Porphyrins incorporating heterocyclic N-oxides: (Oxidopyridyl)porphyrins, porphyrin-N-oxides, and a tirapazamine-porphyrin conjugate

Article abstract of DOI:10.1139/v98-238

Porphyrins containing one to four 4-pyridyl groups as meso-substituents were synthesized via a mixed aldehyde condensation, and then "N-oxidized" with m-chloroperbenzoic acid to produce five novel (oxidopyridyl) porphyrins and seven porphyrin-N-oxides, which were characterized by analysis and spectroscopic methods, especially NMR; an X-ray crystal structure of 5-(1-oxido-4-pyridyl)-10,15,20-triphenylporphyrin was also obtained. Crystals of (oxidopyridyl)triphenylporphyrin are tetragonal, a = b = 15.174(1), c = 13.709(1) A, Z = 4, space group I2d. The structure was solved by direct methods and refined by full-matrix least-squares procedures to R = 0.031 (R_w = 0.026) for 685 reflections with I ≥ 3σ(I). Sulfonation of two of the (oxidopyridyl)porphyrins was achieved readily with no loss of oxygen from the oxidopyridyl groups. Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) was treated with triphosgene to yield the previously reported 2H-[1,2,4]oxadiazolo[3,2-c][1,2,4]benzotriazin-2-one-5-oxide (1); this reacts like an isocyanate and with 5-(4-aminophenyl)-10,15,20-triphenylporphyrin yields a tirapazamine-porphyrin conjugate (2).

Full text of DOI:10.1139/v98-238

182
Porphyrins incorporating heterocyclic N-oxides:
(oxidopyridyl)porphyrins, porphyrin-N-oxides,
and a tirapazamine–porphyrin conjugate
Jeffrey J. Posakony, Russell C. Pratt, Steven J. Rettig, Brian R. James,
and Kirsten A. Skov
Abstract: Porphyrins containing one to four 4-pyridyl groups as meso-substituents were synthesized via a mixed
aldehyde condensation, and then “N-oxidized” with m-chloroperbenzoic acid to produce five novel (oxidopyridyl)
porphyrins and seven porphyrin-N-oxides, which were characterized by analysis and spectroscopic methods, especially
NMR; an X-ray crystal structure of 5-(1-oxido-4-pyridyl)-10,15,20-triphenylporphyrin was also obtained. Crystals of
(oxidopyridyl)triphenylporphyrin are tetragonal, a = b = 15.174(1), c = 13.709(1) Å, Z = 4, space group I2d. The
structure was solved by direct methods and refined by full-matrix least-squares procedures to R = 0.031 (Rw = 0.026)
for 685 reflections with I ≥ 3σ(I). Sulfonation of two of the (oxidopyridyl)porphyrins was achieved readily with no loss
of oxygen from the oxidopyridyl groups. Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) was treated with
triphosgene to yield the previously reported 2H-[1,2,4]oxadiazolo[3,2-c][1,2,4]benzotriazin-2-one-5-oxide (1); this reacts
like an isocyanate and with 5-(4-aminophenyl)-10,15,20-triphenylporphyrin yields a tirapazamine-porphyrin conjugate (2).
Key words: porphyrin-N-oxides, (oxidopyridyl)porphyrins, tirapazamine.
Résumé : Faisant appel à une condensation mixte d’aldéhyde, on a synthétisé des porphyrines contenant d’un à quatre
groupes 4-pyridyles comme substituants méso et on les a ensuite oxydées à l’aide d’acide m-chloroperbenzoïque pour
produire cinq nouvelles (oxydopyridyl)porphyrines et sept N-oxydes de porphyrine que l’on a caractérisés par analyse
et par spectroscopie, principalement la RMN; on a aussi déterminé la structure cristalline de la 5-(1-oxydo-4-pyridyl)-
10,15,20-triphénylporphyrine par diffraction des rayons X. Les cristaux de la (oxydopyridyl)triphénylporphyrine sont té-
tragonaux, groupe d’espace I2d, avec a = b = 15,174(1) et c = 13,709(1) Å et Z = 4. On a résolu la structure par des
méthodes directes et on l’a affinée par la méthode des moindres carrés (matrice entière) jusqu’à des valeurs de R =
0,031 et Rw = 0,026 pour 685 réflexions avec I ≥ 3σ(I). On a effectué la sulfonation de deux des oxydopyridylporphy-
rines facilement, sans perte d’oxygène à partir des groupes oxydopyridyles. On a traité la tirapazamine (le 1,4-dioxyde
de la 3-amino-1,2,4-benzotriazine) avec du triphosgène pour obtenir la 5-oxyde de la 2H-[1,2,4]oxadiazolo[3,2-
c][1,2,4]benzotriazine-2-one (1) rapportée antérieurement; celle-ci réagit comme un isocyanate et, avec la 5-(4-
aminophényl)-10,15,20-triphénylporphyrine, elle fournit une tirapazamine–porphyrine conjuguée.
Mots clés : N-oxyde de porphyrine, (oxydopyridyl)porphyrine, tirapazamine.
[Traduit par la Rédaction] Posakony et al. 198
Heterocyclic N-oxides, most notably aromatic tirapaza-
mine (6) and RB90740 (6, 7) have been investigated as
One advantage of porphyrin-based, cancer therapies is that
neoplastic tissue has been reported to accumulate many por-
phyrins to a greater degree than many normal tissues; this
phenomenon also occurs with other aromatic macrocycles
(e.g., naphthalocyanines, phthalocyanines, and chlorins) (1, 2).
The mechanisms of tumor localization of such aromatic
macrocycles have been discussed and are complex (2–5).
radiosensitizers and hypoxia-selective agents. Thus, because
of our ongoing interest in the development of porphyrin-
based anticancer agents (8–10), we have pursued porphyrins
incorporating heterocyclic N-oxides, as this might improve
the delivery of heterocyclic N-oxides to neoplastic tissue.
Herein, we describe the N-oxidation of several meso-pyridyl
porphyrins to yield their corresponding (oxidopyridyl)por-
phyrins and their corresponding porphyrin-N-oxides, and the
conjugation of tirapazamine to a porphyrin.
Received July 20, 1998.
J.J. Posakony, R.C. Pratt, S.J. Rettig, and B.R. James.1
Department of Chemistry, University of British Columbia,
Vancouver, BC V6T 1Z1, Canada.
K.A. Skov. B.C. Cancer Research Centre, Vancouver,
BC V5Z 1L3, Canada.
All solvents were reagent grade or better. Unless other-
wise indicated, all commercial chemicals were used as pur-
chased from Aldrich Chemical Co., Strem, or Sigma Chemical
Co. Pyrrole was dried over and distilled from CaH2 or vac-
uum-transferred before use. Benzaldehyde, 4-pyridine-
1Author to whom correspondence may be addressed.
Telephone: (604) 822-6645. Fax: (604) 822-2847.
e-mail: brj@chem.ubc.ca
Can. J. Chem. 77: 182–198 (1999)
© 1999 NRC Canada

Posakony et al.
183
Table 1. Abbreviations used for the meso-pyridyl porphyrins, and corresponding (oxidopyridyl)porphyrins and porphyrin-N-oxides.
Porphryin precursor
Abbrev.
Oxidopyridyl abbrev.
Corresponding porphyrin-N-oxide abbrev.
5,10,15-Triphenyl-20-(4-pyridyl)porphyrin
5,15-Diphenyl-10,20-bis(4-pyridyl)porphyrin
5,10-Diphenyl-15,20-bis(4-pyridyl)porphyrin
TrPhPyP
t-DPhBPyPa
c-DPhBPyP
OPyTrPhP
t-BOPyDPhP
c-BOPyDPhP
OPyTrPhP-21O, OPyTrPhP-23O
Not isolated
c-BOPyDPhP-21O, c-BOPyDPhP-22O,
c-BOPyDPhP-24O
5-Phenyl-10,15,20-tris(4-pyridyl)porphyrin
5,10,15,20-Tetrakis(4-pyridyl)porphyrin
PhTrPyP
TPyP
TrOPyPhP
TOPyP
TrOPyPhP-21O, TrOPyPhP-23O
Not isolated
aPresent in small amounts as an impurity in TrPhPyP.
surface of a KBr disk) on an AIT Mattson Genesis Series
FTIR instrument. Elemental analyses were performed by
Mr. P. Borda of the Department of Chemistry (UBC). High-
(HR-MS) and low-resolution (LR-MS) mass spectra were
obtained on a KRATOS MS50 (EI), a KRATOS MS80 (CI),
a KRATOS Concept IIHQ (LSIMS), or a BRUKER Biflex
MALDI-TOF mass spectrometer. When analyzed with EI
techniques, the mass spectra of free-base porphyrins often
showed a peak at 61 m/z higher (M+ – 2H + Cu) than the
parent mass due to a gas phase metallation reaction with Cu
from the ionization source.
Porphyrin precursors
The meso-pyridyl porphyrins were synthesized according
to literature methods (8, 12); the abbreviations used for these
porphyrins and the corresponding (oxidopyridyl)porphyrin
and porphyrin-N-oxide derivatives are given in Table 1.
N-Oxidations
Based on published procedures for the preparation of
pyridine-N-oxide (13), excess meta-chloroperoxybenzoic acid
(m-CPBA, 60–85%, the rest being benzoic acid and H2O)
was added to a stirred solution of the starting porphyrin in
CH2Cl2 or CH2Cl2–MeOH at room temperature (r.t.). Addi-
tion of 1–2 equiv. aliquots of m-CPBA gave the (oxido-
pyridyl)porphyrins, while a single addition of several
equivalents gave the corresponding porphyrin-N-oxides in
low and variable yield. The reactions were monitored by
TLC on silica (CH2Cl2:MeOH 100:1 to 10:1); Et3N was
added to the final reaction mixture, and the product was pu-
rified by chromatography after preadsorbing the product
mixture on silica. With very polar eluent mixtures, metal-
lation (with Zn from the fluorescent indicator in the silica)
occurred on the TLC plate. In such cases, analytical TLC
plates without indicator were used. Tables 2–6 summarize,
carboxaldehyde, and trifluoroacetic acid (TFA) were freshly
distilled before use. Tirapazamine (3-amino-1,2,4-benzotri-
azine-1,4,-di-N-oxide) was a gift from Dr. M. Tracy (SRI In-
ternational, Menlo Park, Calif.). Reaction products were dried
in vacuo at 78°C overnight, unless otherwise noted.
The alumina used for chromatography was Fisher, activity
I, and was deactivated by adding water to give a standard
range of activities (11). Unless otherwise noted, the silica
gel used in the chromatographies was Merck Silica Gel 60,
230–400 mesh, whilst Rf values were measured on Merck
silica TLC Al sheets (silica gel 60 F254). Preparative TLCs
of porphyrin free bases were performed with Merck silica
gel 60, 0.5 mm plates (without indicator, to avoid metal-
lation of the products). In general, the visualization of non-
porphyrinic compounds was achieved with the aid of a UV
lamp. Dialysis tubing (SpectraPor) used in the purification
of the sulfonated porphyrins had a molecular weight cutoff
of 1000.
1
respectively, H NMR data for the (1-oxido-4-pyridyl)por-
1
phyrins, H NMR data for some porphyrin-N-oxides, UV-vis
data, elemental analyses, and mass spectrometry data; the 1H
NMR assignments are considered in Results and Discussion.
IR data for the (oxidopyridyl)porphyrins and the two sulfo-
nated derivatives (Table S1) and the porphyrin-N-oxides
(Table S2) are available as supplementary material.2
1H and 13C NMR spectra were recorded on a BRUKER
AC-200, a Varian XL-300, or a BRUKER WH-400 instru-
ment, and UV-vis spectra on an HP8452A photo-diode array
spectrophotometer (± 2 nm) with molar absorptivity (⑀) values
recorded for analytically pure porphyrins. IR spectra were
recorded as KBr pellets or as a thin film on a KBr disk
(from slow evaporation of a solution of the compound on the
If the porphyrin-N-oxides were not produced or their iso-
lation was not pursued, the product was purified by first re-
moving the solvent under reduced pressure, and the residue
2 Copies of material on deposit may be purchased from: The Depository of Unpublished Data, Document Delivery, CISTI, National Research
Council Canada, Ottawa, Canada, K1A 0S2. Tables of hydrogen atom coordinates have also also been deposited with the Cambridge Crys-
tallographic Data Centre and can be obtained on request from: The Director, Cambridge Crystallographic Data Centre, University Chemical
Laboratory, 12 Union Road, Cambridge, CB2 1EZ, U.K.
© 1999 NRC Canada

184
Table 2. H NMR data for the (1-oxido-4-pyridyl)porphyrins.a
Can. J. Chem. Vol. 77, 1999
1
2,6-OPy
3,5-OPyc
2,6-Ph (or 2,6-SPh)
3,4,5-Ph (or 3,5-SPh)d Pyrrole N-H
β-Pyrroleb
Porphyrin (MHz, solvent)
OPyTrPhP (200, CDCl3)
8.92 d (2), 8.84 s (4), 8.82 d (2)
8.64 dd (2) 8.18 d (6)
8.11 dd (2) 7.76 m (9)
8.62 dd (4) 8.18 d (4)
8.10 dd (4) 7.80 m (6)
–2.82 s (~1.5)
–2.87 s (~1.5)
–2.85 s (~1.5)
–2.88 s (~1.5)
t-BOPyDPhP (200, CDCl3)
c-BOPyDPhP (300, CDCl3)
TrOPyPhP (400, CDCl3)
8.94 d (4), 8.83 d (4)
8.94 d (2), 8.93 s (2), 8.86 s (2), 8.83 d (2) 8.63 dd (4) 8.18 d (4)
8.10 dd (4) 7.78 m (6)
8.95 d (2), 8.94 s (4), 8.83 d (2)
8.63 dd (6) 8.18 d (2)
8.10 dd (6) 7.8 m (3)
8.67 dd (8)
TOPyP (400, CDCl3–CD3OD)
OPyTrSPhP (200, DMSO-d6)
8.95 s br (8)
None observed in
CDCl3–CD3OD
–2.96 s br (1.7)
8.19 dd (8)
9.05 d (2), 8.88 d (2), 8.85 s (4)
8.63 d (2)
8.17 d (2)
8.64 d (4)
8.25 d (4)
8.17 d (6)
8.05 d (6)
8.18 d (4)
8.05 d (4)
c-BOPyBSPhP (200, DMSO-d6)e 9.10 s (2), 9.05 d (2), 8.87 m (4)
–2.95 s br (1.6)
aMeasured at r.t.; δ in ppm, signal pattern (number of protons). An H2O signal was generally observed in the spectra.
bJ values typically ~5 Hz (meso-phenyl derivatives) or ~6 Hz (meso-(4-sulfonatophenyl) derivatives).
cTypically, J1 = 7 Hz, J2 = 1 Hz.
dJ values typically ~8 Hz.
eImpurity peaks appeared near δ ~7.8 and 8.4 (<0.5 H each by integration).
1
Table 3. H NMR data for the porphyrin-N-oxides.a
2,6-OPy
3,5-OPyc
2,6-Ph
β-Pyrroleb
β-Pyrrole-NOd
Porphyrin
3,4,5-Ph
Pyrrole N-H
OPyTrPhP-23O
9.07 d (1), 9.00 d (1), 8.8
overlapping doublets (2), 8.10 d (2)
8.75 d (1), 8.66 d (1)
8.97 overlapping doublets
(2), 8.91 d (1), 8.83 d
(1), 8.65 m (2)
9.06 overlapping doublets
(2), 8.92 d (1), 8.85 d
(1), 8.73 m (2)
8.63 d (2)
8.5 m (6)
7.9 m (9)
7.5 s (2)
1.6 s br (>2)e
OPyTrPhP-21O
TrOPyPhP-21O
Under β-pyrrole signal at 8.65 8.23 d (2),
7.56 d (1),
7.50 d (1)
~1.7 s br (>2)e
8.17 d (4)
Under 2,6-Ph signal at 8.17
8.64 m (6)
8.14 d (2), 8.07 d (4)
7.8 m (9)
8.22 d (2)
7.83 m (3)
7.60 d (1),
7.54 d (1)
N-H peak not
assigned because
of large H2O peak
N-H peak not
assigned because
of large H2O peak
TrOPyPhP-22Of 9.07 d, 9.00 d, 8.94 d,
8.92 d, 8.74 d, 8.63 d
8.6 m
8.14 m, 8.05 d
Under
8.14 m
7.8 m
7.61 s
aMeasured at r.t. in CDCl3 (400 MHz); δ in ppm, signal pattern (number of protons). An H2O signal was observed in all spectra.
bJ values typically ~5 Hz.
cJ values typically ~7 Hz.
dJ values typically ~6 Hz; addition of D2O changed these signals to broad multiplets.
eIntegration includes H2O peak; the peak disappeared when D2O was added.
fIntegrations did not match.
then being washed with acetone. The resulting solid con-
tained mostly the (oxidopyridyl)porphyrin, while the filtrate
contained the corresponding porphyrin-N-oxides and other
reaction byproducts (e.g., large amounts of m-chlorobenzoic
acid (m-CBA)). Excess m-CBA complicated the chromatog-
raphy, and also could be removed by washing the reaction
mixture with dilute aq. NaOH. In some cases, mixtures of
porphyrins (e.g., c-DPhBPyP and PhTrPyP) were treated
with m-CPBA to give mixtures of (oxidopyridyl)porphyrins
and the corresponding porphyrin-N-oxides. The mixtures
were separated by chromatography, but no yield was deter-
mined for the minor components, as the molar ratio of the
compounds was not determined prior to the reaction.
silica. To obtain analytically pure material, the porphyrin
was allowed to crystallize slowly (days to weeks) from a
CH2Cl2:MeOH (~10:1) solution in a vial placed in a cham-
ber containing EtOH.
The (oxidopyridyl)porphyrins were deoxygenated under
the conditions of EI or LSIMS mass spectrometry and had to
be analyzed by MALDI-TOF mass spectrometry. The corre-
sponding porphyrin-N-oxides were deoxygenated to a lesser
degree, and in some cases the parent peaks were intense
enough to be analyzed by HR-MS (EI).
5-(1-Oxido-4-pyridyl)-10,15,20-triphenylporphyrin
(OPyTrPhP)
Because of the polar solvent mixtures used in chromato-
graphy, the porphyrin samples were often contaminated with
TrPhPyP (0.223 g, 0.36 mmol) was dissolved in CH2Cl2
(150 mL), and treated with excess m-CPBA (3 × 0.1 g aliquots)
© 1999 NRC Canada

Posakony et al.
185
Table 4. UV-vis data for the (1-oxido-4-pyridyl)porphyrins and porphyrin-N-oxides.
Porphyrin (solvent)
N-Oxide region (log ⑀)
Soret (log ⑀)
Q bands (log ⑀)
OPyTrPhPa (CH2Cl2:MeOH 20:1)
t-BOPyDPhPb (CH2Cl2:MeOH 10:1)
c-BOPyDPhPa (CH2Cl2:MeOH 10:1)
TrOPyPhPa (CH2Cl2:MeOH 10:1)
TOPyPa (CH2Cl2:MeOH 10:1)
OPyTrPhP-23Oc (CH2Cl2)
274 (4.32)
274, 310 (weak)
272 (4.49)
274 (4.61)
274 (4.66)
420 (5.60)
426
516 (4.35), 552 (3.95), 590 (3.80), 646 (3.67)
516, 556, 602, 648
422 (5.57)
424 (5.65)
424 (5.63)
420 (5.14)
420
518(4.30), 556 (4.00), 592 (3.85), 650 (3.59)
518 (4.33), 554 (4.07), 594 (3.87), 650 (3.65)
518 (4.3), 554 (4.03), 594 (3.85), 650 (3.56)
518 (3.77), 544 (3.76), 594 (3.85), 684 (3.41)
516, 548, 596, 684
282 (4.30)
280
OPyTrPhP-21O (CH2Cl2)
OPyTrPhPZn-23Od,e (CH2Cl2:MeOH 5:1)
OPyTrPhPZn-21O (CH2Cl2:MeOH 5:1)
c-BOPyDPhP-O mixture (CH2Cl2)
TrOPyPhP-21O (CH2Cl2)
TrOPyPhP-22O (CH2Cl2)
OPyTrSPhPf (H2O)
272 (4.35), 322 (4.26)
276, 330
436 (5.27)
438
422
424
424
574 (3.89), 622 (3.91)
574, 622
514, 550, 594, 688
518, 548, 600, 685
518, 552, 598, 685
516 (4.14), 554 (3.84), 580 (3.81), 636 (3.57)
520, 560, 586, 646
280, 342 (weak)
283, 334 (weak)
280, 348 (weak)
258 (4.32), 306 (4.27)
258, 306
414 (5.59)
418
c-BOPyBSPhP (H2O)
aSoret ⑀ determined at 2 × 10–6 M; ⑀ of other bands determined at 2 × 10–5 M.
bThe C, H, and N elemental analyses were ~0.5% too low (see Table 5); ⑀ values were not determined.
cThe C and N elemental analyses were ~0.5 and 1.26% too low, respectively (see Table 5).
dSoret ⑀ determined at 5 × 10–6 M; ⑀ of other bands determined at 5 × 10–5 M.
ePrepared from a known concentration of OPyTrPhP-23O and excess Zn(OAc)2.
f ⑀ determined at 1.0 × 10–6 M.
Table 5. Elemental analyses for the (1-oxido-4-pyridyl)porphyrins and porphyrin-N-oxides.a
Porphyrin (formula)
C (%)
H (%)
N (%)
OpyTrPhP (C43H29N5O)
81.53 (81.75)
77.58 (77.12)
73.67 (73.12)
75.66 (75.83)
72.93 (72.71)
71.09 (71.04)
68.56 (68.55)
45.46 (45.72)
54.02 (54.28)
4.63 (4.88)
4.69 (4.65)
4.71 (4.24)
4.54 (4.41)
4.52 (4.46)
4.37 (4.31)
4.03 (3.98)
4.26 (4.11)
3.78 (3.66)
11.09 (11.10)
10.52 (9.26)
12.27 (11.86)
12.60 (12.73)
12.60 (12.73)
14.15 (13.93)
15.99 (15.75)
6.16 (6.12)
OpyTrPhP-23O (C43H29N5O2·1H2O)
t-BOPyDPhP (C42H28N6O2·2H2O)
c-BOPyDPhP (C42H28N6O2·H2O)
c-BOPyDPhP-O mixture (C42H28N6O3·1.5H2O)
TrOPyPhP (C41H27N7O3·1.5H2O)
TOPyP (C40H26N8O4·2H2O)
OpyTrSPhP (C43H26N5Na3O10S3·11H2O)
c-BOPyBSPhP (C42H26N6Na2O8S2·4.5H2O)
9.00 (8.80)
aCalculated (found); not obtained for OPyTrPhP-23O, TrOPyPhP-21O, and TrOPyPhP-22O.
The porphyrin-N-oxides metallated rapidly when mixed
with Zn(OAc)2 in CH2Cl2–MeOH to give OPyTrPhPZn-21O
and OPyTrPhPZn-23O (see Table 4 for UV-vis data). At-
tempts to grow crystals of the free-base porphyrin-N-oxides
or Zn-porphyrin-N-oxides were unsuccessful.
over 1–2 h while the solution was stirred. Et3N (20 mL) was
then added, and the product was preadsorbed on silica and
chromatographed on silica (CHCl3: pyridine 10:1). The sol-
vent was removed from the main product band to yield
0.128 g (56% yield) of OPyTrPhP. The reaction was scaled
up to ~0.5 g of TrPhPyP with similar results. Slow solvent
evaporation from a CH2Cl2–MeOH solution of the porphyrin
yielded analytically pure crystals which were studied by X-
ray crystallography.
5,15-Bis(1-oxido-4-pyridyl)-10,20-diphenylporphyrin
(t-BOPyDPhP)
t-BOPyDPhP was prepared in the same manner as OPyTrPhP.
The m-CPBA was added in 1–2 equiv. aliquots to a solution
of an impure sample of TrPhPyP containing a small amount
of t-DPhBPyP. The products were purified by column chro-
matography on silica; OPyTrPhP was eluted from the column
with CHCl3:pyridine (10:1), and t-BOPyDPhP was eluted
with CHCl3:pyridine (10:3). The yield was not determined.
Because only small amounts of t-DPhBPyP were obtained
from the mixed-aldehyde synthesis (8, 12), the synthesis of
the corresponding porphyrin-N-oxide was not pursued.
5-(1-Oxido-4-pyridyl)-10,15,20-triphenylporphyrin-21-
oxide (OPyTrPhP-21O), and -23-oxide (OPyTrPhP-23O)
From reactions in which several equivalents of m-CPBA
were added all at once to a solution of TrPhPyP in
CH2Cl2:MeOH, two additional products were isolated (~5%
yield each) after OPyTrPhP was first isolated chromato-
graphically. The two bands following that of OPyTrPhP
were collected; the solvent was removed, and a second
chromatography column silica (CH2Cl2:THF:MeOH
100:5:2.5) was used to separate two isomers. The less polar
band was OPyTrPhP-23O, and the more polar band was
OPyTrPhP-21O.
5,10-Bis(1-oxido-4-pyridyl)-15,20-diphenylporphyrin
(c-BOPyDPhP)
c-BOPyDPhP was synthesized in the same manner as
OPyTrPhP. To a solution of c-DPhBPyP (0.5 g, 0.8 mmol,
© 1999 NRC Canada

186
Can. J. Chem. Vol. 77, 1999
Table 6. Mass spectrometry data for (1-oxido-4-pyridyl)porphyrins and porphyrin-N-oxides.
Formula
Porphyrin
LR-MS, m/za (intensity %, assignment)
HR-MS, calcd. (found)
OPyTrPhPb
632 (88, M+1),
616 (36, M+1 – O)
Not obtained
OPyTrPhP-21Oc
OPyTrPhP-23Oc
692 (2, M+ – O – 2H + Cu),
676 (5, M+ – 2O – 2H + Cu),
647 (4, M+), 631 (10, M+ – O),
615 (100, M+ – 2O)
692 (1, M+ – O – 2H + Cu),
677 (2, M+ –2O – 2H + Cu),
647 (1, M+), 631 (20, M+ – O),
615 (100, M+ – 2O)
650 (100, M+1), 634 (62, M+1 – O),
614 (19, M+1 – 2O)
649 (100, M+1), 633 (86, M+1 – O),
617 (27, M+1 – 2O)
C43H29N5O2
647.232 12 (647.232 07)
C43H29N5O2
647.232 12 (647.229 65)
C43H29N5O
631.237 18 (631.237 01)
Not obtained
t-BOPyDPhPb,d
c-BOPyDPhPb,d
Not obtained
c-BOPyDPhP-NO mixturec
677 (5, M+ – 2O – 2H + Cu),
693 (2, M+ – O –2H + Cu),
664 (1, M+), 648 (5, M+ – O),
632 (29, M+ – 2O),
C42H28O3N6
664.222 29 (664.219 42)
C42H28O2N6
648.227 36 (648.225 16)
616 (100, M+ – 3O)
TrOPyPhPb,d
666 (87, M+1), 650 (100, M+1 – O), 634
(66, M+1 – 2O), 618 (33, M+1 – 3O)
710 (1, M+ – 2O – 2H + Cu),
694 (7, M+ – 3O – 2H + Cu),
678 (8, M+ – 4O – 2H + Cu),
665 (1, M+ – O), 649 (11, M+ – 2O),
633 (80, M+ – 3O),
Not obtained
TrOPyPhP-21Oc
C41H27O3N7
665.217 53 (665.217 19)
C41H27O3N7
649.222 60 (649.222 10)
617 (100, M+ – 4O)
TrOPyPhP-22-NOc
TOPyPb
678 (20, M+ – 4O –2H + Cu),
665 (2, M+ – O), 649 (9, M+ – 2O),
633 (10, M+ – 3O),
C41H27O3N7
665.217 53 (665.215 11)
C41H27O3N7
649.222 60 (649.221 75)
Not obtained
617 (100, M+ – 4O)
683 (51, M+1), 666 (84, M+1 – O),
650 (99, M+1 – 2O),
635 (100, M+1 – 3O),
619 (79, M+1 – 4O)
c-BOPyBSPhPf
875 (0.1, M+1 + Na),
Not obtained
859 (0.1, M+1 + Na – O),
853 (0.1, M+1),
837 (0.1, M+1 – O)
aM+1 implies (M + H)+.
bMALDI-TOF mass spectroscopy.
cEI mass spectroscopy.
dDimer peaks (5–10% intensity, due to combinations of the species listed in the LR-MS column) are observed.
e(+)CI mass spectroscopy.
f(+)LSIMS mass spectroscopy.
but containing
a
small percentage of PhTrPyP) in
followed by a mixture of porphyrin-N-oxides, and then a
small amount (~20 mg) of TrOPyPhP. A c-BOPyDPhP sam-
ple of analytical purity was obtained by slow crystallization
from a solution of the compound in CH2Cl2:MeOH.
CH2Cl2:MeOH (125:25 mL) was added excess m-CPBA (4
× ~1.25 g aliquots), and the reaction monitored by TLC.
Et3N (~10 mL) was added; the product was preadsorbed on
silica and intially chromatographed on silica (CH2Cl2:
pyridine:MeOH 20:1:2 to 20:1:3). The product bands were
poorly resolved, possibly because of large amounts of resid-
ual m-CPBA and m-CBA; the porphyrin-containing fractions
were washed with dilute aq. NaOH. The product mixture
was rechromatographed on silica (CH2Cl2:pyridine:MeOH
10:1:2.5); c-BOPyDPhP eluted first (~0.27 g, ~50% yield),
5,10-Bis(1-oxido-4-pyridyl)-15,20-diphenylporphyrin-21-
oxide (c-BOPyDPhP-21O), -22-oxide (c-BOPyDPhP-
22O), and -24-oxide (c-BOPyDPhP-24O)
A mixture of porphyrin-N-oxides was isolated (~30 mg,
~5%) from the reaction of c-DPhBPyP with a large excess of
m-CPBA (see above). Attempts to separate the mixture on
© 1999 NRC Canada

Posakony et al.
187
(oxidopyridyl)porphyrins and porphyrin-N-oxides can be found
in Tables 2 and 3.
5,10,15-Tris(1-oxido-4-pyridyl)-20-phenylporphyrin
(TrOPyPhP)
TrOPyPhP was produced in the same manner as
OPyTrPhP. PhTrPyP was present in small amounts in the
sample of c-DPhBPyP used above, and TrOPyPhP was iso-
lated from the reaction mixture. The yield was not deter-
mined. A sample of analytical purity was obtained by slow
crystallization from CH2Cl2–MeOH.
5,10,15-Tris(1-oxido-4-pyridyl)-20-phenylporphyrin-21-
oxide (TrOPyPhP-21O), and -22-oxide (TrOPyPhP-22O)
TrOPyPhP-21O and TrOPyPhP-22O were prepared in the
same manner as the porphyrin-N-oxides of OPyTrPhP. The
product was purified by preparative TLC on silica
(CH2Cl2:MeOH 10:1 to 5:1). Only ~1 mg of each porphyrin-
N-oxide was obtained (<5% yield).
5,10,15,20-Tetrakis(1-oxido-4-pyridyl)porphyrin
(TOPyP)
Oxidation of TPyP
TOPyP was prepared in the same manner as OPyTrPhP.
TPyP (0.35 g, 0.57 mmol) was dissolved in CH2Cl2:MeOH
(150:25 mL) and excess m-CPBA (6 × 0.5 g) was added in
aliquots over 3 h to the stirred mixture. TLC showed at least
four intermediates as the reaction progressed. Et3N (~20 mL)
was added to the reaction mixture and the solvent was re-
moved. The residual solid was rinsed with MeOH, acetone,
and H2O, and air-dried. Further purification was achieved by
dissolving the product in H2O:TFA (20:1), neutralizing the
solution (NaOH), and subsequently filtering off the crude
TOPyP precipitate; total yield 0.275 g (0.40 mmol, ~70%).
Analytically pure TOPyP was obtained by slow crystalliza-
tion from CH2Cl2:MeOH. Alternatively, small amounts of
the product could be chromatographed on Al2O3(III)
(CH2Cl2:MeOH 20:1). TOPyP was insoluble in CDCl3, and
thus its 1H NMR spectrum was measured in CDCl3–CD3OD.
silica (CH2Cl2:pyridine:MeOH 20:1:2.5) were unsuccessful.
There are three possible porphyrin-N-oxides isomers: c-
BOPyDPhP-21O, c-BOPyDPhP-22O, and c-BOPyDPhP-24O.
The 1H NMR data given below support the presence of at
least two of these. The two doublets for the β-pyrrole-NO
protons (δ 7.57 and 7.51) indicate the presence of c-
BOPyDPhP-21O, while the corresponding singlet at δ 7.60
indicates the presence of either c-BOPyDPhP-22O or c-
1
BOPyDPhP-24O, or both. H NMR (400 MHz, CDCl3) δ:
9.05 (d, J = 5 Hz), 8.99 (overlapping ds, J = 5 Hz), 8.93 (d,
J = 5 Hz), 8.91 (d, J = 5 Hz), 8.85 (d, J = 5 Hz), 8.74 (d, J =
5 Hz), 8.65 (m), 8.22 (dd, J1 = 6 Hz, J2 = 1.5 Hz), 8.15 (m),
8.08 (d), 7.8 (m), 7.60 (s), 7.57 (d, J = 6 Hz), 7.51 (d, J =
Acid-catalyzed condensation of pyrrole and
4-pyridinecarboxaldehyde-N-oxide
Attempts were also made to prepare TOPyP via acid-catalyzed
condensation of pyrrole and 4-pyridinecarboxaldehyde-N-ox-
ide. Pyrrole (0.3 mL, 0.4 mmol) and 4-pyridinecarboxal-
1
6 Hz). Because the ratio of products is unknown, the H
NMR signals are not assigned; general δH values for the
© 1999 NRC Canada

188
Can. J. Chem. Vol. 77, 1999
dehyde-N-oxide (0.05 g, 0.4 mmol) were dissolved in
propionic acid (2 mL), and the solution was refluxed for
~50 min. UV-vis spectroscopy indicated that a porphyrin
had formed (Soret 418 nm), but TLC analysis of the mixture
on silica (CH2Cl2–MeOH) indicated numerous side products
and so further isolation was not attempted. Attempts to syn-
thesize TOPyP with Lindsey-type conditions in CH2Cl2 with
pyrrole and 4-pyridinecarboxaldehyde (4 or 8 mM), using
TFA or BF3·MeOH and subsequent oxidation with 2,3,5,6-
tetrachloro-1,4-benzoquinone (TCQ), yielded only traces of
porphyrin by UV-vis spectroscopy.
Sodium 5,10-bis(1-oxido-4-pyridyl)-15,20-bis(4-sulfonato-
phenyl)porphyrin (c-BOPyBSPhP)
c-BOPyDPhP (0.10 g, 0.15 mmol) was sulfonated in the
same manner used for OPyTrSPhP. The reaction was moni-
tored by TLC; an intermediate band appeared between that
of the starting material and the baseline, and heating was
continued ~0.5 h after the intermediate had disappeared (6 h
1
total). After dialysis of the worked up product, its H NMR
spectrum revealed the presence of an impurity. Redissolving
the product in conc. H2SO4 and heating at 100°C for another
2 h with the same work-up procedure did not remove the im-
purity (i.e., it is not a monosulfonatophenyl product). At-
tempts to purify by chromatography on silica MeOH or by
recrystallization from MeOH–acetone were unsuccessful.
The results of the elemental analysis (Table 5) suggest that
the composition of the impurity does not differ significantly
from that of c-BOPyBSPhP. Careful TLC analysis of the
sample of c-BOPyDPhP used here showed the presence of
another compound with an Rf nearly identical to that of c-
BOPyDPhP; the presence of the impurity was not evident in
TPyP synthesis via Lindsey conditions
When Lindsey conditions (14) were applied to the synthe-
sis of TPyP using trichloroacetic acid, TFA, or BF3·MeOH
catalysis, TPyP was produced when aliquots of the reaction
mixture were treated with TCQ, which showed that the pre-
cursor porphyrinogen was forming. However, the porphyr-
inogen was eventually consumed by a side reaction that gave
a very polar, unidentified, blue compound. The rate of the
side reaction increased with increasing acid strength and
concentration.
1
the H NMR spectrum (CDCl3).
2H-[1,2,4]Oxadiazolo[3,2-c][1,2,4]benzotriazin-2-one-5-
oxide (1)
Sulfonations
Based on a literature reaction between phosgene and tira-
pazamine (15), a suspension of tirapazamine (0.3 g,
1.7 mmol) in dry toluene (5 mL) was heated to 90°C, and
triphosgene (0.173 g, 0.58 mmol) was added. Within 5 min,
the suspension turned from orange to yellow and TLC indi-
cated near quantitative conversion to 1 (Rf = 0.75 silica
(CH2Cl2:MeOH ~50:1)). (When the solution was heated for
~2 h, TLC indicated that some 1 had been reconverted to
tirapazamine (Rf = 0.18).) The reaction mixture was cooled,
and the precipitate was filtered off and washed with hexanes
to yield 0.324 g (1.59 mmol, 94% yield) of 1 as a yellow–
brown solid, which contained a small amount of tira-
pazamine (by TLC). The product was used without further
purification in subsequent reactions. Compound 1 was re-
converted to tirapazamine when exposed to H2O vapor or
when co-spotted with H2O on a TLC plate, and tirapazamine
was isolated in variable amounts from subsequent reactions
involving 1. IR (KBr pellet): 3460 w br, 3170 w, 3067 w,
1814 vs, 1598 s, 1541 vs, 1437 vw, 1386 s,1344 s, 1226 m,
Sodium 5-(1-oxido-4-pyridyl)-10,15,20-tris(4-sulfonato-
phenyl)porphyrin (OPyTrSPhP)
Based on the literature procedure for the sulfonation of
5,10,15,20-tetraphenylporphyrin (TPhP) (8), OPyTrPhP
(0.535 g, 0.85 mmol) was dissolved in conc. H2SO4 (10 mL),
and the solution was heated at 110°C for 4 h. The reaction
was monitored by first neutralizing an aliquot of the mixture
(using aq. Na2CO3), then analyzing by TLC on silica
(CH2Cl2:MeOH 100:1 to 5:1). The reaction mixture was
cooled with an ice-bath, and cold, distilled H2O (50 mL)
was added, with the pH of the solution being adjusted to 7–8
with aq. NaOH and aq. Na2CO3. The solvent was removed
and the porphyrin washed from the residue with MeOH.
Then the MeOH was removed and the porphyrin subse-
quently dialyzed in dialysis tubing in a beaker of distilled
H2O to yield 0.51 g (~53% yield) of OPyTrSPhP·11H2O.
1
1
The H NMR signals were assigned with the help of a H-
2D-COSY spectrum.
© 1999 NRC Canada

Posakony et al.
189
1
Table 7. H NMR data for the benzotriazine-dioxide compounds.a
Compound (solvent)
-CONH
Benzotriazine ring Hb
Other substituent
Tirapazamine (acetone-d6)c
8.28 t of d (2), 7.98 t of d (2),
7.64 t of d (2)
7.38 s br (2, -NH2)
1 (acetone-d6)
2 (CDCl3)
8.47 d (1), 8.27 t of d (1), 7.97
d of d (1), 7.80 t of d (1)
7.27 t(1), 7.04 m (2), 6.63 t (1) 8.91 s br (4), 8.84 s (4), β-pyrrole H; 8.35 d (2, J 8,
2,6-RNHPh-H); 8.25 m (6, 2,6-Ph-H); 8.00 d (2, J 8,
3,5-RNHPh-H); 7.78 m (9, 3,4,5-Ph-H); –3.19 s br
(2, pyrrole N-H)
9.71 s (1),
8.81 s (1)
aMeasured at r.t. at 400 MHz; δ in ppm, signal pattern (number of protons).
bTypical J values for doublets or triplets 8–9 Hz; for a doublet or triplet of doublets J1 ~ 9, J2 ~ 1 Hz.
1
cThe H NMR data are similar to those reported in the literature (recorded in TFA), ref. 15b.
1196 m, 1149 m, 1110 m, 772 m, 734 m; IR of tirapazamine
(thin film on a KBr disk): 3411 w, 3274 w br, 3098 w, 1594
vs, 1408 vs, 1362 vs, 1105 vs, 1025 w, 828 w, 723 w. For 1,
LR-MS (+CI): 205 (30, M+1), 163 (100, M+1 – NCO), 147
(75, M+1 – NCO – O). HR-MS (+CI) calcd. for C8H5N4O3:
for C52H35N9O3·0.5H2O: C 74.10, H 4.30, N 14.96; found: C
73.96, H 4.18, N 14.41%. The H NMR data for 2 appear in
Table 7.
1
1
205.03616; found 205.03622. The H NMR data for 1 (not
reported previously) and for tirapazamine appear in Table 7.
5-(4-N-(N′-(1,4-dioxido-1,2,4-benzotriazin-3-
yl)aminocarbonyl)aminophenyl)-10,15,20-
triphenylporphyrin (2)
X-ray crystallographic analysis of OPyTrPhP
Crystallographic data appear in Table 8. The final unit-
cell parameters were obtained by least squares on the setting
angles for 25 reflections with 2θ = 45.4 – 80.4°. The intensi-
ties of three standard reflections, measured every 200 reflec-
tions throughout the data collection, showed only small
random fluctuations. The data were processed3 corrected for
Lorentz and polarization effects and absorption (based on
azimuthal scans).
To a solution of 5-(4-aminophenyl)-10,15,20-triphenyl-
porphyrin (APhTrPhP) (8) (31 mg, 0.05 mmol) in dry DMF
(3 mL) was added 1 (11 mg, 0.055 mmol), and the mixture
was stirred at r.t. in the dark for 26 h. TLC (silica
(CH2Cl2:MeOH 100:2–3)) showed a new product (Rf = 0.4),
some APhTrPhP (Rf = 0.65), but little 1; so another 1.0
equiv. of 1 was added, and the mixture was stirred another
24 h. The solvent was removed, and the desired product was
isolated from several minor products by preparative TLC on
silica (CH2Cl2:MeOH 100:2–3). The isolated product was
washed with hexanes, dissolved in CH2Cl2:MeOH (20:1),
and the solution was washed with H2O. The solvent was re-
moved to yield 20 mg of a purple powder (~50%). IR (KBr
pellet, cm–1): 3312 w, 1709 m, 1597 m, 1526 vs, 1405 m,
1332 m, 1311 m, 1225 m, 1181 w, 964 m, 799 m, 727 m,
701 m; UV-vis (2.4 × 10–6 M, CH2Cl2:MeOH (100:1)) corre-
sponding to the tirapazamine moiety: 280 (log ⑀: 4.65), 478
(4.26) nm, corresponding to the porphyrin core: 418 (5.81),
514 (4.54), 550 (4.28), 586 (4.16), 646 (4.03). LR-MS
(+LSIMS): 834 (0.25, M+1), 818 (1, M+1 – O), 802 (0.25
M+1 – 2O), 656 (12, M+1 – tirapazamine). HR-MS (+LSIMS)
calcd. for C52H36N9O3: 834.29411, found: 834.29456; calcd.
for C52H36N9O2: 818.29919, found 818.29953. Anal. calcd.
The space group ambiguity (space groups I41md and I2d
are possible) was resolved by trial and error. The structure
was solved by direct methods, the coordinates of the non-
hydrogen atoms being determined from E-maps or from sub-
sequent difference Fourier syntheses. The molecule is cen-
tred at a point of crystallographic S4 symmetry, thus the
pyridine-N-oxide ligand and the N-H protons are statistically
disordered. Position C(9)/N(2) was refined as 0.75C and
0.25N; the N and C atoms are too close to resolve. The oxy-
gen position is 1/4 occupied and the N-H proton position is
half occupied. All non-hydrogen atoms were refined with
anisotropic thermal parameters. The full-occupancy hydro-
gen atoms were refined isotropically and the N-H and C(9)-
H hydrogen atoms were fixed in calculated positions with
C—H = 0.98 Å, N—H = 0.91 Å and BH = 1.2Bbonded atom. A
correction for secondary extinction was applied (Zachariasen
type, isotropic, Gaussian), the final value of the extinction
3 teXsan: Crystal structure analysis package. Unix version 1.7. Molecular Corporation. The Woodlands, Tex., U.S.A. 1995.
© 1999 NRC Canada

190
Can. J. Chem. Vol. 77, 1999
Table 8. Crystallographic data for OpyTrPhP.a
OEP-O (Ni, Mn, Co, Cu) (19–22), an Fe tetramesityl-
porphyrin-N-oxide complex Fe(TMP-O) (23–26), and a Ti
tetraphenylporphyrin-N-oxide complex Ti(TPhP-O) (27)
have been reported. With OEP-O, the metal was introduced
after N-oxidation, while Fe(TMP-O) was produced by treat-
ing Fe(TMP) with m-CPBA, and Ti(TPhP-O) was prepared
by photolysis of Ti(TPhP)(O2). TMP-O was produced by ox-
idation of TMP, or demetallation of Fe(TMP-O) with HCl–
HOAc (23), and Ti(TPhP-O) spontaneously demetallated in
ethanol (27). In the metal complexes, the oxygen remains on
one face of the porphyrin but, in the free-base porphyrin-N-
oxides, the oxygen rapidly moves through the center of the
porphyrin in a fluxional inversion process (19).
Compound
Formula
fw
Crystal system
Space group
a, Å
c, Å
V, Å3
OPyTrPhP
C43H29N5O
631.73
Tetragonal
I2d (no. 122)
15.174(1)
13.709(3)
3156.3(9)
4
1.329
1320
6.41
0.20 × 0.35 × 0.45
0.87–1.00
ω-2θ
1.00 + 0.20 tan θ
32 (up to 9 scans)
Z
ρcalc, g/cm3
F(000)
µ(Cu Kα), cm–1
Crystal size, mm
Transmission factors
Scan type
In contrast, (oxidopyridyl)porphyrins have not been re-
ported, although a related compound (3) was obtained by
Milgrom et al. (28) from the aerial oxidation of a (1-benzyl-
4-pyridyl)porphyrin, this procedure leading also to further
benzylation of the macrocyclic nitrogens.
Scan range, deg in ω
Scan speed, deg/min
Data collected
2θmax, deg
Crystal decay, %
Total reflections
Total unique reflections
Reflections with I ≥ 3σ(I)
No. of variables
R
Tetraarylporphyrin syntheses
155
Negligible
998
998
685
The precursor meso-phenyl/pyridyl porphyrins and
(aminophenyl)triphenylporphyrin were synthesized accord-
ing to published procedures (8). The synthesis of (oxido-
pyridyl)porphyrins via acid-catalyzed condensation of pyrrole,
benzaldehyde and 4-pyridinecarboxaldehyde-N-oxide was also
attempted, but a significant percentage of the oxidopyridyl
groups was deoxygenated in refluxing propionic acid. How-
ever, acid-catalyzed condensation of pyrrole and 4-pyridine-
carboxaldehyde-N-oxide did produce at least one porphyrin,
probably TOPyP, but this compound was much more conve-
143
0.031
0.026
2.2
0.0008
–0.07 to 0.09
Rw
gof
Max ∆/σ (final cycle)
Residual density, e/Å3
aTemperature 294 K, Rigaku AFC6S diffractometer, Cu Kα radiation (λ
= 1.54178 Å), graphite monochromator, takeoff angle 6.0°, aperture 6.0 ×
6.0 mm at a distance of 285 mm from the crystal, stationary background
counts at each end of the scan (scan/background time ratio 2:1), σ2(F2) =
[S2(C + 4B)]/Lp2 (S = scan rate, C = scan count, B = normalized
background count), function minimized Σw(|Fo| – |Fc|)2 where w =
4Fo /σ2(Fo ), R = Σ||Fo| – |Fc||/Σ|Fo|, Rw = (Σw(|Fo| – |Fc|)2/Σw|Fo|2)1/2, and
gof = [Σw(|Fo| – |Fc|)2/(m – n)]1/2. Values given for R, Rw, and gof are
based on those reflections with I ≥ 3σ(I).
2
2
coefficient being 1.54(5) × 10–6. A parallel refinement of the
mirror image gave marginally larger residuals (by a factor of
1.001). Neutral atom scattering factors for all atoms and
anomalous dispersion corrections for the non-hydrogen at-
oms were taken from ref. 16. Final atomic coordinates and
equivalent isotropic thermal parameters, bond lengths and
bond angles appear in Tables 9–11, respectively. Infrared
spectroscopy data, hydrogen atom parameters, anisotropic
thermal parameters, torsion angles, intermolecular contacts,
and least squares planes are included as supplementary ma-
terial.2
niently prepared by N-oxidation of TPyP.
The synthesis of TPyP, via Lindsey conditions was ham-
pered by a side reaction that produced an unidentified, blue
compound. It has been noted that under Lindsey conditions,
numerous heterocyclic aldehydes fail to give porphyrins but
no explanation was suggested (14). N-Substitution on the
pyridine nitrogen (as in N-oxides, and alkyl or acyl pyridi-
nium salts) greatly activates the pyridine ring to nucleophilic
addition at the α and γ positions (29). Thus, in situ formation
of a pyridinium salt via protonation of (or BF3 coordination
to) the pyridine lone pair under high [H+] and subsequent re-
action (at the pyridine ring and aldehyde substituent) with
pyrrole could explain the results obtained here. The side re-
Porphyrin-N-oxides and (1-oxido-4-pyridyl)porphyrins
Porphyrin-N-oxides have been reported in the literature.
Octaethylporphyrin-N-oxide (OEP-O) was first prepared by
Bonnet et al. (17, 18) by treating OEP with hypofluorous
acid (in 64% yield) or a peroxyacid (26% using m-CPBA, to
68% using permaleic acid). In studies relating to the active
site of cytochrome P-450 enzymes, metal complexes of
© 1999 NRC Canada

Posakony et al.
Table 9. Final atomic coordinates (fractional) and Beq (103 Å2).a
191
Atom
x
y
z
Beq
Occupancy
1/4
O(1)
N(1)
N(2)
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
C(7)
C(8)
C(9)
C(10)
C(11)
0.5590(4)
0.0680(1)
0.4789
0.3378(6)
0.3847(1)
0.3672
0.1417(9)
0.2468(2)
0.1491
8.0(3)
4.68(5)
6.61
1/4
0.0344(2)
0.1059(2)
0.1804(2)
0.1574(2)
0.2170(2)
0.3096(2)
0.3305(2)
0.4147(2)
0.4789(2)
0.4600(2)
0.3762(2)
0.3027(2)
0.2406(2)
0.2851(2)
0.3759(2)
0.4450(2)
0.4202(2)
0.3948(2)
0.3695(3)
0.3672(2)
0.3928(3)
0.4195(3)
0.2670(2)
0.2692(2)
0.2478(3)
0.2349(2)
0.2217(2)
0.1961(2)
0.1022(3)
0.0789(3)
0.1491(3)
0.2407(3)
0.2645(3)
4.78(6)
5.60(8)
5.65(7)
4.71(7)
4.74(7)
4.95(7)
6.10(9)
7.4(1)
6.61(9)
7.2(1)
6.90(9)
3/4
aBeq = (8/3)π2ΣΣ(Uijaiaj(aiaj)).
corresponding porphyrin-N-oxides in low but variable yield,
as pyrrole nitrogens were also oxidized. Use of the same
conditions with TPhP gave no significant amount of TPhP-
oxide, and continued addition of m-CPBA only led to the
degradation of the porphyrin. As an oxidopyridyl group is
more electron-withdrawing than a phenyl group, the (oxido-
pyridyl)porphyrins are more resistant to oxidative degrada-
tion, and thus the (oxidopyridyl)porphyrin-N-oxides could
be isolated.
Table 10. Bond lengths (Å) with estimated standard deviations
in parentheses.a
Bond
Length
Bond
Length
O(1)—N(2)
N(1)—C(4)
N(2)—C(10)
C(1)—C(5)′
C(3)—C(4)
C(5)—C(6)
C(6)—C(11)
C(9)—C(10)
1.299(7)
1.374(3)
1.346(4)
1.398(4)
1.433(4)
1.496(4)
1.378(4)
1.346(5)
N(1)—C(1)
N(2)—C(8)
C(1)—C(2)
C(2)—C(3)
C(4)—C(5)
C(6)—C(7)
C(7)—C(8)
C(10)—C(11)
1.372(3)
1.369(4)
1.438(4)
1.349(4)
1.396(3)
1.381(4)
1.372(4)
1.375(4)
Thermal deoxygenation is commonly observed with heter-
ocyclic N-oxides (13), but no deoxygenation was observed
(by 1H NMR studies) when a solid sample of OPyTrPhP was
heated at 100°C overnight in vacuo. Porphyrin-N-oxides de-
oxygenate at milder conditions (18), and were thus kept be-
low 30°C. Deoxygenation was observed in the mass spectra
of the (oxidopyridyl)porphyrins and their N-oxides. The ele-
mental analyses of these compounds, and the presence of
aSymmetry operation: (′) –1/2 + y, 1/2 – x, 1/2 – z.
action did not occur using weaker acids (e.g., dichloroacetic
acid), but these were not able to catalyze the formation of a
porphyrinogen, and thus no TPyP was observed when TCQ
was added.
1
H2O peaks in their H NMR spectra were consistent with
water being present; of note, pyridine-N-oxide itself is hy-
groscopic and freely soluble in water (13).
The porphyrin-N-oxides are much more soluble in weakly
polar solvents (CH2Cl2, CHCl3) than their parent oxido-
pyridyl compounds, and are soluble in MeOH. OPyTrPhP
has good solubility in CH2Cl2 or CHCl3, but with increasing
numbers of oxidopyridyl groups, increasing amounts of
MeOH are required as a cosolvent to ensure dissolution. For
N-Oxidations
N-Oxidation of the pyridyl substitutents was accomplished
using m-CPBA; the oxidations were sluggish at 0°C, but
proceeded readily at r.t. When the peroxyacid was added in
small aliquots, only the (oxidopyridyl)porphyrins were ob-
tained, while the single addition of m-CPBA generated the
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Can. J. Chem. Vol. 77, 1999
Fig. 1. ORTEP view of OPyTrPhP; 33% probability thermal ellipsoids are shown for the non-hydrogen atoms. The disorder is not
shown for the sake of clarity.
angle of the oxidopyridyl group and the porphyrin plane is
78°. The N(2)—O(1) bond length (1.299(7) Å) is signifi-
cantly shorter than that in pyridine-N-oxide (1.34 Å), but
lies within the range of N—O distances in a variety of sub-
stituted pyridine-N-oxides (13, 19).
Table 11. Bond angles (deg) with estimated standard deviations
in parentheses.a
Bonds
Angle
Bonds
Angle
C(1)-N(1)-C(4)
O(1)-N(2)-C(10)
N(1)-C(1)-C(2)
C(2)-C(1)-C(5)′
C(2)-C(3)-C(4)
N(1)-C(4)-C(5)
107.7(2)
111.8(5)
108.6(2)
126.1(2)
107.7(2)
125.7(2)
125.1(2)
116.8(2)
122.1(3)
120.6(3)
119.8(3)
121.3(4)
O(1)-N(2)-C(8)
C(8)-N(2)-C(10)
N(1)-C(1)-C(5)′
C(1)-C(2)-C(3)
N(1)-C(4)-C(3)
C(3)-C(4)-C(5)
128.3(6)
119.8(2)
125.2(2)
107.4(3)
108.6(2)
125.5(2)
118.1(2)
120.3(3)
117.7(3)
120.4(3)
120.2(3)
1H NMR spectra
1H NMR spectroscopy (Tables 2, 3) was very useful for
characterization of the (oxidopyridyl)porphyrins and enabled
the porphyrin-N-oxide isomers to be identified.
′′
′′
C(1) -C(5)-C(4)
C(1) -C(5)-C(6)
C(4)-C(5)-C(6)
C(5)-C(6)-C(11)
C(6)-C(7)-C(8)
C(8)-C(9)-C(10)
C(6)-C(11)-C(10)
C(5)-C(6)-C(7)
C(7)-C(6)-C(11)
C(9)-C(8)-C(7)
C(9)-C(10)-C(11)
Oxidopyridyl groups
Compared to the δH values for the unoxidized pyridyl
group, the 2-H and 6-H protons of the oxidopyridyl groups
were shifted upfield by ~0.4 ppm, while the 3-H- and 5-H-
oxidopyridyl protons were shifted <0.1 ppm (see Fig. 2).
Similar shift changes, observed for pyridine-N-oxide versus
pyridine (13), were rationalized in terms of additional elec-
tron density at the 2, 4, and 6 positions resulting from meso-
meric structures (Fig. 3). The splitting patterns and δ values
for the phenyl and sulfonatophenyl signals of the (oxido-
pyridyl)porphyrins correspond well to those of the phenyl-
(pyridyl)- and (pyridyl)(sulfonatophenyl)porphyrins, respectively
(8).
aSymmetry operations: ( ′) –1/2 + y, 1/2 – x, 1/2 – z; ( ) 1/2 – y, 1/2 +
′′
x, 1/2 – z.
example, TOPyP is insoluble in CDCl3 but dissolution (re-
quired for 1H NMR measurement) occurs readily on addition
of a few drops of CD3OD. The difference in solubility may
be related to less aggregation in the porphyrin-N-oxides be-
cause of steric effects of the macrocyclic oxygens; also,
these oxygens may well be involved in hydrogen-bonding.
Pyrrole-N-oxides
The β-pyrrole protons of the pyrrole-N-oxide of the
porphyrin-N-oxides are shifted upfield to δ ~7.5 compared
to those of the nonoxidized pyrroles (Fig. 4, ∆ ~ 1.4 ppm).
Similar δ values were noted for the β-pyrrole-oxide positions
in TMP-O (δ 7.49) (23) and Ti(TPhP-O) (δ 7.26) (27). Based
X-ray crystallography
The X-ray structure of OPyTrPhP is shown in Fig. 1. The
morphology of the porphyrin core and the orientation of the
aryl groups do not differ significantly from those of TPhP
(30). The porphyrin ring is nearly planar, and the dihedral
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Posakony et al.
193
1
Fig. 2. The aromatic region of the H NMR spectrum TrPhPyP and OPyTrPhP in CDCl3.
Fig. 3. Mesomeric forms of pyridine-N-oxide.
solely on inductive effects, such shifts are opposite to those
unit is also expected in Ti(TPhP-O) where a ∆δ of ~2 is ob-
served (27).
expected by incorporation of an O-atom, and are unlikely
due to partial negative charges for mesomeric forms (cf.
Fig. 3), as these would tend to be delocalized over the whole
macrocycle. An out-of-plane tilt of the pyrrole-N-oxide could
explain the upfield shift, as the β-pyrrole protons would then
experience less deshielding from the induced ring current.
However, in the solid state, the pyrrole-N-oxide ring in
OEP-O is tipped out of the porphyrin plane only 6.1° (20),
which is similar to that of 6.6° found in two of the pyrrole
rings of TPhP (30), and such a small tilt in solution is un-
likely to explain the large ∆δ. In the dynamic porphyrin in-
version process, in which the oxygen atom moves through
the center of the porphyrin (19), the pyrrole groups may be
tilted far enough for such a loss of deshielding, and in sup-
port of this a similar loss of deshielding is noted for the
methylene groups in the Ni(II) complex of OEP-O, where a
large pyrrole-N-oxide tilt (38.3°) is observed and inversion
does not occur (19, 20). A large tilt of the pyrrole-N-oxide
As in the corresponding pyridyl(phenyl)- and (oxido-
pyridyl)(phenyl)porphyrin series, the β-pyrrole protons show
a singlet when situated between two idendical meso-sub-
stituents, and an AB spectrum when between two different
meso-subsituents (8). The rest of the β-pyrrole region is complex
because of the inequivalence introduced by the proximity of
the oxidopyrrole group and different meso-subsitutents.
The inner N-H protons of the porphyrin-N-oxides appear
as broad singlets at δ ~1.6, which disappear in the presence
of D2O. Compared to the (oxidopyridyl)porphyrins (where
δNH is ~–2.85), the induced ring current is presumably
slightly disrupted because of the out-of-plane pyrrole-N-ox-
ide ring. The inner N-H signals of OEP-O and TMP-O ap-
pear at δ 0.8 (19) and δ 1.75 (23), respectively.
1
In the H NMR spectrum of TOPyP in CDCl3–CD3OD,
the β-pyrrole protons appear as a broad singlet and no N-H
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194
Fig. 4. The aromatic region of the H NMR spectrum of OPyTrPhP-23O and -21O.
Can. J. Chem. Vol. 77, 1999
1
Fig. 5. UV-vis spectrum of TPhPyP (2.3 × 10–6 M) in CH2Cl2.
signals are seen. Similar data were obtained when D2O or
CD3OD was added to the CDCl3 solutions of TrPhPyP,
OPyTrPhP, t-DPBPyP, t-BOPyDPhP, c-DPhBPyP, or c-
BOPyDPhP, and reflect H/D exchange with the N-H pro-
tons. The β-pyrrole protons are weakly coupled to the N-H
protons but rapid tautomerism prevents any observable
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Posakony et al.
195
Fig. 6. UV-vis spectrum of OPyTrPhP (2.6 × 10–6 M) in CH2Cl2.
Fig. 7. UV-vis spectrum of OPyTrPhP-23O (~7.6 × 10–6 M) in CH2Cl2. (At ~2.8 × 10–5 M, an additional, small peak is observed at
518 nm.)
splitting at room temperature; with N-D, the tautomerism is
slower (kH/kD ~ 12.1 at 35°C) (31), and coupling to the
D-atoms broadens the β-pyrrole signals.
absorbance maximum near 350 nm; in polar solvents, the
longer wavelength band is blue-shifted and can be hidden by
the shorter wavelength absorption maximum (13). The
shorter wavelength maximum appears at ~275 nm for the
(oxidopyridyl)porphyrins and the corresponding porphyrin-
N-oxides (see Table 4); and the corresponding longer wave-
length band is sometimes observed in the ~310–350 nm
UV-vis spectroscopy
Pyridine-N-oxide displays an absorbance maximum (log ⑀
> 4) in the 255–285 nm region and a weaker (log ⑀ ~ 2)
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Can. J. Chem. Vol. 77, 1999
Fig. 8. UV-vis spectrum of OPyTrPhPZn-23O (~1.0 × 10–5 M) in CH2Cl2.
region, although this is usually masked by the “background”
absorbance from the porphyrin moiety (see Figs. 5 and 6).
With the exception of the UV region, the spectra of the
(oxidopyridyl)porphyrins are similar to those of free-base
tetraarylporphyrins.
this could be observed using MALDI-TOF mass spectrome-
try (using a dihydroxybenzoic acid matrix) (Table 6). Some
of the parent peaks for the porphyrin-N-oxides were ob-
served under EI ionization conditions (see Fig. 9), and HR-
MS analyses were obtained for these species (Table 6).
The presence of the Soret bands for the porphyrin-N-ox-
ides suggests that the conjugation of the macrocycle is unin-
terrupted (18); however, the Soret bands are broadened, and
the Q bands are not as intense as those in the parent
(oxidopyridyl)porphyrins (Fig. 7). The Soret and Q-bands in
the spectra of OPyTrPhPZn-21O and -23O are similarly broad
and diminished in intensity, but the peaks at 320–330 nm are
more prominent (see Fig. 8). There is no evidence for a band
assignable to any electronic transition of the pyrrole-N-oxide
group.
Sulfonation reactions
Sulfonation of the phenyl groups of OPyTrPhP and c-
BOPyDPhP was achieved readily in hot, conc. H2SO4. Like
the pyridyl subsituent, the oxidopyridyl group was not
sulfonated under these conditions, and the N-oxide was re-
tained. The pyridine nitrogen (or oxygen in the oxidopyridyl
group) is certain to be protonated under the sulfonation con-
ditions, thus making the pyridyl group unreactive towards
electrophilic addition.
The sulfonated porphyrins, when isolated by solvent re-
moval from an aqueous solution, retained several mole
equivalents of water as judged by elemental analysis. This
behavior for sulfonated porphyrins has been noted previ-
ously by our group (9).
Infrared spectroscopy
The N-oxide moiety exhibits an intense, characteristic
band in the 1200–1350 cm–1 region attributed to ν(N-O)
(13), and the IR spectra of the (oxidopyridyl)porphyrins
show an intense band in the 1221–1261 cm–1 region due to
the pyridyl-N-oxide stretch; for the sulfonated derivatives,
the values are about 20 cm–1 lower than for the correspond-
ing nonsulfonated porphyrins (see Table S1)2. Porphyrin- N-
oxide stretches for OEP-O (18) and TMP-O (23) appear at
1265 and 1273 cm–1, respectively. Intense bands for the
porphyrin-N-oxides synthesized here are in the range 1238–
1261 cm–1, and these likely result from overlap of the
porphyrin- and pyridine-N-oxide stretches. The other spec-
tral bands common to the porphyrin ring structure can be as-
signed according to the literature (32).
Tirapazamine chemistry
Patents and publications on the biological activity of
tirapazamine have appeared (33–35), and the desire to incor-
porate tirapazamine into a porphyrin was noted in the intro-
duction. The use of triphosgene, a crystalline compound
(36), in place of the highly toxic, gaseous phosgene, for re-
action with tirapazamine, provided an effective, rapid route
to 1, although the product contained a small amount of
tirapazamine. Compound 1 had previously been made in a
4 h reaction using phosgene, and its structure proposed (15)
on IR data: ν(CϭO) 1814 cm–1, ν(CϭN) 1541 cm–1, and the
absence of ν(NCO) bands at 2273–2000 cm–1 (37). Reaction
of an amine with phosgene typically generates an isocya-
nate, and despite its cyclic structure, 1 does react like an
Mass spectrometry
The oxidopyridyl groups were deoxygenated using EI and
LSIMS techniques and no moleular ion peak was seen, but
© 1999 NRC Canada

Posakony et al.
197
Fig. 9. Low-resolution mass spectrum (EI) of OpyTrPhP-21O.
isocyanate and produce ureas and carbamates when mixed
with amines and alcohols, respectively, undergoing a ring-
opening process (15, 33). Also typical of an isocyanate, 1 re-
acts with water (even on a TLC plate) to regenerate the
amine tirapazamine.
The porphyrin bearing an amino group, APhTrPhP, was
mixed with 1 to yield 2 in ~50% isolated yield. An IR peak
seen at 1225 cm–1 due to ν(NO) was not observed in the
aminophenyl porphyrin (38). Peaks attributed to tirapaz-
amine appeared in the UV-vis spectrum, but these were in a
region of relatively high background absorbance from the
porphyrin core.
We thank the Natural Sciences and Engineering Research
Council and Medical Research Council of Canada for finan-
cial support, and Dr. M. Tracy for the sample of tirapa-
zamine.
1. R. Boyle and D. Dolphin. Photochem. Photobiol. 64, 469 (1996).
2. J. Moan and K. Berg. Photochem. Photobiol. 55, 931 (1992).
3. M.R. Hamblin and E.L. Newman. J. Photochem. Photobiol. B,
23, 3 (1994).
4. H. Pass. J. Nat. Cancer Inst. 85, 443 (1993).
5. M. Ochsner. J. Photochem. Photobiol. B, 39, 1 (1997).
6. P. Wardman, K.I. Priyadarsini, M.F. Dennis, S.A. Everett,
M.A. Naylor, K.B. Patel, I.J. Stratford, M.R.L. Stratford, and
M. Tracy. Br. J. Cancer, 74, S70 (1996).
7. W.A. Denny, W.R. Wilson, and M.P. Hay. Br. J. Cancer, 74,
S32 (1996).
8. G.G. Meng, B.R. James, and K.A. Skov. Can. J. Chem. 72,
1894 (1994).
9. G.G. Meng, B.R. James, K.A. Skov, and M. Korbelik. Can. J.
Chem. 72, 2447 (1994).
10. B.R. James, G.G. Meng, J.J. Posakony, J.A. Ravensbergen,
C.J. Ware, and K.A. Skov. Met.-Based Drugs, 3, 85 (1996).
11. D.C. Harris. Quantitative chemical analysis. W.H. Freeman,
New York. 1987. p. 644.
Porphyrins containing one to four pyridyl groups were
synthesized and N-oxidized with m-CPBA to produce five
novel (oxidopyridyl)porphyrins and seven porphyrin-N-ox-
ides (including three from a c-BOPyBPhP-O mixture). Only
three free-base porphyrin-N-oxides have been reported pre-
viously (OEP-O, TMP-O, and TPhP-O). The (oxidopyri-
dyl)porphyrins and porphyrin-N-oxides were well character-
ized and an X-ray crystal structure of OPyTrPhP was ob-
tained. Sulfonation of OPyTrPhP and c-BOPyDPhP was
achieved readily under standard conditions with no loss of
oxygen from the oxidopyridyl groups.
The product 1 from tirapazamine and triphosgene reacts
like an isocyanate, reacting with porphyrins bearing amines
and alcohols; the best results were obtained using APhTrPhP
to produce the novel porphyrin conjugate 2. The electro-
chemical and in vitro evaluation of these porphyrins incor-
porating N-oxide functionalities (39) will be published
elsewhere.
12. R.G. Little, J.A. Anton, P.A. Loach, and J.A. Ibers. J.
Heterocycl. Chem. 12, 343 (1975).
13. A. Albini and S. Pietra. Heterocyclic N-oxides. CRC Press,
Boca Raton. 1991. pp. 7–31.
14. J.S. Lindsey. In Metalloporphyrins catalyzed oxidations.
Edited by F. Montanari and L. Casella. Kluwer Academic Pub-
lishers, Dortdrecht. 1994. p. 49.
© 1999 NRC Canada

198
Can. J. Chem. Vol. 77, 1999
15. (a) F. Seng and K. Ley. Angew. Chem. Int. Ed. Eng. 11, 1009
(1972); (b) J.C. Mason and G. Tennant. J. Chem. Soc. B, 911
(1970).
16. (a) International tables for X-ray crystallography. Vol. 4. Ky-
noch Press, Birmingham, U.K. (present distributor Kluwer Ac-
ademic Publishers, Boston, Mass., U.S.A.). 1974. pp. 99–102;
(b) International tables for crystallography. Vol. C. Kluwer Ac-
ademic Publishers, Boston, Mass., U.S.A. 1992. pp. 200–206.
17. R. Bonnet and R. Ridge. J. Chem. Soc. Chem. Commun. 310
(1978).
18. L.E. Andrews, R. Bonnet, R. Ridge, and E.H. Appelman. J.
Chem. Soc. Perkin Trans. 1, 1983, 103 (1983), and refs. therein.
19. A.L. Balch, C.Y.-W., M. Olmstead, and M.W. Renner. J. Am.
Chem. Soc. 107, 2393 (1985).
20. A.L. Balch, Y.-W. Chan, and M.M. Olmstead. J. Am. Chem.
Soc. 107, 6510 (1985).
28. L.R. Milgrom, J.P. Hill, and P.J.F. Dempsey. Tetrahedron, 47,
13 477 (1994).
29. J.A. Joule, K. Mills, and G.F. Smith. Heterocyclic chemistry.
Chapman and Hall, London. 1995. p. 95.
30. S.J. Silvers and A. Tulinsky. J. Am. Chem. Soc. 89, 3331
(1967).
31. H. Scheer and H. Katz. In Porphyrins and metalloporphyrins.
Edited by K.M. Smith. Elsevier, Amsterdam. 1975. p. 399.
32. J.O. Alben. In The porphyrins. Edited by D. Dolphin. Aca-
demic Press, New York. 1978. Vol. 3. p. 323.
33. (a) F. Seng, K. Ley, and K.G. Metzger. U.S. Patent 3,957,779
(1976) and 4,027,022 (1977); (b) F. Seng, K. Ley, B. Ham-
burger, and B. Franz. U.S. Patent 3,991,189 (1976).
34. E.M. Zeman, M.A. Baker, M.J. Lemmon, C.I. Pearson, J.A.
Adams, J.M. Brown, W.W. Lee, and M. Tracy. Int. J. Radiat.
Oncol. Biol. Phys. 16, 977 (1989).
21. A.L. Balch and Y.W. Chan. Inorg. Chim. Acta, 115, L45 (1986).
22. R.D. Arasasingham, A.L. Balch, M.M. Olmstead, and M.W.
Renner. Inorg. Chem. 26, 3562 (1987).
35. A.I. Minchinton, M.J. Lemmon, M. Tracy, D.J. Pollart, A.P.
Martinez, L.M. Tosto, and M.J. Brown. Int. J. Radiat. Oncol.
Biol. Phys. 22, 701 (1992).
23. J.T. Groves and Y. Watanabe. J. Am. Chem. Soc. 108, 7836
(1986).
36. H. Eckert and B. Foster. Angew. Chem. Int. Ed. Engl. 26, 894
(1987).
24. J.T. Groves and Y. Watanabe. J. Am. Chem. Soc. 110, 8443
(1988).
25. Y. Mizutani, Y. Watanabe, and T. Kitagawa. J. Am. Chem.
Soc. 116, 3439 (1994), and refs. therein.
26. K. Rachlewicz and L. Latos-Grazynski. Inorg. Chem. 35, 1136
(1996).
37. R.M. Silverstein, G.C. Bassler, and T.C. Morrill. Spectrometric
identification of organic compounds. John Wiley and Sons,
New York. 1991. p. 419.
38. G.G. Meng. Ph.D. thesis, University of British Columbia. 1993.
39. J.J. Posakony. Ph.D. thesis, University of British Columbia.
1998.
27. M. Hoshino, K. Yamamoto, J.P. Lillis, T. Chijimatsu, and J.
Uzawa. Inorg. Chem. 32, 5002 (1993).
© 1999 NRC Canada