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Novel α-carboranyl-α-acyloxy-amides were prepared as potential BNCT agents utilizing three component Passerini reaction. Preliminary cytotoxicity of the representative compounds on two brain tumor cell lines (U-87 and A-172) showed no effect on cell viability; an essential requirement for utility as potential BNCT agents.
Boron Neutron Capture Therapy (BNCT) is a binary treatment method in which, the cancer cells are loaded with 10B atoms, followed by bombardment with low energy neutrons. The resulting excited 11B nuclei produces high linear energy transfer species causing cell death. Since the range of these particles is one cell diameter, the neighboring healthy cells are usually spared of damage. The success of this modality depends on the preferential accumulation of boron atoms in to the cancer cells. Hence it is very important to synthesize polyboronated molecules that could be selectively delivered to tumor site.1
Multicomponent coupling is an extremely important tool in organic and medicinal chemistry towards the synthesis of structurally diverse scaffolds of biological interest. The isocyanide based Passerini and Ugi coupling reactions offer an easy access to a diverse range of peptidomimetic analogs under mild reaction conditions (Figure 1).2
Low density lipoprotein (LDL) contains about 1500 molecules of cholesterol esters per LDL particle and functions as a main carrier of cholesteryl esters in blood circulation. Several cancers such as malignant human gliomas overexpress LDL receptors and thus consume high levels of LDL derived cholesteryl esters for the cell membrane biosynthesis via receptor mediated endocytosis.3 The evidence that rapidly dividing cancer cells have an elevated requirement for cholesterol can be observed by the 100 fold increase in cholesteryl ester concentration as well as the increased LDL receptor related apoprotein in the vicinity of the glioma cells.3
Liposomes have been extensively studied as carrier molecules due to their ability to deliver a wide range of substrates to tumor sites in a targeted way. The biphasic nature of liposomes facilitates the transportation of lipophilic and hydrophilic compounds readily. This procedure also offers advantages in terms of therapeutic efficacy at a lower dosage, minimal side effects and protection of the structural integrity in blood.4 Currently there are several drugs in clinical usage that are being delivered to the cancer cells via liposomes.5
Carboranes and other polyhedral boranes have been extensively studied as BNCT agents.6 Our continued interest on the functionalization of carboranes7 prompted us to utilize the 3-component Passerini reaction to synthesize α-carboranyl-α-acyloxy-amides as valuable intermediates for BNCT applications. Since the success of BNCT requires transporting boronated molecules in a targeted way into the cancer cells, we also synthesized lipophilic carboranes based on cholesterol and long chain fatty acids as substrates for LDL reconstitution and liposomal encapsulation.
We initiated the synthesis of acyloxyamide-carborane conjugates via the formylation of o-carborane 1. Lithiation of 1 with n-BuLi followed by the addition of methyl formate led to the formation of o-carborane aldehyde 2.8 Three component Passerini reaction of the aldehyde 2 with benzoic acid and benzyl isonitrile in water provided N-benzyl-α-carboranyl-α-benzoyloxy-acetamide 4.9 The reaction mixture was worked up with ethyl acetate the crude product was purified by trituration with hexane and diethyl ether. Similarly, the multicomponent reaction of o-carborane aldehyde with four carboxylic acids (benzoic acid, phenyl acetic acid, isobutyric acid, and pivalic acid) and three isonitriles (benzyl, isopropyl, and t-butyl isonitrile) afforded the carboranyl acyloxyamides 4-15 in good yields (Scheme 1).
After synthesizing the carborane conjugates 4-15, we ventured into the synthesis of few biologically relevant carrier linked carborane conjugates as potential substrates for LDL reconstitution, and liposomal encapsulation for targeted delivery to tumor sites. In this regard, we synthesized cholesterol and long alkyl chain carborane conjugates using Passerini reaction. Succinic acid moiety was chosen as a linker because of its relative non-toxic nature and also to create structural mimics of the native cholesteryl esters.
The cholesterol carborane conjugates 18a-b were prepared in two steps starting from cholesterol. Succinylation of cholesterol afforded the monosuccinate ester, that upon reaction with carborane aldehyde 2 and benzyl or isopropyl isonitriles provided the cholesterol carborane conjugates 18a10 and 18b respectively (Scheme 2).
Similarly, the long chain alkyl carborane conjugates 20a-b were envisioned as the potential substrates for liposomal encapsulation. Thus the reaction of bishexadecyl substituted alcohol 19 with succinic anhydride provided the monosuccinate ester that upon reaction with carborane aldehyde 2 and benzyl or isopropyl isonitrile, provided the lipophilic carboranes 20a11 and 20b respectively (Scheme 3).
After synthesizing various carboranyl acyloxy amides, we carried out the cytotoxicity studies of the representative molecules 4-15. Since the BNCT modality works better on localized cancers (such as brain tumors) than systemic treatment, we chose two human brain cancer cell lines A-172 and U-87 for the current studies. Cells were treated with compounds at a high concentration (50 μM), dissolved in DMSO for 18 h. Cell viability was determined using a colorimetric MTS assay. All the compounds tested were found to be non-toxic12 to both the cancer cell lines thus fulfilling the primary criteria as potential BNCT agents. Future studies would include advanced biological studies especially involving LDL and liposomal encapsulation studies to determine the efficacy of these molecules as potential BNCT agents.
In conclusion, we have synthesized several α-carboranyl-α-acyloxy-amides as valuable intermediates for potential BNCT applications. We have also prepared cholesterol and bishexadecyloxyglyceryl carboranes as targeted molecules for LDL receptor and liposomal encapsulation. Some of these molecules were evaluated for cytotoxicity in two brain tumor cell lines, and found to be non-cytotoxic even at high concentration (50μM), thus fulfilling the basic requirement for utility as BNCT agents. The present work should be of interest to organic, inorganic and medicinal chemists due to the flexibility of the multi-component coupling reactions in providing wide array of carboranyl structural entities.
We thank the Departments of Chemistry and Biochemistry, Rowan University, and University of Minnesota Duluth for the resources and funding. Partial support for this work was provided by research grants from the National Institutes of Health (CA129993) (VRM) and Whiteside Institute for Clinical Research (VRM).
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