

26 (A)

27 (A)

28 (A)

29 (A)

30 (A)

31 (A)

32 (A)

33 (A)

34 (A)

35 (A)

36 (A)

37 (A)

38 (A)

39 (A)

40 (A)

41 (A)

42 (A)

43 (A)

44 (A)

45 (B)

46 (B)

Cidofovir^c

H

CH₃

H

–

Cl

H

0.00 ± 0.00

59.84 ± 29.71

75.08 ± 15.71

0.00 ± 0.00

26.47 ± 8.32

49.70 ± 6.77

60.22 ± 8.43

31.00 ± 10.43

40.25 ± 5.27

53.07 ± 16.18

4.43 ± 6.26

40.25 ± 5.27

21.79 ± 30.81

47.01 ± 8.79

81.29 ± 1.10

0.00 ± 0.00

7.30 ± 11.04

18.51 ± 1.92

40.41 ± 13.31

2.12 ± 3.00

26.83 ± 24.33

46.69 ± 3.36

57.85 ± 22.21

20.29 ± 2.82

99.86 ± 0.13

0.00 ± 0.00

3.51 ± 4.97

–

Cl

H

CH₃

H

Cl

CH₃

H

28.70 ± 3.10

Cl

H

OCH₃

CN

H

–

Cl

H

–

Cl

H

NO₂

CF₃

OCH₃

CH₃

H

–

Cl

H

–

Cl

OCH₃

H

–

Cl

OCH₃

97.25 ± 16.56

CH₃

CF₃

H

–

CH₃

H

51.33 ± 19.04

H

OCH₃

CN

H

–

H

–

H

NO₂

CF₃

OCH₃

CH₃

H

–

H

–

OCH₃

H

–

OCH₃

175.16 ± 2.97

H

–

CH₃

H

–

H

OCH₃

CN

H

–

H

–

H

NO₂

CF₃

OCH₃

–

H

–

H

–

OCH₃

H

–

OCH₃

–

H

OCH₃

–

OCH₃

Cl

H

OCH₃

136.62 ± 6.54

–

CH₃

–

179.09 ± 35.10

a

b

c

Percentage of control HAdV5-GFP inhibition in a plaque assay at 10 μM using the 293 β5 cell line. See Experimental Section

Cytotoxic concentration 50%. The results represent means ± SD of triplicate samples from three independent experiments.

Data of cidofovir, as positive clinical drug candidate, have been listed.

2D NMR experiments were performed with compounds 74 and 84, both

4-ﬂuorophenyl triazole derivatives, as models [34].

at room temperature (87). For the preparation of alkynyl phosphonate

88 the propargyl alcohol reacted with the diethyl (tosyloxy)methyl-

phosphonate in basic condition of NaH at 25 ^◦C (Scheme 5). Both

compounds were characterized by NMR spectroscopy (see Experimental

Section).

The formation of the triazole ring was conﬁrmed through the pres-

ence of a single signal of the triazolyl proton at δ = 8.52 ppm (74) and

8.65 ppm (84). Firstly, the presence of the 1,4-adduct was conﬁrmed

using the heteronuclear multiple bond correlation (HMBC) experiment

to identify long-range coupling (three bonds) between protons and

carbons. In compound 74, the 1,4-substitution was proven because the

terminal CH₂signals (δ 4.71 and 4.32 ppm) showed a cross-peak with

the CH of the triazole ring (δ 122.1 ppm). Compound 84 showed similar

coupling proﬁle, between CH signal (4.69–4.62 ppm) of the central

chain with the CH-triazole (120.8 ppm) (Fig. 4 A). To conﬁrm the 1,4-

adduct formation, NOESY experiments were performed. The NOESY

spectrum showed correlation between the proton of the triazole ring and

both CH₂groups, those next to the triazole ring (blue arrows) and those

next to the urea function (little correlation, green arrows); and between

CH protons (red arrow) of the aliphatic chain for compound 74. For

compound 84, correlation between the triazole proton and the CH₂

terminal (blue arrows), CH (red arrow) and one of the CH₂linked to the

urea group (green arrow) was observed (Fig. 4 B) (see Supplementary

Material).

2.2. Biological evaluation

2.2.1. In vitro evaluation of HAdV infection inhibition and cellular viability

As the ﬁrst step in the biological evaluation, the anti-HAdV activity

of the new derivatives from the three routes was evaluated in plaque

assay at 10 μM, quantifying HAdV plaque formation in the presence of

the candidate molecules. Their effect on cellular viability was also car-

ried out and the 50% cytotoxic concentration (CC₅₀) determined for

those compounds that reached a percentage of inhibition in the plaque

assay > 60% (Tables 1-3). Since CDV currently represents the drug of

choice to treat HAdV infections, it was also evaluated following our own

methodologies and compared to our results.

The analysis of the evaluation of the diester derivatives (18–46,

Table 1) showed, from a structure–activity relationship (SAR) point of

view, that the impact of electron-withdrawing substituents of the aro-

matic acyl moiety (CN, NO₂and CF₃) was generally detrimental for the

antiviral activity. For p-Cl substituted phenyl urea derivatives (22–24)

the plaque formation inhibition range was from 0% to 25.83%, slightly

higher than p-CF₃phenyl ureas (38–40) and p-CH₃analogues (30–32),

with 2.12% to 44.65% range values. Compounds 31 (p-NO₂with p-CH₃

substituted phenyl urea) and 38 (p-CN with p-CF₃substituted phenyl

For the synthesis of 1,2,3-triazole derivatives (1,4 adduct), six

different terminal alkynes were employed but only four of them were

commercially available (propargyl alcohol, 4-ethynylanisole, 1-ethynyl-

4-ﬂuorobenzene and 2-ethynylbenzaldehyde). With the aim to intro-

duce an aromatic acid function, the ring-opening of phthalic anhydride

were performed by an aminolysis reaction with propargyl amine, in THF

5

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Percentage of plaque formation inhibition (%)^a

Scheme 2. Synthesis of N-phenylaminocarbonyl monoester and monocarbamate derivatives 59–65.

Table 2

Benzoic monoacylated derivatives 47–49 and 59–66 from route B.

Compound

CC₅₀(μ

M)^b

R²

R³

R⁴

R⁵

R⁶

47 (A)

48 (A)

49 (A)

59 (B)

60 (B)

61 (B)

62 (B)

63 (B)

64 (B)

65 (C)

66 (D)

Cidofovir^c

Cl

H

CH₃

H

–

H

CH₃

H

15.20 ± 1.39

25.99 ± 3.65

5.70 ± 8.06

20.92 ± 10.17

100.00 ± 0.00

0.00 ± 0.00

48.03 ± 6.79

0.00 ± 0.00

40.21 ± 7.11

19.57 ± 2.84

75.04 ± 9.54

3.51 ± 4.97

–

Cl

H

Cl

H

CF₃

CH₃

CF₃

NO₂

OCH₃

CF₃

–

H

Cl

H

Cl

H

> 200

Cl

H

–

Cl

OCH₃

CH₃

Cl

OCH₃

H

–

H

–

Cl

174.32 ± 4.52

–

179.09 ± 35.10

a

Percentage of control HAdV5-GFP inhibition in a plaque assay at 10

μ

M using the 293 β5 cell line. See Experimental Section.

b

c

Cytotoxic concentration 50%. The results represent means ± SD of triplicate samples from three independent experiments.

Data of cidofovir, as positive clinical drug candidate, have been listed.

urea) showed a moderate activity (44.65% and 40.41%, respectively).

For electron donor groups at para position of the acyl function (methyl

and methoxy) the levels of inhibition depended on the urea. p-CF₃

substituted ureas (35–37) showed almost no activity (0.00–18.51%),

similarly to p-Cl phenyl urea derivatives (19 and 21, no inhibition).

Higher levels of inhibition (31.00–60.70%) were found with p-CH₃

phenyl urea derivatives (27–29). Speciﬁcally, compounds with a methyl

group in ortho position on the aromatic ester with p-Cl and p-CH₃phenyl

urea (20 and 28), demonstrated signiﬁcant antiviral activity (75.08%

and 60.70%, respectively). The dimethoxybenzoyl derivatives (25, 33

and 41) gave similar percentage of plaque formation inhibition

(29.70%, 47.01%, 46.69%, respectively), regardless of the type of sub-

stituent on the phenylaminocarbonyl function, indicating the relevance

of this group for the activity. The introduction of an additional methoxy

group (26, 34 and 42, trimetoxybenzoyl derivatives) provided an

improved antiviral activity, inhibiting HAdV plaque formation from

6

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Scheme 3. Synthetic route for the preparation of N-phenylaminocarbonyl-1,2,3-triazole derivatives at primary position (71–76).

Scheme 4. Synthetic route for the preparation of N-phenylaminocarbonyl 1,2,3-triazole derivatives at secondary position (81–86).

57.85% to 81.29%, higher than the corresponding dimethoxy ana-

logues. Compound 34 (p-methyl phenyl urea) resulted the most active

diester derivative, with 81.29% inhibition of HAdV plaque formation. As

p-Cl and p-CH₃urea derivatives gave, in general terms, higher activity

than p-CF₃, they were employed for the preparation of the trimethox-

ycinnamoyl derivatives 45 and 46, which did not show activity (0%

plaque formation inhibition). This modiﬁcation did not lead to poten-

tially interesting analogues. Finally, the unsubstituted benzoyl diester

derivative (18) was also not active.

evaluations, together with compound 17, to gain some mechanistic

understanding for their mode of action.

The evaluation of the antiviral activity of the monoester derivatives

at the primary alcohol showed that they lacked signiﬁcant anti-HAdV

activity, giving percentages of plaque formation inhibition from 0% to

25.99%, (compounds 47–49). It is worth to mention the drastic reduc-

tion on antiviral activity for the o-methyl benzoyl derivative 48

(25.99%) compared to its diester analogue 20 (75.08%) (Table 2)

The urea 13 gave the monoesters at secondary position 59 and 60

with p-CH₃and p-CF₃in order to compare their biological results with

the analogues 47 and 49, displaying a slight higher inhibition for the p-

CH₃benzoyl derivative (20.92% inhibition for compound 59 versus

15.20% inhibition for compound 47). However, for p-CF₃analogue 60, a

complete plaque formation inhibition (100%) was observed, unlike the

regioisomer 49 (5.70%). As CF₃is an electron withdrawing group, p-NO₂

benzoyl analogue (61) was prepared and evaluated, in order to explore

the role of this electronic property (no inhibition observed). In the same

way, the trimethoxy derivative was also synthetized (62), showing a

reduced antiviral activity compared to the diester analogue (26)

(48.03% versus 60.22%). Same activity behaviour was observed for

monoester of p-methyl urea derivative 63 (no inhibition) in contrast to

its diester analogue (34) with 81.29% of inhibition (Table 2).

Considering the in vitro inhibition demonstrated by compound 34,

the preparation of analogues with different substituted phenyl urea was

the natural evolution. Two modiﬁcations were designed: the position

isomer analogue (o-methyl substituted urea, 43) and another electron

donor group at para position (p-OCH₃phenyl urea, 44). They gave

20.29% and 99.86% of inhibition, respectively. These results suggest the

relevance of para substitution for the biological activity.

At this point, it is important to note that the urea derivatives 13–16

used as precursors were also evaluated, yielding percentages inhibition

values between 0.00% and 59%. However, compound 17 (o-CH₃phenyl

urea) gave 82.52% of inhibition, becoming an interesting potential anti-

HAdV agent. For this compound the CC₅₀was 139.95 μM.

As can be seen in Table 1, for compounds 20, 26, 34 and 44, the CC₅₀

values were determined and they were selected for further biological

Other attempt to obtain active compounds started from compound

7

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

speciﬁcity through the selectivity index (SI), measured as the ratio be-

tween their half maximal inhibitory concentration (IC₅₀) and their 50%

cytotoxic concentration (CC₅₀).

Table 3

1,2,3-Triazole

derivatives

71–76

and

81–86

from

route

All compounds showed a dose-dependent inhibition of HAdV5

infection (Figure S62, Supplementary Material), showing IC₅₀values

signiﬁcantly lower than CDV [42]. These IC₅₀values ranged from 2.47

C.

μ

M to 5.75 μM. Their CC₅₀were at least 10 times over their IC₅₀values

Comp.

R

Percentage of plaque

formation inhibition

(%)^a

CC₅₀

with SI between 11.63 and 66.23 (Table 4). We next evaluated the effect

of these compounds on HAdV replication measuring their effectiveness

in blocking the production of new virus particles using a virus burst size

assay. Treatment with the compounds was associated with overall re-

ductions in virus yield in the range 1.52–48.18-fold, but only com-

pounds 34 (194.61) and 44 (285.65) showed a signiﬁcantly reduction

(Table 4).

(μ

M)^b

71 (A)

81 (B)

72 (A)

82 (B)

10.77 ± 15.23

5.80 ± 15.48

4.78 ± 74.93

0.00 ± 0.00

–

In our previous work we identiﬁed a set of serinol derivatives with

signiﬁcant anti-HAdV activity [25]. Compound 11b (Fig. 1) was found

73 (A)

83 (B)

33.33 ± 6.10

0.00 ± 0.00

91.24 ±

4.89

–

to show high antiviral potency (IC₅₀3.67 μm) and safety (SI 17.37). In

the present work, aminoglycerol derivatives 26, 34 and 44, also being

trimethoxy benzoyl diesters as 11b, not only provided the same antiviral

potency, but also improved the safety (in consequence the SI) and

increased the reduction in virus yield. The combination of electron-

releasing groups (p-OCH₃and p-CH₃) with the presence of trimethoxy

benzoyl groups in compounds 34 and 44 respectively seems to be

associated with dramatic reductions in virus yield compare with the rest

of the selected aminoglycerol derivatives or those previously described

serinol derivatives.

74 (A)

84 (B)

21.54 ± 26.11

37.45 ± 0.56

174.34 ±

15.57

30.93 ±

2.08

75 (A)

85 (B)

0.00 ± 0.00

–

21.57 ± 4.21

175.46 ±

2.97

76 (A)

86 (B)

0.00 ± 0.00

–

12.63 ± 7.36

2.2.2. Antiviral mechanism of action studies

Cidofovir^c

3.51 ± 4.97

179.09 ±

2.2.2.1. Time of addition assay. HAdV replicative cycle is a well-known

coordinated process, beginning with the binding of HAdV ﬁber to its

cellular receptors in the plasma membrane of the cell-host and subse-

quent entry into the cell by clathrin-mediated endocytosis or macro-

pynocitosis taking 5 min after binding [43–45]. Using the endocytic

network, HAdV virions force the lysis of the endosomal membrane after

15 min and are transported along the microtubules network to the nu-

clear pore complex (NPC) where they attach after 35–45 min and the

viral genome is released into the nucleus [45,46].

35.10

a

Percentage of control HAdV5-GFP inhibition in a plaque assay at 10 μM

using the 293β5 cell line. See Experimental Section.

b

Cytotoxic concentration 50%. The results represent means ± SD of triplicate

samples from three independent experiments.

c

Data of cidofovir, as positive clinical drug candidate, have been listed.

60, which demonstrated the highest level of inhibition. Two structural

modiﬁcations were introduced on it. Related to the urea function, the

preparation of the analogue with p-CH₃phenyl urea (64) gave less ac-

tivity than 60 (40.21%). The second one was the replacement of the

ester function with a carbamate one (65), which also resulted in a

decreased antiviral activity (19.57%) (Table 2).

In order to determine at what point, during the HAdV replicative

cycle, these compounds could be exerting their antiviral activity, we

next evaluated their antiviral activity in a time of addition assay (Fig. 5).

All compounds showed a time-decrease inhibitory effect, following a

similar pattern with minimal differences. Compounds 20 and 60 showed

an inhibition > 60% when they were either preincubated with the virus

prior to their addition to cells or added at the beginning of the incuba-

tion (60 min and 0 min), but they lacked that activity at further time

points. Compounds 17, 26, 34 and 66 did not show a signiﬁcant

decrease in their inhibition effect during the whole time. Compound 44

did not show an inhibition higher than 50% at any time point, however,

a decrease in that effect was observed from the 5 min time-point to the

120 min time-point.

The evaluation of the trimethoxycinnamic monoester 66 gave

75.04% of plaque inhibition and CC₅₀of 174.32 μM. From this group,

compounds 60 and 66 (as prototypes of monoester at secondary and

primary position respectively) were selected for further biological

studies.

The biological evaluation of the synthesized 1,2,3-triazole de-

rivatives at both positions, 71–76 and 81–86, did not offer a suitable

inhibitory activity (Table 3). Among triazole derivatives at the primary

position, compound 73 (phosphonate group) showed moderate activity,

with 33.33% of plaque formation inhibition (the highest one), while its

analogue at secondary position was not active. For triazole derivatives

with a phenyl ring, those with the heterocycle at secondary position

displayed higher values of inhibition than those with the triazole at

primary one, p-ﬂuorophenyl ring derivatives (37.45% compound 84

versus 21.54% compound 74), p-methoxyphenyl ring triazole derivatives

(21.57% compound 85 versus 0% compound 75). Their CC₅₀was also

determined and high values were observed. They become interesting

compounds for further optimization process, in the search for new

scaffolds, by acting on the urea portion of the main chain, and also by

the generation of different substituted phenyl ring at the triazole moiety.

From this screening we selected 7 compounds which were able to

inhibit HAdV infection in more than 60% compared to a control infec-

tion. First, we assayed these compounds with the aim to determine their

Based on these results, it was difﬁcult to establish the speciﬁc step in

the HAdV replicative cycle at which these molecules could be acting.

Thus, our next step was to evaluate the effect of these compounds on the

viral DNA access to the nucleus cell (Fig. 6).

Once HAdV particles leave the endosome after the lysis mediated by

HAdV protein VI, the partially uncoated capsids are transported to the

nuclear membrane via the microtubule network. Then, the HAdV

genome is imported into the nucleus via the nuclear pore complex [47].

Our hypothesis was that if these derivatives inhibited any of the steps of

the HAdV entry, this would be reﬂected in the quantiﬁcation of HAdV

genomes at the host nucleus after a synchronized infection.

As shown in Fig. 6, we did not ﬁnd statistical differences in the

percentage of nuclear-associated HAdV genomes between the control

sample with DMSO and evaluated compounds. Together, these results

8

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Fig. 4. A) ¹H, ¹³C-HMBC correlations for compounds 74 and 84, B) ¹H, ¹H-NOESY correlations for compounds 74 and 84.

Table 4

IC₅₀, CC₅₀, SI, and virus yield reduction values for selected compounds and for

cidofovir as approved clinical drug^a.

Comp.

IC₅₀

CC₅₀

(μ

M)^c

SI^d

Yield reduction (fold-

reduction)^e

(

μ

M)^b

17

5.75 ±

1.01

139.95 ±

31.13

24.34

11.63

23.20

62.68

29.25

66.23

55.87

7.5

48.18 ± 25.03

11.54 ± 4.23

19.46 ± 3.69

194.61 ± 36.89

285.65 ± 54.15

17.80 ± 5.35

1.52 ± 0.79

20

2.47 ±

0.07

28.70 ± 3.10

26

4.19 ±

2.59

97.25 ±

16.56

34

2.79 ±

0.29

175.16 ±

2.97

44

4.67 ±

0.05

136.62 ±

6.54

60

3.02 ±

0.08

>200

66

3.12 ±

0.44

174.32 ±

4.52

Scheme 5. Synthesis of terminal alkynes 87 and 88.

Cidofovir

24.06 ±

5.9

179.09 ±

35.10

82.5 ± 21.4

suggested that any of these compounds seemed to be blocking any of the

steps covering from the binding of the viral particles to the host cell until

the arrival of HAdV genomes into the nucleus. This result agrees on the

result obtained for the serinol derivative, where none of them showed a

signiﬁcant inhibition of the HAdV genomes accessibility into the nucleus

[25].

a

All results are shown as means ± SD of duplicate samples from two inde-

pendent experiments.

b

c

Inhibitory concentration 50 obtained in a plaque assay.

Cytotoxic concentration 50.

d

Selectivity index value was determined as the ratio of IC₅₀and CC₅₀

.

e

Fold-reduction in virus yield as the ratio of particles produced in the pres-

ence of DMSO divided by the yield in the presence 10-fold IC₅₀concentration

obtained in the plaque assay of each compound.

2.2.2.2. Impact on HAdV DNA replication. We next considered if they

might be acting at the HAdV DNA replication process once the HAdV

genomes get into the cell nucleus. In this sense we quantiﬁed the HAdV

DNA replication in the presence of the compounds by quantitative real-

time PCR after extracting the DNA from cells after 16 h of incubation as a

measure of the DNA replication efﬁciency.

of the HAdV DNA replication comparing to the DMSO control (Fig. 7),

their mechanism of action must be related with viral processes beyond

the HAdV DNA replication as for example, viral proteins maturation or

viral capsid assembly. On the other hand, the presence of compounds

26, 44 and 66 resulted in a signiﬁcant reduction in the number of HAdV

Since compounds 17, 20, 34 and 60 showed no signiﬁcant inhibition

9

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Fig. 5. Time-dependence curve assay. Effect of selected compounds on HAdV infection at different time points at 10-fold the IC₅₀concentration obtained in the

plaque assay of each compound. The line chart represents mean ± SD of duplicate samples.

derivatives, we infected A549 cells in the presence of these compounds

at the speciﬁed concentrations for 6 h. Then, we quantiﬁed the mRNA

copy number of the HAdV immediately early genes E1A and E2B, using

quantitative reverse transcription (RT-PCR). As shown in Fig. 8, only

compound 66 reduced signiﬁcantly the copy number of the E1A mRNA

at 6 h post-infection compared to the DMSO control. The ability of

compound 66 to reduce the expression of HAdV E1A gene transcription

would explain the inhibitory effect observed on HAdV DNA replication.

However, in case of compounds 26 and 44, the reduction of HAdV DNA

quantiﬁed by qPCR seemed to be related to a direct inhibition of the

HAdV DNA replication process rather than to the inhibition of either

E1A or E2B genes transcription.

It is worth it to mention that in our previous work any of the

monoester derivatives were found to have anti-HAdV activity while

compound 66, not only showed signiﬁcant antiviral activity at low

micromolar concentration but also, this activity seems to be associated

with the inhibition of the HAdV E1A gene expression [25].

2.2.3. Synergistic activity of the selected derivatives

Fig. 6. Percentage of the nuclear-associated HAdV genome of selected com-

As three different potential mechanisms of action were identiﬁed, we

pounds. Compounds were added at 10-fold the IC₅₀concentration obtained in

decided to evaluate the antiviral activity of the combination of three

the plaque assay of each compound. Each bar represents mean ± SD of dupli-

representative compounds for each mechanism with the hypothesis that

cate samples.

their combination would result in an improved antiviral activity due to

their synergistic effect activity. The selected compounds for this assay

copies comparing with DMSO. Thus, since they did not block genome

were compound 44, targeting speciﬁcally the HAdV DNA replication

accessibility to the nucleus we value two possible scenarios for this re-

process, compound 66 that targeted HAdV E1A mRNA transcription,

sults; i) these three compounds could be targeting any of the viral pro-

and compound 20 whose mechanism of action was associated with later

teins involved in the HAdV DNA replication, such as the terminal protein

steps after HAdV DNA replication. We employed a combination study

(pTP), the HAdV DNA polymerase or the DNA-binding protein (DBP); ii)

based on the Chou ꢀ Talalay method for drug combination using Cal-

they could be targeting the transcription of HAdV immediately early

cuSyn software [48] and plaque assay starting from 2xIC₅₀and in serial

proteins, a process occurring once the HAdV genomes access the cell

2-fold dilutions. The constant ratio for each combination was selected

nucleus and essential for the subsequent initiation of the HAdV DNA

based on the IC₅₀values for each drug. The data for all the combinations

replication.

showed good conformity with the mass action law principle (r =

To assay the inhibition of HAdV mRNA transcription by these three

10

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Fig. 7. Effect of selected compounds on HAdV DNA replication. Compounds were added at 10-fold the IC₅₀concentration obtained in the plaque assay of each

compound. Results are expressed as the relative copy number of HAdV DNA normalized to GAPDH copy number. Each bar represents mean ± SD of duplicate

samples. Statistical signiﬁcance is pointed as **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

0.97–0.99). Only the combination between compounds 66 and 44 (ratio

1:1.498) resulted in a nearly additive effect at all effective dose values.

The combinations that included compounds 20 and 44 (ratio 1:1.891)

were classiﬁed as slightly antagonistic at their ED₅₀, antagonistic at the

ED₇₅and strongly antagonistic at their ED₉₀. As for the combinations

between compounds 20 and 66 (ratio 1:1.263) they resulted in all cases

as slightly antagonistic. In case of combination between 20 and 66, all

ED values maintained a slight antagonistic effect (Table 5).

= 109-(0.345 TPSA) [51]. Not only those compounds that conﬁrmed

Lipinski’s rule exhibited high oral absorption (%ABS of 85.82, 83.15,

84.44 and 76.57, respectively), but also compounds 26, 34 and 44

(those failed Lipinski’s rule) showed moderate-high %ABS (in an

accepted value range 65–67%). Drug likeness is a complex balance be-

tween various molecular properties like molecule size, hydrogen

bonding characters, electronic distribution and hydrophobicity [52].

The more positive drug likeness score values, the more likely the com-

pound was expected to be as a promising therapeutic pharmacophore

[53]. A positive model score was attributed for all compounds excepting

17, which stranded for good drug likeness behaviour, especially com-

pounds 20, 26, 44 and 66 (score ≥ 1) (Supplementary Material, S63).

Secondly, predicted absorption, distribution, metabolism and

excretion (ADME) data were calculated for the most active compounds

using preADMET software (https://preadmet.bmdrc.kr) and results are

presented in Table 7.

2.3. In silico prediction of drug-likeness and pharmacokinetics properties

Since many potential therapeutic candidates fail to reach the clinic

because of their unfavorable ADME behaviours, the estimation of

physicochemical and pharmacokinetics properties in early stages of the

drug discovery process is an important tool for decreasing the fraction of

these failures in later phases of development.

Firstly, the physico-chemical parameters of the selected compounds

were predicted by the use of Molsoft software (https://www.molsoft.

com) [49,50] to assess their compliance with the Lipinski’s rule of

ﬁve, the theoretical agreement for a molecule to become an oral drug

with respect to bioavailability.

Absorption refers to the process by which the drug can go to the

systemic circulation through the organs of the body. Several routes for

absorption such as oral absorption (human intestinal absorption, HIA),

skin permeability (SP, logKp), and permeability through certain cells

such as Caco2 (a human colon epithelial cancer cell line that differen-

tiates to form tight junctions between cells to serve as a model of par-

acellular movement of compounds across the monolayer) and MDCK

cells (MDCK permeability coefﬁcient provided helpful in vitro tools to

assess a drug’s permeability and transporter interactions during drug

discovery and development) were measured [53].

The obtained results illustrated in Table 6, showed that compounds

17, 20, 60 and 66 are in agreement with Lipinski’s rule (with only one

violation Log P > 5 for compound 20). However, compounds 26, 34 and

44 failed with two violations, LogP and Molecular Weight values,

exceeding the accepted ranges. In addition, topological polar surface

area PSA (a criteria that allows the prediction of transport properties of

polar atoms through membranes) was also predicted (all the tested

compounds had TPSA values < 140 Å²), and used to calculate the

percentage of oral absorption (%ABS) by the following equation: (%ABS

By inspecting results recorded in Table 7, it was found that all

compounds showed good intestinal absorption, six out of seven > 90%

(normal range for well absorbed compounds 70–100% abs). Moderate

permeability through in vitro Caco2 cells ranged from 15.40 to 40.88

11

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Fig. 8. Effect of the selected compounds on HAdV E1A (a) and E2B (b) gene expression. Compounds were tested at 10-fold the IC₅₀concentration obtained in the

plaque assay of each compound. Results are expressed as the relative copy number of HAdV E1A or E2B mRNA normalized to GAPDH copy number. Each bar

represents mean ± SD of duplicate samples. Statistical signiﬁcance is pointed as *p ≤ 0.05.

Table 5

IC₅₀values in a plaque assay for each compound and its combination with the combination index value calculated with CalcuSyn software.

Combinatory index (CI) values in Plaque assay

Combination (Ratio)

Compound

IC₅₀

IC₅₀combination

ED₅₀

ED₇₅

ED₉₀

r

single

20 & 44

(1:1.891)

20 & 66

(1:1.263)

66 & 44

(1:1.498)

20

44

20

66

44

2.47 ± 0.07

4.67 ± 0.05

2.47 ± 0.07

3.12 ± 0.44

2.47 ± 0.07

2.1 ± 0.04

3.96 ± 0.07

2.08 ± 0.37

2.26 ± 0.43

1.65 ± 0.09

2.46 ± 0.14

1.19

1.15

0.97

1.46

1.12

1.03

1.81

1.11

1.10

0.99

0.98

0.97

Table 6

Calculated parameters of Lipinski’s rule for selected compounds.

Compound

Parameter

MW

LogP

HBD

HBA

nVs

MolPSA

%ABS

Drug-likeness score

AR

17

20

26

34

44

60

66

≤500

≤5

4

≤100

3

≤1

0

≤140

–

224.12

480.15

632.18

612.23

628.23

416.08

464.14

0.32

6.11

4.74

4.59

4.10

4.25

3.78

67.17

74.92

120.88

128.42

71.18

94.00

85.82

83.15

67.29

64.69

84.44

76.57

ꢀ 0.08

1.27

1.20

0.88

1.00

0.26

0.93

2

5

1

2

11

12

4

2

3

0

3

7

0

AR: accepted range; MW, Molecular weight; LogP, lipophilicity (O/W); HBD, Number of hydrogen bond donors; HBA, Number of hydrogen bond acceptors; nVs,

Number of Lipinski rule violations; MolPSA, molecular polar surface area (PSA) (Å2); %ABS, percentage of oral absorption.

12

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Table 7

Predicted ADMET properties of selected compounds.

Compound

17

Parameter

20

26

34

44

60

66

Absorption

HIA (%)

72.97

15.40

ꢀ 4.16

12.84

95.62

26.19

ꢀ 2.16

0.04

96.73

33.73

ꢀ 2.90

0.04

95.86

39.62

ꢀ 2.89

0.04

94.63

40.88

ꢀ 3.05

0.04

91.65

18.80

ꢀ 2.74

0.12

92.40

22.07

ꢀ 3.17

0.06

Caco2 (nm/sc)

SP LogP (cm/h)

MDCK (nm/sc)

Distribution

BBB

0.27

1.03

0.02

0.03

1.16

0.11

PPB (%)

20.26

89.45

89.63

89.75

88.01

86.46

84.31

Metabolism (CYP) and excretion

CYP2C19 inhibitor

CYP2D6 inhibitor

CYP3A4 inhibitor

Pgp inhibitor

no

yes

no

yes

HIA: Intestinal absorption; Caco2: permeability through cells derived from human colon adenocarcinoma; SP LogP: skin permeability; MDCK: permeability through

Madin-Darby canine kidney cells; BBB: Blood-Brain Barrier; PPB: Plasma-protein binding; CYP: cytochrome P450; Pgp: P-glycoprotein.

nm/sc was observed (normal range 4–70 nm/s); the skin permeability

was found to be slightly less than acceptable range (-2.5 logKp), while

low values were detected for in vitro MDCK cells.

acceptable physicochemical properties, favourable oral bioavailability

and good drug-likeness scores (most of them).

Although further rounds of optimization as well as a better under-

standing of their mode of action will be required, these molecules,

inspired by the aminoglycerol backbone, represent a promising starting

point for the development of a new class of antiviral compounds.

Two parameters, blood brain barrier (BBB) and plasma binding

protein (PBP), were measured to predict the distribution of molecules

from one organ to another. BBB permits the diffusion to the brain for

small hydrophobic molecules; this parameter evaluates whether com-

pounds pass across the blood–brain barrier. The measurement of the

percentage of a molecule bound to plasma protein (PPB%) is also helpful

in estimation of the novel target compounds distribution (only the un-

bound fraction is available to pass across membranes and for the

interaction with the target). Results showed that most of the tested

compounds displayed high PPB values (84.31–89.75), quite close to that

what it is consider strong bound to plasma proteins (>90%). Only

compound 17 showed weak bound (PPB 20.26%). BBB was low (<0.1)

for compounds 26, 34 and 44, and medium (normal medium range ≤

0.1–2) for 17, 20, 60 and 66. Two compounds, 20 and 60, are in the

acceptable range to be central nervous systems active compounds (>0.4)

with BBB values 1.03 and 1.16 respectively.

4. Experimental Section

4.1. Chemistry

4.1.1. General chemistry methods

All reagents, solvents, and starting materials were obtained from

commercial suppliers and were used without further puriﬁcation. The

crude reaction mixtures were concentrated under reduced pressure by

removing the organic solvents in a rotary evaporator. Reactions were

monitored by thin layer chromatography (TLC) using Kieselgel 60 F₂₅₄

(E. Merck) plates and UV detector for visualization. Flash column

chromatography was performed on Silica Gel 60 (E. Merck). All reported

yields are of puriﬁed products. Melting points were obtained on a Stuart

Melting Point Apparatus SMP 10 and are uncorrected. Mass spectra were

recorded on a Micromass AUTOSPECQ mass spectrometer: CI at 150 eV,

HR mass measurements with resolutions of 10,000. FAB mass spectra

were recorded using a thioglycerol matrix. NMR spectra were recorded

at 25 ^◦C on a Bruker AV500 spectrometer at 500 MHz for ¹H and 125

MHz for ¹³C. COSY, DEPT, HSQC, and HMBC experiments were per-

formed to assign the signals in the NMR spectra. The chemical shifts (δ)

reported are given in parts per million (ppm) on the δ scale relative to

TMS, and the coupling constants (J) are in hertz (Hz). ¹H chemical shift

values (δ) are referenced to the residual nondeuterated components of

the NMR solvents (δ = 2.54 ppm for DMSO, δ = 7.26 ppm for CDCl₃).

The ¹³C chemical shifts (δ) are referenced to deuterated solvent (central

peak, δ = 39.5 ppm for DMSO and 49.15 ppm for MeOD) as the internal

standard. The spin multiplicities are reported as s (singlet), d (doublet), t

(triplet), q (quadruplet), m (multiplet), or br s (broad singlet). The purity

of ﬁnal compounds was evaluated by elemental analysis (C, H, and N).

The purity of all the ﬁnal compounds was conﬁrmed to be ≥ 95% by

combustion.

Cytochrome P450 isoforms predicts the ability of compounds to be

inhibitors for drug metabolizing enzymes such as CYP2C19, CYP2D6

and CYP3A4. In this study, the tested compounds did not show inhibi-

tion behaviour for CYP2C19 and CYP2D6. An inhibitory effect on Pgp

was detected in four compounds (20, 26, 34 and 44), while inhibitory

behaviour on CYP3A4 was detected for compounds 26, 34 and 44.

3. Conclusion

To sum up, a set of new compounds derived from 3-amino-1,2-pro-

panediol, in which the nitrogen was functionalized as phenyl urea and

hydroxyl groups as substituted benzoyl esters or triazoles, has been

designed, synthesized, and the antiviral activity against HAdV was

evaluated. Seven selected compounds showed signiﬁcant anti-HAdV

activity within low micromolar ranges, with low cytotoxicity. Our

mechanistic studies suggest that compounds 26 and 44 target the

replication of HAdV DNA by interfering with some of the viral proteins

involved in this process. Compound 66 would block the transcription of

the HAdV E1A gene which is essential to subsequently carry out HAdV

DNA replication, and compounds 17, 20, 34 and 60 would interfere with

later steps of HAdV life cycle after DNA replication, although further

evaluations will be needed for deeper characterization of their precise

mechanism of action. Physicochemical properties, drug likeness scores

and pharmacokinetic proﬁles were predicted for the seven selected

compounds. While compounds 17, 20, 60 and 66 resulted to be in

agreement with Lipinski’s rule of ﬁve (showing high oral absorption),

compounds 26, 34 and 44 failed with two violations (moderate-high

oral absorption). In general, all of the selected compounds exhibited

4.1.2. General procedure for the synthesis of urea derivatives (13–17, and

68)

To a solution of the appropriate isocyanate (3.6 mmol) in dry DCM

(20 mL), a solution of the aminoalcohol 12 (3.0 mmol) in methanol (1

mL) was added dropwise. A white precipitate appeared that was ﬁltered

at vacuum and washed with fresh DCM to give urea derivative.

4.1.2.1 N-(4-Chlorophenyl)-N’-(2,3-dihydroxypropyl)urea (13). The

product was obtained as a white solid (690 mg, 94% yield), mp

13

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

160–161 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ8.76 (s, 1H, NHAr),

7.37–7.35 (m, 2H, Ar), 7.25–7.23 (m, 2H, Ar), 6.19 (t, J = 5.5 Hz, 1H,

CH₂NH), 5.01 (d, J = 4.9 Hz, 1H. CHOH), 4.82 (t, J = 5,2 Hz, 1H, CH₂OH),

3.56–3.50 (m, 2H, CH₂OH), 3.38–3.30 (m, 2H, CH₂NH), 3.03–2.98 (m,

1H, CH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 155.6, 139.1, 128.4, 124.8,

119.4, 70.5, 63.5, 42.4. HRMS (m/z): calcd for C₁₀H₁₃ClN₂O₃Na 267.0507

[M + Na]⁺; found 267.0501. Anal. Calcd for C₁₀H₁₃ClN₂O₃: C, 49.09; H,

5.36; N, 11.45. Found: C, 49.51; H, 5.66; N, 11.34.

126.5, 122.2, 120.8, 71.3, 64.1, 42.9, 18.4. HRMS (m/z): calcd for

C₁₁H₁₆N₂O₃Na 247.1053 [M + Na]⁺; found 247.1055. Anal. Calcd for

C₁₁H₁₆N₂O₃: C, 58.91; H, 7.19; N, 12.49. Found: C, 58.67; H, 7.21; N,

12.49.

4.1.3. General procedure for the synthesis of diester derivatives by reaction

with acyl chlorides (18–44)

To a solution of the urea derivative (13–17) (0.54 mmol) in dry DCM

(20 mL) and DMAP (1.3 mmol), the appropriate acylating agent (1.1

mmol) in dry DCM (5 mL) was added. The reaction mixture was stirred

at rt until TLC showed that all the starting material had reacted (24 h),

then was washed with 1 N aqueous solution of HCl (2 × 20 mL), satu-

rated solution of NaHCO₃(2 × 20 mL) and brine (20 mL). The organic

layer was dried (Na₂SO₄), ﬁltered and evaporated under reduced pres-

sure. The compound was further puriﬁed by ﬂash column chromatog-

raphy on silica gel using the appropriate eluent.

4.1.2.2 N-(2,3-Dihydroxypropyl)-N’-(4-methylphenyl)urea (14). The

product was obtained as a white solid (615 mg, 91% yield), mp

147–148 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.48 (s, 1H, NHAr), 7.26

(d, J = 8.5 Hz, 2H, Ar), 7.03 (d, J = 8.2 Hz, 2H, Ar), 6.10 (t, J = 5.6 Hz,

1H, CH₂NH), 4.85 (d, J = 4.9 Hz, 1H, CHOH), 4.60 (t, J = 5.7 Hz, 1H,

CH₂OH), 3.51 (sex, J = 5.6 Hz, 1H, CH), 3.32–3.27 (m, 2H, CH₂OH),

3.01–2.95 (m, 2H, CH₂NH), 2.22 (s, 3H, CH₃). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 155.6, 138.1, 129.6, 129.0, 117.6, 70.7, 63.7, 42.5, 20.3.

HRMS (m/z): calcd for C₁₁H₁₆N₂O₃Na 247.1064 [M + Na]⁺; found

247.1056. Anal. Calcd for C₁₁H₁₆N₂O₃: C, 58.91; H, 7.19; N, 12.49.

Found: C, 58.53; H, 7.03; N, 12.16.

4.1.3.1. N-[2,3-Bis(benzoyloxy)propyl]-N’-(4-chlorophenyl)urea (18).

The product was obtained as a white solid and puriﬁed by column

chromatography using hexane–ethyl acetate (1:1) as eluent (160 mg,

70% yield), mp 130–131 ^◦C. NMR (500 MHz, DMSO‑d₆) δ8.72, (s, 1H,

NHAr), 8.02 (d, J = 7.5 Hz, 2H, Ar), 7.95 (d, J = 7.5 Hz, 2H, Ar),

7.60–7.52 (m, 2H, Ar), 7.55–7.49 (m, 4H, Ar), 7.43 (d, J = 8.8 Hz, 2H,

Ar), 7.28 (d, J = 8.7 Hz, 2H, Ar), 6.50 (t, J = 5.9 Hz, 1H, CH₂NH),

5.52–5.45 (m, 1H, CH), 4.67 (dd, J = 3.4 Hz, J = 12.0 Hz, 1H, CH₂O),

4.52 (dd, J = 6.6 Hz, J = 12.0 Hz, 1H, CH₂O), 3.70–3.58 (m, 2H,

CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.4, 165.3, 155.2, 139.3,

133.4,129.6, 129.4, 129.3, 129.1, 128.7, 128.6, 128.4, 124.7 119.2,

710.6, 63.7, 39.5. HRMS (m/z): calcd for C₂₄H₂₁ClN₂O₅Na 475.1031 [M

+ Na]⁺; found 475.1026.

4.1.2.3 N-(2,3-Dihydroxypropyl)-N’-[4-(triﬂuoromethyl)phenyl]urea

(15). The product was obtained as a white solid (673 mg, 80% yield) ,

mp 135–136 ^◦C ¹H NMR (500 MHz, DMSO‑d₆) δ 9,04 (s, 1H, NHAr),

7.60–7.57 (m, 4H, Ar), 6.30 (t, J = 5,3 Hz, 1H, CH₂NH), 4.88 (d, J = 5.0

Hz, 1H, CHOH), 4.61 (t, J = 5,6 Hz, 1H, CH₂OH), 3.57–3.51 (m, 1H, CH),

3.35–3.30 (m, 2H, CH₂OH), 3.04–2.99 (m, 2H, CH₂NH). ¹³C NMR (125

MHz, DMSO‑d₆) δ 155.0, 144.2, 125.9, 121.0, 120.8, 117.1, 70.5, 63.7,

42.5. HRMS (m/z): calcd for C₁₁H₁₃F₃N₂O₃Na 301.0770 [M + Na]⁺;

found 301,0769. Anal. Calcd for C₁₁H₁₃F₃N₂O₃: C, 47.49; H, 4.71; N,

10.07. Found: C, 49.51; H, 5.66; N, 11.34.

4.1.2.4. N-(2,3-Dihydroxypropyl)-N’-(4-methoxyphenyl)urea (16).

The product was obtained as a white solid (635 mg, 88% yield), mp

155–156 ^◦C. ¹H NMR (300 MHz, DMSO‑d₆) δ 8.39 (s, 1H, NHAr), 7.28

(d, J = 6.8 Hz, 2H, Ar), 6.81 (d, J = 6.8 Hz, 2H, Ar), 6.03 (t, J = 5.5 Hz,

1H, CH₂NH), 4.85 (d, J = 4.8 Hz, 1H, CHOH), 4.60 (t, J = 5.7 Hz, 1H,

CH₂OH), 3.69 (s, 3H, OCH₃), 3.53–3.44 (m, 1H, CH), 3.38–3.23 (m, 2H,

CH₂OH), 3.02–2.92 (m, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

156.2, 154.1, 134.2, 119.7, 114.3, 71.2, 64.1, 55.6, 43.1. HRMS (m/z):

calcd for C₁₁H₁₆N₂O₄Na 263.1002 [M + Na]⁺; found 263.1003.

4.1.2.5. N-Allyl-N’-(4-chlorophenyl)urea (68). The product was ob-

tained as a white solid and puriﬁed by column chromatography using

dichloromethane-methanol (140:1) as eluent (570 mg, 90% yield). ¹H

NMR (500 MHz, DMSO‑d₆) δ 8.66 (s, 1H, NHAr), 7.46 (d, J = 8.3 Hz, 2H,

Ar), 7.30 (d, J = 8.7 Hz, 2H, Ar), 6.32 (t, J = 5.3 Hz, 1H, CH₂NH),

5.91–5.87 (m, 1H, CH₂= CH), 5.23–5.18 (m, 1H, CH₂= CH), 5.13–5.08

(m, 1H, CH₂= CH), 3.77 (t, J = 5.4 Hz, 1H, CH₂NH). ¹³C NMR (125

MHz, DMSO‑d₆) δ 154.9, 139.4, 136.2, 128.6, 128.4, 124.5, 119.8,

119.1, 114.7, 41.4. HRMS (m/z): calcd for C₁₀H₁₂ClN₂O 211.0633 [M +

H]⁺; found 211.0633. Anal. Calcd for C₁₀H₁₁ClN₂O: C, 57.02; H, 5.26; N,

13.30. Found: C, 56.79; H, 5.03; N, 12.86.

4.1.3.2. N-[2,3-Bis(4-methylbenzoyloxy)propyl]-N’-(4-chlorophenyl)

urea (19). The product was obtained as a colourless oil and puriﬁed by

column chromatography using hexane–ethyl acetate (1.5:1) as eluent

(205 mg, 85% yield). MS (FAB): m/z 503 (100%) [M + Na]⁺. ¹H NMR

(500 MHz, DMSO‑d₆) δ 8.72, (s, 1H, NHAr), 7.89–7.87 (m, 2H, Ar),

7.83–7.81 (m, 2H, Ar), 7.42–7.40 (m, 2H, Ar), 7.33–7.29 (m, 4H, Ar),

7.27–7.25 (m, 2H, Ar), 6.28 (t, J = 5.8 Hz, 1H, CH₂NH), 5.46–5.41 (m,

1H, CH), 4.61 (dd, J = 4.1 Hz, J = 12.0 Hz, 1H, CH₂O), 4.46 (dd, J = 6.0

Hz, J = 11.6 Hz, 1H, CH₂O), 3.62–3.54 (m, 2H, CH₂NH), 2.38 (s, 3H,

CH₃), 2.37 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.4, 165.2,

155.1, 143.8, 143.6, 139.3, 129.4, 129.3, 129.2, 129.1, 128.4, 126.8,

124.4, 119.2, 71.3, 65.7, 30.7, 21.2, 21.1. Anal. Calcd for C₂₆H₂₅ClN₂O₅:

C, 64.93; H, 5.24; N, 5.82. Found: C, 64.82; H, 5.76; N, 5.74.

4.1.3.3. N-[2,3-Bis(2-methylbenzoyloxy)propyl]-N’-(4-chlorophenyl)

urea (20). The product was obtained as a colourless oil and puriﬁed by

column chromatography hexane–ethyl acetate (1.5:1) as eluent (216

mg, 90% yield). MS (FAB): m/z 503 (100%) [M + Na]⁺. ¹H NMR (500

MHz, DMSO‑d₆) δ 8.72, (s, 1H, NHAr), 7.88–7.86 (m, 1H, Ar), 7.83–7.81

(m, 1H, Ar), 7.50–7.46 (m, 2H, Ar), 7.43–7.41 (m, 2H, Ar), 7.43–7.27

(m, 6H, Ar), 6.48 (t, J = 6.0 Hz, 1H, CH₂NH), 5.50–5.45 (m, 1H, CH),

4.63 (dd, J = 3.4 Hz, J = 12.0 Hz, 1H, CH₂O), 4.48 (dd, J = 6.5 Hz, J =

12.0 Hz, 1H, CH₂O), 3.64–3.55 (m, 2H, CH₂NH), 2.49 (s, 3H, CH₃), 2.48

(s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 167.0, 166.8, 155.6,

139.8, 139.7, 139.6, 132.8, 132.7, 132.1, 132.0, 130.7, 130.6, 129.9,

129.5, 128.9, 126.5, 126.3, 125.2, 119.8, 71.8, 64.1, 31.2, 21.5, 21.4.

Anal. Calcd for C₂₆H₂₅ClN₂O₅: C, 64.93; H, 5.24; N, 5.82. Found: C,

64.75; H, 5.58; N, 5.60.

4.1.2.6. N-(2,3-Dihydroxypropyl)-N’-(2-methylphenyl)urea (17). Ac-

cording to the published procedure [54], a solution of o-methyl aniline

(1.2 mmol) in DCM (25 mL) was added to a solution of Na₂CO₃(1.9

mmol) in water (25 mL) and the reaction mixture was vigorously stirred

for 5 min at rt; then triphosgene (0.39 mmol) was added and the solution

was stirred for additional 30 min. After this time, 3-amino-1,2-propane-

diol (11, 1.8 mmol) was added dropwise to the ﬂask and the solution

was stirred at rt until TLC showed the full consumption of the starting

material (2 h). The mixture was separated and the inorganic layer was

extracted with ethyl acetate (3 × 40 mL). The combined organic layers

were dried (MgSO₄), ﬁltered and evaporated in vacuo to obtain the

compound as a white resin. (455 mg, 67% yield). ¹H NMR (300 MHz,

DMSO‑d₆) δ 7.87–7.77 (m, 2H, NHAr, Ar), 7.13–7.04 (m, 1H, Ar), 6.85

(t, J = 7.3 Hz, 2H, Ar), 6.63 (t, J = 5.2 Hz, 1H, CH₂NH), 4.89–4.84 (m,

1H, CHOH), 4.63–4.56 (m, 1H, CH₂OH), 3.55–3.46 (m, 1H, CH),

3.33–3.25 (m, 2H, CH₂OH), 3.03–2.93 (m, 2H, CH₂NH), 2.18 (s, 3H,

CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 156.2, 138.7, 130.5, 127.0,

4.1.3.4. N-[2,3-Bis(4-methoxybenzoyloxy)propyl]-N’-(4-chlorophenyl)

urea (21). The product was obtained as a white solid and puriﬁed by

column chromatography using hexane–ethyl acetate (1:1) as eluent

(190 mg, 74% yield), mp 202–203 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

8.71, (s, 1H, NHAr), 7.96–7.94 (m, 2H, Ar), 7.90–7.88 (m, 2H, Ar),

7.44–7.42 (m, 2H, Ar), 7.27–7.25 (m, 2H, Ar), 7.04–7.01 (m, 4H, Ar),

6.48 (t, J = 5.7 Hz, 1H, CH₂NH), 5.45–5.41 (m, 1H, CH), 4.59 (dd, J =

3.5 Hz, J = 12.1 Hz, 1H, CH₂O), 4.46 (dd, J = 6.7 Hz, J = 11.8 Hz, 1H,

CH₂O), 3.63–3.55 (m, 2H, CH₂NH), 3.83 (s, 3H, OCH₃), 3.82 (s, 3H,

OCH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ165.1, 165.0, 163.3, 163.2,

14

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

155.1, 139.3, 131.4, 131.3, 128.4, 124.7, 121.8, 121.5, 119.2, 114.0,

113.9, 71.2, 63.4, 55.5, 55.4, 31.2. HRMS (m/z): calcd for

C₂₆H₂₅ClN₂O₇Na 535.1242 [M + Na]⁺; found 535.1247.

3.66–3.55 (m, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.0,

164.9, 155.2, 152.7, 152.6, 141.9, 141.8, 139.2, 129.2, 128.4, 124.7,

124.6, 124.3, 119.2, 106.8, 106.4, 71.7, 63.6, 60.1, 55.9, 55.8. HRMS

(m/z): calcd. for C₃₀H₃₃ClN₂O₁₁Na 655.1665 [M + Na]⁺; found

655.1653. Anal. Calcd for C₃₀H₃₃ClN₂O₁₁: C, 56.92; H, 5.25; N, 4.43.

Found: C, 56.80; H, 5.25; N, 4.41.

4.1.3.5.

N-[2,3-Bis(4-cyanobenzoyloxy)propyl]-N’-(4-chlorophenyl)

urea (22). The product was obtained as a white solid and puriﬁed by

column chromatography using hexane–ethyl acetate (1.5:1) as eluent

(150 mg, 60% yield), mp 205–206 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

8.70, (s, 1H, NHAr), 8.13–8.12 (m, 2H, Ar), 8.08–8.06 (m, 2H, Ar),

8.01–7.99 (m, 4H, Ar), 7.40–7.38 (m, 2H, Ar), 7.26–7.23 (m, 2H, Ar),

6.51 (t, J = 5.7 Hz, 1H, CH₂NH), 5.50–5.47 (m, 1H, CH), 4.70 (dd, J =

3.3 Hz, J = 12.4 Hz, 1H, CH₂O), 4.56 (dd, J = 6.6 Hz, J = 12.0 Hz, 1H,

CH₂O), 3.67–3.60 (m, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

164.3, 164.2, 155.2, 139.2, 133.5, 133.2, 132.8, 132.7, 129.9, 129.8,

128.4, 124.7, 119.3, 118.0, 117.9, 115.6, 7.23, 64.3. HRMS (m/z): calcd

for C₂₆H₁₉ClN₄O₅Na 525.0936 [M + Na]⁺; found 525.0933. Anal. Calcd

for C₂₆H₁₉ClN₄O₅: C, 62.09; H, 3.82; N, 11.14. Found: C, 62.25; H, 3.81;

N, 11.00.

4.1.3.10. N-[2,3-Bis(4-methylbenzoyloxy)propyl]-N’-(4-methylphenyl)

urea (27). The product was obtained as a white solid and puriﬁed by

column chromatography using hexane–ethyl acetate (2.5:1) as eluent

(175 mg, 84% yield), mp 95–96 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.44

(s, 1H, NHAr), 7.89 (d, J = 8.2 Hz, 2H, Ar), 7.82 (d, J = 8.2 Hz, 2H, Ar),

7.31 (t, J = 6.7 Hz, 4H, Ar), 7.27 (d, J = 8.4 Hz, 2H, Ar), 7.02 (d, J = 8.3

Hz, 2H, Ar), 6.39 (t, J = 6.0 Hz, 1H, CH₂NH), 5.44 (quint, J = 5.2 Hz, 1H,

CH), 4.62 (dd, J = 3,5 Hz, J = 8,6 Hz, 1H, CH₂O), 4.47 (dd, J = 6,8 Hz, J

= 5,2 Hz, 1H, CH₂O), 3.63–3.54 (m, 2H, CH₂NH), 2.37 (d, J = 4,0 Hz,

6H, CH₃), 2.22 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.4,

165.3, 155.3, 143.8, 143.7, 137.7, 129.9, 129.4, 129.3, 129.2, 129.1

(2C), 128.9, 126.9, 126.7, 117.9, 71.4, 63.6, 26.8, 21.1, 20.3. HRMS (m/

z): calcd for C₂₇H₂₈N₂O₅Na 483.1890 [M + Na]⁺; found 483.1884. Anal.

Calcd for C₂₇H₂₈N₂O₅: C, 70.42; H, 6.13; N, 6.08. Found: C, 69.40; H,

5.96; N, 5.89.

4.1.3.6. N-[2,3-Bis(4-nitrobenzoyloxy)propyl]-N’-(4-chlorophenyl)urea

(23). The product was obtained as a yellow solid and puriﬁed by column

chromatography using hexane–ethyl acetate (1:1) as eluent (244 mg,

90% yield), mp 216–217 ^◦C. mp 216–217 ^◦C. MS (FAB): m/z 565 (100%)

[M + Na]⁺. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.70, (s, 1H, NHAr),

8.35–8.33 (m, 4H, Ar), 8.23–8.21 (m, 2H, Ar), 8.18–8.16 (m, 2H, Ar),

7.41–7.38 (m, 2H, Ar), 7.26–7.23 (m, 2H, Ar), 6.53 (t, J = 6.01 Hz, 1H,

CH₂NH), 5.55–5.51 (m, 1H, CH), 4.74 (dd, J = 3.3 Hz, J = 12.2 Hz, 1H,

CH₂O), 4.60 (dd, J = 6.7 Hz, J = 12.2 Hz, 1H, CH₂O), 3.70–3.60 (m, 2H,

CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 164.1, 163.0, 155.2, 150.4,

150.3, 139.2, 134.9, 134.7, 130.8, 130.6, 128.4, 124.7, 123.9, 123.8,

119.2, 72.5, 64.4.

4.1.3.11. N-[2,3-Bis(2-methylbenzoyloxy)propyl]-N’-(4-methylphenyl)

urea (28). The product was obtained as a white solid and puriﬁed by

column chromatography using hexane–ethyl acetate (2.5:1) as eluent

(168 mg, 74% yield), mp 116–117 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

8.45 (s, 1H, NHAr), 7.87 (d, J = 7.7 Hz, 2H, Ar), 7.83 (d, J = 7.7 Hz, 2H,

Ar),7.51–7.46 (m, 2H, Ar), 7.34–7.25 (m, 6H, Ar), 7.03 (d, J = 8.0 Hz,

2H, Ar), 6.39 (t, J = 5.9 Hz, 1H, CH₂NH), 5.50–5.44 (m, 1H, CH), 4.63

(dd, J = 3.3 Hz, J = 12.1 Hz, 1H, CH₂O), 4.48 (dd, J = 6.8 Hz, J = 12.1

Hz, 1H, CH₂O), 3.63–3.53 (m, 2H, CH₂NH), 2.49 (s, 6H, CH₃), 2.22 (s,

3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 166.5, 166.3, 155.3, 139.2,

139.1, 137.7, 132.3, 132.2, 131.6, 131.5, 130.2, 130.1, 129.9, 129.3,

129.1, 129.0, 125.9, 125.8, 117.2, 71.3, 63.5, 21.0, 20.8, 20.2. HRMS

(m/z): calcd for C₂₇H₂₈N₂O₅Na 483.1890 [M + Na]⁺; found 483.1886.

4.1.3.7.

N-{2,3-Bis[(4-triﬂuoromethyl)benzoyloxy]propyl}-N’-(4-

chlorophenyl)urea (24). The product was obtained as a white solid and

puriﬁed by column chromatography using hexane–ethyl acetate (2:1) as

eluent (198 mg, 67% yield), mp 156–157 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 8.73, (s, 1H, NHAr), 8.21 (d, J = 8.53 Hz, 2H, Ar), 8.16 (d, J

= 8.62 Hz, 2H, Ar), 7.93–7.88 (m, 4H, Ar), 4.43–4.41 (m, 2H, Ar),

7.28–7.24 (m, 2H, Ar), 6.54 (t, J = 5.90 Hz, 1H, CH₂NH), 5.57–5.52 (m,

1H, CH), 4.74 (dd, J = 3.6 Hz, J = 12.3 Hz, 1H, CH₂O), 4.61 (dd, J = 6.6

Hz, J = 12.0 Hz, 1H, CH₂O), 3.72–3.62 (m, 2H, CH₂NH). ¹³C NMR (125

MHz, DMSO‑d₆) δ 164.4, 164.3, 155.2, 139.2, 133.3, 133.1, 133.0,

132.9, 130.2, 130.0, 128.4, 125.8, 125.7, 125.6, 124.7, 122.6, 122.5,

119.2, 72.2, 64.2. HRMS (m/z): calcd for C₂₆H₁₉Cl F₆N₂O₅Na 611.0779

[M + Na]⁺; found 611.0771. Anal. Calcd for C₂₆H₁₉ClF₆N₂O₅: C, 53.03;

H, 3.25; N, 4.76. Found: C, 52.75; H, 3.36; N, 4.77.

4.1.3.12.

N-[2,3-Bis(4-methoxybenzoyloxy)propyl]-N’-(4-methyl-

phenyl)urea (29). The product was obtained as a solid and puriﬁed by

column chromatography using hexane–ethyl acetate (2:1) as eluent

(150 mg, 61% yield), mp 144–145 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

8.42 (s, 1H, NHAr), 7.95 (d, J = 7.5 Hz, 2H, Ar), 7.89 (d, J = 9.1 Hz, 2H,

Ar), 7.27 (d, J = 8.0 Hz, 2H, Ar), 7.05–7.00 (m, 6H, Ar), 6.37 (t, J = 6.0

Hz, 1H, CH₂NH), 5.43–5.39 (m, 1H, CH), 4.58 (dd, J = 3.8 Hz, J = 11.5

Hz, 1H, CH₂O), 4.45 (dd, J = 3.8 Hz, J = 11.5 Hz, 1H, CH₂O), 3.83 (s,

6H, OCH₃), 3.62–3.53 (m, 2H, CH₂NH), 2.22 (s, 3H, CH₃). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 165.1, 164.9, 163.3, 155.3, 137.7, 131.4, 131.3,

129.9, 128.9, 121.8, 121.5, 117.9, 114.0, 113.9, 71.3, 63.5, 55.5, 55.4,

20.2. HRMS (m/z): calcd for C₂₇H₂₈N₂O₇Na 515.1789 [M + Na]⁺; found

515.1783.

4.1.3.8.

N-[2,3-Bis(2,4-dimethoxybenzoyloxy)propyl]-N’-(4-chlor-

ophenyl)urea (25). The product was obtained as a white solid and pu-

riﬁed by column chromatography using hexane–ethyl acetate (1:1.5) as

eluent (235 mg, 82% yield), mp 113–114 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 8.75, (s, 1H, NHAr), 7.78–7.72 (m, 2H, Ar), 7.45 (d, J = 8.5

Hz, 2H, Ar), 7.28 (d, J = 8.9 Hz, 2H, Ar), 6.65 (s, 2H, Ar), 6.62–6.57 (m,

2H, Ar), 6.42 (t, J = 5.7 Hz, 1H, CH₂NH), 5.35–5.30 (m, 1H, CH), 4.47

(dd, J = 3.2 Hz, J = 12.1 Hz, 1H, CH₂O), 4.37 (dd, J = 6.5 Hz, J = 12.2

Hz, 1H, CH₂O), 3.85 (d, J = 3.3 Hz, 6H, OCH₃), 3.80 (d, J = 4.5 Hz, 6H,

OCH₃), 3.55 (t, J = 5.5 Hz, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆)

δ 164.5, 164.1, 164.0, 163.9, 161.0, 160.9, 155.1, 139.3, 133.2, 133.1,

128.4, 124.6, 119.2, 111.6, 111.3, 105.3, 105.2, 98.9, 70.5, 62.9, 55.7,

55.5. HRMS (m/z): calcd for C₂₈H₂₉ClN₂O₉Na 595.1454 [M + Na]⁺;

found 595.1447. Anal. Calcd for C₂₈H₂₉ClN₄O₉: C, 58.69; H, 5.10; N,

4.89. Found: C, 58.25; H, 5.23; N, 4.44.

4.1.3.13. N-[2,3-Bis(4-cyanobenzoyloxy)propyl]-N’-(4-methylphenyl)

urea (30). The product was obtained as a solid and puriﬁed by column

chromatography using hexane–ethyl acetate (1:1) as eluent (155 mg,

64% yield), mp 181–182 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.40 (s, 1H,

NHAr), 8.13 (d, J = 8.2 Hz, 2H, Ar), 8.07 (d, J = 8.2 Hz, 2H, Ar),

8.03–7.98 (m, 4H, Ar), 7.23 (d, J = 8.4 Hz, 2H, Ar), 7.02 (d, J = 8.3 Hz,

2H, Ar), 6.40 (t, J = 6.0 Hz, 1H, CH₂NH), 5.51–5.47 (m, 1H, CH), 4.69

(dd, J = 3,5 Hz, J = 8,6 Hz, 1H, CH₂O), 4.56 (dd, J = 6,8 Hz, J = 5,2 Hz,

1H, CH₂O), 3.66–3.57 (m, 2H, CH₂NH), 2.22 (s, 3H, CH₃). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 164.3, 164.2, 155.3, 137.6, 133.5, 133.2, 129.9,

129.8, 129.0, 117.9, 115.6, 72.4, 64.3, 39.5, 20.2. HRMS (m/z): calcd

for C₂₇H₂₂N₄O₅Na 505.1482 [M + Na]⁺; found 505.1476. Anal. Calcd

4.1.3.9. N-[2,3-Bis(3,4,5-trimethoxybenzoyloxy)propyl]-N’-(4-chlor-

ophenyl)urea (26). The product was obtained as a white solid and pu-

riﬁed by column chromatography using hexane–ethyl acetate (1:1.5) as

eluent (248 mg, 78% yield), mp 163–164 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 8.73 (s, 1H, NHAr), 7.41 (d, J = 8.7 Hz, 2H, Ar),7.28–7.25

(m, 4H, Ar), 7.22 (s, 2H, Ar), 6.49 (t, J = 8.2 Hz 1H, CH₂NH), 5.48–5.42

(m, 1H, CH), 4.65 (dd, J = 3.4 Hz, J = 11.9 Hz, 1H, CH₂O), 4.49–4.42

(m, 1H, CH₂O), 3.81–3.76 (m, 12H, OCH₃), 3.74–3.71 (m, 6H, OCH₃),

for C

H N₄O₅: C, 67.21; H, 4.60; N, 11.61. Found: C, 66.22; H, 4.47; N,

27 22

11.14.

4.1.3.14. N-[2,3-Bis(4-nitrobenzoyloxy)propyl]-N’-(4-methylphenyl)

urea (31). The product was obtained as a light yellow solid and puriﬁed

by column chromatography using hexane–ethyl acetate (2:1) as eluent

(162 mg, 62% yield), mp 214–215 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

8.45 (s, 1H, NHAr), 8.38–8.33 (m, 4H, Ar), 8.24 (d, J = 8.4 Hz, 2H, Ar),

15

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

8.19 (d, J = 8.3 Hz, 2H, Ar), 7.26 (d, J = 6.0 Hz, 2H, Ar), 7.03 (d, J = 7.0

Hz, 2H, Ar), 6.45 (t, J = 5 Hz, 1H, CH₂NH) 5.56–5.52 (m, 1H, CH), 4.75

(dd, J = 3,5 Hz, J = 8,6 Hz, 1H, CH₂O), 4.62 (dd, J = 6,8 Hz, J = 5,2 Hz,

1H, CH₂O), 3.71–3.61 (m, 2H, CH₂NH), 2.24 (s, 3H, CH₃). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 164.0, 163.9, 155.3, 150.3, 137.6, 134.9, 134.6,

130.7, 130.6, 129.9, 128.9, 123.8, 117.8, 72.5, 64.4, 39.5, 20.2. HRMS

(m/z): calcd for C₂₅H₂₂N₄O₉Na 545.1279 [M + Na]⁺; found 545.1274.

Anal. Calcd for C₂₅H₂₂N₄O₉: C, 57.47; H, 4.24; N, 10.72. Found: C,

56.99; H, 4.20; N, 10.68.

phenyl] urea (36). The product was obtained as a white solid and puriﬁed

by column chromatography using hexane–ethyl acetate (2.5:1) as eluent

(188 mg, 73% yield), mp 112–113 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

9.05 (s, 1H, NHAr), 7.90 (d, J = 8.2 Hz, 1H, Ar), 7.85 (d, J = 8.2 Hz, 1H,

Ar), 7.61 (q, J = 8.7 Hz, 4H, Ar), 7.52–7.47 (m, 2H, Ar), 7.35–7.28 (m,

4H, Ar), 6.63 (t, J = 6.1 Hz, 1H, CH₂NH), 5.54–5.50 (m, 1H, CH), 4.67

(dd, J = 3,5 Hz, J = 11,8 Hz, 1H, CH₂O), 4.52 (dd, J = 6,8 Hz, J = 11,9

Hz, 1H, CH₂O), 3.68–3.61 (m, 2H, CH₂NH), 2.51 (s, 3H, CH₃). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 166.5, 166.3, 154.9, 143.9, 139.3, 139.2, 132.3,

132.2, 131.6, 131.5, 130.2, 130.1, 129.3, 128.9, 125.9, 123.5, 121.3,

117.4, 71.2, 63.5, 39.5, 21.0, 20.9. HRMS (m/z): calcd for

4.1.3.15. N-{2,3-Bis[4-(triﬂuoromethyl)benzoyloxy]propyl}-N’-(4-methyl-

phenyl) urea (32). The product was obtained as a white solid and puriﬁed

by column chromatography using hexane–ethyl acetate (2:1) as eluent

(179 mg, 63% yield), mp 137–138 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.43

(s, 1H, NHAr), 8.20 (d, J = 8.2 Hz, 2H, Ar), 8.15 (d, J = 8.2 Hz, 2H, Ar),

7.92 (d, J = 7.0 Hz, 4H, Ar), 7.26 (d, J = 8.4 Hz, 2H, Ar), 7.04 (d, J = 8.3

Hz, 2H, Ar), 6.43 (t, J = 6.1 Hz, 1H, CH₂NH), 5.55–5.51 (m, 1H, CH), 4.74

(dd, J = 3,4 Hz, J = 8,7 Hz, 1H, CH₂O), 4.60 (dd, J = 6,7 Hz, J = 5,4 Hz,

1H, CH₂O), 3.70–3.60 (m, 2H, CH₂NH), 2.24 (s, 3H, CH₃). ¹³C NMR (125

MHz, DMSO‑d₆) δ 164.4, 164.3, 155.4, 137.6, 133.3, 133.1, 133.0, 132.8,

132.6, 130.2, 130.0, 129.9, 129.0, 125.9, 125.8, 125.7, 125.6, 124.7,

122.6, 117.9, 72.3, 64.2, 20.2. HRMS (m/z): calcd for C₂₇H₂₂F₆N₂O₅Na

C

₂₇H₂₅F₃N₂O₅Na 515.1788 [M + Na]⁺; found 515.1782. Anal. Calcd for

₂₇H₂₅F₃N₂O₅: C, 63.03; H, 4.90; N, 5.44. Found: C, 62.52; H, 4.92; N,

5.49.

4.1.3.20. N-[2,3-Bis(4-methoxybenzoyloxy)propyl]-N’-[4-(triﬂuoromethyl)

phenyl] urea (37). The product was obtained as a white solid and puriﬁed by

column chromatography using hexane–ethyl acetate (1:1) as eluent (195

mg, 71% yield), mp 128–129 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ 9.01 (s,

1H, NHAr), 7.98–7.96 (m, 2H, Ar), 7.94–7.89 (m, 2H, Ar) 7.60 (q, J = 8.0

Hz, 4H, Ar), 7.07–7.03 (m, 4H, Ar), 6.60 (t, J = 6.9 Hz, 1H, CH₂NH),

5.47–5.43 (m, 1H, CH), 4.61 (dd, J = 3.6 Hz, J = 11.8 Hz, 1H, CH₂O), 4.48

(dd, J = 7.0 Hz, J = 11.9 Hz, 1H, CH₂O), 3.85 (s, 6H, OCH₃), 3.65–3.60 (m,

2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.1, 164.9, 163.3, 163.2,

154.9, 143.9, 132.6, 131.4, 131.2, 123.5, 121.7, 121.5, 117.3, 114.1, 114.0,

113.9, 113.8, 71.1, 63.4, 55.5, 39.5. HRMS (m/z): calcd for

681.4719 [M + Na]⁺; found 681.4715. Anal. Calcd for C₂₇

H F₆N₂O₅: C,

22

57.05; H, 3.90; N, 4.93. Found: C, 56.80; H, 3.88; N, 4.90.

4.1.3.16. N-[2,3-Bis(2,4-dimethoxybenzoyloxy)propyl]-N’-(4-methyl-

phenyl)urea (33). The product was obtained as a white resin and puriﬁed

by column chromatography using hexane–ethyl acetate (1:1.5) as eluent

(169 mg, 61% yield). ¹H NMR (500 MHz, DMSO‑d₆) ϖ8.45 (s, 1H,

NHAr), 7.75 (q, J = 8.6 Hz, 2H, Ar), 7.29 (d, J = 8.0 Hz, 2H, Ar), 7.04 (d,

J = 8.1 Hz, 2H, Ar), 6.64 (s, 2H, Ar), 6.59 (t, J = 8.5 Hz, 2H, Ar),

6.32–6.28 (m, 1H, CH₂NH), 5.36–5.29 (m, 1H, CH), 4.51–4.43 (m, 1H,

CH₂O), 4.37 (dd, J = 6.5 Hz, J = 9.5 Hz, 1H, CH₂O), 3.84–3.80 (m, 12H,

OCH₃), 3.55–3.50 (m, 2H, CH₂NH), 2.24 (s, 3H, CH₃). ¹³C NMR (125

MHz, DMSO‑d₆) δ 164.1, 164.0, 163.9, 160.9, 155.3, 137.8, 133.2,

133.1, 129.9, 129.0, 117.8, 111.7, 111.4, 105.3, 105.2, 98.9, 98.8, 70.6,

63.0, 55.5, 55.4, 20.2. HRMS (m/z): calcd for C₂₉H₃₂N₂O₉Na 575.2000

[M + Na]⁺; found 575.1993. Anal. Calcd for C₂₉H₃₂N₂O₉: C, 63.04; H,

5.84; N, 5.07. Found: C, 62.97; H, 5.81; N, 4.99.

C

₂₇H₂₅F₃N₂O₇Na 569.1506 [M + Na]⁺; found 569.1497. Anal. Calcd for

₂₇H₂₅F₃N₂O₇: C, 59.34; H, 4.61; N, 5.13. Found: C, 59.18; H,4.57; N, 5.51.

4.1.3.21. N-[2,3-Bis(4-cyanobenzoyloxy)propyl]-N’-[4-(triﬂuoromethyl)

phenyl]urea (38). The product was obtained as a white solid and puriﬁed

by column chromatography using hexane–ethyl acetate (2:1) as eluent

(205 mg, 76% yield), mp 170–171 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

9.00, (s, 1H, NHAr), 8.14 (d, J = 8.2 Hz, 2H, Ar), 8.08 (d, J = 8.2 Hz, 2H,

Ar), 8.02–7.99 (m, 4H, Ar), 7.56 (m, 4H, Ar), 6.63 (t, J = 5.9 Hz, 1H,

CH₂NH), 5.53–5.49 (m, 1H, CH), 4.71 (dd, J = 3.5 Hz, J = 12.1 Hz, 1H,

CH₂O), 4.57 (dd, J = 6.7 Hz, J = 12.1 Hz, 1H, CH₂O), 3.69–3.62 (m, 2H,

CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 164.3, 164.2, 155.0, 143.9,

133.4, 133.2, 132.8, 132.7, 130.0, 129.8, 125.9, 117.9, 117.4, 115.6,

72.2, 64.3. HRMS (m/z): calcd for C₂₇H₁₉F₃N₄O₅Na: 559.1200 [M +

Na]⁺; found 559.1192 Anal. Calcd for C₂₇H₁₉F₃N₄O₅: C, 60.45; H, 3.57;

N, 10.44. Found: C, 59.85; H, 3.56; N, 10.42.

4.1.3.17. N-[2,3-Bis(3,4,5-trimethoxybenzoyloxy)propyl]-N’-(4-meth-

ylphenyl)urea (34). The product was obtained as a white solid and pu-

riﬁed by column chromatography using hexane–ethyl acetate (1:1.5) as

eluent (267 mg, 87% yield), mp 151–152 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 8.43 (s, 1H, NHAr), 7.26–7.24 (m, 4H, Ar), 7.22 (s, 2H, Ar),

7.02 (d, J = 8.1 Hz, 2H, Ar), 6.38 (t, J = 8.5 Hz 1H, CH₂NH), 5.47–5.42

(m, 1H, CH), 4.65 (dd, J = 3.6 Hz, J = 8.5 Hz, 1H, CH₂O), 4.45 (dd, J =

3.5 Hz, J = 8.4 Hz, 1H, CH₂O), 3.82–3.72 (m, 18H, OCH₃) 3.63–3.55 (m,

2H, CH₂NH), 2.22 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.0,

164.9, 155.4, 152.7, 152.6, 142.0, 141.9, 137.7, 130.0, 128.9, 124.7,

124.4, 117.9, 106.8, 106.6, 71.8, 63.7, 60.1, 55.9, 55.8, 39.5, 20.2.

HRMS (m/z): calcd for C₃₁H₃₆N₂O₁₁Na 635.2211 [M + Na]⁺; found

635.2202. Anal. Calcd for C₂₅H₂₂N₄O₉: C, 60.78; H, 5.92; N, 4.57.

Found: C, 60.90; H, 5.94; N, 4.58.

4.1.3.22. N-[2,3-Bis(4-nitrobenzoyloxy)propyl]-N’-[4-(triﬂuoromethyl)

phenyl]urea (39). The product was obtained as a yellow solid and pu-

riﬁed by column chromatography using hexane–ethyl acetate (1:1) as

eluent (195 mg, 68% yield), mp 186–187 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 9.00, (s, 1H, NHAr), 8.35–8.32 (m, 4H, Ar), 8.23 (d, J = 8.7

Hz, 2H, Ar), 8.17 (d, J = 8.6 Hz, 2H, Ar), 7.56 (m, 4H, Ar), 6.65 (t, J =

5.4 Hz, 1H, CH₂NH), 5.54 (m, 1H, CH), 4.74 (dd, J = 2.8 Hz, J = 12.1 Hz,

1H, CH₂O), 4.61 (dd, J = 6.4 Hz, J = 12.1 Hz, 1H, CH₂O), 3.71–3.63 (m,

2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 164.0, 163.9, 155.0,

150.4, 150.3, 134.9, 134.7, 130.8, 130.6, 125.9, 123.9, 123.8, 117.3,

129.1, 129.0, 126.8, 126.6 125.8, 125.7, 117.3, 72.4, 64.4. HRMS (m/z):

calcd for C₂₅H₁₉F₃N₄O₉Na: 599.0996 [M + Na]⁺; found 599.0988. Anal.

Calcd for C₂₅H₁₉F₃N₄O₉: C, 52.09; H, 3.32; N, 9.72. Found: C, 51.63; H,

3.22; N, 9.78.

4.1.3.18. N-[2,3-Bis(4-methylbenzoyloxy)propyl]-N’-[4-(triﬂuoromethyl)

phenyl] urea (35). The product was obtained as a white solid and puriﬁed

by column chromatography using hexane–ethyl acetate (3:1) as eluent

(217 mg, 84 %yield), mp 111–112 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

9.00, (s, 1H, NHAr), 7.89 (d, J = 8.2 Hz, 2H, Ar), 7.83 (d, J = 8.2 Hz, 2H,

Ar), 7.61–7.55 (m, 4H, Ar), 7.32–7.29 (m, 4H, Ar), 6.59 (t, J = 5.9 Hz, 1H,

CH₂NH), 5.48–5.44 (m, 1H, CH), 4.63 (dd, J = 3.3 Hz, J = 12.1 Hz, 1H,

CH₂O), 4.48 (dd, J = 6.9 Hz, J = 12.1 Hz, 1H, CH₂O), 3.67–3.58 (m, 2H,

CH₂NH), 2.37, 2.36 (2 s, 6H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ

165.4, 165.2, 154.9, 144.0, 143.8, 143.7, 129.4, 129.3, 129.2, 129.1,

129.0, 126.8, 126.6 125.8, 125.7, 117.3, 71.3, 63.5, 21.1, 21.0. HRMS (m/

z): calcd for C₂₇H₂₅F₃N₂O₅Na: 537.1608 [M + Na]⁺; found 537.1600

Anal. Calcd for C₂₇H₂₅F₃N₂O₅: C, 63.03; H, 4.90; N, 5.44. Found: C, 62.88;

H, 4.87; N, 5.38.

4.1.3.23. N-{2,3-Bis[4-(triﬂuoromethxyl)benzoyloxy]propyl}-N’-[4-(tri-

ﬂuoromethyl) phenyl]urea (40). The product was obtained as a white solid

and puriﬁed by column chromatography using hexane–ethyl acetate

(2.5:1) as eluent (236 mg, 76% yield), mp 135–136 ^◦C. ¹H NMR (500

MHz, DMSO‑d₆) δ 9.01 (s, 1H, NHAr), 8.17 (d, J = 8.2 Hz, 2H, Ar), 8.11

(d, J = 8.2 Hz, 2H, Ar), 7.90–7.86 (m, 4H, Ar), 7.58–7.52 (m, 4H, Ar),

6.63 (t, J = 6.0 Hz, 1H, CH₂NH), 5.55–5.50 (m, 1H, CH), 4.52 (dd, J =

3.4 Hz, J = 12.1 Hz, 1H, CH₂O), 4.58 (dd, J = 6.6 Hz, J = 12.2 Hz, 1H,

CH₂O), 3.70–3.61 (m, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

164.9, 164.7, 155.5, 144.4, 133.8, 133.6, 133.3, 130.7, 130.5, 127.4,

126.4, 126.3, 126.2, 125.2, 123.9, 117.8, 72.6, 64.7. HRMS (m/z): calcd.

for C₂₇H₁₉F₉N₂O₅Na 645.1042 [M + Na]⁺; found 645.1035.

4.1.3.19. N-[2,3-Bis(2-methylbenzoyloxy)propyl]-N’-[4-(triﬂuoromethyl)

16

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

4.1.3.24.

N-[2,3-Bis(2,4-dimethoxybenzoyloxy)propyl]-N’-[4-(tri-

aqueous solution of NaHCO₃and brine, then it was dried (Na₂SO₄),

ﬁltered and evaporated in vacuo. The compound was further puriﬁed

through ﬂash column chromatography using the appropriate mixture

hexane–ethyl acetate as eluent.

ﬂuoromethyl) phenyl]urea (41). The product was obtained as a white solid

and puriﬁed by column chromatography using hexane–ethyl acetate

(1:1.5) as eluent (169 mg, 56% yield), mp 101–102 ^◦C. ¹H NMR (500

MHz, DMSO‑d₆) δ 9.04 (s, 1H, NHAr), 7.77 (d, J = 8.6 Hz, 1H, Ar), 7.74 (d,

J = 8.6 Hz, 1H, Ar) 7.61 (q, J = 8.0 Hz, 4H, Ar), 6.67–6.64 (m, 2H, Ar),

6.62–6.56 (m, 2H, Ar), 6.53 (t, J = 5.5 Hz, 1H, CH₂NH), 5.39–5.33 (m, 1H,

CH), 4.50 (dd, J = 3.5 Hz, J = 12.3 Hz, 1H, CH₂O), 4.39 (dd, J = 6.5 Hz, J

= 12.3 Hz, 1H, CH₂O), 3.87–3.84 (m, 6H, OCH₃), 3.83–3.78 (m, 6H,

OCH₃), 3.59 (t, J = 8.6 Hz, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

164.4, 164.1, 164.0, 163.8, 160.9, 154.9, 144.0, 133.2, 125.9, 125.7,

123.5, 120.9, 117.3, 111.7, 111.4, 105.3, 105.2, 98.9, 70.4, 63.0, 55.7,

55.5, 26.7. HRMS (m/z): calcd for C₂₉H₂₉F₃N₂O₉Na 629.1717 [M + Na]⁺;

found 629.1709. Anal. Calcd for C₂₉H₂₉F₃N₂O₉: C, 57.43; H, 4.82; N, 4.62.

Found: C, 57.22; H, 4.79; N, 4.55.

4.1.4.1. N-{2,3-Bis[(E)-(3,4,5-trimethoxyphenyl)acryloxy]prop-2-yl}-

N’-(4- chlorophenyl)urea (45). The product was obtained as a white solid

and puriﬁed by column chromatography using using hexane–ethyl ac-

etate (2:1) as eluent (376 mg, 69% yield), mp 120–121 ^◦C. ¹H NMR (500

MHz, DMSO‑d₆) δ 8.73, (s, 1H, NHAr), 7.67–7.58 (m, 2H, CH = CHCO),

7.46–7.40 (m, 2H, Ar), 7.28–7.23 (m, 2H, Ar), 7.07 (s, 4H, Ar),

6.62–6.54 (m, 2H, CH = CHCO), 6.45 (t, J = 5.7 Hz, 1H, CH₂NH),

5.29–5.23 (m, 1H, CH), 4.48 (dd, J = 3.3 Hz, J = 12.0 Hz, 1H, CH₂O),

4.33 (dd, J = 6.5 Hz, J = 12.2 Hz, 1H, CH₂O), 3.88–3.78 (m, 12H,

OCH₃), 3.72–3.68 (m, 6H, OCH₃), 3.58–3.41 (m, 2H, CH₂NH). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 166.1, 165.9, 155.1, 153.0, 145.3, 139.7, 139.6,

139.2, 129.4, 128.4, 124.6, 119.2, 117.1, 116.7, 106.1, 106.0, 70.8,

60.0, 56.0, 44.8, 30.6. HRMS (m/z): calcd for C₃₄H₃₇ClN₂O₁₁Na

707.1978 [M + Na]⁺; found 707.1971.

4.1.3.25. N-[2,3-Bis(3,4,5-trimethoxybenzoyloxy)propyl]-N’-[4-(tri-

ﬂuoromethyl) phenyl]urea (42). The product was obtained as a white

solid and puriﬁed by column chromatography using hexane–ethyl ace-

tate (1:1) as eluent (276 mg, 82% yield), mp 185–186 ^◦C. ¹H NMR (500

MHz, DMSO‑d₆) δ 9.03 (s, 1H, NHAr), 7.62–7.57 (m, 4H, Ar), 7.28 (s,

2H, Ar), 7.24 (s, 2H, Ar), 6.62 (t, J = 5.6 Hz, 1H, CH₂NH), 5.50–5.46 (m,

1H, CH), 4.68 (dd, J = 3.2 Hz, J = 12.2 Hz, 1H, CH₂O), 4.49 (dd, J = 6.4

Hz, J = 12.2 Hz, 1H, CH₂O), 3.82–3.73 (m, 18H, OCH₃) 3.67–3.61 (m,

2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.0, 155.0, 152.7,

152.6, 143.9, 142.0, 141.9, 127.5, 125.9, 125.8, 124.6, 124.3, 121.0,

117.3, 106.8, 106.6, 71.6, 63.6, 60.1, 55.9, 55.8, 39.5. HRMS (m/z):

calcd for C₃₁H₃₃F₃N₂O₁₁Na: 689.1229 [M + Na]⁺; found 689.1229.

Anal. Calcd for C₃₁H₃₃F₃N₂O₁₁: C, 55.86; H, 4.99; N, 4.20. Found: C,

55.41; H, 5.05; N, 4.21.

4.1.4.2. N-{2,3-Bis[(E)-(3,4,5-trimethoxyphenyl)acryloxy]prop-2-yl}-

N’-(4-methyl phenyl)urea (46). The product was obtained as a white solid

and puriﬁed by column chromatography using using hexane–ethyl ac-

etate (2:1) as eluent (306 mg, 60% yield), mp 106–107 ^◦C. ¹H NMR (500

MHz, DMSO‑d₆) δ 8.44, (s, 1H, NHAr), 7.67–7.59 (m, 2H, CH = CHCO),

7.28 (d, J = 8.2 Hz, 2H, Ar), 7.10–7.00 (m, 6H, Ar), 6.73–6.64 (m, 2H,

CH = CHCO), 6.33 (t, J = 5.7 Hz, 1H, CH₂NH), 5.28–5.22 (m, 1H, CH),

4.47 (dd, J = 3.1 Hz, J = 12.1 Hz, 1H, CH₂O), 4.32 (dd, J = 6.5 Hz, J =

12.1 Hz, 1H, CH₂O), 3.85–3.74 (m, 12H, OCH₃), 3.72–3.66 (m, 6H,

OCH₃), 3.58–3.52 (m, 1H, CH₂NH), 3.47–3.42 (m, 1H, CH₂NH), 2.22 (s,

3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 166.1, 165.9, 155.2, 153.0,

145.3, 139.6, 137.7, 129.9, 129.5, 129.4, 117.8, 117.1, 116.7, 106.1,

106.0, 70.9, 63.1, 60.1, 56.0, 20.2. HRMS (m/z): calcd for

4.1.3.26. N-[2,3-Bis(3,4,5-trimethoxybenzoyloxy)propyl]-N’-(2-meth-

ylphenyl)urea (43). The product was obtained as a white solid and pu-

riﬁed by column chromatography using hexane–ethyl acetate (1:1) as

eluent (254 mg, 77% yield), mp 103–104 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 7.74–7.68 (m, 2H, NHAr, Ar), 7.27 (s, 2H, Ar), 7.23 (s, 2H,

Ar), 7.13–7.05 (m, 2H, Ar), 6.90 (t, J = 7.4 Hz, 1H, Ar), 6.77(t, J = 5.9

Hz, 1H, CH₂NH), 5.48–5.43 (m, 1H, CH), 4.67 (dd, J = 3.6 Hz, J = 11.8

Hz, 1H, CH₂O), 4.47 (dd, J = 7.3 Hz, J = 11.8 Hz, 1H, CH₂O), 3.82–3.72

(m, 18H, OCH₃), 3.64–3.58 (m, 2H, CH₂NH). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 165.1, 165.0, 155.6, 152.7, 142.0, 141.9, 137.8, 130.0,

127.4, 125.9, 124.7, 124.4, 122.3, 121.2, 106.8, 71.8, 63.7, 60.1, 55.8,

17.7. HRMS (m/z): calcd for C₃₁H₃₆N₂O₁₁Na 635.2211 [M + Na]⁺;

found 635.2207.

C

₃₅H₄₀N₂O₁₁Na 687.2508 [M + Na]⁺; found 687.2510.

4.1.5. General procedure for the synthesis of monoester derivatives by

chemoselective O-acylation reaction of the primary hydroxyl (47–51 and

77)

The urea derivatives 13 or 14 (0.7 mmol) was suspended in dry DCM

(15 mL), then pyridine was added until dissolution (5 mL) and the re-

action was cooled to –40 ^◦C. A solution of the appropriate acylating

agent (0.6 mmol) in dry DCM (5 mL) was added dropwise and the re-

action mixture was kept with stirring for 2 h. The reaction mixture was

co-evaporated with toluene to remove the pyridine residue. The com-

pound was further puriﬁed by ﬂash column chromatography on silica

gel using the appropriate eluent.

4.1.3.27. N-[2,3-Bis(3,4,5-trimethoxybenzoyloxy)propyl]-N’-(4-methox-

yphenyl) urea (44). The product was obtained as a white solid and pu-

riﬁed by column chromatography using hexane–ethyl acetate (1:1) as

eluent (300 mg, 90% yield), mp 137–138 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 8.35 (s, 1H, NHAr), 7.28 (d, J = 8.0 Hz, 2H, Ar), 7.23 (s, 4H,

Ar), 6.81 (d, J = 9.0 Hz, 2H, Ar), 6.33 (t, J = 6.1 Hz, 1H, CH₂NH),

5.48–5.42 (m, 1H, CH), 4.66 (dd, J = 3.5 Hz, J = 11.9 Hz, 1H, CH₂O–),

4.45 (dd, J = 7.3 Hz, J = 11.9 Hz, 1H, CH₂O), 3.83–3.77 (m, 12H,

OCH₃), 3.76–3.69 (m, 9H, OCH₃), 3.65–3.53 (m, 2H, CH₂NH). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 165.0, 164.9, 155.5, 154.0, 152.8, 152.7, 142.0,

141.9, 133.3, 124.7, 124.3, 119.6, 113.8, 106.8, 106.6, 71.8, 63.7, 60.1,

55.9, 55.8, 55.1, 36.2. HRMS (m/z): calcd for C₃₁H₃₆N₂O₁₂Na 651.2160

[M + Na]⁺; found 651.2154. Anal. Calcd for C₃₁H₃₆N₂O₁₂: C, 59.23; H,

5.77; N, 4.20. Found: C, 59.40; H, 5.75; N, 4.23.

4.1.5.1.

N-(4-Chlorophenyl)-N’-[2-hydroxy-3-(4-methylbenzoyloxy)

propyl]urea (47). The product was obtained as a white solid and puriﬁed

by column chromatography using using hexane–ethyl acetate (1:2) as

eluent (125 mg, 57% yield), mp 155–156 ^◦C. MS (CI): m/z 369 (100%)

[M + Na]⁺. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.74, (s, 1H, NHAr),

7.93–7.89 (m, 2H, Ar), 7.43–7.39 (m, 2H, Ar), 7.36–7.32 (m, 2H, Ar),

7.27–7.23 (m, 2H, Ar), 6.32 (t, J = 5.5 Hz, 1H, CH₂NH), 5.35 (d, J = 4.9

Hz, 1H, OH), 4.99–4.96 (m, 1H, CH), 4.23–4.20 (m, 2H, CH₂O),

3.43–3.39 (m, 1H, CH₂NH), 3.22–3.17 (m, 1H, CH₂NH), 2.40 (s, 3H,

CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.8, 155.2, 143.6, 139.5,

129.3, 129.2, 129.1, 128.5, 127.0, 124.4, 119.0, 67.6, 66.4, 42.2, 21.1.

Anal. Calcd for C₁₈H₁₉ClN₂O₄: C, 59.59; H, 5.28; N, 7.72. Found: C,

59.24; H, 5.25; N, 7.69.

4.1.4. General procedure for the synthesis of diester derivatives by reaction

with carboxylic acid (45 and 46)

4.1.5.2.

N-(4-Chlorophenyl)-N’-[2-hydroxy-3-(2-methylbenzoyloxy)

propyl]urea (48). The product was obtained as a white resin and puriﬁed

by column chromatography using hexane–ethyl acetate (1:2) as eluent

(131 mg, 60% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.73, (s, 1H,

NHAr), 7.92–7.87 (m, 1H, Ar), 7.51–7.38 (m, 3H, Ar), 7.34–7.23 (m, 4H,

Ar), 6.35–6.29 (m, 1H, CH₂NH), 5.33 (d, J = 5.2 Hz, 1H, OH), 5.08–5.03

(m, 1H, CH), 4.23–4.17 (m, 1H, CH₂O), 3.92–3.87 (m, 1H, CH₂O),

3.63–3.52 (m, 1H, CH₂NH), 3.21–3.16 (m, 1H, CH₂NH), 2.54 (s, 3H,

CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 166.8, 155.2, 139.4, 139.0,

According with reported procedure [55], the carboxylic acid (2.87

mmol) was dissolved in DCM and EDCI (3.55 mmol) was added. The

mixture was stirred for 1 h at rt (mixture 1). At the same time, to a

suspension of urea derivative (13 or 14, 0.78 mmol) in DCM was added

DMAP (0.78 mmol) and the mixture was stirred for 1 h at rt (mixture 2);

then the mixture 2 was added dropwise into the mixture 1. The reaction

was stirred for 24 h at rt. The organic layer was washed with saturated

17

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

132.1, 131.5, 130.2 129.5, 128.4, 125.9, 124.4, 119.2, 74.4, 67.6, 66.4,

60.6, 42.3, 21.1. HRMS (m/z): calcd for C₁₈H₁₉ClN₂O₄Na 385.0926 [M

+ Na]⁺; found 385.0921.

119.2, 71.1, 64.3, 40.9, 21.1.

4.1.6.2. N-{3-(2-Chloroacetoxy)-2-[4-(triﬂuoromethyl)benzoyloxy]

propyl}-N’-(4-chlorophenyl)urea (53). The product was obtained as a

colourless oil (235 mg, 68% yield). MS (FAB): m/z 515 (100%) [M +

Na]⁺. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.68 (s, 1H, NHAr), 8.20–8.13 (m,

2H, Ar), 7.93–7.87 (m, 2H, Ar), 7.44–7.37 (m, 2H, Ar), 7.28–7.22 (m,

2H, Ar), 6.47 (t, J = 5.9 Hz, 1H, CH₂NH), 5.38–5.32 (m, 1H, CH), 4.52

(dd, J = 3.4 Hz, J = 12.2 Hz 1H, CH₂O), 4.45–4.37 (m, 3H, ClCH₂,

CH₂O), 3.60–3.48 (m, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

167.2, 164.2, 155.1, 139.2, 133.3, 130.2, 130.1, 128.4, 125.6, 125.5,

124.7, 119.2, 72.0, 64.2, 40.9. HRMS (m/z): calcd. for

4.1.5.3. N-(4-Chlorophenyl)-N’-{2-hydroxy-3-[4-(triﬂuoromethyl)ben-

zoyloxy] propyl}urea (49). The product was obtained as a white solid and

puriﬁed by column chromatography using hexane–ethyl acetate (1:1.5)

as eluent (120 mg, 48% yield), mp 137–138 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 8.74, (s, 1H, NHAr), 8.23–8.18 (m, 2H, Ar), 7.93–7.88 (m,

2H, Ar), 7.43–7.36 (m, 2H, Ar), 7.28–7.23 (m, 2H, Ar), 6.32 (t, J = 5.7

Hz, 1H, CH₂NH), 5.44 (bs, 1H, OH), 4.33–4.24 (m, 2H, CH₂O),

3.96–3.91 (m, 1H, CH), 3.37–3.31 (m, 1H, CH₂NH), 3.26–3.19 (m, 1H,

CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 164.7, 155.2, 139.4, 133.5,

130.2, 130.1, 128.4, 128.4, 125.7, 124.5, 119.2, 119.1, 67.5, 67.2, 42.1.

HRMS (m/z): calcd for C₁₈H₁₆ClF₃N₂O₄Na 439.0643 [M + Na]⁺; found

439.0639.

C

₂₀H₁₇Cl₂F₃N₂O₅Na 515.0359 [M + Na]⁺; found 515.0359.

4.1.6.3. N-[3-(2-Chloroacetoxy)-2-(4-nitrobenzoyloxy)propyl]-N’-(4-

chlorophenyl)urea (54). The product was obtained as a light yellow oil

(298 mg, 91% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.69 (s, 1H,

NHAr), 8.35 (d, J = 8.8 Hz, 2H, Ar), 8.23–8.19 (m, 2H, Ar), 7.46–7.38

(m, 2H, Ar), 7.30–7.23 (m, 2H, Ar), 6.47 (t, J = 5.9 Hz, 1H, CH₂NH),

5.40–5.33 (m, 1H, CH), 4.53 (dd, J = 3.3 Hz, J = 12.2 Hz 1H, CH₂O),

4.47–4.36 (m, 3H, ClCH₂, CH₂O), 3.62–3.48 (m, 2H, CH₂NH). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 167.2, 163.7, 155.0, 150.3, 139.1, 134.9, 130.7,

128.4, 124.6, 123.7, 119.2, 72.3, 64.2, 40.9, 26.7. HRMS (m/z): calcd

for C₁₉H₁₇Cl₂N₃O₇Na 492.0336 [M + Na]⁺; found 492.0331.

4.1.5.4. N-[3-(2-Chloroacetoxy)-2-hydroxypropyl]-N’-(4-chlorophenyl)

urea (50). The product was obtained as a white resin (380 mg, 51% yield).

MS (FAB): m/z 343 (100%) [M + Na]⁺. ¹H NMR (500 MHz, DMSO‑d₆) δ

8.75, (s, 1H, NHAr), 7.44 (d, J = 8.5 Hz, 2H, Ar), 8.16 (d, J = 8.6 Hz, 2H,

Ar), 6.27 (t, J = 5.8 Hz, 1H, CH₂NH), 5.30 (d, J = 5.1 Hz 1H, OH), 4.44 (s,

2H, ClCH₂), 4.12–4.09 (m, 2H, CH₂O), 3.83–3.79 (m, 1H, CH), 3.30–3.24

(m, 1H, CH₂NH), 3.16–3.10 (m, 1H, CH₂NH). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 167.3, 155.1, 139.4, 128.5, 124.5, 119.0, 67.4, 67.3, 42.0,

41.1. HRMS (m/z): calcd for C₁₂H₁₄Cl₂N₂O₄Na 343.0223 [M + Na]⁺;

found 343.0220.

4.1.6.4. N-[3-(2-Chloroacetoxy)-2-(3,4,5-trimethoxybenzoyloxy)pro-

pyl]-N’-(4-chlorophenyl)urea (55). The product was obtained as a col-

ourless oil (260 mg, 72% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.74 (s,

1H, NHAr), 7.46–7.39 (m, 2H, Ar), 7.30–7.23 (m, 4H, Ar), 6.47–6.40 (m,

1H, CH₂NH), 5.36–5.40 (m, 1H, CH), 4.57–4.52 (m, 1H, CH₂O),

4.47–4.38 (m, 2H, ClCH₂), 4.34–4.28(m, 1H, CH₂O), 3.87–3.80 (m, 6H,

OCH₃), 3.78–3.72 (m, 3H, OCH₃), 3.57–3.50 (m, 1H, CH₂NH),

3.48–3.40 (m, 1H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 167.0,

164.9, 155.1, 152.7, 152.6, 141.9, 139.2, 128.4, 124.7, 124.3, 119.3,

106.9, 106.7, 72.4, 63.7, 60.1, 55.9, 41.2, 40.9. HRMS (m/z): calcd for

4.1.5.5. N-[3-(2-Chloroacetoxy)-2-hydroxypropyl]-N’-(4-methylphenyl)

urea (51). The product was obtained as a white resin (540 mg, 49% yield).

¹H NMR (500 MHz, DMSO‑d₆) δ 8.45, (s, 1H, NHAr), 7.27 (d, J = 8.3 Hz,

2H, Ar), 7.03 (d, J = 8.62 Hz, 2H, Ar), 6.16 (t, J = 5.7 Hz, 1H, CH₂NH),

5.27 (bs, 1H, OH), 4.42 (s, 2H, ClCH₂), 4.12–4.04 (m, 2H, CH₂O),

3.81–3.76 (m, 1H, CH), 3.27–3.21 (m, 1H, CH₂NH), 3.13–3.07 (m, 1H,

CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 167.3, 155.4, 137.9, 129.7,

129.0, 117.7, 67.5, 67.4, 42.0, 41.3, 20.2. HRMS (m/z): calcd for

C

₂₂H₂₄ClN₂O₈Na 537.0802 [M + Na]⁺; found 537.0795.

C

₁₃H₁₇ClN₂O₄Na 323.0769 [M + Na]⁺; found 323.0766.

4.1.6.5. N-{3-(2-Chloroacetoxy)-2-[(4-triﬂuoromethyl)benzoyloxy]pro-

4.1.5.6. N-[3-(Benzoyloxy)-2-hydroxypropyl]-N’-(4-chlorophenyl)urea

pyl}-N’-(4-methylphenyl)urea (56). The product was obtained as a colour-

less oil (249 mg, 75% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.43 (s, 1H,

NHAr), 8.21–8.50 (m, 2H, Ar), 7.94–7.90 (m, 2H, Ar), 7.29–7.21 (m, 2H,

Ar), 7.06–6.99 (m, 2H, Ar), 6.28 (t, J = 6.0 Hz, 1H, CH₂NH), 5.17–5.12 (m,

1H, CH), 4.52 (dd, J = 3.5 Hz, J = 12.0 Hz 1H, CH₂O), 4.45–4.34 (m, 3H,

ClCH₂, CH₂O), 3.59–3.47 (m, 2H, CH₂NH), 2.22 (s, 3H, CH₃). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 167.2, 164.4, 155.3, 137.8, 130.2, 130.1, 129.1,

125.6, 118.0, 72.3, 64.1, 40.1, 40.0, 20.2. HRMS (m/z): calcd for

(77). The product was obtained as a white solid and puriﬁed by column

chromatography using hexane–ethyl acetate (1.5:1) as eluent (122 mg,

58% yield), mp 155–156 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ 8.73 (s, 1H,

NHAr), 8.04–8.01 (m, 2H, Ar), 7.70–7.63 (m, 1H, Ar), 7.57–7.52 (m, 2H,

Ar), 7.41 (t, J = 8.8 Hz, 2H, Ar), 7.28–7.24 (m, 2H, Ar), 6.33 (m, 1H,

CH₂NH), 5.36 (d, J = 4.9 Hz, 1H, OH) 5.08–5.05 (m, 1H, CH), 4.27–4.17

(m, 2H, CH₂O), 3.45–3.39 (m, 1H, CH₂NH), 3.23–3.17 (m, 1H, CH₂NH).

¹³C NMR (125 MHz, DMSO‑d₆) δ 165.7, 155.2, 139.4, 133.3, 129.3,

129.2, 128.7, 128.4, 124.5, 119.1, 119.0, 67.6, 66.6, 42.2. HRMS (m/z):

calcd for C₁₇H₁₇ClN₂O₄Na 371.0769 [M + Na]⁺; found 371.0763.

C

₂₁H₂₀ClCF₃N₂O₅Na 495.0905 [M + Na]⁺; found 495.0898.

4.1.6.6. N-[3-(2-Chloroacetoxy)-2-(3,4,5-trimethoxybenzoyloxy)pro-

pyl]-N’-(4-methylphenyl)urea (57). The product was obtained as a col-

ourless oil (274 mg, 80% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 7.98 (s,

1H, NHAr), 7.32–7.21 (m, 4H, Ar), 7.08–6.99 (m, 2H, Ar), 6.40–6.32 (m,

1H, CH₂NH), 5.28–5.21 (m, 1H, CH), 4.53–4.48 (m, 1H, CH₂O),

4.45–4.35 (m, 2H, ClCH₂), 4.30–4.25 (m, 1H, CH₂O), 3.89–3.80 (m, 6H,

OCH₃), 3.78–3.72 (m, 3H, OCH₃), 3.57–3.44 (m, 2H, CH₂NH), 2.21 (s,

3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 167.3, 162.3, 155.3, 152.7,

141.9, 137.7, 129.9, 129.0, 124.7, 117.9, 107.0, 106.5, 72.3, 64.1, 60.1,

56.0, 35.7, 20.3. HRMS (m/z): calcd for C₂₃H₂₇ClN₂O₈Na 517.1348 [M

+ Na]⁺; found 517.1341.

4.1.6. General procedure for O-acylation reaction of the secondary

hydroxyl of N-[3-(2-chloroacetoxy)-2-hydroxypropyl]-N’-(substituted)

phenylureas (52–57)

To a solution of chloroacetyl derivative (50 or 51) (0.7 mmol) in dry

DCM (10 mL) and DMAP (1.4 mmol), a solution of the appropriate

acylating agent (or isocyanate) (1.0 mmol) in dry DCM (2 mL) was

added. The reaction mixture was stirred at rt until TLC showed that the

starting material had reacted (12–24 h). The organic layer was washed

with 1 N aqueous solution HCl (2 × 20 mL), saturated NaHCO₃(2 × 20

mL), brine (20 mL); then, it was dried (MgSO₄), ﬁltered and evaporated

in vacuo. The product was used without further puriﬁcation.

4.1.7. General procedure for the deprotection reaction of N-[3-(2-

Chloroacetoxy)-2-(acetyl)propyl]-N’-(substituted)ureas (59–65)

To a solution of chloroacetyl derivative (52–58, 0.4 mmol) in ace-

tonitrile–water (3:1, 10 mL), thiourea (1.2 mmol) was added and the

reaction mixture was stirred at 60 ^◦C until TLC showed the full con-

sumption of the starting material (12 h). After this time, a saturated

solution of NaHCO₃was added to the ﬂask and the reaction mixture was

extracted with chloroform (2 × 10 mL). The organic layer was washed

with brine, dried (MgSO₄), ﬁltered and evaporated to dryness to obtain a

crude product. The compound was puriﬁed by ﬂash column chroma-

tography on silica gel using the appropriate eluent.

4.1.6.1. N-[3-(2-Chloroacetoxy)-2-(4-methylbenzoyloxy)propyl]-N’-(4-

chlorophenyl)urea (52). The product was obtained as a colourless oil

(230 mg, 76% yield). MS (FAB): m/z 461 (100%) [M + Na]⁺. ¹H NMR

(500 MHz, DMSO‑d₆) δ 8.68 (s, 1H, NHAr), 7.91–7.83 (m, 2H, Ar),

7.43–7.39 (m, 2H, Ar), 7.36–7.31 (m, 2H, Ar), 7.28–7.24 (m, 2H, Ar),

6.42 (t, J = 6.0 Hz, 1H, CH₂NH), 5.32–5.27 (m, 1H, CH), 4.50–4.46 (m,

1H, CH₂O), 4.40–4.32 (m, 3H, ClCH₂, CH₂O), 3.57–3.44 (m, 2H,

CH₂NH), 2.41–2.37 (m, 3H, CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ

167.2, 165.3, 155.0, 143.8, 139.2, 129.4, 129.1, 128.4, 126.8, 124.6,

18

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Bioorganic Chemistry 114 (2021) 105095

4.1.7.1.

N-(4-Chlorophenyl)-N’-[3-hydroxy-2-(4-methylbenzoyloxy)

164.6, 155.4, 137.8, 137.7, 130.2, 129.8, 129.7, 128.9, 125.6, 125.5,

117.9, 117.7, 75.6, 60.5, 42.2, 20.2. HRMS (m/z): calcd for

C₁₉H₁₉F₃N₂O₄Na 419.1240 [M + Na]⁺; found 419.1237.

propyl]urea (59). The product was obtained as a white solid and puriﬁed

by column chromatography using hexane–ethyl acetate (2:1) as eluent

(110 mg, 76% yield), mp 140–141 ^◦C. ¹H NMR (500 MHz, DMSO‑d₆) δ

8.67, (s, 1H, NHAr), 7.94–7.88 (m, 2H, Ar), 7.44–7.38 (m, 2H, Ar),

7.37–7.32 (m, 2H, Ar), 7.29–7.22 (m, 2H, Ar), 6.37–6.29 (m, 1H,

CH₂NH), 5.07–4.98 (m, 2H, CH, OH), 3.62 (t, J = 5.5 Hz, 2H, CH₂NH),

3.56–3.50 (m, 1H, CH₂OH), 3.43–3.37 (m, 1H, CH₂OH), 2.39 (s, 3H,

CH₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 165.4, 155.1, 143.5, 139.3,

129.4, 129.1, 128.4, 127.2, 124.5,119.1, 74.4, 60.5, 42.1, 21.1. HRMS

(m/z): calcd for C₁₈H₁₉ClN₂O₄Na 385.0922 [M + Na]⁺; found 385.0920.

4.1.7.2.N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[(4-triﬂuoromethyl)ben-

zoyloxy] propyl} urea (60). The product was obtained as a white solid

and puriﬁed by column chromatography using hexane–ethyl acetate

(1.5:1) as eluent (120 mg, 73% yield), mp 175–176 ^◦C. ¹H NMR (500

MHz, DMSO‑d₆) δ 8.73, (s, 1H, NHAr), 8.23–8.18 (m, 2H, Ar), 7.94–7.89

(m, 2H, Ar), 7.43–7.36 (m, 2H, Ar), 7.28–7.22 (m, 2H, Ar), 6.32 (t, J =

5.7 Hz, 1H, CH₂NH), 5.38 (bs, 1H, OH), 5.13–5.07 (m, 1H, CH),

4.32–4.23 (m, 2H, CH₂NH), 3.69–3.62 (m, 1H, CH₂OH), 3.26–3.19 (m,

1H, CH₂OH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 164.6, 155.2, 139.4,

139.3, 133.5, 130.2, 130.1, 128.4, 125.7, 124.8, 119.2, 119.0, 75.5,

67.4, 42.1. HRMS (m/z): calcd for C₁₈H₁₆ClF₃N₂O₄Na 439.0643 [M +

Na]⁺; found 439.0637. Anal. Calcd for C₁₈H₁₆ClF₃N₂O₄: C, 51.87; H,

3.87; N, 6.72. Found: C, 51.85; H, 3.88; N, 6.74.

4.1.7.7. N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[(4-triﬂuoromethyl) phe-

nylaminocarboxy]propyl}urea (65). The product was obtained as an

amorphous solid and puriﬁed by column chromatography using hex-

ane–ethyl acetate (1:2) as eluent (112 mg, 64% yield). ¹H NMR (500 MHz,

DMSO‑d₆) δ 10.11 (s, 1H, NHCOO), 8.70 (s, 1H, NHAr), 8.72–8.62 (m, 4H,

Ar), 7.45–7.39 (m, 2H, Ar), 7.29–7.22 (m, 2H, Ar), 6.32 (t, J = 5.75 Hz,

1H, CH₂NH), 4.96 (t, J = 5.60 Hz, 1H, OH), 4.86–4.70 (m, 1H, CH),

3.62–3.49 (m, 4H, CH₂NH, CH₂OH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

155.1, 153.1, 142.9, 139.3, 128.4, 126.0, 125.5, 124.6, 122.5, 122.2,

119.1, 119.0, 117.9, 74.5, 67.7, 60.7. HRMS (m/z): calcd for

C

₁₈H₁₇ClF₃N₃O₄Na 454.0752 [M + Na]⁺; found 454.0748.

4.1.8. Oleﬁn oxidation reaction of N-Allyl-N’-(4-chlorophenyl)urea N-

(4-chlorophenyl)-N’-(2,3-epoxypropyl)urea (69). According to reported

procedure [56], a solution of meta-chloroperoxybenzoic acid (m-CPBA)

(15 mmol), previous dried (MgSO₄) in anhydrous THF (10 mL), was

added to a solution of N-allyl-N’-(4-chlorophenyl)urea (67, 3 mmol) in

THF (30 mL) and the reaction mixture was stirred at rt until TLC showed

that all the starting material had reacted (24 h). After that, ethyl acetate

(30 mL) and a saturated solution of K₂CO₃was added to the ﬂask and the

mixture was stirred for 5 min, then the phases were separated. The

organic layer was washed with K₂CO₃(3 × 20 mL) and brine, then dried

(MgSO₄) and evaporated to dryness. The compound was obtained as a

light yellow solid and puriﬁed by precipitation in toluene (542 mg,

80%). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.70 (s, 1H, NHAr), 7.42 (d, J =

8.8 Hz, 2H, Ar), 7.27 (d, J = 8.4 Hz, 2H, Ar), 6.32 (t, J = 5.0 Hz, 1H,

CH₂NH), 3.22–3.17 [m, 2H, CH₂(O)CH], 3.07–3.04 [m, 1H, CH₂(O)CH],

2.73 (t, J = 4.7 Hz, 1H, CH₂NH), 2.57–2.55 (m, 1H, CH₂NH). ¹³C NMR

(125 MHz, DMSO‑d₆) δ 155.0, 139.3, 128.5, 124.6, 119.2, 50.6, 44.6,

40.4. HRMS (m/z): calcd for C₁₀H₁₂ClN₂O₂227.0587 [M + H]⁺; found

227.0582.

4.1.7.3. N-(4-Chlorophenyl)-N’-[3-hydroxy-2-(4-nitrobenzoyloxy)pro-

pyl]urea (61). The product was obtained as a light yellow resin and

puriﬁed by column chromatography using hexane–ethyl acetate (1:2) as

eluent (116 mg, 73% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.63, (s,

1H, NHAr), 8.38–8.33 (m, 2H, Ar), 8.27–8.22 (m, 2H, Ar), 7.43–7.37 (m,

2H, Ar), 7.28–7.23 (m, 2H, Ar), 6.38 (t, J = 5.9 Hz, 1H, CH₂NH),

5.14–5.08 (m, 1H, CH), 5.03 (s, J = 5.9 Hz, 1H, OH), 3.71–3.62 (m, 2H,

CH₂NH), 3.59–3.53 (m, 1H, CH₂OH), 3.47–3.40 (m, 1H, CH₂OH). ¹³C

NMR (125 MHz, DMSO‑d₆) δ 164.0, 155.2, 150.2, 139.4, 133.5, 130.7,

128.4, 124.6, 123.7, 119.2, 75.8, 67.5, 60.4. HRMS (m/z): calcd for

C

₁₇H₁₆ClN₃O₆Na 416.0620 [M + Na]⁺; found 416.0617.

4.1.8. Epoxide ring opening of N-(4-chlorophenyl)-N’-(2,3-epoxypropyl)

urea in acid condition

4.1.7.4.

N-(4-Chlorophenyl)-N’-[3-hydroxy-2-(3,4,5-trimethox-

ybenzoyloxy)propyl]urea (62). The product was obtained as a white

solid and puriﬁed by column chromatography using hexane–ethyl

acetate (1.5:1) as eluent (135 mg, 77% yield), mp 155–156 ^◦C. ¹H

NMR (500 MHz, DMSO‑d₆) δ 8.67, (s, 1H, NHAr), 7.43–7.38 (m, 2H,

Ar), 7.29 (s, 2H, Ar), 7.26–7.23 (m, 2H, Ar), 6.33 (t, J = 5.9 Hz, 1H,

CH₂NH), 5.04–4.98 (m, 2H, CH, OH), 3.83–3.79 (m, 6H, OCH₃),

3.75–3.72 (m, 3H, OCH₃), 3.65–3.61 (m, 2H, CH₂NH), 3.57–3.52 (m,

1H, CH₂OH), 3.47–3.41 (m, 1H, CH₂OH). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 165.3, 155.2, 152.7, 141.9, 139.4, 128.4, 125.1, 124.8,

119.1, 106.9, 74.9, 60.4, 60.1, 56.0, 42.2. HRMS (m/z): calcd for

C₂₀H₂₃ClN₂O₇Na 461.1086 [M + Na]⁺; found 461.1084.

N-(3-Azido-2-hydroxypropyl)-N’-(4-chlorophenyl)urea (70). Accord-

ing to reported procedure [57], to a suspension of oxirane (69, 1.0

mmol) in ethanol (20 mL), were added sodium azide (1.2 mmol) and

NH₄Cl (1.2 mmol) and the mixture was heated at 60 ^◦C and stirred until

TLC showed the full consumption of the starting material (15 h). The

reaction was washed with water (10 mL) and brine (10 mL), then dried

(MgSO₄) and evaporated under reduced pressure. The compound was

obtained as a light yellow resin and puriﬁed by ﬂash column chroma-

tography on silica gel using hexane–ethyl acetate (1:1) as eluent (215

mg, 79% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.70 (s, 1H, NHAr),

7.41 (d, J = 9.0 Hz, 2H, Ar), 7.27 (d, J = 8.5 Hz, 2H, Ar), 6.25 (t, J = 5.8

Hz, 1H, CH₂NH), 5.40 (d, J = 5.1 Hz, 1H, OH), 6.76–6.71 (m, 1H, CH),

3.30–3.26 (m, 2H, CH₂NH), 3.25–3.17 (m, 1H, N₃CH₂), 3.11–3.06 (m,

1H, N₃CH₂). ¹³C NMR (125 MHz, DMSO‑d₆) δ 155.1, 139.4, 128.5,

124.5, 119.1, 69.1, 54.0, 42.8. HRMS (m/z): calcd for C₁₀H₁₂ClN₅O₂Na

292.0572 [M + Na]⁺; found 292.0574.

4.1.7.5. N-[3-Hydroxy-2-(3,4,5-trimethoxybenzoyloxy)propyl]-N’-(4-

methylphenyl)urea (63). The product was obtained as a white solid and

puriﬁed by column chromatography using hexane–ethyl acetate (1:2) as

eluent (123 mg, 73% yield), mp 170–171 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 8.39, (s, 1H, NHAr), 7.33–7.22 (m, 4H, Ar), 7.02 (d, J = 8.2

Hz, 2H, Ar), 6.23 (t, J = 5.9 Hz, 1H, CH₂NH), 5.03–4.98 (m, 2H, CH,

OH), 3.88–3.80 (m, 6H, OCH₃), 3.78–3.72 (m, 3H, OCH₃), 3.66–3.61 (m,

2H, CH₂NH), 3.57–3.51 (m, 1H, CH₂OH), 3.47–3.40 (m, 1H, CH₂OH).

¹³C NMR (125 MHz, DMSO‑d₆) δ 165.2, 155.4, 152.6, 141.8, 137.7,

129.8, 128.9, 125.2, 117.8, 106.9, 75.0, 60.4, 60.1, 56.0, 20.2. HRMS

(m/z): calcd for C₂₁H₂₆N₂O₇Na 441.1632 [M + Na]⁺; found 441.1629.

4.1.7.6. N-{3-Hydroxy-2-[(4-triﬂuoromethyl)benzoyloxy]propyl}-N’-

(4-methylphenyl) urea (64). The product was obtained as a white resin

and puriﬁed by column chromatography using hexane–ethyl acetate

(1.5:1) as eluent (128 mg, 80% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ

8.48 (s, 1H, NHAr), 8.25–8.20 (m, 2H, Ar), 7.85–7.79 (m, 2H, Ar),

7.29–7.21 (m, 2H, Ar), 7.14–7.00 (m, 2H, Ar), 6.29 (m, 1H, CH₂NH),

5.12–5.06 (m, 1H, CH), 5.03 (t, J = 5.9 Hz, 1H, OH), 4.32–4.23 (m, 2H,

CH₂NH), 3.69–3.52 (m, 2H, CH₂OH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

4.1.9. Mesylation reaction of N-[3-(benzoyloxy)-2-hydroxypropyl]-N’-(4-

chlorophenyl)urea

N-[3-Benzoyloxy-2-(methylsulfonyloxy)propyl]-N’-(4-chlorophenyl)

urea (78). According to reported procedure [58], to a solution of ben-

zoyl derivative 77 (1.0 mmol) and triethylamine (2.0 mmol) in dry THF

(7 mL) mesyl chloride (2.0 mmol) was added dropwise at 0 ^◦C. The re-

action mixture was heated to room temperature and stirred for 5 h, then

NH₄Cl (5% water solution) and DCM were added. The phases were

separated and the organic layer was washed with saturated NaHCO₃,

brine and dried (MgSO₄); then ﬁltered and evaporated in vacuo to give

compound as colourless oil, that was used without further puriﬁcation.

(220 mg, 92% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 9.01 (s, 1H,

NHAr), 8.09–7.99 (m, 2H, Ar), 7.74–7.68 (m,1H, Ar), 7.61–7.52 (m, 2H,

19

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Ar), 7.48–7.39 (m, 2H, Ar), 7.30–7.23 (m, 2H, Ar), 6.71–6.64 (m, 1H,

CH₂NH), 5.08–4.98 (m, 1H, CH), 4.64–4.39 (m, 2H, CH₂O), 3.60–3.52

(m, 2H, CH₂NH), 3.26 (s, 3H, CH₃). HRMS (m/z): calcd. for

Ar), 7.43–7.39 (m, 2H, Ar), 7.28–7.24 (m, 2H, Ar), 6.22 (t, J = 5.6 Hz, 1H,

CH₂NH), 5.10 (d, J = 5.1 Hz, 1H, OH), 4.86 (s, 2H, ArCONHCH₂),

4.49–2.22 (m, 2H, CH₂N), 3.95–3.88 (m, 1H, CH), 3.20–3.05 (m, 2H,

CH₂NH).¹³C NMR (125 MHz, DMSO‑d₆) δ 168.4, 167.3, 155.2, 141.9,

139.4, 134.5, 131.6, 130.6, 128.4, 124.5, 123.9, 123.2, 119.1, 68.3, 59.4,

53.3, 42.8. HRMS (m/z): calcd. for C₂₁H₂₁ClN₆O₅Na 495.1049 [M + Na]⁺;

found 495.1044.

C

₁₈H₁₉ClN₂O₆SNa 449.0545 [M + Na]⁺; found 449.0541.

4.1.10. Nucleophilic substitution reaction with sodium azide

N-[2-Azido-(3-benzoyloxy)propyl]-N’-(4-chlorophenyl)urea (79). Ac-

cording to reported procedure [58], to a solution of mesyl derivative

(77, 0.5 mmol) in DMF (3 mL), was added sodium azide (2.5 mmol) at rt

and the mixture was heated to 85 ^◦C and stirred overnight. The reaction

was diluted with water and extracted with ethyl acetate. The organic

phase was washed with saturated solution of NaHCO₃and brine, then

was dried (MgSO₄) and ﬁltered. The solvent was removed under reduced

pressure to give compound as colourless oil, that was used without

further puriﬁcation (150 mg, 80% yield). ¹H NMR (500 MHz, DMSO‑d₆)

δ 8.78 (s, 1H, NHAr), 8.06–7.99 (m, 2H, Ar), 7.73–7.66 (m, 1H, Ar),

7.60–7.51 (m, 2H, Ar), 7.47–7.39 (m, 2H, Ar), 7.31–7.23 (m, 2H, Ar),

6.51 (t, J = 5.9 Hz, 1H, CH₂NH), 4.53 (dd, J = 3.5 Hz, J = 11.8 Hz 1H,

CH₂O), 4.35 (dd, J = 7.5 Hz, J = 11.9 Hz 1H, CH₂O), 4.12–4.06 (m, 1H,

CH), 3.52–3.37 (m, 2H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ

165.4, 155.0, 139.2, 133.5, 129.4, 129.3, 129.2, 128.7, 124.7, 119.3,

64.7, 60.2, 36.2.

4.1.13.3.

N-(4-Chlorophenyl)-N’-{2-hydroxy-3-[4

(dieth-

ylphosphonatemethoxymethyl) triazolyl]propyl}urea (73). The product

was obtained as a colourless oil and puriﬁed by column chroma-

tography using ethyl acetate–methanol (15:1, 0.5% Et₃N) as eluent

(193 mg, 81% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.78 (s, 1H,

NHAr), 8.17 (s, 1H, CH triazole), 7.42 (d, J = 8.9 Hz, 2H, Ar), 7.27

(d, J = 8.9 Hz, 2H, Ar), 6.33 (t, J = 5.7 Hz, 1H, CH₂NH), 5.46 (d, J

= 5.5 Hz, 1H, OH), 4.63 (s, 2H, OCH₂), 4.43 (dd, J = 13.9 Hz, J =

3.9 Hz, 1H, NCH₂), 4.29 (dd, J = 13.9 Hz, J = 7.5 Hz, 1H, NCH₂),

4.08–4.00 (m, 4H, OCH₂CH₃), 3.98–3.91 (m, 1H, CHOH), 3.85–3.82

(m, 2H, PCH₂O), 3.22–3.17 (m, 1H, CH₂NH), 3.13–3.07 (m, 1H,

CH₂NH), 1.27–1.21 (m, 6H, OCH₂CH₃). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 155.2, 142.6, 139.3, 128.4, 125.1, 124.6, 119.2, 68.6,

65.1, 63.5, 61.8, 53.2, 42.8, 16.2. HRMS (m/z): calcd for

C

₁₈H₂₇ClN₅O₆PNa 498.1280 [M + Na]⁺; found 498.1271.

4.1.13.4. N-(4-Chlorophenyl)-N’-{2-hydroxy-3-[4-(4-ﬂuorophenyl)tri-

4.1.11. Deprotection reaction of N-[2-azido-(3-benzoyloxy)propyl]-N’-(4-

chlorophenyl)urea

azolyl]propyl}urea (74). The product was obtained as a white solid and

puriﬁed by column chromatography hexane–ethyl acetate (1:2.5, 0.5%

Et₃N) as eluent (117 mg, 60% yield), mp 230–231 ^◦C. ¹H NMR (500

MHz, DMSO‑d₆) δ 8.80 (s, 1H, NHAr), 8.52 (s, 1H, CH triazole),

7.92–7.89 (m, 2H, Ar), 7.43 (d, J = 9.2 Hz, 2H, Ar), 7.31–7.26 (m, 4H,

Ar), 6.36 (t, J = 5.9 Hz, 1H, CH₂NH), 5.47 (d, J = 5.5 Hz, 1H, OH), 4.71

(dd, J = 13.2 Hz, J = 3.5 Hz, 1H, NCH₂), 4.32 (dd, J = 14.0 Hz, J = 7.5

Hz, 1H, NCH₂), 4.04–3.97 (m, 1H, CHOH), 3.29–3.24 (m, 1H, CH₂NH),

3.17–3.12 (m, 1H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 162.7,

160.7, 155.2, 145.2, 139.4, 128.5, 127.5, 127.1, 124.5, 122.1, 119.1,

115.9, 115.7, 68.7, 53.6, 42.9. HRMS (m/z): calcd for C₁₈H₁₈ClFN₅O₂

390.1128 [M + H]⁺; found 390.1121.

N-(2-Azido-3-hydroxypropyl)-N’-(4-chlorophenyl)urea (80). Accord-

ing to reported procedure [59] compound 79 (0.5 mmol) was dissolved

in methanol and a 0.5 M sodium hydroxide solution (0.65 mmol) was

added. The reaction mixture was heated to 55 ^◦C and stirred for 1 h (TLC

revealed the full consumption of the starting material). The reaction was

evaporated to dryness and the residue was crystallized in DCM, ﬁltered

and washed with fresh DCM to give compound a white solid (120, 88%

yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 9.22 (s, 1H, NHAr), 7.50–7.42

(m, 2H, Ar), 7.30–7.23 (d, 2H, Ar), 6.98–6.89 (m, 1H, CH₂NH),

3.75–3.71 (m, 2H, CH₂NH), 3.63–3.45 (m, 2H, CH₂OH), 3.26–3.15 (m,

1H, CHN₃). ¹³C NMR (125 MHz, DMSO‑d₆) δ 155.4, 139.7, 128.4, 124.4,

119.1, 63.2, 61.8, 42.8. HRMS (m/z): calcd for C₁₀H₁₂ClN₅O₂Na

292.0572 [M + Na]⁺; found 292.0573.

4.1.13.5. N-(4-Chlorophenyl)-N’-{2-hydroxy-3-[4-(4-methoxyphenyl)

triazolyl] propyl}urea (75). The product was obtained as a light yellow

solid and puriﬁed by column chromatography using hexane–ethyl ace-

tate (1:2, 0.5% Et₃N) as eluent (140 mg, 71% yield), mp 218–219 ^◦C. ¹H

NMR (500 MHz, DMSO‑d₆) δ 8.80 (s, 1H, NHAr), 8.41 (s, 1H, CH tri-

azole), 7.79 (d, J = 8.8 Hz, 2H, Ar), 7.44 (d, J = 8.9 Hz, 2H, Ar), 7.28 (d,

J = 8.8 Hz, 2H, Ar), 7.02 (d, J = 8.8 Hz, 2H, Ar), 6.37 (t, J = 6.7 Hz, 1H,

CH₂NH), 5.47 (d, J = 5.5 Hz, 1H, OH), 4.48 (dd, J = 13.9 Hz, J = 3.8 Hz,

1H, CH₂N), 4.32 (dd, J = 14.3 Hz, J = 7.6 Hz, 1H, CH₂N), 4.04–3.98 (m,

1H, CHOH), 3.81 (s, 3H, OCH₃), 3.27–3.20 (m, 1H, CH₂NH), 3.17–3.11

(m, 1H, CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 158.9, 155.2, 145.9,

139.4, 128.4, 126.4, 124.5, 123.5, 121.2, 119.1, 114.3, 68.7, 55.1, 53.5,

42.8. HRMS (m/z): calcd for C₁₉H₂₁ClN₅O₃Na 402.1327 [M + H]⁺;

found 402.1321.

4.1.12. Synthesis of 1,2,3-triazole derivative by Cu(I)-catalyzed 1,3-

dipolar cycloaddition (CuAAC) reaction (71–76, 81–86)

According to reported procedure [60], the appropriate azide deriv-

ative (70 or 80, 0.5 mmol) was dissolved in tert-butanol-water (1:1, 15

mL). The appropriate acetylene (0.6 mmol), sodium ascorbate (0.020

mmol) and CuSO₄(0.010 mmol) were added to the ﬂask and the mixture

was stirring at 25 ^◦C. The reaction was monitored by TLC until all the

starting material had reacted, then it was evaporated under vacuum. The

product was puriﬁed by ﬂash column chromatography on silica gel using

the appropriate eluent.

4.1.13.1. N-(4-Chlorophenyl)-N’-{2-hydroxy-3-[4-(hydroxymethyl)tri-

azolyl]propyl}urea (71). The product was obtained as a light yellow solid

and puriﬁed by column chromatography using dichloromethane-

methanol (15:1, 0.5% Et₃N) as eluent (130 mg, 79% yield), mp

131–132 ^◦C. ¹H NMR (500 MHz, CD₃OD) δ 7.95 (s, 1H, CH triazole),

7.39 (d, J = 9.2 Hz, 2H, Ar), 7.26 (d, J = 8.5 Hz, 2H, Ar), 4.83 (s, 2H,

HOCH₂), 4.71 (s, 1H, CHOH), 4.58–4.53 (m, 1H, NCH₂), 4.43–4.37 (m,

4.1.13.6. N-(4-Chlorophenyl)-N’-{2-hydroxy-3-[4-(2-formylphenyl)tri-

azolyl] propyl}urea (76). The product was obtained as a white solid and

puriﬁed by column chromatography using hexane–ethyl acetate (1:2,

0.5% Et₃N) as eluent (122 mg, 62% yield), mp 198–199 ^◦C. ¹H NMR

(500 MHz, DMSO‑d₆) δ 10.51(s, 1H, COH), 8.87 (s, 1H, NHAr), 8.66 (s,

1H, Ar), 7.94 (d, J = 7.5 Hz, 1H, Ar), 7.83–7.78 (m, 2H, Ar, CH triazole),

7.62–7.59 (m, 1H, Ar), 7.50–7.45 (m, 2H, Ar), 7.32–7.29 (m, 2H, Ar),

6.43 (t, J = 5.8 Hz, 1H, CH₂NH), 5.22 (d, J = 5.5 Hz, 1H, OH), 4.60 (dd,

J = 13.9 Hz, J = 3.7 Hz, 1H, CH₂N), 4.42 (dd, J = 13.9 Hz, J = 7.8 Hz,

1H, CH₂N), 4.11–4.05 (m, 1H, CHN), 3.32–3.28 (m, 1H, CH₂NH),

3.22–3.18 (m, 1H, CH₂NH).¹³C NMR (125 MHz, DMSO‑d₆) δ 193.1,

155.7, 143.9, 139.8, 133.8, 133.7, 130.0, 128.9, 128.8, 127.9, 125.9,

125.0, 119.5, 69.1, 54.2, 43.3.

1H, NCH₂), 3.29–3.24 (m, 1H, CH₂NH), 3.21–3.16 (m, 1H, CH₂NH). ¹³

C

NMR (125 MHz, CD₃OD) δ 158.2, 139.8, 132.4, 129.7, 128.3, 125.3,

121.4, 70.7, 56.5, 54.9, 47.9. HRMS (m/z): calcd for C₁₃H₁₆ClN₅O₃Na

348.0834 [M + Na]⁺; found 348.0834.

4.1.13.2. N-(4-Chlorophenyl)-N’-{2-hydroxy-3-[4-(phtaloylaminomethyl)

triazolyl] propyl}urea (72). The product was obtained as a white solid and

puriﬁed by column chromatography using hexane–ethyl acetate (1:4, 0.5%

Et₃N) as eluent (146 mg, 82% yield), mp 222–223 ^◦C. ¹H NMR (500 MHz,

DMSO‑d₆) δ 13.20 (bs, 1H, COOH), 8.75 (s, 1H, ArCONHCH₂), 8.03 (s, 1H,

NHAr), 7.94–7.84 (m, 3H, Ar), 7.79 (s, 1H, CH triazole), 7.60–7.56 (m, 1H,

4.1.13.7. N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[4-(hydroxymethyl)tri-

azolyl] propyl}urea (81). The product was obtained as a light yellow

solid and puriﬁed by column chromatography using dichloromethane-

methanol (15:1, 0.5% Et₃N) as eluent (104 mg, 64% yield), mp

20

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

125–126 ^◦C. ¹H NMR (500 MHz, CD₃OD) δ 8.02 (s, 1H, CH triazole),

7.36–7.32 (m, 2H, Ar), 7.28–7.23 (m, 2H, Ar), 4.83 (s, 2H, HOCH₂),

4.15–4.10 (m, 1H, CHN), 4.02–3.97 (m, 2H, CH₂NH), 4.83 (dd, J = 5.1

Hz, J = 14.3 Hz, 1H, CH₂OH), 3.74 (dd, J = 8.4 Hz, J = 14.4 Hz, 1H,

CH₂OH). ¹³C NMR (125 MHz, CD₃OD) δ 158.2, 148.9, 139.8, 129.7,

128.4, 125.3, 124.2, 121.4, 70.6, 64.7, 56.6, 44.3. HRMS (m/z): calcd.

for C₁₃H₁₆ClN₅O₃Na 348.0834 [M + Na]⁺; found 348.0831.

Hz, 1H, CH₂NH), 5.23 (t, J = 5.5 Hz, 1H, OH), 4.53 (dd, J = 13.9 Hz, J =

3.8 Hz, 1H, CH₂OH), 4.38 (dd, J = 13.9 Hz, J = 7.8 Hz, 1H, CH₂OH),

4.09–4.01(m, 1H, CHN), 3.29–3.23 (m, 1H, CH₂NH), 3.19–3.13 (m, 1H,

CH₂NH). ¹³C NMR (125 MHz, DMSO‑d₆) δ 193.2, 155.7, 143.9, 139.8,

134.4, 133.8, 133.7, 130.0, 128.9, 128.8, 127.9, 125.9, 124.7, 119.5,

69.1, 54.2, 43.3.

4.1.13.8.

N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[4-(phtaloylamino-

4.1.13. Synthesis of terminal alkynes

methyl)triazolyl] propyl}urea (82). The product was obtained as a white

solid and puriﬁed by column chromatography using hexane–ethyl ace-

tate (1:4, 0.5% Et₃N) (162 mg, 69% yield), mp 212–213 ^◦C. ¹H NMR

(500 MHz, DMSO‑d₆) δ 8.65 (s, 1H, , ArCONHCH₂), 8.10 (s, 1H, NHAr),

7.93–7.87 (m, 5H, Ar, CH triazole), 7.38–7.34 (m, 2H, Ar), 7.28–7.22

(m, 2H, Ar), 6.22 (t, J = 5.8 Hz, 1H, CH₂NH), 5.10 (t, J = 5.4 Hz, 1H,

OH), 4.86 (s, 2H, ArCONHCH₂), 4.71–4.65 (m, 1H, CHN), 3.74 (t, J =

5.5 Hz, 2H, CH₂OH), 3.69–3.63 (m, 1H, CH₂NH), 3.57–3.49 (m, 1H,

CH₂NH).¹³C NMR (125 MHz, DMSO‑d₆) δ 167.3, 154.9, 141.9, 139.1,

134.5, 131.6, 128.4, 124.6, 123.2, 122.6, 119.1, 69.8, 62.8, 61.5, 33.0.

HRMS (m/z): calcd. for C₂₁H₂₁ClN₆O₅Na 495.9001 [M + Na]⁺; found

495.8999.

4.1.14.1. N-propargylphtalylmonoamide (87) [61]. To a stirred solu-

tion of propargylamine (2.2 mmol) in THF (4 mL), phthalic anhydride

(2.0 mmol) was added and the mixture was stirred at rt until TLC

showed that all the starting material has reacted (18 h). The formed solid

was ﬁltered, dissolved in water and 1 N solution of HCl was added until

pH 5. The reaction was extracted with ethyl acetate and the organic

layer was washed with brine, dried (MgSO₄) and ﬁltered. The solvent

was removed under reduced pressure to give the compound that was

used without further puriﬁcation (170 mg, 83% yield). ¹H NMR (300

MHz, DMSO‑d₆) δ 9.91 (s, 1H, NH), 7.67–7.61 (m, 1H, Ar), 7.58–7.52

(m, 1H, Ar), 7.48–7.38 (m, 2H, Ar), 3.54 (m, 2H, CH₂), 3.90 (t, J = 2.49

Hz, 1H, CH).

4.1.13.9.

N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[4

(dieth-

4.1.14.2. Propargyloxymethylphosphonate (88). A Solution of prop-

argyl alcohol (1.5 mmol) in THF (3 mL) was added slowly to a solution of

NaH (1.6 mmol, 60% dispersion in mineral oil) in THF (2 mL). The

mixture was stirred for 1 h and then cooled to 0 ^◦C. Diethyl (tosyloxy)

methylphosphonate (2.0 mmol) was added to the ﬂask and the reaction

was stirred for 3 h at 0 ^◦C and then for 24 h at rt. When TLC revealed the

full consumption of the starting material, the reaction was evaporated to

dryness and the residue was dissolved in ethyl acetate and washed with

water (2 × 10 mL). The organic phase was dried (MgSO₄) ﬁltered, and

the solvent was removed under vacuum to give compound as a colour-

less oil. The compound was used without further puriﬁcation (230 mg,

74% yield). MS (FAB): m/z 229 (100%) [M + Na]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 4.28–3.22 (m, 2H, OCH₂), 4.12–3.95 (m, 4H, OCH₂CH₃),

3.86–3.78 (m, 2H, PCH₂O), 3.33 (s, 1H, CH), 1.30–1.13 (m, 6H,

OCH₂CH₃).

ylphosphonatemethoxymethyl) triazolyl]propyl}urea (83). The product

was obtained as a colourless oil and puriﬁed by column chroma-

tography using ethyl acetate–methanol (15:1, 0.5% Et₃N) as eluent

(173 mg, 72% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ 8.71 (s, 1H,

NHAr), 8.18 (s, 1H, CH triazole), 7.38 (d, J = 8.9 Hz, 2H, Ar), 7.25

(d, J = 8.9 Hz, 2H, Ar), 6.27 (t, J = 5.7 Hz, 1H, CH₂NH), 5.16 (t, J =

5.4 Hz, 1H, OH), 4.77–4.68 (m, 1H, CHN), 4.63(s, 2H, OCH₂),

4.08–3.98 (m, 4H, OCH₂CH₃), 3.87–3.82 (m, 2H, PCH₂O), 3.77 (t, J

= 5.4 Hz, 2H, CH₂OH), 4.71–4.64 (m, 1H, CH₂NH), 4.61–4.53 (m,

1H, CH₂NH), 1.25–1.20 (m, 6H, OCH₂CH₃). ¹³C NMR (125 MHz,

DMSO‑d₆) δ 155.5, 143.0, 139.7, 128.9, 125.1, 124.4, 119.5, 65.7,

64.0, 63.3, 62.7, 62.2, 62.0, 16.7. HRMS (m/z): calcd. for

C

₁₈H₂₇ClN₅O₆PNa 498.1280 [M + Na]⁺; found 498.1276.

4.1.13.10. N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[4-(4-ﬂuorophenyl)

triazolyl]propyl} urea (84). The product was obtained as a white solid

and puriﬁed by column chromatography using hexane–ethyl acetate

(1:2, 0.5% Et₃N) as eluent (163 mg, 84% yield), mp 227–228 ^◦C. ¹H

NMR (500 MHz, DMSO‑d₆) δ 8.70 (s, 1H, NHAr), 8.65 (s, 1H, CH tri-

azole), 7.94–7.88 (m, 2H, Ar), 7.42–7.37 (m, 2H, Ar), 7.33–7.23 (m, 4H,

Ar), 6.30 (t, J = 5.8 Hz, 1H, CH₂NH), 5.21 (t, J = 5.5 Hz, 1H, OH),

4.69–4.62 (m, 1H, CHN), 3.83 (t, J = 5.5 Hz, 2H, CH₂OH), 3.79–3.73 (m,

1H, CH₂NH), 3.63–3.56 (m, 1H, CH₂NH).¹³C NMR (125 MHz, DMSO‑d₆)

δ 160.7, 155.0, 145.2, 139.1, 128.4, 127.5, 127.4, 124.6, 120.8, 119.1,

115.8, 115.7, 63.1, 61.6, 40.0. HRMS (m/z): calcd for C₁₈H₁₈ClFN₅O₂Na

390.1128 [M + H]⁺; found 390.1125.

4.1.15. Characterization data of compound 66 (N-(4-Chlorophenyl)-N’-

{2-hydroxy-3-[(E)-(3,4,5-trimethoxyphenyl)acryloxy]propyl}urea). Com-

pound was isolated during the puriﬁcation of compound 45 by column

chromatography, using hexane–ethyl acetate (1:2) as eluent and was

obtained as a white solid (55 mg, 15% yield), mp 144–145 ^◦C. ¹H NMR

(500 MHz, DMSO‑d₆) δ 8.74, (s, 1H, NHAr), 7.67–7.59 (m, 1H, CH =

CHCO), 7.42 (d, J = 8.8 Hz, 2H, Ar), 7.29–7.23 (m, 2H, Ar), 7.09–7–05

(m, 2H, Ar), 6.70–6.64 (m, 1H, CH = CHCO), 6.32–6.26 (m, 1H,

CH₂NH), 5.27 (d, J = 5.2 Hz, 1H, OH), 4.97–4.92 (m, 1H, CH), 4.11 (d, J

= 5.4 Hz, 2H, CH₂O), 3.87–3.80 (m, 6H, OCH₃), 3.73–3.69 (m, 3H,

OCH₃), 3.60–3.48 (m, 1H, CH₂NH), 3.19–3.12 (m, 1H, CH₂NH). ¹³C

NMR (125 MHz, DMSO‑d₆) δ 166.3, 155.1, 153.0 , 144.8, 139.5, 139.4,

129.6, 128.4, 124.5, 119.1, 119.0, 117.6, 105.9, 105.8, 67.6, 66.0, 60.0,

56.0, 42.3. HRMS (m/z): calcd. for C₂₂H₂₅ClN₂O₇Na 487.1242 [M +

Na]⁺; found 487.1238. Anal. Calcd for C₂₂H₂₅ClN₂O₇: C, 56.84; H, 5.42;

N, 6.03. Found: C, 56.80; H, 5.40; N, 5.05.

4.1.13.11. N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[4-(4-methoxyphenyl)

triazolyl]propyl} urea (85). The product was obtained as a light yellow

solid and puriﬁed by column chromatography using hexane–ethyl ace-

tate (1:1.5, 0.5% Et₃N) (104 mg, 64% yield), mp 206–207 ^◦C. ¹H NMR

(500 MHz, DMSO‑d₆) δ 8.70 (s, 1H, NHAr), 8.51 (s, 1H, CH triazole),

7.81–7.76 (m, 2H, Ar), 7.45–7.37 (m, 2H, Ar), 7.30–7.23 (m, 2H, Ar),

7.06–6.98 (m, 2H, Ar), 6.30 (t, J = 5.8 Hz, 1H, CH₂NH), 5.22 (t, J = 5.2

Hz, 1H, OH), 4.78–4.72 (m, 1H, CHN), 4.56–4.46 (m, 1H, CH₂OH),

4.36–4.29 (m, 1H, CH₂OH), 3.28–3.21 (m, 1H, CH₂NH), 3.16–3.11 (m,

1H, CH₂NH).¹³C NMR (125 MHz, DMSO‑d₆) δ 166.7, 158.8, 155.2,

155.0, 145.9, 139.1, 128.3, 127.3, 126.4, 124.7, 123.5, 121.3, 119.9,

114.2, 68.7, 62.9, 55.1, 43.5. HRMS (m/z): calcd. for C₁₉H₂₁ClN₅O₃

402.8890 [M + H]⁺; found 402.8889.

4.2. Biological evaluation

4.2.1. Cell lines

Human A549 and 293 cell lines were obtained from the American

Type Culture Collection (ATCC, Manassas, VA). The 293β5 stable cell

line overexpressing the human β5 integrin subunit was kindly provided

by Dr. Glen Nemerow [62]. The cell lines were propagated in Dulbecco’s

modiﬁed Eagle medium (DMEM, Life Technologies/Thermo Fisher)

supplemented with 10% fetal bovine serum (FBS) (Omega Scientiﬁc,

Tarzana, CA), 10 mM HEPES, 4 mM L-glutamine, 100 units/ml peni-

cillin, 100 mg/mL streptomycin, and 0.1 mM non-essential amino acids

(complete DMEM).

4.1.13.12. N-(4-Chlorophenyl)-N’-{3-hydroxy-2-[4-(2-formylphenyl)

triazolyl]propyl} urea (86). The product was obtained as a white resin

and puriﬁed by column chromatography using hexane–ethyl acetate

(1:2, 0.5% Et₃N) (120 mg, 60% yield). ¹H NMR (500 MHz, DMSO‑d₆) δ

10.37 (s, 1H, COH), 8.82 (s, 1H, NHAr), 8.73 (s, 1H, Ar), 8.62 (s, 1H, CH

triazole), 7.92–7.89 (m, 1H, Ar), 7.80–7.73 (m, 1H, Ar), 7.59–7.55 (m,

1H, Ar), 7.45–7.36 (m, 2H, Ar), 7.28–7.23 (m, 2H, Ar), 6.39 (t, J = 5.4

Wild-type HAdV-5 were obtained from the ATCC. The HAdV-5-GFP

used in this study is a replication-defective virus containing a CMV

21

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

promoter-driven enhanced green ﬂuorescent protein (eGFP) reporter

gene cassette in place of the E1/E3. HAdV were propagated in 293β5

cells and isolated from the cellular lysate by cesium chloride density

centrifugation. Virus concentration, in mg/ml, was calculated with the

Bio-Rad Protein Assay (Bio-Rad Laboratories) and converted to virus

particles/ml (vp/ml) using 4 × 10¹²vp/mg.

The human glyceraldehyde-3-phosphate dehydrogenase GAPDH was

used as an internal control. Oligonucleotide sequences for GAPDH and

the conditions were those previously reported [64]. For quantiﬁcation,

gene fragments from hexon and GAPDH were cloned into the pGEM-T

Easy vector (Promega), and known concentrations of template were

used to generate a standard curve in parallel for each experiment. All

assays were performed in a thermal cycler LightCycler 96 System

(Roche).

4.2.2. Plaque assay

Compounds were tested using low MOI infections (0.03 vp/cell) at

concentrations of 10

μ

M. If the percentage of inhibition was upper than

4.2.5. Quantiﬁcation of Nuclear-Associated HAdV genomes

60%, compounds was also tested in a dose–response assay ranging from

A549 cells (1 × 10⁶cells/well) in 6-well plates were infected with

wild-type HAdV5 at MOI 2,000 in the presence of 10-fold IC₅₀concen-

tration obtained in the plaque assay of the compounds or the same

volume of DMSO (positive control). Forty-ﬁve minutes after infection,

A549 cells were trypsinized and collected and then washed twice with

PBS. Then, cytoplasmic and nuclear fractions were separated using a

hypotonic buffer solution and NP-40 detergent. The cell pellet was

10 to 0.375 μM in a plaque assay. For that, 293β5 cells were seeded in 6-

well plates at a density of 4 × 10⁵cells per well in duplicate for each

condition. When cells reached 80–90% conﬂuency, they were infected

with HAdV5-GFP (0.06 vp/cell) and rocked for 2 h at 37 ^◦C. After the

incubation, the inoculum was removed and the cells were washed once

with PBS. The cells were then carefully overlaid with 2 mL/well of equal

parts of 1.6% (water/vol) Difco Agar Noble (Becton, Dickinson & Co.,

Sparks, MD), 2x EMEM (Minimum EssentialMedium Eagle, Bio-

Whittaker) and the compounds. EMEM was supplemented with 2x

penicillin/streptomycin, 2x ^L-glutamine, and 10% FBS. Compounds

resuspended in 500

μL of 1x hypotonic buffer (20 mM Tris-HCl pH 7.4,

10 mM NaCl, 3 mM MgCl₂) and incubated for 15 min at 4 ^◦C. Then, 25

μL

of NP-40 was added, and the samples were vortexed. The homogenates

were centrifuged for 10 min at 835g at 4 ^◦C. Following supernatant

contained the cytoplasmic fraction and the pellet contained the nuclear

fraction. HAdV DNA was isolated from both fractions using the E.Z.N.A.

Tissue DNA Kit (Omega Biotek, Norcross, GA). HAdV genomes was

quantiﬁed by real-time PCR following the same protocol we described

above.

were then added in concentrations ranging from 10 to 0.375

μM.

Following incubation for 7 days at 37 ^◦C, plates were scanned with a

Typhoon FLA 9000 imager (GE Healthcare Life Sciences) and plaques

were quantiﬁed with ImageJ [63].

4.2.3. Cytotoxicity assay

The cytotoxicity of the compounds was analyzed by commercial kit

AlamarBlue (Invitrogen, ref DAL1025). Brieﬂy, A549 cells at a density of

5 × 10³cells per well in 96-well plates were seeded. Decreasing con-

4.2.6. Virus yield reduction

A549 cells (1.5 × 10⁵cells/well in a 24-well plate) were incubated

24 h in 500 μL of complete DMEM, and they were infected with wild-

centrations of each derivative from 200

μ

M to 2.5

μ

M were diluted in

type HAdV5 (100 vp/cell) when >90% of conﬂuency was observed.

100

μ

L of Dulbecco’s Modiﬁed Eagle Medium (DMEM), added to the

Infected cells were incubated for 48 h at 37 ^◦C in 500

μL of complete

cells and incubated at 37 ^◦C for 48 h following the kit protocol. The

cytotoxic concentration 50 (CC₅₀) value was obtained using the statis-

tical package GraphPad Prism. This assay was performed in duplicate.

DMEM containing 10-fold IC₅₀concentration obtained in the plaque

assay of either compounds or the same volume of DMSO (positive con-

trol). After 48 h, cells were harvested and subjected to three rounds of

freeze/thaw. Serial dilutions of clariﬁed lysates were titrated on A549

cells (3 × 10⁴cells/well in a 96-well plate), and TCID₅₀values were

calculated using an end-point dilution method [65].

4.2.4. Quantiﬁcation of HAdV DNA, E1A mRNA and E2B mRNA

A549 cells (1.5 × 10⁵cells/well in a 24-well plate) were incubated

for 24 h in 500 μL of complete DMEM, and they were infected with wild-

type HAdV5 (100 vp/cell) when 80–90% conﬂuency was observed.

4.2.7. Time of addition curve

For DNA quantiﬁcation, infected cells were incubated for 16 h at

The anti-HAdV effect of derivatives at different points of time was

measured in a time-curve assay using 293β5 cells (3 × 10⁵cells/well in

corning black wall, clear bottom 96-well plates) infected with HAdV5-

GFP (2,000 vp/cell) in the presence of 10-fold IC₅₀concentration ob-

tained in the plaque assay of the compounds or the same volume of

DMSO (positive control). Parallel samples of HAdV-5 were incubated

with or without the selected derivatives on ice for 1 h. The virus was

then added to 293β5 cells and the plate were incubated 1 h on ice. After

that, the inoculum was removed and the derivatives were added at the

indicated time points before or during this incubation. After a total of 2 h

at 37 ^◦C, cells were incubated for an additional 48 h at 37 ^◦C and 5 %CO₂

before being analyzed for GFP expression using the Typhoon 9410

imager (GE Healthcare Life Sciences), as above.

37 ^◦C in 500

μL of complete DMEM containing 10-fold IC₅₀concentra-

tion obtained in the plaque assay of the compounds or the same volume

of DMSO (positive control). All samples were done in duplicate.

After incubation at 37 ^◦C and 5% CO₂, DNA was puriﬁed from the

cell lysate with the E.Z.N.A. Tissue DNA Kit (Omega Biotek, Norcross,

GA) following the manufacturer’s instructions. TaqMan primers and

probes for a common region of the HAdV5 were designed with the

GenScript Real-Time PCR (TaqMan) Primer Design software (Gen-

Script). Oligonucleotide sequences were as follows: AQ1: 5^′-GCC ACG

GTG GGG TTT CTA AAC TT-3^′; AQ2: 5^′-GCC CCA GTG GTC TTA CAT

GCA CAT-3^′; Probe: 6-FAM-5^′-TGC ACC AGA CCC GGG CTC AGG TAC

TCC GA-3^′-TAMRA’. Real-time PCR mixtures consisted of 9.5

μ

L of the

puriﬁed DNA, AQ1 and AQ2 at a concentration of 200 nM each, and

probe at a concentration of 50 nM in a total volume of 12.5 L mixed

μ

4.2.8. Synergy assay

with 12.5

μ

L of FastGene 2x PROBE Universal (NIPPON Genetics). The

For this assay, the same conditions of the plaque assay were used

(293β5 cells were seeded at a density of 4 × 10⁵cells/well in 6 well-plate

and infected at MOI 0.06 with HAdV5-GFP) but the compounds added

were a combination of two compounds in a dose–response assay ranging

from 2xIC₅₀to 1/8xIC₅₀. After incubation for 7 days at 37 ^◦C, plates were

scanned with a Typhoon FLA 9000 imager (GE Healthcare Life Sciences)

and plaques were quantiﬁed with ImageJ [63].

PCR cycling protocol was 95 ^◦C for 3 min followed by 40 cycles of 95 ^◦C

for 10 s and 60 ^◦C for 30 s.

For E1A and E2B mRNA quantiﬁcation, infected cells were incubated

for 6 h at 37 ^◦C in 500

μL of complete DMEM containing 10-fold IC₅₀

concentration obtained in the plaque assay of the compounds or the

same volume of DMSO (positive control). All samples were done in

duplicate. RNA was puriﬁed with the RNeasy Mini Kit (QIAGEN,

Valencia, CA) following the manufacturer’s instructions. Quantiﬁcation

of RNA copy numbers was performed using primers and conditions

previously reported for E1A and E2B [64].

Data obtained was analysed with the CalcuSyn software (Biosoft)

and the output obtained was used to deﬁne the synergistic or antago-

nistic effect of combinations, according to Chou-Talalay method.

22

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

Organ Transplant. 23 (2018) 395–399, https://doi.org/10.1097/

4.2.9. Statistical analyses

MOT.0000000000000542.

One-way ANOVA tests (Dunnet method) were carried out using the

GraphPad Prism 6. We considered a statistical signiﬁcance with a p ≤

0.05. This statistical signiﬁcance was pointed out with asterisk in

graphs, and the numbers of them indicate the level of signiﬁcance (*p ≤

0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

[11] M.M.Y. Waye, C.W. Sing, Anti-viral drugs for human adenoviruses,

Pharmaceuticals. 3 (2010) 3343–3354, https://doi.org/10.3390/ph3103343.

[12] L. Lenaerts, L. Naesens, Antiviral therapy for adenovirus infections, Antiviral Res.

71 (2006) 172–180, https://doi.org/10.1016/j.antiviral.2006.04.007.

¨

[13] K. Schaar, C. Roger, T. Pozzuto, J. Kurreck, S. Pinkert, H. Fechner, Biological

antivirals for treatment of adenovirus infections, Antivir. Ther. 21 (2016) 559–566,

https://doi.org/10.3851/IMP3047.

[14] U. Yusuf, G.A. Hale, J. Carr, Z. Gu, E. Benaim, P. Woodard, K.A. Kasow, E.

M. Horwitz, W. Leung, D.K. Srivastava, R. Handgretinger, R.T. Hayden, Cidofovir

for the treatment of adenoviral infection in pediatric hematopoietic stem cell

transplant patients, Transplantation 81 (2006) 1398–1404, https://doi.org/

10.1097/01.tp.0000209195.95115.8e.

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

[15] G. Lugthart, M.A. Oomen, C.M. Jol-van der Zijde, L.M. Ball, D. Bresters, W.J.

W. Kollen, F.J. Smiers, C.L. Vermont, R.G.M. Bredius, M.W. Schilham, M.J.D. van

Tol, A.C. Lankester, The Effect of Cidofovir on Adenovirus Plasma DNA Levels in

Stem Cell Transplantation Recipients without T Cell Reconstitution, Biol. Blood

Marrow Transplant. 21 (2015) 293–299, https://doi.org/10.1016/j.

bbmt.2014.10.012.

Acknowledgements

[16] A.E. Caruso Brown, M.N. Cohen, S. Tong, R.S. Braverman, J.F. Rooney, R. Giller,

M.J. Levin, Pharmacokinetics and safety of intravenous cidofovir for life-

threatening viral infections in pediatric hematopoietic stem cell transplant

recipients, Antimicrob. Agents Chemother. 59 (2015) 3718–3725, https://doi.org/

10.1128/AAC.04348-14.

´

This work has been supported by Ministerio de Ciencia, Innovacion y

Universidades, Plan Estatal 2017-2020 Retos - Proyectos I + D + i

(PID2019-104767RB-I00); Ministerio de Economía y Competitividad,

Plan Nacional I + D + i 2013-2016, Retos-Proyectos (CTQ2016-78580-

´

[17] J.A. Marrugal-Lorenzo, A. Serna-Gallego, J. Berastegui-Cabrera, J. Pachon,

´

C2-2-R); Instituto de Salud Carlos III, Subdireccion General de Redes y

´

J. Sanchez-Cespedes, Repositioning salicylanilide anthelmintic drugs to treat

adenovirus infections, Sci. Rep. 9 (2019) 1–10, https://doi.org/10.1038/s41598-

018-37290-3.

´

Centros de Investigacion Cooperativa, Ministerio de Economía, Industria

y Competitividad, Spanish Network for Research in Infectious Diseases

(REIPI RD16/0016/0009), coﬁnanced by European Development

Regional Fund “A way to achieve Europe”, the Instituto de Salud Carlos

[18] C.C. Colpitts, L.M. Schang, A Small Molecule Inhibits Virion Attachment to

Heparan Sulfate- or Sialic Acid-Containing Glycans, J. Virol. 88 (2014) 7806–7817,

https://doi.org/10.1128/jvi.00896-14.

´

[19] N.A. Nikitenko, E.S. Gureeva, A.A. Ozerov, A.I. Tukhvatulin, F.M. Izhaeva, V.

S. Prassolov, P.G. Deryabin, M.S. Novikov, D.Y. Logunov, 1-(4-Phenoxybenzyl) 5-

aminouracil derivatives and their analogues - novel inhibitors of human adenovirus

replication, Acta Naturae. 10 (2018) 58–64, https://doi.org/10.32607/20758251-

2018-10-2-58-64.

III, Proyectos de Investigacion en Salud (PI17/01055; PI18/01191) and

´

Proyectos de Desarrollo Tecnologico en Salud (DTS17/00130), the

Spanish Adenovirus Network (AdenoNet, BIO2015/68990-REDT), and

´

the program “Nicolas Monardes” (C-0059-2018), Servicio Andaluz de

´

[20] J. Xu, J. Berastegui-Cabrera, H. Chen, J. Pachon, J. Zhou, J. Sanchez-Cespedes,

Structure-Activity Relationship Studies on Diversiﬁed Salicylamide Derivatives as

Potent Inhibitors of Human Adenovirus Infection, J. Med. Chem. 63 (2020)

3142–3160, https://doi.org/10.1021/acs.jmedchem.9b01950.

Salud, Junta de Andalucía. Authors thank CITIUS (Centro de Inves-

´

tigacion, Tecnología e Innovacion de la Universidad de Sevilla) for its

contribution in recording NMR and Mass spectra and for elemental

analysis determination. The authors are especially grateful to Dr. Man-

[21] D. Zhang, Qian; Xiang, Dehu; Zhang, Ling; Wang, Xia; Li, Application of aromatic

ester compounds in preparing drugs for resisting Adenovirus-7 (ADV-7), CN 2017-

11446779, 2018. https://sciﬁnder–cas–org.us.debiblio.com/sciﬁnder/view/

sciﬁnder/sciﬁnderExplore.jsf.

´

uel Angulo Alvarez for his assistance with heteronuclear 2D NMR and

NOESY studies.

´

[22] S. Mazzotta, J.A. Marrugal-Lorenzo, M. Vega-Holm, A. Serna-Gallego, J. Alvarez-

´

Vidal, J. Berastegui-Cabrera, J. Perez del Palacio, C. Díaz, F. Aiello, J. Pachon,

Appendix A. Supplementary material

´

F. Iglesias-Guerra, J.M. Vega-Perez, J. Sanchez-Cespedes, Optimization of

piperazine-derived ureas privileged structures for effective antiadenovirus agents,

Eur. J. Med. Chem. 185 (2020), https://doi.org/10.1016/j.ejmech.2019.111840.

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.105095.

´

[23] J. Sanchez-Cespedes, P. Martínez-Aguado, M. Vega-Holm, A. Serna-Gallego, J.

´

I. Candela, J.A. Marrugal-Lorenzo, J. Pachon, F. Iglesias-Guerra, J.M. Vega-Perez,

New 4-Acyl-1-phenylaminocarbonyl-2-phenylpiperazine Derivatives as Potential

Inhibitors of Adenovirus Infection. Synthesis, Biological Evaluation, and Structure-

activity Relationships, J. Med. Chem. 59 (2016) 5432–5448, https://doi.org/

10.1021/acs.jmedchem.6b00300.

References

[1] T. Lion, Adenovirus infections in immunocompetent and immunocompromised

patients, Clin. Microbiol. Rev. 27 (2014) 441–462, https://doi.org/10.1128/

CMR.00116-13.

´

´˜

´

[24] S. Sanchez Cespedes, Javier; Pachon Ibanez , María Eugenia; Pachon Díaz,

´

Jeronimo; Martínez Aguado, Pablo; Cebrero Cangueiro, Tania; Vega Perez, Jose

Manuel; Iglesias Guerra, Fernando; Vega Holm, Margarita; Candela Lena, Ignacio;

Mazzotta, Piperazine Derivatives as Antiviral Agents with Increased Therapeutic

Activity, US 2019 / 0308956, 2019. https://patents.google.com/patent/

US20190308956A1/en.

[2] M. Echavarría, Adenoviruses in immunocompromised hosts, Clin. Microbiol. Rev.

21 (2008) 704–715, https://doi.org/10.1128/CMR.00052-07.

[3] E. Hage, U.G. Liebert, S. Bergs, T. Ganzenmueller, A. Heim, Human mastadenovirus

type 70: A novel, multiple recombinant species D mastadenovirus isolated from

diarrhoeal faeces of a haematopoietic stem cell transplantation recipient, J. Gen.

Virol. 96 (2015) 2734–2742, https://doi.org/10.1099/vir.0.000196.

[4] M.A. Prusinkiewicz, J.S. Mymryk, Metabolic reprogramming of the host cell by

human adenovirus infection, Viruses. 11 (2019) 1–21, https://doi.org/10.3390/

v11020141.

[25] S. Mazzotta, J. Berastegui-Cabrera, G. Carullo, M. Vega-Holm, M. Carretero-

´

Ledesma, L. Mendolia, F. Aiello, F. Iglesias-Guerra, J. Pachon, J.M. Vega-Perez,

´

J. Sanchez-Cespedes, Serinol-based benzoic acid esters as new scaffolds for the

development of adenovirus infection inhibitors: design, synthesis, and in vitro

biological evaluation, ACS Infect. Dis. 7 (2021) 1433–1444, https://doi.org/

10.1021/acsinfecdis.0c00515.

[5] C.S. Martín, Latest insights on adenovirus structure and assembly, Viruses. 4 (2012)

847–877, https://doi.org/10.3390/v4050847.

[26] P. Sikka, Role of aryl urea containing compounds in medicinal chemistry, Med.

Chem. 5 (2015) 479–483, https://doi.org/10.4172/2161-0444.1000305.

[27] L. Chiummiento, M. Funicello, P. Lupattelli, F. Tramutola, F. Berti, F. Marino-

Merlo, Synthesis and biological evaluation of novel small non-peptidic HIV-1 PIs:

The benzothiophene ring as an effective moiety, Bioorg. Med. Chem. Lett. 22

(2012) 2948–2950, https://doi.org/10.1016/j.bmcl.2012.02.046.

[6] M.K. Scott, C. Chommanard, X. Lu, D. Appelgate, L. Grenz, E. Schneider, S.

I. Gerber, D.D. Erdman, A. Thomas, Human Adenovirus Associated with Severe

Respiratory Infection, Emerg. Infect. Dis. 22 (2016) 2013 –2014 .

[7] S. Abbas, J.E. Raybould, S. Sastry, O. de la Cruz, Respiratory viruses in transplant

recipients: more than just a cold. Clinical syndromes and infection prevention

principles, Int. J. Infect. Dis. 62 (2017) 86–93, https://doi.org/10.1016/j.

ijid.2017.07.011.

[28] F. Tramutola, M.F. Armentano, F. Berti, L. Chiummiento, P. Lupattelli, R. D’Orsi,

R. Miglionico, L. Milella, F. Bisaccia, M. Funicello, New heteroaryl carbamates:

Synthesis and biological screening in vitro and in mammalian cells of wild-type

and mutant HIV-protease inhibitors, Bioorg. Med. Chem. 27 (2019) 1863–1870,

https://doi.org/10.1016/j.bmc.2019.03.041.

[8] D. Garcia-Zalisnak, C. Rapuano, J.D. Sheppard, A.R. Davis, Adenovirus Ocular

Infections: Prevalence, Pathology, Pitfalls, and Practical Pointers, Eye Contact

Lens. 44 (2018) 1–7, https://doi.org/10.1097/ICL.0000000000000226.

[29] D. Kang, H. Zhang, Z. Zhou, B. Huang, L. Naesens, P. Zhan, X. Liu, First discovery of

novel 3-hydroxy-quinazoline-2,4(1H,3H)-diones as speciﬁc anti-vaccinia and

adenovirus agents via ‘privileged scaffold’ reﬁning approach, Bioorg. Med. Chem.

Lett. 26 (2016) 5182–5186, https://doi.org/10.1016/j.bmcl.2016.09.071.

[30] E. Rivero-Buceta, P. Carrero, E.G. Doyagüez, A. Madrona, E. Quesada, M.

´

[9] K. Kosulin, E. Geiger, A. Vecsei, W.D. Huber, M. Rauch, E. Brenner, F. Wrba,

¨

K. Hammer, A. Innerhofer, U. Potschger, A. Lawitschka, S. Matthes-Leodolter,

G. Fritsch, T. Lion, Persistence and reactivation of human adenoviruses in the

gastrointestinal tract, Clin. Microbiol. Infect. 22 (381) (2016), https://doi.org/

10.1016/j.cmi.2015.12.013.

´

J. Camarasa, M.J. Perez-Perez, P. Leyssen, J. Paeshuyse, J. Balzarini, J. Neyts,

[10] S.M.C. Lopez, M.G. Michaels, M. Green, Adenovirus infection in pediatric

transplant recipients: Are effective antiviral agents coming our way? Curr. Opin.

´

A. San-Felix, Linear and branched alkyl-esters and amides of gallic acid and other

23

S. Mazzotta et al.

Bioorganic Chemistry 114 (2021) 105095

(mono-, di- and tri-) hydroxy benzoyl derivatives as promising anti-HCV inhibitors,

based interactivity calculations, PLoS ONE 12 (2017) 1–19, https://doi.org/

10.1371/journal.pone.0173303.

Eur. J. Med. Chem. 92 (2015) 656–671, https://doi.org/10.1016/j.

ejmech.2015.01.033.

[49] N.H. Amin, M.T. El-Saadi, A.A. Ibrahim, H.M. Abdel-Rahman, Design, synthesis

and mechanistic study of new 1,2,4-triazole derivatives as antimicrobial agents,

Bioorg. Chem. 111 (2021), 104841, https://doi.org/10.1016/j.

[31] S. Saul, S.Y. Pu, W.J. Zuercher, S. Einav, C.R.M. Asquith, Potent antiviral activity of

novel multi-substituted 4-anilinoquin(az)olines, Bioorg. Med. Chem. Lett. 30

(2020), 127284, https://doi.org/10.1016/j.bmcl.2020.127284.

[32] Z. Zhao, H. Song, J. Xie, T. Liu, X. Zhao, X. Chen, X. He, S. Wu, Y. Zhang, X. Zheng,

Research progress in the biological activities of 3,4,5-trimethoxycinnamic acid

(TMCA) derivatives, Eur. J. Med. Chem. 173 (2019) 213–227, https://doi.org/

10.1016/j.ejmech.2019.04.009.

bioorg.2021.104841.

[50] T.S. Ibrahim, A.J. Almalki, A.H. Moustafa, R.M. Allam, G.E.-D.A. Abuo-Rahma, H.

I. El Subbagh, M.F.A. Mohamed, Novel 1,2,4-oxadiazole-chalcone/oxime hybrids

as potential antibacterial DNA gyrase inhibitors: Design, synthesis, ADMET

prediction and molecular docking study, Bioorg. Chem. 111 (2021), 104885,

https://doi.org/10.1016/j.bioorg.2021.104885.

´

[33] I.E. Głowacka, J. Balzarini, A.E. Wroblewski, The synthesis, antiviral, cytostatic

and cytotoxic evaluation of a new series of acyclonucleotide analogues with a

1,2,3-triazole linker, Eur. J. Med. Chem. 70 (2013) 703–722, https://doi.org/

10.1016/j.ejmech.2013.10.057.

[51] R. Wang, Y. Fu, L. Lai, A new method for calculating partition coefﬁcients of

organic compounds, Acta Phys. - Chim. Sin. 13 (1997) 615–621, https://doi.org/

10.3866/pku.whxb19970101.

´

[34] T. Van Nguyen, A. Hospital, C. Lionne, L.P. Jordheim, C. Dumontet, C. Perigaud,

[52] W.P. Walters, A. Murcko, M.A. Murcko, Recognizing molecules with drug-like

properties, Curr. Opin. Chem. Biol. 3 (1999) 384–387, https://doi.org/10.1016/

S1367-5931(99)80058-1.

L. Chaloin, S. Peyrottes, Beta-hydroxyphosphonate ribonucleoside analogues

derived from 4-substituted-1,2,3-triazoles as IMP/GMP mimics: Synthesis and

biological evaluation, J. Org. Chem. 12 (2016) 1476–1486, https://doi.org/

10.3762/bjoc.12.144.

[53] P.F. Lamie, J.N. Philoppes, 2-Thiopyrimidine/chalcone hybrids: design, synthesis,

ADMET prediction, and anticancer evaluation as STAT3/STAT5a inhibitors,

J. Enzyme Inhib. Med. Chem. 35 (2020) 864–879, https://doi.org/10.1080/

14756366.2020.1740922.

[35] S.K.V. Vernekar, L. Qiu, J. Zhang, J. Kankanala, H. Li, R.J. Geraghty, Z. Wang, 5^′-

silylated 3^′-1,2,3-triazolyl thymidine analogues as inhibitors of West Nile Virus and

Dengue virus, J. Med. Chem. 58 (2015) 4016–4028, https://doi.org/10.1021/acs.

jmedchem.5b00327.

[54] B.M. Minrovic, D. Jung, R.J. Melander, C. Melander, new class of adjuvants enables

lower dosing of colistin against acinetobacter baumannii, ACS Infect. Dis. 4 (2018)

1368–1376, https://doi.org/10.1021/acsinfecdis.8b00103.

[36] E. Bonandi, M.S. Christodoulou, G. Fumagalli, D. Perdicchia, G. Rastelli,

D. Passarella, The 1,2,3-triazole ring as a bioisostere in medicinal chemistry, Drug

Discov. Today. 22 (2017) 1572–1581, https://doi.org/10.1016/j.

drudis.2017.05.014.

[55] M.A. Brimble, C. Brocke, Efﬁcient synthesis of the azabicyclo[3.3.1]nonane ring

system in the alkaloid methyllycaconitine using bis(alkoxymethyl)alkylamines as

aminoalkylating agents in a double Mannich reaction, Eur. J. Org. Chem. (2005)

2385–2396, https://doi.org/10.1002/ejoc.200500003.

[37] D.B. Goetz, M.L. Varney, D.F. Wiemer, S.A. Holstein, Amides as bioisosteres of

triazole-based geranylgeranyl diphosphate synthase inhibitors, Bioorg. Med. Chem.

28 (2020), 115604, https://doi.org/10.1016/j.bmc.2020.115604.

[38] I. Mohammed, I.R. Kummetha, G. Singh, N. Sharova, G. Lichinchi, J. Dang,

M. Stevenson, T.M. Rana, 1,2,3-triazoles as amide bioisosteres: Discovery of a new

class of potent HIV-1 Vif antagonists, J. Med. Chem. 59 (2016) 7677–7682, https://

doi.org/10.1021/acs.jmedchem.6b00247.

[56] C. Fehr, Diastereoface-selective epoxidations: dependency on the reagent

electrophilicity, Angew. Chemie - Int. Ed. 37 (1998) 2407–2409, https://doi.org/

10.1002/(SICI)1521-3773(19980918)37:17 <2407::AID-ANIE2407 >3.0.CO;2-W .

[57] B. Zhao, Z. Gao, Y. Zheng, C. Gao, Scalable synthesis of positively charged

sequence-deﬁned functional polymers, J. Am. Chem. Soc. (2019) 1–52, https://doi.

org/10.1021/jacs.9b00172.

[39] Q. Qiu, J. Zhu, Q. Chen, Z. Jiang, J. Xu, X. Jiang, W. Huang, Z. Liu, J. Ye, X. Xu,

Discovery of aromatic amides with triazole-core as potent reversal agents against

P-glycoprotein-mediated multidrug resistance, Bioorg. Chem. 90 (2019), 103083,

https://doi.org/10.1016/j.bioorg.2019.103083.

[58] E.W. Sugandhi, J.O. Falkinham, R.D. Gandour, Synthesis and antimicrobial activity

of symmetrical two-tailed dendritic tricarboxylato amphiphiles, Bioorg. Med.

Chem. 15 (2007) 3842–3853, https://doi.org/10.1016/j.bmc.2007.03.017.

[59] J. Nieschalk, S. Laboratories, S. Road, A short synthesis of (1S,2R)- and (1R,2R)-[1-

ZH]-glycerols, Tetrahedron Asymmetry 8 (1997) 2325 –2330 .

[40] S.G. Agalave, S.R. Maujan, V.S. Pore, Click chemistry: 1,2,3-triazoles as

pharmacophores, Chem. - An Asian J. 6 (2011) 2696–2718, https://doi.org/

10.1002/asia.201100432.

´

[60] M. Martínez-Bailen, A.T. Carmona, E. Moreno-Clavijo, I. Robina, D. Ide, A. Kato, A.

J. Moreno-Vargas, Tuning of β-glucosidase and α-galactosidase inhibition by

[41] L. Liang, D. Astruc, The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC)

“click” reaction and its applications. An overview, Coord. Chem. Rev. 255 (2011)

2933–2945. https://doi.org/10.1016/j.ccr.2011.06.028.

generation and in situ screening of a library of pyrrolidine-triazole hybrid

molecules, Eur. J. Med. Chem. 138 (2017) 532–542, https://doi.org/10.1016/j.

ejmech.2017.06.055.

[42] L. Ganapathi, A. Arnold, S. Jones, A. Patterson, D. Graham, M. Harper, O. Levy, Use

of cidofovir in pediatric patients with adenovirus infection, F1000Research 5

(2016) 758, https://doi.org/10.12688/f1000research.8374.1.

[61] L. Liu, C. Li, S. Cochran, S. Jimmink, V. Ferro, Synthesis of a heparan sulfate

mimetic library targeting FGF and VEGF via click chemistry on a monosaccharide

template, ChemMedChem 7 (2012) 1267–1275, https://doi.org/10.1002/

cmdc.201200151.

[43] C.J. Burckhardt, U.F. Greber, Virus movements on the plasma membrane support

infection and transmission between cells, PLoS Pathog. 5 (2009), https://doi.org/

10.1371/journal.ppat.1000621.

[62] R.R. Nepomuceno, L. Pache, G.R. Nemerow, Enhancement of gene transfer to

human myeloid cells by adenovirus-ﬁber complexes, Mol. Ther. 15 (2007)

571–578, https://doi.org/10.1038/sj.mt.6300048.

[44] O. Meier, U.F. Greber, Adenovirus endocytosis, J. Gene Med. 6 (2004) 152–163,

https://doi.org/10.1002/jgm.553.

[63] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of

image analysis, Nat. Methods. 9 (2012) 671–675, https://doi.org/10.1038/

nmeth.2089.

[45] U.F. Greber, M. Willetts, P. Webster, A. Helenius, Stepwise dismantling of

adenovirus 2 during entry into cells, Cell 75 (1993) 477–486, https://doi.org/

10.1016/0092-8674(93)90382-Z.

[64] A.A. Rivera, M. Wang, K. Suzuki, T.G. Uil, V. Krasnykh, D.T. Curiel, D.

M. Nettelbeck, Mode of transgene expression after fusion to early or late viral genes

of a conditionally replicating adenovirus via an optimized internal ribosome entry

site in vitro and in vivo, Virology 320 (2004) 121–134, https://doi.org/10.1016/j.

virol.2003.11.028.

[46] M. Charman, C. Herrmann, M.D. Weitzman, Viral and cellular interactions during

adenovirus DNA replication, FEBS Lett. 593 (2019) 3531–3550, https://doi.org/

10.1002/1873-3468.13695.

[47] E.J. Kremer, G.R. Nemerow, Adenovirus tales: from the cell surface to the nuclear

pore complex, PLoS Pathog. 11 (2015) 1–8, https://doi.org/10.1371/journal.

ppat.1004821.

[65] H.M.L.J. Reed, A Simple Method Of Estimating Fifty Per Cent Endpoints, Am. J.

Hyg. 27 (1938) 546–558, https://doi.org/10.7723/antiochreview.72.3.0546.

[48] H. Matthews, J. Deakin, M. Rajab, M. Idris-Usman, N.J. Nirmalan, Investigating

antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-

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