Analysis of cytotoxic activity at short incubation times reveals profound differences among Annonaceus acetogenins, inhibitors of mitochondrial Complex I

Annonaceous acetogenins are potent cytotoxic agents against tumor cell lines as well as potent inhibitors of mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116–120, 1994; Degli Esposti et al. Biochem J 301(Pt 1):161–167, 1994; Tormo et al. Arch Biochem Biophys 369:119–126, 1999). Eighteen different ACGs belonging to seven structural sub-families were tested against six tumor and two non tumor cell lines in a MTT cytotoxicity assay to evaluate the correlation between mitochondrial Complex I inhibition and cytotoxic activity potency and selectivity. The results showed a substantial heterogeneity in potency and selectivity among the different compounds tested, although no clear overall structure-activity relationships could be established. To further characterize the biological activity of these compounds, four ACGs were selected based on their inhibition binding sites to Complex I, to evaluate their cytotoxic activity over a 15-minute to 48-hour period using a more sensitive time-course LDH cytotoxicity assay. Our results indicate that, although all of the ACGs were highly cytotoxic in HepG2 cell lines at 24 h, each sub-class behaves rather differently at shorter times. Perhaps other aspects related to how these compounds reach or bind to their target sites, or differences in their ability to cross the cell and/or the mitochondrial membranes, could help explain their different activities. This different behavior between ACGs may provide new clues for a better understanding of their potential antitumor properties.     

Synthesis and antitumor activity of duocarmycin derivatives: A-ring pyrrole compounds bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group

A series of A-ring pyrrole derivatives of duocarmycin bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. New Seg-B analogues bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group containing double bond as spacer had lower peripheral blood toxicity than the derivatives bearing 5',6',7'-trimethoxyindole-2'-carboxyl group in Seg-B of the natural type. Moreover, most of them exhibited potent antitumor activity against in vivo murine tumor models.     

Synthesis and antitumor activity of duocarmycin derivatives: modification at the C-7 position of segment-A of A-ring pyrrole compounds

A series of the C7-substituted A-ring pyrrole derivatives of duocarmycin were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. All of the C7-substituted A-ring pyrrole compounds decreased potency in vitro and in vivo. However, some showed strong antitumor activity with T/C values less than 0.3. Among them, the 7-formyl compound 5d showed remarkable potent in vivo antitumor activity and low peripheral blood toxicity, which were equal to 2c.     

三种药用植物内生菌的分离及其抗肿瘤活性菌株的筛选

对204株分离自长春花、海南粗榧和见血封喉的内生菌进行了抗肿瘤活性筛选。结果显示,有19株内生菌的发酵液至少对一种指示瘤株具有细胞毒活性,其中4株菌的发酵液对S180的抑制率较强,均在70%以上。研究表明菌株PA09006和PA09009发酵液中的抗肿瘤活性物质,具有一定的酸碱耐受性,并且对温度和紫外线具有一定的稳定性。     

Dynamic function of the spacer region of acetogenins in the inhibition of bovine mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Studies of the action mechanism of acetogenins, the most potent and structurally unique inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), are valuable in characterizing the inhibitor binding site in this enzyme. Our previous study deepened our understanding of the dynamic function of the spacer region of bis-THF acetogenins [Abe, M., et al. (2005) Biochemistry 44, 14898-14906] but, at the same time, posed new important questions. First, while the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings) span a distance shorter than that of the extended 13 carbon atoms [-(CH 2) 13-], what is the apparent optimal length of the spacer for the inhibition of 13 carbon atoms? In other words, what is the functional role of the additional methylene groups? Second, why was the inhibitory potency of the mono-THF derivative, but not the bis-THF derivative, drastically reduced by hardening the spacer covering 10 carbon atoms into a rodlike shape [-CH 2-(C identical withC) 4-CH 2-]? This study was designed not only to answer these questions but also to further disclose the dynamic functions of the spacer. We here synthesized systematically designed acetogenins, including mono- and bis-THF derivatives, and evaluated their inhibitory effects on bovine complex I. With regard to the first question, we demonstrated that the additional methylenes enhance the hydrophobicity of the spacer region, which may be thermodynamically advantageous for bringing the polar gamma-lactone ring into the membrane-embedded segment of complex I. With regard to the second question, we observed that a decrease in the flexibility of the spacer region is more adverse to the action of the mono-THF series than that of the bis-THF series. As a cause of this difference, we suggest that for bis-THF derivatives, one of the two THF rings, being adjacent to the spacer, is capable of working as a pseudospacer to overcome the remarkable decrease in the conformational freedom and/or the length of the spacer. Moreover, using photoresponsive acetogenins that undergo drastic and reversible conformational changes with alternating UV-vis irradiation, we provided further evidence that the spacer region is free from steric congestion arising from the putative binding site probably because there is no receptor wall for the spacer region.     

Synthesis of C4-fluorinated solamins and their growth inhibitory activity against human cancer cell lines

C4-Fluorinated analogues of solamin, an antitumor acetogenin, were synthesized and investigated for their antitumor activities against 39 tumor cell lines. C4-Fluorinated solamins showed more potent growth inhibitory activity against cancer cell lines than solamin.     

Synthesis of annonaceous acetogenins from muricatacin

Synthetic studies of annonaceous acetogenins starting from (-)-muricatacin (1a) or (+)-muricatacin are described, involving (-)-muricatacin (1a), mono-THF acetogenin, solamin (2), reticulatacin (3), (15R, 16R, 19S, 20S)-cis-solamin (4a) and (15S, 16S, 19R, 20R)-cis-solamin (4b), non-adjacent bis-THF acetogenin, 4-deoxygigantecin (5), and epoxide-bearing acetogenin, (15S, 16R, 19S, 20R)-diepomuricanin (6a).     

Synthesis and antitumor activity of 7-ethyl-9-alkyl derivatives of camptothecin

A series of new camptothecin derivatives, as topoisomerase I inhibitor, were synthesized to identify potent antitumor agents. The synthesis method was based on the Claisen rearrangement of 10-allyloxy-7-ethylcamptothecin. All of the compounds were assayed for cytotoxicity against two human tumor cell lines, Bel7402, HCT116, and showed good potency in vitro. Compounds 2, 4, 9, were assessed for the stability of lactone in human plasma. And then compound 2 was tested for antitumor activity in vitro against mouse tumor sarcoma-180. The results suggested that the small alkyl groups in the both 7- and 9-positions of camptothecin could promote liposolubility, antitumor activity in vitro and vivo, though did not bring much increase of the stability of lactone.     

Synthesis and antitumor activity of duocarmycin derivatives: modification at C-8 position of A-ring pyrrole compounds bearing the simplified DNA-binding groups

A series of the 8-O-substituted A-ring pyrrole derivatives of duocarmycin bearing the simplified DNA-binding moieties such as cinnamoyl or heteroarylacryloyl groups were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. In addition, the stability of the 8-O-substituted analogues in aqueous solution and the conversion to their active form (cyclopropane compound) from the 8-O-substituted analogues in mice or human serum were examined. The 8-O-substituted A-ring pyrrole derivatives bearing the simplified DNA-binding moieties showed remarkably potent in vivo antitumor activity and low peripheral blood toxicity compared with the 8-O-substituted A-ring pyrrole derivatives having the trimethoxyindole skeleton in segment-B (Seg-B), which were equal to 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4'-methoxycinnamates and 4'-methoxy-beta-heteroarylacrylates. Moreover, among 8-O-substituted analogues, several compounds can be chemically or enzymatically converted to their active form in human serum. This result indicated that new 8-O-substituted derivatives were different prodrugs from KW-2189 and 8-O-substituted analogues being the same type of prodrug as KW-2189.     

Structure–activity relationships of diverse annonaceous acetogenins against human tumor cells

Twelve annonaceous acetogenins (ACGs) with different stereochemical structures and configuration were selected to test for their inhibitions on the growth of Hela, SMMC-7541, SGC-7901, MCF-7 and A-5408 tumor cell lines using MTT method. This was the first to simultaneously investigate effects of structural factors of stereochemical structures and configuration on cytotoxicities with structure–activity relationship. The present study showed that cytotoxic selectivities of ACGs with threo/trans/threo/trans/erythro stereochemical arrangement were gently more active than those with threo/trans/threo/trans/threo stereochemical arrangement, and ACGs with cis THF ring partly produced notable cytotoxic selectivities. Furthermore, ACGs with S configuration at C-24 exhibited gently more cytotoxic selectivities potency than those with R configuration at C-24.     

Synthesis, characterization, and antitumor activity of 5-iodouracil complexes.

Complexes of 5-iodouracil (5IU) with Mn(II), Co(II), Cu(II), Zn(II), and Cd(II) ions have been prepared, characterized, and subjected to a screening system for evaluation of antitumor activity against Sarcoma-180 (S-180) and L 929 tumor cells. The complexes were characterized by their elemental analysis, infrared spectra, electronic spectra, magnetic measurements, and powder x-ray diffraction. The antitumor activity results indicate that some complexes have good antitumor activity both in vivo and in vitro against S-180 and L 929 tumor cells.     

Two bioactive mono-tetrahydrofuran acetogenins, annoglacins A and B, from Annona glabra

Two new bioactive mono-THF Annonaceous acetogenins, annoglacins A and B, have been isolated from the fractionated ethanolic extracts of the leaves of Annona glabra, directing the fractionation with the brine shrimp lethality test (BST). Their structures were elucidated based on spectroscopic and chemical methods and the absolute stereochemistries were determined by the advanced Mosher ester method. Annoglacins A and B were selectively 1000 and 10,000 times, respectively, more potent than adriamycin against the human breast carcinoma (MCF-7) and pancreatic carcinoma (PACA-2) cell lines in our panel of six human solid tumor cell lines.     

Specific interactions of monotetrahydrofuranic annonaceous acetogenins as inhibitors of mitochondrial complex I

Annonaceous acetogenins (ACG) are a wide group of cytotoxic compounds isolated from plants of the Annonaceae family. Some of them are promising candidates to be a future new generation of antitumor drugs due to the ability to inhibit the NADH:ubiquinone oxidoreductase of the respiratory chain (mitochondrial complex I), main gate of the energy production in the cell. ACG are currently being tested on standard antitumor trials although little is known about the structure activity relationship at the molecular level. On recent studies, the relevance of several parts of the molecule for the inhibitory potency has been evaluated. Due to the great diversity of skeletons included in this family of natural products, previous studies on the presence and distribution of oxygenated groups along the alkyl chain only covered the compounds with different bis-tetrahydrofuranic (bis-THF) relative configurations. Therefore, we have investigated the inhibitory action of all the mono-tetrahydrofuranic (mono-THF) acetogenins available, which differ in the oxygenated arrangements along the molecule. Our results show that the hydroxyl and carbonyl groups, placed in the aliphatic chain that links the initial gamma-lactone moiety with the dihydroxylated tetrahydrofuranic ring system, significantly contribute for modulating the inhibitory potency of the ACG through specific effects.     

Allosensitization induced suppression of various murine tumors: role of non-H-2 antigens in antitumor immunity

Presence of alloantigens on various murine tumors was tested by tumor rejection in allosensitized Swiss mice. The results indicated the presence of alloantigen on immunogenic tumors like chemically induced fibrosarcoma (FS), ascitic sarcoma 180 (S 180) and immunogenic variant of lymphosarcoma (LS-A) in Swiss mice, while these antigens could not be detected by this procedure on spontaneous lymphosarcoma (LS). Allosensitization with skin graft was found to offer quantitatively higher antitumor resistance than the allosensitization achieved by allogeneic lymphocytes. Antitumor effect was not seen when tumor cells were inoculated earlier than day 3 of grafting. Further, host immunosuppression with whole body irradiation up to day of 3 of skin grafting abrogated the antitumor effect. H-2 compatible and non-H-2 incompatible skin graft sensitization of host could offer resistance against both S 180 and LS-A. Further, tumor immune mice rejected H-2 compatible, non-H-2 incompatible skin graft significantly earlier.     

Synthesis and biological activities of 2'-deoxy-2'-fluoro-4'-thioarabinofuranosylpyrimidine and -purine nucleosides

As part of our ongoing investigation of the synthesis of biologically interesting 2'-modified-4'-thionucleosides, we synthesized 2'-deoxy-2'-fluoro-4'-thioarabinofuranosylpyrimidine and -purine nucleosides, and evaluated their antiviral and antitumor activities. In the pyrimidine series, beta-anomers of 5-ethyluracil, 5-iodouracil, 5-chloroethyluracil, and 5-iodocytosine derivatives showed potent and selective anti-HSV-1 and HSV-2 activities in vitro. In the purine series, guanine and 2,6-diaminopurine derivatives showed prominent antiviral activities with slight cytotoxicity. On the other hand, the 5-fluorocytosine derivative (5F-4'-thioFAC) showed potent antitumor activity against both leukemia and solid tumor. Its antitumor spectrum against 14 human solid tumor and one leukemic cell lines was compared with that of 4'-thioFAC. The results showed that 5F-4'-thioFAC had an antitumor spectrum similar to that of 4'-thioFAC. However, 5F-4'-thioFAC was about 10 times less active than 4'-thioFAC.     

小鼠腹腔巨噬细胞被S-O_2—1激活后的抑瘤作用 Ⅰ.小鼠腹腔巨噬细胞的体外细胞毒作用及扫描电镜观察

在六十年代Mathe就用卡介苗来治疗小儿白血病,并获得了良好的效果。以后人们开始重视如何利用微生物佐剂刺激机体的防御机制,增强机体对肿瘤的排斥能力,来达到提高肿瘤治疗效果的目的。     

Antioxidant activity and antitumor activity (in vitro) of xyloglucan selenious ester and surfated xyloglucan

Two kinds of xyloglucan derivatives (xyloglucan selenious ester and sulfated xyloglucan) were prepared and evaluated on antioxidant activity and antitumor activity. Compared with xyloglucan, xyloglucan derivatives have new bioactivity against oxidative damage and tumor. Furthermore, xyloglucan selenious ester is more potent than sulfated xyloglucan at antioxidant activity and antitumor activity in vitro. The current data suggest for the first time that selenition of xyloglucan significantly increases its bioactivity and the chemical modification of polysaccharide may allow the preparation of derivatives with new properties and a variety of applications.     

Studies on Chemical‐Structure Modification and Structure?Activity Relationship of Gambogic Acid Derivatives at Carbon(34)

Gambogic acid (GA), a natural product, was identified as a promising antitumor agent. To further explore the structure? activity relationship of GA and discover novel GA derivatives as antitumor agents, 19 novel GA derivatives modified at C(34) were synthesized and evaluated against A549, BGC‐823, U251, HepG2, and MB‐231 cancer cell lines by cellular assays. Among them, 15 compounds were found to be more potent than GA against some cancer cell lines. Notably, compound 3 possessed potent inhibitory activities against five cell lines with IC₅₀ values ranging between 0.24 and 1.09 μM . Compounds 9 and 18 were seven to eightfold more active than GA against A549 cell line. Chemical modification at C(34) of GA by introducing of hydrophilic aliphatic amines resulted in increased activity and improved drug‐like properties. These findings will enhance our understanding of the SAR of GA and can lead to the discovery of novel GA derivatives as potential antitumor agents.     

Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-benzodioxan fragment as a novel class of potent methionine aminopeptidase type II inhibitors

A series of 1,2,4-triazole derivatives containing 1,4-benzodioxan (5a-5q) have been designed, synthesized, structurally determined, and their biological activities were evaluated as potential MetAP2 inhibitors. All the synthesized compounds were first reported. Among the compounds, compound 5k showed the most potent biological activity against HEPG2 cancer cell line (IC(50)=0.81 μM for HEPG2 and IC(50)=0.93 μM for MetAP2), which was comparable to the positive control. Docking simulation by positioning compound 5k into the MetAP2 structure active site was performed to explore the possible binding model. The results of apoptosis and Western-blot assay demonstrated that compound 5k possessed good antitumor activity against HEPG2 cancer cell line. Therefore, compound 5k with potent inhibitory activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell.     

Definition of crucial structural factors of acetogenins, potent inhibitors of mitochondrial complex I

Some natural acetogenins are the most potent inhibitors of bovine heart mitochondrial complex I. These compounds are characterized by two functional units (i.e. hydroxylated tetrahydrofuran (THF) and alpha,beta-unsaturated gamma-lactone ring moieties) separated by a long alkyl spacer. To elucidate which structural factors of acetogenins including their active conformation are crucial for the potent inhibitory effect, we synthesized a series of novel acetogenin analogues possessing bis-THF rings. The present study clearly demonstrated that the natural gamma-lactone ring is not crucial for the potent inhibition, although this moiety is the most common structural unit among a large number of natural acetogenins and has been suggested to be the only reactive species that directly interacts with the enzyme (Shimada et al., Biochemistry 37 (1998) 854-866). The presence of free hydroxy group(s) in the adjacent bis-THF rings was favorable, but not essential, for the potent activity. This was probably because high polarity (or hydrophilicity), rather than hydrogen bond-donating ability, around the bis-THF rings is required to retain the inhibitor in the active conformation. Interestingly, length of the alkyl spacer proved to be a very important structural factor for the potent activity, the optimal length being approximately 13 carbon atoms. The present study provided further strong evidence for the previous proposal (Kuwabara et al., Eur. J. Biochem. 267 (2000) 2538-2546) that the gamma-lactone and THF ring moieties act in a cooperative manner on complex I with the support of some specific conformation of the spacer.