Synthesis and in vitro cytotoxicity and antibacterial activity of novel1,2,3-triazol-5-yl-phosphonates

molecules

Article

Synthesis and In Vitro Cytotoxicity and Antibacterial

Activity of Novel 1,2,3-Triazol-5-yl-Phosphonates

Anna Tripolszky ¹, Emese Tóth ¹, Pál Tamás Szabó ², László Hackler Jr. ³, Beáta Kari ³,

László G. Puskás ^3,* and Erika Bálint ^1,

*

1

Department of Organic Chemistry and Technology, Budapest University of Technology and Economics,

H-1521 Budapest, Hungary; tripolszky.anna@mail.bme.hu (A.T.); emesetoth92@gmail.com (E.T.)

MS Metabolomics Laboratory, Instrumentation Center, Research Centre for Natural Sciences, Hungarian

Academy of Sciences, Magyar tudósok krt. 2., 1117 Budapest, Hungary; szabo.pal@ttk.mta.hu

Avidin Ltd., Alsó kiköto˝ sor 11/D, H-6726 Szeged, Hungary; l.hackler@avidinbiotech.com (L.H.J.);

b.kari@avidinbiotech.com (B.K.)

2

3

*

Correspondence: laszlo@avidinbiotech.com (L.G.P.); ebalint@mail.bme.hu (E.B.);

Tel.: +36-62-202-107 (L.G.P.); +36-1-463-3653(E.B.)

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Academic Editor: Pierre-Antoine Bouit

Received: 9 May 2020; Accepted: 4 June 2020; Published: 6 June 2020

Abstract: Novel 1,2,3-triazol-5-yl-phosphonates were prepared by the copper(I)-catalyzed domino

reaction of phenylacetylene, organic azides and dialkyl phosphites. The process was optimized on

the synthesis of the dibutyl (1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phosphonate in respect of the

catalyst, the base and the solvent, as well as of the reaction parameters (molar ratio of the starting

materials, atmosphere, temperature and reaction time). The method elaborated could be applied to a

range of organic azides and dialkyl phosphites, which conﬁrmed the large scope and the functional

group tolerance. The in vitro cytotoxicity on diﬀerent cell lines and the antibacterial activity of the

synthesized 1,2,3-triazol-5-yl-phosphonates was explored. According to the IC₅₀values determined,

only modest antibacterial eﬀect was detected, while some derivatives showed moderate activity

against human promyelocytic leukemia HL-60 cells.

Keywords: 1,2,3-triazoles; triazolyl phosphonates; copper(I) catalyst; domino reaction; multicomponent

reaction; anticancer activity

1. Introduction

Heterocyclic scaﬀolds bearing phosphonate moieties have been attracting continuous interest due

to their importance in synthetic- and medicinal chemistry, as well as in the agriculture and plastic

industry [

phosphonates are multicomponent reactions [

and meet several criteria of a theoretical “ideal synthesis”, such as high atom economy, fast and simple

1

–

4]. One of the most convenient and eﬃcient tools for the preparation of heterocyclic

5

], which usually utilize simple starting materials,

accomplishment, ability to save time and energy and being as environmentally-friendly as possible [

6,7].

They may also be feasible for the creation of large libraries of structurally related compounds.

In the last decades, 1,2,3-triazoles have received signiﬁcant attention due to the wide range of their

possible applications in materials science, biochemistry or even in drug design [ 10]. Compounds

containing the 1,2,3-triazole scaﬀold may have versatile biological activities, such as antibacterial,

antiviral, antifungal, anticancer or anti-inﬂammatory eﬀects [11 13]. Some novel triazole-based

heterocycles showed activity against bacterial strains (Bacillus subtilis and Escherichia coli) [14 16],

and in vitro cytotoxicity against tumor cell lines HL-60 (myeloid leukemia), MCF-7 (breast cancer),

8–

–

A549 (lung cancer) and HCT-116 (colon cancer) [17

–19]. Furthermore, the unique structural properties

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Molecules 2020, 25, 2643

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of 1,2,3-triazoles enable the introduction of various functional groups obtaining new biologically

active molecules.

1,2,3-Triazolyl phosphonates combine the advantages of the triazole- and the phosphonate

moieties [20]. Several derivatives showed anti-HIV eﬀect [21

assays [23] or could be appropriate for bioconjugation [24,25].

The Huisgen 1,3-dipolar cycloaddition [26 27] between azides and alkynyl phosphonates is one

,22], were investigated in protein binding

,

of the most common synthetic routes towards 1,2,3-triazol-5-yl-phosphonates (Table 1). Hall and

Trippett studied the cycloaddition of ethyl (diethoxyphosphinyl)propynoate and methyl azidoacetate

in the absence of any catalyst in boiling toluene (Table 1, Entry 1) [28]. The reaction was not

selective; two regioisomers, 1,2,3-triazol-5-yl-phosphonate (

1) and 1,2,3-triazol-4-yl-phosphonate

(2

), were formed in a ratio of ca. 2:1, respectively. Triﬂuoromethylated triazolyl phosphonates

were also synthesized by the catalyst-free addition of tert-butyl azidoacetate to diisopropyl

3,3,3-triﬂuoroprop-1-ynylphosphonate at room temperature (Table 1, Entry 2) [29]. The click reaction

of benzyl azide and diethyl (ethynyl)phosphonates was also carried out in the presence of copper(II)

sulfate pentahydrate and sodium ascorbate in DMF (Table 1, Entry 3) [30]. The corresponding

1,2,3-triazol-5-yl-phosphonates (

performed the cycloaddition of

1

ω

) were obtained selectively at 170 ^◦C after 12 h. Odinets and co-workers

-phosphorylalkyl azide to tetramethoxy acetylenediphosphonate

in water (Table 1, Entry 4) [31]. A microwave-assisted reaction was also reported, in which diethyl

(azidomethyl)phosphonate was reacted with diethyl prop-1-ynylphosphonate under catalyst- and

solvent-free conditions (Table 1, Entry 5) [32].

Table 1. Cycloaddition of azides and alkynyl phosphonates.

Product Ratio (%)

Y¹

Y²

Entry

T, t

Yield (%)

R

Cat.

Solvent

Ref.

1

2

1

2

CH₂COOMe

CH₂COO^tBu

COOEt

CF₃

Et

-

toluene

DEE

110 ^◦

25 ^◦C, 20 h

C

64

75

36

25

-

[28 ]

[29 ]

ⁱPr

90

Ph, 4-MeC₆H₄,

4-FC₆H₄, etc.

3

4

5

Bn

Et

Me

Et

CuSO₄

·

5H₂O/NaAsc DMF

170 ^◦C, 12 h

100

0

83–92

95

[30 ]

[31 ]

[32 ]

25 ^◦C, 36 h

(EtO)₂P(O)(CH₂)₂

(EtO)₂P(O)CH₂

(MeO)₂P(O)

Me

-

water

-

100

or 60 ^◦C, 2 h

MW, 90 ^◦C,

20 min

34

66

78

Although the 1,3-dipolar cycloaddition is an important synthetic possibility of triazolyl

phosphonates, the preparation of the starting alkynyl phosphonate derivatives is a real challenge.

By the development of multicomponent approaches for this compound family, utilization of simple

building blocks as starting materials may become available.

Several examples were reported on the preparation of 1,2,3-triazol-4-yl-phosphonates by domino

or three-component reactions [33,34], however, to the best of our knowledge, only one example

mentions the multicomponent synthesis of 1,2,3-triazol-5-yl-phosphonates [20]. Li and co-workers

carried out the domino reaction of terminal alkynes, azides, and diethyl-, diisopropyl- or dibenzyl

phosphite in the presence of copper(I) chloride as the catalyst (Scheme 1).

The mechanism of the three-component reaction has also been investigated [20]. A detailed

proposal in Scheme 2 indicates the three possible pathways (

, the acetylene reacts with the dialkyl phosphite by an oxidative coupling to give alkynylphosphonate

, which may react with the azide by click reaction to generate the 1,2,3-triazol-5-yl-phosphonate ( ).

A–C) of the reaction. According to pathway

A

5

3

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Based on pathway

B, the aerobic oxidative coupling of the dialkyl phosphite with the in situ-formed

organocopper intermediate (

reaction of acetylene with azide provides triazole

the dialkyl phosphite resulting in product

6

) may lead to triazol-5-yl-phosphonate (

, which undergoes an oxidative coupling with

. It was concluded that the reaction took place through

3). While in pathway C, the click

7

3

pathway B.

Scheme 1. Synthesis of 1,2,3-triazol-5-yl-phosphonates (3).

Scheme 2. Proposed reaction mechanism for the Cu(I)-catalyzed domino reaction [20].

In this paper, we report on the synthesis of novel 1,2,3-triazol-5-yl-phosphonates by the

copper(I)-catalyzed three-component domino reaction of phenylacetylene, various organic azides

and dialkyl phosphites. The biological activity of the compounds synthesized was also studied in

antibacterial activity and in vitro cytotoxicity assays.

2. Results and Discussion

In our prior work, the copper(I)-catalyzed domino reaction of phenylacetylene, benzyl azide

and diethyl phosphite was investigated [35]. The results reported herein include a comprehensive

optimization of the domino reaction using dibutyl phosphite (DBP) as a phosphorus reagent, in respect

of the molar ratio of the starting materials, the atmosphere, the additives (catalyst, base and solvent),

and of the temperature and the reaction time as well (Table 2).

At ﬁrst, the multicomponent reaction was performed in the presence of 10 mol% of CuCl as a

catalyst and two equivalents of TEA (triethylamine) as a base at room temperature in acetonitrile under

diﬀerent atmospheres. Reacting the three components at a molar ratio of 1:1.1:1.1 under nitrogen

atmosphere, the 1-benzyl-4-phenyl-1H-1,2,3-triazole (

236.1188} was obtained as the main product instead of the desired triazol-5-yl-phosphonate (

Entry 1). Repeating the reaction using continuous air bubbling, the triazol-5-yl-phosphonate (

formed in 55%, along with 42% of the “click product” ( ) and 3% of dibutyl (phenylethynyl)phosphonate

9

) {[M + H]⁺

= 236.1165, C₁₅H₁₄N₃requires

found

8) (Table 2,

8) was

9

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(

10) {³¹P NMR (CDCl₃) δ −5.8; [M + H]⁺

= 295.1440, C₁₆H₂₄O₃P requires 295.1463} (Table 2 , Entry

found

2). Applying a continuous oxygen bubbling, the proportion of alkynylphosphonate (10) signiﬁcantly

increased and the 1,2,3-triazol-5-yl-phosphonate (8) was obtained only in 24% (Table 2, Entry 3). After

that, the eﬀect of various bases was investigated, thus the domino reaction was also carried out in the

presence of DPA (dipropylamine), DIPA (diisopropylamine) or DIPEA (N,N-diisopropylethylamine)

(Table 2, Entries 4–6). In terms of the formation of the desired 1,2,3-triazol-5-yl-phosphonate (8),

TEA was proved to be the most eﬃcient. The eﬀect of the solvent was also studied, from among

the solvents tried out (MeCN, DMF, DCM, THF and EtOH), MeCN turned out as the most suitable

(Table 2, Entries 2 and 7–10). In the next step, the counterion of the copper catalyst was varied.

The reaction was performed with CuCl, CuBr, CuI or CuSO₄

achieved using CuCl (Table 2 , Entries 2,11–13). Increasing the amount of dibutyl phosphite to two

equivalents, the proportion of the desired product ( ) could be further improved (59%) (Table 2, Entry

14). Applying a larger excess of dibutyl phosphite or more CuCl did not favor the formation of the

triazol-5-yl-phosphonate ( ) (Table 2, Entries 15 and 16). Finally, the eﬀect of the temperature was

investigated. Neither lowering (0 ^◦C) nor raising (50 ^◦C) the temperature caused improvement in the

ratio of 1,2,3-triazol-5-yl-phosphonate ( ) (Table 2, Entries 17 and 18). The best result was obtained

·

5H₂O/NaAsc, and the best result was

8

using two equivalents of dibutyl phosphite in the presence of two equivalents of TEA and 10 mol% of

CuCl at room temperature in acetonitrile and continuous air bubbling (Table 2, Entry 14).

Table 2. Optimization of the domino reaction of phenylacetylene, benzyl azide and dibutyl phosphite.

Product Composition (%) ^a

Catalyst (10

Entry

DBP (Equiv.)

Base (2 Equiv.)

Condition

Solvent

8

9

10

mol%)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16 ^b

17 ^c

18 ^d

1.1

2

N₂bubbling

air bubbling

O₂bubbling

air bubbling

CuCl

CuBr

CuI

TEA

DPA

DIPA

DIPEA

TEA

MeCN

DMF

DCM

THF

EtOH

MeCN

22

55

24

46

54

35

41

43

21

39

42

26

0

77

42

70

49

44

63

57

56

78

59

56

72

100

37

45

49

62

75

1

3

6

5

2

1

2

0

4

2

CuSO₄5H₂O/NaAsc

·

CuCl

59

51

49

36

23

3

2

a

b

c

Based on HPLC (222 nm). The reaction was performed using 15 mol% of CuCl. The reaction was carried out at

0 ^◦C. ^dThe reaction was carried out at 50 ^◦C.

Applying the optimal parameters, the reaction time was also investigated. It was found that, during the

first eight hours, the proportion of the desired dibutyl (1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phosphonate

(8) increased, and the proportion of the 1-benzyl-4-phenyl-1H-1,2,3-triazole (9) continuously decreased

(Figure 1 ). It should be noted that the dibutyl (phenylethynyl)phosphonate (10) did not form in a significant

amount during the observed 24 h. Based on Figure 1 , a reaction time of eight hours was considered as

optimal for the domino reactions.

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100

90

80

70

60

50

40

30

20

10

0

8

9

10

0

2

4

6

8

10

12

14

16

18

20

22

24

Time [h]

Figure 1. Time dependence of the product composition in the model reaction of phenylacetylene, benzyl

azide and dibutyl phosphite. Conditions: phenylacetylene (1 equiv.), benzyl azide (1.1 equiv.), dibutyl

phosphite (2 equiv.), CuCl (0.1 equiv.) and TEA (2 equiv.) were stirred in MeCN with continuous air

a

bubbling at 25 ^◦C for 24 h. Based on HPLC (222 nm).

In the next series of experiments, the three-component reaction of phenylacetylene, various benzyl

azides and dialkyl phosphites was investigated under the optimized conditions (Scheme 3). First, the

domino reaction of phenylacetylene and benzyl azide was carried out with a wide range of dialkyl

phosphites. Using dimethyl phosphite, the dimethyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate

(11) was isolated in a yield of 53%. Performing the reactions with diethyl- or dipropyl phosphite,

the product 12 and 13 was prepared in a yield of 62% and 43%, respectively. Changing for a

dialkyl phosphite containing branched alkyl chains, such as diisopropyl phosphite, the desired

triazol-5-yl-phosphonate (14) was obtained in a yield of 40% after column chromatography. The dibutyl

(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phosphonate (8) was isolated from the experiments of Table 2,

Entry 14 in a yield of 49%. Applying a dialkyl phosphite incorporating long alkyl chains, such as

dipentyl phosphite, the corresponding dipentyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate

(15) was prepared in a yield of 34%. After that, the domino reaction was performed with substituted

benzyl azides (4-methylbenzyl azide 2-, 3- or 4-ﬂuorobenzyl azide or 4-(triﬂuoromethyl)benzyl azide).

Carrying out the reaction of phenylacetylene and 4-methylbenzyl azide with dimethyl-, diethyl- or

dibutyl phosphite, the desired triazol-5-yl-phosphonates (16–18) could be isolated in yields of 40%,

52% and 42%, respectively. Finally the domino reaction of phenylacetylene, and dibutyl phosphite

was also carried out with 2-, 3- or 4-ﬂuorobenzyl azide or 4-(triﬂuoromethyl)benzyl azide, and the

corresponding dibutyl (1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phosphonate derivatives (19–22) were

synthesized in yields of 30–39%.

The CuCl-catalyzed domino reaction was also carried out with aliphatic azides, such

as octyl or iso-octyl azide using the optimized conditions (Scheme 4). In the reactions of

phenylacetylene, octyl azide and dimethyl-, diethyl- or dibutyl phosphite, the corresponding

1-octyl-4-phenyl-1H-1,2,3-triazolyl phosphonates (23–25) were prepared in yields of 43–58%. Finally,

iso-octyl azide was reacted with phenylacetylene and dibutyl phosphite, and the desired product (26

)

was obtained in a yield of 28%, possibly due to the steric hindrance of the 2-ethylhexyl group.

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Scheme 3. Domino reaction of phenylacetylene, substituted benzyl azides and dialkyl phosphites.

a

Isolated yield.

The multicomponent approach developed is a fast and simple methodology for the synthesis of

1,2,3-triazol-5-yl-phosphonates, which utilizes simple starting materials and applies milder reaction

conditions compared to the preparation of these compounds by 1,3-dipolar cycloaddition. In contrast

to a previous report dealing with a similar topic [20], besides a comprehensive optimization, we have

provided exact product compositions, including the amount of the “click product” and the amount of

the (phenylethynyl)phosphonate, respectively. The scope of the reaction was extended for 15 novel

derivatives. (Copies of ³¹P, ¹H, and ¹³C NMR spectra for all compounds synthesized are presented in

Supplementary Materials).

Finally, the biological activity of the synthesized 1,2,3-triazol-5-yl-phosphonates (8, 11–26) was

investigated. On the one hand, the antibacterial activity of the compounds was tested on GFP

producing Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells. The GFP

producing bacteria are an eﬀective tool for screening for antibacterial activity since GFP signal

measured via ﬂuorimetry is proportional to the number of bacterial cells. Active compounds

would kill bacterial cells, which results in the decrease in GFP ﬂuorescence signal, therefore

it is suitable for evaluating the antimicrobial eﬀect of diﬀerent agents. On the other hand,

in vitro cytotoxicity assessments were performed on three diﬀerent cell lines, such as human lung

adenocarcinoma (A549), mouse ﬁbroblast (NIH/3T3) as healthy cell line and human promyelocytic

leukemia (HL-60). Table 3 contains the determined IC₅₀values in antimicrobial and cytotoxicity

assays. According to the results, only modest antibacterial activity was detected against the more

sensitive Bacillus subtilis strain. The IC₅₀values of triazolyl phosphonates (8, 13, 15, 16, 19–22

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and 26), which showed some activity against NIH/3T3 and/or HL-60 cells fall in the range of

9.7 and 27.5 M. Dimethyl [1-(4-methylbenzyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (16) and

µ

dibutyl [1-(4-triﬂuoromethyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (22) proved to be the most active

compounds, which showed activity in the 10 micromolar range against HL-60 cells.

Scheme 4. Domino reaction of phenylacetylene, aliphatic azides and dialkyl phosphites. ^aIsolated yield.

Table 3. Summary of biological activity measurements.

Antibacterial Activity (IC₅₀, µM)

In Vitro Cytotoxicity (IC₅₀, µM)

R¹

R²

Compound

B. subtilis

E. coli

A549

NIH/3T3

HL-60

8

Bn

Bu

Me

Et

>30

–

27.5 ± 1.4

>30

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

>30

26.2 ± 1.1

19.8 ± 1.2

>30

Pr

29.3 ± 1.2

12.6 ± 1.7

ⁱPr

Pn

Me

Et

>30

Bn

23.9 ± 1.0

>30

18.2 ± 1.0

19.3 ± 1.0

>30

15.4 ± 1.2

11.0 ± 1.2

>30

4-MeC₆H₄

2-FC₆H₄

3-FC₆H₄

4-FC₆H₄

4-CF₃C₆H₄

Oct

Bu

Me

Et

>30

27.5 ± 1.1

11.7 ± 1.2

15.4 ± 1.2

16.6 ± 1.3

9.7 ± 1.1

>30

23.2 ± 1.1

>30

23.1 ± 1.2

>30

Oct

>30

–

>30

Oct

Bu

ⁱOct

29.6 ± 1.0

0.04 ± 0.01

0.49 ± 0.14

–

26.7 ± 1.0

Doxycycline

Gentamicin

Doxorubicin

0.10 ± 0.02

4.23 ± 0.99

–

0.31 ± 0.24 5.65 ± 0.81

–

7.42

2.60

×

10⁻³

Bortezomib

–

×

Data were expressed as mean ± standard deviation.

Molecules 2020, 25, 2643

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3. Materials and Methods

3.1. General

The reactions under conventional heating were carried out in an oil bath. The microwave-assisted

experiments were performed in a 300 W CEM Discover focused microwave reactor (CEM Microwave

Technology Ltd., Buckingham, UK) equipped with a pressure controller using 5–10 W irradiation

under isothermal conditions.

HPLC-MS measurements were performed with an Agilent 1200 liquid chromatography system

coupled with a 6130 quadrupole mass spectrometer equipped with an ESI ion source (Agilent

Technologies, Palo Alto, CA, USA). The analysis was performed at 40 ^◦C on a Gemini C18 column (150

mm

×

4.6 mm, 3 µm; Phenomenex, Torrance, CA, USA) with a mobile phase ﬂow rate of 0.6 mL/min.

The composition of eluent A was 0.1% (NH₄)(HCOO) in water; eluent B was 0.1% (NH₄)(HCOO) and

8% water in acetonitrile. 0–3 min. 5% B, 3–13 min. gradient, 13–20 min. 100% B. The injection volume

was 5 µL. The chromatographic proﬁle was registered at 222 nm. The MSD operating parameters were

as follows: positive ionization mode, scan spectra from m/z 120 to 1200, drying gas temperature 300 ^◦C,

nitrogen ﬂow rate 10 L/min, nebulizer pressure 60 psi, capillary voltage 4000 V.

High resolution mass spectrometric measurements were performed using a Sciex 5600+ Q-TOF

mass spectrometer in positive electrospray mode.

The ³¹P, ¹H, ¹³C, NMR spectra were taken in CDCl₃solution on a Bruker AV-300 spectrometer

operating at 121.5, 300 and 75.5 MHz, respectively. Chemical shifts are downﬁeld relative to 85%

H₃PO₄and TMS.

3.2. General Procedure for the Synthesis of Organic Azides

3.2.1. Method A

To a stirred solution of 10.0 mmol alkyl halides (1.19 mL of benzyl bromide, 1.85 g of 4-methylbenzyl

bromide, 1.21 mL of 2-ﬂuorobenzyl bromide, 1.23 mL of 3-ﬂuorobenzyl bromide, 1.25 mL of

4-ﬂuorobenzyl bromide or 1.55 mL of 4-(triﬂuoromethyl)benzyl bromide) in 100 mL of acetone/H₂O

4:1 (v/v) was added 15.0 mmol (0.98 g) of sodium azide. The reaction mixture was stirred at room

temperature for 24 h. After that, the reaction was extracted with Et₂O (3

and concentrated under reduced pressure to give benzyl azides as pale yellow oils.

×

50 mL), dried over Na₂SO₄,

3.2.2. Method B

To a stirred solution of 10.0 mmol alkyl halides (1.76 mL of octyl bromide or 1.78 mL of iso-octyl

bromide) in 20 mL of DMF, 12.0 mmol (0.78 g) of sodium azide was added. The reaction mixture was

stirred at 70 ^◦C for 24 h in an oil bath. After, the reaction was extracted with Et₂O (3

over Na₂SO₄, and concentrated under reduced pressure to give alkyl azides as pale yellow oils.

The following organic azides were prepared using method A or method B (Table 4):

×

50 mL), dried

Table 4. Yield and mass data for the organic azides prepared.

R¹N₃

Yield

[M + H]⁺

found

[M + H]⁺

requires

BnN₃[36]

4-Me-BnN₃[37]

2-F-BnN₃[38]

3-F-BnN₃[39]

4-F-BnN₃[37]

4-CF₃-BnN₃[40]

OctN₃[41]

93% (1.24 g)

80% (1.18 g)

68% (1.02 g)

76% (1.14 g)

83% (1.25 g)

86% (1.72 g)

67% (1.04 g)

55% (0.85 g)

134.0725

148.0880

152.0632

152.0633

152.0632

202.0601

156.1514

134.0718

148.0874

152.0624

202.0592

156.1501

ⁱOctN₃[42]

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3.3. General Procedure for the Synthesis of Dipropyl and Dipentyl Phosphite

A mixture of 5.0 mmol (0.64 mL) of diethyl phosphite and 25 equivalents, and 150.0 mmol of

alcohol (10.0 mL n-propanol or 14.0 mL n-pentanol) was heated at 175 ^◦C in a vial in the MW reactor for

120–135 min. The volatile components were removed in vacuum and the crude product was puriﬁed

by column chromatography using silica gel as the solid phase and ethyl acetate as the eluent. The

following dialkyl phosphites were thus prepared (Table 5):

Table 5. Yield, ³¹P NMR and MS data for the dialkyl phosphites prepared.

(R²O)₂P(O)H

Yield

δ

in CDCl₃

δ

[lit.] in CDCl₃

[M + H]⁺

found

[M + H]⁺

requires

P

(PrO)₂P(O)H

(PentO)₂P(O)H

65% (0.54 g)

68% (0.75 g)

7.9

7.8

7.8 [43 ]

8.1 [43 ]

167.0833

223.1462

167.0837

223.1463

3.4. General Procedure for the Synthesis of 1,2,3-Triazol-5-yl-Phosphonates

In combination, 1.0 mmol phenylacetylene (0.12 mL), 1.1 mmol organic azide (0.14 g of benzyl

azide, 0.17 g of 4-methylbenzyl azide, 0.17 g of 2-ﬂuorobenzyl azide, 0.17 g of 3-ﬂuorobenzyl azide,

0.17 g of 4-ﬂuorobenzyl azide, 0.22 g of 4-(triﬂuoromethyl)benzyl azide, 0.18 g of octyl azide or 0.18 g

of iso-octyl azide) and 2.0 mmol dialkyl phosphite (0.18 mL dimethyl phosphite, 0.26 mL diethyl

phosphite, 0.33 g dipropyl phosphite, 0.33 mL diisopropyl phosphite, 0.40 mL dibutyl phosphite,

0.44 g dipentyl phosphite) were dissolved in acetonitrile (20 mL). To this was added 2 mmol (0.28

mL) triethylamine and 0.10 mmol (0.02 g) CuCl. The mixture was stirred at room temperature with

continuous air bubbling for 8 h. The resulting solution was extracted with ethyl acetate (3

×

30 mL) and

the combined organic layers were dried over Na₂SO₄. After evaporating the solvent, the crude product

was puriﬁed by column chromatography using silica gel and ethyl acetate/hexane 1:1 as the eluent and,

if it was necessary, by reversed-phase column chromatography using C₁₈-reversed-phase silica gel and

the composition of eluent A was 0.1% (NH₄)(HCOO) in water; eluent B was 0.1% (NH₄)(HCOO) and

8% water in acetonitrile. The following products were thus prepared:

Dimethyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (11): yield: 53% (0.18 g), colorless oil; ³¹P NMR

(CDCl₃)

δ δ

7.5; ¹H NMR (CDCl₃) 3.40 (d, 6H, ³J_HP= 11.8, OCH₃), 6.01 (s, 2H, CH₂Ph), 7.26–7.36 (m,

3H, C_3’H, C_4’H), 7.36–7.45 (m, 5H, C₃H, C₄H, C_2’H), 7.74 (dd, 2H, ³J_HH= 7.4, J_HH= 2.2, C₂H); ¹³

C

NMR (CDCl₃) δ

53.0 (d, ²J_CP= 5.1, OCH₃), 54.3 (CH₂Ph), 120.5 (d, ¹J_CP= 219.4, P(O)C=), 128.2 (C₂),

128.3 (C_2’), 128.5 (C_4’), 128.8 (C_3’), 129.1 (C₃), 129.2 (C₁), 130.0 (C₄), 135.9 (C_1’), 153.9 (d, ²J_CP= 19.5,

PhC=); [M + H]⁺_found= 344.1161, C₁₇H₁₉N₃O₃P requires 344.1164.

Diethyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (12): yield: 62% (0.23 g), colorless oil; ³¹P NMR

(CDCl₃)

(m, 2H, OCH₂CH₃), 3.82–3.95 (m, 2H, OCH₂CH₃), 6.03 (s, 2H, CH₂Ph), 7.26–7.36 (m, 3H, C_3’H, C_4’H),

7.37–7.44 (m, 5H, C₃H, C₄H, C_2’H), 7.76 (dd, 2H, ³J_HH= 7.4, J_HH= 2.2, C₂H);

20] (CDCl₃) 0.96 (t, 6H,

J = 7.2 Hz), 3.57–3.67 (m, 2 H), 3.81–3.91 (m, 2 H), 6.01 (s, 2 H), 7.30–7.40 (m, 8 H), 7.73–7.75 (m, 2 H);

δ 4.4; δ [20] (CDCl₃) δ δ

4.53; ¹H NMR (CDCl₃) 0.98 (t, 6H, ³J_HH= 7.1, OCH₂CH₃), 3.58–3.72

δ

[

¹³C NMR (CDCl₃)

δ

15.9 (d, ³J_CP= 7.0, OCH₂CH₃), 54.2 (CH₂Ph), 63.1 (d, ²J_CP= 5.2, OCH₂CH₃), 121.6

(d, ¹J_CP= 217.5, P(O)C=), 128.2 (C₂), 128.3 (C_2’), 128.4(C_4’), 128.7 (C_3’), 129.1 (C₁), 129.3 (C₃), 130.3

2

(C₄), 135.9 (C_1’), 153.6 (d, J_CP= 19.0, PhC=);

127.2, 127.3, 127.4, 127.7, 128.1, 128.3, 129.2, 134.9, 152.6; [M + H]⁺

δ [20] (CDCl₃) 14.8, 14.9, 53.2, 62.0, 62.1, 119.4, 121.6,

= 372.1479, C₁₉H₂₃N₃O₃P

found

requires 372.1477.

Dipropyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (13): yield: 43% (0.17 g), colorless oil; ³¹P

1

NMR (CDCl₃)

δ

4.6; H NMR (CDCl₃)

δ

0.68 (t, 6H, ³J_HH= 7.4, OCH₂CH₂CH₃), 1.27–1.42 (m, 4H,

OCH₂CH₂CH₃), 3.42–3.60 (m, 2H, OCH₂CH₂CH₃), 3.70–3.84 (m, 2H, OCH₂CH₂CH₃), 6.04 (s, 2H,

CH₂Ph), 7.25–7.35 (m, 3H, C_3’H, C_4’H), 7.35–7.45 (m, 5H, C₃H, C₄H, C_2’H), 7.77 (dd, 2H, ³J_HH= 7.2,

J_HH= 2.5, C₂H); ¹³C NMR (CDCl₃)

δ

9.9 (OCH₂CH₂CH₃)_,23.4 (d, ³J_CP= 7.1, OCH₂CH₂CH₃), 54.2

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(CH₂Ph), 68.4 (d, ²J_CP= 5.6, OCH₂CH₂CH₃), 121.6 (d, ¹J_CP= 217.9, P(O)C=), 128.2 (C₂), 128.4 (C_2’),

128.4(C_4’), 128.7 (C_3’), 129.1 (C₁), 129.2 (C₃), 130.3 (C₄), 136.0 (C_1’), 153.6 (d, ²J_CP= 19.0, PhC=); [M +

H]⁺

= 400.1778, C₂₁H₂₇N₃O₃P requires 400.1790.

found

Diisopropyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (14): yield: 40% (0.16 g), colorless oil; ³¹

NMR (CDCl₃) 2.0;

20] (CDCl₃) 2.19; ¹H NMR (CDCl₃) 0.83 (d, 6H, ³J_HH= 6.2, OCH(CH₃)₂), 1.19

(d, 6H, ³J_HH= 6.2, OCH(CH₃)₂), 4.23–4.44 (m, 2H, OCH(CH₃)₂), 6.06 (s, 2H, CH₂Ph), 7.24–7.35 (m,

3H, C_3’H, C_4’H), 7.36–7.45 (m, 5H, C₃H, C₄H, C_2’H), 7.81 (dd, 2H, ³J_HH= 7.6, J_HH= 2.2, C₂H);

20

(CDCl₃) 0.80 (d, 6H, J = 6.4 Hz), 1.17 (d, 6H, J = 6.0 Hz), 4.27–4.35 (m, 2 H), 6.05 (s, 2 H), 7.27–7.39 (m, 8

P

δ

[

δ

[ ]

H), 7.79 (d, 2 H, J = 5.6 Hz); ¹³C NMR (CDCl₃) 23.2 (d, ³J_CP= 5.6, OCH(CH₃)₂), 23.9 (d, ³J_CP= 3.9,

δ

OCH(CH₃)₂), 54.2 (CH₂Ph), 72.6 (d, ²J_CP= 5.5, OCH(CH₃)₂), 122.6 (d, ¹J_CP= 218.6, P(O)C=), 128.1 (C₂),

128.29 (C_4’), 128.32 (C_2’), 128.7 (C_3’), 128.9 (C₁), 129.3 (C₃), 130.6 (C₄), 136.1 (C_1’), 153.2 (d, ²J_CP= 18.9,

PhC=);

δ

[

20] (CDCl₃) 22.1, 22.2, 22.8, 22.9, 53.1, 71.6, 120.5, 122.7, 127.1, 127.3, 127.6, 127.9, 128.3, 129.5,

135.1, 152.2; [M + H]⁺

= 400.1777, C₂₁H₂₇N₃O₃P requires 400.1790.

found

Dibutyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (

(CDCl₃)

8

): yield: 49% (0.20 g), colorless oil; ³¹P NMR

δ 2.2; H NMR (CDCl₃) δ 0.76 (t, 6H, J_HH= 7.3, OCH₂CH₂CH₂CH₃), 0.99–1.19 (m, 4H,

1

3

OCH₂CH₂CH₂CH₃), 1.22–1.38 (m, 4H, OCH₂CH₂CH₂CH₃), 3.46–3.62 (m, 2H, OCH₂CH₂CH₂CH₃),

3.72–3.91 (m, 2H, OCH₂CH₂CH₂CH₃), 6.03 (s, 2H, CH₂Ph), 7.25–7.34 (m, 3H, C_3’H, C_4’H), 7.36–7.43

3

(m, 5H, C₃H, C₄H, C_2’H), 7.76 (dd, 2H, J_HH= 6.6, J_HH= 2.9, C₂H); ¹³C NMR (CDCl₃)

δ 13.5

(OCH₂CH₂CH₂CH₃), 18.5 (OCH₂CH₂CH₂CH₃), 32.0 (d, ³J_CP= 7.0, OCH₂CH₂CH₂CH₃), 54.2 (CH₂Ph),

66.7 (d, ²J_CP= 5.5, OCH₂CH₂CH₂CH₃), 121.6 (d, ¹J_CP= 217.8, P(O)C=), 128.2 (C₂), 128.3 (C_2’), 128.4

2

(C_4’), 128.7 (C_3’), 129.1 (C₁), 129.3 (C₃), 130.4 (C₄), 136.0 (C_1’), 153.6 (d, J_CP= 19.1, PhC=); [M +

H]⁺

= 428.2086, C₂₃H₃₁N₃O₃P requires 428.2103.

found

Dipentyl (1-benzyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (15): yield: 34% (0.15 g), colorless oil; ³¹P

NMR (CDCl₃)

2.4; ¹H NMR (CDCl₃) 0.80 (t, 6H, ³J_HH= 7.1, OCH₂CH₂CH₂CH₂CH₃), 1.00–1.13

δ

(m, 4H, OCH₂CH₂CH₂CH₂CH₃), 1.13–1.25 (m, 4H, OCH₂CH₂CH₂CH₂CH₃), 1.25–1.40 (m, 4H,

OCH₂CH₂CH₂CH₂CH₃), 3.47–3.62 (m, 2H, OCH₂CH₂CH₂CH₃), 3.74–3.89 (m, 2H, OCH₂CH₂CH₂CH₃),

6.03 (s, 2H, CH₂Ph), 7.27–7.37 (m, 3H, C_3’H, C_4’H), 7.37–7.47 (m, 5H, C₃H, C₄H, C_2’H), 7.76

3

(dd, 2H, J_HH= 6.6, J_HH= 3.0, C₂H); ¹³C NMR (CDCl₃)

δ 14.0 (OCH₂CH₂CH₂CH₂CH₃), 22.2

(OCH₂CH₂CH₂CH₂CH₃), 27.4 (OCH₂CH₂CH₂CH₂CH₃), 29.7 (d, ³J_CP= 7.1, OCH₂CH₂CH₂CH₂CH₃),

54.2 (CH₂Ph), 67.0 (d, ²J_CP= 5.5, OCH₂CH₂CH₂CH₂CH₃), 121.6 (d, ¹J_CP= 217.8, P(O)C=), 128.2 (C₂),

128.3 (C_2’), 128.4 (C_4’), 128.7 (C_3’), 129.1 (C₁), 129.3 (C₃), 130.3 (C₄), 136.0 (C_1’), 153.6 (d, ²J_CP= 19.1,

PhC=); [M + H]⁺_found= 456.2398, C₂₅H₃₅N₃O₃P requires 456.2416.

Dimethyl [1-(4-methylbenzyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (16): yield: 40% (0.14 g), colorless oil;

³¹P NMR (CDCl₃) 7.5; ¹H NMR (CDCl₃) 2.32 (s, 3H, PhCH₃), 3.43 (d, 6H, ³J_HP= 11.7, OCH₃), 5.96 (s,

δ δ

2H, CH₂Ph), 7.15 (d, 2H, ³J_HH= 7.8, C_3’H), 7.30 (d, 2H, ³J_HH= 7.9, C_2’H), 7.40–7.44 (m, 3H, C₃H, C₄H),

7.73 (dd, 2H, ³J_HH= 7.3, J_HH= 2.4, C₂H); ¹³C NMR (CDCl₃)

δ

21.3 (CH₃Ph), 53.0 (d, ²J_CP= 5.3, OCH₃),

54.1 (CH₂Ph), 120.4 (d, ¹J_CP= 219.5, P(O)C=), 128.3 (C₂, C_2’), 129.15 (C_3’), 129.17 (C₁), 129.4 (C₃), 130.1

2

(C₄), 132.8 (C_1’), 138.3 (C_4’), 153.8 (d, J_CP= 19.4, PhC=); [M + H]⁺

= 358.1315, C₁₈H₂₁N₃O₃P

found

requires 358.1315.

Diethyl [1-(4-methylbenzyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (17): yield: 52% (0.20 g), colorless oil;

³¹P NMR (CDCl₃) 4.3; ¹H NMR (CDCl₃) 0.99 (t, 6H, ³J_HH= 7.0, OCH₂CH₃), 2.32 (s, 3H, PhCH₃),

δ δ

3.58–3.72 (m, 2H, OCH₂CH₃), 3.85–3.95 (m, 2H, OCH₂CH₃), 6.00 (s, 2H, CH₂Ph), 7.14 (d, 2H, ³J_HH= 7.8,

C_3’H), 7.30 (d, 2H, ³J_HH= 8.0, C_2’H), 7.38–7.44 (m, 3H, C₃H, C₄H), 7.74 (dd, 2H, ³J_HH= 7.7, J_HH= 2.0,

C₂H); ¹³C NMR (CDCl₃) 15.9 (d, ³J_CP=7.0, OCH₂CH₃), 21.2 (CH₃Ph), 54.0 (CH₂Ph), 63.1 (d, ²J_CP=5.1,

δ

OCH₂CH₃), 121.5 (d, ¹J_CP= 217.7, P(O)C=), 128.2 (C₂), 128.3 (C_2’), 129.0 (C₁), 129.3 (C_3’), 129.4 (C₃),

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130.4 (C₄), 132.9 (C_1’), 138.2 (C_4’), 153.5 (d, ²J_CP= 19.1, PhC=); [M + H]⁺_found= 386.1633, C₂₀H₂₅N₃O₃P

requires 386.1628.

Dibutyl [1-(4-methylbenzyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (18): yield: 42% (0.19 g), colorless oil;

³¹P NMR (CDCl₃)

δ δ

3.1; ¹H NMR (CDCl₃) 0.69 (t, 6H, ³J_HH= 7.3, OCH₂CH₂CH₂CH₃), 0.93–1.10 (m,

4H, OCH₂CH₂CH₂CH₃), 1.11–1.13 (m, 4H, OCH₂CH₂CH₂CH₃), 2.25 (s, 3H, PhCH₃), 3.41–3.57 (m, 2H,

OCH₂CH₂CH₂CH₃), 3.67–3.83 (m, 2H, OCH₂CH₂CH₂CH₃), 5.91 (s, 2H, CH₂Ph), 7.06 (d, 2H, ³J_HH= 7.8,

C_3’H), 7.22 (d, 2H, ³J_HH= 7.7, C_2’H), 7.27–7.38 (m, 3H, C₃H, C₄H), 7.67 (dd, 2H, ³J_HH= 6.6, J_HH= 3.0,

C₂H); ¹³C NMR (CDCl₃)

δ 13.6 (OCH₂CH₂CH₂CH₃), 18.5 (OCH₂CH₂CH₂CH₃), 21.3 (CH₃Ph), 32.0

(d, ³J_CP= 7.1, OCH₂CH₂CH₂CH₃), 54.1 (CH₂Ph), 66.7 (d, ²J_CP= 5.5, OCH₂CH₂CH₂CH₃), 121.5 (d,

¹J_CP= 218.5, P(O)C=), 128.2 (C₂), 128.4 (C_2’), 129.0 (C₁), 129.3 (C_3’), 129.4 (C₃), 130.4 (C₄), 133.1 (C_1’),

138.2 (C_4’), 153.5 (d, ²J_CP= 19.3, PhC=); [M + H]⁺_found= 442.2242, C₂₄H₃₃N₃O₃P requires 442.2260.

Dibutyl [1-(2-ﬂuorobenzyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (19): yield: 30% (0.13 g), pale yellow oil;

³¹P NMR (CDCl₃)

δ δ

4.6; ¹H NMR (CDCl₃) 0.79 (t, 6H, ³J_HH= 7.3, OCH₂CH₂CH₂CH₃), 1.07–1.22 (m,

4H, OCH₂CH₂CH₂CH₃), 1.29–1.42 (m, 4H, OCH₂CH₂CH₂CH₃), 3.59–3.76 (m, 2H, OCH₂CH₂CH₂CH₃),

3.83–3.99 (m, 2H, OCH₂CH₂CH₂CH₃), 6.10 (s, 2H, CH₂Ph), 7.03–7.15 (m, 3H, C_3’H, C_5’H, C_6’H),

7.24–7.35 (m, 1H, C_4’H), 7.38–7.47 (m, 3H, C₃H, C₄H), 7.79 (dd, 2H, ³J_HH= 7.2, J_HH= 2.6, C₂H); ¹³C NMR

(CDCl₃) δ

13.5 (OCH₂CH₂CH₂CH₃), 18.6 (OCH₂CH₂CH₂CH₃), 32.0 (d, ³J_CP= 6.9, OCH₂CH₂CH₂CH₃),

48.2 (d, ³J_CP=4.7, CH₂Ph), 66.8 (d, ²J_CP= 5.7, OCH₂CH₂CH₂CH₃), 115.7 (d, ²J_CF= 21.1, C_3’), 122.1 (d,

¹J_CP= 218.6, P(O)C=), 123.4 (d, ²J_CF= 14.3, C_1’), 124.4 (d, ²J_CF= 3.7, C_5’), 128.2 (C₂), 129.1 (C₁), 129.3

(C₃), 129.4 (d, ³J_CF= 3.2, C_4’), 130.1 (d, ³J_CF= 8.1, C_6’), 130.2 (C₄), 153.5 (d, ²J_CP= 19.2, PhC=), 160.4 (d,

¹J_CF= 248.8, C_2’); [M + H]⁺

= 446.1975, C₂₃H₃₀N₃O₃FP requires 446.2008.

found

Dibutyl [1-(3-ﬂuorobenzyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (20): yield: 34% (0.15 g), pale yellow oil;

³¹P NMR (CDCl₃) 2.1; ¹H NMR (CDCl₃) 0.77 (t, 6H, ³J_HH= 7.3, OCH₂CH₂CH₂CH₃), 1.03–1.22 (m,

δ

4H, OCH₂CH₂CH₂CH₃), 1.22–1.42 (m, 4H, OCH₂CH₂CH₂CH₃), 3.53–3.69 (m, 2H, OCH₂CH₂CH₂CH₃),

3.81–3.94 (m, 2H, OCH₂CH₂CH₂CH₃), 6.03 (s, 2H, CH₂Ph), 6.95–7.05 (m, 1H, C_4’H), 7.06–7.22 (m, 2H,

C_2’H, C_6’H), 7.25–7.34 (m, 1H, C_5’H), 7.36–7.47 (m, 3H, C₃H, C₄H), 7.76 (dd, 2H, ³J_HH= 6.4, J_HH= 3.4,

C₂H); ¹³C NMR (CDCl₃)

δ

13.5 (OCH₂CH₂CH₂CH₃), 18.5 (OCH₂CH₂CH₂CH₃), 32.0 (d, ³J_CP= 7.0,

OCH₂CH₂CH₂CH₃), 53.5 (CH₂Ph), 66.8 (d, ²J_CP= 5.6, OCH₂CH₂CH₂CH₃), 115.3 (d, ²J_CF= 21.8, C_2’,

C_4’), 121.6 (d, ¹J_CP= 218.0, P(O)C=), 124.0 (d, J_CF= 3.1, C_6’), 128.2 (C₂), 129.1 (C₁), 129.2 (C₃), 130.2 (C₄),

130.3 (d, ³J_CF= 8.1, C_5’), 138.3 (d, ³J_CF= 7.5, C_1’), 153.5 (d, ²J_CP= 18.9, PhC=), 162.3 (d, ¹J_CF= 247.0,

C_3’); [M + H]⁺_found= 446.1969, C₂₃H₃₀N₃O₃FP requires 446.2008.

Dibutyl [1-(4-ﬂuorobenzyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (21): yield: 36% (0.16 g), pale yellow oil;

³¹P NMR (CDCl₃)

δ δ

2.3; ¹H NMR (CDCl₃) 0.77 (t, 6H, ³J_HH= 7.3, OCH₂CH₂CH₂CH₃), 1.04–1.19 (m,

4H, OCH₂CH₂CH₂CH₃), 1.22–1.41 (m, 4H, OCH₂CH₂CH₂CH₃), 3.49–3.68 (m, 2H, OCH₂CH₂CH₂CH₃),

3

3.77–3.96 (m, 2H, OCH₂CH₂CH₂CH₃), 6.00 (s, 2H, CH₂Ph), 7.03 (t, 2H, J_HH= J_HF= 8.7, C_2’H),

7.34–7.47 (m, 5H, C₃H, C₄H, C_3’H), 7.75 (dd, 2H, ³J_HH= 6.7, J_HH= 3.0, C₂H); ¹³C NMR (CDCl₃)

δ

3

13.5 (OCH₂CH₂CH₂CH₃), 18.5 (OCH₂CH₂CH₂CH₃), 32.0 (d, J_CP= 7.0, OCH₂CH₂CH₂CH₃), 53.4

2

1

(CH₂Ph), 66.7 (d, J_CP= 5.6, OCH₂CH₂CH₂CH₃), 115.6 (d, J_CF= 21.7, C_3’), 121.4 (d, J_CP= 218.0,

P(O)C=), 128.2 (C₂), 129.1 (C₁), 129.2 (C₃), 130.4 (d, ³J_CF= 8.3, C_2’), 130.2 (C₄), 131.7 (d, J_CF= 3.1, C_1’),

153.5 (d, J_CP= 19.0, PhC=), 162.8 (d, J_CF= 247.4, C_4’); [M + H]⁺

= 446.1972, C₂₃H₃₀N₃O₃FP

2

1

found

requires 446.2008.

Dibutyl [1-(4-triﬂuoromethyl)-4-phenyl-1,2,3-triazol-5-yl]phosphonate (22): yield: 39% (0.19 g), pale yellow

oil; ³¹P NMR (CDCl₃) 2.0; ¹H NMR (CDCl₃) 0.76 (t, 6H, ³J_HH= 7.3, OCH₂CH₂CH₂CH₃), 0.97–1.18 (m,

δ

4H, OCH₂CH₂CH₂CH₃), 1.20–1.40 (m, 4H, OCH₂CH₂CH₂CH₃), 3.53–3.67 (m, 2H, OCH₂CH₂CH₂CH₃),

3.77–3.95 (m, 2H, OCH₂CH₂CH₂CH₃), 6.10 (s, 2H, CH₂Ph), 7.37–7.46 (m, 3H, C₃H, C₄H), 7.51

3

(d, 2H, J_HH= 8.0, C_2’H), 7.61 (d, 2H, J_HH= 8.1, C_3’H), 7.76 (dd, 2H, J_HH= 6.8, J_HH= 2.9,

Molecules 2020, 25, 2643

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C₂H); ¹³C NMR (CDCl₃)

δ

13.4 (OCH₂CH₂CH₂CH₃), 18.5 (OCH₂CH₂CH₂CH₃), 31.9 (d, ³J_CP= 7.0,

OCH₂CH₂CH₂CH₃), 53.6 (CH₂Ph), 66.8 (d, ²J_CP= 5.7, OCH₂CH₂CH₂CH₃), 124.0 (q, ¹J_CF= 272.2, CF₃),

121.7 (d, ¹J_CP= 218.2, P(O)C=), 125.7 (q, ³J_CF= 3.8, C_3’), 128.2 (C₂), 128.7 (C₃), 129.2 (C₁, C_2’), 130.1

2

(C₄), 130.7 (q, J_CF= 32.6, C_4’), 139.9 (C_1’), 153.6 (d, J_CP= 18.9, PhC=); [M + H]⁺

= 496.1936,

found

C₂₄H₃₀N₃O₃F₃P requires 496.1976.

Dimethyl (1-octyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (23): yield: 43% (0.16 g), colorless oil; ³¹P

1

3

NMR (CDCl₃)

δ

8.0; H NMR (CDCl₃)

δ

0.81 (t, 3H, J_HH= 6.9, NCH₂(CH₂)₆CH₃), 1.12–1.37 (m,

3

10H, NCH₂CH₂(CH₂)₅CH₃), 1.82–2.02 (m, 2H, NCH₂CH₂(CH₂)₅CH₃), 3.58 (d, 6H, J_HP= 11.6,

3

OCH₃), 4.68 (t, 2H, J_HH= 7.5, NCH₂(CH₂)₆CH₃), 7.29–7.45 (m, 3H, C₃H, C₄H), 7.65 (dd, 2H,

³J_HH= 6.5, J_HH= 3.3, C₂H); ¹³C NMR (CDCl₃)

δ 14.2 (NCH₂(CH₂)₆CH₃), 22.7 (N(CH₂)₆CH₂CH₃), 26.7

(N(CH₂)₅CH₂CH₂CH₃), 29.16 (N(CH₂)₃CH₂CH₂(CH₂)₂CH₃), 29.20 (N(CH₂)₃CH₂CH₂(CH₂)₂CH₃),

31.1 (N(CH₂)₂CH₂(CH₂)₄CH₃), 31.8 (NCH₂CH₂(CH₂)₅CH₃), 51.4 (NCH₂CH₂(CH₂)₅CH₃), 53.2 (d,

²J_CP= 5.6, OCH₃), 120.6 (d, ¹J_CP= 221.5, P(O)C=), 128.2 (C₂), 129.1 (C₁), 129.3 (C₃), 130.2 (C₄), 153.5 (d,

²J_CP= 20.1, PhC=); [M + H]⁺_found= 366.1930, C₁₈H₂₉N₃O₃P requires 366.1946.

Diethyl (1-octyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (24): yield: 58% (0.23 g), colorless oil; ³¹P

1

3

NMR (CDCl₃)

δ 5.0; H NMR (CDCl₃) δ 0.88 (t, 3H, J_HH= 6.6, NCH₂(CH₂)₆CH₃), 1.16 (t, 6H,

³J_HH= 7.1, OCH₂CH₂CH₂CH₃), 1.22–1.47 (m, 10H, NCH₂CH₂(CH₂)₅CH₃), 1.93–2.06 (m, 2H,

NCH₂CH₂(CH₂)₅CH₃), 3.83–4.01 (m, 2H, OCH₂CH₃), 4.01–4.19 (m, 2H, OCH₂CH₃), 4.76 (t, 2H,

³J_HH= 7.5, NCH₂(CH₂)₆CH₃), 7.34–7.48 (m, 3H, C₃H, C₄H), 7.75 (dd, 2H, ³J_HH= 6.4, J_HH= 2.8, C₂H);

¹³C NMR (CDCl₃) 14.2 (NCH₂(CH₂)₆CH₃), 16.0 (d, ³J_CP= 7.0, OCH₂CH₃), 22.7 (N(CH₂)₆CH₂CH₃),

δ

26.7 (N(CH₂)₅CH₂CH₂CH₃), 29.19 (N(CH₂)₃CH₂CH₂(CH₂)₂CH₃), 29.21 (N(CH₂)₃CH₂CH₂(CH₂)₂CH₃),

31.1 (N(CH₂)₂CH₂(CH₂)₄CH₃), 31.8 (NCH₂CH₂(CH₂)₅CH₃), 51.3 (NCH₂CH₂(CH₂)₅CH₃), 63.1 (d,

²J_CP= 5.5, OCH₂CH₃), 121.7 (d, ¹J_CP= 220.2, P(O)C=), 128.1 (C₂), 129.0 (C₁), 129.5 (C₃), 130.5 (C₄),

153.2 (d, ²J_CP= 20.0, PhC=); [M + H]⁺

= 394.2241, C₂₀H₃₃N₃O₃P requires 394.2259.

found

Dibutyl (1-octyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (25): yield: 50% (0.22 g), colorless oil; ³¹P NMR

(CDCl₃) 0.75–0.97 (m, 9H, OCH₂CH₂CH₂CH₃, NCH₂(CH₂)₆CH₃), 1.13–1.55

5.3; ¹H NMR (CDCl₃)

δ

(m, 18H, OCH₂CH₂CH₂CH₃, NCH₂CH₂(CH₂)₅CH₃), 1.89–2.07 (m, 2H, NCH₂CH₂(CH₂)₅CH₃),

3.75–3.95 (m, 2H, OCH₂CH₂CH₂CH₃), 3.95–4.14 (m, 2H, OCH₂CH₂CH₂CH₃), 4.77 (t, 2H, ³J_HH= 7.4,

3

NCH₂(CH₂)₆CH₃), 7.34–7.51 (m, 3H, C₃H, C₄H), 7.74 (dd, 2H, J_HH= 6.7, J_HH= 3.1, C₂H); ¹³C

NMR (CDCl₃)

δ 13.6 (OCH₂CH₂CH₂CH₃), 14.2 (NCH₂(CH₂)₆CH₃), 18.7 (OCH₂CH₂CH₂CH₃),

22.8 (N(CH₂)₆CH₂CH₃), 26.7 (N(CH₂)₅CH₂CH₂CH₃), 29.3 (N(CH₂)₃CH₂CH₂(CH₂)₂CH₃), 29.8

3

(N(CH₂)₂CH₂(CH₂)₄CH₃), 31.2 (N(CH₂CH₂(CH₂)₅CH₃), 32.2 (d, J_CP= 6.9, OCH₂CH₂CH₂CH₃),

51.4 (N(CH₂CH₂(CH₂)₅CH₃), 66.8 (d, ²J_CP= 5.8, OCH₂CH₂CH₂CH₃), 121.6 (d, ¹J_CP= 220.2, P(O)C=),

128.2 (C₂), 129.0 (C₁), 129.4 (C₃), 130.5 (C₄), 153.2 (d, ²J_CP= 19.9, PhC=); [M + H]⁺

= 450.2862,

found

C₂₄H₄₁N₃O₃P requires 450.2885.

Dibutyl (1-isooctyl-4-phenyl-1,2,3-triazol-5-yl)phosphonate (26): yield: 28% (0.13 g), colorless

oil; ³¹P NMR (CDCl₃) 3.0; ¹H NMR (CDCl₃)

0.77–0.96 (m, 12H, OCH₂CH₂CH₂CH₃,

δ

NCH₂CHCH₂CH₃, NCH₂CH(CH₂)₃CH₃), 1.16–1.53 (m, 16H, OCH₂CH₂CH₂CH₃, NCH₂CHCH₂CH₃,

NCH₂CH(CH₂)₃CH₃), 2.06–2.20 (m, 1H, NCH₂CHCH₂CH₃), 3.77–3.94 (m, 2H, OCH₂CH₂CH₂CH₃),

3

3.97–4.11 (m, 2H, OCH₂CH₂CH₂CH₃), 4.69 (d, 2H, J_HH= 7.5, NCH₂CHCH₂CH₃), 7.34–7.45

3

(m, 3H, C₃H, C₄H), 7.75 (dd, 2H, J_HH= 6.4, J_HH= 3.3, C₂H); ¹³C NMR (CDCl₃)

δ 10.5

(NCH₂CHCH₂CH₃), 13.5 (OCH₂CH₂CH₂CH₃), 14.1 (NCH₂CH(CH₂)₃CH₃), 18.7 (OCH₂CH₂CH₂CH₃),

23.0 (NCH₂CH(CH₂)₂CH₂CH₃), 23.7 (NCH₂CHCH₂CH₃), 28.5 (NCH₂CHCH₂CH₂CH₂CH₃), 30.4

(NCH₂CHCH₂(CH₂)₂CH₃), 32.1 (d, ³J_CP= 6.9, OCH₂CH₂CH₂CH₃), 40.5 (NCH₂CHCH₂CH₃), 54.8

(NCH₂CHCH₂CH₃), 66.7 (d, ²J_CP= 5.8, OCH₂CH₂CH₂CH₃), 121.9 (d, ¹J_CP= 221.1, P(O)C=), 128.1 (C₂),

128.9 (C₁), 129.4 (C₃), 130.5 (C₄), 153.2 (d, ²J_CP= 19.8, PhC=); [M + H]⁺

= 450.2855, C₂₄H₄₁N₃O₃P

found

requires 450.2885.

Molecules 2020, 25, 2643

13 of 16

3.5. Biological Evaluation

3.5.1. Cell Culture

Cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Human lung adenocarcinoma A549 cells were maintained in Dulbecco s Modiﬁed Eagle Medium

'

(DMEM), while mouse ﬁbroblast NIH/3T3 and human promyelocytic leukemia HL-60 cells were

maintained in Roswell Park Memorial Institute 1640 medium (RPMI-1640) containing 10% FCS. Media

were supplemented with 2 mM GlutaMAX, 100 U/mL penicillin, and 100

Technologies, Carlsbad, California, USA). Cell cultures were maintained at 37 C in a humidiﬁed

incubator in an atmosphere of 5% CO₂(Sanyo, Japan).

µ

g/mL_◦streptomycin (Life

3.5.2. Antibacterial Activity

All reagents were purchased from Sigma-Aldrich (Hungary, Budapest). E. coli-GFP and

B. subtilis-GFP bacteria were grown in Lysogeny Broth (Luria-Bertani broth, LB, 10 g tryptone, 5

g yeast extract, 10 g/L NaCl, pH 7.0) medium overnight (ON) at 37 ^◦C in an incubator with continuous

shaking [44

medium containing 10

,

45]. The ON bacterial culture was diluted (E. coli-GFP: 1:10000, B. subtilis-GFP: 1:1000) in LB

g/mL ampicillin. Two hundred microliters of diluted E. coli suspension per well

µ

were transferred to 96-well plates and diﬀerent agents were added in the appropriate concentration,

then incubated in a shaker (600 rpm) for 3 h at 37 ^◦C. Test compounds were dissolved in 3-fold amounts

of dimethyl sulfoxide (DMSO). Cells were treated with an increasing concentration of test compounds

(1–30

(IC₅₀= 4.23

µ

M). Doxycyclin (IC₅₀= 0.10

±

0.02 and 0.04

±

0.01 µM for E. coli and B. subtilis) and gentamicin

±

0.99 and 0.49 0.14

±

µ

M for E. coli and B. subtilis) were used as positive controls. For

induction of the GFP expression IPTG (0.1 mM ﬁnal concentration) was added to each well and

incubated for 3 h at 37 ^◦C.

The following incubation samples were centrifuged at 2750 rpm at room temperature (RT) for 5

min and washed twice with 200

third centrifugation step, the bacterial cells were suspended in 100

was estimated according to the ﬂuorimetric measurements. The ﬂuorescence of the bacterial cells was

measured by the Victor 1420 Multilabel Counter (Perkin Elmer, Waltham, MA, USA) at 485/535 nm

µL PBS, with a centrifugation step between the two washes. After a

µL PBS. The number of bacteria

™

for 1 sec per well controlled by Wallac 1420 Manager software. Each treatment was repeated in at least

2 wells per plate during the experiments. Percentage ﬂuorescence intensity based on the quantity of

E. coli-GFP cells was calculated using untreated control values as 100%. Error was represented by

standard deviation (SD). IC₅₀values (50% inhibiting concentration) were calculated by GraphPad

Prism^®5 (La Jolla, CA, USA).

3.5.3. Cytotoxicity Assay

Cytotoxicity of the synthesized molecules was determined on A549, NIH/3T3 and HL-60 cells

using the ﬂuorescent Resazurin assay as described previously [46].

Brieﬂy, cells (A549 and NIH/3T3: 6000, HL-60: 120.000 cells/well) were seeded into 96-well plates

(Corning Life Sciences) in media and incubated overnight. Test compounds were dissolved in three-fold

amounts of dimethyl sulfoxide (DMSO). Cells were treated with an increasing concentration of test

compounds (1–30

µ

M). Positive controls were doxorubicin for A549 and NIH/3T3 (IC₅₀= 0.31

±

0.24 µM

and 5.65 0.81 M, respectively), and bortezomib for HL60 (IC₅₀= 7.42

±

µ

±

2.60 nM). Cell viability was

determined after 72 h incubation. Resazurin reagent (Sigma-Aldrich) was added at a ﬁnal concentration

◦

of 25

µg/mL. After a 2 h incubation at 37 C in 5% CO₂, ﬂuorescence (530 nm excitation/580 nm

emission) was recorded on a multimode microplate reader (Cytoﬂuor4000, PerSeptive Biosytems).

Viability was calculated with relation to untreated control cells and blank wells containing media

without cells. IC₅₀values (50% inhibiting concentration) were calculated by GraphPad Prism^®5. A

Student’s t test was performed for individual test compound concentrations with relation to untreated

control cell viability values.

Molecules 2020, 25, 2643

14 of 16

4. Conclusions

In summary, the synthesis of 1,2,3-triazol-5-yl-phosphonates was studied by the

copper(I)-chloride-catalyzed three-component domino reaction of phenylacetylene, azides and dialkyl

phosphites. The model reaction of phenylacetylene, benzyl azide and dibutyl phosphite was optimized

in details. The eﬀect of the atmosphere, the type of base, the catalyst and the solvent, as well as the

molar ratio of the starting materials, the temperature and the reaction time were studied. Applying the

approach developed, altogether 17 triazol-5-yl-phosphonate derivatives were synthesized and fully

characterized, except two (12 and 14), all of them are new compounds. The biological activity of the

compounds prepared was investigated in antibacterial activity and in vitro cytotoxicity assays. None

of the synthesized 1,2,3-triazol-5-yl-phosphonates were active against selected Gram-negative bacteria,

while the growth of Gram-positive bacteria was somewhat reduced. Several derivatives showed low

or modest activity against the tested cell lines, two of them have the IC₅₀value in the 10 micromolar

range against HL-60 cells.

Supplementary Materials: Supplementary data associated with this article are available online. Copies of ³¹P,

¹H, and ¹³C NMR spectra for all compounds synthesized are presented.

Author Contributions: E.B. and A.T. planned the experiments, A.T. and. E.T. carried out the experiments,

P.T.S. performed the high-resolution mass spectrometric measurements, B.K. and L.H.J. performed the biological

evaluation (screening), E.B., A.T., L.H.J. and L.G.P. wrote the paper. All authors have read and agreed to the

published version of the manuscript.

Funding: The project was supported by the Hungarian Research Development and Innovation Oﬃce (FK123961).

E.B. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00278/17/7).

A.T. was supported by the

and Technology.

ÚNKP-19-3-I-BME-391 New National Excellence Program of the Ministry for Innovation

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds 8 and 11–26 are available from the authors.

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Synthesis and in vitro cytotoxicity and antibacterial activity of novel1,2,3-triazol-5-yl-phosphonates

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