(Amino)cyclophosphazenes as Multisite Ligands for the Synthesis of Antitumoral and Antibacterial Silver(I) Complexes

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(Amino)cyclophosphazenes as Multisite Ligands for the Synthesis of

Antitumoral and Antibacterial Silver(I) Complexes

́

Elena Gascon, Sara Maisanaba, Isabel Otal, Eva Valero, Guillermo Repetto, Peter G. Jones,

and Jose ﬁ na Jimenez *

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ABSTRACT: The reactivity of the multisite (amino)cyclotriphosphazene ligands,

[N₃P₃(NHCy)₆] and [N₃P₃(NHCy)₃(NMe₂)₃], has been explored in order to

obtain silver(I) metallophosphazene complexes. Two series of cationic silver(I)

metallophosphazenes were obtained and characterized: [N₃P₃(NHCy)₆{AgL}_n]_-

(TfO)_n[n = 2, L = PPh₃(2), PPh₂Me (4); n = 3, L = PPh₃(3), PPh₂Me (5), TPA

(TPA = 1,3,5-triaza-7-phosphaadamantane, 6)] and nongem-trans-

[N₃P₃(NHCy)₃(NMe₂)₃{AgL}_n](TfO)_n[n = 2, L = PPh₃(7), PPh₂Me (9); n =

3, L = PPh₃(8), PPh₂Me (10)]. 5, 7, and 9 have also been characterized by single-

crystal X-ray diﬀraction, thereby allowing key bonding information to be obtained.

Compounds 2−6, 9, and 10 were screened for in vitro cytotoxic activity against

two tumor human cell lines, MCF7 (breast adenocarcinoma) and HepG2

(hepatocellular carcinoma), and for antimicrobial activity against ﬁve bacterial

species including Gram-positive, Gram-negative, and Mycobacteria strains. Both

the IC₅₀and MIC values revealed excellent biological activity for these metal

complexes, compared with their precursors and cisplatin and also AgNO₃and silver

sulfadiazine, respectively. Both IC₅₀and MIC values are among the lowest values

found for any silver derivatives against the cell lines and bacterial strains used in

this work. The structure−activity relationships were clear. The most cytotoxic and antimicrobial derivatives were those with the

triphenylphosphane and [N₃P₃(NHCy)₆] ligands. A signiﬁcant improvement in the activity was also observed upon a rise in the

number of silver atoms linked to the phosphazene ring.

nontoxic to humans in minute concentrations.⁸The most

widely used silver reagents have been in the form of inorganic

1. INTRODUCTION

Over the years, there has been continuous interest in the

salts or complexes, such as silver nitrate and silver sulfadiazine,

biological activity of metal complexes. Among them, silver has

and in combination with proteins. Actually, silver sulfadiazine

been used for numerous medical conditions, mostly because of

its antibacterial activity.¹The antibacterial, antifungal, and

works as a broad-spectrum antibiotic used mainly in the

treatment of burn wounds. However, almost all of the current

antiviral properties of silver ions, silver compounds, and,

clinical silver agents have their own disadvantages, limiting

recently, silver nanoparticles have been extensively studied, and

these topics have been thoroughly reviewed.^2,3Nowadays the

their clinical usefulness. Therefore, on the basis of the beneﬁts

of silver, the pressing interest in ﬁnding new silver

pharmaceuticals promoted the development of more eﬃcient

and convenient silver agents, in which employing ligands that

can strongly coordinate to the active silver(I) ions is essential.^2f

In recent years, the systematic discovery and development of

silver complexes has yielded a large number of promising

antibacterial, antifungal, and even anticancer compounds, and

several reviews on the topics have been reported. Recently,

preparation of novel antibacterial agents has become a priority

because of the presence of multidrug-resistant pathogens.⁴

Mycobacterium tuberculosis (the organism causing tuberculosis

that still wreaks havoc in both developed and undeveloped

countries) aside, Staphylococcus aureus (resistant to methicillin,

MRSA), Escherichia coli, and Pseudomonas aeruginosa, among

others, are becoming more and more resistant, and the lack of

eﬃcient treatment is one of the leading causes of high

mortality rates.⁵Although the antimicrobial activity mecha-

nism of silver(I) complexes has not been well established,^2a,6

microorganisms are unlikely to develop resistance against silver

compared to antibiotics because silver attacks a broad range of

targets in the microbes, and only a few accounts of resistance

have been reported.⁷What is more, silver is also found to be

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Lobana et al. performed an in vitro antimicrobial potential and

biosafety evaluation of silver(I) derivatives of several thio

ligands, some of which have shown signiﬁcant antimicrobial

activity and low cellular toxicity with a high percent of cell

viability.^9a−cBanti and Hadjikakou reviewed the antiprolifer-

ative activity of silver(I) compounds^9din comparison with that

of cisplatin, a clinical chemotherapeutic drug currently in use.

Liang et al. also recently reviewed the advances in the medical

use of silver complexes.^2fOther more general reviews

concerning metallodrugs have also recently been reported.^5,10

These surveys show that silver(I) complexes exhibit selectivity

against a variety of cancer cells and bacterial strains with regard

to the kinds of ligands coordinated to silver(I) ions. However,

the research in this ﬁeld is still limited at present. Most studies

focus on silver(I) N-heterocyclic carbene complexes.¹¹New

ligands for better activity and lower toxicity need to be studied

further. Besides, more research is still required to fully

elucidate the mechanism of action of silver(I) complexes

against both microorganism and tumor cells.⁵

rise to a wide variety of branched molecules that have precise

structure and are also monodisperse.¹⁸Over the past few years,

CPZs have been developed in several pharmacological

domains,¹⁹and the use of dendrimers, in general, and

dendrimers based on the CPZ core, in particular, has been

said to represent a new strategy in nanomedicine. Recent

developments of CPZs for major pharmaceutical applications

have been reviewed by Majoral et al.^13cBesides, both PPZs and

CPZs can be used as scaﬀolds for the design and construction

of a variety of ligands,^12c,20to coordinate to metallic drugs. The

facile substitution of P−Cl bonds in the cyclic trimer

hexachlorocyclotriphosphazene, [N₃P₃Cl₆], allows the ready

construction of cyclophosphazenes carrying additional exocy-

clic donor functions, giving a library of multisite coordination

ligands. Furthermore, the ring nitrogen atoms have suﬃcient

basicity to coordinate to metals depending on the electronic

properties of the exocyclic substituents at phosphorus. Alkyl,

aryl, and primary and secondary organoamino substituents

enhance the donor ability of ring nitrogen atoms.^20c

Since its synthesis for the ﬁrst time in 1834, hexachlor-

ocyclotriphosphazene, [N₃P₃Cl₆], has been an important

compound of phosphorus chemistry as a scaﬀold, and a large

number of cyclotriphosphazene (CPZ) derivatives and their

analogue polymers, polyphosphazenes (PPZs), have been

synthesized and applied in various ﬁelds such as biology,

catalysis, ﬂuorescence, nanomaterials, etc.¹²Today, one of the

most important uses concerns its biomedical applications.¹³

Phosphazenes comprise a broad class of molecules based on

the repeating unit [NPR₂] and include cyclic or linear

oligomers and polymers. The most striking characteristic of

this type of compound is its associated synthetic versatility,

which enables the introduction of almost any substituent group

R at phosphorus and allows its properties to be tailored by the

choice of appropriate functional groups.¹²Notably, biocompat-

ibility and biodegradability can be regulated by the

introduction of speciﬁc side groups, R.¹⁴The degradation

rate can also be controlled by external stimuli, such as the

temperature, radiation, pH of the degradation medium, and

degradation product solubility.¹⁴Nowadays, the use of PPZs in

drug-delivery applications, especially proteins and anticancer

drug delivery, has become a signiﬁcant focus.^13b,15The eﬀects

of various side groups on the properties of the resultant PPZs

and their use in diﬀerent approaches, such as drug delivery,

have recently been reviewed.^13bMacromolecular prodrugs are

known to show excellent tumor-targeting properties by their

enhanced permeability and retention eﬀect¹⁶and exhibit

improved body distribution and prolonged blood circulation

because of the dominant pharmacokinetic properties of the

macromolecular carrier.¹⁷In this respect, drugs can be

physically encapsulated by liposomes, nanocapsules, or

polymeric micelles or vesicles and, alternatively, conjugated

to linear polymers or dendrimers by covalent bonding. Drug−

polymer conjugates are an emerging area of drug delivery in

order to combat the hurdles related to the delivery of drugs

such as low solubility, protection against degradation by

various factors, low bioavailability, and high dose toxicity.

Thus, some antibiotic, antiviral, antitumoral, or antimalarial

drugs have been conjugated to PPZs and CPZs.¹³CPZs are

even in a much better position than PPZs because the

phosphazene trimer backbone is monodisperse and it is much

easier to control the purity and molecular weight of these

molecules,^13awhich both play a critical role for its clinical use.

Besides, CPZs can be used as the core of dendrimers, giving

Several authors have prepared and studied the tumoral

properties of cyclophosphazene- or polyphosphazeneplatinum-

(II) conjugates.²¹Recently, Henke et al. introduced PPZ-based

anticancer prodrug conjugates in which cisplatin and

oxaliplatin were ﬁrst converted into prodrugs and then

attached to the PPZ through a linker.²²Silver derivatives of

phosphazenes (cyclic or polymers) are also known.²³The

Steiner group has published interesting work using (amino)-

cyclophosphazenes to prepare structurally characterized silver-

(I) coordination polymers by the formation of linear N−Ag−

N connections via nitrogen centers of the phosphazene ring.²⁴

We have also contributed to this ﬁeld by reporting CPZs and

PPZs containing gold or silver and their thermolytic trans-

formation into nanostructured materials or into metallic micro-

and nanostructures deposited on silicon and silica surfaces.²⁵

PPZs and CPZs decorated with silver nanoparticles have also

been synthesized.²⁶However, to the best of our knowledge,

there are no reports of silver phosphazenes with biological

properties, such as the antimicrobial or antitumoral ones. Thus,

the interesting biological properties of both silver(I) complexes

and phosphazenes, discussed above, together with our

experience in the chemistry of both silver and phosphazenes,

prompted us to prepare new silver cyclotriphosphazenes and

study their antimicrobial and antitumoral properties.

Herein, the reactivity of the multiside (amino)-

cyclotriphosphazene ligands, [N₃P₃(NHCy)₆] and

[N₃P₃(NHCy)₃(NMe₂)₃], has been explored in order to

obtain silver(I) metallophosphazene complexes. For this

purpose, the simple strategy used was to employ silver(I)

precursors bearing a phosphane ligand and a labile triﬂate

(OTf) to be displaced by the phosphazene ligand. Thus, two

series of silver(I) metallophosphazenes were obtained, and

studies of their anticancer activity in the human breast

adenocarcinoma (MCF7) and human hepatocellular carcino-

ma (HepG2) cell lines were carried out. The antibacterial

activity was also tested against the Gram-negative strains E. coli

and P. aeruginosa, against the Gram-positive strain S. aureus,

and against two M. tuberculosis complex (MTBC) strains, M.

tuberculosis H37Rv and M. bovis BCG Pasteur. Comparisons

with the corresponding antiproliferative and antimicrobial

activity of cisplatin and silver(I) nitrate, respectively, were also

performed.

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i.e., 2,2,4- and 2,4,6- (numbering starts at the nitrogen atom),

which are also referred to as geminal and nongeminal isomers,

respectively, but also di- and tetrasubstituted derivatives as

minor products. Besides, the nongeminal materials can exist in

two stereoisomeric forms, either with the three substituents on

the same side of the mean plane of the phosphazene ring

(nongeminal cis-2,4,6) or with two on one side and the third

on the other (nongeminal trans-2,4,6) (see Chart 1).

2. RESULTS AND DISCUSSION

2.1. Synthesis and Characterization of the Starting

CPZs. Syntheses of the starting CPZs are outlined in Scheme

1.

Scheme 1. Syntheses of the Starting CPZs

As reported, some of the variations in the aminolysis

patterns may be associated with the experimental conditions,

such as the temperature, solvent, and stoichiometry, which in

some circumstances may determine the presence or absence of

a particular isomer or at least its relative proportions.³⁰All

three compounds that we obtained, phos-1 to phos-3, were

1

completely characterized by elemental analysis, IR, H, ¹³C-

{¹H}, and ³¹P{¹H} NMR spectroscopy, and mass spectrometry

(MS).³²All of these data are given in the Experimental Section

and are consistent with the formulas and structures indicated.

Speciﬁcally, phos-2 has a nongeminal trans trisubstituted

conﬁguration. That nongeminal trans conﬁguration is main-

tained in the new phosphazene phos-3 after complete

substitution of the chlorine atoms by the cyclohexylamino

units. Elemental analysis and MS indicated that the

compositions of phos-2 and phos-3 are [N₃P₃Cl₃(NMe₂)₃]

and [N₃P₃(NHCy)₃(NMe₂)₃], respectively, and ¹H and

³¹P{¹H} NMR spectroscopy indicated that both have a

nongeminal trans conﬁguration. NMR spectroscopy is one of

the most powerful tools for the structural characterization of

cyclophosphazenes because the most critical structural

information is available from the chemical shifts and spin−

spin coupling data of their ³¹P{¹H} NMR spectra.^30,33,34Thus,

the ³¹P{¹H} NMR spectra of phos-2 and phos-3 showed a

multiplet from a spin system AB₂, which indicates that both of

them are nongeminal trans (see Figure 1a for phos-2 and

Figure 2a for phos-3). The geminal trisubstituted derivative (I

in Chart 1) would have exhibited a spin system AX₂, i.e., a

doublet and a triplet (both phosphorus atoms would have

markedly diﬀerent environments), and the nongeminal cis

trisubstituted derivative (II in Chart 1) a spin system A₃, i.e., a

Speciﬁcally, the starting CPZs, a single-substituent trimer,

[N₃P₃(NHCy)₆] (phos-1), and a mixed-substituent trimer,

nongem-trans-[N₃P₃(NHCy)₃(NMe₂)₃] (phos-3), were both

prepared in high yield (>80%) from [N₃P₃Cl₆] and nongem-

trans-[N₃P₃Cl₃(NMe₂)₃] (phos-2), respectively, using tetrahy-

drofuran as a solvent and cyclohexylamine as a nucleophile and

a base (see the Experimental Section). The syntheses of phos-

1 and phos-2 were described previously,²⁷⁻²⁹starting from

hexachlorocyclotriphosphazene, [N₃P₃Cl₆], but involving less

eﬃcient methods than those used by us. The trimer phos-1

was prepared by Chandrasekhar et al. using diazabicyclounde-

cane as a base and chloroform as a solvent.^27,28In this reaction,

the major product was the incompletely substituted derivative

[N₃P₃Cl₂(NHCy)₄] and only prolonged reaction conditions (8

days of heating under reﬂux) led to the fully single-substituted

derivative phos-1, which was formed in only minor yield

(6.8%).²⁷Compound phos-2 was synthesized by Ford et al.

using diethyl ether as a solvent at room temperature (RT; yield

27%) and by Keat and Shaw at −78 °C (yield 49%).²⁹Starting

also from [N₃P₃Cl₆] but using tetrahydrofuran as a solvent and

with the slow dropwise addition of dimethylamine as a

nucleophile and a base at 0 °C, we obtained phos-2 in high

yield (84%; see the Experimental Section for details). The

substitution reaction of [N₃P₃Cl₆] has been extensively

studied, with primary interest in the regio- and stereochemical

pathways.^30,31In particular, partial substitution of hexachlor-

ocyclotriphosphazene usually results not only in stoichiometri-

cally diﬀerent products but also in various geometrical and

positional isomers that are not easy to separate. Thus, reactions

leading to the trisubstituted material [N₃P₃Cl₃X₃] usually

result in the formation of not only trisubstituted regioisomers,

1

singlet. The H NMR spectra of all of the dimethylaminolysis

products of [N₃P₃Cl₆] have been examined elsewhere,²⁹as

have those of the trisubstituted derivatives including phos-2.

As the authors commented,²⁹detailed interpretation of the ¹H

NMR spectra is diﬃcult because of their great complexity. At

their simplest, when they contain only one type of

dimethylamino environment, the spectrum consists of one

doublet centered at approximately 2.50−2.70 ppm (with an

additional broadened structure between the major peaks),³⁵

with a separation (apparent coupling constant, J′(P−H) or N)

varying between 11 and 18 Hz. The N value is dependent on

the nature of the second substituent bonded directly to the

phosphorus carrying the −NMe₂group. A P(Cl)(NMe₂)

group gives an N value of about 18 Hz, and a P(NMe₂)₂or

P(NR₂)(NMe₂) group gives about 11 Hz.²⁹Thus, the coupling

Chart 1. Trisubstituted CPZ Isomers

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1

Figure 1. (a) ³¹P{¹H} and (b) H NMR spectra of compound phos-2 in CDCl₃.

1

Figure 2. (a) ³¹P{¹H} and (b) H NMR spectra of compound phos-3 in CDCl₃.

Scheme 2. Synthesis of New Metallophosphazenes

1

constants indicate the groups attached to the phosphorus

atoms, and the number of doublets indicates diﬀerent sets of

environments. The H NMR spectrum of phos-2 showed two

pseudodoublets, each with N of approximately 18 Hz (see

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Figure 1b), as reported in the literature.^29aThis N value is in

accordance with the fact that the −NMe₂groups are each

located on a separate phosphorus atom (nongeminal

derivative), i.e., P(Cl)(NMe₂) groups. The presence of two

pseudodoublets conﬁrms that phos-2 is the trans isomer. The

¹H NMR spectrum of the new phosphazene phos-3 is shown

in Figure 2b, which presents the signals corresponding to the

cyclohexylamino and dimethylamino units. For dimethyamino

protons, a multiplet centered at 2.62 ppm is observed with N =

13.6 Hz. As mentioned before, this N value is in accordance

with the presence of P(NR₂)(NMe₂) groups.

2.2. Synthesis and Characterization of the Metal-

lophosphazenes. The reaction of phos-1 or phos-3 with the

silver complexes [Ag(OTf)L] (OTf = OSO₂CF₃; L = PPh₃,

PPh₂Me, or TPA, where TPA = 1,3,5-triaza-7-phosphaada-

mantane) in dichloromethane and in diﬀerent molar ratios of

1:2 or 1:3 led to two series of cationic metallophosphazenes,

[N₃P₃(NHCy)₆{AgL}_n](TfO)_n[n = 2, L = PPh₃(2), PPh₂Me

(4); n = 3, L = PPh₃(3), PPh₂Me (5), TPA (6)] and nongem-

trans-[N₃P₃(NHCy)₃(NMe₂)₃{AgL}_n](TfO)_n[n = 2, L = PPh₃

(7), PPh₂Me (9); n = 3, L = PPh₃(8), PPh₂Me (10)]. In all of

them, the silver groups “AgL” are coordinated to the nitrogen

atoms of the phosphazene ring, whereby their number (2 or 3)

depends on the molar ratio used (see Scheme 2). The reaction

of phos-1 with the same starting complexes, [Ag(OTf)L] (L =

PPh₃or PPh₂Me), but in a molar ratio of 1:1 evolved in a more

complex way, giving a mixture of compounds in which the

major product was [N₃P₃(NHCy)₆Ag(TfO)]_n(1). The

identiﬁcation of this derivative 1 was conﬁrmed by the

reaction of phos-1 with AgTfO in a molar ratio of 1:1 (see the

Experimental Section).

However, all of the 1−10 complexes have very similar bands in

shape and position in the stretching region of the triﬂate

(1280−1000 cm⁻¹, ν[SO₃(E)], ν[SO₃(A₁)], ν[CF₃(A₁)], and

ν[CF₃(E)]; see the Experimental Section). This is consistent

with the presence of ionic triﬂuoromethanesulfonate, which

was clearly conﬁrmed in 7 and 9 by X-ray structural analyses.

Besides, a single peak at approximately −78 ppm appears in the

¹⁹F NMR spectra of all synthesized complexes. The bands of

the characteristic phosphazene absorptions in the IR spectra,

such as PN and C−N (at 1186 and 1177 cm⁻¹in phos-1

and 1180, 1171, and 1140 cm⁻¹in phos-3), change after

coordination of the metal, as previously observed in other

metallophosphazenes with silver coordinated to the backbone

nitrogen atoms.^23cHowever, new bands corresponding to

these peak frequencies could not be assigned because of

overlap with CF₃, SO₃, and phosphane vibrational modes. The

bands in the 3000−3400 cm⁻¹region, which are assigned to

the N−H stretching, are also diﬀerent from those of the

starting phosphazenes. Upon complexation, only one band at

approximately 3300 cm⁻¹is observed for all complexes.

1

The ³¹P{¹H} and H NMR spectra in solution are also

consistent with coordination of the metal fragments to the ring

nitrogen atoms. The signals observed for 2−6, with the single-

substituent trimer phos-1, in their ³¹P{¹H} NMR spectra at

RT, are collected in Table 1. A single peak, shifted downﬁeld

Table 1. ³¹P{¹H} NMR Spectroscopic Data for 2−6 and

a

Starting Products

δ[PR₃]

b

[¹J(¹⁰⁹Ag−P)],

b

compound

δ[N−P−N] [¹J(¹⁰⁷Ag−P)]

All new metallophosphazenes 2−10 were obtained in high

yield and could be handled and stored protected from light

under ambient conditions for large periods of time. They are

soluble in dichloromethane, acetone, chloroform, and dimethyl

sulfoxide (DMSO) and only slightly soluble or insoluble in

hexane or pentane. Their solutions must also be handled and

stored protected from light, and they were stable for at least 1

week at RT under these conditions, as was proven by ³¹P{¹H}

and ¹H NMR spectroscopy. All of them are insoluble in water,

even compound 6, bearing the water-soluble phosphane ligand

TPA, which was prepared in an unsuccessful attempt to obtain

a water-soluble metallophosphazene. Compound 1 is only

sparingly soluble in dichloromethane, chloroform, or acetone

and insoluble in water, diethyl ether, ethanol, hexane, or

pentane. All of the compounds 1−10 were characterized by

elemental analysis, IR, ¹H, ¹³C{¹H}, and ³¹P{¹H} NMR

spectroscopy, and MS. All of these data are given in the

Experimental Section and are consistent with the formulas and

structures indicated and, speciﬁcally, with the coordination of

metals to the backbone nitrogen atoms. In addition, X-ray

structural analyses of 5, 7, and 9 conﬁrmed the proposed

structures.

IR spectra of all of these complexes clearly evidence

coordination of the metal fragments, AgL, to phos-1 or

phos-3. Thus, the IR spectra show absorptions attributable to

triﬂuoromethanesulfonate (triﬂate) units and to the phosphane

ligands, which are all shifted from those in the starting

complexes [Ag(OTf)L] (L = PPh₃, PPh₂Me, or TPA). The

triﬂate peaks, which could, in principle, be used to distinguish

covalent and ionic triﬂuoromethanesulfonate,³⁶are not very

useful because an unambiguous assignment is hindered by the

overlap of CF₃, SO₃, and phosphane vibrational modes.³⁷

[N₃P₃(NHCy)₆] (phos-1)

[Ag(OTf)PPh₃]

[N₃P₃(NHCy)₆{AgPPh₃}₂](TfO)₂(2)

14.44 (s)

16.79 (br)

16.90 (br) 17.12 (dd)

[746.1],

[653.4]

[N₃P₃(NHCy)₆{AgPPh₃}₃](TfO)₃(3)

18.38 (s)

16.53 (dd)

[761.7],

[662]

−3.13 (br)

[Ag(OTf)PPh₂Me]

[N₃P₃(NHCy)₆{AgPPh₂Me}₂](TfO)₂(4)

16.22 (br) −1.90 (dd)

[757.6],

[651.4]

[N₃P₃(NHCy)₆{AgPPh₂Me}₃](TfO)₃(5)

18.57 (s)

15.93 (s)

−2.78 (d, br)

[718]

[Ag(OTf)TPA]

[N₃P₃(NHCy)₆{AgTPA}₃](TfO)₃(6)

−85.82 (br)

−85.46 (br)

a

Data taken at RT in CDCl₃, except for 6 and [Ag(OTf)TPA], whose

data are in DMSO. Values in parts per million. Values in hertz.

b

from the peak of the parent phosphazene, is shown by the

phosphazene phosphorus atoms in all complexes 2−6. This

downﬁeld shift can be ascribed to deshielding by the silver ion

coordinated to the adjacent nitrogen atoms, which increases

with the number of metals linked to the adjacent nitrogen

atoms. This shift has also been observed in other metal-

lophosphazenes in which metals (such as silver or lithium) are

coordinated to the backbone nitrogen atoms.^23b,cThe signal

observed at RT is a single peak even for 2 and 4, for which a

AX₂spin system corresponding to two types of phosphorus

atoms would be expected. This is attributable to a ﬂuxional

process, which is typical in the coordination chemistry of silver

and can be attributed to exchange phenomena involving all of

the phosphazene nitrogen atoms. The ﬂuxionality can be

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quenched at low temperature. Thus, the single broad peak

observed for the phosphazene phosphorus atoms in 2 and 4 at

RT is split into two signals at −80 °C (as shown in Figure 3 for

ﬂuxional process. For 2 and 4, though, two types of NHCy

units were observed when the spectra were collected at −80

°C, as a consequence of the nonequivalence of all of the amino

units. Thus, the single broad peak corresponding to the NH

protons, which was observed at δ 4.03 (br, 6H) and 4.0 (br,

6H) for 2 and 4, respectively, in (CD₃)₂CO at RT, was split

into two signals at −80 °C [δ 4.65 (t, 2H) and 4.40 (br, 4H)

for 2; δ 4.55 (br, 2H) and 4.20 (br, 4H) for 4; see

Experimental Section]. As can be observed in Table 3, which

1

presents the H NMR data in CDCl₃at RT, coordination at

phosphazene leads in all complexes to a deshielding of the

protons NH, with the rise in the number of metals coordinated

to the adjacent nitrogen atoms.

The signals for the phosphazene phosphorus atoms for 7−

10 also shift downﬁeld relative to the parent phosphazene in

their ³¹P{¹H} NMR spectra at RT (see Table 4). However,

even in 8 and 10 (both with three silver atoms), several signals

are observed because of the nongeminal trans conﬁguration of

the starting phosphazene phos-3, which is expected to be

maintained in all metallophosphazenes 7−10. Thus, the part of

the spectrum corresponding to the phosphazene phosphorus

atom in the ³¹P{¹H} NMR spectra at RT shows a single set of

resonances of an AB₂spin system for 10, with the peaks

centered at δ 24.07 (d, 2P) and 23.08 (t, 1P), ²J(P−P) = 33.1

Hz, and a broad signal at 24.27 ppm for 8. For 7 and 9, this

part of the spectrum (between 24 and 19 ppm) is even more

complex, as is shown in Figure 4b for 9 as an example, with

signals that are broad as a consequence of the above-

mentioned ﬂuxional process. In these compounds, 7 and 9,

two diastereomers A and B are expected (see Chart 2), namely,

a pair of R_P2,R_P3- or S_P2,S_P3-conﬁgured enantiomers (B) and

the diastereomer R_P1,R_P2,S_P3(A). At −80 °C, two sets of

resonances of two AB₂spin systems are observed for 9 in this

part of the spectrum when the ﬂuxional process is quenched

(see Figure 4a), which indicates that both stereoisomers A and

B are present in solution.⁴⁰The part of the spectrum

corresponding to the phosphane ligands (see also Figure 4a)

shows two set of two doublets, consistent with the presence of

both diastereomers.⁴¹

Figure 3. ³¹P{¹H} NMR spectra of compound 4 in (CD₃)₂CO at (a)

RT and (b) −80 °C.

4; see also Table 2). The sparingly soluble compound 1 was

not ﬂuxional in solution, and two signals were observed for the

phosphazene phosphorus atoms in its ³¹P{¹H} NMR spectrum

at RT [10.39 (t, 1P) and 8.43 (d, 2P), AB₂system, ²J_AB= 45.1

Hz]. Both the poor solubility and nonﬂuxionality might be

associated with the oligomeric or polymeric nature of 1.

However, single crystals suitable for X-ray diﬀraction analysis

could not be obtained.

The ³¹P{¹H} NMR spectra of 2−6 also show signals from

the phosphane ligands (see Tables 1 and 2), which are all

shifted from those in the starting complexes.^37,38For 6, the

phosphane resonates at −85.46 (s) ppm, which is consistent

with a silver(I) compound containing TPA acting as a

phosphorus-donor ligand.³⁹Unlike 2−5, this signal for 6

consists of a broad singlet at RT, and coupling with the two

silver isotopomers (¹⁰⁷Ag and ¹⁰⁹Ag) is not observed, which

indicates that the ﬂuxional process in this compound also

involves the TPA ligand. The ¹H NMR spectra of 2−6 (see the

Experimental Section) also show the signals corresponding to

the phosphane ligands and the signals from the cyclo-

hexylamino units (Table 3). These signals, speciﬁcally those

of NH and NH−CH, were veriﬁed by two-dimensional

heteronuclear (HSQC ¹H−¹³C) correlations. Most signiﬁ-

1

In the H NMR spectra of 7−10, the NH−CH protons

resonate as a single broad signal at approximately 2.9 ppm and

the NH protons resonate as two broad signals even at RT, as a

result of the nonequivalence of all of the amino groups due to

the nongeminal trans conﬁguration of the phosphazene ring.

The NH protons are also shielded upon coordination. All of

these data are collected in the Experimental Section, as are the

¹³C{¹H} NMR, MS, and microanalytical data. It is worth

1

cantly, the H NMR spectra of 2−6 all show a unique type of

cyclohexylamino unit at RT, as a result of the above-mentioned

Table 2. ³¹P{¹H} NMR Spectroscopic Data for 2 and 4 at RT and −80 °C

a

b

Data taken in (CD₃)₂CO. Values in parts per million. Values in hertz.

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a

1

Table 3. H NMR Spectroscopic Data for phos-1 and Complexes 2−6

compound

δ[NH] δ[NH−CH]

δ[NH(C₆H₁₁)]

δ[PPR₃]

[N₃P₃(NHCy)₆] (phos-1)

2.0 (br,

6H)

3.05 (m,

6H)

1.94, 1.65, 1.50, 1.26, 1.10 (m,

60H)

[N₃P₃(NHCy)₆{AgPPh₃}₂](TfO)₂(2)

[N₃P₃(NHCy)₆{AgPPh₃}₃](TfO)₃(3)

3.20 (br, 3.0 (br, 6H) 1.90, 1.87, 1.66, 1.54, 1.40,

7.54−7.40 (m, 30H; Ph)

7.49−7.42 (m, 45H; Ph)

6H)

1.26−1.07 (m, 60H)

4.20 (br, 3.02 (br,

1.86, 1.46, 1.34, 1.25,

1.04−0.85 (m, 60H)

6H)

2

[N₃P₃(NHCy)₆{AgPPh₂Me}₂](TfO)₂(4) 3.5 (br,

2.95 (br,

6H)

1.85, 1.59, 1.42, 1.21, 1.06 (m, 7.60−7.41 (m, 20H; Ph) 2.11 (d, 6H, J_P−H= 5.9 Hz;

6H)

60H)

Me)

2

[N₃P₃(NHCy)₆{AgPPh₂Me}₃](TfO)₃(5) 4.27 (br, 2.96 (br,

6H) 6H)

1.85, 1.50, 1.39, 1.24,

1.06−0.88 (m, 60H)

7.58−7.44 (m, 30H; Ph) 2.11 (d, 9H, J_P−H= 7.6 Hz;

Me)

2

[N₃P₃(NHCy)₆{AgTPA}₃](TfO)₃(6)

3.41 (br, 2.86 (br,

6H) 6H)

1.85, 1.65, 1.53, 1.15 (m, 60H) 4.59 (d), 4,42 (d) (AB system, 18H, J_H−H= 12.6 Hz;

NCH₂N), 4.24 (br, 18H; PCH₂N)

a

Data taken at RT in CDCl₃, except for 6, whose data are in DMSO. Values in parts per million.

a

Table 4. ³¹P{¹H} NMR Spectroscopic Data for 7−10 and Starting Products

a

b

a

b

compound

δ[N−P−N] [²J(P−P)]

δ[PR₃] [¹J(¹⁰⁹Ag−P)], [¹J(¹⁰⁷Ag−P)]

[N₃P₃(NHCy)₃(NMe₂)₃] (phos-3)

[Ag(OTf)PPh₃]

20.96, 20.70 [44.3]

16.79 (br)

[N₃P₃(NHCy)₃(NMe₂)₃{AgPPh₃}₂](TfO)₂(7)

[N₃P₃(NHCy)₃(NMe₂)₃{AgPPh₃}₃](TfO)₃(8)

[Ag(OTf)PPh₂Me]

24.20 (d, 2P), 23.0 (t, 1P) [36.2]

24.27 (br)

16.58 (dm) [671.5]

16.19 (br)

−3.13 (br)

[N₃P₃(NHCy)₃(NMe₂)₃{AgPPh₂Me}₂](TfO)₂(9)

[N₃P₃(NHCy)₃(NMe₂)₃{AgPPh₂Me}₃](TfO)₃(10)

23.55 (d, br, 2P), 21.05 (t, br, 1P) [40]

24.07 (d, 2P), 23.08 (t, 1P) [33.1]

−2.13 (dd) [765.0], [665.2]

− 3.10 (br)

a

b

Data taken at RT in CDCl₃. Values in parts per million. Values in hertz.

Figure 4. ³¹P{¹H} NMR spectra of compound 9 in CD₂Cl₂at (a) −80 °C and (b) RT.

noting that the molecular cations of almost all complexes 2−10

are observed in the MS spectra.

structure was only conﬁrmed qualitatively but could not be

reﬁned satisfactorily because of severely disordered anions and

unidentiﬁed solvent regions. Selected bond lengths and angles

and details of the data collection and reﬁnement for 7 and 9

are given in tables in the Supporting Information. In both

compounds 7 and 9, the silver atoms show a nearly linear

coordination with angles close to 180° [167.97(8) and

165.57(8)° for 7; 168.26(4) and 177.26(4)° for 9]. The

Ag−N and Ag−P bond lengths are typical.^{23g,24a,37a,42}There

are also short Ag···O contacts: for 7, Ag2···O1 2.704(4) Å; for

9, Ag1···O1 2.781(2) Å and Ag2···O5 2.870(3) Å. Metalation

The single-crystal X-ray diﬀraction analyses of the racemic

complexes 7 and 9 show that in both cases the R_P2,R_P3-

conﬁgured (and S_P2,S_P3-conﬁgured) diastereomer is present

(see Figures 6 and 7). Single crystals of 5, 7·2CH₂Cl₂, and

9·¹/₂C₆H₁₄were grown by the slow diﬀusion of hexane into a

solution of the complex in dichloromethane. The X-ray

analysis conﬁrmed not only the presence of ionic triﬂuor-

omethanesulfonate but also the coordination of metals to the

ring nitrogen atoms (see Figures 5−7). In the case of 5, the

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Chart 2. Diastereomers Expected for Compounds 7 and 9

P−N(ring) bonds at the noncoordinating nitrogen center [av.

1.583(3) Å for 7 and 1.5861(15) Å for 9] and also the P−

N(ring) bond in the starting phosphazene phos-1 (1.598

Å).^28,43Such an N−P bond length increase ﬂanking the site of

coordination (or protonation or alkylation) is known from

studies on CPZs.^{23g,24a,44,45}This is consistent with the bonding

model of Craig and Paddock.⁴⁶Accordingly, in such situations,

the lone pair on the ring nitrogen atom of the cyclo-

phosphazenes is not available for π-bonding interactions within

the ring, causing an increase in the aﬀected bond distance. The

exocyclic P−N bond lengths [av. 1.632(3) Å for P−NHCy and

1.649(3) Å for P−NMe₂in 7; 1.6341(16) Å for P−NHCy and

1.6530(16) Å for P−NMe₂in 9] are longer than the P−

N(ring) bond lengths but are shorter than the ideal P−N

single-bond value of 1.77 Å.^27,30cWhile the phosphazene rings

of the free ligands, i.e., phos-1, are planar or close to

planarity,⁴³the coordination to silver causes the rings to

pucker, which is evident from the ring torsion angles

[maximum absolute values of 24.5(3)° in 7 and 32.9(1)° in 9].

The NH groups of the ligands form classical hydrogen bonds

to the triﬂate anions: for 7, N4···O1 2.964(4) Å, N6···O4

2.968(4) Å, and N8···O2 3.135(5) Å; for 9, N4···O4 2.991(3)

Å, N6···O2 3.020(2) Å, and N8···O4 3.374(3) Å.

Figure 5. Molecular structure of complex 5 determined by single-

crystal X-ray diﬀraction. Hydrogen atoms, anions, and solvent

molecules have been omitted for clarity. The radii are arbitrary.

2.3. Biological Evaluation: Cytotoxic and Antibacte-

rial Activity. All of the complexes are insoluble in water but

soluble in DMSO and in the DMSO/water mixtures used in

the biological tests (cytotoxic and antibacterial ones). In the

cytotoxicity tests, the mixtures used contain a minimal amount

causes distortion of the cyclophosphazene ring skeleton. The

P−N(ring) bonds associated with metal coordination are

longer [av. 1.627(3) Å for 7 and 1.6270(15) Å for 9] than the

Figure 6. Molecular structure of complex 7 determined by single-crystal X-ray diﬀraction. Hydrogen atoms, anions, and solvent molecules have

been omitted for clarity. The radii are arbitrary.

H

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Figure 7. Molecular structure of complex 9 determined by single-crystal X-ray diﬀraction. Hydrogen atoms, anions, and solvent molecules have

been omitted for clarity. The radii are arbitrary.

Table 5. IC₅₀Obtained by AB and NRU in (a) MCF7 and (b) HepG2 Cell Lines Exposed to the New Metallophosphazenes

a

and Their Precursors after 48 h

IC₅₀(μM)

compound

N₃P₃(NHCy)₆(phos-1)

[N₃P₃(NHCy)₆{Ag(PPh₃)}₂](TfO)₂(2)

[N₃P₃(NHCy)₆{Ag(PPh₃)}₃](TfO)₃(3)

[Ag(OTf)PPh₃]

[N₃P₃(NHCy)₆{Ag(PPh₂Me)}₂](TfO)₂(4)

[N₃P₃(NHCy)₆{Ag(PPh₂Me)}₃](TfO)₃(5)

[Ag(OTf)PPh₂Me]

[N₃P₃(NHCy)₆{Ag(TPA)}₃](TfO)₃(6)

[Ag(OTf)TPA]

AB

NRU

(a) MCF7

14.80 6.10

2.87 0.15

1.60 0.05

5.67 0.57

4.89 1.08

3.88 0.11

11.76 2.88

4.46 0.01

59.93 11.36

16.58 4.76

5.48 0.18

4.18 0.18

56.82 4.23

>25

2.34 0.28

2.14 0.65

8.09 1.39

2.45 0.66

3.64 0.05

14.02 1.05

3.32 0.12

30.74 3.06

7.81 0.02

5.95 0.49

3.44 0.10

23.71 1.24

N₃P₃(NMe₂)₃(NHCy)₃(phos-3)

[N₃P₃(NMe₂)₃(NHCy)₃{Ag(PPh₂Me)}₂](TfO)₂(9)

[N₃P₃(NMe₂)₃(NHCy)₃{Ag(PPh₂Me)}₃](TfO)₃(10)

cisplatin

(b) HepG2

N₃P₃(NHCy)₆(phos-1)

>25

[N₃P₃(NHCy)₆{Ag(PPh₃)}₂](TfO)₂(2)

[N₃P₃(NHCy)₆{Ag(PPh₃)}₃](TfO)₃(3)

[Ag(OTf)PPh₃]

[N₃P₃(NHCy)₆{Ag(PPh₂Me)}₂](TfO)₂(4)

[N₃P₃(NHCy)₆{Ag(PPh₂Me)}₃](TfO)₃(5)

[Ag(OTf)PPh₂Me]

[N₃P₃(NHCy)₆{Ag(TPA)}₃](TfO)₃(6)

[Ag(OTf)TPA]

N₃P₃(NMe₂)₃(NHCy)₃(phos-3)

[N₃P₃(NMe₂)₃(NHCy)₃{Ag(PPh₂Me)}₂](TfO)₂(9)

[N₃P₃(NMe₂)₃(NHCy)₃{Ag(PPh₂Me)}₃](TfO)₃(10)

cisplatin

1.40 0.23

0.93 0.37

4.45 0.37

2.38 0.12

2.16 0.13

11.02 0.09

2.02 0.34

45.88 3.34

8.80 0.69

2.82 0.20

2.76 0.22

11.32 1.11

2.41 0.18

1.37 0.55

7.69 1.96

2.61 0.43

2.63 0.39

14.03 1.04

5.48 0.23

29.31 4.09

10.25 2.05

4.05 0.24

3.20 0.24

6.94 0.72

a

All the compounds analyzed were dissolved in DMSO, not exceeding 0.1%, except cisplatin, which was dissolved in water.

of DMSO (≤0.1%). In the antibacterial tests, the solvent

DMSO was also used as a control (in the same percentage

used to dissolve the compounds) so as to conﬁrm that it did

not inhibit bacterial growth. While the tests were performed,

no precipitation of any compound at the concentration ranges

assayed was observed. The colorless DMSO solutions were

very stable at RT. The stability of compounds in DMSO was

provided by ³¹P and ¹H NMR spectroscopy. In the Supporting

Information, we have included these spectra for compound 2,

as an example, measured over 48 h, which is the time for the

biological assays. These spectra showed the expected signals

according to the proposed structure for compound 2, slightly

displaced with respect to those observed for the same complex

in other solvents such as acetone, which remain exactly the

same for more than 48 h. Among the new metallophospha-

zenes 1−10, we have selected 2−6, 9, and 10 to carry out the

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Table 6. MICs (μM) Obtained for Silver Phosphazenes and Their Precursors against Gram-Positive, Gram-Negative, and

MTBC Strains

S. aureus (Gram-

E. coli (Gram-

negative)

P. aeruginosa (Gram-

M. bovis

BCG

M. tuberculosis

compound

positive)

negative)

H37Rv

[N₃P₃(NHCy)₆] (phos-1)

250

≤0.12

0.24

0.97

1.95

0.97

7.8

125

0.49

0.97

3.9

1.95

0.97

3.9

1.95

62.5

125

3.9

125

3.9

15.6

7,8

3.9

15.6

3.9

62.5

7.8

≤0.12

15.6

7.8

3.9

15.6

7.8

31.25

3.9

0.97

31.25

3.9

31.25

3.9

[N₃P₃(NHCy)₆{AgPPh₃}₂](TfO)₂(2)

[N₃P₃(NHCy)₆{AgPPh₃}₃](TfO)₃(3)

[Ag(OTf)PPh₃]

[N₃P₃(NHCy)₆{AgPPh₂Me}₂](TfO)₂(4)

[N₃P₃(NHCy)₆{AgPPh₂Me}₃](TfO)₃(5)

[Ag(OTf)PPh₂Me]

[N₃P₃(NHCy)₆{AgTPA}₃](TfO)₃(6)

[Ag(OTf)TPA]

[N₃P₃(NHCy)₃(NMe₂)₃] (phos-3)

3.9

250

62.5

7.8

250

125

15.6

≤0.12

31.25

15.6

3.9

[N₃P₃(NHCy)₃(NMe₂)₃{AgPPh₂Me}₂](TfO)₂

(9)

[N₃P₃(NHCy)₃(NMe₂)₃{AgPPh₂Me}₃](TfO)₃

7.8

1.95

15.6

≤0.12

3.9

(10)

a

AgNO₃

31.25

44.8

15.6

31.25

b

c

d

AgSD

22.4

13−90

nd

a

b

c

Data measured by us, under the same conditions as those for the other compounds. Data taken from ref 60. Data taken from ref 61, where

d

authors studied several strains of P. aeruginosa. MICs were transformed by us from milligrams per milliliter to micromolars. Not determined.

biological studies so as to evaluate the structure−activity

relationship. Under the same conditions, cisplatin and silver(I)

nitrate, two well-known chemotherapeutic and antimicrobial

drugs, respectively, have been tested and compared to all of the

compounds studied.

was more sensitive in comparison to NRU. Once more, all

metallophosphazenes showed lower IC₅₀values than their

precursors and cisplatin, with values of 11.32 and 6.94 μM in

the AB and NRU assays, respectively, with the new silver

phosphazenes being extraordinarily eﬀective as cytotoxic

agents in vitro. Regarding the cytotoxicity of the precursors

in the HepG2 cells, only [Ag(OTf)PPh₃] was just as or even

more cytotoxic than cisplatin but also much less cytotoxic than

their silver phosphazenes, 2 and 3.

Cytotoxic Activity. The in vitro cytotoxicity of the new

complexes 2−6, 9, and 10 and their precursors (the

phosphazene ligands phos1 and phos3 and the silver starting

complexes) has been evaluated against two tumor human cell

lines, MCF7 and HepG2, by two diﬀerent biomarkers, Alamar

Blue (AB) and Neutral Red Uptake (NRU) after 48 h of

exposure. At 24 h, the exposed cells were checked under an

optical microscope, and the damage was observed at the similar

range, which has been calculated after 48 h (see the Supporting

Information). By using both viability assays, median inhibitory

concentration (IC₅₀) values (Table 5a,b) were calculated from

the dose−response curves obtained by nonlinear regression

analysis. IC₅₀values are the concentrations of a drug required

to inhibit tumor cell proliferation by 50% compared to

nontreated cells.

All of the tested metallophosphazenes (2−6, 9, and 10)

showed excellent antitumor activities toward the MCF7 cell

line with IC₅₀values lower than 5.5 μM (see Table 5a), and

the best of these were complexes 2 and 3 with IC₅₀values with

both biomarkers lower than 3 μM. Except [Ag(OTf)TPA], all

of the compounds that we tested were more cytotoxic than

cisplatin toward MCF7. Besides, all of the new metal-

lophosphazenes (2−6, 9, and 10) were more cytotoxic than

their phosphazene ligands and silver precursors and much

more cytotoxic than cisplatin, which showed IC₅₀values of

56.82 and 23.71 μM in the AB and NRU assays, respectively.

In general, NRU proved to be a more sensitive assay for MCF7

cells than AB except for compound 3, in which both of them

are similar.

As for the structure−activity relationships toward both cell

lines in these metallophosphazenes 2−6, 9, and 10, the

following can be concluded: (1) the silver atom exerts the

cytotoxic activity, which is signiﬁcantly enhanced by the rise in

the number of silver atoms linked to the phosphazene ring (see

Table 5a,b; 3 is more toxic than 2, 5 more than 4, and 10 more

than 9); (2) the ligands coordinated to the silver atom also

have an inﬂuence in the cytotoxicity, which is higher in

triphenylphosphane derivatives than in those with diphenyl-

methylphosphane or TPA (see Table 5a,b; 3 is more toxic than

5 or 6). Moreover, the cytotoxicity was also higher in those

with the phosphazene ligand phos-1 than those with phos-3

(see Table 5a,b; 4 is more toxic than 9 and 5 more than 10).

Thus, 3 is the most toxic compound, followed by 2.

Remarkably, both metallophosphazenes 2 and 3 were just as

toxic as auranoﬁn, a well-known cytotoxic gold(I) compound

that is even more toxic than cisplatin for MCF7 and HepG2

cells. The IC₅₀values reported for auranoﬁn are 1.1 μM for

MCF7 cells⁴⁷and 2.0 μM for HepG2 cells.⁴⁸The anticancer

activity of silver(I) complexes has been summarized in recent

reviews,^2f,9,11cand the IC₅₀values obtained for all of the tested

silver phosphazenes are among the lowest found for any silver

derivatives against MCF7 and HepG2 cell lines, taking into

account the experimental conditions (measured at 48 h). To

the best of our knowledge, the lowest IC₅₀values found in the

literature for silver complexes against the mentioned cell lines

have been described by Hadjikakou et al. (IC₅₀values range

between 1.6 and 2.5 μM in MCF7, for acetyl salicylate or

hydroxybenzoic acid derivatives),⁴⁹by Galal et al. (IC₅₀value

of 2.0 μM in MCF7, for a complex of 2-methyl-1H-

benzimidazole-5-carboxylic acid hydrazide),⁵⁰by Gautier et

The cytotoxicity of all metallophosphazenes acquired in

HepG2 cells was even more drastic than that in MCF7 (Table

5b). In the AB assay, the obtained IC₅₀values were lower than

3 μM, with 2 and 3 being the most cytotoxic compounds, with

values of 1.40 and 0.93 μM, respectively. In this case, in

contrast to the results we found for MCF7, the biomarker AB

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al.,⁵¹Hague et al.,⁵²or Tacke et al.⁵³(IC₅₀values of <0.4 μM,

0.9 μM, or ranging from 1.4 to 5.8 μM in MCF7, respectively,

all of them for carbene complexes), by Yilmaz et al. (IC₅₀

values ranging from approximately 1 to 5 μM in MCF7, for

saccharinate complexes with mono-⁵⁴and diphosphane

ligands),⁵⁵by Gimeno et al. (IC₅₀values ranging between

2.81 and 25.42 μM in MCF7 and HepG2 cells, for complexes

with modiﬁed amino acid esters and phosphane ligands),⁵⁶and

by McCann, Egan, and co-workers (IC₅₀values between 0.9

and 3.8 μM in HepG2, for 1,10-phenanthroline-5,6-dione,⁵⁷6-

hydroxycoumarin-3-carboxylato,⁵⁸and 4-oxy-3-nitrocoumarin

complexes).⁵⁹

Antibacterial Activity. The antibacterial activities of the

new complexes 2−6, 9, and 10 and their precursors

(phosphazene ligands phos-1 and phos-3 and silver starting

compounds) were tested against Gram-negative strains E. coli

ATCC 10536 and P. aeruginosa ATCC 15442, Gram-positive

strain S. aureus ATCC 11632, and two MTBC strains, M.

tuberculosis H37Rv ATCC 27294 and M. bovis BCG Pasteur.

The minimum inhibitory concentrations (MICs) obtained and

those of AgNO₃and silver sulfadiazine (AgSD) are listed in

Table 6.

10; there is an exception regarding M. bovis and M. tuberculosis

H37Rv, which can be attributed to the fact that the free

phosphazene ligand phos-3 was also active against the two

MTBC strains). (3) There is not such a clear inﬂuence of the

number of silver atoms linked to the phosphazene ring as that

observed for the cytotoxic activity (see Table 6; 5 is more toxic

than 4, but the activity of 9 and 10 is similar against all

bacterial strains used, and 2 is even more toxic than 3 against

Gram-positive and Gram-negative strains). Remarkably, as in

the case of the cytotoxic properties, both metallophosphazenes

2 and 3 showed outstanding antibacterial activity against all

strains studied. The broad spectral antimicrobial eﬃcacy of all

of these metallophosphazenes may be explained by the lability

of the ligands in these silver(I) complexes. It was concluded

that the antimicrobial properties of silver(I) complexes depend

upon the fast rate of ligand exchange of the metal ion in the

biological system (rather than the solubility, charge, or

chirality), which correlates to the nature of the donor atoms

coordinated to the silver atom. Weak Ag−O and Ag−N bonds

play a key role in determining the wide spectral antibacterial

activity of silver(I) complexes because of the easy substitution

of the corresponding labile ligands with the sulfur- or nitrogen-

donor sites of amino acids or nucleotides in bacteria.⁶²

The MIC values of the tested metallophosphazenes (2−6, 9,

and 10) indicated that all of them exhibited excellent

antibacterial activity against all bacterial strains used in this

work, being much higher than those of AgNO₃and AgSD for

the entire range of bacteria studied, except 3, 9, and 10, which

had a activity similar to that of AgNO₃against P. aeruginosa.

The MICs of all metallophosphazenes range from 0.12 to 15.6

μM. All metallophosphazenes exhibited much better activity

than their phosphazene ligands and also than their silver

precursors except 9 and 10, which were as eﬀective as their

silver precursor [Ag(OTf)PPh₂Me] against S. aureus, E. coli,

and P. aeruginosa. 3 also exhibited an activity similar to that of

its silver precursor, [Ag(OTf)PPh₃], against P. aeruginosa. The

phosphazene ligands phos-1 and phos-3 were not active

against Gram-positive and Gram-negative strains, but they

were against the two MTBC strains, M. tuberculosis H37Rv and

M. bovis BCG Pasteur, especially phos-3. Moreover, all

metallophosphazenes and silver precursors showed better

bactericidal properties against S. aureus and E. coli than against

P. aeruginosa and, in general, also better than those against the

two MTBC strains. The best of these complexes were 2, 3, and

5 with MIC values for S. aureus and E. coli between 0.12 and

0.97 μM (or 0.2−2.2 μg/mL if the molar mass is taken into

account), which are among the lowest values found for any

silver derivatives (see ref 2f and references cited therein). 3, 9,

and 10 also exhibited an outstanding activity against M. bovis

BCG Pasteur (see Table 6); the MICs for the three complexes

are ≤0.12 μM (0.3 μg/mL for 3, 0.2 μg/mL for 9, and 0.2 μg/

mL for 11). 3 also exhibited excellent activity against M.

tuberculosis H37Rv (see Table 6; 0.97 μM or 2.2 μg/mL).

As for the structure−activity relationships toward all

bacterial strains in these metallophosphazenes, it can be said

that the general trends observed for the cytotoxic activity have

been followed, in spite of not being so clear in some cases. The

following can be concluded: (1) The antibacterial activity is

higher in the triphenylphosphane derivatives than those in

diphenylmethylphosphane or TPA (see Table 6; 3 is more

toxic than 5 or 6; there is an exception regarding P.

aeruginosa). (2) The antibacterial activity is also higher in

those metallophosphazenes with phos-1 than in those with

phos-3 (see Table 6; 4 is more toxic than 9 and 5 more than

3. CONCLUSIONS

Two multisite (amino)cyclotriphosphazene ligands, phos-1

and phos-3, were both prepared in high yield (>80%) from

[N₃P₃Cl₆] and phos-2, respectively. The reaction of phos-1 or

phos-3 with the silver complexes [Ag(OTf)L] (OTf =

OSO₂CF₃; L = PPh₃, PPh₂Me, or TPA, where TPA= 1,3,5-

triaza-7-phosphaadamantane) in dichloromethane and in

diﬀerent molar ratios of 1:2 or 1:3 led to two series of cationic

metallophosphazenes, [N₃P₃(NHCy)₆{AgL}_n](TfO)_n[n = 2,

L = PPh₃(2), PPh₂Me (4); n = 3, L = PPh₃(3), PPh₂Me (5),

TPA (6)] and nongem-trans-[N₃P₃(NHCy)₃(NMe₂)₃{AgL}_n]_-

(TfO)_n[n = 2, L = PPh₃(7), PPh₂Me (9); n = 3, L = PPh₃

(8), PPh₂Me (10)]. In all of them, the silver groups “AgL” are

coordinated to the nitrogen atoms of the phosphazene ring,

whereby their number (2 or 3) depends on the molar ratio

used. This type of coordination was conﬁrmed by NMR

spectroscopy and also, more speciﬁcally, by single-crystal X-ray

diﬀraction for complexes 5, 7, and 9.

The cytotoxic activity of 2−6, 9, and 10 has been studied

against two tumor human cell lines, MCF7 (breast

adenocarcinoma) and HepG2 (hepatocellular carcinoma).

The IC₅₀values, which range from 1.6 to 5.5 μM for MCF7

and from 0.9 to 2.8 μM for HepG2, reveal excellent cytotoxic

activity for these metal complexes compared with their

precursors and much higher than that of cisplatin, with their

IC₅₀values being among the lowest found in silver complexes.

The structure−activity relationships toward both cell lines

were clear. The cytotoxicity is higher in triphenylphosphane

derivatives than in those with diphenylmethylphosphane or

TPA and is higher in those with the phosphazene ligand phos-

1 than in those with the phosphazene phos-3. A signiﬁcant

improvement in the activity was also observed upon a rise in

the number of silver atoms linked to the phosphazene ring.

The same complexes were also tested against Gram-negative

strains E. coli and P. aeruginosa, Gram-positive strain S. aureus,

and two MTBC strains, M. tuberculosis H37Rv and M. bovis

BCG Pasteur. The MIC values, which range from 0.12 to 15.6

μM (some of them being the lowest found in silver

complexes), indicated that all of them also exhibited excellent

K

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Yield: 3.13 g, 84%. Anal. Calcd for C₆H₁₈Cl₃N₆P₃(373.53): C,

19.29; H, 4.86; N, 22.50. Found: C, 19.50; H, 4.90; N, 22.40. IR

(ATR, cm⁻¹): 1193 (vs), 1172 (vs), 1146 (s, sh) (PN and C−N);

1059 (m) (P−NR₂); 595 (s), 512 (vs), 495 (vs, br) (P−Cl). ³¹P{¹H}

antibacterial activity against all bacterial strains used, with their

activity being much higher than those of AgNO₃and AgSD. As

for the structure−activity relationships, the general trends

observed for the cytotoxic activity are also followed for the

bacterial growth inhibition of these new metallophosphazenes.

Thus, 2 and 3 are the most eﬀective bacterial and cell growth

inhibitors, which showed outstanding antitumoral and

antibacterial activity against all cell lines and bacterial strains

studied. 9 and 10 also exhibited remarkable activity against the

two MTBC strains, especially for M. bovis BCG Pasteur. The

outstanding biological activities of these complexes are worth

studying further to determine action mechanisms and to

elucidate the possibility of new biological targets.

2

NMR (CDCl₃): δ 28.65 (1P), 28.18 (2P) (AB₂system, J(P−P) =

40.6 Hz; N₃P₃ring). ¹H NMR (CDCl₃): δ 2.72 (m, N = 17.6 Hz, 6H;

N(CH₃)₂), 2.70 (m, N = 18.0 Hz, 12H; N(CH₃)₂). ¹³C{¹H} NMR

(CDCl₃): δ 36.40 (s, 2C; N(CH₃)₂), 36.19 (s, 4C; N(CH₃)₂). MS

(FAB⁺): m/z 373 (100%; [M]⁺); and peaks derived from the

sequential loss of NMe₂and Cl.

Synthesis of nongem-trans-[N₃P₃(NHCy)₃(NMe₂)₃] (phos-3). To a

solution of phos-2 (0.374 g, 1 mmol) in dry tetrahydrofuran (20 mL)

was added slowly and dropwise a solution of NH₂Cy (4.6 mL, 40

mmol) in dry tetrahydrofuran (10 mL). The mixture was stirred at

RT for 12 h and then reﬂuxed for another 48 h. The precipitate of

amine hydrochloride was ﬁltered oﬀ, and the solvent was removed

from the ﬁltrate in a vacuum. The addition of light petroleum (40−60

°C) led to the precipitation of phos-3 as a white solid. Successive

additions of petroleum ether led to the precipitation of phos-3 as a

white solid.

4. EXPERIMENTAL SECTION

4.1. General Data. IR spectra were recorded in the range 4000−

250 cm⁻¹on a PerkinElmer Spectrum-100 (ATR mode) Fourier

transform infrared spectrometer. Carbon, hydrogen, nitrogen, and

sulfur analyses were performed using a PerkinElmer 240 B

microanalyzer. NMR spectra were recorded on a Bruker AV 400

spectrometer. Chemical shifts are quoted relative to SiMe₄(TMS; ¹H

and ¹³C NMR, external) and H₃PO₄(85%; ³¹P NMR, external) and

given in parts per million (ppm). Fast-atom-bombardment (FAB) MS

spectra were recorded using a Micromass Autospec spectrometer in

positive-ion mode with m-nitrobenzyl alcohol as the matrix.

Hexachlorocyclotriphosphazene, [N₃P₃Cl₆] (Stream Chemicals),

was puriﬁed by recrystallization from hot hexane and dried in a

vacuum. Metal complexes [Ag(OTf)L] (L = PPh₃, PPh₂Me, or TPA;

OTf = OSO₂CF₃) were prepared according to published procedur-

es.^37a,38,63The culture medium, fetal bovine serum, and cell culture

reagents were obtained from Gibco and Corning (Biomol, Spain).

Chemicals for the diﬀerent assays were provided by VWR

International Eurolab and Merck. Plastic materials for the cytotoxicity

assays were supplied by Fisher Scientiﬁc.

4.2. Synthesis and Spectroscopic Characterization Data.

Synthesis of [N₃P₃(NHCy)₆] (phos-1). To a solution of [N₃P₃Cl₆]

(0.348 g, 1 mmol) in dry tetrahydrofuran (20 mL) was added slowly

and dropwise a solution of NH₂Cy (4.6 mL, ρ = 0.867 g/mL, 40

mmol) in dry tetrahydrofuran (10 mL). The mixture was stirred at

RT for 12 h and then reﬂuxed for 48 h. The precipitate of amine

hydrochloride was ﬁltered oﬀ, and the solvent was removed from the

ﬁltrate in a vacuum. The addition of light petroleum (40−60 °C) led

to the precipitation of phos-1 as a white solid. Successive additions of

petroleum ether led to the precipitation of phos-1 as a white solid.

Yield: 579 mg, 80%. Anal. Calcd for C₃₆H₇₂N₉P₃(723.94): C,

59.73; H, 10.02; N, 17.41. Found: C, 59.60; H, 9.90; N, 17.32. IR

(ATR, cm⁻¹): 3405 (w), 3360 (w), 3224 (w, br) (N−H); 1186 (s,

sh), 1178 (vs) (PN and C−N); 1092 (vs), 1080 (s) (P−NHR).

³¹P{¹H} NMR (CDCl₃): δ 14.44 (s, 3 P; N₃P₃ring). ¹H NMR

Yield: 477.4 mg, 85%. Anal. Calcd for C₂₄H₅₄N₉P₃(561.67): C,

51.32; H, 9.69; N, 22.44. Found: C, 51.30; H, 10.0; N, 22.0. IR (ATR,

cm⁻¹): 3418 (w), 3367 (w), 3214 (w, br) (N−H); 1180 (s, sh), 1171

(vs), 1140 (m) (PN and C−N); 1111 (m, sh), 1096 (s), 1084 (vs)

(P−NHR). ³¹P{¹H} NMR (CDCl₃): δ 20.96 (1P), 20.70 (2P) (AB₂

2

1

system, J(P−P) = 44.3 Hz; N₃P₃ring). H NMR (CDCl₃): δ 3.01

(br, 3H; NH−CH), 2.62 (m, N = 13.6 Hz, 18H; N(CH₃)₂), 1.96 (m,

br, 9H; NH and NH(C₆H₁₁)), 1.64 (br, 6H; NH(C₆H₁₁)), 1.53 (br,

3H; NH(C₆H₁₁)), 1.26 (br, 6H; NH(C₆H₁₁)), 1.09 (br, 9H;

1

NH(C₆H₁₁)). H NMR ((CD₃)₂CO): δ 2.98 (br, 3H; NH−CH),

2.57 (m, N = 14 Hz, 18H; N(CH₃)₂), 2.41 (br, 3H; NH), 1.95 (m, br,

6H; NH(C₆H₁₁)), 1.67 (m, 6H; NH(C₆H₁₁)), 1.54 (m, 3H;

NH(C₆H₁₁)), 1.27 (m, 6H; NH(C₆H₁₁)), 1.15 (m, 9H; NH(C₆H₁₁)).

2

¹³C{¹H} NMR ((CD₃)₂CO, APT): δ 50.38 (d, J(P−C) = 10.3 Hz,

2

2C; NH−CH), 50.28 (d, J(P−C) = 10.3 Hz, 1C; NH−CH), 37.60

(s, 6C; N(CH₃)₂), 36.81 (d, ³J(P−C) = 8.4 Hz, 4C; CH₂), 36.73 (d,

³J(P−C) = 11.3 Hz, 2C; CH₂), 26.58, 26.24, 26.17 (s, 9C; CH₂). MS

(FAB⁺): m/z 562.5 (100%; [M + H]⁺); and peaks derived from the

sequential loss of NMe₂and NHCy.

Synthesis of [N₃P₃(NHCy)₆Ag]_n(TfO)_n(1). To a solution of phos-1

(144.8 mg, 0.2 mmol) in acetone (20 mL) was added AgTfO (51.4

mg, 0.2 mmol). The mixture was stirred at RT for 30 min protected

from light. The solution was evaporated to ca. 1 mL. The addition of

hexane led to the precipitation of 1 as a white solid.

Yield: 129.5 mg, 66%. Anal. Calcd for C₃₇H₇₂AgF₃N₉O₃P₃S

(980.88): C, 45.31; H, 7.40; N, 12.85; S, 3.27. Found: C, 45.15; H,

8.01; N, 12.85; S, 3.60. IR (ATR, cm⁻¹): 3308 (m, br) (N−H); 1076

(s) (P−NHR). Other bands: 1297 (s), 1226 (s, br), 1165 (m), 1081

(s), 1030 (vs). ³¹P{¹H} NMR (CDCl₃): δ 10.39 (t, 1P), 8.43 (d, 2P)

2

1

(AB₂system, J_AB= 45.1 Hz; N₃P₃ring). H NMR (CDCl₃): δ 3.15

(br, 6H; NH), 3.04 (br, 6H; NH−CH), 1.90 (m, 12H; NH(C₆H₁₁)),

1.68 (br, 12H; NH(C₆H₁₁)), 1.56 (m, 6H; NH(C₆H₁₁)), 1.30−1.19

(m, 30H; NH(C₆H₁₁)). ¹⁹F{¹H} NMR (CDCl₃, RT): δ −78.17 (s;

SO₃CF₃). MS (FAB⁺): m/z 1556 (3%; [[N₃P₃(NHCy)₆]₂Ag]⁺), 831

(3%; [M − SO₃CF₃]⁺), 725 (100%; [M − AgSO₃CF₃+ H]⁺); and

peaks derived from the sequential loss of NH₂Cy and Cy.

(CDCl₃): δ 3.05 (m, 6H; NH−CH), 2.0 (br, 6H; NH), 1.94 (m,

12H; NH(C₆H₁₁)), 1.65 (m, 12H; NH(C₆H₁₁)), 1.50 (m, 6H;

NH(C₆H₁₁)), 1.26 (m, 12H; NH(C₆H₁₁)), 1.10 (m, 18H; NH-

1

(C₆H₁₁)). H NMR ((CD₃)₂CO): δ 3.03 (m, 6H; NH−CH), 2.36

(br, 6H; NH), 1.97 (m, 12H; NH(C₆H₁₁)), 1.67 (m, 12H;

NH(C₆H₁₁)), 1.54 (m, 6H; NH(C₆H₁₁)), 1.28 (m, 12H; NH-

(C₆H₁₁)), 1.14 (m, 18H; NH(C₆H₁₁)). ¹³C{¹H} NMR ((CD₃)₂CO,

APT): δ 50.45 (s, 6C; NH−CH), 36.99, 26.6, 26.17 (s, 30C; CH₂).

MS (FAB⁺): m/z 725 (100%; [M + H]⁺); and peaks derived from the

sequential loss of NH₂Cy and Cy.

Synthesis of [N₃P₃(NHCy)₆{AgL}₂](TfO)₂[L = PPh₃(2), PPh₂Me

(4)]. To a solution of phos-1 (72.4 mg, 0.1 mmol) in dichloromethane

(15 mL) was added [Ag(OTf)L] (0.2 mmol, 103.8 mg for 2 or 91.4

mg for 4), and the mixture was stirred at RT for 30 min protected

from light. The solution was evaporated to ca. 1 mL. The addition of

hexane led to the precipitation of 2 or 4 as a white solid.

Synthesis of nongem-trans-[N₃P₃Cl₃(NMe₂)₃] (phos-2). To a

solution of [N₃P₃Cl₆] (3.48 g, 10 mmol) in dry tetrahydrofuran (30

mL) was added slowly and dropwise at 0 °C 60 mmol of NHMe₂(30

mL of a 2.0 M solution in tetrahydrofuran). The mixture was stirred

for 1 h. When the mixture had reached RT, the precipitate of amine

hydrochloride was ﬁltered oﬀ, and the solvent was removed from the

ﬁltrate in a vacuum, aﬀording an oil. The addition of hexane led to the

precipitation of phos-2 as a white solid.

Compound 2. Yield: 130.4 mg, 74%. Anal. Calcd for

C₇₄H₁₀₂Ag₂F₆N₉O₆P₅S₂(1762.39): C, 50.43; H, 5.83; N, 7.15; S,

3.64. Found: C, 50.00; H, 5.30; N, 6.95; S, 3.60. IR (ATR, cm⁻¹):

3285 (m, br) (N−H); 1074 (s) (P−NHR). Other bands: 1284 (m),

1237 (s), 1221 (vs), 1157 (m), 1095 (s), 1025 (vs). ³¹P{¹H} NMR

(CDCl₃, RT): δ 16.90 (br s, 3P; N₃P₃ring), 17.12 (dd, ¹J(¹⁰⁹Ag−P) =

746.1 Hz, ¹J(¹⁰⁷Ag−P) = 653.4 Hz, 2P; PPh₃). ³¹P{¹H} NMR

((CD₃)₂CO, RT): δ 16.11 (br s, 3P; N₃P₃ring), 17.48 (dd, ¹J(¹⁰⁹Ag−

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1

P) = 748.9 Hz, J(¹⁰⁷Ag−P) = 652.6 Hz, 2P; PPh₃). ³¹P{¹H} NMR

addition of hexane led to the precipitation of 3 or 5 as a white solid.

The addition of diethyl ether led to the precipitation of 6.

((CD₃)₂CO, −80 °C): δ 17.54 (t, 1P), 13.23 (d, 2P) (AB₂system,

²J(P−P) = 36.3 Hz; N₃P₃ring), 17.51 (dd, J(¹⁰⁹Ag−P) = 740.6 Hz,

Compound 3. Yield: 178.0 mg, 78%. Anal. Calcd for

C₉₃H₁₁₇Ag₃F₉N₉O₉P₆S₃(2281.61): C, 48.96; H, 5.17; N, 5.53; S,

4.22. Found: C, 49.00; H, 5.35; N, 4.95; S, 4.10. IR (ATR, cm⁻¹):

3267 (m, br) (N−H); 1074 (vs) (P−NHR). Other bands: 1282 (m),

1236 (s), 1219 (s), 1154 (s), 1095 (s, sh), 1023 (vs). ³¹P{¹H} NMR

1

¹J(¹⁰⁷Ag−P) = 641.3 Hz, 2P; PPh₃). ¹H NMR (CDCl₃, RT): δ 7.54−

7.40 (m, 30H; C₆H₅), 3.40 (br, 6H; NH), 3.00 (br, 6H; NH−CH),

1.90 (br, 12H; NH(C₆H₁₁)), 1.66 (br, 12H; NH(C₆H₁₁)), 1.54 (br,

1

6H; NH(C₆H₁₁)), 1.26−1.09 (m, br, 30H; NH(C₆H₁₁)). H NMR

1

(CDCl₃, RT): δ 18.38 (s, 3P; N₃P₃ring), 16.53 (dd, J(¹⁰⁹Ag−P) =

((CD₃)₂CO, RT): δ 7.67−7.54 (m, 30H; C₆H₅), 4.03 (br, 6H; NH),

3.20 (br, 6H; NH−CH), 2.00 (br, 12H; NH(C₆H₁₁)), 1.63 (br, 12H;

NH(C₆H₁₁)), 1.49 (br, 6H; NH(C₆H₁₁)), 1.31 (m, br, 12H;

NH(C₆H₁₁)), 1.11 (m, br, 18H; NH(C₆H₁₁)). ¹H NMR

((CD₃)₂CO, −80 °C): δ 7.72−7.55 (m, 30H; C₆H₅), 4.65 (t, br,

2H; NH), 4.40 (br, 4H; NH), 3.13 (br, 6H; NH−CH), 2.00 (br,

12H; NH(C₆H₁₁)), 1.65 (br, 6H; NH(C₆H₁₁)), 1.53−1.41 (br, 12H;

NH(C₆H₁₁)), 1.29−0.56 (m, br, 30H; NH(C₆H₁₁)). ¹³C{¹H} NMR

((CD₃)₂CO, APT, RT): δ 134.96 (d, ²J(P−C) = 16.1 Hz, 12C;

1

761.7 Hz, J(¹⁰⁷Ag−P) = 662.0 Hz, 3P; PPh₃). H NMR (CDCl₃,

RT): δ 7.49−7.42 (m, 45H; C₆H₅), 4.20 (br, 6H; NH), 3.02 (br, 6H;

NH−CH), 1.86 (m, 12H; NH(C₆H₁₁)), 1.46 (m, 12H; NH(C₆H₁₁)),

1.34 (m, 6H; NH(C₆H₁₁)), 1.25 (m, 12H; NH(C₆H₁₁)), 1.04−0.85

(m, 18H; NH(C₆H₁₁)). ¹³C{¹H} NMR (CDCl₃, APT, RT): δ 134.07

(d, ²J(P−C) = 11.0 Hz, 18C; PPh₃), 131.40 (s, 9C; PPh₃), 130.03 (s,

1

9C; PPh₃), 129.40 (s, 18C; PPh₃), 120.53 (q, J(C−F) = 320.2 Hz,

3C; SO₃CF₃), 51.89 (s, 6C; NH−CH), 36.60, 25.37, 25.12 (s, 30C;

CH₂). ¹⁹F{¹H} NMR (CDCl₃, RT): δ −77.86 (s; SO₃CF₃). MS

(FAB⁺): m/z 2132 (1%; [M − SO₃CF₃]⁺), 1613 (3%; [M − 2SO₃CF₃

− PPh₃− Ag]⁺), 1464 (1%; [M − 3SO₃CF₃− PPh₃− Ag + H]⁺),

1202 (1%; [M − 3SO₃CF₃− 2PPh₃− Ag + H]⁺), 1094 (100%; [M −

3SO₃CF₃− 2PPh₃− 2Ag]⁺), 830 (20%; [M − 3SO₃CF₃− 3PPh₃−

2Ag]⁺), 725 (35%; [M − 3SO₃CF₃− 3PPh₃− 3Ag + H]⁺); and peaks

derived from the sequential loss of NH₂Cy.

1

PPh₃), 132.58 (s, 6C; PPh₃), 130.46 (d, J(P−C) = 41.9 Hz, 6C;

PPh₃), 130.36 (d, ³J(P−C) = 11.0 Hz, 12C; PPh₃), 121.99 (q, ¹J(C−

F) = 321.8 Hz, 2C; SO₃CF₃), 52.12 (s, 4C; NH−CH), 52.01 (s, 2C;

NH−CH), 37.22, 26.14, 26.03 (s, 30C; CH₂). ¹⁹F{¹H} NMR (CDCl₃,

RT): δ −77.84 (s; SO₃CF₃). MS (FAB⁺): m/z 1613 (10%; [M −

SO₃CF₃]⁺), 1464 (3%; [M + H − 2SO₃CF₃]⁺), 1202 (8%; [M + H −

2SO₃CF₃− PPh₃]⁺), 1093 (30%; [M − 2SO₃CF₃− PPh₃− Ag]⁺),

831 (7%; [M − 2SO₃CF₃− 2PPh₃− Ag]⁺), 724 (100%; [M −

2SO₃CF₃− 2PPh₃− 2Ag]⁺); and peaks derived from the sequential

loss of NH₂Cy.

Compound 5. Yield: 136.2 mg, 65%. Anal. Calcd for

C₇₈H₁₁₁Ag₃F₉N₉O₉P₆S₃(2095.40): C, 44.71; H, 5.34; N, 6.02; S,

4.59. Found: C, 44.45; H, 5.70; N, 5.85; S, 4.40. IR (ATR, cm⁻¹):

3281 (m, br) (N−H); 1076 (vs) (P−NHR). Other bands: 1281 (m),

1239 (s), 1221 (s), 1156 (s), 1096 (m, sh), 1024 (vs). ³¹P{¹H} NMR

Compound 4. Yield: 142.5 mg, 87%. Anal. Calcd for

C₆₄H₉₈Ag₂F₆N₉O₆P₅S₂(1638.25): C, 46.92; H, 6.03; N, 7.69; S,

3.91. Found: C, 46.45; H, 6.40; N, 7.35; S, 3.70. IR (ATR, cm⁻¹):

3301 (m, br) (N−H); 1075 (vs) (P−NHR). Other bands: 1275 (m),

1239 (s), 1221 (vs), 1155 (s), 1095 (s, sh), 1025 (vs). ³¹P{¹H} NMR

(CDCl₃, RT): δ 16.22 (br s, 3P; N₃P₃ring), −1.90 (dd, ¹J(¹⁰⁹Ag−P)

1

(CDCl₃, RT): δ 18.57 (s, 3P; N₃P₃ring), −2.78 (d, br, J(Ag−P) =

1

718.0 Hz, 3P; PPh₂Me). H NMR (CDCl₃, RT): δ 7.58−7.44 (m,

30H; PPh₂Me), 4.27 (br, 6H; NH), 2.96 (br, 6H; NH−CH), 2.11 (d,

²J(H−P) = 7.6 Hz, 9H; PPh₂Me), 1.85 (m, 12H; NH(C₆H₁₁)), 1.50

(m, 12H; NH(C₆H₁₁)), 1.39 (m, 6H; NH(C₆H₁₁)), 1.24 (m, 12H;

NH(C₆H₁₁)), 1.06−0.88 (m, 18H; NH(C₆H₁₁)). ¹H NMR

((CD₃)₂CO, RT): δ 7.77−7.51 (m, 30H; PPh₂Me), 4.38 (br, 6H;

NH), 3.15 (br, 6H; NH−CH), 2.20 (d, ²J(H−P) = 7.6 Hz, 9H;

PPh₂Me), 2.0 (m, 12H; NH(C₆H₁₁)), 1.61 (m, 12H; NH(C₆H₁₁)),

1.46 (m, 6H; NH(C₆H₁₁)), 1.40−1.27 (m, 12H; NH(C₆H₁₁)), 1.10−

1.05 (m, 18H; NH(C₆H₁₁)). ¹³C{¹H} NMR ((CD₃)₂CO, APT, RT):

δ 133.56 (d, ²J(P−C) = 15.2 Hz, 12C; PPh₂Me), 132.79 (d, ¹J(P−C)

= 757.6 Hz, J(¹⁰⁷Ag−P) = 651.4 Hz, 2P; PPh₂Me). ³¹P{¹H} NMR

1

((CD₃)₂CO, RT): δ 16.24 (br s, 3P; N₃P₃ring), −1.25 (dd,

¹J(¹⁰⁹Ag−P) = 757.8 Hz, ¹J(¹⁰⁷Ag−P) = 653.5 Hz, 2P; PPh₂Me).

³¹P{¹H} NMR ((CD₃)₂CO, −80 °C): δ 17.20 (t, 1P), 13.50 (d, 2P)

(AB₂system, ²J(P−P) = 36.9 Hz; N₃P₃ring), 0.64 (dd, ¹J(¹⁰⁹Ag−P) =

753.9 Hz, ¹J(¹⁰⁷Ag−P) = 652.4 Hz, 2P; PPh₂Me). ¹H NMR (CDCl₃,

RT): δ 7.60−7.41 (m, 20H; PPh₂Me), 3.5 (br s, 6H; NH), 2.95 (br,

2

6H; NH−CH), 2.11 (d, J(H−P)= 7.6 Hz, 6H; PPh₂Me), 1.85 (m,

3

= 39.3 Hz, 6C; PPh₂Me), 132.16 (s, 6C; PPh₂Me), 130.15 (d, J(P−

12H; NH(C₆H₁₁)), 1.59 (m, 12H; NH(C₆H₁₁)), 1.42 (m, 6H;

NH(C₆H₁₁)), 1.21 (m, 12H; NH(C₆H₁₁)), 1.06 (m, 18H; NH-

(C₆H₁₁)). ¹H NMR ((CD₃)₂CO, RT): δ 7.80−7.51 (m, 20H;

PPh₂Me), 4.00 (br, 6H; NH), 3.13 (br, 6H; NH−CH), 2.20 (d,

²J(H−P) = 7.6 Hz, 6H; PPh₂Me), 1.99 (m, 12H; NH(C₆H₁₁)), 1.64

(m, 12H; NH(C₆H₁₁)), 1.48 (m, 6H; NH(C₆H₁₁)), 1.32 (m, 12H;

NH(C₆H₁₁)), 1.21−1.05 (m, 18H; NH(C₆H₁₁)). ¹H NMR

((CD₃)₂CO, −80 °C): δ 7.85−7.54 (m, 20H; PPh₂Me), 4.55 (br,

2H; NH), 4.20 (br, 4H; NH), 3.06 (br, 6H; NH−CH), 2.21 (br, 6H;

PPh₂Me), 2.12 (br, 6H; NH(C₆H₁₁)), 1.90 (br, 6H; NH(C₆H₁₁)),

1.63 (br, 12H; NH(C₆H₁₁)), 1.51 (br, 6H; NH(C₆H₁₁)), 1.29−0.66

(m, br, 30H; NH(C₆H₁₁)). ¹³C{¹H} NMR ((CD₃)₂CO, APT, RT): δ

1

C) = 10.4 Hz, 12C; PPh₂Me), 121.95 (q, J(C−F) = 312.6 Hz, 3C;

SO₃CF₃), 52.35 (s, 6C; NH−CH), 37.28, 26.15, 25.95 (s, 30C; CH₂),

12.95 (d, ¹J(P−C) = 23.7 Hz, 3C; PPh₂CH₃). ¹⁹F{¹H} NMR (CDCl₃,

RT): δ −77.87 (s; SO₃CF₃). MS (FAB⁺): m/z 1598 (5%; [M −

2SO₃CF₃− PPh₂Me + H]⁺), 1031 (5%; [M − 3SO₃CF₃− 2PPh₂Me

− 2Ag]⁺), 725 (100%; [M − 3SO₃CF₃− 3PPh₂Me − 3Ag + H]⁺);

and peaks derived from the sequential loss of NH₂Cy.

Compound 6. Yield: 137.6 mg, 70%. Anal. Calcd for

C₅₇H₁₀₈Ag₃F₉N₁₈O₉P₆S₃(1966.21): C, 34.82; H, 5.54; N, 12.82; S,

4.89. Found: C, 34.35; H, 5.80; N, 12.50; S, 4.55. IR (ATR, cm⁻¹):

3324 (m, br) (N−H); 1080 (m) (P−NHR). Other bands: 1267 (m,

sh), 1238 (s), 1221 (s), 1158 (m), 1096 (m), 1026 (vs). ³¹P{¹H}

NMR (DMSO, RT): δ 15.75 (s, 3P; N₃P₃ring), −85.51 (s, br, 3P;

TPA). ¹H NMR (DMSO, RT): δ 4.59, 4.42 (AB system, ²J(H−H) =

12.6 Hz, 18H; NCH₂N), 4.24 (s, br, 18H; NCH₂P), 3.41 (br, 6H;

NH), 2.86 (br, 6H; NH−CH), 1.85 (m, br, 12H; NH(C₆H₁₁)), 1.65

(m, br, 12H; NH(C₆H₁₁)), 1.53 (m, br, 6H; NH(C₆H₁₁)), 1.15 (m,

br, 30H; NH(C₆H₁₁)). ¹⁹F{¹H} NMR (DMSO, RT): δ −77.72 (s;

SO₃CF₃). MS (MALDI, ditranol): m/z 724 (100%; [M − 3SO₃CF₃−

3TPA − 3Ag⁺]⁺); and peaks derived from the sequential loss of

NH₂Cy.

Synthesis of nongem-trans-[N₃P₃(NHCy)₃(NMe₂)₃{AgL}₂](TfO)₂[L

= PPh₃(7), PPh₂Me (9)]. To a solution of phos-3 (56.1 mg, 0.1

mmol) in dichloromethane (15 mL) was added [Ag(OTf)L] (0.2

mmol, 103.8 mg for 7 or 91.4 mg for 9). The mixture was stirred at

RT for 30 min protected from light. The solution was evaporated to

ca. 1 mL. The addition of hexane led to the precipitation of 7 or 9 as a

white solid.

2

1

133.63 (d, J(P−C) = 15.1 Hz, 8C; PPh₂Me), 132.70 (d, J(P−C) =

40.5 Hz, 4C; PPh₂Me), 132.20 (s, 4C; PPh₂Me), 130.16 (d, ³J(P−C)

= 10.4 Hz, 8C; PPh₂Me), 122.03 (q, ¹J(C−F) = 310.7 Hz, 2C;

SO₃CF₃), 51.96 (s, 6C; NH−CH), 37.13, 26.21, 26.10 (s, 30C; CH₂),

12.94 (d, ¹J(P−C) = 23.4 Hz, 2C; PPh₂CH₃). ¹⁹F{¹H} NMR (CDCl₃,

RT): δ −77.77 (s; SO₃CF₃). MS (FAB⁺): m/z 1489 (2%; [M −

SO₃CF₃]⁺), 1340 (1%; [M + H − 2SO₃CF₃]⁺), 1140 (2%; [M + H −

2SO₃CF₃− PPh₂Me]⁺), 1031 (90%; [M − 2SO₃CF₃− PPh₂Me −

Ag]⁺), 831 (25%; [M − 2SO₃CF₃− 2PPh₂Me − Ag]⁺), 725 (100%;

[M − 2SO₃CF₃− 2PPh₂Me − 2Ag + H]⁺); and peaks derived from

the sequential loss of NH₂Cy.

Synthesis of [N₃P₃(NHCy)₆{AgL}₃](TfO)₃[L = PPh₃(3), PPh₂Me (5),

TPA (6)]. To a solution of phos-1 (72.4 mg, 0.1 mmol) in

dichloromethane (15 mL) (for 3 or 5) or methanol for 6 was

added [Ag(OTf)L] (0.3 mmol, 155.7 mg for 3, 137.1 mg for 5, or

124.2 mg for 6), and the mixture was stirred at RT for 30 min

protected from light. The solution was evaporated to ca. 1 mL. The

M

https://dx.doi.org/10.1021/acs.inorgchem.9b03334

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

pubs.acs.org/IC

Article

Compound 7. Yield: 100.8 mg, 63%. Anal. Calcd for

C₆₂H₈₄Ag₂F₆N₉O₆P₅S₂(1600.12): C, 46.54; H, 5.29; N, 7.88; S,

4.01. Found: C, 46.95; H, 5.45; N, 7.70; S, 4.00. IR (ATR, cm⁻¹):

3283 (m, br) (N−H); 1078 (vs) (P−N). Other bands: 1284 (m),

1237 (s), 1221 (vs), 1157 (m), 1095 (s), 1025 (vs). ³¹P{¹H} NMR

3257 (m, br) (N−H); 1079 (s) (P−N). Other bands: 1282 (m),

1236 (s), 1219 (s), 1154 (s), 1095 (s, sh), 1023 (vs). ³¹P{¹H} NMR

1

(CDCl₃, RT): δ 24.27 (br, 3P; N₃P₃ring), 16.19 (br, J(Ag−P) =

671.5 Hz, 3P; PPh₃). ¹H NMR (CDCl₃, RT): δ 7.46−7.42 (m, 45H;

PPh₃), 4.76 (br, 1H; NH), 4.54 (br, 2H; NH) 2.85 (br, 3H; NH−

CH), 2.75 (m, 18H; N(CH₃)₂), 2.0−1.84 (m, 6H; NH(C₆H₁₁)), 1.74

(m, 3H; NH(C₆H₁₁)), 1.50−1.27 (m, 12H; NH(C₆H₁₁)), 1.10−0.95

(m, 9H; NH(C₆H₁₁)). ¹⁹F{¹H} NMR (CDCl₃, RT): δ −77.84 (s;

SO₃CF₃). MS (FAB⁺) m/z 1450 (1%; [M − 2SO₃CF₃− PPh₃−

Ag]⁺), 1301 (1%; [M − 3SO₃CF₃− PPh₃− Ag + H]⁺), 1188 (1%;

[M − 2SO₃CF₃− 2PPh₃− Ag]⁺), 1038 (1%; [M − 3SO₃CF₃−

2PPh₃− Ag + H]⁺, 931 (100%; [M − 3SO₃CF₃− 2PPh₃− 2Ag]⁺),

561 (90%; [M − 3SO₃CF₃− 3PPh₃− 3Ag]⁺); and peaks derived

from the sequential loss of NMe₂and NHCy.

2

(CDCl₃, RT): δ 24.20 (d, 2P), 23.0 (t, 1P) (AB₂system, J(P−P) =

1

36.2 Hz; N₃P₃ring), 16.58 (d, br, J(Ag−P) = 671.5 Hz. H NMR

(CDCl₃, RT): δ 7.51−7.48 (m, 30H; PPh₃), 4.62 (br, 1H; NH), 4.44

(br, 2H; NH), 2.86 (br, 3H; NH−CH), 2.76−2.71 (m, 18H;

N(CH₃)₂), 1.99−1.85 (m, 6H; NH(C₆H₁₁)), 1.70 (m, 6H; NH-

(C₆H₁₁)), 1.57−1.27 (m, 9H; NH(C₆H₁₁)), 1.13−0.97 (m, 9H;

NH(C₆H₁₁)). ¹⁹F{¹H} NMR (CDCl₃, RT): δ −78.01 (s; SO₃CF₃).

MS (FAB⁺) m/z 1600 (1%; [M]⁺), 1451 (1%; [M − SO₃CF₃]⁺),

1302 (1%; [M − 2SO₃CF₃+ H]⁺), 1189 (1%; [M − SO₃CF₃−

PPh₃]⁺), 1040 (1%; [M − 2SO₃CF₃− PPh₃+ H]⁺), 933 (25; [M −

2SO₃CF₃− PPh₃− Ag]⁺), 562.5 (100%; [M − 2SO₃CF₃− 2PPh₃−

2Ag + H]⁺); and peaks derived from the sequential loss of NMe₂and

NHCy.

Compound 10. Yield: 135.3 mg, 70%. Anal. Calcd for

C₆₆H₉₃Ag₃F₉N₉O₉P₆S₃(1933.13): C, 41.01; H, 4.85; N, 6.52; S,

4.98. Found: C, 41.40; H, 5.13; N, 6.96; S, 4.80. IR (ATR, cm⁻¹):

3269 (m, br) (N−H); 1079 (s, br) (P−N). Other bands: 1279 (m),

1237 (s), 1221 (s), 1151 (s), 1095 (s, sh), 1025 (vs). ³¹P{¹H} NMR

Compound 9. Yield: 106.3 mg, 72%. Anal. Calcd for

C₅₂H₈₀Ag₂F₆N₉O₆P₅S₂(1475.98): C, 42.31; H, 5.46; N, 8.54; S,

4.35. Found: C, 42.73; H, 5.60; N, 8.32; S, 4.16. IR (ATR, cm⁻¹):

3293 (m, br) (N−H); 1077 (s) (P−N). Other bands: 1281 (m),

1235 (s), 1221 (s), 1155 (s), 1100 (m, sh), 1026 (vs). ³¹P{¹H} NMR

(CDCl₃, RT): δ 23.55 (br, 2P), 21.05 (br, 1P), (AB₂system; N₃P₃

2

(CDCl₃, RT): δ 24.07 (d, 2P), 23.08 (t, 1P) (AB₂system, J(P−P)=

1

33.1 Hz; N₃P₃ring), −3.10 (br, 3P; PPh₂Me). H NMR (CDCl₃,

2

RT): δ 7.55−7.41 (m, 30H; PPh₂Me), 4.55 (t, J(P−H) = 10.3 Hz,

2

1H; NH), 4.36 (t, br, J(P−H) = 9.3 Hz, 2H; NH) 2.87 (br, 3H;

NH−CH), 2.72 (m, N = 11.2 Hz, 18H; N(CH₃)₂), 2.09 (d, ²J(H−P)

= 7.6 Hz, 9H; PPh₂Me), 1.94 (m, 3H; NH(C₆H₁₁)), 1.82 (m, 3H;

NH(C₆H₁₁)), 1.71 (m, 3H; NH(C₆H₁₁)), 1.56 (m, 6H; NH(C₆H₁₁)),

1.43−1.26 (m, 6H; NH(C₆H₁₁)), 1.12−0.95 (m, 9H; NH(C₆H₁₁)).

¹H NMR ((CD₃)₂CO, RT): δ 7.75−7.51 (m, 30H; PPh₂Me), 4.43

(m, 3H; NH), 3.02 (br, 3H; NH−CH), 2.82 (m, N = 11.6 Hz, 18H;

1

ring), −2.13 (dd, J(¹⁰⁹Ag−P) = 765.0 Hz, J(¹⁰⁷Ag−P) = 665.2 Hz,

2P; PPh₂Me). ³¹P{¹H} NMR (CD₂Cl₂, −80 °C): δ 24.80 (t, 1P),

21.13 (d, 2P) (AB₂system, ²J(P−P) = 30.1 Hz; N₃P₃ring); 24.80 (t,

2

1P), 21.03 (d, 2P), (AB₂system, J(P−P) = 34.8 Hz; N₃P₃ring),

−0.81 (dd, J(¹⁰⁹Ag−P) = 728.6 Hz, J(¹⁰⁷Ag−P) = 696.0 Hz, 2P;

1

PPh₂Me), −1.23 (dd, J(¹⁰⁹Ag−P) = 728.9 Hz, J(¹⁰⁷Ag−P) = 695.9

1

2

N(CH₃)₂), 2.18 (d, J(H−P) = 7.2 Hz, 9H; PPh₂Me), 2.00 (m, 3H;

1

Hz, 2P; PPh₂Me). H NMR (CDCl₃, RT): δ 7.56−7.41 (m, 20H;

NH(C₆H₁₁)), 1.89 (m, 3H; NH(C₆H₁₁)), 1.65 (m, 6H; NH(C₆H₁₁)),

1.50 (m, 3H; NH(C₆H₁₁)), 1.34 (m, 6H; NH(C₆H₁₁)), 1.22−1.03

(m, 9H; NH(C₆H₁₁)). ¹³C{¹H} NMR ((CD₃)₂CO, APT): δ 133.43

2

PPh₂Me), 4.24 (t, br, J(P−H) = 9.6 Hz 1H; NH), 4.03 (br, 2H;

NH), 2.84 (br, 3H; NH−CH), 2.71−2.66 (m, 18H; N(CH₃)₂), 2.11

(d, ²J(P−H) = 7.6 Hz, 6H; PPh₂Me), 1.98−1.83 (m, 6H;

NH(C₆H₁₁)), 1.67 (m, 6H; NH(C₆H₁₁)), 1.50−1.43 (br, 3H;

NH(C₆H₁₁)), 1.36−1.21 (m, 6H; NH(C₆H₁₁)), 1.13−1.03 (m, 9H;

2

(d, J(P−C) = 15.2 Hz, 12C; PPh₂Me), 133.05 (s, 6C; PPh₂Me),

131.95 (s, 6C; PPh₂Me), 130.09 (d, ³J(P−C) = 10.2 Hz, 12C;

1

PPh₂Me), 122.06 (q, J(C−F) = 321.2 Hz, 3C; SO₃CF₃), 51.95 (s,

1

NH(C₆H₁₁)). H NMR (CD₂Cl₂, −80 °C): δ 7.61−7.45 (m, 20H;

2C; NH−CH), 51.87 (s, 1C; NH−CH), 38.12 (s, 4C; N(CH₃)₂),

2

3

PPh₂Me), 4.31 (m, 2H; NH), 4.02 (t, J(P−H) = 9.0 Hz, 1H; NH)

38.06 (s, 2C; N(CH₃)₂), 37.15 (s, 2C; CH₂), 36.68 (d, J(P−C) =

1

2.74 (br, 3H; NH−CH), 2.68−2.56 (m, 18H; N(CH₃)₂), 2.07 (d,

²J(P−H) = 7.2 Hz, 6H; PPh₂Me), 2.07 (m, 3H; NH(C₆H₁₁)), 1.60

(m, 6H; NH(C₆H₁₁)), 1.49 (m, 6H; NH(C₆H₁₁)), 1.35−0.93 (m,

15.5 Hz, 4C; CH₂), 26.28, 26.09 (s, 9C; CH₂), 12.72 (d, J(P−C) =

26.0 Hz, 3C; PPh₂CH₃). ¹⁹F{¹H} NMR (CDCl₃, RT): δ −77.80 (s;

SO₃CF₃). MS (FAB⁺) m/z 1733 (1%; [M − PPh₂Me]⁺), 1327 (1%;

[M − 2SO₃CF₃− PPh₂Me − Ag]⁺), 1178 (1%; [M − 3SO₃CF₃−

PPh₂Me − Ag + H]⁺), 1127 (1%; [M − 2SO₃CF₃− 2PPh₂Me −

Ag]⁺), 977 (1%; [M − 3SO₃CF₃− 2PPh₂Me − Ag + H]⁺), 869

(40%; [M − 3SO₃CF₃− 2PPh₂Me − 2Ag]⁺), 562 (100%; [M −

3SO₃CF₃− 3PPh₂Me − 3Ag + H]⁺); and peaks derived from the

sequential loss of NMe₂and NHCy.

1

15H; NH(C₆H₁₁)). H NMR ((CD₃)₂CO, RT): δ 7.75−7.52 (m,

20H; PPh₂Me), 4.27 (br, 3H; NH), 3.02 (br, 3H; NH−CH), 2.81 (m,

2

N = 11.6 Hz, 18H; N(CH₃)₂), 2.20 (d, J(P−H) = 7.2 Hz, 6H;

PPh₂Me), 1.99−1.89 (m, 6H; NH(C₆H₁₁)), 1.67 (m, 6H; NH-

(C₆H₁₁)), 1.54−1.49 (m, 3H; NH(C₆H₁₁)), 1.37−1.26 (m, 6H;

NH(C₆H₁₁)), 1.24−1.03 (m, 9H; NH(C₆H₁₁)). ¹³C{¹H} NMR

((CD₃)₂CO, APT, RT): δ 133.44 (d, ²J(P−C) = 15.0 Hz, 8C;

4.3. X-ray Structure Determinations. Crystals were mounted

on glass ﬁbers and transferred to the cold gas stream of a Bruker

SMART 1000 CCD diﬀractometer. Measurements were made at

−130 °C. Absorption corrections were based on multiscans.

Structures were reﬁned anisotropically using the program SHELXL-

2017.⁶⁴Hydrogen atoms of the NH groups were reﬁned freely but

with N−H distance restraints (“SADI”). Methyl groups were reﬁned

as idealized rigid groups allowed to rotate but not tip. Other hydrogen

atoms were included using a riding model starting from calculated

positions. Special features: For 7, two dichloromethane sites were

identiﬁed and could be reﬁned but were not entirely satisfactory (high

U values; signiﬁcant residual electron density). Attempts to reﬁne

disordered models were unsuccessful. Accordingly, the program

SQUEEZE (part of the PLATON suite)⁶⁵was used to remove

mathematically the eﬀects of the solvent. The chemical formula and

associated parameters were calculated using an idealized composition

of two dichloromethane molecules per asymmetric unit. For 9, the

cyclohexyl group C51−56 is disordered over two positions.

Appropriate restraints were employed to improve the reﬁnement

stability, but the dimensions of the disordered groups should be

interpreted with caution. The dimensions of the hexane molecule,

which lies across an inversion center, are not entirely satisfactory,

despite the use of restraints.

1

PPh₂Me), 132.91 (d, J(P−C) = 38.8 Hz, 4C; PPh₂Me), 132.10 (s,

3

4C; PPh₂Me), 130.16 (d, J(P−C) = 10.3 Hz, 8C; PPh₂Me), 122.10

1

(q, J(C−F) = 315.8 Hz, 2C; SO₃CF₃), 51.87 (s, 3C; NH−CH),

3

38.05 (s, 6C; N(CH₃)₂), 37.11 (s, 2C; CH₂), 36.71 (d, J(P−C) =

1

13.2 Hz, 4C; CH₂), 26.27, 26.09 (s, 9C; CH₂), 12.72 (d, J(P−C) =

23.0 Hz, 2C; PPh₂CH₃). ¹⁹F{¹H} NMR (CDCl₃, RT): δ −77.88 (s;

SO₃CF₃). MS (FAB⁺): m/z 1327 (1%; [M − SO₃CF₃]⁺), 1178 (1%;

[M − 2SO₃CF₃+ H]⁺), 1127 (1%; [M − SO₃CF₃− PPh₂Me]⁺), 978

(1%; [M − 2SO₃CF₃− PPh₂Me + H]⁺), 869 (30%; [M − 2SO₃CF₃

− PPh₂Me − Ag]⁺), 562 (100% [M − 2SO₃CF₃− 2PPh₂Me −

2Ag]⁺); and peaks derived from the sequential loss of NMe₂and

NHCy.

Synthesis of nongem-trans-[N₃P₃(NHCy)₃(NMe₂)₃{AgL}₃](TfO)₃[L

= PPh₃(8), PPh₂Me (10)]. To a solution of phos-3 (56.1 mg, 0.1

mmol) in dichloromethane (15 mL) was added [Ag(OTf)L] (0.3

mmol, 155.7 mg for 8 or 137.1 mg for 10). The mixture was stirred at

RT for 30 min protected from light. The solution was evaporated to

ca. 1 mL. The addition of hexane led to the precipitation of 8 or 10 as

a white solid.

Compound 8. Yield: 158.9 mg, 75%. Anal. Calcd for

C₈₁H₉₉Ag₃F₉N₉O₉P₆S₃(2119.34): C, 45.90; H, 4.71; N, 5.95; S,

4.54. Found: C, 46.14; H, 5.05; N, 6.23; S, 4.32. IR (ATR, cm⁻¹):

N

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Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

pubs.acs.org/IC

Article

CCDC 1946587 and 1946588 contain the supplementary crystallo-

graphic data for this paper (for 7 and 9, respectively).

For both biomarkers, the cell viability was calculated using the ratio

of the ﬂuorescence/absorbance (depending on the biomarker) of

treated cells to the ﬂuorescence/absorbance of nontreated cells

(control cells). All of the results are expressed as mean standard

deviation of the three independent experiments.

4.6. Antimicrobial Activity Assays. Determination of the MICs

for Gram-Positive and Gram-Negative Strains. The antibacterial

activities of all compounds were tested against the Gram-negative

strains E. coli ATCC 10536 and P. aeruginosa ATCC 15442 and the

Gram-positive strain S. aureus ATCC 11632. Bacteria were stored as

glycerol stocks at −80 °C and streaked onto Luria−Bertani (LB)

plates prior to each experiment. Colonies from these newly prepared

plates were inoculated into 5 mL of LB media, and the tubes were

incubated at 37 °C. The overnight cultures were diluted to obtain a

ﬁnal concentration in the experiment of approximately 5 × 10⁵cfu/

mL. Stock solutions of each compound in DMSO were prepared at a

concentration of 1 mM.

For quantitative determination of the susceptibility to all

compounds, the MICs were calculated by using 2-fold serial dilutions

of the compounds in a 96-well microplate. The MICs were performed

according to the Clinical Laboratory Standards Institute (CLSI)

methods, for Antimicrobial Susceptibility Testing.⁷⁰The range of ﬁnal

concentrations tested spanned from 0.12 to 250 μM. Bacterial growth

inhibition was assessed using 0.01% resazurin added at the ﬁfth hour

of incubation at 37 °C. When a color change from blue to pink was

seen in the antibiotic-free control wells, the MIC values were

determined. The MIC is the lowest concentration of antimicrobial

agent that prevents a color change. Each experiment was performed

twice.

4.4. Cell Cultures. To assess the cellular cytotoxicity and

antitumoral potential, human breast adenocarcinoma (MCF7) and

human hepatocellular carcinoma (HepG2) epitelial cell lines were

used. Both of them are commonly used in toxicology and in tumoral

studies. Both were recognized as good experimental models, the ﬁrst

one, MCF7 cell line, because of its exquisite hormone sensitivity

through expression of the estrogen receptor, making it an ideal model

to study hormone response and a great breast cancer representative.⁶⁶

The second one, HepG2 cells, retained inducibility and activities of

several phase I and II xenobiotic metabolizing enzymes.⁶⁷Both cell

lines were obtained from the American Type Culture Collection

(ATCC, Manassas, VA). MCF7 cells were cultured in a monolayer in

Dulbecco’s minimum essential medium, supplemented with 10% of

fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL

streptomycin (1%) and 2 mM ^L-glutamine (1%). The passages used

for MCF7 are between 7 and 12. For the HepG2 cells, a minimum

essential medium was used as the main culture medium with similar

nutrients and proportions as those mentioned above for MCF7. The

passages we used were from 9 to 14. Cells were routinely grown at 37

°C and 5% CO₂in a humidiﬁed atmosphere.

4.5. Cytotoxicity Assays. For cytotoxicity assays, all of the

compounds analyzed were dissolved in DMSO. In any case, the

DMSO concentration did not exceed 0.1%, and appropriate controls

of solvent were tested in all of the experiments performed. Under the

same conditions, but in water, cisplatin was tested and compared to all

of the compounds studied.

The exposure concentrations for the silver complexes (2−6, 9, and

10) were set from 0 to 8 μM, prior to the wide range assayed (0−200

μM), to determine the speciﬁc spectrum to test and the compound

precipitation (data not shown). Diﬀerent ranges of concentrations

were tested based on their self-precipitation for phosphazene ligands

phos-1 and phos-2 and the silver precursors. Thus, phos1 was tested

from 0 to 25 μM, and [Ag(OTf)L] (L = PPh₃, PPh₂Me, TPA) and

phos3 were assayed from 0 to 80 μM. The MCF7 and HepG2 cells

were seeded at densities of 8 × 10⁴and 1 × 10⁵cells/mL in 96-well

microplates and allowed to attach for 24 h prior to the addition of

compounds under study. In each assay, all of the compounds were

tested in sextuplicate/microplate, with three independent experiments

being conducted to check the reproducibility and repetitivity of the

results. The time of exposure of the silver complexes to the cell lines

selected was 48 h at 37 °C and 5% CO₂. At the same conditions,

cisplatin (0−80 μM) was also tested.

To evaluate the cell viability, two diﬀerent biomarkers were carried

out, AB and NRU assays. In the AB assay, the system incorporates an

oxidation−reduction (REDOX) indicator, assessing the mitochon-

drial ability to reduce resazurin into the ﬂuorescent product

resoruﬁn.⁶⁸Brieﬂy, after 48 h of compound exposure, the AB

medium was prepared by mixing a cell culture medium with a AB

stock solution [resazurin sodium salt (5 mg/mL) in phosphate-

buﬀered saline] in a 10:1 ratio (10% of the ﬁnal volume). Once

prepared, 100 μL of the AB medium was added to each well

containing the cells exposed to the studied compounds. The

microplates were incubated at 37 °C for 2−3 h, and ﬂuorescence

was measured with a multimodal Variouskan Lux spectrophotometer

(Thermo Scientiﬁc) with an excitation wavelength of 560 nm and an

emission wavelength of 585 nm. With regard to NRU, it is a suitable

end point to determine viable cells. It is based on the ability of viable

cells to incorporate and bind the supravital dye neutral red in the

lysosomes. This assay was performed according to Repetto et al.⁶⁹

Brieﬂy, after a 48 h of exposure, the medium with the complexes was

removed and neutral red (NR) in a fresh medium (1:100) was

incorporated into each well (100 μL) to be absorbed and

concentrated in the cell lysosomes. After 3 h, the NR medium was

removed, a formol-Ca₂solution was added for 2 min, and, afterward,

an acetic acid−ethanol−water mixture was also added for 15 min with

continuous shaking, prior to quantitative measurement at 540 nm

with a Variouskan Lux spectrometer (Thermo Scientiﬁc).

Determination of the MICs for M. tuberculosis Complex (MTBC)

Strains. The compounds were assayed against two MTBC strains, M.

tuberculosis strain H37Rv ATCC 27294 and M. bovis BCG Pasteur. In

this work, we also utilized M. bovis BCG, which is commonly used as a

model organism for the study of M. tuberculosis because it is not a

virulent vaccine strain and the BCG genome shares a very high degree

of similarity with that of M. tuberculosis. The anti-MTBC activity of all

compounds was determined by the REMA (Resazurine Microtiter

Assay) method according to Palomino et al.⁷¹Stock solutions of the

tested compounds were prepared in DMSO and diluted in

Middlebrook 7H9 (Difco), supplemented with oleic acid, albumin,

dextrose, and catalase (OADC enrichment-BBL/Becton−Dickinson),

to obtain the ﬁnal drug concentration range of 0.12−250 μM of the

M. tuberculosis strain H37Rv ATCC 27294, cultured in Middlebrook

7H9 broth, supplemented with OADC and 0.05% Tween 80. The

concentration was adjusted by 5 × 10⁵UFC/mL, and 100 μL of the

inoculum was added to each well of the 96-well microtiter plate,

together with 100 μL of the compounds. Samples were set up in

duplicate. The plate was incubated at 37 °C under a normal

atmosphere. After 7 days of incubation, 30 μL of 0.01% resazurin

(solubilized in water) was added to each well, and the plate was

reincubated for 24 h. A change in color from blue to pink indicated

the growth of bacteria, and the MIC was deﬁned as the lowest

concentration of drug that prevented this change in color. For the

resazurin solution, a resazurin sodium salt (Sigma-Aldrich) stock

solution of 0.01 g was dissolved in 100 mL of sterile distilled water

and sterilized by ﬁltration.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.inorgchem.9b03334.

Tables containing details of data collection and structure

reﬁnement (Tables S1 and S2) and selected bond

lengths and angles (Tables S3 and S4) for compounds 7

1

and 9, ³¹P and H NMR spectra of compound 2 in

DMSO measured over 48 h, which is the time for the

biological assays (Figures S1 and S2), and a table

containing the expected IC₅₀after 24 h for metal-

O

https://dx.doi.org/10.1021/acs.inorgchem.9b03334

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

lophosphazenes and their precursor exposure on MCF7

pubs.acs.org/IC

Article

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AUTHOR INFORMATION

■

Corresponding Author

́

Joseﬁna Jimenez − Departamento de Quimica Inorganica,

́

Facultad de Ciencias, Instituto de Sintesis Quimica y Catalisis

́

Homogenea, Universidad de Zaragoza-CSIC, 50009 Zaragoza,

Spain; orcid.org/0000-0002-3444-0851; Email: jjimvil@

unizar.es

Authors

Elena Gascon − Departamento de Quimica Inorganica,

́

Facultad de Ciencias, Instituto de Sintesis Quimica y Catalisis

́

Homogenea, Universidad de Zaragoza-CSIC, 50009 Zaragoza,

Spain

́

Sara Maisanaba − Departamento de Biologia Molecular e

́

Ingenieria Bioquimica, Area de Toxicologia, Universidad Pablo

de Olavide, 41013 Sevilla, Spain

Aslan, T.; Ustun, Y.; Yilmaz, E. Ş.; Katı, A.; Demirbas, A.; Mandal, A.

́

Isabel Otal − Grupo de Genetica de Micobacterias,

Departamento de Microbiologia, Medicina Preventiva y Salud

K.; Ocsoy, I. Biosynthesis of silver nanoparticles and their versatile

antimicrobial properties. Mater. Res. Express 2019, 6, 012001.

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2010, 8, 836.

́

Publica, Universidad de Zaragoza, Zaragoza 50009, Spain;

Instituto de Salud Carlos III, CIBER de Enfermedades

Respiratorias, E-28029 Madrid, Spain

́

Eva Valero − Departamento de Biologia Molecular e Ingenieria

(5) Sierra, M. A.; Casarrubios, L.; de la Torre, M. C. Bio-

Organometallic Derivatives of Antibacterial Drugs. Chem. - Eur. J.

2019, 25, 7232−7242 and references cited therein..

́

Bioquimica, Area Nutricion y Bromatologia, Universidad Pablo

de Olavide, 41013 Sevilla, Spain

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Guillermo Repetto − Departamento de Biologia Molecular e

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Ingenieria Bioquimica, Area de Toxicologia, Universidad Pablo

de Olavide, 41013 Sevilla, Spain

Peter G. Jones − Institut fur Anorganische und Analytische

̈

Chemie, Technische Universitat Braunschweig, D-38106

Braunschweig, Germany

̈

Complete contact information is available at:

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by Ministerio de Economia y

Competitividad (MINECO)-FEDER, under Project

MAT2017-84838-P, and Gobierno de Aragon-FEDER

(Groups E47_17R and B25_17R, FEDER 2014-2020

́

“Construyendo Europa desde Aragon”), Proyecto Puente

(Grant CTM2016-76304-C2-1-R), Consejeria de Economia y

Conocimiento de la Junta de Andalucia 2017 (Grant CTS996

́

PAIDI 2018), and Project 2019-PPI1901 (VPPI-UPO). We

́

thank Lucia Vallejo Valero for her contribution to the image

design of the Table of Contents graphic.

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T

https://dx.doi.org/10.1021/acs.inorgchem.9b03334

Inorg. Chem. XXXX, XXX, XXX−XXX

Article Doi

(Amino)cyclophosphazenes as Multisite Ligands for the Synthesis of Antitumoral and Antibacterial Silver(I) Complexes

DOI: 10.1021/acs.inorgchem.9b03334

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Full text of DOI:10.1021/acs.inorgchem.9b03334

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