VI. Conclusion and outlook
6.2 Outlook
6.2.1 Hetero-MCO for the synthesis of collections RGD-containing macrocycles
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Chapter VI
VI. Conclusion and outlook
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With the goal to develop a hetero-MCO in mind, we have begun to develop an approach around the incorporation of the tripeptide Arginine-Glycine-Aspartic Acid (RGD), also known as the universal cell recognition sequence.217 The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix (ECM), blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize the RGD sequence in their adhesion protein binding sites.217 As the integrin-mediated cell attachment influences and regulates cell migration, growth, differentiation, and apoptosis, synthetic RGD-containing small molecules can be used to probe integrin functions in various biological systems, and provide a basis for drug design targeting diseases such as cancer and thrombosis.218 Additionally, specific integrins are overexpressed in diseased cells, making the RGD-binding site a direct target for drug design. For example, αvβ3 and αvβ5 integrins mediate the binding of endothelial cells during tumor angiogenesis, and are overexpressed in solid tumors.219 Merck used this receptor specificity to their advantage to design a more specific and less toxic chemotherapeutic. The resulting lead compound, Cilengitide©,220 was an RGD-containing cyclic pentapeptide, which made it to Phase III clinical trials221 before it was discontinued. The rigid structure of cyclic RGD-containing peptides222 has proven useful in the development of Cilengitide© and other analogs. Furthermore, studies that examine the effects of amide to ester substitution resulted in an analog three times as potent,223 which highlights the importance of RGD-containing depsipeptides to medicinal chemistry.
In addition to the usual challenges associated with traditional depsipeptide synthesis, there are a number of challenges associated with the incorporation of arginine into a peptide sequence.
The two main challenges are unwanted deprotection of the delta N-protecting group under basic conditions, and deamidation via nucleophilic attack on the guanidine.224 These two challenges occur more when Cbz protecting groups are used, and can potentially be avoided with the use of Boc protecting groups.224 However, the harsh acidic conditions necessary to remove Boc-groups at the end of the synthesis would likely result in decomposition of the macrocyclic products. In
217 Ruoslahti, E. Annu. Rev. Cell Dev. Biol. 1996, 12, 697.
218 Thundimadathil, J. J. Amino Acids 2012, 2012, 967347.
219 Chen, Z.; Deng, J.; Zhao, Y.; Tao, T. Int. J. Nanomedicine 2012, 7, 3803.
220 Dechantsreiter, M. A.; Planker, E.; Mathä, B.; Lohof, E.; Hölzemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. J. Med. Chem. 1999, 42, 3033.
221 Carlos, M.-M.; Florian, R.; Horst, K. Anti-Cancer Agents Med. Chem. 2010, 10, 753.
222 Haubner, R.; Schmitt, W.; Hölzemann, G.; Goodman, S. L.; Jonczyk, A.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 7881.
223 Cupido, T.; Spengler, J.; Ruiz-Rodriguez, J.; Adan, J.; Mitjans, F.; Piulats, J.; Albericio, F. Angew. Chem. Int.
Ed. 2010, 49, 2732.
224 Bastiaans, H. M. M.; van der Baan, J. L.; Ottenheijm, H. C. J. J. Org. Chem. 1997, 62, 3880.
Scheme 51. Hetero-MCO synthesis of collections of RGD macrocycles
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this work, the challenges associated with Cbz-Arginine incorporation were avoided with the use of a truncated ornithine (Orn)-containing monomer, which can undergo a late-stage guanidinylation to provide the desired arginine.225
Monomer 138, alone, rapidly undergoes macrolactonization when subjected to standard Mitsunobu MCO conditions. However, when subjected to MCO conditions in the presence of excess 74, 18-membered ring 139 was not observed. Instead, formation of RGD-containing oligomers with mono (140) and di-incorporation (141) of 74 were identified by LCMS.226 This preliminary result is promising for our goal of developing a controlled hetero-MCO. Further optimization of conditions, screening of templates, and ITC analysis, is necessary to develop this reaction to its full potential.
225 Wohlrab, A.; Lamer, R.; VanNieuwenhze, M. S. J. Am. Chem. Soc. 2007, 129, 4175.
226 1H NMR spectra of 140 and 141 were very broad, so LCMS is the basis for proposed ring sizes. This reaction was run with 7.0 mg of 138 and 3.6 mg of 74. Isolated approximately 1-2 mgs of 140 and 141.
Scheme 52. Preliminary RGD macrocycle synthesis results: homo-MCO vs hetero-MCO
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A major barrier to this project is the current synthesis of RGD-containing monomer 138, and challenges associated with the compatibility of sidechain protecting groups with the hydroxy terminal-MOM group. The acidic conditions required to remove the MOM group, also result in partial removal of the Orn-Cbz group. Despite much effort in attempting to develop new MOM- deprotection conditions, the yield of the final deprotection step remains low. Therefore, attempting the synthesis with a different hydroxyl protecting group may be the best option, since the Bn and Cbz sidechain protecting groups are necessary to ensure that the macrocyclic products remain intact upon their deprotection.
The MOM group is used in our synthesis of α-oxy bromonitroalkanes because it is easily installed under mild acidic conditions (dimethoxymethane, P2O5). The use of basic conditions to install the hydroxyl protecting group are not tolerated, and result in decomposition through a retro- Henry pathway. However, a cursory protecting group screen revealed that the MOM-group is not the only viable hydroxy protecting group, especially because nitroalkanes are viable donors in UmAS. Bromonitroalkanes bearing 1-ethoxyethyl (EE) or 2-methoxy-2-propyl (MOP) groups spontaneously deprotect/decompose (in my hands). However, nitroalkanes bearing EE or MOP protecting groups can be installed without decomposition, but only EE survived UmAS reasonably well. The EE group can be removed using PPTS (1 equiv), which will not deprotect the Cbz, Bn, or even the highly acid-labile PMB ester. The downside of using the EE group is that it adds a chiral center to all of the EE-containing intermediates, and therefore adds a significant element of complexity227 to reaction monitoring by TLC, purification steps, and subsequent characterization by NMR spectroscopy.