Cyclic RGD peptide and method for preparing the same

Abstract

A method for preparing a cyclopeptide and a cyclopeptide preparing by the method are disclosed. The method includes the following steps: (A) providing compounds represented by the following formulas (I-1) and (I-2): ##STR00001## wherein, G, R.sub.a, R.sub.b, R.sub.c, R.sub.d, and R.sub.e are defined in the specification; (B) performing a reaction between the compounds of formulas (I-1) and (I-2) to obtain a compound represented by the following formula (I-3): ##STR00002##
and (C) performing a cyclization reaction of the compound of formula (I-3) with a catalyst of formula (II) and deprotection to obtain a compound represented by the following formula (III): ##STR00003## wherein, G′, Q, M, L.sup.1, L.sup.2, m, y, and z are defined in the specification.

Claims

1. A method for preparing a cyclopeptide, comprising the following steps: (A) providing compounds represented by the following formulas (I-1) and (I-2): ##STR00032## wherein, R.sub.a, R.sub.b and R.sub.e are each independently a protection group; R.sub.c and R.sub.d are each independently alkyl, cycloalkyl, aryl or heteroaryl; G is H, or O-t-Bu; and R.sub.1 is ##STR00033## in which R.sub.2 and R.sub.3 are each independently H or C.sub.1-6 alkyl; X is O, S, CH.sub.2, or N—R.sub.4, in which R.sub.4 is H, C.sub.1-6 alkyl, (CH.sub.2CH.sub.2O).sub.nH, —C(═O)—C.sub.1-15 alkyl, —C(═O)CH.sub.2(OC.sub.2H.sub.4).sub.nOR′ or C(═O)(C.sub.2H.sub.4).sub.2C(═O)O(C.sub.2H.sub.4O).sub.nR′, in which n=1-3 and R′ is H or CH.sub.3; (B) performing a reaction between the compounds of formulas (I-1) and (I-2) to obtain a compound represented by the following formula (I-3): ##STR00034## and (C) performing a cyclization reaction of the compound of formula (I-3) with a catalyst of formula (II) and deprotection to obtain a compound represented by the following formula (III): ##STR00035##
M(O).sub.mL.sup.1.sub.yL.sup.2.sub.z  (II) wherein G′ is H or OH; Q is halogen, OC(O)CF.sub.3 or OC(O)CH.sub.3; M is a metal selected from the group consisting of IVB, VB, VIB and actinide groups; L.sup.1 and L.sup.2 respectively is a ligand; m and y are integers greater than or equal to 1; and z is an integer greater than or equal to 0.

2. The method of claim 1, wherein L.sup.1 is selected from the group consisting of Cl, OTf, OTs, NTf.sub.2, halogen, RC(O)CH.sub.2C(O)R, OAc, OC(O)CF.sub.3, OEt, O-iPr and O-t-butyl, in which R is alkyl.

3. The method of claim 1, wherein L.sup.2 is selected from the group consisting of Cl, H.sub.2O, CH.sub.3OH, EtOH, THF, CH.sub.3CN, and ##STR00036##

4. The method of claim 1, wherein R.sub.a and R.sub.e are fluorenylmethyloxycarbonyl, and R.sub.b is 2,3,6-trimethyl-4-methoxylbenzenesulphonyl.

5. The method of claim 1, wherein M is a group IVB transition element, m is 1 and y is 2.

6. The method of claim 1, wherein M is a group VB transition element, m is 1 and y is 2 or 3.

7. The method of claim 1, wherein M is a group VIB transition element, m is 1 and y is 4.

8. The method of claim 1, wherein M is a group VIB transition element, m is 2 and y is 2.

9. The method of claim 1, wherein M is selected from the actinide group, m is 2 and y is 2.

10. The method of claim 1, wherein the catalyst of formula (II) is MoO.sub.2Cl.sub.2, V(O)Cl.sub.2, V(O)(OAc).sub.2, V(O)(O.sub.2CCF.sub.3).sub.2, Ti(O)(acac).sub.2, Zr(O)Cl.sub.2, Hf(O)Cl.sub.2, Nb(O)Cl.sub.2, MoO.sub.2(acac).sub.2, V(O)(OTs).sub.2, VO(OTf).sub.2, or V(O)(NTf.sub.2).sub.2.

11. The method of claim 1, wherein z is 0.

12. The method of claim 1, wherein the compound of formula (III) is any one of formulas (III-1) to (III-5): ##STR00037## ##STR00038## wherein R.sub.5 is C.sub.1-15 alkyl.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the relationship between the inhibition rate and concentration of cyclopeptide (III-3).

(2) FIG. 2 is a graph showing the relationship between the inhibition rate and concentration of cyclopeptide (III-1).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(3) The present disclosure has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present disclosure are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

(4) The cyclopeptide of one preferred embodiment of the present disclosure can be prepared as follows.

(5) ##STR00022##

(6) To a solution of Fmoc-Gly-OH (5.866 g, 20 mmol, 1.0 equiv) in CH.sub.3CN (200 mL) was added catalyst ZrOCl.sub.2 (5 mol %), N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide HCl (EDCI.HCl) (4.970 g, 26 mmol, 1.3 equiv) and N-Hydroxysuccinimide (NHS) (2.53 g, 22 mmol, 1.1 equiv) at room temperature under N.sub.2 atmosphere and the reaction was monitored by TLC analysis. The reaction was stirred at room temperature for 4 h till the starting Fmoc-Gly-OH was totally consumed and cooled to 0° C. A solution of H-Asp(O.sup.tBu)-OH (3.98 g, 21 mmol, 1.05 equiv) and NaHCO.sub.3 (1.77 g, 21 mmol, 1.05 equiv) in 100 mL H.sub.2O was added to the above solution via syringe at room temperature. The reaction mixture was stirred at room temperature for 16 h. Solvent was evaporated, and the remaining residue was acidified to pH 3.2-3.4 with diluted aqueous HCl (0.1 N) and the white solid was precipitated out from the aqueous solution. The white solid was filtered and washed with H.sub.2O and the crude product was recrystallized in 60% aqueous ethanol to afford Fmoc-Gly-Asp(O.sup.tBu)-OH as a white solid (8.521 g, 91% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.74 (d, 2H, J=7.6 Hz), 7.56 (d, 2H, J=5.0 Hz), 7.36 (d, 3H, J=7.5 Hz), 7.28 (d, 2H, J=7.4 Hz), 5.83 (s, 1H), 4.83 (q, 1H, J=4.5 Hz), 4.34 (q, 2H, J=6.5 Hz), 4.18 (t, 2H, J=6.5 Hz), 4.02 (d, 1H, J=16 Hz), 3.88 (d, 1H, J=16.6 Hz), 2.94 (dd, 1H, J=16.7, 3.1 Hz), 2.76 (dd, 1H, J=16.8, 4.2 Hz), 1.38 (s, 9H); HRMS (ESI), calculated for C.sub.25H.sub.28N.sub.2NaO.sub.7 ([M+Na]+): 491.1794, found: 491.1797.

(7) ##STR00023##

(8) A dry microwave vial was added Fmoc-Pip(Boc)-OH (2.33 g, 5 mmol, 1.0 equiv), H-Arg(Mtr)-OMe (2.2 g, 5.5 mmol, 1.0 equiv), catalyst ZrOCl.sub.2 (5 mol %), and N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate (TCFH) (1.995 g, 6 mmol, 1.20 equiv) in dry CH.sub.3CN (2 mL/mmol) under argon. Subsequently, 1-Methyl imidazole (NMI) (0.837 mL, 10.5 nmol, 2.1 equiv) was added and the vial was sealed and heated in an oil bath at 70° C. for 12 h (CAUTION: Heating CH.sub.3CN causes pressure increase in the reactor). The reaction mixture was cooled to room temperature and diluted with water, saturated aqueous NaHCO.sub.3 and extracted with ethyl acetate. The collected organic phases were combined and washed with brine, dried over Na.sub.2SO.sub.4 and concentrated.

(9) The crude product was purified by flash column chromatography under the conditions indicated give Fmoc-Pip(Boc)-Arg(Mtr)-OMe as a light yellow solid (3.648 g, 86% yield). .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.73 (d, 2H, J=7.5 Hz), 7.51 (t, 2H, J=3.0 Hz), 7.37 (t, 3H, J=7.0 Hz), 7.26-7.05 (m, 10H), 7.05 (br, 1H), 6.52 (s, 1H), 5.94 (br, 1H), 4.44-4.39 (m, 2H), 4.31 (q, 1H, J=5.2 Hz), 4.13 (t, 1H, J=6.5 Hz), 3.77 (s, 3H), 3.82-3.77 (m, 2H), 3.59 (s, 3H), 3.59 (m, 2H), 3.27-3.10 (m, 3H), 3.08-2.76 (m, 1H), 2.78-2.61 (m, 1H), 3.77 (s, 3 h), 2.56 (s, 3H), 2.00-1.84 (m, 4H), 1.82 (t, 1H, J=6.3 Hz), 1.77-1.54 (m, 2H), 1.40 (s, 9H); HRMS (ESI), calculated for C.sub.43H.sub.56N.sub.6NaO.sub.10S ([M+Na]+): 871.3676, found: 871.3672.

(10) ##STR00024##

(11) To a MeOH solution (100 mL) of Fmoc-Pip(Boc)-Arg(Mtr)-OMe (2.24 g, 2.64 mmol, 1.0 equiv) was added 20 equivalents of piperidine. The reaction mixture was then stirred until complete consumption of the Fmoc-Pip(Boc)-Arg(Mtr)-OMe as confirmed by TLC (1 h) and the reaction mixture was then evaporated to dryness under reduced pressure at room temperature. A piperidine adduct of dibenzofulvene was washed out with hexane and the resulting H-Pip(Boc)-Arg(Mtr)-OMe was dried in vacuo and used directly for the next step. To a dry microwave vial was added Fmoc-D-Phe-OH (1.123 g, 2.9 mmol, 1.1 equiv), H-Pip(Boc)-Arg(Mtr)-OMe (1.0 equiv), catalyst ZrOCl.sub.2 (5 mol %), and TCFH (1.02 g, 3.17 mmol, 1.2 equiv) in dry CH.sub.3CN (2 mL/mmol) under argon. Subsequently, 1-Methyl imidazole (NMI) (0.443 mL, 5.55 mmol, 2.1 equiv) was added and the vial was sealed and then heated in an oil bath at 70° C. for 12 h (CAUTION: Heating CH.sub.3CN causes pressure increase in the reactor). The reaction mixture was cooled to room temperature and diluted with water, saturated aqueous NaHCO.sub.3 and extracted with ethyl acetate. The collected organic phases were combined and washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash column chromatography under the conditions indicated give Fmoc-Phe-Pip(Boc)-Arg(Mtr)-OMe as a light yellow solid (1.865 g, 71% yield relative to the amount of Fmoc-Pip(Boc)-Arg(Mtr)-OMe). .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.75 (d, 2H, J=7.0 Hz), 7.56 (t, 2H, J=7.5 Hz), 7.39 (t, 3H, J=7.4 Hz), 7.30-7.17 (m, 10H), 6.51 (s, 1H), 6.05 (br, 1H), 4.46-4.42 (m, 2H), 4.42-4.23 (m, 2H), 4.20 (t, 1H, J=6.7 Hz), 3.88-3.78 (m, 2H), 3.80 (s, 3H), 3.66 (s, 3H), 3.38-3.34 (m, 1H), 3.19-2.97 (m, 3H), 32.92-2.76 (m, 2H), 2.75-2.61 (m, 2H), 2.67 (s, 3H), 2.57 (s, 3H), 2.11 (s, 3H), 1.96-1.77 (m, 4H), 1.76-1.67 (m, 1H), 1.67-1.54 (m, 1H), 1.42 (t, 1H, J=6.3 Hz), 1.37 (s, 9H); HRMS (ESI), calculated for C.sub.52H.sub.65N.sub.7NaO.sub.11S ([M+Na]+): 1018.4361, found: 1018.4356.

(12) ##STR00025##

(13) To a stirred MeOH solution (10 mL) of Fmoc-D-Phe-Pip(Boc)-Arg(Mtr)-OMe (996 mg, 1.0 mmol, 1.0 equiv) was added 20 equivalents of piperidine, and the reaction mixture was stirred until complete consumption of the Fmoc-D-Phe-Pip(Boc)-Arg(Mtr)-OMe as confirmed by TLC (1 h) and the reaction mixture was then evaporated to dryness under a reduced pressure at room temperature. A piperidine adduct of dibenzofulvene was washed out with hexane and the resulting H-D-Phe-Pip(Boc)-Arg(Mtr)-OMe was dried in vacuo and used directly for the next step. To a dry vial was added Fmoc-Gly-Asp(O.sup.tBu)-OH (535 mg, 1.1 mmol, 1.1 equiv), H-D-Phe-Pip(Boc)-Arg(Mtr)-OMe (1.0 equiv), catalyst ZrOCl.sub.2 (5 mol %), and 1-hydroxy-7-azabenzotriazole (HOAt) (1.2 equiv) in dry CH.sub.3CN (2 mL/mmol) under argon and stirred at room temperature for 10 minutes. EDCI.HCl (229 mg, 1.2 mmol, 1.2 equiv) was then added in four portions followed by the addition of N-methylmorpholine (NMM) (167 μL, 2.1 mmol, 2.1 equiv) and the reaction mixture was stirred at room temperature for 12 hours. The reaction was diluted with ethyl acetate and washed with diluted HCl (0.1 N), water, saturated aqueous NaHCO.sub.3 and extracted with ethyl acetate. The collected organic phase was dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash column chromatography under the conditions indicated give Fmoc-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr)-OMe as a light yellow solid (1.016 g, 83% yield relative to the amount of Fmoc-D-Phe-Pip(Boc)-Arg(Mtr)-OMe). .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.74 (d, 2H, J=7.0 Hz), 7.55 (t, 2H, J=7.5 Hz), 7.37 (t, 3H, J=7.5 Hz), 7.30-7.17 (m, 10H), 6.52 (s, 1H), 6.05 (br, 1H), 4.61-4.41 (m, 2H), 4.41-4.21 (m, 2H), 4.21 (t, 1H, J=6.7 Hz), 3.91-3.72 (m, 2H), 3.82 (s, 3H), 3.67 (s, 3H), 3.39-3.32 (m, 1H), 3.33-3.21 (m, 2H), 3.18-2.98 (m, 3H), 2.73-2.86 (m, 2H), 2.82-2.67 (m, 2H), 2.66 (s, 3H), 2.56 (s, 3H), 2.25-2.03 (m, 2H), 2.11 (s, 3H), 1.84-1.82 (m, 4H), 1.74-1.61 (m, 3H), 1.42 (s, 9H), 1.37 (s, 9H); HRMS (ESI), calculated for C.sub.62H.sub.81N.sub.9NaO.sub.15S ([M+Na]+): 1246.5471, found: 1246.5477.

(14) ##STR00026##

(15) Fmoc-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr)-OMe (568 mg, 0.462 mmol, 1.0 equiv) was dissolved in 20 ml of methanol, lithium hydroxide (12 mg, 0.485 mmol, 1.05 equiv) was added and the mixture was stirred for 2.5 hours at 25° C. After evaporation, the residue was dissolved in water, acidified to pH=3 with dilute HCl (0.1 N) and extracted with ethyl acetate. The extract was dried over Na.sub.2SO.sub.4, evaporated again and the obtained Fmoc-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr)-OH was stirred at 25° C. for 1 hour with piperidine (15 equiv) in MeOH (10 mL). The mixture was evaporated and the piperidine adduct of dibenzofulvene was washed out with diethyl ether. The obtained H-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr)-OH was dissolved in a mixture of dichloromethane (2 mL) and CH.sub.3CN (8 mL), and cooled to 0° C. To this solution was added catalyst ZrOCl.sub.2 (5 mol %), 1-hydroxy-7-azabenzotriazole (HOAt) (1.1 eq) and N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide.HCl (EDC.HCl) (102 mg, 0.531 mmol, 1.15 eq) at 0° C. under N.sub.2 atmosphere and stirred at 0° C. for 20 minutes. N-Methylmorpholine (107 μL, 0.97 mmol, 2.0 eq) was slowly added via syringe at 0° C., the temperature of the reaction was gradually warmed to room temperature, and then it was stirred at room temperature for 16 hours. Solvent was evaporated, and the remaining residue was suspended in EtOAc (25 mL), and the pH value was adjusted to 3.5-4.0 with 0.1 N HCl. The organic layer was separated and washed with H.sub.2O (10 mL), saturated aqueous NaHCO.sub.3 (10 mL), brine (10 mL), and dried over Na.sub.2SO.sub.4. After the evaporation of solvent, the remaining residue was purified by silica gel flash chromatography under the conditions indicated to provide cyclo-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr) (184 mg, 41% yield relative to the amount of Fmoc-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr)-OMe). .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.70 (br, 1H), 7.40-7.38 (m, 5H), 6.56 (s, 1H), 6.05 (br, 1H), 4.46-4.23 (m, 2H), 3.94-3.77 (m, 2H), 3.84 (s, 3H), 3.28-3.21 (m, 1H), 3.20-3.08 (m, 3H), 2.92-2.71 (m, 4H), 2.21-2.08 (m, 3H), 2.12 (s, 3H), 1.88-1.83 (m, 3H), 1.72-1.69 (m, 2H), 1.62-1.60 (m, 1H), 1.52-1.47 (m, 1H), 1.44 (s, 9H), 1.38 (s, 9H), 1.29-1.25 (m, 2H); HRMS (ESI), calculated for C.sub.46H.sub.67N.sub.9NaO.sub.2S ([M+Na]+): 992.4527, found: 992.4521.

(16) ##STR00027##

(17) The protected cyclopeptide cyclo-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr) (120 mg, 0.124 mmol) was treated with 2 mL of a solution of TFA (80%), phenol (5%), water (2.5%), thioanisole (5%), triisopropylsilane (2.5%) and 1,2-ethanedithiol (5%) at ambient temperature. After 9 hours, the solvent was evaporated in vacuo. The residue was dissolved in 0.1 N HCl and freeze-dried after each dissolving operation six times. The resulting solid was precipitated with cold diethyl ether (5 mL×2) and centrifuged. The resulting pellet was washed several times with cold diethyl ether to provide cyclo-Gly-Asp-D-Phe-Pip-Arg.2HCL as pale yellow solid (78 mg, 94%, relative to the amount of cyclo-Gly-Asp(O.sup.tBu)-D-Phe-Pip(Boc)-Arg(Mtr). .sup.1H NMR (500 MHz, D.sub.2O): δ 7.26-7.08 (m, 5H), 4.51 (t, 1H, J=6.7 Hz), 4.47-4.23 (m, 2H), 4.41-4.37 (m, 1H), 3.84-3.79 (m, 1H), 3.78-3.76 (m, 1H), 3.70-3.67 (m, 1H), 3.54-3.50 (m, 1H), 3.19-3.17 (m, 311), 3.06-3.02 (m, 4H), 3.02-2.97 (m, 3H), 2.75-2.72 (m, 2H), 2.66-2.63 (q, 2H, J=7.9 Hz), 2.46-2.39 (m, 4H), 2.14-2.10 (m, 2H), 2.09-2.01 (m, 4H), 1.82-1.77 (m, 2H), 1.68-1.61 (m, 2H), 1.50-1.46 (m, 2H), 1.44-1.40 (m, 2H), 1.29-1.25 (m, 2H); HRMS (ESI), calculated for C.sub.27H.sub.43Cl.sub.2N.sub.9NaO.sub.7 ([M+Na+2H+]): 698.25602, found: 698.2572.

(18) The cyclopeptide of a preferred embodiment of the present disclosure can be prepared by the following method.

(19) ##STR00028##

(20) To a stirred CH.sub.2Cl.sub.2 or THF solution (3.5 mL) of C5P-1 (740 mg, 0.76 mmol, 1.0 equiv) was added 10-15 equivalents of piperidine (8.4 mmol), and the reaction mixture was stirred until complete consumption of the C5P-1 confirmed by TLC (0.5-1 hour). The reaction mixture was then evaporated to dryness with toluene under reduced pressure at 60° C.

(21) A piperidine adduct of dibenzofulvene was washed out with hexane and the resulting C5P-2 (LC-MS: t.sub.R 5.2 minutes for 91% purity) was dried in vacuo and used directly for the next step. To a dry microwave vial was added C5P-2 (900 mg, 1.2 mmol, 1.0 equiv), Fmoc-Asp(O.sup.tBu)-OH (986 mg, 2.4 mmol, 2.0 equiv), catalyst ZrOCl.sub.2 (5 mol %), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI) (344 mg, 1.8 mmol, 1.5 equiv), and hydroxybenzotriazole (HOBt) (278 mg, 1.8 mmol, 1.5 equiv) in dry CH.sub.3CN (6 mL) under argon. N,N-Diisopropylethylamine (DIPEA) (232 mg, 1.8 mmol, 1.5 equiv) was subsequently added and the vial was sealed at 20-25° C. for 0.5 hour. The reaction mixture was diluted with water, saturated aqueous NaHCO.sub.3 and extracted with ethyl acetate. The collected organic phases were combined and washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash column chromatography under the conditions indicated to give C5P-3 as a light yellow solid (1.370 g, 72.2% conversion yield; LC-MS: t.sub.R 7.15 minutes).

(22) ##STR00029##

(23) In a solution of EtOAc/MeOH (25/25 mL) was placed C5P-3 (685 mg, 0.6 mmol, 1 equiv) and Pd/C (10% wt, 70 mg). Hydrogenolysis was carried out at ambient temperature for 18 hours. The reaction mixture was filtered and concentrated to give crude C5P-4 (645 mg, 71.6% conversion yield; LC-MS: t.sub.R 5.50 minutes). The crude acid C5P-4 (645 mg, 0.61 mmol, 1.0 equiv) obtained after debenzylation was treated with glycine methyl ester hydrochloride (152 mg, 1.22 mmol, 2 equiv), catalyst ZrOCl.sub.2 (5 mol %), EDCI (174 mg, 0.91 mmol, 1.5 equiv), and HOBt-H.sub.2O (142 mg, 0.91 mmol, 1.5 equiv) in dry CH.sub.3CN (6 mL) under argon. DIPEA (275 mg, 2.13 mmol, 3.5 equiv) was subsequently added and the reaction was stirred at 20-25° C. for 2.5 hours. The reaction mixture was diluted with water (4 mL), saturated aqueous NaHCO.sub.3 (4 mL) and extracted with ethyl acetate. The collected organic phases were combined and washed with brine, dried over Na.sub.2SO.sub.4 and concentrated.

(24) The crude product was purified by flash column chromatography under the conditions indicated to give C5P-5 as a light yellow oil (417 mg, 97.53% conversion yield; LC-MS: t.sub.R 6.56 min).

(25) ##STR00030##

(26) CP5-5 (834 mg, 0.76 mmol, 1.0 equiv) was dissolved in CH.sub.2Cl.sub.2 or THF solution (3.5 mL), the solution was stirred, followed by adding 10-15 equivalents of piperidine (8.14 mmol). The reaction mixture was stirred until complete consumption of the C5P-5 confirmed by TLC (0.5-1 h) and the reaction mixture was then evaporated to dryness under a reduced pressure at ambient temperature. A piperidine adduct of dibenzofulvene was washed out with hexane and the resulting C5P-6 (LC-MS: t.sub.R 5.06 min for 92.84% purity) was dried in vacuum and used directly for the next step. To a dry reaction sealed vial was added C5P-6 (200 mg, 0.22 mmol, 1.0 equiv), catalyst V(O)Cl.sub.2, Ti(O)(acac).sub.2 or ZrOCl.sub.2 (5-10 mol %) in dry CH.sub.3CN, cyclopentyl methyl ether (CPME), or toluene (1.6-2.2 mL). Subsequently, 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (10 mg, 0.066 mmol, 0.3 equiv) was added and the vial was sealed at 80-110° C. for 7-23.5 hours. The reaction mixture was concentrated and then diluted with water, saturated aqueous NaHCO.sub.3 and extracted with ethyl acetate. The collected organic phases were combined and washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash column chromatography under the conditions indicated to give C5P-7 as a light yellow solid (0.1370 g, 71.55% conversion yield; LC-MS: t.sub.R 4.98 min).

(27) ##STR00031##

(28) C5P-7 (300 mg, 0.34 mmol, 1 equiv) was dissolved in TFA (15 mL) and stirred at ambient temperature for 18-19 hours. The reaction mixture was concentrated to give crude C5P (˜200 mg, 94.4% conversion yield; LC-MS: t.sub.R 2.03 min). The crude C5P was washed with IPA/IPE (2/1) to get pure C5P and stored as a TFA salt with addition of 1 equiv of TFA.

(29) Anti-aging Evaluation Type I collagen is the primary component of the skin dermis. Both the quantity and quality of extracellular collagen are primarily related to skin ageing. The present embodiment was examined, by using Procollagen Type I C-Peptide (PIP) EIA Kit, effects of induction of procollagen secretion and cytotoxicity in respect of hs68 human fibroblasts. The results are shown in the following Table 1.

(30) TABLE-US-00001 TABLE 1 TGFβ 0.83 — — — — — — — Conc. (μM) Cyclopeptide — 0.0008 0.004 0.02 0.1 0.25 0.5 — Conc..sup.a (μM) Procollagen 320 235 225 220 208 208 184 90 Type I (ng/mL) Cell viability.sup.b 95 92 97 100 97 99 100 100 (%) .sup.a(III-5) was under test, in which Q is Cl. .sup.bcell viability (%) = (sample/control) × 100%

(31) It can be found in the experimental results that each of the tested cyclopeptides in different concentrations can promote the secretion of procollagen type I. When the cyclopeptide concentration is as low as 0.0008 μM, it can increase the secretion amount of procollagen type I by 2.5 times. It is therefore predicted that the cyclopeptide can repair skin aging. In addition, all the cell viabilities are greater than 80%, indicating that the cyclopeptide has no cytotoxicity.

(32) MMP-1 Inhibition Test

(33) Matrix Metalloproteinase-1 (MMP-1) is one of collagenases, and is involved in the degradation of the extracellular matrix (ECM). In details, fibroblasts result in the overexpression of MMP-1 after exposure to UV-containing sunlight, so that the ECM is then degraded by MMP-1.

(34) To examine the effect of the cyclopeptide of the present disclosure, a series of MMP-1 inhibition test was conducted.

(35) Matrix metalloproteinases (MMPs) are involved in skin physiological functions such as wound healing, aging, and inflammatory responses. The MMPs plays an important role in maintaining normal physiological functions or pathological phenomena of skin. In addition, skin aging results in various impacts on the skin, including wrinkles, dryness, anetoderma, inhibition of collagen production, and promotion of MMP, which accelerates the degradation of the extracellular matrix (ECM). As a result, skin elasticity and skin water-holding capacity are lost.

(36) In this test, test compound and human fibroblasts were co-cultured, and cytokine tumor necrosis factor-α (TNF-α) was added as an inducer to induce the expression of MMP-1 to a high level. Then, the test compound was evaluated for the ability of reducing the MMP-1 level induced by TNF-α.

(37) Material and Method

(38) Test compound: cyclopeptide (III-3) of the present disclosure, in which Q is Cl.

(39) Cell line: skin fibroblast Hs68

(40) First, the seeding of skin fibroblast Hs68 cells onto culture medium dish was conducted overnight, and then different concentrations (0.05 μM, 0.25 μM, 1.25 μM, 2.5 μM and 5 μM) of cyclopeptide (III-3) were added to the skin fibroblast cells for 6 hours, which was further incubated with TNF-α (20 ng/mL) for 42 hours. Afterwards, the concentrations of MMP-1 in each group were measured by ELISA, and the results are shown in Table 2.

(41) TABLE-US-00002 TABLE 2 Test Test Test Test Test Positive Control.sup.c TNF-α 1 2 3 4 5 control.sup.d TNF-α — 20  20 20 20 20 20 20 Conc. (ng/mL) Cyclopeptide — — 0.05 0.25 1.25 2.5 5 — Conc. (μM) RA — — — — — — — 3.3 Conc. (μM) MMP-1 243.8 604.4 337.8 244.2 212.7 194.2 168.6 155.9 Conc. (pg/mL) .sup.ccontrol group is the group without adding TNF-α, cyclopeptide and retinoic acid (RA). .sup.dRA is a known MMP-1 inhibitor, and the positive control is the group added with RA.

(42) According to the results above, compared to 3.3 μM of RA (set as 100%), 0.05 μM of cyclic peptide (III-3) could inhibit MMP-1 by about 60%, and 0.25 μM of cyclopeptide (III-3) could inhibit about 80% of MMP-1; 1.25 μM of cyclopeptide (III-3) could almost inhibit 87% of MMP-1; 2.5 μM cyclopeptide (III-3) could almost inhibit 91% of MMP-1.

(43) In addition, the result shown in FIG. 1 was obtained by measuring the inhibition concentration of cyclopeptide (III-3), thereby calculating the IC.sub.50 of cyclopeptide (III-3), which was 0.02 μM.

(44) Likewise, the aforementioned MMP-1 inhibition test was conducted with different concentrations (0.05 μM, 0.25 μM, 1.25 μM, 2.5 μM and 5 μM) of the cyclopeptide (III-1) (Q is Cl), and the results were shown in the following Table 3. It could be found from Table 3 that 0.05 μM of cyclopeptide (III-1) could inhibit 12% of MMP-1, 0.25 μM of cyclopeptide (III-1) could inhibit 38% of MMP-1, and 1.25 μM of cyclopeptide (III-1) could inhibit 90% of MMP-1.

(45) TABLE-US-00003 TABLE 3 Test Test Test Test Positive Control.sup.c TNF-α 6 7 8 9 control.sup.d TNF-α — 20  20 20 20 20 20 Conc. (ng/mL) Cyclo- — — 0.05 0.25 1.25 2.5 — peptide Conc. (μM) RA — — — — — — 3.3 Conc. (μM) MMP-1 304.2 442.2 422.3 378.2 288.0 271.8 271.3 Conc. (pg/mL) .sup.ccontrol group is the group without adding TNF-α, cyclopeptide and retinoic acid (RA). .sup.dRA is a known MMP-1 inhibitor, and the positive control is the group added with RA.

(46) In addition, the result shown in FIG. 2 was obtained by measuring the inhibition concentration of cyclopeptide (III-1), thereby calculating the IC.sub.50 of cyclopeptide (III-1), which is 0.7 μM.

(47) Although the present disclosure has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the disclosure as hereinafter claimed.