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

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 (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).

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 R_w= 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 CaH₂or 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)

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-DPhBPyP^a

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

CH₂Cl₂or 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 R_fvalues 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

²Copies 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.

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-OPy^c

2,6-Ph (or 2,6-SPh)

3,4,5-Ph (or 3,5-SPh)^dPyrrole N-H

β-Pyrrole^b

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)^e9.10 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-OPy^c

2,6-Ph

β-Pyrrole^b

β-Pyrrole-NO^d

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-22O^f9.07 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)

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 ⑀)

OPyTrPhP^a(CH₂Cl₂:MeOH 20:1)

t-BOPyDPhP^b(CH₂Cl₂:MeOH 10:1)

c-BOPyDPhP^a(CH₂Cl₂:MeOH 10:1)

TrOPyPhP^a(CH₂Cl₂:MeOH 10:1)

TOPyP^a(CH₂Cl₂:MeOH 10:1)

OPyTrPhP-23O^c(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-23O^d,e(CH₂Cl₂:MeOH 5:1)

OPyTrPhPZn-21O (CH₂Cl₂:MeOH 5:1)

c-BOPyDPhP-O mixture (CH₂Cl₂)

TrOPyPhP-21O (CH₂Cl₂)

TrOPyPhP-22O (CH₂Cl₂)

OPyTrSPhP^f(H₂O)

272 (4.35), 322 (4.26)

276, 330

436 (5.27)

438

422

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^–6M; ⑀ of other bands determined at 2 × 10^–5M.

^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^–6M; ⑀ of other bands determined at 5 × 10^–5M.

^ePrepared from a known concentration of OPyTrPhP-23O and excess Zn(OAc)₂.

^f⑀ determined at 1.0 × 10^–6M.

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)

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)₂in 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,

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/z^a(intensity %, assignment)

HR-MS, calcd. (found)

OPyTrPhP^b

632 (88, M⁺¹),

616 (36, M⁺¹– O)

Not obtained

OPyTrPhP-21O^c

OPyTrPhP-23O^c

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-BOPyDPhP^b,d

c-BOPyDPhP^b,d

Not obtained

c-BOPyDPhP-NO mixture^c

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)

TrOPyPhP^b,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-21O^c

C₄₁H₂₇O₃N₇

665.217 53 (665.217 19)

C₄₁H₂₇O₃N₇

649.222 60 (649.222 10)

617 (100, M⁺– 4O)

TrOPyPhP-22-NO^c

TOPyP^b

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-BOPyBSPhP^f

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⁺¹implies (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

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 δ_Hvalues for the

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 CH₂Cl₂with

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. H₂SO₄and 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 R_fnearly 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 (R_f= 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 (R_f= 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

The H NMR signals were assigned with the help of a H-

2D-COSY spectrum.

Posakony et al.

189

1

Table 7. H NMR data for the benzotriazine-dioxide compounds.^a

Compound (solvent)

-CONH

Benzotriazine ring H^b

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

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 processed³corrected 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 (R_f= 0.4),

some APhTrPhP (R_f= 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^–6M, 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 S₄symmetry, 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 B_H= 1.2B_{bonded atom}. A

correction for secondary extinction was applied (Zachariasen

type, isotropic, Gaussian), the final value of the extinction

³teXsan: Crystal structure analysis package. Unix version 1.7. Molecular Corporation. The Woodlands, Tex., U.S.A. 1995.

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

ρ_calc, g/cm³

F(000)

µ(Cu K_α), 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

2θ_max, deg

Crystal decay, %

Total reflections

Total unique reflections

Reflections with I ≥ 3σ(I)

No. of variables

R

Tetraarylporphyrin syntheses

155

Negligible

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

R_w

gof

Max ∆/σ (final cycle)

Residual density, e/Å³

^aTemperature 294 K, Rigaku AFC6S diffractometer, Cu K_αradiation (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(|F_o| – |F_c|)²where w =

4F_o/σ²(F_o), R = Σ||F_o| – |F_c||/Σ|F_o|, R_w= (Σw(|F_o| – |F_c|)²/Σw|F_o|²)^1/2, and

gof = [Σw(|F_o| – |F_c|)²/(m – n)]^1/2. Values given for R, R_w, and gof are

based on those reflections with I ≥ 3σ(I).

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 BF₃coordination

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

Posakony et al.

Table 9. Final atomic coordinates (fractional) and B_eq(10³Å²).^a

191

Atom

x

y

z

B_eq

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

^aB_eq= (8/3)π²ΣΣ(U_ija_ia_j(a_ia_j)).

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 CH₂Cl₂or 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

192

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 δ_Hvalues 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 CDCl₃but 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

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

δ_NHis ~–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

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^–6M) in CH₂Cl₂.

signals are seen. Similar data were obtained when D₂O or

CD₃OD was added to the CDCl₃solutions 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

Posakony et al.

195

Fig. 6. UV-vis spectrum of OPyTrPhP (2.6 × 10^–6M) in CH₂Cl₂.

Fig. 7. UV-vis spectrum of OPyTrPhP-23O (~7.6 × 10^–6M) in CH₂Cl₂. (At ~2.8 × 10^–5M, an additional, small peak is observed at

518 nm.)

splitting at room temperature; with N-D, the tautomerism is

slower (k_H/k_D~ 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)

196

Can. J. Chem. Vol. 77, 1999

Fig. 8. UV-vis spectrum of OPyTrPhPZn-23O (~1.0 × 10^–5M) 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^–1region attributed to ν(N-O)

(13), and the IR spectra of the (oxidopyridyl)porphyrins

show an intense band in the 1221–1261 cm^–1region due to

the pyridyl-N-oxide stretch; for the sulfonated derivatives,

the values are about 20 cm^–1lower 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

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^–1due 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.

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Article Doi

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

DOI: 10.1139/v98-238

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