Charles Conway Hanson Lab Group Fall 2012 Synthesis of Bi-functional Linkers for Paclitaxel INTRODUCTION The usage

of a linker between the anti-cancer drug paclitaxel and various targeting groups can allow for localized drug delivery as the conjugate can localize to a tumor and release the drug through cleavage of the linker from the paclitaxel. The linkers chosen for this research use the enol-ether group as a possible linking group to the drug due to its ability to readily conjugate with the hydroxyl group present in the paclitaxels structure. This linkage can then be cleaved at a moderately low pH in the body releasing the paclitaxel to the targeted area. The candidate for conjugation used here connects the enol-ether to a group which can be modified to fit the criteria of the desired linkage through an aliphatic chain. The linker has potential to be interchanged with different groups based on what targeting groups are needed for the drug delivery. When coupled to these targeting groups and the anti-cancer drug, the linker may have potential for targeted drug delivery in cancer patients. EXPERIMENTAL SECTION Synthesis of 6-Bromo-1-hexanal (2) PCC (5.57g, 1.5 eq, 16.6mmol) was suspended in 200mL DCM at room temperature. 6-bromo-1-hexanol (1) (2g, 1.0eq, 11.0mmol) was added drop-wise to the suspension and stirred overnight. The crude product was filtered and concentrated in vacuo. The product was purified on a silica gel column using a gradient of hexanes/EtOAc (100% to 9:1). The product was collected yielding 1.400g (70% yield). Synthesis of 1-methoxy-8-bromo-1-heptene (3) Methoxy methyl triphenyl phosphonium chloride (1.192g, 3.48mmol, 1.5eq) was dissolved in 20mL THF under a dry ice/acetonitrile bath and argon gas. 1.6M n-BuLi (1.45mL, 1.0eq, 2.32mmol) was added to the phosphonium salt solution and stirred for 1h producing a deep red solution. 6-bromo-1-hexanal (.416g, 1.0eq, 2.32g) was dissolved in 5mL THF and added drop-wise to the solution producing a light orange color. The reaction was stirred for 10min before bringing to room temperature. The reaction mixture was dissolved in ether and the salt was removed with water. The ether layer was collected and the aqueous layer was extracted with water two more times. The ether layers were combined and dried using MgSO4 for 1h. The product was filtered and evaporated in vacuo. The crude product was purified using silica gel chromatography with hexanes and 2% triethylamine. The product was collected yielding 119mg (23.2% yield) of product with an enol ether impurity.

Synthesis of phthalimide (4) 1-methoxy-8-bromo-1-octene(113mg, 1.0eq, .51mmol) was dissolved in 2mL DMF and potassium phthalimide(289mg, 3.0eq, 1.53mmol) was dissolved in 2mL DMF at room temperature. The two mixtures were combined and stirred at room temperature under argon gas for 72 hours. The crude product was diluted in DCM and washed with water 3 times to remove the DMF. The product was dried using MgSO4 for 1 hour. The product was purified using silica gel column chromatography using 95:5 Hexane/EtOAc with 2% triethylamine. The product was isolated to yield 30mg of the phtalimide linker with an enol ether impurity. Conjugation with Paclitaxel Paclitaxel (281.7mg, 3eq, .330mmol) was dissolved in 3mL THF with 2mg p-toluenesulfonic acid (2mg, 0.1eq, .011mmol). The phthalimide linker (30mg, 1 eq, .110mmol) was added and stirred with the paclitaxel for 72 hours under argon gas and covered with aluminum foil. The reaction was quenched with 300L TEA. Analyzed by TLC: best separation under 9:1 hexanes/EtOAc.

RESULTS AND DISCUSSION The synthesis of the linker 4 was verified to be viable with a reasonable yield and purity that could potentially be used for synthesis of the paclitaxel conjugate. The first step of the synthesis, the oxidation of the alcohol to produce an aldehyde was a very successful and reproducible reaction with a high yield and purity which was determined by 1H-NMR, 13C-NMR and IR. The product separated from the starting material using column chromatography at approximately 9:1 hexane/EtOAc. The spectra for the product from the first reaction are shown below.

Both analyses show a high purity and positive identification of the compound. The 1H-NMR shows the presence of the aldehyde in the high 9 range while the IR shows the presence of the aldehyde in the 1700 range. The synthesis of the enol-ether 3 through Wittig reaction had a much lower yield than the

first step of the reaction, however the desired product was produced and isolated. The Wittig reaction produced a mixture of 2 isomers of the enol-ether in approximately 2:1 ratio, most likely having a higher concentration of the E isomer due to the higher stability of this isomer. The characterization and identification of this compound was done by 1H-NMR which shows the presence of the enol-ether in two separate isomers.

The stability of this compound was not very high due to the presence of both the bromo- group and the enol-ether in solution. The enol ether group is also very sensitive to low pH, one of the desired features of the paclitaxel, which is why the column was treated with triethylamine before purification. Therefore, the third step must be done relatively quickly after completion of the second step. The third step produced a relatively stable product 4 with a fairly decent yield and relatively high purity. The characterization of this compound was done by 1H-NMR and showed the desired enol-ether phthalimide linker. The spectrum contained the presence of the aromatic protons in the pthalimide group as well as the protons from the two isomers of the enol-ether group in approximately 2:1 ratio as shown in the second step of the synthesis as well.

After verification of the identity of compound 4, the linker was attempted to be coupled to the drug paclitaxel through a reaction of the drug with the linker in the presence of 10% p-toluenesulfonic acid in THF. This reaction scheme was previously shown to conjugate the linker to the hydroxyl group in estradiol. However this seemed to not be viable reaction conditions for the drug paclitaxel. The crude product from the reaction was a brown sticky oil which did not seem to be the product we were looking for. A product was isolated that seemed to be the linker with the enol-ether group cleaved off because it contained the characteristics of the phthalimide as well as an aliphatic carbon chain when analyzed by 1H-NMR. In a separate reaction, a small amount of the paclitaxel was reacted with 10% ptoluenesulfonic acid and a similar product was formed as a brown sticky oil leading to the assumption that the acid denatures the drug to produce an undesirable compound. Therefore, a different conjugation method must be used to attach this linker to the paclitaxel. CONCLUSIONS The synthesis of this enol-ether linker yielded a pure and active compound that could potentially be used to couple with the anti-cancer drug paclitaxel. Although the drug did not hold up well to the reaction with p-toluenesulfonic acid, there is still a possibility that this enol-ether could conjugate to the drug. However, a new coupling reaction must be used to produce the desired linker. Despite the negative result of the last step of this reaction, this linker shows promise as a candidate for targeted drug delivery. REFERENCES Gillies, E., Goodwin, A., Frchet, J. 2004. Acetals as pH-Sensitive Linkages for Drug Delivery. Bioconjugate Chem. 15, 1254-1263. Hideki, O., Tomonori, M., Yoko, S., Masaya, N. 2009. Formation of -hydroxyketones via irregular Wittig reaction. Tetrahedron Letters. 50, 1276-1278. Yu, Y., Zou, J., Yu, L., Ji, Wei., Li, Y., Law, W., Cheng, C. 2011. Functional Polyactide-g-PaclitaxelPoly(ethylene glycol) by Azide-Alkyne Click Chemistry. Macromolecules. 44, 4793-4800.