Synthesis of two acridine conjugates of the bis(phenanthroline) ligand 'Clip-Phen' and evaluation of the nuclease activity of the corresponding copper complexes

Article abstract of DOI:10.1002/(SICI)1099-0682(199903)1999:3<557::AID-EJIC557>3.0.CO;2-Y

The synthesis of three novel derivatives [N-acetyl-Clip-Phen (1), Clip- Phen-hexylaminoacridine (4) and Clip-Phen-hexylchloromethoxyaminoacridine (5)] of the bis(phenanthroline) ligand 'Clip-Phen', are described. Complexation of these ligands with copper(II) afforded the 1:1 complexes as hexafluorophosphate salts. The relaxation of φX 174 DNA was used as a DNA cleavage assay, with the following results. [Cu(1)]²⁺ was found to show a diminished activity relative to the parent complex [Cu(Clip-Phen)]²⁺. However, the acridine-containing complexes [Cu(4)H]³⁺ and [Cu(5)H]³⁺ exhibited significantly enhanced cleavage efficiencies, which has been attributed to increased affinity of the complexes for DNA. UV/Vis-spectral data of the complexes in the presence of calf-thymus DNA was consistent with an intercalative mode of binding.

Full text of DOI:10.1002/(SICI)1099-0682(199903)1999:3<557::AID-EJIC557>3.0.CO;2-Y

FULL PAPER
Synthesis of Two Acridine Conjugates of the Bis(phenanthroline) Ligand
“Clip-Phen” and Evaluation of the Nuclease Activity of the
Corresponding Copper Complexes
[a]
[a]
[a]
´
Steven A. Ross, Marguerite Pitie, and Bernard Meunier*
Keywords: Bioinorganic chemistry / Copper / DNA cleavage / Intercalation
The synthesis of three novel derivatives [N-acetyl-Clip-Phen
(1), Clip-Phen-hexylaminoacridine (4) and Clip-Phen-
hexylchloromethoxyaminoacridine (5)] of the bis(phen-
anthroline) ligand “Clip-Phen”, are described. Complexation
of these ligands with copper(II) afforded the 1:1 complexes
as hexafluorophosphate salts. The relaxation of φΧ 174 DNA
was used as a DNA cleavage assay, with the following
results. [Cu(1)]2+ was found to show a diminished activity
relative to the parent complex [Cu(Clip-Phen)]2+. However,
the acridine-containing complexes [Cu(4)H]3+ and [Cu(5)H]3+
exhibited significantly enhanced cleavage efficiencies,
which has been attributed to increased affinity of the
complexes for DNA. UV/Vis-spectral data of the complexes
in the presence of calf-thymus DNA was consistent with an
intercalative mode of binding.
calation of polyaromatic species between adjacent base
pairs was first proposed by Lerman[8] in 1961. Efficient in-
tercalators must be planar and often contain three fused
six-membered rings. The presence of a positive charge usu-
ally aids in the binding process.[9] Although intercalation
has been studied by various groups, the overall binding pro-
cess is complex, partly due to ambiguities in the actual
binding mode (intercalation, minor-groove binding, or elec-
trostatic interactions) and difficulties in measuring associ-
ation constants.[10] Certain intercalators, such as daunomy-
cin, adriamycin, and m-AMSA exhibit anti-tumour activity,
in a process which is believed to involve the disruption of
topoisomerase activity in vivo.[11] Association constants for
intercalation are broadly comparable, normally between 105
and 106 Ϫ1, and there is a general lack of correlation be-
tween affinity and pharmacological efficacy. Overall, there
is little sequence specificity observed in the interactions of
intercalators with DNA.[9]
We wished to quantify the nuclease efficiency of an inter-
calatorϪClip-Phen hybrid, such that the reactive Clip-Phen
fragment would be anchored to the DNA. We chose to at-
tach acridine derivatives for several reasons. Firstly, acri-
dines are one of the most studied of the intercalating
groups. Secondly, acridines are known to rapidly enter cells
and accumulate in the nucleus,[12] which is an important
consideration with regard to in vivo activity. For example,
a netropsinϪacridine hybrid has been reported to accumu-
late in the cell nucleus, using the acridine moiety as the cell-
penetration vector,[13] whereas netropsin itself exhibits slow
kinetics of cell penetration and poor uptake by the nu-
cleus.[14] Finally, conjugation to acridines was considered to
be the most straightforward and least expensive synthetic
route available. Synthetic strategies to oligonucleotide-inter-
calator hybrids[15] and minor-groove binder-intercalator hy-
brids[16] have been reviewed recently in the literature.
Introduction
The artificial nuclease capability of redox-active copper
complexes of the chelating ligand 1,10-phenanthroline
(Phen) is well established.[1][2] Single-strand cleavage of nu-
cleic acids is observed in the presence of dioxygen and a
reductant, by oxidative attack on deoxyribose units from
the DNA minor groove.[1] The reactive species (Phen)2CuI
is significantly more active than the (Phen)CuI species,[3]
although the association constant for the second Phen li-
gand[4] is only 105.5

Ϫ1. Competitive binding of copper
can thus be a problem in cleavage experiments, and may
hamper the use of (Phen)copper compexes in vivo. With
these concepts in mind, we recently reported the synthesis
of the novel bis(phenanthroline) ligand “Clip-Phen”,
whereby two Phen ligands are linked at the 2Ј position by
a serinol bridge.[5] The artificial nuclease activity of this
new ligand was found to be higher than Phen itself (by a
factor of 2) and, in addition, the serinol bridge contains an
exogenous amine group, which allows further functionali-
sation.
Attachment of the natural polyamine spermine to Clip-
Phen also afforded a new conjugate with even greater nucle-
ase efficiency.[6] This was attributed to the high affinity of
the spermine moiety for nucleic acids, through electrostatic
interactions with the anionic phosphate backbone.[7] Cleav-
age reactions on restriction fragment DNA showed that
there was very little sequence specificity of this Clip-Phen-
spermine bioconjugate.
Attachment of an intercalating agent to Clip-Phen was
the motivation for the current work. DNA binding by inter-
[a]
Laboratoire de Chimie de Coordination du CNRS,
205 route de Narbonne, F-31077, Toulouse cedex 4, France
E-mail: bmeunier@lcc-toulouse.fr
Eur. J. Inorg. Chem. 1999, 557Ϫ563
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ1948/99/0303Ϫ0557 $ 17.50ϩ.50/0
557

´
S. A. Ross, M. Pitie, B. Meunier
FULL PAPER
raphic separation. 1H-NMR data from side-products ob-
tained suggested that this was due to decomposition of the
Clip-Phen fragment in hot phenol, possibly due to nucleo-
philic attack of phenol at the C-2Ј position of the phen-
anthroline. We chose, therefore, to isolate the 9-phenoxyac-
ridine derivatives prior to reaction with 3. Reaction of 9-
phenoxyacridine with 3 in refluxing acetonitrile afforded
the Clip-Phen-hexylaminoacridine (4) in 30Ϫ40% yield
after chromatography. However, the corresponding reaction
between 6-chloro-9-phenoxy-2-methoxyacridine and 3 pro-
ceeded in very poor yield. Addition of 5Ϫ10 equivalents of
acid (usually acetic acid) was found to greatly improve the
yield by increasing the electrophilicity of the C-9ЈЈ carbon
atom.[19] Subsequent addition of acid to the reaction be-
tween 9-phenoxyacridine and 3 also led to a significant en-
hancement in yield (to 74%). Products 4 and 5 (see Scheme
1) were bright yellow, fluorescent solids, which made chro-
Results and Discussion
Ligand Synthesis
Clip-Phen was readily prepared according to the pre-
viously reported procedure.[5] The primary amine of Clip-
Phen presents a convenient site for further functionali-
sation. However, previous reports[17] have suggested that
the reactive fragments in intercalator hybrids show greater
efficacy if separated by a polymethylene linker. We chose,
therefore, to protect the amine function of 6-aminohexanoic
acid (with the BOC group) allowing facile attachment of
the carboxylate to Clip-Phen by amide bond formation. De-
protection of Clip-Phen-hexyl-BOC (2) proceeded smoothly
to afford Clip-Phen-hexylamine (3) in 80% yield (see
Scheme 1).
1
matographic purification simple, and gave satisfactory H-
NMR and mass spectra and analytical data.
Since the Clip-Phen fragments of both 4 and 5 contain
an amide function in place of the primary amine, we also
wished to prepare an analogous Clip-Phen amide derivative
(without an intercalating group) to allow direct comparison
of reactivities. This was readily achieved by reaction of
acetic anhydride with Clip-Phen in CH2Cl2, to afford N-
acetyl Clip-Phen (1) (see Figure 1) in 94% yield. The DNA
cleavage reactivity of (phenathroline)CuII derivatives
necessitates reduction to CuI, and thus formation of a tetra-
hedral intermediate. By converting the amine of Clip-Phen
to the amide in 1, we would thus avoid any differences in
the coordination sphere or any steric constraints upon re-
duction.
Figure 1. N-Acetyl Clip-Phen (1)
Complex Formation with CuII Salts
The coordination chemistry of the Clip-Phen family of
ligands (L) with CuII is not straightforward.[20] The 5-atom
bridge between the two phenanthrolines leads to little
thermodynamic stability of the mononuclear species
Scheme 1. Synthesis of ligands 4 and 5, and their corresponding
Ϫ
CuII/PF6 complexes; conditions: (i) 6-BOC-amino-1-hexanoic
[Cu(L)]2ϩ
, with respect to higher nuclearity species
acid, EtOCOCl; (ii) 25% CF3COOH in CH2Cl2; (iii) appropriate 9-
phenoxyacridine, CH3CN, 10 equiv. CH3COOH; (iv) Cu(OAc)2 · 2
H2O, MeOH, then NH4PF6
[Cun(L)n]2nϩ, in a process that may be dependent upon con-
centration and solvent. Complexes with a Cu/L ratio of 2:1
Primary amines are known to react with 9-chloroacridine have also been observed when the CuII concentration is
derivatives in hot phenol (by in situ 9-phenoxyacridine for- higher than that of the ligand. In general, we found that
mation) to give the 9-amino acridines in high yield.[18] We isolation of solid products of overall stoichiometry 1:1 (L/
encountered several problems with this reaction, only being Cu) was problematic when CuCl2 · 2 H2O or Cu(SO4) · 4
able to isolate products in very low yield after chromatog- H2O were used as the starting copper salts (data not
558
Eur. J. Inorg. Chem. 1999, 557Ϫ563

Synthesis of Two Acridine Conjugates of the Bis(phenanthroline) Ligand “Clip-Phen”
FULL PAPER
shown). We found that a reproducible procedure for com-
plex formation with 1, 4, and 5 was to treat one equivalent
of Cu(OAc)2 · H2O with one equivalent of ligand in warm
methanol for two hours. Addition of an excess of NH4PF6
gave a precipitate which was redissolved by addition of ace-
tone. Upon concentration of this solution, the products pre-
cipitated and could be isolated by centrifugation. Analytical
data and mass spectra were consistent with the formation
of L/CuII 1:1 complexes. The pKa of the acridine fragment
(> 10) is sufficiently high to allow proton transfer from the
ammonium ion of NH4PF6 (pKa ϭ 9.3), such that the inter-
calator is present as an acridinium hexafluorophophate in
the complexes of both 4 and 5 (see Scheme 1). This did not
present a problem, however, since the intercalators are also
protonated under biological conditions, and under the con-
ditions used for the DNA experiments described (phosphate
buffer, pH ϭ 7.2). The UV/Vis spectra of the complexes
exhibited the expected ligand absorptions between 200 and
450 nm, in addition to a broad CuII d-d band at circa 810
nm (ε ഠ 150 Ϫ1 cmϪ1).
The 1:1 copper complex of Clip-Phen was prepared by
using the previously reported synthesis of [Cu(Clip-
Phen]Cl2.[5] After equimolar quantities of Clip-Phen and
anhydrous CuCl2 were stirred in DMF for 4 h, the chloride
salt was precipitated with ether. This was redissolved in
MeOH and an excess of NH4PF6 was added to afford a
green precipitate, which was isolated by centrifugation. The
analytical data was consistent with the overall stoichi-
ometry [Cu(Clip-Phen)](PF6)2. The absence of a third hexa-
fluorophosphate anion in this complex indicates that the
exogenous primary amine of Clip-Phen is not protonated.
Figure 2. Comparison of Φx 174 cleavage efficiency between Clip-
Phen, 1, 4, and 5 (1 µ) in the presence of CuCl2 (1 µ) and MPA
(5 m); lane 1: control, no complex, no MPA; lane 2: control, no
complex, with MPA; lanes 3, 5, 7, and 9: Clip-Phen, 1, 4, and 5,
respectively, without addition of Cu; lanes 4, 6, 8, and 10: Clip-
Phen, 1, 4 and 5, respectively, in the presence of Cu
Figure 2 shows a representative gel illustrating the rela-
tive reactivities of the 4 complexes at 1 µ concentration.
The reactivity of Clip-Phen-copper (1.4 ± 0.1 strand breaks
per plasmid) is consistent with the previously reported
data,[5] showing a cleavage activity which is approximately
twice that of phenanthroline itself. However, N-acetyl Clip-
ϪPhenϪcopper shows a diminished reactivity (0.9 ± 0.1
strand breaks per plasmid), either due to the lack of a pri-
mary amine to coordinate or due to the steric effects of the
bulkier amide group. The lack of protonation of the pri-
mary amine in the [Cu(Clip-Phen)](PF6)2 complex is con-
sistent with coordination to the CuII centre, although it is
unlikely to be coordinated upon reduction to CuI. The cop-
per complexes of the acridine-containing ligands 4 and 5
both exhibit cleavage activities of circa 4 ± 1, which is sig-
nificantly higher than the two non-acridine-containing li-
gands. A direct comparison between 1 and 4 suggests an
approximate fourfold increase in reactivity. Since the esti-
mation of cleavage efficiency is less accurate after the disap-
pearance of form 1, the variation in reactivity with concen-
tration was also measured for 1 and 4, this data being pre-
sented in Figure 3. Enhanced reactivity is thus observed at
all concentrations.
DNA Cleavage Activity
Relaxation of supercoiled circular Φx 174 DNA (form I)
into relaxed circular (form II) and linear (form III) confor-
mations was the assay used to assess the relative cleavage
efficiency of the four complexes. The redox activity of the
CuII centres was initiated by the addition of 5 m mercap-
topropionic acid (MPA) in the presence of air.
Reactions were carried out by using either the isolated
hexafluorophosphate salts described, or by premetallating
the ligands with CuCl2 in DMF/H2O (40:60, 1 m in li-
gand, 1 m in CuCl2) for 2 h prior to dilution and addition
to DNA. The DNA/complex mixtures were allowed to equi-
librate for 30 min at room temperature prior to addition
of MPA. Several reactions were also carried out by pre-
equilibration of the ligand (1 µ, 15 min) with DNA, fol-
lowed by CuCl2 (1 µ, 15 min), or by pre-equilibration of
CuCl2 (1 µ, 15 min) with DNA, followed by ligand (1 µ,
15 min). The choice of counterion (chloride or hexafluoro-
phosphate) or order of addition was found to have a negli-
gible effect upon the degree of reactivity (within experimen-
tal error). Since stock solutions of the hexafluorophosphate
salts appeared to decompose over time (becoming an or-
Figure 3. Concentration dependence of Φx 174 cleavage efficiency
for 1 (solid) and 4 (hatched) in the presence of equimolar CuCl2
Spectrophotometric Studies of DNA Binding
The binding of the complexes and ligands described
ange-brown colour), we chose to freshly prepare our com- herein to DNA is expected to be complex, since both moie-
plexes by metallation with CuCl2 prior to reaction.
ties (the acridine fragment and the CuϪClip-Phen frag-
Eur. J. Inorg. Chem. 1999, 557Ϫ563
559

´
S. A. Ross, M. Pitie, B. Meunier
FULL PAPER
ment) each have a separate affinity for DNA. Intercalative Table 1. UV/Vis data in the region 370Ϫ470 nm for 4, 5, 4 ϩ CuCl2,
5 ϩ CuCl2, 6, and 7 (10 µ) in phosphate buffer (pH ϭ 7.2): εf
binding of acridine derivatives is well established, and gives
and λf are the values in the absence of calf-thymus DNA; εb and
rise to small (but significant) bathochromic and hypo-
chromic shifts in the visible spectrum.[21] This technique
was therefore used as a guide to whether intercalation was
taking place with derivatives 4 and 5. The alkylamino de-
rivatives 9-(3-methoxyprop-1-ylamino)acridine (6) and 6-
chloro-2-methoxy-9-(3-methoxyprop-1-ylamino)acridine
(7) (see Figure 4) were also prepared and studied, since
these analogues should have the same chromophores as 4
and 5, without containing the clip-phen fragment.
λb are the values in the presence of 20 base-pair equivalents of calf-
thymus DNA
λf (εf)
λb (εb)
4
396 (7000)
415 (8600)
437 (8100)
398 (4400)
419 (5700)
440 (5400)
4 ϩ CuCl2
394 (4200)
414 (6800)
437 (5500)
397 (2700)
417 (4100)
440 (3700)
6
395 (4500)
410 (8100)
432 (6900)
398 (2900)
416 (5800)
436 (5000)
5
433 (7800)
455 (7700)
428 (9700)
450 (9500)
5 ϩ CuCl2
426 (2900)
444 (2700)
427 (8100)
451 (7700)
7
420 (13300)
443 (12700)
429 (6800)
451 (6900)
Figure 4. Alkylaminoacidire derivatives 6 and 7
The spectrophotometric studies were carried out under
identical buffer and salt conditions to the DNA cleavage
experiments. Spectral shifts were recorded for DNA base-
pair ratios of 0, 1, 4, 12, and 20 equivalents. We did not
attempt to interpret this data in terms of binding constants,
since the CuϪClip-Phen fragment may also be binding in
the minor groove.
Compounds 4, 4 ϩ CuCl2, and 6 all gave bathochromic
shifts of around 4 nm and hypochromic shifts of around
30% upon the addition of calf-thymus DNA (see Table 1),
which were in good agreement with data previously re-
ported in the literature.[21] We conclude from these results
that the binding process of the free ligand (4), its copper
complex (4 ϩ CuCl2) and a related alkylaminoacridine (6)
all involve intercalative binding of the acridine fragment.
The spectral shifts of 4 ϩ CuCl2 and 6 upon titration with
calf-thymus DNA are shown in Figure 5.
The data obtained from the 9-amino-6-chloro-2-meth-
oxyacridine derivatives was more complex, as can be evi-
denced from the data in Table 1. Compounds 5, 5 ϩ CuCl2,
and 7 all show similar λb and εb in the presence of excess
DNA, suggesting that the acridine fragment is in a similar
environment for all three upon DNA binding. However, in
the absence of DNA, whilst the free acridine compound 7
exhibits the expected behaviour, 5 and its copper complex
show anomalous behaviour. This is probably due to stack-
Figure 5. UV/Vis titration of 6 (top) and 4 ϩ CuCl2 (bottom) (all
10 µ) against calf-thymus DNA (0, 1, 4, 12, and 20 base-pair
equivalents) in phosphate buffer (pH ϭ 7.2) in the presence of
NaCl (50 m) and MgCl2 (10 m)
Conclusions
We have described the synthesis of two novel conjugate
ing interactions of the acridine fragment with the phen- ligands (4 and 5) and their copper(II) complexes, which
anthroline rings in the absence of DNA, particularly with contain a reactive DNA cleavage unit attached to an inter-
free 5, which shows a 12-nm red-shift, relative to its Clip- calating DNA recognition unit. The presence of the inter-
Phen-free analogue 7.
calator has been shown to significantly enhance the DNA
The in vivo activity of compounds 4 and 5 is currently cleavage activity, in spite of the diminished activity which is
being investigated and will be reported in due course.
560
found upon blockage of the exogenous primary amine of
Eur. J. Inorg. Chem. 1999, 557Ϫ563

Synthesis of Two Acridine Conjugates of the Bis(phenanthroline) Ligand “Clip-Phen”
FULL PAPER
(609 mg, 5.62 mmol). After stirring for 30 min, the solvent was
removed in vacuo, the residue dissolved in CH2Cl2, Clip-Phen (1
g, 2.23 mmol) added, and the solution stirred at room temperature
for 3 h. The solvent was removed in vacuo, the residue redissolved
in CH2Cl2 (50 mL), and the solution was washed with aqueous
KHCO3 (5%, 3 ϫ 50 mL), H2O (3 ϫ 50 mL), dried (MgSO4), and
concentrated in vacuo to afford the product as an oily solid (1.22
the parent Clip-Phen from which they are derived. Thus,
these species are of potential interest for in vivo appli-
cations.
Experimental Section
Warning: Acridines and ethidium bromide are mutagenic and
1
g, 83%). Ϫ H NMR (CD2Cl2): δ ϭ 1.0Ϫ1.2 (m, 4 H, CH2CH2),
should always be handled with appropriate precautions.
1.38 [s, 9 H, C(CH3)3] 1.41 (m, 2 H, CH2), 2.24 [t, 2 H, J ϭ 7.5
Hz, CH2C(O)NH], 2.88 [m, 2 H, CH2NHC(O)O], 4.55 [br. s, 1 H,
OC(O)NH ], 4.84 (m, 3 H, serinol bridge), 5.06 (m, 2 H, serinol
bridge), 7.14 (d, 2 H, J ϭ 8.5 Hz, 3Ј-H), 7.62 (dd, 2 H, J ϭ 8, 4.25
Hz, 8Ј-H), 7.71 (d, 2 H, J ϭ 8.75 Hz, 6Ј-H) 7.77 (d, 2 H, J ϭ 8.75
Hz, 5Ј-H), 8.14 (d, 2 H, J ϭ 8.5 Hz, 4Ј-H), 8.27 (dd, 2 H, J ϭ 1.5,
8 Hz, 7Ј-H), 8.69 [d, 1 H, J ϭ 5.8 Hz, CH2C(O)NH], 9.10 (dd, 2
H, J ϭ 1.5, 4.25 Hz, 9Ј-H). Ϫ DCI MS; m/z (%): 661 (72) [MHϩ].
Ϫ C38H40N6O5 (660).
General: Reactions were monitored by thin-layer chromatography
using Macherey-Nagel Alugram Sil G/UV 254 plates and spots
were visualized with UV light. Ϫ Proton NMR spectra were re-
corded with a Bruker 250 MHz instrument and referenced to deut-
erated solvent. Aromatic proton signals due to phenanthroline are
denoted by a single prime (Ј), those due to acridine are denoted by
double prime (ЈЈ). Ϫ UV/Vis spectra were recorded with a Hewlett-
Packard 8452A diode-array spectrophotometer in cuvettes of 1 cm
path length. Ϫ Electrospray mass spectra were recorded with a Per-
kin-Elmer SCIEX API 100 instrument. Ϫ Ligand metallation and
DNA reactions were carried out in Nanopure deionized water.
DMF was dried with calcium sulfate. Other commercial reagents
were used without purification.
Clip-Phen-hexylamine (3): 2 (1 g, 1.52 mmol) was dissolved in 25%
(v/v) of CF3COOH/CH2Cl2 (40 mL) and the solution stirred for 1
h at room temperature. The solvent was then removed in vacuo, the
residue dissolved in H2O, and the pH adjusted to 12 (1  NaOH).
Extraction of this solution with CH2Cl2, followed by drying
(MgSO4) and concentration in vacuo afforded the product as a pale
yellow solid (823 mg, 97%). Ϫ 1H NMR (CD2Cl2): δ ϭ 1.0Ϫ1.2
(m, 4 H,CH2CH2), 1.41 (m, 2 H, CH2), 2.05 (br. s, 2 H, CH2NH2),
2.21 [t, 2 H, J ϭ 7.8 Hz, CH2C(O)NH], 2.44 (t, 2 H, J ϭ 6.5 Hz,
CH2NH2), 4.85 (m, 3 H, serinol bridge), 5.06 (m, 2 H, serinol
bridge), 7.14 (d, 2 H, J ϭ 8.5 Hz, 3Ј-H), 7.58 (dd, 2 H, J ϭ 8, 4.25
Hz, 8Ј-H), 7.69 (d, 2 H, J ϭ 8.75 Hz, 6Ј-H), 7.76 (d, 2 H, J ϭ 8.75
Hz, 5Ј-H), 8.13 (d, 2 H, J ϭ 8.5 Hz, 4Ј-H), 8.26 (dd, 2 H, J ϭ 1.5,
8 Hz, 7Ј-H), 8.69 [d, 1 H, J ϭ 5.8 Hz, CH2C(O)NH], 9.06 [dd, 2
H, J ϭ 1.5, 4.25 Hz, 9Ј-H]. Ϫ DCI MS; m/z (%): 561 (100) [MHϩ].
Syntheses
2-Amino-1,3-bis(1Ј,10Ј-phenanthrolin-2Ј-yloxy)propane (Clip-Phen)
was prepared by reaction of 2-chloro-1,10-phenathroline with seri-
nol according to the method described previously.[5]
6-(Butoxycarbonylamino)-1-hexanoic Acid: 6-Aminohexanoic acid
(1 g, 7.6 mmol), MgO (296 mg, 7.6 mmol), and NaOH (305 mg,
7.6 mmol) were added to 40 mL of dioxane/H2O (6:1). Di-tert-
butyl dicarbonate (1.83 g, 8.39 mmol) was then added in portions
over 20 min, the solution was stirred for 18 h at room temperature,
filtered, and concentrated in vacuo. The residue was dissolved in
H2O (20 mL), washed with diethyl ether (20 mL), acidified to pH ϭ
3 with acetic acid (10%), and extracted with ethyl acetate (2 ϫ 50
mL). The organic was then dried (MgSO4) and concentrated to
afford the product as a colourless oil (1.56 g, 89%). Ϫ 1H NMR
(CD2Cl2): δ ϭ 1.41 [s, 9 H, C(CH3)3], 1.3Ϫ1.5 (m, 4 H, CH2CH2),
1.59 (m, 2 H, CH2), 2.34 (t, 2 H, J ϭ 7.5 Hz, CH2COOH), 3.06
[m, 2 H, CH2NHC(O)O], 4.67 (br. s, 1 H, CH2NH). Ϫ DCI MS;
m/z (%): 232 (63) [MHϩ], 249 (56) [MNH4ϩ]. Ϫ C11H21NO4
(231.3): calcd. (ϩ 0.5 H2O) C 54.9, H 9.15, N 5.82; found C 55.5,
H 8.91, N 5.84.
9-Chloroacridine: Acridone (2 g, 10.3 mmol) and POCl3 (20 mL)
were placed in a round-bottomed flask and heated at reflux for 1
h. The unused POCl3 was then removed by distillation under vac-
uum, the residue cooled to 50°C, diluted with CHCl3 (20 mL), and
poured into aqueous ammonia (30%, 10 mL) and ice (25 g). After
the organic layer was separated and the aqueous further extracted
with CHCl3 (3 ϫ 50 mL), the organic layers were combined, dried
(MgSO4), and concentrated to 10 mL, and then hexane was added.
Filtration afforded the product as a yellow solid (1.99 g, 91%). Ϫ
1H NMR (CD2Cl2): δ ϭ 7.67 (dd, 2 H, J ϭ 9.9 Hz, 2ЈЈ-H or 3ЈЈ-
H), 7.82 (dd, 2 H, J ϭ 9.9 Hz, 2ЈЈ-H or 3ЈЈ-H), 8.20 (d, 2 H, J ϭ
9 Hz, 1ЈЈ-H or 4ЈЈ-H), 8.44 (d, 2 H, J ϭ 9 Hz, 1ЈЈ-H or 4ЈЈ-H). Ϫ
DCI MS; m/z (%): 214 (100) [MHϩ]. Ϫ C13H8ClN (213): calcd. C
73.0, H 3.77, N 6.56; found C 72.7, H 3.35, N 6.41.
N-Acetyl Clip-Phen (1): Clip-Phen (447 mg, 1 mmol) was dissolved
in CH2Cl2, to which was added Et3N (1 mL) and acetic anhydride
(1 mL). Upon standing for 2 h at room temperature, the solvent
was removed in vacuo, the residue redissolved in CH2Cl2 (10 mL),
washed with aqueous NaOH (10 mL, 1 ), and dried (MgSO4).
The solution was then concentrated to ca. 2 mL, and hexane was
added to precipitate the product as a white solid (460 mg, 94%),
9-Phenoxyacridine: 9-Chloroacridine (680 mg, 3.2 mmol) was ad-
ded to a solution of NaOH (200 mg, 5 mmol) in phenol (3 g, 32
mmol), the mixture was stirred at 100°C for 2 h, then poured into
NaOH solution (2 , 50 mL). The solution was then extracted with
CHCl3 (3 ϫ 50 mL) and the solution dried (MgSO4) and concen-
trated in vacuo. Recrystallisation of the residue from hot MeOH/
H2O (50:50) afforded the product as pale yellow needles (770 mg,
89%). Ϫ 1H NMR (CD2Cl2): δ ϭ 6.87 (m, 2 H, phenoxy 2-H), 7.05
(m, 1 H, phenoxy 4-H), 7.27 (m, 2 H, phenoxy 3-H) 7.47 (dd, 2 H,
J ϭ 9.9 Hz, 2ЈЈ-H or 3ЈЈ-H), 7.78 (dd, 2 H, J ϭ 9, 9 Hz, 2ЈЈ-H or
3ЈЈ-H), 8.11 (d, 2 H, J ϭ 9 Hz, 1ЈЈ-H or 4ЈЈ-H), 8.25 (d, 2 H, J ϭ
9 Hz, 1ЈЈ-H or 4ЈЈ-H). Ϫ DCI MS; m/z (%): 272 (100) [MHϩ].
C19H13NO (271).
1
which was filtered off and dried in vacuo. Ϫ H NMR (CD2Cl2):
δ ϭ 2.03 (s, 3 H, CH3), 4.84 (m, 3 H, serinol bridge), 5.04 (m, 2
H, serinol bridge), 7.15 (d, 2 H, J ϭ 8.5 Hz, 3Ј-H), 7.59 (dd, 2 H,
J ϭ 8, 4.25 Hz, 8Ј-H), 7.70 (d, 2 H, J ϭ 8.75 Hz, 6Ј-H), 7.77 (d, 2
H, J ϭ 8.75 Hz, 5Ј-H), 8.14 (d, 2 H, J ϭ 8.5, 4Ј-H), 8.27 (dd, 2 H,
J ϭ 1.5, 8 Hz, 7Ј-H), 9.07 [br. s, 1 H, CH3C(O)NH], 9.07 (dd, 2
H, J ϭ 1.5, 4.25 Hz, 9Ј-H). Ϫ DCI MS; m/z (%): 490 (100) [MHϩ].
Ϫ C29H23N5O3 (489): calcd. (ϩ 0.33 CHCl3, 0.5 H2O) C 65.4, H
4.43, N 13.1; found C 65.4, 4.49, N 13.4.
Clip-Phen-hexyl-BOC (2): 6-Butoxycarbonylamino-1-hexanoic acid
(1.3 g, 5.62 mmol) was dissolved in CH2Cl2 (10 mL) at 0°C, to
6-Chloro-2-methoxy-9-phenoxyacridine: 6,9-Dichloro-2-methoxy-
acridine (2 g, 7 mmol) was added to a solution of NaOH (400 mg,
which was then added Et3N (567 mg, 5.62 mmol) and EtOC(O)Cl 10 mmol) in phenol (10 g, 106 mmol), the mixture was stirred at
Eur. J. Inorg. Chem. 1999, 557Ϫ563 561

´
S. A. Ross, M. Pitie, B. Meunier
FULL PAPER
100°C for 2 h and then poured into NaOH solution (2 , 50 mL). 1 H, NHCO), 9.00 (dd, 2 H, J ϭ 1.5, 4.25 Hz, 9Ј-H). Ϫ DCI MS;
After this solution had been extracted with CHCl3 (3 ϫ 50 mL),
m/z (%): 802 (1.5) [MHϩ], 606 (100) [MHϩ Ϫ phenanthrolyloxy],
the combined organic layers were dried (MgSO4) and concentrated 197 (89) [hydroxyphenathrolineHϩ]. Ϫ C47H40ClN7O3 (801.5):
in vacuo. Recrystallisation of the residue from hot MeOH afforded
calcd. (ϩ CH2Cl2) C 64.9, H 4.73, N 11.1; found C 65.1, H 4.50,
the product as yellow needles (2.02 g, 84%). Ϫ 1H NMR (CD2Cl2): N 11.2.
δ ϭ 3.79 (s, 3 H, OCH3), 6.86 (d, 2 H, J ϭ 12 Hz, phenoxy 2-H),
9-(3-Methoxyprop-1-ylamino)acridine (6): 3-Methoxypropylamine
7.06 (t, 1 H, J ϭ 7 Hz, phenoxy 4-H), 7.16 (d, 1 H, J ϭ 2.75 Hz,
1ЈЈ-H or 5ЈЈ-H), 7.26 (m, 2 H, phenoxy 3-H), 7.37 (dd, 1 H, J ϭ
9.25, 2 Hz, 3ЈЈ-H or 7ЈЈ-H), 7.45 (dd, 1 H, J ϭ 2.75, 9.5 Hz, 3ЈЈ-H
or 7ЈЈ-H), 7.97 (d, 1 H, J ϭ 9.25 Hz, 4ЈЈ-H or 8ЈЈ-H), 8.08 (d, 1 H,
J ϭ 9.5 Hz, 4ЈЈ-H or 8ЈЈ-H), 8.17 (d, 1 H, J ϭ 2 Hz, 1ЈЈ-H or 5ЈЈ-
H). Ϫ DCI MS; m/z (%): 336 (100) [MHϩ].
(50 mg, 0.56 mmol), 9-phenoxyacridine (140 mg, 0.52 mmol), and
acetic acid (100 mg) were added to CH3CN (5 mL) and the solution
was heated at reflux for 1 h. The solvent was then removed in vacuo
and the residue purified by column chromatography (CH2Cl2/
MeOH/NH4OH, 90:9:1) on silica gel to afford the product as a
bright yellow solid (104 mg, 76%). Ϫ 1H NMR (CD2Cl2): δ ϭ 2.15
(m, 2 H, CH2), 3.41 (s, 3 H, CH3), 3.62 (t, 2 H, J ϭ 5.5 Hz, CH2),
3.99 (t, 2 H, J ϭ 7 Hz, CH2), 7.09 (dd, 2 H, J ϭ 9, 9 Hz, 2ЈЈ-H or
3ЈЈ-H), 7.41 (dd, 2 H, J ϭ 9, 9 Hz, 2ЈЈ-H or 3ЈЈ-H), 7.72 (d, 2 H,
J ϭ 9 Hz, 1ЈЈ-H or 4ЈЈ-H), 8.09 (d, 2 H, J ϭ 9 Hz, 1ЈЈ-H or 4ЈЈ-
H). Ϫ DCI MS; m/z (%): 331 (100) [MHϩ].
Clip-Phen-hexylaminoacridine (4): Compound 3 (200 mg, 0.36
mmol), 9-phenoxyacridine (195 mg, 0.72 mmol), and acetic acid
(216 mg , 3.6 mmol) were added to CH3CN (15 mL) and the solu-
tion was heated at reflux for 18 h. The solvent was then removed
in vacuo, the residue was dissolved in H2O and the pH adjusted to
12 (1  NaOH). The solution was extracted with CHCl3 (2 ϫ 50
mL), the combined organic layers dried (MgSO4), concentrated in
vacuo, and the residue was purified by column chromatography
(CH2Cl2/MeOH/Et3N, 92:7:1) on silica gel to afford the product
(Rf ϭ 0.3) as a bright yellow solid (194 mg, 74%). Ϫ UV/Vis (phos-
phate buffer, pH ϭ 7.2): λmax (ε Ϫ1cmϪ1) ϭ 226 (87500), 268
(90600), 396 (7000), 415 (8600) and 437 (8100). Ϫ 1H NMR
(CD2Cl2): δ ϭ 1.23 (m, 2 H, NHCH2CH2CH2CH2CH2), 1.51 (m,
4 H, NHCH2CH2CH2CH2CH2), 2.31 (t, 2 H, J ϭ 7.25 Hz,
NHCH2CH2CH2CH2CH2), 3.57 (t, 2 H, J ϭ 7 Hz, NHCH2-
CH2CH2CH2CH2), 4.84 (m, 3 H, serinol bridge), 4.99 (m, 3 H,
serinol bridge), 7.08 (d, 2 H, J ϭ 8.5 Hz, 3Ј-H), 7.25 (m, 2 H, 2ЈЈ-
H or 3ЈЈ-H), 7.51 (dd, 2 H, J ϭ 8, 4.25 Hz, 8Ј-H), 7.58 (d, 2 H,
J ϭ 8.75 Hz, 6Ј-H), 7.59 (m, 2 H, 2ЈЈ-H or 3ЈЈ-H), 7.65 (d, 2 H,
J ϭ 8.75 Hz, 5Ј-H), 7.96 (d, 2 H, J ϭ 8.75 Hz, 1ЈЈ-H or 4ЈЈ-H),
6-Chloro-2-methoxy-9-(3-methoxyprop-1-ylamino)acridine (7): 3-
Methoxypropylamine (50 mg, 0.56 mmol), 6-chloro-2-methoxy-9-
phenoxyacridine (100 mg, 0.30 mmol), and acetic acid (100 mg)
were added to CH3CN (5 mL) and the solution was heated at reflux
for 1 h. The solvent was then removed in vacuo and the residue
was purified by column chromatography (CH2Cl2/MeOH/NH4OH,
90:9:1) on silica gel to afford the product as a bright yellow solid
(80 mg, 81%). Ϫ 1H NMR (CD2Cl2): δ ϭ 1.97 (m, 2 H, CH2), 3.45
(s, 3 H, CH3), 3.67 (t, 2 H, J ϭ 5.5 Hz, CH2), 3.88 (t, 2 H, J ϭ 6
Hz, CH2), 3.93 (s, 3 H, OCH3), 7.27 (dd, 1 H, J ϭ 2, 9 Hz, 3ЈЈ-H
or 7ЈЈ-H), 7.30 (d, 1 H, J ϭ 2 Hz, 1ЈЈ-H or 5ЈЈ-H), 7.38 (dd, 1 H,
J ϭ 2, 9 Hz, 3ЈЈ-H or 7ЈЈ-H), 7.92 (d, 1 H, J ϭ 9 Hz, 4ЈЈ-H or 8ЈЈ-
H), 7.99 (d, 1 H, J ϭ 2 Hz, 1ЈЈ-H or 5ЈЈ-H) and 8.12 (d, 1 H, J ϭ
9 Hz, 4ЈЈ-H or 8ЈЈ-H). Ϫ DCI MS; m/z (%): 267 (100) [MHϩ].
8.04 (d, 2 H, J ϭ 8.5 Hz, 4Ј-H), 8.06 (d, 2 H, J ϭ 8.75 Hz, 1ЈЈ-H [(Clip-Phen)CuII](PF6)2: CuCl2 (3 mg, 0.022 mmol) and Clip-Phen
or 4ЈЈ-H), 8.16 (dd, 2 H, J ϭ 1.5, 8 Hz, 7Ј-H), 8.92 (br. d, 1 H, J ϭ
(10 mg, 0.022 mmol) were dissolved in DMF (2 mL) and the solu-
5.75 Hz, NHCO), 9.02 (dd, 2 H, J ϭ 1.5, 4.25 Hz, 9Ј-H). Ϫ DCI tion was stirred for 4 h. Diethyl ether was then added and the
MS; m/z (%): 738 (18) [MHϩ], 542 (38) [MHϩ Ϫ phenanthrolyl-
oxy], 197 (100) [hydroxyphenathrolineHϩ]. Ϫ C46H39N7O3 (737):
calcd. (ϩ 2 H2O) C 71.4, H 5.56, N 12.6; found C 71.6, H 5.39,
N 12.5.
solution left at Ϫ20°C for 18 h. The green precipitate which had
formed was isolated by centrifugation, washed with ether, and
dried. This solid was then dissolved in MeOH (2 mL) and a solu-
tion of NH4PF6 (15.9 mg, 0.1 mmol) in MeOH/H2O (2 mL, 50:50)
was added to give immediately a green precipitate. This was isolated
by centrifugation, washed with H2O and dried in vacuo (11.8 mg,
78%). Ϫ UV/Vis (CH3CN): λmax (ε Ϫ1cmϪ1) ϭ 226 (71800), 272
(49800), 810 (132). Ϫ ES MS; m/z (%): 510 (26) [Cu(Clip-Phen)]ϩ.
Ϫ C27H21CuF12N5O2P2 (800.5): calcd. (ϩ 2 H2O) C 38.7, H 2.98,
N 8.37; found C 38.7, H 2.61, N 8.21.
Clip-Phen-hexylchloromethoxyaminoacridine (5): Compound 3 (200
mg, 0.36 mmol), 6-chloro-2-methoxy-9-phenoxyacridine (241 mg,
0.72 mmol), and acetic acid (216 mg , 3.6 mmol) were added to
CH3CN (15 mL) and the solution was heated at reflux for 18 h.
The solvent was then removed in vacuo, the residue was dissolved
in H2O and the pH was adjusted to 12 (1  NaOH). The solution
was extracted with CHCl3 (2 ϫ 50 mL), the combined organic lay-
ers dried (MgSO4), concentrated in vacuo, and the residue was
purified by column chromatography (CH2Cl2/MeOH/Et3N, 92:7:1)
on silica gel to afford the product (Rf ϭ 0.35) as a bright yellow
solid (189 mg, 66%). Ϫ UV/Vis (phosphate buffer, pH ϭ 7.2): λmax
(ε Ϫ1cmϪ1) ϭ 228 (112000), 272 (101400), 433 (7800) and 455
(7700). Ϫ 1H NMR (CD2Cl2): δ ϭ 1.25 (m, 2 H, NHCH2CH2-
CH2CH2CH2), 1.51 (m, 4 H, NHCH2CH2CH2CH2CH2), 2.32 (t, 2
H, J ϭ 7.25 Hz, NHCH2CH2CH2CH2CH2), 3.45 (t, 2 H, J ϭ 7
Hz, NHCH2CH2CH2CH2CH2), 3.83 (s, 3 H, OCH3), 4.84 (m, 3 H,
serinol bridge), 4.98 (m, 3 H, serinol bridge), 7.05 (d, 2 H, J ϭ 8.5
Hz, 3Ј-H), 7.12 (dd, 1 H, J ϭ 2.25, 9.25 Hz, 3ЈЈ-H or 7ЈЈ-H), 7.20
(d, 1 H, J ϭ 2.5 Hz, 1ЈЈ-H or 5ЈЈ-H), 7.30 (dd, 1 H, J ϭ 2.5, 9.25
Hz, 3ЈЈ-H or 7ЈЈ-H), 7.49 (dd, 2 H, J ϭ 8, 4.25 Hz, 8Ј-H), 7.54 (d,
2 H, J ϭ 8.75 Hz, 6Ј-H), 7.61 (d, 2 H, J ϭ 8.75 Hz, 5Ј-H), 7.86 (d,
1 H, J ϭ 9.25 Hz, 4ЈЈ-H or 8ЈЈ-H), 7.91 (d, 1 H, J ϭ 2 Hz, 1ЈЈ-H
Complex Formation with Ligands 1, 4, and 5: Molar equivalents of
ligand and Cu(OAc)2 · 2 H2O were dissolved separately in a small
volume (1Ϫ2 mL) of warm methanol, then the solutions were
mixed and left to stand for 2 h. A fivefold excess of NH4PF6 was
then added as an aqueous solution to afford a precipitate. Acetone
was slowly added until the solid dissolved and the solution was left
open to the air for 24Ϫ48 h. Evaporation of the acetone afforded
the product as a precipitate, which was isolated by centrifugation,
washed with water and dried in vacuo.
[(1)CuII](PF6)2: Reaction of 1 and Cu(OAc)2 · H2O on a 0.024-
mmol scale afforded 14.9 mg (74%) of product as a pale green solid.
Ϫ UV/Vis (CH3CN): λmax (ε Ϫ1cmϪ1) ϭ 226 (55900), 276 (44400),
818 (135). Ϫ ES MS; m/z (%): 550.9 (100) [Cu(1)]ϩ. Ϫ C29H23Cu-
F12N5O3P2 (842.5): calcd. (ϩ 0.5 CH3COCH3) C 41.9, H 2.97, N
8.03; found C 42.2, H 3.00, N 8.31.
or 5ЈЈ-H), 7.96 (d, 1 H, J ϭ 9.25 Hz, 4ЈЈ-H or 8ЈЈ-H), 8.01 (d, 2 H, [(4)CuII](PF6)2 · HPF6: Reaction of 4 and Cu(OAc)2 · H2O on a
J ϭ 8.5 Hz, 4Ј-H), 8.13 (dd, 2 H, J ϭ 1.5, 8 Hz, 7Ј-H), 8.99 (br. s,
0.025-mmol scale afforded 17.0 mg (55%) of product as a bright
562
Eur. J. Inorg. Chem. 1999, 557Ϫ563

Synthesis of Two Acridine Conjugates of the Bis(phenanthroline) Ligand “Clip-Phen”
FULL PAPER
[1]
D. S. Sigman, Biochemistry 1990, 29, 9097; D. S. Sigman, T. W.
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yellow solid. Ϫ UV/Vis (phosphate buffer, pH ϭ 7.2): λmax (ε

Ϫ1cmϪ1) ϭ 226 (97600), 274 (89900), 394 (4200), 414 (6800), 437
(5500) and 814 (180). Ϫ ES MS; m/z (%): 799.3 (73) [Cu(4)]ϩ. Ϫ
C46H40CuF18N7O3P3 (1236.5): calcd. C 44.6, H 3.23, N 7.88; found
C 45.0, H 3.22, N 7.92.
[(5)CuII](PF6)2 · HPF6: Reaction of 5 and Cu(OAc)2 · H2O on a
0.025-mmol scale afforded 20.2 mg (62%) of product as a bright
yellow solid. Ϫ UV/Vis (phosphate buffer, pH ϭ 7.2): λmax (ε
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and 818 (146). Ϫ ES MS; m/z (%): 865.3 (100) [Cu(5)]ϩ. Ϫ
C47H41ClCuF18N7O4P3 (1301): calcd. (ϩ CH3COCH3) C 44.2, H
3.45, N 7.21; found C 44.2, H 3.30, N 7.46.
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DNA Cleavage Experiments: Reactions were carried out in a total
volume of 20 µL (10 µL DNA and buffer ϩ 5 µL complex ϩ 5 µL
MPA) containing 250 ng of Φx174 viral DNA (7n, 19 µ in base
pairs) in 40 m sodium phosphate (pH ϭ 7.2), 50 m NaCl and
10 m MgCl2 (final concentrations). Complexes were prepared as
1m stock solutions in DMF, which were then diluted to 4 µ with
water, prior to being added to DNA solutions for 30 min at room
temperature. DNA cleavage was initiated by addition of mercapto-
propionic acid (5 µL, 20m, final concentration 5mM) and samples
were incubated at 37°C for 1 h. 7.5 µL of a solution of 50% (v/v)
glycerol/40 m Tris. HCl buffer (pH ϭ 8) and 0.05% bromophenol
blue (w/v) was then added and the samples were immediately
loaded on agarose gel (0.8%) containing 1 µg/mL of ethidium bro-
mide. Electrophoresis was carried out at constant current (25 mA)
for 15 h in TBE buffer. Bands were located by UV light, photo-
graphed, and quantified by microdensity, rates being averaged from
at least 3 different sets of experimental data. The correction coef-
ficient 1.47 was used for the decrease in stainability of form I DNA
versus forms II and III.[22] The average number of single-strand
scissions per DNA molecule, expressed as µ, was considered to be
equal to Ϫln(fraction of form I) according to a Poisson distri-
bution.[23]
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M. Pitie, B. Meunier, Bioconj. Chem. 1998, 9, 604.
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UV/Vis-Spectrophotometric Titrations: Solutions were prepared
with the appropriate acridine derivative (4 µL of a 2.5 m solution
in DMF), calf-thymus DNA (diluted from a 1 mg/mL solution),
NaCl (final concentration 50 m), MgCl2 (final concentration 10
m) and phosphate buffer (pH ϭ 7.2, final concentration 40 m)
and diluted with H2O to a total volume of 1 mL. After equili-
bration for 24 h, spectra were recorded against an analogous blank
solution containing the same concentration of DNA/NaCl/MgCl2
and phosphate buffer. The CuCl2 complexes of 4 and 5 were pre-
formed for 30 min (4 µL of 2.5 m ligand solution and 1 µL of 10
m CuCl2 solution) prior to dilution with the appropriate salts
and DNA.
[18]
A. Albert, The Acridines, 2nd ed., E. Arnold, London, 1966,
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[19]
[20]
[21]
Caudrey, I. C. I., B. Pat. 583,220 (Chem. Abstr. 1947, 41, 330).
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Received September 21, 1998
Acknowledgments
We thank the European Commission for
a Marie Curie
Fellowship (Training and Mobility of Researchers: Grant No.
ERBFMBICT972447) to SAR.
[I98320]
Eur. J. Inorg. Chem. 1999, 557Ϫ563
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