U.S. patent application number 12/912033 was filed with the patent office on 2011-04-21 for water soluble tetrapyrollic photosensitizers for photodynamic therapy.
This patent application is currently assigned to Health Research, Inc.. Invention is credited to Lalit Goswami, Amy Gryshuk, Allan Oseroff, Ravindra K. Pandey, William Potter.
Application Number | 20110091390 12/912033 |
Document ID | / |
Family ID | 30000878 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110091390 |
Kind Code |
A1 |
Pandey; Ravindra K. ; et
al. |
April 21, 2011 |
WATER SOLUBLE TETRAPYROLLIC PHOTOSENSITIZERS FOR PHOTODYNAMIC
THERAPY
Abstract
A tetrapyrollic photosensitizer compound having at least one
pendant
--CH.sub.2CH.sub.2CON(CH.sub.2CON(CH.sub.2COOH).sub.2).sub.2 or
--N(CH.sub.2COOH).sub.2 group or esters thereof said tetrapyrollic
compound being a chlorin, bacteriochlorin, porphyrin,
pyropheophorbide, purpurinimide, or bacteriopurpurinimide.
Desirably the compound has the formula: ##STR00001## or a
pharmaceutically acceptable derivative thereof, wherein
R.sub.1--R.sub.8 and R.sub.10 are various substituents and R.sub.9
is substituted or unsubstituted
--CH.sub.2CH.sub.2CON(CH.sub.2CON(CH.sub.2COOH).sub.2).sub.2; or
--N(CH.sub.2COOH).sub.2. The invention also includes a method of
treatment by photodynamic therapy by treatment with light after
injecting the compound and a method of imaging by fluorescence
after injection of the compound.
Inventors: |
Pandey; Ravindra K.;
(Williamsville, NY) ; Gryshuk; Amy; (Pleasanton,
CA) ; Goswami; Lalit; (Amherst, NY) ; Potter;
William; (Grand Island, NY) ; Oseroff; Allan;
(Buffalo, NY) |
Assignee: |
Health Research, Inc.
Buffalo
NY
|
Family ID: |
30000878 |
Appl. No.: |
12/912033 |
Filed: |
October 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12378751 |
Feb 19, 2009 |
7820143 |
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12912033 |
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11452511 |
Jun 14, 2006 |
7501509 |
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12378751 |
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10607922 |
Jun 27, 2003 |
7166719 |
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11452511 |
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60392473 |
Jun 27, 2002 |
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Current U.S.
Class: |
424/9.61 ;
424/9.6 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 41/0076 20130101; C09B 69/108 20130101; C09B 47/00 20130101;
A61K 41/0071 20130101; C07D 471/22 20130101 |
Class at
Publication: |
424/9.61 ;
424/9.6 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61P 35/00 20060101 A61P035/00 |
Claims
1-24. (canceled)
25. A method for detecting the presence of a hyperproliferative
tissue in a subject comprising: (i) administering to the subject a
sufficient quantity of a compound or a pharmaceutically acceptable
derivative thereof, said compound or pharmaceutically acceptable
derivative thereof being fluorescent or magnetically resonant and
that preferentially associates with the hyperproliferative tissue;
and (ii) visualizing the compound within the patient by fluorescent
spectroscopy when the compound is fluorescent or MRI when the
compound is magnetically resonant; where the compound is a
tetrapyrollic photosensitizer compound having at least one pendant
--CH.sub.2CH.sub.2CON(CH.sub.2CON(CH.sub.2COOH).sub.2).sub.2 or
--N(CH.sub.2COOH).sub.2 group or esters thereof said tetrapyrollic
compound being a chlorin, bacteriochlorin, porphyrin,
pyropheophorbide, purpurinimide, or bacteriopurpurinimide.
26. The method of claim 25 where the compound has the formula:
##STR00009## or a pharmaceutically acceptable derivative thereof,
wherein: R.sub.1 and R.sub.2 are each independently substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
--C(O)R.sub.a or --COOR.sub.a or --CH(CH.sub.3)(OR) or
--CH(CH.sub.3)(O(CH.sub.2).sub.nXR) where R.sub.a is hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, or substituted or
unsubstituted cycloalkyl where R.sub.2 may be CH.dbd.CH.sub.2,
CH(OR.sub.20)CH.sub.3, C(O)Me, C(.dbd.NR.sub.21)CH.sub.3 or
CH(NHR.sub.21)CH.sub.3; where X is an aryl or heteroaryl group; n
is an integer of 0 to 6; R and R' are independently H or lower
alkyl of 1 through 8 carbon atoms; where R.sub.20 is methyl, butyl,
heptyl, docecyl or 3,5-bis(trifluoromethyl)-benzyl; and R.sub.21 is
3,5-bis(trifluoromethyl)benzyl; R.sub.1a and R.sub.2a are each
independently hydrogen or substituted or unsubstituted alkyl, or
together form a covalent bond; R.sub.3 and R.sub.4 are each
independently hydrogen or substituted or unsubstituted alkyl;
R.sub.3a and R.sub.4a are each independently hydrogen or
substituted or unsubstituted alkyl, or together form a covalent
bond; R.sub.5 is hydrogen or substituted or unsubstituted alkyl;
R.sub.6 and R.sub.6a are each independently hydrogen or substituted
or unsubstituted alkyl, or together form .dbd.O; R.sub.7 is a
covalent bond, alkylene, azaalkyl, or azaaraalkyl or .dbd.NR.sub.20
where R.sub.20 is hydrogen or lower alkyl of 1 through 8 carbon
atoms or --CH.sub.2-3,5-bis(tri-fluoromethyl)benzyl or
--CH.sub.2X--R.sup.1 or --YR.sup.1 where Y is an aryl or heteroaryl
group; R.sub.8 and R.sub.8a are each independently hydrogen or
substituted or unsubstituted alkyl or together form .dbd.O; R.sub.9
is --CH.sub.2CH.sub.2CON(CH.sub.2CON(CH.sub.2COOH).sub.2).sub.2; or
--N(CH.sub.2COOH).sub.2 R.sub.10 is hydrogen, or substituted or
unsubstituted alkyl and; each of R.sub.1-R.sub.10, when
substituted, is substituted with one or more substituents each
independently selected from Q, where Q is alkyl, haloalkyl, halo,
pseudohalo, or --COOR.sub.b where R.sub.b is hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, araalkyl, or
OR.sub.c where R.sub.c is hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, or aryl or CONR.sub.dR.sub.e where R.sub.d and R.sub.e
are each independently hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, or aryl, or NR.sub.fR.sub.g where R.sub.f and R.sub.g
are each independently hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, or aryl, or .dbd.NR.sub.h where R.sub.h is hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or is an amino acid
residue; each Q is independently unsubstituted or is substituted
with one or more substituents each independently selected from
Q.sub.1, where Q.sub.1 is alkyl, haloalkyl, halo, pseudohalo, or
--COOR.sub.b where R.sub.b is hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, heteroaryl, araalkyl, or OR.sub.c where R.sub.c
is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl or
CONR.sub.dR.sub.e where R.sub.d and R.sub.e are each independently
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or
NR.sub.fR.sub.g where R.sub.f and R.sub.g are each independently
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or
.dbd.NR.sub.h where R.sub.h is hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, or aryl, or is an amino acid residue.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a division of U.S. patent application Ser. No.
12/378,751 to Pandey et al. filed Feb. 19, 2009 for WATER SOLUBLE
TETRAPYROLLIC PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY, which is a
division of U.S. patent application Ser. No. 11/452,511 to Pandey
et al. filed Jun. 14, 2006 for WATER SOLUBLE TETRAPYROLLIC
PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY which is a
continuation-in-part of U.S. patent application Ser. No. 10/607,922
to Pandey et al. filed Jun. 27, 2003 entitled FLUORINATED
PHOTOSENSITIZERS RELATED TO CHLORINS AND BACTERIOCHLORINS FOR
PHOTODYNAMIC THERAPY which in turn claims priority from Provisional
Application Ser. No. 60/392,473 to Pandey et al. filed Jun. 27,
2002 entitled FLUORINATED PHOTOSENSITIZERS RELATED TO CHLORINS AND
BACTERIOCHLORINS FOR PHOTODYNAMIC THERAPY.
[0002] The above applications are incorporated herein by reference
in their entirety.
BACKGROUND OF THE INVENTION
[0003] For a number of years, attempts have been underway in
various laboratories to replace Photofrin.RTM. with new
porphyrin-based photosensitizers (PS). To date, most PS are
amphiphilic in nature in that they contain both hydrophilic and
hydrophobic substituents. Due to their 7-conjugated systems, a
phenomenon known as aggregation has become a concern such that it
can: "decrease fluorescence quantum yields, shorten a
photosensitizer's triplet excited state lifetime or reduce its
photosensitizing efficiency". Most of these compounds, therefore,
are visibly aggregated in solution, so the challenge remains to be
the synthesis of effective water-soluble photosensitizers that
accumulate in the tumor, yet clear at a suitable time as to limit
toxicity. Several researchers have either incorporated sugar
residues on the periphery or ionic groups such as pyridinium,
sulfonato or carboxylate groups as a means to enhance
photosensitizers' aqueous solubility. The 5, 10, 15,
20-tetrakis(4-sulfonatophenyl)-porphyrin (TPPS.sub.4) is a known
tetrasodium salt that although soluble in water still absorbs
weakly at .about.630 nm. Core modifications have been made to
TPPS.sub.4 in which chalcogen atoms such as sulfur, selenium and
tellurium have aided in the water solubility of the PS, as well as,
increasing the wavelength maximum to .about.695 nm. Unfortunately,
these compounds were found to be toxic Therefore, the aim of the
present invention was to synthesize effective and non-toxic
water-soluble long wavelength absorbing photosensitizers with high
singlet oxygen ability, singlet oxygen being a key cytotoxic agent
for PDT. Tetrapyrollic compounds, especially porphyrin related
compounds, have played a key role in developing a variety of
photosensitizers. Inventors herein have recently shown that
porphyrin-based compounds can also be used (i) as PET and SPECT
imaging agents and (ii) as vehicles to deliver the required
contrast agents (MRI, Fluorescence etc.) to image tumors. These
approaches have been extremely useful in developing multimodality
agents. However, one major drawback with most of these compounds is
their limited solubility in water. Therefore, most of the
formulations require a biocompatible surfactant, e.g. such as those
commonly sold under the trademarks TWEEN-80 or CREMOPHORE. At low
concentrations, such formulations are approved by FDA for clinical
use, but to avoid a number of disadvantages with such formulations,
it would be `ideal` to design water soluble compounds for tumor
imaging and therapy.
[0004] An approach for increasing the water solubility is to
introduce hydrophilic substituents (e.g., --COOH, PEG, amino acids,
charged species etc.) in the desired molecules. Unfortunately such
incorporation can limit biological efficacy.
[0005] The following references are incorporated by reference as
background art. [0006] 1. R. K. Pandey, G. Zheng The Porphyrin
Handbook (Eds: Kadish, Rodgers and Smith), vol. 6, Academic Press,
Boston, 2000. [0007] 2. Suresh K. Pandey, Amy L. Gryshuk, Munawwar
Sajjad, Xiang Zheng, Yihui Chen, Mohei M. Abouzeid, Janet Morgan,
Ivan Charamisinau, Hani A. Nabi, Allan Oseroff and Ravindra K.
Pandey, Multiomodality Agents for Tumor Imaging (PET, Fluorescence)
and Photodynamic Therapy: A Possible See and Treat Approach. J.
Med. Chem. 2005, 48, 6286-6295. [0008] 3. Ravindra K. Pandey et
al., Chlorophyll-a Analogs Conjugated with Aminophenyl-DTPA as
Potential Bifunctional Agents for Magnetic Resonance Imaging and
Photodynamic Therapy. Bioconjugate Chem. 2005, 16, 32-42. [0009] 4.
Ravindra K. Pandey, A. B. Sumlin, W. R. Potter, D. A. Bellnier, B.
W. Henderson, S. Constantine, M. Aoudia, M. R. Rodgers, K. M. Smith
and T. J. Dougherty, Structure and Photodynamic Efficacy Among
Alkyl Ether Analogues of Chlorophyll-a Derivatives. Photochem.
Photobiol. 1996, 63, 194-205. [0010] 5. Gang Zheng, Susan Camacho,
William Potter, David A. Bellnier, B. W. Henderson, Thomas J.
Dougherty and Ravindra K. Pandey, Synthesis, tumor uptake and in
vivo photosensitizing efficacy of a homologous series of the
3-(1'-alkoxy)ethyl-purpurin-18-N-alkylimides, J. Med. Chem., 2001,
44, 1540-1559. [0011] 6. Yihui Chen, Andrew Graham, William Potter,
Janet Morgan, Lurine Vaughan, David A. Bellnier, Barbara W.
Henderson, Allan Oseroff, Thomas J. Dougherty and Ravindra K.
Pandey, J. Med. Chem. (Rapid Communication), 2002, 45, 255-258.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A shows a graph of In vivo photosensitizing efficacy
of PS 15 and the corresponding water-soluble analog PS 16 (24 h
p.i.) BALB-C mice were implanted with Colo-26 tumors. The tumors
were exposed with laser light (135 J/cm.sup.2, 75 mW/cm.sup.2 for
30 min) 24 h post injection.
[0013] FIG. 1B shows a schematic preparation of compound 16 from
compound 15.
[0014] FIG. 2 shows a graph of In vivo photosensitizing efficacy of
compounds 9, 12 and 14. BALB-C mice were implanted with Colo-26
tumors. The tumors were exposed with laser light (135 J/cm.sup.2,
75 mW/cm.sup.2 for 30 min) 24 h post injection.
[0015] FIG. 3A shows a BALB/c Colon-26 background fluorescence
image Prior to PS injection.
[0016] FIG. 3B is a graph showing fluorescence emissions with
respect to FIG. 3A.
[0017] FIG. 3C shows an In vivo fluorescence image of PS 16 (24 h
p.i.). of an intact tumor;
[0018] FIG. 3D is a graph showing fluorescence emissions with
respect to FIG. 3C.
[0019] FIG. 3E shows an In vivo fluorescence image of PS 16 (24 h
p.i.) skin flap with tumor removed so that PS fluorescence could be
imaged on underside.
[0020] FIG. 3F is a graph showing fluorescence emissions with
respect to FIG. 3E.
[0021] FIG. 4A shows an In vivo fluorescence image of PS 16 in
various organs (24 h p.i.). A: Colon-26 tumor; B: skin over tumor;
C: large intestine; D: liver; E: stomach.
[0022] FIG. 4B is a graph showing fluorescence emissions with
respect to FIG. 4A
[0023] FIG. 5 is a graph showing In vivo quantitation of PS 16
fluorescence normalized to controls (ex: 417 nm; em: .about.710
nm).
BRIEF DESCRIPTION OF THE INVENTION
[0024] In accordance with the present invention, a series of water
soluble purpurinimides were prepared and some of these compounds
were found to be quite effective both for PDT efficacy and tumor
imaging (fluorescence).
[0025] The photosensitizers are tetrapyrollic photosensitizers
having at least one pendant
--CH.sub.2CH.sub.2CON(CH.sub.2CON(CH.sub.2COOH).sub.2).sub.2 or
--N(CH.sub.2COOH).sub.2 group or esters thereof. The substituted
tetrapyrollic compound is usually a chlorin, bacteriochlorin,
porphyrin, pyropheophorbide, purpurinimide, or
bacteriopurpurinimide.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In a preferred embodiment the compound of the invention has
the formula:
##STR00002##
or a pharmaceutically acceptable derivative thereof.
[0027] R.sub.1 and R.sub.2 are each independently substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
--C(O)R.sub.a or --COOR.sub.n or --CH(CH.sub.3)(OR) or
--CH(CH.sub.3)(O(CH.sub.2).sub.nXR) where R.sub.a is hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, or substituted or
unsubstituted cycloalkyl where R.sub.2 may be CH.dbd.CH.sub.2,
CH(OR.sub.20)CH.sub.3, C(O)Me, C(.dbd.NR.sub.2i)CH.sub.3 or
CH(NHR.sub.21)CH.sub.3.
[0028] X is an aryl or heteroaryl group.
[0029] n is an integer of 0 to 6.
[0030] R and R' are independently H or lower alkyl of 1 through 8
carbon atoms.
[0031] R.sub.20 is methyl, butyl, heptyl, docecyl or
3,5-bis(trifluoromethyl)-benzyl.
[0032] R.sub.21 is 3,5-bis(trifluoromethyl)benzyl.
[0033] R.sub.1a and R.sub.2a are each independently hydrogen or
substituted or unsubstituted alkyl, or together form a covalent
bond.
[0034] R.sub.3 and R.sub.4 are each independently hydrogen or
substituted or unsubstituted alkyl.
[0035] R.sub.3a and R.sub.4a are each independently hydrogen or
substituted or unsubstituted alkyl, or together form a covalent
bond.
[0036] R.sub.5 is hydrogen or substituted or unsubstituted
alkyl.
[0037] R.sub.6 and R.sub.6a are each independently hydrogen or
substituted or unsubstituted alkyl, or together form .dbd.O.
[0038] R.sub.7 is a covalent bond, alkylene, azaalkyl, or
azaaraalkyl or .dbd.NR.sub.20 where R.sub.20 is hydrogen or lower
alkyl of 1 through 8 carbon atoms or
--CH.sub.2-3,5-bis(tri-fluoromethyl)benzyl or --CH.sub.2X--R.sup.1
or --YR.sup.1 where Y is an aryl or heteroaryl group.
[0039] R.sub.8 and R.sub.8a are each independently hydrogen or
substituted or unsubstituted alkyl or together form .dbd.O.
[0040] R.sub.9 is
--CH.sub.2CH.sub.2CON(CH.sub.2CON(CH.sub.2COOA).sub.2).sub.2 or
--N(CH.sub.2COOH).sub.2; where A is --OH or -lower alkyl.
[0041] R.sub.10 is hydrogen, or substituted or unsubstituted
alkyl.
[0042] Each of R.sub.1-R.sub.10, when substituted, is substituted
with one or more substituents each independently selected from Q,
where Q is alkyl, haloalkyl, halo, pseudohalo, or --COOR.sub.b
where R.sub.b is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
aryl, heteroaryl, araalkyl, or OR.sub.c where R.sub.c is hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl, or aryl or --CONR.sub.dR.sub.e
where R.sub.d and R.sub.e are each independently hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, or aryl, or --NR.sub.fR.sup.g, where
R.sub.f and R.sup.g are each independently hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, or aryl, or .dbd.NR.sub.h where
R.sub.h is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl,
or is an amino acid residue;
[0043] each Q is independently unsubstituted or is substituted with
one or more substituents each independently selected from Q.sub.1,
where Q.sub.1 is alkyl, haloalkyl, halo, pseudohalo, or
--COOR.sub.b where R.sub.b is hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, heteroaryl, araalkyl, or OR.sub.c where R.sub.c
is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl or
CONR.sub.dR.sub.e where R.sub.d and R.sub.e are each independently
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or
NR.sub.fR.sub.g where R.sub.f and R.sub.g are each independently
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, or
.dbd.NR.sub.h where R.sub.h is hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, or aryl, or is an amino acid residue.
[0044] Synthetic details for the preparation of examples of water
soluble photosensitizers of the invention are depicted in Schemes
1-4 as follow:
##STR00003##
##STR00004##
##STR00005## ##STR00006##
##STR00007## ##STR00008##
[0045] All the intermediates and the final products were
characterized by NMR and mass spectrometry analyses. The purity was
ascertained by analytical TLC. The starting photosensitizers (e.g.
HPPH, fluorinated purpurinimide 7 and the N-butyl-purpurinimide 10
were synthesized by following published methodologies that were
developed in our laboratory) The Synthetic details are as
follows:
Compound No. 2
[0046] Iminodiacetic acid (5.0 gm, 0.03756 mole) was taken in a 500
ml RBF, water (150 ml) and THF (50 ml) were added to it. Resultant
mixture was cooled to 0.degree. C. using an ice bath.
K.sub.2CO.sub.3 (25.9 gm, 0.187 mole) was added to it in portions
keeping temperature of reaction mixture below 10.degree. C. After
10 min of stirring at the same temperature Cbz-Cl (7.9 ml, 0.056
mole) was added to it drop wise. Resultant mixture was stirred for
6 hr at room temperature, concentrated partially to remove THF.
Reaction mixture was washed with ether to remove excess of Cbz-Cl,
aq layer was separated, acidified with dil HCl and extracted with
EtOAc (100 ml.times.3). Organic layers were separated, combined and
washed with H.sub.2O (100 ml), dried over sodium sulfate and
concentrated to give 2 as viscous oil in quantitative yield.
[0047] Yield: 9.6 gm (95.7%).
[0048] .sup.1HNMR (400 MHz, CDCl.sub.3): .delta. 7.36-7.30 (m, 5H,
Ph), 5.16 (s, 2H, Ph*CH.sub.2O), 4.15 (s, 2H, CH2), 4.12 (s, 2H,
CH.sub.2). EIMS: 267(m).
Compound No. 3
[0049] Di-acid 2 (0.5 gm, 1.88 mmol), Di-tert-butyl iminodiacetate
(0.92 gm, 3.77 mmol), EDCI (1.0 gm, 5.6 mmol) and DMAP (0.36 gm,
5.6 mmol) were dissolved in dry DCM (30 ml). Resultant mixture was
stirred at room temperature for 16 hr under N.sub.2 atm, diluted
with DCM (100 ml) and washed with brine (50 ml). Organic layer was
separated, dried over sodium sulfate and concentrated. Crude was
purified on silica gel column using EtOAc/hexane (20-40%) as eluent
to give product 3. Yield: 1.0 gm (75%).
[0050] .sup.1HNMR (400 MHz, CDCl.sub.3): .delta. 7.34-7.28 (m, 5H,
Ph), 5.12 (s, 2H, PhCH.sub.2O), 4.28 (d, 1H, J=6.4 Hz), 4.24 (d,
1H, J=6.8 Hz), 4.18-4.14 (m, 1H), 4.05 (m, 4H), 3.91 (m, 1H), 3.74
(d, 1H, J=8.0 Hz), 3.67 (d, 1H, J=10.8 Hz), 1.47 (s, 9H,
CO.sub.2Bu.sup.t), 1.45 (s, 9H, CO.sub.2Bu.sup.t), 1.44 (s, 9H,
CO.sub.2Bu.sup.t), 1.40 (s, 9H, CO.sub.2Bu.sup.t). SIMS:
744(m+Na.sup.+).
Compound No. 4
[0051] Compound 3 (0.9 gm, 1.24 mmol), Pd/C (10%, 1.0 gm), MeOH (60
ml) were stirred together under H.sub.2 atm for 2 hr. Reaction
mixture was filtered over celite, filtrate was concentrated and
chromatographed over silica get using MeOH/DCM (1-3%) as eluent.
Yield: 0.6 gm (82.5%).
[0052] .sup.1HNMR (400 MHz, CDCl.sub.3): .delta. 4.06 (s, 4H,
CH.sub.2), 4.01 (s, 4H, CH.sub.2), 3.46 (s, 4H, CH.sub.2), 1.46 (s,
36H, CO.sub.2Bu.sup.t). SIMS: 587(m.sup.+).
Compound No. 5
[0053] HPPH (100.0 mg, 0.157 mmol), amine 4 (184.5 mg, 0.314 mmol),
EDCI (90.4 mg, 0.471 mmol) and DMAP (57.5 mg, 0.471 mmol) were
dissolved in dry DCM (30 ml). Resultant mixture was stirred at room
temperature for 16 hr under N.sub.2 atm, diluted with DCM (100 ml)
and washed with brine (50 ml). Organic layer was separated, dried
over sodium sulfate and concentrated. Crude was purified on silica
gel column using MeOH/DCM (1-3%) as eluent to give product 5.
Yield: 120.0 mg (63.35%). UV-vis (.lamda.max cm.sup.-1,
dichloromethane): 409, 505, 535, 606 & 661.
[0054] .sup.1HNMR (400 MHz, CDCl.sub.3): .delta. 9.74 (s, 1H,
meso-H), 9.51 (s, 1H, meso-H), 8.52 (s, 1H, meso-H), 5.91 (m, 1H,
CH.sub.3*CHOhexyl), 5.35 (d, 1H, 15.sup.1*CH, J=20.0 Hz), 5.13 (d,
1H, 15.sup.1*CH, J=20.0 Hz), 4.52-4.49 (m, 2H, H-17 & H-18),
4.29-4.27 (m, 4H), 4.11 (m, 2H), 4.09-4.04 (m, 4H), 3.88-3.85 (m,
2H, CH.sub.2), 3.74-3.72 (m, 2H, O*CH.sub.2 hexyl), 3.67 (s, 3H,
ring-CH.sub.3), 3.66-3.59 (m, 2H, 8.sup.1-CH.sub.2), 3.36 (s, 3H,
ring-CH.sub.3), 3.26 (s, 3H, ring-CH.sub.3), 2.78-2.66 (m, 2H,
17.sup.2-CH.sub.2), 2.53-2.49 (m, 1H, 17.sup.1-CH), 2.15 (m, 1H,
17.sup.1-CH), 2.11 (d, 3H, CH.sub.2*CH.sub.3 CHOhexyl, J=6.8 Hz),
1.79 (d, 3H, 18-CH.sub.3, J=7.6 Hz), 1.74 (t, 3H,
8-CH.sub.2*CH.sub.3, J=7.6 Hz) 1.63 (m, 4H, CH.sub.2-hexyl),
1.47-1.43 (four singlets each for CO.sub.2Bu.sup.t, 36H), 1.20 (m,
4H, CH.sub.2-hexyl), 0.77 (t, 3H, CH.sub.3-hexyl, J=6.4 Hz), 0.37
(brs, 1H, NH), -1.82 (brs, 1H, NH). EIMS: 1206 (m.sup.+).
Compound No. 6
[0055] Compound 5 (70.0 mg) was stirred in 5 ml of 70% TFA/DCM for
3 hr at room temperature. The reaction mixture was concentrated and
dried under high vacuum to give 6 in quantitative yield.
[0056] Yield: 50.0 mg (87.7%). UV-vis (.lamda.max cm.sup.-1, THF):
408, 505, 538, 605 & 660. SIMS: 983 (m.sup.++1).
Compound No. 8
[0057] Acid 7 (100.0 mg, 0.115 mmol), amine 4 (136.0 mg, 0.231
mmol), EDCI (44.4 mg, 0.231 mmol) and DMAP (28.27 mg, 0.231 mmol)
were dissolved in dry DCM (30 ml). Resultant mixture was stirred at
room temperature for 16 hr under N.sub.2 atm, diluted with DCM (100
ml) and washed with brine (50 ml). Organic layer was separated,
dried over sodium sulfate and concentrated. Crude was purified on
silica gel column using MeOH/DCM (1-3%) as eluent to give product
8. Yield: 80.0 mg (48%). UV-vis (.lamda.max cm.sup.-1,
dichloromethane): 365, 414, 548 & 701. .sup.1HNMR (400 MHz,
CDCl.sub.3): .delta. 9.74 (s, 1H, meso-H), 9.60 (s, 1H, meso-H),
8.51 (s, 1H, meso-H), 8.20 (s, 2H, bis-CF.sub.3C.sub.6H.sub.3),
7.79 (s, 1H, bis-CF.sub.3C.sub.6H.sub.3), 5.79 (s, 2H, benzylic
CH.sub.2), 5.75 (m, 1H, CH.sub.3*CHObutyl), 5.19-5.16 (m, 1H,
H-17), 4.60-4.49 (m, 2H, CH.sub.2), 4.40-4.31 (m, 2H, CH.sub.2),
4.18-3.96 (m, 8H, 4-CH.sub.2), 3.62 (s, 3H, ring-CH.sub.3),
3.61-3.60 (m, 4H, 2CH.sub.2), 3.26 (s, 3H, ring-CH.sub.3), 3.16 (s,
3H, ring-CH.sub.3), 2.94-2.87 (m, 1H, 17.sup.2-CH), 2.76-2.69 (m,
1H, 17.sup.2-CH), 2.40-2.34 (m, 1H, 17.sup.1-CH), 2.05 (d, 3H,
CH.sub.3CHObutyl, J=10.2 Hz), 1.77-1.64 (m, 11H, 17.sup.1-CH,
18-CH.sub.3, 2CH.sub.2butyl, 8-CH.sub.2*CH.sub.3), 1.48 (s, 9H,
CO.sub.2Bu.sup.t), 1.46 (s, 9H, CO.sub.2Bu.sup.t), 1.39 (s, 9H,
CO.sub.2Bu.sup.t), 1.38 (s, 9H, CO.sub.2Bu.sup.t), 0.89-0.85
(spitted t, 3H, CH.sub.3-butyl), 0.21 (brs, 1H, NH), 0.07 (brs, 1H,
NH). SIMS: 1403 (m.sup.1).
Compound No. 9
[0058] Compound 8 (60.0 mg) was stirred in 5 ml of 70% TFA/DCM for
3 hr at room temperature. Reaction mixture was concentrated and
dried under high vacuum to give 9 in quantitative yield.
[0059] Yield: 40.0 mg (77.36%). UV-vis (.lamda.max cm.sup.-1, THF):
363, 414, 546 & 699. SIMS: 211 (m.sup.++1).
Compound No. 11
[0060] Acid 10 (50.0 mg, 0.072 mmol), amine 4 (84.7 mg, 0.144
mmol), EDCI (34.5 mg, 0.18 mmol) and DMAP (22.0 mg, 0.18 mmol) were
dissolved in dry DCM (30 ml). Resultant mixture was stirred at room
temperature for 16 hr under N.sub.2 atm, diluted with DCM (100 ml)
and washed with brine (50 ml). Organic layer was separated, dried
over sodium sulfate and concentrated. Crude was purified on silica
gel column using MeOH/DCM (1-2%) as eluent to give product 11.
[0061] Yield: 65.0 mg (71.42%). UV-vis (.lamda.max cm.sup.1,
dichloromethane): 363, 415, 508, 547 & 701.
[0062] .sup.1HNMR (400 MHz, CDCl.sub.3): .delta. 9.72 (s, 1H,
meso-H), 9.63 (s, 1H, meso-H), 8.52 (s, 1H, meso-H), 5.79 (m, 1H,
CH.sub.3*CHObutyl), 5.22 (m, 1H, H-17), 4.66 (m, 2H, CH.sub.2),
4.45 (t, 2H, OCH.sub.2butyl, J=7.6 Hz), 4.33 (m, 1H, H-18),
4.18-4.00 (m, 4H, 2CH.sub.2), 3.97-3.95 (m, 4H, 2CH.sub.2), 3.84
(s, 3H, ring-CH.sub.3), 3.68-3.61 (m, 4H, 8*CH.sub.2CH.sub.3,
CH.sub.2), 3.30 (s, 3H, ring-CH.sub.3), 3.18 (s, 3H,
ring-CH.sub.3), 3.00-2.90 (m, 1H, 17.sup.2-CH), 2.74-2.69 (m, 1H,
17.sup.2-CH), 2.45-2.39 (m, 1H, 17.sup.1-CH), 2.06 (d, 3H,
CH.sub.3CHObutyl, J=6.8 Hz), 2.01-1.96 (m, 2H, NCH.sub.2-butyl),
1.70 (m, 1H, 17.sup.1-CH), 1.68-1.61 (m, 10H, 18-CH.sub.3,
2CH.sub.2butyl, 8-CH.sub.2*CH), 1.51, 1.49, 1.37 & 1.36 (each
singlet for 36H, CO.sub.2Bu.sup.t), 1.10 (t, 3H, CH.sub.3-Obutyl,
J=7.6 Hz), 0.87 (t, 3H, CH.sub.3-Nbutyl, J=7.4 Hz), -0.02 (brs, 1H,
NH), -0.12 (brs, 1H, NH). SIMS: 1263 (m.sup.+).
Compound No. 12
[0063] Compound 11 (60.0 mg) was stirred in 5 ml of 70% TFA/DCM for
3 hr at room temperature. Reaction mixture was concentrated and
dried under high vacuum to give 12 in quantitative yield.
[0064] Yield: 42.0 mg (85.19%). UV-vis (.lamda.max cm.sup.-1,
dichloromethane): 363, 415, 508, 547 & 701. EIMS: 1039
(m.sup.+).
In Vivo Photosensitizing Efficacy
[0065] The experiments were performed in female BALB/c mice (6-8
weeks of age) purchased from Clarence Reeder (National Cancer
Institute Fredrick Cancer Research Facility, Fredrick, Md.). The
mice were injected s.c. in the axilla with 10.sup.6 Colo-26 cells
in 50 .mu.L complete RPMI-1640 and were used for experimentation
when the tumors reached 5-6 mm All experiments were performed under
the approved protocols of the RPCI Animal Care and Use Committee
and followed DLAR regulations.
[0066] (a) Comparative Photosensitizing Efficacy of 15 vs its water
soluble analog 16:
BALB/c mice inoculated with Colon-26 tumors were injected with 0.7
.mu.moles/kg of either PS 15 or 16 and at .about.24 h p.i., the
mice were treated with PDT for a total fluence of 135 J/cm.sup.2 at
75 mW/cm.sup.2 (30 minute treatment). Preliminary studies had shown
that PS 15 was only 30% effective using the 135 J/cm.sup.2 at 75
mW/cm.sup.2 (30 minute) PDT regimen. However, when its
water-soluble analog was tested, the PDT response enhanced to 70%
mice tumor-free by day 90.
[0067] Three explanations for this may be that (1) the slight
charge from the carboxylate groups may be contributing to differing
localization sites of PS 16 in comparison to 15 (as mentioned
above), (2) the PDT-induced mechanism of action may differ in
comparison to 16 or (3) the increased PS uptake in the tumor
compared to the skin of 16 could be contributing to the enhanced
PDT response. The main purpose of these experiments was to
determine if the water-soluble PS could be utilized as both a PDT
agent and diagnostic imaging tool. The initial in vivo experiments
displayed the advantage of the water-soluble PS over its parent
compound, 15.
[0068] Comparative Photosensitizing Efficacy Water-soluble
Photosensitizers 9 and 12:
[0069] The in vivo photosensitizing efficacy of water-soluble
photosensitizers 9 and 12 was determined in BALB-C mice bearing
Colo-26 tumors at similar treatment conditions. At 24 h
postinjction of the photosensitizer (i.v., 0.5 .mu.mol/Kg), the
tumors were exposed to laser light (at the photosensitizer's
longest wavelength absorption (135 J/cm.sup.2, 75 mW/cm.sup.2 for
30 min) and the tumor regrowth was measured daily. The results are
summarised in FIG. 2. As can be seen among the three candidates,
compared to 14, compounds 9 and 12 were found to be more
effective.
[0070] In Vivo Fluorescence Imaging with the Water-Soluble Analog
16.
[0071] Measurement of PS accumulation in the tumor and skin via
fluorescence measurements using a non-invasive optical imaging
camera system was performed. When tumors reached 4-5 mm in
diameter, the BALB/c mice were imaged prior to PS injection (using
body weight of Ketamine Xylazine or 80 mg/kg of Pentobarbital
Sodium anesthesia) to make certain that no endogenous chromophores
were excited at the particular wavelengths utilized (425/50 nm or
540/40 nm excitation filters). Background fluorescence measurements
had been a concern for previous researchers because it was found
that the current diet of the mice contained chlorophyll
(.lamda..sub.max fluorescence=676 nm). When evaluating a
photosensitizer such as HPPH, the PS emission peak at .about.668 nm
overlapped with that of chlorophyll. Therefore, the fluorescence
images obtained were not particularly specific for only PS
fluorescence. For instance, when the background mice were imaged
(No PS) using an excitation wavelength of 425/50 nm the chlorophyll
from the diet was present in both the hair (yellow) and BALB/c skin
(red) exhibiting an emission peak at .about.676 nm. For the
experiments with PS 15 and 16, there was no concern that the
emission peak of chlorophyll would overlap with that of the PS
(emission at .about.710 nm). See FIGS. 3A-3F
[0072] For non-invasive in vivo imaging of PS fluorescence, the
Nuance.TM. Imaging Camera was beneficial in that once anesthetized
the whole body of the mouse could be placed into the imaging
LT-9CABINET, which provided the proper light insulation required
for measurement and the ILLUMATOOL low power light source necessary
for keeping the amount of light delivered to each mouse constant (3
mice per time point). This imaging technology was quite beneficial
due to the fact that it was minimally invasive, so that there was
no need to sacrifice the animal in order to obtain information
about where the PS was localized. Previous studies have involved
invasive procedures in which a mouse was sacrificed, the tumor or
skin was excised and histological staining was performed on the
paraffin blocks. Below are fluorescence images of PS 16 excited
using the 425/50 nm filter and collected via the non-invasive CCD
Nuance Imaging Camera (Princeton Instruments Inc.). This system was
capable of taking qualitative hyperspectral images in the specific
range of 650-720 nm focused on 710 nm.
[0073] From FIG. 3, it can be seen that PS 16 showed a significant
selectivity for tumors (peak fluorescence at .about.710 nm), but
when the skin flap was performed there appeared to be a noticeable
amount of PS remaining in the underside of the skin after tumor
removal. It is important to remember that these are qualitative
images of PS accumulation in the tumor and skin. As a means to
determine the exact uptake of the PS in the tumor versus the skin
and other organs, a skin-flap excision, as well as, an ex vivo
biodistribution study were performed. Once removed, the organs
(tumor, skin, heart, spleen, muscle, kidney, stomach, intestine,
lung and liver) were placed on a plexiglass plate and their
fluorescence was collected (425/50 nm excitation). The fluorescence
image displayed fluorescence peaks at .about.675 (yellow spectrum
characteristic of chlorophyll-a from diet) and .about.710 nm (red
spectrum characteristic of PS 16) with visible fluorescence in the
tumor, skin, large intestine, liver and stomach. The organs were
homogenized, dissolved in Solvable and read on the Fluoromax II
Fluorimeter at 417 nm. After reading the fluorescence of all the
organ samples, it was determined that the tumor and liver retained
PS 16 (peak emission .about.710 nm), while the skin, stomach and
intestine retained material characteristic of chlorophyll-a (peak
emission .about.676 nm). The average fluorescence per mg/mL of
protein was normalized to background mice (no PS) and plotted for
each organ (avg. of 3 samples per organ).
[0074] This invention describes the successful synthesis of a new
long wavelength water-soluble PS. The in vitro and in vivo PDT
photosensitizing experiments indicated that PS 16 was superior to
its parent compound, 15
[0075] At its therapeutic PDT dose of 0.7 .mu.moles/kg (70% mice
were tumor-free by day 60, 7/10 mice), PS 16 displayed selective
tumor uptake at 24 h p.i. as visualized by Nuance.TM. imaging and
confirmed by the fluorescence extraction experiments. This is the
first report of a water-soluble fluorinated purpurinimide being
utilized as a dual PDT-imaging agent.
* * * * *