Conjugate of a photosensitiser and chitosan and uses thereof
09901636 ยท 2018-02-27
Assignee
Inventors
- Kristian Berg (Heggedal, NO)
- Anders Hogset (Oslo, NO)
- Mar Masson (Reykjavik, IS)
- Vivek S. Gaware (Reykjavik, IS)
Cpc classification
C08B37/003
CHEMISTRY; METALLURGY
A61K41/0057
HUMAN NECESSITIES
A61K47/61
HUMAN NECESSITIES
A61K41/0076
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to novel chitosan-based conjugates, e.g. nanocarriers, comprising a derivative of the biocompatible polymer chitosan conjugated to a photosensitizing agent, and uses thereof in photochemical internalization (PCI) and photodynamic therapy (PDT). The invention also relates to the use of the novel conjugates of the invention in treatment or prevention of diseases, particularly cancer, and for vaccination purposes.
Claims
1. A compound comprising a conjugate of a photosensitiser and chitosan, wherein said compound is a compound of Formula (I): ##STR00048## wherein n is an integer greater than or equal to 3, R appears n times in said compound and in 0.5%-99.5% of said total Rn groups, each R is a group A selected from: H, ##STR00049## wherein a is 1, 2, 3, 4 or 5; and X is Br, Cl or OH; ##STR00050## wherein each R.sub.1, which may be the same or different, is selected from H, CH.sub.3 and (CH.sub.2).sub.cCH.sub.3; b is 1, 2, 3, 4 or 5; and c is 0, 1, 2, 3, 4 or 5; ##STR00051## wherein Y is O; S; SO.sub.2, NCH.sub.3; or N(CH.sub.2).sub.eCH.sub.3; d=1, 2, 3, 4 or 5; and e=1, 2, 3, 4 or 5; ##STR00052## wherein R.sub.2 is (CH.sub.2).sub.hCH.sub.3 or CO(CH.sub.2).sub.hCH.sub.3; f is 1, 2, 3, 4 or 5; g is 1, 2, 3, 4 or 5; and h is 0, 1, 2, 3, 4 or 5; ##STR00053## wherein R.sub.3 is (CH.sub.2).sub.jCH.sub.3, i is an integer from 1 to 200; j is 0, 1, 2, 3, 4 or 5; and k is 1, 2, 3, 4 or 5; ##STR00054## wherein R.sub.3 is (CH.sub.2).sub.jCH.sub.3, i is an integer from 1 to 200; and j is 0, 1, 2, 3, 4 or 5; ##STR00055## wherein R.sub.3 is (CH.sub.2).sub.jCH.sub.3, i is an integer from 1 to 200; j is 0, 1, 2, 3, 4 or 5; and each R.sub.1, which may be the same or different, is selected from H, CH.sub.3 and (CH.sub.2).sub.cCH.sub.3; and c is 0, 1, 2, 3, 4 or 5; ##STR00056## wherein R.sub.3=(CH.sub.2).sub.jCH.sub.3, i is an integer from 1 to 200; and j is 0, 1, 2, 3, 4 or 5; ##STR00057## wherein R.sub.3=(CH.sub.2).sub.jCH.sub.3, i is an integer from 1 to 200; L is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and j is 0, 1, 2, 3, 4 or 5; ##STR00058## wherein m is 1, 2, 3, 4 or 5; wherein each R group may be the same or different; and in 0.5%-99.5% of said total Rn groups, each R is a group B selected from: ##STR00059## wherein p is 0, 1, 2, 3, 4 or 5; q is 1, 2, 3, 4 or 5; and r is 1, 2, 3, 4 or 5; R.sub.4 is a group selected from: ##STR00060## W is a group selected from O, S, NH or N(CH.sub.3); R.sub.5 is a group selected from: (CH.sub.2).sub.sCO; (CH.sub.2).sub.sZ(CH.sub.2).sub.tCO and (CH.sub.2).sub.sZ(CH.sub.2).sub.tZCO; wherein s is 0, 1, 2, 3, 4 or 5; t is 0, 1, 2, 3, 4 or 5; Z is NH, O, S, or SO.sub.2, R.sub.6 is a group selected from CN and CH.sub.3, R.sub.7 is a group selected from: ##STR00061## V is a group selected from CO, SO.sub.2, PO, PO.sub.2H or CH.sub.2; and R.sub.8 is a group (substituted in the o, m or p position), which may be the same or different, selected from H, OH, OCH.sub.3, CH.sub.3, COCH.sub.3, C(CH.sub.3).sub.4, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2 and NCOCH.sub.3, wherein each R group may be the same or different.
2. A compound as claimed in claim 1 wherein n is an integer from 10 to 100.
3. A compound as claimed in claim 1 wherein R.sub.4 is selected from ##STR00062##
4. A compound as claimed in claim 1 wherein R.sub.7 is selected from ##STR00063##
5. A compound as claimed in claim 3 wherein R.sub.4 is TPCa.sub.1 or TPCa.sub.2.
6. A compound as claimed in claim 1 wherein group A provides 70 to 95% of the total Rn groups and group B provides 5 to 30% of the total Rn groups.
7. A compound as claimed in claim 1 wherein each group A R group is selected from: ##STR00064## wherein each R.sub.1 is CH.sub.3 and b is 1; ##STR00065## wherein Y is NCH.sub.3 and d is 1; ##STR00066## wherein preferably j is 0 or 1; i is 3 or 6 and k is 1; ##STR00067## wherein j is 1 and i is 2; ##STR00068## wherein j is 0 or 1 and i is 2, 4 or 5 and L is 1; ##STR00069## wherein m is 1, and each R group may be the same or different.
8. A compound as claimed in claim 1 wherein each group B R group is selected from: ##STR00070## wherein p is 1; ##STR00071## wherein p is 1 and q is 1; ##STR00072## wherein p is 1; and ##STR00073## wherein p is 1, and each R group may be the same or different.
9. A compound as claimed in claim 1 wherein said compound is selected from the following compounds ##STR00074## ##STR00075##
10. A compound as claimed in claim 4 wherein R.sub.7 is TPCc.sub.1 or TPCc.sub.2.
11. The compound of claim 1, wherein when group A is ##STR00076## i is an integer from 1-10.
12. The compound of claim 1, wherein when group A is ##STR00077## i is an integer from 1-10.
13. The compound of claim 1, wherein when group A is ##STR00078## i is an integer from 1-10.
14. The compound of claim 1, wherein when group A is ##STR00079## i is an integer from 1-10.
15. The compound of claim 1, wherein when group A is ##STR00080## i is an integer from 1-10.
16. A pharmaceutical composition comprising a compound as defined in claim 1 and one or more pharmaceutically acceptable diluents, carriers or excipients, and optionally a molecule to be internalized.
17. A kit comprising a compound as defined in claim 1, or a composition comprising said compound, and a molecule to be internalized.
18. A method for introducing a molecule into the cytosol of a cell, comprising contacting said cell with the molecule to be introduced and a compound as defined in claim 1, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent of the compound thereby releasing the molecule into the cytosol.
19. A method of achieving death of a cell comprising contacting said cell with a compound as defined in claim 1, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent of the compound to generate reactive oxygen species which cause death of said cell.
20. A method of expressing an antigenic molecule or a part thereof on the surface of a cell, comprising contacting said cell with said antigenic molecule and a compound as defined in claim 1, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent of the compound, wherein said antigenic molecule is released into the cytosol of the cell and the antigenic molecule or a part thereof of sufficient size to stimulate an immune response is presented on the cell's surface.
21. A cell or a population of cells obtainable by a method as defined in claim 18.
22. A method of achieving death of a cell in a patient comprising introducing a compound as defined in claim 1, or a composition comprising said compound, and a cytotoxic molecule to be internalized, into one or more cells in vivo by a method comprising contacting said cells with said compound and said cytotoxic molecule to be introduced, and irradiating the cells with light of a wavelength effective to activate the photosensitising agent of the compound thereby generating reactive oxygen species which cause death of the cell and/or releasing said cytotoxic molecule to be introduced into the cytosol.
23. A cell or a population of cells obtainable by a method as defined in claim 20.
24. A method of stimulating an immune response in a subject, comprising introducing a compound as defined in claim 1, or a composition comprising said compound, and an antigenic molecule to be internalized, into one or more cells in vitro, in vivo or ex vivo by a method comprising contacting said cells with said compound and said antigenic molecule to be introduced, and irradiating the cells with light of a wavelength effective to activate the photosensitising agent of the compound thereby releasing said antigenic molecule to be introduced into the cytosol and presenting the antigenic molecule or a part thereof of sufficient size to stimulate an immune response, and where necessary administering said cells to said patient, whereby an immune response is stimulated in said subject.
25. A method as claimed in claim 18 wherein said method is carried out in vivo.
26. A method as claimed in claim 19 wherein said method is carried out in vivo.
27. A method as claimed in claim 20 wherein said method is carried out in vivo.
28. The method of claim 22, wherein the cell is a cancer cell.
Description
(1) The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:
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Example 1Synthesis of Meso-Tetraphenylporphyrin-Chitosan-Based Nanocarriers
(30) Highly water soluble chitosan nanocarriers tethered with the photosensitizer meso-tetraphenylporphyrin (TPP) were synthesized, in a 7 step procedure, from 3,6-di-O-tert-butyldimethylsilyl-chitosan (DiTBDMS-CS) and 5-(p-aminophenyl)-10,15,20-triphenylporphyrin [TPP(p-NH.sub.2).sub.1] as starting materials. DiTBDMS-CS is highly soluble in CH.sub.2Cl.sub.2 and the highly lipophilic photosensitizer could therefore be introduced in a quantitative reaction to give 0.1 and 0.25 degree of substitution. This was followed by introduction of trimethylammoniumyl and or 1-methylpiperazinyl groups onto the polymer backbone in order to increase aqueous solubility of the final deprotected carriers. It was shown that the method is highly reproducible and that the obtained material could be fully characterized by solid state NMR, FT-IR, and .sup.1H NMR. UV-Vis, fluorescence and NMR investigations showed that the carriers are dynamic structures, which form nanoparticle-like structures in aqueous solution with a core of semi-solid m-stacked photosensitizers. In lipophilic environments it is probable that these structures can unfold and the photosentizer moiety can thus be inserted into the cell membrane.
(31) General Materials and Methods
(32) The chitosan polymer was donated by Genis EHF, Iceland and was used for the synthesis [chitosan polymer GO 30626-2 (95% DD, 95 cp)]. All solvents and reagents were purchased commercially and used without further purification. NMR spectra were recorded on a DRX 400 MHz Bruker NMR spectrometer at 298 K and chemical shifts were reported relative to the deuterated NMR solvent used [.sup.1H NMR: CDCl.sub.3 (7.26 ppm), DMSO-d.sub.6 (2.50 ppm); .sup.13C NMR: CDCl.sub.3 (77.16 ppm), DMSO-d.sub.6 (39.52 ppm)]. The Acetone peak (2.22 ppm) was used as the internal reference for D.sub.2O as solvents. The protons (ortho, meta, para) on the phenyl rings of porphyrins are identified with respect to their positions relative to the porphyrin ring system and not with respect to the substituent on the phenyl ring.
(33) Solid-state .sup.13C NMR of compounds 17B and 19B was obtained from the Department of Chemistry, Durham University. These spectra were obtained using a Varian VNMRS spectrometer operating at 100.56 MHz for .sup.13C. Cross-polarization magic-angle spinning experiments were carried out with a 6 mm (rotor o.d.) probe. The spectra were recorded at a spin-rate of 6.8 kHz, with a 1 ms contact time, a 1.5 s recycle delay and with TOSS spinning sideband suppression. Spectral referencing is with respect to an external sample of neat tetramethylsilane carried out indirectly by setting the high-frequency signal from adamantane to 38.5 ppm.
(34) Mass spectra were recorded on Bruker Autoflex III or a Bruker micrOTOF-Q11. FTIR-measurements were performed with an AVATAR 370 FT-IR instrument (Thermo Nicolet Corporation, Madison, USA). Samples (2-3 mg) were kneaded thoroughly with KBr. The sample was pressed into pellets with a Specac compressor (Specac Inc., Smyrna, USA). Melting Points were recorded on Buchi Melting Point B-540. Polymer samples were dialyzed using Spectra/Por Dialysis Membrane (MWCO: 3500).
(35) Absorption and Steady-State Fluorescence Spectra.
(36) UV-Vis measurements were recorded on a Perkin-Elmer Lambda 25 UV-Vis. spectrometer equipped with a Peltier Temperature Programmer. Fluorescence emission spectra were obtained using a SPEX FluoroMax spectrometer, using a cell with spectral range 170-2200 nm (Spectrocell Corporation, Oreland, Pa., USA). Absorption spectra were recorded at 20? C. and fluorescence emission spectra were recorded at ambient temperature, using quartz cuvette with a 10 mm path length. All the fluorescence spectra were recorded with constant slit widths, 1 nm for excitation and 1 nm for emission and fluorescence spectra were averaged over three scans for quantum yield study. However, for
(37) Fluorescence quantum yields of compounds 3, 5, 16A-19B (all excited at ?ex.=?max=419 nm) were determined relative to a dilute solution of standard anthracene (?.sub.F=0.27, ?ex=365.5 nm) in absolute ethanol by using the steady-state comparative method. using the following equation:
?.sub.X=?.sub.ST(Grad.sub.X/Grad.sub.ST)(?.sub.S.sup.2,?.sub.ST.sup.2)
where, the subscripts ST and X denote standard and test respectively, ? is the fluorescence quantum yield, Grad the gradient from the plot of integrated fluorescence intensity versus absorbance, and ? the refractive index of the solvent.
(38) Stability Study:
(39) For the physical stability study 17A was dissolved in H.sub.2O (1 mg/mL), sonicated for 30 minutes, centrifuged (on HERMLE Z-320 4000 rpm for 10 min), decanted and wrapped with aluminium foil. UV-Vis absorbance were measured at ?.sub.max=419 nm in H.sub.2O over the period of 0-90 days.
(40) Determination of the Degree of Substitution (DS) by .sup.1H NMR.
(41) In order to calculate the substitution degrees of the TPPNH-Pip, we used 1H NMR of the final compounds. For calculating DS from Final compounds 16A-19B, we used integration values of TPP peaks (from TPPNH-Pip part) and that of H-1 (from chitosan part) peaks. The integral of TPP (four peaks from aromatic region) was considered, while the integrals of H-1 group was calculated and used in the following equations:
DS=(?Aromatic TPP peaks/27)/(?H?1 peak/1)
(42) Under this condition the polymer backbone is partially overlapped by the HDO peak but the DS can be determined with good accuracy from the relative ratio of the integrals of the H?1 peak and the TPP peaks.
(43) For calculating the DS of TPCNH-Pip AND TPCCO-Pip we used intermediate compounds 29 & 34 and integration values of TPC peaks (from TPCNH-Pip or TPCCO-Pip in the aromatic region as well as ?-pyrrole NH peak integrations) and that of TBDMS peaks integration (from chitosan) in the following equations:
DS=(?Aromatic TPC peaks+? pyrrole NH peak/27)/(?TBDMS peak/30)
(44) For calculating the DS of the TEGylation for various chitosan derivatives we used corresponding .sup.1H NMR. We considered the integration values of the H?1 peak of chitosan and the integration value of CH.sub.3 (of terminal ethyl peak of TEG) on a chitosan backbone The integration value for the -ethyl end triplet would be equal to 3 if the substitution degree is 100%. Therefore, the equation is following:
DS(%)(?Ethyl peak(end triplet,CH3)/3)/(?H?1 peak/1)*100%
(45) SEM and Elipsometry.
(46) Solutions were spin-coated at 1000 rpm onto pristine silicon <100> substrates (15 mm?15 mm) using a conventional spinner in a Class-100 clean room environment. The silicon has a layer of native oxide of approximately 15 ? thickness. Furthermore, 10 ?l of the same solutions were pipetted directly onto silicon substrates and allowed to dry in air at room temperature. The coated substrates were imaged in a Zeiss LEO 1550 scanning electron microscope at 10 keV acceleration voltage and 2 mm working distance using an in-lens detector.
(47) Water Contact Angle Measurements.
(48) Water contact angles were determined using a KSV CAM 200 optical contact angle meter (KSV Instruments). A 5 ?l deionized water droplet was dropped on the centre of the silicone wafer and water contact angles determined based on the Laplace & Young equation. Measurements were done at room temperature and ambient humidity.
(49) Synthesis
(50) See scheme 1 in
(51) Meso-Tetraphenylporphyrin (1). Following the literature procedure (Adler, J Organic Chem 1967, 32:476).
(52) 5-(4-Nitrophenyl)-10,15,20-triphenylporphyrin [TPP(p-NO.sub.2).sub.1] (2).
(53) Following the literature procedure (Luguya R et al. Tetrahedron 2004, 60:2757).
(54) 5-(4-Aminophenyl)-10,15,20-triphenylporphyrin [TPP(p-NH.sub.2).sub.1] (3).
(55) Following the literature procedure (Luguya R et al. Tetrahedron 2004, 60:2757).
(56) 5-(4?-Bromoacetylamidophenyl)-10,15,20-triphenylporphyrin (4).
(57) Compound 3 (500 mg, 0.793 mmol) was dissolved in CH.sub.2Cl.sub.2 (15 mL) and stirred under an N.sub.2 atmosphere. Triethylamine (0.24 mL, 1.75 mmol) was added followed by drop wise addition of bromoacetyl bromide (0.097 mL, 1.11 mmol) at rt and the stirring continued at it for 1 h. The reaction mixture was diluted into CH.sub.2Cl.sub.2(45 mL), washed with water (2?25 mL) and brine (20 mL). The organic layer was then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Crude product was purified by silica gel column chromatography, using CH.sub.2Cl.sub.2 and hexane as eluent, which yielded 385 mg (64%) of desired product 4. TLC (Hexane/CH.sub.2Cl.sub.2 3:7): Rf=0.17; FT-IR, v cm.sup.?1: 3313 (NH), 3049, 3019 (aryl CH), 1687, 1594 (CONH), 1556, 1514, 1471, 1439, 1399, 1348, 1177, 1153, 964, 798, 726, 699; .sup.1H NMR (CDCl.sub.3): ?=8.88-8.91 (m. 8H, ?-pyrrole H), 8.41 (br s, 1H, TPPNHCO), 8.22-8.27 (m, 8H, tetraphenyl H.sub.O), 7.91 (d, J=8 Hz, 2H, CONH-phenyl-Hm), 7.75-7.80 (m, 9H, triphenyl-Hm,p), 4.15 (s, 2H, COCH.sub.2Br), ?2.70 (br s, 2H, ?-pyrrole NH) ppm; .sup.13C NMR (CDCl.sub.3): ?=163.78, 142.26, 139.20, 136.78, 135.25, 134.68, 131.23, 127. 87, 126.83, 120.42, 120.38, 119.24, 118.34, 29.72 ppm; MS (ESI): m/z calcd. for C.sub.46H.sub.33BrN.sub.5O ([M+H].sup.+) 750.1863 found 750.1864; UV-vis (DMSO): ?.sub.max: 417, 517, 542, 597, 650 nm.
(58) 5-(4?-Piperazineacetylamidophenyl)-10,15,20-triphenylporphyrin [TPPNH-Pip[(5).
(59) Compound 4 (275 mg, 0.366 mmol) and excess piperazine (158 mg, 1.83 mmol) was mixed together in CH.sub.2Cl.sub.2 (10 mL) and stirred at rt for 1 h under an N.sub.2 atmosphere. After completion of the reaction, the reaction mixture was diluted with CH.sub.2Cl.sub.2 (85 mL) and washed with water (2?40 mL) and brine (35 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated under vacuo. The crude product was purified by silica gel column chromatography using 1:12 MeOH: CH.sub.2Cl.sub.2 as eluent to afford titled compound 5 (260 mg, 94%) as a purple solid. TLC (CH.sub.2Cl.sub.2/MeOH, 9:1): Rf=0.15; FT-IR (KBr): v 3442 (pip. NH), 3312 (aryl NH), 3052, 3022 (aryl CH), 2903, 2816 (aliphatic CH2), 1691, 1596 (CONH), 1557, 1517, 1471, 1439, 1400, 1349, 1309, 1179, 1153, 1071, 1001, 965, 799, 728, 700 cm.sup.?1; .sup.1H NMR(CDCl.sub.3): ?=9.51 (br s, 1H, TPPNHCO), 8.89-8.93 (m, 8H, ?-pyrrole H), 8.23-8.26 (m, 8H, tetraphenyl H.sub.O), 8.01 (d, J=8.0 Hz, 2H, Pip-NH-phenyl-Hm), 7.75-7.81 (m, 9H, triphenyl-Hm,p), 3.31 (s, 2H, COCH.sub.2-Pip.), 3.11 (t, 4H), 2.77 (br t, 4H), 2.60 (br s, 1H, piperazine NH), ?2.71 (br s, ?-pyrrole 2H) ppm; .sup.13C NMR (CDCl.sub.3): ?=168.75, 142.30, 138.26, 137.44, 135.33, 134.68, 131.26, 127.86, 126.83, 120.32, 119.66, 117.89, 62.87, 54.53, 46.24 ppm; MS (ESI): m/z calcd. for C.sub.50H.sub.42N.sub.7O ([M+H].sup.+) 756.3445 found 756.3467; UV-vis (DMSO): ?.sub.max: 417, 517, 542, 597, 650 nm.
(60) See scheme 2 in
(61) Chitosan mesylate (7).
(62) Synthesized according to our previously published procedure (Song et al. Carbohydrate Polymers 2010, 81:140).
(63) 3,6-O-di-tert-butyldimethylsilyl chitosan [DiTBDMS-CS] (8).
(64) Synthesized according to our previously published procedure (Song et al. Carbohydrate Polymers 2010, 81:140).
(65) N-bromoacetyl-3,6-O-DiTBDMS-CS [BrA-DiTBDMS-CS] (9).
(66) DiTBDMS-CS 8 (1 g, 2.60 mmol) was dissolved in dry CH.sub.2Cl.sub.2 (15 mL) in a round bottom flask under an N.sub.2 atmosphere. Then the reaction mixture was cooled to ?20? C. with an ice/salt mixture. Et.sub.3N (1.81 mL, 13 mmol) was added followed by slow drop wise addition of bromoacetyl bromide (0.91 mL, 10 mmol). Stirred for 1 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 and concentrated in vacuo. The crude product was stirred in acetonitrile, filtered and washed with fresh acetonitile. Dry material was dissolved and extracted in CH.sub.2Cl.sub.2, washed with water and brine, and dried over Na.sub.2SO.sub.4, concentrated under vacuo. Faint yellow powdered product 9 was obtained 1.2 g (92% yield). FT-IR (KBr): v 3402 (br, NH), 2957, 2931, 2886, 2858 (s, CH TBDMS), 1682 (vs, C?O amide I), 1530 (vs, C?O amide II), 1473, 1391, 1362, 1311, 1259, 1101, 1005, 837, 777 (SiC), 669 cm.sup.?1; NMR (CDCl.sub.3) ? ppm: 4.40 (br s, H-1), 4.02-3.26 (m, H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and 2H GluNHC?OCH.sub.2Br), 0.90 and 0.88 (br s, (CH.sub.3).sub.3C), 0.13 and 0.07 (br s, (CH.sub.3).sub.2Si) ppm.
(67) (N-TPPNH-Pip-acetyl).sub.0.1-(N-bromoacetyl).sub.0.9-DiTBDMS-CS [TPPp.sub.0.1-BrA.sub.0.9-DiTBDMS-CS] (10A).
(68) Compound 9 (700 mg, 1.38 mmol) and compound NH-pip-TPP 5 (105 mg, 0.138 mmol) were dissolved in CH.sub.2Cl.sub.2 under an N.sub.2 atmosphere. Exact equimolar quantity of Et.sub.3N (19.3 ?L, 0.130 mmol) with respective to 5 was added and reaction mixture was stirred at rt for 24 h. Total consumption of starting material was confirmed by TLC. The reaction mixture was diluted with CH.sub.2Cl.sub.2, extracted, and washed with water and brine. The organic layer was dried over Na.sub.2SO.sub.4, concentrated under vacuo to yield 730 mg (92%) product 10A. FT-IR (KBr): v 3324 (br, NH), 2955, 2929, 2884, 2856 (s, CH TBDMS), 1678 (vs, C?O amide 1), 1600, 1524 (vs, C?O amide II), 1472, 1403, 1361, 1311, 1256, 1098, 1004, 966, 837, 801, 778, 701, 670, 550 cm.sup.?1; .sup.1H NMR (CDCl.sub.3) ? ppm: 9.30(s, TPPNHCO), 8.85 (m, ?-pyrrole H), 8.23-8.20 (m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 7.97 (d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.79-7.73 (m, triphenyl-Hm,p), 4.41 (br s, H-1), 4.13-3.50 (m, H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and 2H GluNH-C?O, TPPNHCOCH.sub.2pip, CH.sub.2CONGlc and (CH.sub.2).sub.2 of piperazine), 2.81-2.86 (m, piperazine-H), 0.92, 0.89 (br s, (CH.sub.3).sub.3C), 0.14, 0.07 (br s, (CH.sub.3).sub.2Si), ?2.77 (br s, ?-pyrrole NH) ppm; UV-vis (DMSO): ?.sub.max: 417, 517, 542, 597, 650 nm.
(69) TPPp.sub.0.1-BrA.sub.0.9-DiTBDMS-CS (10B):
(70) Compound 10B (1.3 mg, 91%) was prepared exactly as the above procedure using intermediate 5 (180 mg, 0.24 mmol) and 9 (1.2 g, 2.4 mmol).
(71) TPPp.sub.0.25-BrA.sub.0.75-DiTBDMS-CS (11A). Compound 9 (550 mg, 1.09 mmol) and compound NH-pip-TPP 5 (206 mg, 0.273 mmol) were dissolved in CH.sub.2Cl.sub.2 under an N.sub.2 atmosphere. An exact equimolar quantity of Et.sub.3N (38 ?L, 0.27 mmol) was added with respect to 5 and the reaction mixture was stirred at rt for 24 h. Total consumption of starting material was confirmed by TLC. The reaction mixture was diluted with CH.sub.2Cl.sub.2, extracted, and washed with water and brine. The organic layer was dried over Na.sub.2SO.sub.4, and concentrated under vacuo to yield 670 mg (91%) of product 11A. FT-IR (KBr): v 3317 (br, NH), 2952, 2926, 2883, 2855 (s, CH TBDMS), 1680 (vs, C?O amide I), 1598, 1520, 1471, 1440, 1402, 1361, 1309, 1254, 1096, 1002, 966, 837, 800, 778, 730, 701, 667, 558 cm.sup.?1; .sup.1H NMR (CDCl.sub.3) ? ppm: 9.30(s, TPPNHCO), 8.85 (m, ?-pyrrole H), 8.22-8.20 (m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 7.97 (d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.80-7.75 (m, triphenyl-Hm,p), 4.40 (br s, H-1), 4.06-3.63 (m, H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and 2H GluNHC?O, TPPNHCOCH.sub.2pip, CH.sub.2CONGlc and (CH.sub.2).sub.2 of piperazine), 2.81-2.86 (m, piperazine-H), 0.92, 0.89 (br s, (CH.sub.3).sub.3C), 0.14, 0.10, 0.07 and 0.02 (br s, (CH.sub.3).sub.2Si), ?2.77 (br s, ?-pyrrole NH) ppm; UV-vis (DMSO): ?.sub.max: 417, 517, 542, 597, 650 nm.
(72) TPPp.sub.0.25-BrA.sub.0.75-DiTBDMS-CS (11B):
(73) Compound 11B (1.35 g, 92%) was prepared exactly as the above procedure using intermediate 5 (415 mg, 0.55 mmol) and 9 (1.1 g, 2.18 mmol).
(74) General procedure A for Synthesis of compounds 12A, 12B, 13A and 13B (N-TPPNH-Pip-acetyl).sub.0.1-(N(N,N,N-trimethylammoniumyl)acetyl).sub.0.9-DiTBDMS-CS [TPPp.sub.0.1-DiTBDMS-CS-TMA.sub.0.9] (12A).
(75) Compound 10A (350 mg, 0.61 mmol) was dissolved in CH.sub.2Cl.sub.2 under N.sub.2 atmosphere. An excess amount of trimethylamine solution was added and the reaction mixture was stirred at rt for 24 h. Solvent was removed in vacuo. The crude product was dried completely under high vacuum yielding crude product 12A (420 mg, 99%) as a purple solid. FT-IR (KBr): v 3426, 3021, 3011, 2962, 2855, 27.7, 2560, 2438, 1749, 1689, 1599, 1563, 1482, 1443, 1401, 1360, 1288, 1254, 1053, 1010, 966, 922, 901, 837, 797, 779, 744, 701, 671, 552 cm.sup.?1.
(76) TPPp.sub.0.1-DiTBDMS-CS-TMA.sub.0.9 (12B).
(77) The general procedure A was followed using 10B (600 mg, 1.04 mmol) and NMe.sub.3 to give 12B as a crude solid (720 mg, 99%).
(78) TPPp.sub.0.25-DiTBDMS-CS-TMA.sub.0.75 (13A).
(79) The general procedure A was followed using 11A (300 mg, 0.44 mmol) and NMe.sub.3 to give 13A crude solid (340 mg, 98%). FTIR (KBr): v 3415, 3021, 3011, 2962, 2854, 1749, 1686, 1598, 1522, 1482, 1442, 1402, 1360, 1311, 1288, 1252, 1053, 1010, 966, 922, 901, 837, 798, 779, 744, 701, 671, 558 cm.sup.?1.
(80) TPPp.sub.0.25-DiTBDMS-CS-TMA.sub.0.75 (13B).
(81) The general procedure A was followed using 11B (600 mg, 0.89 mol) and NMe.sub.3 to give 13B as a crude solid (685 mg, 99%)
(82) General procedure B for compounds 14A, 14B, 15A & 15B (N-TPPNH-Pip-acetyl).sub.0.1-(N(N-methylpiperazinyl)acetyl).sub.0.9-DiTBDMS-CS [TPPp.sub.0.1-DiTBDMS-CS-MP.sub.0.9] (14A).
(83) Compound 10A (350 mg, 0.61 mmol) was dissolved in CH.sub.2Cl.sub.2 under an N.sub.2 atmosphere. An excess amount of 1-methylpiperizine was added and the reaction mixture was stirred at room temperature for 24 h. Solvent was removed in vacuo. Then crude product was dried completely under high vacuum yielding corresponding crude product 14A (425, 105%). FT-IR (KBr): v 3378, 2950, 2930, 2884, 2854, 2798, 2694, 2608, 2477, 2223, 1678, 1617, 1519, 1461, 1394, 1371, 1293, 1253, 1168, 1092, 1051, 1003, 966, 939, 920, 837, 801, 779, 701, 671, 612 cm.sup.?1.
(84) TPPp.sub.0.1-DiTBDMS-CS-MP.sub.0.9 (14B).
(85) The general procedure B was followed using 10B (600 mg, 1.04 mol) and 1-methylpiperazine to give 14B as crude solid (710 mg, 102%).
(86) TPPp.sub.0.25-DiTBDMS-CS-MP.sub.0.75 (15A).
(87) The general procedure B was followed using 11A (250 mg, 0.37 mmol) and 1-methylpiperazine to give 15A as a crude solid (290 mg, 104%). FT-IR (KBr): v 3380, 2950, 2930, 2884, 2854, 2800, 2480, 1677, 1598, 1519, 1461, 1400, 1394, 1371, 1284, 1252, 1168, 1089, 1050, 1003, 966, 939, 920, 837, 801, 779, 732, 701, 671, 591 cm.sup.?1.
(88) TPP.sub.0.25-DiTBDMS-CS-MP.sub.0.75 (15B).
(89) The general procedure B was followed using 11B (650 mg, 0.96 mol) and 1-methylpiperazine to give 15B as a crude solid (735 mg, 102%).
(90) General TBDMS Deprotection Procedure C for Compounds 16A, 17A, 18A & 19A (1.sup.st Batch Compounds).
(91) TPPp.sub.0.1-CS-TMA.sub.0.9 (16A).
(92) The material 12A (350 mg, 0.50 mmol) was dissolved in MeOH (5-10 mL) followed by addition of concentrated HCl (2-3 mL). The reaction mixture was stirred for 12 h at rt. An excess amount of deionised water was added into the reaction mixture and stirred for 30 minutes before dialysis against 8% NaCl for one day, and against deionised water for 3 days. During this time the colour of the solution changed gradually from dark green to red. The red colour solution was then freeze-dried to yield a brown sponge-like material. The materials were again deprotected, ion exchanged, dialyzed and freeze-dried. The procedure was repeated using the same conditions in order to remove trace amount of TBDMS to obtain 16A (210 mg, 89%) brown sponge material. FT-IR (KBr): v 3419 (br, OH), 3064 (CH), 1683, 1558, 1506, 1488, 1473, 1403, 1296, 1153, 1112, 1068, 1032, 966, 911, 800, 730, 701, 618 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 96:4) ? ppm: 8.81 (br m, ?-pyrrole H), 8.12-8.16 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.04 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.75-7.84 (m, triphenyl-Hm,p), 4.66 (br s, H-1), 4.21 (br s, BrCH.sub.2C?O), 3.83-3.54 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.32 (s, .sup.+N(CH.sub.3).sub.3)) ppm.
(93) TPPp.sub.0.25-CS-TMA.sub.0.75 (17A).
(94) The general procedure C was followed using 13A (240 mg, 0.30 mmol) and conc.HCl/MeOH to give 17A as a purple solid (120 mg, 71%). FT-IR (KBr): v 3392, 3061, 2950, 1683, 1559, 1506, 1489, 1473, 1402, 1350, 1296, 1154, 1112, 1068, 1032, 966, 911, 800, 730, 701, 619 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 98:2) ? ppm: 8.81 (br m, ?-pyrrole H), 8.12-8.16 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.04 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.79-7.87 (m, triphenyl-Hm,p), 4.50 (br s, H-1), 4.15 (br s, BrCH.sub.2C?O), 3.27-3.65 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.24 (s, .sup.+N(CH.sub.3).sub.3)) ppm.
(95) TPPp.sub.0.1-CS-MP.sub.0.9 (18A).
(96) The general procedure C was followed using 14A (350 mg, 0.52 mmol) and conc.HCl/MeOH to give 18A as a purple solid (200 mg, 87%). FT-IR (KBr): v 3419 (br, OH), 3057 (CH), 1683, 1558, 1474, 1403, 1296, 1234, 1154, 1112, 1068, 1032, 966, 930, 911, 799, 730, 701 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 96:4) ? ppm: 8.83 (br m, ?-pyrrole H), 8.13-8.19 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.08 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.79-7.87 (m, triphenyl-Hm,p), 4.48 (br s, H-1), 3.24-3.78 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.92 (dd, J=12 Hz, Pip-CH.sub.2C?O), 2.30-2.67 (m, piperazine (CH.sub.2).sub.4), 2.48 (br s, piperazine, NCH.sub.3) ppm.
(97) TPPp.sub.0.25-CS-MP.sub.0.75 (19A).
(98) The general procedure C was followed using 15A (200 mg, 0.26 mmol) and 1-methylpiperazine to give 19A as a purple solid (80 mg, 58%). FT-IR (KBr): v 3392, 3056, 2947, 1683, 1558, 1520, 1489, 1472, 1458, 1400, 1349, 1309, 1248, 1154, 1068, 1031, 1001, 966, 911, 800, 729, 701, 658 cm.sup.?1. .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 96:4) ? ppm: 8.83 (br m, ?-pyrrole H), 8.14-8.20 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.08 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.78-7.86 (m, triphenyl-Hm,p), 4.50 (br s, H-1), 3.24-3.78 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.92 (dd, J=12 Hz, Pip-CH.sub.2C?O), 2.28-2.67 (m, piperazine (CH.sub.2).sub.4), 2.48 (br s, piperazine, NCH.sub.3) ppm.
(99) General TBDMS deprotection procedure D for Final compounds 16B, 17B, 18B & 19B (2.sup.nd Batch Compounds). (Compound 16B as Representative). TPPp.sub.0.1-CS-TMA.sub.0.9 (16B): The material 12B (600 mg, 0.86 mmol) was dissolved in N-Methyl-2-pyrrolidone (NMP) (5-10 mL) followed by addition of an excess amount of tetra-n-butylammoniumfluoride (TBAF). The reaction mixture was stirred for 24 h at 55? C. and cooled and acidified with dilute HCl and stirred for 30 minutes before dialysis against 8% NaCl (w/v) in deionised water for two days and against deionised water for 3 days. During this time the colour of the solution changed gradually from grey to red. The red colour solution was then freeze-dried to yield a brown sponge-like material. The materials were again deprotected, ion exchanged, dialyzed and freeze-dried. The procedure was repeated using the same conditions in order to remove the remaining trace amount of TBDMS to obtain 16B (350 mg, 87%) brown sponge material. FTIR (KBr): v 3405, 2943, 2817, 1655, 1528, 1459, 1401, 1375, 1308, 1248, 1153, 1111, 1068, 1031, 966, 832, 799, 729, 702 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 96:4) ? ppm: 8.82 (br m, ?-pyrrole H), 8.12-8.16 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.04 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.75-7.84 (m, triphenyl-Hm,p), 4.47 (br s, H-1), 4.04 (br s, BrCH.sub.2C?O), 3.24-3.55 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.19 (br s, .sup.+N(CH.sub.3).sub.3)) ppm.
(100) TPPp.sub.0.25-CS-TMA.sub.0.75 (17B):
(101) The general procedure C was followed using 13B (650 mg, 0.84 mmol) and TBAF/NMP to give 17B as a purple solid (400 mg, 87%). FTIR(KBr):v 3396, 2942, 2829, 1662, 1526, 1458, 1441, 1401, 1310, 1249, 1068, 1032, 1002, 966, 800, 730, 701 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 98:2) ? ppm: 8.81 (br m, ?-pyrrole H), 8.12-8.16 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.08 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.74-7.86 (m, triphenyl-Hm,p), 4.51 (br s, H-1), 4.10 (br s, BrCH.sub.2C?O), 3.26-3.55 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.22 (br s, .sup.+N(CH.sub.3).sub.3)) ppm; Solid-state .sup.13C NMR (100.56 MHz): ? 170.85, 164.68, 128.11, 119.45, 101.53, 75.39, 61.09, 55.47.
(102) TPPp.sub.0.1-CS-MP.sub.0.9 (18B):
(103) The general procedure C was followed using 14B (600 mg, 0.90 mmol) and TBAF/NMP to give 18B as a purple solid (315 mg, 80%). FTIR (KBr): v 3396, 3315 (br, OH, NH), 2941, 1655 (vs, C?O amide I), 1534 (vs, C?O amide II), 1522, 1471, 1440, 1401, 1375, 1310, 1244, 1069, 1030, 1001, 966, 800, 729, 701 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 98:2) ? ppm: 8.81 (br m, ?-pyrrole H), 8.13-8.19 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.07 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.79-7.85 (m, triphenyl-Hm,p), 4.48 (br s, H-1), 3.24-3.72 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.92 (dd, J=12 Hz, Pip-CH.sub.2C?O), 2.33-2.67 (m, piperazine (CH.sub.2).sub.4), 2.48 (br s, piperazine, NCH.sub.3) ppm.
(104) TPPp.sub.0.1-CS-MP.sub.0.9 (19B):
(105) The general procedure C was followed using 15B (700 mg, 0.93 mmol) and TBAF/NMP to give 19B as a purple solid (415 mg, 85%). FTIR (KBr): v 3396, 3315 (br, OH, NH), 2941, 1655 (vs, C?O amide I), 1534 (vs, C?O amide II), 1522, 1471, 1440, 1401, 1375, 1310, 1244, 1069, 1030, 1001, 966, 800, 729, 701 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 95:5) ? ppm: 8.82 (br m, ?-pyrrole H), 8.13-8.19 (br m, phenyl-Ho & Pip-NHTPP-phenyl-Hm), 8.07 (br d, J=8.0 Hz, RNTPP-phenyl-Ho), 7.79-7.87 (m, triphenyl-Hm,p), 4.49 (br s, H-1), 3.24-3.78 (m, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6), 3.92 (dd, J=12 Hz, Pip-CH.sub.2C?O), 2.30-2.67 (m, piperazine (CH.sub.2).sub.4), 2.40 (br s, piperazine, NCH.sub.3) ppm; Solid-state .sup.13C NMR (100.56 MHz): ? 171.63, 138.74, 127.97, 119.74, 101.93, 75.54, 61.54, 55.42, 45.34 ppm.
(106) Results and Discussion
(107) Nucleophilic TPP intermediate 5.
(108) In the current study, TPP 1 has been synthesized on a large scale and used as the starting material. The mono-nitro TPP(p-NO.sub.2).sub.1 2 functionality was introduced regioselectively using 1.8 equivalent NaNO.sub.2 in TFA, followed by reduction with SnCl.sub.2.2H.sub.2O to obtain the mono-aminoporphyrin TPP(p-NH.sub.2).sub.1 3 The previously reported procedure was simplified by avoiding time consuming purification of the crude material 2 before the reduction step. Aminoporphyrin 3 was then obtained after purification without compromising its overall yield (54%).
(109) The aminoporphyrin TPP(p-NH.sub.2).sub.1 3 is known to be weakly nucleophilic and attempts to link this compound to electrophilic BrA-DiTBDMS-CS 9 by SN.sup.2 attack did not give desired results in our initial study, even under harsh conditions. Thus, in order to convert this photosensitizer derivative into a more potent nucleophile, TPP(p-NH2).sub.1 was first acylated to give bromoacyl-TPP 4 followed by nucleophilic substitution with excess piperazine to give nucleophilic porphyrin intermediate TPPNH-Pip 5. The piperazine motif has positive charge under physiological conditions (aqueous pH 7.4). The piperazine moiety is also suitable as a spacer due to low toxicity and biotransformations that involves several well-known metabolic reactions. The overall synthetic route for synthesis of TPPNH-Pip is shown in Scheme 1 in
(110) Electrophilic chitosan intermediate 9.
(111) Chitosan was modified, as previously reported to obtain DiTBDMS-CS 8. After protection of hydroxyl groups, solubility of the biopolymer dramatically changes and it becomes freely soluble in common organic solvents like CH.sub.2Cl.sub.2 which facilitates modification with highly lipophilic moieties. Thus the electrophilic intermediate BrA-DiTBDMS-CS 9 was prepared by reacting 8 with 2-bromoacetyl bromide (Scheme 2 in
(112) TPP derivatives of Chitosan (16A-19B).
(113) A total of eight TPP derivatives of chitosan compounds were synthesized in two separate batches of four different compounds. The first batch (labeled A) was synthesized on an 80-200 mg scale and the second batch (labeled B) was synthesized on a slightly larger 300-450 mg scale in order to confirm the reproducibility and consistency of the procedure.
(114) The lipophilic PS was attached covalently to the polymer backbone by reaction of BrA-DiTBDMS-CS 9 with the nucleophilic TPPNH-Pip 5 intermediate. The reaction was quantitative which facilitated good control of the degree of substitution in the resulting material. Preliminary investigations showed that carriers with a high degree of substitution (DS) of porphyrin were insoluble in aqueous solution and the modification was therefore limited to 0.1 and 0.25 DS (per glucosamine monomer unit). Thus, TPPNH-Pip 5 was reacted in a controlled manner at 0.1 or 0.25 equivalents per monomer units of electrophilic chitosan intermediate 9 to obtain desired products TPPp.sub.0.1-BrA.sub.0.9-DiTBDMS-CS (10A/10B) or TPPp.sub.0.25-BrA.sub.0.75-DiTBDMS-CS (11A/11B) respectively. Progress of the reaction was monitored by TLC and the reaction was stopped when 5 was no longer present in order to avoid side reactions. .sup.1H NMR analysis of these materials was consistent with the quantitative reaction in the covalent linkage of TPP to chitosan.
(115) Cationic moieties were then introduced on to the TPP-substituted chitosan backbone in order to enhance the aqueous solubility of the carriers and to provide affinity to the endocytic membrane. Therefore, compounds 10A/10B and 11A/11B were reacted with an excess amount of Me.sub.3N (TMA) to afford TPPp.sub.0.1DiTBDMS-CS-TMA.sub.0.9 (12A/12B) and TPPp.sub.0.25DiTBDMS-CS-TMA.sub.0.75 (13A, 13B) respectively with a fixed cationic charge. Similarly, compounds 10A/10B and 11A/11B were reacted with an excess amount of 1-methyl piperazine (MP) to afford TPPp.sub.0.1-DiTBDMS-CS-MP.sub.0.9 (14A/14B) and TPPp.sub.0.25DiTBDMS-CS-MP.sub.0.75 (15A, 15B) respectively. The crude materials were used directly for the final deprotection steps.
(116) Finally, crude materials from the first batch 12A-15A were deprotected by conc. HCl in MeOH at rt, 12 h where as crude materials from second batch 12B-15B were deprotected by TBAF/NMP at 60? C., 24 h methods to give final products 16A-19A and 16B-19B respectively. In both the cases the deprotection step was repeated in order to remove some trace amount of TBDMS (1.5-7%) that was still present after the first deprotection step. Deprotection with conc. HCl in MeOH has been used previously, but, recently the milder TBAF/NMP deprotection of DiTBDMS chitosan derivatives has been introduced in order to avoid highly acidic conditions which may contribute to the degradation of the polymer backbone. However, the disadvantage of the latter method is that it requires a lengthy dialyzing process in order to remove the NMP solvent. Trimethylammoniunm (Me.sub.3N.sup.+) derivatives [TPPp.sub.0.1-CS-TMA.sub.0.9 (16A, 16B) and TPPp.sub.0.25-CS-TMA.sub.0.75 (17A, 17B)] solubilized faster into water than the 1-methyl-piperazine derivatives [TPPp.sub.0.1-CS-M P.sub.0.9 (18A, 18B), TPPp.sub.0.25-CS-MP.sub.0.75 (19A, 19B)]. This may be due to the presence of the fixed ionic charge in the former carrier compounds.
(117) Characterization of the Nano-Carriers.
(118) FT-IR Analysis:
(119) The comparison of FT-IR spectra of various intermediates in the synthesis of the representative final compound TPPp.sub.0.1-CS-MP.sub.0.9 (18A) is shown in
(120) The main characteristic peak in the BrA-DiTBDMS-CS intermediate 9 is observed at 2858-2957 cm.sup.?1 showing CH stretching of SiCH.sub.3, the amide peaks at 1682 and 1530 cm.sup.?1. Also, peaks at 836 and 777 cm.sup.?1 represents SiC stretching and CH.sub.3 rocking (These last two peaks are indicated by arrows in
(121) The appearance of characteristic TPP peaks in the spectra of intermediate TPPp.sub.0.1-BrA.sub.0.9-DiTBDMS-CS 10A (
(122) .sup.1H and .sup.13C (Solid State) Nuclear Magnetic Resonance Spectroscopy (NMR) analysis:
(123) .sup.1H NMR Analysis:
(124) The representative example of the overlay of the .sup.1H NMR spectra of all intermediates and the final product in the synthesis of TPP.sub.0.1CS-MP.sub.0.9 18A is shown in
(125) Bromoacetylation of DiTBDMS-CS to give BrA-DiTBDMS-CS 9 is marked by a downfield shift of the H-2 (GIuN) peak. Also, all chitosan backbone peaks H-1 to H-6 along with COCH.sub.2Br peaks come together at 4.40-3.26 ppm, while TBDMS peaks show no significant change in their positions (
(126) The .sup.1H NMR spectra of the TPPp.sub.0.1-BrA.sub.0.9-DiTBDMS-CS 10A intermediate (
(127) In the TPPp.sub.0.1-DiTBDMS-CS-MP.sub.0.9 14A spectra, new peaks for NCH.sub.3 of the 1-methylpiperazine moiety (
(128) After the final deprotection step a material with excellent aqueous solubility was obtained. In the .sup.1H NMR spectra of final compound TPPp.sub.0.1-CS-MP.sub.0.9 18A all peaks of the chitosan backbone along with the 1-methyl piperazine peaks can be clearly identified; however there is no observable NMR signal of the TPP moiety (
(129) Solid State .sup.13C NMR analysis:
(130) The carbon peaks of final carriers could be observed in solid state NMR (
(131) Degree of Substitution (DS) and Conversion Efficiency.
(132) Table 1 below shows the DS for the final carrier compounds. The aim was to control the DS with an equivalent ratio between compounds 5 and 9 in reaction step e (Scheme 2 in
(133) TABLE-US-00001 TABLE 1 Degree of substitution (DS) for the TPP modified chitosan carriers 16A-19B TPP-NH-Pip DS* (eq. per (linked TPP Chitosan sugar moieties Entry Derivatives Compound unit used) per sugar unit) 1. TPPp.sub.0.1-CS-TMA.sub.0.9 16A 0.10 0.10 2. TPPp.sub.0.1-CS-TMA.sub.0.9 16B 0.10 0.10 3. TPPp.sub.0.25-CS-TMA.sub.0.75 17A 0.25 0.23 4. TPPp.sub.0.25-CS-TMA.sub.0.75 17B 0.25 0.25 5. TPPp.sub.0.1-CS-MP.sub.0.9 18A 0.10 0.09 6. TPPp.sub.0.1-CS-MP.sub.0.9 18B 0.10 0.10 7. TPPp.sub.0.25-CS-MP.sub.0.75 19A 0.25 0.25 8. TPPp.sub.0.25-CS-MP.sub.0.75 19B 0.25 0.25 *DS determined by .sup.1H NMR
Analysis of the Self Aggregation of Carriers to Form Nanoparticles and Unfolding of Carrier Nanopaticles.
(134) Aromatic porphyrins can form ?-? stacking systems which are defined as J (red-shifted) or H (blue-shifted) type aggregates. Peripheral substituent groups can contribute to aggregation mechanisms. This aggregation can be observed by NMR, UV-Vis and Fluorescence spectroscopy. Thus, extreme broadening of peaks and loss of peaks in .sup.1H NMR has been reported for carboxyphenyl-porphyrin (TCPP) aggregates, p-sulfonatophenyl and phenyl meso-substituted porphyrins and three kinds of cationic porphyrins (TOPyP, TMPyP and APP). Similar observations of loss of signal due to immobilization have been made in dispersion copolymerization of lipophilic n-butylmethancrylate with a poly(ethylene oxide) macro monomer.
(135) In the current work the lack of peaks in the aromatic region, in the .sup.1H NMR in D.sub.2O of final compounds 16A-19B suggested aggregation of the TPP moieties in the D.sub.2O due to t-m stacking and hydrophobic interaction. NMR study of the representative TPPp.sub.0.1-CS-TMA.sub.0.9 16B compound, in a DMSO-d.sub.6: D.sub.2O mixture, is shown in
(136) The nanocarriers dissolved in water show a broad Soret band for the ?-? stacked photosensitizer with an absorption maxima at 417 nm (
(137) In pure water the fluorescence is almost fully quenched which is also consistent with aggregation of the photosensitizer moieties. The fluorescence intensity dramatically increases but as DMSO content increased to 50% there is a sharp increase in the fluorescence intensity with further gradual increase to 100% DMSO concentration observed (FIG. 8III).
(138) An attempt was made to further characterize the nanocarrier particles by scanning electron microscopy. Droplets of nanocarrier solution were pipetted onto a silicone wafer surface and allowed to dry (
(139) Nanoparticle aggregation and physical instability leading to formation of insoluble aggregates is commonly observed. Physical stability of a 1 mg/ml aqueous solution of TPPp.sub.0.25-CS-MP.sub.0.75 (17A) was monitored over a period of three months. No precipitation of this compound was observed.
(140) Fluorescence Quantum Yields.
(141) Herein, the fluorescence quantum yields of TPP(p-NH.sub.2).sub.1, TPPNH-Pip and chitosan derivatives of TPP (16A-19B) were investigated in DMSO (excited at 419 nm). The quantum yield of TPP(p-NH.sub.2).sub.1 was less than that of its derivative TPPNH-Pip as well as TPPCS-TMA & TPPCS-MP derivatives. This demonstrated a high degree of excited state quenching of TPP(p-NH.sub.2).sub.1 as compared to its derivatives. ?.sub.F is almost equal for all carrier compounds (16A-19B) with a minor fluctuation?0.004 around the value obtained for the intermediate TPPNH-Pip.
(142) However, ?.sub.F values of TPP(p-NH.sub.2).sub.1 (0.0002) are lower as compared to some literature published data. Bhaumik et.al. (J Org Chem 2009, 74:5894) reported it as 0.05 when excited at 418 nm in DMF. They reported fluorescence emission maxima at 664 nm which is different from our current finding of 650 nm. TPP was used as a standard, whereas we used anthracene as the standard. Low ?.sub.F in the current work may be due to self-association of the photosensitizers. Table 2 below shows the Fluorescence quantum yield (?.sub.F) of TPP modified chitosan carriers 16A-19B.
(143) TABLE-US-00002 TABLE 2 Chitosan Com- ?.sub.abs* ?.sub.em* Quantum Entry Derivatives pound (nm) (nm) Yield (?.sub.F) 1. TPP(p-NH.sub.2).sub.1 3 419 651 0.0002 2. TPP-NH-Pip 5 419 651 0.0036 3. TPPp.sub.0.1-CS-TMA.sub.0.9 16A 419 651 0.0032 4. TPPp.sub.0.1-CS-TMA.sub.0.9 16B 419 651 0.0034 5. TPPp.sub.0.25-CS- 17A 419 651 0.0036 TMA.sub.0.75 6. TPPp.sub.0.25-CS- 17B 419 651 0.0032 TMA.sub.0.75 7. TPPp.sub.0.1-CS-MP.sub.0.9 18A 419 651 0.0036 8. TPPp.sub.0.1-CS-MP.sub.0.9 18B 419 651 0.0033 9. TPPp.sub.0.25-CS-MP.sub.0.75 19A 419 651 0.0036 10. TPPp.sub.0.25-CS-MP.sub.0.75 19B 419 651 0.0033 *All data collected in DMSO at room temperature ?-wavelength
Conclusion
(144) We have shown that DiTBDMS chitosan can be used for highly efficient synthesis of meso-Tetraphenylporphyrin tethered chitosan based nano-carriers. The synthesis of these carriers was fully reproducible and the method allowed precise control of the degree of substitution for highly lipophilic photosensitizer. The NMR, florescence, and UV-Vis studies were consistent with self-association of the photosensitizer moieties. The carriers are polar and show good aqueous solubility and physical stability. In DMSO the photosensitizer dissociates from the self-association and therefore become fluorescent. In aqueous solution the carriers will assemble into nanoparticle-like structures with the cationic group and polymer backbone forming an outer shell around a core composed of the aggregated (t-t stacked) lipophilic TPP moieties. The cationic groups and polymer backbone have relatively free movement in the liquid and can therefore be observed by NMR. In contrast the TPP core is semi-solid with very limited movement and can therefore only be detected in solid state NMR. This structure is in dynamic equilibrium with the unfolded, and fluorescent, form which becomes dominant in DMSO allowing detection of the TPP by NMR. When the carrier is in contact with the cell or the endocytic membrane, the structure unfolds and lipophilic moieties are inserted into the endocytic membrane allowing for the excitation and photosensitization which leads to PDT and PCI effects.
Example 2Synthesis of TPC-Chitosan-Based Nanocarriers
(145) General Materials and Methods were as for Example 1, where appropriate.
(146) Synthesis
(147) See scheme 3 in
(148) Meso-Tetraphenylporphyrin (1).
(149) Compound 1 (TPP) was prepared by the procedure described in Journal of Organic Chemistry 1967, 32, 476.
(150) 5-(4-Aminophenyl)-10,15,20-triphenylporphyrin [TPP(p-NH.sub.2).sub.1] (3).
(151) Compound 3 was prepared following the literature procedure in Tetrahedron 2004, 60, 2757.
(152) 5-(4-Aminophenyl)-10,15,20-triphenylchlorin: TPC(p-NH.sub.2).sub.1 (20).
(153) Compound 3 (1.5 g, 2.38 mmol) was dissolved in pyridine under N.sub.2 and dark atmosphere. K.sub.2CO.sub.3 2.96 g, 21.5 mmol) and p-toluenesulfonylhydrazide (0.887 g, 4.77 mmol) were added and the resulting reaction mixture was heated to reflux temperature. Further quantities of p-toluenesulfonylhydrazide (0.887 g, 4.77 mmol) were added after an interval of 2, 4, 6 and 8 h. Stirring was continued at reflux temperature for 24 h. The reaction mixture was added to a 1:1 mixture of EtOAc:H.sub.2O (2:1, 900 mL) and refluxed for 1 h. After cooling to room temperature, the organic phase was separated and washed with 2N HCl (3?200 mL) followed by washing with water (2?100 mL) and saturated aqueous NaHCO.sub.3 (2?150 mL) The organic phase was then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford a 1.3 g crude mixture. Analysis of the visible spectrum of crude product showed it to be a mixture of chlorine and bacterochlorin (band at 651 and 738 respectively). Also, analysis by .sup.1H NMR spectra showed that there was no trace amount of the starting porphyrin material left.
(154) Crude material (1.3 g) (chlorine/bacterochlorin mixture) obtained from the above reaction step was dissolved in CH.sub.2Cl.sub.2 (100 mL). ortho-Chloranil (420 mg, 2.7 mmol) was then added in one portion to the stirring organic solution at room temperature and the progress of the reaction was simultaneously monitored by UV-vis. Immediately after the peak of bacterochlorin (738 nm) was completely diminished, the reaction mixture was washed with 5% aqueous sodium bisulfite (2?125 mL), followed by washing with water (100 mL), 5% NaOH (2?150 mL), and finally with water (150 mL). The organic phase was then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford exclusively the titled chlorin compound 20 (1.2 g, 80%) as a brown colour solid. Compound 3 seems to be exists in more than one isomer. TLC (Hexane/CH.sub.2Cl.sub.2 3:7): Rf=0.23, .sup.1H NMR (CDCl.sub.3): ?=7.86-8.66 (m, 14H, ?-pyrrole-H & phenyl-Ho), 7.63-7.73 (m, 9H, triphenyl-Hm,p), 7.00 (d, J=8 Hz, 2H, NH.sub.2-phenyl-Hm), 4.14-4.23 (m, 4H, chlorin ?-pyrrole-CH.sub.2), 3.95 (br s, 2H, NH.sub.2), ?1.38 and ?1.46 (br s, 2H, ?-pyrrole-NH) ppm; MS (ESI): m/z calcd. for C.sub.44H.sub.34N.sub.5 ([M+H].sup.+) 632.2809, found 632.2792.; UV-vis (DMSO): ?.sub.max: 422, 524, 553, 600, 652 nm.
(155) Synthesis of intermediate TPCNH-pip (21).
(156) The compound 20 (600 mg, 0.95 mmol) was dissolved in CH.sub.2Cl.sub.2 (15 mL) and stirred under an N.sub.2 atmosphere. Et.sub.3N (0.32 mL, 2.27 mmol) was added followed by dropwise addition of chloroacetyl chloride (0.092 mL, 1.15 mmol) at room temperature and continued stirring at room temperature. After 2 h in situ an excess amount of piperazine (0.328 g, 3.8 mmol) was added and stirring was continued overnight. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (85 mL), extracted, washed with water (3?35 mL), brine (35 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Crude product was then purified by silica gel column chromatography. The desired product was isolated in MeOH: CH.sub.2Cl.sub.2 (8:92) as eluent to afford the titled intermediate 21 (440 mg, 61%) as a brown solid.
(157) TLC (CH.sub.2Cl.sub.2: MeOH, 9:1): R.sub.f=0.15; .sup.1H NMR (CDCl.sub.3): ?=9.34, 9.39 (s, 1H, TPCNH), 7.86-8.65 (m, 16H, ?-pyrrole-H, phenyl-Ho & R-NHTPC-phenyl-Ho,m), 7.66-7.73 (m, 9H, triphenyl-Hm,p), 4.18-4.19 (br s, 4H, chlorin ?-pyrrole-CH.sub.2), 3.30 (s, 2H, ArNHCOCH.sub.2-pip), 3.17 (br m, 4H, piperazine ring-CH.sub.2), 2.81 (br m, 4H, piperazine ring-CH.sub.2), ?1.37 (br s, 2H, ?-pyrrole-NH); .sup.13C NMR (CDCl.sub.3): ?=168.37, 167.48, 152.61, 143.14, 142.22, 140.86, 139.20, 138.32, 137.19, 136.99, 135.33, 134.64, 133.98, 133.01, 132.37, 132.12, 131.96, 128.17, 127.69, 126.81, 123.56, 123.38, 122.79, 122.08, 119.22, 117.94, 112.41, 111.65, 62.63, 53.50, 45.59, 35.90 ppm; UV-vis (DMSO): ?.sub.max: 421, 521, 549, 598, 651 nm.
(158) See scheme 4 in
(159) Synthesis of tert-Butyl piperazine-1-carboxylate (22).
(160) Piperazine (6 g, 69.6 mmol) was dissolved in CH.sub.2Cl.sub.2 (120 mL). The solution was cooled to 0? C. and Boc.sub.2O (7.6 g, 34.8 mmol) in CH.sub.2Cl.sub.2 (80 mL) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirring was continued for 24 h. The precipitate was filtered off and washed with CH.sub.2Cl.sub.2 (2?20 mL) and the combined filtrate was separated and washed with water (3?40 mL), brine (30 mL) and dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the titled compound 22 (6.5 g, 50%) as a white solid.
(161) Mp 44-46? C. (lit. mp 46-47? C.); .sup.1H NMR (CDCl3): ?=3.32 (t, J=4 Hz, 4H), 2.74 (t, J=4 Hz, 4H), 1.39 (s, 9H); .sup.13C NMR (CDCl.sub.3): ?=154.85, 80.00, 79.52, 45.96, 44.45, 28.45 ppm; MS (ESI): m/z calcd. for C.sub.9H.sub.19N.sub.2O.sub.2 ([M+H].sup.+) 187.1441 found 187.1412.
(162) Synthesis of p-(Methoxycarbonyl)benzaldehyde (23).
(163) 4-Carboxybenzaldehyde (4 g, 26.6 mmol) was suspended in 60 mL of anhydrous MeOH and stirred under N.sub.2. The reaction mixture was cooled to 0? C. and acetyl chloride (9.5 mL, 133 mmol) was added dropwise. The reaction mixture was stirred for 12 h at room temperature. The MeOH was removed in vacuo, and the crude mixture was diluted with EtOAc (120 mL). The organic phase was washed with aqueous 1N NaOH (5?30 mL) and brine (2?25 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Crude solid was then recrystallised by using EtOAc and Petroleum ether to afford ester 23 (3.8 g, 87%) as a white solid.
(164) TLC (Hexane: CH.sub.2Cl.sub.2 3:7): R.sub.f=0.36; Mp: 61-63? C. (lit. mp 59-64? C.); .sup.1H NMR (CDCl.sub.3): ?=10.06 (s, 1H, CHO), 8.15 (d, J=8.4 Hz, 2H), 7.91 (d, J=8.4 Hz, 2H), 3.92 (s, 3H) ppm; .sup.13C NMR (CDCl.sub.3): ?=191.66, 166.07, 139.21, 135.13, 130.23, 129.55, 52.62 ppm.
(165) Synthesis of 5-(4-Methoxycarbonylphenyl)-10,15,20-triphenylporphyrin TPP(p-CO.sub.2Me).sub.1 (24).
(166) Following the literature method, (J. Am. Chem. Soc. 2008, 130, 4236-4237).
(167) Synthesis of 5-(4-Carboxyphenyl)-10,15,20-triphenylporphyrin TPP(p-CO.sub.2H).sub.1 (25)
(168) Compound 24 (1.2 g, 1.78 mmol) was dissolved in a mixture of THF: Pyridine (10:1, 100 mL). 2N methanolic KOH (120 mL) was added and the reaction mixture was refluxed for 24 h. Then the reaction mixture was cooled down to room temperature and neutralized with saturated aqueous citric acid solution. Subsequently the reaction mixture was concentrated in vacuo until removal of MeOH and THF as completed. The crude mixture was then diluted with CH.sub.2Cl.sub.2 (150 mL) and water (120 mL) and the aqueous phase was extracted with CH.sub.2Cl.sub.2 (3?50 mL). The combined organic phase was washed with water (2?40 mL) and brine (35 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography using MeOH: CH.sub.2Cl.sub.2 (100:0 to 96:4 as eluent) to afford the title acid 25 (0.83 g, 71%) as a purple solid.
(169) TLC (CH.sub.2Cl.sub.2: MeOH 95:5): R.sub.f=0.54; .sup.1H NMR (DMSO-d.sub.6): ?=8.84 (br s, 8H, ?-pyrrole-H), 8.33-8.39 (m, 4H, RCOTPP-phenyl-Ho,m), 8.21-8.23 (m, 6H, triphenyl-Ho), 7.81-7.88 (m, 9H, triphenyl-Hm,p), ?2.92 (s, 2H, NH) ppm; MS (ESI): m/z calcd. for C.sub.45H.sub.31N.sub.4O.sub.2 ([M+H].sup.+) 659.2442, found 659.2420.
(170) Synthesis of 5-(4-Carboxyphenyl)-10,15,20-triphenylchlorin TPC(p-CO.sub.2H).sub.1 (26)
(171) Compound 25 (600 mg, 0.9 mmol) and anhydrous K.sub.2CO.sub.3 (1.13 g, 8.2 mmol) was dissolved in pyridine (42 mL) under N.sub.2 and dark atmosphere. Toluene-4-sulfonylhydrazide (340 mg, 1.8 mmol) was then added and the mixture was stirred at reflux temperature. Further quantities of toluene-4-sulfonylhydrazide (340 mg, 1.8 mmol) in 3 mL of pyridine were added after an interval of 2, 4, 6, 8 and 10 h reaction. The stirring was continued at reflux temperature for 24 h. After cooling to room temperature, EtOAc (500 mL) and deionised H.sub.2O (250 mL) were added and the reaction mixture was again refluxed for 1 h. After cooling to room temperature, the organic phase was separated and washed with 2N HCl (2?150 mL) and then with water (2?150 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford 565 mg of the crude mixture. Analysis of the visible spectrum of crude product showed it to be a mixture of chlorin and bacterochlorin (band at 651 and 738 respectively). Also, analysis by .sup.1H NMR spectra showed that there was no trace amount of starting porphyrin material left.
(172) Crude material (chlorin/bacterochlorin mixture, 565 mg) obtained from the above reaction step, was completely dissolved in a mixture of CH.sub.2Cl.sub.2: MeOH (75:25). ortho-Chloranil (180 mg, 0.7 mmol) was then added in one portion to the stirred organic solution at room temperature and the progress of the reaction was simultaneously monitored by UV-Vis. Immediately after the absorption peak of bacterochlorin (738 nm) diminished, the organic phase was washed with 5% aqueous sodium bisulfite solution (2?150 mL), followed by washing with water (100 mL), then by 5% aqueous NaOH (2?150 mL) and finally with water (120 mL). If an emulsion was observed the organic phase was washed with saturated aqueous citric acid solution. The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford exclusively the titled chlorin compound 26 (420 mg, 70%) as a brown solid. Compound 9 is present in more than one isomer.
(173) TLC (CH.sub.2Cl.sub.2: MeOH, 95:5): R.sub.f=0.54, .sup.1H NMR (DMSO-d.sub.6): ?=7.91-8.58 (m, 16H, ?-pyrrole-H, phenyl-Ho & RCOTPC-phenyl-Ho,m), 7.68-7.77 (m, 9H, triphenyl-Hm,p), 4.12-4.13 (m, 4H, chlorin ?-pyrrole-CH.sub.2), ?1.53 and ?1.60 (2 brs, 2H, ?-pyrrole-NH); .sup.1H NMR (CDCl.sub.3): ?=7.87-8.60 (m, 16H, ?-pyrrole-H, phenyl-Ho & RCOTPC-phenyl-Ho,m), 7.64-7.74 (m, 9H, triphenyl-Hm,p), 4.16-4.18 (m, 4H, chlorin ?-pyrrole-CH.sub.2), ?1.39 and ?1.49 (2 br s, 2H, ?-pyrrole-NH) ppm; MS (ESI) calcd. for C.sub.45H.sub.33N.sub.4O.sub.2 ([M+H].sup.+) 661.2598, found 661.2566; UV-vis (DMSO): ?.sub.max: 420, 520, 547, 599, 651 nm.
(174) Synthesis of intermediate tert-Butyl N-[piperazine-1-carboxylate]-5-(4-carboxyphenyl)-10,15,20-triphenylchlorin (27).
(175) Chlorin compound 26 (500 mg, 0.76 mmol) and tert-butyl piperazine-1-carboxylate 22 (155 mg, 0.83 mmol) was dissolved in DMF (4 mL) under N.sub.2 and in the dark. To the reaction mixture, EDCI-HCl (174 mg, 0.91 mmol) and HOBT (123 mg, 0.91 mmol) were added followed by addition of Et.sub.3N (0.26 mL, 1.82 mmol) at room temperature. The reaction mixture was then stirred overnight at room temperature before it was slowly poured into stirring water (100 mL). The solid material was filtered off, washed with plenty of water, and dried well. The crude product was purified by silica gel column chromatography (CH.sub.2Cl.sub.2: MeOH 100:0 to 99:1) to afford titled amide compound 27 (340 mg, 54%) as a brown solid.
(176) TLC (CH.sub.2Cl.sub.2: MeOH 99:1): Rf=0.74; .sup.1H NMR (CDCl.sub.3): ?=7.74-8.59 (m, 16H, ?-pyrrole-H, phenyl-Ho & RCOTPC-phenyl-Ho,m), 7.65-7.72 (m, 9H, triphenyl-Hm,p), 4.16-4.17 (m, 4H, chlorin ?-pyrrole-CH.sub.2), 3.78-3.86 (br m, 4H, piperazine ring-CH.sub.2), 3.63 (br m, 4H, piperazine ring-CH.sub.2), 1.53 (s, 9H, boc-(CH.sub.3).sub.3), ?1.39 and ?1.47 (2 brs, 2H, ?-pyrrole-NH) ppm.
(177) Synthesis of intermediate TPCCO-pip (28).
(178) The compound 27 (320 mg, 0.39 mmol) was dissolved in CH.sub.2Cl.sub.2 (8 mL) under N.sub.2 in the dark. CH.sub.2Cl.sub.2: TFA (1:1, 4 mL) was added and stirred at rt for 1 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (40 mL) and washed with water (2?15 mL), saturated aqueous NaHCO.sub.3 (2?15 mL) and brine (15 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude product was then purified by silica gel column chromatography (CH2Cl.sub.2: MeOH 100:0 to 92:8 as eluent) to afford the titled intermediate 28 (250 mg, 89%) as a brown solid.
(179) TLC (CH.sub.2Cl.sub.2: MeOH, 9:1): R.sub.f=0.35, .sup.1H NMR (CDCl.sub.3): ?=7.74-8.59 (m, 16H, ?-pyrrole-H, phenyl-Ho & RCOTPC-phenyl-Ho,m), 7.64-7.72 (m, 9H, triphenyl-Hm,p), 4.16-4.17 (m, 4H, chlorin ?-pyrrole-CH.sub.2), 3.73-3.90 (br m, 4H, piperazine ring-CH.sub.2), 3.04 (br m, 4H, piperazine ring-CH.sub.2), ?1.40 and ?1.47 (2 brs, 2H, ?-pyrrole-NH) ppm; MS (ESI) calcd. for C.sub.49H.sub.42N.sub.6O ([M+2H].sup.+/2) 365.1705, found 365.1707; UV-vis (DMSO): ?.sub.max: 420, 520, 546, 599, 651 nm.
(180) Chitosan mesylate salt (7).
(181) Synthesized according to our previously published procedure (Carbohydrate Polymers 2010, 81:140-144).
(182) N-bromoacetyl-3,6-O-DiTBDMS-CS (BrA-DiTBDMS-CS, 9).
(183) DiTBDMS-CS 8 (1 g, 2.60 mmol) was dissolved in dry CH.sub.2Cl.sub.2 (15 mL) in a round bottom flask under an N.sub.2 atmosphere. The reaction mixture was cooled to ?20? C. with an ice/salt mixture. Et3N (1.81 mL, 13 mmol) was added followed by slow dropwise addition of bromoacetyl bromide (0.91 mL, 10 mmol). Stirring was continued for 1 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 and concentrated in vacuo. The crude product was stirred in acetonitrile, filtered and washed with fresh acetonitrile. The dry material was dissolved and extracted in CH.sub.2Cl.sub.2, washed with water and brine, dried over Na.sub.2SO.sub.4, concentrated in vacuo to afford the titled bromo compound 26 (1.2 g, 92%) as a faint yellow powdered solid.
(184) FT-IR (KBr): v 3402 (br, NH), 2957, 2931, 2886, 2858 (s, CH TBDMS), 1682 (vs, C?O amide I), 1530 (vs, C?O amide II), 1473, 1391, 1362, 1311, 1259, 1101, 1005, 837, 777 (SiC), 669 cm.sup.?1; .sup.1H NMR (CDCl.sub.3) ? ppm: 4.40 (br s, H-1), 4.02-3.26 (m, H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and 2H GluNH-C?OCH.sub.2Br), 0.90 and 0.88 (br s, (CH.sub.3).sub.3CSi), 0.13 and 0.07 (br s, (CH.sub.3).sub.2Si) ppm.
(185) See Scheme 6A in
(186) Synthesis of Intermediate 29
(187) (N-TPCNH-Pip-acetyl).sub.0.1-(N-bromoacetyl).sub.0.9-DiTBDMS-CS (TPC.sub.NP0.1-BrA.sub.0.9-DiTBDMS-CS, 29).
(188) Compound 9 (800 mg, 1.58 mmol) and compound TPCNH-Pip 21 (120 mg, 0.158 mmol) were dissolved in CH.sub.2Cl.sub.2 (25 mL) under N.sub.2 and in the dark. An exact equimolar quantity of Et.sub.3N (22 ?L, 0.158 mmol) with respect to 21 was added and the reaction mixture was stirred at rt for 24 h. Total consumption of starting material was confirmed by TLC. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (55 mL) and washed with water (2?25 mL) and brine (25 mL). The organic phase was dried over Na.sub.2SO.sub.4, concentrated in vacuo to afford the compound 29 (700 mg, 78%).
(189) .sup.1H NMR (CDCl.sub.3): ?=9.21, 9.25 (s, TPCNHCO), 7.86-8.60 (m, ?-pyrrole-H, phenyl-Ho & RNHTPC-phenyl-Ho,m), 7.65-7.73 (m, triphenyl-Hm,p), 3.35-4.50 [br m, chitosan (H-1, H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and 2H GluNH-C?O, CH.sub.2CONGlc), TPCNHCOCH.sub.2-pip, piperazin ring-CH.sub.2 and chlorin ?-pyrrole-CH.sub.2], 2.77-2.83 (m, piperazine ring-CH.sub.2), 0.88-0.89 [br s, (CH.sub.3).sub.3CSi], 0.02-0.13 [(br m, (CH.sub.3).sub.2Si], ?1.44 (br s, 2H, ?-pyrrole-NH) ppm.
(190) See Scheme 6B in
(191) Synthesis of Intermediate 34
(192) (N-TPCCO-Pip-acetyl).sub.0.1-(N-bromoacetyl).sub.0.9-DiTBDMS-CS (TPC.sub.CP0.1BrA.sub.0.9-DiTBDMS-CS, 34).
(193) Compound 9 (800 mg, 1.58 mmol) was dissolved in NMP (25 mL) under N.sub.2 and in the dark. TPCCO-Pip 28 (125 mg, 0.173 mmol) and NaHCO.sub.3 (0.29 g, 3.45 mmol) were added at room temperature. The reaction mixture was then heated at 75? C. and stirred overnight before it was cooled down and poured into stirring water. The solid was filtered off, washed with plenty of water, and dried under suction. The solid obtained was then dissolved in CH.sub.2Cl.sub.2, filtered and dried over Na.sub.2SO.sub.4, and the solvent removed in vacuo to afford compound 34 (810 mg, 89%) as a brown solid.
(194) .sup.1H NMR (CDCl.sub.3): ?=7.75-8.60 (m, ?-pyrrole-H, phenyl-Ho & RCOTPC-phenyl-Ho,m), 7.64-7.71 (m, triphenyl-Hm,p), 3.38-4.5 [br m, chitosan (H-1, H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and 2H GluNHC?O, CH.sub.2CONGlc), piperazin ring-CH.sub.2 and chlorin ?-pyrrole-CH.sub.2], 2.76-2.84 (m, piperazin ring-CH.sub.2), 0.89-0.92 [br s, (CH.sub.3).sub.3CSi], 0.02-0.10 [(br m, (CH.sub.3).sub.2Si)], ?1.40 and ?1.48 (br s, ?-pyrrole-NH) ppm.
(195) General Procedure A for Compounds 30 & 35
(196) (N-TPCNH-Pip-acetyl).sub.0.1-(N(N,N,N-trimethylammoniumyl)acetyl).sub.0.9-DiTBDMS-CS (TPC.sub.NP0.1-DiTBDMS-CS-TMA.sub.0.9, 30).
(197) Compound 29 (350 mg, 0.61 mmol) was dissolved in CH.sub.2Cl.sub.2 (15 mL) under N.sub.2 and in the dark. An excess amount of trimethylamine solution was added and the reaction mixture was stirred at rt for 24 h. Solvent was removed in vacuo. The crude product was dried completely under a high vacuum yielding crude product 30 (355 mg, 94%) as a brown solid. The crude compound was used as it was for the next step.
(198) (N-TPCCO-Pip-acetyl).sub.0.1-(N(N,N,N-trimethylammoniumyl)acetyl).sub.0.9-DiTBDMS-CS (TPC.sub.PP0.1-DiTBDMS-CS-TMA.sub.0.9, 35).
(199) The general procedure A was followed using 34 (350 mg, 0.61 mol) and trimethylamine solution to give 35 as a crude solid (360 mg, 94%). The crude compound was used as it was for the next step.
(200) General Procedure B for Compounds 31 & 36
(201) (N-TPCNH-Pip-acetyl).sub.0.1-(N(N-methylpiperazinyl)acetyl).sub.0.9-DiTBDMS-CS (TPC.sub.NP0.1-DiTBDMS-CS-MP.sub.0.9, 31).
(202) Compound 29 (350 mg, 0.61 mmol) was dissolved in CH.sub.2Cl.sub.2 (15 mL) under N.sub.2 and in the dark. An excess amount of 1-methylpiperazine was added and the reaction mixture was stirred at room temperature for 24 h. Solvent was removed in vacuo. Then crude product was dried completely under a high vacuum yielding corresponding crude product 31 (330, 89%). The crude compound was used as it was for the next step.
(203) (N-TPCCO-Pip-acetyl).sub.0.1-(N(N-methylpiperazinyl)acetyl).sub.0.9-DiTBDMS-CS (TPC.sub.CP0.1-DiTBDMS-CS-MP.sub.0.9, 36).
(204) The general procedure B was followed using 34 (250 mg, 0.38 mol) and 1-methylpiperazine to give 36 as a crude solid (265 mg, 93%). The crude compound was used as it was for the next step.
(205) Synthesis of Final Products (32, 33, 37 & 38)
(206) Final deprotection was achieved by following general procedure C:
(207) Compounds (30/31/35/36) were dissolved in MeOH under N.sub.2 and in the dark. The reaction mixture was degassed by purging with N.sub.2 for 5 minutes and subsequently cooled to 0? C. before addition of 4 mL of conc. HCl. The reaction mixture was allowed to warm to room temperature and stirred for 12 h before it was concentrated completely in vacuo. Crude residue was again dissolved in MeOH and degassed under N.sub.2 and in the dark. The reaction mixture was cooled to 0? C. before addition of 2 mL conc. HCl. The reaction mixture was allowed to warm to room temperature and stirred for 12 h. The reaction mixture was then diluted and ion exchanged by addition of 5% aqueous NaCl for 1 h, before it was dialyzed against 8% aqueous NaCl for 24 h and then against deionised water for 3 days. The clean brown solution was subsequently freeze-dried overnight to afford the final products (32/33/37/38 respectively) as a brown fluffy material.
(208) TPC.sub.NP0.1CS-TMA.sub.0.90 (32).
(209) The general procedure C was by followed using 30 (325 mg, 0.52 mmol) and conc.HCl/MeOH to give 32 as a brown solid (175 mg, 85%). .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 98:2): ?=7.83-8.62 (m, ?-pyrrole-H, phenyl-Ho & RNHTPC-phenyl-Ho,m), 7.69-7.77 (m, triphenyl-Hm,p), 4.52 (br s, H-1), 4.11-4.14 (m, CH.sub.2CONGlc and chlorin ?-pyrrole-CH.sub.2), 3.26-3.67 (br m, partially overlapped with HDO peak, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6, 2H GluNH-C?O, TPCNHCOCH.sub.2-pip, piperazine ring-CH.sub.2] 3.24 (s, .sup.+N(CH.sub.3).sub.3)) ppm; UV-vis (DMSO): ?.sub.max: 421, 520, 549, 599, 651 nm.
(210) TPC.sub.NP0.1-CS-MP.sub.0.90 (33).
(211) The general procedure C was followed using 31 (300 mg, 0.45 mmol) and conc.HCl/MeOH to give 33 (165 mg, 84%) as a brown solid. .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 98:2): ?=7.83-8.62 (m, ?-pyrrole-H, phenyl-Ho & RNHTPC-phenyl-Ho,m), 7.66-7.75 (m, triphenyl-Hm,p), 4.50 (br s, H-1), 4.10-4.14 (m, chlorin ?-pyrrole-CH.sub.2), 2.92-3.55 (m, partially overlapped with HDO peak, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6, 2H GluNH-C?O, CH.sub.2CONGlc, TPCNHCOCH.sub.2-pip), 2.33-2.63 (m, partially overlapped with DMSO-d6 peak, piperazine ring-CH.sub.2, piperazine-NCH.sub.3) ppm; UV-vis (H.sub.2O): ?.sub.max: 412, 430, 531, 560, 611, 664 nm; UV-vis (DMSO): ?.sub.max: 421, 521, 548, 596, 651 nm.
(212) TPC.sub.CP0.1CS-TMA.sub.0.90 (37).
(213) The general procedure C was followed using 35 (300 mg, 0.48 mmol) and conc.HCl/MeOH to give 37 as a brown solid (170 mg, 89%).
(214) FT-IR (KBr): v 3353, 3061, 2950, 1683, 1580, 1473, 1440, 1376, 1291, 1154, 1112, 1067, 1032, 970, 911, 794, 703 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 98:2): ?=7.89-8.62 (m, ?-pyrrole-H, phenyl-Ho & RCOTPC-phenyl-Ho,m), 7.67-7.76 (m, triphenyl-Hm,p), 4.50 (br s, H-1), 4.06-4.16 (m, CH.sub.2CONGlc and chlorin ?-pyrrole-CH.sub.2), 3.26-3.75 (m, partially overlapped with HDO peak, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6, 2H GluNH-C?O, piperazine ring-CH.sub.2), 3.24 (s, .sup.+N(CH.sub.3).sub.3)) ppm; UV-vis (DMSO): ?.sub.max: 420, 520, 547, 599, 651 nm.
(215) TPC.sub.CP0.1-CS-MP.sub.0.90 (38).
(216) The general procedure C was followed using 36 (240 mg, 0.38 mmol) and conc.HCl/MeOH to give 38 as a brown solid (85 mg, 52%). FT-IR (KBr): v 3349, 2927, 1644, 1580, 1461, 1440, 1374, 1285, 1070, 1043, 985, 945, 794719, 703 cm.sup.?1; .sup.1H NMR (DMSO-d.sub.6: D.sub.2O 98:2): ?=7.86-8.63 (m, ?-pyrrole-H, phenyl-Ho & RCOTPC-phenyl-Ho,m), 7.67-7.76 (m, triphenyl-Hm,p), 4.50 (br s, H-1), 4.08-4.14 (m, chlorin ?-pyrrole-CH.sub.2), 2.92-3.55 (m, partially overlapped with HDO peak, H-2 GlcNAc, H-3, H-4, H-5, H-6, H-6, 2H GluNH-C?O, CH.sub.2CONGlc) 2.27-2.63 (m, partially overlapped with DMSO-d6 peak, piperazine ring-CH.sub.2, piperazine-NCH.sub.3) ppm; UV-vis (DMSO): ?.sub.max: 421, 520, 547, 599, 651 nm.
(217) TPP Analogues of Compounds 32, 33, 37 and 38
(218) Unexpected results (Back-oxidation of TPC compounds to TPP compounds by TBAF/NMP) were observed when used following the general TBDMS deprotection procedure D for final compounds 32, 33, 37 and 38).
Example: TPP Analogue of Compound 32 (TPPNP0.1CS-TMA0.9)
(219) The material 30 (600 mg, 0.86 mmol) was dissolved in N-Methyl-2-pyrrolidone (NMP) (5-10 mL) followed by addition of an excess amount of tetra-n-butylammoniumfluoride (TBAF). The reaction mixture was stirred for 24 h at 55? C. and cooled and acidified with dilute HCl, and stirred for 30 minutes before it was dialyzed against 8% NaCl (w/v) in deionised water for two days and against deionised water for 3 days. During this time the colour of the solution changed gradually from grey to red. The red coloured solution was then freeze-dried to yield a brown sponge-like material. The materials were again deprotected, ion exchanged, dialyzed and freeze-dried. However, surprisingly due to back-oxidation the compounds were converted back to their TPP analogues which was confirmed by UV-Vis (as characteristic peak at 650 diminished almost completely). (Data not shown)
(220) Results
(221) Table 3, below, shows the DS for the final carrier compounds. Table 4 shows the fluorescence quantum yields (?.sub.F) of the TPC modified chitosan carriers.
(222)
(223) TABLE-US-00003 TABLE 3 DS (linked TPC-NH- TPC-CO- TPP Pip Pip moieties (eq. per (eq. per per Chitosan sugar sugar sugar Entry Derivatives Compound unit used) unit used) unit) 1. TPC.sub.NP0.1-CS-TMA.sub.0.9 32 0.10 0.10* 2. TPC.sub.NP0.1-CS-MP.sub.0.9 33 0.10 0.10* 3. TPC.sub.CP0.1-CS-TMA.sub.0.9 37 0.11 0.13** 4. TPC.sub.CP0.1-CS-MP.sub.0.9 38 0.11 0.13** *DS determined by .sup.1H NMR of intermediate 29 **DS determined by .sup.1H NMR of intermediate 34
(224) TABLE-US-00004 TABLE 4 Chitosan ?.sub.abs* ?.sub.em* Quantum Entry Derivatives Compound (nm) (nm) Yield (?.sub.F) 1. TPC(p-NH.sub.2).sub.1 20 420 653 0.00246 2. TPC-NH-Pip 21 420 653 0.01355 3. TPC.sub.NP0.1-CS-TMA.sub.0.9 32 420 653 0.01383 4. TPC.sub.NP0.1-CS-MP.sub.0.9 33 420 653 0.01353 5. TPC(p-CO.sub.2H).sub.1 26 420 653 0.01331 6. TPC-CO-Pip 28 420 653 0.01366 7. TPC.sub.CP0.1-CS-TMA.sub.0.9 37 420 653 0.01270 8. TPC.sub.CP0.1-CS-MP.sub.0.9 38 420 653 0.01275 *All data collected in DMSO at room temperature ?-wavelength
Example 3In Vitro and In Vivo Studies
(225) Materials
(226) The HCT116/LUC human colon carcinoma cell line (permanently transfected with a gene encoding luciferase) was kindly provided by Dr. Mohammed Amarzguioui, siRNAsense, Oslo, Norway. MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was from Sigma-Aldrich (MO, USA; cat. no. M 2128), dissolved in PBS to a concentration of 5 mg/ml, sterile filtered and stored at 4? C.
(227) The plasmid pEGFP-N1 was purchased from Clontech Laboratories Inc. (CA, USA; Cat. No. 6085-1), produced by ELIM Biopharmaceuticals, Inc. (CA, USA) (lot #1002) and delivered at a concentration of 2 mg/ml in sterile water. This stock solution was aliquoted and kept at ?20? C. Poly-L-Lysine HBr (MW 15000-30000) was from Sigma-Aldrich (MO, USA; cat. no. P 7890). Poly-L-Lysine HBr was dissolved and diluted in distilled water, sterilized by filtration and stored at ?20? C.
(228) In Vitro Studies
(229) Cell Cultivation.
(230) HCT116/LUC were cultured in DMEM medium (Lonza, Veviers, Belgium) supplemented with 10% fetal calf serum (PAA Laboratories GmbH, Pasching, Austria) 100 U/ml penicillin and 100 ?g/ml streptomycin (Sigma-Aldrich, Mo., USA) at 37? C. and 5% CO.sub.2 in a humid environment.
(231) Treatment of the Cells.
(232) HCT116/LUC cells (1.5?10.sup.5 cells per well for the transfection measurements, 3.75?10.sup.5 cells per well for the MTT assay) were seeded into 6-well (transfection) and 24-well (MTT) plates (Nunc, Roskilde, Denmark) and incubated for 24 h (5% CO.sub.2, 37? C.). The photosensitizer TPCS.sub.2a or the chitosan conjugates (16A, 16B, 17A, 19A, 37, 38, 32 or 33) were then added to the cells and the cells were incubated for 18 h (5% CO.sub.2, 37? C.). The cells were then washed three times with cell culture medium and incubated for 4 h (5% CO.sub.2, 37? C.) in medium containing the plasmid complex. Then the cells were washed once. After addition of fresh medium the cells were illuminated with different light doses. After 48 h of incubation the expression of EGFP (Enhanced Green Fluorescent Protein) was analyzed by flow cytometry. Cell survival was measured by the MTT assay in parallel experiments. The cells were exposed to light from LumiSource? (PCI Biotech, Oslo, Norway). LumiSource? is delivered with a bank of 4 light tubes (4?18 W Osram L 18/67, Blue) emitting mainly blue light with a peak wavelength in the region of 420-435 nm.
(233) Preparation of Plasmid/Poly-L-Lysine Complexes.
(234) Plasmid/poly-L-lysine complexes with charge ratio 2.2 were formed by gentle mixing of plasmid DNA and poly-L-lysine solutions. 2.5 ?l of DNA (stock solution 2 ?g/?l) was diluted with 47.5 ?l water, and 6.92 ?l poly-L-lysine (1 ?g/?l) was diluted with 43.08 ?l water. After mixing, the solution was incubated at room temperature for 30 min, then diluted with culture medium to a final volume of 1 ml and added to the cells (1 ml per well).
(235) Measurement of Transfection.
(236) The cells were trypsinized in 100 ?l trypsin (Trypsin-EDTA, Sigma-Aldrich, Mo., USA), resuspended in 500 ?l cell culture medium and filtered through a 5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap (BD Falcon) (50 ?m mesh nylon filter) before analysis in a BD LSR flow cytometer (Becton Dickinson, Calif., USA). EGFP was measured through a 425-475 nm filter after excitation at 488 nm, and propidium iodide (Calbiochem Corporation, CA, USA) was measured through a 600-620 nm filter after excitation at 561 nm. Propidium iodide (1 ?g/ml) was used to discriminate dead cells from viable cells, and pulse-processing was performed to discriminate cell doublets from single cells. 10000 events were collected for each sample, and the data was analyzed with BD FACSDiva Software (Becton Dickinson, Calif., USA).
(237) Measurement of Cell Survival.
(238) Cell survival was measured by a method based on reduction of a water-soluble tetrazolium salt (MTT) to a purple, insoluble formazan product by mitochondrial dehydrogenases present in living, metabolically active cells. 0.5 ml medium containing 0.125 mg MTT was added to the cells, followed by a 2 h incubation at 37? C., 5% CO.sub.2. The resulting formazan crystals were dissolved by adding 500 ?l DMSO (Sigma-Aldrich, Mo., USA) per well. The plates were read by a PowerWave XS2 Microplate Spectrophotometer (BioTek Instruments, VT, USA). Cell survival was calculated as percent of controls (parallels with no light).
(239) In Vivo Studies
(240) Animals.
(241) Hsd:Athymic nude-Foxn1.sup.nu female mice were bred at the animal department at the Norwegian Radium Hospital. The mice were kept under specific pathogen-free conditions. Water and food was given ad libitum. All procedures involving mice were carried out in agreement with the protocols approved by the animal care committee at the Norwegian Radium Hospital, under the National Ethical Committee's guidelines on animal welfare.
(242) The mice were 22-25 g (5-8 weeks old) when included in the experiment. The HCT116/LUC cells were cultured at 37? C. and 5% CO.sub.2 in a humid environment before transplantation. 1.5?10.sup.6 cells were injected subcutaneously on the right hip of each mouse. The tumor size was measured two or three times per week by measuring two perpendicular diameters. Tumour volume was calculated using the following formula:
V=(W.sup.2?L)/2
where W is the width and L the length diameters of the tumour measured.
Treatment.
(243) The chitosans were diluted to 1.25 mg/ml TPC in PBS (compound 37) and 3% Tween 80 (compounds 38 and 33). 88-100 ?l was injected intravenously in the tail vein (final dose 5 m/kg) when the tumours had reached a volume of 60-100 mm.sup.3. The TPCS.sub.2a was diluted to 1.25 mg/ml in 3% Tween 80 and 88-100 ?l was injected intravenously in the tail vein (final dose 5 m/kg) when the tumours had reached a volume of 60-100 mm.sup.3. 96 h after the injection of photosensitizer the tumours were illuminated with a 652 nm diode laser (Ceramoptec GmbH, Bonn, Germany) at an irradiance of 90 mW/cm.sup.2 and a 15 J/cm.sup.2 light dose. For animals receiving PCI+Bleomycin treatment, 1500 IU Bleomycin (European units) in 100 ?l was injected intraperitoneally. The tumours were illuminated 30 min after BLM injection with a 652 nm diode laser (Ceramoptec GmbH, Bonn, Germany) at an irradiance of 90 mW/cm.sup.2. The animals were covered with aluminum foil except the tumour area where a hole in the foil was made with a diameter 2 mm larger than the tumour area.
(244) In Vivo Imaging System.
(245) The bioluminescence was measured with an IVIS Lumina 100 Series from Caliper Life Sciences, MA, USA. The animals were anesthetized (Zoletil) and injected with 200 ?l D-Luciferin (Caliper Life Sciences) (20 mg/ml in PBS) intraperitoneally. The images were taken 10 min after D-Luciferin injection. The bioluminescence was measured approximately once a week from day 11 after PS injection. The animals were sacrificed when the tumour reached a volume>1000 mm.sup.3 or when the animal was showing signs of pain or abnormal behaviour.
(246) The data was analyzed with Living Image 4.2 Software (Caliper Life Sciences).
(247) The biological effect of the TPP-chitosan conjugates 16A and 16B was tested in experiments where the conjugates were used as photosensitising agents in photochemical internalisation to enhance gene delivery. The experimental details are described under Materials and Methods. As can be seen in
(248) Similar experiments were performed with the TPP-chitosans 17A and 19A (
(249) As can be seen from
(250) As can be seen from
(251) TPC-conjugates have also been explored in in vivo experiments, investigating whether the conjugates are active in PDT- and PCI-based therapeutic approaches.
(252) In
Example 4
(253) General materials and methods were as for Example 1, where appropriate.
(254) See Scheme 7 in
(255) Synthesis of triethyleneglycol monomethyl ether tosylate (40) (also named 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate)
(256) Triethylene glycol monomethyl ether 39 (4.87 mL, 30.43 mmol) was dissolved in THF (25 mL). An aqueous solution (25 mL) of potassium hydroxide (3.7 g, 65.95 mmol) was added and the resulting mixture was cooled down to 0? C. Then, p-toluenesulfonyl chloride (6.86 g, 48.77 mmol) dissolved in THF (50 mL) was added dropwise via dropping funnel over a period of 30 minutes. The reaction mixture was stirred for 2 h more at 0? C. and then allowed to stir at rt overnight. The reaction mixture was concentrated in vacuo to remove THF before it was diluted with EtOAc (40 mL) and water (30 mL) and extracted with EtOAc (2?75 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford compound 40 (7.13 g) as a gray-cloudy oily material.
(257) FT-IR: 2878, 1598, 1453, 1356, 1177, 1097 cm.sup.?1; .sup.1H NMR (400 MHz, CDCl.sub.3): ?=7.80 (d, J=8 Hz, 2H), 7.34 (d, J=8 Hz, 2H), 4.16 (t, 2H, CH.sub.2OTs), 3.67-3.70 (m, 2H, CH.sub.2CH.sub.2OTs), 3.58-3.62 (m, 6H, TEG OCH.sub.2's), 3.37 (s, 3H, OCH.sub.3), 2.44 (s, 3H, ArCH.sub.3) ppm; .sup.13C NMR (400 MHz, CDCl.sub.3): ?=144.91, 133.19, 129.94, 128.12, 72.05, 70.90, 70.71, 69.36, 68.83, 59.17, 21.78 ppm.
(258) Synthesis of (triethyleneglycol monoethyl ether tosylate)(42) (also named 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate)
(259) Triethylene glycol monoethyl ether 41 (4.9 mL, 28.1 mmol) was dissolved in THF (30 mL). An aqueous solution (25 mL) of potassium hydroxide (3.7 g, 65.95 mmol) was added. The reaction mixture was then cooled down to 0? C. Then, p-toluenesulfonyl chloride (6.86 g, 48.77 mmol) dissolved in THF (50 mL) was added dropwise via dropping funnel over a period of 30 minutes. The reaction mixture was stirred for 2 h more at 0? C. and then allowed to stir at rt overnight. The reaction mixture was concentrated in vacuo to remove THF before it was diluted with EtOAc (40 mL) and water (30 mL) and extracted with EtOAc (2?75 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford compound 42 (6.83 g) as a gray-cloudy oily material.
(260) FT-IR: 2870, 2975, 1598, 1453, 1358, 1177, 1110 cm.sup.?1; .sup.1H NMR (400 MHz, CDCl.sub.3): ?=7.77 (d, J=8 Hz, 2H), 7.31 (d, J=8 Hz, 2H), 4.13 (t, 2H, CH.sub.2OTs), 3.64-3.67 (m, 2H, CH.sub.2CH.sub.2OTs), 3.52-3.59 (m, 8H, TEG OCH.sub.2's), 3.49 (q, J=8 Hz, 2H, OCH.sub.2CH.sub.3), 2.42 (s, 3H, ArCH.sub.3), 1.17 (t, J=8 Hz, 3H, OCH.sub.2CH.sub.3) ppm.
(261) Synthesis of Methoxy Polyethyleneglycol Tosylate (44)
(262) Polyethylene glycol monomethyl ether 43 (5 g, 14.29 mmol, average MW: 350 Da) was dissolved in THF (50 mL). An aqueous solution (25 mL) of potassium hydroxide (1.76 g, 31.44 mmol) was added. The reaction mixture was then cooled down to 0? C. Then, p-toluenesulfonyl chloride (3.27 g, 17.14 mmol) dissolved in THF (50 mL) was added dropwise via a dropping funnel over a period of 30 minutes. The reaction mixture was stirred for 2 h more at 0? C. and then allowed to stir at rt overnight. The reaction mixture was concentrated in vacuo to remove THF before it was diluted with EtOAc (40 mL) and water (30 mL) and extracted with EtOAc (2?75 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford compound 44 (5.91 g) as a gray-cloudy oily material.
(263) .sup.1H NMR (400 MHz, CDCl.sub.3): ?=7.74 (d, J=8 Hz, 2H), 7.29 (d, J=8 Hz, 2H), 4.11 (br t, 2H, CH.sub.2OTs), 3.49-3.65 (m, 28H, CH.sub.2CH.sub.2OTs & PEG OCH.sub.2's), 3.32 (s, 3H, OCH.sub.3), 2.39 (s, 3H, ArCH.sub.3) ppm.
(264) Synthesis of triethyleneglycol monoethylether piperazine (46) (also named 1-(2-(2-(2-ethoxyethoxy)ethoxy)ethyl)piperazine (46) (TEG-Pip)
(265) Piperazine (10.4 g, ?120 mmol) was dissolved in acetonitrile (150 mL) under nitrogen atmosphere. Compound 42 (5 g, 15.04 mmol) dissolved in acetonitrile (30 mL) was added dropwise. The resulting mixture was stirred at rt for 12 h before it was concentrated in vacuo to remove acetonitrile. The crude product was purified by flash silica gel column chromatography using MeOH: CH.sub.2Cl.sub.2 (8:92) as eluent to afford pure compound 46 (1.52 g, 41%) as a colourless liquid.
(266) .sup.1H NMR (400 MHz, CDCl.sub.3): ?=3.54-3.64 (m, 10H, TEG OCH.sub.2's), 3.50 (q, J=8 Hz, 2H, OCH.sub.2CH.sub.3), 3.43 (s, 1H, NH), 2.88 (t, 4H, Piperazine ring-CH.sub.2), 2.55 (t, 2H, OCH2-CH.sub.2-Piperazine), 2.46 (br m, 4H, Piperazine ring-CH.sub.2), 1.18 (t, J=8 Hz, 3H, OCH.sub.2CH.sub.3) ppm; .sup.13C NMR: 70.76, 70.70, 70.47, 69.92, 68.86, 66.73, 58.47, 54.89, 50.55, 45.99, 15.25 ppm; MS (ESI) calcd. for C.sub.12H.sub.27N.sub.2O.sub.3 ([M+H].sup.+) 247.2016, found 247.2014.
(267) Synthesis of 2-(2-(2-methoxyethoxy)ethoxy)acetaldehyde (45)
(268) Oxalyl chloride (5 mL, 58.24 mmol) was dissolved in CH.sub.2Cl.sub.2 (75 mL) under nitrogen atmosphere. The resulting mixture was cooled down to ?78? C. using dry-ice/acetone mixture before careful drop-wise addition of DMSO (5 mL) diluted in CH.sub.2Cl.sub.2 (15 mL). After complete addition, the reaction mixture was stirred for 10 minutes more before dropwise addition of triethyleneglycol monomethyl ether 39 (5 mL, 31 mmol) diluted in CH.sub.2Cl.sub.2 (30 mL). The reaction mixture was stirred for 15 minutes after complete addition. Then, Et.sub.3N (20 mL, 143 mmol) diluted in CH.sub.2Cl.sub.2 (20 mL) was added drop-wise over a period of 20 minutes and stirred for 30 minutes more to ?78? C. before allowing it to reach rt. The white milky coloured reaction mixture was then washed with water (2?45 mL) and with brine (40 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The resulting crude product was purified by silica gel column chromatography using MeOH/EtOAc (0:100-10:90) as eluent to afford aldehyde 45 (2.40 g). 1H NMR analysis revealed that compound 47 was contaminated with some inseparable impurity.
(269) FT-IR: 3436, 2879, 1734, 1454, 1353, 1108, 1028 cm.sup.?1; .sup.1H NMR (400 MHz, CDCl.sub.3) ? ppm: 9.73 (s, 1H, CHO), 4.16 (s, 2H), 3.54-3.76 (m, 10H), 3.38 (s, 3H) ppm. (Product is contaminated with starting material. Product is approximately 50%.)
(270) Synthesis of 2-(2-(2-ethoxyethoxy)ethoxy)acetaldehyde (47)
(271) Same procedure as used for above compound; except the starting material used was triethyleneglycol monoethyl ether (41) instead of triethyleneglygol monomethyl ether (39). Compound 47 (Yield: 2.34 g, ?42%) contaminated with some inseparable impurity.
(272) .sup.1H NMR (400 MHz, CDCl.sub.3) ? ppm: 9.67 (s, 1H, CHO), 4.10 (s, 2H), 3.52-3.69 (m, 10H), 3.45 (q, 2H), 1.15 (t, 3H) ppm. (Product is contaminated with starting material. Product is approximately 42%).
(273) TEGylation of Chitosan by Direct N-Modifications of 3,6-di-O-TBDMS-Chitosan See scheme 8 in
(274) Synthesis of N-(2-(2-(2-ethoxyethoxy)ethoxy)ethylamino chitosan TEG.sub.0.41-CS (49a)
(275) DiTBDMS-chitosan 8 (300 mg, 0.77 mmol) was dissolved in NMP (5 mL) and heated to 50? C. in a reaction vial. Cs.sub.2CO.sub.3 (1 g, 3.07 mmol) and a catalytic amount of potassium iodide was added. Then, the reaction vial was sealed and the reaction mixture stirred for 2 h before addition of compound 42 (767 mg, 2.31 mmol) via a syringe. The reaction mixture was stirred for 48 h before it was cooled down and poured into ice-water and the precipitate obtained was filtered off, and dried using a vacuum oven. The crude product 48a (270 mg) was obtained as a yellow powder and used as it was for the next deprotection step as described below.
(276) For deprotection of hydroxyl groups (removal of the silyl groups), the crude compound 48a was suspended in MeOH (15 mL). Concentrated HCl (2 mL) was added slowly at rt and the resulting mixture was stirred for 12 h before it was concentrated in vacuo. The crude product obtained was again suspended in MeOH (15 mL) and conc. HCl (2 mL) was added and stirred for 12 h before it was diluted and ion exchanged with aqueous NaCl (5%, 35 mL) solution and then dialyzed against deionized water for 3 days. After completion of dialysis, the water soluble material was freeze-dried to afford 49a (125 mg) as a white sticky material.
(277) FT-IR: 3418, 2874, 1712, 1631, 1536, 1378, 1077 cm.sup.?1; .sup.1H NMR (400 MHz, D.sub.2O) ?=4.68 and 4.27 (s, 1H, chitosan H-1 & H-1), 3.14-3.97 (br m, 10H, chitosan H-2 to H-6, TEG (?41% DS) OCH.sub.2's and OCH.sub.2CH.sub.3), 2.96-3.14 (br m, 1H, H-2 & H-2), 1.21 (t, 1.24H (?41% DS) TEG OCH.sub.2CH.sub.3) ppm.
(278) Synthesis of N-(2-(2-(2-ethoxyethoxy)ethoxy)ethylamino chitosan TEG.sub.0.27-CS (51b) (27% DS)
(279) Compound 49b was prepared by using same procedure as described above, except 1.5 equivalents of the triethylene glycol tosylate (42) reagent was used instead of 3 equivalents. Compound 49b (27 mg) was obtained as a white sticky material.
(280) FT-IR: 3417, 2876, 1602, 1382, 1259, 1078, 599 cm.sup.?1; .sup.1H NMR (400 MHz, D.sub.2O: DCI, 95:5) ?=5.13 and 4.95 (s, 1H, chitosan H-1 & H-1), 3.24-3.99 (br m, 12H, chitosan H-2 to H-6, H-2, TEG (27% DS) OCH.sub.2's and OCH.sub.2CH.sub.3), 1.21 (t, 0.89H, TEG OCH.sub.2CH.sub.3) ppm.
(281) Synthesis of N-acetylbromo-3,6-O-diTBDMS-Chitosan (9)
(282) DiTBDMS-chitosan 8 (3 g, 7.70 mmol) (previously synthesized by Ing?lfur Magnusson) was dissolved in CH.sub.2Cl.sub.2 (30 mL) under nitrogen atmosphere. The resulting mixture was cooled down to ?20? C. before addition of triethylamine (5.30 ml, 38 mmol) using a syringe. After 10 minutes of stirring, bromo acetylbromide (2.65 ml, 30.51 mmol) diluted in CH.sub.2Cl.sub.2 (2.5 mL) was added dropwise using a syringe. The reaction mixture was then stirred for 1 hour at ?20? C. before it was diluted with CH.sub.2Cl.sub.2 (70 mL) and immediately concentrated in vacuo. Then, the thick brown material was triturated and washed with acetonitrile (3?35 mL), dried before it was re-dissolved in CH.sub.2Cl.sub.2 (40 mL) and placed in a separatory funnel where it was washed with water (2?25 mL) and with brine (35 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford bromoacyl intermediate 9 (2.57 g) as a brown solid material.
(283) FT-IR (KBr): v 3404 (br, NH), 2956-2858 (s, CH TBDMS), 1682 (vs, C?O amide I), 1530 (vs, C?O amide II), 1473, 1391, 1362, 1311, 1259, 1101, 1005, 837, 777 (SiC), 669 cm.sup.?1; .sup.1H NMR (CDCl.sub.3) ? ppm: 4.40 (br s, 1H, H-1), 3.26-4.02 (m, 8H, H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and GluNHC?OCH.sub.2Br), 0.90 and 0.88 (br s, 18H, (CH.sub.3).sub.3CSi), 0.13 and 0.07 (br s, 12H, CH.sub.3-Si) ppm.
(284) PEGylation of Chitosan by Nucleophilic Substitution on N-Acetyl Bromo-3,6-diTBDMS-Chitosan
(285) N-(acetyl piperazine-TEG)-Chitosan (51)
(286) (i) Acetyl bromochitosan 9 (200 mg, 0.397 mmol) was dissolved in CH.sub.2Cl.sub.2 (25 mL) under a nitrogen atmosphere. Compound 46 (204 mg, 0.828 mmol) diluted in CH.sub.2Cl.sub.2 (10 mL) was added dropwise at rt. The resulting mixture was stirred for 10 minutes before addition of triethylamine (115 ?L, 0.828 mmol). Stirring was continued at rt for 24 h and the total consumption of starting material 46 was confirmed by checking TLC in MeOH: CH.sub.2Cl.sub.2 (1:9). Then the reaction mixture was placed in a separatory funnel and the organic phase was washed with water (2?35 mL) and brine (35 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford compound 50 (214 mg) as a crude liquid which was used as it is for the next deprotection step.
(287) (ii) For deprotection of hydroxyl groups (removal of the silyl groups), the crude compound 50 was suspended in MeOH (15 mL). Concentrated HCl (2 mL) was added slowly at rt and the resulting mixture was stirred for 12 h before it was concentrated in vacuo. The crude product obtained was again suspended in MeOH (15 mL) and conc. HCl (2 mL) was added and stirred for 12 h before it was diluted and ion exchanged with aqueous NaCl (5%, 35 mL) solution and then dialyzed against deionized water for 3 days. After completion of dialysis, the water soluble material was dried in vacuo to afford 51 (112 mg) as a yellowish clear sticky material.
(288) .sup.1H NMR (400 MHz, D.sub.2O) ?=4.63 (s, 1H, chitosan H-1), 2.74-3.76 (m, 30H, chitosan H-2 to H-6, GlcNHCOCH.sub.2-Pip-TEG, TEG OCH.sub.2's & OCH.sub.2CH.sub.3, piperazine ring-CH.sub.2's), 1.21 (t, 3H, TEG OCH.sub.2CH.sub.3) ppm; DS=100%.
(289) Synthesis of N-acetyl-piperazine tetraphenylporphyrin (TPP-NH-Pip) (5)
(290) This was performed as described above in Example 1 and Scheme 1.
(291) Synthesis of TPPNHCOCH.sub.2-TBDMS-chitosan (55)
(292) See Scheme 9 in
(293) DiTBDMS-chitosan (100 mg, mmol) was dissolved in NMP (10 mL) at 55? C. Cesium carbonate (500 mg, 1.54 mmol) was added and the reaction mixture was stirred for 15 minutes before addition of compound 4 (72.2 mg, 0.096 mmol). A catalytic amount of potassium iodide was added and stirring continued for 24 h before a catalytic amount of DMAP was added and stirred for 24 h before being cooled down and poured into water. The precipitate was filtered off, dried in a vacuum oven to afford compound 55 as a crude solid. However, a red colour of the water solution was observed indicating that some of the compound was wasted in water. That might be because of NMP; thus dialysis at this stage might be useful.
(294) .sup.1H NMR (400 MHz, D.sub.2O) ? ppm: 8.84-8.86 (br m, ?-pyrrole H), 8.68 (s, TPPNHCO), 8.21-8.22 (m, tetraphenyl-Ho), 7.99 (d, J=8.0 Hz, RNHTPP-phenyl-Hm), 7.72-7.79 (m, triphenyl-Hm,p), 2.72-4.80 (m, chitosan H-2-H6, TPPNHCOCH.sub.2), 0.89-0.90 (br s, (CH.sub.3).sub.3CSi), 0.05-0.07 (br s, 12H, CH.sub.3Si). ?2.78 (s, ?-pyrrole NH); ((DS of TPP-NHCOCH.sub.2=?5%)
(295) Synthesis of TPPp.sub.0.1-BrA.sub.0.9-DiTBDMS-CS (52)
(296) Compound 9 (800 mg, 1.587 mmol) was dissolved in CH.sub.2Cl.sub.2 (40 mL) under a nitrogen atmosphere at room temperature. TPP-NH-Pip 5 (120 mg, 0.158 mmol) was added followed by addition of triethylamine (30 ?l, 0.216 mmol). The reaction mixture was stirred for 24 h at rt. Complete consumption of starting material 5 was confirmed by TLC (MeOH: CH.sub.2Cl.sub.2, 1:9). The reaction mixture was diluted with CH.sub.2Cl.sub.2 (75 mL) and washed with water (2?35 mL) and brine (25 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 52 (yield: 716 mg) as a purple solid.
(297) .sup.1H NMR (CDCl.sub.3) ? ppm: 9.31 (s, TPPNHCO), 8.84-8.86 (m, ?-pyrrole H), 8.20-8.23 (m, tetraphenyl-Ho), 7.97 (d, J=8.0 Hz, RNHTPP-phenyl-Hm), 7.74-7.79 (m, triphenyl-Hm,p), 4.43 (br s, H-1), 3.50-4.14 (m, chitosan H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and H-2 GluNHCO, TPPNHCOCH.sub.2-Pip, CH.sub.2CONGlc and Piperazine ring-CH.sub.2's), 2.81-2.86 (m, piperazine ring-CH.sub.2's), 0.91, 0.87 (br s, (CH.sub.3).sub.3CSi), 0.07, 0.14 (br s, CH.sub.3-Si), ?2.77 (br s, ?-pyrrole NH) ppm; (DS of TPPNH-Pip=?10%).
(298) TEGylation and Deprotection of TPP-Conjugated DiTBDMS-CS
(299) Synthesis of TEGylated N-(TPPNHCOCH.sub.2)-chitosan (57)
(300) (i) Crude compound 55 (350 mg, 0.833 mmol) was dissolved in NMP (10 mL) at 50? C. in a reaction vial. Cs.sub.2CO.sub.3 (1.09 g, 3.33 mmol) was added and the reaction mixture stirred for 30 minutes before addition of compound 42 (1.108 g, 4.16 mmol) and a catalytic amount of potassium iodide. Stirring was continued for 12 h before the reaction mixture was cooled down, diluted with water (30 mL) and dialyzed against deionized water for 3 days before being freeze-dried to afford compound 56 which was used as it was for the next deprotection step.
(301) (ii) The crude compound 56 was suspended in MeOH (15 mL). Concentrated HCl (2 mL) was added slowly at rt and the resulting mixture was stirred for 12 h before it was concentrated in vacuo. The crude product obtained was again suspended in MeOH (15 mL) and conc. HCl (2 mL) was added and stirred for 12 h before it was diluted and ion-exchanged with aqueous NaCl (5%, 35 mL) solution and then dialyzed against deionized water for 3 days. After completion of dialysis, the partly water soluble material was dried in vacuo to afford 57 (115 mg) as a brown solid.
(302) Synthesis of TEGylated TPPNH-Pip-chitosan TPPp.sub.0.1-CS-TEG.sub.0.9 (54)
(303) (i) Compound 52 (400 mg, 0.799 mmol) was dissolved in CH.sub.2Cl.sub.2 (35 mL) under a nitrogen atmosphere. Compound 46 (394 mg, 1.56 mmol) was added followed by addition of Et.sub.3N (222 ?l, 1.56 mmol) and the reaction mixture was stirred for 24 h at rt. Then, TLC was checked and Et.sub.3N (222 ?l, 1.56 mmol) added in order to complete consumption of starting material 46 and stirring was continued for 12 h more. Then, the reaction mixture was concentrated in vacuo to afford 53 (417 mg) as a crude material which was used as it was for next the deprotection step.
(304) (ii) The crude compound 53 was suspended in MeOH (15 mL). Conc. HCl (2 mL) was added slowly at rt and the resulting mixture was stirred for 12 h before it was concentrated in vacuo. The crude product obtained was again suspended in MeOH (15 mL) and conc. HCl (2 mL) was added and stirred for 12 h before it was diluted and ion-exchanged with aqueous NaCl (5%, 35 mL) solution and then dialyzed against deionized water for 3 days. After completion of dialysis, the water soluble material was freeze-dried to afford final compound 54 (252 mg) as a purple-red-brown solid.
(305) FT-IR: 3431, 2869, 1665, 1529, 1442, 1308, 1109, 1070, 1029, 800, 701, 559 cm.sup.?1; .sup.1H NMR (DMSO-d6: D.sub.2O 96:4) ? ppm: 8.83 (br m, ?-pyrrole H), 8.15-8.22 (m, tetraphenyl-Ho), 8.11 (d, J=8.0 Hz, RNHTPP-phenyl-Hm), 7.80-7.88 (m, triphenyl-Hm,p), 4.55 (br s, H-1), 2.54-3.65 (br m, partially overlapped with HDO peak, chitosan H-2 GlcN, H-3, H-4, H-5, H-6, H-6 and H-2 GluNHCO, TPPNHCOCH.sub.2-pip, CH.sub.2CONGlc, piperazine ring-CH.sub.2's TEG OCH.sub.2's and TEG OCH.sub.2CH.sub.3), 1.09 (t, TEG OCH.sub.2CH.sub.3) ppm.; (DS of TPPNH-Pip=?10% and DS of TEG=?90%)
(306) Structural data was confirmed by NMR, FT-IR and Mass analysis (data not shown). A representative NMR spectrum for compound 54 is shown in
Example 5
(307) In Vitro T Cell Activation Assay
(308) For testing the effect of chitosan-conjugates 32 and 38 on MHC class I-restricted antigen-presentation and CD8+ T cell activation, murine primary macrophages were incubated with conjugates 32 and 38 and the ovalbumin OVA 257-264 peptide antigen in an antigen-specific T cell setting with an ovalbumin-specific (OVA 257-264) CD8+ T cell clone. IL-2 production from activated CD8+ T cells was analyzed by an ELISA.
(309) Bone-marrow derived macrophages (BMDMs) were used as antigen-presenting cells (APCs) in an antigen-specific T cell setting with ovalbumin-specific T cell hybridomas. BMDMs were generated by cultivating mouse bone-marrow cells for at least 5 days in medium supplemented with 20% L-292 cell line supernatant.
(310) 30,000 APCs per well were incubated overnight in 96-well plates with or without chitosan-conjugates 32 and 38 at a concentration of conjugates giving a TPC concentration of 0.05 ?g/ml. The next day the APCs were incubated with 2 ?g/ml of antigenic peptide (OVA 257-264, from Anaspec) for 4 h (all stimulations in triplicate).
(311) Cells were washed and exposed to different doses of blue light (0; 30, 60, 90, 180 sec) before 100,000 ovalbumin-specific T cells per well were added and co-cultured with the APCs overnight. The CD8+ T cell clone RF33.70 (MHC I-restricted recognition of OVA 257-264) was used.
(312) After overnight co-culture of CD8+ T cells and APCs, supernatants from the cell culture were harvested. The supernatants were analyzed for interleukin (IL)-2 production from activated T cells by use of a standard mouse IL-2 ELISA (IL-2 ELISA Duoset, RnD Systems, analysis of duplicates from each well of T cell culture, 25 ?l of undiluted supernatant was analyzed in each ELISA well).
(313) The results (