Heteroditopic P,N ligands in gold(I) complexes: Synthesis, structure and cytotoxicity

DOI: 10.1016/j.jinorgbio.2015.01.014

Source and publish data:

Journal of Inorganic Biochemistry p. 108 - 120 (2015)

Update date:2022-09-26

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Authors:

Traut-Johnstone, Telisha

Kanyanda, Stonard

Kriel, Frederik H.

Viljoen, Tanya

Kotze, P.D. Riekert

Van Zyl, Werner E.

Coates, Judy

Rees, D. Jasper G.

Meyer, Mervin

Hewer, Raymond

Williams, D. Bradley G.

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Article abstract of DOI:10.1016/j.jinorgbio.2015.01.014

New heteroditopic, bi- and multidentate imino- and aminophosphine ligands were synthesised and complexed to [AuCl(THT)] (THT = tetrahydrothiophene). X-ray crystallography confirmed Schiff base formation in three products, the successful reduction of the imino-group to the sp³-hybridised amine in several instances, and confirmed the formation of mono-gold(I) imino- and aminophosphine complexes for four Au-complexes. Cytotoxicity studies in cancerous and non-cancerous cell lines showed a marked increase in cytotoxicity upon ligand complexation to gold(I). These findings were supported by results from the 60-cell line fingerprint screen of the Developmental Therapeutics Programme of the National Institutes of Health for two promising compounds. The cytotoxicity of some of these ligands and gold(I)complexes is due to the induction of apoptosis. The ligands and gold(I)complexes demonstrated selective toxicity towards specific cell lines, with Jurkat T cells being more sensitive to the cytotoxic effects of these compounds, while the non-cancerous human cell line KMST6 proved more resistant when compared to the cancerous cell lines. Results from the NIH DTP 60 cell-line fingerprint screen support the observed enhancement of cytotoxicity upon gold(I) complexation. One gold(I)complex induced high levels of apoptosis at concentrations of 50 μM in all the cell lines screened in this study, while some of the other compounds selectively induced apoptosis in the cell lines. These results point towards the potential for selective toxicity to cancerous cells through the induction of apoptosis.

Full text of DOI:10.1016/j.jinorgbio.2015.01.014

Journal of Inorganic Biochemistry 145 (2015) 108 –120

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry

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

Heteroditopic P,N ligands in gold(I) complexes: Synthesis, structure

and cytotoxicity

Telisha Traut-Johnstone ^a,b, Stonard Kanyanda ^c,1, Frederik H. Kriel ^b, Tanya Viljoen ^a, P.D. Riekert Kotze ^a,

Werner E. van Zyl ^a,d, Judy Coates ^b,2, D. Jasper G. Rees ^c,3, Mervin Meyer ^c,

Raymond Hewer ^b, D. Bradley G. Williams ^a,e,

⁎

Research Centre for Synthesis and Catalysis, Department of Chemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa

Advanced Materials Division, Mintek, 200 Malibongwe Drive, Strijdom Park 2125, South Africa

Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville, Cape Town 7535, South Africa

School of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4000, South Africa

Ferrier Research Institute, Victoria University of Wellington, 69 Graceﬁeld Rd, Lower Hutt 5010, New Zealand

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 8 October 2014

Received in revised form 20 January 2015

Accepted 21 January 2015

Available online 30 January 2015

New heteroditopic, bi- and multidentate imino- and aminophosphine ligands were synthesised and complexed

to [AuCl(THT)] (THT = tetrahydrothiophene). X-ray crystallography conﬁrmed Schiff base formation in three

products, the successful reduction of the imino-group to the sp³-hybridised amine in several instances, and con-

ﬁrmed the formation of mono-gold(I) imino- and aminophosphine complexes for four Au-complexes. Cytotox-

icity studies in cancerous and non-cancerous cell lines showed a marked increase in cytotoxicity upon ligand

complexation to gold(I). These ﬁndings were supported by results from the 60-cell line ﬁngerprint screen of

the Developmental Therapeutics Programme of the National Institutes of Health for two promising compounds.

The cytotoxicity of some of these ligands and gold(I)complexes is due to the induction of apoptosis. The ligands

and gold(I)complexes demonstrated selective toxicity towards speciﬁc cell lines, with Jurkat T cells being more

sensitive to the cytotoxic effects of these compounds, while the non-cancerous human cell line KMST6 proved

more resistant when compared to the cancerous cell lines. Results from the NIH DTP 60 cell-line ﬁngerprint

screen support the observed enhancement of cytotoxicity upon gold(I) complexation. One gold(I)complex

induced high levels of apoptosis at concentrations of 50 μM in all the cell lines screened in this study, while

some of the other compounds selectively induced apoptosis in the cell lines. These results point towards the

potential for selective toxicity to cancerous cells through the induction of apoptosis.

Keywords:

Gold(I) complexes

Metal-based drugs

P,N ligands

Cytotoxicity

1. Introduction

arthritis [17,18]. Gold(I) mostly forms linear, two-coordinate complexes

with so-called “soft” ligands such as sulfur and phosphorus [19,20]. Con-

The recent interest in the biochemistry and bio-inorganic chemistry

of gold has triggered a ﬂood of reports on the use of gold complexes as

potential treatment for a variety of disease models, including rheuma-

toid arthritis [1–3] and other chronic inﬂammatory and auto-immune

diseases [4–6], various cancers [7–10], as well as bacterial, fungal and

viral infections [11–13]. Most of these reports focus on gold in either

+I or +III oxidation states, as these forms are comparatively stable in

aqueous solution, facilitating their use in biological research [14–16].

Several gold compounds are in clinical for the treatment of rheumatoid

versely, gold(III) is isoelectronic with Pt(II) and typically forms square-

planar, 4-coordinate complexes [21,22]. Due to this similarity, many

gold(III) complexes have been synthesised and tested as analogues for

Pt(II) drugs such as the highly effective anticancer drug cisplatin [23,

24]. Despite their structural similarities, different mechanisms of biolog-

ical action for gold(III) and platinum(II) complexes have been proposed.

While platinum(II) complexes have been shown to intercalate into DNA

strands, inhibiting the function of various DNA and RNA enzymes [25],

the mode of action of gold(I) and gold(III) complexes has not been con-

clusively determined. Recent studies have indicated that the cytotoxic

anti-cancer mechanisms of gold complexes appear to be distinct from

cisplatin and unrelated to DNA intercalation [26,27]. Several mecha-

nisms have been proposed that involve protein inhibition and/or inter-

action [27–32], and involve the induction of cellular apoptosis via

alterations to the intracellular mitochondrial membrane potential,

stimulating the release of pre-apoptotic factors and the subsequent

downstream activation of caspases [33].

⁎

Corresponding author at: Ferrier Research Institute, Victoria University of Wellington,

69 Graceﬁeld Rd, Lower Hutt 5010, New Zealand.

E-mail addresses: bwilliams@uj.ac.za, Bradley.williams@vuw.ac.nz (D.B.G. Williams).

Deceased.

Present address: Technology Innovation Agency, P.O Box 172, Pretoria 0063, South

Africa.

Present address: Biotechnology Platform, Agricultural Research Council, Private Bag

X05, Onderstepoort 0110, South Africa.

http://dx.doi.org/10.1016/j.jinorgbio.2015.01.014

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

109

Heteroditopic multidentate P,N ligands [34–37] show differential

binding ability between the P and N atoms, a characteristic put to use in

homogeneous catalysts [38–40]. Investigations into the structure and re-

activity of this class of compounds have intensiﬁed, including their poten-

tial use in various medicinal applications such as anticancer activity [41,

42]. The complexation of P,N ligands to transition metals such as Pd(II)

and Pt(II) centres has been well studied. However, similar investigations

into the gold(I) complexation of the P,N ligand class are limited [43]. We

have previously communicated the differential gold(I) binding afﬁnity of

the P and N donor atoms in bi- and multidentate bi-metallic binding sys-

tems [44], where the reactivity of the ligand may be tailored for speciﬁc

applications. As a part of our continued interest in the ﬁeld of coordina-

tion complexes and their applications [45], a range of new bi- and

multidentate heteroditopic P,N ligands has been prepared representing

the nitrogen in both sp²and sp³hybridisation states. The complexation

of the ligands to monovalent gold(I) and the structures of the resultant

imino- and aminophosphine gold(I) complexes were investigated with

the aid of single crystal X-ray analysis. In this paper, we report on the syn-

thesis, structure evaluation and promising in vitro cytotoxicity proﬁles

observed for a family of ﬁfteen structurally related imino- and

aminophosphine P,N ligands and their corresponding gold(I) complexes

in a panel of thirteen cell lines which includes ten human cancer cell

lines and one non-cancerous human cell line and two rodent cell lines.

Furthermore, we demonstrate the selective cytotoxicity displayed by

the compound family towards cancerous cell lines over the non-

cancerous human cell line KMST6, as well as the enhanced cytotoxic po-

tential demonstrated upon complexation of the free ligands to the

gold(I) metal centre. Finally, we report on the results obtained for two

compounds (one ligand and one gold(I)complex) screened through

the 60-cell line ﬁngerprint screen of the Developmental Therapeutics

Program (DTP) of the National Institutes of Health (NIH).

Scheme 1. Facile one-pot synthesis and gold(I) complexation of imino- and

aminophosphine ligands. 4: R = methyl; 5, 20: R = butyl; 6, 13, 22, 29: R = benzyl; 7,

14, 23, 30: R = phenyl; 8, 15, 24, 31: R = isopropyl; 9, 17, 25, 33: R = 2-pyridylethyl;

10, 18, 26, 34: R = 3-dimethylamino-propyl; 11, 19, 27, 35: R = 2-thiophenethyl; 12,

28: R = tert-butyl; 16, 32: R = 2-pyridylmethyl; 21: R = phenylethyl.

these steps complexed to the gold(I) starting material in a similar way

as observed for the gold(I) complexation of triphenylphosphine (36;

Scheme 2B) [46], resulting in the corresponding P,N gold(I) complexes

20–35 (Scheme 2A). The ¹H NMR spectra of the imine ligands 4–12 dis-

play a characteristic doublet signal for the imine-H group at around 8.7–

8.9 ppm with J_H,P= 4.2–5.4 Hz, accompanied by a ¹³C NMR signal at

154–161 ppm with J_C,P= 20.4–22.4 Hz and a ³¹P NMR signal at around

−13 ppm. In contrast, the amine ligands 13–19 show a diagnostic ben-

zylic CH₂moiety in the ¹H NMR spectra, which presents as a two proton

singlet at 3.9–4.4 ppm, accompanied by a ¹³C NMR signal presenting as a

doublet at 47.0–52.4 ppm with J_C,P= 20.6–23.5 Hz, while the ³¹P NMR

spectrum is characterised by a shifted signal at around −16 ppm. The

main feature differentiating the free ligands from their

gold(I) complexes is the signals in the ³¹P NMR spectra, which shift sig-

niﬁcantly downﬁeld to 30.8–32.6 ppm for P,imine complexes 20–18 and

to 25.7–26.3 ppm for P,amine complexes 29–35.

2. Results and discussion

2.1. Synthesis

With our previous work as a springboard [44], we synthesised a col-

lection of P,N ligands and their corresponding gold(I) complexes with

the general structures 2 and 3, respectively (Fig. 1). The amine starting

materials were chosen so that a diverse series of products could be

generated based on R-group structure, with some measure of series

correlation between the products. Synthesis of the P,N ligand library

was accomplished by coupling a variety of primary amines with 2-

(diphenylphosphino)benzaldehyde (1) in a Schiff base condensation

reaction facilitated by the azeotropic removal of water from the system

(Scheme 1). This strategy allowed the one-pot synthesis of

iminophosphine ligands 4–12 as well as their reduced aminophosphine

counterparts 13–19. Accordingly, 1 equivalent of aldehyde 1 was

reacted with 1.1 M equivalents of amine in toluene under reﬂux and

the product puriﬁed by employing Kugel–Rohr bulb-to-bulb vacuum

distillation. The puriﬁcation of the iminophosphine ligands was per-

formed under an inert (nitrogen) atmosphere to prevent the oxidation

of the P atom, preserving its metal-binding afﬁnity. A reduction step

using 1.1 equivalents of sodium borohydride in methanol was required

to obtain the aminophosphine ligands. Ligands obtained from either of

2.2. Structural analysis

2.2.1. Crystallography of ligands 4 and 14

Several X-ray crystal structures were obtained to conﬁrm the pro-

posed structure, in agreement with related studies [47]. Speciﬁcally,

Scheme 2. A: Gold(I) complexation of imino- and aminophosphine ligands to afford

the corresponding gold(I) complexes 20–35. B: Binding mode reported for

(triphenylphosphine)gold(I) chloride 36 [33].

Fig. 1. Structural backbone of the P,N ligands and corresponding gold(I) complexes.

110

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

we report here single crystal analysis that represents each of the four

product types explored in our synthesis: iminophosphine ligands;

aminophosphine ligands; iminophosphine gold(I) complexes and

aminophosphine gold(I) complexes. The single crystal structure obtained

for iminophosphine ligand 4 (Fig. 2) conﬁrmed the proposed Schiff base

condensation between C17 of 2-(diphenylphosphino)benzaldehyde and

N1 of the primary amine, methylamine (C\N bond length of

1.246(3) Å). The hydrogen atoms riding on C18 show some disorder

about the freely rotating methylamine single bond between N1 and

C18. The molecular packing shows reciprocal hydrogen bonding between

N1 and H26 (2.662 Å) of two adjacent molecule pairs of 4 (Supplementa-

ry Fig. S1). In the single crystal structure obtained for aminophosphine li-

gand 14 (Fig. 3), the most obvious feature was the reduced single bond

between N1 and C37. The bond length of 1.486(11) Å is typical of singly

bonded C\N species (C_N bonds are typically in the order of 1.2 Å, as

illustrated for iminophosphine ligand 4). This is clear evidence that the

reduction of the original iminophosphine ligands proceeds to yield the

corresponding range of aminophosphine ligands.

Fig. 3. ORTEP diagram of the crystal structure obtained for aminophosphine ligand 14,

drawn with 50% probability ellipsoids.

2.2.2. Crystallography of gold complexes 21, 23, 30a and 30b

Gold(I) complexation facilitated the formation of suitable single

crystals and several examples of both imino- and aminophosphine

gold(I) complexes were obtained. In all cases mono-gold(I) complexes

resulted with the P\Au\Cl bond angle approaching linearity, falling

in the range 175.5–179.6° (Table 1). In the crystal structures of

iminophosphine gold(I) complexes 21 and 23, we not only conﬁrmed

the proposed bond formation between the P and Au(I) atoms (Figs. 4

and 5) but the characteristic C_N double bond was also evident in

these structures. The structure of 21 (Fig. 4) shows some disorder of

the ﬂexible phenylethyl moiety, due mainly to the inherent ﬂexibility

of the aliphatic carbon chain. The molecular packing shows the forma-

tion of reciprocal H–Cl short contacts between Cl1 and H35 (2.858 Å)

and Cl1 and H37 (2.903 Å) of two adjacent molecule pairs of 21, respec-

tively, as well as demonstrating a third hydrogen–chloride short contact

between Cl1 and H24 (2.924 Å) of a third molecule (Supplementary

Fig. S2). The crystal packing also illustrates the head-to-head staggered

aromatic stacking interactions between the disordered phenylethyl

moieties of two adjacent molecules of 21. A similar three pronged hy-

drogen–chloride short contact network (Supplementary Fig. S3) was

noted in the single crystal structure of 23 (Fig. 5), with two H–Cl short

contacts forming between Cl1 and H33 (2.869 Å) and Cl1 and H34

(2.918 Å) on an adjacent molecule, respectively, while the third short

contact can be observed between Cl1 and H50A (2.580 Å) located on a

disordered dichloromethane molecule.

Crystallisation of aminophosphine gold(I) complex 30 afforded two

different structures (Figs. 6 and 7), depending on the processing to

which the compound had been subjected. While 30a crystallised as

the neutral gold(I) complex from a layered solution of diethyl ether

and hexane, 30b crystallised as the hydrochloride salt derivative from

a layered solution of chloroform and n-heptane. Compound 30b had

been recovered from an aqueous HCl-acidiﬁed solution of 30a. Although

the crystal structures of 30a and 30b show considerable differences in

terms of the interaction networks formed and the packing of the respec-

tive crystals, both crystals clearly displayed the reduced single bond

C\N that is characteristic of the aminophosphine ligands, as discussed

for ligand 14. In addition, both aminophosphine gold(I) complexes

exhibited exclusively mono-gold(I) structures and adopted the linear

geometry expected, with P\Au\Cl bond angles showing only slight

distortion from linearity (176.77° and 175.48° for 30a and 30b, respec-

tively). Furthermore, Au\P and Au\Cl bond lengths are in good agree-

ment with the corresponding bond lengths reported for 36 [AuCl(PPh₃)]

(Table 1; Figs. 6 and 7). Complex 30a (Fig. 6) crystallised as the neutral

aminophosphine gold(I) complex without any solvent inclusion. The

structure of 30a displays one reciprocal HCl short contact between Cl1

of the AuCl moiety and H15 (2.714 Å) of an adjacent molecule (Supple-

mentary Fig. S4). Complex 30b (Fig. 7) crystallised as its hydrochloride

salt with the inclusion of two chloroform solvent molecules. In contrast

to the simple crystal packing observed for 30a, an elaborate network of

interactions is formed between adjacent molecules of 30b and the

solvent-derived chloroform molecules (Supplementary Fig. S5). Specif-

ically, Cl5 forms intramolecular hydrogen–chloride interactions with

H1B (2.214 Å) situated on the positively charged N1, and intermolecular

interactions with H1A (2.176 Å) situated on the positively charged N1 of

an adjacent molecule. A third hydrogen–chloride short contact can be

observed between Cl5 and H50 (2.687 Å) situated on one of the

solvent-derived chloroform molecules. Furthermore, an intermolecular

Table 1

Comparison of selected bond angles (°) and bond lengths (Å) observed in prepared com-

pounds and [AuClPPh₃], compound 36.

Crystal

Bond angle

Bond lengths (Å)

P\Au\Cl (°)

N\C

Au\P

Au\Cl

N/A

1.246(3)

1.486(11)

1.257(5)

1.278(5)

1.426(8)

1.486(11)

N/A

30a

30b

177.33(3)

177.81(3)

175.48(4)

176.77(4)

179.63(8)

2.239(1)

2.236(1)

2.228(2)

2.239(2)

2.23(5)

2.295(1)

2.279(2)

2.281(2)

2.27(9)

Fig. 2. ORTEP diagram of the crystal structure obtained for iminophosphine ligand 4,

drawn with 50% probability ellipsoids. Disorder of the hydrogens on C20 is displayed.

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

111

Fig. 4. ORTEP diagram of the crystal structure obtained for iminophosphine

gold(I) complex 21, drawn with 50% probability ellipsoids. Disorder of the phenylethyl

moiety has been omitted for clarity.

chloride–chloride short contact can be distinguished between Cl5 and

Cl2 of two separate solvent derived chloroform molecules (3.316 Å).

The chloride from the AuCl moiety participates in an intermolecular hy-

drogen–chloride short contact between Cl1 and H15 (2.949 Å) of an ad-

jacent molecule (similar to the interaction observed in the crystal

structure of 30a), as well as forming a chloride–chloride short contact

between Cl1 and Cl4 (3.401 Å) situated on a solvent-derived chloroform

molecule (Supplementary Fig. S6). In the crystallography, some level A

and B alerts remain. These stem from residual electron density present

around the gold atoms present in the structures. Absorption corrections

have been made to compensate for most of the electron density of gold,

but these have been unable to compensate completely for the electron

density of the heavy gold element and thus the “unaccounted” for elec-

tron density around the gold atoms still present as level A and B alerts.

Fig. 6. ORTEP diagram of the crystal structure obtained for aminophosphine

gold(I) complex 30a, drawn with 50% probability ellipsoids.

and their corresponding gold(I) P,N complexes in a more extensive range

of cell lines, both cancerous and non-cancerous. In this study the cytotox-

ic effects of the ligands and their corresponding gold(I) complexes were

evaluated on a panel of thirteen cell lines. This study was performed on

ten cancerous human cell lines (A549J, Caski, HepG2, HeLa, HT29,

H157, Jurkat T, MCF7, MG-63 and Hek293T), one non-cancerous

human cell line (KMST6) and two non-cancerous rodent cell lines

(CHO and NIH 3T3). Two in vitro bioassays (the MTT colorimetric end-

point assay [49–53] and the APOPercentage™ apoptosis assay [54])

were used to assess cytotoxic effects of the thirty compounds.

The MTT assay was used to determine the IC₅₀values for all thirty

compounds on the thirteen cell lines used in this study. The IC₅₀values

for the positive control, cisplatin, were more than 100 μM for all the cell

lines tested in this study. For the purposes of the MTT cytotoxicity assay,

compounds with IC₅₀values of 50 μM or lower were considered to be bi-

ologically active. In comparing the cytotoxic potential of the free ligands

(Table 2) and the gold(I) complexes (Table 3), it is immediately evident

that a generally higher level of cell lethality resulted from the inclusion

of gold to the ligand system, in line with previously published reports

[55,56]. Furthermore, the cytotoxicity results of the most active

2.3. In vitro evaluation of cytotoxicity

Four closely related iminophosphine gold(I) complexes were shown

recently to have low micro-molar in vitro anticancer activity against the

human oesophageal cancer cell lines WHCO1 and KYSE450 as measured

via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) colorimetric assay [48]. No similar results have been reported

for the P,N ligands or aminophosphine gold(I) complexes. It was there-

fore of interest to evaluate the activity of our varied family of P,N ligands

Fig. 5. ORTEP diagram of the crystal structure obtained for iminophosphine

gold(I) complex 23, drawn with 50% probability ellipsoids. The dichloromethane

molecule has been omitted for clarity.

Fig. 7. ORTEP diagram of the crystal structure obtained for aminophosphine

gold(I) complex 30b, drawn at a 50% probability level. The chloroform molecules

have been omitted for clarity.

112

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

Table 2

Summary of the cytotoxic IC₅₀values (μM) after treatment with P,N ligands.^a

Sample

KMST6

A549J

Caski

HepG2

HeLa

HT29

H157

Hek293T

Jurkat T

MCF7

MG63

CHO

3T3

‡

b10

‡

b10

‡

‡ indicates an IC₅₀of more than 100 μM; values reported are the average of at least triplicate experiments and standard deviations did not exceed 5%. The IC₅₀values for the positive

control, cisplatin, were more than 100 μM for all the cell lines tested in this study.

gold(I) complexes detailed here correspond well to the ﬁndings report-

ed for three recent studies on gold(I) cytotoxicity [57–59], with IC₅₀

values in the low- to sub-micromolar range. While the free P,N ligands

showed cytotoxic IC₅₀values under 50 μM in 26 of the 195 assays, the

gold(I) complexes proved much more toxic, with IC₅₀values below

50 μM in 105 of the 195 assays performed. Based on the MTT assay, 14

was more cytotoxic compared to the other ligands, while 5 and 6 dem-

onstrated the lowest cytotoxicity. The IC₅₀value for 14 was less than

50 μM in seven of the cell lines tested in this study, while the IC₅₀values

for 5 and 6 were higher than 50 μM in all cell lines screened in this study.

In general, Hek293T cells were more sensitive to the effects of ligands,

while Caski cells were more resistant. The IC₅₀values for nine of the li-

gands were below 50 μM in Hek293T cells, while the IC₅₀values for all

the ligands were higher than 50 μM in Caski cells. Based on the MTT

assay, 22, 29 and 35 were the most cytotoxic gold(I) complexes, while

30 demonstrated the lowest cytotoxicity. The IC₅₀value for 22, 29 and

35 was below 50 μM in twelve of the cell lines tested in this study,

while the IC₅₀values for 30 were higher than 50 μM in twelve of the

cell lines screened in this study. Nine of the gold(I) complexes (22, 23,

26, 27, 29, 30, 33, 34 and 35) had IC₅₀values below 10 μM at least one

cell line. This is consistent with values obtained for platinum(II) and

gold(I) iminophosphine complexes in a related study [48]. In other

work [60], Auranoﬁn was found to have activity against MCF7 cells

with an IC₅₀value of 11.4 μM. In the present study, the Hek293T cell

line was overall more sensitive to the effects of gold(I) complexes,

while the Caski cell line was the most resistant cell line.

be more active than the imines. Nevertheless, within the imines, simple

aliphatic R groups tended to lower activity. More notable activity was

achieved with R = phenyl (23), 3-dimethylaminopropyl (26) and 2-

thiophenylethyl (27) substituents, these giving IC₅₀values of ≤15 μM

in three or more cell lines. Particularly good activity was achieved

with 26 and 27, with IC₅₀values of ≤10 μM in three cell lines each. Of

these, there was overlap in the HT29 cell line and in the Hek293T cell

line. For the amines, aromatic functionality tended to improve activity.

Most notable activity was identiﬁed for R = benzyl (29), phenyl (30),

2-pyridylethyl (33) and 2-thiophenylethyl (35), all of which contain ar-

omatic functional groups. Interestingly, compound 34 with its 3-

dimethylaminopropyl group also afforded good activity. Of these, all

but compound 30 gave IC₅₀values of ≤10 μM in three or more cell

lines, with complexes 29 and 30 giving these values in six and ﬁve

lines, respectively, with cell line overlap in four of these cases (HepG2,

Hek293T, MCF7 and 3T3). The differential activity of 32 and 33 is re-

markable. In the former, low overall activity is noted while in the latter,

with its slightly longer chain, rather signiﬁcant activity was realised. To-

gether with the similar observation concerning 29 and 30 (both showed

good activity but the benzyl group with its additional chain length was

better), this aspect may lead to improved activity in future studies if the

chain length is manipulated.

The MTT assay measures cell viability as a function of the cell's met-

abolic activity. However, there are several forms of cell death that can

lead to loss of cell viability. Apoptosis is one of these forms of cell

death. One of the hallmarks of cancer cells is that these cells are resistant

to this form of cell death [61]. Apoptosis is therefore a therapeutic target

for the development of anticancer drugs [62] and compounds with

Given the low activity of the ligands, no structure–activity analysis

was performed. For the gold complexes, overall, the amines tended to

Table 3

Summary of the cytotoxic IC₅₀values (μM) after treatment with P,N gold(I) complexes.^a

Compound

KMST6

A549J

Caski

HepG2

HeLa

HT29

H157

Hek293T

Jurkat T

MCF7

MG63

CHO

3T3

‡

b10

‡

b10

‡

b10

‡

b10

‡

180

b10

‡

b10

‡ indicates an IC₅₀of more than 100 μM; values reported are the average of at least triplicate experiments and standard deviations did not exceed 5%. The IC₅₀values for the positive

control, cisplatin, were more than 100 μM for all the cell lines tested in this study.

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

113

pro-apoptotic activity are potential leads for the development of an-

ticancer drugs. Hence, we also investigated whether the free ligands

and the gold(I) complexes can induce apoptosis in the cell lines used

in this study. The cells were treated for 24 h with 50 μM of the com-

pounds and apoptosis was quantiﬁed by ﬂow cytometry using the

APOPercentage™ assay (Tables 4 and 5). For the purpose of this

study, treatments that caused apoptosis in more than 50% of the

cells were considered to be signiﬁcant. Compared to the other li-

gands, 7 demonstrated the highest pro-apoptotic activity (Table 4).

Ligand 7 induced signiﬁcant levels of apoptosis in ﬁve of the cell

lines screened in this study, while most of the other ligands induced

signiﬁcant levels of apoptosis in only one cell line. Ligands 16, 17 and

18 did not induce signiﬁcant levels of apoptosis in any of the cell

lines screened in this study. Compared to the other cell lines, Jurkat

T cells were more susceptible to the pro-apoptotic effects of the li-

gands. Eleven of the ligands induced signiﬁcant levels of apoptosis

in Jurkat T cells. None of the ligands was able to induce signiﬁcant

levels of apoptosis in six of the cell lines (Caski, HeLa, HT29, MG-

63, Hek293T and NIH 3T3) screened in this study. Amongst the

gold(I) complexes, 29 demonstrated the highest pro-apoptotic activ-

ity inducing signiﬁcant levels of apoptosis in all thirteen cell lines

screened in this study (Table 5). Complex 35 demonstrated the low-

est pro-apoptotic activity, inducing signiﬁcant levels of apoptosis in

only one cell line, Jurkat T. Complexes 20, 22, 31, 32, and 34 induced

signiﬁcant levels of apoptosis in between 7 and 10 of the cell lines,

respectively. Jurkat T cells were more sensitive to the pro-apoptotic

activity of the gold(I) complexes. All ﬁfteen gold(I) complexes

induced signiﬁcant levels of apoptosis in these cells. The non-

cancerous cell line, KMST6 was the most resistant cell line with

only one of the gold(I) complexes, 29 inducing signiﬁcant levels of

apoptosis in KMST6 cells. With 10 and 9 of the gold(I) complexes

inducing signiﬁcant levels of apoptosis in CHO and HeLa cells,

respectively, these two cell lines were also very susceptible to the

pro-apoptotic activity of the gold(I) complexes. Interestingly, while

both the MTT assay and the APOPercentage™ assay showed that

22 and 29 are the most cytotoxic compounds, it is only the

APOPercentage™ assay that shows that 7, 20, 31, 32 and 34 are cyto-

toxic. In addition, while the MTT assay showed that 35 is one of the

most cytotoxic compounds, the APOPercentage™ assay showed that

35 is one of the least cytotoxic compounds. This apparent contradic-

tion can be explained by the fact that the two assays use very differ-

ent test principles. The MTT assay is an enzyme dependent assay and

although this assay is routinely used to assess toxicity, it is known

that test compounds that inhibit enzyme activity can affect the

MTT assay [63].

As with the MTT assay, the ligands showed low overall cytotoxicity

and their data were therefore not interpreted for a structure activity

point of view. In the gold complexes, good cytotoxicity was noted for

several imine and amine compounds, as opposed to the MTT assay

where principal activity was found in the amines. Nevertheless, the

amine complexes were more active than the imines. Within the imines

(20–28), greatest activity tended to be found in non-polar substituents

with lower steric hindrance (compounds 20 and 22), but 25 (R = 2-

pyridylethyl), 26 (R = 3-dimethylaminopropyl) and 27 (R = 2-

thiophenylethyl) induced N95% apoptosis in two cell lines. In all cases,

CHO and Jurkat cell lines were susceptible. In the amine complexes,

greatest cytotoxicity was achieved with 29 (R = benzyl), 30 (R =

phenyl), 31 (R = isopropyl), 32 (R = 2-pyridylethyl) and 34 (R =

3-dimethylaminopropyl). When using a low hurdle (≥50% apopto-

sis), there is little to learn about structure activity relationships.

With a higher bar (≥90% apoptosis), it becomes evident that 29,

30, 31 and 34 presented the highest levels of cytotoxicity. In all

cases, Jurkat cells were found to be susceptible. Across all cell lines

and all complexes tested, CHO and Jurkat cells were most suscepti-

ble. Of all of the compounds tested, 29 is the most promising and

would be a good candidate for additional structural manipulations.

Based on the cytotoxicity results from the MTT and apoptosis assays,

the most promising ligand and gold(I) complex (7 and 29, respectively)

were submitted to the 60-cell line screen of the NIH DTP [64]. Results of

the initial one-dose screen highlighted a 17.3% mean growth inhibition

for iminophosphine ligand 7 at a one-dose concentration of 10 μM

(range of 61.12 and delta value of 35.77), while 29 proved much more

potent, with a mean cell lethality of 40.5% at a one-dose concentration

of 10 μM (range of 119.78 and delta value of 35.20). While

gold(I) complex 29 progressed to the more in-depth ﬁve-dose screen,

the promising cytotoxic effects could not be reproduced in the dose–re-

sponse assay. Although neither of the two compounds submitted was

found to be sufﬁciently potent anticancer agents to warrant further

screening by the NCI, valuable data could be gathered from the NCI

COMPARE program in terms of the proposed mechanism of action of

the gold(I)-based test compound 29. Speciﬁcally, a correlation of

0.505 could be found between the cell line ﬁngerprints of test com-

pound 29 and cytembena, a cytostatic agent found in the DTP database,

that has been reported to interfere with the synthesis of cellular DNA

[65]. Furthermore, a 0.356 correlation could be found between 29 and

L-buthionine sulfoximine, another compound from the DTP database,

reported to inhibit glutathione synthase and the formation of glutathi-

one from gamma-glutamylcysteine, resulting in a depletion of cellular

glutathione and leading to cell death (apoptosis) due to the unrestricted

formation of reactive oxygen species (ROS) [66]. It is thus conceivable

Table 4

Levels of apoptosis (% apoptosis) as determined by the APOPercentage™ technique after treatment with the P,N ligands.^a

Phosphine ligand

A549J

Caski

CH0

HepG2

HeLa

HT29

H157

Jurkat

KMST6

MCF7

MG-63

Hek

3T3

60 10

Values reported are the average of at least triplicate experiments.

114

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

Table 5

Levels of apoptosis (% apoptosis) as determined by the APOPercentage™ technique after treatment with the P,N gold(I) complexes.^a

Complexes

A549J

Caski

CH0

HepG2

HeLa

HT29

H157

Jurkat

KMST6

MCF7

MG-63

Hek

3T3

100

Values reported are the average of at least triplicate experiments.

that complex 29 may act on one or both of these pathways. It is interest-

ing to note that the biologically active compounds (ligands and gold-

complexes alike) showed potential for selective cytotoxicity in the

range of cancerous cell lines tested. The differences observed in selectiv-

ity and potency proﬁles of the free ligands vs the gold(I) complexes,

coupled with the variable cytotoxic effect displayed by the biologically

active compounds across the panel of cell lines, illustrate the possibility

of tailoring the potency of the ligands, either through the inclusion of

gold(I) to the ligand systems, or by incorporating variability into the

molecular structure through changing the nitrogen-borne substituent

of both imino- and aminophosphine analogues (Fig. 8).

4. Experimental section

4.1. General methods and materials

Chemical ionisation mass spectrometry (CIMS) was performed on a

Finnigan-Matt 8200 spectrometer at a 70 eV ionisation potential. Fast

atom bombardment mass spectra (FAB-MS) were acquired with a

VG70SE mass spectrometer under the control of a MS Services data sys-

tem, with glycerol matrices. ¹H-, ¹³C- and ³¹P NMR data were obtained

on a Varian Gemini-300 Hz spectrometer. The data are reported as

parts per million relative to TMS and referenced to CDCl₃. The peak

multiplicities are abbreviated as follows: s — singlet; d — doublet;

dd — doublet of doublets; t — triplet; tt — triplet of triplets; p — pentet;

sx — sextet; and m — multiplet. The coupling constant (J) is calculated in

Hertz and reported to the nearest 0.1 Hz [67]. Where appropriate, J_C,P

values are given as observed in the ¹³C NMR spectra of the compounds.

Infrared spectroscopy (IR) was performed on a Perkin-Elmer 881 spec-

trometer in CHCl₃solvent or KBr pellets. The characteristic peaks are re-

ported in wavenumber (cm⁻¹). Melting points were collected on a

DSC822^eMettler Toledo differential scanning calorimetry instrument.

All solvents used were chemically pure or pre-puriﬁed [68,69]. Hexane

was puriﬁed by distillation. Toluene and diethyl ether were pre-dried

over an Al₂O₃column and then dried over sodium-benzophenone

under nitrogen until the dark blue colour of the benzophenone ketyl

persisted. Solvents were removed by rotary evaporation under reduced

pressure and puriﬁcation performed by bulb-to-bulb vacuum distil-

lation using Kugel–Rohr distillation apparatus. All other reagents

were analytically and synthetically pure. All reagents and starting

materials were degassed before use to minimise the oxidation of

the iminophosphine products. All experiments were performed in

oven-dried, ﬂamed out ﬂasks under an atmosphere of UHP argon. A

Dean–Stark apparatus was used in all cases for the azeotropic remov-

al of water.

3. Conclusions

A range of new P,N ligands has been synthesised that represent the

nitrogen in sp²and sp³hybridisation states. Complexation to

Au(I) allowed the formation of several new gold(I) complexes of

which representative examples were characterised by single crystal X-

ray crystallography. Biological evaluations into the cytotoxicity of the

free ligands and gold(I) complexes on a range of cancerous and non-

cancerous human and rodent cell lines show that the complexes are

more highly cytotoxic than their corresponding ligands towards the

cancer cell lines tested, and display lower cytotoxicity towards healthy

cells. The enhanced cytotoxicity of the gold(I) complexes as compared

to the free ligands was conﬁrmed in the 60 cell-line ﬁngerprint screen

of the NIH DTP. Additionally, the high pro-apoptotic activity of 29 as

determined in the APOPercentage assay was supported by correlations

between the characteristic 60 cell-line ﬁngerprint of 29 and that of ^L-

buthionine sulfoximine, a known pro-apoptotic agent. While no conclu-

sive structure–activity relationships could be established for this group

of compounds, the differences in activity due to the R-group structure

are notable and point towards the value of additional studies in this

area, which would include in vitro investigations to elucidate the mech-

anism by which the observed pro-apoptotic activity is conferred. These

studies may prove to add to the currently limited knowledge-base on

biologically active gold(I) compounds.

4.2. General procedure for the synthesis of iminophosphine ligands (4–12).

Procedure A

The iminophosphine ligands were prepared according to the

method reported by Shirakawa and co-workers [70]. To 2-

(diphenylphosphino)benzaldehyde (1) (200 mg, 0.689 mmol)

0.758 mmol (1.1 M equivalent) of the corresponding amine and

10 mL of freshly distilled toluene were added. The mixture was

stirred under reﬂux (150–160 °C oil bath temperature) for 6 h.

The solvent was removed in vacuo and the crude product was puri-

ﬁed by bulb-to-bulb vacuum distillation (170 °C at 0.05 mm Hg,

consistently used for all products) using a Kugel Rohr apparatus

into which argon was continuously piped to prevent the ingress

Fig. 8. Molecular structures of cytembena and L-buthionine sulfoximine.

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

115

of oxygen. Since the iminophosphine products were unstable on

4.2.5. (2-Diphenylphosphanyl-benzylidene)-isopropyl amine (8)

silica, no R_f-values are included for the iminophosphine ligands.

Isopropylamine (0.758 mmol, 0.065 mL) was added to the 2-

(diphenylphosphino) benzaldehyde mixture and treated according to

general procedure A to provide 8 as a viscous light yellow oil after distil-

lation (0.668 mmol, 0.221 g, 97%). ¹H NMR: (300 MHz, CDCl₃) δ_H8.71

(d, J = 4.5 Hz, 1H), 7.81 (dd, J = 7.4 and 3.8 Hz, 1H), 7.23–7.07 (m,

12H), 6.64 (dd, J = 7.5 and 4.8 Hz, 1H), 3.23 (sp, J = 6.3 Hz, 1H), and

0.89 (d, J = 3.2 Hz, 6H); ¹³C NMR: (75 MHz, CDCl₃) δ_C156.9 (d, J =

20.9 Hz), 139.6 (d, J = 16.9 Hz), 137.0 (d, J = 18.6 Hz), 136.4 (d, J =

9.5 Hz), 134.0 (d, J = 20.0 Hz), 132.9, 129.9, 128.8, 128.7, 128.5 (d,

J = 7.2 Hz), 127.4 (d, J = 4.0 Hz), 61.2, and 23.9; ³¹P NMR: (121 MHz,

CDCl₃) δ_P−13.0; IR: ν_max(CHCl₃)/cm⁻¹2940 and 1642; CIMS: m/z

332 ([M]⁺, 100%), 288 ([M−C₃H₇]⁺, 82%).

4.2.1. (Z)-N-(2-(Diphenylphosphino)benzylidene)-methanamine (4)

Methylamine (0.758 mmol, 0.712 mL) was added to the 2-

(diphenylphosphino) benzaldehyde mixture and treated according to

general procedure A to provide 4 as white powder after distillation

(0.659 mmol, 0.200 g, 87%). Colourless single crystals were grown

from a layered solution of chloroform and n-hexane. mp: 117.5–

119.0 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.93 (dq, J = 4.5 and 1.5 Hz,

1H), 7.98 (ddd, J = 7.7, 4.1 and 1.4 Hz, 1H), 7.40–7.24 (m, 12H), 6.89

(ddd, J = 7.6, 4.7 and 1.3 Hz, 1H), and 3.38 (d, J = 1.5 Hz, 3H); ¹³C

NMR: (75 MHz, CDCl₃) δ_C160.8 (d, J = 22.4 Hz), 139.5 (d, J =

17.4 Hz), 137.0 (d, J = 19.1 Hz), 136.3 (d, J = 9.7 Hz), 133.8 (d, J =

19.7 Hz), 133.3 (d, J = 10.8 Hz), 130.1, 128.9, 128.7, 128.5 (d, J =

7.1 Hz), 127.0 (d, J = 14.3 Hz), and 47.9 (d, J = 11.7 Hz); ³¹P NMR:

(121 MHz, CDCl₃) δ_P−14.1; IR: ν_max(CHCl₃)/cm⁻¹2879 and 1641;

HRMS calcd for C₂₀H₁₉NP ([M + H]⁺): m/z 304.1255; found:

304.1253. Crystal data are available in the Supplementary Information.

4.2.6. (2-Diphenylphosphanyl-benzyl)-(2-pyrid-2-yl-ethyl)-amine (9)

2-(Aminoethyl)pyridine (0.758 mmol, 0.091 mL) was added to the

2-(diphenylphosphino) benzaldehyde mixture and treated according

to general procedure A to provide 9 as a viscous yellow oil after distilla-

tion (0.634 mmol, 0.250 g, 92%). ¹H NMR: (300 MHz, CDCl₃) δ_H8.76 (d,

J = 4.5 Hz), 8.37 (d, J = 4.2 Hz, 1H), 7.84 (dd, J = 7.2 and 3.3 Hz, 1H),

7.36 (td, J = 7.7 and 1.7 Hz, 1H), 7.28–7.13 (m, 12H), 6.93 (d, J =

7.2 Hz, 2H), 6.77 (dd, J = 7.2 and 5.1 Hz, 1H), 3.79 (t, J = 7.2 Hz, 2H),

and 2.88 (t, J = 7.2 Hz, 2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C160.1 (d,

J = 20.9 Hz), 159.7, 149.1, 139.4 (d, J = 17.2 Hz), 137.2 (d, J =

19.4 Hz), 136.5 (d, J = 9.8 Hz), 135.9, 133.8 (d, J = 19.7 Hz), 133.3,

130.0, 128.7, 128.6, 128.4 (d, J = 7.2 Hz), 127.5 (d, J = 4.3 Hz), 123.3,

121.0, 60.7, and 39.4; ³¹P NMR: (121 MHz, CDCl₃) δ_P−13.5; IR: ν_max

(CHCl₃)/cm⁻¹2963 and 1657; CIMS: m/z 395 ([M]⁺, 100%) and 288

([M−C₇H₈N₂]⁺, 62%).

4.2.2. N-(2-(Diphenylphosphino)phenyl)-methylene-butylamine (5)

n-Butylamine (0.758 mmol, 0.078 mL) was added to the 2-

(diphenylphosphino) benzaldehyde mixture and treated according to

general procedure A to provide 5 as a viscous orange oil after distillation

(0.668 mmol, 0.231 g, 97%). ¹H NMR: (300 MHz, CDCl₃) δ_H8.71 (d, J =

4.2 Hz, 1H), 7.88 (dd, J = 3.9 Hz, 1H), 7.30–7.14 (m, 12H), 6.77 (dd, J =

7.7 and 4.7 Hz, 1H), 3.38 (t, J = 6.8 Hz, 2H), 1.37 (p, J = 7.1 Hz, 2H), 1.02

(sx, J = 7.4 Hz, 2H), and 0.71 (t, J = 7.2 Hz, 3H); ¹³C NMR: (75 MHz,

CDCl₃) δ_C159.3 (d, J = 21.5 Hz), 139.6 (d, J = 16.9 Hz), 137.1 (d, J =

18.9 Hz), 136.4 (d, J = 9.5 Hz), 134.0 (d, J = 19.7 Hz), 133.1, 130.0,

128.8, 128.7, 128.5 (d, J = 7.2 Hz), 127.5 (d, J = 4.4 Hz), 61.2, 32.6,

20.1, and 13.8; ³¹P NMR: (121 MHz, CDCl₃) δ_P−13.3; IR: ν_max

(CHCl₃)/cm⁻¹2964 and 1647; CIMS: m/z 345 ([M]⁺, 86%) and 288

([M−C₄H₉]⁺, 100%).

4.2.7. N′-(2-Diphenylphosphanyl-benzylidene)-N,N-dimethyl-propane-

1,3-diamine (10)

3-Dimethylamino-propyl amine (0.758 mmol, 0.095 mL) was added

to the 2-(diphenylphosphino) benzaldehyde mixture and treated ac-

cording to general procedure A to provide 10 as a viscous yellow oil

after distillation (0.634 mmol, 0.237 g, 92%). ¹H NMR: (300 MHz,

CDCl₃) δ_H8.78 (d, J = 4.5 Hz, 1H), 7.87 (dd, J = 7.1 and 3.5 Hz, 1H),

7.28–7.14 (m, 12H), 6.76 (dd, J = 6.9 and 5.1 Hz, 1H), 3.40 (t, J =

6.8 Hz, 2H), 2.08–1.97 (m, 8H), and 1.56 (p, J = 7.1 Hz, 2H); ¹³C NMR:

(75 MHz, CDCl₃) δ_C159.4 (d, J = 20.9 Hz), 139.4 (d, J = 17.2 Hz),

137.0 (d, J = 20.9 Hz), 136.4 (d, J = 9.8 Hz), 133.8 (d, J = 20.0 Hz),

133.1, 129.9, 128.7, 128.6, 128.4 (d, J = 7.1 Hz), 127.5 (d, J = 4.4 Hz),

59.3, 57.3, 45.3, and 28.6; ³¹P NMR: (121 MHz, CDCl₃) δ_P−13.1; IR:

4.2.3. N-(2-(Diphenylphosphino)phenyl)-methylene benzylamine (6)

Benzylamine (0.758 mmol, 0.082 mL) was added to the 2-

(diphenylphosphino) benzaldehyde mixture and treated according to

general procedure A to provide 6 as a viscous yellow oil after distillation

that was precipitated as a white powder from methanol (0.661 mmol,

0.251 g, 96%). mp: 105–106.5 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.94

(d, J = 5.4 Hz, 1H), 7.97 (dd, J = 7.4 and 3.8 Hz, 1H), 7.32–7.08 (m,

15H), 6.97–6.94 (m, 2H), 6.80 (dd, J = 6.8 and 5.3 Hz, 1H), and 4.59

(s, 2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C160.5 (d, J = 22.3 Hz), 139.3 (d,

J = 17.2 Hz), 138.9, 137.5 (d, J = 18.9 Hz), 136.3 (d, J = 9.5 Hz),

134.0 (d, J = 20.0 Hz), 133.2, 130.4, 128.9, 128.6 (d, J = 7.1 Hz),

128.3, 128.0, 127.6 (d, J = 4.1 Hz), 126.7, and 65.1; ³¹P NMR:

(121 MHz, CDCl₃) δP −13.6; IR: νmax (CHCl₃)/cm⁻¹2845 and 1640;

CIMS: m/z 379 ([M]⁺, 12%) and 288 ([M−C₇H₇]⁺, 100%).

_max(CHCl₃)/cm⁻¹2949 and 1643; CIMS: m/z 375 ([M]⁺, 100%) and

303 ([M−C₅H₁₂N]⁺, 20%).

4.2.8. (2-Diphenylphosphanyl-benzylidene)-(2-thiophen-2-yl-ethyl)-

amine (11)

2-Thiophenethylamine (0.758 mmol, 0.088 mL) was added to the 2-

(diphenylphosphino) benzaldehyde mixture and treated according to

general procedure A to provide 11 as a viscous rich-yellow oil after dis-

tillation (0.648 mmol, 0.259 g, 94%). ¹H NMR: (300 MHz, CDCl₃) δ_H8.78

(d, J = 4.8 Hz), 7.90 (dd, J = 7.5 and 3.6 Hz, 1H), 7.33–7.15 (m, 12H),

6.97 (d, J = 5.4 Hz, 1H), 6.82–6.75 (m, 2H), 6.65 (d, J = 3.3 Hz, 1H),

3.66 (t, J = 7.4 Hz, 2H), and 2.90 (t, J = 7.4 Hz, 2H); ¹³C NMR:

(75 MHz, CDCl₃) δ_C160.4 (d, J = 21.2 Hz), 142.3, 139.4 (d, J =

17.2 Hz), 137.4 (d, J = 19.2 Hz), 136.5 (d, J = 9.5 Hz), 133.9 (d, J =

20.0 Hz), 133.4, 130.2, 128.9, 128.8, 128.6 (d, J = 7.1 Hz), 127.7 (d,

J = 4.3 Hz), 126.6, 124.8, 123.4, 62.5, and 31.3; ³¹P NMR: (121 MHz,

CDCl₃) δ_P−13.4; IR: ν_max(CHCl₃)/cm⁻¹3051, 3026, 2939, 2837, and

1635; CIMS: m/z 400 ([M]⁺, 100%) and 288 ([M−C₆H₇S] ⁺, 22%).

4.2.4. N-(2-(Diphenylphosphino)phenyl)-methylene benzenamine (7)

Aniline (0.758 mmol, 0.069 mL) was added to the 2-

(diphenylphosphino) benzaldehyde mixture and treated according to

general procedure A to provide 7 as a viscous light yellow oil after dis-

tillation (0.668 mmol, 0.244 g, 97%). ¹H NMR: (300 MHz, CDCl₃) δ_H

8.98 (d, J = 5.4 Hz), 8.12 (dd, J = 7.1 and 3.8 Hz, 1H), 7.36 (t, J =

7.6 Hz, 1H), 7.27–7.14 (m, 14H), 7.09–7.04(m, 1H), and 6.87–6.81 (m,

2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C158.8 (d, J = 21.8 Hz), 151.6, 139.1

(d, J = 16.4 Hz), 138.6 (d, J = 20.0 Hz), 136.4 (d, J = 9.5 Hz), 134.0

(d, J = 19.8 Hz), 133.5, 130.8, 128.9 (d, J = 2.9 Hz), 128.7 (d, J =

7.1 Hz), 128.1 (d, J = 4.1 Hz), 125.9, and 120.9; ³¹P NMR: (121 MHz,

CDCl₃) δ_P−12.9; IR: ν_max(CHCl₃)/cm⁻¹3019 and 1626; CIMS: m/z

365 ([M]⁺, 89%) and 288 ([M−C₆H₅]⁺, 100%).

4.2.9. tert-Butyl-(2-diphenylphosphanyl-benzylidene)-amine (12)

tert-Butylamine (0.758 mmol, 0.079 mL) was added to the 2-

(diphenylphosphino) benzaldehyde mixture and treated according to

general procedure A to provide 12 as a creamy white solid after

116

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

distillation (0.661 mmol, 0.228 g, 96%). Colourless, needle-like single

crystals were grown from ethanol. mp: 115–116.5 °C; ¹H NMR:

(300 MHz, CDCl₃) δ_H8.67 (d, J = 5.1 Hz, 1H), 7.82 (dd, J = 7.1 and

3.5 Hz, 1H), 7.28–7.10 (m, 12H), 6.72 (dd, J = 6.8 and 5.3 Hz, 1H), and

0.95 (s, 9H); ¹³C NMR: (75 MHz, CDCl₃) δ_C154.3 (d, J = 20.3 Hz),

139.9 (d, J = 15.8 Hz), 137.3 (d, J = 18.3 Hz), 136,5 (d, J = 9.5 Hz),

134.1 (d, J = 20.0 Hz), 132.7, 129.6, 128.7, 128.5 (d, J = 7.1 Hz), 127.3

(d, J = 3.7 Hz), 57.6, and 29.4; ³¹P NMR: (121 MHz, CDCl₃) δ_P−11.6;

IR: ν_max(CHCl₃)/cm⁻¹2961 and 1638; CIMS: m/z 346 ([M]⁺, 26%)

and 288 ([M−C₄H₉]⁺, 100%).

0.181 g, 79%). ¹H NMR: (300 MHz, CDCl₃) δ_H7.44 (ddd, J = 7.3, 4.5

and 1.2 Hz, 1H), 7.30–7.22 (m, 11H), 7.09 (td, J = 7.5 and 1.4 Hz, 1H),

6.88 (ddd, J = 7.6, 3.8 and 1.4 Hz, 1H), 3.87 (s, 2H), 2.56 (sp, J =

6.6 Hz, 1H), and 0.79 (d, J = 6.6 Hz, 6H); ¹³C NMR: (75 MHz, CDCl₃)

δ_C144.4 (d, J = 23.6 Hz), 136.5 (d, J = 9.9 Hz), 135.4 (d, J = 13.7 Hz),

133.6 (d, J = 19.5 Hz), 133.3, 129.1 (d, J = 5.5 Hz), 128.7, 128.4, 128.3

(d, J = 6.8 Hz), 126.9, 49.9 (d, J = 20.6 Hz), 47.7, and 22.5; ³¹P NMR:

(121 MHz, CDCl₃) δ_P−15.7; IR: ν_max(CHCl₃)/cm⁻¹2945 and 1438;

CIMS: m/z 334 ([M]⁺, 100%).

4.3.4. (2-Diphenylphosphanyl-benzyl)-pyrid-2-ylmethyl amine (16)

2-(Aminomethyl)-pyridine (0.758 mmol, 0.078 mL) was used as the

amine starting material and the iminophosphine intermediate prepared

as per procedure A above. Immediate reduction as per the general

aminophosphine synthesis (Procedure B) above, afforded the pure

aminophosphine product 16 as a creamy white oil after distillation

(0.400 mmol, 0.153 g, 58%). ¹H NMR: (300 MHz, CDCl₃) δ_H8.48 (d,

J = 4.5 Hz, 1H), 7.52 (d, J = 6.0 Hz, 3H), 7.34–7.06 (m, 14H), 6.89 (t,

J = 6.0 Hz, 1H), 4.05 (s, 2H), and 3.82 (s, 2H); ¹³C NMR: (75 MHz,

CDCl₃) δ_C159.7, 149.0, 144.2 (d, J = 24.1 Hz), 136.7 (d, J = 9.8 Hz),

136.2, 135.7 (d, J = 13.7 Hz), 133.8 (d, J = 19.5 Hz), 133.6, 129,0 (d,

J = 5.5 Hz), 128.9, 128.6, 128.4 (d, J = 7.1 Hz), 127.2, 121.9, 121.6,

54.5, and 51.9 (d, J = 20.9 Hz); ³¹P NMR: (121 MHz, CDCl₃) δ_P−15.5;

IR: ν_max(CHCl₃)/cm⁻¹2992 and 1431; CIMS: m/z 383 ([M]⁺, 30%)

and 290 ([M−C₆H₆N]⁺, 20%).

4.3. General procedure for the preparation of aminophosphine ligands (13–

19). Procedure B

To 2-(diphenylphosphino)-benzaldehyde (200 mg, 0.689 mmol)

0.758 mmol (1.1 M equivalents) of the corresponding amine and

10 mL of freshly distilled toluene were added. The mixture was stirred

under reﬂux for 6 h. The toluene was removed in vacuo and anhydrous

MeOH and 3 equivalents NaBH₄were added to the residue under argon

ﬂow. The reactions were left to stir at room temperature for 12 h,

quenched with distilled water and extracted with CH₂Cl₂(×3). The

organic phase was dried over anhydrous Na₂SO₄. The aminophosphine

ligands were obtained in pure form following bulb-to-bulb vacuum

distillation (170 °C, 0.05 mm Hg).

4.3.1. N-(2-Diphenylphosphinobenzyl)benzylamine (13)

Benzylamine (0.758 mmol, 0.082 mL) was used as the amine

starting material to prepare iminophosphine intermediate 6, and imme-

diately reduced as per the general aminophosphine synthesis (Proce-

dure B) above. The pure aminophosphine product 13 was obtained as

a light yellow oil after distillation (0.537 mmol, 0.205 g, 78%). ¹H

NMR: (300 MHz, CDCl₃) δ_H7.55 (dd, J = 7.2 and 4.2, 1H), 7.41–7.19

(m, 17H), 6.98 (dd, J = 7.4 and 4.7 Hz, 1H), 4.09 (s, 2H), 3.73 (d, J =

2.7 Hz, 2H), and 1.69 (s, 1H); ¹³C NMR: (75 MHz, CDCl₃) δ_C144.4 (d,

J = 23.6 Hz), 140.2, 136.6 (d, J = 9.5 Hz), 135.7 (d, J = 13.5 Hz),

133.8 (d, J = 19.6 Hz), 133.6, 129.2 (d, J = 5.5 Hz), 128.9, 128.6, 128.5

(d, J = 7.1 Hz), 128.1, 128.0, 127.2, 126.6, 53.2, and 52.0 (d, J =

20.6 Hz); ³¹P NMR: (121 MHz, CDCl₃) δ_P−15.6; IR: ν_max(CHCl₃)/

4.3.5. (2-Diphenylphosphanyl-benzyl)-2-(pyrid-2-yl)-ethyl amine (17)

2-(Aminoethyl)pyridine (0.758 mmol, 0.091 mL) was used as the

amine starting material to prepare iminophosphine intermediate 9,

and immediately reduced as per the general aminophosphine synthesis

(Procedure B) above. The pure aminophosphine product 17 was obtain-

ed as a creamy white oil after distillation (0.489 mmol, 0.194 g, 71%). ¹H

NMR: (300 MHz, CDCl₃) δ_H8.40 (d, J = 4.8 Hz, 1H), 7.43 (td, J = 7.7 and

1.9 Hz, 1H), 7.35 (dd, J = 7.1 and 4.7 Hz, 1H), 7.24–7.12 (m, 12H), 7.04

(td, J = 7.5 and 1.2 Hz, 1H), 6.96 (dd, J = 7.5 and 0.9 Hz, 1H), 6.78 (dd,

J = 6.5 and 4.7, 1H), 3.91 (s, 2H), 2.82 (t, J = 6.6 Hz, 2H), 2.71 (t, J =

6.6 Hz, 2H), and 1.42 (s, 1H); ¹³C NMR: (75 MHz, CDCl₃) δ_C160.2,

149.2, 144.5 (d, J = 23.6 Hz), 136.7 (d, J = 10.2 Hz), 136.1, 135.6 (d,

J = 14.0 Hz), 133.7 (d, J = 19.7 Hz), 133.5, 128.9 (d, J = 4.3 Hz),

128.5, 128.5 (d, J = 14.8 Hz), 127.1, 123.1, 121.0, 52.2 (d, J =

20.6 Hz), 48.7, and 38.5; ³¹P NMR: (121 MHz, CDCl₃) δ_P−15.7; IR:

cm⁻¹3026 and 1438; CIMS: m/z 381 ([M]⁺

([M−C₇H₈N]⁺, 83%).

, 41%) and 275

4.3.2. N-(2-Diphenylphosphinobenzyl)phenylamine (14)

_max(CHCl₃)/cm⁻¹3026 and 1438; CIMS: m/z 397 ([M]⁺, 100%) and

Aniline (0.758 mmol, 0.069 mL) was used as the amine starting ma-

terial to prepare iminophosphine intermediate 7, and immediately re-

duced as per the general aminophosphine synthesis (Procedure

B) above. The pure aminophosphine product 14 was obtained as a ﬁne

white powder after distillation (0.558 mmol, 0.205 g, 81%). Colourless

single crystals were grown from a layered solution of chloroform and

n-hexane. mp: 143–145 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H7.39–7.37

(m, 1H), 7.26–7.14 (m, 11H), 7.08 (t, J = 7.4 Hz, 1H), 6.99 (t, J =

7.5 Hz, 2H), 6.83 (t, J = 5.4 Hz, 1H), 6.56 (t, J = 6.9 Hz, 1H), 6.30 (d,

J = 8.1 Hz, 2H), 4.40 (s, 2H), and 3.82 (br s); ¹³C NMR: (75 MHz,

CDCl₃) δ_C147.6, 143.2 (d, J = 23.2 Hz), 136.2 (d, J = 9.7 Hz), 135.6 (d,

J = 14.6 Hz), 133.9 (d, J = 19.7 Hz), 133.5, 129.0, 128.8, 128.6 (d, J =

7.1 Hz), 128.1 (d, J = 5.2 Hz), 127.4, 117.3, 112.8, and 47.0 (d, J =

23.5 Hz); ³¹P NMR: (121 MHz, CDCl₃) δ_P−15.5; IR: ν_max(CHCl₃)/

318 ([M−C₅H₄N]⁺, 20%).

4.3.6. N′-(2-Diphenylphosphanyl-benzyl)-N,N-dimethyl-propane-1,3-

diamine (18)

3-Dimethylamino-1-propylamine (0.758 mmol, 0.095 mL) was used

as the amine starting material to prepare iminophosphine intermediate

10, and immediately reduced as per the general aminophosphine syn-

thesis (Procedure B) above. The pure aminophosphine product 18 was

obtained as a creamy white oil after distillation (0.475 mmol, 0.179 g,

69%). ¹H NMR: (300 MHz, CDCl₃) δ_H7.33 (dd, J = 4.8 and 6.9 Hz, 1H),

7.16–7.13 (m, 11H), 7.99 (t, J = 7.4 Hz, 1H), 6.76 (dd, J = 7.2 and

4.5 Hz, 1H), 3.85 (s, 2H), 2.40 (t, J = 7.1 Hz, 2H), 2.03 (m, 8H), 1.41 (s,

1H), and 1.34 (p, J = 7.2 Hz, 2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C144.4

(d, J = 23.8 Hz), 136.5 (d, J = 10.3 Hz), 135.4 (d, J = 13.5 Hz), 133.6

(d, J = 19.4 Hz), 133.4, 128.9 (d, J = 5.2 Hz), 128.7, 128.5, 128.3 (d,

cm⁻¹3026 and 1219; CIMS: m/z 367 ([M]⁺

, 24%) and 275

([M−C₆H₆N]⁺, 100%). Crystal data are available in the Supplementary

J = 6.9 Hz), 126.9, 57.7, 52.4 (d, J = 20.6 Hz), 47.5, 45.3, and 27.8; ³¹

Information.

NMR: (121 MHz, CDCl₃) δ_P−15.7; IR: ν_max(CHCl₃)/cm⁻¹2956, 2831,

and 1438; CIMS: m/z 377 ([M]⁺, 100%) and 298 ([M−C₅H₁₃N₂]⁺, 30%).

4.3.3. (2-Diphenylphosphanyl-benzyl)-isopropyl amine (15)

Isopropyl amine (0.758 mmol, 0.065 mL) was used as the amine

starting material to prepare iminophosphine intermediate 8, and imme-

diately reduced as per the general aminophosphine synthesis (Proce-

dure B) above. The pure aminophosphine product 15 was obtained as

a semi-translucent viscous white oil after distillation (0.544 mmol,

4.3.7. N-(2-Diphenylphosphino)benzyl-2-(thiophen-2-yl)ethanamine

(19)

2-Thiophenethylamine (0.758 mmol, 0.088 mL) was used as the

amine starting material to prepare iminophosphine intermediate 11,

and immediately reduced as per the general aminophosphine synthesis

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

117

(Procedure B) above. The pure aminophosphine product 19 was obtain-

ed as a viscous light yellow oil after distillation (0.537 mmol, 0.216 g,

78%). ¹H NMR: (300 MHz, CDCl₃) δ_H7.34 (dd, J = 7.5 and 4.2 Hz, 1H),

7.20–7.14 (m, 11H), 7.03 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 4.8 Hz, 1H),

6.80–6.75 (m, 2H), 6.62 (d, J = 3.0 Hz, 1H), 3.91 (s, 2H), 2.68 (s, 4H),

and 1.51 (s, 1H); ¹³C NMR: (75 Hz, CDCl₃) δ_C144.2 (d, J = 23.5 Hz),

142.5, 136.6 (d, J = 10.0 Hz), 135.6 (d, J = 13.7 Hz), 133.7 (d, J =

19.7 Hz), 133.6, 128.9 (d, J = 5.5 Hz), 128.8, 128.6, 128.4 (d, J =

7.2 Hz), 127.1, 126.6, 124.7, 123.2, 52.1 (d, J = 20.6 Hz), 50.3, and

30.2; ³¹P NMR: (121 MHz, CDCl₃) δ_P−15.7; IR: ν_max(CHCl₃)/cm⁻¹

3053, 3031, 3003, 2916, and 2825; CIMS: m/z 402 ([M]⁺, 100%) and

304 ([M−C₅H₅S]⁺, 85%).

4.4.3.

gold(I) chloride (22)

N-(2-(Diphenylphosphino)phenyl)-methylene-benzylamine

To a stirred solution of 6 (0.057 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 22 was obtained as a ﬁne white

powder after washing and dried thoroughly in vacuo (0.102 mmol,

0.062 g, 68%). mp: 154–156 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.69 (s,

1H), 7.90 (dd, J = 7.1 and 4.7 Hz, 1H), 7.59 (t, J = 7.7 Hz, 2H),

7.52–7.33 (m, 13H), 7.17–7.15 (m, 2H), 6.85–6.81 (m, 1H), and 4.68

(s, 2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C158.5 (d, J = 5.7 Hz), 138.7 (d,

J = 7.1 Hz), 137.7, 134.3 (d, J = 5.7 Hz), 134.1 (d, J = 14.0 Hz), 131.6

(d, J = 2.3 Hz), 131.5 (d, J = 2.5 Hz), 131.4 (d, J = 8.3 Hz), 130.1 (d,

J = 10.1 Hz), 129.2, 129.1 (d, J = 12.0 Hz), 128.3 (d, J = 9.2 Hz),

127.7 (d, J = 55.8 Hz), 126.8, and 64.0; ³¹P NMR: (121 MHz, CDCl₃) δ_P

30.8; IR: ν_max(KBr)/cm⁻¹3059 and 1631; LRFAB: m/z 612

([M + H]⁺, 25%) and 576 ([M−Cl]⁺, 100%).

4.4. General procedure for the preparation of iminophosphine

gold(I) complexes (20–28). Procedure C

[AuCl(THT)] (THT = tetrahydrothiophene) was prepared according

to literature methods [71]. To a stirred solution of the iminophosphine

ligand (0.150 mmol) in 5 mL diethyl ether, 1 equivalent of [AuCl(THT)]

(0.150 mmol, 0.048 g) dissolved in 2 mL chloroform was added. The ad-

dition was done in a dropwise manner. The mixture was stirred at room

temperature for 3 min after which two thirds of the solvent was re-

moved in vacuo. The product precipitate was washed with 10 mL

diethyl ether (×5) and 10 mL hexane (×3) to remove the displaced

THT. The pure product was dried thoroughly in vacuo.

4.4.4.

gold(I) chloride (23)

N-(2-(Diphenylphosphino)phenyl)-methylene-benzenamine

To a stirred solution of 7 (0.055 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 23 was obtained as a bright

green powder after washing and dried thoroughly in vacuo

(0.108 mmol, 0.065 g, 72%). This colour is thought to arise from the

delocalised π-system that stretches throughout the molecule, enhanced

by the unique ﬂuorescent properties that gold is known to exhibit when

complexed to certain ligands [72,73]. Green single crystals were grown

from a layered solution of dichloromethane, diethyl ether and n-

hexane. mp: 187–189 °C; ¹H NMR (CDCl₃) δ_H8.53 (s, 1H), 7.87 (dd,

J = 6.8 and 4.4 Hz, 1H), 7.50–7.25 (m, 12H), 7.08–7.03 (m, 2H),

6.95 (t, J = 7.4 Hz, 1H), 6.72 (dd, J = 13.2 and 7.8 Hz, 1H), and 6.62

(d, J = 7.5 Hz, 2H); ¹³C NMR (CDCl₃) δ_C156.5 (d, J = 5.2 Hz), 138.3

(d, J = 6.8 Hz), 134.4 (d, J = 7.1 Hz), 134.1 (d, J = 14.0), 132.2 (d,

J = 8.0 Hz), 131.7, 131.6 (d, J = 2.6 Hz), 130.6 (d, J = 10.1 Hz),

130.0, 129.4, 129.1 (d, J = 12.0 Hz), 128.9, 128.3 (d, J = 54.9 Hz),

and 126.4; ³¹P NMR (CDCl₃) δ_P32.0; IR: ν_max(KBr)/cm⁻¹3056 and

1624; LRFAB: m/z 598 ([M + H]⁺, 21%) and 562 ([M−Cl]⁺, 38%).

Crystal data are available in the Supplementary Information.

4.4.1.

N-(2-(Diphenylphosphino)phenyl)-methylene-butylamine

gold(I) chloride (20)

To a stirred solution of 5 (0.0518 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 20 was obtained as a ﬁne white

powder after washing and dried thoroughly in vacuo (0.104 mmol,

0.060 g, 69%). mp: 136–138 °C; ¹H NMR: (300 MHz, CDCl3) δ_H8.53 (s,

1H), 7.80 (dd, J = 7.2 and 4.8 Hz, 1H), 7.60–7.30 (m, 12H), 6.80 (dd,

J = 13.1 and 7.7 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 1.21 (m, 2H), 0.93

(sx, J = 7.5 Hz, 2H), and 0.71 (t, J = 7.4 Hz, 3H); ¹³C NMR: (75 MHz,

CDCl₃) δ_C157.7 (d, J = 4.1 Hz), 138.7 (d, J = 6.5 Hz), 134.4 (d, J =

7.5 Hz), 134.1 (d, J = 14.0 Hz), 131.6 (d, J = 8.5 Hz), 131.6, 131.4 (d,

J = 2.5 Hz) 129.8 (d, J = 9.9 Hz), 129.0 (d, J = 12.0 Hz), 127.4 (d, J =

56.1 Hz), 60.5, 32.2, 20.3, and 13.7; ³¹P NMR: (121 MHz, CDCl₃) δ_P

32.0; IR: ν_max(KBr)/cm⁻¹2952 and 1653; LRFAB: m/z 578

([M + H]⁺, 15%).

4.4.5.

gold(I) chloride (24)

(2-Diphenylphosphanyl-benzylidene)-isopropyl-amine

To a stirred solution of 8 (0.050 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 24 was obtained as a ﬁne white

powder after washing and dried thoroughly in vacuo (0.104 mmol,

0.058 g, 69%). mp: 172–174 °C; ¹H NMR: (300 MHz, CDCl3) δH 8.62

(s, 1H), 7.85–7.81 (dd, J = 6.6 and 4.2 Hz, 1H), 7.63–7.43 (m, 11H),

7.35 (t, J = 7.5 Hz, 1H), 6.80 (dd, J = 13.1 and 7.7 Hz, 1H), 3.40 (p,

J = 6.2 Hz, 1H), and 0.95 (d, J = 6.3 Hz, 6H); ¹³C NMR: (75 MHz,

CDCl₃) δ_C155.9 (d, J = 4.6 Hz), 138.8 (d, J = 7.1 Hz), 134.2 (d, J =

8.1 Hz), 134.2 (d, J = 14.0 Hz), 131.7, 131.6, 131.5 (d, J = 2.3 Hz),

130.6, 129.9, 129.7, 129.0 (d, J = 11.9 Hz), 127.2 (d, J = 55.0 Hz),

61.5, and 23.9; ³¹P NMR: (121 MHz, CDCl₃) δ_P31.7; IR: ν_max(KBr)/

cm⁻¹2964 and 1644; LRFAB: m/z 565.4 ([M + H]⁺, 26%) and 528.4

([M−Cl]⁺, 30%).

4.4.2.

N-(2-(Diphenylphosphino)phenyl)-methylene-2-(phenyl)

ethylamine gold(I) chloride (21)

Phenylethyl amine (0.758 mmol, 0.095 mL) was used as the amine

starting material to prepare the iminophosphine intermediate as per

the general iminophosphine synthesis (Procedure A) above. To a stirred

solution of the pure iminophosphine intermediate (0.059 g, 0.150 mmol)

in diethyl ether (5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in

chloroform (2 mL) was added. The pure product 21 was obtained as a

ﬁne white powder after washing and dried thoroughly in vacuo

(0.106 mmol, 0.067 g, 71%). Colourless crystals were obtained from a

hexane solution layered with chloroform. mp: 160–162 °C; ¹H NMR:

(300 MHz, CDCl₃) δ_H8.22 (s, 1H), 7.55 (dd, J = 7.5 and 3.9 Hz, 1H),

7.43–7.24 (m, 11H), 7.17 (t, J = 7.5 Hz, 1H), 7.07–6.96 (m, 3H), 6.81

(d, J = 7.8 Hz, 2H), 6.67 (dd, J = 13.1 and 7.7 Hz, 1H), 3.48 (t, J =

7.7 Hz, 2H), and 2.39 (t, J = 7.7 Hz, 2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C

158.4 (d, J = 3.7 Hz), 139.5, 138.59 (d, J = 6.8 Hz), 134.5 (d, J =

7.1 Hz), 134.1 (d, J = 14.0 Hz), 131.8 (d, J = 8.3 Hz), 131.6, 131.4 (d,

J = 2.3 Hz), 130.7, 130.0, 129.8 (d, J = 3.2 Hz), 129.1 (d, J = 11.8 Hz),

128.8, 128.2, 127.6 (d, J = 55.8 Hz), 126.0, 61.8, and 36.7; ³¹P NMR:

(121 MHz, CDCl₃) δ_P32.6; IR: ν_max(KBr)/cm⁻¹3020 and 1644; LRFAB:

m/z 591 ([M−Cl]⁺, 25%). Crystal data are available in the Supplementa-

ry Information.

4.4.6. (2-Diphenylphosphanyl-benzyl)-(2-pyrid-2-yl-ethyl) amine gold(I)

chloride (25)

To a stirred solution of 9 (0.059 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 25 was obtained as a ﬁne white

powder after washing and dried thoroughly in vacuo (0.102 mmol,

0.064 g, 68%). mp: 160–162 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.45 (d,

J = 4.5 Hz, 1H), 8.42 (br s, 1H), 7.70 (dd, J = 7.8 and 3.6 Hz, 1H),

7.59–7.41 (m, 11H), 7.33 (t, J = 7.7 Hz, 2H), 7.06–6.98 (m, 2H), 6.82

(dd, J = 13.2 and 7.8 Hz, 1H), 3.80 (t, J = 7.5 Hz, 2H), and 2.80 (t, J =

118

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

7.5 Hz, 2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C159.4, 158.8 (d, J = 3.1 Hz),

149.1, 138.6 (d, J = 6.8 Hz), 136.2, 134.6 (d, J = 6.9 Hz), 134.0 (d, J =

13.7 Hz), 132.0 (d, J = 8.9 Hz), 131.6 (d, J = 2.3 Hz), 131.4 (d, J =

2.3 Hz), 130.8, 130.0 (d, J = 4.1 Hz), 129.8, 129.1 (d, J = 12.0 Hz),

127.6 (d, J = 55.7 Hz), 123.7, 121.2, 59.8, and 38.8; ³¹P NMR:

(121 MHz, CDCl₃) δ_P32.5; IR: ν_max(KBr)/cm⁻¹2835 and 1651;

LRFAB: m/z 627 ([M + H]⁺, 4%) and 591 ([M−Cl]⁺, 31%).

4.5. General procedure for the preparation of aminophosphine gold(I)

complexes (29–35). Procedure D

[AuCl(THT)] was prepared from literature methods [71]. To a stirred

solution of aminophosphine ligand (0.50 mmol) in 20 mL of diethyl

ether 0.95 equivalents of [AuCl(THT)] dissolved in 2 mL of chloroform

were added. The solution was stirred at room temperature for 5 min.

In most cases the product precipitated out of solution within 2 min

after addition. When this was not the case, some of the solvent was

evaporated in vacuo to ca. 5 mL and the product was precipitated out

of the solution by the addition of 10 mL of cold hexane (×3).

4.4.7. N′-(2-Diphenylphosphanyl-benzylidene)-N,N-dimethyl-propane-

1,3-diamine gold(I) chloride (26)

To a stirred solution of 10 (0.056 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 26 was obtained as a ﬁne white

powder after washing and dried thoroughly in vacuo (0.101 mmol,

0.061 g, 67%). mp: 139–141 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.47 (s,

1H), 7.73 (dd, J = 7.2 and 4.2 Hz, 1H), 7.55–7.32 (m, 11H), 7.27 (t,

J = 7.6 Hz, 1H), 6.75 (dd, J = 13.2 and 7.8 Hz, 1H), 3.37 (t, J = 7.1 Hz,

4.5.1. N-(2-Diphenylphosphinobenzyl)benzylamine gold(I) chloride (29)

The pure aminophosphine ligand 13 (0.50 mmol, 0.191 g) was dis-

solved in 20 mL of diethyl ether. To this solution a solution of

[AuCl(THT)] (0.95 equivalents, 0.475 mmol, 0.152 g) dissolved in chlo-

roform (2 mL) was added. The pure gold complex 29 was obtained as

a ﬁne white powder after washing and drying (0.325 mmol, 0.200 g,

65%). mp: 174–177 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H7.62 (dd, J =

7.5 and 4.8 Hz, 1H), 7.48–7.33 (m, 11H), 7.19–7.11 (m, 4H), 7.05–7.02

(m, 2H), 6.76 (ddd, J = 11.4, 7.8 and 0.9 Hz, 1H), 4.11 (s, 2H), 3.49 (s,

2H), and 1.43 (br s); ¹³C NMR: (75 MHz, CDCl₃) δ_C144.2 (d, J =

11.0 Hz), 139.6, 134.2 (d, J = 14.0 Hz), 133.7 (d, J = 7.5 Hz),

131.8–131.7 (m), 130.5 (d, J = 9.0 Hz), 129.6, 129.2 (d, J = 11.6 Hz),

128.7, 128.1 (d, J = 16.0 Hz), 127.4 (d, J = 10.1 Hz), 126.8, 126.7 (d,

J = 59.1 Hz), 53.2, and 52.2 (d, J = 11.0 Hz); ³¹P NMR: (121 MHz,

CDCl₃) δ_P26.1; IR: ν_max(KBr)/cm⁻¹3279 and 1433; LRFAB: m/z 614

([M]⁺, 8%) and 578 ([M−Cl]⁺, 14%).

2H), 2.05 (s, 6H), 1.97 (t, J = 7.4 Hz, 2H), and 1.46–1.39 (m, 2H); ¹³

NMR: (75 MHz, CDCl₃) δ_C158.2 (d, J = 3.8 Hz), 138.6 (d, J = 6.9 Hz),

134.5 (d, J = 7.1 Hz), 134.0 (d, J = 14.0 Hz), 131.8 (d, J = 8.3 Hz),

131.6 (d, J = 2.3 Hz), 131.4 (d, J = 2.3 Hz), 130.7, 129.9, 129.8 (d, J =

4.6 Hz), 129.0 (d, J = 12.0 Hz), 127.4 (d, J = 55.5 Hz), 58.67, 57.16,

45.3, and 28.1; ³¹P NMR: (121 MHz, CDCl₃) δ_P32.1; IR: ν_max(KBr)/

cm⁻¹2937 and 1650; LRFAB: m/z 607 ([M + H]⁺, 87%) and 571

([M−Cl]⁺, 62%).

4.4.8. (2-Diphenylphosphanyl-benzylidene)-(2-thiophen-2-yl-ethyl)-

amine gold(I) chloride (27)

4.5.2. N-(2-Diphenylphosphinobenzyl)phenylamine gold(I) chloride (30)

The pure aminophosphine ligand 14 (0.50 mmol, 0.184 g) was dis-

solved in 20 mL of diethyl ether. To this solution a solution of

[AuCl(THT)] (0.95 equivalents, 0.475 mmol, 0.152 g) dissolved in chlo-

roform (2 mL) was added. The pure gold complex 30 was obtained as

a blue powder after washing and drying (0.405 mmol, 0.243 g, 81%).

The formation of two crystal forms was possible. In the ﬁrst, the free

base 30 could be crystallised to form crystals 30a as the neutral

gold(I) complex from a layered solution of diethyl ether and hexane.

In contrast, if 30 was ﬁrst treated with aqueous HCl, 30b as the hydro-

chloride salt crystal form could be produced from a layered solution of

chloroform and n-heptane. For compound 30: mp: 223–225 °C; ¹H

NMR: (300 MHz, CDCl₃) δ_H7.60 (m, 1H), 7.52–7.39 (m, 11H), 7.17 (m,

1H), 6.94 (t, J = 7.5 Hz, 2H), 6.77 (dd, J = 13.1 and 7.7 Hz, 1H), 6.57

(t, J = 7.2 Hz, 1H), 6.17 (d, J = 7.8 Hz, 2H), and 4.61 (s, 2H); ¹³C

NMR: (75 MHz, CDCl₃) δ_C146.7, 143.3, 134.4 (d, J = 13.7 Hz), 133.7

(d, J = 7.4 Hz), 132.1 (d, J = 2.6 Hz), 132.1, 129.3 (d, J = 12.0 Hz),

129.1, 129.0, 128.4, 127.5 (d, J = 9.2 Hz), 127.3, 126.1 (d, J =

57.5 Hz), 118.0, 112.8, and 47.6 (d, J = 12.6 Hz); ³¹P NMR: (121 MHz,

CDCl₃) δ_P25.7; IR: ν_max(KBr)/cm⁻¹3414 and 1435; LRFAB: m/z 564.5

([M−Cl]⁺, 33%). Crystal data for the neutral gold(I) complex 30a and

the gold(I) hydrochloride salt 30b are available in the Supplementary

Information.

To a stirred solution of 11 (0.060 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 27 was obtained as a ﬁne white

powder after washing and dried thoroughly in vacuo (0.108 mmol,

0.068 g, 72%). Single crystals were grown from a chloroform solution

left at room temperature overnight. Due to the rapid crystallisation pro-

cess, the crystals obtained are not of exceptionally high quality. mp:

138–140 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.38 (s, 1H), 7.73 (dd, J =

7.5 and 3.9 Hz, 1H), 7.60–7.39 (m, 10H), 7.34 (t, J = 7.5 Hz, 2H), 7.03

(d, J = 5.1 Hz, 1H), 6.87–6.80 (m, 2H), 6.60 (d, J = 2.4 Hz, 1H), 3.68

(t, J = 7.1 Hz, 2H), and 2.75 (t, J = 7.2 Hz, 2H); ¹³C NMR: (75 MHz,

CDCl₃) δ_C158.8 (d, J = 3.8 Hz), 141.9, 138.4 (d, J = 6.8 Hz), 134.5 (d,

J = 7.1 Hz), 133.97 (d, J = 13.7 Hz), 131.9 (d, J = 8.3 Hz), 131.6 (d,

J = 2.3 Hz), 131.4 (d, J = 2.5 Hz), 130.4, 129.9 (d, J = 10.1 Hz), 129.8,

129.0 (d, J = 12.0 Hz), 127.5 (d, J = 55.5 Hz), 126.6, 125.0, 123.3,

61.6, and 30.5; ³¹P NMR: (121 MHz, CDCl₃) δ_P32.3; IR: ν_max(KBr)/

cm⁻¹3055 and 1645; LRFAB: m/z 596 ([M−Cl]⁺, 21%) and 487

([M−C₆H₅S]⁺, 37%).

4.4.9. tert-Butyl-(2-diphenylphosphanyl-benzylidene)-amine gold(I)

chloride (28)

To a stirred solution of 12 (0.052 g, 0.150 mmol) in diethyl ether

(5 mL), [AuCl(THT)] (0.048 g, 0.150 mmol) dissolved in chloroform

(2 mL) was added. The pure product 28 was obtained as a ﬁne

white powder after washing and dried thoroughly in vacuo

(0.105 mmol, 0.061 g, 70%). Single crystals were grown from chloro-

form. mp: 192–194 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.67 (d, J =

1.8 Hz, 1H), 7.86 (ddd, J = 7.6, 4.4 and 1.4 Hz, 1H), 7.61–7.38 (m,

11H), 7.31 (tt, J = 7.7 and 1.7 Hz, 1H), 6.77 (ddd, J = 13.2, 7.8 and

1.2 Hz, 1H), and 0.99 (s, 9H); ¹³C NMR: (75 MHz, CDCl₃) δ_C153.7

(d, J = 6.5 Hz), 139.3 (d, J = 7.1 Hz), 134.3 (d, J = 12.7 Hz), 134.1

(d, J = 7.1 Hz), 131.7 (d, J = 2.6 Hz), 131.5 (d, J = 2.6 Hz), 131.2

(d, J = 8.6 Hz), 129.7 (d, J = 10.5 Hz), 129.0 (d, J = 12.0 Hz), 127.2

(d, J = 55.1 Hz), 58.6, and 29.6; ³¹P NMR: (121 MHz, CDCl₃) δ_P

31.0; IR: ν_max(KBr)/cm⁻¹2958 and 1644; LRFAB: m/z 578

([M + H]⁺, 13%) and 542 ([M−Cl]⁺, 17%).

4.5.3. (2-Diphenylphosphanyl-benzyl)-isopropyl-amine gold(I) chloride

(31)

The synthesis and structure of 31 have already been published [44].

4.5.4. (2-Diphenylphosphanyl-benzyl)-pyrid-2-ylmethyl-amine gold(I)

chloride (32)

The structure of 32 has already been published [47].

4.5.5. (2-Diphenylphosphanyl-benzyl)-(2-pyrid-2-yl-ethyl)-amine gold(I)

chloride (33)

The pure aminophosphine ligand 17 (0.50 mmol, 0.198 g) was dis-

solved in 20 mL of diethyl ether. To this solution a solution of

[AuCl(THT)] (0.95 equivalents, 0.475 mmol, 0.152 g) dissolved in

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

119

chloroform (2 mL) was added. The pure gold complex 33 was obtained

4.7. Cell lines and culture conditions

as a ﬁne brown powder after washing and drying (0.315 mmol, 0.198 g,

63%). mp: 135–136 °C; ¹H NMR: (300 MHz, CDCl₃) δ_H8.43 (dd, J = 6.0

and 1.8 Hz, 1H), 7.63 (dd, J = 6.6 and 5.4 Hz, 1H), 7.57–7.40 (m, 11H),

7.20 (t, J = 7.5 Hz, 2H), 7.08–7.04 (m, 2H), 6.79 (dd, J = 12.8 and

7.7 Hz, 1H), 4.15 (s, 2H), 2.84 (t, J = 6.9 Hz, 2H), 2.72 (t, J = 6.6 Hz,

2H), and 1.83 (br s); ¹³C NMR: (75 MHz, CDCl₃) δ_C159.9, 149.0, 143.9

(d, J = 12.8 Hz), 136.4, 134.2 (d, J = 14.0 Hz), 133.8 (d, J = 7.4 Hz),

131.9–131.8 (m), 130.4 (d, J = 9.2 Hz), 129.6, 129.2 (d, J = 12.0 Hz),

128.7, 127.4 (d, J = 9.4 Hz), 126.6 (d, J = 59.2 Hz), 123.5, 121.2, 52.5,

48.6, and 37.4; ³¹P NMR: (121 MHz, CDCl₃) δ_P26.1; IR: ν_max(KBr)/

cm⁻¹3283 and 1435; LRFAB: m/z 593 ([M−Cl]⁺, 25%).

3T3 (mouse embryonic ﬁbroblast) cells and CHO (Chinese hamster

ovary) cells were obtained from the Sir William Dunn School of Pathol-

ogy, University of Oxford, South Parks Road, Oxford, United Kingdom.

A549J (human lung adenocarcinoma epithelial) cells, Caski (human cer-

vical cancer) cells, HepG2 (human hepatocellular liver carcinoma) cells,

HeLa (human cervical adenocarcinoma) cells, Hek 293-T (human em-

bryonic kidney) cells, HT-29 (human colon adenocarcinoma grade II)

cells, H157 (human non-small cell lung carcinoma) cells, Jurkat T

(human leukaemia) cells, MCF7 (human breast adenocarcinoma) cells,

MG-63 (human osteogenic sarcoma) cells and KMST-6 (non-cancer

human ﬁbroblast) cells were kindly provided by Prof D. Hendricks, De-

partment of Clinical and Laboratory Medicine, University of Cape Town,

South Africa. Cells were cultured in CELLSTAR® Cell Culture Flasks

(Greiner Bio-One GmbH, Frickenhausen, Germany). All cell culture re-

agents were obtained from Invitrogen Ltd. (Carlsbad, California, USA).

Jurkat T cells were cultured in Roswell Park Memorial Institute (RPMI)

medium; CHO cells were cultured in Ham's F12 nutrient mixture; all

other cell lines were cultured in Dulbecco's Minimal Essential Medium

(DMEM). The cell culture medium for all cell lines was supplemented

with 10% heat-inactivated foetal calf serum, 1 mM sodium pyruvate,

2 mM ^L-glutamine and 1% Penicillin/Streptomycin. Cultures were main-

tained at 37 °C in a humidiﬁed atmosphere containing 5% CO₂and 95%

air.

4.5.6. N′-(2-Diphenylphosphanyl-benzyl)-N,N-dimethyl-propane-1,3-di-

amine gold(I) chloride (34)

The pure aminophosphine ligand 18 (0.50 mmol, 0.188 g) was dis-

solved in 20 mL of diethyl ether. To this solution a solution of

[AuCl(THT)] (0.95 equivalents, 0.475 mmol, 0.152 g) dissolved in chlo-

roform (2 mL) was added. The pure gold complex 34 was obtained as

a viscous yellow oil (0.240 mmol, 0.146 g, 48%). ¹H NMR: (300 MHz,

CDCl₃) δ_H7.57 (dd, J = 7.4 and 4.6 Hz, 1H), 7.52–7.35 (m, 11H), 7.15

(t, J = 7.7 Hz, 1H), 6.73 (dd, J = 12.8 and 8.0 Hz, 1H), 4.05 (s, 2H),

2.37 (t, J = 7.2 Hz, 2H), 2.11–2.07 (m, 8H), 1.67 (br s), and 1.30 (p,

J = 7.2 Hz, 2H); ¹³C NMR: (75 MHz, CDCl₃) δ_C144.4 (d, J = 10.8 Hz),

134.1 (d, J = 14.0 Hz), 133.6 (d, J = 7.4 Hz), 131.7 (d, J = 2.3 Hz),

130.3 (d, J = 9.1 Hz), 129.7, 129.1 (d, J = 11.9 Hz), 128.8, 127.2 (d,

J = 9.9 Hz), 126.5 (d, J = 58.7 Hz), 57.7, 53.1 (d, J = 10.9 Hz), 48.0,

45.4, and 27.5; ³¹P NMR: (121 MHz, CDCl₃) δ_P26.2; IR: ν_max(NaCl)/

cm⁻¹2817 and 1437; LRFAB: m/z 609 ([M + H]⁺, 20%) and 573

([M−Cl]⁺, 23%).

4.8. Cytotoxicity tests in cell lines

Cell proliferation was determined using the MTT assay following

methods previously described [49–53]. Speciﬁcally, adherent cells

were trypsinised and both adherent and suspension cells were plated

in 96-well cell culture plates (24 000 cells per well; 100 μL/well) and

cultured at 37 °C in a humidiﬁed CO₂incubator until 90% conﬂuence

was reached. The culture medium was removed and replaced with

100 μL of fresh medium, which contained various concentrations of

the test compounds (ranging from 10 μM to 100 μM). As a positive con-

trol, cells were treated with 100 μM cisplatin, for all cell lines used in this

study. All treatments were performed in triplicate. The cells were incu-

bated at 37 °C in a humidiﬁed CO₂incubator for 20 h. After incubation,

10 μL of a 5 mg/mL solution of MTT was added to each well and the

plates were incubated for a further 4 h at 37 °C. At the end of the

24 hour incubation period, the media was removed and DMSO

(100 μL) was added to each well. The plates were placed on a rotating

shaker for 10 min, and the optical density (OD) was determined at

560 nm using a LabSystems Multiscan Plus microplate reader. Cell via-

bility was determined using the following formula: (Viable cells)% =

(OD560 of drug-treated sample/OD560 of untreated sample) × 100.

IC₅₀values (the half maximal inhibitory concentration) were calculated

using Graph Pad Prism (GraphPad Software, Inc.). Triplicate experi-

ments were conducted and the results were expressed as mean SD.

4.5.7. N-(2-Diphenylphosphanyl-benzyl)-(2-thiophen-2-yl-ethyl)-amine

gold(I) chloride (35)

The pure aminophosphine ligand 19 (0.50 mmol, 0.184 g) was dis-

solved in 20 mL of diethyl ether. To this solution a solution of

[AuCl(THT)] (0.95 equivalents, 0.475 mmol, 0.152 g) dissolved in chlo-

roform (2 mL) was added. The pure gold complex 35 was obtained as

a ﬁne white powder (0.335 mmol, 0.212 g, 67%). ¹H NMR: (300 MHz,

CDCl₃) δ_H7.55–7.35 (m, 11H), 7.16 (t, J = 7.1 Hz, 2H), 7.01 (dd, J =

5.4 and 1.2 Hz, 1H), 6.81–6.72 (m, 2H), 6.59 (d, J = 3.3 Hz, 1H), 4.10

(s, 2H), 2.59 (d, J = 3.3 Hz, 4H), and 1.21 (br s); ¹³C NMR: (75 MHz,

CDCl₃) δ_C144.3 (d, J = 10.9 Hz), 142.3, 134.2 (d, J = 13.7 Hz), 133.9

(d, J = 7.1 Hz), 131.7 (d, J = 2.6 Hz), 130.4 (d, J = 9.1 Hz), 129.9,

129.2 (d, J = 11.9 Hz), 129.1, 127.4 (d, J = 10.0 Hz), 126.7, 126.7 (d,

J = 58.9 Hz), 125.0, 123.4, 52.9 (d, J = 10.2 Hz), 50.5, and 29.9; ³¹P

NMR: (121 MHz, CDCl₃) δ_P26.3; IR: ν_max(KBr)/cm⁻¹3053 and 1435;

LRFAB: m/z 634 ([M + H]⁺, 6%) and 598 ([M−Cl]⁺, 25%).

4.6. Crystallographic structure determination

The intensity data for all compounds were collected at 173 K on a

Bruker SMART 1K CCD diffractometer with area detector using graphite

monochromated Mo–K_αradiation (λ = 0.71073 Å, 50 kV, 30 mA). Data

reduction was carried out using the program SAINT+ [73] and face

indexed absorption corrections were made using the program XPREP

[74]. The structures were solved by direct methods using SHELXTL

[75]. Non-hydrogen atoms were ﬁrst reﬁned isotropically followed by

anisotropic reﬁnement by full matrix least-squares calculations based

on F²using SHELXTL. Hydrogen atoms were ﬁrst located in the differ-

ence map then positioned geometrically and allowed to ride on their re-

spective parent atoms. Diagrams and publication material were

generated using SHELXTL and Mercury [76]. Selected bond lengths

and angles for compounds 4, 14, 21, 23, 30a and 30b are summarised

in Table 1 in comparison to triphenylphosphine gold(I) chloride

(AuClPPh₃; 36).

4.9. APOPercentage™ apoptosis assay

The assay was performed as described in the literature [54]. In brief,

the cells were plated in 24 well cell culture plates at a cell density of

2.5 × 10⁴cells/mL and incubated for 24 h at 37 °C in a humidiﬁed CO₂

incubator. The cells were treated for 24 h with the test compounds at

50 μM concentrations. Stock solutions of the compounds were prepared

in DMSO and working concentrations were prepared in cell culture

media. The ﬁnal DMSO concentration in the treated wells was less

than 0.1%. Following the 24 hour treatment, the cells were recovered

by trypsinisation and stained with APOPercentage™ dye. The cells

were analysed on a FACScan™ (Becton Dickson) instrument equipped

with a 488 nm argon laser. APOPercentage™ ﬂuorescence was measured

using the FL3 channel.

120

T. Traut-Johnstone et al. / Journal of Inorganic Biochemistry 145 (2015) 108–120

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