CN1329398C - Dihydroporphine E6 zinc compound photosensitizer and its preparation method and application - Google Patents

Abstract

The present invention discloses a dihydroporphine E6 zinc compound photosensitizer and a preparation method and application thereof. A dihydroporphine E6 zinc compound of the present invention is characterized in that zinc atoms are introduced in a dihydroporphine E6 mixed with a zinc compound in pH >8 water solution, polyvinylpyrrolidone solution is added, and thus, the dihydroporphine E6 zinc compound is obtained. The dihydroporphine E6 zinc compound photosensitizer can be obtained by oral application or intravenous injection, can not cause skin photosensitive problems, and does not need being protected form light. In addition, the dihydroporphine E6 zinc compound photosensitizer can perform local treatment by the activation of a laser illumination instrument, but a luminous tube array instrument (light bed) with low power can be used for whole body treatment. Compared with other photosensitizers, the dihydroporphine E6 zinc compound photosensitizer has the advantages of practicability and safety, and the over-all performance of the dihydroporphine E6 zinc compound photosensitizer is superior to that of other photosensitizers.

Description

Dihydroporphine E 6 zinc compound photosensitizer and preparation method thereof and application

Technical field

The present invention relates to photon dynamics therapy field, be specifically related to a kind of Dihydroporphine E 6 zinc compound photosensitizer and preparation method thereof and application.

Background technology

Photon dynamics therapy (PDT) is a promising cancer therapy method, has comprised the use of optical photon and photosensitizers.Each element of photosensitizers all is harmless, but in case will produce fatal cytotoxic factor when combining with oxygen element, is initially the singlet oxygen form, can stop the development of tumour cell.Owing to have only the sick cell that is acted on photosensitizers, photon and oxygen simultaneously just can be attacked, thereby this photosensitizers has higher selectivity to pathological tissues by cytotoxin.The present pathological tissues of double selection gonosome of PDT therapy is to the preferential picked-up of photosensitizers and illumination is only acted on these photosensitizerss that rest on specific diseased tissue area.Porphyrin and relevant ring family are used for photon dynamics therapy (PDT) synthetics of broad research the most.Porphyrin is the aromatic series macrocylc compound with 18p electronics, and it shows distinctive spectrum and is accompanied by intensive p-p transition and 4 Q band absorption peaks are arranged in visibility region near (Soret band) about 400nm.Nature obtains sun power by these optical characteristics of porphyrin, carries out photosynthesis with chlorophyll and bacteriochlorophyll as antenna pigment and reactive center pigment.The long wave absorption characteristic of this nature chromophore makes them can be used as photosensitizers in the photon dynamics therapy (PDT).

In sum, photon dynamics therapy (PDT) be with rest on the tumor tissues photosensitizers and with the effect that reacts to each other of optical photon, thereby cause producing singlet oxygen ( ¹O2) this generally acknowledged destructive medium that has.The result who reaches thorough destruction tumor tissues needs high singlet oxygen output.Even lacking under the situation that heavy atom replaces and transition metal ion is coordinated, the porphyrin system generally also can satisfy these requirements.Why Here it is all is porphyrin for the employed sensitizer of clinical assessment of photon dynamics therapy (PDT) recently or has been the molecule of base with the porphyrin.Therefore, a lot of photosensitizerss relevant with chlorin arranged in recent years, picture bacteriochlorophyll (bacteriochlorins), porphycenes, titanium cyanines color (phthalocyanines), naphthalene titanium cyanines (naphthalocyanines) and expansion porphyrin (expanded porphyrins) all are used to curative effect synthetic and assessment photon dynamics therapy (PDT).But, design meet photosensitizers that photon dynamics therapy (PDT) uses also needs consider following problem, for example whole lipotropys, (being the balance of satisfactory wetting ability and hydrophobe), pH value, lymph Excretory system and lipoprotein binding, these factors all can influence distribution and the location of sensitizer in pathological tissues and tumour.Except that synthetic fine water-soluble porphyrin, research usually all can be paid attention to those molecules aspect the science of promotion and the practical application of humanistic aspect.Can stay in a certain privileged site of health as porphines, just reflect its application medically.Haematoporphyrin IX can send fluorescence in malignant tissue, the sulfonation tetraphenylporphyrin then can accumulate on the tumor tissues.The main chlorin and the generation of bacteriochlorophyll have three kinds of approach.First kind is to improve to form from the porphyrin of moulding; Second kind of chlorin and bacteriochlorophyll that is to use chlorophyll a as synthetic other kind of starting material.The third approach is to utilize unsettled bacteriochlorophyll a as the bacteriochlorophyll of substratum as synthesizing stable.Each approach all can successfully be used as prepares the requirement that photon dynamics therapy (PDT) photosensitizers uses, and each approach all is to carry out according to synthetic scientific approach and meaning biologically.

Porphyrin is easy to combine with multiple metallic element with chlorine system; Metalloporphyrin is numerous transition metals, lanthanon, and actinide elements and main group element are familiar with.These metal composites not only can protect inner nitrogen-atoms away from electrophilic reagent and powerful matrix.And can produce significant big ring reaction effect.The chemical of the metalloporphyrin of rear transition metal element is known the most.The modal geometry of this synthetics is octahedra, and metal ion is positioned at N4 porphyrin planar mid-way, and metal ligand body is positioned at the horizontal position.Recently, the chemical of early transition metal element porphyrin is looked back again.

Metalation is attended by the reaction (for example halogenide, hydride, acetylacetonate and carbonic acyl radical) of metallic salt or synthetics usually in organizing solvent, chloroform for example, toluene or N, dinethylformamide (DMF).The de-metallization reaction needed is carried out in sour environment, and the requirement of acid concentration is by the stability decision of metal composite.

Having now at the photosensitizers of usefulness such as the happiness pool branch in Canadian Photofrin and Chinese Chongqing all is the haematoporphyrin goods, and this type of photosensitizers adopts quiet notes, needs lucifuge 30-40 days.Activation need be used the bigger laser device of power as the photosensitizers of hematoporphyrin derivative, and only can be used for the partial irradiation treatment, otherwise the danger of burned skin is arranged.

Summary of the invention

The objective of the invention is to overcome the deficiency that existing photosensitizers exists, provide a kind of easy to use, the Dihydroporphine E 6 zinc compound photosensitizer that toxic side effect is little.

Another object of the present invention provides the preparation method of above-mentioned Dihydroporphine E 6 zinc compound photosensitizer.

Further purpose of the present invention provides the application of above-mentioned Dihydroporphine E 6 zinc compound photosensitizer in preparation photon dynamics therapy photosensitizer.

Dihydroporphine E 6 zinc compound structural formula of the present invention is shown in (I).

M represents zinc atom; R ¹, R ², R ³... R ¹⁴Be selected from hydrogen atom, (CH respectively ₂) n-CHO, (CH ₂) n-CO ₂R ¹⁵Or one arbitrarily by one or more-OH and-NH ₂Saturated or the insatiable hunger alkyl group of C1-C6 that replaces; N represents 0,1,2 or 3; R ¹⁵Be selected from hydrogen, lithium, sodium, potassium, magnesium, calcium, one arbitrarily by one or more-OH and-NH ₂The C1-C6 that replaces saturated or insatiable hunger alkyl group or an amino acid that generates naturally.

The preferred substance of above-mentioned Dihydroporphine E 6 zinc compound is: R ¹, R ³, R ⁴, R ⁵, R ⁸, R ¹¹All represent hydrogen atom, R ²Expression-CH ₂-CH ₂-COOH, R ⁶, R ⁹, R ¹²All represent methyl, R ⁷Expression-CH=CH ₂, R ¹⁰Expression-CH ₂-CH ₃, R ¹³Expression-COOH, R ¹⁴Expression-CH ₂-COOH, M represents zinc atom.

The structural formula of chlorin E6 is shown in (II).

The preparation method of above-mentioned Dihydroporphine E 6 zinc compound is: with the compound of chlorin E6 and zinc, add polyvinylpyrrolidonesolution solution again in the aqueous solution of pH＞8, can obtain Dihydroporphine E 6 zinc compound.Concrete preparation process is:

(1) the not stopping reaction of chlorin E6: sodium bicarbonate is dissolved in the deionized water, adds chlorin E6, slowly stir; Polyvinylpyrrolidone is dissolved in the deionized water, then it is joined in the chlorin E6 test solution, slowly stir down, obtain unstabilized chlorin E6 at 40 ℃;

(2) Dihydroporphine E 6 zinc compound is synthetic: zinc nitrate hexahydrate is dissolved in the deionized water, under the room temperature zinc nitrate hexahydrate solution is joined among the unstabilized chlorin E6, and slowly stir synthesizing dihydro porphines E6 zinc compound;

(3) stabilizing step of Dihydroporphine E 6 zinc compound: polyvinylpyrrolidone is dissolved in the deionized water, adds Dihydroporphine E 6 zinc compound again, slowly stir down at 60 ℃.

Above-mentioned Dihydroporphine E 6 zinc compound photosensitizer can be used as the medicine in the optical dynamic therapy cancer.Be used for the treatment of uterine cervix, tracheae, larynx, segmental bronchus, bronchiole, lung, skin, bladder, oesophagus, stomach, rectum, colon, prostate gland, hollow organ, bile duct, urethra, kidney, uterus, cancers such as vagina.

Compared with prior art, the present invention has following beneficial effect:

1. easy to use: Dihydroporphine E 6 zinc compound photosensitizer can not cause the photosensitive problem of skin by oral or intravenous injection, does not need lucifuge.In addition, Dihydroporphine E 6 zinc compound photosensitizer can activate according to instrument by laser light and carry out topical therapeutic, but also available low power luminous tube array instrument (light bed) is used as whole body therapeutic simultaneously;

2. toxic side effect is little;

Therefore, contrast other photosensitizers, Dihydroporphine E 6 zinc compound photosensitizer has more practicality and security, and overall performance is better than other photosensitizers.

Description of drawings

Fig. 1 is the abosrption spectrogram of chlorin E6;

Fig. 2 is the Dihydroporphine E 6 zinc compound abosrption spectrogram;

Fig. 3 is a Dihydroporphine E 6 zinc compound activity time graphic representation.

Embodiment

The preparation of embodiment 1 Dihydroporphine E 6 zinc compound

The not stabilizing step of flow process 1. chlorin E6

Reagent:

Chlorin E6; (C ₃₄H ₃₆N ₄O ₆, m.w.596.68),

Sodium bicarbonate, (NaHCO ₃, m.w.84.01),

Polyvinylpyrrolidone (PVP), (m.w.10,000),

Step:

A. the sodium bicarbonate of 15M is dissolved in the deionized water that concentration is 7mg/ml;

B. add the chlorin E6 of 1M, slowly stir solvent, the absorption spectrum of chlorin E6 is seen Fig. 1;

C. the PVP of 1M (10000) is dissolved in the deionized water that concentration is 80mg/ml; Then it is joined in the chlorin E6 test solution, slowly stir down at 40 ℃;

D. finish the stable of first step and under 40 ℃ state, kept 2 hours slowly stirring.

The synthesis step of flow process 2. Dihydroporphine E 6 zinc compounds

Reagent:

Unstabilized chlorin E6 (seeing flow process 1)

Zinc nitrate hexahydrate Zn (NO ₃) ₂-6H ₂O, (m.w.189),

Step:

A. the zinc nitrate hexahydrate of 1M is dissolved in the deionized water that concentration is 1mg/ml;

B. under the room temperature zinc nitrate hexahydrate solution is joined in (chlorin E6+PVP), and slowly stir, synthesizing dihydro porphines E6 zinc compound, the Dihydroporphine E 6 zinc compound absorption spectrum is seen Fig. 2;

C. finish and waited at least 3 hours after synthetic, (Dihydroporphine E 6 zinc compound activity curve figure sees Fig. 3) is put in this synthetics among the refrigerator then, and keeps 24 hours under 2～8 ℃ of temperature.

The stabilizing step of flow process 3. Dihydroporphine E 6 zinc compounds

Reagent:

Dihydroporphine E 6 zinc compound (referring to flow process 1,2)

Polyvinylpyrrolidone (PVP), (m.w.10,000),

Step:

A. the PVP of 4M (10000) is dissolved in the deionized water that concentration is 70mg/ml;

B. add Dihydroporphine E 6 zinc compound and advance in polyvinylpyrrolidone (PVP) solution, slowly stir down at 60 ℃;

C. cooling is kept at 20 ℃ or following temperature, keeps in Dark Place until arriving requirement to some extent.

Purify by gel chromatography, and carry out sterilization by antibiotic millipore filtration.

Embodiment 2 pharmacokinetics

Medicine (metabolism) kinetics is distributed in specific organ, tissue, last 30 hours of body fluid and tumor tissues (embriocarcinome).Test mouse: female BALB/c mouse gross weight 20～21 grams.This research is to inject in the capable abdomen of Zu Zhi Jin for organ and tumour on the basis of Percin-elmer spectrophotofluorometer, and dosage is the Dihydroporphine E 6 zinc compound of 2.5 mg/kg (body weight).

Injection the results are shown in Table 1, this explanation:

A. animal can be born the belly injection that injected dose is the Dihydroporphine E 6 zinc compound of 2.5 mg/kg well, any signs of toxicity do not occur, and after 30 hours, the behavior reaction of mouse does not all have to influence in injection back and injection immediately.

B. the dosage of being prepared can promptly absorb in blood from belly, and is stored in liver in injection in back one hour.It will exceed 10 to 14 times in its level in blood of the volume ratio in the liver organization.

C. preceding 12 hours after being injected into priming dose, kidney have stored enough dosage (only lacking 2～2.5 times than the quantity of liver the inside), yet in fact do not exist in urine.Among the dosage of preparing in ensuing 18 hours was scavenged into blood from renal tissue fully, simultaneously, in whole viewing duration, the secreting function of animal was not affected.

D. in accepting preceding 8 hours of injection back, photosensitizers farthest accumulates on the liver organization, and residuum was absorbed rapidly in ensuing 24 hours in the small intestine.The dynamically lucky gang of the curve of liver curve dynamic and small intestine.Therefore, farthest gather on the tumor tissues for making injected dose, it is just enough that suggestion injection lack 5～10 times amount than priming dose.

E. in the quantity of spleen and lung tissue than lacking 5～8 times in the aggregate amount of liver and tumour, and had data readings (phone reading) to produce at preceding 24 hours.

F. the aggregate amount curve on skin and muscle is dynamically about the same, and except a difference: the dosage content in preceding 15 hours in the muscle exceeds 1.5 times.

Just progressively rise when G. tumour-the accumulate in dosage on the tumour is from injection and after 15 hours, reach maximum value.And the time of maximal dose accumulative steady state (12～20 hours) many than the length of having injected chlorine e6 synthetics.After the obvious decline at preceding 24 hours ends, the steady state time that reaches maximum gathering reading for the second time is noted as 24 to 30 hours.In the test of liver and tumour, " intercepting " effect has been recorded twice.This is an index (reading of tumour rises, and the reading of liver descends) in 12 hours, has more obvious contrast after 24 hours.

Conclusion: after belly absorbs, redistributing and the removing of unnecessary dosage in first 24 hours from blood to the organ, Dihydroporphine E 6 zinc compound accumulates in the amount of tumor tissues than high 2.5 times at liver organization, than high 6 times at integumentary musculature and other soft tissue organs.Compare with the dynamic (dynamical) dipolymer Dihydroporphine E 6 zinc compound of medicine (metabolism), the monomer in the demonstration has more activity to tumor tissues and in in-house steady chemical structure.

Table 1

Sequence number. organ	1 hour	5 hours	15 hours	24 hours	30 hours
						1. blood	4	9	6	1	1
2. urine	7	0	0	0	0
						3. small intestine	98	175	136	90	46
4. liver	86	147	66	49	33
						5. spleen	11	17	17	12	13
6. kidney	38	64	25	11	8
						7. lungs	18	15	26	11	8
8. tumour	8	40	99	67	79
						9. skin	9	8	19	7	15
10. muscle	25	7	33	6	18

Annotate: reading and the Fl1 unanimity relevant in the table with unit.

Embodiment 3 toxicity tests

Be defined parameters LD10 and LD50, specially prepared three kinds of dosage: 100,125 and 150 mg/kg are used for injection in the one abdomen.Chlorine e6 synthetics reading comparing as original shape and LD10-119 mg/kg and LD50-160.Ford China photosensitizers has only the 3rd group dosage (150 mg/kg) injection that corresponding reaction is arranged after the above-mentioned dosage of injection, and its injection volume is 3 milliliters, makes animal temporary static owing to belly swelling produces.After having absorbed unnecessary dosage, the reflection of this treated animal and other action of two groups reaction is as good as (it is movable to move, opposing behavior, to the reaction of food, fur situation).(being slow in action recess side, diarrhoea, passivity ability and reactionless to food) do not appear in toxic susceptibility again in ensuing 72 hours.Continue these animals are studied observation at the fortnight of following, to carry out the observational study of possibility genotoxic potential.

Second group-175,200 and 225 mg/kg were carried out from 1 to 7 July in 2004, did not have toxicity.

There are not toxicity in the 3rd group-300,350 and 450 mg/kg from 10 to 20 July in 2004.

Embodiment 4 Dihydroporphine E 6 zinc compounds produce the comparative experiments of singlet oxygen

Dihydroporphine E 6 zinc compound photosensitizer of the present invention can produce singlet oxygen under illumination, the singlet oxygen output of itself and existing photosensitizers is as shown in table 2.

Table 2

Photosensitizers Photosensitiser	Output f Δ
		Protoporphyrin Protoporphyrin dme	0.57
Haematoporphyrin Haemotoporphyrin	0.65
		Uroporphyrin IIIUroporphyrinIII	0.52
TPP	0.63
		Four (3-hydroxy phenyl) porphyrin m-THPP	0.46
Bacteriochlorophyll Bacteriochlorophyll a	0.32
		Porphycene	0.30
Chlorin e 6 Chlorin e6	0.32
		Zinc (II) chlorin e 6 Zinc (II) Chlorin e6	0.46

Dihydroporphine E 6 zinc compound photosensitizer demonstrates luminous because of singlet oxygen.It has two kinds of luminous forms, but all more weak in the aqueous solution.Monomolecular luminous zone is positioned at 1270mn.

¹O2-> ³O2+hv(1270nm)

But bimolecular can interact and be luminous in higher energy range

2 ¹O2->2 ³O2+hv(634nm；704nm)

Claims (1)

1, a kind of preparation method of Dihydroporphine E 6 zinc compound photosensitizer is characterized in that comprising the steps:

(1) the not stopping reaction of chlorin E6: sodium bicarbonate is dissolved in the deionized water, adds chlorin E6, slowly stir; Polyvinylpyrrolidone is dissolved in the deionized water, then it is joined in the chlorin E6 test solution, slowly stir down, obtain unstabilized chlorin E6 at 40 ℃;

(2) Dihydroporphine E 6 zinc compound is synthetic: zinc nitrate hexahydrate is dissolved in the deionized water, under the room temperature zinc nitrate hexahydrate solution is joined among the unstabilized chlorin E6, and slowly stir synthesizing dihydro porphines E6 zinc compound;

(3) stabilizing step of Dihydroporphine E 6 zinc compound: polyvinylpyrrolidone is dissolved in the deionized water, adds Dihydroporphine E 6 zinc compound again, slowly stir down at 60 ℃.