The American mayapple (Podophyllum) has been a source of bio-active lignans for many years. The most studied one of these is podophyllotoxin (1) Figure 1(a).

This natural compound 1 presents a chemical structure consisting of four fused planar rings with consecutive chiral centers [1] . Original uses for podophyllotoxin since 1942 (still in use) include topical applications [2] . Among the various medical applications of podophyllotoxin or its derivatives are skin disorders, periodontal disease, parasitic infections and a number of anti-viral applications among others [3] . Podophyllotoxin was antimitotic and tubulin inhibitor by itself [4] but later it was developed as a topoisomerase-II inhibitor. Several semisynthetic derivatives of podophyllotoxin have been used as chemotherapeutic drugs. These current derivatives are etoposide (2), etopophos (3) and teniposide (4) [5] (Figure 1(b)).

Arguably the most important derivative of these has been etoposide (also known as VP-16). This drug has been widely studied and has been classified since the 1980’s as a topoisomerase II inhibitor [6] .

The success of etoposide has led to a great deal of work with podophyllotoxin derivatives, much of it studying a variety of biological effects and anti-cancer activities [7] [8] .

Synthesis of podophyllotoxin in laboratory is still quite challenging chemistry [9] . To fulfill the industrial demand of 1 we are still dependent on the endangered natural sources of this compound. 1 is also the precursor to a new derivative CPH 82 (5) (Figure 1(c)) which has been tested for rheumatoid arthritis in Europe [10] , and it is the precursor to other derivatives used for the treatment of psoriasis [11] and malaria.

In our previous work we explored the synthesis and application of novel aza-podophyllotoxin derivative compounds which led to several new analogs with anti-cancer potential [12] . Using a one pot method, synthesis of an aza-podophylltoxin derivative is less complicated and results in a high yield. This method also, allows us to create extensively modified libraries of several novel derivatives, which were previously impossible to synthesize from podophyllotoxin [13] . These new aza-podophyllotoxin derivative compounds have only two chiral centers at the 1 and 4 positions of ring “C” as compared to 4 chiral centers in ring “C” of podophyllotoxin. Elimination of chirality at C2 and C3 by double bond in aza-podophyllotoxin is helping all the 4 A, B, C, and D fused rings in one plane, better than podophyllotoxin. Probably this is one of the reasons for enhanced activity of aza-podophyllotoxin derivatives against tumor cells [14] . Most of the drugs from our library showed excellent activity against 60 types of human cancer cell line panels performed at the United States National Cancer Institute (NCI) [15] . Two AZP derivatives 8a (4-(2-Hydroxyethyl)-10-(3,4,5-trimethoxyphenyl)-3,4,6,7,8,10-hexahydro-1H-yclopenta[g]furo[3,4-b]quinolin-1-one) (Figure 1(c)) and 9a (6-(2-Hydroxyethyl)-10-(3,4,5-trimethoxyphenyl)-2,3,7,10-tetrahydro-[1,4]dioxino[2,3-g]furo[3,4-b]quinolin-9(6H)-one) (Figure 1(c)) were found very active against COLO 205 colon cancer cell line. Compound 8a does not contain an oxygen atom in ring A, while compound 9a contains 2 oxygen atoms and expanded ring with 1 carbon in ring A. Here we are presenting the major apoptotic associated activities of 8a and 9a in COLO 205 human cancer cell line directed to determine the mechanism of action of these novel podophyllotoxin derivatives. This information will establish the background biological activities necessary for conducting further in vivo and gene expression studies.

Expression of phosphatidylserine was measured as an apoptosis marker. Both tested compounds presented statistical significance (P < 0.05) for apoptosis induction. Compound 8a presented 44% of apoptotic cells while 9a presented 59.5%. Figure 2(a) presents the values obtained. Compound 9a presented a majority of cells at a late

apoptotic stage (50%) shown on Figure 2(b). Both compounds are comparable to the positive controls etoposide (32%) and podophyllotoxin (27%) clearly showing apoptotic activity.

In this study we screened the biological activity and apoptosis induction of two novel aza-podophyllotoxin derivatives. These compounds were previously assayed for toxicity against a number of cell lines and were determined to be active for growth inhibition upon COLO 205 colorectal adenocarcinoma cells among other cancer cell types. Both of our tested compounds presented different biological activities on the COLO 205 cell line. Com-

pound 8a presented an unexpected apoptotic activity profile. This compound presented PS migration to the exterior of cells as evidenced by the annexin V staining. Migration of PS is usually indicative of an apoptotic cell death mechanism. Additionally this compound presented a significant cell cycle arrest at the S phase and as well as DNA fragmentation; however none of the traditional apoptosis hallmarks such as mitochondrial membrane permeabilization and caspase 8, 9, 3 and 7 activation were detected in a significant manner.

This type of apoptosis sometimes referred to as atypical apoptosis, has been observed after activation of intra-S DNA damage checkpoints [26] , or p27 mediated autophagy [27] . Intra-S checkpoint effects involve the phosphorylation of proteins such as the retinoblastoma tumor suppression protein (pRB) causing accumulation of cells at the S phase to permit reparation of any DNA damage. P27 mediated autophagy results from inhibition of cyclin dependent kinases (CDK) which regulate cell cycle progression [28] [29] . Other types of similar effects include inhibition of the topoisomerases in which cell cycle arrest with DNA fragmentation have been documented [30] in this case however, evidence of caspase 3 activation was observed. Given our compounds close structural relationship with the known topoisomerase II inhibitor, etoposide; we deduced that biological activities could be similar to this chemotherapeutic drug.

Our results for compound 8a are similar to previous work [31] in which etoposide induces autophagic cell death through a BCL-2 dependent mechanism. The activity of compound 9a included the same positive parameters as in compound 8a such as PS migration, DNA fragmentation and cell cycle arrest at S phase. This compound however did cause activation of caspases 3, 8 and 9 in a significant manner. Generally the type of activated caspase can give insight to the cell death mechanism that is occurring. The death receptor pathway (also known as extrinsic) is usually associated with activation of caspase 8 [32] . Other evidence exists in which this caspase can be activated by other mechanisms such as p53 [33] . Intrinsic mediated pathways include the liberation of cytochrome C after mitochondrial membrane permeabilization. This pathway can cause activation of the apoptosome complex involving caspase 9 [34] . It is this caspase in its activated form that cleaves effector caspase 3 which acts upon numerous cell substrates that can cause the characteristic DNAse mediated fragmentation [35] [36] . If we consider this information in the context of compound 9a we can see that the classical apoptosis effects are present. Work by Liu et al. [37] demonstrates that etoposide can cause p53/p73 apoptosis through a caspase 8 signal amplification of the intrinsic mitochondrial pathway. This suggests a similar mechanism for 9a in which we evidenced mitochondrial permeability as well as caspase activation including the classic hallmarks of apoptotic cell death.

Although a complete mechanistic description was beyond the scope of this work, we found interesting evidence which suggests a structure-activity difference for our aza-podophyllotoxin derivatives. Compound 8a presents similar core structure to the podophyllotoxin, precursor of etoposide. The only difference is substitution of nitrogen in ring “C” at position 4, elimination of two chiral centers and elimination of 2 oxygen atoms from ring A. Substitution of nitrogen in ring C might not make significant difference in biological activity [38] and elimination of chirality from ring C probably enhancing its biological activity by increasing planarity in one plane of all four fused rings [39] [40] . Elimination of 2 oxygen atoms from ring A is probably playing a key role in the biological activity of these compounds. 8a does not have the two oxygen atoms in ring A appearing to have activity similar to etoposide inducing autophagy [31] , whereas compound 9a has minor structural difference from compound 8a except that it has two oxygens and ethylene group on ring A presented typical apoptosis cell death with the hallmarks of etoposide induced apoptosis. Overall, this preliminary biological screening for these novel substances provides the ground work for further insight on these promising substances.

This project was supported by the National Center for Research Resources and the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 8 P20 GM 103475.