Xingjie Zhng,Zhi Meng,Zhiqing M,Junhong Liu,Guiyn Hn,Fuji M,Ningyng Ji,Zhenyun Mio,Wnnin Zhng,*,Chunqun Sheng,*,Jinzhong Yo,*

a Department of Medicinal Chemistry,School of Pharmacy,Second Military Medical University,Shanghai 200433,China

b Department of Pharmacy,455th Hospital of Chinese People’s Liberation Army,Shanghai 200052,China

c Department of Radiology,Shanghai Eastern Hepatobiliary Surgery Hospital,Second Military Medical University,Shanghai 200438 China

Key words:Photodynamic therapy(PDT)Photosensitizer Silkw orm excrement Chlorin p6 Antitumor

ABSTRACT Eight new w ater-soluble amino acid conjugates 6a-h of chlorin p 6 ethers(5a-d)were synthesized and preliminarily investigated for theirin vitro PDT antitumor activity and structure-activity relationship(SAR).The results showed that all compounds exhibited much higher phototoxicity against tumor cells than talapor fi n.SARanalysis indicated that PDTantitumor effect enhanced with the increase of carbon chain length of alkoxyl ether bonds at 31- position,and L-aspartic acid was superior to L-glutamic acid.In particular,the IC50 values of most phototoxic compound 6d were 0.20 m mol/L against A549 cell and 0.41 m mol/L against B16-F10 cell,which individually represented 31-and 24-fold increase of antitumor potency compared to talapor fi n,suggesting that it was a promising candidate photosensitizer(PS)for PDT applications due to its strong absorption at long w avelength,high phototoxicity,low dark cytotoxicity and good w ater-solubility.

Photodynamic therapy(PDT)now is an attractive approach to innovative cancer therapy involving combined use of visible light and a photosensitizer(PS)[1].PDT relies on the interaction between light and PS in tum or tissues to generate superoxide anions and radicals(type I reaction)or highly cytotoxic singlet oxygen(type II reaction)with the ultimate formation of reactive oxygen species(ROS)to inactivate the tumor cells[2].

Por fi mer sodium,the fi rst generation of porphyrin-type PS,has achieved enormous clinical ef fi cacy for the treatment of bladder cancer in the w orld.It also has suffered from some serious draw backs such as complex component,poor tissue penetration due to its limited maximum absorption w avelength of 630 nm,inefficient absorption(e=1170 Lmol?1cm?1)at 630 nm,and prolonged cutaneous phototoxicity up to 4-6 weeks after treatment caused by its slowelimination in skin tissues[3].

A great number of so-called the second generation of PSs especially related to chlorins such as chlorophyll-a derivatives etc.,are becoming more and more concerned ow ing to rapid clearance from tissues and intense absorption in near-infrared region(>650 nm,also called “phototherapeutic w indow”),which are relatively harmless and penetrate deeply in biological tissues[4–6].Among them,talapor fi n[7],vertepor fi n(BPD-MA)[8]and temopor fi n(m-THPC)[9]were clinically approved for PDT applications(Fig.1).

Chlorin p6(3),the one of chlorophyll-a derivatives as chlorintype PS,has poor stability to hamper its clinical development because it is easily converted into stable purpurin-18(2)with poor w ater solubility by its automatically intramolecular dehydration in theneutralcondition.Chlorin p6trimethylester(4),which isformed by methylation of 3,has good stability but poor w ater solubility.Because introducing amino acid was reported to be an effective strategy to improve the w ater-solubility and the biological activity of chlorin-and porphyrin-based derivatives[10–12],we previously synthesized some chlorin p6-and pyropheophorbide-a-based w ater-soluble amino acid derivatives and obtained a candidate PSwith a better ef fi cacy than vertepor fi n[13,14].Considering that alkoxylether derivativesof chlorin at 31-position exhibited stronger photosensitiveactivity than parent compound[15],a seriesof novel w ater-soluble amino acid conjugates 6a-h of chlorine p6ethers(5a-d)were further designed,synthesized and preliminarily investigated their photodynamic antitumor activity against melanoma B16-F10 and mammary carcinoma cells(Scheme 1).

Fig.1.Three clinical available chlorin-type photosensitizers.

As show n in Scheme 1,all intermediates pheophorbide-a(1),purpurin-18(2)and chlorin p6trimethylester(4)were obtained via acid and base degradation of chlorophyll-a followed by carboxyl methylation according to our previous methodology developed in our laboratory using crude chlorophyll extracts in Chinese traditional herb named silkw orm excrements[16,17].Brie fl y,intermediate 1 was gotvia cond.aqueous HCl degradation of chlorophyll-a in Et2O.Treatment of 1 in Et2O with KOH-i-Pr OH under an atmosphere of O2gave 2 in 34.4%yield.13,15-Anhydride ring of 2 was hydrolyzed in the presence of tetrahydrofuran(THF)and CH3OH using NaOH as the base to form unstable chlorin 3,which was rapidly methylated in Et2O with CH2N2to give 4 in 83.6%yield from 2.

In this paper,the details of the synthesis of key intermediate 5a-d and target compounds 6a-h from initial intermediate 4 were also given in Supporting information.Brie fl y,addition of 4 with 33%HBr in HOAc followed by substitution with excessive alcohol donors(ROH)in the presence of K2CO3produced chlorin p6ether derivatives 3-devinyl-3-(1-(R/S)-alkoxy)ethyl-chlorin p6-13,15-dimethylester(5a-d)in modest yields ranged from 26.7%to 32.7%.Obviously,intermediate 5 consisted of tw o epimerides of R-and S-con fi guration at 31-alkoxyl.High performance liquid chromatography(HPLC)analysis with chiral column showed that the general peak area ratio of the tw o epimerides was individually 22.5%vs.77.5%for 5a,35.9%vs.64.1%for 5b,31.3%vs.68.7%for 5c,33.3%vs.65.7%for 5d(Figs.S1–S4 in Supporting information).Then,key intermediate 5a-d was each coupled with L-(S)-(+)-aspartic acid hydrochlorate or L-(S)-(+)-glutamic acid hydrochlorate w hose carboxyl protected by tert-butyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochlorate(EDC?HCl),1-hydroxybenzotriazole(HOBt)and N,N-diisopropylethylamine(DIPEA)followed by removal of tert-butyl with tri fl uoroacetic acid(TFA)to generate the target compounds N-(3-devinyl-3-(1-(R/S)-alkoxy)ethyl-chlorin p6-13,15-dimethylester-173-acyl)-L-(S)-(+)-aspartic acid(6a-e)and N-(3-devinyl-3-(1-(R/S)-alkoxy)ethyl-chlorin p6-13,15-dimethyl ester-173-acyl)-[13_TD DIFF]L-(S)-(+)-glutamic acid(6f-h)in receivable yields ranged from 50.0%to 77.1%.Similarly,target compound 6 also was composed of tw o anisometric epimerides of R-and S-con fi guration at 31-alkoxyl,which possessed characteristic of dextral rotation(Supporting information),and its general peak area ratio of tw o epimerides analyzed by HPLC with chiral column was 35.2%vs.64.8%for 6c,35.3%vs.64.7%for 6d,41.8%vs.58.2%for 6 g,48.2%vs.51.8%for 6 h,respectively(Figs.S5–S8 in Supporting information).It is limited to our technical conditions that a pair of epimers in both 5 and 6 was even failure to complete effective separation and preparation.In general,the change of minor group con fi guration has generally little effect on the PDT antitumor activity as PS belongs to structural nonspecific drugs without specific drug target such as enzymes,receptors and proteinsetc.The structures of key intermediate 5a-d and target compounds 6a-h were identi fied by1H NMR,13C NMR,ESIMS and elemental analysis data(Supporting information).In addition,the UV-visible spectral data showed that they possessed more efficient absorption(e=1.67?105?2.42?105Lmol?1cm?1)at longer maximum absorption w avelength of 660–662 nm than por fi mer sodium(e=1170 Lmol?1cm?1)at 630 nm(Table 1),suggesting their greater tissue penetration[4–6].

Schem e 1.Synthetic route for the titled compounds 6a-h.Reagents and conditions:(a)cond.aqueous HCl-Et2O,0–5?C,30 min;(b)KOH,i-Pr OH-Et2O,12 h,34.4%;(c)THFCH3OH-aqueous NaOH(0.5 mol/L)(1:4:5,v/v/v),r.t.,1 h;(d)0.5 mol/Laqueous HCl to adjust p H value to 5-6,dilution with H2O,extraction with Et2O,dried by Na2SO4 for 1 h;(e)CH2N2,83.6%(from c to e);(f)33%HBr-HOAc,r.t.,36 h;(g)alcohol,CH2Cl2,K2CO3,r.t.,2.5 h.Alcohol donors:R=CH3(5a),n-C3H7(5b),n-C4H9(5c),n-C5H11(5d);(h)L-(S)-(+)-Asp(OBu t)2?HCl(n=1)or L-(S)-(+)-Glu(OBu t)2?HCl(n=2),EDC?HCl,HOBt,DIPEA,CH2Cl2,r.t.,12 h;(i)CH2Cl2-TFA(3:1),r.t.,6 h.Alcohol donors and amino acid residues:n=1 and R=CH3(6a),n-C3H7(6b),n-C4H9(6c),n-C5H11(6d);n=2 and R=CH3(6e),n-C3H7(6f),n-C4H9(6 g),n-C5H11(6 h).

Table 1 UV-vis data of the synthetic titled compounds.

In order to evaluate the effect of the introduced different amino acid moiety at 173-carboxyl and alkoxyl at 31-alkoxyl on the grow th inhibitory activity of the target compounds against human cancer cell lines and to clarify the SAR,the dark toxicity and phototoxicity of all the target compounds 6a-h were measured by the CCK-8 assay against human mammary carcinoma and murine melanoma B16-F10 cells using talapor fi n as positive control(Supporting information).To eliminate the experimental error caused by solvent,all tested compounds and talapor fi n were both made into w ater-soluble sodium salt.As show n in Table 2,allthe compounds exhibited better phototoxicity and considerable dark toxicity against tw o tested tumor cell lines compared to talapor fi n.SAR analysis showed that their PDT antitumor activity enhanced with the increase of carbon chain length of alkoxyl ether bonds at 31-position,and L-aspartic acid was superior toL-glutamic acid.Among them,compound 6d exhibited the bestin vitro PDT antitumor ef fi cacy and its IC50values against A549 and B16-F10 cells were individually 0.20 m mol/L and 0.41 m mol/L,which represented 31-and 24-fold increase of antitumor potency compared to talapor fi n,respectively.

In summary,8 new w ater-soluble amino acid conjugates 6a-h of 31-alkoxyl ethers(5a-d)for chlorin p6trimethylester(4)were synthesized and evaluated for their preliminary in vitro photodynamic antitumor activity against A549 and B16-F10 cells.All target compounds exhibited much stronger phototoxicity against tested tumor cell lines than talapor fi n.In particular,compounds 6d wasthe most effective,which individually showed 31-and 24-fold antitumor potency on A549 and B16-F10 cells compared to talapor fi n.As a result,compound 6d represents a promising PS for PDT applications ow ing to its strong absorption at long w avelength,high phototoxicity,low dark cytotoxicity and good w ater-solubility.Moreover,its more extensive and deeper physical and biological study including singlet oxygen quantum yield,in vivo PDTantitumor ef fi cacy,subcellular localization and tumor cell apoptosis detectionetc.are ongoing.

Table 2 Cytotoxicity(IC50,m mol/L)for the titled compounds against tumor cells.

Acknow ledgm ents

This work was supported by grants from the 3IF]National Natural Science Foundation of China(Nos.81172950 and 81671739),and the Project of Science and Technology Commission of Shanghai(No.11431920401)and the College Students’Innovation Ability Training Project of Second Military Medical University(No.MS2017040).

Appendix A.Supplem entary data

Supplementary data associated with thisarticle can be found,in the online version,at https://doi.org/10.1016/j.cclet.2018.04.029.References

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