Innovative three-step microwave-promoted synthesis of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXA) inhibitors: Drug design, synthesis, and biological evaluation.

molecules

Article

Innovative Three-Step Microwave-Promoted

Synthesis of N-Propargyltetrahydroquinoline and

1,2,3-Triazole Derivatives as a Potential Factor Xa (FXa)

Inhibitors: Drug Design, Synthesis, and

Biological Evaluation

Fabián Santana-Romo ¹, Carlos F. Lagos ², Yorley Duarte ³, Francisco Castillo ^1,4

,

Yanina Moglie ⁵, Miguel A. Maestro ⁶, Nitin Charbe ¹and Flavia C. Zacconi ^1,4,

*

1

Departamento de Química Orgánica, Facultad de Química y de Farmacia, Pontiﬁcia Universidad Católica de

Chile, Av. Vicuña Mackenna 4860, Macul, Santiago 7820436, Chile; fmsantana@uc.cl (F.S.-R.);

fjcastil@uc.cl (F.C.); nitinunimi@gmail.com (N.C.)

Chemical Biology & Drug Discovery Laboratory, Facultad de Medicina y Ciencia, Universidad San Sebasti

Lota 2465, Providencia 7510157, Santiago de Chile, Chile; carlos.lagos@uss.cl

Center for Bioinformatics and Integrative Biology, Facultad de Ciencias de la Vida, Universidad Andrés

Bello, Santiago 8370146, Chile; yorley.duarte@unab.cl

Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences,

Pontiﬁcia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Macul, Santiago 7820436, Chile

Departamento de Química, Instituto de Química del Sur (INQUISUR-CONICET), Universidad Nacional del

2

3

4

5

6

án,

Sur Avenida Alem 1253, Bahía Blanca B8000CPB, Argentina; ymoglie@uns.edu.ar

Department of Chemistry—CICA, University of A Coruña, Campus da Zapateira, 15008A A Coruña, Spain;

miguel.maestro@udc.es

*

Correspondence: fzacconi@uc.cl; Tel.: +56-2354-1150

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

Academic Editor: Derek J. McPhee

Received: 2 January 2020; Accepted: 20 January 2020; Published: 23 January 2020

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: The coagulation cascade is the process of the conversion of soluble ﬁbrinogen to insoluble

ﬁbrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved

in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence

which ends with the thrombus production. Thrombosis is a common causal pathology for three

widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism

(VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole

derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated

for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design

for the desired novel molecules was performed through protein-ligand complexes selection and

ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out

by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar

cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be

an eﬃcient way to obtain all novel compounds in high yields (73–93%). Furthermore, a thermochemical

analysis, optimization and reactivity indexes such as electronic chemical potential (

hardness ( ), and electrophilicity ( ) were performed to understand the relationship between the

structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds

27 29 31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal

µ), chemical

η

ω

,

–

inhibitory concentration (IC₅₀) values were calculated. Next, a cell viability assay in HEK293 and

HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial

Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the

corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that

compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic

Molecules 2020, 25, 491; doi:10.3390 /molecules25030491

ww w .mdpi.com /journal /molecules

Molecules 2020, 25, 491

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pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common

pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa

and coagulation parameters did not show toxicity at the performed coagulation assay concentrations.

Finally, docking studies conﬁrmed the preferential binding mode of N-propargyltetrahydroquinoline

and 1,2,3-triazole derivatives inside the active site of FXa.

Keywords: factor Xa inhibitors; microwave-assisted synthesis; N-propargyltetrahydroquinoline;

1,2,3-triazole; cell viability assay; coagulation parameters

1. Introduction

In the last few decades, statistics relating to a high number of cardiovascular syndromes that

are a leading cause of heart problems and increasing death rates in Europe and the US have been

published. A recent study carried out in Chile found the incidence risk rate for thromboembolic

diseases among patients under general surgery is 55% and the principal cause of death in Chile

is related to cardiovascular diseases [1,2]. With more than 24,000 deaths annually, cerebrovascular

accidents (CVA) represent almost a third of all deaths [3,4] in Chile.

Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute

coronary syndrome (ACS), venous thromboembolism (VTE), and strokes [5,6]. Furthermore, the latest

statistical study from the Global Burden of Diseases, Injuries and Risk Factors (GBD) showed that 25%

of people worldwide die from thrombosis related events [7].

The coagulation cascade is the process of the conversion of soluble ﬁbrinogen to insoluble ﬁbrin

that terminates in production of a clot. This process is the property of plasma where factor X (FX), which

is a serine endopeptidase enzyme, plays a vital role in the conversion of prothrombin to thrombin [8].

The clot composition is composed of activated platelets, red blood cells, and cross-linked ﬁbrin protein.

A clot, which blocks the bloodstream, can produce acute myocardial infarction (AMI), an ischemic

stroke, and deep vein thrombosis (DVT) [9–11].

In the normal circulation system, blood remains in liquid form and does not coagulate. It is

now well documented that endothelium cells have an important role in homeostasis maintenance

and various pathological conditions. Under healthy conditions, it not only degrades thrombus and

downregulates the overall process of thrombosis, but it also promotes thrombosis when damage to the

endothelium of vessels occurs. The endothelial cell produces and releases certain factors such as PGI2

(Prostaglandin I2/Prostacycline), nitric oxide (NO), and adenosine diphosphate (ADP), which acts as

the antiplatelet aggregating factors [12]. Thrombomodulin or CD141 is another important integral

protein expressed in the membrane of the endothelial cells. This integral protein acts as a cofactor in

the activation of protein C [13]. As the name suggests it modulates the activity of thrombin to work as

the protein C activator. Once activated, protein C inhibits factor Va and VIIIa and thereby inhibits

the platelet aggregation and clot formation [14]. Heparan sulphate a highly sulfated polysaccharide

is also expressed on the surface of endothelial cells. Its analog heparin is the most commonly use

anticoagulant drug in hospitals [15]. Binding of antithrombin with heparan sulfate leads to the

conformational changes in the antithrombin. These changes in the antithrombin, generates the active

form of antithrombin which not only inhibits the IXa and Xa, but also inhibits the thrombin, i.e., factor

IIa. Tissue plasminogen activator is another protein expressed on the endothelial cells which line the

internal lumen of blood vessels [16].

During an injury, at the site of action, endothelial cells lose the ability to act as an antiplatelet

aggregating activity and became pro-coagulant. Injured endothelial cells release the Von Willebrand

factor or factor VIII (FVIII), which is a large glycoprotein complex continuously produced by the

endothelial cells [17]. VW in particular binds to the platelets to activate it and to factor VIII to begin

the process of the intrinsic coagulation cascade [17–20]. Activated platelets activate the factor XII to

Molecules 2020, 25, 491

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XIIa. XIIa then activates the factor XI to XIa, which then activates IX to IXa. IXa along with Ca ions,

phospholipids, and factor VIII activates factor X to Xa (FXa). FXa again in the presence of Ca ions,

phospholipids, and factor V catalyzes the conversion of prothrombin to thrombin [21]. Thrombin then

converts the ﬁbrinogen to ﬁbrin monomers [22]. Fibrin monomer is then deposited on the primary

hemostatic plug, which then cross-links and stabilizes itself with the help of factor XIIIa. Tissue factor

produced by the damaged endothelial cells also activates factor VII to VIIa leading to the extrinsic

coagulation cascade. Factor VIIa activates factor IX and IX to IXa and Xa which are the common factors

involved in the intrinsic coagulation pathway [21,22] (Figure 1).

Figure 1. Blood coagulation cascade: extrinsic, intrinsic, and common pathway.

Moreover, Factor Xa is noteworthy as the sole and common factor of intrinsic and extrinsic

pathways that are involved in thrombin activation. Considering that one molecule of FXa catalyzes

the formation of 1000 thrombin molecules, this enzyme plays a central role in the coagulation process

of blood. Finally, this serine protease enzyme generates the main catalyzing reaction that results in

clot production and wound closure [23,24]. Because of its vital role in the coagulation cascade, its

inhibition is an important approach for the development of anticoagulant/antithrombic agents.

Mortality associated to thrombin is very high, and the currently available prescription drugs

to prevent thrombosis in cardiovascular patients include heparins and warfarin. Rivaroxaban and

Apixaban are the recently approved orally active agents available for FXa inhibition, whereas Dabigatran

inhibits the thrombin directly [25]. Warfarin is the vitamin K antagonist, which, if taken incorrectly,

increases the chances of dangerous bleeding. Warfarin, which is available in orally active form, has

several disadvantages. It is associated with drug interactions with over the counter drugs and has

been associated with severe side eﬀects such as blood in the urine, severe stomach and joint pain,

vomiting of blood, and dizziness, among others [26]. Heparins on the other hand, which can only

Molecules 2020, 25, 491

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be administered parenterally, have high interpatient variability in metabolism, and hence, require

therapeutic drug monitoring for dose adjustment. Due to its short half-life and low bioavailability,

frequent dosing of heparin is also essential [27,28]. Both of these agents required monitoring of clotting

time and dose adjustments to prevent bleeding.

As discussed earlier, FXa is the point of junction of the intrinsic and extrinsic coagulation pathways.

Inhibition of FXa could therefore be useful to inhibit intrinsic and extrinsic pathways. Furthermore,

unlike warfarin, FXa inhibitors directly inhibit the critical factor involved in the cascade. The coagulation

cascade is the process whereby ampliﬁcation of the enzymes takes place at each step, which means

the concentration of the FXa is always lower than the concentration of the thrombin. This allows for

the eﬃcient and potent antithrombotic activity of such inhibitors at very low concentrations. On the

other hand, several preclinical and clinical investigations have already demonstrated a lesser risk

of bleeding with the direct inhibition of FXa [22,29]. Nevertheless, the latest clinical studies have

just demonstrated that Rivaroxaban and Apixaban dose interruption could produce thromboembolic

events. Moreover, the utilization of warfarin combined with Rivaroxaban heightens the chance of

hemorrhage in non-valvular atrial ﬁbrillation patients [30–32].

In recent years, several direct FXa inhibitors have become available in the marketplace, and

others are in active development (Figure 2) [33

more eﬃcacious than warfarin for the prevention of strokes and systemic embolism in patients

with atrial ﬁbrillation, the reversal agent Andexanet alfa has only recently been approved [35 36].

,34]. Although these new oral anticoagulants are

,

The development of antidotes for oral direct FXa inhibitors are still in the pipeline, but their expected

approval for therapeutic purposes will be further beneﬁcial to anticoagulation therapy [37,38].

Figure 2. Currently clinically used FXa Inhibitors and the Food and Drug Administration (FDA) year

of approval.

Novel FXa inhibitors under development are mainly structural bioisosteres of existing drugs

(Figure 3 ). They show better eﬃcacy and have a higher safety proﬁle. Several amidines, peptide,

pyrrolidines, azetidines, and triazoles are among the newly reported compounds [39,40].xd

Figure 3. (a) Apixaban and (b) Rivaroxaban as gold standard reference compounds. The S₄binding

scaﬀolds phenyloxopiperidine or phenyloxomorphonilo are highlighted in blue.

Within the approved FXa inhibitors, the phenyloxomorphonilo or phenyloxopiperidine scaﬀolds

present in Apixaban and Rivaroxaban are important anchoring moieties because along with the S₁site,

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S₄is the most important binding region for factor Xa inhibitors [39]. These groups have also shown to

be pivotal for potency in that these two drugs were the ﬁrst approved FXa inhibitors [33,34].

Consequently, FXa inhibitors are the critical milestones to be achieved in the clinical management

of thrombosis. In recent years, various pharmacological activities of N-heterocyclic compounds

including tetrahydroquinoline (Figure 4d) have received immense importance because of their various

pharmacological activities. Heterocyclic compounds containing nitrogen not only play a crucial role in

system biology but are the scaﬀold essential for the development of new drugs. Tetrahydroquinolines

(THQs) are found to be the basic skeleton of numerous natural and synthetic compounds with various

biological properties. For example, alkyl- and arylalkylderivatives of THQ such as (

)-cuspareine, ( )-galipeine, and ( )-galipinine, which were extracted from the angostura (Galipea

oﬃcinalis, Angostura trifoliata), tree have a variety of medicinal properties [41 43]. Diﬀerent

−

)-angustureine,

(−

−

–

1,2,3,4-tetrahydroquinolines with long alkyl chains at C-5 (Figure 4a) were extracted from a combined

culture of Streptomyces nigrescens HEK616 and Tsukamurella pulmonis TP-B0596. These compounds

were found to inhibit the growth of microorganisms [44]. Several 4-dioxygenated 3,4-dihydro

4-aryl-1,2,3,4-tetrahydroquinolin-2-(1H)-ones have been extracted and isolated from a variety of

plants and fungi with a variety of medicinal properties [45]. For example, aﬂaquinolones A–G

(Figure 4b), scopuquinolone B, aniduquinolones A–C, 6-deoxyaﬂaquinolone F, isoaﬂaquinolone E,

14-hydroxyaﬂaquinolone E, aspoquinolones A–D were also isolated from the various fungus belonging

to the specie Aspergillus. These compounds have shown to have useful medicinal properties [46–48].

Moreover, some 1,4-disubstituted 1,2,3,4-tetrahydroquinoline derivatives (Figure 4c) were tested

against HIV and few N-substituted tetrahydroquinoline derivatives were found to inhibit the reverse

transcriptase enzyme of HIV-1 virus [49–52]. Previously, Quan et al. reported the synthesis of

tetrahydroquinoline derivatives compounds having reversible factor XIa inhibitor activity in rats and

rabbit thrombosis animal modes, whereas tetrahydroquinoline scaﬀold (Figure 4d) was assayed for

its direct Factor Xa inhibition activity [52,53]. Similarly, 1,4-disubstituted-1,2,3-triazole derivatives

(Figure 4e) have become an important pharmacophore due to its various biological properties

and excellent stability. More recently, triazole has also been studied for its anticoagulant activity.

For example, the hydrazone and sulfonamide derivatives of triazole were studied for their antiplatelet

activity [54,55].

Figure 4.

(

a) 5-heptyl-1,2,3,4-tetrahydroquinoline [44]. (b) Aﬂaquinolone A [46]. (c) 1,4-disubstituted

1,2,3,4-tetrahydroquinoline [50] (

triazole chemical structure.

d

) Tetrahydroquinoline chemical structure. ( ) 1,4-disubstituted-1,2,3-

e

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The synthesis of current antithrombotic agents is highly ineﬃcient given that it has high costs

and involves several synthetic steps, and speciﬁc catalysts. Considering the multiple-step synthesis

development of commercially available FXa inhibitors [56,57], it makes the synthesis of these kind of

inhibitors a challenge for the medicinal chemistry ﬁeld.

Since the ﬁrst reported microwave utilization techniques in organic synthesis in 1986, microwave

techniques have become a recognized laboratory system and they have an important synthetic approach

in the ﬁeld of chemistry [58]. Moreover, these methodologies are therefore being used as an important

synthetic tool [59–63]. For these reasons, our group has been interested in using microwave approaches

to achieve our synthetic goals [64–67] in the last few years.

To this end, we have reported a modiﬁed Ullmann type procedure for related main core structures

using microwave-assisted methodologies [67]. Currently, the utilization of microwave-assisted

methodologies allows for the desired organic molecules in only three steps to be reached which

involves Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar

cycloaddition type reactions.

Hence, in our continuous search for decisive FXa inhibitors, several approaches in designing

synthesis and characterization novel FXa inhibitors [68] have been undertaken.

The present work includes the synthesis of novel tetrahydroquinoline and triazole derivatives by

using microwave-assisted synthetic methodologies along with modern computer-aided drug designing

and docking studies. Finally, in vitro and ex vivo assays have been performed in order to analyze the

bioactivity of the molecules FXa.

2. Results and Discussion

2.1. Protein-Ligand Complexes Selection and Ligand Clustering

First, the Protein Data Bank (PDB) (ww w .rcsb.org) [69] was searched for human FXa-ligand

complexes available crystal structures, using the appropriate Uniprot accession number (P00742). A set

of 130 enzyme-ligand reported complexes were found for humans, and after substructure search using

the phenyloxomorphonilo or phenyloxopiperidine scaﬀolds present in Apixaban and Rivaroxaban,

a set of 5 FXa-ligand complexes was compiled and their structures retrieved from the PDB (Figure 5).

Figure 5. Virtual screening of interest fragment in FXa PDB crystallography data base.

After structural alignment against the protein corresponding to the cluster center (Root-mean-

square deviation connected component, RMSD CC), the root-mean square deviation for the α-carbon

for all residues and residues within 6 Å from the center of mass of all co-crystallized ligands was

measured (Table 1).

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Table 1. Summary of crystal structures of Factor Xa-ligand complexes used in this study.

1

2

PDB ID

(Reference)

RMSD Cluster

Center (Å)

RMSD Binding

Site (6Å)

Tanimoto

Distance

Resolution (Å)

4BTI [70]

4BTU [70]

4BTT [70]

2.30

2.37

2.59

2.08

2.30

—–

0.222

0.381

0.207

0.381

2

0.000

0.426

0.582

0.618

0.689

0.440

0.530

0.276

0.290

2W26 Rivaroxaban [34

2P16 Apixaban [71]

1

]

All atoms within 6 Å from the center of mass of ligand alignment. Tanimoto coeﬃcient.

The alpha-carbon root-mean square deviation (CαRMSD) against the low-resolution FXa-ligand

structure (4BTI) ranged from 0.276 to 0.530 Å, while the all-atom RMSD within 6 Å from the center of

mass of all aligned ligands ranged from 0.207 to 0.381 Å, indicating that proteins have no signiﬁcant

conformational diﬀerences within their binding sites in comparison with the reference structure.

The three-dimensional (3D) alignment of the ligands was used to generate a shape-based query,

which was validated using a library of decoy [72,73]. The enrichment curve plots the number of

active compounds recovered versus the proportion of the database screened. The AUC (area under

the curve of the Receiver Operating Characteristic (ROC) plot) is deﬁned as the probability that a

randomly-chosen active compound has a higher score than a randomly-chosen inactive compound.

The AUC of the probability obtained for the hypotheses is higher than 99% at 95% conﬁdence

±

(Figure 6), suggesting the shape query hypothesis can be considered highly selective when using

the actives that correspond to each cluster. However, when using a more diverse collection of FXa

inhibitors from the extended database of useful decoys (DUD) [74], the AUC probability falls to near

77%. Although most actives rank higher than most of the decoy molecules, the query is considered

mildly selective and only 5% of the top scoring solutions were retrieved.

Figure 6. Shape-based query for phenyloxomorphonilo or phenyloxopiperidine containing FXa

inhibitors.

(

a) Three-dimensional (3D) structural alignment of ligands after protein complexes

superposition. (

b

) Shape-based query derived with color representing the combined shape with

chemical features represented as spheres (hydrophobic = brown; rings = green; HB acceptor = red; HB

donor = blue). (c) ROC area under the curve. HB: hydrogen bonding

After the generation of the shape-based query, the virtual screening protocol was applied to an in

house developed library. The shape similarity between the screened compounds was evaluated by the

Tanimoto Combo score method, which consists of the Tanimoto coeﬃcient and the score retrieved from

the ROCS color force ﬁeld, which represents the structural complementarity between the template and

the screened molecules. A ﬁnal set of 28 molecules was ﬁnally prioritized and selected for synthesis

(Scheme 1).

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Scheme 1. General synthetic pathway for selected compounds. (

a

) N,N⁰-dimethylethylenediamine

) propargylbromide, K₂CO₃,

(DMEDA), CuI, K₃PO₄_◦, PhMe, microwave irradiation (MW), 160 ^◦C. (

◦

KI, CH₃CN, MW, 160 C. (c) formaldehyde, 1-vinyl-2-pyrrolidinone, InCl₃, CH₃CN, MW, 160 C.

b

(d e) (11–14), (24–26), copper nanoparticles supported on ZnO

) HCl, NaNO₂, NaNO₃, H₂O, 40 ^◦C. (

(CuNPs/ZnO), Et₃N, tetrahydrofuran (THF), 160 ^◦C.

2.2. Synthesis of Novel Derivatives

The synthetic strategy applied for preparation of selected tetrahydroquinolines and 1H-1,2,3-triazole

molecules was performed by using Ullmann-Goldberg, N-propargylation, Mannich addition,

Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions (Scheme 1).

2.2.1. Synthetic Methodology Improvements for Aniline Precursors

Through the application of a methodology study, the performance of the Ullmann-type reaction

has been improved. In previous research of our group, where the ﬂuorine atom was not included, we

observed a predictable behavior in the reaction and obtained a moderate yield, without the need to

exceed the solvent boiling point [75]. In this case, applying our knowledge of the Arrhenius equation, in

which the energy transmitted by microwaves aﬀects the parameters of pressure, heating and especially

time. The reaction time in all C-N couplings could be reduced for the synthesis of anilines when we

increase the temperature to 160 ^◦C [76–78].

In a theoretical study of C-arylations with aryl halides [79], the authors have explained the reaction

reactivity between the corresponding lactams and 4-iodoaniline compound [67]. According to this, the

reaction conditions were optimized by using lactam

1 (1.2 equivalents), aniline 5 (1 equivalent), CuI as

catalyst and N,N⁰-dimethylethylenediamine (DMEDA) as ligand, in toluene as solvent using diﬀerent

heating sources (Table 2). The corresponding isolated compound

(Scheme 2).

6 was obtained in good yield [80]

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1,2.

Table 2. Optimization of reaction conditions for the synthesis of compound 6.

DMEDA

(equiv.)

Heating

Source

Entry

CuI (equiv.)

T (^◦C)

Time (h)

Yield (%)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19³

0.05

0.1

0.05

0.1

0.05

0.1

0.05

0.1

0.15

0.25

0.5

1.00

2.00

0.1

0.2

0.5

0.1

0.2

0.5

0.1

0.2

0.5

0.1

0.2

0.3

0.5

1.0

2.0

20

96

72

48

6

2

1.5

r.t.

-

1

-

1

4

8

-

5

20

6

8

11

17

19

26

46

50

62

100

120

60

conventional

sonication

60

sonication

120

160

microwave

1

3

Conditions: lactam (1.2 equiv.), aniline (1 equiv.), K₃PO₄(2 equiv.), toluene (3.5 mL). ²Yield of isolated compound.

Reaction carried out using 1 equiv. of lactam and 1.2 equiv. of aniline. r.t.: room temperature.

Scheme 2. Synthesis of aniline derivative 6 [80].

When one of the proposed mechanisms for Ullmann-Goldberg type reaction was analyzed, it was

noticed that both the catalyst and the ligand bind directly to the lactam in the intermediate activation

step [81]. This premise allowed us to change the reaction stoichiometry, changing the limiting reagent to

lactam 1, obtaining satisfactorily 62% yield of isolated compound under microwave heating conditions.

This modiﬁcation resulted in a 12% higher yield of the starting materials than previously reported

yields (Table 1, entry 19).

Although for piperazinone, morpholinone and thiomorpholinone derivatives, the synthesis was

carried out with a lower catalyst loading (0.5 equiv.) and ligand (0.5 equiv.) (Scheme 3).

Scheme 3. Synthesis of Anilines Derivatives (7–9) [80].

The aniline derivatives

of isolated products under microwave reaction conditions in 1–2 h. However, the coupling reaction

for compound

was carried out at 90 ^◦C, due to the deprotection side reaction. This methodological

7, 8, and 9 were obtained in excellent yield (90%, 86%, and 94%, respectively)

7

improvement allowed us to produce these interesting block buildings anilines in excellent yields,

reducing reaction time and minimizing the energy consumption by using microwave technologies.

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2.2.2. Synthesis of N-propargylanilines

In the next step, N-propargyl anilines were synthesized according to the procedure developed

by Rasool et al. with minor modiﬁcations such as temperature, time reaction and heating source

(Scheme 4) [82]. The reaction conditions were optimized using diﬀerent heating sources (Table 3).

The best results were achieved using the aniline derivatives (

6–9), propargyl bromide, K₂CO₃as base,

KI as additive, in acetonitrile as a solvent under microwave irradiation at 160 ^◦C. N-propargyl aniline

derivatives (11–14) were obtained in good yields of isolated products (62–72%). However, the synthesis

of compound 13 had to be carried out at 90 ^◦C, due to the deprotection side reaction [83–86].

Scheme 4. Synthesis of N-allyl/propargyl aniline derivatives (11–14) [80].

Table 3. Optimization of reaction conditions for the synthesis of compounds 11–14.^1,2.

Yield (%)

T (^◦C)

Time (h)

Entry

Heating Source

11

12

13

14

1

2

3

4

5

6

7

r.t.

20

80

90

40

96

24

72

24

15

4

13

31

36

37

43

55

69

19

40

47

49

57

67

72

10

29

34

37

45

51

70

9

conventional

sonication

microwave

15

30

34

39

43

67

80

160

0.5

1

Conditions: aniline derivatives (1.2 equiv.), propargyl bromide (1 equiv.), K₂CO₃(1.5 equiv.), KI (0.1 equiv.), in

acetonitrile (3.5 mL). ²Yield of isolated compounds. r.t.: room temperature

Noteworthy, the propargyl derivatives were obtained with the lower yields of isolated compounds

using room temperature (96 h, 9–19% yield), sonication (40–80 ^◦C, 4–15 h, 39–67%), and conventional

heating sources (80–90 ^◦C, 24–72 h, 15–49%). In contrast, the microwave procedure showed the highest

products yields (67–72%) within 30 min of reaction at 160 ^◦C. As a second reaction step, the microwave

methodology allowed us to produce the propargyl derivatives in an eﬃcient and environmentally

friendly approach.

2.2.3. Synthesis of N-Propargyl Tetrahydroquinolines

Diﬀerent N-propargyl tetrahydroquinolines (17–20) were synthesized using acid-catalyzed

three-component cationic imino Diels–Alder reaction [87–89] (Scheme 5).

.

Scheme 5. Synthesis of N-propargyl tetrahydroquinoline derivatives (17–20).

Molecules 2020, 25, 491

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The reaction conditions optimization involved the modiﬁcation of the heating source [90,91].

Thus, the total reaction time was reduced from 96 h to 15 min (Table 4) through microwave-assisted

methodology and compound yields were 2.5-fold increase in comparison with conventional procedures.

It must be considered that compound 18 was not obtained, only decomposition products were

obtained [87]. Novel tetrahydroquinolines derivatives 17, 19, and 20 were obtained in excellent yields

(83–89%) of isolated products.

Table 4. Optimization of reaction conditions for the synthesis of compounds 17–20.^1,2.

Yield (%)

T (^◦C)

Time (h)

Entry

Heating Source

17

18

19

20

1

2

3

4

5

6

7

r.t.

20

80

90

40

96

24

72

24

8

32

47

53

59

69

72

85

-

34

45

54

56

64

72

89

36

52

59

62

67

73

83

conventional

sonication

microwave

80

160

2

0.5

1

Conditions: N-propargyl aniline derivatives (1 equiv.), formaldehyde 30% in methanol (1.1 equiv.), InCl₃(0.2

equiv.), KI (0.1 equiv.), in acetonitrile (3 mL). ²Yield of isolated compounds.

2.2.4. Synthesis of Aryl Azides

The synthesis of the aryl azides were based on the reaction of diazonium salts with sodium azide

in water under sonication at 40 ^◦C (Scheme 6 ) [92]. The corresponding products were obtained in

quantitative yields and they were used without further puriﬁcation.

Scheme 6. Synthesis of aryl azides derivatives (24–26).

2.2.5. Synthesis of 1H-1,2,3-triazole derivatives

The synthesis of novel triazoles were prepared by the 1,3-dipolar cycloaddition between the

N-propargyl amines (11–14) and the organic azide (24–26) catalyzed by copper nanoparticles supported

on ZnO in tetrahydrofuran as solvent, triethylamine as base at 160 ^◦C under microwave irradiation

(Scheme 7) [93].

Scheme 7. Synthesis of 1H-1,2,3-triazole derivatives (27

respectively); 30 32 (X = N-Boc; R = H, Cl, F, respectively); 33

36 38 (X = O; R = H, Cl, F, respectively). Novel 1H-1,2,3-triazole derivatives (27

–

38). Compounds 27

35 (X = S; R = H, Cl, F, respectively);

38) were obtained in

–29 (X = CH₂; R = H, Cl, F,

–

good to excellent yield (73–93%) using a lower loading of catalyst and in shorter reaction time (15 min)

than previously reported by assisted microwave methodologies.

Molecules 2020, 25, 491

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2.3. Theoretical Study

In order to understand the relationship between the structure and the energetic behavior of all the

series, including a thermochemical analysis associated to the yield of the reactions, an optimization

and reactivity indexes calculation of electronic chemical potential (µ), chemical hardness (η), and

electrophilicity (ω) was performed.

The electronic chemical potential (µ) has the same signiﬁcance as the classic thermodynamics

chemical potential but characterizes the tendency to escape of electrons from the equilibrium. This index

its associated to the ionization potential and the electron aﬃnity, and to the energies of Frontier Molecular

Orbitals (FMOs) based on the Koopman’s theorem.

The chemical hardness (η) is associated to the stability of the systems and the resistance of a

molecule to exchange electron density. The same approximations as for electronic chemical potential

were made.

For electrophilicity (ω), a higher index value indicates a more electrophilic character for the

molecule, as a lower index value, a more nucleophilic character has the molecule. Electrophilicity can

also be associated with the energy stabilization of a molecule [79,94].

2.3.1. X-Ray and Theoretical Optimization Correlation

Structural parameters of molecules

data (Tables 5 –7 ). The structures are well refined, with final R indices (I > 2

for , R₁= 0.0343, wR₂= 0.0944 for and R₁= 0.0518, wR₂= 0.1552 for 20. The crystallographic data and

6,

9, and 20 were optimized and compared with crystallographic

σ(I)) of R₁= 0.0461, wR₂= 0.1292

6

9

structure refinement can be found in the Supplementary Material (Tables S27 to S29).

Table 5. Selected bond lengths and angles for molecule 6.

Bond Length

CALC EXP

Crystal Structure ¹

F(1)-C(10)

O(1)-C(1)

N(1)-C(1)

N(1)-C(6)

N(1)-C(5)

N(2)-C(9)

C(2)-C(3)

C(3)-C(4)

1.366

1.220

1.382

1.435

1.477

1.391

1.529

1.301

1.339

1.232

1.354

1.438

1.480

1.382

1.540

1.497

Bond Angles

CALC EXP

109.2 109.4

115.9 115.24

116.3 115.67

118.8 119.09

119.2 119.26

C(4)-C(3)-C(2)

C(3)-C(2)-C(1)

C(5)-N(1)-C(6)

C(1)-N(1)-C(6)

C(11)-C(10)-F(1)

C(11)-C(6)-N(1)-C(1) 112.7 116.25

C(4)-C(5)-C(1)-C(2) 14.8 9.1

ORTEP [95] drawing with thermal ellipsoids drawn at the 50% probability level. ORTEP: Oak Ridge Thermal

1

Ellipsoid Plot.

For the all studied molecules, bond angles and distances show a good correlation between

calculated and experimental, suggesting good robustness for the calculated structure and parameters,

with no signiﬁcative diﬀerence between bond lengths.

Regarding bond angles for molecule

6 (Table 5), the most signiﬁcant diﬀerence occurs in the

dihedral angles, with a diﬀerence between 3 to 5 degrees, showing that the molecule has a slightly

diﬀerent conformations around the central C(6)-N(1) atoms, and a slightly more complex twist in the

non-aromatic ring.

Molecules 2020, 25, 491

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Table 6. Selected bond lengths and angles for molecule 9.

Bond Length

CALC

EXP

Crystal Structure ¹

F(1)-C(7)

O(2)-C(2)

N(1)-C(2)

N(1)-C(5)

N(1)-C(3)

N(2)-C(8)

C(1)-O(1)

O(1)-C(4)

1.365

1.219

1.377

1.433

1.474

1.390

1.418

1.417

1.364

1.231

1.352

1.436

1.479

1.383

1.418

1.430

Bond Angles

CALC

EXP

C(4)-O(1)-C(1)

O(1)-C(1)-C(2)

C(3)-N(1)-C(5)

C(2)-N(1)-C(5)

C(6)-C(7)-F(1)

C(6)-C(5)-N(1)-C(2)

C(4)-C(3)-C(2)-C(1)

C(10)-C(5)-N(1)-C(2)

1

110.6

116.7

117.6

120.8

119.0

50.0

109.4

115.6

117.2

120.7

118.3

50.2

18.7

50.2

16.0

131.7

ORTEP [95] drawing with thermal ellipsoids drawn at the 50% probability level.

Table 7. Selected bond lengths and angles for molecule 20.

Bond Length

CALC

EXP

Crystal Structure ¹

F(1)-C(4)

O(1)-C(10)

N(1)-C(2)

N(1)-C(10)

N(1)-C(13)

N(2)-C(5)

N(2)-C(6)

N(3)-C(8)

N(3)-C(18)

N(3)-C(15)

O(3)-C(15)

O(2)-C(12)

O(2)-C(11)

C(8)-N(3)

1.365

1.219

1.432

1.378

1.474

1.393

1.465

1.474

1.464

1.372

1.219

1.417

1.418

1.474

1.363

1.222

1.436

1.368

1.475

1.411

1.474

1.457

1.471

1.356

1.231

1.426

1.421

1.457

Bond Angles

CALC

EXP

C(12)-O(2)-C(11)

O(2)-C(11)-C(10)

C(13)-N(1)-C(2)

C(10)-N(1)-C(2)

C(3)-C(4)-F(1)

110.7

116.5

117.6

120.5

116.5

122.1

126.8

18.2

108.3

115.1

117.4

120.1

115.8

117.8

137.7

13.1

C(5)-N(2)-C(19)

C(3)-C(2)-N(1)-C(10)

C(12)-C(13)-C(10)-C(11)

C(5)-N(2)-C(19)-C(20)

C(9)-C(8)-N(3)-C(15)

C(21)-N(2)-C(8)-N(3)

1

128.5

153.8

108.7

55.3

111.2

158.0

ORTEP [95] drawing with thermal ellipsoids drawn at a 50% probability level.

For molecule 9, in addition to the good length correlation, between theoretical and experimental,

there was a favorable correspondence between bond angles (Table 6). The main diﬀerence takes place

in the O(1) oxygen atom position, the calculations showed that this atom is facing the same plane as the

Molecules 2020, 25, 491

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ﬂuorine F(1) atom. On the other hand, the crystal shows that the atoms are opposite from each other.

The C(10)-C(5)-N(1)-C(2) dihedral angle was measured in order to visualize the diﬀerence between

experimental and calculated structures (Figure S30, Supplementary Material). This behavior could be

explained due to the hydrogen bonds interactions generated between the F(1) and O(2) atoms with the

opposite hydrogen atoms in the molecule (Figure S31, Supplementary Material).

Bond angles for molecule 20 also shows that the most signiﬁcant diﬀerence between calculated

and experimental occurred in the dihedral angles with a slight diﬀerence of 5 degrees in the twist

structure on the non-aromatic ring but a large diﬀerence between the propargyl moiety position and

the tetrahydroquinoline cycle. The last three angles shown (Table 7) were measured to demonstrate

the diﬀerent positioning of the tetrahydroquinoline cycle and the propargyl moiety. This could be due

to the interactions generated between the propargyl and the carbonyl groups in the crystal (Figure S32,

Supplementary Material).

2.3.2. Energetic Behavior and Thermochemistry

The electronic chemical potential for propargyl precursors 11

with a maximum of 2.889 eV for molecule 11 and a minimum of

arylazide compounds (24 26), the mean is 4.4049 eV, with a maximum of

and a minimum of 4.490 eV for molecule 26. This implies that the reaction between precursors 11

and 24 26 present a medium polar character, and the electron density ﬂux will take place from the

–

−

14 shows a mean of

3.169 for molecule 13. For the

4.277 eV for molecule 24

14

−

3.0471 eV,

−

–

−

–

propargylated molecule to the arylazide [94].

The Density Functional Theory (DFT) study for 1H-1,2,3-triazole derivatives showed that the

highest chemical hardness is obtained in the molecules synthetized from precursor 24, thus being the

more stable ones (average 2.013 eV). It is worth to notice that these derivatives (27

from precursor 24) had the highest yield of all synthesized compounds (91.11 ± 1.55%).

For the electrophilicity index, propargyl molecules 11 14 showed mean of 1.8301 eV, with a

maximum of 1.908 eV for molecule 14 and a minimum of 1.667 for molecule 11. For arylazide

compounds 24 26, the mean is 3.4799 eV, with a maximum of 3.657 eV for molecule 27 and a minimum

, 30, 33, and 36, all

–

of 3.234 eV for molecule 25. According to these values, propargyl precursors acts as nucleophiles and

the arylazide precursors acts as electrophiles. This approach may not be entirely correct because the

arylazide is a dipolar compound and the propargyl is a dipolarophile center. The reaction should go

through a Huisgen cycloaddition, following a concerted mechanism with a nucleophilic attack to and

from the propargylated molecule [96]. All energies for reactivity indexes and FMOs are summarized in

Table 8.

In order to understand the electrophilic behavior of arylpropargylated and arylazide precursors,

the Fukui functions for molecules 13 and 24 were calculated. These molecules were chosen as an

example due therefrom compound 33 was obtained. This reaction presented the best performance

according to the products yield.

Fukui functions helped us to understand the behavior of chemical reactions providing information

on the local reactivity of atoms within the molecule, as shown in the Supplementary Material (Table S33

and S34). According to the f ⁺function, N(9) nitrogen atom (molecule 24) was the most susceptible

atom to experience a nucleophilic attack. However, the most susceptible atom for compound 13 was

0

the C(18) carbon. For neutral or radical reagents or reactions, the f function can be used. In this

case, with a concerted mechanism, the function shows the same behavior previously discussed [97].

The calculations predicted that the mesomeric structure that reacts is the one with a triple bond between

the nitrogen atoms (Figure S35, Supplementary Material).

DFT calculations have been previously performed on similar systems, showing that the mechanism

for the copper (I) catalyzed cycloaddition are in concordance with experimental and theoretical ﬁndings

reported in this work. Moreover, previously published calculations also agrees with the mesomeric

structure that actually reacts, showing that the reaction with and without catalyst should undergo the

same mechanism, with the same sites being susceptible to nucleophilic attack [98].

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Table 8. Calculated energies (eV) for FMOs and reactivity indexes, for precursors and ﬁnal compounds.

Compound

HOMO

LUMO

GAP

HARD. (η)

C. POT. (µ)

ELEC. (ω)

11

12

13

14

27

30

33

36

28

31

34

37

29

32

35

38

24

25

26

−5.3917

−5.5550

−5.8366

−5.5593

−5.4418

−5.5892

−5.8339

−5.5982

−5.5354

−5.6674

−5.9278

−5.6973

−5.5147

−5.6524

−5.9082

−5.6750

−7.1049

−7.2350

–7.2467

−0.3859

−0.5519

−0.5010

−0.5954

−1.5467

−1.5913

−1.6205

−1.6003

−1.8583

−1.8931

−1.9249

−1.9111

−1.7138

−1.7557

−1.7843

−1.7682

−1.4487

−1.6602

–1.7339

5.0058

5.0031

5.3357

4.9639

3.8951

3.9979

4.2134

3.9979

3.6771

3.7742

4.0028

3.7862

3.8009

3.8967

4.1239

3.9068

5.6562

5.5748

5.5128

2.5029

2.5016

2.6678

2.4820

1.9475

1.9989

2.1067

1.9990

1.8386

1.8871

2.0014

1.8931

1.9005

1.9484

2.0620

1.9534

2.8281

2.7874

2.7564

−2.8888

−3.0534

−3.1688

−3.0774

−3.4942

−3.5903

−3.7272

−3.5993

−3.6968

−3.7802

−3.9264

−3.8042

−3.6142

−3.7040

−3.8462

−3.7216

−4.2768

−4.4476

–4.4903

1.6671

1.8635

1.8819

1.9078

3.1347

3.2242

3.2970

3.2404

3.7167

3.7862

3.8514

3.8222

3.4367

3.5209

3.5872

3.5452

3.2339

3.5483

3.6575

The thermochemistry of the series was studied taking into consideration the sum of electronic

and thermal enthalpies for both precursors and ﬁnal compounds, in order to calculate the H for each

reaction (Table S36, Supplementary Material). All reactions have shown a highly exothermic behavior

with a reaction enthalpy value of between 61.765 0.295 kcal/mol. These energies were in agreement

∆

−

±

with previously calculated similar compounds [98]. It is important to note the most exothermic reaction

is the one to obtain 33 (from 13 and 24) in accordance to its highest yield of reaction.

2.4. Biological Analysis

2.4.1. FXa Inhibition in Vitro Assay

By using SensoLyte^®520 Factor Xa Assay Kit “Fluorimetric” FXa (bovine isolated enzyme)

inhibition was measured. Upon FXa protease cleavage, this substrate generates the Rh110 (rhodamine

110) as free ﬂuorophore, which can be detected at excitation/emission of 490 nm/520 nm, where

the following values of inhibition FXa percentage and IC₅₀from the representative compounds of

each series (compounds

were obtained.

6–38, Table 9), as well as for all ﬁnal 1H-1,2,3-triazole derivatives [68,99]

The assay showed that only ﬁve of the novel compounds (27, 30, 31, and 34) exhibited inhibitory

activity higher than 50% against FXa at 100 M. According to these results, IC₅₀values were calculated

µ

using corresponding dilutions of each sample (Materials and Methods). It is important to note that

none of the evaluated compounds showed ﬂuorescence quench at the assayed wavelength avoiding

the possibility to present a potential pan-assay interference compounds (PAINS) fragment [100–102].

Molecules 2020, 25, 491

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Table 9. FXa inhibition and IC₅₀values of novel compounds 6–38.

Compound^a

IC₅₀± SD [µM]^c

Inhibition FXa (%)^b

6

19

18

20

15

17

14

25

24

25

27

15

52

33

46

63

73

21

41

60

28

34

39

b

-

7

8

-

10

11

12

13

14

17

19

20

27

28

29

30

31

32

33

34

35

36

37

38

-

102.10 ± 0.14

-

41.71 ± 0.07

29.73 ± 0.09

-

67.92 ± 0.08

-

It was not possible to synthesize Compound 18. Inhibition percentage at 100 µ

M. ^cApixaban 97% inhibition,

a

IC₅₀= 2.8 nM. Rivaroxaban 94% inhibition, IC₅₀= 0.70 nM.

2.4.2. Anti FXa assay, Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) ex

Vivo Assay

Ex vivo anticoagulation eﬃcacy assays of compounds 27, 30, 31, 34, Apixaban, and Rivaroxaban

(control samples) were evaluated by anti FXa (Table 10), activated partial thromboplastin time (aPTT)

and prothrombin time (PT) assays by using a human plasma pool.

Table 10. Anti FXa assay values of selected compound.

Compound

Anti-FXa [IU/mL] ^a

27

30

31

34

0.29 ± 0.10

0.00

0.05 ± 0.09

0.42 ± 0.18

a

IU/mL: International Units per milliliter. Concentration 1 mM. Apixaban (500 nM, 1.50

±

0.36 IU/mL) and

Rivaroxaban (300 nM, 1.20 ± 0.23 IU/mL), respectively.

We know that chromogenic FX assay measures the amount of FX activity in a patient, however,

chromogenic anti-FXa activity assay does not measure the patient’s FX. What it does instead is that

it measures the ability of the patient’s plasma to repress exogenous FXa. The chromogenic anti-FXa

assay is therefore used to determine the concentration of anticoagulants that inhibit FXa.

What will need to be taken into consideration in the future is how anti-FXa monitoring will need

to be increased. As more anticoagulants that are FX inhibitors are more freely available in the market,

therapeutic compliance, together with diagnosing the causes of bleeding in a patient being treated with

these anticoagulants, will need to be done much more often [103]. Widespread use of FXa inhibitors

may require performing the ant-FXa assay as a complementary assay. If FXa inhibitors are more

commonly used, this may mean that the anti-FXa assay as complementary assay will need to be done.

Molecules 2020, 25, 491

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As an example, high anti-FXa levels demonstrated the increasing risk of bleeding and low levels

of anti-FXa established potential thromboembolic future events.

According to the therapeutic range of the anti-FXa obtained values (0.3–0.7 IU/mL), selected

compounds 27 and 34 were assay for PT and aPTT coagulation parameters (Figure 7).

Figure 7. (a) PT and (b) aPTT coagulation parameters for compounds 27 and 34.

PT parameter evaluated the extrinsic and common coagulation pathway. On the contrary,

aPTT parameter evaluated the intrinsic pathway and its less sensitive to the ﬁnal common pathway.

Compound 27 simultaneously prolonged aPTT and PT in a dose-dependent manner for concentrations

up to 50

µM (Figure 6a,b). Meanwhile, compound 34 only prolonged PT in a dose-dependent manner

(Figure 6a) for concentrations up to 50

µM. Apixaban and Rivaroxaban prolonged both coagulation

parameters in a dose-dependent manner for concentrations up to 0.1 nM [104,105].

These interesting results suggest that compounds 27 inhibited FXa targeting this coagulation

factor through both pathways in order to increase the possible potency of their derivatives. Finally,

compound 34 may target coagulation FXa mainly by the extrinsic and common pathway.

2.4.3. Cell Viability Assays

Cell viability assay was performed in HEK293 and HepG2 cells using the 3-(4,5-dimethylthiazol-2-yl)

-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method (Figure 8 ). The compounds

were assayed under increasing concentrations, namely, 1 nM (a), 10 nM (b), 50 nM (c), 100 nM (d), 10 mM

(e), and 100 mM (f). In HEK293 cells, most of the assayed compounds only shows signs of toxicity (defined

as the value obtained after subtracting two standard deviations from the average value of control wells

treated only with vehicle) when concentrations are higher than 10 mM, such as compounds 30 and 31

.

Compound 31 showed a greater toxic effect in HepG2, reducing cell viability to 40% and 20% of control at

10mM and 100mM, respectively, while the rest of the compounds did not affect HepG2 cell viability.

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Figure 8. Eﬀect of selected compounds on cell viability using the MTS assay in (

HepG2 cell lines. Concentrations used were 100 mM (a), 10 mM (b), 100 nM (c), 50 nM (d), 10 nM (e),

and 1 nM (f). Blue and red lines deﬁnes 2 SD limits from the average value of control wells treated

a) HEK293 and (b)

±

only with vehicle (green line). All values are means ± S.D (n = 6) vs. control. Api: Apixaban and Riv:

Rivaroxaban. SD: standard deviation.

Interestingly, the most active compounds in relation to the inhibition activity against FXa

and coagulation parameters 27 and 34, did not show toxicity at the performed coagulation assays

concentrations (1–500 µM).

2.5. Computational Analysis

2.5.1. Docking Studies

Molecular docking and MM-GBSA studies (Molecular Mechanics-Generalized Born Surface Area)

were performed to get insight into their binding modes and aﬃnity of the most active compounds of

each series (8, 19, 27, 30, 31, and 34) against factor Xa enzyme (FXa), In this study, the Glide module of

Schrödinger suite was used to ﬁnd the suitable orientation of the most active compounds in the active

site FXa. Comparison of the docking poses of compounds of each series and apixaban showed some

similar interactions.

An overlay of the Apixaban (green) and N-propargyl- tetrahydroquinoline (THQ) (compound 19

on the active site of FXa can be seen in Figure 8a. The Apixaban ﬁt in the FXa binding site, standing

between Y99 and F174 so that the phenyl lactam portion can be parallel to residue W215. Carbonyl

group of the 2-oxopiperidine ring in Apixaban presents HB interaction with F174 through the water

molecule. Docking analysis also revealed that the central pyridine ring of FXa inhibitor is located

strategically to form HB with G216 and Y99; this last interaction is water-mediated.

)

On the other hand, the electron-rich nature of the phenyl ring of the aromatic residues F174, Y99,

and W215 surrounded the ring 6-membered of the molecules

stacking interaction with residue Y99. (Figure 9a,b). This orientation is similar to the benzene ring of

apixaban, which forms the well-deﬁned stacking interaction with the phenyl ring. Similarly, the

oxygen of the lactam group of thiomorpholine moiety of compounds 19 and forms HB interaction

8 and 19, forming a well-deﬁned π-π

π-π

8

with a water molecule close to residue F174 at 3.00 Å (Figure 10a). The interaction with F174 is

comparable to presented by 2-oxopiperidine ring of apixaban. Additionally, near the bottom of the

FXa active site, the propargyl group of THQ 19 forms two HB interactions, one of them with residue

Molecules 2020, 25, 491

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G216 at 3.22 Å, and other with the residue Y99 water-mediated. This interaction is like the interaction

of the carbonyl group of the piperidine ring of apixaban (Figure 10b).

Figure 9.

(a) Overlay of Apixaban (green) and compound 19 over active site of FXa. (b) Aniline

derivative 8 on FXa.

Figure 10.

(a) Interaction diagram of Compound 19 in the FXa active site. (b) Apixaban interactions

with diﬀerent residues in the FXa active site.

In the complex FXa-compound 19, it was possible to observe one water-mediated halogen bond

(dotted line in magenta) between the ﬂuorine atom of THQ with the aromatic residue Y99 at a distance

of 3.02 Å [106,107]. Although ﬂuorine is a highly electronegative element, it could form a noncovalent

bond with another electron donor when ﬂuorine itself is linked with the suﬃciently electron attracting

group. In such an interaction, ﬂuorine acts as an electrophilic group. Such interactions are known as

the halogen bonding or ﬂuorine bond. The halogen (ﬂuorine) bonding has the property of stabilizing

inter- and intramolecular interactions that can preserve ligand interactions and can aﬀect molecular

folding [108]. In pharmacology research, several halogenated compounds as drugs have been included,

but this interaction is rarely considered necessary for the rational drug design process [109]. Thereby,

potential interactions as described above could explain the inhibitory potency of the compound 19

.

The structural diﬀerence of compound 19 with respect to the other THQ derivatives was the presence

of sulfur atom on the morpholine scaﬀolds. The sulfur atom on the thiomorpholine ring did not appear

to interact with any speciﬁc residue in this region of the enzyme, and it was oriented to the exposed

hydrophobic region solvent.

On closer examination of the docking binding modes, binding diﬀerences between N-propargyl-

THQ and apixaban inhibitors (Figure 10a) were better understood. Compound 19 lacked some

fundamental intermolecular interactions that were established by Apixaban, which could explain the

diﬀerence in activity. As an example, apixaban formed an H-bond with negative charged residue E146

through its amine terminal group, while compound 19 was incapable of forming such an interaction.

Moreover, the docking poses show that a methoxyphenyl ring of Apixaban spread through the active

Molecules 2020, 25, 491

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site cavity close to residues C191 and G216, while THQ was not large enough to reach the site of the

enzyme. Other interactions that compound 19 were missing was the HB interaction of the pyrazole

ring of the Apixaban with residue G216 at distance 2.9 Å [71]. Unlike that of Apixaban and compound

19, the aniline derivative

cover of the binding site, evidencing the low biological activity against FXa enzyme.

Unlike compounds and 19, four triazole derivatives (31, 30, 34 and 27) showed a marked

8 presented a small chemical structure that did not allow for it to build a good

8

biological activity against FXa enzyme. These compounds displayed a similar orientation to

Apixaban in the binding site and similar molecular interactions dominated by CH···O hydrogen

bonds (HB) and π-stacking interactions with residues Y99, G218, D189, and G216, as shown in Figure 11.

CH···O hydrogen bonds are well-recognized interactions in protein structures. These interactions are

energetically stabilizing and play a fundamental role in several biological processes such as in the

protein structure and their folding until enzyme catalysis [109]. Several studies have shown that carbon

atoms can form weak hydrogen bonds, which are denoted as CH···O hydrogen bonds. Due to the

increase of polarization by the adjacent atoms, carbon atoms can theoretically participate in hydrogen

bonds as strong as those formed by conventional donors, like oxygen or nitrogen, thus, this type of

interaction is relevant to the study of molecular docking [110,111].

Figure 11. Molecular docking of structures 31, 30, 34, and 27 ((a), (b), (c), and (d), respectively) into

the active site of FXa enzyme (PDB: 2HYY). The dotted lines show the most common ligand-protein

interactions in all ligand binding poses, HB in yellow, π-π stacking in orange, halogen bond in magenta

and CH···O HB in green.

At the molecular level, compound 31 presented the highest biological activity (29.7 µM), as it

occupied most of the active site of FXa exhibiting several molecular interactions. Speciﬁcally, the

triazole and chlorobenzene rings showed CH···O HB interactions with G218 and D189 residues with

distances of less than 3.71 Å. The ﬂuorobenzene central ring established CH···O HB interactions with

G218, and the Boc terminal moiety formed the same kind of interactions with the Y99 residue, with

distances of 2.47 and 3.90 Å, respectively. The second and fourth most active compounds, 30 and 27

in the series, presented similar molecular interactions to compound 31, highlighting the interactions

with G218 and G216 residues, mainly mediated by CH···O HB interactions. Furthermore, the aromatic

interaction with Y99 residue was maintained with a Boc moiety of compound 30. Compound 34, the

third in biological activity, was oriented entirely into the active site of the enzyme, presenting molecular

interactions not shown by the other compounds of the same series. The structural characteristics of

this molecule, such as the absence of the Boc protective group, allowed it to interact by well-deﬁned

Molecules 2020, 25, 491

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π-π staking between the ﬂuorobenzene ring and the Y99 residue. At the same time, the terminal

chlorobenzene ring was positioned in a way that allowed it to establish a halogen bond with A190 at

3.33 Å.

The predicted Glide Emodel values obtained from molecular docking for compounds 8, 19,

27, 30, 31, and 34 are listed in Table 6 . Emodel energy values agree with the values inhibitory

activities [112

than the triazole derivatives (

77.10 to 82.94 kcal/mol for triazole derivatives). The inspection of the Glide SP scoring terms

,

113]. Compound 19 bound more strongly to FXa than compound

8, but it was weaker

−

56.62 and 45.66 kcal/mol for 19 and , respectively, and a range of

−

8

−

revealed that the Van der Waals (VdW) interaction energy was the major contributor to the inhibitor

binding. The VdW contribution had higher values for triazole derivatives than for compounds 19 and

8. Glide Emodel and VdW contribution predicted the correct ranking of the compounds.

MM-GBSA calculations using the module Prime MM/GBSA [114,115] were also performed.

This was done to get an estimation of the energetic diﬀerences observed in the binding aﬃnity and to

understand better the thermodynamic factors involved in diﬀerential FXa biological activities of the

more active compounds. The MM/GBSA results showed that compound 19 and

of 53.15 and 49.49kcal/mol, respectively, while the calculated values for triazole derivatives were

in the range of 52.84 to 67.39 kcal/mol. The G_{bind VdW}term often dominates the net G_bind, and

8 have energy values

−

∆

its contribution in this study is relevant in that it is the highest value for compound 31, which was

the compound that showed the highest biological activity. This result indicated that VdW interaction

is the driving force for the inhibitor binding, and the Boc group interactions of compound 31 has

contributed to this term. Unfortunately, the ∆G_bindcalculations contained one vague approximation

that has aﬀected our results.

The approximation is related to the lack of conformational entropy and information about the

number and free energy of water molecules in the binding site [114]. The water molecules were key

in many of the electrostatic and H-bond interactions in enzyme-ligands complexes, and the lack of

inclusion of their contribution has aﬀected the ∆G_bindresults.

The molecular docking and MM/GBSA calculations are an interesting approach to try to explain

or predict the activity of the diﬀerent chemical derivatives in a FXa enzyme. Moreover, the diﬀerent

binding patterns of the best molecules proposed in this study can help us to better understand at a

molecular level the modes of interaction of the new series of synthesized compounds (Table 11).

Table 11. Predicted Glide SP score values obtained from molecular docking for selected compounds 8,

19, 27, 30, 31, and 34.

Glide Emodel

(kcal/mol)

Glide EVdW

(Kcal/mol)

∆G_{bind VdW}

(Kcal/mol)

Compound

∆G_bind(Kcal/mol)

8

−56.62

−45.66

−79.77

−77.10

−82.94

−82.89

−36.62

−27.25

−44.64

−49.62

−54.62

−51.19

−53.15

−49.49

−67.39

−52.84

−65.81

−64.28

−40.49

−26.66

−49.17

−46.57

−58.48

−53.24

19

27

30

31

34

Emodel is a speciﬁc combination of Docking Score, CVdW (CVdW = Coul + VdW is the non-bonded interaction

energy between the ligand and the receptor) and the internal torsional energy of the ligand conformer. G_{bind VdW}

is the VdW interaction energy between ligand and receptor calculated using Glide SP. G_bindand G_{bind VdW}were

∆

obtained through a MM-GBSA approach implemented in the Prime module of the Schrödinger suite. CVdW:

Coulomb-van der Waals interaction-energy score. MM-GBSA: Molecular Mechanics-Generalized Born Surface Area.

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2.5.2. Molecular Dynamics Simulations

The generated protein-ligand complexes from more active molecular docking compounds

(N-propargyl-THQ, 19 and 1,4-disubstituted-1,2,3-triazole derivative, 31) were subsequently submitted

to MD simulations to optimize the ligand position into the FXA binding site using Schrödinger

software suite [116]. In comparison with the initial docking position of compound 19, it showed

an accommodation slightly diﬀerent in the binding pocket throughout the MD simulation time.

In particular, the initial

of the simulation. Indeed, the aromatic ring of the THQ compound showed an accommodation parallel

to Y99 and W215 settling two stables stacking interactions with both residues. The interaction

π-π staking interaction with Y99 was maintained for a signiﬁcant part of the time

π-π

with W215 was the most frequent. The pyrrolidone ring kept in constant rotation, established an

interaction network of hydrogen bonds with residues E146 and G216. Similarly, the morpholine moiety

presented some unstable hydrophobic interactions with F174 as well as aromatic-HB interactions with

Y99. Despite the high degree of rotation of the THQ molecule, the strong hydrophobic interactions

with the aromatic residues W215, Y99, and F174 did not allow this molecule to disperse from this part

of the active site, demonstrating some stability in this region of the protein. On the other hand, the

analysis of the MD trajectory of compound 31 revealed π-stacking interactions between the Boc moiety

and Y99 residue for much of the trajectory time. In addition, a moderately frequent hydrophobic

interaction was observed between the same Boc moiety and the ring of F174. The morpholine fragment

displayed a high-frequency of hydrophobic interaction with W215, while chlorobenzene ring revealed

low-frequency of CH···O HB interactions with D189 and G218, as well as a low presence of HB

interactions in the protein-compound 31 complex. MD analysis shows that Y99, W215, and F274

residues contribute signiﬁcantly to the stabilization of compound 31 within the FXa binding site.

The RMSD values of the backbone atoms in the protein show a convergence in the ﬁrst nanoseconds

(ns) of simulation, showing ﬂuctuations between 1.0–1.8 Å. The low RMSD indicates that the FXA

protein in complex with compounds 19 and 31 presents a stable conformation during the MD simulation.

2.5.3. In Silico Pharmacokinetics Prediction

The potential of novel compounds to cross the blood–brain barrier via passive diﬀusion was

evaluated by molecular descriptors: the topological polar surface area (TPSA), the calculated logarithm

of the octanol/water partition coeﬃcient (logP) (Table 12) [117,118]. On the other hand, several

parameters were calculated in parallel, the molecular weight (MW), the number of hydrogen bond

acceptors (HBA), the number of hydrogen bond donors (HBD), and the number of rotatable bonds (RB).

Molecular descriptors were determined using QikProp of Schrödinger [119]. QuikProp allows you to

compute physicochemical descriptors as well as to predict absorption, distribution, metabolism, and

excretion (ADME) parameters, pharmacokinetic properties, druglike nature, and medicinal chemistry

friendliness of one or multiple small molecules to support drug discovery [120].

Taking into account the in silico pharmacokinetics results for compounds 27 and 34, they showed

logP < 5, appropriate logBB, logS and PHOA values that encourage us to utilize these novel molecules

as scaﬀolds for potential anticoagulant oral administration drugs.

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Table 12. Drug likeness score of synthesized compounds.

Molecular

Weight

H-Bonds

Donors

H-Bonds

Acceptors

Compound

logPo/w

logS

logBB

PHOA

7

8

9

2.30

1.71

0.76

2.81

3.58

2.83

1.94

2.83

3.37

2.75

1.79

4.02

4.51

4.25

4.51

4.99

3.94

4.74

4.44

4.18

2.97

3.46

3.21

309.34

226.27

210.21

246.28

347.39

264.32

248.26

369.44

470.54

387.47

371.41

365.41

399.85

383.40

466.51

500.96

383.44

484.50

417.89

401.43

367.38

401.83

385.37

−3.48

−2.60

−2.02

−3.91

−5.82

−4.01

−3.00

−3.92

−5.80

−3.99

−2.90

−6.25

−6.99

−6.62

−7.63

−8.35

−6.17

−7.99

−6.91

−6.54

−5.01

−5.75

−5.43

1.5

0.5

1.0

6.5

4.5

5.7

4.0

6.5

4.5

5.7

7.0

9.5

7.5

8.7

6.0

8.5

6.5

8.5

6.5

7.7

0.80

0.12

0.21

0.10

0.57

0.08

0.04

0.22

0.85

88.62

91.48

85.49

100.00

96.68

11

12

13

14

17

18

19

20

27

28

29

30

31

33

34

35

36

37

38

93.68

96.52

89.95

0.10

0.25

0.63

100.00

90.07

100.00

−0.48

−0.53

−1.26

−1.11

−0.51

−1.15

−0.35

−0.40

−0.63

−0.48

−0.54

QPlogPo/w = Predicted octanol / water partition coeﬃcient. Recommended values 2.0–6.5. donor HB = Estimated

−

number of hydrogen bonds that would be donated in an aqueous solution. Values are averages taken over a number

of conﬁgurations, thus, they can be non-integer. Acceptors HB = Estimated number of hydrogen bonds that would

be accepted in an aqueous solution. Values are averages taken over a number of conﬁgurations, thus, they can be

non-integer. QPlogS = Predicted aqueous solubility, log S. Recommended values

−

6.5 to 0.5. QPlogBB = Predicted

brain/blood partition coeﬃcient. Recommended values 3.0 to 1.2. logBB = Passive blood-brain partitioning values

−

expressed in terms of logarithm. PHOA= Predicted human oral absorption on 0 to 100% scale. Recommended

values >80% is high <25% is poor.

3. Materials and Methods

3.1. FXa-Ligand Structure Retrieval and Standardization

The reference sequence for human FXa was obtained from the Uniprot database (entry code

P00742). The protein databank PDB advanced search tool was used to BLAST the sequence and identify

all available human FXa crystal structures. All small co-crystallized ligands were retrieved in Simpliﬁed

Molecular-Input Line-Entry System (SMILES) format for the substructure search. It was done as a

pdb ﬁle for further clustering by chemical similarity according to the Tanimoto distance by using

the FCFP_4 (functional-class extended-connectivity ﬁngerprint count up to diameter 4) ﬁngerprint

descriptors set available within Discovery Studio v2.1 suite (Accelrys Inc., San Diego, CA, USA).

Protein preparation, editing, atom typing, and structural alignment were performed using

Accelrys Discovery Studio v2.1 (Accelrys Inc., San Diego, CA, USA). Co-crystallized ligands plus

crystallographic waters found within 4 Å from the center of mass of the ligand structure were retained.

Visual inspection and manual modiﬁcation of the ligand structure valences were performed to ensure

a proper state in the protein, and the 20 crystal structures of the selected human FXa-ligand complexes

structures were aligned. After superimposition, CαRMSD against the lower resolution structure, was

calculated. In addition, an all atom RMSD calculation, including only residues within 6Å from the

center of mass of all crystallized ligands, was also performed. All FXa-ligand complexes atoms were

typed with the CHARMM force ﬁeld for further experiments [121].

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3.2. Shape-Based Query Generation and Validation

The 3D alignment of the ligands was used to generate shape-based queries with vROCS v3.3.1.2

(OpenEye Scientiﬁc Software, Santa Fe, NM, USA) [122]. The queries were validated using the

active compounds. The FXa inhibitors dataset from the extended database of useful decoys (DUD-E)

database [74], which contains 792 actives and 24,017 decoy molecules, were also used. The in-house

library was generated and standardized using InstantJChem v19.17.0 (ChemAxon Inc., Budapest,

Hungary) and a multiple conformer database was generated by OMEGA v2.5.1.4 (OpenEye Scientiﬁc

Software, Santa Fe, NM, USA) [123]. Shape-based screening was performed by using ROCS v3.3.1.2

using the Tanimoto Combo metric (OpenEye Scientiﬁc Software, Santa Fe, NM, USA) [72].

3.3. Synthesis of Derivatives

3.3.1. Instrumentation and Chemicals

Sensitive reactions were carried out under a nitrogen atmosphere. Anhydrous solvents

(tetrahydrofuran (THF), acetonitrile, and toluene) were dried over anhydrous calcium chloride

and then stored over activated 0.3 nm molecular sieves. Other solvents (ethyl acetate, AcOEt; n-hexane,

or others) were treated prior to their use by standard methods [124].

All starting materials were of the best available grade (Sigma-Aldrich, Merck, AK Scientiﬁc)

and were used without further puriﬁcation. Commercially available copper(II) chloride dihydrate

was dehydrated upon heating in an oven (150 ^◦C, 45 min) prior to use by the preparation of CuNPs.

Column chromatography was performed with Merck silica gel 60 (0.040–0.063 mm, 240–400 mesh).

Reactions were monitored by thin-layer chromatography on silica gel plates (60F-254) and visualized

under UV.

Reactions were performed in a microwave Synthesis Reactor Monowave 200, Anton Paar.

The melting points of synthesized derivatives were measured using an Electrothermal IA9100 digital

melting point apparatus (Staﬀordshire, UK). Sonication was performed using a Hinotek SB-120DT

Ultrasonic Cleaner ultrasound device. The nuclear magnetic resonance (NMR) spectra were recorded

on Bruker Advance III HD 400 NMR spectrometer (400 MHz) with CDCl₃as a solvent, except where

indicated. The NMR data were reported in δ (ppm) from tetramethylsilane (TMS). Multiplicities are

abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet; brs = broad

singlet. Coupling constants (J) were reported in Hz. The FT-IR spectra were recorded in a KBr pellet,

on a Bruker VECTOR 22 spectrophotometer. High-resolution mass spectra (HRMS) were recorded

on a Thermo Scientiﬁc Exactive Plus Orbitrap. Enzymatic activity was measured a Cytation^TM5 Cell

Imaging Multi-Mode Reader (Winooski, VT, USA) programmed to read continuously for 30 min at

37 ^◦C, at a wavelength of Ex/Em 490 nm/520 nm.

3.3.2. General Procedure for the Synthesis of Aniline Derivatives (6–9)

Microwave-assisted Ullmann-Goldberg coupling of 1.0 equivalent of the corresponding lactams

(compound 1–4) with 1.2 equiv. of 2-fluor-4-iodoaniline (5), 0.5 equiv. of CuI (except for compound 1

where 2 equivalents of catalyst were used), 2 equivalents of N,N⁰-dimethlyethylenediamine (DMEDA),

and 2 equivalents of K₃PO₄in 3.5 mL of dry toluene. The reaction mixture was irradiated in a microwave

oven with 6–7 bar, 850 watts for 2 h at 160 ^◦C (except for compound 7, where 90 ^◦C was used).

The resulting solution was passed through a pad of silica and Celite with ethyl acetate and the

crude mixture was puriﬁed by ﬂash column chromatography (silica gel, n-hexane/AcOEt, 5:5) [80].

3.3.3. General Procedure for the Synthesis of N-propargyl Aniline Derivatives (11–14)

The synthesis of N-propargyl aniline derivatives was carried out using 1 equivalent of aniline

(compounds 6–9), 1.5 equiv. K₂CO₃in 3 mL of dry acetonitrile. The mixture was stirred in a G-30

microwave instrument vessel at 500 rpm under an inert atmosphere of nitrogen for 20 min. In addition,

in another vessel 0.1 equivalent of KI and 1 equivalent of propargyl bromide (10) solution 80% wt

Molecules 2020, 25, 491

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in toluene and 1 mL of acetonitrile were added. This mixture was added to the previous mixture

reaction and irradiated with 7–8 bar, 850 watts at 160 ^◦C for 30 min. The resulting solution was passed

through a pad of silica and Celite with ethyl acetate and the crude mixture was puriﬁed by ﬂash

column chromatography (silica gel, n-hexane/AcOEt, 8:2) [80].

3.3.4. General Procedure for the Synthesis of N-propargyl tetrahydroquinoline Derivatives (17–20)

1 equiv. of N-propargyl aniline derivative (compounds 11–14), 1.1 equiv. of formaldehyde 30%

in methanol (15) and 0.2 equiv. of InCl₃were mixed into a G-30 vessel. The reaction mixture was

stirred at 500 rpm for 20 min under an inert atmosphere. Then, 1.1 equiv. of 1-vinyl-2-pyrrolidinone

(

16) were added to the mixture and irradiated with 7–8 bar, 850 watts of power at 160 ^◦C for 15 min.

The resulting solution was passed through a pad of silica and celite with ethyl acetate and the crude

mixture was puriﬁed by ﬂash column chromatography (silica gel, n-hexane/AcOEt, 9:1) [87,89].

1-(8-Fluoro-4-(2-oxopyrrolidin-1-yl)-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl)piperidin-2-one (17).

Brown oil; 85% yield; FT-IR (KBr)

(400 MHz, CDCl₃) 6.70 (dd, J = 14.2, 2.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 5.17 (dd, J = 9.8, 5.7 Hz, 1H),

ν

/ cm⁻¹2924, 2098, 1743, 1681, 1604, 1489, 1381, 1134, 795; ¹H NMR

δ

3.92 (qd, J = 18.0, 2.4 Hz, 2H), 3.40 (ddt, J = 17.8, 11.5, 6.1 Hz, 2H), 3.20 (dtt, J = 17.2, 8.4, 4.1 Hz, 2H),

3.10 (dt, J = 9.8, 7.2 Hz, 2H), 3.01 (dt, J = 9.7, 6.6 Hz, 2H), 2.33 (d, J = 7.1 Hz, 2H), 2.30 (dd, J = 8.1, 3.6

Hz, 2H), 2.09 (t, J = 2.2 Hz, 1H), 1.84 (dt, J = 12.5, 6.3 Hz, 2H), 1.73 (dt, J = 6.4, 3.1 Hz, 4H); ¹³C NMR

(101 MHz, CDCl₃) δ 175.6, 169.9, 154.3, 151.8, 133.8, 126.8, 120.5, 113.1, 79.8, 72.5, 51.1, 48.1, 47.5, 43.1,

43.1, 32.3, 31.0, 24.3, 23.0, 20.8, 17.9; ¹⁹F NMR (376 MHz, CDCl₃) δ −120.9; ESI-HRMS (m/z): calcd. for

C₂₁H₂₄FN₃O₂[M + H] ⁺: 369.1853, found 369.1847.

4-(8-Fluoro-4-(2-oxopyrrolidin-1-yl)-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl)thiomorpholin-3-one

(

19). Brown oil; 89% yield; FT-IR (KBr)

ν

/ cm⁻¹3533, 3224, 3055, 2854, 2106, 1705, 1573, 1504, 1118,

1064, 1987, 933, 856, 817, 786, 740, 632; ¹H NMR (400 MHz, CDCl₃)

δ

6.81 (dd, J = 14.0, 2.8 Hz, 1H), 6.58

(d, J = 2.6 Hz, 1H), 5.28 (dd, J = 9.9, 5.0 Hz, 1H), 4.15–3.94 (m, 2H), 3.83 (tt, J = 5.2, 2.6 Hz, 2H), 3.34 (q,

J = 1.9 Hz, 2H), 3.29 (td, J = 3.9, 2.1 Hz, 2H), 3.19 (qd, J = 7.2, 3.5 Hz, 2H), 3.11 (tt, J = 9.8, 5.1 Hz, 2H),

2.93 (dtd, J = 5.9, 4.2, 1.8 Hz, 2H), 2.42 (tq, J = 7.8, 4.9, 3.4 Hz, 2H), 2.15 (s, 1H), 1.95 (ddt, J = 9.7, 7.6, 2.8

Hz, 2H); ¹³C NMR (101 MHz, CDCl₃)

δ 175.7, 166.8, 154.5, 152.1, 134.0, 127.4, 120.7, 113.4, 80.0, 72.7,

52.1, 48.5, 47.8, 43.5, 43.3, 31.3, 30.5, 26.6, 24.7, 18.3; ¹⁹F NMR (376 MHz, CDCl₃) δ −120.3; ESI-HRMS

(m/z): calcd. for C₂₀H₂₂FN₃O₂S [M + H]⁺: 387.1417, found 388.1410.

4-(8-Fluoro-4-(2-oxopyrrolidin-1-yl)-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl)morpholin-3-one (20

Brown oil; 83% yield; FT-IR (KBr)

/ cm⁻¹3317, 3240, 3070, 2939, 2885, 2368, 2106, 1697, 1573, 1504,

1111, 1056, 995, 956, 925, 864, 786, 725, 648; ¹H NMR (400 MHz, CDCl₃)

6.91 (dd, J = 14.2, 2.5 Hz,

).

ν

δ

1H), 6.69 (dt, J = 2.5, 1.2 Hz, 1H), 5.32 (dd, J = 9.9, 5.7 Hz, 1H), 4.23 (s, 2H), 4.18–3.95 (m, 2H), 3.94 (td,

J = 4.8, 4.2, 1.2 Hz, 2H), 3.71–3.55 (m, 2H), 3.42–3.27 (m, 2H), 3.18 (ddt, J = 37.6, 9.7, 7.0 Hz, 2H), 2.44

(td, J = 7.9, 2.4 Hz, 2H), 2.16 (s, 1H), 2.15 (qd, J = 10.8, 10.3, 5.2 Hz, 2H), 1.97 (q, J = 7.6 Hz, 2H); ¹³

C

NMR (101 MHz, CDCl₃)

δ 175.8, 166.6, 154.6, 152.1, 133.3, 127.4, 120.0, 112.8, 80.0, 72.8, 68.5, 64.0, 49.4,

48.6, 47.9, 43.5, 43.4, 31.3, 24.8, 18.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −120.2; ESI-HRMS (m/z): calcd. for

C₂₀H₂₂FN₃O₃[M + H]⁺: 371.1645, found 371.1644.

3.3.5. General Procedure for the Synthesis of Azides (24–26)

A 100-mL round bottom ﬂask was charged with 4 equiv. of aniline (compounds 21–23), water

(4 mL) as a solvent, and 2.6 equiv. of concentrated HCl were added to the vigorously stirred reaction

mixture. Then, 4.2 equiv. of NaNO₂in water (6.0 mL) were added dropwise into the reaction mixture.

Next, the mixture was sonicated at 40 ^◦C for an additional 10 min. Then, a freshly prepared solution of

4.2 equiv. of NaN₃in water (12.0 mL) was added dropwise into the reaction mixture. Upon complete

addition of the sodium azide solution, the reaction mixture was stirred for 1 h. The reaction mixture

was extracted with EtOAc (3

×

10 mL) and washed with water (10 mL). The organic layer was dried

Molecules 2020, 25, 491

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with MgSO₄, then ﬁltered and the solvent removed in vacuo to aﬀord the corresponding arylazide a

quantitative yield.

3.3.6. General Procedure for the synthesis of 1H-1,2,3-triazole Derivatives (27–38)

In a G-30 vessel, 1 equiv. of N-propargyl amines (compounds 17–20), 1 equiv. of aryl azide (compounds

24–26), 0.85 mol% of copper nanoparticles supported on ZnO (CuNPs/ZnO, synthesis detailed in the next

section) as a catalyst, 1 equiv. of Et₃N in THF were added. The reaction mixture was heated at 160 ^◦C

under microwave irradiation for 15 min with a pressure of 7.7 bar at 850 W. The corresponding triazoles

were purified by flash column chromatography (silica gel, n-hexane/AcOEt, 3:7).

Synthesis of Copper Nanoparticles Supported on ZnO

The catalyst used in the synthesis of diﬀerent triazoles were prepared according to the procedure

reported in the literature [125].

Anhydrous copper(II) chloride (135 mg, 1 mmol) was added to a suspension of lithium (14 mg,

2 mmol) and 4,4⁰-di-tert-butylbiphenyl (DTBB, 27 mg, 0.1 mmol) in THF (2 mL) at room temperature

under a nitrogen atmosphere. The reaction mixture, which was initially dark blue, rapidly changed to

black, indicating that the suspension of copper nanoparticles was formed. This suspension was diluted

with THF (8 mL) followed by the addition of the zinc oxide (800 mg). The resulting mixture was stirred

for 1 h at room temperature, ﬁltered, and the solid successively washed with ethanol (20 mL) and then

dried under vacuum.

1-(3-Fluoro-4-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)piperidin-2-one (27

)

.

Brown solid; 92%

yield; m.p. 146–148 ^◦C; FT-IR,

λm

áx (KBr),

ν

/ cm⁻¹3418, 3132, 2855, 1651, 1528, 1466, 1420, 1342, 1273,

1227, 1173, 1119, 1049, 926, 856, 826, 756, 687; ¹H NMR (400 MHz, DMSO) δ 8.68 (s, 1H), 8.00–7.81 (m,

2H), 7.63–7.51 (m, 2H), 7.50–7.42 (m, 1H), 6.99 (dd, J = 13.0, 2.2 Hz, 1H), 6.90–6.72 (m, 2H), 6.04 (td,

J = 6.0, 2.1 Hz, 1H), 4.46 (d, J = 5.9 Hz, 2H), 3.49 (t, J = 5.6 Hz, 2H), 2.32 (t, J = 6.2 Hz, 2H), 1.79 (qt,

J = 8.4, 4.2 Hz, 4H); ¹³C NMR (101 MHz, DMSO)

δ 168.7, 151.3, 148.9, 146.7, 136.7, 133.4, 129.8 (2C),

128.5, 122.3, 121.1, 119.9 (2C), 113.2, 111.6, 51.1, 38.3, 32.5, 23.0, 20.9; ¹⁹F NMR (376 MHz, DMSO)

δ

−133.5; ESI-HRMS (m/z): calcd for C₂₀H₂₀FN₅O [M + H]⁺: 365,1652; found 366,1713.

1-(4-(((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-3-ﬂuorophenyl) piperidin-2-one (28

)

.

Brown

/ cm⁻¹3402, 2955, 1643, 1520, 1412, 1273,

7.89 (s, 1H), 7.73–7.61 (m,

◦

solid; 90% yield; m.p. 180–181 C; FT-IR,

1227, 1173, 1111, 1088, 1042, 988, 926, 826; H NMR (400 MHz, CDCl₃)

λ

1

m

á

x (KBr),

ν

δ

2H), 7.52–7.39 (m, 2H), 6.94 (d, J = 2.3 Hz, 1H), 6.85 (dd, J = 8.6, 2.3 Hz, 1H), 6.72 (t, J = 9.0 Hz, 1H),

4.57 (s, 1H), 4.56 (s, 2H), 3.55 (h, J = 3.7 Hz, 2H), 2.57–2.45 (m, 2H), 1.90 (p, J = 3.3 Hz, 4H); ¹³C NMR

(101 MHz, CDCl₃)

δ 170.4, 152.4, 150.0, 147.2, 135.6, 134.8, 134.6, 133.3, 130.0, 122.5, 121.8, 120.0, 113.7,

113.5, 112.3, 52.1, 39.7, 32.9, 23.6, 21.5; ¹⁹F NMR (376 MHz, CDCl₃) δ −133.9; ESI-HRMS (m/z): calcd for

C₂₀H₁₉ClFN₅O [M + H]⁺: 399,1262; found 400.1334.

1-(3-Fluoro-4-(((1-(4-ﬂuorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl) piperidin-2-one (29). Brown

solid; 76% yield; m.p. 139–140 ^◦C; FT-IR, λ

m

á

x (KBr),

1412, 1335, 1273, 1227, 1180, 1119, 1049, 980, 934, 841, 625; ¹H NMR (400 MHz, CDCl₃)

7.65 (ddt, J = 6.6, 4.5, 2.5 Hz, 2H), 7.14 (t, J = 8.6 Hz, 2H), 6.89 (dd, J = 12.3, 2.4 Hz, 1H), 6.82 (dd, J = 8.6

2.4 Hz, 1H), 6.69 (t, J = 8.8 Hz, 1H), 4.61 (q, J = 6.1 Hz, 1H), 4.50 (d, J = 5.6 Hz, 2H), 3.52 (t, J = 5.5

Hz, 2H), 2.48 (t, J = 5.8 Hz, 2H), 1.87 (p, J = 3.4 Hz, 4H); ¹³C NMR (101 MHz, CDCl₃)

170.3, 163.6,

ν

/ cm⁻¹3318, 3140, 3071, 2947, 2862, 1643, 1520,

δ

7.88 (s, 1H),

,

δ

161.1, 152.2, 149.8, 146.9, 134.8, 133.2, 122.5, 122.3, 120.3, 116.6, 113.4, 112.1, 52.0, 39.5, 32.8, 29.7, 23.5,

21.4; ¹⁹F NMR (376 MHz, CDCl₃) δ −112.3,

−

133.9; ESI-HRMS (m/z): calcd for C₂₀H₁₉F₂N₅O [M + H]⁺:

383,1558; found 384,1771.

Tert-butyl 4-(3-ﬂuoro-4-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-3-oxopiperazine-1-carboxylate

(30). Brown solid; 90% yield; m.p. 90–92 ^◦C; FT-IR, / cm⁻¹3348, 2924, 2855, 1528, 1420,

x (KBr),

1327, 1242, 1042; ¹H NMR (400 MHz, CDCl₃)

7.91 (s, 1H), 7.69 (dd, J = 7.8, 1.6 Hz, 2H), 7.48 (t, J = 7.8

λm

á

ν

δ

Molecules 2020, 25, 491

27 of 39

Hz, 2H), 7.46–7.36 (m, 1H), 6.95 (dd, J = 12.1, 2.4 Hz, 1H), 6.94–6.80 (m, 1H), 6.75 (t, J = 8.9 Hz, 1H), 4.65

(d, J = 15.1 Hz, 1H), 4.56 (s, 2H), 4.20 (s, 2H), 3.73 (t, J = 5.3 Hz, 2H), 3.63 (dd, J = 6.2, 4.4 Hz, 2H), 1.47

(s, 9H); ¹³C NMR (101 MHz, CDCl₃)

δ 166.0, 153.9, 152.2, 149.8, 146.6, 137.0, 135.3, 131.4, 129.8, 128.9,

122.0, 120.6, 120.0, 113.1, 112.3, 81.0, 50.2, 48.3, 40.4, 39.6, 29.8, 28.4 (3C); ¹⁹F NMR (376 MHz, CDCl₃)

−133.5; ESI-HRMS (m/z): calcd for C₂₄H₂₇FN₆O₃[M + H]⁺: 466,2129; found 466,2097.

δ

Tert-butyl 4-(4-(((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-3-ﬂuorophenyl)-3-oxopiperazine-1-

carboxylate (31). Brown solid; 80% yield; m.p. 129–130 ^◦C; FT-IR, / cm⁻¹3433, 3140, 3109,

x (KBr),

2978, 2847, 1697, 1535, 1404, 1335, 1234, 1173, 1126, 1088, 1049, 1018, 988, 941, 864, 833, 795, 764, 733,

656; ¹H NMR (400 MHz, CDCl₃)

7.89 (s, 1H), 7.71–7.60 (m, 2H), 7.52–7.41 (m, 2H), 6.96 (dd, J = 12.1,

λmá

ν

δ

2.3 Hz, 1H), 6.87 (ddd, J = 8.6, 2.4, 1.1 Hz, 1H), 6.73 (t, J = 9.0 Hz, 1H), 4.62 (s, 1H), 4.56 (d, J = 5.1 Hz,

2H), 4.20 (s, 2H), 3.74 (dd, J = 6.4, 4.3 Hz, 2H), 3.63 (dd, J = 6.3, 4.3 Hz, 2H), 1.48 (s, 9H); ¹³C NMR (101

MHz, CDCl₃)

δ 165.9, 153.8, 152.1, 149.8, 146.9, 135.5, 135.2, 134.6, 131.4, 129.9, 122.0, 121.7, 119.9, 113.2,

113.0, 112.2, 81.0, 50.2, 48.2, 40.4, 39.5, 28.4 (3C); ¹⁹F NMR (376 MHz, CDCl₃) δ −133.5; ESI-HRMS

(m/z): calcd for C₂₄H₂₆ClFN₆O₃[M + H]⁺: 500,1739; found 501,1817.

Tert-butyl 4-(3-ﬂuoro-4-(((1-(4-ﬂuorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-3-oxopiperazine-1-

carboxylate (32). Brown solid; 73% yield; m.p. 80–82 ^◦C; FT-IR,

λ

m

á

x (KBr),

3078, 2978, 2924, 2862, 2778. 2361, 1705, 1528, 1481, 1412, 1366, 1327, 1288, 1234, 1173, 1126, 1042, 1018,

988, 941, 841, 756, 656; ¹H NMR (400 MHz, CDCl₃)

7.88 (s, 1H), 7.68–7.59 (m, 2H), 7.19–7.08 (m, 2H),

ν

/ cm⁻¹3588, 3341, 3140,

δ

6.92 (dd, J = 12.1, 2.2 Hz, 1H), 6.83 (ddd, J = 8.6, 2.4, 1.1 Hz, 1H), 6.70 (t, J = 9.0 Hz, 1H), 4.68 (td, J = 5.9,

2.7 Hz, 1H), 4.50 (d, J = 5.8 Hz, 2H), 4.17 (s, 2H), 3.71 (dd, J = 6.4, 4.2 Hz, 2H), 3.60 (dd, J = 6.2, 4.4

Hz, 2H), 1.45 (s, 9H); ¹³C NMR (101 MHz, CDCl₃)

δ

165.9, 163.6, 161.1, 153.8, 152.1, 149.7, 146.7, 135.2,

133.2, 131.2, 122.4, 121.9, 120.2, 116.6, 113.0, 112.1, 80.9, 50.1, 48.2, 40.6, 39.3, 28.3 (3C); ¹⁹F NMR (376

MHz, CDCl₃) δ −112.2,

−

133.5; ESI-HRMS (m/z): calcd for C₂₄H₂₆F₂N₆O₃[M

−

H]⁺: 484,2034, found

483,1983.

4-(3-Fluoro-4-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)thiomorpholine-3-one (33). Brown solid;

◦

93% yield; m.p. 178–180 C; FT-IR,

λm

á

x (KBr),

ν

/ cm⁻¹3140, 2924, 1659, 1528, 1466, 1427, 1327,

1

1273, 1219, 1111; H NMR (400 MHz, CDCl₃)

δ

7.91 (s, 1H), 7.70 (dd, J = 7.5, 1.6 Hz, 2H), 7.49 (t, J

= 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 6.94 (dd, J = 12.0, 2.3 Hz, 1H), 6.86 (dd, J = 8.5, 2.4 Hz, 1H),

6.75 (t, J = 8.9 Hz, 1H), 4.59 (s, 1H), 4.56 (d, J = 4.7 Hz, 2H), 3.92–3.84 (m, 2H), 3.41 (s, 2H), 2.98 (dd, J

= 6.8, 4.7 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃)

δ 167.1, 152.2, 149.8, 146.6, 137.1, 135.3, 132.4, 129.8,

128.9, 122.4, 120.6, 120.0, 113.5, 112.3, 52.8, 39.6, 30.7, 29.8, 26.8; ¹⁹F NMR (376 MHz, CDCl₃) δ −133.6;

ESI-HRMS (m/z): calcd for C₁₉H₁₈FN₅OS [M + H]⁺: 383,1216; found 384,1278.

4-(4-(((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-3-ﬂuorophenyl) thiomorpholine-3-one (34).

Brown solid; 88% yield; m.p. 240–242 ^◦C; FT-IR,

λ

m

á

x (KBr),

1528, 1504, 1404, 1342, 1273, 1234, 1188, 1126, 1088, 1042, 988, 957, 895, 864, 826, 718, 687, 648, 617;

¹H NMR (400 MHz, DMSO)

8.71 (s, 1H), 7.93 (d, J = 8.9 Hz, 2H), 7.64 (d, J = 8.9 Hz, 2H), 7.01 (dd,

J = 12.8, 2.3 Hz, 1H), 6.87 (dd, J = 8.7, 2.3 Hz, 1H), 6.79 (t, J = 9.1 Hz, 1H), 6.12 (td, J = 6.1, 2.1 Hz, 1H),

4.47 (d, J = 5.9 Hz, 2H), 3.85 (t, J = 5.8 Hz, 2H), 3.34 (d, J = 11.2 Hz, 2H), 2.99 (t, J = 5.8 Hz, 2H); ¹³

ν

/ cm⁻¹3426, 3132, 3063, 1659, 1589,

δ

C

NMR (101 MHz, DMSO)

δ 166.9, 151.2, 148.8, 146.9, 135.4, 134.7, 132.8, 131.4, 129.8, 122.2, 122.2, 121.6,

121.2, 113.0, 111.6, 51.4, 38.2, 29.5, 26.1; ¹⁹F NMR (376 MHz, DMSO) δ −133.3; ESI-HRMS (m/z): calcd

for C₁₉H₁₇ClFN₅OS [M + H]⁺: 417,0826; found 418,0906.

4-(3-Fluoro-4-(((1-(4-ﬂuorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl) thiomorpholine-3-one (35).

Brown solid; 75% yield; m.p. 161–163 ^◦C; FT-IR,

λ

m

á

x (KBr),

ν

/ cm⁻¹3356, 3240, 1651, 1528, 1420, 1319,

1281, 1242, 1211, 1188, 1126, 1111, 1026, 964, 910, 864, 818, 772, 702, 648; ¹H NMR (400 MHz, CDCl₃)

δ

7.88 (s, 1H), 7.69–7.61 (m, 2H), 7.20–7.11 (m, 2H), 6.90 (dd, J = 12.1, 2.3 Hz, 1H), 6.83 (dt, J = 8.5, 1.6 Hz,

1H), 6.70 (t, J = 8.9 Hz, 1H), 4.64 (td, J = 6.0, 2.7 Hz, 1H), 4.51 (d, J = 5.8 Hz, 2H), 3.89–3.81 (m, 2H), 3.38

(s, 2H), 2.97–2.94 (m, 2H); ¹³C NMR (101 MHz, CDCl₃)

δ 167.1, 163.6, 161.1, 152.1, 149.7, 146.7, 135.1,

Molecules 2020, 25, 491

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133.2, 132.3, 122.5, 122.3, 120.2, 116.7, 113.4, 112.1, 52.7, 39.4, 30.6, 26.7; ¹⁹F NMR (376 MHz, CDCl₃)

−112.1, −133.6; ESI-HRMS (m/z): calcd for C₁₉H₁₇F₂N₅OS [M + H]⁺: 401,1122; found 401,1901.

δ

4-(3-Fluoro-4-(((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)morpholin-3-one (36). Brown solid;

90% yield; m.p. 168–169 ^◦C; FT-IR, / cm⁻¹3426, 1651, 1535, 1474, 1420, 1342, 1234, 1119;

x (KBr),

¹H NMR (400 MHz, CDCl₃)

7.91 (s, 1H), 7.68 (dd, J = 7.7, 1.8 Hz, 2H), 7.51–7.43 (m, 2H), 7.40 (dd,

λmá

ν

δ

J = 8.5, 6.4 Hz, 1H), 6.99 (dd, J = 12.2, 2.4 Hz, 1H), 6.90 (dd, J = 8.8, 2.4 Hz, 1H), 6.74 (t, J = 9.0 Hz, 1H),

4.66 (d, J = 7.1 Hz, 1H), 4.54 (d, J = 4.5 Hz, 2H), 4.27 (s, 2H), 3.96 (dd, J = 6.1, 4.1 Hz, 2H), 3.65 (dd,

J = 6.0, 4.1 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃)

δ 166.9, 152.2, 149.8, 146.6, 137.0, 135.2, 130.9, 129.8,

128.8, 121.8, 120.5, 120.0, 113.0, 112.8, 112.2, 68.6, 64.1, 50.1, 39.5; ¹⁹F NMR (376 MHz, CDCl₃) δ −133.5;

ESI-HRMS (m/z): calcd for C₁₉H₁₈FN₅O₂[M − H]⁺: 367,1445; found 366,1372.

4-(4-(((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-3-ﬂuorophenyl) morpholin-3-one (37). Brown

solid; 86% yield; m.p. 235–237 ^◦C; FT-IR,

λ

m

á

x (KBr),

ν

/ cm⁻¹3372, 3102, 2862, 1667, 1620, 1520, 1466,

1427, 1319, 1281, 1219, 1180, 1111, 1049, 980, 895, 864, 671, 625; ¹H NMR (400 MHz, DMSO)

δ

8.71 (s,

1H), 8.04–7.86 (m, 2H), 7.73–7.60 (m, 2H), 7.13 (dd, J = 13.0, 2.3 Hz, 1H), 6.99–6.91 (m, 1H), 6.81 (dd,

J = 9.8, 8.7 Hz, 1H), 6.14 (td, J = 6.0, 2.1 Hz, 1H), 4.47 (d, J = 5.9 Hz, 2H), 4.14 (s, 2H), 3.97–3.88 (m, 2H),

3.69–3.59 (m, 2H); ¹³C NMR (101 MHz, DMSO)

δ 165.9, 151.2, 148.8, 146.9, 135.5, 134.8, 132.8, 130.2,

129.8, 121.8, 121.6, 121.2, 112.6, 111.6, 67.7, 63.5, 54.9, 49.3, 38.2; ¹⁹F NMR (376 MHz, DMSO) δ −133.2;

ESI-HRMS (m/z): calcd for C₁₉H₁₇ClFN₅O₂[M + H]⁺: 401,1055; found 402,1119.

4-(3-Fluoro-4-(((1-(4-ﬂuorophenyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl) morpholin-3-one (38). Brown

solid; 78% yield; m.p. 153–154 ^◦C; FT-IR,

λ

m

á

x (KBr),

1466, 1427, 1319, 1281, 1227, 1180, 1157, 1111, 1049, 980, 941, 895, 856, 818, 671, 625; ¹H NMR (400 MHz,

DMSO) 8.67 (s, 1H), 7.93 (dd, J = 9.0, 4.7 Hz, 2H), 7.42 (t, J = 8.8 Hz, 2H), 7.13 (dd, J = 12.9, 1.9 Hz, 1H),

6.96 (dd, J = 8.7, 2.5 Hz, 1H), 6.81 (t, J = 9.2 Hz, 1H), 6.13 (td, J = 5.9, 2.1 Hz, 1H), 4.47 (d, J = 6.0 Hz, 2H),

4.14 (s, 2H), 3.92 (t, J = 5.1 Hz, 2H), 3.62 (t, J = 5.0 Hz, 2H); ¹³C NMR (101 MHz, DMSO)

166.4, 163.2,

ν

/ cm⁻¹3364, 3125, 3094, 2862, 1667, 1620, 1520,

δ

160.8, 151.7, 149.3, 147.2, 135.3, 133.7, 130.7, 122.8, 122.3, 121.8, 117.1, 113.1, 112.1, 68.2, 64.0, 49.78, 38.7.

¹⁹F NMR (376 MHz, DMSO) δ −113.4,

−

133.2; ESI-HRMS (m/z): calcd for C₁₉H₁₇F₂N₅O₂[M + H]⁺

:

385,1350; found 386,1413.

3.4. Theoretical Study

The Gaussian 09 computational package was used. It was used to perform ground-state geometry

optimization and reactivity parameters calculations, without symmetry constraints, employing Becke’s

three-parameter hybrid functional combined with Lee-Yang-Parr’s nonlocal correlation functional

B3LYP. A 6-311G basis set was employed [126].

The FMOs GAP, reactivity indexes and Fukui functions were calculated as follows [97,127]:

GAP = ε_LUMO− ε_HOMO

(1)

(2)

(3)

(4)

1

2

Chemical Hardness η = (ε_LUMO− ε_HOMO

)

1

2

Chemical Potential µ = (ε_LUMO+ ε_HOMO

2µ

Electrophilicity ω =

2η

ꢀ

f⁺= q_N+1− q_N

(5)

(6)

ꢀ

f⁻= q_N− q_N−1

ꢀ

1

2

0

f = (q_N+1− q_N−1

)

(7)

ꢀ

Molecules 2020, 25, 491

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3.5. Biological Analysis

3.5.1. FXa in Vitro Inhibition Assay

All compounds were evaluated in vitro for their FXa enzyme inhibitory activity using as the

positive control Rivaroxaban (gold standard inhibitor) and Gabexate mesylate (FXa kit inhibitor).

First, all the proposed compounds were screened at 100 µM to obtain a preliminary result of the

potential inhibitory activity of the novel molecules. The enzymatic assay was prepared according

to the manufacturer’s instructions (Sensolyte^®Rh 110 factor Xa Assay Kit *Fluorometric*, Anaspec,

Fremont, CA, USA). Inhibition control 1 mM was diluted 1:10 with the prepared buﬀer 1x ibtaining of

the 0.1 mM inhibition control. The reagents containing compound dilutions, buﬀers, and enzymes were

mixed, centrifuged, and incubated for 30 min at 37 ^◦C in 96-well microtiter plates. The enzyme reaction

was initiated by adding an appropriate substrate (FXa, S-2222). The 96-well plate for ﬂuorescence

was prepared with 40

50 L of the FXa substrate was added to each well. In the second trial, the selected compounds (27

30 31, and 34) were prepared to span ﬁve diﬀerent concentrations: 0.1–100 M. All inhibitory assays

µL of enzyme and 1 µL of each compound. To initiate the enzymatic reaction

µ

,

µ

were performed three times in triplicate. For compounds with FXa inhibition over 50%, respectively,

IC₅₀values were calculated from the mean of triplicates from a dilution series of the compound with

GraphPad Prism v. 8.0.2 (GraphPad Inc., MA, USA).

3.5.2. Coagulation Parameters Analyses: ex Vivo Assay

All measurements were read on the ACL TOP 500 CTS spectrophotometer equipment (Instrumental

Laboratory, Milano, Italy), as it can perform medium to high volume recesses. To measure, monitor,

and analyze Prothrombin time (PT), activated partial thromboplastin time (aPTT), anti-FXa activity

were used. All methodologies were used with their corresponding calibrators and controls. Statistical

analysis was expressed as a mean

test. Two-sided p < 0.05 was considered statistically signiﬁcant.

±

SD. Statistical comparison was performed by using the Student t

Anti-FXa Assay

The anti-FXa assay was performed by using the Hemosil Liquid anti-FXa kit (IL^®LAX,

Instrumentation Laboratory, Orangeburg, NY, USA) and the measures were done through ACL

Top 500 CTS spectrophotometer instrument (Instrumental Laboratory, Italy). The assay was prepared

according to the manufacturer’s instructions. The solution anti-Xa kit is a one-stage chromogenic assay

based on a synthetic chromogenic substrate and an FXa inactivation. FXa is neutralized by the tested

compounds. Residual FXa is quantiﬁed with a synthetic chromogenic substrate. The p-nitroaniline

released is monitored kinetically at 405 nm and is inversely proportional to the compounds in the

sample (27, 30, 31, 34, Apixaban, Rivaroxaban). The anti-FXa activity measurement was expressed as

heparin activity (IU/mL). Anti-FXa analysis was performed in triplicate.

Prothrombin Time (PT) Test

It is the most important clinical screening test in the evaluation of disorders of the extrinsic

coagulation pathway. A highly sensitivity thromboplastin reagent, based on recombinant human tissue

factor (RTF), was done for the quantitative determination in human citrated plasma of PT. Pathway

and domino activation reactions are the target of this assay, which is widely used in hemostasis ﬁeld

(RecombiPlasTin 2G-0020003050, HemosIL^®, Milano, Italy). The assay was prepared according to the

manufacturer’s instructions. PT analysis was performed in triplicate.

Activated Partial Thromboplastin Time (aPTT) Test

A contact activator was used to stimulate the production of Factor XIIa by providing a surface for

the function of high molecular weight kininogen, kallikrein, and Factor XIIa. This contact activation

Molecules 2020, 25, 491

30 of 39

proceeds at 37 ^◦C for a couple of minutes. Calcium is then added to trigger further reactions and the

time required for clot formation is measured. Phospholipids are necessary to form complexes that

activate Factor X. Assay provides a highly reproducible and stable response. Prolonged clotting times

are observed in the presence of heparin, or another inhibitor.

aPTT results may be reported in seconds in plasma pool assays; it is not aﬀected by hemoglobin up

to 100 mg/dL or triglycerides up to 1200 mg/dL. The ﬁnal step was to remove the tube and tilt once per

second and stop the stopwatch when the ﬁrst vestige of clot formation (APTT-SP (liquid)-0020006300,

HemosIL^®) was noticed. aPTT analysis was performed in triplicate.

3.5.3. Cell Viability Assays

The human cell lines (HEK293 and HepG2) used in the cell viability assays were purchased from

American Type Culture Collection (Manassas, VA, USA). HEK293 cell lines were cultured in Dulbecco’s

modiﬁed Eagle medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin and

100 mg/mL streptomycin and kept at 37 ^◦C in a humidiﬁed atmosphere containing 5% CO₂. A 100-

µL

aliquot of adherent cells was used to seed 96-well cell culture plates at 30,000 cells/well and allowed to

adhere for 16 h prior to the addition of the compounds.

HepG2 cells were maintained in Dulbecco’s Modiﬁed Eagle Medium (DMEM-F12) with 5% fetal

bovine serum (FBS), and penicillin/streptomycin 1%, under standard cell culture conditions (37 ^◦C,

humidiﬁed, 5% CO₂). Cells were plated at a density of 25,000 cells/well in a 96-well plate and incubated

in a growth medium for 16 h. Cells were treated with each compound in DMEM containing 10% FBS

for 48 h. Subsequently, 20 µL MTS reagent was added into each well and incubated for 4 h. The cell

viability was detected by the CellTiter 96 AQueous One solution Cell Proliferation Assay kit (Promega

Corporation, Madison, WI, USA). All experiments were done in sextuplicate.

3.6. Computational Analysis

3.6.1. Docking Studies

FXa consists of 254 amino acid catalytic domains and the substrate cleaves at its active site in

FFNPR-TF and YIDGR-IV in prothrombin to form thrombin. Basically, FXa has four binding pockets

that are involved in the substrate-speciﬁc cleavage making them an important target for ligands

designed for inhibition activity. These active sites host diﬀerent amino residues, which play a vital

role in ligand binding through hydrogen bonding. The four binding pockets present in the active site

are S1, S2, S3, and S4. The synthesized compounds found to interact with the S1 residues make them

potential inhibitors of FXa. S1 pocket is symmetrical, and U shaped, whereas this site in thrombin is

less symmetrical and shallower due to the presence of L99 in place of Y99 in FXa [128].

FXa S1 pocket is hydrophobic in nature and contains Y228, D189, and S195 residues. Y228 of S1

pockets plays a vital role in forming the important hydrogen bonds with the ligands. S4 is another

hydrophobic site present on FXa and has residues like F174, Y99, and W215. These residues form the

aromatic box capable of being bound to ligands having aliphatic, aromatic, and positively charged

components. Because of its aﬃnity towards the positively charged moieties, it is also known as a

cation hole. S4 site of FXa makes its distinct form from the thrombin in terms of ligand speciﬁcity.

This is because S4 site of FXa is perfectly symmetrical when compared to thrombin and another site

of diﬀerentiation in the presence of Y99 and F174 residues in FXa and L99 and I174 amino residue

in thrombin. Among the residues of site S4, residue Y99 is the most crucial for selectivity towards

FXa [128]. Similarly, W215 formed the basis of the U shape pocket and played a vital role in binding

with the synthesized compounds. Residue C191, which has the disulﬁde linkage with C220, is present

at the bottom of the S1 site, is highly conserved, and hence, was expected to play an important role in

FXa inhibition.

Basically, the S4 site of FXa is aromatic in nature due to the presence of aromatic residues and

mostly host hydrophobic and positively charged substrates. On the other hand, S4 site of thrombin

Article Doi

Innovative three-step microwave-promoted synthesis of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXA) inhibitors: Drug design, synthesis, and biological evaluation.

DOI: 10.3390/molecules25030491

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Article abstract of DOI:10.3390/molecules25030491

Full text of DOI:10.3390/molecules25030491

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