Antibacterial activity of amidodithiophosphonato nickel(II) complexes: An experimental and theoretical approach

Article abstract of DOI:10.3390/molecules25092052

The reactions of 2,4-bis(4-methoxyphenyl)-1,3-dithio-2,4-diphosphetane-2,4-disulfide (Lawesson’s Reagent, LR) with benzylamine (BzNH₂) and 4-phenylbutylamine (PhBuNH₂) yield benzylammonium P-(4-methoxyphenyl)-N-benzyl-amidodithiophosphonate (BzNH₃)(BzNH-adtp) and 4-phenylbutylammonium P-(4-methoxyphenyl)-N-(4-phenylbutyl)-amidodithiophosphonate (PhBuNH₃)(PhBuNH-adtp). The relevant nickel complexes [Ni(BzNH-adtp)₂] and [Ni(PhBuNH-adtp)₂] and the corresponding hydrolysed derivatives (BzNH₃)₂[Ni(dtp)₂] and (PhBuNH₃)₂[Ni(dtp)₂] were prepared and fully characterized. The antimicrobial activity of the aforementioned amidodithiophosphonates against a set of Gram-positive and Gram-negative pathogen bacteria was evaluated, and [Ni(BzNH-adtp)₂] and [Ni(PhBuNH-adtp)₂] showed antiproliferative activity towards Staphylococcus aureus and Staphylococcus haemolyticus strains. density functional theory (DFT) calculations were performed to shed some light on the activity of reported compounds related to their tendency towards P–N bond cleavage.

Full text of DOI:10.3390/molecules25092052

molecules
Article
Antibacterial Activity of Amidodithiophosphonato
Nickel(II) Complexes: An Experimental and
Theoretical Approach
Enrico Podda 1 , Massimiliano Arca , Giulia Atzeni , Simon J. Coles , Antonella Ibba ,
1
1
2
3
1
1
3,4
2
1
Francesco Isaia , Vito Lippolis , Germano Orrù , James B. Orton , Anna Pintus ,
Enrica Tuveri 3 and M. Carla Aragoni *
1,
1
Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Cagliari, Cittadella Universitaria,
SS. 554 bivio Sestu, 09042 Monserrato–Cagliari, Italy; e.podda@unica.it (E.P.); marca@unica.it (M.A.);
giulia.atzeni86@gmail.com (G.A.); isaia@unica.it (F.I.); lippolis@unica.it (V.L.); apintus@unica.it (A.P.)
UK National Crystallography Service, School of Chemistry, Faculty of Engineering and Physical Sciences,
University of Southampton, Southampton SO17 1BJ, UK; S.J.Coles@soton.ac.uk (S.J.C.);
J.B.Orton@soton.ac.uk (J.B.O.)
Department of Surgical Sciences, University of Cagliari, 09042 Cagliari, Italy; antonella.i.1977@live.it (A.I.);
orru@unica.it (G.O.); enricatuveri@gmail.com (E.T.)
National Research Council of Italy, ISPA-CNR, 07100 Sassari, Italy
2
3
4
*
Correspondence: aragoni@unica.it; Tel.: +39-070-675-4491; Fax: +39-070-675-4456
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Franco Bisceglie
Received: 13 March 2020; Accepted: 24 April 2020; Published: 28 April 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: The reactions of 2,4-bis(4-methoxyphenyl)-1,3-dithio-2,4-diphosphetane-2,4-disulfide
Lawesson’s Reagent, LR) with benzylamine (BzNH ) and 4-phenylbutylamine (PhBuNH ) yield
(
2
2
benzylammonium P-(4-methoxyphenyl)-N-benzyl-amidodithiophosphonate (BzNH )(BzNH-adtp)
3
and 4-phenylbutylammonium P-(4-methoxyphenyl)-N-(4-phenylbutyl)-amidodithiophosphonate
(
PhBuNH )(PhBuNH-adtp). The relevant nickel complexes [Ni(BzNH-adtp) ] and [Ni(PhBuNH-
3
2
adtp) ] and the corresponding hydrolysed derivatives (BzNH ) [Ni(dtp) ] and (PhBuNH ) [Ni(dtp) ]
2
3 2
2
3 2
2
were prepared and fully characterized. The antimicrobial activity of the aforementioned
amidodithiophosphonates against a set of Gram-positive and Gram-negative pathogen bacteria
was evaluated, and [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] showed antiproliferative activity
2
2
towards Staphylococcus aureus and Staphylococcus haemolyticus strains. density functional theory (DFT)
calculations were performed to shed some light on the activity of reported compounds related to their
tendency towards P–N bond cleavage.
Keywords: amidodithiophosphonate; nickel complexes; antibacterial activity; density functional
theory (DFT); X-ray diffraction; P–N cleavage
1
. Introduction
Phosphorus-1,1-dithiolates such as dithiophosphates, dithiophosphinates, dithiophosphonates,
and amidodithiophosphonates (I, II, III and IV, respectively, see Scheme 1), are important classes of
sulfur-donor anionic ligands that display a multiplicity of coordination patterns with transition metal
−
ions and main group elements [
1–
3
]. The PS2 moiety can coordinate in monodentate, bidentate (with
either symmetric or asymmetric bonding), and polydentate modes. A huge variety of both discrete
and polymeric structures are prevalent in the literature [ ]. Since the early 1960s, dithiophosphates and
dithiophosphinates gained increasing importance due to their applications as pesticides and extracting
agents in mineral ores, and a large amount of information describing the reactivity of compounds and
II was reported [ ]. On the contrary, due to synthetic difficulties, the chemistry of dithiophosphonate
4
I
5
Molecules 2020, 25, 2052; doi:10.3390/molecules25092052
www.mdpi.com/journal/molecules

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complexes (III) became relevant only after the turn of the new century, with the development of a novel
synthetic route starting from 1,3-dithiadiphosphetane-2,4-disulfides (such as Lawesson’s Reagent) [ ].
3,6
A similar synthetic route was used to prepare amidodithiophosphonates (IV in Scheme 1), a class of
phosphorus-1,1-dithiolates, featuring a P–N bond, that still remains largely unexplored.
Scheme 1. Phosphorus-1,1-dithiolates: dithiophosphate (I), dithiophosphinate (II), dithiophosphonate
(
III), and amidodithiophosphonate (IV) anions.
The tendency to undergo cleavage of the P–N bond to give the corresponding dithiophosphonic
acid may explain the low occurrence of structurally characterized amidodithiophosphonates (Figure 1).
The majority of known structures are in an anionic form, with the released protonated amine acting as
a counterion [7–10].
Figure 1. Number of structures per year published for compounds
.0.4–CSDS 2020–V. 5.41).
I–IV since 1960 (ConQuest
2
Some examples are also reported where the complete hydrolysis of P–N and P–S bonds in
amidodithiophosphonates yields phosphonates, with a concurrent loss of the amine and hydrogen
sulphide [11,12].
Recently, the antiproliferative and antibacterial activity of this class of compounds was reported
to be related to the slow release of H S [11
,
12]. This aspect is of particular interest as antimicrobial
resistance is becoming one of the principal public health problems of the 21st century [13 15]. In the
search for novel antimicrobial agents, coordination compounds containing transition metal ions
represent a promising avenue for drug development [16 17]. Complexes of metals such as Au, Ir, Co,
and Cu have demonstrated an excellent activity against aerobic Gram-positive pathogenic bacteria,
such as Staphylococcus spp. [17 20]. This bacterial group, in particular S. aureus, can cause many forms
of infections in different organs and is one of the major causes of nosocomial infections of surgical
2
–
,
–

Molecules 2020, 25, 2052
3 of 18
wounds and in indwelling medical devices [21
,
22]. Methicillin-resistant S. aureus (MRSA) is solely
responsible for many life-threatening nosocomial infections in humans, causing an increase both in
the treatment duration and medical costs [23]. The problem of resistance is amplified by the ability
of S. aureus to form biofilms on biotic and abiotic surfaces and is of particular concern with several
implanted medical devices [24–26]. Bacteria in these biofilms are stubbornly difficult to treat because
such microbial aggregates are traditionally considered impervious to drug diffusion [27,28].
8
Given the scarcity of data reported on square-planar complexes of d metal ions with potential
antimycotic and antimicrobial activity [
3,11,12,18,29,30] and the different hydrolytic products
described [ 12], we report here the synthesis, characterization and activity (against a set of
7–
Gram-positive and Gram-negative pathogenic bacteria), of the novel benzylammonium P-(4-
methoxyphenyl)-N-benzyl-amidodithiophosphonate (BzNH )(BzNH-adtp), 4-phenylbutylammonium
3
P-(4-methoxyphenyl)-N-(4-phenylbutyl)-amidodithiophosphonate (PhBuNH )(PhBuNH-adtp), their
3
nickel complexes [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] and the corresponding hydrolytic
2
2
products (BzNH ) [Ni(dtp) ] and (PhBuNH ) [Ni(dtp) ] (Scheme 2).
3
2
2
3 2
2
Ar
S
S
S
S
(
CH ) NH
2 n 2
P
P
Ar
BzNH2 (n=1) - PhBuNH2 (n=4)
S -
i
NH Pr
2
LR
S -
+
i
+
Ar
Ar
NH Pr
NH (CH )
3 2 n
3
P
S
P
S
iPrHN
(CH ) HN
(BzNH3)(BzNH-adtp) (n=1)
2
n
(iPrNH )( Pr-adtp)
i
3
(PhBuNH )(PhBuNH-adtp) (n=4)
3
.
.
NiCl 6H O
NiCl 6H O
2
2
2
2
i
Ar
S
S
S
NH Pr
Ar
S
S
S
S
NH(CH2)n
Ar
P
Ni
P
P
Ni
P
iPrHN
S
Ar
(CH ) HN
2 n
i
[
Ni( Pr-adtp) ]
[Ni(BzNH-adtp) ] (n=1)
2
2
[
Ni(PhBuNH-adtp) ] (n=4)
2
H2O
2-
Ar
P
O
S
S
S
S
O
+
NH (CH )
Ni
P
3
2 n
Ar
2
(BzNH ) [Ni(dtp) ] (n=1)
3 2 2
(
PhBuNH ) [Ni(dtp) ] (n=4)
3
2
2
Scheme 2. Preparation scheme for amidodithiophosphonate salts and related NiII complexes.
i
Synthetic procedures are detailed in the experimental part. LR = Lawessons’ Reagent; PrNH =
2
isopropylamine; BzNH2
= benzylamine; PhBuNH = phenylbutylamine; Ar = 4-methoxyphenyl;
2
i
Pr-adtp = 4-methoxyphenyl-isopropyl-amidodithiophosphonate; BzNH-adtp = 4-methoxyphenyl-benzyl-
amidodithiophosphonate; PhBuNH-adtp = 4-methoxyphenyl-phenylbutyl-amidodithiophosphonate;
2
−
dtp = 4-methoxyphenyl-dithiophosphonate.
2
. Results
The reaction between 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide
i
(
Lawesson’s Reagent, LR) and primary amines PrNH , BzNH and PhBuNH (isopropylamine,
2
2,
2
benzylamine, and 4-phenylbutylamine, respectively) in toluene gave rise to the corresponding
i
i
amidodithiophosphonate ammonium salts ( PrNH )( PrNH-adtp), (BzNH )(BzNH-adtp),
3
3
and (PhBuNH )(PhBuNH-adtp) (Scheme 2). The compounds were characterized by m.p.
3

Molecules 2020, 25, 2052
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1
31
1
determination, FT-IR, H-NMR, and P{ H} NMR spectroscopies (see experimental for details).
The FT-IR spectra of (BzNH )(BzNH-adtp) and (PhBuNH )(PhBuNH-adtp) (Figures S11 and S14)
3
3
−1
show the N–H stretching frequency as a strong single peak at around 3300 cm . The bands peculiar to
the asymmetric and symmetric P–S stretching vibrations can be envisaged at about 624 and 550 cm
respectively [30]. The H-NMR spectra of (BzNH )(BzNH-adtp) and (PhBuNH )(PhBuNH-adtp)
−1
,
1
3
3
in DMSO-d solutions are shown in Figures S1 and S2 and are consistent with those reported
6
for the amidodithiophosphonate salt prepared from benzylamine and phenylethylamine and LR,
32]. The 31P{ H} NMR
1
corroborating the formation of the P–N bond and the proposed structure [31
,
spectra of (BzNH )(BzNH-adtp) and (PhBuNH )(PhBuNH-adtp) show a singlet at 79.7 and 93.1 ppm,
3
3
i
i
respectively, in agreement with what it was previously reported for ( PrNH )( PrNH-adtp) and similar
3
known compounds [7–10,30–32].
The reaction of the amidodithiophosphonate salts with NiCl
·
6H O afforded the corresponding
2
2
i
nickel(II) complexes [Ni( PrNH-adtp) ], [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] (Scheme 2),
2
2
2
as purple solids. The obtained compounds were fully characterized by elemental analysis, m.p.
determination, FT-IR and 1H and P{ H} NMR spectroscopies, confirming their nature of
31
1
amidodithiophosphonato nickel complexes. The FT-IR spectra of the compounds (Figures S12 and S15
show the N–H stretching vibration as a strong single peak falling at around 3250 cm , and the
bands for asymmetric and symmetric P–S stretching modes are found, as expected, around 660
)
−1
−
1
and 560 cm , respectively [29]. Due to the peak broadening encountered in DMSO-d solutions,
6
1
H-NMR spectra of [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] were recorded in CDCl solution.
H-NMR data are consistent with the proposed structure (Scheme 2). In particular, the H-NMR
spectra of [Ni(BzNH-adtp) ] (Figure S3) show three signals in the aliphatic region at 3.34–3.20, 3.86,
2
2
3
1
1
2
and 4.35–4.21 ppm, assigned to the protons of NH, OCH and CH moieties, respectively. The aromatic
3
2
portion of the spectrum displays two signals at 8.04–7.87 and 7.05–6.93 ppm, attributed to the protons
of the aryl ring directly bonded to the phosphorus atom. The amine aromatic protons are observed at
1
7
.40–7.16 ppm. The H-NMR spectrum of [Ni(PhBuNH-adtp) ] (Figure S4) shows the signal that can be
2
assigned to CH protons overlapped with the water residue; the signals at 2.62, and 3.17–2.99 ppm can
2
be attributed to the protons of the aliphatic chain of the amine, and the broad signal at around 2.91 ppm
can be assigned to the NH proton, similar to that found for (BzNH )(BzNH-adtp) and [Ni(BzNH-adtp) ];
3
2
the singlet at 3.86 ppm is attributed to the protons of the OCH group. The protons of the aromatic
3
portion display signals at 7.90 and 6.97 ppm, assigned to the methoxyphenyl P-substituent, and signals
between 7.3–7.1 ppm can be assigned to the aromatic protons of the amine. The 31P{ H} NMR spectra
1
of [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] recorded in DMSO-d show only a singlet at 75.8 and
2
2
6
31
7
6.2 ppm, respectively, and are comparable with the CP-MAS P value of 71.7 ppm measured for of
Ni( PrNH-adtp) ] and consistent with the presence of the P–N bond.
i
[
[
2
The attempts of growing single crystals suitable for X-ray analysis for [Ni(BzNH-adtp) ] and
2
Ni(PhBuNH-adtp) ] from either acetonitrile or chloroform solutions, revealed that hydrolysis occurred,
2
resulting in the formation of compounds (BzNH ) [Ni(dtp) ] 2H O and (PhBuNH ) [Ni(dtp) ],
·
3
2
2 2 3 2 2
2–
featuring the anionic dithiophosphonato Ni complex [Ni(dtp) ] counterbalanced by two
2
BzNH3+ and PhBuNH3+ cations, respectively (Scheme 2; Figures 2 and 3, Tables 1 and S1).
The obtained hydrolytic products were afterwards deliberately synthesized and fully characterized
(
see experimental). It is interesting to note that, notwithstanding the similar constituent moieties,
several differences can be evidenced between the neutral amidodithiophosphonato nickel complexes
Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] and the corresponding Ni-dithiophosphonate ammonium
[
2
2
salts (BzNH ) [Ni(dtp) ] and (PhBuNH ) [Ni(dtp) ], mainly regarding the melting points and the IR
3
2
2
3 2
2
+
and NMR signals relative to the ammonium -NH3 groups (Figures S9, S10 and S13–S16).

Molecules 2020, 25, 2052
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Figure 2. Ellipsoid plot (50% probability level) and numbering scheme for (BzNH ) [Ni(dtp) ] 2H O.
·
3
2
2 2
0
Only one cation and one water molecule are depicted for clarity. Symmetry code: = 1-x, -y, 2-z.
II
The asymmetric unit of the (BzNH ) [Ni(dtp) ] 2H O comprises half a molecule, with a Ni
·
3
2
2 2
−
ion lying about a crystallographic inversion center, one dithiophosphonato ligand (ArPOS ) ,
2
one benzylammonium cation, and one water molecule. The metal center is tetracoordinated in
a square-planar geometry by four sulphur atoms belonging to two isobidentate ligands with Ni–S
◦
bond lengths of 2.2255(4) and 2.2232(4) Å, respectively, and an S–Ni–S angle of 87.92(2) . The P–S1 and
P–S2 bond lengths show very similar values [2.0409(5) and 2.0387(5) Å respectively], indicating an
−
◦
electron delocalization over the whole PS2 fragment and an S–P–S angle of 98.39(2) ; the P–O bond
exhibits a length of 1.5094(10) Å (Table 1).
◦
Table 1. Selected bond lengths (Å) and angles ( ) for compounds (BzNH ) [Ni(dtp) ]
·
2H O,
3
2
2
2
(
PhBuNH ) [Ni(dtp) ], and (PhBuNH ) [(pdtp) O].
3
2
2
3 2
2
[a]
[b]
(
BzNH ) [Ni(dtp) ]·2H O
(PhBuNH ) [Ni(dtp) ]
(PhBuNH ) [(pdtp) O]
3 2 2
3
2
2
2
3 2
2
Ni–S1
Ni–S2
P1–S1
P1–S2
P1-C1
2.2255(4)
2.2232(4)
2.0409(5)
2.0387(5)
1.7955(14)
1.5094(10)
87.92(2)
2.218(6)
2.216(7)
2.029(8)
2.029(2)
1.797(5)
1.511(2)
87.8(6)
-
-
1.98(1)
1.97(1)
1.79(1)
1.61(3)
-
P1-O1
S1–Ni–S2
S1–P1–S2
C1-P1-O1
98.39(2)
108.24(6)
98.7(7)
107 (1)
116.3(6)
99 (1)
[
a]
Average values calculated on the four crystallographic independent units, in parentheses the corresponding
standard deviation. [ Average values calculated on the two crystallographic independent units, in parentheses the
b]
corresponding standard deviation; for PS2 groups, the average was calculated on all independent fragments.
The structure of (PhBuNH ) [Ni(dtp) ] (Figure 3) displays four nickel metal centers in the
3
2
2
asymmetric unit, each sitting on a special position with its occupancy necessarily set to 0.5. One half of
each metal complex is crystallographically unique, with the other chelated ligand being generated
through the symmetry of the space group. There are also four crystallographically unique protonated
organic amine counter ions, three of which are disordered (Figure S6). The nickel coordination closely
resembles that found in (BzNH ) [Ni(dtp) ] 2H O (see above) with similar bond lengths and angles
·
3
2
2 2
(
Table 1), and an average P–O bond length of 1.511(2) Å.

Molecules 2020, 25, 2052
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Figure 3. Ellipsoid plot (50% probability level) and numbering scheme for (PhBuNH ) [Ni(dtp) ].
3
2
2
0
Only one cation is depicted for clarity. Symmetry codes: = -x, -y, 1-z; ” = 1-x, 1-y, 1-z.
It is interesting to note that the bond lengths and angles of the –POS moiety are comparable
2
with those found in similar neutral and anionic dithiophosphonato nickel complexes described as
bearing either a P=O or P–OH bond [
of the crystallographic P–O and/or P–S bond lengths alone) [
7
–
10
,
33
–
40]. It is therefore very difficult (from comparison
10 33 40], to discriminate between
7
– , –
purely single and double P–O bonds in this class of compounds, or to confidently assign the negative
−
charge on either the oxygen or on the sulphur atoms. Additionally, the [ArPOS ] fragments are
2
often engaged in strong H-bonds with counterions, which affects the bond lengths between the atoms
involved. A better understanding of the nature of the P–O bond (and its charge distribution)
may be gleaned if the bond lengths involved in the –POS fragment are considered together.
2
The correlation reported in Figure 4 suggests that purely double P=O bonds may only be found
on P(O)S fragments not directly bearing a negative charge (empty green circles, CCDC ref-codes
2
IDUNEC, NEYLUA, NEYMAH, and YABQEZ) [7–10,33–40]. Similarly, pure single P–O bonds are
detected in neutral O-alkyl-dithiophosphonates (III in Scheme 1, yellow circles in Figure 4). All the
fragments bearing a negative charge fall in the same area, notwithstanding the attributions, as single
P–OH (blue squares) or double P=O bonds (full green circles) reported for the deposited structures.
Compounds (BzNH ) [Ni(dtp) ] 2H O (black triangle in Figure 4) and (PhBuNH ) [Ni(dtp) ] (red
·
3
2
2 2 3 2 2
triangle in Figure 4) lie in the same region as the anionic fragments. It is worth noting the three
blue squares lying in the same region as the yellow dots: these data refer to structures (IKOSUX
and LIFGAJ) [10
proposed correlation.
The crystal packing of both (BzNH ) [Ni(dtp) ]
governed by the strong H-bonds involving the anionic complexes, the ammonium BzNH and
PhBuNH3+ cations and the crystallized water molecules in (BzNH ) [Ni(dtp) ]
,33–40] containing single P–OH bonds in neutral fragments, thus confirming the
·
2H O and (PhBuNH ) [Ni(dtp) ] are mainly
2 3 2 2
3
2
2
+
3
·
2H O (Figure S5,
3 2
2 2
Tables S2 and S3 in ESI).
An attempt at crystallizing (PhBuNH )(PhBuNH-adtp) in toluene yielded a few crystals of
3
the salt (PhBuNH ) [(ArPS ) O] (Figure 5 and Figure S7; Table 1, Tables S1 and S4) containing
3
2
2 2
2
−
the bis(4-methoxyphenyl)tetrathiodiphosphonate anion [(ArPS ) O] counterbalanced by two
2
2
4-phenylbutylammonium cations. The structure was solved in the space group P-1, and the main
structural and refinement parameters are reported as Supporting Information (Tables S1 and S4,

Molecules 2020, 25, 2052
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Figure S7). There is a large extent of disorder in the crystal. The disordered atoms were modelled and
refined over two or four positions using a combination of thermal and geometric parameter restraints
and/or constraints where necessary (see Experimental).
Figure 4. Diagram reporting P–O vs. the average of the two P–S bond lengths in the –P(O)S fragment in
2
dithiophosphonates or their metal complexes found in the CDS. Green circles refer to structures reporting
a double P=O bond (refcodes: EANIPS [33], IDUNEC [34], IKOVAG [35], NEYLUA [37], NEYMAH [37],
NIBKOZ [
refer to structures reporting a P–OH bond (refcodes: FIDWEV [40], IKOSUX [35], IKOVAG [35],
LIGFAJ [10], XIXTON [ ]); yellow circles refer to the P–O single bonds in the 229 structures featuring
a single P–OR bond (R = alkyl/aryl; compounds III in Scheme 1); triangles refer to the data for
BzNH ) [Ni(dtp) ]·2H O (black triangle) and (PhBuNH ) [Ni(dtp) ] (red triangle).
9], NIBKUF [9], NOPHIJ [36], TOSNUM [38], YABQEZ [39], PEKXOV [8]); blue squares
7
(
3
2
2
2
3 2
2
Figure 5 shows the ellipsoid plot (50% probability level) and numbering scheme of one
2
−
[
(ArPS ) O] anion and one PhBuNH3+ cation; the complete content of the asymmetric unit with
2 2
2
−
disorder shown is reported in Figure S7 in ESI. Bond lengths and angles in the (S P–O–PS ) fragment
2
2
are similar to those previously reported for the thionated(naphthalene-1,8-diyl)bis(phosphonic) acid
monoanhydride [41]. The anions interact with ammonium cations through strong N–H···S hydrogen
bonds (Table S4 in Supplementary Material).
The isolation of compound (PhBuNH ) [(ArPS ) O] confirms that the hydrolytic process involves
3
2
2 2
the cleavage of P–N bond in amidodithiophosphonates, while retaining the P–S, as already observed
in (BzNH ) [Ni(dtp) ]·2H O and (PhBuNH ) [Ni(dtp) ].
3
2
2
2
3 2
2

Molecules 2020, 25, 2052
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Figure 5. Ellipsoid plot (50% probability level) and numbering scheme of part of the asymmetric unit
of (PhBuNH ) [(ArPS ) O].
3
2
2 2
2
.1. Antibacterial Activity
A set of different tests were performed to evaluate the antimicrobial activity of
i
i
amidodithiophosphonate salts ( PrNH )( PrNH-adtp), (BzNH )(BzNH-adtp), and (PhBuNH )(PhBuNH-
adtp) and the relevant nickel(II) complexes [Ni( PrNH-adtp) ], [Ni(BzNH-adtp) ] and [Ni(PhBuNH-
3
3
3
i
2
2
adtp) ] against different Gram-positive and Gram-negative bacterial species, both in planktonic and
2
in sessile life. In this context, microbial species described as being commensals or pathogens in
humans were assayed, namely Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and
two strains of Pseudomonas aeruginosa, PA-01 and PA-02, that showed a different susceptibility pattern
to disinfectants [42]. In addition, three different clinical isolates of Candida spp. were assayed, namely
Candida albicans, Candida kruseii, and Candida glabrata. The antimicrobial activity of the ligand salts
and nickel complexes was measured by the Agar diffusion method against the mentioned strains.
These tests revealed that, while none of the tested Gram-negative bacteria or fungi were sensitive to any
of the compounds examined, complexes [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] are active against
2
2
both Staphylococcus spp. (Figure S8 in Supplementary Material). In particular, a growth inhibition (Ø) of
2 and 8 mm was exerted on S. aureus and of 17 and 15 mm against S. haemolyticus by [Ni(BzNH-adtp)2]
1
and [Ni(PhBuNH-adtp) ], respectively (Figure S8 in Supplementary Material). The fact that there
2
was activity against Gram-positive bacteria and not against Gram-negative may be related to the
increased difficulty of these compounds to penetrate the cell wall of the Gram-negatives [43
Notably, no inhibitory activity towards S. aureus and S. haemolyticus was observed for the complexes’
ligand precursors (BzNH )(BzNH-adtp), (PhBuNH )(PhBuNH-adtp), and NiCl 6H O, showing that
–45].
·
3
3
2
2
the coordination compounds are responsible for the antimicrobial activity. In contrast, the inability
i
of [Ni( PrNH-adtp) ] to inhibit bacterial growth suggests that the tendency towards hydrolysis of
2
the complexes [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] could play an important role in their
2
2
antimicrobial activity. The tendency to hydrolysis could be tentatively ascribed to the different nature
of the alkyl/aryl amine substituents, as evidenced by the slight elongation of the P–N bond on passing
i
from [Ni( PrNH-adtp) ] to [Ni(PhEtNH-adtp) ] (1.619(5) and 1.641(4) Å, respectively) [3,31,32].
2
2

Molecules 2020, 25, 2052
9 of 18
Minimum inhibitory concentration (MIC) represents the lowest concentration of an antimicrobial
that inhibits the visible growth of a microorganism after an appropriate incubation time. Evaluating the
MIC confirmed the activity against Staphylococci.
Both [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] were observed to inhibit the growth of S. aureus
2
2
−
4
−4
up to just a 2-fold dilution of the stock solution (200
µ
g/mL; 5.95
·
10 and 5.00 10 M respectively).
·
S. haemolyticus showed a MIC up to a 32-fold dilution in the case of [Ni(BzNH-adtp) ], corresponding
2
−
g/mL (1.56 10 M). In contrast, [Ni(PhBuNH-adtp) ] lost the ability to inhibit the bacterial
2
5
to 6.25
µ
·
growth after dilution (MIC > 100
µ
g/mL). Moreover, the bactericidal activity was assessed by
evaluating the minimum bactericidal concentration (MBC), i.e., the lowest concentration of an
antimicrobial required to kill a particular bacterium life in suspension (planktonic status). This approach
is established when the substance under investigation can inactivate bacterial contamination in a
fluid, such as water, saliva and urine. Neither [Ni(BzNH-adtp) ] nor [Ni(PhBuNH-adtp) ] showed
2
2
any bactericidal activity (MBC > 100 µg/mL; Table 2) against these strains. Microorganisms living
within a structured biofilm cause many human infections. Such a sessile structure is generally
more resistant to various antimicrobial treatments [28]. For this reason, we measured the influence
of complexes [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] on biofilm formation by evaluating the
2
2
minimum concentration required to inhibit the formation of the biofilm in vitro, i.e., the minimum
biofilm inhibitory concentration (MBIC). Both complexes showed the ability to inhibit the biofilm
growth, however they required relatively high concentrations in the case of S. aureus (MBIC = 100
µg/mL).
Notably, for both complexes, lower MBICs were observed for the biofilms of S. haemolyticus (MBIC = 50.0
−
4
and 12.5
and 3.12
out on NiCl2
µ
g/mL for [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ], respectively, corresponding to 1.49
·
10
10 M). For the sake of comparison, MIC, MBC, and MBIC measurements were carried
6H O under the same experimental conditions, on the same strains of microrganisms.
2
2
−
5
·
·
2
As expected, nickel chloride did not show any antibacterial activity towards S. aureus and S. haemolyticus
(Tables S6 and S7).
Table 2. Minimum inhibitory concentration (MIC), minumum bactericidal concentration (MBC),
and minimum biofilm inhibitory concentration (MBIC) values (µg/mL) towards sensitive strains
determined for compounds [Ni(BzNH-adtp) ] (A) and [Ni(PhBuNH-adtp) ] (B).
2
2
Strain
MIC
MBC
MBIC
A
B
A
B
A
B
Staphylococcus aureus
Staphylococcus haemolyticus
100
6.25
100
>100
>100
>100
>100
>100
100
50
100
12.5
2
.2. DFT Calculations
Following recent studies on different complexes featuring chalcogen donors [18,46–49], the electronic
i
i
structures of salts ( PrNH )( PrNH-adtp), (BzNH )(BzNH-adtp), and (PhBuNH )(PhBuNH- adtp),
and the corresponding Ni complexes were investigated by theoretical calculations carried out at
the density functional theory (DFT) [50] in order to theorize why complexes [Ni(BzNH-adtp)2]
3
3
3
II
and [Ni(PhBuNH-adtp) ] undergo hydrolysis and try to explain the different antimicrobial
2
i
activity determined between these complexes and the analogous [Ni( PrNH-adtp) ] and the free
amidodithiophosphonate salts. DFT calculations were carried out on the starting amines PrNH ,
2
i
2
BzNH , PhBuNH , the corresponding ammonium cations, the relevant amidodithiophosphonate
2
2
i
−
−
−
i
anions ( PrNH-adtp) , (BzNH-adtp) , and (PhBuNH-adtp) , and the complexes [Ni( PrNH-adtp) ],
2
[
Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ]. As expected, all the starting amines feature a Kohn–Sham
2 2
(
KS) HOMO corresponding to the lone pair (LP) of electrons localized on the nitrogen atoms (with
i
eigenvalues of
−
6.612, 6.898, and
−
6.579 eV, for PrNH , BzNH , PhBuNH , respectively). This LP is
2
2
2
therefore available to react with Lawesson’s Reagent, which features a positive charge on the P atom
Q = 0.878 |e|) accompanied by a low energy ( 2.075 eV) KS-LUMO, antibonding in nature with respect
(
−

Molecules 2020, 25, 2052
10 of 18
i
−
−
to the P–S bonds of the P S 1,3-dithia-2,4-diphosphetane ring. The ( PrNH-adtp) , (BzNH-adtp) ,
2
2
−
and (PhBuNH-adtp) anions feature strongly polarized P–N bonds (NBO charges Q : 1.269, 1.262,
P
1
.264; QN:
−
1.012,
−
1.002, 1.000 |e|, respectively). This results in Wiberg bond indices (WBI) sensibly
−
lower than unity (WBI = 0.721, 0.697, 0.707, respectively), reflected in optimized P–N bond lengths
PN
(
dPN) in the range between 1.741 and 1.749 Å. All the anions feature virtual MOs antibonding with
II
respect to the P–N bonds. Moving from the anions to the corresponding Ni complexes, the P–N bonds
are slightly strengthened (WBIPN 0.791–0.828; dPN 1.674–1.679 Å). In analogy to the corresponding
ligands, these complexes feature low-lying virtual MOs, antibonding with respect to the P–N bonds.
In addition, the charge on the P atoms, and therefore the polarization of the P–N bonds, increases, thus
indicating a large electrophilic character of the P atoms (Q = 1.365, 1.361, 1.361; Q
=
−
1.010, 1.006,
−
P
N
i
−
1.007 |e| for [Ni( PrNH-adtp) ], [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ], respectively). As a result,
2
2
2
the eigenvalues of the virtual antibonding MOs (with respect to the P–N bonds) are more stable than
those of the corresponding free anionic ligands (0.672, 0.211, 0.712 eV, respectively) and result in an
−
increased electrophilic character of the P atom. Therefore, the susceptibility to the hydrolysis of related
II
amidodithiophosphonato Ni complexes is expected to be larger than that of the starting ligands.
These results are in line with the hypothesis that the antibacterial activity could be related to the
hydrolytic process. The subsequent cleavage of the P–N bond in the neutral [Ni(adtp) ] complexes in
2
turn leads to the formation of the complexes (BzNH ) [Ni(dtp) ] and (PhBuNH ) [Ni(dtp) ] that have
3
2
2
3 2
2
been isolated and structurally characterized (Scheme 2; Figures 2 and 3, Tables 1 and 2). This hypothesis
i
would also be consistent with the lack of antibacterial activity determined for [Ni( PrNH-adtp) ],
2
which has proved experimentally to be less prone to hydrolysis, as confirmed by the stability in solution
and isolation in the solid state [3]. It is interesting to note that no virtual MOs (antibonding with
respect to the P–S bonds) can be found at energies close to, or lower than, those of the aforementioned
−
antibonding P–N MOs, either in the free R-adtp anions or in the corresponding [Ni(R-adtp)2]
complexes. This indicates that hydrolysis of the compounds should be expected to occur through P–N
bond breaking and dithiophosphonate anion formation, causing some doubt regarding the previously
hypothesized emission of dihydrogen sulfide as the first step of the hydrolysis [51]. The hydrolysis
of P–N was also confirmed by the isolation of a few crystals of the 4-phenylbutylammonium salt of
bis(4-methoxyphenyl)-tetrathiodiphosphonate (PhBuNH ) [(ArPS ) O] (Figure 5) during an attempt
3
2
2 2
at crystallizing (PhBuNH )(PhBuNH-adtp). A similar salt was hypothesized as the intermediate in the
3
in situ formation of a mixed cymene-ferrocenylphosphonodithiolate ruthenium complex, obtained by
the hydrolysis of 2,4-diferrocenyl-1,3-dithiadiphosphetane 2,4-disulfide in the presence of ammonium
hydroxide [35].
3
. Materials and Methods
Starting materials and solvents were purchased from commercial sources TCI (Tokio, Japan)
and Aldrich (Darmstadt, Germany) and, when necessary, the solvents have been distilled
and dried according to the standard literature techniques. Melting point measurements were
◦
determined in capillaries, using melting point apparatus BUCHI M-560 (30–240 C, Flawil, Svizzera).
Elemental analyses were performed with an EA1108 CHNS-O Fisons instrument (Thermo Fisons,
1
31
◦
Okehampton, EX20 1UB, UK). H and P NMR measurements were carried out at 25 C using
a Bruker Avance 300 MHz (7.05 T, Billerica, MA, USA) spectrometer at operating frequencies of
1
3
00.13 and 121.49 MHz, respectively. Chemical shifts for H-NMR are reported in parts per million
(
ppm), calibrated to the residual solvent peak set, with coupling constants J reported in Hertz (Hz).
Chemical shifts for 31P NMR are reported in parts per million (ppm), calibrated to the external reference
TPP 48.5 mM in acetone-d . Infrared (IR) spectra were recorded on a Thermo Nicolet 5700 FT-IR
6
−1
spectrophotometer (Waltham, MA, USA) using KBr pellets and reported in wavenumbers (cm ).
Single-crystal X-ray diffraction data were collected at 100 K on a Rigaku FRE+ equipped with
VHF Varimax confocal mirrors and an AFC12 goniometer (Tokio, Japan) and HyPix 6000 detector
diffractometer (Tokio, Japan) [52]. The structures were solved with the ShelXT [53] structure solution

Molecules 2020, 25, 2052
11 of 18
program using the Intrinsic Phasing solution method, using Olex2 [54] as the graphical interface.
The model was refined with version 2018/3 of ShelXL [55] using Least Squares minimization.
All hydrogen atoms were added in calculated positions and refined in riding positions relative
to the parent atom. CCDC deposition numbers: 1944063–1944065.
3
.1. Theoretical Calculations
Quantum-mechanical calculations were carried out at density functional theory (DFT) [50] level
(
mPW1PW functional) [56] with the Gaussian 16 (rev B.01, Gaussian Inc., Wallingford, CT, USA) [57]
commercial suite of computational software. All calculations were performed by adopting the
i
def2-SVP [58] basis sets for all atomic species. The calculations were carried out on the amines PrNH ,
2
BzNH , PhBuNH , the corresponding ammonium cations, the relevant amidodithiophosphonate
2
2
i
−
−
−
i
anions ( PrNH-adtp) , (BzNH-adtp) , and (PhBuNH-adtp) , and nickel complexes [Ni( PrNH-adtp) ],
2
[
Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ]. For all the investigated compounds the geometries were
2 2
optimized, starting from structural data when available, and symmetrized to achieve the highest
possible point group. For all the neutral, anionic, and cationic species tight convergence criteria were
−
5
−1
−5
−1
adopted (maximum force 1.5
·
10 Ha Bohr , RMS force 1.0
·
10 Ha Bohr , maximum displacement
−
5
−5
6.0
·
10 Å, and RMS displacement 4.0
geometries were verified by a vibrational analysis, computed by determining the second derivatives
of the energy with respect to the Cartesian atomic coordinates and subsequently transforming to
·
10 Å). The nature of the energy minima at the optimized
mass-weighted coordinates. Natural bonding orbitals [59], natural charges and Wiberg bond indices [60
]
were calculated at the optimized geometries. The programs Chemissian [61], GaussView 6.0 [62],
and Molden 5.9 [63] were used to analyze optimized geometries and Kohn–Sham molecular orbitals.
3
.2. Microbiological Assays
The following species were used: (i) Gram-positive bacteria, Staphylococcus aureus ATCC 6538
(
American Type Culture Collection), Staphylococcus haemolyticus clinical isolate NC1; (ii) Gram-negative
bacteria, Escherichia coli ATCC 7075, and two strains of Pseudomonas aeruginosa: P. aeruginosa ATCC
5442 (PA-1) recommended for disinfectant testing by official methods was used as the high
1
biocide-resistant strain; P. aeruginosa ATCC 27853 (PA-2) was used as the susceptible disinfectant
strain. In addition, the clinical isolates of Candida spp. were assayed: C. albicans BF1, C. kruseii BF2 and
i
i
C. glabrata BF3. In vitro susceptibility testing was carried out on compounds ( PrNH )( PrNH-adtp),
3
i
(
BzNH )(BzNH-adtp), (PhBuNH )(PhBuNH-adtp), NiCl
·
H O, and complexes [Ni( PrNH-adtp) ],
Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] using: (a) the agar diffusion method, (b) minimum
3
3
2
2
2
[
2
2
inhibitory concentration (MIC) and (c) minimum bactericidal concentration (MBC), determined in
accordance with the Clinical Laboratory Standard Institute CLSI [64 65]. The Agar diffusion method
,
was performed by using the Kirby–Bauer procedure [66], and it was employed to reveal the entire
7
antimicrobial susceptibility profile for the examined compounds. 1
·
10 cells/mL were inoculated
onto the surface of an agar plate containing one of the subsequent bacterial growth agar media
manufactured by Microbiol (Uta, Italy): (i) Muller–Hinton agar was used for aerobic bacteria, (ii) Fungi
on Sabouraud agar. Each agar plate contained a central circular cavity, Ø = 10 mm, able to contain
i
i
−3
5
0
µ
L volume of the solution of the examined compounds (C: ( PrNH )( PrNH-adtp) = 1.28
·
10 ;
·
3
−
4
−4
−4
(BzNH )(BzNH-adtp) = 7.93
·
10 ; (PhBuNH )(PhBuNH-adtp) = 6.17
·
10 , NiCl2
·
6H O = 5.00
10 ,
3
3
2
i
−4
−4
−4
[
Ni( PrNH-adtp) ] = 8.33
·
10 , [Ni(BzNH-adtp) ] = 5.95
·
10 and [Ni(PhBuNH-adtp) ] = 5.00
in 1/10 v/v DMSO/water). The antimicrobial activity was expressed as mm of inhibition diameter
around the cavity, after the microbial growth at 37 C. All experiments were performed in
·
10
M
2
2
2
◦
triplicate and the values shown are reported as the average
diameter. MIC and MBC were determined only against the microbial strains susceptible to
Kirby–Bauer assay, according to the micro-broth dilution method [67 68], by using a 1/2 serial
dilution, from 100 to 0.20 g/mL of the compounds under study in the previously described
−6
liquid growth mediums (C: ( PrNH )( PrNH-adtp) = 2.56 10 , (BzNH )(BzNH-adtp) = 1.59 10 ,
±
standard deviation of the inhibition
,
µ
i
i
−6
·
·
3
3

Molecules 2020, 25, 2052
12 of 18
−
6
−6
i
−6
(
PhBuNH )(PhBuNH-adtp) = 1.23
·
10 , NiCl
·
6H O = 1.00
·
10 , Ni( PrNH-adtp) = 1.67
·
10 ,
3
2
2
2
−6
−6
Ni(BzNH-adtp) = 1.19
·
10 and Ni(PhBuNH-adtp) = 1.00
used the protocol described by Montana University’s Center for Biofilm Engineering [69]. A microplate
containing serial concentrations of the compound, inoculated with the bacterial strains as previously
described for MIC and MBC evaluation, was incubated at 37 C for 48 h, to permit the biofilm formation.
The plate samples were subsequently washed three times with phosphate-buffered saline GIBCO PBS
·
10 M). For the biofilm evaluation, we
2
2
◦
®
(
Thermo Fisher, Waltham, MA, USA) to eliminate planktonic cells; thus, the biofilm was stained with
◦
1
00
washes with PBS solution, 200
from the bacterial biomass. The biofilm amount was measured with a plate reader spectrophotometer
SLT-Spectra II, SLT Tecan Instruments, Männedorf, Switzerland) at 620 nm.
µL of 0.1% w/v of crystal violet solution (Microbial, Uta, Italy) for 10 min at 25 C; after three
µL of 30% v/v acetic acid was added in every well to solubilize the dye
(
3
.3. Synthesis
(iPrNH )( PrNH-adtp), (BzNH )(BzNH-adtp) were synthesized as reported previously [3,31,32,70]
i
.
3
3
3
.3.1. Synthesis of (PhBuNH )(PhBuNH-adtp)
3
−
3
Lawesson’s Reagent (0.972 g; 2.40
×
10 mol) was suspended in dry toluene (25 mL) in a sealed
flask and stirred under nitrogen atmosphere. After few minutes 4-phenylbutylamine (PhBuNH )
2
−2
(
1.90 mL; 1.20
×
10 mol) was added dropwise. The reaction mixture was stirred vigorously for 5 h and
10 C; the precipitate was then filtered under reduced pressure and washed several times
with cold toluene. The solid was then suspended in diethyl ether for 30 min, filtered and dried under
vacuum (0.427 g; Y = 18%). M.p. 143 C (dec.) Elemental analysis calculated (%) for C H N OPS :
C 64.77; H 7.45; N 5.59; S 12.81; found C 65.01; H 7.53; N 5.61, S 12.65. FT-IR (KBr, 4000–400 cm ,
Figure S14): 2928br, 1594s, 1569ms, 1492s, 1462ms, 1403w, 1301ms, 1253s, 1173m, 1137w, 1105s, 1028ms,
89w, 855vs, 800m, 749m, 717w, 697s, 658vs νasym(P–S), 624s, 551ms νsym(P–S), 516m, 439m cm .
◦
cooled at
−
◦
27
37
2
2
1
−
−1
9
1
H-NMR (300 MHz, DMSO-d , Figure S2) δ(ppm) 8.01–7.87 (m, 4H), 7.36–7.24 (m, 4H), 7.24–7.12
6
(
(
m, 9H), 6.71 (d, J = 8.3 Hz, 4H), 3.73 (s, 6H), 2.81 (t, J = 7.2 Hz, 4H), 2.58 (t, J = 7.1 Hz, 4H), 1.71–1.45
m, 8H). 31P{ H} NMR (121 MHz, DMSO-d ) δ(ppm) 93.1 (s).
1
6
3
.3.2. Synthesis of [Ni(BzNH-adtp)2]
−
5
NiCl2
·
6H O (12.0 mg; 5.05
×
10 mol) was dissolved in MeOH (5 mL) and added dropwise to a
2
−4
suspension of (BzNH )(BzNH-adtp) (42.2 mg; 1.01
was then stirred at room temperature for 8 h. The purple solid was filtered and washed with acetonitrile
12.3 mg; Y = 36%) M.p. 162 C (dec.) Elemental analysis calculated (%) for C H N NiO P S :
C 49.79; H 4.48; N 4.15; S 18.99; found C 49.81; H 4.55; N 4.21, S 18.75. FT-IR (KBr, 4000–400 cm ,
×
10 mol) in MeCN (5 mL). The reaction mixture
3
◦
(
28
30
2
2
2
4
1
−
Figure S12): 3251m
ν
(N-H), 3026w, 2926w, 2837w, 1951ms, 1570w, 1498ms, 1462m, 1454m, 1441w,
1
8
4
406m, 1306mw, 1294m, 1428vs, 1209w, 1178m, 1113s, 1061s, 1022m, 968mw, 916w, 872m, 823ms,
16mw, 800m, 744ms, 688ms, 658m νasym(P–S), 636w, 609m, 588w, 557ms νsym(P–S), 523w, 496w,
−1
1
22w cm . H-NMR (300 MHz, CDCl , Figure S3)
δ
7.89 (dd, J = 12.5, 8.6 Hz, 2H), 7.31–7.09 (m,
3
1
0H), 6.81–6.76 (m, 2H), 3.73 (s, 3H), 2.78 (t, J = 7.4 Hz, 2H), 2.68 (br, 1H, NH), 2.64–2.54 (m, 4H), 2.45
31
1
(t, J = 7.6 Hz, 2H), 1.66–1.40 (m, 6H), 1.35–1.26 (m, 2H). P{ H} NMR (121 MHz, DMSO-d ) δ 75.8 (s).
6
3
.3.3. Synthesis of (BzNH ) [Ni(dtp) ]
3
2
2
−
5
NiCl2
·
6H O (12.2 mg; 5.13
×
10 mol) was dissolved in H O (5 mL) and added dropwise to a
2
2
−4
suspension of (BzNH )(BzNH-adtp) (41.9 mg; 1.00
was stirred at room temperature for 1 h, filtered and left to rest for overnight (20.4 mg; Y = 57%).
M.p. > 240 C (dec). Elemental analysis calculated (%) for C H N NiO P S : C 44.99; H 5.15; N 3.75;
S 17.16; found C 50.06; H 5.23; N 3.77, S 17.05. FT-IR (KBr, 4000–400 cm , Figure S13): 3473m
×
10 mol) in MeCN (5 mL). The purple solution
3
◦
28
38
2
6
1
2 4
−
ν
(N-H),
3034w, 2603w, 1620mw, 1595ms, 1570mw, 1497ms, 1456m, 1441w, 1402w, 1385w, 1296w, 1292m, 1252s,
1213w, 1174m, 1119s, 1076vs, 1026ms, 922w, 829m, 812w, 798m, 741m, 694m, 656m νasym(P–S), 627w,

Molecules 2020, 25, 2052
13 of 18
−
1
5
8
96ms, 548ms νsym(P–S), 523w cm . 1H-NMR (600 MHz, DMSO-d , Figure S9)
δ
8.25 (dd, J = 11.5,
6
.6 Hz, 4H), 8.21 (br, NH, 6H), 7.53–7.46 (m, J = 7.2 Hz, 4H), 7.46–7.41 (m, 4H), 7.41–7.36 (m, 2H), 6.92
(
d, J = 7.0 Hz, 4H), 4.07 (s, CH2, 4H), 3.80 (s, OCH , 6H).
3
3
.3.4. Synthesis of [Ni(PhBuNH-adtp)2]
−
5
NiCl2
·
6H O (12.0 mg; 5.05
×
10 mol) was dissolved in MeOH (5 mL) and added dropwise to a
2
−4
suspension of (PhBuNH )(PhBuNH-adtp) (50.1 mg; 1.00
mixture was then stirred at room temperature for 8 h. The purple solid was filtered and washed with
chloroform (14.0 mg; Y = 37%). M.p. 147 C. Elemental analysis calculated (%) for C H N NiO P S :
C 53.76; H 5.75; N 3.62; S 16.88; found C 53.81; H 5.85; N 3.61, S 16.75. FT-IR (KBr, 4000–400 cm ,
×
10 mol) in MeCN (5 mL). The reaction
3
◦
34
42
2
2
2
4
1
−
Figure S15): 3261m
ν
(N-H), 3022w, 2937w, 2854w, 1591ms, 1568m, 1498ms, 1454m, 1439m, 1404m,
1
7
306mw, 1292m, 1252vs, 1176ms, 1115s, 1080s, 1051m, 1024ms, 970m, 920m, 868m, 825ms, 798m, 750m,
−
1 1
35m, 698m, 660m νasym(P–S), 633mw, 606ms, 573w, 548s νsym(P–S), 525m, 469w, 401w cm . H-NMR
(ppm) 7.90 (dd, 4H), 7.33–7.11 (m), 6.97 (d, J = 7.1 Hz, 4H), 3.86 (s,
H, OCH ), 3.17–2.99 (m, 4H, -CH -NH), 2.97–2.83 (m, 2H, NH), 2.62 (d, J = 7.1 Hz, 4H, -CH -Ph),
(300 MHz, CDCl , Figure S4) δ
3
6
3
2
2
31
1
1
.77–1.61 (m). P{ H} NMR (121 MHz, DMSO-d ) δ(ppm) 76.2 (s).
6
3
.3.5. Synthesis of (PhBuNH ) [Ni(dtp) ]
3 2 2
−
10 mol) was dissolved in H O (5 mL) and added dropwise to a
2
5
NiCl2
·
6H O (12.1 mg; 5.09
×
2
−4
suspension of (PhBuNH )(PhBuNH-adtp) (50.5 mg; 1.01
solution was stirred at room temperature for 1 h, filtered and left to rest overnight. The solid was then
×
10 mol) in MeCN (5 mL). The purple
3
◦
filtered and recrystallized from chloroform (24.8 mg; Y = 62%). M.p. > 240 C (dec). Elemental analysis
calculated (%) for C H N NiO P S : C 51.33; H 5.83; N 3.52; S 16.12; found C 51.41; H 5.86; N 3.54, S
34
46
2
4 2 4
−1
1
2
1
5
7
6.15. FT-IR (KBr, 4000–400 cm , Figure S16): 3248m
515w, 1953s, 1566m, 1498s, 1456m, 1454m, 1404mw, 1300m, 1296m, 1254s, 1180m, 1115s, 1063ms,
022m, 970w, 920mw, 870w, 835m, 800m, 742m, 698m, 654m νasym(P–S), 629w, 607ms, 544ms νsym(P–S),
ν(N-H), 3024vs, 2972vs, 2931vs, 2858s, 2607w,
−
1 1
23w, 467mw, 407m cm . H-NMR (600 MHz, DMSO-d , Figure S10)
δ
8.30–8.20 (m, 4H), 7.80 (s, 6H),
6
.28 (t, J = 7.3 Hz, 4H), 7.23–7.14 (m, 6H), 6.94 (d, J = 7.5 Hz, 4H), 3.80 (s, OCH , 6H), 2.82 (m, 4H), 2.60
3
(t, J = 7.1 Hz, 4H), 1.67–1.51 (m, 8H).
3
.3.6. Synthesis of (PhBuNH ) [(ArPS ) O]
3
2
2 2
Few crystals of bis(4-methoxyphenyl)-tetrathiodiphosphonate (PhBuNH ) [(ArPS ) O] were
3
2
2 2
obtained during an attempt at crystallizing (PhBuNH )(PhBuNH-adtp) by slow diffusion of ethyl
3
ether in a toluene solution of the salt. The very exiguous amount of compound prevented us from
further characterizations.
4
. Conclusions
The reaction between Lawesson’s Reagent (LR) and isopropylamine ( PrNH ), benzylamine
i
2
(
BzNH ), and 4-phenylbutylamine (PhBuNH ) in toluene gave rise to the corresponding
2
2
i
i
amidodithiophosphonate ammonium salts ( PrNH )( PrNH-adtp), (BzNH )(BzNH-adtp),
3
3
and (PhBuNH )(PhBuNH-adtp) that were reacted with nickel chloride hexahydrate, yielding
3
i
the corresponding amidodithiophosphonato complexes [Ni( PrNH-adtp) ], [Ni(BzNH-adtp) ] and
2
2
[
Ni(PhBuNH-adtp) ]. All the compounds were tested against a library of bacteria and fungi of clinical
2
importance belonging to the genera Staphylococcus, Escherichia, and Pseudomonas, and Candida, but only
the complexes [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] demonstrated some antimicrobial activity
2
2
that was tentatively ascribed to their tendency towards hydrolysis. Theoretical and experimental
results evidenced that [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] undergo hydrolysis and that
2
2
during the hydrolytic process a cleavage of the polarized P–N bond occurs with consequent
formation of a P–O bond and retaining of the two P–S bonds in the amidodithiophosphonate
moiety. Even if hydrolysis was proven to occur both in the amidodithiophosphonate salts and in

Molecules 2020, 25, 2052
14 of 18
the corresponding nickel complexes, an increased polarization of the P–N bond was calculated for
the latter, suggesting a higher tendency to undergo hydrolysis. The antibacterial inactivity of the
salts can be tentatively explained by taking into account their high hydrophilicity associated with
their ionic nature, which circumvents the penetration of the cellular membrane. On the contrary,
the neutral complexes [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] can pass the cellular membrane
2
2
i
and thus exploit their activity. The inactivity of the analogous [Ni( PrNH-adtp) ], can be explained,
2
taking into account its higher resistance to hydrolysis, demonstrated by its higher stability both
in solution and in the solid state, also confirmed by a P–N bond that is slightly shorter than those
determined for analogous phenyl-alkyl-amidodithiophosphonato complexes. Further studies are
ongoing in order to better understand the role of the alkyl-aryl substituents of the amines in the final
amidodithiophosphonato complexes.
Supplementary Materials: The following are available online. Table S1: Crystal data and refinement parameters for
compounds (BzNH ) [Ni(dtp) ] 2H O, (PhBuNH ) [Ni(dtp) ], and (PhBuNH ) [(ArPS ) O]. Table S2: Hydrogen
·
3
2
2 2 3 2 2 3 2 2 2
Bond information for (BzNH ) [Ni(dtp) ] 2H O. Table S3: Hydrogen Bond information for (PhBuNH ) [Ni(dtp) ].
·
3
2
2 2 3 2 2
Table S4: Hydrogen Bond information for (PhBuNH ) [(ArPS ) O]. Table S5: Drug susceptibility profile of the
3
2
2 2
bacterial strains used in this work. Table S6 MIC, MBC, and MBIC values (
determined for NiCl2 6H O. Table S7 MIC, MBC, and MBIC values (mol/L) towards bacterial strains determined
for NiCl2 6H O. Figure S1: H-NMR spectrum of (BzNH )(BzNH-adtp) in DMSO-d at 25 C. Figure S2: H-NMR
spectrum of (PhBuNH )(PhBuNH-adtp), in DMSO-d at 25 C. Figure S3: H-NMR spectrum of [Ni(BzNH-adtp) ]
µg/mL) towards bacterial strains
·
2
1
◦
1
·
2
3
6
◦
1
3
6
2
◦
1
◦
in CDCl at 25 C. Figure S4: H-NMR spectrum of [Ni(PhBuNH-adtp) ] in CDCl at 25 C. Figure S5: Packing
3
2
3
view for (BzNH ) [Ni(dtp) ] 2H O along the 010 direction. Figure S6: Contents and numbering scheme of the
·
3
2
2 2
asymmetric unit of (PhBuNH ) [Ni(dtp) ] thermal ellipsoids drawn at the 50% probability level, disorder shown.
3
2
2
Figure S7: Contents and numbering scheme of the asymmetric unit of (PhBuNH ) [(ArPS ) O] thermal ellipsoids
3
2
2 2
drawn at the 50% probability level, disorder shown. Figure S8: Antimicrobial activity, as inhibition diameter
ø of [Ni(BzNH-adtp) ] and [Ni(PhBuNH-adtp) ] against eight different microbial species, C = [200 g/mL].
The bars represent the mean and lines indicate the standard error of the mean. Figure S9: H-NMR spectrum of
µ
2
2
1
◦
BzNH ) [Ni(dtp) ] in DMSO-d at 25 C. Figure S10: H-NMR spectrum of (PhBuNH ) [Ni(dtp) ] in DMSO-d
3 2 2 6 3 2 2 6
1
(
◦
at 25 C. Figure S11: FT-IR spectrum of (BzNH )(BzNH-adtp). Figure S12: FT-IR spectrum of [Ni(BzNH-adtp) ].
3
2
Figure S13: FT-IR spectrum of (BzNH ) [Ni(dtp) ]. Figure S14: FT-IR spectrum of (PhBuNH )(PhBuNH-adtp).
3
2
2
3
Figure S15: FT-IR spectrum of [Ni(PhBuNH-adtp) ]. Figure S16: FT-IR spectrum of (PhBuNH ) [Ni(dtp) ].
2
3 2
2
Figure S17: FT-IR spectrum of (PhBuNH ) [(ArPS ) O]. CheckCIF/PLATON reports for (PhBuNH ) [(ArPS ) O],
3
2
3 2
2 2
3 2
2 2
(
BzNH ) [Ni(dtp) ]·2H O, and (PhBuNH ) [Ni(dtp) ].
3
2
2
2
2
Author Contributions: E.P. and G.A. synthesized and characterized all the new compounds; S.J.C. and J.B.O.
collected and resolved structural data; M.A. and A.P. carried out DFT calculations; A.I., G.O., and E.T. carried
microbiological assays; M.C.A., M.A., F.I., and V.L. conceptualization and funding acquisition; E.P. and M.C.A.
wrote the article. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Universit
annualit 2018-2019), Fondazione di Sardegna (FdS) and Regione Autonoma della Sardegna (Progetti Biennali di
Ateneo FdS/RAS annualità 2016).
à degli Studi di Cagliari (Fondo Integrativo per la Ricerca, FIR,
à
Acknowledgments: CESAR (Centro Servizi di Ateneo per la Ricerca) is kindly acknowledged for technical support.
Conflicts of Interest: The authors declare no conflict of interest.
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