New polyethyleneglycol-functionalized texaphyrins: Synthesis and in vitro biological studies

Article abstract of DOI:10.1039/b515636j

The synthesis of four new analogues of motexafin gadolinium (MGd), a gadolinium(iii) texaphyrin complex in clinical trials for its anticancer properties, is described. These new derivatives contain either 1,2-diaminobenzene or 2,3-diaminonaphthalene subun

Full text of DOI:10.1039/b515636j

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PAPER
www.rsc.org/dalton | Dalton Transactions
New polyethyleneglycol-functionalized texaphyrins: synthesis and in vitro
biological studies†‡
a
Wen-Hao Wei,a Zhong Wang,b Toshihisa Mizuno,§ Cecilia Cortez,b Lei Fu,b Mint Sirisawad,b
Louie Naumovski,b Darren Magda*b and Jonathan L. Sessler*a
Received 4th November 2005, Accepted 17th January 2006
First published as an Advance Article on the web 24th January 2006
DOI: 10.1039/b515636j
The synthesis of four new analogues of motexafin gadolinium (MGd), a gadolinium(III) texaphyrin
complex in clinical trials for its anticancer properties, is described. These new derivatives contain either
1,2-diaminobenzene or 2,3-diaminonaphthalene subunits as the source of the imine nitrogens and bear
multiple 2-[2-(2-methoxyethoxy)ethoxy]ethoxy (PEG) groups, on either meso aryl or beta-pyrrolic
substituents, to increase their water solubility. All four analogues were found to be more active in vitro
than the parent system MGd as judged from cell proliferation assays using the PC3 and A549 cell lines.
Introduction
Results and discussion
R
Xcytrinꢀ (MGd, motexafin gadolinium, 1; Fig. 1) has completed
Synthesis
two Phase III clinical trials as an agent for treating brain
metastases in conjunction with whole brain radiation therapy,
with strong positive trends being seen in both trials for patients
with non-small cell lung cancer.1–3 Recent preclinical and early
stage clinical trials have provided support for the notion that
this gadolinium texaphyrin derivative may also have utility as
a stand-alone anticancer agent.4 One proposed mechanism of
action involves the concept of redox cycling,5–7 wherein a reduced
texaphyrin species, formed by reaction with endogenous electron
rich species such as ascorbate or glutathione, serves to form
reactive oxygen species including superoxide and peroxide that
are known to act as apoptosis triggering agents.8–10 A similar
process of electron capture followed by air-based oxidation could
also contribute to the induced expression of metallothioneins
and altered zinc homeostasis.11 To the extent such redox-based
modes of action are important, it provides an incentive to
make and test new texaphyrin derivatives whose electronic prop-
erties are modified compared to 1. In this paper, we report
the synthesis and preliminary in vitro biological analysis of a
new series of gadolinium texaphyrins in which the electron-
rich dialkoxydiaminobenzene derivative used in 1 is replaced by
diaminonaphthalene and diaminobenzene subunits (compounds
2–5; Fig. 1). The diaminonaphthalene-derived systems 2 and 4
were found to display similar in vitro activity in PC3 and A549 cell
models, compared to the corresponding diaminobenzene systems
3 and 5. Both sets of new compounds were found to be more active
in vitro than the parent system 1.
A naphthalene-containing texaphyrin was synthesized previously
via the direct condensation of a tripyrrane dialdehyde with
diaminonaphthalene.12–13 The product, however, proved to have
very low solubility in aqueous media. Thus, a new analogue,
2, bearing multiple 2-[2-(2-methoxyethoxy)ethoxy]ethoxy (PEG)
groups to increase water solubility, was targeted for synthesis. Its
preparation is summarized in Schemes 1 and 2. The first stage
in the synthesis consisted of preparing the diformyltripyrrane
7. Towards this end, meso-diaryltripyrrane 6 was prepared
(Scheme 1) by condensing pyrrole (3–5 equiv.) with 3,5-bis[2-
[2-(2-methoxyethoxy)ethoxy]ethoxy]-benzaldehyde (1 equiv.)14 in
the presence of trifluoroacetic acid (TFA, 5% equiv.) according
to the procedure of Lee and Lindsey.15,16 After purification,
the product was isolated in 15–23% yield. While this purified
tripyrrane proved stable when stored in the freezer for over two
weeks, it darkened rapidly, turning from pink to dark brown,
when dissolved in common organic solvents and left to stand on
the bench top unprotected from air or light. Thus, as a general
rule, this intermediate was converted directly into the more stable
diformyl precursor 7; this was performed by subjecting it to
Vilsmeier formylation (DMF/POCl3) in accordance with standard
protocols.17
Once in hand, intermediate 7 was subject to acid-catalyzed
condensation with 2,3-diaminonaphthalene under conditions of
high dilution. This gave the so-called “sp3-texaphyrin” 8. The
structure of this nonaromatic macrocycle was confirmed by CI-MS
and HRMS. Unfortunately, compound 8 proved to be unstable,
decomposing completely when attempts were made to purify it
by column chromatography over silica gel or alumina, or using
a tC18 cartridge. This instability stands in contrast to what is
true for most meso-free sp3-texaphyrins, which are generally quite
stable and which can be separated by column chromatography over
various supports with nearly quantitative recovery.18,19 A possible
explanation for the instability of 8 is that the two meso-aryl groups
introduce strain that make the imine bonds more susceptible to
solvolysis. While not an exact analogy, it is noteworthy that several
aDepartment of Chemistry and Biochemistry, Institute for Cellular and
Molecular Biology, 1 University Station-A5300, The University of Texas
at Austin, Austin, TX 78712-0165. E-mail: Sessler@mail.utexas.edu
bPharmacyclics, Inc., 995 East Arques Avenue Sunnyvale, CA 94085, E-mail:
dmagda@pcyc.com
† Electronic supplementary information (ESI) available: Cyclic voltammo-
grams and X-ray experimental data for 14. See DOI: 10.1039/b515636j
‡ Dedicated to the memory of Ian Rothwell.
§ Current address: Department of Material Science, Graduate School of
Engineering, Nagoya Institute of Technology, 466-8555 Japan.
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Fig. 1 Motexafin gadolinium (MGd, 1) and new targets prepared in the course of the present study.
Scheme 1 Reagents and conditions: (a) 5% TFA, rt, 0.5 h, 23% for 6, 24% for 11; (b) 1) DMF/POCl3, rt, 1 h, under Ar, 2) 0.5 N aq. NaOH, 85 ◦C, 8 h,
72% for 7, 38% for 12.
Scheme 2 Reagents and conditions: (a) 37% HCl, MeOH, reflux, overnight; (b) Et3N, Gd(OAc)3·4H2O, air [O2], MeOH, reflux, 9% without DBU and
23% with DBU for 2.
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meso-aryl substituted porphyrins have been reported that have
distorted non-planar conformations.20,21
This intermediate was then formylated to yield the corresponding
tripyrrane dialdehyde 12 (Scheme 1). This dialdehyde was then
condensed with 4,5-dimethoxy-1,2-phenylenediamine to give 13
(Scheme 3). Following standard oxidative metal insertion and
purification, target 10 was obtained in the form of its acetate
salt. The nitrate form 14, which we considered more suitable for
growing X-ray diffraction quality single crystals based on our
prior experience,19 was obtained via anion exchange with nitrate
(Scheme 3). Both derivatives (i.e., 10 and 14) were characterized
by ESI-HRMS and UV/Vis spectroscopy, while complex 14 was
further characterized by single-crystal X-ray diffraction analysis.
The two water soluble meso-substituted texaphyrins 2 and 3 were
tested in an in vitro assay using the A549 cell line, and compared
with MGd, 1. The results are shown in Fig. 2. Compounds 2 and
3 showed dramatically higher activities than MGd towards A549
cells. Compound 2 was also tested in Ramos lymphoma cell line
in tissue culture. It induced apoptosis as demonstrated by annexin
V-positive cells at 2.5 lM but not 1 lM (Fig. 3). Compound 2
also activated caspases at 2.5 lM, demonstrating that compound
2 induced apoptosis (data not shown).
Fortunately, we found that intermediate 8 could be used directly
in the next step without a need for purification. Specifically,
treatment of 8 with 2 equiv. of Gd(OAc)3·4H2O and 7.5 equiv.
of Et3N in the presence of air in methanol under reflux was
found to effect ring oxidation and metal insertion. This produced
a green solution of the crude gadolinium texaphyrin complex,
2. This target species was purified by column chromatography
over neutral alumina followed by passage through a reversed-
phase tC18 cartridge column (Waters) to afford the desired meso-
diaryl substituted gadolinium texaphyrin 2 in pure form. The
overall yield was 23% based on 7 with DBU and 9% without
DBU. The UV-vis spectrum of compound 2 recorded in methanol
is characterized by a Soret-like band at 466 nm and Q-type band
at 840 nm. Thus, compared to MGd 1, the Soret-type band is blue-
shifted, while the Q-type band is red-shifted (the kmax of MGd, 1,
are 470 nm and 740 nm, respectively, in this same solvent).
An analogous compound 3, which has the same meso-PEG
substitution, but a simple benzene ring in place of the naphthalene
structure, was also synthesized; it was prepared from dialdehyde
7 and 1,2-phenylenediamine via intermediate 9 as shown in
Scheme 3. The overall yield for the two steps was 26% without
DBU. As might be expected, the spectral features of compound 3
(kmax = 451 and 747 nm in methanol) are blue shifted compared
to those of 2 (kmax = 466 and 840 nm, also in methanol).
To the best of our knowledge, compounds 2 and 3 repre-
sent the first texaphyrins bearing meso substitutents. In both
cases, the associated meso-3,5-bis[2-[2-(2-methoxyethoxy)ethoxy]-
ethoxy]phenyl functionality imparts sufficient water-solubility to
allow for testing under typical in vitro assay conditions (vide
infra). Such solubility features were considered critical since they
would allow for direct comparisons with MGd 1 in cell culture.
Nonetheless, it was also recognized that the presence of these
meso substituents might make compounds 2 and 3 less than ideal
electronic analogues of 1; they could engender deviations from
ring planarity,20–23 which, in turn, could lead to altered interactions
with biological targets.24–26
In light of this potential concern, we prepared a simpler
analog of 3, namely, the meso-phenyl substituted system 10.
The thought was that this model system would prove amenable
to characterization by single crystal X-ray diffraction analysis,
allowing the structural effect of the meso-aryl substitution to be
more accurately assessed. Compound 10 was synthesized using a
procedure analogous to that used to prepare 2 and 3. Specifically,
a TFA catalyzed condensation between benzaldehyde and pyrrole
was used to obtain the meso-diphenylsubstituted tripyrrane 11.
Fig. 2 Antiproliferative activity of motexafin gadolinium (MGd, 1) and
analogues 2–5 in A549 lung cancer cells. A549 cultures were treated with
texaphyrins for 24 h, medium was replaced with fresh medium, and viable
cells were quantified after 72 h by tetrazolium salt reduction.
These promising results prompted our interest to synthesize
other related Gd-Tex derivatives, and try to understand the key
structural characteristics as well as physicochemical properties
that lead to the increased biological activity.
Thus, compounds 4 and 5 were synthesized utilizing a slightly
different strategy starting from tripyrrane dialdehyde 15, an
intermediate used in the synthesis of MGd. As shown in Scheme 4,
reaction of 15 with disuccinimidyl carbonate in the presence of
diisopropylethylamine (DIEA), followed by reaction with bis[2-
[2-(2-methoxyethoxy)ethoxy]ethyl]amine, gave the side-chain
Scheme 3 Reagents and conditions: (a) 37% HCl, MeOH, reflux, overnight; (b) Et3N, Gd(OAc)3·4H2O, air [O2], MeOH, reflux, 26% for 3, 38% for 10,
(c) anion exchange with NaNO3, 100%.
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Fig. 3 Cytotoxicity of compound 2 as determined by annexin V-positive
cells. Ramos cells were treated with compound 2 at 1 or 2.5 lM
for 24 h. Annexin V-positive cells were quantified by flow cytometry.
Similar cytotoxic effects were seen when normalized caspase-3 activity
was monitored (data not shown).
Fig. 4 (a) Top (a) and side views of 14 showing the atom labeling scheme.
Displacement ellipsoids are scaled to the 50% probability level. The Gd3+
˚
ion is 0.698(1) A out of the plane defined by the five nitrogen atoms of the
texaphyrin macrocycle.
the nitrogen atoms present within the pentaaza texaphyrin core;
four donor atoms come from the coordinated bidentate nitrate
anions, while the last donor atom is derived from a methanol
solvent molecule. The two phenyl groups linked to the meso-
positions adopt a nearly orthogonal orientation with regard to
the plane of the five nitrogen atoms. Presumably as a consequence
of the resulting steric interactions, the pyrrole ring between the
two phenyl substituents deviates from planarity, being bent “up”
toward the two NO3 anions. The two imine nitrogens are also
raised such that the macrocycle as a whole exists in a “ruffled”
non-planar conformation.
CCDC reference number 284060.
For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b515636j
Scheme 4 Reagents and conditions: (a) N,Nꢀ-disuccinimidyl carbonate,
N,N-diisopropylethylamine (DIEA), DMSO, rt, then diPEG amine, 62%;
(b) 2,3-diaminonaphthalene, HCl, MeOH, 45 ◦C; (c) Gd(OAc)3·4H2O,
DIEA, air [O2], MeOH, reflux, 36%.
−
PEGylated tripyrrane dialdehyde 16. Column chromatography
over silica gel yielded this intermediate in 62% yield in purified
form. An HCl-catalyzed condensation of dialdehyde 16 with
2,3-diaminonaphthalene gave the non-aromatic macrocycle 17,
which was used directly without purification. Finally, Gd inser-
tion/oxidation in methanol at reflux in the presence of DIEA and
O2 furnished the Gd complex 4. The overall yield from 16 was
36%. Using the diformyl tripyrrane 16 as a common intermediate,
compound 5 was also readily synthesized in 55% yield. As with 2
and 3, compounds 4 and 5 displayed good solubility in aqueous
media.
In vitro biological study
Once in hand, the four new Gd-Tex derivatives 2–5 were tested
for anti-proliferative activity in vitro using two cell lines (PC3
and A549), with representative data being shown in Fig. 2 and 5.
From these data, we infer that the new naphthalene and benzene
ring-containing texaphyrins all display higher in vitro activity than
MGd. These compounds displayed significant activity in the 10–
20 lM concentration range, while MGd, 1, showed little activity
even at 30 lM. In preliminary studies, analogue 4 was also found
to display significant cytotoxicity in Ramos cells at concentrations
of 5 lM and above (data not shown).
Crystal structure of 14
The single crystal X-ray structure of 14 is shown in Fig. 4,
with important bond lengths and angles listed in Table 1. As
can be inferred from an inspection of Fig. 4, 14 consists of a
1 : 1 metal–ligand complex in which the Gd(III) cation is found
to be 10-coordinate. Five of the donor atoms are provided by
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◦
a
˚
Table 1 Selected bond lengths (A) and angles ( ) for 14
Gd–N1
Gd–N8
Gd–N13
2.486(2)
2.4142(19)
2.540(2)
Gd–N20
Gd–N23
2.565(2)
2.424(2)
Gd–O1A
Gd–O2A
Gd–O1B
2.484(2)
2.786(3)
2.5500(19)
Gd–O2B
Gd–O1C
2.5529(18)
2.5498(18)
N1–Gd–N8
N8–Gd–N13
N13–Gd–N20
75.68(6)
66.02(7)
61.96(6)
N20–Gd–N23
N23–Gd–N1
65.33(6)
74.46(7)
O1A–Gd1–O2A
O1A–Gd1–O1B
O1B–Gd1–O2B
47.15(8)
67.09(7)
49.90(6)
O2B–Gd1–O2A
O1A–Gd1–O1C
63.59(7)
138.61(7)
a The pyrrolic nitrogens are numbered N1, N8 and N23; and the imine nitrogens are numbered N13 and N20. X-ray Experimental for
(C36H26N5O2)Gd(NO3)2-CH3OH-0.5C6H6. Green prisms were grown by slow evaporation fro◦m methanol–benzene, monoclinic, P21/c (No. 2), Z =
3
4 in a cell of dimensions: a = 17.9235(2) A, b = 11.9870(1) A, c = 18.7840(2) A, b = 114.244(1) , V = 3679.79(6) A , Mr = 912.98, qcalc = 1.648 g cm−3
,
˚
˚
˚
˚
l = 1.871 mm−1, F(000) = 1832. A total of 14460 reflections were measured, 8372 unique (Rint = 0.017), on a Nonius Kappa CCD using graphite
◦
monochromatized Mo Ka radiation (k = 0.71073 A) at −120 C. The structure was refined on F2 to an Rw = 0.0741, with a conventional R = 0.0278
˚
(6989 reflections with Fo > 4[r(Fo)]), and a goodness of fit = 1.14 for 491 refined parameters. A molecule of benzene was found to be disordered about
1
2
a crystallographic inversion center at 0,
,
12 . The molecule was poorly resolved and its contribution to the scattering was removed using the utility
SQUEEZE in PLATON98. For full details see the Supporting Information.
Comparing the order of biological activity to that of the redox
potentials, one can see that both the antiproliferative activity and
redox potentials of the new derivatives fall in a relatively narrow
range, which is distinct from that of MGd. Moreover, the new
derivatives with their more positive redox potentials display a
higher level of in vitro antiproliferative activity. By contrast, no
clear correlation between the presence or absence of the meso
substitutents was observed. On this basis we conclude that limited
differences in planarity do not have a direct effect on the biological
activity. However, since such distortions could affect the observed
redox behavior, an indirect effect cannot be ruled out.
Since MGd has been shown to redox cycle in the presence of
ascorbate to generate cytotoxic H2O2,6 we sought to determine if
compound 2 would display enhanced cytotoxicity in the presence
of ascorbate. As shown in Fig. 6, treatment of Ramos cells with
sub-toxic doses of 2 and ascorbate resulted in elevated levels of
annexin V-positive cells and decreased growth. Cytotoxicity and
growth inhibition was reversible in the presence of catalase, leading
us to propose, in analogy to MGd, that the combination of 2 and
ascorbate serves to generate H2O2 via redox cycling.
Fig. 5 Antiproliferative activity of motexafin gadolinium (MGd, 1) and
analogues 2–5 in PC3 prostate cancer cells. PC3 cultures were treated with
texaphyrins for 24 h, medium was replaced with fresh medium, and viable
cells were quantified after 72 h by tetrazolium salt reduction.
Electrochemical study
Because it is believed that Gd-Tex derivatives affect cell
metabolism, at least in part, through redox cycling, we became
interested in measuring the redox potentials of this new series
of texaphyrins, and in establishing a possible correlation between
their biological activity and electrochemical properties. Accord-
ingly, the redox potentials of 2–5 were determined in DMF
solution using tetrabutylammonium perchlorate as the supporting
electrolyte. The E1/2 values were calculated from cyclic voltammo-
grams and are summarized in Table 2. The redox potential of
MGd, 1, was also determined under the same conditions and is
included for comparison.
Conclusion
In summary, we have developed two general synthetic methods
that can be applied to prepare a wide range of water-soluble,
bioassay-compatible texaphyrin derivatives by simply using differ-
ent aromatic diamines. Employing these two methods, four novel
texaphyrins were synthesized, and cell culture studies showed these
compounds possessed higher in vitro antiproliferative activity
than MGd. Such preliminary structure–activity analyses lead
us to suggest that the biological activity of MGd (1) and its
analogues (e.g., 2–5) is closely correlated with the redox potential
of the compounds. Detailed studies to clarify the mechanism of
texaphyrin activity and efforts to assess these compounds in animal
tumor models are currently underway.
Table 2 Electrochemical reduction potentials of gadolinium texaphyrin
complexes 1–5 in DMF solution (reference: Ag/AgCl)a
1 (MGd)
2
3
4
5
1E1/2/mV
2E1/2/mV
−329
−802
−130
−559
−122
−597
−107
−443
−203
−678
Experimental
All starting materials were purchased and used without further pu-
rification unless otherwise noted. 3,5-Di[2-[2-(2-methoxyethoxy)-
ethoxy]ethoxy]benzaldehyde,14 5,10-diphenyltripyrrane 11,15,16
a Recorded in anhydrous DMF using 0.2 M tetrabutylammonium perchlo-
rate as the supporting electrolyte.
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meso-diarytripyrrane 6 (1.08 g, 23%). FAB-MS m/z 1026 [M]+;
FAB-HRMS [M]+ calcd for C54H79N3O18: 1025.546034 found:
1
1025.547545; H NMR (400 Hz, CDCl3): d 8.07 (bs, 2H, NH),
7.83 (bs, 1H, NH), 6.64 (s, 2H, b-H), 6.35 (t, J = 2.4 Hz, 4H), 6.09
(q, J = 3.2 Hz, 2H), 5.85 (bs, 2H), 5.74 (d, J = 2.4 Hz, 2H), 5.25
(d, J = 3.2 Hz), 4.00 (t, J = 4.0 Hz, 4H, CH2), 3.77 (t, J = 4.4 Hz,
4H, CH2), 3.69–3.67 (m, 4H, CH2), 3.65–3.60 (m, 8H, CH2),
3.52–3.50 (m, 4H, CH2), 3.34 (s, 12H, CH3). 13C NMR (63 MHz,
CDCl3): d 159.8, 144.4, 132.1, 131.8, 116.9, 107.9, 107.3, 107.1,
106.8, 99.8, 71.7, 70.68, 70.4, 70.3, 69.5, 67.2, 58.8, 44.1.
1,14-Bisformyl-5,10-diphenyltripyrrane (12). A pink solution
of tripyrrane 11 (94 mg, 0.25 mmol) in dry DMF (2 ml) was cooled
in an ice-bath and stirred for 5 min under argon. At this point,
freshly distilled POCl3 (116 ll, 1.25 mmol) was added dropwise
and the reaction mixture was stirred for 1 h at room temperature.
The mixture was hydrolyzed with 0.5 M NaOH (15.0 ml) at 80–
90 ◦C for 8 h under air. To the cooled mixture, was added 30 ml of
CH2Cl2. The resulting solution was washed three times with water
and dried over Na2SO4 overnight. After the solvent was removed
under reduced pressure, column chromatography over silica gel
(MeOH–CH2Cl2) gave 37 mg of the desired tripyrrane dialdehyde,
1
12 (37 mg, 38%). CI-MS m/z 434 [M + H]+; H NMR (250 Hz,
CDCl3): d 10.10 (bs, 2H, NH), 9.04 (bs, 1H, NH), 8.95 (d, J =
2.5 Hz, 2H, CHO), 7.23–7.08 (m, 10H, Ph–H), 6.75 (J = 2.5 Hz,
2H), 6.04 (t, J = 2.5 Hz, 2H), 5.76 (J = 2.5 Hz, 2H), 5.44 (s, 2H).
13C NMR (62 MHz, CDCl3): d 178.5 (CHO), 143.0, 140.5, 140.4,
132.2, 131.2, 128.6, 128.2, 127.2, 122.6, 44.0.
Fig. 6 Effect of compound 2 on annexin V-binding and cell growth in
Ramos. (A) Cells were treated with ascorbate (asc) 25 lM, compound
(Cpd) 2 at 1 lM, the combination or the combination with catalase (Cat)
260 units ml−1 for 24 h. Apoptotic cells were determined by annexin
V-binding. (B) Cells were treated as above for 4 days and cell numbers
were measured by a Coulter counter.
1,14-Bisformyl-5,10-bis{3,5-bis[2-[2-(2-methoxyethoxy)ethoxy]-
ethoxy]phenyl}tripyrrane (7). A pink solution of the diaryl-
tripyrrane 6 (0.230 g, 0.224 mmol) in dry DMF (1.4 ml) was
cooled in an ice-bath and stirred for 5 min under argon. As
above, freshly distilled POCl3 (52 ll, 0.56 mmol) was added
drop-wise and the resulting solution was allowed to stir for 1.5 h
at room temperature. The mixture was hydrolyzed with 0.5 M
NaOH (6.8 ml) at 80–90 ◦C for 8 h under air. To the cooled
mixture, was added 30 ml of CH2Cl2. The resulting solution was
washed three times with water, dried over Na2SO4 overnight.
After the solvent was removed under reduced pressure, tripyrrane
dialdehyde 7 was obtained in purified form in 175 mg, 72% yield by
column chromatography over silica gel (eluent: MeOH–CH2Cl2).
MS (FAB-MS) m/z 1083 [M + H]+; FAB-HRMS [M + H]+
calculated for C56H80N3O18: 1082.543687 found: 1082.542574; 1H
NMR (500 Hz, CDCl3): d 9.32 (bs, 2H, NH), 9.29 (d, J = 2.1 Hz,
2H, CHO), 8.18 (d, J = 10 Hz, 1H, NH), 6.78 (p, J = 2.05, 2H,
b-H), 6.29 (t, J < 2.0 Hz, 2H, 4-Ar–H), 6.27 (t, J < 2 Hz, 4H,
2,6-Ar–H), 5.98 (p, J = 2.03 Hz, 2H, b-H), 5.72 (t, J = 3.1 Hz,
2H, b-H), 5.25 (d, J = 5.62, 2H, meso-H), 3.95–3.92 (m, 4H,
CH2), 3.72–3.70 (m, 4H, CH2), 3.62–3.60 (m, 4H, CH3), 3.58–3.54
1,2-diamino-4,5-dimethoxybenzene,19 and bis[2-[2-(2-methoxy-
ethoxy)ethoxy]ethyl]amine27 were prepared according to literature
methods. Proton and 13C NMR spectra were recorded using a
250 MHz Varian, 300 MHz GE Tacmag spectrometer, 400 MHz
Varian MERCURY or 500 MHz Varian INOVA. UV/Vis
spectra were taken on Beckman DU-640B or Agilent 8453
Spectrophotometer. Column chromatography was run using ICN-
˚
Silitech 32–63 D60 A silica gel or Sorbent Technologies std.
activity 50–200 l neutral alumina. Sep-pak reverse-phase tC18
cartridge columns were purchased from Waters.
Syntheses
5,10-Bis{3,5-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]phenyl}-
tripyrrane (6). A mixture of 3,5-di[2-[2-(2-methoxyethoxy)-
ethoxy]ethoxy]benzaldehyde (4.30 g, 10 mmol)14 and pyrrole
(3.35 g, 50 mmol) was stirred for 5 min at room temperature
under argon. To the pale yellow solution, was added TFA (39 ll,
0.5 mmol). The resulting pink mixture was stirred for an additional
30 min, and then CH2Cl2 (30 ml) and 0.5 M aqueous NaOH
(1.5 ml) were added to quench the reaction and the mixture was
washed three times with H2O. The pale yellow organic solution
was separated and dried over Na2SO4 overnight. The solvent was
removed with water aspirator and excess pyrrole was distilled off
under vacuum. The brown viscous residue was then purified by
column chromatography on silica gel (MeOH–CH2Cl2) to give
(m, 8H, CH2), 3.46–3.44 (m, 4H, CH2), 3.28 (s, 12H, OCH3). 13
C
NMR (126 MHz, CDCl3): d 178.5 (CHO), 160.0, 142.7, 142.6 (d,
J = 7.3 Hz), 142.3 (d, J = 7.3 Hz), 132.1 (d, J = 4.5 Hz), 130.8 (d,
J = 5.4 Hz), 121.9, 113.9, 110.7, 107.9, 107.8, 107.5, 107.4, 100.2,
71.8, 70.7, 70.7, 70.5, 70.4, 69.7, 67.3, 58.9, 44.2, 29.6.
meso-Diarylsubstituted gadolinium-texaphyrin (2). To a solu-
tion of tripyrrane dialdehyde 7 (112 mg, 0.103 mmol) and 2,3-
diaminonaphthalene (16.4 mg, 0.103 mmol) in MeOH (50 ml),
was added 37% HCl (25.9 ll 0.309 mmol) at room temperature
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under argon. The solution was heated at reflux for 10 h. CI-
MS showed that the sp3-texaphyrin 8 was formed; CI-MS: m/z
1204 [M + 1]+, FAB-HRMS [M + 1]+ calculated for C66H86N5O16:
1204.6069; found: 1204.6049. At this point, Gd(OAc)3·4H2O
(84 mg, 206 mmol), triethylamine (108 ll, 0.772 mmol), and
1.8-diazabicyclo[5.4.0]undecene (DBU, 31 ll, 0.206 mmol) were
added. The reaction mixture was then stirred while heating at
reflux for an additional 24 h under air. UV/vis spectra were
taken to monitor the progress of the reaction. When the ratio
of absorbance at 324 nm to absorbance at 432 nm reached a
minimum, the reaction was deemed completed. The green solution
was cooled to room temperature, and the solvent removed under
reduced pressure. The resulting green solid was then dried in vacuo
for several hours. The crude complex obtained in this way was
purified by column chromatography over neutral alumina. After
the initial red/brown fraction was discarded, the green complex
was gradually eluted using 10–30% MeOH–CH2Cl2 as the eluent.
These combined green fractions were washed with 3 × 50 ml of
0.1 M aqueous NaOAc and the organic layer was collected. After
evaporating off the solvent under reduced pressure, the complex
was further purified using a tC18 cartridge column (Waters) to
afford pure 11 in 23% yield (the yield was only 9% when DBU was
not used). ESI-MS: m/z 1415 [M − 59]+; FAB-HRMS [M − 59 +
H]+ calcd, for C68H84Gd[158]N5O18: 1415.5052; found: 1415.5071;
UV/Vis in MeOH [kmax, nm]: 316, 466, 765, 840.
[M − 59]+ calculated for C38H29Gd[158]N5O4: 777.146066; found:
777.146103; UV/Vis in MeOH [kmax, nm]: 322, 411 (shoulder), 468
and 716.
Preparation of (14). A green solution of 0.100 g 10 in 10 ml of
MeCN was added into a solution of 0.1 N aq. NaNO3 (100 ml).
The mixture solution was loaded on a 10 g tC18 cartridge column.
Another 100 ml of 0.1 N aq. NaNO3 was passed through the
column to achieve effective anion exchange. The excess NaNO3
was removed by washing with 200 ml deionized water. The pure
salt 14 was eluted off the column using MeOH as the eluent.
Finally, MeOH was removed under reduced pressure, to give 14
as a green solid in quantitative yield.
Preparation of (16). In a 100 ml Schlenk flask were placed
tripyrrane 15 (2.415 g, 5 mmol, 1 equiv.), N,Nꢀ-disuccinimidyl
carbonate (5.12 g, 20 mmol, 4 equiv.), and a magnetic stir bar. The
system was dried in vacuum at rt for 2 h. Under a stream of N2, an-
hydrous DMSO (50 ml) and diisopropylethylamine (DIEA, 5.16 g,
40 mmol, 8 equiv.) were added. The reaction mixture was stirred
at rt for 4 h. Then bis[2-[2-(2-methoxyethoxy)ethoxy]ethyl]amine
(9.30 g, 30 mmol, 6 equiv.),27 dissolved in 20 ml anhydrous DMSO,
was added, and the resulting mixture was stirred for another
1 h. The reaction mixture was diluted with deionized (DI) water,
loaded onto a pre-conditioned tC18 cartridge column and eluted
with DI water to remove DMSO. After removing the DMSO, the
product fraction was eluted from the column with MeOH. After
concentrating the MeOH fraction, the crude product was purified
by column chromatography over neutral alumina (eluent: 0.5–
1.0% MeOH in CH2Cl2) to yield 16 as an orange oil (3.57 g,
62%). ESI-MS: m/z 1152.4 [M + 1]+; FAB-HRMS: calculated
for C58H97N5O18 [M]+: 1151.6829; found: 1151.6810. 1H NMR
(300 Hz, CDCl3): d 9.41 (s, 2H, CHO), 4.04 (t, J = 6.0 Hz, 4H,
CH2OCO), 3.79 (s, 4H, Pyr–CH2–Pyr), 3.65–3.42 (m, 48H, CH2 in
PEG groups), 3.36 (s, 6H, CH3O), 3.33 (s, 6H, CH3O), 2.49 (t, J =
6.5 Hz, 4H, Py–CH2CH2), 2.41 (q, J = 7.3 Hz, 4H, Py–CH2CH3),
2.24 (s, 6H, Py–CH3), 1.80–1.70 (m, 4H, Py–CH2CH2), 1.09 (t, J =
7.3 Hz, 6H, CH2CH3) ppm. 13C NMR (75 MHz, CDCl3): d 175.6,
156.3, 137.7, 132.0, 131.9, 128.0, 121.4, 120.7, 71.81, 70.50, 69.5,
69.4, 64.7, 58.8, 47.9, 47.7, 29.6, 22.6, 20.0, 17.6, 16.4, 8.9 ppm.
Elemental calcd for C58H97N5O18: C, 60.45%; H, 8.48%; N, 6.08%;
found C, 60.09%; H, 8.52%; N, 6.00%.
General procedure for the preparation of meso-diarylsubstituted
gadolinium texaphyrins 3 and 10
To a solution of tripyrrane dialdehyde 7 or 12 (1 equiv.) and
the appropriate diamino precursor (1 equiv.) in MeOH (1 mmol
500 ml−1), was added 37% HCl (3 equiv.) at room temperature
under argon. The solution was heated at reflux for 10 h. At this
juncture, CI-MS analysis showed that the sp3-texaphyrin 9 or 13
was present as the dominant organic species. CI-MS: m/z 1155
[M + 1]+ for 9 and 565 [M + 1]+ for 13. Thus, Gd(OAc)3·4H2O
(1.5 equiv.) and triethylamine (7.5 equiv.) were added to the
reaction mixtures, which were then heated to reflux with stirring
for an additional 24 h under air. UV/vis spectra were taken,
and once the expected aromatic texaphyrin-like spectral features
were observed, the respective reactions were deemed complete.
The green solutions obtained in this way were allowed to cool to
room temperature before the solvent was removed under reduced
pressure. The resulting green solids were then dried in vacuo for
several hours. The targeted complexes, either 3 or 10, were purified
by column chromatography over neutral alumina. After the initial
red/brown fraction was discarded, the green complexes were
gradually eluted off the respective chromatography columns using
10–30% MeOH–CH2Cl2 as the eluent. The appropriate fractions
were combined and washed with 3 × 50 ml of 0.1 M aqueous
NaOAc with the organic layers being saved. After concentrating
the organic solutions, the desired complexes were further purified
using a tC18 cartridge column. Without DBU, this afforded pure
meso-diarylsubstituted gadolinium-texaphyrin 3 and 10 in yields
of 24 mg (26%) and 28 mg (38%), respectively.
Preparation of PEGylated naphthalene Gd-Tex (4). TetraPEG
tripyrrane 16 (115 mg, 0.1 mmol) and 2,3-diaminonaphthalene
(16 mg, 0.1 mmol) were dissolved in 5 ml MeOH. To the solution
was added HCl (0.2 ml of a 1 M MeOH solution, 0.2 mmol).
◦
The resulting mixture was stirred at 45 C under N2 for 2 h. At
this point, UV-vis spectroscopic analysis revealed the presence of
a major absorption band around 370 nm. The reaction mixture
was evaporated to dryness and further dried in vacuum at room
temperature to give 17. Without further purification, intermediate
17 was dissolved in 50 ml MeOH and transferred to a 100 ml three-
neck round bottom flask. To the flask were added Gd(OAc)3·4H2O
(0.2 mmol), and diisopropylethylamine (52 mg, 0.4 mmol). The
reaction mixture was stirred and heated to reflux under air. The
reaction progress was monitored by UV-vis spectroscopy, which
revealed the absorption band at ∼342 nm decreasing and a new
strong absorption appearing at ∼445 nm (as well as two other
bands in the ∼800 and ∼876 nm range). The reaction mixture
Texaphyrin (3). FAB-MS: m/z 1366 [M − 59]+; FAB-HRMS
[M − 59 + H]+ calculated for C64H82Gd[158]N5O18: 1366.4896;
found: 1366.4889; UV/Vis in MeOH [kmax, nm]: 322, 451, 685,
747. Texaphyrin (10): CI-MS: m/z 776 [M − 60]+; FAB-HRMS
1940 | Dalton Trans., 2006, 1934–1942
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was then concentrated on a rotary evaporator and thoroughly
dried in vacuum overnight. The crude product 4 was then purified
using a reversed-phase tC18 cartridge column and an aqueous
ammonium acetate buffer (pH 4.3)/MeOH mixture (eluent: 50–
60% MeOH/buffer). The product fraction (red in color) was
collected, concentrated on a rotary evaporator to remove MeOH,
and extracted with CH2Cl2 (3 × 50 ml). The CH2Cl2 solutions were
combined, washed with 50 ml DI water, dried with anhydrous
MgSO4, and concentrated to give the final product 4 (56 mg,
36%). ESI-MS: m/z 1485.7 [M − OAc]+; FAB-HRMS: calculated
for C70H102Gd[158]N7O18 ([M − OAc + H]+): 1486.6522; found:
1486.6507; UV/Vis in MeOH [kmax, nm]: 342, 445, 795, 876;
Elemental calcd for C70H90GdN5O22 (M + 2H2O): C, 55.65%; H,
6.00%; N, 4.64%; found C, 55.34%; H, 6.09%; N, 4.54%.
Caspase-3 Assay Kit #2 (Molecular Probes, Eugene, OR) as per
the manufacturer’s protocol.
Electrochemistry
Electrochemical measurements were carried out at room tem-
perature with a Cypress System 1090 apparatus operated in
cyclic voltammetry mode. All reported electrochemical half-wave
potentials were measured using a glassy carbon working electrode
and a Ag/AgCl reference electrode and referenced to such.
Platinum wire (0.5 mm diameter) was employed as an auxiliary
electrode. Each experiment was performed on a 0.5–1.5 mM
solution of texaphyrin complex in 1 mL of anhydrous DMF,
using 0.2 M tetrabutylammonium perchlorate as the supporting
electrolyte.
The potential was scanned between 0 and −1100 mV. The
electrochemical potentials presented in this paper were obtained
using a 50 mV s−1 scan rate.
Preparation of bisPEGylated texaphyrin (5). Compound 5 was
prepared from 16 and 1,2-phenylenediamine following the same
procedure used to prepare 4 (see above). The yield was 55% based
on 16. For 5: ESI-MS: m/z 1435.7 [M − OAc]+; FAB-HRMS:
calculated for C66H100Gd[158]N7O18 ([M − OAc + H]+): 1436.6366;
found: 1436.6377; UV/Vis in MeOH [kmax, nm]: 332, 431, 707, 771;
Elemental calcd for C66H88GdN5O22 (M + 2H2O): C, 54.27%; H,
6.07%; N, 4.79%; found C, 53.99%; H, 6.16%; N, 4.65%.
Acknowledgements
We would like to thank Pharmacyclics, Inc. and the NIH (grant
CA 68682 to J. L. S.) for support of this work.
In vitro biological activity
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