CN104892724B - Fischer carbene compounds stable small numerator modified miscellaneous N of dipeptides and preparation method thereof - Google Patents
Fischer carbene compounds stable small numerator modified miscellaneous N of dipeptides and preparation method thereof Download PDFInfo
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Abstract
The invention discloses Fischer carbene compounds stable a kind of small numerator modified miscellaneous N of dipeptides and its preparation method and application, the structural formula of the compound is
Description
Technical field
The invention belongs to synthesize pharmaceutical technology field, and in particular to a kind of dipeptides with light-operated release carbon monoxide performance
Fischer carbene compounds stable small numerator modified miscellaneous N, and the compound irrigate in anti-ischemic and damage aspect again
Using.
Background technology
CO is a kind of hypertoxic gas, and the injury to human body is very big, but Recent study shows, CO is a kind of non-as NO
It is often important and the molecule of bio signal can be discharged.In fact, can be continuous during blood in human body in heme oxygenase degraded ferroheme
Produce a small amount of CO, it show cytoprotection, anti-inflammatory, promote vasodilation, prevent ischemical reperfusion injury and its
His therapeutic efficiency.
In order to develop the effective form of CO treatments, a series of carbon monoxide-releasing molecules (CO Releasing
Molecules, CO-RMs) developed.The compound for being determined discharging CO under field conditions (factors) has five kinds.First
It is aldehydes to plant：Preliminary data determines their potential source biomolecule activity, but its CO rate of release is slow, and its toxicology
Matter hampers its development as CO-RMs；Second is oxalic acid class：The speed for discharging CO is too slow；The third is the carbonic acid of boron
Ester：It is a kind of well-known CO releasers, however, the chemical conversion scope of these compounds is limited, and is unsatisfactory for compound
The appropriate pharmacological feature that should have；4th kind be silicon carbonic ester：It is organic solvent of this kind of compound only in heat, strong
In the presence of catalyst, CO can be just discharged, its high temperature, the requirement of strong Basal activity show that they are incompatible with biosystem；
5th kind is metal carbonyl：With stablizing, controlled release and the advantage of targeting delivery are realized, is the great medical science of a class
The CO-RMs of application prospect.
In recent years, with the development of science and technology, the organ transplant such as dermatoplasty, kidney transplant, liver transplant of burn is slow
The problem of can not being captured before slow solution, and larger progress is achieved, but remain such-and-such technology and ask
Topic.Body tissue organ eubolism, the maintenance of function, depend on good blood circulation.Cause local group of a variety of causes
The ischemic of organ is knitted, histocyte is occurred ischemia injury, but, it was also found that one in animal experiment and clinical observation
Recover under fixed condition after blood reperfusion, Some Animals or Patient cells' functional metabolism obstacle and structure destruction do not mitigate anti-not only
And aggravate, thus the phenomenon referred to as ischemical reperfusion injury that ischemia injury after this blood reperfusion is further aggravated.Lack
The mechanism of blood reperfusion injury mainly has：The effect of free radical, the effect of calcium overload, the effect of leucocyte, energy-rich phosphate
Compound shortage, the effect of Endothelin, the effect of angiotensinⅡ etc., and the oxidation of free radical is the most important thing.
The tissue damage that ischemic is drawn is the main cause of fatal disease, cardiac muscle stalk caused by such as coronary arteries hardening
Extremely, cerebral apoplexy etc..In ischemic disease rescue and therapeutic process, physicians are had found gradually, and the master of damage is caused to tissue
Want factor, be not ischemic in itself, but recover blood supply after, excessive free radical attacks this part and regains blood supply
Tissue in cell cause.Having many evidence explanations, only ischemic is also not enough to cause tissue damage, but in ischemic one
Recover just to damage during blood supply (i.e. Reperfu- sion) suddenly again after the section time.Traumatic shock, surgical operation, organ transplant,
During the blood circulation disorders such as burn, frostbite and thrombus, postischemic reperfusion damage can all occur.Caused during ischemic tissue's Reperfu- sion
Capilary and organa parenchymatosum damage mainly as caused by active oxygen radical, this is proven in a variety of organs.
There is the antioxidant reductase synthesis capability for removing free radical to occur obstacle in ischemic tissue, so as to exacerbate free radical to ischemic
The damage of Reperfu- sion tissue afterwards.The CO that carbon monoxide method for releasing molecule is discharged has the antioxidation for removing free radical, and
And there is CO the physiological action of anti-inflammation to further increase carbon monoxide-releasing molecules, particularly metal carbonyl anti-scarce
Courage and uprightness perfusion damages the effect of aspect again, but existing metal carbonyl is present that poorly water-soluble, heat endurance be poor, toxicity
Greatly, biocompatibility is low, carbon monoxide uncontrollable release the shortcomings of.
The content of the invention
The technical problems to be solved by the invention are that there is provided one kind for the shortcoming that overcomes existing metal carbonyl to exist
Thermally-stabilised preferable, toxicity is relatively low, and with preferably water-soluble and biocompatibility, can realize light-operated release carbon monoxide,
It is effectively applied to anti-ischemic and irrigates the stable Fischer carbene compounds of the small numerator modified miscellaneous N of the dipeptides damaged again, with
And the preparation method of the compound.
Solving the technical scheme that is used of above-mentioned technical problem is：The Fischer stable small numerator modified miscellaneous N of the dipeptides
The structural formula of carbene compound is as follows：
M represents Cr, Mo or W, R in formula1、R2、R3、R4Respective independent representative-H, alkyl ,-CnH2n-R5, appointing in benzyl
Meaning is a kind of, wherein R5Representative-H ,-OH ,-COOH ,-NH2、-SH、-SCH3In any one, n be 1~3 integer.
Above-mentioned R1、R2、R3、R4It is preferred that respective independent representative-H, C1~C4Any one in alkyl, benzyl.
The preparation method of Fischer the carbene compounds stable small numerator modified miscellaneous N of dipeptides of the present invention is as follows：
1st, in anhydrous and oxygen-free, under nitrogen protective condition, using absolute methanol as solvent, by the Fischer Cabbeens shown in formula 1,
Amino acid, weak base shown in formula 2 are reacted at room temperature 5~10 hours, and reduced pressure at room temperature rotary evaporation removes solvent, obtains shown in formula 3
Amino acid modified Fischer Cabbeens, reaction scheme is as follows：
2nd, under the conditions of anhydrous and oxygen-free, using dichloromethane as solvent, by the amino acid modified Fischer cards shown in formula 3
Guest and DMAP (DMAP), N, the amino acid methyl ester hydrochloride shown in N'- Dicyclohexylcarbodiimides (DCC), formula 4
Reacted 18~24 hours under condition of ice bath, using pillar layer separation, obtain the small numerator modified miscellaneous N stabilizations of dipeptides
Fischer carbene compounds, reaction scheme is as follows：
Above-mentioned Fischer Cabbeens and amino acid, weak base, DMAP, N, N'- Dicyclohexylcarbodiimides,
The mol ratio of amino acid methyl ester hydrochloride is 1:(1~1.2):(1~6):(0.2~0.3):(1.5~2):(1~1.2), it is described
Weak base be triethylamine, K2CO3Or Na2CO3。
Beneficial effects of the present invention are as follows：
The present invention first passes through amino acid and is coupled the amino acid modified Fischer Cabbeens of generation, Ran Hou with Fischer Cabbeens
Amino acid modified Fischer Cabbeens and amino acid methyl ester hydrochloride are coupled under catalyst action and generate stable amido link,
The Fischer carbene compounds that i.e. the small numerator modified N of dipeptides stablizes, it can be easily separated and purity is higher.
The present invention is modified Fischer Cabbeens by dipeptides small molecule, not only increases the water-soluble of Fischer Cabbeens
Property, biocompatibility, reduce the toxicity of molecule, and substantially increase the heat endurance of molecule.
Fischer the carbene compounds stable small numerator modified N of dipeptides of the present invention can be rapid under ultraviolet light
Carbon monoxide molecule is discharged, there is preferable response to light, and the compound molecular weight is smaller, the CO of carrying is more, each
Molecule can carry five CO, and CO release efficiencies are very high, can realize the controllable of carbon monoxide as light-operated carbon monoxide-releasing molecules
Release, is carbon monoxide-releasing molecules for the treatment of the antimicrobial antiphlogistic and ischemic tissue reperfusion injury of pathological tissues
Targeting delivery opens a new road.
Brief description of the drawings
Fig. 1 is the crystal pattern of the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 1.
Fig. 2 is the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 1 along a directions
One-dimensional stratiform figure.
Fig. 3 is the polyhedron of the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 1
Stacked micros vias figure.
Fig. 4 is the crystal pattern of the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 4.
Fig. 5 is the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 4 along a directions
One-dimensional stratiform figure.
Fig. 6 is the polyhedron of the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 4
The micropore figure of accumulation.
Fig. 7 is the thermogravimetric analysis figure of the Fischer Cabbeens of pentacarbonyl chromium.
Fig. 8 is the thermogravimetric of the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 1~7
Analysis chart.
Fig. 9 is the CO releases of myoglobins method test carbon monoxide-releasing molecules.
Figure 10 is the stable Fischer Cabbeens of the small numerator modified miscellaneous N of dipeptides prepared by 10umol/L embodiments 1~7
CO release dynamics figure of the compound in PBS.
Figure 11 is the stable Fischer Cabbeens of the small numerator modified miscellaneous N of dipeptides prepared by 20umol/L embodiments 1~7
CO release dynamics figure of the compound in PBS.
Figure 12 is that the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides prepared by embodiment 1 are protected in organ
Release dynamics figure in liquid storage.
Figure 13 is the survival condition after the left renal ischaemia different time Reperfu- sion of mouse.
Figure 14 be mouse tail vein inject respectively the compound of embodiment 1 and physiological saline row kidney lethal ischemic after 1 hour/
The survival condition of Reperfu- sion.
Figure 15 be mouse tail vein inject respectively the compound of embodiment 1 and physiological saline of inactivation after 1 hour row kidney it is lethal
The survival condition of property ischemia/reperfusion.
Embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
By taking Fischer the carbene compounds stable small numerator modified miscellaneous N of the following dipeptides of preparation structure formula as an example, it is made
Preparation Method is：
1st, under the conditions of anhydrous and oxygen-free, by the Fischer Cabbeens and 0.1501g of 0.5002g (2mmol) pentacarbonyl chromium
(2mmol) glycine is added in reaction bulb, is added anti-under 15mL absolute methanols and 0.3mL (2mmol) triethylamine, room temperature condition
Answer 5 hours, reduced pressure at room temperature rotary evaporation removes solvent, obtain the Fischer Cabbeens of glycine modification.
2nd, under the conditions of anhydrous and oxygen-free, 0.02510g is added in the Fischer Cabbeens of the glycine modification obtained to step 1
(2mmol) glycine methyl ester hydrochloride, 0.0611g (0.5mmol) DMAP (DMAP), 0.4124g (2mmol) two
Carbodicyclo hexylimide (DCC), 20mL anhydrous methylene chlorides, react 24 hours under condition of ice bath, after reaction terminates, with dichloro
Methane uses pillar layer separation product as eluant, eluent, and desciccate obtains the small numerator modified miscellaneous N of yellow solid dipeptides stable
Fischer carbene compounds, its yield is 77.6%, and structural characterization data are：IR(CH2Cl2, cm-1)：V(CO)=
2056cm-1、1927cm-1, V (COCH3)=1748cm-1, V (CONH)=1697cm-1、1518cm-1；1H-NMR (400MHz,
CDCl3) (Z/E=5/2) δ：9.51 (s, J=96.5Hz, 1H, NH), 6.37 (s, J=134.2Hz, 1H, NH), 4.40 (dd, J
=191.8,3.8Hz, 4H), 3.81 (s, 3H, CH3), 2.75 (d, J=52.3Hz, 3H, CH3)；13C-NMR (101MHz, CDCl3)
δ：217.70 (CO), 217.5 (CO), 169.74 (COCH3), 169.65 (COCH3), 166.80 (CONH), 165.71 (CONH),
53.30(OCH3), 52.83 (CH), 52.78 (CH), 48.37 (=CCH3), 45.36 (=CCH3), 41.42 (CH2), 37.23
(CH2)；ESI-Ms：Theoretical value [M]=364.00, measured value [M]=362.9494.
Using cryogenic fluid diffusion method, the yellow solid that 100mg is obtained is added in bottle, adds dichloromethane to just
Dissolving, is then slowly added to the n-hexane of 1-2 times of methylene chloride volume along walls of beaker, seals, and is put into refrigerator in -18 DEG C of standings
To there is yellow crystals precipitation.The crystal pattern of gained crystal is shown in Fig. 1, sees Fig. 2 along the one-dimensional stratiform figure in a directions, polyhedra packing is micro-
Hole pattern is shown in Fig. 3, and mono-crystalline structures and crystal data are shown in Table 1.
The mono-crystalline structures of table 1 and crystal data
Embodiment 2
By taking Fischer the carbene compounds stable small numerator modified miscellaneous N of the following dipeptides of preparation structure formula as an example, it is made
Preparation Method is：
In embodiment 1, glycine used is replaced with equimolar alanine, and other steps are same as Example 1, obtain
To the Fischer carbene compounds that the small numerator modified miscellaneous N of dipeptides is stable, its yield is 51%, and structural characterization data are：IR
(CH2Cl2, cm-1)：V (CO)=2056cm-1、1927cm-1, V (COCH3)=1748cm-1, V (CONH)=1697cm-1,
1518cm-1；1H-NMR (400MHz, CDCl3)δ：9.66 (s, 1H, NH), 6.35 (s, 1H), 5.30 (s, 1H, H), 4.59-4.47
(m, 1H, CH), 4.12 (d, J=3.7Hz, 2H, CH2), 3.80 (s, 3H, COCH3), 2.75 (d, J=19.5Hz, 3H, CH3),
1.56 (d, J=6.8Hz, 3H, CH3)；13C-NMR (101MHz, CDCl3)δ：217.61 (CO), 169.92 (COCH3), 169.80
(CONH), 55.24 (OCH3), 53.43 (CH), 52.76 (CH2), 41.39 (CH3), 35.96 (CH3), 19.73 (=CCH3)；
ESI-Ms：Theoretical value [M]=378.02, measured value [M+23]=401.0005.
Embodiment 3
By taking Fischer the carbene compounds stable small numerator modified miscellaneous N of the following dipeptides of preparation structure formula as an example, it is made
Preparation Method is：
In embodiment 1, glycine used is replaced with equimolar phenylalanine, and other steps are same as Example 1,
The stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides are obtained, its yield is 78%., and structural characterization data are：
IR(CH2Cl2, cm-1)：V (CO)=2055cm-1、1924cm-1, V (COCH3)=1749cm-1, V (CONH)=1692cm-1、
1515cm-1；1H-NMR (400MHz, CDCl3)δ：9.49 (s, 1H, NH), 7.40-7.29 (m, 3H, Ph), 7.20 (d, J=
6.7Hz, 2H, Ph), 6.11 (s, 1H, NH), 4.61 (d, J=6.1Hz, 1H, CH), 4.09 (d, J=4.1Hz, 2H, CH2),
3.79 (s, 3H, OCH3), 3.15 (ddd, J=21.8,13.7,6.9Hz, 2H, CH2), 2.32 (s, 3H, CH3)；13C-NMR
(101MHz, CDCl3)δ：221.81 (CO), 216.45 (CO), 168.58 (COCH3), 167.43 (CONH), 133.43 (Ph),
128.32 (Ph), 128.28 (Ph), 127.01 (Ph), 60.33 (OCH3), 51.76 (CH), 40.38 (=CCH3), 39.07
(CH2), 34.74 (CH2)；ESI-Ms：Theoretical value [M]=454.05, measured value [M+23]=477.0334.
Embodiment 4
By taking Fischer the carbene compounds stable small numerator modified miscellaneous N of the following dipeptides of preparation structure formula as an example, it is made
Preparation Method is：
In embodiment 1, glycine methyl ester hydrochloride used is replaced with equimolar leucine methyl ester hydrochloride, other
Step is same as Example 1, obtains the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides, and its yield is
73.14%, structural characterization data are：IR(CH2Cl2, cm-1)：V (CO)=2055cm-1、1924cm-1, V (COCH3)=
1743cm-1, V (CONH)=1693cm-1、1516cm-1；1H-NMR (400MHz, CDCl3) (Z/E=7/1) δ：9.44 (d, J=
100.3Hz, 1H, NH), 6.14 (dd, J=112.1,7.3Hz, 1H, NH), 4.61 (ddd, J=43.8,11.8,6.5Hz, 1H,
CH), 4.17-3.86 (m, 2H, CH2), 3.71 (s, 3H, OCH3), 2.67 (d, J=50.4Hz, 3H, CH3), 1.75-1.61 (m,
2H, CH2), 1.61-1.42 (m, 1H, CH), 0.90 (t, J=5.6Hz, 6H, C (CH3)2)；13C-NMR (101MHz, CDCl3)δ：
216.70 (CO), 216.54 (CO), 172.14 (COCH3), 172.05 (COCH3), 164.47 (CONH), 164.35 (CONH),
51.71(OCH3), 51.67 (OCH3), 50.21 (CH2), 50.17 (CH2), 47.49 (=CCH3), 40.54 (=CCH3), 36.23
(CH), 23.93 (CH2), 23.85 (CH2), 21.67 (CH3), 21.63 (CH3), 20.90 (CH3), 20.82 (CH3)；ESI-Ms：
Theoretical value [M]=420.06, measured value [M+23]=443.0494.
Embodiment 5
By taking Fischer the carbene compounds stable small numerator modified miscellaneous N of the following dipeptides of preparation structure formula as an example, it is made
Preparation Method is：
In embodiment 1, glycine used is replaced with equimolar alanine, and glycine methyl ester hydrochloride used is used
Equimolar leucine methyl ester hydrochloride is replaced, and other steps are same as Example 1, obtain the small numerator modified miscellaneous N of dipeptides steady
Fixed Fischer carbene compounds, its yield is 50%, and structural characterization data are：IR(CH2Cl2, cm-1)：V (CO)=
2054cm-1、1924cm-1, V (COCH3)=1743cm-1, V (CONH)=1693cm-1、1516cm-1；1H NMR (400MHz,
CDCl3)δ：9.30 (d, J=126.6Hz, 1H), 5.96 (dd, J=80.7,8.2Hz, 1H), 4.73-4.23 (m, 2H), 3.70
(s, 3H), 2.68 (t, J=6.8Hz, 3H), 1.69-1.54 (m, 3H), 1.48 (dd, J=10.1,5.9Hz, 3H), 1.19 (s,
2H), 0.89 (dt, J=12.9,6.4Hz, 6H)；13C NMR (101MHz, CDCl3)δ：216.57 (CO), 171.86 (COCH3),
168.36 (CONH), 54.46 (OCH3), 51.62 (CH), 50.15 (CH), 40.37 (=CCH3), 35.11 (CH2), 23.94
(CH2), 21.65 (CH3), 20.88 (CH3), 18.61 (CH3)；ESI-Ms：Theoretical value [M]=434.08, measured value [M+23]=
457.0636。
Using cryogenic fluid diffusion method, Fischer the Cabbeens stable small numerator modified miscellaneous N of dipeptides that 100mg is obtained
Compound is added in bottle, is added dichloromethane to just dissolving, is then slowly added to methylene chloride volume 1~2 along walls of beaker
Times n-hexane, sealing is put into refrigerator and stood at -18 DEG C to there is yellow crystals precipitation.The crystal pattern of gained crystal is shown in Fig. 4,
See Fig. 5 along the one-dimensional stratiform figure in a directions, polyhedra packing micropore figure is shown in Fig. 6, and mono-crystalline structures and crystal data are shown in Table 2.
The mono-crystalline structures of table 2 and crystal data
Embodiment 6
By taking Fischer the carbene compounds stable small numerator modified miscellaneous N of the following dipeptides of preparation structure formula as an example, it is made
Preparation Method is：
In embodiment 1, glycine used is replaced with equimolar leucine, and glycine methyl ester hydrochloride used is used
Equimolar phenylalanine methyl ester hydrochloride is replaced, and other steps are same as Example 1, obtain the small numerator modified miscellaneous N of dipeptides
Stable Fischer carbene compounds, its yield is 48%, and structural characterization data are：IR(CH2Cl2, cm-1)：V (CO)=
2055cm-1、1925cm-1, V (COCH3)=1742cm-1, V (CONH)=1686cm-1、1513cm-1；1H-NMR (400MHz,
CDCl3)δ：9.05 (d, J=6.3Hz, 1H, NH), 7.30-7.19 (m, 3H, Ph), 6.98 (dd, J=7.6,1.6Hz, 2H,
Ph), 5.91 (d, J=7.7Hz, 1H, NH), 4.82 (dt, J=7.6,6.0Hz, 1H, CH), 4.20 (dd, J=14.0,7.7Hz,
1H, CH), 3.65-3.75 (s, 3H, CH3), 3.20 (dd, J=14.1,5.7Hz, 1H, CH2), 3.02 (dd, J=14.1,
6.2Hz, 1H, CH2), 2.52 (s, 3H, CH3), 1.61-1.48 (m, 3H, CH-CH2), 0.86 (dd, J=16.0,6.2Hz, 6H, C
(CH3)2)；13C-NMR (101MHz, CDCl3)δ：216.57 (CO), 170.55 (COCH3), 167.62 (CONH), 134.15
(Ph), 128.09 (Ph), 127.78 (Ph), 126.52 (Ph), 66.94 (CH), 58.37 (OCH3), 52.09 (CH), 51.72
(CH), 40.90 (=CCH3), 36.53 (CH), 34.98 (CH2), 23.67 (CH2), 21.55 (CH3), 21.07 (CH3)；ESI-
Ms：Theoretical value [M]=510.11, measured value [M+23]=533.0947.
Embodiment 7
By taking Fischer the carbene compounds stable small numerator modified miscellaneous N of the following dipeptides of preparation structure formula as an example, it is made
Preparation Method is：
In embodiment 1, glycine used is replaced with equimolar phenylalanine, glycine methyl ester hydrochloride used
Replaced with equimolar phenylalanine methyl ester hydrochloride, other steps are same as Example 1, obtain small numerator modified miscellaneous of dipeptides
Fischer carbene compounds stable N, its yield is 40%, and structural characterization data are：IR(CH2Cl2, cm-1)：V (CO)=
2054cm-1、1925cm-1, V (COCH3)=1743cm-1, V (CONH)=1687cm-1、1511cm-1；1H-NMR (400MHz,
CDCl3)δ：9.27 (d, J=7.5Hz, 1H, NH), 7.30-7.22 (m, 3H, Ph), 7.22-7.16 (m, 3H, Ph), 7.09 (dd,
J=7.4,1.7Hz, 2H, Ph), 6.90 (dd, J=6.5,2.9Hz, 2H, Ph), 5.86 (d, J=7.5Hz, 1H, CH), 4.85-
4.74 (m, 1H, NH), 4.38 (td, J=8.1,5.7Hz, 1H, CH), 3.71 (s, 3H), 3.13 (dd, J=14.0,5.8Hz,
1H, CH), 3.09-2.92 (m, 3H, OCH3), 2.21 (d, J=0.5Hz, 3H, CH3)；13C-NMR (101MHz, CDCl3)δ：
216.38 (CO), 170.21 (COCH3), 166.67 (CONH), 133.96 (Ph), 133.30 (Ph), 128.34 (Ph), 128.25
(Ph), 128.03 (Ph), 127.80 (Ph), 126.97 (Ph), 126.52 (Ph), 60.46 (CH), 52.37 (OCH3), 51.70
(CH2), 38.61 (=CCH3), 36.43 (CH2), 34.81 (CH2)；ESI-Ms：Theoretical value [M]=544.09, measured value [M+
23]=567.0784.
In order to prove beneficial effects of the present invention, the small numerator modified miscellaneous N of dipeptides that inventor prepares to embodiment 1~7
Stable Fischer carbene compounds have carried out various performance tests, and specific experiment is as follows：
1st, heat stability testing
Small point of the dipeptides that inventor is prepared using thermogravimetric analyzer to the Fischer Cabbeens and embodiment 1~7 of pentacarbonyl chromium
The thermally-stabilised of Fischer carbene compounds that the miscellaneous N of son modification is stable is tested, and as a result sees Fig. 7~8, from Fig. 7~8,
The Fischer Cabbeens of pentacarbonyl chromium start to decompose at 45 DEG C, and a stage fast decoupled is finished, and illustrates pentacarbonyl chromium
The heat endurance of Fischer Cabbeens is poor, and Fischer the cards stable small numerator modified miscellaneous N of dipeptides prepared by embodiment 1~7
Guest's compound starts to decompose (the wherein He of embodiment 1 respectively in 140 DEG C, 152 DEG C, 148 DEG C, 139 DEG C, 150 DEG C, 129 DEG C, 155 DEG C
Fischer the carbene compounds stable small numerator modified miscellaneous N of 4 dipeptides prepared are in a small amount of weightless the waving for solvent most started
Hair), and 2~3 stage decompositions, heat endurance is significantly improved compared with the Fischer Cabbeens of raw material pentacarbonyl chromium.
2nd, carbon monoxide release performance is tested
(1) release performance experiments of the myoglobins method test CO-RM in PBS
Weigh 10mg myoglobins to be added in 5mL volumetric flasks, then with the phosphate buffer solution (PBS) that pH value is 7.4
Constant volume, is configured to myoglobin solution.1mL myoglobin solutions are drawn with liquid-transfering gun to add in cuvette, then add 25mg
Na2S2O4, at ambient temperature, use ultraviolet-visible spectrophotometer test be reduced myoglobins in wavelength for 500
Ultra-violet absorption spectrum in the range of~600nm, is then passed through CO gases until solution face into the myoglobin solution being reduced
Discoloration is red, and it is the ultra-violet absorption spectrum in the range of 500~600nm to test it in wavelength.As a result Fig. 9 is seen.
The stable Fischer Cabbeens of the small numerator modified miscellaneous N of dipeptides of the preparation of 0.0012g embodiments 1~7 are weighed respectively
Compound, adds 120uL DMSO dissolvings and obtains mother liquor, obtained mother liquor then is configured into 10umol/L and 20umol/ with DMSO
L standard liquid.Take 5uL standard liquids to be added in 1mL myoglobin solutions, add 25mg Na2S2O4Mix, on cuvette
Side adds the sealing of 2 dropstone wax oils, then uses ultraviolet-visible spectrophotometer test in the range of wavelength is 500~600nm
Ultra-violet absorption spectrum.From Figure 10 and 11, sweep test 6 times (1 1min), chemical combination under conditions of initially not light stimulus
The thing almost release without CO；Then 2s is irradiated under 20% transmitance using 365nm uviol lamp, compound is sharper
The irradiation of ultraviolet light is responded, and discharges CO rapidly, after a period of time, compound hardly discharges CO；Continue to use
365nm uviol lamp irradiates 2s under 50% transmitance, and compound discharges rapidly CO again, after a period of time, compound
Hardly discharge CO；2s is irradiated under 100% transmitance using 365nm uviol lamp again, compound continues to discharge rapidly
CO, the uviol lamp that compound hardly discharges after CO again using 365nm irradiates 2s under 100% transmitance；Finally use
365nm uviol lamp irradiates 60s in 100% transmitance, and there is no significant change for compound spectra.
From the above experiments, it was found that Fischer the cards stable small numerator modified miscellaneous N of the dipeptides of the preparation of embodiment 1~7
Guest's compound has an extraordinary optical Response, and with preferable CO release performances, the compound of each molecule discharge compared with
Many CO, almost five CO discharge completely.
(2) release performance experiments of the myoglobins method test CO-RM in organ preservative fluid
The PBS that above-mentioned Lactoferrin method is tested in release performance experiments of the CO-RM in PBS is protected with pH=7.1 organ
Liquid storage is replaced, and Fischer the carbene compounds stable small numerator modified miscellaneous N of dipeptides prepared by testing example 1 are protected in organ
Release performance in liquid storage, concrete operations are identical with experiment (1).As seen from Figure 12, the dipeptides that prepared by embodiment 1 is small numerator modified
The stable Fischer carbene compounds of miscellaneous N there is extraordinary optical Response in organ preservative fluid, and with preferable CO
Release performance, the compound of each molecule discharge more CO, and almost five CO discharge completely.
3rd, anti-ischemic irrigates injury experiment again
(1) kidney lethal ischemia/reperfusion model is set up
Male BALB/c (H-2d) mouse (8~10 weeks, 20~25g), the yellow Jackets of intraperitoneal injection 1% (6mL/kg)
Anesthesia.Left renal ischaemia 30,40 or 50 minutes (5/group), then remove blood vessel clip and cut off contralateral kidney.Observe dead after Reperfu- sion
Situation is died, kidney lethal ischemia/reperfusion model is set up.
Observed 15 days after removing blood vessel clip to the left renal ischaemia of mouse 30, after 40 or 50 minutes respectively and cutting off contralateral kidney,
As a result show 50 minutes group mouse death rate highests, postoperative 1 day dead 4, postoperative 2 days dead 1；And 30 minutes groups and 40 points
Clock group mouse survive to after 15 days (see Figure 13), so determine ischemic after 50 minutes Reperfu- sion be Mouse Kidney lethal ischemic/
Re-perfusion model.
(2) toxic actions of the DMSO for mouse is excluded
Tail vein injection various concentrations DMSO (5%, 10%, 20%, 30%, 200 μ L/ are only) and physiological saline (20mg/
Kg), other processing, influences of the observation DMSO for mouse are not made.As a result do not find that mouse has any adverse reaction, feed water inlet
As usual, the state of mind is good, and death did not occurred in 15 days.Proof DMSO is in above-mentioned concentration to mouse without overt toxicity.
(3) compound of embodiment 1 is examined for the protective effect of Mouse Kidney lethal ischemia/reperfusion
Fischer the Cabbeens stable small numerator modified miscellaneous N of dipeptides that concentration according to 20mg/kg prepares embodiment 1
Compound is dissolved in DMSO, by solution normal saline dilution to 10%, Tail Vein injection Mouse, to mouse row kidney after 1 hour
The processing of lethal ischemia/reperfusion, observes dead mouse situation.Control group tail vein injection saline, row kidney is caused after 1 hour
Dead property ischemia/reperfusion processing, observes dead mouse situation.
Make discovery from observation, mouse tail vein injected the compound of embodiment 1 and physiological saline (control group) after 1 hour respectively
Row kidney lethal ischemia/reperfusion, every group 7, only the 4th day and the 6th day each dead 1, remaining 5 survivals are to after 15 days
(see Figure 14).
(4) compound of embodiment 1 of inactivation is examined for the effect of Mouse Kidney lethal ischemia/reperfusion
According to the Fischer Cabbeens that the small numerator modified miscellaneous N of the dipeptides of the 20mg/kg preparation of concentration embodiment 1 is stable
Compound is dissolved in DMSO, and room temperature is placed 24 hours, the CO in the compound of embodiment 1 is discharged completely, the embodiment 1 inactivated
Compound.The compound of embodiment 1 of inactivation is dissolved in DMSO according to 20mg/kg concentration, solution 10% is diluted to, tail is quiet
Arteries and veins injects 5 mouse, to the processing of mouse row kidney lethal ischemia/reperfusion after 1 hour, observes dead mouse situation.Control group
Tail vein injection saline, row kidney lethal ischemia/reperfusion is handled after 1 hour, observes dead mouse situation.
Make discovery from observation, 1 compound of embodiment row kidney lethal ischemic after 1 hour of mouse tail vein injection inactivation/
Reperfu- sion, find 5 mouse respectively at the 2nd, death in 3,4,7,9 days；Control group mice is the 1st, 2 days all death (see figure
15)。
Claims (2)
1. Fischer the carbene compounds stable a kind of small numerator modified miscellaneous N of dipeptides, it is characterised in that the structure of the compound
Formula is as follows：
M represents Cr or Mo, R in formula1、R2、R3、R4Respective independent representative-H, C1~C4Any one in alkyl, benzyl.
2. the preparation method of Fischer the carbene compounds stable small numerator modified miscellaneous N of dipeptides described in claim 1, its
It is characterised by that it is made up of following step：
(1) under the conditions of anhydrous and oxygen-free, using methanol as solvent, by the Fischer Cabbeens shown in formula 1, the amino acid shown in formula 2,
Weak base is reacted at room temperature 5~10 hours, and reduced pressure at room temperature rotary evaporation removes solvent, obtains amino acid modified shown in formula 3
Fischer Cabbeens；
(2) under the conditions of anhydrous and oxygen-free, using dichloromethane as solvent, by the amino acid modified Fischer Cabbeens shown in formula 3 with
DMAP, N, the amino acid methyl ester hydrochloride shown in N'- Dicyclohexylcarbodiimides, formula 4 are anti-under condition of ice bath
Answer 18~24 hours, pillar layer separation, obtain the stable Fischer carbene compounds of the small numerator modified miscellaneous N of dipeptides；
Above-mentioned Fischer Cabbeens and amino acid, weak base, DMAP, N, N'- Dicyclohexylcarbodiimides, amino
The mol ratio of acid methyl ester hydrochloride salt is 1:(1~1.2):(1~6):(0.2~0.3):(1.5~2):(1~1.2), described is weak
Alkali is triethylamine, K2CO3Or Na2CO3；M represents R in Cr or Mo, formula 2 and formula 3 in formula 1 and formula 31、R2Respective independent representative-H,
C1~C4Any one in alkyl, benzyl, R in formula 43、R4Respective independent representative-H, C1~C4It is any in alkyl, benzyl
It is a kind of.
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