Alkylation of manganese(II) tetraphenylporphyrin by a synthetic antimalarial trioxane

Article abstract of DOI:10.1039/b302835f

The synthesis and the antimalarial activity of a new kind of polycyclic 1,2,4-trioxanes are reported. The alkylation of the heme model Mn^IITPP by the biologically active (IC 50 = 320 nmol L^-1) hemiperketal 2 is presented.

Full text of DOI:10.1039/b302835f

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Alkylation of manganese(II) tetraphenylporphyrin by a synthetic
antimalarial trioxane
a
a
a
b
Jean-François Berrien, Olivier Provot, Joëlle Mayrargue,* Michel Coquillay,
c
c
c
d
d
Liliane Cicéron, Frédérick Gay, Martin Danis, Anne Robert and Bernard Meunier*
UMR 8076 BioCIS, Faculté de Pharmacie, rue J.-B. Clément,
a
F-92296 Châtenay-Malabry Cedex, France. E-mail: Joelle.Mayrargue@chimorg.u-psud.fr
Laboratoire de Physique, Faculté de Pharmacie, rue J.-B. Clément,
F-92296 Châtenay-Malabry Cedex, France
INSERM U511, Immuno-biologie cellulaire et moléculaire des infections parasitaires,
Groupe hospitalier Pitié-Salpêtrière, F-75013 Paris, France
b
c
d
Laboratoire de Chimie de Coordination du CNRS, 205, route de Narbonne,
31077 Toulouse Cedex 4, France
Received 14th March 2003, Accepted 23rd June 2003
First published as an Advance Article on the web 14th July 2003
The synthesis and the antimalarial activity of a new kind of polycyclic 1,2,4-trioxanes are reported. The alkylation of


Ϫ1
the heme model Mn TPP by the biologically active (IC 50 = 320 nmol L ) hemiperketal 2 is presented.
Introduction
Two billion people are now exposed to the risk of malaria and 1
to 3 million die each year from this disease, due to the increas-
ing resistance of the parasite Plasmodium falciparum to the
1,2
classical drug chloroquine. During malaria infection, free
heme is liberated in the parasite food vacuole by digestion of
the host hemoglobin and then polymerized to malaria pig-
3
ment. Heme is therefore an attractive pharmacological target,
since it comes from a metabolic pathway that is unique for
infected erythrocytes.
Artemisinin 1, a peroxide-containing drug extracted from
the Chinese plant Artemisia annua L., is very efficient against
multidrug resistant strains and no case of clinical resistance
has been reported up to now despite more than 20 years of
2
intensive use in Asia. The possible reactivity of artemisinin
Scheme 1 Structures of the antimalarial artemisinin 1, and model
peroxides 2 and 3.
with heme has been considered as a key factor of its pharma-
4,5
6
cological activity, and the alkylation of heme or proteins
was observed after incubation of pharmacologically relevant
concentrations of artemisinin derivatives in cultured para-
7
sites. We have reported the in vitro alkylation of manganese
tetraphenylporphyrin used as a heme model by a C-centred
radical derived from artemisinin or other pharmacologically
8
–10
active trioxanes.
Covalent heme–artemisinin adducts have
11
also been characterized.
In this article, we report a simple access to a new kind of
,2,4-trioxanes, their antimalarial activities and the alkylation
of the heme model Mn TPP by the trioxane 2.
1

Results and discussion
Syntheses
We envisaged a simple access to the polycyclic 1,2,4-trioxane 2
(
Scheme 1) which could be compared with two antimalarial and
natural 1,2,4-trioxanes: artemisinin 1 and the peroxide 3 iso-
1
2
lated from the fruit of Amomum krervanh Pierre (cardamom).
The hetero Diels–Alder dimer 6 of methylene cyclohexanone
Scheme 2 Synthesis of the trioxanes 2, 8, and 9. (Only one enantiomer
is depicted.)
1
3
5
was known to add water in acidic media to give the poly-
14
cyclic hemiacetal 7 (Scheme 2). We thought that hydrogen per-
oxide could react with the dimer 6 in the same manner leading
to a polycyclic peroxide. In fact, acidic 30% aqueous hydrogen
peroxide in methanol converts 6 into the trioxane 2 and two
other compounds, presumably diastereomers, in a 46% yield.
The major one could be obtained in a pure form after careful
chromatography and crystallisation. Its structure was deter-
mined by X-ray diffraction (vide infra). The water addition
product 7 was also isolated from the reaction mixture but in
only 4% yield (Scheme 2). One can note that the reaction gave
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 5 9 – 2 8 6 4
2859

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about ten times more hydrogen peroxide addition product 2
than water addition product 7. However, in the reaction
medium, water was more than four times more concentrated
than hydrogen peroxide. This result can be attributed to the
α effect on H O that makes the oxygen atom of hydrogen per-
Table 1 Selected angles (Њ)
C(7)–C(3)–O(2)
O(1)–C(6)–O(17)
C(13)–C(5)–O(4)
101.8 (2)
102.6 (2)
106.0 (2)
C(10)–C(3)–O(2)
C(5)–C(13)–C(14)
C(5)–C(12)–C(11)
114.8 (2)
114.3 (2)
114.2 (2)
2
2
15
oxide more nucleophilic than that of H O. We wanted to pre-
2
pare the trioxane 12, a pinenic analogue of 2 with a structure
close to cardamom peroxide (Scheme 3). Unfortunately,
cedronellone 11, a natural product isolated from Cedronella
consists of four fused rings of single bonded atoms. The bond
lengths have standard values: [1.513–1.538 Å] for C–C and
[1.429–1.465 Å] for C–O and O–O. Most of the angles are in the
range [108.1–113.9Њ] except those listed in Table 1. There is an
intermolecular H-bond between the hydroxyl of any molecule
with the O(4) of the next b-translated molecule [O(17)–
H ؒ ؒ ؒ O(4), 3.035 Å]. All other contributions to the packing
cohesion are van der Waals interactions. The molecular con-
16
canariensis which was prepared by hetero Diels–Alder dimer-
17
ization of d-pinocarvone 10, did not react with H O under
2
2
these conditions. These results could be attributed to the low
reactivity of the hindered carbonyl function of cedronellone.
Moreover, heating reactions with cedronellone and H O in
2
2
3
sulfuric acid gave only tarry materials.
formation is rigidly fashioned because of the sp hybridisation
of atoms so that we can expect a low molecular compliance
under the packing constraints as suggested by the medium
Ϫ3
value of density (1.293 g cm ) and the low intensity of the
intermolecular H-bond. The graphical examination of the
region surrounding the peroxide shows that the local accessible
surface including O(1), O(2) and the next hydrogen H, is almost
planar so that there is no steric hindrance for an attack of
peroxide.
Antimalarial activity
Trioxanes 2, 8 and 9 were evaluated in vitro against Plasmodium
falciparum (Table 2). The hemiperketal 2 had a substantial
activity with 1/80 of the antimalarial potency of artemether
23
on the P. falciparum “H” strain. The cardamom peroxide 3
12
had an activity one tenth of that of artemisinine and roughly
one fortieth of that of artemether. So, the antimalarial activ-
ities of the natural peroxide 3 and 2 are comparable even if
Scheme 3 Reaction of cedronellone with H O .
2
2
24
other synthetic 1,2,4-trioxanes have a better in vitro activity.
18
Recently, acyclic α-methoxy-substituted dialkyl peroxide
Although the latter showed an interesting activity, its substi-
tuted derivatives 8 and 9 were inactive in the range of concen-
tration tested. Examination of the molecular structure of
these inactive trioxanes could explain the biological results. As
19
and α-methoxyprop-2-yl peroxide derivatives
have been
reported for their powerful antimalarial activity. In this way
and in order to establish structure–activity relationships, two
compounds were synthesized from peroxide 2. To obtain per-
oxides with a different structure, the hydroxyl function of tri-
oxane 2 was substituted by two groups with different electronic
25
we have recently published for substituted trioxanes, we
suppose that compounds 8 and 9 could not approach the iron–
heme nucleus because of steric hindrance. The Fenton-type
cleavage of the peroxide moiety, which affords primarily oxyl
radicals, is inefficient and compounds are devoid of activity.
Moreover, we can suppose that only compound 2 thanks to
its hydroxyl substituent is able to coordinate with heme by
hydrogen bonding unlike methoxy and acetoxy compounds 8
and 9.
effects. Thus, the acetate 8 was prepared with Ac O, pyridine
2
20
and using DMAP as the hydroxyl function was hindered. The
methyl perketal 9 was also synthesized from trioxane 2 using
t-BuOK and subsequent methylation of the so-formed oxanion
with MeI (Scheme 2). In our case, deprotonation using n-BuLi
as previously reported for 3-hydroxy-1,2-dioxolanes gave poor
21
results. Moreover, classical methods of perketal formation
21
22
II
from hemiperketal (MeOH, HCl; MeOH, TsOH ) did not
provide the desired product.
Reaction of the trioxane 2 with Mn TPP
As a model of the possible reaction of the antimalarial agent
2
with the intraparasitic heme, we investigated the activ-
X-Ray analysis of trioxane 2
ation of 2 with manganese() tetraphenylporphyrin generated
in situ from Mn (TPP)Cl and borohydride. The reaction condi-
tions were similar to those previously reported for the alkyl-
ation of Mn TPP by artemisinin, artemether, and several

I
The molecular structure of the title compound is shown in
Scheme 4. Selected angles are listed in Table 1. The molecule


8–10
synthetic antimalarial endoperoxides.
This reaction pro-
duced, after demetallation of the macrocycle under mild condi-
tions, a mixture of the chlorin and porphyrin adducts 15C and
1
5P, respectively, containing the main fragment of compound 2
(
Scheme 5). The chlorin adduct was the major one, as sup-
ported by UV–vis spectroscopy, the band at 652 nm having a
relative intensity of 11% with respect to the Soret band (15%
is expected for a pure chlorin). This indicated a ratio chlorin :
porphyrin close to 80 : 20. The mass spectrometry analysis of
the mixture 15C ϩ 15P exhibited molecular peaks at m/z =
ϩ
ϩ
8
53 (MH for 15C) and 851 (MH for 15P), correspond-
ing to the substitution of one proton of tetraphenylchlorin
or tetraphenylporphyrin, respectively, by a drug-derived frag-
ment without extensive degradation of both partners. The
relative abundance of 853 : 851 was 84 : 16, consistent with the
Scheme 4 Ortep drawing of the molecule 2 at 50% probability level.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 5 9 – 2 8 6 4
2
860

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Table 2 In vitro antimalarial activity of trioxanes 2, 8 and 9 against P. falciparum H strain
Compounds
IC 50/nmol L
2
8
9
Artemether
4.1
Chloroquine
15
Ϫ1
320
> 5000
> 5000
The formation of 15P and 15C can be explained by the fol-
lowing mechanism: the reductive activation of the peroxide
bond of 2 gave rise to the alkoxy radical centred at O2 13,
followed by β-scission of the C3–C10a bond to form the
C-centred radical 14. This reactive radical species generated just
above the metalloporphyrin ring was able to intramolecularly
alkylate a β-pyrrolic position of the macrocycle at C2Ј, gener-
ating a C-centred radical at the adjacent position C3Ј (structure
ؒ
not shown). An intramolecular electron transfer from C3Ј to
the manganese() resulted in a manganese() complex with a
carbocation centred at C3Ј. This cationic species can be
trapped by the borohydride (initially present in the reaction


mixture to reduce the metal centre of Mn (TPP)Cl), leading to
a chlorin macrocycle. After demetallation of the complex, the
chlorin-type adduct 15C was obtained (for a mild demetal-
lation method, see ref. 8b). As a competitive reaction, the
ϩ
manganese()–C3Ј can lose a proton at C2Ј to generate, after
removal of manganese, the porphyrin adduct 15P. Both reac-
tion pathways were observed. It should be mentioned that the
attack of the radical C10a onto a β-pyrrolic position (which
is in fact an intramolecular reaction) is probably more rapid
than the rotation of this radical, leading to retention of con-
figuration at C10a. The stereoselectivity of this alkylation
of MnTPP by an antimalarial trioxane has recently been
9
documented.
In order to confirm this mechanism, a similar reaction was
performed in the presence of borodeuteride instead of boro-
hydride. As expected, the alkylation step produced a mixture of
chlorin (major) and porphyrin adducts, and the subsequent
oxidation by DDQ produced a mixture of porphyrin adducts
ϩ
bearing at C3Ј either a hydrogen atom [15P, m/z = 851 (MH )],
ϩ
or a deuterium atom [15P-d, m/z = 852 (MH )] (Scheme 6). In
1
the H NMR spectrum, the intensity of H3Ј (8.82 ppm) was
Scheme 5 Reductive activation of the trioxane 2 by the heme model

Mn TPP, leading to the covalent adducts 15C and 15P. (Only one
enantiomer is depicted.)
UV–vis analysis, indicating that 15C and 15P have similar
behavior under the MS conditions (allowing, in these peculiar
conditions, quantitative comparison of mass peaks).
All attempts to separate 15C and 15P by column chromato-
graphy or by preparative TLC failed. We therefore decided to
oxidize the mixture 15C ϩ 15P by 2,3-dichloro-5,6-dicyano-
benzoquinone, in order to isolate and characterize the pure
1
porphyrin adduct 15P. The H NMR analysis of pure 15P (less
than 1 mol% of remaining 15C) thus obtained exhibited seven
β-pyrrolic protons, confirming the monoalkylation of the por-
phyrin macrocycle at a β-pyrrolic position. Six of the β-pyrrolic
3
resonances appeared as doublets (1H each, J = 5 Hz), and one
as a broad singlet corresponding to H3Ј (8.86 ppm), adjacent to
the alkylated carbon C2Ј. The proton H10a was detected at
3
.63 ppm, and two different intracyclic NH resonances were
detected at Ϫ2.86 and Ϫ2.67 ppm. However, this analysis per-
formed at 250 MHz did not allow individual attribution of the
2
0 methylene protons that appeared as an unresolved pattern
between 2.63 and 1.20 ppm. Full assignment of these methylene
protons was performed at 500 MHz on the mixture 15P ϩ

Scheme 6 Reductive activation of the trioxane 2 by Mn TPP in the
1
5P-d, and is described in the corresponding paragraph.
presence of borodeuteride. (Only one enantiomer is depicted.)
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 5 9 – 2 8 6 4
2861

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Ϫ
13
1
0
.9H, indicating partial incorporation of deuterium from BD4
at C3Ј, with a molar ratio 15P : 15P-d of 9 : 1 (the ratio 15P :
5P-d calculated from MS analysis was 86 : 14. Owing to the
and C, respectively). H chemical shifts are reported in ppm
from an internal standard TMS or of residual chloroform
13
1
(7.27 ppm). C chemical shifts are reported in ppm from the
central peak of deuteriochloroform (77.1). NMR characteriz-
ation of adduct 15P was performed on a Bruker DMX500
spectrometer equipped with a 5 mm probe operating at
precision of these analyses, both results should be considered as
consistent).
It has been previously reported for a similar reaction dealing
ϩ
1
with artemisinin that the trapping of the manganese()–C3Ј
500.13 MHz for H. The signal assignment was made with the
species by borodeuteride resulted in the incorporation of a
help of 2D TOCSY (mixing time 60 ms), 1D NOE selec-
tive excitation, and heteronuclear H– C HMQC-GS long
1
13
deuterium atom at C3Ј in the trans position with respect to the
8b
drug-derived fragment (15C-d, Scheme 6). The quinone-
mediated oxidation of 15C-d can in principle be performed by
removal of HD, producing the porphyrin 15P, or by removal of
range correlation. Mass-spectral analyses were recorded by
ϩ
DCI/NH3 with a Nermag R10-10H spectrometer. Elemental
analyses were performed with a Perkin-Elmer 240 analyzer.
Analytical TLC was performed on Merck precoated silica gel
60F plates. Merck silica gel 60 (230–400 mesh) was used for
column chromatography.
H , producing the porphyrin 15P-d. In the case of the artemisi-
2
nin–tetraphenylporphyrin adduct, the artemisinin fragment was
connected to the macrocycle via a long –(CH ) – arm and did
2
2
not disturb an extensive π-stacking between the chlorin and the
quinone residues allowing a statistic removal of HD and H2,
and thus formation of equimolecular amounts of porphyrin
adducts bearing H or D at C3Ј. In the present case, the drug
fragment was bound to the β-pyrrolic position via a secondary
carbon that belongs itself to a macrocycle. The upper face of
the chlorin was therefore too crowded to accommodate a quin-
one stacking, consequently the removal of HD (lower face) was
favored, leading to the porphyrin 15P as main product.
Trioxane 2
To a stirred solution of 6 (13.741 g, 0.062 mol) in MeOH
(
100 mL) were added at room temperature, 30% H O (13 mL,
2 2
0
.125 mol) and then, 0.5 M H SO in MeOH (65 mL, 0.032
2 4
mol) over 5 min. The solution was stirred for 10 min, neutral-
ized with aqueous saturated NaHCO and concentrated. The
residue was treated with CH Cl (100 mL), decanted and the
aqueous phase was extracted twice with CH Cl (2 × 50 mL).
The combined organic phases were washed two times with
water (2 × 25 mL), dried over MgSO and evaporated to dry-
3
2
2
2
2
The porphyrin adduct 15P has been completely characterized
(
as a mixture 90 : 10 with 15P-d) using different NMR
4
sequences; in particular, NOE correlations were obtained
between C10a (3.53 ppm) and H3Ј (8.82 ppm) on the one hand,
and between C10a (3.53 ppm) and a meso-phenyl, thus confirm-
ing that the alkylation of the porphyrin was obtained by a
C10a-centred radical derived from the drug. It should be men-
tioned that a β-scission from the alkoxy radical 13, Scheme 5,
may also produce a primary alkyl radical centred at C7 by
cleavage of the C3–C7 bond. In fact, this route was not
observed and such a radical was not trapped.
ness to give a bright yellow oil as a mixture of diastereoisomers
(
11.875 g). Yield 46%. Careful purification by chromatography
on silica gel (eluent: petroleum ether–diethyl ether 8 : 2)
gave the major diastereomer 2 that could be obtained analyti-
cally pure after crystallization in hot heptane. One can note
that the other diastereoisomers have not been isolated with a
satisfactory purity to be fully characterized.
Major diastereomer 2
In addition, we were surprised to isolate a compound bearing
a ketone at C6, instead of the corresponding alcohol, from a
reaction mixture containing borohydride. In fact the low
reactivity of a ketone located in the α-position of a spiro-
carbon with respect to sodium borohydride has already been
(
8
Found C, 66.29; H, 8.76. C H O requires C, 66.12; H,
14 22
4
Ϫ1
.72%). ν (cm ): 3410, 2870, 2830, 1442, 1165.
max
1
H NMR for 2 (CDCl , 400 MHz, 298 K): δ, ppm 2.90–2.60
3
(
m, 1H, CH10a), 2.55 (s, 1H, OH), 2.40–1.90 (m, 2H, –CH –),
2
26
1.80–1.00 (m, 18H, –CH –).
2
13
reported.
C NMR for 2 (CDCl , 100.6 MHz, 298 K): δ, ppm 101.9
3
(
C2 or C9), 101.8 (C2 or C9), 72.6 (C5), 42.7(C10a), 35.6, 35.1,
Experimental
31.7, 31.0, 29.2, 26.5, 25.9, 22.5, 22.3, 19.9.
The two other minor diastereomers were not obtained pure
but in a mixture with the major one.
Materials
Tetrahydrofuran (THF) was distilled from sodium–benzo-
phenone ketyl. 4-Dimethylaminopyridine (DMAP) and pyr-
idine were distilled from potassium hydroxide under argon
X-Ray analysis of 2†
X-Ray data collection. Single crystals of the compound were
obtained from n-heptane solution. Data collection was per-
formed at room temperature. Programs used were SHELX97
to solve and refine the structure, and WinGX package for
crystal structure analysis.
prior to use. Hydrogen peroxide, 30% (H O ) was supplied by
2
2
Acros. Dichloromethane (stabilized with amylene) and hexane
were supplied by Fluka, and had a low content of evaporation
residue (≤ 0.0005%). The tetra-n-butylammonium borodeuter-
ide was prepared by reaction of tetra-n-butylammonium chlor-
ide with sodium borodeuteride 98% D. Isotopic purity of
29
30
Crystal data. C H O , M = 254.32, monoclinic, a = 21.864
14
22
4
ϩ
Ϫ
nBu N BD was 97% D (determined by NMR).
(9), b = 6.091 (3), c = 20.852 (9) Å, β = 109.83 (3)Њ, V = 2612 (2)
4
4
3
Å , T = 293 (2) K, space group C2/c (nЊ 15), Z = 8, µ (Mo-K ) =
α
Ϫ1
Antimalarial tests
0.093 mm , 3849 reflections measured, 2486 unique (R
=
int
2
0
.44). The final wR(F ) was 0.154 for all data.
Antimalarial activity against “H” chloroquine-sensitive
2
3,27
strain
of Plasmodium falciparum was determinated by
Trioxane 8
A solution of 2 (180 mg, 0.71 mmol) and DMAP (20 mg,
0.16 mmol) in CH Cl (5 mL) at 0 ЊC was treated with pyridine
(500 mg, 7.25 mmol) and then Ac O (740 mg, 7.25 mmol). After
3
measuring the incorporation of [ H] hypoxanthine by the
28
method of Desjardins. Only slight modifications were intro-
duced: the parasite cultures with an initial parasitaemia of 0.5%
in a 1.8% erythrocyte-suspension were incubated for 48 hours.
2
2
2
completion of the reaction at RT, the reactant was destroyed
with water (1 mL) over 1 h. CH Cl (10 mL) was then added
Instrumentation
2
2
IR spectra were recorded on a Perkin-Elmer 841 spectrometer.
†
CCDC reference number 206129. See http://www.rsc.org/suppdata/
1
13
H and C NMR spectra of compounds 2, 6, and 7 were meas-
ob/b3/b302835f/ for crystallographic data in .cif or other electronic
format.
1
ured with a Bruker ARX 400 (400 MHz and 100.6 MHz, for H
2
862
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 5 9 – 2 8 6 4

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and the reaction mixture was washed twice with 1 M aqueous
In order to get the single adduct 15P, the mixture 15C ϩ 15P
HCl (2 × 5 mL) and then with saturated aqueous NaHCO (2 ×
was oxidized by 2,3-dichloro-5,6-dicyanobenzoquinone in
refluxing dichloromethane for 20 min. The organic layer was
washed three times with aqueous NaOH (pH 8), and dried with
sodium sulfate. The adduct 15P was purified by chromato-
graphy on neutral alumina grade II–III, with ethyl acetate–
hexane 20 : 80 v/v as eluent.
3
5
mL). The organic layer was dried over MgSO and evaporated
4
to dryness to give an oil (225 mg) which was purified by flash
chromatography on silica gel (eluent: petroleum ether–diethyl
ether 7 : 3). Acetate 8 (152 mg) was obtained as an oil. Yield 72%.
(
Found C, 64.76; H, 8.06. C H O requires C, 64.87; H,
16 24 5
Ϫ1
1
8
.11%). νmax (cm ): 2938, 2865, 1770, 1214, 1196, 1173, 1091,
H NMR for 15P (CD Cl , 250.13 MHz, 298 K): δ, ppm
2
2
3
1
022.
8.92, 8.89, 8.79, 8.75, 8.69, and 8.41 (6 × d, 6 × 1H, J = 5 Hz,
β-pyrrolic-H), 8.86 (br s, 1H, H3Ј), 8.27–8.20 (8H, phenyl-H),
7.85–7.75 (12H, phenyl-H), 3.63 (H10a), 2.63–1.20 (unresolved
1
H NMR for 8 (CDCl , 400 MHz, 298 K): δ, ppm 2.80–2.45
3
(
m, 1H, CH10a), 2.40–2.05 (m, 1H), 2.00 (s, 3H, COCH ),
3
1
.90–1.00 (m, 18H).
m, 20H, –CH –), Ϫ2.86, and Ϫ2.67 (2 × br s, 2 × 1H, NH). For
full NMR characterization of 15P, see next paragraph.
2
13
C NMR for 8 (CDCl , 100.6 MHz, 298 K): δ, ppm 167.4
3
(
3
CO), 106.6 (C6), 102.4 (C3), 71.7 (C5), 42.7 (C10a), 35.7, 35.2,
2.5, 31.5, 29.1, 26.3, 25.9, 25.1, 22.1, 21.7 (CH CO), 18.6.
3
Alkylation of Mn(TPP) by 2 in the presence of borodeuteride
Trioxane 9

Mn (TPP)Cl (37.5 mg, 53.3 µmol, 1 equiv.) and 2 (40 mg, 157.5
To a solution of t-BuOK (440 mg, 3.92 mmol) in anhydrous
THF (6 mL) at Ϫ78 ЊC under argon, was added 2 (345 mg,
µmol, 3 equiv.) were dissolved in dichloromethane (5.5 mL).
This solution was degassed and kept under an argon atmos-
phere. Tetra-n-butylammonium borodeuteride (130 mg, 498
µmol, 9 equiv.) was added as a solid. The mixture was stirred at
room temperature for 2 h. A degassed solution of cadmium()
nitrate tetrahydrate (300 mg, 972 µmol, 18 equiv.) in DMF
(2 mL) was added and stirring was continued for 15 min. Aque-
ous acetic acid (10 vol%, 10 mL) and dichloromethane (20 mL)
were added under air. The organic solution was washed with
water (5 times), and dried on sodium sulfate. As observed when
borohydride was used as reductant, UV–vis analysis and TLC
indicated that the chlorin adduct was the major one. The
dichloromethane solution (3 mL) of adducts was treated with
2,3-dichloro-5,6-dicyanobenzoquinone (117 mg, 515 µmol, 10
equiv.), at reflux for 40 min. The organic layer was washed four
times with aqueous NaOH (pH 8), and dried with sodium sul-
fate. Purification was obtained by chromatography on neutral
alumina grade II–III, with ethyl acetate–hexane from 5 : 95 to
30 : 70 v/v as eluent. The solvents were eliminated under vacuum.
Yield = 8 mg (18% with respect to starting Mn(TPP)Cl).
1
.35 mmol). One hour after the solubilisation of the substrate,
MeI (300 µL, 4.81 mmol) was added and the reaction left over-
night at RT. Aqueous 5% NaHCO (10 mL) was added and the
3
mixture was extracted twice with CH Cl (2 × 15 mL). The
2
2
combined organic layers were washed with water (10 mL), dried
over MgSO and evaporated to dryness to give an oil (330 mg).
4
Purification by flash chromatography on silica gel (eluent: pet-
roleum ether–diethyl ether 95 : 5) gave 9 (102 mg) and unreacted
2
(215 mg). Yield not optimized (28%). An analytically pure
sample of 9 was obtained by vacuum microdistillation.
Found C, 67.26; H, 9.05. C H O requires C, 67.14; H,
(
15
24
4
Ϫ1
9
.01%). ν (cm ): 2938, 2865, 1447, 1089, 1031.
max
ϩ ϩ ϩ
IC (NH ) 286 (25%, MNH ), 269 (100%, MHϩ).
4
4
1
H NMR for 9 (CDCl , 400 MHz, 298 K): δ, ppm 3.32 (s,
3
3
0
H), 2.65–2.40 (m, 1H), 2.35–2.05 (m, 1H), 1.95–1.10 (m, 17H),
.90–0.70 (m, 2H).
13
C NMR for 9 (CDCl , 100.6 MHz, 298 K): δ, ppm 104.2,
3
1
2
01.8, 72.9, 49.5, 37.6, 35.6, 35.1, 31.0, 29.1, 26.4, 25.9, 24.0,
2.5, 21.8, 19.9.
UV–vis in CH
Cl : λmax (relative intensity) 420 (100), 518
2
2
CAUTION: While organic peroxides are potentially
explosive compounds and must be handled with safety, these
trioxanes were particularly stable.
(5.6), 552 (2.8), 596 (2.3), 648 (2).
ϩ
MS (DCI/NH ): m/z (relative intensity) 850 (10), 851 (100,
3
ϩ ϩ
MH for 15P), 852 (84, MH for 15P-d), 853 (43), 854 (25), 855
16). Proportion of 15P-d = 14%, calculated from m/z = 851 and
52 (taking also in account the isotopic contribution of 15P in
the peak at m/z = 852).
(
8
Alkylation of Mn(TPP) by 2 in the presence of borohydride


Mn (TPP)Cl (18 mg, 25.6 µmol, 1 equiv.) and 2 (20 mg, 78.7
µmol, 3 equiv.) were dissolved in dichloromethane (3 mL). This
solution was degassed and kept under an argon atmosphere.
Tetra-n-butylammonium borohydride (78 mg, 303 µmol, 12
equiv.) was added as a solid. The mixture was stirred at room
temperature. After 2 h, the manganese() macrocyclic complex
was demetallated in situ. For this purpose, a degassed solution
of cadmium() nitrate tetrahydrate (212 mg, 687 µmol, 27
equiv.) in DMF (1.8 mL) was added and stirring was continued
for 10 min in order to allow the transmetallation of the com-
plex from manganese() to cadmium(). Dichloromethane
Ϫ1
Ϫ1
IR (KBr pellet): νCO = 1729 cm (band width = 20 cm ); IR
Ϫ1
(
dichloromethane): νCO = 1725, and 1719 cm (poorly defined).
NMR for 15P ϩ 15P-d. For clarity, the signals of the por-
phyrin moiety are described first, and then that of the drug
fragment (see Scheme 5 for the numbering of carbon atoms).
1
H NMR for 15P ϩ 15P-d (CD Cl , 500.13 MHz, 298 K):
2
2
δ, ppm 8.90, 8.87, 8.79, 8.76, 8.69, and 8.39 (6 × d, 6 × 1H,
3
J = 5 Hz, β-pyrrolic-H), 8.82 (br s, 0.9H, H3Ј), 8.25–8.20
(
(
8H, Phenyl-H), 7.83–7.74 (12H, Phenyl-H), Ϫ2.79, and Ϫ2.59
2 × br s, 2 × 1H, NH), 3.53 (m, 1H, H10a), 1.86 (2H, H C10),
2
1
.34 and 1.25 (H C9), 1.90 and 1.50 (H C8), 2.49 and 2.22
(
10 mL) was then added under air, and this solution was treated
2
2
(
H C7), 2.46 and 1.66 (H C12), 2.02 and 1.99 (H C11), 2.44 and
2 2 2
with aqueous acetic acid (10 vol%, 10 mL) to demetallate the
cadmium() complex. The organic layer was washed with 1 M
sodium acetate (3 times), and dried on sodium sulfate. Purifi-
cation on a column of neutral alumina (grade II–III), eluted
with a dichloromethane–hexane mixture (from 50 : 50 to 80 : 20
v/v) afforded a mixture of the chlorin and porphyrin adducts
1
2
.25 (H C13), 1.88 and 1.67 (H C14), 2.12 and 1.66 (H C15),
2 2 2
.59 and 2.34 (H C16). The chlorin adduct 15C was present as
2
contaminant [10 mol%, Ϫ1.41 ppm (NH)].
13
C NMR for 15P ϩ 15P-d (CD Cl , 125.7 MHz, 298 K):
2
2
δ, ppm 150.9 (C2Ј), 34.9 (C10a), 32.8 (C10), 25.9 (C9), 22.9
C8), 37.1 (C7), 174.3 (C3), 86.4 (C5), 26.3 (C12), 30.7 (C11),
7.1 (C13), 21.4 (C14), 29.4 (C15), 39.3 (C16), 209.3 (C6).
(
3
1
5C and 15P, respectively.
Analysis of the mixture 15C ؉ 15P. TLC (SiO , ethyl acetate–
2
hexane 30 : 70 v/v): R 0.48 (15C, major), 0.42 (15P, minor).
f
UV–vis in CH Cl : λ (relative intensity) 372 (14), 420
2
2
max
Acknowledgements
(
100), 518 (6), 546 (4), 598 (2), 652 (11).
ϩ
ϩ
MS (DCI/NH ): m/z (relative intensity) 851 (19, MH for
Dr Yannick Coppel, from the LCC-CNRS, is gratefully
acknowledged for accurate NMR characterization of the
covalent adduct 15P.
3
ϩ
1
(
5P), 852 (18), 853 (100, MH for 15C), 854 (63), 855 (23), 856
6). Proportion of 15P = 19/(100 ϩ 19) × 100 = 16%.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 5 9 – 2 8 6 4
2863

View Article Online
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O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 5 9 – 2 8 6 4