Superoxide dismutase mimetics: synthesis and structure-activity relationship study of MnTBAP analogues.

Article abstract of DOI:10.1016/S0968-0896(02)00153-0

Carboxylic ester and amide-substituted analogues of [5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrinato]manganese(III) chloride (MnTBAP) were synthesized and assayed as potential superoxide dismutase (SOD) mimetics. The tetraester analogues 4a and 4b were

Full text of DOI:10.1016/S0968-0896(02)00153-0

Bioorganic & Medicinal Chemistry 10 (2002) 3013–3021
Superoxide Dismutase Mimetics: Synthesis and Structure–Activity
Relationship Study of MnTBAP Analogues
Polivina Jolicia F. Gauuan,a,* Michael P. Trova,a Livia Gregor-Boros,a
Stephen B. Bocckino,b James D. Crapoc and Brian J. Dayc
aAlbany Molecular Research, Inc., 21 Corporate Circle, PO Box 15098, Albany, NY 12212-5098, USA
bIncara, Inc., PO Box 14287, 3200 East Highway 54, Cape Fear Building, Suite 300, Research Triangle Park, NC 27709, USA
cNational Jewish Medical and Research Center, 706A Goodman Building, 1400 Jackson Street, Denver, CO 80206, USA
Received 19 October 2001; accepted 25 March 2002
Abstract—Carboxylic ester and amide-substituted analogues of [5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrinato]manganese(III)
chloride (MnTBAP) were synthesized and assayed as potential superoxide dismutase (SOD) mimetics. The tetraester analogues 4a
and 4b were found to have comparable SODactivity to the known SODmimetic MnTBAP, while amides 4c–4e exhibited reduced
SODactivity. In the substituted methyl benzoate/acid and disubstituted porphyrin series, analogues 12c, 12f, and 12m were found
to have comparable to improved SODactivity relative to MnTBAP and analogues 12j, 13a, and 13d exhibited improved activity in
both the SODand thiobarbituric acid reactive species (TBARS) assays relative to MnTBAP. # 2002 Elsevier Science Ltd. All
rights reserved.
Introduction
more potent and stable metal chelates has resulted in the
discovery of at least three classes of metal-containing
SODmimetics.
Superoxide radicals (Oꢀ2ꢁ) are produced as part of the
normal metabolism of all aerobic cells. Superoxide is
known to be important in the pathogenesis of many
disease processes including lung, central nervous system
and skeletal muscle diseases1 and, by modulating the
effects of nitric oxide, contributes to the pathogenesis of
inflammatory2 and vascular disorders3 and the aging
process.4 A critical balance of enzymes defending
against antioxidants is therefore required to maintain
normal cell and organ function. Superoxide dismutases
(SODs), a family of metalloenzymes, represent the first
line of defense against the harmful effects of superoxide
radicals by catalyzing the intra and extracellular
These include the salen,6a macro-
6
cyclic,6b and metalloporphyrins.6c The most stable of
these complexes are the metalloporphyrins including
MnTBAP. They have also been shown to be potent
inhibitors of lipid peroxidation.7 Lipid peroxidation is a
natural occurring reaction with polyunsaturated fatty
acids and reactive oxygen species. As the fatty acids
degrade they accumulate as small aldehyde fragments
that are associated with a number of disease processes
and inflammation.8 The stability of metalloporhyrins
compared to other metal chelates made them attractive
basis to develop a new series of Mn(III) porphyrins as
SODmimetics.
ꢀ
ꢁ
conversion of O2 into H2O2 plus O2.
Although many newer SODmimetic have greater in
vitro activity than the MnTBAP (the prototype of this
series), MnTBAP has been shown to possess very potent
effects in vivo against oxidative stress.9a MnTBAP has
an extensive and impressive amount of pharmacology.
In cell cultures models, MnTBAP has been shown to be
effective in micromolar levels against injury produced
by paraquat, hydrogen peroxide, endotoxin, and the
excitotoxic agents NMDA and kainic acid.9a In addi-
tion, MnTBAP is a potent inhibitory of apoptosis
induced by staurosporine, ceramide, and growth factor
It has been recognized for years that simple metal che-
lates could react with superoxide and hydrogen perox-
ide.5 These include the Mn-desferal5a and manganese
complexes of lactate, citrate, or succinate.5b However,
the rates of reaction of these chelates with superoxide
were low and the complexes unstable. The search for
*Corresponding author. Fax: +1-518-464-0289; e-mail: joli@
ablmolecular.com
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00153-0

3014
P. J. F. Gauuan et al. / Bioorg. Med. Chem. 10 (2002) 3013–3021
.
aldehyde 5a or 5b with pyrrole, catalyzed by BF3 OEt2,
withdrawal. MnTBAP also is very effective in animal
models of injury such as paraquat-induced lung injury,
carrageenan-induced lung injury, and bleomycin-
induced lung injury. Recently, it was found to be more
efficacious against acetaminophen toxicity than N-ace-
tyl-l-cysteine.9b One of the most remarkable effects of
MnTBAP was its ability to substitute for mitochondrial
SODin the mitochondrial knockout mouse and correct
the dilated cardiomyopathy phenotype and extend its
life span.9c MnTBAP has a 9.5-h plasma half-life in
mice and was fairly non-toxic to mice with a reported
LD50 of 100 mg/kg body weight.9d These features of
MnTBAP were the incentive to develop a number of
structurally related compounds reported in this paper.
followed by oxidation with p-chloranil provided cyano-
phenyl porphyrin 6a and ketoester porphyrin 6b,
respectively. Porphyrins 6a and 6b were then metalated
to provide porphyrins 7a and 7b, respectively. Further
functionalization of cyanophenyl 6a by treatment with
NaN3 provided tetrazole 8 that was metalated to pro-
vide porphyrin 9. The Mn-tetrazole porphyrin 9 was
transformed to oxadiazole porphyrin 10 by treatment
with acetic anhydride.11 The physical properties of por-
phyrin derivatives 4, 7, 9, and 10 are shown in Table 1.
Porphyrin analogues bearing salicylic acid groups at the
meso positions and tetraphenylporphyrin analogues
with electron donating or electron donating groups in
the pendant aromatic rings were prepared and evaluated
next 11–13 (Fig. 1). Analogues 11a, 11b, 12m, 12n, 12o,
and 12p were prepared from commercially available
aldehydes by methods analogous to those shown in
Scheme 2. However, the requisite aldehydes needed for
the preparation of porphyrin analogues 12a–j and 13a–b
were not commercially available and were prepared
from the corresponding toluic acids as shown in Scheme
3. Thus, esterification of the acid 14 to ester 15, bis-
bromination of 15, followed by hydrolysis provided the
corresponding aldehyde 16.
Results and Discussion
Chemistry
Utilizing MnTBAP (2) as our lead compound, we
investigated the syntheses and biological evaluation of
porphyrin analogues by substituting the carboxylic acid
functionality in MnTBAP with carboxylic acid mimics
(i.e., tetrazole, oxadiazole, etc.) and carboxylic acid
derivatives (i.e., amides and esters). The esters 4a and
4b, amides 4c and 4e, and oxadiazole 4f were prepared
from the commercially available 5,10,15,20-tetrakis
(4-carboxyphenyl)porphyrin (H2TBAP) (Scheme 1).
H2TBAP (1) was metalated10 by treatment with MnCl2
in refluxing DMF to provide MnTBAP. Conversion of
MnTBAP to the acid chloride followed by treatment of
alcohol, amine, or amideoxime provided ester 4b,
amides 4c and 4e, and oxadiazole 4f, respectively. Ester
4a was prepared by converting H2TBAP to the acid
chloride first followed by quenching with anhydrous
methanol and metalation.
Once the aldehydes 16 were on hand, their condensation
with pyrrole provided the corresponding porphyrins
(Scheme 4, Table 2). Metalation of the porphyrins was
accomplished according to the procedure reported by
Longo et al.10 Under these conditions, by products
resulting from the partial hydrolysis of the ester
group(s), 11a, 12c, 12d, 12k, and 13b, were formed.
These by-products were subsequently isolated and their
biological activity was investigated. Porphyrin 12g was
also isolated as a byproduct during metalation of tetra-
methyl 40,400,4000,40000-tetrafluoro-30,300,3000,30000-(21H,23H-
porphine-5,10,15,20-tetrayl)tetrabenzoate.
The preparation of tetrazole11 porphyrin 9 and keto-
ester porphyrin 7b began with the construction of the
porphyrin core 6a and 6b, from aldehydes 5a and 5b,
respectively, according to a general procedure reported
by Lindsey et al.12 (Scheme 2). Thus, condensation of
Scheme 1.
Scheme 2.

P. J. F. Gauuan et al. / Bioorg. Med. Chem. 10 (2002) 3013–3021
Table 1. Physical properties of porphyrins 4, 7, 9, and 10
3015
Compd
R
Mp (ꢂC)
lmax
(nm)
ea ꢃ 105
FABMS
(m/z)
(cmꢁ1Mꢁ1
)
MnTBAP
CO2H
CO2CH3
CO2CH2CH3
CONH(CH2)2NHBoc
>300
>330
—
>375
—
—
468.0
466.0
466.0
466.5
466.5
466.5
466.5
464.5
466.5
467.5
466.5
0.88
1.1
1.5
—
0.50
0.56
1.1
1.4
1.1
843
899
955
–
1012
952
995
767
1012
939
995
4a
4b
4c
4d
4e
4f
7a
7b
9
.
CONH(CH2)2NH2 HCl
CON(CH3)2
3-Methyl-1,2,4-oxadiazol-5-yl
CN
COCO2CH3
Tetrazol-5-yl
2-Methyl-1,3,4-oxadiazol-5-yl
—
>310
>300
>320
>300
0.42
1.6
10
aExtinction coefficient of porphyrins dissolved in ethanol unless otherwise noted.
Biology
monitored as described by Faulkner et al.15 Briefly,
E. coli were cultured in minimal medium contained
0.2% glucose, and 0.5 mM each of leucine, threonine,
proline, arginine, and histidine, plus M9 salts in tap
water, pH adjusted to 7.0. The compounds were asses-
sed for efficacy (X+) or lack of efficacy (Xꢁ) by their
ability to preserve aerobic growth in the SODnull E. coli
(JI132) as assessed by turbidity measurement at 700 nm.
The porphyrin analogues were screened using assays for
SODand peroxidase activity. Superoxide dismutase
activity was measured by cytochrome c reductase as
previously described in the literature.13 Briefly, buffer
consisted of 50 mM potassium phosphate containing
0.1 mM EDTA at pH 7.8. Cytochrome c was added to
give a final concentration of 10 mM of the oxidized
form. Xanthine was added to give a final concentration
of 50 mM. Xanthine oxidase (2 nM) was added to start
the formation of superoxide. Rates of reduction of
Our biological results for the monosubstituted phenyl
derivatives (carboxylic acid mimics and carboxylic acid
derivatives) are summarized in Table 3. The ester ana-
logues 4a and 4b exhibited SODactivities that were
comparable to MnTBAP while the amides generally
showed reduced SODactivities relative to MnTBAP.
The amide analogues 4d and 4e showed improved inhi-
bition in the TBARS assay relative to MnTBAP or the
ester analogues. Of the three heterocyclic analogues
prepared, oxadiazole 10 exhibited comparable SOD
activity and better inhibition in the TBARS assay rela-
tive to MnTBAP. The tetrazole derivative 9 was found
to be highly toxic to cultured cells (results not shown
here).
cytochrome
c were followed at 550 nm spectro-
photometrically for 1 min. The amount of metallopor-
phyrin that inhibited the rate of cytochrome reduction
by one-half was 1 SODunit of activity.
The ability of mimetics to inhibit lipid peroxidation
(TBARS, IC50 mM) was assessed as previously described.7
Iron and ascorbate were used to initiate lipid peroxidation
in tissue homogenates and the formation of thiobar-
bituric acid reactive species (TBARS) was measured.14a,b
The aerobic growth of Escherichia coli strain JI 132,
which lacks superoxide dismutase activity, was
Scheme 3. (a) (COCl)2, cat. DMF, CH2Cl2; CH3OH, Et3N; (b)
(CH3O)2SO2, K2CO3, acetone; (c) NBS, CCl4, initiator; (d) AgNO3,
H2O, acetone.
.
Scheme 4. (a) Pyrrole, BF3 OEt2, CH2Cl2; p-chloranil; (b) MnCl2,
DMF, reflux; (c) Claisen’s base, MeOH.
Figure 1.

3016
P. J. F. Gauuan et al. / Bioorg. Med. Chem. 10 (2002) 3013–3021
The biological data for the substituted MnTBAP ana-
logues are also shown in Table 3. Several novel por-
phyrin derivatives that were synthesized showed
enhanced SODactivity relative to MnTBAP (vide
infra). Relative to MnTBAP, porphyrin analogues 12
and 13a exhibited enhanced SODactivity while por-
phyrin analogues 12a, 12b, 12f, 12g, 12j, 12o, 13a, 13b,
and 13d exhibited enhanced acitivity in the TBARS
assay. The activity of analogues 12c, 12f, 12n, and 13d
in the SODassay was comparable to that of MnTBAP.
Within the same series of compounds, the tetraester
porphyrin analogues 12f, 12j, 13a, and 13d showed
enhanced activity in the SODand TBARS assays rela-
tive to their corresponding acid analogues. In the E. coli
assay, the tetraacids 12e, 12l, and 13c showed activity
while the tetraester analogues 12b, 12j, 13a, and 13d,
respectively, did not.
compounds continue in an attempt to elucidate the rea-
sons for these observations. We are also exploring
additional porphyrin derivatives of differing substitu-
tion in order to further improve these biological find-
ings. This work will be reported in due course.
Experimental
Proton NMR spectra were obtained on a Bruker AC
300 spectrometer at 300 MHz and were referenced to
tetramethylsilane as an internal standard. IR spectra
were prepared in a pressed disc of KBr or as a film on
NaCl plates and acquired with a total of four accumu-
lations at a resolution of 4.00 cmꢁ1 on a single beam
Perkin–Elmer Spectrum 1000 FT-IR. Melting points
were obtained on a Mel-Temp II apparatus and are
uncorrected. FAB mass spectra were performed by M-
Scan. Elemental analyses were performed by Quantita-
tive Technologies, Inc. of Whitehouse, NJ, USA. HLPC
analyses were obtained using a Microsorb C18 Column
with UV detection at 230 nm (unless otherwise speci-
fied). HPLC grade methylene chloride and anhydrous
DMF were used.
Conclusion
Several novel porphyrin derivatives synthesized showed
enhanced activities in the SODand/or TBARS assays
relative to MnTBAP. Derivatives prepared in the first
series, carboxylic acid mimics and derivatives, have
shown comparable to reduced SODactivity but
enhanced activity in the TBARS assay relative to the
lead compound MnTBAP. In the second series of ana-
logues prepared (substituted MnTBAP analogues),
compounds 12j and 13a exhibited enhanced activity in
both the SODand TBARS assays. Analogues 12a, 12b,
12f, 12j, 12o, 13b, and 13d exhibited enhanced activity in
the TBARS assay relative to MnTBAP. SODactivity
was not predictive of lipid peroxidation or rescue of null
SOD E. coli (i.e., analogues 12c, 12e, and 12j). Further
in vitro and in vivo biological evaluations of these
5,10,15,20-Tetrakis(methyl 4-benzoate)porphyrin (3a)
A solution of TBAP (559 mg, 1.32 mmol), thionyl chlo-
ride (5 mL), and a drop of pyridine was heated at reflux
for 4 h. The excess thionyl chloride was evaporated off
then the residue was dissolved in anhydrous MeOH.
The solution was cooled to 0 ꢂC then Et3N (0.4 mL) was
added dropwise to provide, after purification by flash
chromatography, porphyrin 3a as a purple solid
1
(388 mg, 61%): H NMR (CDCl3) d 8.83 (s, 8H), 8.45
(d, 8H), 8.30 (d, 8H), 4.15 (s, 12H), ꢁ2.80 (br s, 2H).
Table 2. Physical properties of porphyrins 11–13
Compd
R1
R2
R3
R4
R5
Mp
(ꢂC)
lmax
(nm)
ea ꢃ 105
FABMS
(m/z)
(cmꢁ1Mꢁ1
)
11a
11b
12a
12b
12c
12d
12e
12f
12g
12h
12i
OH
OH
OCH3
OH
OH
CO2CH3
CO2H
CO2CH3
CO2CH3
CO2H
CO2H
CO2H
F
CO2H
CO2H
CO2CH3
CO2CH3
CO2CH3
CO2H
CO2H
F
CO2H
CO2H
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2H
F
CO2H
CO2H
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2H
F
>300
>300
>300
>300
>300
>300
>320
>300
>300
>300
>300
>300
>300
>300
—
472.0
472.0
467.0
467.5
467.5
468.5
467.5
465.0
468.0
467.5
467.0
464.0
464.0
465.0
465.0
472.5
468.0
469.5
462.5
463.0
463.0
467.0
467.0
0.46
—
1.3
1.1
921
907
—
963
949
935
944
971
996
915
940
1079
1065
1023
—
911
859
803
971
957
915
1019
963
1.3
OH
OH
1.0b
0.66
1.3
CO2CH3
CO2CH3
CO2H
CO2H
NO2
NO2
NO2
NO2
NO2
F
N(CH3)2
F
F
F
F
F
F
F
F
F
F
1.3
1.0
N(CH3)2
CO2CH3
CO2H
CO2H
CH3
OH
OCH3
OH
CO2CH3
CO2H
CO2H
CO2CH3
CO2H
0.94
1.4c
1.2c
1.3c
0.77
1.8
0.90
0.52
1.7
0.39
1.5
1.5
1.5
12j
12k
12l
CO2CH3
CO2CH3
CO2H
CH3
OH
OCH3
OH
CO2CH3
CO2CH3
CO2H
CO2CH3
CO2H
CO2CH3
CO2CH3
CO2H
CH3
OH
OCH3
OH
CO2CH3
CO2CH3
CO2H
CO2CH3
CO2H
CO2CH3
CO2CH3
CO2H
CH3
OH
OCH3
OH
CO2CH3
CO2CH3
CO2H
CO2CH3
CO2H
12m
12n
12o
12p
13a
13b
13c
13d
13e
—
>300
>300
>300
>300
>300
>300
>300
F
F
F
F
OCH3
OCH3
aExtinction coefficient of porphyrins dissolved in ethanol unless otherwise noted.
bCompound dissolved in ethanol with 50 mL 0.1 N NaOH (100 mL total volume).
cCompound dissolved in methanol.

P. J. F. Gauuan et al. / Bioorg. Med. Chem. 10 (2002) 3013–3021
3017
[5,10,15,20-Tetrakis(methyl 4-benzoate)porphyrinato]man-
ganese (III) chloride (4a). A solution of porphyrin 3a
(288 mg, 0.32 mmol), MnCl2 (202 mg, 1.61 mmol) in
DMF (30 mL) was heated at reflux. The extent of reac-
tion was monitored by UV–Vis spectroscopy; the dis-
appearance of the free porphyrin Soret band (typically
around 417 nm) and the emergence of the metalated
porphyrin Soret band (typically around 466 nm) would
indicate that metalation was complete. Once metalation
was complete, the reaction mixture was evaporated off,
adsorbed onto silica gel, and purified by column chro-
matogꢂraphy to provide 4a as a green solid: mp
>330 C; FABMS m/z 899 [C52H36MnN4O8]+. UV
(EtOH) lmax, nm (e) 466.0 (1.1ꢃ105). HPLC 94%.
and quenching the acid chloride with anhydrous etha-
nol: FABMS m/z 955 [C56H44MnN4O8]+; HPLC 93%.
UV (EtOH) lmax, nm (e) 46.0 (1.5 ꢃ 105).
5,10,15,20-Tetrakis(N,N-dimethyl-4-benzamide)porphyri-
nato]manganese(III) chrloride (4e). Following the proce-
dure for the preparation of porphyrin 3a, porphyrin 4e
was prepared by converting MnTBAP (2) to the acid
chloride and quenching the acid chloride with dimethy-
lamine: FABMS m/z 952 [C56H48MnN8O4]+. HPLC
>99%. UV (EtOH) lmax, nm (e) 466.5 (0.56ꢃ105).
{5,10,15,20-Tetrakis[4-(4-methyl-2,3,5-oxadiazolyl)phe-
nyl]porphyrinato}manganese(III) chloride (4f). Por-
phyrin 4f was prepared in a similar manner as porphyrin
3a by converting MnTBAP (2) to the acid chloride and
quenching the acid chloride with N-hydroxy-acetamidine.
FABMS m/z 995 [C56H36MnN12O4]+. HPLC 93%. UV
(EtOH) lmax, nm (e) 466.5 (1.1ꢃ105).
.
Anal. calcd for C48H27ClMnN4O8 H2O: C, 64.30; H,
4.15; N, 5.77. Found: C, 64.41; H, 3.73; N, 5.74.
[5,10,15,20-Tetrakis(ethyl 4-benzoate)porphyrinato]man-
ganese(III) chrloride (4b). Following the procedure for
the preparation of porphyrin 3a, porphyrin 4b was pre-
pared by converting MnTBAP (2) to the acid chloride
5,10,15,20-Tetrakis(4-cyanophenyl)porphyrin (6a). In a
foil-covered 2-L 3-neck flask equipped with a reflux
condenser, magnetic stirrer, and a N2 inlet was added
4-formyl benzonitrile (1.13 g, 98%, 8.46 mmol), pyrrole
(0.6 mL, 98%, 8.46 mmol), and CH2Cl2 (850 mL). The
reaction mixture was stirred for 15 min at room tem-
Table 3. In vitro biological activity data for porphyrins 4–13
Compd
SODa
(units/mg)
TBARSb
(IC50 mM)
E. colic
41 (n=3)d
36 (n=2)
23
8
6.2 (n=3)
45
56 (n=5)
59
X+
Xꢁ, ppte
Xꢁ
.
perature then BF3 OEt2 (105 mL, 0.85 nmol) was added
MnTBAP
4a
4b
4c
4d
4e
4f
7a
and the extent of reaction was followed by UV–Vis
spectrometry. After 2 h, p-chloranil (1.56 g, 6.37 mmol)
was added and the reaction mixture was heated at reflux
for 2 h. The reaction mixture was evaporated to a
volume of 50–100 mL, then adsorbed onto silica gel
(2.8 g). Purification by column chromatography pro-
vided 6a (700 mg) in 46% yield as a violet solid. 1H NMR
(CDCl3) d 8.83 (s, 8H), 8.35 (d, 8H), 8.10 (d, 8H), ꢁ2.85
(br s, 2H).
NAf
4.0 (n=3)
4.9
Xꢁ, ppt
Xꢁ
2
2
Xꢁ
20
0.28
7.2
NA
1.6
NA
X+
Xꢁ
6
NA
Xo Inhg
36
2.9
7b
9
10
Xꢁ
Xꢁ
Xꢁ, ppt
11a
11b
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
13a
13b
13c
13d
13e
X+
7
27
ppt
40 (n=3)
NA
0.85 (n=2)
18
208 (n=3)
54
47 (n=2)
12
29 (n=4)
NA
NA
Xꢄ
Xꢁ, ppt
Xꢁ, ppt
X+
{5,10,15,20 - Tetrakis[4 - cyanophenyl]porphyrinato}man-
ganese (III) chloride (7a). A solution 6a (0.19 g,
0.26 mmol) and MnCl2 (0.18 g, 1.4 mmol) in DMF
(20 mL) was heated at reflux for 4–5 h. The reaction
mixture was allowed to cool to room temperature then
DMF was evaporated under reduced pressure. The
crude product was adsorbed onto silica gel (1.5 g) then
was purified by column chromatography to provide of
7a (0.20 g, 95%) as a black solid: mp >310 ꢂC. FABMS
m/z 767 [C48H24MnN8]+. HPLC 99%. UV (EtOH)
5
3
38
X+
X+
31 (n=2)
NA
NA
58
NA
7.6 (n=2)
56
35 (n=2)
21
Xꢄ
0.6
8.4
NA
NA
NA
6.4
NA
6.9
Xꢄ
X+
X+, Xꢄ
Xꢁ
lmax
C48H24ClMnN8 CH3OH: C, 70.47; H, 3.38; N, 13.42.
,
nm (e) 464.5 (1.4ꢃ105). Anal. calcd for
X+
Xꢁ
.
7
56
31
Xꢁ
Found: C, 70.46; H, 3.85; N, 13.11.
Xꢁ
15
Xꢁ
5,10,15,20-Tetrakis[4-(2,3,4,5-tetrazolyl)phenyl]porphyrin
(8). A solution of tetrabenzonitrile porphine 6a (0.30 g,
0.42 mmol), NaN3 (0.24 g, 3.67 mmol), NH4Cl (0.18 g,
3.37 mmol), and DMF (25 mL) was heated at 120 ꢂC for
3 days. Additional NaN3 (0.17 g, 2.59 mmol) and
NH4Cl (0.11 g, 2.05 mmol) were added to push the
reaction to completion. The DMF was evaporated
under reduced pressure then cold H2O (10 mL) was
added. The resulting solution was acidified with 6 N
HCl then CH2Cl2 (10 mL) was added. The resulting
precipitate was collected and dried under vacuum to
provide 8 (0.37 g) in 99% yield as a green solid, which
2.5 (n=2)
42 (n=2)
NA
69 (n=2)
12
NA
X+ (n=2)
Xꢄ
Xꢄ
aUnits of superoxide dismutase (SOD) activity defined as the amount
of compound that inhibits 50% the reduction of cytochrome c.
bThe concentration of compound that inhibits iron/ascorbate-medi-
ated brain homogenate lipid peroxidation by 50%.
cPositive (X+) or negative (Xꢁ) E. coli aerobic growth.
dn, number of repetitions.
eppt, compound precipitated out of the assay medium.
fNA, not active.
gThe compound interferes with the assay by inhibiting xanthine
oxidase which is utilized in the assay.

3018
P. J. F. Gauuan et al. / Bioorg. Med. Chem. 10 (2002) 3013–3021
was used in the metalation step without further purifica-
tion. 1H NMR (DMSO-d6) d 7.97 (s, 8H), 7.49 (m, 16H).
due was dissolved in acetone/H2O (200 mL, 83:17) then
AgNO3 (47.25 g, 275.2 mmol) was added. The flask was
covered with foil to avoid decomposition of the AgNO3.
The reaction mixture was stirred at room temperature
for 2–3 h then the AgBr salts were filtered off through a
pad of Celite. The filtrate was diluted with EtOAc
(400 mL), transferred to a separatory funnel, then
washed successively with saturated NaHCO3 (300 mL),
H2O (300 mL), and brine (300 mL). The organic extract
was dried (Na2SO4), filtered, and evaporated under
reduced pressure. Chromatography of the residue on
silica gel provided aldehyde 16a (11.92 g) in 42% yield.
1H NMR (CDCl3) d 10.05 (s, 1H), 7.9 (d, 1H, H-4), 7.5
(overlapping s and d, 2H), 3.99 (s, 3H), 3.92 (s, 3H).
{5,10,15,20-Tetrakis[4-(2,3,4,5-tetrazolyl)phenyl]porphyr-
inato}manganese(III) chloride (9). A solution of 8 (0.35 g,
0.4 mmol) and MnCl2 (0.25 g, 2 mmol) in DMF (20 mL)
was heated at reflux for 4–5 h. The reaction mixture was
allowed to cool to room temperature then D MF was eva-
porated under reduced pressure. The crude product was
adsorbed onto silica gel (1.5 g) then was purified by column
chromatography to provide of 9 as a dark green solid; mp
>320 ꢂC. FABMS m/z 939 [C48H28MnN20]+. HPLC
84%. UV (EtOH) lmax, nm (e) 467.5 (0.42 ꢃ 105).
Methyl 2-methoxy-3-methylbenzoate (15a). To a magne-
tically stirred solution of 2-hydroxy-4-methyl benzoic
acid (25.5 g, 168 mmoL), finely ground anhydrous
K2CO3 (60 g, 434 mmol) and acetone (300 mL) was
added (MeO)2SO2 (47.6 mL, 503 mmol). The solution
was stirred at room temperature for 18 h then heated at
reflux until analysis by TLC indicated the reaction was
complete (1–2 h). The reaction mixture was cooled to
room temperature, filtered, and the excess K2CO3 cake
was thoroughly washed with acetone. The filtrate was
evaporated and the residue was redissolved in EtOAc
(300 mL) then Et3N (70 mL) was added. The reaction
mixture was stirred at room temperature for 30 min,
transferred to a separatory funnel and washed successively
with H2O (200 mL), 2 N HCl (until slightly acidic), H2O
(200 mL), and brine (200 mL) then dried (Na2SO4), fil-
tered, and evaporated under reduced pressure. Chromato-
graphy of the residue on silica provided 15a (30 g) in 95%
yield. 1H NMR (CD C3l) d 7.73 (d, 1H), 6.79 (overlapping
d and s, 2H), 3.91 (s, 3H), 3.86 (s, 3H), 2.39 (s, 3H).
Methyl 2-fluoro-5-formylbenzoate (16b). A magnetically
stirred solution of 15b (4.70 g, 27.9 mmol), NBS (10.5 g,
58.9 mmol), AIBN (226 mg, 1.4 mmol), and CCl4
(350 mL) was Heated at reflux for 20 h. Analysis by
TLC indicated that mono-, di- and tribrominated
benzoates were formed and the dibromide was the
major product. The reaction mixture was evaporated
and the residue was redissolved in EtOAc (250 mL),
washed successively with H2O (150 mL), saturated
Na2S2O3 (150 mL), H2O (150 mL), and brine (150 mL).
The organic extract was then dried (Na2SO4), filtered
and evaporated. The residue was dissolved in acetone/
H2O (200 mL, 83:17) then AgNO3 (12.2 g, 71.8 mmol)
was added. The flask was covered with foil to avoid
decomposition of the AgNO3. The reaction mixture was
stirred at room temperature overnight then the AgBr
salts were filtered off from the solution. The filtrate was
diluted with EtOAc (400 mL), transferred to a separa-
tory funnel then, washed successively with saturated
NaHCO3 (200 mL), H2O (200 mL), and brine (200 mL).
The organic extract was dried (Na2SO4), filtered, and
evaporated under reduced pressure. Chromatography of
the residue on silica gel provided aldehyde 16b (2.89 g)
Methyl 2-fluoro-5-methylbenzoate (15b). Following the
procedure for the preparation of 15a and using 2-fluoro-
5-methyl benzoic acid (5.03 g, 32.6 mmol), finely ground
anhydrous K2CO3 (14.2 g, 102.7 mmol), acetone
(150 mL), and (MeO)2SO2 (3.80 mL, 40.1 mmol), ester
15b was isolated in 94% yield (5.41 g). 1H NMR
(CDCl3) d 7.73 (dd, 1H, J=2.31, 6.87 Hz), 7.30 (m, 1H),
7.01 (dd, 1H, J=8.55, 10.65 Hz), 3.93 (s, 3H), 2.35 s).
1
in 57% yield. H NMR (CDCl3) d 10.01 (s, 1H), 8.49
(dd, 1H, J=6.9, 2.16 Hz), 8.09 (ddd, 1H, J=8.5, 4.5,
2.16 Hz), 7.31 (dd, 1H, J=8.5, 9.96 Hz), 3.98 (s. 3H).
Methyl 3-fluoro-4-formylbenzoate (16c). Following the
procedure for the preparation of 16b and using 15c (2.11 g,
12.5 mmol), NBS (6.31 g, 35.4 mmol), AIBN (118 mg,
0.70 mmol), and CCl4 (150 mL). The resulting crude
dibromide was treated with AgNO3 (6.15 g, 36.2 mmol) in
acetone/H2O (100 mL, 83:17). After purification by col-
umn chromatography, compound 16c was obtained in
Methyl 3-fluoro-4-methylbenzoate (15c). Following the
procedure for the preparation of 15a and using 3-fluoro-
4-methyl benzoic acid (9.5 g, 61.6 mmoL), finely ground
anhydrous K2CO3 (25.8 g, 186.7 mmol), acetone
(150 mL), and (MeO)2SO2 (7.0 mL, 74.0 mmol), ester
15c was isolated in 72% yield (7.42 g). 1H NMR
(CDCl3) d 7.72 (dd, 1H, J=1.83, 7.83 Hz), 7.65 (dd, 1H,
J=1.32, 10.11 Hz), 7.26 (dd, 1H, J=5.82, 7.62 Hz), 3.91
(s, 3H), 2.33 (d, 3H, J=1.89 Hz).
1
85% yield (1.94 g). H NMR (CDCl3) d 10.42 (s, 1H),
7.93 (m, 2H), 7.84 (d, 1H, J=11.3 Hz), 3.97 (s, 3H).
5,10,15,20-Tetrakis(4-carboxymethyl-3-methoxyphenyl)-
porphyrin (17a). Following the procedure for the pre-
paration of 6a and using aldehyde 16a (1 g, 5.1 mmol),
porhyrin 17a (102 mg) was isolated in 8% yield as a
Methyl 4-formyl-2-methoxybenzoate (16a). A magneti-
cally stirred solution of 15a (24.43 g, 135.6 mmol), NBS
(54.41 g, 305.7 mmol), and CCl4 (1 L) was exposed to a
100-watt lamp for 6 h. Analysis by TLC indicated that
mono-, di- and tribrominated benzoates were formed
and the dibromide was the major product. The reaction
mixture was quenched with H2O (200 mL), washed with
saturated Na2S2O3 (500 mL), dried over Na2SO4, fil-
tered, and evaporated under reduced pressure. The resi-
1
violet solid. H NMR (CDCl3) d 8.90 (s, 8H), 8.19 (d,
4H), 7.88 (d, 4H), 7.86 (s, 4H), 4.11 (s, 12H), 4.00 (s,
12H), ꢁ2.83 (br s, 2H).
5,10,15,20-Tetrakis(3-carboxymethyl-4-fluorophenyl)por-
phyrin (17b). Following the procedure for the prepara-
tion of 6a and using aldehyde 16b (1.16 g, 6.4 mmol),

P. J. F. Gauuan et al. / Bioorg. Med. Chem. 10 (2002) 3013–3021
3019
porhyrin 17b (580 mg) was isolated in 40% yield as a
1
ification by column chromatography as green solids. For
porphyrin 11a: mp >300 ꢂC; IR (KBr) 3134, 1674, 1624,
1484, 1384, 1290, 1212, 1089, 804 cmꢁ1. FABMS m/z 921
[C49H30MnN4O12]+. HPLC 99%. UV (EtOH) lmax, nm
(e) 472.0 (0.46 ꢃ 105). For porphyrin 11b: mp >300 ꢂC; IR
(KBr) 3422, 3159, 1625, 1558, 1483, 1429, 1342, 1208,
805 cmꢁ1. FABMS m/z 907 [C48H28MnN4O12]+. HPLC
98%. UV (EtOH) lmax, nm (e) 472.0.
violet solid. H NMR (CDCl3) d 8.83 (s, 8H), 8.78 (d,
4H, J=6.78 Hz), 8.34 (m, 4H), 7.57 (dd, 4H, J=8.6,
10.1 Hz), 4.01 (s, 12H), ꢁ2.86 (s, 2H).
5,10,15,20-Tetrakis(4-carboxymethyl-2-fluorophenyl)por-
phyrin (17c). Following the procedure for the prepara-
tion of 6a and using aldehyde 16c (1.16 g, 10.0 mmol),
porhyrin 17c (228 mg) was isolated in 10% yield as a
1
violet solid. H NMR (CDCl3) d 8.83 (s, 4H), 8.25 (m,
[5,10,15,20-Tetrakis(2-carboxymethyl-3-methoxyphenyl)-
porphyrinato]manganese(III) chloride (12a). Following
the procedure for the preparation of 9 and using por-
phyrin 17a (100 mg, 0.103 mmol), porphyrin 12a (59 mg)
was isolated in 54% yield after purification by column
chromatography a green solid: mp >325 ꢂC. HPLC
>99%. UV (EtOH) lmax, nm (e) 467.5 (1.3 ꢃ 105).
8H), 4.15 (s, 12H), ꢁ1.25 (br s, 2H).
5,10,15,20-Tetrakis(3-carboxymethyl-4-hydroxyphenyl)-
porphyrin (17d). Following the procedure for the pre-
paration of 6a and using methyl 3-formyl-6-hydroxy
benzoate (1.65 g, 9.1 mmol), porphyrin 17d (300 mg) was
isolated in 14% yield as a violet solid. 1H NMR
(CDCl3) d 11.16 (4H), 8.87 (s, 8H), 8.67 (s, 4H), 8.30 (d,
4H), 7.39 (d, 4H), 3.96 (2, 12H).
.
Anal. calcd for C56H44ClMnN4O12 H2O: C, 62.66; H,
4.32; N, 5.22. Found: C, 62.21; H, 4.69; N, 5.07.
[5,10,15,20-Tetrakis(4-carboxymethyl-3-hydroxyphenyl)-
porphyrinato]manganese(III) chloride (12b), [5-(4-carboxy-
3-hydroxyphenyl)-10,15,20-tris(4-carboxymethyl-3-hydro-
xyphenyl)porphyrinato]-manganese(III) chloride (12c) and
[5,10-bis(4-carboxy-3-hydroxyphenyl)-15,20-bis(4-car-
boxymethyl - 3 - hydroxyphenyl)porphyrinato]manga-
nese(III) chloride (12d). Following the procedure for
the preparation of 9 and using porphyrin 17e, porphyr-
ins 12b, 12d, and 12d were isolated after purification by
column chromatography as green solids. For porphyrin
12b: mp >300 ꢂC. FABMS m/z 963 [C52H36MnN4O12]+.
HPLC 88%. UV (EtOH) lmax, nm (e) 467.5 (1.1 ꢃ 105).
For porphyrin 12c: mp >300 ꢂC. FABMS m/z 949
[C51H34MnN4O12]+. HPLC 98%. UV (EtOH) lmax, nm
(e) 467.5 (1.3 ꢃ 105). For porphyrin 12d: mp >300 ꢂC.
IR (KBr) 3423, 1677, 1618, 1439, 1341, 1290, 1202,
1094, 948 cmꢁ1. FABMS m/z 935 [C50H32MnN4O12]+.
HPLC 98%. UV (EtOH) lmax, nm (e) 468.5 (1.0ꢃ105).
5,10,15,20-Tetrakis(4-carboxymethyl-3-hydroxyphenyl)-
porphyrin (17e). Following the procedure for the pre-
paration of 6a and using methyl 4-formyl-2-hydroxy
benzoate (1.01 g, 10.0 mmol), porphyrin 17e (554 mg)
was isolated in 42% yield as a violet solid. H NMR
(CDCl3) d 11.13 (4H), 8.90 (s, 8H), 8.20 (d, 4H), 7.89 (s,
4H), 7.73 (d, 4H), 4.15 (s, 12H), ꢁ2.85 (br s, 2H).
1
5,10,15,20-Tetrakis(4-carboxymethyl-3-nitrophenyl)por-
phyrin (17f). Following the procedure for the prepara-
tion of 6a and using methyl 4-formyl-2-nitro benzoate
(1.05 g, 5.0 mmol), porphyrin 17f (678 mg) was isolated
in 56% yield after repeated purification by column
1
chromatography as a violet solid. H NMR (CDCl3) d
8.86 (s, 8H), 8.75 (s, 4H), 8.54 (d, 4H), 8.21 (d, 4H), 4.14
(s, 12H), ꢁ2.89 (br s, 2H).
.
5,10,15,20-Tetrakis(4-fluoro-3-methoxyphenyl)porphyrin
(17i). Following the procedure for the preparation of
6a and using 3-flouro-4-methoxybenzaldehyde (1.50 g,
9.6 mmol), porphyrin 17i (735 mg) was isolated in 38%
yield after purification by column chromatography as a
Anal. calcd for C50H32ClMnN4O12 NH4OH: C, 59.68;
H, 3.71; N, 6.96. Found: C, 59.83; H, 3.80; N, 6.82.
[5,10,15,20 - Tetrakis(4 - carboxy - 3 - hydroxyphenyl)por-
phyrinato]manganese(III) chloride (12e). Saponification
of a mixture of porphyrins 12b–12d provided porphyrin
12e after purification by column chromatography as a
green solid: mp >320 ꢂC. FABMS m/z 944
[C48H28ClMnN4O12+H]+. UV (EtOH) lmax, nm (e)
467.5 (0.66 ꢃ 105).
1
violet solid. H NMR (CDCl3) d 8.87 (s, 8H), 7.96 (dd,
4H, J=1.4, 11.7 Hz), 7.87 (d, 4H, J=8.3 Hz), 7.29 (dd,
4H, J=8.4, 8.6 Hz), 4.12 (s, 12H), ꢁ1.25 (br s, 2H).
5,10,15,20-Tetrakis(4-carboxymethyl-2-methoxyphenyl)-
porphyrin (17k). Following the procedure for the pre-
paration of 6a and using methyl 4-formyl-3-methoxy
benzoate (1.23 g, 6.3 mmol), porphyrin 17k (333 mg)
was isolated in 22% yield after purification by column
[5,10,15,20 -Tetrakis(3 -carboxymethyl- 4- fluorophenyl)-
porphyrinato]manganese(III) chloride (12f) and [5-(4-di-
methylaminophenyl) - 10,15,20 - tris(4 - carboxymethyl - 3 -
methoxyphenyl) porphyrinato]-manganese(III) chloride
(12g). Following the procedure for the preparation of 9
and using porphyrin 17b (470 mg, 0.51 mmol), porphyr-
ins 12f (149 mg, 29%) and 12g were isolated after pur-
ification by column chromatography as green solids.
Porphyrin 12g was a by product resulting from the
nucleophilic substitution the 4-fluoro position in 12f:
For porphyrin 12f: mp >300 ꢂC. FABMS m/z 971
1
chromatography as a violet solid. H NMR (CDCl3) d
8.70 (s, 8H), 8.10 (m, 12H), 4.12 (s, 12H), 3.62–3.61
(four singlets corresponding to rotamers, 12H), ꢁ2.65
(br s, 2H). FABMS m/z 967 [C56H46N4O12+H]+.
[5-(3-Carboxymethyl-4-hydroxyphenyl)-10,15,20-tris(3-
carboxy-4-hydroxyphenyl)porphyrinato]-manganese(III)
chloride (11a) and [5,10,15,20-tetrakis(3-carboxy-4-hy-
droxyphenyl)porphyrinato]-manganese(III) chloride (11b).
Following the procedure for the preparation of 9 and
using porphyrin 17d (100 mg, 0.103 mmol), porphyrins
11a (33 mg) and 11b (121 mg) were isolated after pur-
[C52H32F4MnN4O8]+. HPLC 97%; UV (EtOH) lmax
,
nm (e) 467.0 (1.3ꢃ105). For porphyrin 12g: mp
>300 ꢂC. FABMS m/z 996 [C54H38F3MnN5O8]+.
HPLC 96%. UV (EtOH) lmax, nm (e) 468.0 (1.3 ꢃ 105).

3020
P. J. F. Gauuan et al. / Bioorg. Med. Chem. 10 (2002) 3013–3021
[5,10,15,20-Tetrakis(3-carboxyl-4-fluorophenyl)porphyri-
nato]manganese(III) chloride (12h). To a solution of
porphyrin 12g (130 mg, 0.13 mmol) in MeOH (10 mL)
was added Claisen’s base (0.25 mL, excess) and the
solution was heated at reflux for 3 h. The solution was
then cooled to room temperature then acidified with 6 N
HCl. The reaction mixture was then evaporated off and
the residue was adsorbed onto silica gel. Purification by
column chromatography provided tetraacid porphyrin
12h (80 mg) in 65% yield as a green solid: mp >300 ꢂC.
FABMS m/z 915 [C48H24F4MnN4O8]+. HPLC 96%.
UV (EtOH) lmax, nm (e) 4637.5 (1.0 ꢃ 105).
solids. Porphyrin 13b was a by-product, resulting from
the mono-hydrolysis of the ester functionality. For por-
phyrin 13a: mp >300 ꢂC. IR (KBr) 3436, 2953, 1728,
1437, 1414, 1297, 1221, 1092, 1010, 763 cmꢁ1. FABMS
m/z 971 [C52H32F4MnN4O8]+. HPLC 96%. UV (EtOH)
l
max, nm (e) 462.5 (1.7 ꢃ 105). For porphyrin 13b: mp
>300ꢂ. IR (KBr) 3417. 2950, 1728, 1384, 1298, 1222,
1011, 765 cmꢁ1. FABMS m/z 957 [C51H30F4MnN4O8]+.
HPLC 94%. UV (EtOH) lmax, nm (e) 463.0 (0.39ꢃ105).
5,10,15,20-tetra(4-carboxy-2-fluorophenyl) porphyrinato]
manganese(III) chloride (13c). Saponification of 13a
provided 13c as a green solid: mp >300 ꢂC. FABMS m/
z 915 [C48H24F4MnN4O8]+. HPLC 94%. UV (EtOH)
[5-(4-Dimethylaminophenyl)-10,15,20-tris(4-carboxy-3-
methoxyphenyl)porphyrinato]manganese(III) chloride (12i).
Saponification of 12g provided 12i as a green solid: mp
>300 ꢂC. FABMS m/z 940 [C50H30F3MnN5O8]+.
HPLC 91%. UV (EtOH) lmax, nm (e) 467.0 (0.94ꢃ105).
l
max, nm (e) 463.0 (1.5ꢃ105).
5,10,15,20-Tetrakis(4-carboxymethyl-2-methoxyphenyl)-
porphyrinato]manganese(III) chloride (13d). Following
the procedure for the preparation of 9 and using por-
phyrin 17k (321 mg, 0.33 mmol), porphyrin 13d (294 mg)
was obtained in 47% yield after purification by column
chromatography as a green solid: mp >300 ꢂC. FABMS
m/z 1019 [C56H44MnN4O12]+. HPLC 92%. UV (EtOH)
[5,10,15,20-Tetrakis(4-carboxymethyl-3-nitrophenyl)por-
phyrinato]manganese(III) chloride (12j) and [5-(4-car-
boxy - 3 - nitropheyl) - 10,15,20 - tris(4 - carboxymethyl - 3 -
nitrophenyl)porphyrinato]manganese(III) chloride (12k).
Following the procedure for the preparation of 9 and
using porphyrin 17f (280 mg, 0.27 mmol), porphyrins
12j (210 mg, 69%) and 12k (34 mg, 11%) were isolated
after purification by column chromatography as green
solids. For porphyrin 12j: mp >300 ꢂC. IR (KBr) 3438,
2962, 1736, 1540, 1437, 1353, 1297, 1131, 1071,
l
max, nm (e) 467.0 (1.5ꢃ105).
5,10,15,20 - Tetrakis(4 - carboxy - 2 - methoxyphenyl)por-
phyrinato]manganese(III) chloride (13e). Saponification
of 13d provided 13e as a green solid: mp >300 ꢂC.
FABMS m/z 963 [C52H36MnN4O12]+. HPLC 96%. UV
(EtOH) lmax, nm (e) 467.0 (1.5ꢃ105).
824 cmꢁ1
.
FABMS m/z 1079 [C52H32MnN8O16]+.
HPLC >99%. UV (MeOH) lmax, nm (e) 464.0
(1.4ꢃ105). For porphyrin 12k: mp >300 ꢂC. IR (KBr)
3422, 2923, 1736, 1612, 1540, 1384, 1297, 1131, 1072,
802 cmꢁ1. FABMS m/z 10710659 [C51H30MnN8O16]+.
HPLC 96%. UV (MeOH) lmax, nm (e) 464.0 (1.2ꢃ105).
Acknowledgements
We acknowledge Mark Goldstein of the National Jew-
ish Center and the Analytical Department of Albany
Molecular Research, Inc. for technical assistance.
[5,10,15,20-Tetrakis(4-carboxy-3-nitrophenyl)porphyri-
nato]manganese(III) chloride (12l). Saponification of
porphyrin 12j provided porphyrin 12l as a green solid:
mp >300 ꢂC. IR (KBr) 3424, 1719, 1612, 1535, 1353, 1072,
1013, 802 cmꢁ1. FABMS m/z 1023 [C48H24MnN8O16]+.
HPLC 98%. UV (MeOH) lmax, nm (e) 465.0 (1.3ꢃ105).
References and Notes
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M. Biochem. Pharmacol. 1991, 42, 991.
[5,10,15,20-Tetrakis(4-carboxymethyl-3-nitrophenyl)por-
phyrinato]manganese(III) chloride (12o). Following the
procedure for the preparation of 9 and using porphyrin
17i (192 mg, 0.24 mmol), porphyrin 12o (150 mg) in 71%
yield after purification by column chromatography as a
green solid: mp >300 ꢂC. IR (KBr) 3449, 2932, 1578,
1517, 1304, 1273, 1132, 1016, 943, 804 cmꢁ1. FABMS
m/z 859 [C48H32F4MnN4O4]+. HPLC 94%. UV
(MeOH) lmax, nm (e) 468.0 (0.9ꢃ105). Anal. calcd for
3. (a) Kukreja, R. C.; Hess, M. L. Cardiovasc. Res. 1992, 26,
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Acad. Sci. U.S.A. 1993, 90, 7915.
.
C48H32ClF4MnN4O4 CH3OH: C, 63.47; H, 3.91; N,
6.04. Found: C, 63.70; H, 3.99; N, 5.89.
5. (a) Gray, B.; Carmichael, A. J. Biochem. J. 1992, 281, 795.
(b) Archibald, F. S.; Fridovich, I. Arch. Biochem. Biophys.
1982, 214, 452.
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sen, E.; Malfroy, B. Biochem. Biophys. Res. Commun. 1993,
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[5,10,15,20-Tetrakis(4-carboxymethyl -2-fluorophenyl)-
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after purification by column chromatography as green

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