Note: Descriptions are shown in the official language in which they were submitted.
CA 02414089 2002-12-02
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IMPROVEMENTS IN AND RELATING TO CHROMOPHORES
The present invention relates to novel porphyrin and porphyrin-based
chromophores and sets of porphyrin and porphyrin-based chromophores, which may
be
particularly useful in a range of photodynamic applications, including
photochemotherapy and fluorescence analysis and imaging.
The importance of porphyrin and porphyrin-based chromophores both as research
tools, for example in fluorescence-activated cell sorting (FACS), and as
therapeutic
agents in photodynamic therapy (PDT) fc5i- bringing about the death of
targeted cells in
vivo, is widely recognised in the art. Each of these applications is dependent
on the
ability of the chromophore to be excited by incident light to a singlet
excited state, and to
decay to a lower energy state with the consequent emission of energy. This
energy may
be emitted in the form of fluorescent light at a specific wavelength, thereby
enabling a
cell or biostructure attached to the decaying chromophore to be visualised,
and/or sorted
by FACS. Alternatively, the energy of excitation may be dissipated by initial
conversion
of the singlet chromophore into the triplet excited state, followed by the
transfer of
energy to another triplet such as dioxygen, with the consequent formation of
singlet
oxygen. Singlet oxygen is a powerful cytotoxic agent, and hence where this
latter process
occurs in or in the immediate vicinity of a cell, it will usually result in
the death of that
cell. Accordingly, the chromophore can be exploited both for its fluorescent
properties,
and for its ability to act as a photosensitiser.
Evidently, for the purposes of fluorescence imaging or analysis, or PDT, some
degree of control over the localisation of the chromophore in vitro or in vivo
is a
prerequisite. This is particularly important in photodynamic therapy, as the
typical sphere
of radiation of singlet oxygen produced by decay of a chromophore is no more
than
0. I L~m in diameter, so that in order to bring about the death of a target
cell, the
chromophore must usually be positioned immediately alongside, or preferably
within,
that cell.
Hitherto, however, few attempts have been made to control the targeting of
porphyrin photosensitisers to particular target cells in vivo for the purposes
of PDT.
Instead, reliance has typically been placed on the inherent tendency of
porphyrins to
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accumulate in tumours in the absence of lymphatic drainage from tumour
structures.
Phototi-inOO , a photosensitising agent comprising a mixture of porphyrin
structures
derived from hematoporphyrin-IX by treatment with acids which is commercially
used in
the treatment of carcinomas and sarcomas, is, for example, conventionally
administered
systemically to patients without any targeting vehicle or means. This is
evidently
undesirable, as incorrect localisation of the photosensitiser will not only
decrease the
efficiency of the photochemotherapy, but may also result in the death of
healthy cells.
Efforts have been made to achieve the specific attachment of chromophores to
biological targets in vitro, in particular for the purposes of FACS and
fluorescence
imaging, by covalently conjugating the chromophores to suitable protein
delivery
molecules. This approach has however been subject to various problems.
Firstly, the
degree of background fluorescence caused by non-specific binding of porphyrin
chromophores to cell surfaces has proved difficult to reduce. Secondly, it has
been found
that the attachment of a chromophore to a protein molecule can result in a
significant
decree of excited state quenching by the proximate protein, which will clearly
reduce the
efficacy of the chromophore as a marker or in targeted photodynamic
applications.
A reduction in these effects remains a desirable objective.
According to one aspect of the present invention therefore, there is provided
a
porphyrin chromophore of formula (I) below:
2~ ~ ~ \
s
1/ N 21 2z H \s
R3 1o Rs
19~ N 24 23
N .~ 11
/16 ~ ~ 12
14
17 1g 13
18 RZ (I)
or a chlorin chromophore of any of formulas (II), (III), (IV), or (V) below:
2
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~2 K "~ 1
3 7 7
2 I \ ~ 8 2 ~ \~ ~ ~2
1 /
1 N21 22H 9 1 NH N- 9
R 21 22
Rs R3 3 ~ / 10 Rs
19~ N24 N~ 110 19 N4 HN 11
18 ~ '1 / / 12
16 / ~ 12 16 14
17 15 14 13 17 15 13
18 Rz R
z
( II) (III)
7 7
2~ 2
~
_ 8 8
1 N21 2zN 9 1 NH N19
H 21 22
R3 ~ H ~ 10 R3 ~ ~ 10 R3
R3
19 N24 N 1 24 N
11 11
1 H
16~ ~ 12 X1 X2 / ~ 12
16 v
17 1~ 14 17 Y 14 13
13 1 5
I
(IV) 18 X2 (V) X1 R1
Rz 2
or a bacteriochlorin chromophore of any of formulas (VI) and (VII) below
7 ~ 7
2 ~ \~ ~ ~2 x1 / /
1 NH N_
21 22 9 1 ' N 21 22 H 9
R3 ~ ~ 1o R3 R3 ~ R3
189 N HN 11 19 N24 N ~10
X4 /14 ~ 12 ~ 16~ ~ 12 x
16
17 15 13 17 15 14 13 3
?C3 R2 18 Rz
4
(VI) (VII)
wherein Rl is an aryl moiety which is linked to a conjugating group Z which is
capable of conjugating the chromophore to a polypeptide molecule for
delivering said
chromophore to a specific biological target in vitro or in vivo; RZ is a
hydrophilic aryl
moiety; R3 is H or a hydrophilic aryl or hydrophilic non-aromatic moiety; and
each of Xl,
3
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Xz, X~ and X:, is independently selected from H, OH, halogen, C1_3 alkyl and
OC1_3 alkyl,
or Xl and XZ andlor X3 and X:~ together form a bridging moiety selected from
O, CH2,
CH C1_3 alkyl, or C(C1_3 alkyl)Z, such that XI and X2 and/or X3 and X~ with
the adjacent
C-C bond form an epoxide or cyclopropanyl structure; wherein each of said Rl,
RZ and R~
is optionally further substituted one or more times by -OH, -CN, -NO2,
halogen, -T or -
OT, where T is a Cl-CI; alkyl, cycloalkyl or aryl group or a hydroxylated,
halogenated,
sulphated, sulphonated or aminated derivative thereof or a carboxylic acid,
ester, ether,
polyether, amide, aldehyde or ketone derivative thereof.
It has been found that the inclusion of one or more hydrophilic substituents
around the core of a chromophore in accordance with the invention results in
enhanced
solubility in basic buffer/DMSO or DMF co-solutions which are commonly used in
protein bioconjugation. Increased hydrophilicity also produces a marked
reduction in the
tendency of the chromophore to bind non-covalently to proteins. Where the
chromophore
is to be conjugated to a targeting protein such as a monoclonal antibody for
delivery to
specific cells or tissues, for example for the purposes of PDT or FACS, a
decrease in non-
covalent binding between the chromophore and the protein will reduce the
degree of non-
specific transfer of chromophore to cell surfaces, which will substantially
increase the
accuracy of targeting the chromophore to the cells or tissue of interest.
Accordingly, according to yet another aspect of the present invention there is
provided a method for separating a mixture which comprises one or more
hydrophilic
chromophores each having a hydrophilic or amphiphilic moiety, and a plurality
of less
hydrophilic substances and/or molecules, comprising the step of introducing
said mixture
to a hydrophobic eluting solvent, and passing said mixture and said eluting
solvent over a
hydrophilic or polar solid phase, such that said one or more chromophores are
arrested on
said solid phase whilst said substances and/or molecules are eluted or
substantially eluted
from said solid phase by said eluting solvent.
Said method may, for example, comprise chromatography on a
Sephadex~ (dextran) column, or reverse-phase HPLC. Typically, said mixture of
less
hydrophilic substances and/or molecules may comprise a mixture of cells and/or
membranes. Advantageously, said one or more hydrophilic chromophores include
one or
more chromophores in accordance with the present invention.
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In some embodiments, each or some of Xi- X~ is H. In particularly preferred
embodiments, however, each of Xl - X4 is OH. Accordingly, said chromophore may
be a
dihydroxychlorin of formula (II), (III), (IV) or (~ above or a
tetrahydroxybacteriochlorin
of formula (VI) or (VII) above. The hydrophilicity of dihydroxychlorins and
tetrahydroxybacteriochlorins is found to be greater than that of the
corresponding
porphyrins, owing to the presence of extra hydrophilic hydroxy groups around
the core of
the chromophore.
Preferably, said aryl moiety Rl may comprise a phenyl ring, which phenyl ring
may preferably be linked by a single bond to the macrocyclic core of said
chromophore
or may alternatively be linked thereto by a C1.6 branched or linear alkyl
chain.
Advantageously, said conjugating group Z may be linked to said phenyl ring at
the para
(4') position thereof.
Said conjugating group Z may comprise a group which is capable of bonding
covalently to an amine group on a polypeptide molecule; such as an isocyanate,
isothiocyanate, or NHS ester group. Advantageously, therefore, each of the
meso
substituents around said porphyrin, chlorin or bacteriochlorin should comprise
no NH-,
NH~, -NHZ+- or -NH3+ groups which could become covalently bonded to said
conjugating
group Z. This will serve to reduce the probability of internal crass-linkage
within said
chromophore. Said conjugating group Z may alternatively comprise any other
protein
conjugating group, such as -NH2, -NH(Cl-s alkyl), maleamide, iodoacetamide,
ketone or
aldehyde. Methods for achieving the conjugation of such groups to protein
molecules are
known in the art.
In especially preferred embodiments, said conjugating group Z comprises an
isothiocyanato group. Isothiocyanates react readily with lysine residues to
produce a
stable linkage to proteins, and hence are particularly suitable for
bioconjugation of
chromophores in accordance with the invention.
Said conjugating group Z may be linked directly to said aryl moiety RI by a
single
bond. Alternatively, said conjugating group Z may be linked to said aryl
moiety Rl by a
linking moiety having a relatively high degree of inflexibility and/or steric
hindrance.
Said linking moiety may, for example, comprise a chain of fused or linked
cycloalkyl
and/or cycloaryl ring structures having a total molecular weight no greater
than
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1000gmo1-1. In particular, said linking moiety may comprise an anthracene,
acridine,
anthranil, naphthyl or naphthalene moiety, or a polyacetylene,
phenylacetylene, or
polyphenylacetylene moiety. When said chromophore is conjugated by said
conjugating
group Z to a polypeptide molecule, therefore, said linking moiety can serve to
keep the
photoactive core of said chromophore apart from said polypeptide, thereby
helping to
reduce the degree of fluorescence quenching which may be caused by said
polypeptide
when said chromophore is caused to fluoresce. Said linking moiety may include
a
hydrophilic or amphiphilic moiety of the kind described above, such as a C2-
C3~
polyethylene glycol moiety. This will help to ensure that the hydrophilicity
of the
chromophore is not impaired by the presence of said linking moiety.
Optionally, said aryl moiety Rl may be further substituted by one or more
hydrophilic substituents, such as hydroxy, which will serve to improve the
hydrophilicity
of said chromophore.
Said hydrophilic aryl moiety R2 may comprise a phenyl ring, which phenyl ring
may be substituted one or more times, preferably at least two times, by one or
more
hydrophilic substituents which serve to increase the hydrophilicity of said
aryl moiety R2.
Said phenyl ring may preferably be linked by a single bond to the macrocyclic
core of
said chromophore or may alternatively be linked thereto by a Ci-s branched or
linear alkyl
chain. Alternatively, said hydrophilic aryl moiety R2 may comprise a
heteroaryl ring,
such as a pyridyl or quaternised pyridyl (pyridiniumyl) ring, which heteroaryl
ring may
be substituted one or more times, preferably at least two times, by one or
more
hydrophilic substituents which serve to increase the hydrophilicity of said
aryl moiety Rz.
Said heteroaryl ring may preferably be linked by a single bond to the
macrocyclic core of
said chromophore or may alternatively be linked thereto by a C1_s branched or
linear alkyl
chain. Said one or more hydrophilic substituents may advantageously be
selected from
hydroxy; alkoxy such as methoxy or ethoxy; CZ-C15 polyethylene glycol;
quatenised
pyridyl (pyridiniumyl) such as N-methylpyridiniumyl; mono-, di- or poly-
saccharide; Ci_
~alkylsulfonatea phosphonium group R~P(RS)(R6)(R~), wherein R~ is a single
bond or
C1_G alkyl, and each of R5, R~ and R~ is independently selected from hydrogen,
an aryl
ring such as a phenyl ring, a heteroaryl ring such as a pyridyl ring, and a
C1_6 alkyl chain,
which aryl ring, heteroaryl ring or Ci_s alkyl chain is unsubstituted or is
substituted one or
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more times by hydroxy, C1_~ alkyl or alkoxy, aryl, oxo, halogen, nitro, amino
or cyano; or
a phosphate or phosphonate group RgOP(O)(OR9)(ORI~) or RgP(O)(OR9)(ORI~)
respectively, wherein Rg is a single bond or Cl_~ alkyl, and each of R9 and
Rl~ is
independently selected from hydrogen and C1_6 alkyl. Preferably, each of said
R5, Rs and
R~ may be the same, and may advantageously be unsubstituted phenyl. Suitably,
said Rg
may be methyl. Advantageously, said R9 and said Rl~ may be the same, and/or
may be
methyl or ethyl.
In especially preferred embodiments, said hydrophilic aryl moiety RZ is
selected
from m,m-(dihydroxy)phenyl
HO OH
or a PEGylated derivative thereof; m,m,p-(trihydroxy)phenyl
HO ~ OH
OH
or a PEGylated derivative thereof; o,p,o-(trihydroxy)phenyl
HO ~ OH
OH
or a PEGylated derivative thereof; m- or p-((C1_
r)allcyltriphenylphosphonium)phenyl such as p-
(methyltriphenylphosphonium)phenyl
i
PPh3
m- or p-(C1_6alkylphosphono-di-alkoxy)phenyl such as p-methylphosphono-di-
ethoxy)phenyl
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O~ ~O
,P~
Et0 OEt
m- or p-(C1_~alkylphosphonato-di-alkoxy)phenyl such as p-methylphosphonato-
di-ethoxy)phenyl
i
0
~P~
Et0 OEt
m- or p-(N-methyl-pyridiniumyl)phenyl
(
i w
Me
and meta- or para- sugar-substituted phenyl such as pentose-, hexose- or
disaccharide-substituted phenyl
OH OH
O
OH OH
In other preferred embodiments, said hydrophilic aryl moiety R2 comprises a
quaternised pyridyl (pyridiniumyl) group such as a p-N-(C1_6alkyl)pyridiniumyl
group or
m-N-(C1_calkyl)pyridiniumyl group. Quaternised pyridyl (pyridiniumyl) groups
are
highly hydrophilic and display advantageous properties when incorporated into
chromophores in accordance with the invention. Particularly preferred groups
in this
regard are m- or p-N-((C1_6)alkyl)pyridiniumyl, such as m-N-methylpyridiniumyl
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i N~
CH3
or p-N-methylpyridiniumyl
~+
CH3
In other especially preferred embodiments, said quaternised pyridiniumyl group
may comprise a zwitterionic group, such as p-N-
(C1_6alkylsulfonate)pyridiniumyl or m-
N-(Cl_~alkylsulfonate)pyridiniumyl; in particular, p-N-
(propylsulfonate)pyridiniumyl
w N~S03
or m-N-(propylsulfonate)pyridiniumyl
~ N~\S03
Preferably, the or each quaternised pyridiniumyl group R2 may be associated
with
a halide counterion, such as an iodide counterion .or, in most preferred
embodiments, a
chloride counterion.
In some advantageous embodiments, R3 is H, such that said chromophore
constitutes a S,15-diaryl-porphyrin, -chlorin or -bacteriochlorin. In other
advantageous
embodiments, said R3 is a hydrophilic aryl or non-aromatic moiety. For
example, said R3
may comprise a hydrophilic aryl moiety as defined above in relation to R2.
Said
hydrophilic aryl moiety R3 may be the same as said hydrophilic aryl moiety R2,
such that
the chromophore possesses the same substituents at the 10, 15 and 20 positions
thereof;
or may be different from said hydrophilic aryl moiety R2. Alternatively, said
R3 may
comprise a hydrophilic alkyl moiety, such as a C1_6 alkyl chain which is
substituted one or
more times by one or more hydrophilic substituents such as hydroxy or Cz-is
polyethylene
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glycol. In particularly preferred embodiments, said R3 comprises
polyhydroxy(C1_6 alkyl),
such as 1,2-dihydroxyethyl.
Chromophores in accordance with the invention wherein RZ is the same as R3 may
be synthesised in accordance with methods known in the art, for example by
acid
catalysed condensation of benzaldehydes with pyrrole, or by means of the
"MacDonald
2+3" method for synthesising porphyrins from dipyrromethanes (Arsenault et al,
J.
Chen~. 'foc. 1960, 82:4384-4389 - incorporated herein by reference) .
A generalised scheme for the synthesis of 5-isothiocyanatophenyl-15-pyridinium
porphyrins, chlorins and bacteriochlorins in accordance with the present
invention is set
out as Scheme 1 below, in which "RX" represents a quaternising group such as
C1_6 alkyl
or a hydrophilic substituent as defined above in relation to formulas (I) to
(VII):
(i) OsQi/pyridine
(ii) Hzs (i) osC7~/pyridine
(ii) HzS
(i) Piperidine (i) Piperidine (i) Piperidine
(ii) TDP (ii) TDP (ii) TDP
(iii) RX (iii) RX
(iii) RX
r r
r
R
R
Scheme 1
to
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A generalised scheme for the synthesis of .5-isothiocyanatophenyl-15-
methylphosphoniumphenyl porphyrins, chlorins and bacteriochlorins in
accordance with
the present invention is set out as Scheme 2 below, wherein R represents
hydrogen, C1-s
alkyl, a heterocyclic group or an aromatic group
(i) Os04/pyridine
(ii) HAS (i) OsOqIpYridine
(ii) H~.S
r- >
HO
(i) CBr~PPh3 (i) CBr4IPPh3 (i) CBrqIPPh3
(ii) PR3 ~ (ii) PR3 ~ (i7 PRg
y (iii) TMSI (iii) TMSI (iii) TMSI
r"~ rno ~ (iv) TDP 1' (iv) TDP
rr~3
Scheme 2
HO
Porphyrin, chlorin and bacteriochlorin chromophores in accordance with the
present invention wherein said R2 and optionally said R3 comprises
pyridiniumylphenyl
may be synthesised in accordance with the generalised reaction scheme set out
below as
11
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Scheme 3, wherein "R" represents hydrogen or one or more hydrophilic
substituents as
defined above in relation to formulas (I) to (VII):
Scheme 3
Porphyrin, chlorin and bacteriochlorin chromophores in accordance with the
present invention wherein said RZ and optionally said R3 comprise
alkylphosphonatophenyl or alkylphosphonophenyl may be synthesised in
accordance
12
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with the generalised reaction scheme set out below as Scheme 4, wherein "R"
represents
OH, ONa, or O(C~_~ alkyl):
LiAIH4
Me
COOMe 10~o~q~rrn3
(ii) P(OEt)3
(oi) NaOHaq or TMSBr
(i) CIP(O)(OEt)Z
(ii) NaOHa9
Scheme 4
In a further aspect of the invention, there is provided a novel method for the
synthesis of a 5,10,15,20-tetra-meso-substituted porphyrin, chlorin or
bacteriochlorin
chromophore having selected substituents at the 5, 10, 15 and 20 meso-
positions thereof;
comprising the steps of providing a 5,15-di-meso-substituted porphyrin,
chlorin or
bacteriochlorin chromophore; attaching a leaving group Q to the 10 and 20 meso-
positions of said chromophore, which leaving group Q is selected from halide
and triflate;
providing a coupling reagent (R110)(R120)BR13, wherein Rn and R12 are
independently
selected from H or C1_6 alkyl, or Rll and R12 together constitute a C1_6 alkyl
chain
bridging said two O atoms, and R13 is vinyl or aryl, such as a hydrophilic
aryl moiety as
IJ
CA 02414089 2002-12-02
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hereinbefore defined in relation to R3; and reacting said chromophore with
said coupling
reagent in the presence of a base selected from potassium phosphate, sodium
phosphate,
caesium carbonate and barium hydroxide, and a Pd~ catalyst; such that said R13
replaces
said leaving group Q at the 10- and 20- meso positions of said chromophore.
Pdo-catalysed Suzuki coupling reactions using boronic acid or boronic ester
reagents are known in the art, and are described for example in Miyaura &
Suzuki,
Palladium-catalyzed cross-coupling reactions of organoboron compounds, Chern.
Rev.
( 1995) 95:2457-2483; the disclosure of which is incorporated herein by
reference.
Hitherto, however, attempts to carry out Suzuki-coupling at the meso-positions
of
porphyrins, chlorins or bacteriochlorins, as a means of importing selected
substituents
onto said meso-positions, have failed. The present inventors have found
however that
under the reaction conditions of the invention, Suzuki-coupling proceeds
rapidly and
successfully at the 10- and 20- meso-positions of the starting porphyrin,
chlorin or
bacteriochlorin chromophore. This method thereby enables convenient synthesis
of tetra-
meso-substituted porphyrins, chlorins or bacteriochlorins by Suzuki-coupling.
Said leaving group Q may be chloride, bromide, iodide or triflate
(trifluoromethanesulfonate). Suitably, said leaving group Q may be bromide.
Methods for
the meso-bromination of di-meso-substituted porphrins, chlorins or
bacteriochlorins are
known in the art. For example, said 5,15-di-meso-substituted porphyrin,
chlorin or
bacteriochlorin chromophore may be halogenated at the 10- and 20- meso-
positions
thereof by way of reaction with halosuccinimide, such as bromosuccinimide.
Said coupling reagent may comprise a boronic ester or a boronic acid. In
preferred
embodiments, each of said Rl1 and R12 is H, such that said coupling reagent is
a boronic
acid.
Advantageously, said 5,15-di-meso-substituted porphyrin, chlorin or
bacteriochlorin chromophore is a chromophore in accordance with the invention,
or a
protected form thereof. Thus, said 5,15-di-meso-substituted porphyrin, chlorin
or
bacteriochlorin chromophore may be selected from a porphyrin chromophore of
formula
(VIII) below:
14
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3 7
2 ~ ~ ~ ~ $ ,...
1/ N 21 22 H \ 9 .
\2
N 24 23 10
19 i N ~ 11
/16 / ~ 12
17 4
15 13
18 RS
( VIII)
or a chlorin chromophore of any of formulas (IX), (X), (XI), and (XII) below:
K ,.4
3 7 7
X1 2 ~ ~ ~ $ 2 \ \~ ~ X2
1 N21 22H ,9 21 NH N-'s
2 21 22
/ 10
19~ N24 N~ 110 1 ~ HN 11
18 ~ / ~ 12
/ ~ 12 16 14
16 15 13
17 15 14 13 17
18 RS Rs
SIX) (X)
"4 ..4
7 7
2
1 N 21 22 H ~ $ , 1~ N H N - 9 s
2 2 21 22
H 23 / 10 ~ 24 / 10
19 N24 N 11 19 i HN 11
18
14 12 X1 X2 16 / ~ 12
v
17 15 13 17 15~ 14 13
(~) 18 RS X2 (~I) X1 R5
or a bacteriochlorin chromophore of any of formulas (XIII) and (XIV) below:
IJ
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Ra 1 ~ Ra
7 7
2 ~ ~~ ~ 8 X2 X1 _~ ~ ~ 8
1 NH N19 1 N21 22N 9
2 21 22 2 H
24 / 10 H / 10
1 ~ HN 11 19 N24 N 11
1s
X4 17 16 / ~ 12 ~ ~ 12
16
1Y 14 13 17 15 14 13
)(3 5'RS 18 RS )(4
(XIII) (XIV)
wherein R4 is a group Rl as defined above in relation to formulas (I) to (VII)
or a
protected form thereof or a group convertible thereto; R5 is a group Rz as
defined above
in relation to formulas (I) to (VII) or a protected form thereof or a group
convertible
thereto; and each of Xl, Xz, X3 and X4 is independently selected from H, OH,
halogen,
C1_~ alkyl and OC1_3 alkyl, or Xl and Xz and/or X3 and X4 together form a
bridging
moiety selected from 0, CHz, CH C1_3 alkyl, or C(C1_3 alkyl)z, such that Xl
and Xz and/or
X~ and X4 with the adjacent C-C bond form an epoxide or cyclopropanyl
structure.
Accordingly, where R13 is a hydrophilic aryl substituent as defined above in
relation to R3, said 5,10,15,20-tetra-meso-substituted porphyrin, chlorin or
bacteriochlorin chromophore may also constitute a chromophore in accordance
with the
present invention.
Said Pd~ catalyst may, for example, comprise Pd(PPh3)4, PdClz(PPh3)z, or
Pd(OAc)z. Advantageously, said Pd~ catalyst may comprise Pd(PPh3)4.
Said coupling reaction is performed in a solvent, which may be selected from
toluene or dry THF. It is found that the coupling reaction proceeds swiftly in
dry THF,
and so dry THF is preferred as solvent.
Optionally, where said R13 is vinyl, said 5,10,15,20-tetra-meso-substituted
porphyrin, chlorin or bacteriochlorin chromophore may be subjected following
said
coupling reaction to an osmylation reaction utilising Os04, such as to convert
said 10-
and 20- vinyl substituents to hydroxyalkyl. Said osmylation reaction may be
carried out
under conditions identical to those suitable for converting a porphyrin to a
di-beta-
hydroxy-chlorin and then to a tetra-beta-hydroxy-bacteriochlorin. It is noted
that this step
16
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may be performed in accordance with the invention on 5,10(vinyl),15,20(vinyl)-
meso-
substituted porphyrin, chlorin or bacteriochlorin chromophores which are
obtained
otherwise than in accordance with the method of the invention, such as by way
of Pd-
catalysed Stifle coupling performed on said 5,15-di-meso-substituted
chromophore in
accordance with the method described in DiMagno et al, ,l. Ong. Chena.
1993:58, 5983-
5993, (incorporated herein by reference) wherein vinyl tributyl tin is used as
a coupling
reagent.
Where said tetra-meso-substituted chromophore is a porphyrin or a chlorin'
chromophore, said chromophore may be respectively converted to a chlorin or
bacteriochlorin chromophore or to a bacteriochlorin chromophore in accordance
with
methods known to the man skilled in the art. For example, said porphyrin or
chlorin
chromophore may be osmylated by way of reaction with OsOd, such as to produce
a di-
beta-hydroxy-chlorin or a tetra-beta-hydroxy-bacteriochlorin.
Generalised schemes for reactions in accordance with the present invention are
set
out in Schemes 5 and 6 below. In Scheme 5, "R" and "Rl" each represents one or
more
hydrophilic substituents as defined above in relation to Rz and R3
respectively. In Scheme
G, "R" represents one or more hydrophilic substituents as defined above in
relation to RZ,
and "X" represents a carbon or nitrogen atom.
(i) Pd(PPh3)a~IC3P04
R~
(ii)
R~ \ / B(OH)2
R
R
Scheme 5
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(i) OsO~/pyridine (3 ec OsO~/pyridine (4e~
HzS
HC (ii) HzS HC
R R R
(i) OsO~/pyridine (2eq)
(i) piperidine ~ (ii) I-i~S ~ (i) piperidine
(ii)TDP (iii) piperidine (ii)TDP
1
'I (iv)TDP
HC
HC
K R
Scheme 6
According to another aspect of the present invention, there is provided a 5,15-
diphenylporphyrin, 5,15-diphenylchlorin or 5,15-diphenylbacteriochlorin
chromophore,
wherein each of the ortho-, meta-, and/or para- positions of each of the 5-
and 15- phenyl
groups is substituted by a substituent P1-PS and Q1-Q; respectively which is
independently H or an inert substituent which in combination with the other
substituents
P1-P, and Q1-Q; does not substantially impair the fluorescent properties of
the
chromophore; and the chromophore further comprises a conjugating group Z which
is
capable of conjugating the chromophore to a polypeptide molecule for
delivering said
chromophore to a specific biological target in vitro or in vivo.
Such chromophores are novel, and are each capable on excitation of emitting
fluorescent light at different and substantially non-overlapping wavelengths.
As indicated
above, the provision of conjugating group Z enables a chromophore in
accordance with
the invention to be specifically targetted to a specific biological target,
thus facilitating
is
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control over the localisation of the chromophore in vitro or in vivo.
Chromophores in
accordance with the invention may therefore be usefully employed in
fluorescence
analysis and imaging applications (including FACS), or in PDT.
Advantageously, said fluorochrome is selected from the following compounds:
P3 P3 P3
pz p~ Pz \ p' pz pa
a: \ Pa ( \ ~ ~ \
pt / Ps Pt / ps Pi / ps
Pi / Ps X,
7 7 2
\ \ Xz 2 ~ / / 2 3 \ 7
~_ 8 ~ / 8 X1 ~ ~ 8
t\ NH N-s 7 1 N~1 zzN s 2 1 Nzi zzH s 1/ Nzi zzH s
.t zz ~ \ z
io ~ H ~ to io
t N' HN ~~o is Nza HN it is Nza N 11 is/ N24 N._- 11
t8 ~ '16 /14 ~ 12 ~ ~16 /14 ~ 12 ~ 16\ 14 12 Xi /16 X14 ~ 12
17 15 13 17 15 13~ 17 15 13
t7 15 13 18 18 X2 18
di / ~ Qs Qi ~ ~ Qs Qt ~ ~ Qs Qt / ~ Qs
Qz \ Ds Qz \ Qa Qz \ Qa Qz \ Qa
~7 Q7 Q3 ~.3
P3 P3
Pz \ P4 Pz \ Pa
/ P pt ~ / P
7 7 '1
2 \ \ ~ 8 X. 2 ~ \ \ a X2
i NH N- s 1 NH N
21 22 z zi 22 9
iD
tas N H N it is i4 H N ~ ~o
is
is X14 ~ iz X3 ~4 / .A iz
17 75 13 17 16 15 14 v
Xi X3
Ot ~ ~ Ds 47 ~ ~ Qs
qz Qa Llz Qa
n3 q3 0.,
wherein each of X1, X2, X3 and X4 are as defined above in relation to the
first aspect of
the invention. Optionally, said chromophore may be further substituted at one
or more of
the 2, 3, 7, 8, 12, 13, 17 or 18 positions thereof by a Ci-3 alkyl
substituent.
In the foregoing chemical structures, Z has been omitted for clarity. However,
said Z substituent may be attached to any of the 1-4, 6-14, or 16-20 positions
of each
chromophore, or may be one of the substituents P1-Ps or Q1-Qs, or may be
attached to one
ofthe 5- or 15- phenyl groups through one of said substituents P1-P; or Q1-Qs.
1~
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In some embodiments, each of Pl-P; is the same or substantially the same as
the
corresponding one of QI-Q;, such that said two primary phenyl rings are
symmetrically
substituted. In other embodiments, one or more of P1-P; is not the same as the
corresponding one of Q1-Q;, such that said two primary phenyl rings are not
symmetrically substituted. In particular, all of P1-P; and/or all of Q1-Q; may
comprise H,
such that one or both of said two primary phenyl rings is or are
unsubstituted.
Advantageously, said substituents P1-PS and Q1-Q; collectively provide a
degree
of steric hindrance around the core of said chromophore which is sufficient to
reduce the
rate of spontaneous oxidation of said chromophore, such that said chromophore
is
substantially inert in air, but which does not to a substantial extent inhibit
selective
addition or substitution at the 2, 3, 7, 8, 12, 13, 17 or 18 positions around
the core of said
chromophore. Thus, each of PI, Ps, Q1 and Q; may be H. Typically, the total
cumulative
molecular weight of said substituents P1-P; does not exceed 1000gmol-1, and
the total
cumulative molecular weight of said substituents Q1-Q; does not exceed
1000gmofl.
One or more of said substituents P1-P; and Q1-Q; may comprise -OH, -CN, -NOz,
halogen, -T or-OT, where T is a C1-C1; alkyl, cycloalkyl or aryl group or a
hydroxylated, halogenated, sulphated or aminated derivative thereof or a
carboxylic acid,
ester, ether, polyether, amide, aldehyde or ketone derivative thereof. One or
more of said
substituents P1-P; and Ql-Q; may additionally or alternatively comprise a C3-
Ciz
cycloalkyl and/or aryl ring structures, or between two and six, preferably two
- three,
fused or linked C3-Clz cycloalkyl and/or aryl ring structures, each of which
ring
structures may optionally comprise one or more N, O or S atoms. In particular,
one or
more of said substituents P1-PS and Q1-Q; may comprise a quatenised amine or
pyridyl
group, such as an N-methyl pyridyl (pyridiniumyl) group.
Preferably, one of P1-P; and Q1-Q; is a conjugating substituent which
comprises
said conjugating group Z. In particularly preferred embodiments, said
conjugating
substituent is P3 or Q3, such that said conjugating group Z is provided on the
para-
position of one of the two primary phenyl rings.
Suitably, said conjugating group is as defined above in relation to the first
aspect
of the invention.
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In particular, one or more of said substituents P1-PS and Q1-Q5, not being
said
conjugating substituent, may consist of a member independently selected from
the group
consisting of AlZlAla; wherein Zl is Z2, Z2A5 or Z2ASA6; Al and AS are
independently selected from -(CA2A~)ri , -C(Y)(CA2A;)n-, -C(Y)Y'(CA2A;)ri , -
C(Y)NA4(CA2A~)p , -NA4C(Y)(CA~A;)ri , -NAq.(CA2A;)n, -YC(Y')(CA2A~)ri
and -Y(CA~A~)ri ; n = 0 - 6; Y and Y' are independently O or S; A2, A~ and Aq.
are
independently H or Cl_2 alkyl which is unsubstituted or substituted by one or
more
tluorines; AG = - (C2H40)m or -S(O)p; m = 1 - 12; p = 0 - 2; Z2 is a single
bond or
Z~; Z~ is selected from Zq., ZS and Z6, wherein Z; is unsubstituted or
substituted one or
more times by OH, halo, CN, N02, AlAlO, A6Ag, NAlOAI l, C(Y)AN, C(Y)Y'A~,
Y(CH?)qY'A~, Y(CH2)qA~, C(Y)NAlOAIl, Y(CH2)qC(Y')NAlOAI l~
Y(CH2)qC(Y')A~, NAIpC(Y)NAlOAI l, NAIOC(Y)Al l, NAIOC(Y)Y'A9,
NAIOC(Y)Z~, C(NAlp)NAl0All. C~CN)NA10A11, C(NCN)SA9,
NAIpC(NCN)SAc~, NAIOC(NCN)NAlOAI l, NAlOS(O)2Ag, S(O)rA9,
NAl OC(Y)C(Y')NAlOAI 1, NAIOC(Y)C(Y')Al0 or Z6; q = 0, 1 or 2; r = 0 - 2; A~
is
independently selected from H and A9; Ag is O or A9; A9 is Cl-4 alkyl which is
unsubstituted or substituted by one or more fluorines; Al0 is OA9 or Al l; A11
is A~ or
when Al0 and Al l are as NAlOAI 1 they may together with the nitrogen form a 5
to 7
membered ring comprising only carbon atoms or carbon atoms and at least one
heteroatom selected from O, N and S; Z4 is C6_ 12 aryl or aryloxyC 1 _~alkyl;
ZS is
selected from furanyl, tetrahydrofuranyl, indanyl, indenyl, tetrahydropyranyl,
pyranyl,
thiopyranyl, tetrahydrothiopyranyl, tetrahydrothienyl, thienyl, C;_g
cycloalkyl or Cq.-g
cycloalkyl containing one or two unsaturated bonds, and C~_11 polycycloalkyl;
Z6 is
selected from N-azolyl, dioxadiazinyl, dioxadiazolyl, dioxanyl, 2-N-
dioxatriazinyl,
dioxazinyl, N-dioxazolyl, dioxolyl, dithiadiazinyl, dithiadiazolyl, N-
dithiatriazinyl,
dithiazinyl, N-dithiazolyl, 1-N-imidazolyl, N-morpholinyl, pyrollyl,
tetrazolyl, thiazolyl,
triazolyl, oxazinyl, oxazolyl, naphthydrinyl, oxadiazinyl, oxadiazolyl,
oxatetrazinyl,
oxatriazinyl, oxatriazolyl, oxazinyl, oxazolyl, pentazinyl, phthalazinyl, N-
piperidinyl,
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N,N-piperazinyl, 1-N-pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl,
tetrathiazinyl,
tetrazinyl, 1-N-tetrazolyl, tetroxazinyl, thiadiazinyl, thiadiazoyl,
thiatetrazinyl,
thiatriazinyl, thiatriazolyl, thiazolyl, triazinyl, 1-N-triazolyl,
trioxadazinyl, trioxanyl,
trioxazinyl, trioxazolyl, trithiadiazinyl, trithiazinyl, trithiadiazolyl;
wherein Zq., ZS or Z6
may be fused to one or more other members selected independently from Zq., ZS
and Z6;
AI ~ is hydrogen, methyl, hydroxyl, aryl, halo substituted aryl, aryloxyC l _;
alkyl, halo
substituted aryloxyC 1_; alkyl, indanyl, indenyl, C~_ 11 polycycloalkyl,
tetrahydrofuranyl,
furanyl, tetrahydropyranyl, pyranyl, tetrahydrothienyl, thienyl,
tetrahydrothiopyranyl,
thiopyranyl, C;-( cycloalkyl, or a C4_6 cycloalkyl containing one or two
unsaturated
bonds, wherein the cycloalkyl or heterocyclic moiety is unsubstituted or
substituted by 1
to 3 methyl groups, one ethyl group, or a hydroxyl group.
Said conjugating substituent may consist of a member selected from the group
consisting of A1Z1Z; wherein Z1 is Z2, Z2A5 or Z2ASA6; A1 and AS are
independently
selected from -(CA~A;)ri , -C(Y)(CA2A~)n-, -C(Y)Y'(CA2A~)ri , -
C(Y)NA4(CA~Aj)ri , -NA4C(~')(CA2A3)ri , -NA4(CA2A;)n, -YC(Y')(CA2A3)ri
and -Y(CA~A~)ri ; n = 0 - G; Y and Y' are independently O or S; A2, A3 and A4
are
independently H or C1_2 alkyl which is unsubstituted or substituted by one or
more
fluorines; A~ _ - (C2H40)m or -S(O)p; m = 1 - 12; p = 0 - 2; Z2 is a single
bond or
Z~; Z~ is selected from Zq, ZS and Z6, wherein Z~ is unsubstituted or
substituted one or
more times by OH, halo, CN, NO2, AlAlO, A6Ag, NA10A11, C(Y)AN, C(Y)Y'A~,
Y(CH~)~Y'A~, Y(CH2)qA~, C(Y)NA10A11, Y(CH2)~C(Y')NA10A11~
Y(CH~)~C(Y')Ag, NAIpC(Y)NA10A11, NAlpC(Y)A11, NAlpC(Y)Y'Ag,
NAIOC(Y)Z6, C(NA10)NA10A11~ C~CN)NA10A11, C(NCN)SAg,
NAIOC(NCN)SAg, NAIpC(NCN)NA10A11, NAIpS(O)2Ag, S(O)rAg,
NAIpC(Y)C(Y')NA10A11, NAlpC(Y)C(Y')A10 or Z6; q = 0, 1 or 2; r = 0 - 2; A~ is
independently selected from H and Ag; Ag is O or Ag; Ag is C1_q alkyl which is
unsubstituted or substituted by one or more fluorines; A10 is OAg or Al 1; Al
l is A~ or
when A10 and A11 are as NAlOAI 1 they may together with the nitrogen form a 5
to 7
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membered ring comprising only carbon atoms or carbon atoms and at least one
heteroatom selected from O, N and S; Zq. is C6_12 aryl or aryloxyCl_3alkyl; ZS
is
selected from furanyl, tetrahydrofuranyl, indanyl, indenyl, tetrahydropyranyl,
pyranyl,
thiopyranyl, tetrahydrothiopyranyl, tetrahydrothienyl, thienyl, C;-g
cycloalkyl or Cq._g
cycloalkyl containing one or two unsaturated bonds, and C~_11 polycycloalkyl;
Z6 is
selected from N-azolyl, dioxadiazinyl, dioxadiazolyl, dioxanyl, 2-N-
dioxatriazinyl,
dioxazinyl, N-dioxazolyl, dioxolyl, dithiadiazinyl, dithiadiazolyl, N-
dithiatriazinyl,
dithiazinyl, N-dithiazolyl, 1-N-imidazolyl, N-morpholinyl, pyrollyl,
tetrazolyl, thiazolyl,
triazolyl, oxazinyl, oxazolyl, naphthydrinyl, oxadiazinyl, oxadiazolyl,
oxatetrazinyl,
oxatriazinyl, oxatriazolyl, oxazinyl, oxazolyl, pentazinyl, phthalazinyl, N-
piperidinyl,
N,N-piperazinyl, 1-N-pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl,
tetrathiazinyl,
tetrazinyl, 1-N-tetrazolyl, tetroxazinyl, thiadiazinyl, thiadiazoyl,
thiatetrazinyl,
thiatriazinyl, thiatriazolyl, thiazolyl, triazinyl, 1-N-triazolyl,
trioxadazinyl, trioxanyl,
trioxazinyl, trioxazolyl, trithiadiazinyl, trithiazinyl, trithiadiazolyl;
wherein Zq, ZS or Z6
may be fused to one or more other members selected independently from Z4, ZS
and Z6.
In particular embodiments of the present invention, said chromophore may
comprise a chromophore having a structure set out as (x), (y) or (z) below:
OH
HO
fix) (Y) (z)
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wherein R and R' may be any of the following combinations:
R R'
4-H 4-NC S
'~-IVIe 4-NCS
4-B r 4-NC S
4-CO~Me 4-NCS
3,4,5-tris(OMe)4-NCS
4-NC S 4-OMe
4-NC S 4-Me
4-NCS 4-C02Me
4-NCS 4-Br
4-NC S 4-CN
4-NCS 4-COZMe
In another embodiment of the present invention, said chromophore may comprise
a
porphyrin chromophore having the structure set out below:
s.
2 i yv
1 N --
2 21 22
24 ~ 10
19 N HN 11
18 ~ ~ / ~ 12
16 Y 1q~
1~ 151 13
~\
O~C~NCS
L- J ''P
24
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wherein m = 0-G ; p = 0-15, preferably 0-5; or the corresponding chlorin or
bacteriochlorin chromophore.
According to another aspect of the present invention, there is provided a set
of
fluorochromic markers for multicolour fluorochromic analysis, comprising at
least two
chromophores selected from the group consisting of a porphyrin chromophore, a
chlorin
chromophore and a bacteriochlorin chromophore, each of which chromophores
comprises
the same porphyrin skeleton, each of which chromophores comprises one or more
substituents on said porphyrin skeleton, one of which substituents is a
conjugating
substituent L comprising a conjugating group Z, wherein Z is a conjugating
group
capable of conjugating each of said chromophores to a polypeptide molecule for
delivering each chromophore to one of a plurality of different specific
biological targets.
Preferably, each of the other of said substituents on the skeleton is
independently
H or an inert substituent R which together with said conjugating substituent L
and all of
the other core substituents does not substantially impair the fluorescent
properties of each
chromophore.
It has been found that each of the chromophores in a set in accordance with
the
r~
present invention, on excitation, will emit fluorescent light at a different
discrete
wavelength. Thus, all of the chromophores within the set can be excited by a
single laser,
producing separate emission bands which can be substantially individually
resolved.
Moreover, all of the chromophores provided in said set share substantially the
same
molecular structure, and will accordingly share substantially the same
biochemical and
physicochemical properties, including substantially the same degree of
efficiency of
bioconjugation to a biological target under given conditions. Accordingly, a
set of
chromophores in accordance with the present invention may be usefully employed
in
fluorescence analysis and sorting applications, including FACS, for the
convenient
sorting and analysis of several types of cells or other biological targets.
The components
of such a set may, for example, be introduced to a mixture comprising one or
more of
said different specific biological targets, under conditions which will allow
the delivery
of each chromophore to its respective specific biological target; and said
mixture may be
exposed to light so as to cause said chromophores to fluoresce. A multicolour
analysis
may then be carried out for identifying the different emission bands produced
by each
CA 02414089 2002-12-02
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chromophore, thereby permitting counting and visualisation of the location of
each of the
different biological targets.
Said set of chromophores may in particular comprise two or more of a porphyrin
chromophore in accordance with any aspect of the present invention, the
corresponding
chlorin chromophore, and the corresponding bacteriochlorin chromophore. (By
"corresponding" herein is meant having the same meso-substituents around the
macrocyclic core of the molecule).
In a chromophore in accordance with the present invention, or in each member
of
a chromophore set in accordance with the present invention, said conjugating
group Z
may be conjugated to a binding protein which is adapted to bind specifically
to said
biological target. Alternatively, said conjugating group Z may be conjugated
to a bridging
polypeptide which is adapted to bind to a complementary bridging polypeptide
so as to
couple said chromophore to said complementary bridging polypeptide.
In some embodiments, said bridging polypeptide may be bound to said
complementary bridging polypeptide, and said complementary bridging
polypeptide may
comprise or be coupled to or fused with a binding protein which is adapted to
bind
specifically to said biological target. Accordingly, said chromophore may be
covalently
linked to said binding protein by means of said bridging polypeptide and said
complementary bridging polypeptide.
According to another aspect of the present invention, there is provided a kit
comprising a chromophore in accordance with the present invention or a set of
chromophores in accordance with the present invention, wherein said
chromophore or
each chromophore is conjugated to a bridging polypeptide that is adapted to
bind to a
complementary bridging polypeptide so as to couple the chromophore to said
complementary bridging polypeptide; and a construct or plurality of constructs
each of
which comprises said complementary bridging polypeptide fused or coupled to a
binding
protein which is adapted to bind specifically to said biological target; the
arrangement
being such that said chromophore or each chromophore in the kit is adapted to
bind to a
different construct in the kit with specificity for said specific biological
target, so as to
link said or each chromophore to a binding protein with specificity for said
specific
biological target.
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Said binding protein may, for example, be an antibody such as a monoclonal or
polyclonal antibody or a fragment thereof with specificity for a target
specific molecule
on the surface of said biological target. In particular, said antibody may be
a phage
antibody, that is an antibody expressed on the surface of a bacteriophage.
Alternatively
said binding protein may be a protein which is adapted to bind to one or more
cell surface
molecules or receptors, such as a serum albumin protein. As yet a further
alternative, said
binding protein may comprise a low density lipoprotein, such as a fatty acid
chain, which
is adapted for insertion into a cell membrane. When conjugated to a
chromophore, such a
lipoprotein can serve to anchor the chromophore to a cell membrane.
Said bridging polypeptide may comprise calmodulin, and said complementary
bridging polypeptide may comprise calmodulin binding peptide; or vice versa.
Preferably, however, said bridging polypeptide may comprise avidin or
streptavidin, and
said complementary bridging polypeptide may comprise biotin; or vice versa. In
particular, said or each chromophore in a kit in accordance with the present
invention
may be conjugated to avidin, and said or each construct may comprise a
biotinylated
monoclonal antibody with specificity for a target specific molecule on the
surface of said
biological target. Accordingly, when said avidin-linked chromophore is allowed
to bind
said biotinylated antibody, said chromophore will become firmly linked to said
antibody.
Conveniently, said or each biotinylated monoclonal antibody in the kit may be
selected
and/or readily substituted, so as to enable said or each chromophore to be
delivered to
any desired biological target. Methods for the preparation of monoclonal
antibodies and
for the biotinylation thereof are well known in the art.
According to another aspect of the present invention, there is provided a
method
for attaching a chromophore in accordance with the invention or a set of
chromophores in
accordance with the invention to said specific biological target or targets;
comprising the
steps of providing a kit in accordance with the present invention, and
introducing the
components of said kit into the vicinity of said specific biological target or
targets, under
conditions suitable for enabling the binding of said or each binding protein
to said
specific biological target or targets. Advantageously, the components of said
kit may be
allowed to associate with one another prior to introduction to said target or
targets, so as
to enable the bridging polypeptide conjugated to said or each chromophore to
bind to a
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complementary bridging polypeptide provided on one of said constructs in the
kit. This
will ensure that said or each chromophore in the kit is linked to a binding
protein prior to
introduction of said chromophore to said target or targets. Alternatively, the
components
of said kit may be introduced sequentially to said target or targets.
Typically, said specific biological target may be a cell or a membrane. Said
specific biological target may be in vivo or in vitro (ex vivo). Said
biological target may,
for example, be a cancer cell, a tumour cell, a cell infected with HIV or with
any other
microbe or virus, a cell responsible for detrimental activity in auto-immune
disease, a
foreign or diseased cell, or any other such cell.
In some embodiments of the present invention, said biological target is a cell
in
vitro, and said target specific molecule comprises a molecule exposed on the
surface of
said cell, such as a polypeptide, carbohydrate, fatty acid, lipoprotein,
phospholipid or
other biological molecule. Preferably, said target specific molecule is
specifically
expressed by, or is over-expressed by, said cell. Said target specific
molecule may, for
example, be a T cell marker such as CD4 or CDB. Accordingly, when a
chromophore in
accordance with the present invention or a chromophore forming part of a set
of
chromophores in accordance with the present invention is attached to said
cell, and said
cell is illuminated so as to cause fluorescence of said chromophore, the
fluorescence of
the chromophore will enable said cell to be visualised and counted and/or
sorted by
FRCS.
According to a further aspect of the present invention, therefore, there is
provided
a method for fluorescence-activated sorting of target cells from a mixture of
cells,
comprising the step of attaching to said target cells a chromophore in
accordance with the
invention or a set of chromophores in accordance with the invention,
illuminating said
mixture of cells so as to cause fluorescence of one or more of said
chromophores attached
to said target cells, imparting a charge to the fluorescing cells, and passing
said mixture
of cells through a polarised environment so as to cause or allow said charged
cells to be
separated from said mixture.
According to another aspect of the present invention, there is provided a
method
for the visualisation and/or counting of a plurality of target cells, said
target cells
including cells of two or three different cell types, comprising the steps of
providing a
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chromophore set in accordance with the present invention, which chromophore
set
comprises two or three chromophores each of which is adapted to be delivered
to a
different one of said cell types; attaching said chromophores in the set to
said target cells
in accordance with the method of the present invention; illuminating said
target cells so
as to cause the emission of fluorescence by said chromophores; detecting the
fluorescent
emission bands produced by each of said chromophores; and optionally measuring
for
each of said bands the area under an emission/wavelength curve, so as to
obtain a
measure of the number of fluorescent cells of each respective cell type.
In other embodiments of the present invention, said target cell is a cell in
vivo,
such as a cancer cell, tumour cell, or an infected, foreign or diseased cell,
and said target
specific molecule is a target cell specific molecule which is specifically
expressed by, or
is over-expressed by, or is attached to, and is exposed on, the surface of
said target cell;
such as a target cell specific membrane protein. Accordingly, when a
chromophore in
accordance with the invention is delivered to said target specific molecule,
said
chromophore will be caused to be attached to said cell. If said cell is
subsequently
illuminated with light at a wavelength suitable for causing the excitation of
said
chromophore, said chromophore attached to said cell may be caused to be
excited, and
this may result in the production of singlet oxygen in the immediate vicinity
of said cell,
hence bringing about the death of the cell.
In especially preferred embodiments, said target cell specific molecule
comprises
an internalisation receptor on the surface of said cell, which internalisation
receptor is
capable of binding said binding protein and thereby mediating the
internalisation of said
chromophore within said cell. Accordingly, subsequent illumination of said
cell with
light at a wavelength suitable for causing excitation of said chromophore may
result in
the production of singlet oxygen within said cell, hence bringing about the
death of said
cell.
The present invention therefore comprehends a method for causing the death of
a
target cell, comprising the step of attaching a chromophore in accordance with
the present
invention to said cell and illuminating said cell so as to cause the
production of singlet
oxygen in the vicinity of said cell, thereby causing the death of the cell.
Preferably, said
chromophore is attached to an internalisation receptor on the surface of said
cell, which
29
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internalisation receptor is capable of mediating the internalisation of said
chromophore
within said cell, and said cell is thereafter illuminated such as to cause the
production of
ringlet oxygen within said cell, thereby causing the death of the cell.
Preferably, where said chromophore is adapted to be internalised within the
cell,
said chromophore comprises a cationic group such as a quatenised amine or
pyridyl
(pyridiniumyl) group, or a phosphonium group, so as to promote the
intracellular
accumulation of said chromophore around the mitochondria of the cell, owing to
the net
negative charge on the mitochondria) membrane. This will result in the rapid
and efficient
killing of the cell, on production of ringlet oxygen by decay of the
chromophore.
In accordance with another aspect of the invention, there is provided a method
for
treating a disease or disorder which is characterised by the presence in the
body of
diseased or undesired cells, such as tumours, cancers, viral infections such
as HIV
infection, or autoimmune disorders such as rheumatoid arthritis or multiple
sclerosis,
comprising the step of administering to a patient in need thereof an effective
amount of a
chromophore in accordance with the invention, which chromophore is adapted to
be
targeted to a target cell specific molecule on the surface of said diseased or
undesired
cells for attachment thereto, such that the chromophore is caused to be
attached to said
cells, and illuminating said cells with light so as to cause the production of
singlet oxygen
in the vicinity of said cells, thereby killing said cells. Suitably, said
target cell specific
molecule comprises an internalisation receptor, and said chromophore is
adapted to be
internalised within said cells on delivery to said internalisation receptor,
such as to enable
the production of ringlet oxygen within said cells on illumination thereof.
Said chromophore may be administered topically or systemically to said
patient.
For example, said chromophore may be administered by injection.
In accordance with yet another aspect of the invention, there is provided a
pharmaceutical composition for administration to a patient for the treatment
of a disease
or disorder which is characterised by the presence in the body of diseased or
undesired
cells, such as tumours, cancers, viral infections such as HIV infection, or
autoimmune
disorders such as rheumatoid arthritis or multiple sclerosis, which
composition comprises
a chromophore in accordance with the present invention that is adapted to be
delivered to
said diseased or undesired cells, and a suitable carrier.
CA 02414089 2002-12-02
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Yet another aspect of the invention envisages a chromophore in accordance with
the invention for use in the production of a medicament, for use in the
treatment of
patients suffering from a disease or disorder which is characterised by the
presence in the
body of diseased or undesirable cells, such as tumours, cancers, viral
infections including
HIV infection, and autoimmune disorders including rheumatoid arthritis or
multiple
sclerosis; said chromophore being adapted for delivery to said diseased or
undesired
cells.
Detailed Description of Examples of the Invention
Following are descriptions and examples, by way of illustration only, of
embodiments of
the invention and methods for putting the invention into effect.
Synthesis of Chromophores
Instrumentation and materials
Melting points are uncorrected. IH/I3C NMR spectra were recorded on Jeol JNM
EX270
FT-NMR spectrometer, and are referenced to tetramethylsilane unless otherwise
stated.
LR. spectra were obtained using a series 1600 FT-LR and nominal mass spectra
were
obtained by Kratos Kompact MALDI II spectrometer. Accurate mass were obtained
from
EPSRC Mass Spectrometry Service, Swansea. The electronic spectra were obtained
using
Unicam UV-2 or UV-4 spectrometers and were taken in DCM unless otherwise
stated.
All reagents and solvents were commercially available and of reagent grade or
higher,
and were, unless otherwise specified, used as received. TLC analysis were
performed on
Merck silica-gel 60 plates (F254, 500 p.m thickness). Merck Silica-Gel 60 (230-
400
mesh) was used for flash chromatographic purification.
31
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Descriptions
(1) 5-(4-Acetamidophenyl)-10,15,20-tris(3,S-dimethoxyphenyl)porphyrin
0
M Me
M~ Me
4-Acetamidobenzaldehyde (3.36 g, 0.02 mol) and 3,5-dimethoxybenzaldehyde (10
mL,
0.06 mol) were stirred in propionic acid (300 mL) at 90 °C. Pyrrole
(5.5 mL, 0.08 mol)
was added and the mixture stirred under reflux for 30 min. Upon cooling the
reaction
mixture was evaporated itz vaczco to yield a dark purple solid. The crude
mixture of
porphyrin isomers was purified by flash chromatography (silica, eluent:
CHZC12/EtOAc,
4:1 ). Relevant fractions were combined, dried (NazS04) and evaporated itz
vacuo to yield
1 as a purple solid (1.55 g, 9.1%); Rf= 0.50 (silica, CHZC12/EtOAc, 4:1); mp
>350 °C
decomp.; IH NMR [270 MHz, CDCl3] ~-2.96 (2H, br s, NH), 2.23 (3H, s, NHCOCH3),
3.93 ( 18H, s, 3, 5-OCH3), 6.99 (3H, s, 10, 15, 20-Ar-4-H), 7.07 (2H, m, .I* =
8 Hz, 5-Ar-
3,5-H), 7.38 (6H, s, 10, 15, 20-Ar-2,6-H), 7.44 (2H, m, J* = 8 Hz, 5-Ar-2,6-
H), 8.86-8.93
(8H, m, (3-H), 10.42 (1H, br s, NHCOCH3); isC NMR [67.5 MHz, CDCl3] &20.4,
23.9,
103.9, 113.5, 117.3, 119, 119.4, 119.6, 120, 129, 131.1, 131.3, 131.4, 131.8,
134.5, 135.6,
136.8, 139.3, 143.1, 158.6, 160.3, 167.9, 168.7; UV-vis (CHZC12) ~,m;"; 421,
515, 551,
590, 650 nm; MS (MALDI-TOF) oz/z 852 (M+, 100%).
32
ivicv UWC
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(2) 5-(4-Aminophenyl)-10,15,20-tris(3,5-dimethoxyphenyl)porphyrin
Me
Me
Porphyrin 1 (500 mg, 0.587 mmol) was dissolved in 18% HCl (100 mL) and the
solution
heated for 2 hours under reflux. Upon cooling the reaction mixture was
evaporated in
vaczro to yield a crude green solid. The solid was redissolved in a 9:1
mixture of
dichloromethane/triethylamine (200 mL) and stirred for 10 min at room
temperature.
The solution was then washed with water (3 x 200 mL) and brine (200 mL), the
organic
layer separated and dried (Na2S04). Excess solvent was evaporated in vacuo and
the
crude purple solid purified by flash chromatography (silica, eluent:
CHZC12/EtOAc, 4:1).
Relevant fractions were combined, dried (NaZS04) and evaporated i» vacuo to
yield 2 as
a purple solid (426 mg, 89.7%); Rf = 0.89 (silica, CHZCI2BtOAc, 4:1); mp >350
°C
decamp.; 1H NMR [270 MHz, CDCl3] 8-2.80 (2H, br s, NH), 3.96 (18H, s, 3, 5-
OCH3),
6.90 (3H, s, 10, 15, 20-Ar-4-H), 7.06 (2H, m, J* = 8 Hz, 5-Ar-3,5-H), 7.40
(6H, s, 10, 15,
20-Ar-2,6-H), 7.98 (2H, m, .l* = 8 Hz, S-Ar-2,6-H), 8.93 (8H, m, ~3-H); 13C
NMR [67.5
MHz, CDC13] 8 20.13, 100.6, 113.9, 114.3, 115.7, 119.8, 120.1, 121.4, 130.2,
131.4,
132.7, 136.1, 144.5, 144.6, 146.5, 159.3; UV-vis (CHZCl2) ~,m;,x 422, 517,
553, 593, 651
nm; MS (MALDI-TOF) »a/z 809 (M+, 100%).
33 --
rnCV Vlyle
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(3) ~-(4-Aminophenyl)-10,15,20-tris(3,5-dihydroxyphenyl)porphyrin
To a stirred solution of 2 (1 g, 1.23 mmol) in freshly distilled chloroform
(50 mL) was
added boron tribromide (1.17 mL, 0.012 mol). The reaction was allowed to
proceed
under argon for 17 hours at room temperature. The reaction was subsequently
cooled to
0 °C, water (20 mL) added and the solution stirred for a further 60
min. The reaction was
evaporated to dryness in nacuo and redissolved in a 9:1 mixture of
chloroform/triethylamine (S00 mL). The solution was washed with water (3 x 500
mL)
and brine (500 mL), the organic layer separated, dried (Na2S0~), and
evaporated in vacuo
to yield a crude purple solid. The crude solid was purified by flash
chromatography
(silica. eluent: CHC13/MeOH, 9:1). Relevant fractions were combined, dried
(Na2S04)
and evaporated ire oacuo to yield 3 as a purple solid (667 mg, 74.5%); Rf=
0.19 (silica,
CHCI~/MeOH, 9:1); mp >350 °C decomp.; 'H NMR [270 MHz, (CD3)ZSO] 8-
2.95 (2H,
br s, NH), 5.56 (2H, br s, NHZ), 6.69 (3H, s, 10, 15, 20-Ar-4-H), 7.02 (2H, m,
.I* = 8 Hz,
5-Ar-3,5-H), 7.06 (6H, s, 10, 15, 20-Ar-2,6-H), 7.87 (2H, m, ,J* = 8 Hz, 5-Ar-
2,6-H),
8.94 (8H, s, (3-H), 9.75 (6H, br s, 3, 5-OH); 13C NMR [67.5 MHz, (CD3)2S0] S
102.3,
112.5, 113.9, 114.1, 119.2, 119.7, 121.5, 127.5, 128.3, 130.7-131.3, 135.5,
142.8, 142.9,
148.6, 156.4, 156.5; UV-vis (CHZC12) a.max 422, 517, 553, 592, 649 nm; MS
(MALDI-
TOF) »~% 726 (M+ 100%).
3=~
v vn
CA 02414089 2002-12-02
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(4) ~-(4-Acetamidophenyl)-10,15,20-tris(4-pyridyl)porphyrin
0
N
4-Acetamidobenzaldehyde (3.26 g, 0.02 mol) and 4-pyridinecarboxaldehyde (5.66
mL, 0.06 mol) were stirred in propionic acid (300 mL) at 90 °C. Pyrrole
(5.4 mL, 0.08
mol) was added and the mixture stirred under reflux for 30 min. Upon cooling
the
reaction mixture was evaporated izz vacuo to yield a dark purple solid. The
crude mixture
of porphyrin isomers was purified by flash chromatography (silica, eluent:
CHCl3/IVIeOH,
19:1). Relevant fractions were combined, dried (Na2S0~) and evaporated izz
vacuo to
yield 4 as a purple solid (526 mg, 3.9°f°); Rf= 0.22 (silica,
CHCl3/MeOH, 19:1); mp >350
°C decomp.; 1H NMR [270 MHz, CDC13] 8 -2.79 (2H, br s, NH), 2.49 (3H,
s,
NHCOCH;), 8.07 (2H, m, ,I* = 8 Hz, 5-Ar-3,5-H), 8.21-8.28 (8H, m
(overlapping), 5-Ar-
2,6-H & 10, 15, 20-Py-2,6-H), 8.84-9.06 (8H, m, ~3-H), 9.10-9.15 (6H, m, 10,
15, 20-Py-
3,5-H), 10.35 (1H, br s, NHCOCH3); isC NMR [67.5 MHz, CDCl3] 826.8, 106.9,
110.1,
110.2, 117.9, 121.1, 121.5, 122.1, 122.2, 123.3, 123.8, 123.9, 134.7, 140.1,
142.5, 145.1,
148.2, 149, 149.3, 149.4, 149.6, 150.1, 175.2; UV-vis (CHZCIz) 7~m:~x 418,
514, 548, 587,
644 nm; MS (MALDI-TOF) rn/z 675 (M+, 100%).
JJ
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(~) ~-(4-Aminophenyl)-10,15,20-tris(4-pyridyl)porphyrin
N
Porphyrin 4 (500 mg, 0.74 mmol) was dissolved in 18% HCl (100 mL) and the
solution
heated for 2 hours under reflux. Upon cooling the reaction mixture was
evaporated in
1'CIC;IlI) to yield a crude green solid. The solid was redissolved in a 9:1
mixture of
dichloromethane/triethylamine (200 mL) and stirred for 10 min at room
temperature.
The solution was then washed with water (3 x 200 mL) and brine (200 mL), the
organic
layer separated and dried (NazS04). Excess solvent was evaporated in vacuo and
the
purple crude solid purified by flash chromatography (silica, eluent:
CHCl3/MeOH, 20:1).
Relevant fractions were combined, dried (Na2S0~) and evaporated in vacuo to
yield 5 as
a purple solid (422 mg, 90.1%); Rf = 0.31 (silica, CHC13/MeOH, 20:1); mp >350
°C
decomp.; ~H NMR [270 MHz, CDC13] 8-2.86 (2H, br s, NH), 4.09 (2H, br s, NHZ),
7.08
(2H, m, ,l* = 8 Hz, 5-Ar-3,5-H), 7.98 (2H, m, ,I* = 8 Hz, 5-Ar-2,6-H), 8.16
(6H, m, J* _
Hz, 10, 15, 20-Py-2,6-H), 8.80-9.01 (8H, m, [3-H), 9.04 (6H, m, J* = 5 Hz, 10,
15, 20-
Py-3,5-H); 1~C NMR [67.5 MHz, CDCl3] 8 113.6, 116.7, 117.4, 117.9, 122.7,
129.5,
131.7, 135.9, 146.5, 148.4, 148.5, 149.8, 150.2; UV-vis (CHZCl2) ~,,t,~X 418,
515, 552,
592, 653 nm; MS (FAB) rrzi'~ 633 (M+, 100%).
36
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(6) 5-(4-Acetamidophenyl)-10,15,20-tris(3-pyridyl)porphyrin
4-Acetamidobenzaldehyde (5 g, 0.031 mol) and 3-pyridinecarboxaldehyde (8.67
mL,
0.092 mol) were stirred in propionic acid (300 mL) at 90 °C. Pyrrole
(8.5 mL, 0.123 mol)
was added and the mixture stirred under reflux for 30 min. Upon cooling the
reaction
mixture was evaporated in vcrcuo to yield a dark purple solid. The crude
mixture of
porphyrin isomers was purified by flash chromatography (silica, eluent:
CHCl3/MeOH,
19:1). Relevant fractions were combined, dried (Na2S04) and evaporated ifZ
vacuo to
yield G as a purple solid (0.96 g, 4.6%); Rf= 0.26 (silica, CHC13/MeOH, 19:1);
mp >350
°C decomp.; 1H NMR [270 MHz, GDCl3] 8-2.97 (2H, br s, NH), 2.17 (3H, s,
NHCOCH;), 7.40 (2H, m, .I* = 8 Hz, 5-Ar-3,5-H), 7.49 (3H, m, 10, 15, 20-Py-5-
H), 7.98
(2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.21-8.33 (3H, m, 10, 15, 20-Py-6-H), 8.57-
8.82 (11H,
m (overlapping), 10, 15, 20-Py-4-H & (3-H), 8.99 (1H, br s, NHCOCH3), 9.26
(3H, m, 10,
15, 20-Py-2-H); UV-vis (CH2C12) 7~m~X 419, 516, 552, 592, 648 nm; MS (MALDI-
TOF)
~rz; z 675 (M+, 100%).
37 w
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(7) ~-(4-Aminophenyl)-10,15,20-tris(3-pyridyl)porphyrin
Porphyrin 6 (300 mg, 0.45 mmol) was dissolved in 18% HCl (100 mL) and the
solution
heated for 2 hours under reflux. Upon cooling the reaction mixture was
evaporated in
nacwo to yield a crude green solid. The solid was redissolved in a 9:1 mixture
of
dichloromethane/triethylamine (200 mL) and stirred for 10 min at room
temperature.
The solution was then washed with water (3 x 200 mL) and brine (200 mL), the
organic
layer separated and dried (Na2S0~). Excess solvent was evaporated in vacuo and
the
purple crude solid purified by flash chromatography (silica, eluent:
CHC13/MeOH, 20:1).
Relevant fractions were combined, dried (Na2S0~) and evaporated ire vacz~o to
yield 7 as
a purple solid (206 mg, 68.5%); Rf= 0.38 (silica, CHC13/MeOH, 20:1); mp >350
°C
decomp.; 1H NMR [270 MHz, CDCl3] 8-2.74 (2H, br s, NH), 3.93 (2H, br s, NH2),
6.91
(2H, m, .l* = 8 Hz, 5-Ar-3,5-H), 7.67 (3H, m, 10, 15, 20-Py-S-H), 7.93 (2H, m,
J* = 8
Hz, 5-Ar-2,6-H), 8.48 (3H, m, 10, 15, 20-Py-6-H), 8.79-9.02 (11H, m
(overlapping), 10,
15, 20-Py-4-H & (3-H), 9.47 (3H, s, 10, 15, 20-Py-2-H); 13C NMR [67.5 MHz,
CDC13] 8
113.3, 115.6, 116.1, 116.7, 121.9, 122.3, 131, 131.4, 131.8, 132.1, 132.3,
135.7, 137.5,
137.7, 137.8, 140.8, 146.3, 149, 149.2, 153.5; UV-vis (CHZC12) ~,m;"420, 517,
553, 597,
649 nm; MS (MALDI-TOF) nz/z 632 (M+, 100%).
3S
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(8) 5-(4-Acetamidophenyl)-15-(4-methoxyphenyl)porphyrin,
The DDP was synthesised according to the method of Dolphin et al.(1998 5-
Phenyldipyrromethane and 5, 15-Diphenylporphyrin Org. Synth. 76, 287-293
incorporated herein by reference) The resulting mixture of three porphyrins
was
chromatographed, eluting initially with DCM to allow removal of 5,15-(4-
methoxy)-
DPP, and then continuing with ethyl acetate/DCM ( 1:4) to elute the required
product as
purple crystals (150 mg, 12%); R~= 0.40 (DCM/MeOH, 19:1); mp 305-307°C
(decomposed); UV-vis (DCM) 7~n,~" (relative intensity) 410 (1.0), 502 (0.04),
538 (0.02),
578 (0.015), 630 (0.01) nm; 1H NMR (270 MHz, CDC13) b 10.35 (s, 2H, 10+20-H),
9.43
(d, 4H, J = 4.8 Hz, ,(3 ~, 9.14 (d, 4H, J = 4.8 Hz, ~3 H), 8.65 (m, ZH, J =
7.2 Hz, .5-nz-An),
8.22-8.12 (m, 4H, (overlapping), J = 8.1 Hz, .i+IS-o Ar), 7.56 (m, 2H, J = 8.1
Hz, l~-ni-
Ar), 4.14 (s, 3H, CHj), -3.00 (br s, 2H, NH); MALDI-MS m/z 550.3 (M+, 100%).
(9) 5-(4-Aminophenyl)-15-(4-methoxyphenyl)porphyrin,
5-(4-Acetamido phenyl)-15-(4-methoxyphenyl)porphyrin 8 (1 eq., 100 mg, 0.182
mmol)
was dissolved in 5 M aqueous HCI (100 mL) and the solution heated for 3 h
under reflux.
The hot reaction mixture was concentrated irz vaczco to yield a crude green
solid. The
solid was re-dissolved in a mixture of DCM/triethylamine (9:1) (200 mL) and
stirred for
min at room temperature. The solution was then washed with water (3 x 200 mL),
saturated brine (200 mL) and the organic layer separated and dried (NazSOa),
then
concentrated in vaezio. The crude purple solid was chromatographed, eluting
with DCM,
and gave the desired porphyrin as a purple crystalline solid (51 mg, 54%), Rf=
0.30
(DCM), mp 300°C (decomposed); UV-vis (DCM) ~,t"a~ (relative intensity)
410 (1.0), 503
(0.045), 538(0.02), 578 (0.015), 630 (0.005) nm; Fluorescence (DCM) ~,max 634
nm
(~, excitation = 410 nm); 1H NMR (270 MHz, CDC13) 8 10.30 (s, 2H, 10+,~0-~~
9.39 (d,
4H, J = 4.9 Hz, ~ H), 9.17 (d, 2H, J = 4.9 Hz, ,Q H), 9.10 (d, 2H, J = 4.9 Hz,
~3-H), 8.19
(m, 2H, J = 8.8 Hz, IS-o Ar), 8.07 (m, 2H, J = 8.1 Hz, 5-o An), 7.35 (m, 2H, J
= 8.8 Hz,
L5-nz Ay~), 7.14 (m, 2H, J = 8.1 Hz, 5-m Ar), 4.13 (s, 3H, CHj), 4.08 (br s,
2H, NH), -3.06
(br s, 2H NH); MALDI-MS m/z 508.3 ([M+1]+, 100%). ES-HRMS calcd. for C33HzsNsO
([M+1]T) 508.2137, found 508.2144.
39
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(10) 17,18-Dihydroxy-5-(4-aminophenyl)-15-(4-methoxyphenyl)chlorin and
(11) 7,8-dihydroxy 5-(4-aminophenyl)-15-(4-methoxyphenyl)chlorin regioisomers,
Porphyrin 9 (28 mg, 55.2 p.mol) was converted into the required mixture of
chlorin
regioisomers following the procedure of Sutton et c~l.(2000 Functionalised
diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for
targeted
photodynamic therapy and tumour cell imaging J. Po~ph~rir~ ccnd
Phthalocyaraines 4,
655-658) The crude reaction mixture was then chromatographed, eluting with 1%
MeOH
in DCM. First, some un-reacted starting material was eluted, then the higher
Rf chlorin
isomer 10 as a brown-purple crystalline solid; RJ-= 0.28 (DCM/MeOH, 19:1). The
lower
Rt~ isomer 11 was obtained by further elution with 2.5% MeOH in DCM and gave
also a
brown-purple crystalline solid (Rf= 0.17 (DCM/MeOH, 19:1).
High Rt~ chlorin regioisomer (17,18-dihydroxy-15-(4-methoxy phenyl)-5-(4-
aminophenyl)chlorin assigned previously(26)) (7.0 mg, 24%), mp 165-
167°C
(decomposed); UV-vis (DCM) ~,",ax (relative intensity) 401 (0.99), 414 (1.0),
503 (0.08),
535 (0.07), 582 (0.035), 636 (0.22) nm; Fluorescence (DCM) 7~~"ax 639 nm (~,
excitation
= 412 nm); 1H NMR (270 MHz, 10% CD30D in CDC13) 8 9.95 (s, 1H, 10-I~, 9.42 (s,
1H, 20-I~, 9.17 (d, 1H, J = 4.8 Hz, ~3-I~, 9.03 (d, 1H, J = 4.0 Hz, ,(3-~,
8.97 (s, 2H, /.3 I~
8.78 (d, 1H, J = 4.8 Hz, ,(3-I~, 8.51 (d, 1H, J = 4.8 Hz, ,~3 I~, 8.05 (m, 2H,
J = 8.9 Hz, o-
Ar), 7.94 (m, 2H, J = 8.1 Hz, o'-An), 7.25 (m, 2H, J = 8.9 Hz, na Ar), 7.12
(m, 2H, J = 8.1
Hz, m '-Ar), 6.42 (d, 1H, J = 6.5 Hz, 17-I~, 6.03 (d, 1H, J = 6.5 Hz, 18-I~,
4.08 (s, 3H.,
H; ), (NH'.s exchanged & CFI '.s~ not observed); MALDI-MS m/z 542.2 ([M+H]''-,
100%);
ES-HRMS calcd. for C33H28NSO3 ([M+H]+) 542.2192, found 542.2187.
Low Rt~ chlorin regioisomer (7,8-dihydroxy-5-(4-aminophenyl)-15-(4-
methoxyphenyl)chlorin) (8.5 mg, 30%), mp 168-171°C (decomposed); UV-vis
(DCM)
7~",~" (relative intensity) 401(0.99), 413 (1.0), 507 (0.08), 536 (0.06), 586
(0.025), 637
(0.20) nm; Fluorescence (DCM) ~,m~X 639 nm (~, excitation = 412 nm); 1H NMR
(270
1VJIIz, 10% CD30D in CDC13) d 9.96 (s, 1H, 20-I~, 9.40 (s, 1H, 10-I-~, 9.18
(d, 1H, J =
4.8 Hz, ,Q-I~, 9.05 (d, 1H, J = 4.8 Hz, ~3-I~, 8.98 (d, 1H, J = 4.0 Hz, ~3 I~
8.92 (d, 1H, J
= 4. 0 Hz, ~3-I~, 8. 74 (d, 1 H, J = 4 . 0 Hz, ~3 I~, 8 . 5 8 (d, 1 H, J = 4.
0 Hz, ,Ci I~, 8.13 (m, 1 H,
J = 8.9 Hz, e~ An), 8.08 (m, 1H, J = 8.9 Hz, o-Ar~), 7.95 (m, 1H, J = 8.1 Hz,
o'-Ar), ?.79
o -
CA 02414089 2002-12-02
WO 02/00662 PCT/GBO1/02846
( m, 1 H, J = 8 .1 Hz, o ' Ar), 7. 3 6 (m, 1 H, J = 8. 9 Hz, m-Ar), 7. 3 0 (m,
1 H, J = 8. 9 Hz, m-
Ar), 7.11 (m, 1H, J = 8.1 Hz, m' Ar), 7.05 (m, 1H, J = 8.1 Hz, m' Ar), 6.42
(d, 1H, J = 6.5
Hz, 7-H), 6.09 (d, 1H, J = 6.5 Hz, f-H), 4.11 (s, 3H, CH3), (NH's exchanged &
OH's not
observed); MALDI-MS m/z 542.2 ([M+H]+, 100%) ES-HRMS calcd. for C33HZ8NSO3
([NI+H]T) 542.2192, found 542.2185.
(12) 5-(4-Fluorenylmethylaminophenyl)-15-(4-methoxyphenyl)porphyrin,
To a stirred solution of porphyrin 9 (28 mg, 55 p.mol) in anhydrous 1,4-
dioxane (2.5 mL)
was added solid sodium hydrogen carbonate (6 eq., 28 mg, 0.33 mmol). To this
mixture
was then added a solution of 9-fluorenylmethyl chloroformate (2 eq., 0.11
mmol, 28.5
mg) in 1,4-dioxane (0.5 mL) under N2. The reaction flask was covered with
aluminium
foil to exclude light and stirred at room temperature for a period of 3 h. At
this time the
reaction was complete (as monitored by TLC). The 1,4-dioxane was removed in-
vacuo
and the residue partitioned between water (25 mL) and DCM (2 x 25 mL). The
combined
organic extracts were washed with saturated brine (25 mL) then dried (Na2SOa),
filtered
and concentrated in vacuo. The required porphyrin was obtained by
chromatography,
eluting with DCM. The desired porphyrin was obtained as purple crystals (38
mg, 95%),
R'= 0.39 (DCM), mp 292-295°C (decomposed); UV-vis (DCM) ~,max (relative
intensity)
410 ( 1.0), 505 (0.042), 541 (0.02), 578 (0.015), 633 (0.01) nm; Fluorescence
(DCM) ~,max
635 nm (~, excitation = 410 nm); 1H NMR (270 MHz, CDC13) b 10.35 (s, 2H, 10+20
~,
9.69 (br. s, 1H, NH), 9.44 (d, 4H, J = 4.8 Hz, ,~33-F~, 9.12 (d, 4H, J = 4.8
Hz, ~3-H), 8.20-
8.17 (m (overlapping), 4H, J ='8.1 Hz, .5+IS-o-Ar), 7.85 (m, 4H, .S+IS-nz Ar),
7.76-7.66
(m, 2H,,fZuorerro Ar), 7.51-7.30 (m, 6H, fZuo~°eno Ar), 4.69 (d, 2H, J
= 7.2 Hz, CHI), 4.30
(t, 1H, J = 7.2 Hz, CH), 4.13 (s, 3H, CH3), -3.15 (br. s, 2H, NH); MALD-MS m/z
731.5
([M+H]+, 100°J°), 508.3 ([M-FMOC+2]~, 50%); ES-HRMS calcd. for
C4gH36N5O3
([M+H]~) 730.2818, found 730.2809.
(13, 14) cis/traps-7,8,17,18-Tetrahydroxy-5-(4-fluorenylmethylaminophenyl)-
15-(4-methoxyphenyl) bacteriochtorins
Porphyrin 12 (35 mg, 48.0 pmol) was converted into the required mixture of
bacteriochlorin stereoisomers by minor modification of the procedure of Sutton
et
al. (2000 Functionalised diphenylchlorins and bacteriochlorins - their
synthesis and
bioconjugation for targeted photodynamic therapy and tumour cell imaging J.
Porphyrin
41
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arzcl Phthcrlocyarzines 4, 655-658 - incorporated herein by reference)
(reaction carried out
using 1,4-dioxane (5 mL) to allow dissolution of 12). The crude reaction
mixture was
chromatographed, eluting initially with 1% MeOH in DCM to remove chlorin by-
products. Further elution with 2% MeOH/DCM allowed isolation of both
stereoisomeric
bacteriochlorin tetrols. The higher R~~trans bacteriochlorin isomer 13 was
isolated as a
pink-green crystalline solid, (6 mg, 15%), Rf= 0.25 (DCM/MeOH, 19:1), mp 142-
145oC
(decomposed); UV-vis (DCM) ~.",:1~ (relative intensity) 374 (1.0) 512 (0.23),
702 (0.52)
nm; Fluorescence (DCM) 7~",~, 708 nm (7,, excitation = 512 nm); 1H NMR (270
MHz,
10% CD30D in CDCl3) d 9.20 (s, 2H, 10+?0-H), 8.78 (d, 2H, J = 4.0 Hz, ~3 H),
8.36 (d,
2H, J = 4.0 Hz, ,(3-H), 7.95 (m, ZH, o-Ar), 7.85 (m, 2H, J = 7.3 Hz,
fZzcor~erzo-Ar), 7.79 (m,
2H o '-Ar~), 7.65 (m, 2H, rn ' Ar), 7.47-7.3 8 (m, 6H, fZuor°erzo-Ar),
7.24 (m, 2H, rn-Ar"),
6.27-6.24 (m, 2H, 7+17-H), 5.85 (m, 2H, 8+18-H), 4.65 (d, 2H, J = 7.2 Hz,
CHz), 4.39 (t,
1H, J = 7.2 Hz, CH), 4.06 (s, 3H, CHj), -1.94 (br s (partly exchanged), 2H,
NH), (OH's
not observed); MALDI-MS m/z 800.4 ([M+H]+, 100%); ES-HRMS calcd. for
C~,RH:,«N;O~ ([M+H]+) 798.2927, found 798.2921.
The lower Rt~ cis-bacteriochlorin isomer 14 was isolated as a pink-green
crystalline solid,
(8.5 mg, 21%), Rf= 0.2 (DCM/MeOH, 19:1), mp 148-150oC (decomposed); UV-vis
(DCM) ~,",:" (relative intensity) 374 (1.0) 512 (0.24), 703 (0.54) nm;
Fluorescence
(DCM) ~,",:1~ 708 nm (7~ excitation = 512 nm); 1H NMR (270 MHz, 10% CD30D in
CDC13) 8 9.12 (s, 2H, 10+20-H), 8.76 (d, 2H, J = 4.8 Hz, /3-H), 8.34 (d
(overlapping),
2H, J = 4.8 Hz, ,(3-H), 8.02 (m, 2H, o-Ar), 7.85 (m (obscured), 2H, J = 8.0
Hz, o' AY),
7.83 (m, 2H, J = 7.3 Hz, fZuoYerzo Ar~), 7.76 (m, 2H, J = 8.0 Hz, rn'-Ar),
7.50-7.38 (m, 6H,
f7zcoueroo-Ar~), 7.24 (m, 2H, rn Ar~), 6.27-6.23 (m, 2H, 7+ 17-H), 5.85-5.82
(m, 2H, 8+18-
H), 4.65 (d, 2H, J = 7.2 Hz, CHI), 4.39 (t, 1H, J = 7.2 Hz, CH), 4.05 (s, 3H,
CH3), -1.88
(br. s (partly exchanged), 2H, NH), (OH's not observed); MALDI-MS mlz 800.4
([M+H]T, 100%); ES-HRMS calcd. for Ca$H4~N50~ ([M+H]+) 798.2927, found
798.2921.
(15) 5-(3,4,5-Trismethoxyphenyl)dipyrromethane
3,4,5-Trismethoxybenzaldehyde (5.0 g, 25.5 mmol) was dissolved in freshly
distilled
pyrrole (75 ml) and the solution degassed by bubbling with dry NZ for 10 min.
TFA
(0.075 eq. , 0.15 ml, 1.91 mmol) was added and the mixture stirred under NZ
until no
starting aldehyde could be detected by TLC (ca. 10 min). The reaction mixture
was
42
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concentrated lr2 VaCZlO at water aspirator pressure (evaporator water bath
temp 75°C )
then under high vacuum for 16 h to remove excess pyrrole. The crude product
was
recrystallised from hot ethylacetate/ nHexane and afforded the required
dipyrromethane
as a white solid, ( 5.41 g, 68%): v",a~ (nujol mull)/ cm i 3378 (br. NH), 1594
(C=C),
1233, 1040; UV-VIS (MeOH) ~.",a~/ (rel. intensity) 222 (1.0), 280 (0.75) nm;
c~H(270 MHz; CDC13) 8.07 (2H, br. s, NH), 7.53 (2H, m, I-H), 6.68 (2H, s, 2'-
H), 6.37
(2H, m, ?-H), 5.93 (2H, m, 3-H), 5.38 (1H, s, rr7etharae), 3.80 (3H, s, -~'-
OCH;), 3.73 (6H,
s, 3 '+~ '-UCH;); bC(68 MHz; CDC13) 152.7 (CH, 2'-C), 137.3 (q, 3'+5'-C),
136.1 (q, 4'-
C), 131.8 (q, 4-C), 116.7 (CH, 1-C), 107.9 (CH, 2-C), 106.6 (CH, 3-C), 104.9
(q, 1'-C),
60.3 (CH;), 55.5 (CH;), 43.7 (CH., methane); MS (MALDI) m/e 311.2 (100%, (M-
1)+).
(16) 5-(4-Acetomidophenyl)dipyrromethane
The dipyrromethane was synthesised using the general procedure detailed above
using
the same molar quantity of starting aldehyde. The crude reaction mixture was
chromatographed on flash silica-gel (350 ml), (dry loaded on to 50 ml flash
silica-gel
from ethylacetate) and eluted with 40% ethylacetate/ DCM and afforded the pure
product
as an off white solid, (4.3 g, 50%): vm~,,; (nujol mull)/ cm 1 3409 (NH,
amide), 3248 (br.
NH), 1650 (C=O), 1593 (C=C), 1320, 1009; UV-VIS (MeOH) ~,m~;;/ (rel.
intensity) 224
( 1.0) nm; dH(270 MHz; CDC13) 8.00 (ZH, br. s, NH), 7.40 (2H, d, J = 8.5 Hz, o-
Ar ), 7.30
(1H, br. s, NH-acetotnido), 7.13 (2H, d, J= 8.5 Hz, m-Ar), 6.68 (2H, m, I-H),
6.16 (2H,
m, ?-I~, 5.90 (2H, m, 3-H), 5.42 (1H, s, rnetharze), 2.14 (3H, s, NH~~); 8C(68
MHz;
CDC13) 168.4 (q, LOCH;), 138.2 (q, 4'-C), 136.5 (q, 2'-C), 132.4 (q, 4-C),
128.9 (CH,
2'-C), 120.3 (CH, 3'-C), 117.2 (CH, 1-C), 108.4 (CH, 2-C), 107.1 (CH, 3-C),
43.4 (CH.,
methane), 24.5 (CH3); MS (MALDI) m/e 279.4 ( 100%, (M)~).
(17) ~-(4-Methoxyphenyl)dipyrromethane
The dipyrromethane was synthesised using the general procedure detailed above
using
the same molar quantity of starting aldehyde. The crude reaction mixture was
chromatographed on flash silica-gel (350 ml), (dry loaded on to 50 ml flash
silica-gel
from ethylacetate) and eluted with 30% nHexane/ DCM and afforded the pure
product as
an off white solid, (4.3 g, 50%): vma;; (nujol mull)/ cm 1, 3382 (br. NH),
1598 (C=C),
1300, 1050; UV-VIS (MeOH) 7~",a,~/ (rel. intensity) 224 (1.0) nm; 8H(270 MHz;
CDC13)
43
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7.87 (2H, br. s, NIA, 7.10 (2H, d, J = 8.8 Hz, m-Ar ), 6.83 (2H, d, J = 8.8
Hz, o-An), 6.66
(2H, m, I-I~, 6.14 (2H, m, ?-I~, 5.89 (2H, m, 3-I~, 5.40 (1H, s, rrZethane);
MS (MALDI)
m/e 252.4 (100°J°, (M)+).
Example 1
s-(4-Isothiocyanatophenyl)-10,15,20-tris(3,5-dihydroxyphenyl)porphyrin
H
H~
To a stirred solution of 3 (100 mg, 0.137 mmol) in freshly distilled THF (25
mL) was
added 1,1'-thiocarbonyldi-2(ll~-pyridone (64 mg, 0.276 mmol). The reaction was
allowed to proceed under argon for 4 hours at room temperature. Excess solvent
was
evaporated lf7 VClCZlO to yield a crude purple solid. The solid was dissolved
in a minimal
amount of chloroform/methanol (9:1) and purified by flash chromatography
(silica,
eluent: CHC13/MeOH, 9:1). Relevant fractions were combined, dried (Na2S0a) and
evaporated irZ ocrcuo to yield the above compound as a purple solid (67.5 mg,
63.8%); Rf
= 0.29 (silica, CHCl3/MeOH, 9:1); mp >350 °C decomp.; 1H NMR [270 MHz,
CDC13/CD30D, 3:1] 86.77 (3H, s, 10, 15, 20-Ar-4-H), 7.12 (6H, s, 10, 15, 20-Ar-
2,6-H),
7.64 (2H, m, .I* = 8 Hz, 5-Ar-3,5-H), 8.19 (2H, m, J* = 8 Hz, S-Ar-2,6-H),
8.76-9.0 (8H,
m, (3-H); 13C NMR [67.5 MHz, CDC13/CD30D, 3:1] 8101.9, 107.1, 114.7, 117.6,
119.9,
120, 120.1, 123.9, 130.9, 134, 135.2, 136.1, 141.2, 142, 143.6, 155.8; UV-vis
(MeOH)
44 -
w vn
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~.",~"422, 516, 552, 592, 648 nm; HRMS (ES) m/z calc'd for C45Hz9N5OsS [M+H]+
768.1914, found 768.1908.
Example 2
5-(4-Isothiocyanatophenyl)-10,15,20-tris(4-pyridyl)porphyrin
To a stirred solution of 5 (100 mg, 0.158 mmol) in freshly distilled
dichloromethane (20 mL) was added l,1'-thiocarbonyldi-2(11-pyridone (320 mg,
1.38
mmol). The reaction was allowed to proceed under argon for 4 hours at room
temperature. Excess solvent was evaporated in vacuo to yield a crude purple
solid. The
solid was dissolved in a minimal amount of chloroform and purified by flash
chromatography (silica, eluent: CHCl3/MeOH, 49:1). Relevant fractions were
combined,
dried (NazSO:~) and evaporated in vacuo to yield the above compound as a
purple solid
( 104 mg, 97.5%); Rf= 0.57 (silica, CHCI3IMeOH, 49:1); mp >350 °C
decomp.; 1H NMR
[270 MHz, CDC13] 8-2.91 (2H, br s, NH), 7.65 (2H, m, J* = 8 Hz, 5-Ar-3,5-H),
8.15-
8.21 (8H, m (overlapping), 10, 15, 20-Py-2,6-H & 5-Ar-2,6-H), 8.67 (8H, br s,
(3-H), 9.06
(6H, m, J* = 5 Hz, 10, 15, 20-Py-3,5-H); 13C NMR [67.5 MHz, CDCl3] 8117.4,
117.6,
119.7, 124.7, 129.3, 131.6, 135.4, 136.9, 140.6, 148.4, 149.8; UV-v15 (CHZCIz)
a,max 417,
514, 548, 587, 643 nm; HRMS (ES) m/z calc'd for C4zHz6NsS (M+H) 675.2079,
found
675.2078.
45 -
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Example 3
5-(4-Isothiocylnatophenyl)-10,15,20-tris(3-pyridyl)porphyrin (160)
To a stirred solution of 7 (200 mg, 0.316 mmol) in freshly distilled
dichloromethane (40 mL) was added l,1'-thiocarbonyldi-2(lf~-pyridone (640 mg,
2.76
mmol). The reaction was allowed to proceed under argon for 17 hours at room
temperature. Excess solvent was evaporated in vacz.ro to yield a crude purple
solid. The
solid was dissolved in a minimal amount of chloroform and purified by flash
chromatography (silica, eluent: CHCl3/MeOH, 49:1). Relevant fractions were
combined,
dried (Na2S0:,) and evaporated irz vacuo to yield the above compound as a
purple solid
( 171 mg, 80.3%); Rf= 0.55 (silica, CHCl3/MeOH, 49:1); mp >350 °C
decomp.; 1H NMR
[270 MHz, CDC13] 8-2.83 (2H, br s, NH), 7.65 (2H, m, J* = 8 Hz, 5-Ar-3,5-H),
7.78
(3H, m, 10, 15, 20-Py-5-H), 8.20 (2H, m, J* = 8 Hz, 5-Ar-2,6-H), 8.54 (3H, m,
10, 15,
20-Py-6-H), 8.83-8.88 (8H, m, (3-H), 9.07 (3H, m, 10, 15, 20-Py-4-H), 9.07
(3H, s, 10,
15, 20-Py-2-H); 13C NMR [67.5 MHz, CDCl3] 8116.6, 122.1, 124.2, 131.5, 135.5,
137.7,
140.8, 140.9, 149.2, 153.5; UV-vis (CHZCIz) a,mah 421, 513, 547, 587, 657 rim;
MS
(MALDI-TOF) nZ~s 674 (MT, 100%).
=~6
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Example 4
5-(4-Isothiocyanatophenyl)-10,15,20-tris(4-N methylpyridiniumyl) porphyrin
triiodide
I Me- -Me I
N
I
Me
I
To a solution of Example 2 (50 mg, 0.074 mmol) in anhydrous DMF (10 mL,
distilled from CaHz, 0.1 torr) was added iodomethane (1 mL, 0.016 mol). The
reaction
was stirred under argon for 3 hours at room temperature, monitored by TLC
(normal
phase silica) in a water/saturated aqueous potassium nitrate/acetonitrile
(1:1:8) solvent
system. Upon reaction completion excess DMF was evaporated in vacuo (0.1 torr)
at 30-
40 °C to yield the above compound as a lustrous purple solid (77 mg,
95%); Rf = 0.32
(silica, H20/sat.aq. KN03/MeCN, 1:1:8); mp >350 °C decomp.; 1H NMR [270
MHz,
(CD3)zS0] 8-3.03 (2H, br s, NH), 4.74 (9H, br s, N CH3-pyridine), 7.96 (2H, m,
J* = 8
Hz, 5-Ar-3 , 5-H), 8. 3 2 (2H, m, J* = 8 Hz, 5-Ar-2, 6-H), 9. 03 (6H, m, J* =
6 Hz, 10, 15,
20-Py-2,6-H), 9.16 (8H, m, (3-H), 9.50 (6H, m, J* = 6 Hz, 10, 15, 20-Py-3,5-
H); 13C
NMR [67.5 MHz, (CD3)zSO] X47.9, 114.7, 115.3, 121.1, 124.8, 130.6, 132, 134.7,
135.4,
139.7, 144. l, 156.3, 156.4; UV-vis (Hz0) ~,m;~X 423, 520, 585 nm; MS (FAB)
nalz 719
(MT, 100%), 704 (M-CH3, 26%), 689 (M-2CH3, 20%), 674 (M-3CH3, 5%); HRMS (ES)
nz/z calc'd for C:~;H35N8S (M+H) 719.2705, found 719.2686.
47
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E~cample 5
5-(4-Isothiocyanatophenyl)-10,15,20-tris(3-N methylpyridiniumyl) porphyrin
triiodide
I
~e
ME
I
me
I
To a solution of Example 3 (50 mg, 0.074 mmol) in anhydrous DMF (5 mL,
distilled from CaH, 0.1 torr) was added iodomethane (1 mL, 0.016 mol). The
reaction
was stirred under argon for 4 hours at room temperature, monitored by TLC
(normal
phase silica) in a water/saturated aqueous potassium nitrate/acetonitrile
(1:1:8) solvent
system. Upon reaction completion excess DMF was evaporated irt mcuo (0.1 torr)
at 30-
40 "C to yield the above compound as a lustrous purple solid (72 m~, 89%); Rf
= 0.46
(silica, H~O/sat.aq. KN03/MeCN, 1:1:8); mp >350 °C decomp.; iHNMR [270
MHz,
(CDs)ZSO] ~-3.07 (2H, br s, NH), 4.69 (9H, br s, N CH3-pyridine), 7.97 (2H, m,
,l* = 8
Hz, 5-Ar-3,5-H), 8.31 (2H, m, J* = 8 Hz, S-Ar-2,6-H), 8.64 (3H, m, 10, 15, 20-
Py-5-H),
9.03-9.25 (8H, m, (3-H), 9.35 (3H, m, 10, 15, 20-Py-6-H), 9.57 (3H, m, 10, 15,
20-Py-4-
H), 10.03 (3H, s, 10, 15, 20-Py-2-H); 13C NMR [67.5 MHz, (CD3)ZSO] 848.3,
112.3,
112.9, 120.7, 124.8, 126.3, 126.4, 126.6, 130.6, 132.1, 132.3, 132.4, 132.6,
132.8, 133.1,
133.4, 134.7, 135.4, 139.8, 139.9, 140, 145.5, 145.6, 147.4, 147.5, 147.8,
147.9, 148.5,
155.9; UV-vis (H20) ~,",<«419, 516, 552, 581, 637 nm; MS (MALDI-TOF) m%z 689
([M-
2CH~]T, 100%).
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Example 6
5-(4-Isothiocyanatophenyl)-10,15,20-tris(4-N methylpyridiniumyl) porphyrin
trichloride
Ci Me- ~ Me Cl
Me
C1
To a solution of Example 4 (30 mg, 0.027 mmol) in anhydrous methanol (30 mL)
was added Amberlite° IRA 400 (lg) and the mixture stirred for 1 hour at
room
temperature. Amberlite° IRA 400 resin was filtered under vacuum and the
porphyrin
filtrate recovered, dried (Na2S0~) and evaporated i~z vacuo to yield the above
compound
as a water soluble purple solid (22 mg, 96.4%). Porphyrins of Examples 4 and 6
were
distinguished only by their respective solubility in water.
Example 7
5-(4-Isothiocyanatophenyl)-10,15,20-tris(4-N methylpyridiniumyl) porphyrin
trichloride
49 =
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CI
~e
Me
Cl
IVIC
CI
To a solution of 5 (30 mg, 0.027 mmol) in anhydrous methanol (30 mL) was
added Amberlite° IR.A 400 (lg) and the mixture stirred for 1 hour at
room temperature.
Amberlite° IRA 400 resin was filtered under vacuum and the porphyrin
filtrate
recovered, dried (NaZS04) and evaporated ifZ vacuo to yield the above compound
as a
water soluble purple solid (21 mg, 92.0%). Porphyrins of Examples 5 and 7 were
distinguished only by their respective solubility in water.
Example 8
17,18-Dihydroxy-5-(4-isothiocyanltophenyl)-15-(4-methoxyphenyl)chlorin, The
higher Rt~regioisomeric chlorin 10 (17.5 mg, 23.2 p.mol) was converted into
the
corresponding isothiocyanate according to the following method. To a stirred
solution of
( 1 ec~., 50 mg, 0.099 mmol) in freshly distilled DCM (20 mL) was added 1,1'-
thiocarbonyldi-2(11~-pyridone (2 eq., 46 mg, 0.198 mmol). The reaction was
allowed to
stir under argon for 2 h at room temperature, after which the reaction mixture
was
filtered, and concentrated then chromatographed, eluting with 1% MeOH in DCM
to
afford the title compound. The title compound was isolated as a brown-purple
crystalline
solid, (17 mg, 90%), Rf= 0.36 (DCM/MeOH, 19:1), mp 155-158°C
(decomposed); UV-
vis (DCM) ~.",,1~ (relative intensity) 410 (1.0) 505 (0.09), 534 (0.06), 586
(0.04), 637
(0.18) nm; Fluorescence (DCM) 7~",~~ 639 nm (~, excitation = 412 nm); 1H NNiR
(270
MHz, 10% CD30D in CDC13) 8 10.0 (s, 1H, 10-I~, 9.45 (s, 1H, 20-I~, 9.20 (d,
1H, J =
50 -
CA 02414089 2002-12-02
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4.8 Hz, ,C~H), 9.06 (d, 1H, J = 4.0 Hz, ~ H), 9.02 (d, 1H, J = 4.8 Hz, ~3 H)
8.84 (d, 1H, J =
4.8 Hz, ,C~H), 8.64 (d, 1H, J = 4.0 Hz, ~3 H), 8.55 (d, 1H, J = 4.8 Hz, ~3 H),
8.21 (m, 1H, J
= 8.1 Hz, ~-o-Ar), 8.15 (m, 1H, J = 8.1 Hz, ~-o An), 8.05 (m, 1H, J = 8.9 Hz,
1.5-o-Ar~)
7.93 (m, 1H, J = 8.9 Hz, !~-o-Af~), 7.65 (m, 2H, ~-n~-An), 7.24 (m, 2H, l.iayz-
Ar), 6.43 (d,
1H, J = 6.5 Hz, 17-H), 6.04 (d, 1H, J = 6.5 Hz, 18-H), 4.08 (s, 3H, CH;),
(NH's
exchanged & OH's not observed); MALDI-MS m/z 583.7 ([M=H]+, 100%); ES-HRMS
calcd. for C3aH2~N;03S ([M+H]t) 584.1757, found 584.1756.
Example 9
cis-7,8,17,18-Tetrahydroxy-5-(4-isothiocyanatophenyl)-15-(4-
methoxyphenyl)bacteriochlorin
The ci.s-bacteriochlorin 14 (8.5 mg, 10.7 p.mol) in 25% MeOH in DCM (1.25 mL)
was
treated with piperidine (.SO eq., 53 p.l , 0.53 mmol) and left to stir for a
period of 3 h at
room temperature under N2 with light excluded. The reaction mixture was
concentrated
ira vcrc~ro (0.1 torr) to remove all traces of piperidine. The crude amine was
then converted
into the reduired isothiocyanate following the procedure described above The
cis-
bacteriochlorin_isothiocyanate was isolated as a pink-green crystalline solid
(5.0 mg,
76%), Rf~= 0.40 (DCMIMeOH, 19:1), mp 132-135°C (decomposed); UV-vis
(DCM) ~,max
(relative intensity) 375 (1.0) 516 (0.22), 702 (0.48) nm; Fluorescence ~,max
709 nm (~,
excitation = 516 nm); 1H NMR (270 MHz, 10% CD30D in CDC13) 8 9.20 (s, 1H, meso-
H), 9.18 (s, 1H, n2eso-H), 8.77 (d, 2H, J = 4.8 Hz, ,l3-H), 8.40 (d, 1H, J =
4.8 Hz, ,<3 H),
8.34 (d, 1H, J = 4.8 Hz, ,Q-H), 8.14 (m, 2H, o Af~), 8.05 (m, 2H, o' An), 7.42-
7.08 (m, 4H,
~+l~-m-Ar), 6.20 (m, 2H, 7+17-H), 5.98 (m, 1H, 8-H), 5.93 (m, 1H, 18 I~, 4.04
(s, 3H,
CH;), -1.80 (br s, 2H, partly exchanged-NH), (OH's not observed); MALDI-MS m/z
618.9 ([M+H]+, 100%); ES-HRMS calcd. for C3~IZgN;OSS ([M+H]+) 618.1815, found
618.1810.
Examples 10, 11, 12, 13
17,18-Dihydroxy-5-(4-acetamidophenyl)-15-(4-methoxyphenyl)chlorinl7,8-
dihydroxy-5-(4-acetamidophenyl)-15-(4-methoxyphenyl)chlorin regioisomers, and
51
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cis/trap.s-7,8,17,18-tetrahydroxy-5-(4-acetamidophenyl)-15-(4-
methoxyphenyl)bacteriochlorin stereoisomers,
Porphyrin 8 (100 mg, 0.18 mmol) was converted, in a single reaction, to a
mixture of
chlorin diols/bacteriochlorin tetrols following the procedure of Sutton et al.
After 38 h the
reaction was stopped. The crude reaction mixture was then chromatographed,
eluting
with 2% MeOH in DCM to give first, some un-reacted starting material then the
higher
Ri~ chlorin isomer of Example 10 as a brown-purple crystalline solid (5 mg,
5%). The
lower Rt~ isomer of Example 11 was obtained by further elution with 3.5% MeOH
in
DCM and gave also a brown-purple crystalline solid (7.0 mg, 7%). Further
elution with
5% MeOH in DCM afforded the required tj~ar~sicis-bacteriochlorin tetrols of
Examples 12
and 13 respectively as pink/green solids (5.0 mg, 5%) and (7.0 mg, 7%)
respectively.
High Rt~ chlorin regioisomer of Example 10 (17,18-dihydroxy-15-(4-
methoxyphenyl)-5-
(4-acetamidophenyl) chlorin assigned on the basis of past data)(~6) R~= 0.40
(DCM/MeOH, 37:3), mp 186-188°C (decomposed); UV-vis (DCM) ~,max
(relative
intensity) 410(1.0), 505.5 (0.12), 535 (0.08), 585.5 (0.05), 637 (0.18) nm;
Fluorescence
(DCM) 7~",;,~ 639 nm (~, excitation = 410 nm); 1H NMR (270 MHz, CDCl3) 8 9.97
(s, 1H,
10-I~, 9.42 (s, 1H, 20-H), 9.19 (d, 1H, J = 4.0 Hz, ~3-H), 9.03 (d, 1H, J =
4.0 Hz, ,(3-H),
8.98 (d, 1H, J = 4.8 Hz, /3-H) 8.89 (d, 1H, J = 4.0 Hz, ,~3-H), 8.70 (d, 1H, J
= 4.8 Hz, /3
H), 8.52 (d, 1H, J = 4.8 Hz, ,(3-H), 8.14-8.10 (m, 3H, S-olna At~), 7.96-7.82
(m, 3H, ~+IS-
o%jn-Ar), 7.50 (s, 1H, NH), 7.34 (m, 2H, IS-na Af°), 6.48 (m, 1H, 17-
H), 6.20 (m, 1H, I~-
H), 4.08 (s, 3H, CH;), 2.38 (s, 3H, CH;), -1.89, -2.20 (s, 2H, NH); MALDI-MS
miz 582.6
([M+H]~, 100%); ES-HRMS calcd. for C3sHzsNsOa ([M+H]+) 582.2141, found
582.2137.
Low Rt~ chlorin regioisomer of Example 11 (7,8-dihydroxy-5-(4-aminophenyl)-15-
(4-
methoxyphenyl)chlorin) Rf= 0.35 (DCM/MeOH, 37:3), mp 182-185°C
(decomposed);
UV-vis (DCM) ~,",a,; (relative intensity) 410(1.0), 505.5 (0.1), 535 (0.07),
585 (0.04), 636
(0.19) nm; Fluorescence (DCM) ~,",ax 639 nm (~, excitation = 410 nm); 1H NMR
(270
MHz, 10% DMSO-d~ in CDC13) 8 9.96 (s, 1H, 10 ~, 9.92 (s, 1H, NH), 9.42 (s, 1H,
20-
H), 9.22 (m, 1H, ~H), 9.02 (d, 1H, J = 4.8 Hz, ,(3-H), 9.00 (m, 1H, ,l3-I~
8.92 (m, 1H, ,l3
H), 8.70 (d, 1H, J = 4.8 Hz, ,(3-FI), 8.53 (m, 1H, ,(3 I~, 8.18-7.91 (m, 6H,
5+1.5-olm Ar),
7.33-7.28 (m, 2H, L5-n~ Ar), 6.36 (m, 1H, 7-H), 5.96 (m, 1H, 8 II), 4.10 (s,
3H, CHj),
52
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2.30 (s, 3H, CH;), -1.74, -2.17 (s, 2H, NH); MALDI-MS m/z 582.6 ([M+H]+,
100%). ES-
HRMS calcd. for C35H28N;Oa ([M+H]+) 582.2141, found 582.2135.
High Rt~trccrts bacteriochlorin of Example 12; Rf= 0.29 (DCM/MeOH, 37:3:1), mp
152-
155°C (decomposed); UV-vis (DCM) ~.max (relative intensity) 373.5 (1.0)
514 (0.25), 702
(0.49) nm; Fluorescence (DCM) ~,max 708 nm (~, excitation = 514 nm); 1H NMR
(270
MHz, DMSO-d~) 8 10.27 (s, 1H, NH), 9.16 (s, 2H, 10+20-H), 8.96 (d, 2H, J = 4.0
Hz, ~
H), 8.24 (d, 2H, J = 4.0 Hz, /.~H), 7.99-7.89 (m, 6H, 5+1 S-olm Ar), 7.25 (m,
2H, I S-m-
An), 6.30 (m, 2H, 7+17-H), 6.15 (m, 2H, 8+18 l~, 5.63 (m, 2H, Ol~, 5.32 (m,
2H, OH),
3.99 (s, 3H, CH3), 2.20 (s, 3H, CH3), -1.87 (br s, 2H, NH); MALDI-MS m/z 616.3
([M+H]1, 100%). ES-HRMS calcd. for C35H3oNsOs ([M+H]+) 616.2196, found
616.2192.
Low Rt~ cis-bacteriochlorin 13; Rf= 0.24 (DCM/MeOH, 19:1), mp 148-
151°C
(decomposed); UV-vis (DCM) ~,maX (relative intensity) 373.5 (1.0) 514.5
(0.24), 703
(0. SO) nm; Fluorescence (DCM) ~,max 708 nm (~, excitation = 514 nm); 1H NMR
(270
MHz, 20% CD30D in CDC13) 8 9.20 (s, 2H, 10+20-H), 8.78 (m, 2H, ,Q H), 8.35 (m,
2H,
/.3 H), 8.05 (m, 2H, 5-~ AY), 7.89-7.86 (m, 3H, 5+IS-olm Ar), 7.75 (m, 1H, IS-
o Ar),
7.20-7.17 (m, 2H, 15-m Ar), 6.25 (m, 2H, 7+17-F~, 5.86 (m, 2H, 8+18-H), 4.06
(s, 3H,
C:H;), 2.31 (s, 3H, CHj), (NH's exchanged); MALDI-MS m/z 616.4 ([M+H]+, 100%).
ES-HRMS calcd. for C35H30N506 ((M+H]~) 616.2196, found 616.2192.
Example 16 5-(4-Isothiocyanatophenyl)-10,15,20-tri(4-
methylphosphoniumphenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-(4-
methylphosphoniumphenyl)- porphyrin - General Synthetic Procedure
Boc N-protected 5-(4-aminophenyl)-10,25,20-tri-(4-carbomethoxyphenyl)
porphyrin and
5-(4-aminophenyl)-15-(4-carbomethoxyphenyl) porphyrin were synthesised by
mixed
condensation using Lindsey conditions (Lindsey, J.S., Schreiman, LC., Hsu,
H.C.,
I~earney, P.C., Marguerettaz, A.M. (1987) .I. Org. Che»a. 52, 827) or by 2+2
condensation methodology via the appropriately substituted 5-
phenyldipyrromethanes as
described by Boyle et al (Boyle, R.W., Bruckner, C., Posakony, J., James,
B.R., Dolphin,
D. ( 1999) Ongarzic Sy~ztheses. 76, 287 - incorporated herein by reference)
respectively.
The (4-carbomethoxyphenyl) groups on these porphyrins were then converted to
(4-(1-
bromomethyl)phenyl) groups using the following standard procedure: the
porphyrin (0.2
53 =
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mmol) was dissolved in dry THF (25 ml) at 0°C and stirred under argon
for 10 minutes.
Lithium aluminium hydride (0.7 mmol) was added and the stirring continued for
24
hours. The reaction was monitored by TLC and, when the reaction was complete
ethyl
acetate (2 ml) was added and the mixture washed with aqueous HCl (0.2 M, 20
ml),
saturated sodium bicarbonate solution (30 ml) and finally, brine (20 ml). The
organic
layer was dried (MgSO~) and evaporated to dryness to yield the corresponding
(4-(1-
hydroxymethyl)phenyl) substituted porphyrins, bearing three or one reduced
carbomethoxy groups respectively. (4-(1-Hydroxymethyl)phenyl) substituted
porphyrins
(0.2 mmol) were dissolved in dry chloroform (40 ml) and stirred under argon
while
triphenylphosphine (1.0 mmol) and carbon tetrabromide (1.6 mmol) were added.
The
reaction was stirred, in the dark, for 24 hours and then monitored by TLC.
Once all the
hydroxymethyl groups had been converted to bromomethyl groups the reaction
mixture
was diluted with dichloromethane (40 ml), washed with saturated sodium
bicarbonate (2
x 20 ml) then brine (2 x 20 ml) and the organic layer dried (MgSO~,). Removal
of solvent
by evaporation in vacuo afforded the corresponding bromomethyl porphyrins as
purple
crystalline solids.
Boc N-protected 5-(aminophenyl)-10,25,20-tri-(4-bromomethylphenyl) porphyrin
and 5-
(aminophenyl)-15-(4-bromomethylphenyl) porphyrin (0.75 mmol) were dissolved in
dry
dichloromethane (50 ml) under an atmosphere of argon at 25°C. Triaryl
or
trialkylphosphine (7.5 mmol) dissolved in dry dichloromethane (10 ml) was
injected by
syringe and the progress of the reaction was followed by TLC. Upon completion
the
solvent was evaporated from the reaction in vacuo and the crude product was
purified by
flash column chromatography (silica; gradient elution: dichloromethane to
methanol) to
give the required Boc-N-protected-5-(aminophenyl)-methylphosphonium-meso-aryl
porphyrins as lustrous purple crystalline solids. The Boc protecting group was
removed
by dissolution of the porphyrin in chloroform or acetonitrile, depending upon
solubility,
and addition of trimethylsilyl iodide (5.0 equivalents), after 30 minutes the
reaction was
quenched with methanol (10 ml). Removal of solvent by evaporation, followed by
purification by flash column chromatography (silica; gradient: dichloromethane
to
methanol) gave the 5-(aminophenyl)-methylphosphonium-meso-aryl porphyrins
which
were converted to the required mono-4-(isothiocyanatophenyl) compounds by
treatment
~4 --
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with 1,1'-thiocarbonyldi-2(1H)-pyridone using standard procedures (Clarke,
O.J. and
Boyle, R.W. (1999.LC.S. Chem. Cornmicn. 2231).
Example 17
5-(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylphosphono-di-ethoxy)phenyl)-
porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylphosphono-di-
ethoxy)phenyl)- porphyrin - General Synthetic Procedure
Boc N-protected 5-(4-aminophenyl)-10,15,20-tri-(4-bromomethylphenyl) porphyrin
and
5-(aminophenyl)-15-(4-bromomethylphenyl) porphyrin (0.75 mmol) were dissolved
in a
mixture of triethyl phosphite (15 mmol) and dry acetonitrile (50 ml). A reflux
condenser
was fitted and the reaction was refluxed under argon. The reaction was
followed by TLC
and upon completion was washed with saturated sodium
bicarbonate (2 x 20 ml), water (2 x 20 ml) and brine (2 x 20 ml). The organic
layer was
then dried (MgSO:~) and the solvent evaporated in vacuo. The crude product was
then
purified by flash column chromatography (silica; gradient elution:
dichloromethane to
ethyl acetate) to give the title compounds as purple crystalline solids. The
methylphosphono-di-ethoxy groups were then deprotected to either
methylphosphono-
mono-ethoxy sodium groups by sonication in aqueous sodium hydroxide for 1 hour
followed by reversed phase medium pressure chromatography (C18; gradient
elution 0.1%
aqueous TFA to methanol) (Boyle, R.W. and van Lier, J.E. (1993) Synlett 351),
or to the
fully deprotected methylphosphonic acids by treatment with
bromotrimethylsilane (2
equivalents per methylphosphono-di-ethoxy group) for 2 hours followed by
reversed
phase chromatographic purification chromatography (CIB; gradient elution 0.1%
aqueous
TFA to methanol) (McI~enna, C.E., Higa, M.T., Cheung, N.H., McI~enna, M-C.
(1977)
2, 155). Boc deprotection (see above) followed by conversion of the unmasked 4-
(aminophenyl) group to its isothiocyanato analogue was performed using
standard
procedures (Clarke, O.J. and Boyle, R.W. (1999 J. C. S. Chena. Com~rrura.
2231)_
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Example 18
5-(4-Isothiocyanatophenyl)-10,15,20-tri((4-methylphosphonato-di-ethoxy)phenyl)-
porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-methylphosphonato-di-
ethoxy)phenyl)- porphyrin - General Synthetic Procedure
Boc N-protected 5-(aminophenyl)-10,15,20-tri-(4-hydroxymethylphenyl) porphyrin
and
5-(aminophenyl)-15-(4-hydroxymethylphenyl) porphyrin (0.75 mmol) were
dissolved in
a mixture of dry dichloromethane and pyridine (4:1) under an atmosphere of
argon.
Diethyl chlorophosphate (2 equivalents per hydroxymethyl group) was injected
and the
mixture was stirred for 16 hours. Evaporation of solvent from the reaction
mixture
followed by chromatographic purification gave the corresponding tri or mono
((4-
methylphosphonato-di-ethoxy)phenyl) porphyrins. Treatment with aqueous sodium
hydroxide ( 1M) gave the sodium salts of tri or mono ((4-
methylphosphonatoethoxy)phenyl) porphyrins (Boyle, R.W. and van Lier, J.E.
(1995)
Synthesis 1079). Boc deprotection and generation of the isothiocyanato group
were
performed as described above.
Example 19 5-(4-Isothiocyanatophenyl)-10,15,20-tri((4-
methylpyridiniumyl)phenyl)- porphyrin and 5-(4-isothiocyanatophenyl)-15-((4-
methylpyridiniumyl)phenyl)- porphyrin - General Synthetic Procedure
Boc N-protected 5-(aminophenyl)-10,25,20-tri-(4-bromomethylphenyl) porphyrin
and 5-
(aminoophenyl)-15-(4-bromomethylphenyl) porphyrin (0.75 mmol) were dissolved
in
dichloromethane (50 ml) and pyridine (15 mmol), or substituted pyridine (15
mmol), as
required, were added. A reflux condenser was fitted and the reaction was
refluxed under
argon. The reaction was followed by TLC and, upon completion, was evaporated
to
dryness in vacuo. The residue was purified by reversed phase medium pressure
chromatography (C1R; gradient elution 0.1% aqueous TFA to methanol) to yield
the N-
Boc protected 4-aminophenyl compounds. Deprotection of the aminophenyl groups)
and
conversion to the isothiocyanato analogues) were conducted using the standard
protocols
(see above).
~6 =
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Example 20 5-(4-Isothiocyanatophenyl)-15-axyl-10,20-(1,2-dihydroxyethyl)-
porphyrin - General Synthetic Procedure
The Fmoc protected 5-(4-aminophenyl)-15-aryl porphyrin (0.8 mmol) was
dissolved in
dry chloroform (300 ml) under an atmosphere of argon. Freshly recrystallised N-
bromosuccinimide (1.8 mmol) in dry chloroform (20 ml) was injected by syringe
and the
mixture was stirred for 30 min. The solvent was then evaporated in vacuo and
the crude
product purified by flash column chromatography (silica; gradient elution:
hexane to
ethyl acetate) to give the required 5, 15-dibromo-10, 20-diarylporphyrin as a
purple
crystalline solid. The product was then metallated by refluxing in a
chloroform/methanol
(9:1 ) solution of zinc acetate dihydrate (80 mmol). The metallation was
followed by
visible spectroscopy and, upon completion, was passed through a short column
of neutral
alumina to remove uncoordinated zinc. The zinc 5,15-dibromo-10, 20-
diarylporphyrin
(0,6 mmol) was dissolved in dry THF to which had been added
tetrakis(triphenylphosphine)-palladium(0) (0.6 mmol) and vinyltributyltin (1.4
mmol).
The mixture was refluxed under nitrogen for 48 hours after which the solvent
was
evaporated in vacuo and the residue chromatographed by flash column (silica;
gradient
elution: dichloromethane to ethyl acetate) to give zinc 5-(Fmoc aminophenyl)-
15-aryl-
10,20-diethenyl porphyrin as a purple crystalline solid. Zinc 5-(Fmoc
aminophenyl)-15-
aryl-10,20-diethenyl porphyrin was demetallated by dissolution in a solution
of
trifluoroacetic acid in dichloromethane (1% vlv) to give 5-(Fmoc aminophenyl)-
15-aryl-
10,20-diethenyl porphyrin after extracting with water and evaporation of
solvent from
the organic layer in vacuo. Finally the 10 and 20 ethenyl groups were
hydroxylated by
osmium tetroxide as described (Sutton J, Fernandez N, Boyle RW (2000) J.
Porphyrins
and Phthalocyanines 4, 655), however due to the rapidity of the reaction
between the
ethenyl groups and osmium tetroxide it was possible to selectively hydroxylate
these
groups by control of reaction time and stoichiometry. In a typical set of
conditions the 5-
(Fmoc aminophenyl)-15-aryl-10,20-diethenyl porphyrin, when treated with osmium
tetroxide (5 equivalents) in 10°f° pyridine/chloroform for 24 -
48 hours, gave the desired
5-(Fmoc aminophenyl)-15-aryl-10,20-bis(1,2-dihydroxyethyl) porphyrin, while if
longer
reaction times (72 hours) and higher molar ratios of osmium tetroxide (7.5 or
10
equivalents) are used under the same conditions 5-(Fmoc aminophenyl)-15-aryl-
10,20-
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bis( 1,2-dihydroxyethyl) 7,8-dihydroxychlorin and 5-(Fmoc aminophenyl)-15-aryl-
10,20-
bis( 1,2-dihydroxyethyl) 7,8,17,18-tetrahydroxybacteriochlorin respectively
are obtained.
All the above products are converted cleanly to the corresponding
isothiocyanates upon
piperidine mediated deprotection of the amino group (see above) and treatment
with l, l'-
thiocarbonyldi-2(1H)-pyridone using standard procedures (Clarke, O.J. and
Boyle, R.W.
( 1999 ,I. C. S. Chena. Common. 2231 ).
Example 21 5-(4-Isothiocyanatophenyl)-15-phenyl-10,20-(diaryl)-porphyrins -
Synthesis from 5,15-diphenyl porphyrins by Pd° mediated Suzuki
coupling
Boc N-protected 5-(aminophenyl)-15-phenyl porphyrin was brominated at the 10
and 20
meso positions as described above. The meso-10,20-dibrominated product (0.75
mmol)
was dissolved in dry THF (50 ml) or toluene (50 ml), depending upon the
boronic acid
used in the coupling reaction, tetrakis-(triphenylphosphine) palladium (0)
(0.75 mmol)
and anhydrous potassium phosphate (0.75 mmol) were added, a reflux condenser
was
then fitted to the flask and the whole apparatus was placed under an
atmosphere of argon.
The required aryl or heterocyclic boronic acid was then added as a solution in
the
appropriate solvent (10 ml) by injection. The reaction was brought to reflux
and followed
to completion by TLC. On completion the crude reaction mixture was diluted
with
dichloromethane (100 ml) and extracted with saturated sodium bicarbonate (2 x
50 ml),
water (2 x 50 ml) and brine (2 x 50 ml). The organic phase was dried (Mg SOa)
and
concentrated by evaporation in vacuo. Finally, the residue was purified by
flash column
chromatography (silica; gradient elution: dichloromethane to methanol) to give
the Boc
N-protected 5-(aminophenyl)-15-phenyl-10,20-(diaryl)-porphyrin as a purple
crystalline
solid. The Boc protecting group was removed by dissolution of the porphyrin in
chloroform or acetonitrile, depending upon solubility, and addition of
trimethylsilyl
iodide (1.2 equivalents), after 30 minutes the reaction was quenched with
methanol (10
ml). Removal of solvent by evaporation followed by purification by flash
column
chromatography (silica; gradient: dichloromethane to methanol) gave the 5-
(aminophenyl)-15-phenyl-10,20-(diaryl)-porphyrin which was converted to the
title
compound by treatment with 1,1'-thiocarbonyldi-2(1H)-pyridone using standard
procedures (Clarke, O.J. and Boyle, R.W. (1999 J. C. S. Chem. Conamun. 2231).
58 _
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Example 22 5-(4-lsothiocyanatophenyl)-10,15,20-tri(4-glycosylphenyl)-
porphyrin
and 5-(4-isothiocyanatophenyl)-15-(4-glycosylphenyl)- porphyrin - General
Synthetic Procedure
4-(2',3',4',6'-tetra-O-acetyl-(3-D-glucopyranosyloxy)benzaldehyde was
condensed with
4-nitrobenzaldehyde and pyrrole using Lindsey conditions (Sol, V., Blais,
J.C., Carre, V.,
Granet, R., Guilloton, M., Spiro, M., I~rausz, P. (1999) J. Org. Chenz. 64,
4431) and the
crude reaction mixture purified by flash column chromatography to give 5-(4-
nitrophenyl)-10,15,20-tris[4-(2',3',4',6'-tetra-O-acetyl-(3-
glucopyranosyloxy)phenyl]
porphyrin. Alternatively, 4-(2',3',4',6'-tetra-O-acetyl-(3-D-
glucopyranosyloxy)benzaldehyde was used to synthesise 5-(4-(2',3',4',6'-tetra-
O-acetyl-
(3-D-glucopyranosyloxy)phenyl) dipyrromethane using the method of Boyle
(Boyle,
R.W., Bruckner, C., Posakony, J., James, B.R., Dolphin, D. (1999) Organic
Syntheses.
76, 287) which was then condensed to give 5-(4-nitrophenyl)-15,-[4-
(2',3',4',6'-tetra-O-
acetyl-[i-glucopyranosyloxy)phenyl] porphyrin. Reduction of the nitro group of
these
porphyrins was performed by dissolution in THF and addition of 10% palladium
on
carbon. Stirring of the mixture under HZ for 5 hours followed by filtration
through Celite
and purification by flash column chromatography gave the corresponding amino
porphyrins, which were N-protected by reaction with Fmoc chloride (2
equivalents) in
anhydrous 1,4-dioxane in the presence of sodium bicarbonate (6 equivalents)
under
argon. The reaction was monitored by TLC and, upon completion, diluted with
dichloromethane and washed with water then brine before drying the organic
layer
(MgSO~). Purification by flash column chromatography gave the Fmoc N-protected
5-(4-
aminophenyl)-10,15,20-tris[4-(2',3',4',6'-tetra-O-acetyl-(3-
glucopyranosyloxy)phenyl]
porphyrin or 5-(4-aminophenyl)-15,-[4-(2',3',4',6'-tetra-O-acetyl-(3-
glucopyranosyloxy)phenyl] porphyrin. N and O protecting groups were removed by
dissolution of the porphyrin in dichloromethane/morpholine (1:1) and stirring
for 1 hour.
Removal of solvent by evaporation in vacuo was followed by redissolution of
the residue
in a mixture of dichloromethane and methanol (4:1). Sodium methanolate in dry
methanol(1.5 equivalents per OAc group) was added and the mixture stirred for
1 hour.
The fully deprotected porphyrin was recovered by precipitation with hexane.
Finally, the
59 =
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5-(4-aminophenyl) porphyrin was dissolved in dry methanol and l, l'-
thiocarbonyldi-
2(1H)-pyridone (2 equivalents) was added. The reaction was stirred under argon
for 2
hours and monitored by TLC, upon completion, solvent was evaporated in
vacuo~and the
crude product was purified by preparative medium pressure reversed phase
chromatography (Clg;gradient elution: 0.1% aqueous TFA to methanol).
Example 23 : Symmetrical Porphyrin/ Chlorin Diol/ Bacteriochlorin Tetrol
Series
~,1~-(3,4,~-Tris»zetlzoxyphenyl)porphyrin (,general procedure)
To a 3 L round bottom flask was added 5-(3,4,5-
trismethoxyphenyl)dipyrromethane (1.86
g, 6 mmol), then DCM (1L) under NZ. To this stirred solution was added
trimethylorthoformate (48 ml, mmol). A pressure equalizing dropping funnel
containing a
solution of trichloroacetic acid (23.0 g, mmol) in DCM (500 ml) was then
fitted to the
flask and the solution added dropwise to the reaction mixture over a period of
10 min.
The reaction vessel was covered in aluminium foil to exclude light and allowed
to stir
under NZ for a period of 3.5 h. Pyridine was then added to the reaction
mixture, rapidly
with stirring, and the reaction allowed to stir for a further 16 h. at room
temperature under
NZ with the light excluded. The aluminium foil was removed and air was bubbled
through
the solution for a period of 20 min. After this the reaction was left to stir
unstoppered for
a further period of 3 h. at room temperature with the aluminium foil removed.
The
reaction mixture was then concentrated in vacuo to remove DCM and remaining
pyridine
by evaporator, then high vacuum. The crude reaction mixture was then
chromatographed
on flash silica-gel (250 ml), (dry loaded on to 50 ml flash silica-gel from
DCM and a
little methanol to ensure complete solubility) eluting with chloroform . The
title
compound was obtained as purple crystalline solid (347 mg, 18%); ~,max/
(relative
intensity) 410 (1.0), 502 (0.04), 538 (0.02), 578 (0.015), 630 (0.01) nm; UV-
VIS
(CH~CIz) (fluorescence) 7~""t, = 634 nm (7~ excitation = 408 nm); (270 MHz,
CDCl3)
10.32 (2H, s, I Q-H, ?D-H), 9.40 (4H, d, J = 4.8 Hz, ~3 H), 9.18 (4H, d, J =
4.8 Hz, ,(3 H),
7.52 (4H, s, o An), 4.20 (6H, s, CHj), 4.00 (12H, s, CHj), -3.10 (2H, br. s,
NH); MS
(MALDI) m/z = 643.4 (100%, M+).
CA 02414089 2002-12-02
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7, 8-Dihplroxy ~,1 ~-(3, 4, ~-trismethoxyphenyl) clzlorin (A General
procedure)
To a stirred solution of 5,15-(3,4,5-trismethoxyphenyl)porphyrin (50 mg, 77.8
p.mol) in
HPLC grade chloroform (5.0 ml) was added a solution, in anhydrous pyridine
(0.5 ml), of
osmium tetroxide (~..5 eq., 0.195 mmol, 49 mg). The reaction vessel was
flushed with NZ
and sealed with a lightly greased glass stopper, then covered in aluminium
foil to exclude
the light and left to stir for 72 h at room temperature. After this period the
reaction
vessels glass stopper was replaced With a plastic stopper and a continuous
stream of
hydrogen sulfide gas was bubbled through the reaction mixture for 5 min., (a
gas outlet
needle was attached and allowed excess hydrogen sulfide gas to escape into a
series of
Dreshel bottles filled with mineral oil and a bleach solution respectively).
After this time
the reaction mixture was filtered through Celite~ and then concentrated in
vacuo. Any
excess pyridine was removed under high vacuum. The crude reaction mixture was
then
chromatographed on flash silica-gel (100 ml), (dry loaded on to 20 ml flash
silica-gel
from DCM and a little methanol to ensure complete solubility) eluting with 1%
methanol
in DCM. Some starting material was recovered (15%) and the title compound was
obtained as browny-purple crystalline solid , (26 mg, 50%); m.p. 170 °C
(decomposed);
UV-VIS (CHZC12) 7~ma~ (relative intensity) 410 (1.0) 504 (0.09), 534 (0.06),
582 (0.04),
636 (0.18) nm; UV-VIS (CHZC12) (fluorescence) ~,max 639 nm (~, excitation 410
nm);
SH(270 MHz, CDC13) 9.98 (1H, s, 10-H), 9.42 (1H, s, ?0-H), 9.20 (1H, m,,QH),
9.04
( 1 H, d, J = 4. 0 Hz, /.3 I~, 8 . 99 (2H, s, ~3 H), 8. 79 ( 1 H, d, J = 4. 0
Hz, ,(3 H), 8. 66 ( 1 H, m, ,~i
H), 7.45 (1H, d, J = 1.6 Hz, l~-o Ar), 7.42 (1H, d, J = 1.6 Hz, l.i-o-Ar),
7.40 (1H, d, J =
1.6 Hz, ~-o Ar), 7.19 ( 1 H, d, J = 1.6 Hz, 15-o Ar), 6.49 ( 1 H, d, J = 7. 3
Hz, 7-H), 6.23
(1H, d, J = 7.3 Hz, 8-H), 4.17 (3H, s, GH;), 4.15 (3H, s, CH3), 4.04 (3H, s,
CH3), 4.00
(3H, s, CHI), 3.98 (3H, s, CH3), 3.91(3H, GH3), -1.80 (1H, br. s, NH), -2.19
(1H, br. s,
NH), (OH'.s not observed); MS (MALDI) m/z = 677.3 (100%, M+); HRMS calcd. for
C3RH~~N:~Og: 676.2533. Found: 676.2587.
7,8,17,18-Tetrahydroxy~,l~-(3,4,x-trismethoxyphenyl) bacteriochlorin ( n r 1
r ce ure)
To a stirred solution of 5,15-(3,4,5-trismethoxyphenyl)porphyrin (50 mg, 77.8
pmol) in
HPLC grade chloroform (5.0 ml) was added a solution, in anhydrous pyridine
(0.5 ml), of
61
CA 02414089 2002-12-02
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osmium tetroxide (~.0 ed., 0.39 mmol, 49 mg). The reaction vessel was flushed
with NZ
and sealed with a lightly greased glass stopper, then covered in aluminium
foil to exclude
the light and left to stir for 72 h at room temperature. After this period the
reaction
vessels glass stopper was replaced with a plastic stopper and a continuous
stream of
hydrogen sulfide gas was bubbled through the reaction mixture for 5 min., (a
gas outlet
needle was attached and allowed excess hydrogen sulfide gas to escape into a
series of
Dreshel bottles filled with mineral oil and a bleach solution respectively).
After this time
the reaction mixture was filtered through Celite~ and then concentrated in
vcrcuo. Any
excess pyridine was removed under high vacuum. The crude reaction mixture was
then
chromatographed on flash silica-gel (100 ml), (dry loaded on to 20 ml flash
silica-gel
from DCM and a little methanol to ensure complete solubility) eluting
initially with 1%
methanol in DCM to elute chlorin by-product then 2.5 % methanol in DCM to
elute the
major bacteriochlorin isomer (assumed as tr~ans form Bruckner et cal (1995)
Tetrahedron
Lett. 3G, 9425). The title compound was obtained as a greeny-pink crystalline
solid, (20
mg, 3G%); m.p. 135°C (decomposed); UV-VIS (CH2Clz) ~.max (relative
intensity) 374
(1.0) 512 (0.23), 702 (0.52) nm; UV-VIS (CHZCIz) (fluorescence) 7~,na,; 708 nm
(~,
excitation 512 nm); 8H(270 MHz, CDCl3) 9.23 (2H, s, 10-H, 20-H), 8.79 (2H, d,
J = 3.2
Hz, ~3-H), 8.44 (2H, d, J = 3.2 Hz, ,~ 1~, 7.37 (2H, s, 5+15-o-Ar), 7.13 (2H,
s, S + 15-0-
Ar~), G.31 (2H, d, J = 6. 5 Hz, 7-H, 17-H), 6.01 (2H, d, J = 6.5 Hz, ~-H, I ~-
H), 4.12 (6H, s,
CH;), 3.92 (GH, s, CH;), 3.89 (6H, s, CH3), -1.97 (2H, br. s, NH), (OH's not
observed);
MS (1VIALDI) m/z = 712.4 (100%, (M+1)~'); HRMS calcd. for C3gH36N~O1~:
710.2590.
Found: 710.2607.
Example 24 : Unsymmetrical Porphyrin/ Chlorin Diol/ Bacteriochlorin Tetrol
Fluorochrome Sets for Bioconjugation
~-(4-AcetomidoplZenyl)-1 ~-(4-naetlaoxyplzenyl)porplZyrin
The required unsymmetrical diphenylporphyrin was synthesised using the general
procedure outlined earlier, but with only slight modification. In this example
a mixture of
dipyrromethanes were used. Due to the different reactivities of the respective
dpyrromethanes, the amounts needed for optimisation of mixed porphyrin were
different.
For the same scale reaction 5-(4-methoxyphenyl)dipyrromethane (505 mg, 2 mmol)
and
62
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5-(4-acetomidophenyl)dipyrromethane (838 mg, 3 mmol) were used. The porphyrin
mixture was chromatographed on silica-gel (400 ml), (dry loaded on to 20 ml
flash silica-
gel from DCM and a little methanol to ensure complete solubility) eluting
initially with
DCM ( 1 glass pipette full of triethylamine was added to 500 ml of eluent to
aid elution)
to remove 5,15-(methoxyphenyl)porphyrin byproduct. After separation of this
component
the elution was continued with chloroform to allow 5-(4-Acetomidophenyl)-15-(4-
methoxyphenyl)porphyrin collection. The desired porphyrin was obtained as
purple
crystals; (150 mg, 12%); 7~r"~,/ (relative intensity) 410 (1.0), 502 (0.04),
538 (0.02), 578
(0.015), 630 (0.01) nm; 8H(270 MHz, CDC13) 10.35 (2H, s, 10-H, 20-H), 9.43
(4H, d, J =
4. 8 Hz, ,(3-H), 9.14 (4H, d, J = 4. 8 Hz, f3 H), 8.65 (2H, d, J = 7.2 Hz, 5-
»a-Ar), 8.22-8.12
(4H, d (overlapping), J= 8.1 Hz, .5-o Ar+IS-o Ar), 7.56 (2H, d, J= 8.1 Hz, IS-
rrz Ar),
4.14 (3H, s, C:H;), -3.00 (2H, br. s, NH); MS (MALDI) m/z = 550.3 (100%, M~).
~-(4-Anzinophenyl)-1 ~-(4-i~iethoxyphenyl)porphyrin
5-(4-Acetomidophenyl)-15-(4-methoxyphenyl)porphyrin (100 mg, 0.182 mmol) was
treated with 18% hydrochloric acid (200 ml) and fitted with an air condenser.
The green
solution was left to warm to 85oC for a period of 3 h. Prior to cooling, the
reaction
mixture was concentrated irz vacuo (water aspirator; evaporator water bath at
75°C) to
remove excess hydrochloric acid then treated carefully with a solution of
triethylamine
(50 ml) in DCM. The organic extract was washed with water (100 ml) then
saturated
brine ( 100 ml) prior to drying (arzhyd. Na2S0~), filtering via Buckner funnel
and finally
concentration ifz vacuo. The required porphyrin was obtained by chromatography
on
silica-gel (100 ml), (liquid loaded in 10 ml DCM) eluting with DCM (1 glass
pipette full
of triethylamine was added to 500 ml of eluent to aid elution). The desired
porphyrin was
obtained as purple crystals; (150 mg, 12%); ~,",a,I (relative intensity) 410
(1.0), 503
(0.045), 538(0.02), 578 (0.015), 630 (0.005) nm; UV-VIS (CHZC12)
(fluorescence) ~,m~X=
634 nm (~, excitation = 410 nm); dH(270 MHz, CDC13) 10.30 (2H, s, 10-H, 20-H),
9.39
(4H, d, J = 4.9 Hz, ,l3-H), 9.17 (2H, d, J = 4.9 Hz, ,(3 H), 9.10 (2H, d, J =
4.9 Hz, ,l3 H),
8.19 (2H, d, J = 8.8 Hz, LS-o Ar), 8.07 (2H, d, J = 8.1 Hz, 5-o Ar), 7.35 (2H,
d, J = 8.8
Hz, I~-na Ar), 7.14 (2H, d, J = 8.1 Hz, S-m Ar), 4.13 (3H, s, CH3), 4.08 (2H,
br. s, NH), -
3.06 (2H, br. s, NH); MS (MALDI) m/z = 508.3 (100%, (M+1)~).
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~-(~-Fluorenonzetlzylanzinoplzenyl)-I'-(9-nze'tlzoxyphenyl)porphyrin
To a stirred solution of 5-(4-aminophenyl)-15-(4-methoxyphenyl)porphyrin (28
mg, 55
ymol) in anhydrous 1,4-dioxane (2.5 ml) was added solid sodium hydrogen
carbonate (6
ed., 28 mg, 0.33 mmol). To this mixture was then added a solution of 9-
fluorenomethylchloroformate (2 eq., 0.11 mmol, 28.5 mg) in 1,4-dioxane (0.5
ml) under
N~. The reaction flask was covered with aluminium foil to exclude light and
stirred at
room temperature for a period of 3 h. At this time the reaction had gone to
completion (as
monitored by TLC). The 1,4-dioxane was removed irz-vacuo and the residue
partitioned
between water (25 ml) and DCM (2 x 25 ml). The combined organic extracts were
washed with saturated brine (25 ml) then dried (anlzyd. Na2S04), filtered and
concentrated irmacuo. The required porphyrin was obtained by chromatography on
silica-gel ( 100 ml), (dry loaded on to 10 ml flash silica-gel from DCM and a
little
methanol for solubility) eluting with DCM. The desired porphyrin was obtained
as purple
crystals; ( 38 mg, 95%); 7~",a,I (relative intensity) 410 (1.0), 505 (0.042),
541 (0.02), 578
(0.015), 633 (0.01) nm; UV-VIS (CH2C12) (fluorescence) 7~",ax = 635 nm (~,
excitation =
410 nm); bH(270 MHz, CDC13) 10.35 (2H, s, I D-H, 20-H), 9.69 (1H, br. s, NH),
9.44
(4H, d, J = 4. 8 Hz, ,(3-H), 9.12 (4H, d, J = 4.8 Hz, /3 H), 8.20-8.17 (4H, 2
x d
(overlapping), J = 8.1 Hz, 5+I~-o Ar), 7.85 (4H, m, 5+15-»z Ar), 7.76-7.66
(2H, m,
fTll()7'E'll0-Ar), 7.51-7.30 (6H, m, fZurerzo Ar), 4.69 (2H, d, J = 7.2 Hz,
CH2), 4.30 (1H, t, J
= 7.2 Hz, CH), 4.13 (3H, s, CH3), -3.15 (2H, br. s, NH); MS (MALDI) mlz =
731.5
(100%, (M+1)+), 508.3 (52%, (M-FMOC+1)'~).
17,18-Dilzydroxy-S-(~ anzinoplzenyl)-l~-(4-nzethoxyplzenyl) chlorin and 7,8-
dihydroxy
~-(~l-caminopfzenyl)-1 ~-(4-nzetlzoxyplzenyl) chlorin regioisomers
5-(4-aminophenyl)-15-(4-methoxyphenyl)porphyrin (28 mg, 55.2 p,mol) was
converted to
a mixture of chlorin diol regioisomers using the general chlorin formation
procedure
given earlier. The crude reaction mixture was then chromatographed on flash
silica-gel
(200 ml), (dry loaded on to 20 ml flash silica-gel from DCM and a little
methanol to
ensure complete solubility) eluting with 1% methanol in DCM to elute first
some
unreacted starting material then the higher Rf chlorin isomer as a browny-
purple
64
CA 02414089 2002-12-02
WO 02/00662 PCT/GBO1/02846
crystalline solid. The lower Rt~ isomer was obtained by further elution with
2.5%
methanol in DCM and gave also a browny-purple crystalline solid.
~Hiah t chlorin regioisomer (2,3-dihydroxy-5-(4-methoxyphenyl)-15-(4-
aminophenyl)
chlorin, from nOe measurements and JPP paper): (8.5 mg, 30%); m.p.
165°C
(decomposed); UV-VIS (CHZC12) Amax (relative intensity) 401 (0.99), 414 (1.0),
503
(0.08), 535 (0.07), 582 (0.035), 636 (0.22) nm; UV-VIS (CHZC12) (fluorescence)
7~max
639 nm (~, excitation 412 nm); 8H(270 MHz, 10% MeOH-d4 in CDC13) 9.95 (1H, s,
10-
H), 9.42 ( 1 H, s, 20-H), 9.17 ( 1 H, d, J =4. 8 Hz, ~3 H), 9.03 ( 1 H, d,
J=4.0 Hz, ~3-H), 8. 97
(2H, s, ,(3-H) 8.78 (1H, d, J = 4.8 Hz, ,(3-H), 8.51 (1H, d, J = 4.8 Hz, ,f3-
H), 8.05 (2H, d, J =
8.9 Hz, v Ar), 7.94 (2H, d, J = 8.1 Hz, o' Ar), 7.25 (2H, d, J = 8.9 Hz, m
Ar), 7.12 (2H, d,
J = 8 .1 Hz, na '-Ar), 6.42 ( 1 H, d, J = 6. 5 Hz, 17-H), 6. 03 ( 1 H, d, J =
6. 5 Hz, I ~-H), 4.08
(3H, s, CH;), (NH's exchanged), (OH's not observed).; MS (MALDI) m/z = 642.2
( 100%, (M+1 )~).
Low Rt~ chlorin regioisomer (2,3-dihydroxy-5-(4-aminophenyl)-15-(4-
methoxyphenyl)
chlorin from nOe measurements and JPP paper): (8.5 mg, 30%); m.p. 168°C
(decomposed); UV-VIS (CHZCl2) ~.max (relative intensity) 401(0.99), 413 (1.0),
507
(0.08), 536 (0.06), 586 (0.025), 637 (0.20) nm; UV-VIS (CH2C12) (fluorescence)
7~max
639 nm (7~ excitation 412 nm); 8H(270 MHz, 10% MeOH-d4 in CDC13) 9.96 (1H, s,
20-
H), 9. 40 ( 1 H, s, I 0-H), 9.18 ( 1 H, d, J =4. 8 Hz, /.3 H), 9. 05 ( 1 H, d,
J=4. 8 Hz, ~3 H), 8. 9 8
( 1 H, d, J = 4. 0, ,~3 H) 8. 92 ( 1 H, d, J = 4. 0 Hz, /.~H), 8 . 74 ( 1 H,
d, J = 4. 0 Hz, ~3-H), 8. 5 8
( 1 H, d, J = 4. 0 Hz, (3-H), 8.13 ( 1 H, d, J = 8. 9 Hz, o Ar), 8. 08 ( 1 H,
d, J = 8. 9 Hz, o Ar),
7. 9 5 ( 1 H, d, J = 8 .1 Hz, o ' Ar), 7. 79 ( 1 H, d, J = 8.1 Hz, o '-Ar), 7.
3 6 ( 1 H, d, J = 8 . 9 Hz,
nz-Ar), 7. 3 0 ( 1 H, d, J = 8. 9 Hz, m Ar), 7.11 ( 1 H, d, J = 8.1 Hz, n2 '
Ar), 7.05 ( 1 H, d, J =
8 .1 Hz, ni '-Ar), 6. 42 ( 1 H, d, J = 6. 5 Hz, 7-H), 6. 09 ( 1 H, d, J = 6. 5
Hz, ~-H), 4.11 (3 H, s,
CH;), (NH'.s exchanged), (OH's not observed); MS (MALDI) m/z = 642.2 (100%,
(M+1 )T).
CA 02414089 2002-12-02
WO 02/00662 PCT/GBO1/02846
17,18-1)ihydrowy-~-(4-isotlaiocycttcatophenyl)-1~-(4 tnetho~yplaenyl)chloritt
(higherRf
regioi,sosoer)
To a stirred solution of l, l'-thiocarbonyldi-2(1 H)-pyridone (1.07 eq., 7.6
mg, 43.5
mmol) in DCM (25 ml), (dried via passage through an activated alumina column)
was
added a solution of 17,18-dihydroxy-5-(4-methoxyphenyl)-15-(4-
aminophenyl)chlorin
(17.5 mg, 23.2 Omol) in DCM (10 ml). The reaction flask was covered with
aluminium
foil to exclude light and left to stir under NZ for 2 h, at which time TLC
indicated
complete loss of starting material. The reaction mixture was then washed with
water (2 x
50 ml) and saturated brine (50 ml) then dried (anhyd. Na2S0:~). The organic
extract was
then filtered and concentrated on to 10 ml flash silica-gel and
chromatographed on flash
silica-gel ( 100 ml), eluting with 1 % methanol in DCM to elute the required
isothiocyanato chlorin diol (NB. traces~of all TDP must be removed prior to
concentration
of product from column chromatography otherwise some decomposition to 3 higher
Rf
byproducts occurs. These have not been identified at this time). The title
compound was
isolated as a browny-purple crystalline solid (17 mg, 90%); m.p. 155°C
(decomposed);
UV-VIS (CHZCl2) a,may (relative intensity) 410 (1.0) 505 (0.09), 534 (0.06),
586 (0.04),
637 (0.18) nm; UV-VIS (CHZC12) (fluorescence) ~,ma~ 639 nm (~, excitation 412
nm);
bH(270 MHz, 10% MeOH-d~ in CDC13) 10.0 (1H, s, I D-H), 9.45 (1H, s, 20-H),
9.20 (1H,
d, J =4. 8 Hz, ,(3-H), 9. 06 ( 1 H, d, J=4. 0 Hz, ~3-H), 9.02 ( 1 H, d, J = 4
. 8 Hz, ~3 I~ 8 . 84 ( 1 H,
d, J = 4. 8 Hz, ,Q-H), 8. 64 ( 1 H, d, J = 4. 0 Hz, ,!1--I~, 8 . 5 5 ( 1 H, d,
J = 4. 8 Hz, ,Q H), 8. 21
( 1 H, d, J = 8.1 Hz, v Ar), 8.15 ( 1 H, d, J = 8 .1 Hz, o-Ar), 8. 05 ( 1 H,
d, J = 8. 9 Hz, o '-Ar)
7.93 (1H, d, J = 8.9 Hz, a' An), 7.65 (2H, m, »z-AY), 7.24 (2H, m, »~' Ar),
6.43 (1H, d, J =
6.5 Hz, 17-H), 6.04 (1H, d, J = 6.5 Hz, 18-I~, 4.08 (3H, s, CH;), (NH's
exchanged),
(OH'.s not observed); MS (MALDI) m/z = 583.7 (100%, M+); HRMS calcd. for
C~:~HZ~N;03S: 584.1757. Found: 584.1756 ((M+1)*).
7, 8,17.18-Tetrahydrowy-~-(4-f luoretzomethyla»zinophenyl)-1 ~-(4-
nzetlzovyplaetzyl)
b~ccteriochloritc (cisltratzs stereoisotners)
5-(4-Fluorenomethylaminophenyl)-15-(4-methoxyphenyl)porphyrin (35 mg, 48.0
~mol)
was converted to a mixture of bacteriochlorin stereoisomers using the general
bacteriochlorin formation procedure given earlier. The crude reaction mixture
was then
66
CA 02414089 2002-12-02
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chromatographed on flash silica-gel (200 ml), (dry loaded on to 20 ml flash
silica-gel
from DCNI and a little methanol to ensure complete solubility) eluting
initially with 1%
methanol in DCM to elute the higher Rf chlorin byproducts, then 2% methanol/
DCM to
elute separately the two stereoisomeric bacteriochlorins. The higher R~-
tnafi,s
bacteriochlorin isomer was isolated as a pinky-green crystalline solid, (6 mg,
15%); m.p.
142"C (decomposed); UV-VIS (CH2C12) ~.ma;; (relative intensity) 374 (1.0) 512
(0.23),
702 (0.52) nm; UV-VIS (CHZC12) (fluorescence) ~nlax 708 nm (~, excitation 512
nm);
8H(270 MHz, 10 % MeOH-da in CDC13) 9.20 (2H, s, 10-H, 20-I~, 8.78 (2H, d, J
=4.0
Hz, (3-I~, 8.36 (2H, d, J = 4.0 Hz, ,(3 H), 7.95 (2H, m, o-Aj~), 7.85 (2H, d,
J = 7.3 Hz,
.flure~r~ern~-Ar), 7.79 (2H, m, o'-Ar), 7.65 (2H, m, na'-An), 7.47-7.38 (6H,
m,.fhaoretzo-Ar),
7.24 (2H, m, rn-Ar), 6.27-6.24 (2H, 2 x d (overlapping), J = 6.5 Hz, 7-H, 17-
I~, 5.85
(2H, d, J = 6.5 Hz, 8-H, 18-I~, 4.65 (2H, d, J = 7.2 Hz, CHI), 4.39 (1H, t, J
= 7.2 Hz,
C.I~, 4.06 (3H, s, CH;), -1.94 (2H, br. s (partly exchanged), NH), (OH's not
observed);
MS (MALDI) m/z = 800.4 (100%, (M+1)+).
The lower Rt~ ci.s-bacteriochlorin isomer was isolated as a pinky-green
crystalline solid,
(8.5 mg, 21%); m.p. 148°C (decomposed); UV-VIS (CH2C12) ?~max (relative
intensity)
374 (1.0) 512 (0.24), 703 (0.54) nm; UV-VIS (CHZC12) (fluorescence) ~,max 708
nm (~,
excitation 512 nm); bH(270 MHz, 10 % MeOH-d4 in CDCl3) 9.12 (2H, s, 10-H, 20-
H),
8.76 (2H, d, J =4.8 Hz, (3-HJ, 8.34 (2H, 2 x d (overlapping), J = 4.8 Hz, ,(3
I~, 8.02 (2H,
m, o-Ar), 7.85 (2H, d (obscurred), J = 8.0 Hz, o' Ar), 7.83 (2H, d, J = 7.3
Hz,, fZuorefao-
Ar), 7.76 (2H, d, J = 8.0 Hz, na'-An), 7.50-7.38 (6H, m, fluof~er2o AY), 7.24
(2H, m, m At~),
G.27-6.23 (2H, 2 x d (overlapping), J = 6.5 Hz, 7-H, 17-H), 5.85-5.82 (2H, 2 x
d
(overlapping), J = 6.5 Hz, 8-H, 18-H), 4.65 (2H, d, J = 7.2 Hz, CHI), 4.39 (
1H, t, J = 7.2
Hz, CH), 4.05 (3H, s, CH3), -1.88 (2H, br. s (partly exchanged), NH), (OH's
not
observed); MS (MALDI) m/z = 800.4 (100%, (M+1)+).
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7, 8.17.18-Tetralzydroxy-S-(4-isothiocytcnatoplzenyl)-1 ~-(4-rnethoxyplzenyl)
bacterioclzlorin (lower Rf cis stereoisomer)
~1 solution of 7,8,17,18-tetrahydroxy-5-(4-fluorenomethyaminophenyl)-15-(4-
methoxyphenyl) bacteriochlorin (lower Rf cis stereoisomer), (8.5 mg, 10.7
p,mol) in 25%
methanol in DCM (1.25 ml) was treated with piperidine (~0 eq., 53 p,l , 0.53
mmol) and
left to stir for a period of 3 h. at room temperature under NZ with the light
excluded. The
reaction mixture was concentrated irmcrcuo to remove all traces of piperidine
(high
vacuum needed). To a stirred solution of 1,1'-thiocarbonyldi-2(1 H)-pyridone
(1.07 eq.,
7.6 mg, 43.5 mmol) in DCM (25 ml), (dried vicr passage through an activated
alumina
column) was added a solution of 2,3,12,13-tetrahydroxy-5-(4-aminophenyl)-15-(4-
methoxyphenyl)bacteriochlorin (6.1 mg, 10.7 ymol) in DCM (10 ml). The reaction
flask
was covered with aluminium foil to exclude light and left to stir under NZ for
2 h, at
which time TLC indicated complete loss of starting material. The reaction
mixture was
then washed with water (2 x 50 ml) and saturated brine (50 ml) then dried
(arzhyd.
Na~SO:~). The organic extract was thenfiltered and concentrated on to 10 ml
flash silica-
gel and chromatographed on flash silica-gel (100 ml), eluting with 2% methanol
in DCM
to elute the required isothionato bacteriochlorin tetrol (NB. traces of all
TDP must be
removed prior to concentration of product from column chromatography otherwise
some
decomposition occurs). The Lower Rf cis-bacteriochlorin isomer was isolated as
a pinky-
green crystalline solid, (5.0 mg, 76%); m_p. 132°C (decomposed); UV-VIS
(CHzCl2) ?~max
(relative intensity) 375 (1.0) 516 (0.22), 702 (0.48) nm; UV-VIS (CHzCl2)
(fluorescence)
~,",as 709 nm (~, excitation 516 nm); 8H(270 MHz, 10 % MeOH-d;~ in CDC13) 9.20
(1H,
s, n~esn-H), 9.18 ( 1 H, s, me.so '-H), 8. 77 (2H, d, J =4. 8 Hz, /3-H), 8.40
( 1 H, d, J = 4. 8 Hz,
,(I-H), 8.34 ( 1H, d, J = 4.8 Hz, ~-H), 8.14 (2H, m, c~-Ar), 8.05 (2H, m, o'
An), 7.42-7.08
(4H, m, .5~-1.5-n? Ar), 6.20 (2H, 2 x d (overlapping), J = 6.5 Hz, 7-H, 17-H),
5.98 (1H, d, J
= 6.5 Hz, 8-H), 5.93 (1H, d, J = 6.5 Hz, 18-H), 4.04 (3H, s, CH3), -1.80 (2H,
br. s (partly
exchanged), NH), (pH's not observed); MS (MALDI) m/z = 618.9 (100%, (M+1)T);
HRMS calcd. for C3~IIZgNsOSS: 618.1815. Found: 618.1810 ((M+1)''-).
Further synthetic protocols and methodology protocols are also described in
Sutton et al, Por~h~ Chlorin and Bacteriochlorin I~~th;~~yanates Synthesis and
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Potential Applications in Fluorescence Ima~in,g and Photod;rnamic Ther~v
(Journal of
Phthalocyaniraes ~ Photosensitisers - in press) and in Oliver J Clarke,
Isothioc ay nato
P r h. rip f r i n' i n h n i i h n
~.
fluorescence imagi~r~(PhD thesis, April 2001, University ofEssex); the entire
contents of
each of which are incorporated herein by reference.
Methodology Description 1 ~ General Bioconjug~,ation rotocol
Hexahydroxy PITC + Antibody
A stock solution of hexahydroxy PITC in DMSO was prepared to a molarity of
0.027, this solution was desiccated and stored at 0°C until required. A
solution of
antibody was extensively dialysed against sterilised PBS to remove any trace
of azide.
The dialysed antibody solution was then adjusted to a concentration of 10
mg/mL via
centrifugal concentration and separated into 250 ~,L aliquots.
A 1 M solution of sodium bicarbonate was prepared and adjusted to pH 9.0 with
2
M sodium hydroxide.
To a 250 p,L aliquot of antibody was added 30 pL of 1 M sodium bicarbonate. A
predetermined volume of hexahydroxy PITC stock solution was then added to give
a
desired molar ratio (MR) of porphyrin to antibody. For example an MR of 20 was
achieved via the addition of 10 p.L of stock solution to 250 p.L of antibody
at 10 mg/mL.
In order to maintain a constant concentration of DMSO in the bioconjugation
reaction
mixture, all aliquots of stock solution were diluted to 25 ~.L with further
portions of
DMSO.
DesiredVol. of (C] of Vol, of Vol. of Vol.
1 M of
MR antibody antibody sodium PITC stockextra
solution solution bicarbonatesolution DMSO
2 0 25 0 p,L 10 mg/mL 3 0 p,L 10 p,L 15
p,L
250 ~.L 10 mglmL 30 ~,L 5 ~L 20
p.L
25 0 p,L 10 mg/mL 3 0 p,L 2. 5 ~L 22.
S
~,L
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250 yL 10 mglmL 30 yL 1.25 p,L 23.75 ~,L
Table 1.0 Quantities of reagents for bioconjugation
Following addition of PITC the bioconjugation reaction was agitated gently for
1
hour at 25°C. After 1 hour the crude bioconjugation reaction mixture
was loaded directly
onto the top of a prepacked PD10 size exclusion column pre-equilibrated with
sterile PBS
(25 mL). The column was eluted with sterile PBS. Antibody-porphyrin conjugate
was
eluted in the first coloured band/fraction. The antibody-porphyrin conjugate
concentration following dilution during chromatography was determined as 1.25
mg/mL.
The degree of labelling (DOL) of porphyrin to antibody was calculated via
standard
spectroscopic methods using known constants of molar absorptivity for both
porphyrin
and protein.
Antibody-porphyrin conjugates were stored, without further concentration, in
PBS
+ azide at 0°C unless otherwise stated.
1V lVlethylpyridinium chloride PITC + Antibody
A stack solution of N methylpyridinium chloride PITC in DMSO was prepared to
a molarity of 0.027, this solution was desiccated and stored at 0°C
until required. A
solution of antibody was extensively dialysed against sterilised PBS to remove
any trace
of azide. The dialysed antibody solution was then adjusted to a concentration
of 10
mg/mL vicr centrifugal concentration and separated into 250 1.~L aliquots.
A 1 NI solution of sodium bicarbonate was prepared and adjusted to pH 9.0 with
2
M sodium hydroxide.
To a 250 ~,L aliquot of antibody was added 250 p,L of sterile PBS then 60 yL
of 1
M sodium bicarbonate. A predetermined volume of N methylpyridinium chloride
PITC
stock solution was then added to give a desired molar ratio (MR) of porphyrin
to
antibody. For example an MR of 20 was achieved via the addition of 10 p,L of
stock
solution to 500 p,L of antibody at 5 mg/mL. In order to maintain a constant
concentration
of DMSO in the bioconjugation reaction mixture, all aliquots of stock solution
were
diluted to 25 p,L with further portions of DMSO,
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DesiredVol. of [C] of Vol. of Vol. of Vol.
1 M of
MR antibody antibody sodium PITC stockextra
solution solution bicarbonatesolution DMSO
30 500 l.~L 5 mg/mL 60 ~.L 10 ~,L 15 p,L
500 ~.L 5 mg/mL 60 ~.L 5 p,L 20 p,L
5 500 p.L 5 mg/mL 60 p.L 2.Sp.L 22.5
~,L
2.5 500 1~L 5 mg/mL 60 yL 1.25 yL 23.75
~,L
Table 2.0 Quantities of reagents for bioconjugation
Following addition of PITC the bioconjugation reaction was agitated gently for
1
hour at 25°C. After 1 hour the crude bioconjugation reaction mixture
was loaded directly
onto the top of a prepacked PD10 size exclusion column pre-equilibrated with
sterile PBS
(25 mL). The column was eluted with sterile PBS. Antibody-porphyrin conjugate
was
eluted in the first coloured band/fraction. The antibody-porphyrin conjugate
concentration following dilution during chromatography was determined as 1.25
mg/mL.
The degree of labelling (DOL) of porphyrin to antibody was calculated via
standard
spectroscopic methods using known constants of molar absorptivity for both
porphyrin
and protein.
Antibody-porphyrin conjugates were stored, without further concentration, in
PBS
+ azide at 0°C unless otherwise stated.
Methodology Description 2 ~ Standard Photocytotoxi i~Y
Cells are grown to confluence or appropriate density then washed 2 times with
PBS
(phosphate buffered saline) to eliminate all trace of FBS (foetal bovine
serum). Cell
density is adjusted to 1.5x106 cells/ml in medium without FBS and these are
then
incubated for 1 hour in the dark (37 degrees C, 5% C02 ) with a range of
photosensitiser/conjugate concentrations. Post incubation, cells are washed
further with
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medium (without FBS )to eliminate unbound photosensitiser, then resuspended
and
seeded in 96 wells plates (1x10' cells/well) in quadruplate.Plates are then
either irradiated
(3.GJ/cm2 of filtered red light 0600nm ) or left in the dark as "dark toxicity
controls" for
the same period of time (~14 minutes).Five microliters (5%/well) of FBS is
added after
the irradiation/dark period and the plates are returned to the incubator
overnight. Twenty
to 24 hours after treatment, 10 pl of MTT solution (Sigma Thiazolyl blue,
4.8x10-4M in
PBS)is added per well and the plates are returned to the incubator until color
develops
(between 1 and 4 hours). A solution of acid-alcohol (100p1/well of 0.04N HCL
in
isopropanol) is the added and mixed thoroughly to dissolve the dark blue
crystals. Plates are then read at 570nm in a microplate reader and the % cell
survival
calculated against controls.
Methodology Description 3 : Initial Flow~vtometrY Chromouhore Anal, i~
The two fluorochromic probes were generated from separate reactions of 2,3-
dihydroxy-5-(4-methoxyphenyl)-15-(4-isothiocyanatophenyl)chlorin (higher Rf
regioisomer) and 2,3,12,13-tetrahydroxy-5-(4-isothionatophenyl)-15-(4-
methoxyphenyl)
bacteriochlorin (lower Rf ci.r stereoisomer) with avidin under the standard
bioconjugation
protocols given earlier. An initial flow experiment has been undertaken
utilising these
separate avidin conjugates with RAJI cells and biotin monoclonal antibodies
(HLA-DR1,
L243), (laser excitation 488 nm, collecting emissions at < 640 nm (FL2) > 670
nm
(FL3)). Data indicated that the signals from the DPBC samples were much higher
due to
good match to emission filter (FL3). Samples containing avidin DPCH or. DPBC
conjugates with L243 antibodies indicated modest increases in fluorescence
compared to
controls. Using higher concentrations of avidin-DPCH/DPBC the peak
fluorescence
increased, which may either be due to the initial concentrations of conjugates
being too
Low to saturate receptors or to a lesser extent to some non-covalent binding.
Control
samples with avidin-DPCH/DPBC (no antibody) showed some background
fluorescence
in the absence of L243 antibodies, suggesting that some non-specific binding
of the
conjugates to the RAJI cells had occurred or that a small quantity of non-
covalently
bound fluorophore had transferred from the protein to the cell surface. A FITC-
avidin
control indicated that a slightly higher signal was present in FL2 which
appears also in
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FL3 due to a broad emission band. In the presence of L243 antibody the mean
signal
increased by 150%. This indicates that non-covalent binding is less
significant with
FITC-avidin conjugates.
Experiments have been undertaken to determine the level of non-covalent
binding
of fluorophore to the protein surface (BSA and avidin). 'Blank'
bioconjugations using
mixtures of the unreactive DPCH and DPBC derivatives 2,3-dihydroxy-5-(4-
methoxyphenyl)-15-(4-acetomidophenyl)chlorin (higher Rf regioisomer) and
2,3,12,13-
tetrahydroxy-5-(4-acetomidophenyl)-15-(4-methoxyphenyl) bacteriochlorin
(higher Rr
trar~s stereoisomer) with both BSA and avidin have been carried out and the
resultant
protein solutions have been purified by gel filtration (PD-10) as described
for the reactive
probes described earlier. UV analysis indicated that approximately similar
amounts of
unreactive probes non-covalently bind to the proteins. For BSA or avidin, 1
unreactive
DPCH binds to each protein molecule, whereas DPBC is less than 1 due probably
to its
increased polarity and non-amphiphilic nature.
Initial studies have been undertaken to remove non-covalently bound
fluorophore
from the protein (BSA and avidin) using SDS-PAGE. When the 'blank'
bioconjugation
mixtures were subjected to SDS-PAGE separation of all non-covalently bound
fluorophore was achieved (UV/fluorescence of a solubilised gel segment at
66000 D for
BSA and 16500 D for avidin monomer indicated no signal). Further to these
investigations, we have been able to show that fluorophore which is non-
covalently
bound to BSA (or avidin) transfers to the surface of HeLa cells. When HeLa
cells were
added to solutions of the non-covalent fluorophore-protein complexes, and
incubated for
20 min, fluorescence was removed from the solution with removal of the HeLa
cells. This
effect was much more marked with 2,3-dihydroxy-5-(4-methoxyphenyl)-15-(4-
acetomidophenyl)chlorin (higher Rf regioisomer) than with 2,3,12,13-
tetrahydroxy-5-(4-
acetomidophenyl)-15-(4-methoxyphenyl) bacteriochlorin (higher Rt trar2s
stereoisomer).
Re-suspension of the cells and measurement of the fluorescence indicated a 10-
fold
increase in fluorescence in the case of the DPCH, whereas the DPBC only showed
a
modest increase. These measurements suggest that there is significant
fluorescence
quenching of both DPCH and DPBC by the protein and that the DPCH's amphiphilic
nature has allowed incorporation into the HeLa cell membrane resulting in
restoration of
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almost complete fluorescence. The DPBC, being non-amphiphilic, may complex to
the
surface of the HeLa cell in a similar manner as it does to the protein
resulting in similar
fluorescence quenching.
Since the fluorophore conjugates can be purified by SDS-PAGE we have
investigated the use of preparative electrophoresis as a technique for removal
of non-
covalently bound fluorophore. To this end we have used a Centrilutor~ micro-
electroeluter bought from Millipore. This device has allowed recovery of pure
protein
fluorophore conjugates from SDS gels.
VIethodologv Description 4 : Elution of Conjugates from SDS PAGE Utili ing
Micro-Electroeluter
~ Working in greatly subdued lighting, the SDS-PAGE of the required protein
conjugate
was cut into small strips and added to the centrilutor sample tubes and the
tops closed
(no more than half full, 3-4 sample tube used).
~ The lower buffer chamber of the electroeluter was filled with degassed SDS
running
buffer up to the level of the first electrode.
~ 3 to 4 Centricon~ centrifugal devices ( -30 used for BSA conjugates and YM-
33 for
avidin conjugates) from Millipore were inserted firmly into the holes in the
upper
buffer chamber rack of the electroeluter from below (with filter membrane
lowest) and
the vacant holes of the rack were stoppered with stoppers provided, from the
underside
of the rack.
~ The upper buffer chamber was placed into the lower buffer chamber with both
electrodes aligned on the same side of the electroelutor.
~ The upper buffer chamber was then filled with degassed SDS running buffer
(as
before) until all Centricon~ unit tops were completely immersed. If no leaks
were
detected the air bubbles trapped below the Centricon~ units were removed vicz
an
angled plastic pipette(reinforced with paper clip).
~ The centrilutor sample tubes were then placed into the top of the Centricon~
units,
ensuring the sample tube fitted snugly and filled completely with sample
buffer (air
bubbles were removed as described earlier).
CA 02414089 2002-12-02
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~ The safety cover of the electroelutor was added and the power supply
connected (200
V, 50 mA used).
~ After a period of 2-3 h. the power supply was removed and the Centricon~
filter
extracted from the upper buffer chamber of the electroelutor.
~ The filtrate vial was added to the filter unit and a retentate top added.
The excess
buffer was then removed by centrifugation at SOOOG (BSA) and 7,SOOG (avidin)
for 2
h. Fresh 0.5 M phosphate buffer (pH 7.0) was added to the Centricon~ unit and
the
procedure was repeated to ensure all SDS was removed.
~ The concentrated purified conjugates were then collected in the retentate
vials of the
filter units by inversion and centrifugation. Sodium azide (2 M, 20 ml) was
added and
the conjugates were stored at 4°C.
Methodolo~,y Descri~ion 5 ~ FACS Conjugate Bindin"
a Protocol
Wash flask of cells with phosphate buffered saline (PBS) pH 7.3. Treat with
SmM EDTA in PBS for 10 min at 37C. Tap flask to dislodge cells, place in SOmL
polypropylene tube and pellet at 400g 3 min. Resuspend in 10 mL PBS and count
cells.
Place 2 x 10' in FRCS tube (Falcon 2054) and wash with 1mL PBS by
centrifugation
(4008 3min) and resuspension by agitation
Block cells in 500 ~L 2% Marvel milk powder in PBS, 1% BSA 30 min RT
Wash cells in 1 mL PBSBSA/Azide centifuge and resuspend pellet (as above)
Add 10 p.L appropriate antibody dilution. Incubate on ice lh
Wash cells in 1 mL PBS/BSA/Azide centifuge and resuspend pellet (as above)
Add 50 pL Rabbit anti-mouse:FITC (Serotec, 1/100 dilution) and incubate on ice
in the dark 1 h
Wash cells in 1 mL PBS/BSA/Azide centifuge (as above) and resuspend pellet in
400 pL PBS/BSA/Azide.
Run samples through FACS machine using CellQuest acquisition software to
collect data.
PB S/B SA/AZIDE
250 mL PBS
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0.6258 BSA
1.56 mL Sodium Azide (1.6M)
Methodologw Descrip tion 6 : SDS-PAGE
Separating gel
Component % of gel 5 20
Acrylamide/Bis (40% w/v) 1.67mL 6.66mL
I.SM Tris-HCl (pH 8.8) 2.SmL 2.SmL
Water 5.67mL 0.7mL
TEMED 10 pL 10 pL
10% Ammonium persulphate 50 pL 50 pL
SDS 100 ~L 100 pL
For gradient gel 5-20%
a gradient mixer connected
to a peristaltic pump
is used.
Stacking gel (3%)
Component mL
Acrylamide/Bis (40% w/v) 1.3
1M Tris-HC1 (pH 6.8) 1.25
Water 7.4
TEMED 20 pL
10,/ Ammonium persulphate50 ~tL
SDS 100 pL
Running buffer
0.0251VI Tris, 0.192M 0.1% SDS, pH8.3 in water.
glycine,
Sample buffer
1M Tris-HC1 pH 6.8 l3mL ,
20% SDS 6.SmL
Glycerol 5.2mL
0.5% Bromophenol blue 0.26mL
Biorad Protean 2 equipment
was used in accordance
with manufacturer's
instructions
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Samples (total volume 15-20 11L containing 1-10 pg sample protein) were loaded
onto a gel.
Gels were run at 200V for approximately lh. Gels were then scanned by light,
after which they were stained using Coomassie blue stain and subsequently
destained
using acetic acid/methanol.
Further exemplification of the invention
It has been demonstrated in our original work, described inter alia in Sutton,
J.,
Fernandez, N. and Boyle, R.W. (2000) Functionalised Diphenylchlorins and
Bacteriochlorins - Their Synthesis and Biocon~L,gation for Targeted
Photodynamic
Therapy and Tumour Cell Ima,g'~ng, J. Porphyrit~s and Phthalocyanines 4, 655-
658; and
Clarke, O.J. and Boyle, R.W. (1999) Isothioc, a~~o~~ r~ ins useful
intermediates fir
the con~iuaation of ~r_phyrins with biomolecules anr~ solid supports. ,L C. S.
Chem.
C:omnaura. 2231-2232, each of which is incorporated herein by reference, that
a set of
porphyrin, chlorin and bacteriochlorin molecules can be efficiently conjugated
to proteins
via a stable thiourea bond, and that these conjugates have potential as
fluorescence
imaging agents.
As exemplification of the present invention, we now describe the use of this
method to form conjugates between monoclonal antibodies having high
specificity for
human cancer cells, and our set of porphyrin based photosensitisers.
Conjugates formed
in this way have been assayed for photodynamic activity against the
corresponding
carcinoma cells, and also for their ability to selectively bind to, and
photosensitise, these
target cells in the presence of non-target cells. We also demonstrate the
specific
internalisation of porphyrin-BSA conjugates into HeLa cells.
Our examples utilise 5,10,15-tris(3,5-dihydroxyphenyl)-20-(4-
isothiocyanatophenyl) porphyrin (OH6) and 5,10,15-tris(pyridyl)-20-(4-
isothiocyanatophenyl)porphyrin (PYR), as we have found from our previous
studies that
the pattern of hydrophilic substituents around the photoactive porphyrin core
of each of
these chromophores leads to efficient conjugation with proteins; hydrophilic
substituents
also minimise non-covalent binding of photosensitiser to protein, often found
with more
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hydrophobic porphyrins. Synthetic protocols for these chromophores are
described in
Examples 1 and 2 above respectively.
(OH6) (PYR)
Example 25 - stable con~iugation to antibodie
OHG and PYR were prepared as described in Examples 1 and 2 above
respectively. Antibody 17. lA was selected for the bioconjugation procedure.
17. lA is an
antibody which reacts specifically with a receptor that is over-expressed on
colorectal
cancer cells, in particular Colo 320 cells (ECACC, deposit no. 87061205).
However, any
antibody which reacts against any antigen that is over-expressed on a suitable
cell line
may be utilised in accordance with the invention. Examples of such antibodies
include
Ber-EP4 and MOK-31, each of which is commercially available from DAKO Ltd,
Ely,
Cambridgeshire, and each of which is reactive against an antigen that is over-
expressed
on epithelial cells.
To increase the buffer pH of the antibody preparation to approximately pH9,
prior
to and for the purposes of the bioconjugation procedure, the monoclonal
antibody
preparation was either buffer-exchanged from a phosphate to an acetate buffer
using a
Centricon centrifuge or was subjected to dialysis so as to exchange the
phosphate buffer
for an acetate buffer.
Each of OH6 and PYR was separately conjugated with l7.lA monoclonal
antibody in accordance with the method described in Methodology Description 1,
to
obtain a range of conjugation dilutions having respective MRs of 2.5, 5, 10
and 20..
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The acetate-buffered antibody preparation and range of conjugation dilutions
obtained therefrom were subjected to SDS-PAGE in accordance with the method
described in Methodology Description 6. The results are shown in Figures 1-3
respectively. Figure 1 shows a gel loaded with buffer-exchanged l7.lA antibody
(lane 1),
and buffer-exchanged antibody/OH6 conjugations at MRs 2.5 (lanes 2, 3), 5
(lanes 4, 5),
(lanes G, 7) and 20 (lanes 8, 9) and molecular weight markers (lane 10).
Figure 2
shows a gel loaded with dialysed 17. lA antibody (lane 1), and dialysed
antibody/OH6
conjugations at MRs 2.5 (lanes 2, 3), 5 (lanes 4, 5), 10 (lanes 6, 7) and 20
(lanes 8, 9) and
molecular weight markers {lane 10). Figure 3 shows a gel loaded with buffer-
exchanged
17. lA antibody (lane 1), and buffer-exchanged antibody/PYR conjugations at
MRs 2.5
(lanes 2, G), 5 (lanes 3, 7), 10 (lanes 4, 8) and 20 (lanes 5, 9) and
molecular weight
markers {lane 10).
As seen in these Figures, neither the buffer-exchange nor dialysis procedures
disrupt the antibody structure, the light and heavy chains remaining
associated with one
another and migrating together on each of the gels (lane 1). Conjugation of
OH6 and
PYR at each of the MRs can also be seen on the;gels (lanes 2-9).
Example 26 - FACS anal
FACS analyses were run in accordance with Methodology Description 5.
Figure 4 shows results derived utilising FITC-labelled l7.lA and Colo 320
cells
(3 repeats) and indicates that binding of the antibody to the cells has
occurred (ie the Colo
320 cells express the antigen specific to 17. lA).
Figure 5 shows results derived utilising OH6/l7.lA conjugate and Colo 320
cells
with a FITC-labelled anti-17. lA antibody for detection (3 repeats) and
indicates that the
OHG/l7.lA conjugate has bound to the cells.
Figure 6 shows results derived utilising PYR/l7.lA conjugate and Colo 320
cells
with a FITC-labelled anti-17. lA antibody for detection (3 repeats) and
indicates that the
PYR/17. lA conjugate has bound to the cells.
Figure 7 shows results derived utilising FITC-labelled OX-34 which is an
antibody of the same class (IgG2a) as 17. lA but with a different antigen
specificity (3
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repeats). The results indicate that OX-34 has not bound to the Colo 320 cells
and hence
that there are no binding sites for OX-34 on Colo 320 cells.
Example 27 - Photocytotoxicitv experiments
Photocytotoxicity tests in accordance with the method described in Methodology
Description 2 were performed on Colo 320 cells utilising various antibody
conjugates.
Figures 8 and 9 show the results of control experiments performed using
OHG/OX-34 and PYR/OX-34 conjugates respectively. As described in Example 16 0X-
34 has been found to lack specificity for any antigens expressed on the
surface of Colo
320 cells. Accordingly, as expected these control experiments show no
photocytotoxicity
following irradiation.
Figures 10 and 11 show the results of further control experiments performed
using
"capped" OHG and PYR respectively. The "capping" procedure involved reacting
the
NCS group on each chromophore with propylamine, so as to block serum protein
conjugation. Figure 10 shows no cytotoxicity in the dark, indicating that OH6
is non-
toxic to Colo 320 cells. On irradiation, however, some photocytotoxicity is
observed,
indicating that an amount of the capped OH6 has been transferred to the
surface of the
Colo 320 cells. Figure 11 meanwhile shoves some cytotoxicity in the dark,
suggesting that
PYR is to some extent cytotoxic to Colo 320 cells, and increased
photocytotoxicity on
irradiation, which again indicates that an amount of the capped PYR has been
transferred
to the surface of the Colo 320 cells.
In the absence of any antibody, transfer of the capped chromophores to the
cell
membrane is probably attributable to the amphiphilic nature of the capped
chromophores,
which possess both hydrophilic groups around the porphyrin core and a
hydrophobic
propylamine "capping" group. This renders them particularly susceptible to
becoming
embedded in a lipid membrane such as the Colo 320 cell membrane.
Figures 12 and 13 show results obtained using OH6/l7.lA and PYR/l7.lA
conjugates respectively, at various conjugation dilutions (2.5, 5, 10, 20 for
OH6/l7.lA;
and 20 for PYR/17, lA). The results indicate a significant increase in
cytotoxicity on
irradiation, indicating that the binding of the bioconjugates to the cell
surface confers
photosensitivity upon the cells. Hence, these species are suitable candidates
for PDT.
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Example 28 - hotod;~namic theraw in vivo
Protocols for performing and assessing photodynamic therapy in vivo, utilising
the conjugates of the invention, are variously described in R Boyle et al, Br.
J. Cancer
( 1992) 65:813-817; R Boyle et al, Br. .I. Cancer (1993) 67:1177-1181; R Boyle
et al, Br.
.l. Cancer ( 1996) 73 :49-53; and Lapointe et al, .I. Nuclear Medicine, Vol.
40, No. 5 (May
1999) 876-882; the contents of each of which are incorporated herein by
reference.
As described in these papers, tumours may be induced or transplanted into
animals such as mice, and the animal may then be injected with a quantity of
photosensitiser in accordance with the invention conjugated to an antibody
with
specificity for an antigen which is specifically expressed or over-expressed
on the surface
of the tumour cells. Thereafter, the animal may be subjected to irradiation,
and the effects
on the tumour assessed, qualitatively or metrically, with reference to tumour
metabolism
(as described in Lapointe et al, J: Nuclear Medicine, Vol. 40, No. 5 (May
1999) 876-
882). As described in R Boyle et al, Bj-. J. Cancer (1996) 73:49-53, the
distribution of the
photosensitiser in vivo may also be measured, by biodistribution and/or
vascular stasis
assays.
Example 29 - Confocal Laser Scanning Microsco~"Y
A preliminary examination of the intracellular localisation of a conjugate of
10,15,20-tris(3,5-dihydroxyphenyl)5-isothiocyanatophenylporphyrin (OH6-NCS)
with
BSA was carried out using confocal laser scanning microscopy. The readily
available
epithelial human carcinoma cell line HeLa was selected for incubation with the
conjugate. All incubations were performed' in triplicate with sub-confluent
cultures of
HeLa cells, including a series of control solutions of unlabelled BSA,
10,15,20-tris(3,5-
dihydroxyphenyl)5-aminophenylporphyrin porphyrin (OH6-NH2, amino precursor of
OH6-NCS), and PBS on its own. Cells were seeded onto coverslips in 35 mm
dishes.
Fluorescence images of cells were obtained with a Bio-Rad Radiance2000
confocal laser scanning microscope (Bio-Rad Microscience, Cambridge, MA) on an
inverted Olympus IX70 microscope using a 60x (NA 1.4) oil immersion objective
lens.
The illumination source was the 514 nm line from a 25 mW argon ion laser.
Porphyrins
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WO 02/00662 PCT/GBO1/02846
were visualised with a 514 nm band-pass excitation filter, a 510 nm dichroic
mirror, and
a 570 nm long-pass emission filter.
Each field of cells was sectioned 3-dimensionally by recording images from a
series of focal planes. Movement from one focal plane to another was achieved
by a
stepper motor attached to the fine focus control of the microscope, the step
sizes (in the
range 0.5 l.~m to 1.25 ~~m) being chosen with regard to the aperture size
being used, so
that there would be some overlap between adjacent sections. Enough vertical
sections
were taken so that the tops and bottoms of all the cells in each field would
be recorded.
Each image collected was the average of four scans at the confocal
microscope's normal
scan rate. During each imaging session calibration images were taken of: (i) a
microscope slide containing medium, in order to measure background levels;
(ii) a slide
containing ITC porphyrin OH6-NCS dissolved in DMSO; and (iii) a slide bearing
only
un-probed HeLa cells.
Image data acquisition and remote microscope operation was carried out using
the
Bio-Rad Lasersharp2000 software. All images were managed using Confocal
Assistant
version 4.02, (build 101) 1994-1996 Todd Clark Brelje. Artificial colour was
applied
using standard Bio-Rad look-up tables (LUT).
A preliminary evaluation of the fluorescence of OH6-NCS at each of the
excitation laser lines available on the CLSM set-up was carried out for a 0.01
mM
solution of OH6 in DMSO. Figure 14 shows the UV-visible spectrum of OH6-NCS
identifying its principal absorption bands. Unfortunately, no laser line was
available in
order to excite OH6-NCS at its Soret band ~,max. Figure 15 demonstrates the
relative
intensities of fluorescence emission for OH6-NCS when excited at 422 nm
(optimal), and
at the four wavelengths of the argon ion laser, 457, 476, 488, and 514 nm.
It was determined that the intensity of fluorescence emitted by a solution of
OH6-
NCS when excited at 514 nm was roughly three times greater than fluorescence
emission
at excitation wavelengths of 457, 476, and 488 nm. The UV-visible absorption
spectrum
of OH6-NCS showed that the 516 nm argon-ion laser line was the only excitation
source
compatible with OH6-NCS. The three strongest laser lines, 457, 476, and 488 nm
all
excited in the region between the Soret and first Q band of OH6-NCS, whereas
the 514
line overlapped well with the Q band at 516 nm.
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WO 02/00662 PCT/GBO1/02846
Cell cultures separately incubated with conjugate OH6-NCS-BSA and each of the
three controls, were subsequently washed and fixed. Coverslips containing the
incubated
cells were then cautiously mounted onto standard glass microscope slides ready
to be
imaged. All four argon-ion laser lines were tested, but, as expected
satisfactory
resolution of fluorescence could only be achieved using the 514 nm laser line.
A Z-series fluorescence image of HeLa cells incubated with OH6-NCS-BSA is
shown in Figure 1G (this Figure should be viewed from top left to bottom
right).
Consecutive sections were scanned with a 2l.tM step between each focal plane
resolved
by the microscope, thus enabling three dimensional visualisation of the
localisation of the
conjugate within the cell. Clearly the conjugate OH6-NCS-BSA had entered the
cell, no
studies of the nature of cellular uptake were conducted, however it is most
likely that
uptake had taken place via endocytosis. It can be seen that the conjugate has
not entered
the nucleus and appears to be largely distributed throughout the cytoplasm.
When imaged, cells incubated with the BSA control or the PBS control, showed
only very low, barely detectable levels of fluorescence, attributed to normal
levels of
cellular autofluorescence. The localisation of OH6-NHZ (unconjugated porphyrin
control), is shown in Figure 17, which shows a CLSM image of porphyrin control
cells
with zoom view. No fluorescence was found to emanate from inside the cells,
instead it
appeared that the majority of OH6-NH? had become localised on the plasma
membrane.
Evidently the BSA component of the conjugate is required in order to
facilitate the
transport of porphyrin to the interior of the cell.
In summary, it has been shown that the cellular localisation of porphyrin-BSA
conjugates, constructed via the formation of covalent thiourea linkages, can
be imaged
using conventional CLSM techniques. Unconjugated porphyrin OH6-NH2 was not
found to penetrate the cellular membrane, whereas a significant level of
fluorescence was
detected from inside cells incubated with OH6-NCS-BSA, indicating good
conjugate
penetration.
~J
