Phosphorus–nitrogen compounds. Part 29. Syntheses, crystal structures, spectroscopic and stereogenic properties, electrochemical investigations, antituberculosis, antimicrobial and cytotoxic activities and DNA interactions of ansa-spiro-ansa cyclotetraphosphazenes

European Journal of Medicinal Chemistry 87 (2014) 662e676

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Phosphorusenitrogen compounds. Part 29. Syntheses, crystal structures, spectroscopic and stereogenic properties, electrochemical investigations, antituberculosis, antimicrobial and cytotoxic activities and DNA interactions of ansa-spiro-ansa cyclotetraphosphazenes
Okumus¸ a, L.Yasemin Koç b, Hossien Soltanzade c, Zeynel Kılıç a, *, Gamze Elmas a, Aytug d
f, Devrim Dündar g, € kelek , Hakan Dal e, Leyla Açık c, Zafer Üstündag Tuncer Ho g Makbule Yavuz a

Department of Chemistry, Ankara University, 06100 Ankara, Turkey Department of Biology, Ankara University, 06100 Ankara, Turkey c Department of Biology, Gazi University, 06500 Ankara, Turkey d Department of Physics, Hacettepe University, 06800 Ankara, Turkey e Department of Chemistry, Anadolu University, 26470 Eskis¸ehir, Turkey f Department of Chemistry, Dumlupınar University, 43270 Kütahya, Turkey g Department of Clinical Microbiology, Kocaeli University, 41380 Kocaeli, Turkey b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 May 2014 Received in revised form 29 September 2014 Accepted 2 October 2014 Available online 5 October 2014

A number of novel ansa-spiro-ansa (asa) cyclotetraphosphazenes (1ae5b) was prepared in the range of 63e90 % yields. The structures of the compounds were verified by MS, FTIR, 1H, 13C{1H} and 31P{1H} NMR, heteronuclear single quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) techniques. The crystal structures of 1b, 2c and 5a were determined by X-ray crystallography. The compound 2c was analyzed by the changes in the 31P{1H}NMR spectrum in addition of the chiral solvating agent; (R)-(þ)-2,2,2-trifluoro-1-(90 -anthryl)-ethanol (CSA), to investigate its stereogenic properties. The result supports that compound 2c was found to be in the racemic mixture. Cyclic voltammetric and chronoamperometric data of the mono-ferrocenyl-spiro-asa-cyclotetraphosphazenes exhibited electrochemically reversible one-electron oxidation of Fe redox centres. The mono-ferrocenyl-spiro-asa compounds (3ae5b) were evaluated for antituberculosis activity against reference strain Mycobacterium tuberculosis H37Rv and M. tuberculosis clinical strain, which is resistant to rifampicin and isoniazid. These compounds appear not to be good candidates for being antituberculosis agents to clinical strains. All of the compounds were screened for antibacterial activities against G(þ) and G() bacteria, and for antifungal activities against yeast strains. They seem to be more active against Gram positive bacteria than Gram negative. The interactions of the phosphazenes with plasmid DNA and the evaluations for cytotoxic activity against MCF-7 breast cancer cell lines were investigated. The compounds 1b, 2b, 3a and 4a were found to be more effective than Cisplatin against MCF-7 breast cancer cell lines at lower concentrations. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Asa-cyclotetraphosphazenes Chirality Crystal structure Cytotoxic activity Antituberculosis activity Biological activity

1. Introduction The cyclophosphazenes, (NPX2)n, are an important family of inorganic heterocylic ring systems constituting a regular and homologous series. The ring sizes vary significantly from n ¼ 3e40 [1]. The best known members of the cyclophosphazenes are

* Corresponding author. E-mail address: [email protected] (Z. Kılıç). http://dx.doi.org/10.1016/j.ejmech.2014.10.005 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

hexachlorocyclotriphosphazene (trimer, N3P3Cl6) and octachlorocyclotetraphosphazene (tetramer, N4P4Cl8). They serve as the starting materials for the rich and attractive chemistry of the phosphazenes [2]. The reactions of the chlorocyclotetraphosphazenes are studied much less than the reactions of chlorocyclotriphosphazenes [3]. Although there is plenty of work on the reactions of cyclophosphazenes with monodentate ligands in the literature, the studies with the multidentate ligands (diols, diamines, polyamines etc.) are very limited [4,5]. Furthermore, it is mentioned that one or several of the spiro-, ansa-, bino-, di-spiro-,

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

tri-spiro-, spiro-ansa- and spiro-ansa-spiro- derivatives can occur from the reactions of trimer with the multidentate ligands [6]. It is also indicated that tetramer with bidentate ligands can afford similar compounds. But, to the best of our knowledge, we are aware of only one paper on the reactions of cyclotetraphosphazenes with tetradentate polyamines [7], but no report was found with the ferrocenyldiamines in the literature. Recently, the chiral properties of cyclotriphosphazene derivatives have been discovered as an attractive topic [8]. The chiralities of the cyclotriphosphazenes are studied using 31P{1H} NMR spectroscopy in the presence of (R)(þ)-2,2,2-trifluoro-1-(90 -anthryl)-ethanol (CSA) and HPLC techniques [9,10]. Meanwhile, there is also a report investigating the chirality of the morpholine substituted ansa-spiro-ansa (asa) cyclotetraphosphazene using CSA and HPLC techniques [11]. As it is known, cancer causes unregulated cell growth and may spread into different parts of the body. Thus, a great deal of research is being carried out to design new agents for preventing and curing

663

cancer. Aminocyclotriphosphazenes especially have attracted a great deal of attention for their potential as anti-cancer agents [12]. They were found to be very active on many series of tumor cells [13]. It was observed that some amino cyclotriphosphazenes exhibited cytotoxic activity against HT-29 (human colon adenocarcinoma), Hep2 (Human epidermoid larynx carcinoma), Vero (African green monkey kidney) and HeLa cancer cells, and they stimulate apoptosis [14,15]. In contrast to the cyclotriphosphazenes, there are very limited investigations about cyclotetraphosphazene derivatives as antimicrobial and anticancer agents. For instance, octapyrrolidinocyclotetraphosphazene was mentioned as a significant anticancer agent [16], and the Cu(II) complex of the fully-phenoxy-substituted star-branched cyclotetraphosphazene was found out to be active in the oxidative cleavage of DNA [17]. As the biological activities of the ferrocenyldiamines and their cyclotriphosphazene derivatives against Mycobacterium tuberculosis H37Rv are known [18] by our groups,

Scheme 1. The Chloride Replacement Reaction Pathway of N4P4Cl8 with Aminopodands, Piperidine, DASD and Mono-ferrocenyldiamines.

664

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

we decided to investigate antituberculosis activity of the ferrocenyl tetrameric phosphazene derivatives. The present work, in particular, focuses on the Cl replacement reactions of N4P4Cl8 with N2O2 donor-type ligands and ferrocenyl diamines (Scheme 1) with the aim to investigate the antimicrobial, antituberculosis, and cytotoxic activities against MCF-7 cancer cell lines, and the DNA interactions of the cyclotetraphosphazene derivatives. 2. Results and discussion 2.1. Chemistry: syntheses and characterizations of the compounds All aminopodands [(HOPhCH2NH)2R, R](CH2)n, n ¼ 2 (1) and n ¼ 3 (2)] were prepared by the reduction of bis-iminopodands with the NaBH4/borax system [19]. The reactions of N4P4Cl8 with equimolar amounts of the dipotassium salts, (KOPhCH2NH)2R, of aminopodands (1 and 2) afford the asa (1a and 2a) and spiro-ansaspiro (sas) tetrachlorocyclotetraphosphazenes in THF, respectively [11]. The yields of asa products are higher than those of sas ones. Hence, asa derivatives which were almost the major products, were used for the starting compounds containing four active Cl atoms. The reactions of the partly substituted asa phosphazenes (1a and 2a) with excess piperidine and 1,4-dioxa-8-azaspiro[4,5] decane (DASD) in THF produce solely 2-trans-6-dichloro geminal asa [(1b and 2b) and (1c and 2c)] phosphazene derivatives. At this stage, the geminal products seem to be formed according to the SN1(P) pathway [6,11]. As an example, the decoupled and coupled 31 P NMR spectra of geminal product 2b are depicted in Fig. 1a and b. The 31P{1H} NMR spectrum of the reaction mixture of 2a with piperidine (Fig. 1c), indicates that the geminal 2b and 2-trans-6,8trichloro asa products are present in the reaction mixture. But both of the products were not separated from the mixture. Hence, pure geminal products are obtained from the reactions of equimolar amounts of asa compounds (1a and 2a) with excess amines. The replacement reactions of 2-trans-6-dichloro asa compounds (1b,

Fig. 1. (a) The

31

P{1H} NMR spectrum of pure 2b, (b) 1H-coupled

31

2b, 1c and 2c) with excess piperidine, DASD and sodium 4nitrophenoxide in boiling THF for six days, were not achieved, indicating that both Cl atoms are highly inert. As an example, the chloride replacement reactions of 1a with amines were made separately in THF, toluene, and o-xylene by microwave-assisted experiments. Even then, no fully substituted product was obtained; instead the compound 1b was isolated. Alternatively, both of the two Cl atoms of 1b were reacted with excess piperidine by microwave-assisted experiments, but even so, fully substituted products were not obtained. The last two Cl atoms in the asa derivatives 1be2c and 3ae5b are highly inert against the replacement reactions by secondary amines, as a result of both of the endocyclic PN bond strengthening and steric hindrances of the substituents. This is supported by the structural data of the asa compounds (1b, 2c and 5a). The values of the P1eN1 and P3eN2 bond lengths are 1.563(2) Å and 1.566(1) Å (for 1b), 1.554(2) Å and 1.565(2) Å (for 2c), and 1.554(2) Å and 1.561(2) Å (for 5a), respectively (Table 1). It could be seen that these endocyclic PN bonds are systematically short, indicating that the electron densities on the P1 and P3 atoms increase, which can be attributed to the presence of the negative hyperconjugation [20]. Nevertheless, when the reaction of 1b is carried out by using microwave irradiation, reaction time reduces and the yield of the compound increases. In addition, when the replacement reactions of equimolar amounts of asa compounds (1a and 2a) with mono-ferrocenyldiamines (3, 4 and 5) were carried out, the spirocyclo-asa products (3ae5a and 3be5b) were isolated regioselectively. The expected ansa and bino products were not obtained. The reaction mechanism for this stage is likely to be the SN1(P). Data from the microanalyses, FTIR, APIES-MS and 1H, 13C{1H} and 31P{1H} NMR, heteronuclear single quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) are consistent with the proposed structures of the compounds. The 1H and 13C{1H} NMR results together with the decoupled 31 P-NMR data from the cyclotetraphosphazenes indicate that all the compounds have the asa architectures, and they are preserved in all

P NMR spectrum of pure 2b, (c)

31

P{1H} NMR spectrum of the reaction mixture of 2b and piperidine (1:1).

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

665

Table 1 31 P-NMR (decoupled) Spectral Data of the Compounds. [Chemical shifts (d) are reported in ppm, J values in Hz].a

R1= (CH2)2 ; R2= CH3 R= (CH2)2

N P

N N

X

N

(N

P

N R

AB2X (1b, 2b, 1c, 2c, 4a and 4b)

N

P

N Cl

P

(

O

Fc CH2

(

Cl

B

O

X'

P

R1 O

Fc CH2

N X

N

O

B

N R

ABXX' (3a)

Cl

M(C)

(N

P

N

A

P

N Cl

P

N

NR2

P O

O

N B

N

X

P

N

(

P

A

(

N

Cl

(

P

Y

B

R1

(

A

NR2

(

Cl

Y

R1= (CH2)2 or (CH2)3; R2= CH3 R= (CH2)2 or (CH2)3

N R

(

Y= Piper, DASD or Mono-ferrocenyl diamine (4) R= (CH2)2 or (CH2)3

ABMX (5a and 5b) ABCX (3b)

Compound

Spin system

d(ppm) NPN

POCl

2

1b

AB2X

12.33 (spiro) 6.22 (pip) 5.96 (spiro) 0.72 (pip) 12.34 (spiro) 6.21 (DASD) 4.59 (spiro) 0.45 (DASD) 12.99 (ferrocenyl) 12.51 (spiro) 10.11 (ferrocenyl) 1.13 (spiro) 11.89 (ferrocenyl) 2.96 (spiro) 8.80 (ferrocenyl) 1.09 (spiro) 11.51 (ferrocenyl) 8.93 (spiro) 5.93 (ferrocenyl) 0.08 (spiro)

8.15

2

2b

AB2X

1c

AB2X

2c

AB2X

3a

ABXX0

3b

ABCX

4a

AB2X

4b

AB2X

5a

ABMX

5b

ABMX

JPP (Hz)

JAB: 63.6 JBx: 31.8 2 JAB: 56.3 2 JBx: 35.5 2 JAB: 63.5 2 JBx: 37.4 2 JAB: 59.7 2 JBx: 34.0 2 JAB: 52.0 2 Jxx’: 31.7 2 JAB: 51.6 2 JCx: 31.2 2 JAB: 55.5 2 JBx: 31.1 2 JAB: 51.4 2 JBx: 30.4 2 JAB: 51.1 2 JMx: 32.7 2 JAB: 51.5 2 JMx: 33.1 2

0.84 7.12 1.34 9.04 7.96 2.90 2.41 8.45 0.88 6.48 8.08 1.97 0.93

a 31

P NMR measurements for all the compounds in CDCl3 solutions at 293 K.

the phosphazene derivatives. All the d P shift values are undoubtedly interpreted from the 1H-coupled 31P NMR spectrum of 2b (Fig. 1b), indicating three sets of multiplets corresponding to the P spiro (Px), POCl (PB) and P (pip)2 (PA) groups. The d P and the coupling constants (2JPP) of the other asa derivatives are also assigned as 2b (Table 1). The 31P{1H} NMR results showed that piperidine and DASD bonded to the phosphorus atoms with geminal fashion; similarly the mono-ferrocenyldiamines (3, 4 and 5) bonded to the phosphorus atoms with spiro fashion by replacing both geminal Cl atoms in PCl2. As an example, the 1H-decoupled and coupled 31P NMR spectra of 5a are depicted in Fig. 2a and b, respectively. All the phosphorus atoms are distinguished from the 1H-coupled 31P NMR spectrum of 5a (Fig. 2b), indicating two sets of triplets and two sets of doublets corresponding to the POCl (PA, d 6.48 ppm), POCl (PB, d 8.08 ppm), FcCH2NPNCH3 spiro (PM, d 8.93 ppm) and NPN spiro (Px, d 11.51 ppm) groups. Both of the triplets at the 1H-coupled 31P NMR spectrum are broadened, which indicates the existence of the d PNN phosphorus atom and the signals of POCl groups remain unchanged. The chirality of the cyclotriphosphazenes and cyclotetraphosphazenes is an interesting new subject that has been studied during the last decade [21]. There is only one paper about the chirality of cyclotetraphosphazenes [11]. Some of the phosphorus atoms of the tetrameric phosphazene derivatives obtained

in this study are stereogenic, because they have four different substituents. The asa phosphazene derivatives (1b, 2b, 1c and 2c) which have the trans structures with respect to the Cl atoms have two equivalent stereogenic P-atoms. Table 2 shows stereoisomeric assignments for compounds 1b, 2c and 5a. The stereoisomer distributions can be rationalized with the tentative reaction route given in Fig. S1 (S designates supplementary material). According to this stick diagram the limited number of stereoisomers can be obtained via the reactions of tetramer and N2O2 donor type ligands. In other words, tetradentate symmetric ligands arrange the configurations of tetracoordinated P and tricoordinated N atoms, which form stereogenic centres. The compounds 1b and 2c appear to have racemate (trans) and meso (cis) forms. Nevertheless, the X-ray crystallographic data indicate that only racemic (trans) mixtures are present (Table 2). It is understood that, the bulky substituents appear to take a trans arrangement with respect to the ring system and steric crowding might prevent the substituents from taking cis configurations. In addition, the mono-ferrocenyl-spiro-asa phosphazenes (3a-5a and 3b-5b) have three stereogenic P atoms. As an example, in order to predict the stereogenic properties of 5a, two equivalent chiral centres P(ArOCl) is labeled as RR or SS, and the other stereogenic centres are labeled as R0 /S0 . The compound 5a has four stereoisomers (RRR0 , SSR0 , RRS0 and SSS0 ), since the two Cl atoms are in trans conformation

666

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

Fig. 2. (a) 1H-decoupled and (b) 1H-coupled

according to the X-ray crystal structure analysis result (See Crystallographic Part). Due to the presence of the stereogenic centres in all the phosphazene derivatives, one would expect the occurrence of diastereomers, which should give rise to distinguishable NMR signals. Table 1 displays only a single set of signals, indicating that all isolated cyclotetraphosphazenes are obtained as single diastereomers. The crude reaction mixture NMRs exhibit that only one diastereomer occurs during synthesis. The chirality of the cyclotetraphosphazene derivatives can be verified by 31P{1H} NMR spectroscopy in the presence of CSA. The 31P{1H} NMR signals of the chiral compounds may split into two lines pointing out that they exist as racemate [22]. The stereogenic property of 2c is investigated by 31P{1H} NMR spectroscopy in the presence of CSA (Fig. 3).

31

P NMR spectra of 5a.

The 31P{1H} NMR signals of this compound split into two lines (for d PNNDASD) exhibiting that it exists as racemate. In addition, the chemical shift changes as a result of the equilibrium between the compound and its ligand-complexed form, and the changes (in ppb) at a mole ratio of CSA:compound of 10:1 are presented in Table 3. The interpretations of the 13C{1H} and 1H NMR signals of all the asa derivatives are carried out unambiguously by HSQC and HMBC using values corresponding to 1J(CH), and 2J(CH), 3J(CH) and 4J(CH) between the protons and carbons, respectively. As an example, the HMBC and HSQC spectra of 2c are depicted in Figs. S2a and S2b. The two bond-coupling constants, 2JPNC, for the NeCH2 carbons of the five-membered spiro rings of asa compounds, (1b, 1c and 3ae5a) are in the ranges of 9.6e13.1 Hz. The average values of

Table 2 Theoretical stereoisomer distributions and expected geometrical isomers of the compounds 1b, 2c and 5a. Compound

Centre of chirality

Stereogenic P atoms (n)

Stereoisomersa(2n) (expected)

Chirality (expected)

Chirality (found)

Geometrical isomerb

1b

Two equivalent

2

Racemic (lines 1/4) Meso (lines 2 ¼ 3)

Racemic (lines 1/4)

Cl1Cl2(trans)

2c

Two equivalent

2

Racemic (lines 1/4) Meso (lines 2 ¼ 3)

Racemic (lines 1/4)

Cl1Cl2(trans)

5a

Two equivalent One different

3

1 2 3 4 1 2 3 4 1 2 3 4 5 6 7 8

Racemic (lines 1/8) Racemic (lines 4/5)

Racemic (lines 1/8) Racemic (lines 4/5)

Cl1Cl2(trans) Cl1Cl2(trans)

a b

RR RS SR SS RR RS SR SS RRR0 RSR0 SRR0 SSR0 RRS0 RSS0 SRS0 SSS0

P(ArOCl) as labeled R/S for 1b, 2c and 5a, and P(NRN) as labeled R0 /S0 for 5a. Cis/trans isomerism is labeled explicitly according to Figs. 4e6.

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

Fig. 3. (a)

31

P{1H} NMR spectrum of 2c. (b) The addition of CSA at ca. 10:1 mol ratio showing the doubling of PA signals, indicating the characteristic of the racemate.

3

JPNCC, for the NeCH2eCH2 carbons of piperidino asa (1b and 2b) derivatives are 7.3 Hz. The piperidine and DASD substituted cyclotetraphosphazenes (1b, 2b, 1c and 2c) are symmetric structures in solution according to 13C{1H} NMR spectra, whereas in mono-ferrocenyl-spiro-asa phosphazenes (3ae5a and 3be5b), generally all the C atoms are distinguishable. Since all of the aliphatic protons are diastereotopic, all the cyclotetraphosphazenes give very complex 1H NMR spectra. The benzylic Ar-CH2 protons are separated from each other and one of the peak groups is in the range of 4.88e4.64 ppm, whereas the other one is in the range of 3.68e3.3.49 ppm. The spin system of benzylic protons is an ABX, depending on the geminal protoneproton coupling and vicinal coupling with the phosphorus-31 nucleus. The average 2JHH and 3JPH coupling constants of all the compounds are 9.5 and 15.1 Hz, respectively. Additionally, the geminal Fc-CH2-N protons of the mono-ferrocenyl-spiro-asa phosphazenes (3ae5a and 3be5b) give rise to doublets indicating that these protons are equivalent to each other, and the average 3JPH value is 13.7 Hz. The NeCH3 protons of the mono-ferrocenyl-spiroasa phosphazenes (3ae5a and 3be5b) are observed at ca. 2.76 ppm and give rise to doublets and the average coupling constant, 3JPH, is 12.8 Hz. The characteristic nNH bands of aminopodands (1 and 2) at 3276 and 3289 cm1, respectively, are not present in the FTIR spectra of the asa cyclotetraphosphazenes. All the phosphazenes exhibit strong absorptions in the ranges of 1284e1242 cm1 and 1178e1167 cm1, attributed to the nP]N bands of phosphazene rings [23]. The FTIR spectra of the asa cyclotetraphosphazenes

Table 3 31 P NMR Parameters of 2c and Effect of CSA on31P NMR Chemical Shifts.a Compounds

d/ppm >P(NN)

JPP/Hz >P(OCl)

31

P NMR chemical shifts (ppm) and geminal PNP coupling constants (Hz) 2 2c 4.59 (spiro) 1.34 JAx: 59.7 2 0.45 (DASD) JBx: 34.0 ii) Effect of CSA on31P NMR chemical shifts (ppb) at a 10:1 mol ratio 2 2c 175 (spiro) 180 JAx: 61.1 2 670 (DASD) JBx: 32.0 iii) Separation of enantiomeric signals (ppb) at a 10:1 mol ratio of CSA:molecule b b 2c (spiro) 60 (DASD) i)

a 31 b

667

P NMR measurements in CDCl3 solutions at 293 K. Magnitude of effect too small to observe up to a 10:1 mol ratio.

exhibit two medium-intensity absorption signals between 3087 and 3059 cm1 and between 3041 and 3024 cm1, ascribed to the asymmetric and symmetric stretching bands of the AreH bonds. Suitable crystals of 1b, 2c and 5a were crystallized from acetonitrile at ambient temperature. The structure analyses were made to figure out the influences of the bulky side groups and of the steric and electronic factors on the cyclotetraphosphazene rings. The crystallographic data, and the selected bond lengths and angles are listed in Table 4 and S1, respectively. The molecular and solid-state structures of 1b, 2c, and 5a, along with the atom-numbering schemes, are illustrated in Figs. 4e6 respectively. The X-ray crystallographic data of 1b and 2c indicate that they do not have symmetric structures in the solid state. The four P atoms of 1b, 2c and 5a are non-coplanar, and the four N atoms are displaced above (þ) and below () their best least-squares planes through the P atoms by the following distances: N1 [e 0.649(1) Å], N2 [þ 0.620(1) Å], N3 [e 0.166(2) Å] and N4 [þ 0.591(2) Å] (for 1b), N1 [þ 0.576(2) Å], N2 [e 0.543(2) Å], N3 [þ 0.376(2) Å] and N4 [e 0.485(2) Å] (for

Table 4 Crystallographic details.

Empirical formula Fw Crystal system Space group a () b () c () a ( ) b ( ) g ( ) V (3) Z m (mm1) r (calcd) (g cm3) Number of reflections total Number of reflections unique Rint 2qmax ( ) Tmin/Tmax Number of parameters R [I > 2s(I)] wR

1b

2c

5a

C26H36O2N8P4Cl2 687.41 Monoclinic P 21/c 14.8175(4) 8.9546(2) 23.9043(6) 90 98.515(3) 90 3136.77(14) 4 0.451(Mo Ka) 1.456 28,998

C31H42N8O6P4Cl2 817.53 Triclinic P 1 11.7222(3) 12.4413(3) 13.9565(4) 69.444(2) 85.080(4) 77.638(3) 1861.50(9) 2 0.401(Mo Ka) 1.459 31,589

C31H36O2N8P4FeCl2 803.31 Monoclinic P 21/c 17.3996(6) 15.4573(5) 13.2613(4) 90 106.544(3) 90 3419.0(2) 4 0.829(Mo Ka) 1.561 32,891

7762

9355

8564

0.0249 56.72 0.7959/0.8728 379

0.0672 57.08 0.9099/0.9497 460

0.0541 56.94 0.828/0.890 434

0.0315 0.1223

0.0626 0.1860

0.0405 0.1023

668

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

Fig. 4. An ORTEP-3 [31] drawing of 1b with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.

2c) and N1 [e 0.649(1) Å], N2 [þ 0.620(1) Å], N3 [e 0.166(2) Å] and N4 [þ 0.591(2) Å] (for 5a). The conformations of the cyclotetraphosphazene rings are depicted by the torsion angles of the ring bonds (Fig. S3), indicating that all the cyclotetraphosphazene rings are not planar. The structures of 1b, 2c, and 5a, exhibit that the tetradentate ligands (1 and 2) are bonded to the phosphorus atoms in ansa-spiro-ansa (2,4,6-asa) fashion, allowing the trans orientation of both of the Cl atoms (Figs. 4e6). Thereby, it is understood that the substituents; piperidine, DASD, and N-alkly-Nmono-ferrocenyldiamines, FcCH2NH(CH2)nNHR1 [n ¼ 2, R1 ¼ Me (3); n ¼ 2, R1 ¼ Et (4) and n ¼ 3, R1 ¼ Me (5)], prefer only the geminal bonding. In 1b, 2c and 5a, the three eight-membered rings (Figs. S4b, S5b and S6b) are not planar having total puckering amplitudes, QT [24], of 1.066(1) Å (for tetrameric N4P4 ring of 1b), 1.118(1) Å [for ansa (P1/N1/P2/N5/C7/C6/C1/O1) ring of 1b] and 3.623(1) Å [for ansa (P2/N2/P3/O2/N6/C10/C11/C16) ring of 1b], 1.034(1) Å [for tetrameric N4P4 ring of 2c], 1.299(10) Å [for ansa (P1/N1/P2/O1/N5/C1/ C6/C7) of 2c] and 3.660(3) Å [for ansa (P2/N2/P3/O2/N6/C11/C12/

Fig. 5. An ORTEP-3 [31] drawing of 2c with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.

Fig. 6. An ORTEP-3 [31] drawing of 5a with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.

C17) ring of 2c], 1.104(2) Å (for tetrameric N4P4 ring of 5a), 1.204(9) Å [for ansa (P1/N1/P2/O1/N5/C1/C6/C7) of 5a] and 3.594(2) Å (for ansa (P2/N2/P3/O2/N6/C10/C11/C16) of 5a]. The tetrameric N4P4 rings (Figs. S4a, S5a and S6a) for 1b, 2c and 5a, the six-membered spiro ring [P2/N5/N6/C8/C9/C10, Fig. S5c; QT ¼ 0.995(6) Å, 42 ¼ 140.0(3) , and q2 ¼ 41.5(2) , for 2c], all the eight-membered ansa rings and the five-membered spiro rings [P2/N5/N6/C8/C9, (Fig. S4c; 42 ¼  111.0(1) for 1b, and Fig. S6c; 42 ¼  111.4(1) for 5a] have a boat, chair and twisted conformations, respectively. The PN bond lengths of N4P4 rings of 1b, 2c and 5a are in the ranges of 1.548(1)e1.603(1) Å, 1.547(1)e1.602(1) Å and 1.554(2)e 1.594(2) Å, and the average values are 1.577(1), 1.574(1) and 1.573(2) Å, respectively (Table S1). They are shorter than the average exocyclic PN bond lengths (with respect to N4P4 rings) of 1b, 2c and 5a; 1.648(2), 1.646(2) and 1.652(2) Å, respectively, indicating that the bulky substituents are releasing electrons to the tetrameric phosphazene rings. All the exocyclic PN and PO bond lengths in 1b, 2c and 5a are almost equal among themselves. Besides, the PeNeP and NePeN bond angles are more susceptible than that of the PN bond lengths. The PeNeP bond angles of 1b, 2c and 5a are found to be in the ranges of 127.57(9)e 138.53(10) ; 129.74(12)e138.29(12) and 126.10(12)e135.08(14) , respectively. Furthermore, the variations in the endocyclic NePeN bond angles also spread, ranging from 113.54(8) to 125.04(8) (for 1b), from 116.15(10) to 125.04(9) (for 2c) and from 113.84(11) to 125.69(11) (for 5a). In the literature [25], similar spreads of PeNeP and NePeN bond angles are observed in some cyclotetraphosphazene derivatives. The endocyclic N1eP1eN2 (a) bond angles are 113.54(8) (for 1b), 116.15(10) (for 2c) and 113.84(11) (for 5a). The a bond angles of 1b, 2c and 5a are considerably affected by the N2O2 donor-type tetradentate ligands (1 and 2) bonded to the P atoms with spiro fashion. While the exocylic N5eP2eN6 (a0 ) bond angles of 1b [95.10(7) ] and 5a [95.53(10) ] are highly narrowed, the N5eP2eN6 (a0 ) bond angle of 2c [105.41(9) ] is slightly expanded, compared to the corresponding angle in the standard compound, N4P4Cl8. The endocyclic NePeN, PeNeP and exocylic ClePeCl bond angles of N4P4Cl8 are 121.2 , 131.3 and 102.8 , respectively [26]. The variations of PeNeP and NePeN angles of 1b, 2c, and 5a, may possibly be due to the steric hindrances and electronic properties of the bulky side groups [27].

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

On the other hand, in cyclophosphazenes, the PN single and double bonds are generally observed in the ranges of 1.628e1.691 Å and 1.571e1.604 Å, respectively [28]. In recent years, particularly, the effects of the steric hindrances, electronic factors, and negative hyperconjugation on the bond lengths and angles were discussed in cyclotriphosphazenes [29,30]. In addition, compounds 1b, 2c and 5a have intramolecular CeH$$$N and intermolecular CeH$$$O hydrogen bonds (Table S2). The p ∙∙∙ p contact between the rings, Cg2 ∙∙∙ Cg3 [where Cg2 and Cg3 are the centroids of the rings (C22eC26) and (C27eC31), respectively] (for 5a) may further stabilize the crystal structure with centroidecentroid distance of 3.288(2) Å. Electrochemistry of the ferrocenyl-cyclotetraphosphazenes were investigated by using cyclic voltammetry (CV) on polished glassy carbon electrode surface with acetonitrile (0.1 M TBATFB) solution vs. Ag/Agþ (0.01 M). The electrochemical data of the compounds 3ae5a and 3be5b are listed in Table S3. Anodic peak currents (ipa), the ratio of the anodic and cathodic peak currents (ipc/ipa), peak potentials (Ep) and their half-wave potentials (E1/ 2 ¼ [(Epa e Epc)/2]), differences between anodic and cathodic peak potentials (DEp) slope of log(ipa) e log(v), and diffusion coefficient (D) of the ferrocenyl-cyclotetraphosphazenes are measured and evaluated as important electrochemical data [32]. The typical CVs of the compounds which contain electroactive ferrocenyl group in asa rings are given in Fig. S7. All of the ferrocenylcyclotetraphosphazenes have reversible CVs with one-electron anodic and cathodic peaks corresponding to the redox-responsible moieties. The size of the alkyl groups of R, R1, [(-CH2)n], and R2 (-CH3 or -C2H5) has no significant effect on the peak separations, which means that it does not affect the reversibility of the ferrocenyl electrochemistry in the molecules. The values of cathodic-to-anodic peak current ratios are very close to 1.0 for all the compounds, indicating that there are no solubility differences between the cyclotetraphosphazenes and their reduced forms and the high electron-transfer rate of the iron centre as observed in spirocyclic monoferrocenyl and bisferrocenylphosphazenes [33]. The values of the slope of logipa vs logv plots, being about 0.5, also verify that CVs are not influenced much because of the adsorption of either the compounds or their reduced forms. As can be seen from Table S3, there are no significant differences in the diffusion coefficients of 3ae5a and 3be5b as expected, because the molecular sizes and weights are almost the same for all the molecules. It appears that the oxidation potentials of the ferrocenylcyclotetraphosphazenes are very close to the oxidation potential of ferrocene (Fcþ/Fc ¼ 640 mV). As a result, there seems to be no electronic contact between the ferrocenyl units and the rest of the molecule. As well, there is no p ∙∙∙ p communication between the ferrocene and phosphazene rings with respect to the crystallographic data.

669

2.2. Biological section 2.2.1. The cytotoxic effects of cyclotriphosphazene compounds In this study, MCF-7 cancer cell lines were used to determine the cytotoxic effects of 1be5b, 1c, 2c and 3ae5a on mammalian cells. The cell viability has been investigated following exposure of MCF7 breast cancer cell treatment of 1be5b, 1c, 2c and 3ae5a by 4,5dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay [34]. The cisplatin was used as reference in order to compare the in vitro cytotoxic activities of the compounds. As shown in Fig. 7, compounds 1b and 2b decreases the cell viability about 40% following exposure of MCF-7 breast cancer cell lines at the concentration of 1 mg/mL, while cisplatin decreases 35% at the similar concentration. The other compounds were not very effective at 1 mg/mL. In conclusion, compounds 1b and 2b exhibit almost the cytotoxic effect as cisplatin against MCF-7 cancer cell lines at the concentration of 1 mg/mL. 2.2.2. Antituberculosis activity M. tuberculosis H37Rv reference strain and drug resistant M. tuberculosis clinical strain were treated with different concentrations of compounds 3ae5b in the range 1000e5000 mM. The compounds were active against H37Rv strain above 1000 mM and were not active up to the 5000 mM against drug resistant strain. The susceptibility results of the compounds for M. tuberculosis strains were proved that the compounds are not very effective against clinical M. tuberculosis. 2.2.3. Antimicrobial activities Pathogen bacteria cause damage to humans. There are many antibiotics to treat bacteria diseases, however microorganisms develop drug resistance. Thus, there is an urgent need to discover new substances to treat bacterial diseases. The newly prepared cyclotetraphosphazene derivatives were tested for their antimicrobial activity against Staphylococcus aureus ATCC 25923 (Gþ), Pseudomonas aeruginosa ATCC 27853 (G), Escherichia coli ATCC 35218 (G), E. coli ATCC 25922 (G), Bacillus subtilis ATCC 6633 (Gþ), Bacillus cereus NRRL-B-3711 (Gþ) and Enterococcus faecalis ATCC 292112 (Gþ), and antifungal activity against Candida albicans ATCC 10231 and Candida tropicalis ATCC 13803 (Table 5a). Furthermore, the starting compound (N4P4Cl8), piperidine, DASD, and aminopodands 1 and 2 are checked for the same bacteria and fungi. The results are given in Table 5b for comparison to the cyclotetraphosphazene derivatives. One of the bacteria that showed a susceptibility to compounds 2b, 2c, and 5a was B. subtilis. Compounds 3b, 4b, and 5b were determined to have weak antibacterial activity against B. cereus. Compound 5a was also found to be a weak antibacterial activity against E. coli ATCC 35218 (G). The results showed that the compounds seem to be active only against

Fig. 7. Relative cell viability (%) of compounds 1be4b, 1c, 2c, 3a and 4a in MCF-7 cells following exposure of cells with various concentrations between 0.1 ng/mL 1 mg/mL for 24 h.

670

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

Table 5 Antimicrobial activities of (a) the compounds 2b, 2c, 3b, 4b, 5a, 5b and (b) starting compound (N4P4Cl8), piperidine, DASD, aminopodand 2 (Antibiotics; Amp ¼ Ampisilin and C]Chloramphenicol, Antifungal; Keto ¼ Ketoconazole). (a) Inhibition zone diameter (mm)a Agar well diffusion

b

b

Antibiotics

Antifungal

Test bacteria/compounds

2b

2c

3b

4b

5a

5b

Amp

C

S. aureus ATCC 25923 B. subtilis ATCC 6633 E. coli ATCC 35218 B. cereus NRRL-B-3711 C. tropicalis ATCC 13803 C. albicans ATCC 10231

e 9.0 ± 0.7 e e e e

e 7.0 ± 0.0 e e e e

e e e 8.7 ± 0.6 e e

9.3 ± 0.6 e e 9.7 ± 1.5 e e

e 9.3 ± 1.2 7.7 ± 0.6 e e e

e e e 7.7 ± 0.6 e e

35.0 ± 0.0 30.3 ± 0.5 R 10.0 ± 0.0 NS NS

29.6 31.0 10.5 23.4 NS NS

Keto ± ± ± ±

0.5 1.0 0.7 0.3

NS NS NS NS 29.0 ± 0.5 30.0 ± 0.0

(b) Inhibition zone diameter (mm)a b

Agar well diffusion

Antibiotics

Test bacteria/compounds

N4P4Cl8

Piperidine

DASD

2

Amp

E. coli ATCC 25922 P. aeruginosa ATCC 27853 B. cereus NRRL-B-3711 B. subtilis ATCC 6633

e 9.7 ± 0.6 9.0 ± 1.7 e

11.0 ± 0.0 e 13.0 ± 1.0 e

11.0 ± 1.0 e 13.0 ± 1.0 12.0 ± 0.0

10.3 ± 0.6 e 11.0 ± 1.7 e

20.3 28.0 10.0 30.3

b

Antifungal

C ± ± ± ±

0.0 0.0 0.0 0.5

26.6 21.9 23.4 31.0

Keto ± ± ± ±

0.0 0.9 0.3 1.0

NS NS NS NS

a Values represent average ± standard deviations for triplicate experiments, and the deviation value is 0.0 when three measurements exactly same (NS: Not Studied, R: Resistant). b Whereas Ampisilin (10 mg) and Chloramphenicol (30 mg) were used as standard antibacterial agents, Ketoconazole (50 mg) was used as antifungal control.

Gram positive bacteria except compound 5a. When we compared the positive control activity with the compounds, the control antibiotics demonstrated higher antibacterial activity, except 4b, piperidine, DASD, and 2 for B. cereus; meanwhile, none of the compounds were effective on the studied yeast strains. It was observed that the pyrrolidino and morpholino asa phosphazenes, which were the analogous compounds of 1be2c, did not exhibit any antibacterial activity against the same bacteria. But, the pyrrolidino asa compounds exhibit mild antifungal activity against C. tropicalis [11]. On the other hand, sufficient data of tetramer derivatives for making structureeactivity relationship comparison were not available in the literature. 2.2.4. Interaction with pBR322 plasmid DNA As known, the activity of a compound like cisplatin is believed to be related to its binding/cleavage with nucleobases with DNA, causing changes in DNA conformation and/or damage to DNA. The results of the changes in DNA conformation can affect the rate of migration of DNA in an electric field [35]. Since the cytotoxic potency of cisplatin has been ascribed to the different capacities of forming particular DNA cross-links DNA interactions of the starting compound (N4P4Cl8), piperidine, DASD, 1, 2, and cyclotetraphosphazene derivatives 1be5b, 1c, 2c, 3ae5a with supercoiled pBR322 DNA were investigated to find hints for possible reasons for their different activities. The plasmid DNA was exposed to compounds for 24 h and then the mobility of three forms of DNA was studied in an agarose gel electrophoresis. The retardation of the supercoiled form I DNA in the gel means decrease of positive charge or untwisting of the supercoiled form of DNA due to formation of adducts. Fig. 8 gives the electrophoretograms applied to the incubated mixtures of pBR322 DNA at varying concentrations (5000 mMe312 mM) of these compounds. Lane 1 applies to the untreated pBR322 plasmid DNA (control DNA), showing the major supercoiled circular form I and minor singly nicked relaxed circular form II of the plasmid DNA. Lanes 2e6 apply to pBR322 plasmid DNA incubated with compounds ranging from 5000 mM to 312 mM. Further evaluation of the compounds with plasmid DNA has demonstrated that 3a and 4a promoted the formation of strand

breaks in DNA. Among tested compounds most cytotoxic compounds 1b and 2b causes slight retardation of electrophoretic migration of supercoiled form I DNA suggests weak untwisting. The results show that the compounds 3ae5a alter the electrophoretic mobility of plasmid significantly, suggesting that the compounds interact with DNA, causing DNA cleavage (existence of form III linear DNA). This is not consistent with cytotoxicity of the compounds. Generally, there is no direct correlation between the cytotoxicity and the alteration of dsDNA for most of the drugs [35]. All other compounds cause a slight delay in the mobility of the supercoiled circular plasmid DNA (the mobility of the supercoiled circular plasmid DNA decreased). The mobility of form I DNA decreases sharply with the interactions of the starting compound N4P4Cl8; however, the mobility of the supercoiled circular plasmid DNA decreases with the control compounds, piperidine and DASD. The compounds 3ae5a and 3be5b include the pendant ferrocenly groups. It is strongly emphasized that the biological activity of these conjugates was expected due to possible oxidative DNA damage by the redox active ferrocene unit.

3. Conclusions In this study, geminal substituted 2-trans-6-dichloro asa (1b, 2b, 1c and 2c) and 2-trans-6-dichloro mono-ferrocenyl-spiro-asa cyclotetraphosphazenes were synthesized to determine their potential as anti-cancer agents and to evaluate the antituberculosis activity of ferrocenyl cyclotetraphosphazenes. The structures of the compounds were unambiguously determined using 1D and 2D NMR techniques in solution. The crystal structures of 1b, 2c, and 5a were determined. The cyclotetraphosphazene derivatives have chiral-P centres, and they are likely to be useful compounds for preparing biologically active materials. The cytotoxic effect of the cyclotetraphosphazenes on MCF-7 cell lines was investigated. Consequently, the cytotoxicity results highlight the anticancer potency of cyclotetraphosphazenes against breast cancer cell lines. The compounds 1b and 2b decrease the cell viability about 40% following exposure of MCF-7 breast cancer cell lines.

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

671

Fig. 8. Gel electrophoretic mobility of pBR322 plasmid DNA, when incubated with different concentrations of starting compound (N4P4Cl8), piperidine, DASD, 1, 2 and cyclotetraphosphazene derivatives 1be5b, 1c, 2c, 3ae5a. Concentrations (in mM) are as follows: lanes 1 untreated pBR322 plasmid DNA; lanes 2: 5000, lanes 3: 2500, lanes 4: 1250, lanes 5: 625, lanes 6: 312.

On the other hand, the compounds seem to be slightly active against Gram positive bacteria. They have no antifungal activities against C. tropicalis and C. albicans. The interactions of the compounds with supercoiled pBR322 DNA were investigated using the agarose gel electrophoresis. It was observed that compounds 3ae5a and 3be5b have DNA cleavage ability. The presence of the linear form III in the DNA-compound mixtures indicates the conformational changes of DNA. 4. Experimental 4.1. Chemistry N4P4Cl8 (a gift from the Otsuka Chemical Co. Ltd.), ferrocenecarboxaldehyde (Aldrich), CSA (Aldrich), aliphatic amines (Fluka),

salicylaldehyde (Fluka), piperidine (Fluka) and DASD (Fluka) were purchased. The reaction solvents were dried and distilled by standard methods before use. All reactions were monitored using thinlayer chromatography (TLC) in different solvents and chromatographed by column chromatography using silica gel. TLC was performed with Merck DC Alufolien Kiesegel 60 B254 sheets. Column chromatography was performed on Merck Kiesegel 60 (230e400 mesh ATSM) silica gel. All experiments were carried out under argon atmosphere. Melting points were measured with a Gallenkamp apparatus using a capillary tube. 1H, 13C{1H}, and 31P{1H} NMR, HSQC and HMBC spectra were recorded on a Bruker DPX FTNMR (500 MHz) spectrometer (SiMe4 as internal and 85% H3PO4 as external standards). The spectrometer was equipped with a 5 mm PABBO BB inverse gradient probe. Standard Bruker pulse programs [36] were used in the experiment. IR spectra were recorded on a

672

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

Mattson 1000 FTIR spectrometer in KBr discs and were reported in cm1 units. Microanalyses were carried out by the microanalytical service of TUBITAK Turkey. APIES mass spectra were recorded on the AGILENT 1100 MSD spectrometer. Microwave-assisted experiments were performed with a Milestone Start S system by using weflon™ magnet for toluene and xylene solvents. Experiments involving the chiral solvating agent (CSA) were carried out by the addition of small aliquots of a concentrated solution of CSA in the solvent used in NMR spectroscopy and the proton-decoupled 31P NMR spectra were recorded at each addition. Crystallographic data were recorded on a Bruker Kappa APEXII CCD area-detector diffractometer using Mo Ka radiation (l ¼ 0.71073 Å) at T ¼ 100(2) K. Absorption corrections by multi-scan [37] were applied. Structures were solved by direct methods and refined by full-matrix least squares against F2 using all data [38]. All non-H atoms were refined anisotropically. The H atom positions were calculated geometrically at distances of 0.93 Å (CH) and 0.97 Å (CH2) (for compounds 1b and 2c) and 0.95 Å (CH) and 0.99 Å (CH2) (for compound 5a) from the parent C atoms; a riding model was used during the refinement process and the Uiso(H) values were constrained to be 1.2Ueq(carrier atom). Starting compounds; 2,20 -[1,2Ethanediylbis(iminomethanediyl)]diphenol (1), 2,20 -[1,3propanediylbis(imino methanediyl)]diphenol (2) [18], N-(1ferrocenylmethyl)-N-methly-ethylenediamine (3) and N-(1ferrocenylmethyl)-N-ethly-ethylenediamine (4) were synthesized according to the methods reported in the literature [18]. Also compounds 1a and 2a were obtained from the reaction of 1 and 2 with N4P4Cl8 according to the reported procedure, respectively [11]. 4.1.1. N-(1-ferrocenylmethyl)-N-methly-propylenediamine (5) A solution of ferrocenecarboxaldehyde (3.00 g, 14.02 mmol) in methanol (100 mL) was added to N-ethyl-ethylenediamine (1.24 g, 14.02 mmol) in methanol (50 mL). The mixture was refluxed for 3 h, and then the excess sodium borohydride (1.59 g, 42.06 mmol) was added. The crude product was extracted with dichloromethane (150 mL, three times). The solvent was evaporated at reduced pressure and the oily product was obtained. Yield: 3.44 g (12.04 mmol, 90%). Anal. Calcd. for C15H22N2Fe: C, 62.95; H, 7.75; N, 9.79. Found: C, 62.61; H, 7.70; N, 9.53. FTIR (KBr, cm1): n 3344; 3221 (NeH), 3089; 3075 (CeH arom.), 2958; 2810 (CeH aliph). 4.1.2. Asa 1b A solution of piperidine (1.00 mL, 10.20 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.60 mL) and 1a (0.60 g, 1.00 mmol) in dry THF (100 mL) at room temperature. The solution was heated to reflux for 35 h under Ar being passed over the mixture. The precipitated triethylaminehydrochloride was filtered off and the solvent was evaporated. The product was purified by column chromatography with benzene, and a white powder was crystallized from acetonitrile. Yield: 0.58 g (0.84 mmol, 83%). mp > 320  C. Anal. Calcd. for C26H36O2N8P4Cl2: C, 45.35; H, 5.27; N, 16.28. Found: C, 45.39; H, 5.06; N, 16.33. APIES-MS (fragments are based on 35Cl, Ir %, Ir designates the fragment abundance percentage): m/z 687 ([MH]þ, 100). FTIR (KBr, cm1): n 3070 (asymm.), 3024 (symm.) (CeH arom.), 2958, 2844 (CeH aliph.), 1583 (C]C), 1245 (asymm.), 1176 (symm.) (P]N), 551 (PCl). 1 H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.36 (m, 4H, H3 and H5), 7.19 (d, 4H, H2 and H4), 4.69 (dd, 2H, 3JHH ¼ 9.4 Hz, 2JPH ¼ 15.2 Hz, Ar-CH2-N), 3.62 (dd, 2H, 3 JHH ¼ 13.7 Hz, 2JPH ¼ 15.3 Hz, AreCH2eN), 3.36 and 3.21 [m, 8H, NeCH2 (pip)], 2.95 [m, 4H, NeCH2 (spiro)], 1.60 [m, 8H, NeCH2eCH2 (pip)], 1.52 [m, 4H, NeCH2eCH2eCH2 (pip)]. 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.71 (d, 2JPC ¼ 6.1 Hz, C6), 132.21 (s, C2), 129.57 (s, C4), 128.12

(t, 3JPC ¼ 3.4 Hz, C1), 125.13 (s, C3), 123.40 (t, 3JPC ¼ 3.7 Hz, C5), 45.34 [s, NeCH2 (pip)], 44.25 [d, 2JPC ¼ 11.9 Hz, NeCH2 (spiro)], 43.58 (s, AreCH2eN), 26.32 [d, 3JPC ¼ 7.1 Hz, NeCH2eCH2 (pip)], 24.91 [s, NeCH2eCH2eCH2 (pip)]. Microwave-assisted synthesis of 1b: Piperidine (1.00 mL, 10.20 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.60 mL) and 1a (0.60 g, 1.00 mmol) in dry THF (100 mL). The mixture was refluxed for 0.50 h, and triethylaminehydrochloride was filtered off. THF was evaporated and the crude product was purified by column chromatography with benzene. Compound 1b was crystallized from acetonitrile. Yield: 0.66 g (0.96 mmol, 95%). Compound 1b was also synthesized in toluene and o-xylene. The work-up procedure was similar to that of THF solvent. Whereas the mixture was refluxed for 0.75 h in toluene, Yield: 0.62 g (0.90 mmol, 89%), and in o-xylene for 1 h, Yield: 0.54 g (0.78 mmol, 77%). 4.1.3. Asa 2b The work-up procedure was similar to that of compound 1b, using 2a (0.50 g, 0.80 mmol), piperidine (0.80 mL, 8.30 mmol) and triethylamine (0.50 mL). The product was purified by column chromatography with benzene, and a white powder was crystallized from acetonitrile. Yield: 0.50 g (0.71 mmol, 86%). mp: 250  C. Anal. Calcd. for C27H38O2N8P4Cl2. C6H6 (benzene): C, 50.78; H, 5.68; N, 14.35. Found: C, 49.50; H, 6.09; N, 14.79. APIES-MS (fragments are based on 35Cl, Ir %): m/z 701 ([MH]þ, 100). FTIR (KBr, cm1): n 3059 (asymm.), 3039 (symm.) (CeH arom.), 2922, 2850 (CeH aliph.), 1583 (C]C), 1271 (asymm.), 1176 (symm.) (P]N), 546 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.32 (m, 6H, H3, H4, H5), 7.18 (t, 2H, H2), 4.78 (dd, 2H, 3 JHH ¼ 10.7 Hz, 2JPH ¼ 15.3 Hz, AreCH2eN), 3.54 (dd, 2H, 3 JHH ¼ 8.6 Hz, 2JPH ¼ 15.3 Hz, AreCH2eN), 3.24 [m, 8H, NeCH2 (pip)], 3.08 [m, 4H, NeCH2 (spiro)], 1.76 [m, 2H, NeCH2eCH2 (spiro)], 1.59 [m, 8H, NeCH2eCH2 (pip)], 1.51 [m, 4H, NeCH2eCH2eCH2 (pip)]. 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.26 (d, 2 JPC ¼ 8.3 Hz, C6), 131.23 (s, C2), 130.65 (t, 3JPC ¼ 3.6 Hz, C1), 129.70 (s, C4), 125.72 (s, C3), 123.01 (s, C5), 128.10 (s, C6H6), 48.92 [s, NeCH2 (spiro)], 47.14 (d, 2JPC ¼ 3.6 Hz, Ar-CH2-N), 45.26 [s, NeCH2 (pip)], 29.88 [s, NeCH2eCH2 (spiro)], 26.28 [d, 3JPC ¼ 7.5 Hz, NeCH2eCH2 (pip)], 25.03 [s, NeCH2eCH2eCH2 (pip)]. 4.1.4. Mono-ferrocenyl-spiro-asa 3a A solution of 3 (0.40 g, 1.45 mmol) in dry THF (50 mL) was slowly added to a stirred solution of triethylamine (0.65 mL) and 1a (0.70 g, 1.20 mmol) in dry THF (100 mL) at 10  C for over 2 h. After the mixture had been allowed to warm to room temperature, it was stirred for 72 h. The precipitated triethylaminehydrochloride was filtered off and the solvent was evaporated. The product was purified by column chromatography with toluene-THF (1:1), and was crystallized from acetonitrile. Yield: 0.63 g (0.80 mmol, 67%). mp: 278  C. Anal. Calcd. for C30H36O2N8P4FeCl2: C, 45.57; H, 4.56; N, 14.18. Found: C, 45.50; H, 5.16; N, 13.90. APIES-MS (fragments are based on 35Cl and 56Fe, Ir %): m/z 790 ([M]þ, 100). FTIR (KBr, cm1): n 3077 (asymm.), 3033 (symm.) (CeH arom.), 2954, 2850 (CeH aliph.), 1581 (C]C), 1242 (asymm.), 1171 (symm.) (P]N), 552 (PCl). 1 H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.32e7.38 (m, 4H, H4 and H5), 7.19e7.21 (m, 4H, H2 and H3), 4.77 (dd, 1H, 3JPH ¼ 14.8 Hz, 2JHH ¼ 6.9 Hz, AreCH2eN), 4.70 (dd, 1H, 3JPH ¼ 12.4 Hz, 2JHH ¼ 7.2 Hz, AreCH2eN), 4.37 (d, 1H, 3 JHH ¼ 1.2 Hz, H20 ), 4.33 (d, 1H, 3JHH ¼ 1.2 Hz, H20 ), 4.10 (m, 5H, H40 ), 4.03 (m, 2H, H30 ), 3.72 (d, 2H, 3JPH ¼ 15.2 Hz, H50 ), 3.68 (dd, 1H, 3 JPH ¼ 15.2 Hz, 2JHH ¼ 7.6 Hz, AreCH2eN), 3.61 (dd, 1H, 3 JPH ¼ 13.2 Hz, 2JHH ¼ 7.6 Hz, AreCH2eN), 2.97e3.17 (m, 2H,

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

FceCH2eNeCH2), 2.97e3.17 (m, 2H, CH3eNeCH2), 2.97e3.17 [m, 4H, NeCH2 (spiro)], 2.75 (d, 3H, 3JPH ¼ 11.6 Hz, NeCH3). 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.74 (d, 2JPC ¼ 10.0 Hz, C6), 149.62 (d, 2JPC ¼ 10.0 Hz, C6), 132.37 (d, 4JPC ¼ 2.5 Hz, C2), 132.35 (d, 4JPC ¼ 2.5 Hz, C2), 129.94 (d, 3JPC ¼ 3.1 Hz, C5), 129.87 (d, 3JPC ¼ 2.3 Hz, C5), 128.29 (t, 3 JPC ¼ 3.8 Hz, C1), 128.20 (t, 3JPC ¼ 3.8 Hz, C1), 125.56 (s, C4), 125.50 (s, C4), 123.96 (s, C3), 123.90 (s, C3), 83.60 (d, 3JPC ¼ 9.2 Hz, C10 ), 70.34 (s, C20 ), 69.99 (s, C20 ), 68.78 (s, C40 ), 68.36 (s, C30 ), 68.31 (s, C30 ), 48.10 (d, 2JPC ¼ 12.2 Hz, FceCH2eNeCH2), 45.38 (d, 2JPC ¼ 4.6 Hz, C50 ), 44.70 [d, 2JPC ¼ 13.0 Hz, NeCH2 (spiro)], 44.56 [d, 2JPC ¼ 13.0 Hz, NeCH2 (spiro)], 44.36 (d, 2JPC ¼ 13.0 Hz, CH3eNeCH2), 43.65 (s, AreCH2eN), 43.53 (s, AreCH2eN), 32.58 [d, 2JPC ¼ 3.0 Hz, NeCH3). 4.1.5. Mono-ferrocenyl-spiro-asa 3b The work-up procedure was similar to that of compound 3a, using 2a (0.70 g, 1.15 mmol), 3 (0.40 g, 1.45 mmol) and triethylamine (0.65 mL). The residue was purified by column chromatography with toluene, and was crystallized from acetonitrile. Yield: 0.60 g (0.75 mmol, 64%). mp: 235  C. Anal. Calcd. for C31H38O2N8P4FeCl2: C, 46.27; H, 4.73; N, 13.93. Found: C, 45.75; H, 4.68; N, 13.84. APIES-MS (fragments are based on 35Cl and 56Fe, Ir %): m/z 804 ([M]þ, 100). FTIR (KBr, cm1): n 3075 (asymm.), 3037 (symm.) (CeH arom.), 2927, 2843 (CeH aliph.), 1583 (C]C), 1255 (asymm.), 1173 (symm.) (P]N), 550 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.29e7.35 (m, 4H, H4 and H5), 7.18 (m, 4H, H2 and H3), 4.88 (dd, 1H, 3JPH ¼ 16.4 Hz, 2 JHH ¼ 10.4 Hz, AreCH2eN), 4.82 (dd, 1H, 3JPH ¼ 16.0 Hz, 2 JHH ¼ 6.9 Hz, AreCH2eN), 4.36 (d, 1H, 3JHH ¼ 1.3 Hz, H20 ), 4.27 (d, 1H, 3JHH ¼ 1.3 Hz, H20 ), 4.08 (m, 5H, H40 ), 3.95 (m, 2H, H30 ), 3.67 (d, 2H, 3JPH ¼ 14.8 Hz, H50 ), 3.60 (dd, 1H, 3JPH ¼ 15.2 Hz, 2JHH ¼ 8.8 Hz, AreCH2eN), 3.51 (dd, 1H, 3JPH ¼ 15.2 Hz, 2JHH ¼ 8.8 Hz, AreCH2eN), 2.90e3.19 (m, 2H, FceCH2eNeCH2), 2.90e3.19 (m, 2H, CH3eNeCH2), 2.90e3.19 [m, 4H, NeCH2 (spiro)], 2.73 (d, 3H, 3 JPH ¼ 12.0 Hz, NeCH3), 1.79 [m, 2H, NeCH2eCH2 (spiro)]. 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.47 (d, 2JPC ¼ 10.0 Hz, C6), 149.30 (d, 2JPC ¼ 8.5 Hz, C6), 131.64 (d, 4JPC ¼ 2.3 Hz, C2), 131.50 (d, 4JPC ¼ 2.3 Hz, C2), 130.61 (t, 3JPC ¼ 3.8 Hz, C1), 130.32 (t, 3JPC ¼ 3.8 Hz, C1), 129.78 (d, 4 JPC ¼ 2.3 Hz, C4), 129.73 (d, 4JPC ¼ 2.3 Hz, C4), 125.66 (d, 3 JPC ¼ 8.5 Hz, C5), 125.57 (d, 3JPC ¼ 8.5 Hz, C5), 123.34 (s, C3), 123.31 (s, C3), 83.82 (d, 3JPC ¼ 10.1 Hz, C10 ), 70.15 (s, C20 ), 70.09 (s, C20 ), 68.77 (s, C40 ), 68.39 (s, C30 ), 68.23 (s, C30 ), 49.07 [m, NeCH2 (spiro]), 48.87 [m, NeCH2 (spiro)], 48.03 (d, 2JPC ¼ 12.3 Hz, FceCH2eNeCH2), 47.60 (d, 2JPC ¼ 3.8 Hz, Ar-CH2-N), 47.46 (d, 2JPC ¼ 3.7 Hz, AreCH2eN), 45.32 (d, 2JPC ¼ 4.6 Hz, C50 ), 44.60 (d, 2JPC ¼ 13.1 Hz, CH3eNeCH2), 32.50 [d, 2JPC ¼ 3.0 Hz, NeCH3), 26.41 [d, 3JPC ¼ 3.1 Hz, NeCH2eCH2 (spiro)]. 4.1.6. Mono-ferrocenyl-spiro-asa 4a The work-up procedure was similar to that of compound 3a, using 1a (0.70 g, 1.20 mmol), 4 (0.40 g, 1.40 mmol) and triethylamine (0.65 mL). The residue was purified by column chromatography with toluene-THF (1:1), and was crystallized from hexane. Yield: 0.65 g (0.81 mmol, 68%). mp: 150  C. Anal. Calcd. for C31H38O2N8P4FeCl2. C6H12 (hexane): C, 50.05; H, 5.63; N, 12.61. Found: C, 50.68; H, 5.44; N, 11.98. APIES-MS (fragments are based on 35Cl and 56Fe, Ir %): m/z 804 ([M]þ, 100). FTIR (KBr, cm1): n 3070 (asymm.), 3026 (symm.) (CeH arom.), 2924, 2862 (CeH aliph.), 1581 (C]C), 1272 (asymm.), 1172 (symm.) (P]N), 565 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.32e7.39 (m, 4H, H4 and H5), 7.16e7.23 (m, 4H, H2 and H3), 4.77 (dd, 1H, 3JPH ¼ 16.8 Hz, 2JHH ¼ 7.2 Hz, AreCH2eN), 4.70 (dd, 1H, 3JPH ¼ 16.0 Hz, 2JHH ¼ 7.2 Hz, AreCH2eN), 4.37 (d, 1H, 3 JHH ¼ 1.2 Hz, H20 ), 4.35 (d, 1H, 3JHH ¼ 1.3 Hz, H20 ), 4.11 (m, 5H, H40 ),

673

4.01 (m, 2H, H30 ), 3.73 (d, 2H, 3JPH ¼ 15.2 Hz, H50 ), 3.68 (dd, 1H, 3 JPH ¼ 15.2 Hz, 2JHH ¼ 8.4 Hz, AreCH2eN), 3.58 (dd, 1H, 3 JPH ¼ 15.2 Hz, 2JHH ¼ 7.6 Hz, AreCH2eN), 2.96e3.31 (m, 2H, FceCH2eN-CH2), 2.96e3.31 (m, 2H, C2H5eNeCH2), 2.96e3.31 [m, 4H, NeCH2 (spiro)], 2.96e3.31 (m, 2H, NeCH2eCH3), 1.22 (t, 3H, 3 JHH ¼ 8.8 Hz, NeCH2eCH3). 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.50 (d, 2 JPC ¼ 9.5 Hz, C6), 149.45 (d, 2JPC ¼ 10.0 Hz, C6), 132.09 (s, C2), 132.03 (s, C2), 129.67 (d, 4JPC ¼ 3.1 Hz, C4), 129.64 (d, 4JPC ¼ 2.2 Hz, C4), 128.33 (t, 3JPC ¼ 3.8 Hz, C1), 128.04 (t, 3JPC ¼ 3.8 Hz, C1), 125.30 (s, C3), 125.20 (s, C3), 123.76 (d, 3JPC ¼ 3.9 Hz, C5), 123.70 (d, 3 JPC ¼ 3.8 Hz, C5), 83.10 (d, 3JPC ¼ 9.5 Hz, C10 ), 70.35 (s, C20 ), 69.95 (s, C20 ), 68.76 (s, C40 ), 68.34 (s, C30 ), 68.21 (s, C30 ), 45.15 (d, 2JPC ¼ 4.6 Hz, C50 ), 44.51 (d, 2JPC ¼ 13.1 Hz, FceCH2eNeCH2), 44.29 [d, 2 JPC ¼ 13.2 Hz, NeCH2 (spiro]), 44.22 [d, 2JPC ¼ 13.1 Hz, NeCH2 (spiro)], 43.43 (s, Ar-CH2-N), 40.34 (d, 2JPC ¼ 3.1 Hz, C2H5eNeCH2), 30.33 [s, NeCH2eCH3), 13.73 (d, 3JPC ¼ 6.9 Hz, NeCH2eCH3). 4.1.7. Mono-ferrocenyl-spiro-asa 4b The work-up procedure was similar to that of compound 3a, using 2a (0.70 g, 1.15 mmol), 4 (0.40 g, 1.40 mmol) and triethylamine (0.65 mL). The residue was purified by column chromatography with toluene, and was crystallized from acetonitrile. Yield: 0.60 g (0.73 mmol, 63%). mp: 188  C. Anal. Calcd. for C32H40O2N8P4FeCl2: C, 46.94; H, 4.89; N, 13.69. Found: C, 46.29; H, 5.08; N, 12.94. APIES-MS (fragments are based on 35Cl and 56Fe, Ir %): m/z 818 ([M]þ, 100). FTIR (KBr, cm1): n 3078 (asymm.), 3026 (symm.) (CeH arom.), 2917, 2850 (CeH aliph.), 1583 (C]C), 1284 (asymm.), 1174 (symm.) (P]N), 553 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.26e7.33 (m, 4H, H4 and H5), 7.19 (m, 4H, H2 and H3), 4.87 (dd, 1H, 3JPH ¼ 15.3 Hz, 2 JHH ¼ 14.0 Hz, AreCH2eN), 4.80 (dd, 1H, 3JPH ¼ 15.3 Hz, 2 JHH ¼ 13.6 Hz, AreCH2eN), 4.37 (d, 1H, 3JHH ¼ 1.2 Hz, H20 ), 4.35 (d, 1H, 3JHH ¼ 1.2 Hz, H20 ), 4.12 (m, 5H, H40 ), 3.96 (m, 2H, H30 ), 3.65 (d, 2H, 3JPH ¼ 15.2 Hz, H50 ), 3.60 (dd, 1H, 3JPH ¼ 15.2 Hz, 2JHH ¼ 8.2 Hz, AreCH2eN), 3.49 (dd, 1H, 3JPH ¼ 15.2 Hz, 2JHH ¼ 8.8 Hz, AreCH2eN), 2.90e3.47 (m, 2H, FceCH2eNeCH2), 2.90e3.47 (m, 2H, C2H5eNeCH2), 2.90e3.47 [m, 4H, NeCH2 (spiro)], 2.90e3.47 (m, 2H, NeCH2eCH3), 1.80 [m, 2H, NeCH2eCH2 (spiro)], 1.21 (t, 3H, 3 JHH ¼ 8.9 Hz, NeCH2eCH3). 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.40 (d, 2 JPC ¼ 9.2 Hz, C6), 149.10 (d, 2JPC ¼ 9.7 Hz, C6), 131.32 (s, C2), 131.72 (s, C2), 130.24 (t, 3JPC ¼ 3.9 Hz, C1), 129.79 (t, 3JPC ¼ 3.9 Hz, C1), 129.49 (d, 4JPC ¼ 3.1 Hz, C4), 129.03 (d, 4JPC ¼ 2.3 Hz, C4), 125.58 (d, 3 JPC ¼ 9.7 Hz, C5), 125.35 (s, C5), 123.11 (s, C3), 122.94 (s, C3), 83.15 (d, 3 JPC ¼ 9.7 Hz, C10 ), 69.98 (s, C20 ), 69.88 (s, C20 ), 68.53 (s, C40 ), 68.14 (s, C30 ), 67.96 (s, C30 ), 48.69 [m, NeCH2 (spiro]), 48.39 [m, NeCH2 (spiro)], 47.29 (d, 2JPC ¼ 4.1 Hz, AreCH2eN), 47.09 (d, 2JPC ¼ 4.2 Hz, AreCH2eN), 45.10 (d, 2JPC ¼ 4.7 Hz, C50 ), 44.61 (d, 2JPC ¼ 13.1 Hz, FceCH2eNeCH2), 40.42 (s, C2H5eNeCH2), 29.43 [s, NeCH2eCH3), 26.16 [d, 3JPC ¼ 3.2 Hz, NeCH2eCH2 (spiro)], 13.48 (d, 3JPC ¼ 4.6 Hz, NeCH2eCH3). 4.1.8. Mono-ferrocenyl-spiro-asa 5a The work-up procedure was similar to that of compound 3a, using 1a (0.70 g, 1.20 mmol), 5 (0.40 g, 1.40 mmol) and triethylamine (0.65 mL). The residue was purified by column chromatography with toluene, and was crystallized from acetonitrile. Yield: 0.66 g (0.82 mmol, 69%). mp: 252  C. Anal. Calcd. for C31H38O2N8P4FeCl2: C, 46.27; H, 4.73; N, 13.93. Found: C, 46.17; H, 4.53; N, 14.07. APIES-MS (fragments are based on 35Cl and 56Fe, Ir %): m/z 804 ([M]þ, 100). FTIR (KBr, cm1): n 3080 (asymm.), 3033 (symm.) (CeH arom.), 2931, 2856 (CeH aliph.), 1583 (C]C), 1270 (asymm.), 1174 (symm.) (P]N), 557 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.32e7.41 (m, 4H, H4

674

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

and H5), 7.18 (m, 4H, H2 and H3), 4.77 (dd, 1H, 3JPH ¼ 15.2 Hz, 2 JHH ¼ 9.6 Hz, AreCH2eN), 4.69 (dd, 1H, 3JPH ¼ 15.2 Hz, 2 JHH ¼ 9.6 Hz, AreCH2eN), 4.37 (d, 1H, 3JHH ¼ 1.2 Hz, H20 ), 4.31 (d, 1H, 3JHH ¼ 1.2 Hz, H20 ), 4.17 (d, 2H, 3JPH ¼ 7.6 Hz, H50 ), 4.08 (m, 5H, H40 ), 4.02 (m, 2H, H30 ), 3.67 (dd, 1H, 3JPH ¼ 15.2 Hz, 2JHH ¼ 13.2 Hz, AreCH2eN), 3.60 (dd, 1H, 3JPH ¼ 14.4 Hz, 2JHH ¼ 14.0 Hz, AreCH2eN), 2.95e3.26 (m, 2H, FceCH2eNeCH2), 2.95e3.26 (m, 2H, CH3eNeCH2), 2.95e3.26 [m, 4H, NeCH2 (spiro)], 2.77 (d, 3H, 3 JPH ¼ 13.6 Hz, NeCH3), 1.72 (m, 2H, CH3eNeCH2eCH2). 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.54 (d, 2JPC ¼ 8.9 Hz, C6), 149.45 (d, 2JPC ¼ 8.8 Hz, C6), 132.09 (s, C2), 129.66 (d, 4JPC ¼ 3.1 Hz, C4), 129.61 (d, 4 JPC ¼ 2.3 Hz, C4), 128.08 (t, 3JPC ¼ 4.6 Hz, C1), 128.00 (t, 3JPC ¼ 4.6 Hz, C1), 125.31 (s, C3), 123.86 (s, 3JPC ¼ 3.9 Hz, C5), 84.64 (d, 3 JPC ¼ 10.7 Hz, C10 ), 70.10 (s, C20 ), 69.76 (s, C20 ), 68.54 (s, C40 ), 67.90 (s, C30 ), 67.86 (s, C30 ), 50.64 (d, 2JPC ¼ 4.6 Hz, C50 ), 47.02 (s, FceCH2eNeCH2), 45.58 (s, CH3eNeCH2), 44.32 [d, 2JPC ¼ 12.2 Hz, NeCH2 (spiro)], 43.34 (s, Ar-CH2-N), 36.43 (s, NeCH3), 23.84 (d, 3 JPC ¼ 3.0 Hz, CH3eNeCH2eCH2). 4.1.9. Mono-ferrocenyl-spiro-asa 5b The work-up procedure was similar to that of compound 3a, using 2a (0.70 g, 1.15 mmol), 5 (0.40 g, 1.40 mmol) and triethylamine (0.65 mL). The residue was purified by column chromatography with toluene, and was crystallized from acetonitrile. Yield: 0.62 g (0.76 mmol, 65%). mp: 198  C. Anal. Calcd. for C32H40O2N8P4FeCl2: C, 46.94; H, 4.89; N, 13.69. Found: C, 46.64; H, 4.74; N, 13.44. APIES-MS (fragments are based on 35Cl and 56Fe, Ir %): m/z 818 ([M]þ, 100). FTIR (KBr, cm1): n 3087 (asymm.), 3037 (symm.) (CeH arom.), 2929, 2854 (CeH aliph.), 1581 (C]C), 1273 (asymm.), 1174 (symm.) (P]N), 553 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.26e7.37 (m, 4H, H4 and H5), 7.16e7.20 (m, 4H, H2 and H3), 4.87 (dd, 1H, 3 JPH ¼ 15.2 Hz, 2JHH ¼ 8.4 Hz, AreCH2eN), 4.83 (dd, 1H, 3 JPH ¼ 15.2 Hz, 2JHH ¼ 8.8 Hz, AreCH2eN), 4.21 (d, 1H, 3JHH ¼ 1.3 Hz, H20 ), 4.19 (d, 1H, 3JHH ¼ 1.2 Hz, H20 ), 4.17 (d, 2H, 3JPH ¼ 14.0 Hz, H50 ), 4.07 (m, 5H, H40 ), 4.02 (m, 2H, H30 ), 3.59 (dd, 1H, 3JPH ¼ 14.8 Hz, 2 JHH ¼ 10.0 Hz, AreCH2eN), 3.53 (dd, 1H, 3JPH ¼ 14.4 Hz, 2 JHH ¼ 8.8 Hz, AreCH2eN), 3.00e3.18 (m, 2H, FceCH2eNeCH2), 3.00e3.18 (m, 2H, CH3eNeCH2), 3.00e3.18 [m, 4H, NeCH2 (spiro)], 2.77 (d, 3H, 3JPH ¼ 14.0 Hz, NeCH3), 1.79 [m, 2H, NeCH2eCH2 (spiro)], 1.71 (m, 2H, CH3eNeCH2eCH2). 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.44 (d, 2JPC ¼ 8.5 Hz, C6), 149.29 (d, 2JPC ¼ 8.5 Hz, C6), 131.66 (d, 4 JPC ¼ 2.3 Hz, C2), 131.51 (d, 4JPC ¼ 2.3 Hz, C2), 130.52 (t, 3JPC ¼ 3.8 Hz, C1), 130.37 (t, 3JPC ¼ 4.6 Hz, C1), 129.74 (d, 4JPC ¼ 2.3 Hz, C4), 129.68 (d, 4JPC ¼ 2.3 Hz, C4), 125.60 (s, C3), 125.53 (s, C3), 123.40 (d, 3 JPC ¼ 9.2 Hz, C5), 123.31 (d, 3JPC ¼ 9.2 Hz, C5), 84.82 (d, 3 JPC ¼ 11.6 Hz, C10 ), 70.47 (s, C20 ), 70.15 (s, C20 ), 68.81 (s, C40 ), 68.21 (s, C30 ), 68.03 (s, C30 ), 50.95 (d, 2JPC ¼ 4.8 Hz, C50 ), 48.94 [m, NeCH2 (spiro]), 48.81 [m, NeCH2 (spiro)], 47.45 (d, 2JPC ¼ 5.3 Hz, AreCH2eN), 47.40 (d, 2JPC ¼ 5.2 Hz, AreCH2eN), 47.05 (s, FceCH2eNeCH2), 45.85 (s, CH3eNeCH2), 36.55 (s, NeCH3), 26.42 [d, 3JPC ¼ 3.1 Hz, NeCH2eCH2 (spiro)], 24.26 (d, 3JPC ¼ 2.2 Hz, CH3eNeCH2eCH2). 4.1.10. Asa 1c The work-up procedure was similar to that of compound 1b, using 1a (0.60 g, 1.00 mmol), DASD (1.30 mL, 10.20 mmol) and triethylamine (0.60 mL). The oily residue was purified by column chromatography with benzene-THF (1:1), and was crystallized from acetonitrile. Yield: 0.72 g (0.90 mmol, 88%). mp: 285  C. Anal. Calcd. for C30H40O6N8P4Cl2: C, 49.06; H, 5.26; N, 12.71. Found: C, 48.66; H, 5.49; N, 12.93. APIES-MS (fragments are based on 35Cl, Ir %): m/z 803 ([MH]þ, 100). FTIR (KBr, cm1): n 3085 (asymm.), 3041

(symm.) (CeH arom.), 2956, 2861 (CeH aliph.), 1583 (C]C), 1263 (asymm.), 1167 (symm.) (P]N), 555 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.36 (m, 4H, H4, H5), 7.19 (m, 4H, H2, H3), 4.64 (dd, 2H, 3JPH ¼ 15.3 Hz, 2 JHH ¼ 8.9 Hz, AreCH2eN), 4.11 (s, 8H, OeCH2), 3.63 (dd, 2H, 3 JPH ¼ 15.3 Hz, 2JHH ¼ 13.7 Hz, AreCH2eN), 3.48 [m, 4H, NeCH2 (DASD)], 3.36 [m, 4H, NeCH2 (DASD)], 3.23 [m, 2H, NeCH2 (spiro)], 2.97 [m, 2H, NeCH2 (spiro)], 1.76 [m, 4H, NeCH2eCH2 (DASD)], 1.45 [m, 4H, NeCH2eCH2 (DASD)]. 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 149.40 (d, 2 JPC ¼ 6.7 Hz, C6), 132.98 (s, C2), 130.40 (s, C4), 129.80 (t, 3 JPC ¼ 8.2 Hz, C1), 125.62 (s, C3), 125.23 (t, 3JPC ¼ 11.1 Hz, C5), 107.14 (s, OeCeO), 64.12 (s, OeCH2), 44.27 [d, 2JPC ¼ 9.6 Hz, NeCH2 (spiro)], 43.41 (s, Ar-CH2-N), 42.70 [s, NeCH2 (DASD)], 30.40 [s, NeCH2eCH2 (DASD)]. 4.1.11. Asa 2c The work-up procedure was similar to that of compound 1b, using 2a (0.50 g, 0.80 mmol), DASD (1.10 mL, 8.30 mmol) and triethylamine (0.50 mL). The oily residue was purified by column chromatography with benzene-THF (1:1), and was crystallized from acetonitrile. Yield: 0.61 g (0.75 mmol, 90%). mp: 243  C. Anal. Calcd. for C31H42O6N8P4Cl2: C, 45.55; H, 5.14; N, 13.70. Found: C, 45.45; H, 5.27; N, 13.50. APIES-MS (fragments are based on 35Cl, Ir %): m/z 817 ([MH]þ, 100). FTIR (KBr, cm1): n 3068 (asymm.), 3028 (symm.) (CeH arom.), 2964, 2864 (CeH aliph.), 1583 (C]C), 1267 (asymm.), 1178 (symm.) (P]N), 551 (PCl). 1H NMR (500 MHz, CDCl3, ppm, numberings of protons are given in Scheme 1): d 7.25 (dd, 2H, H4), 6.95 (d, 2H, H5), 6.83 (d, 2H, H2), 6.77 (dd, 2H, H3), 4.74 (dd, 2H, 3JPH ¼ 12.4 Hz, 2JHH ¼ 8.5 Hz, AreCH2eN), 4.01 (s, 8H, OeCH2), 3.65 (dd, 2H, 3JPH ¼ 15.2 Hz, 2JHH ¼ 8.5 Hz, AreCH2eN), 3.53 [m, 8H, NeCH2 (DASD)], 3.09 [m, 4H, NeCH2 (spiro)], 1.79 [m, 2H, NeCH2eCH2 (spiro)], 1.79 [m, 8H, NeCH2eCH2 (DASD)]. 13C{1H} NMR (500 MHz, CDCl3, ppm, numberings of carbons are given in Scheme 1): d 150.37 (d, 2JPC ¼ 7.1 Hz, C6), 131.16 (s, C2), 130.09 (t, 3 JPC ¼ 9.6 Hz, C1), 129.75 (s, C4), 125.64 (s, C3), 123.87 (s, C5), 108.18 (s, OeCeO), 64.60 (s, OeCH2), 49.12 [s, NeCH2 (spiro)], 47.89 (s, AreCH2eN), 43.85 [s, NeCH2 (DASD)], 30.72 [s, NeCH2eCH2 (DASD)], 26.87 [s, NeCH2eCH2 (spiro)]. 4.2. Biological assays 4.2.1. Cytotoxicity of the compounds Cell Lines and culture. The following in vitro human cancer cell line was used: MCF-7 cancer cells were grown in RPMI-1640, which was supplemented with 10% of fetal calf serum, 5 mL of penicillin/ streptomycin (50 IU/mL and 50 mg/mL respectively), MTT assay. The cytotoxic effects of compounds were investigated by MTT assay using a colorimetric substrate tetrazolium salt, 3-[4,5-dimethylthiazollyl-2-yl]-2,5-diphenyltetrazolium bromide (Appli-Chem, Darmstadt, Germany) [39]. Control drug cisplatin, which is commonly used to treat cancer, was used as positive control. The cells are adjusted to 10.000 cells/well and exposed to 1be5b, 1c, 2c and 3ae5a at various concentrations (0.1 ng/mL, 1 mg/mL, 10 mg/mL, 100 mg/mL and 1 mg/mL) for 24 h. Following exposure of cells with MTT dye for 4 h, formazan products are dissolved in DMSO, and then the absorbance of the solution was determined by spectroscopy at a wavelength of 540 nm. 4.2.2. Determination of antituberculosis activity Susceptibility testing. M. tuberculosis H37Rv reference strain and M. tuberculosis clinical strain which is resistant to rifampicin and isoniazid were used for susceptibility testing by Mycolor TK (Salubris, Turkey) automatized system. Strains were inoculated to

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

TK medium containing serial dilutions (1000, 1250, 2500 and 5000 mM) of compounds. 4.2.3. Determination of antimicrobial activity Antibacterial susceptibility testing was performed by the BACTEC MGIT 960 (Becton Dickinson, Sparks, MD) systems using the agar-well diffusion method [40]. In this study, the organisms used in antimicrobial screening covered seven bacteria and two fungi given in Section 2.2.3. Bacterial strains were grown in nutrient agar medium and incubated at 37  C for 24 h. The yeast cells were cultured on Sabouraud dextrose agar medium and incubated at 30  C for 72 h. Chloramphenicol (30 mg) and ampicillin (10 mg) were used as controls. The solutions (2500 mM) of the compounds were obtained in DMSO. The experiments were made three times, and the mean values were used. Cultures were grown in exponential phase in nutrient broth at 37  C for 18 h, adjusted to a final concentration of 108 cfu/mL by diluting fresh cultures, and compared to McFarland density. The medium was prepared, mixed with culture suspension, and poured into plates. Wells with a 6.0 mm diameter were prepared and the solution (50 mL) of the 2500 mM test compound was added to the well. After incubation, the diameter of the inhibition zone was measured in millimeters. 4.2.4. Determination of the DNA interaction with the compounds The interactions of the tetrameric phosphazenes with the DNA were investigated using agarose gel electrophoresis [41]. The compounds were incubated with plasmid DNA in an incubator at 37  C for 24 h in the dark. The mixtures were loaded onto the 1% agarose gel with a loading buffer (0.1% bromophenol blue, 0.1% xylene cyanol). Electrophoresis was made in 0.05 M Tris base, 0.05 M glacial acetic acid and 1 mM EDTA (TAE buffer, pH ¼ 8.0) for 3 h at 60 V [42]. Eventually, the gel was stained with ethidium bromide (0.5 mg/mL), visualized under UV light using a transilluminator (BioDoc Analyzer, Biometra), photographed with a video-camera, and saved as a TIFF file. The experiments were repeated three times. Acknowledgments The authors thank the “Scientific and Technical Research Council of Turkey” (Grant No. 211T019), “Medicinal Plants and Medicine Research Center of Anadolu University, Eskis¸ehir, Turkey” for the use of their X-ray facilities, and Z. K. thanks Turkish Academy of Sciences (TÜBA) for partial support. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.10.005. References [1] V. Chandrasekhar, P. Thilagar, B.M. Pandian, Cyclophosphazene-based multisite coordination ligands, Coord. Chem. Rev. 251 (2007) 1045e1074. [2] Y. Kondo, “Phosphazene: Preparation, Reaction and Catalytic Role”, Superbases for Organic Synthesis: Guanidines, Amidines, Phosphazenes and Related Organocatalysts, John Wiley & Sons, Ltd., Wiltshire, 2009, pp. 145e185. [3] E. Okutan, B. Ços¸ut, S. Beyaz Kayıran, M. Durmus¸, A. Kılıç, S. Yes¸ilot, Synthesis of a dendrimeric phenoxy-substituted cyclotetraphosphazene and its noncovalent interactions with multiwalled carbon nanotubes, Polyhedron 67 (2014) 344e350. _ ¸ og
lu, Synthesis and characterization of some fused tricyclic spermidine [4] H. Ibis derivatives of cyclotriphosphazene, Heterocycles 71 (2007) 2173e2181. _ ¸ og _ Ün, F. Yüksel, Spiro, ansa-derivatives of cyclo
lu, A. Kılıç, I. [5] S. Bes¸li, H. Ibis tetraphosphazenes with a tetrafluorobutane-1,4-diol, Polyhedron 29 (2010) 3220e3228. [6] M. Gleria, R. De Jaeger, Phosphazenes: a Worldwide Insight, Nova Publishers, 2004.

675

_ ¸ og _ Ün, H. Dal, T. Ho
lu, Ç.G. Yenilmez, A. Kılıç, E. Tanrıverdi, I. € kelek, [7] H. Ibis Formation of novel spiro, spiroansa and dispiroansa derivatives of cyclotetraphosphazene from the reactions of polyfunctional amines with octachlorocyclotetraphosphazatetraene, J. Chem. Sci. 121 (2009) 125e135. [8] S. Bes¸li, S.J. Coles, D.B. Davies, R.J. Eaton, M.B. Hursthouse, A. Kılıç, R.A. Shaw, G.Y. Çiftçi, S. Yes¸ilot, The anomalous NMR behavior of meso compounds with remote stereogenic centers on addition of chiral shift or chiral solvating agents, J. Am. Chem. Soc. 125 (2003) 4943e4950. _ ¸ og
lu, A. Kılıç, S. Yes¸ilot, Synthesis and enantiomeric analysis of [9] B. Ços¸ut, H. Ibis cyclotriphosphazene derivatives with one centre of chirality, Inorg. Chim. Acta 362 (2009) 4931e4936. [10] S¸. S¸ahin Ün, Cyclotriphosphazenes having stereogenic phosphorus atoms: synthesis, characterization and their stereogenic properties, Polyhedron 70 (2014) 148e154. € kelek, L. Açık, H. Dal, [11] G. Elmas (nee Egemen), A. Okumus¸, Z. Kılıç, T. Ho
lu, L.Y. Koç, Phosphorusnitrogen compounds. Part 24. SynN. Ramazanog theses, crystal structures, spectroscopic and stereogenic properties, biological activities, and DNA interactions of novel Spiro-ansa-spiro- and ansaspiroansa-cyclotetraphosphazenes, Inorg. Chem. 51 (2012) 12841e12856. [12] I. Porwolik-Czomperlik, M. Siwy, D. S¸ek, B. Kaczmarczyk, A. Nasulewicz, I. Jaroszewicz, M. Pelczynska, A. Opolski, Synthesis and in vitro cytostatic activity of some new 1,3-(oxytetraethylenoxy)-cyclotrophosphazatriene derivatives, Acta Pol. Pharm. 61 (4) (2004) 267e272. [13] K. Brandt, T.J. Bartczak, R. Kruszynski, I. Porwolik-Czomperlik, AIDS-related lymphoma screen results and molecular structure determination of a new crown ether bearing aziridinylcyclophosphazene, potentially capable of ionregulated DNA cleavage action, Inorg. Chim. Acta 322 (2001) 138e144.
, L. Tomak, [14] T. Yıldırım, K. Bilgin, Y. Çiftçi, E.T. Eçik, E. S¸enkuytu, Y. Uludag A. Kılıç, Synthesis, cytotoxicity and apoptosis of cyclotriphosphazene compounds as anticancer, Eur. J. Med. Chem. 52 (2012) 213e220. €kelek, Y. Süzen, L.Y. Koç, L. Açık, Z.B. Çelik, [15] H. Akbas¸, A. Okumus¸, Z. Kılıç, T. Ho Phosphorusenitrogen compounds part 27. Syntheses, structural characterizations, antimicrobial and cytotoxic activities, and DNA interactions of new phosphazenes bearing secondary amino and pendant (4-fluorobenzyl)spiro groups, Eur. J. Med. Chem. 70 (2013) 294e307. [16] J.O. Bovin, J. Galy, J.F. Labarre, F. Sournies, Cyclophosphazenes as novel potential antitumor agents: x-ray crystal structure of the octapyrrolidinocyclotetraphosphazene, N4P4(NC4H8)8, J. Mol. Struct. 49 (1978) 421e423. [17] X. Zhu, Y. Liang, D. Zhang, L. Wang, Y. Ye, Y. Zhao, Synthesis and characterization of side groupemodified cyclotetraphosphazene derivatives, Phosphorus, Sulfur, Silicon Relat. Elem. 186 (2011) 281e286. _ [18] E.E. Ilter, N. Asmafiliz, Z. Kılıç, L. Açık, M. Yavuz, E.B. Bali, A.O. Solak, €kelek, Phosphorus-nitrogen compounds: part 19. F. Büyükkaya, H. Dal, T. Ho Syntheses, structural and electrochemical investigations, biological activities and DNA interactions of new spirocyclic monoferrocenylcyclotriphosphazenes, Polyhedron 29 (2010) 2933e2944. €kelek, Phosphorus[19] S. Bilge, A. Natsagdorj, S¸. Demiriz, N. Çaylak, Z. Kılıç, T. Ho nitrogen compounds: novel spirocyclic phosphazene derivatives. Structure of 0 0 0 0 0 3,3 '-Propane-1,3-diylbis[4 ,4 ,6 ,6 -tetracholoro-3,4-dihydrospiro[1,3,2benzoxazaphosphorine-2,20 l5-[4l5,6l5][1,3,5,2,4,6] triazatriphosphorine], Helv. Chim. Acta 87 (2004) 2088e2099. [20] A.B. Chaplin, J.A. Harrison, P. Dyson, Revisiting the electronic structure of phosphazenes, J. Inorg. Chem. 44 (2005) 8407e8417. € kelek, L.Y. Koç, L. Açık, M.L. Yola, [21] Y. Tümer, N. Asmafiliz, Z. Kılıç, T. Ho € A.O. Solak, Y. Oner, D. Dündar, M. Yavuz, Phosphorusenitrogen compounds: part 28. Syntheses, structural characterizations, antimicrobial and cytotoxic activities, and DNA interactions of new phosphazenes bearing vanillinato and pendant ferrocenyl groups, J. Mol. Struct. 1049 (2013) 112e124. € _ [22] M. Is¸ıklan, N. Asmafiliz, E.E. Ozalp, E.E. Ilter, Z. Kılıç, B. Ços¸ut, S. Yes¸ilot, A. Kılıç, € €kelek, L.Y. Koç, L. Açık, E. Akyüz, Phosphorusnitrogen comA. Oztürk, T. Ho pounds. 21. Syntheses, structural investigations, biological activities, and DNA interactions of new N/O spirocyclic phosphazene derivatives. The NMR behaviors of chiral phosphazenes with stereogenic centers upon the addition of chiral solvating agents, Inorg. Chem. 49 (2010) 7057e7071. [23] G.A. Carriedo, F.G. Alonso, P.A. Gonzalez, J.R. Menendez, Infrared and Raman spectra of the phosphazene high polymer [NP(O2C12H8)]n, J. Raman Spectrosc. 29 (1998) 327e330. [24] D. Cremer, J.A. Pople, General definition of ring puckering coordinates, J. Am. Chem. Soc. 97 (1975) 1354e1358. € kelek, Phosphorus-nitrogen compounds. [25] M. Is¸ıklan, Z. Kılıç, N. Akduran, T. Ho Part VI. Aminolysis of octachlorocyclotetraphosphazatetraene and the crystal structure of 2-trans-6-bis(n-propylamino)-2,4,4,6,8,8-hexakis-tert-butylaminocyclo-2l5, 4l5, 6l5, 8l5-tetraphosphazatetraene, J. Mol. Struct. 660 (2003) 167e179. [26] A.J. Wagner, Aafje Vos, The crystal structure of compounds with (N-P)n rings. IV. The stable modification (T form) of tetrameric phosphonitrilic chloride, N4P4Cl8, Acta Crystallogr. B24 (1968) 707e713. [27] E.W. Ainscough, A.M. Brodie, A.B. Chaplin, A. Derwahl, J.A. Harrison, C.A. Otter, Conformational flexibility in 2,20 -dioxybiphenyl-chloro-cyclotetraphosphazenes and its relevance to polyphosphazene analogues, Inorg. Chem. 46 (2007) 2575e2583. [28] F.H. Allen, O. Kennard, D.G. Watson, L. Brammer, G. Orpen, R. Taylor, Tables of bond lengths determined by X-ray and neutron diffraction. Part 1. Bond lengths in organic compounds, J. Chem. Soc. Perkin Trans. 2 (12) (1987) 1e19.

676

G. Elmas et al. / European Journal of Medicinal Chemistry 87 (2014) 662e676

[29] R.J. Davidson, E.W. Ainscough, A.M. Brodie, J.A. Harrison, M.R. Waterland, The nature of the phosphazene nitrogenemetal bond: DFT calculations on 2(Pyridyloxy)cyclophosphazene complexes, Eur. J. Inorg. Chem. (2010) 1619e1625. € €kelek, H. Dal, L. Açık, Y. Oner, [30] A. Okumus¸, Z. Kılıç, T. Ho L.Y. Koç, Phosphorusenitrogen compounds part 22. Syntheses, structural investigations, biological activities and DNA interactions of new mono and bis (4-fluorobenzyl) spirocyclophosphazenes, Polyhedron 30 (2011) 2896e2907. [31] L.J. Farrugia, ORTEP-3 for Windows e a version of ORTEP-III with a Graphical User Interface (GUI), J. Appl. Crystallogr. 30 (1997) 565e566. [32] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, John Wiley and Sons, Inc, Hoboken, 2001, pp. 230e231. € € Kısa, A. Albay, € kelek, L.Y. Koç, L. Açık, O. [33] N. Asmafiliz, Z. Kılıç, A. Oztürk, T. Ho
, A.O. Solak, Phosphorus-nitrogen compounds. 18. Syntheses, Z. Üstündag stereogenic properties, structural and electrochemical investigations, biological activities, and DNA interactions of new spirocyclic mono- and bisferrocenylphosphazene derivatives, Inorg. Chem. 48 (2009) 10102e10116. [34] A.M. Escalante, R.T. McGrath, M.R. Karolak, R.T. Dorr, R.M. Lynch, T.H. Landowski, Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo, Cancer Chemother. Pharmacol. 71 (2013) 1567e1576.

[35] A.A. Legin, M.A. Jakupec, N.A. Bokach, M.R. Tyan, V.Y. Kukushkin, B.K. Keppler, Guanidine platinum(II) complexes: synthesis, in vitro antitumor activity, and DNA interactions, J. Inorg. Biochem. 133 (2014) 33e39. [36] Bruker program 1D WIN-NMR (release 6.0) and 2D WIN-NMR (release 6.1). [37] Bruker, SADABS, Bruker AXS Inc, Madison, Wisconsin, USA, 2005. [38] G.M. Sheldrick, A short history of SHELX, Acta Crystallogr. Sect. A. 64 (2008) 112e122. [39] T. Mossmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55e63. [40] Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobia Susceptibility Testing Sixteenth Informational Supplement, CLSI Document M100eS16, Pennsylvania, 2006. € €
lu, A. Okumus¸, Z. Kılıç, A. Oztürk, €kelek, Y. Oner, [41] S.B. Koçak, S. Koçog T. Ho L. Açık, Syntheses, spectroscopic properties, crystal structures, biological activities, and DNA interactions of heterocyclic amine substituted spiro-ansa-spiro- and spirobino-spiro-phosphazenes, Inorg. Chim. Acta 406 (2013) 160e170. [42] H. Cheng, F. Huq, P. Beale, K. Fisher, Synthesis, characterisation, activities, cell uptake and DNA binding of a trinuclear complex: [{trans-PtCl(NH3)}2m-{transPd(NH3)(2-hydroxypyridine)-(H2N(CH2)6NH2)2]Cl4, Eur. J. Med. Chem. 41 (2006) 896e903.