Synthesis of 8-aminoquinolines as inhibitors of tyrosine kinases

Chronic myelogenous leukemia (CML), characterized by unregulated proliferation of myeloid cells in the bone marrow, accounts for 15 to 20 % of all adult leukemia cases in the Western population. The molecular cause for the disease is the characteristic translocation between chromosome 9 and 22 which results in the so called Philadelphia chromosome (Ph) and in the formation of the chimeric Bcr-Abl gene. In CML the protein product of this hybrid gene is a constitutively active protein kinase. Bcr-Abl kinase drives the pathogenesis of CML through the phosphorylation and activation of a broad range of downstream substrates playing a critical role in cellular signal transduction and cell transformation. ABL tyrosin kinase therefore is an interesting therapeutic target and many potent inhibitors have been developed and brought to the clinic in recent years, including Imatinib, Bosutinib, Nilotinib and Dasatinib. However, the T315I mutant form of Bcr-Abl, which is frequently found in CML patients, mediates complete resistance to Imatinib and all of the next generation Abl kinase inhibitors. Therefore, there is an eminent need for the development of drugs which are active against the T315I mutant of Bcr-Abl. Bosutinib and its quinoline scaffold have been chosen as a template for the construction of a new drug scaffold potentially able to inhibit the mutated Bcr-Abl. Starting from the model of Bosutinib bound to wild-type Abl kinase domain and the model of T315I Abl kinase domain it was apparent that in order to get potent inhibitors of Abl T315I the unfavorable interaction caused by the bulkiness of isoleucine had to be avoided and another strong favorable interaction should be added. The strategy to accomplish this goal was to remove the cyano group at 3 position of the quinoline and strengthen the interaction with the protein by adding an amino group at position 8 to establish an additional hydrogen bond with the backbone carbonyl of M318. Based on this rational, the scaffold of 8-aminoquinolines resulted to be promising for potential Bcr-Abl T315I inhibitors. Three main lines of compounds have been synthetized: 4-substituted-8-aminoquinolines, 3-substituted-8-aminoquinolines, 3,4-disubstituted-8-aminoquinolines. All of these molecules have an oxygen-linked alkyl group at position 7. Different groups have been employed to functionalize the position 7. 4-Substituted-8-aminoquinolines were synthetized starting from a 4,7-dihaloquinoline. Nitration of this molecule yielded the 8-nitro derivative which was then successfully functionalized firstly at position 7, and then at the position 4. Reduction of the nitro group allowed to obtain the desired 4-substituted-8-aminoquinolines. On the contrary, 3-substituted 8-aminoquinolines have been prepared starting from commercially available m-aminophenol. Nitration of this molecule yielded 2-nitro-3-aminophenol which was alkylated with an appropriate side chain at the phenolic oxygen and subsequently cyclized with -bromoacrolein to give the 3-bromo-7-oxyalkyl-8-nitroquinoline. This precursor has been employed for functionalization at position 3. As for the 4-substituted derivatives, the last step was the transformation of nitro group into amino group. 3,4-Disubstituted-8-aminoquinolines, after a first attempt of synthesis from a substituted aniline, were synthetized starting from 4,7-dichloro-8-nitroquinoline. Position 4 was firstly functionalized with an electron donating group such as methoxy or pyrrolidino and bromine was successively introduced at position 3 by an electrophilic aromatic substitution. Attachment of the side chain at position 7 was followed by reduction from 8-nitro to 8-aminoquinoline. For all the synthetized molecules, different groups have been attached at the specific positions either via an oxygen or a nitrogen or a carbon linker. Oxygen and nitrogen linked groups have been introduced in the quinoline scaffold with an aromatic nucleophilic substitution using the corresponding alcoholate or amide. Carbon linked groups have been introduced through palladium catalyzed cross coupling reactions involving the corresponding aryl or alkyl boronic acids. Two alkynyl groups have been introduced under the classic Sonogashira reaction conditions coupling and then reduced to alkyl with hydrogen. All of the molecules synthetized have been tested in biological assays in order to verify their activity toward the enzyme in solution and toward cells expressing the oncogenic enzyme. It was discovered that as hydrochloride salt, the inhibitors were more active than as free base, therefore many inhibitors have been tested as water-soluble salts. Good results have been obtained, since the scaffold resulted active in inhibiting the enzyme. Some of the compounds exhibited inhibitory activity in the nanomolar range. Seven of the most active and specific compounds have also been tested toward a panel of 85 protein kinases (Cohen Lab, Dundee) of different classes in order to asses their selectivity profile. Three compounds (CPD 131, 106 and 148) having nanomolar activity on T315I Abl resulted to have low selectivity on the panel, inhibiting respectively 10, 40 and 11 kinases by more of 50% at a concentration of 10 M. In contrast, CPD 142, 163, 167 and 138 resulted very selective. Among them, CPD 138 appears to be the most selective one because it inhibits only the Aurora A kinases by more than 50% at the tested 10 M concentration. All other compounds inhibited strongly PKB (AKT2) which is a Serine/Threonine kinase, while enzymes more related to Abl, e.g. LCK, SRC and FGFR1, are less targeted by the selected compounds. Insulin receptor kinase, a clear anti-target for protein kinase inhibitors, is not inhibited by these compounds. Interestingly there is an inverse correlation between the selectivity on cells (transduced cells versus non-transduced cells) and the PKB inhibitory activity. The higher the PKB inhibitory activity is the less selective are the compounds. This indicates that PKBis a target for substituted 8-aminoquinolines. In conclusion, a novel active scaffold for the inhibition of Abl WT and Abl T315I has been developed. Very active compounds both on the cellular and enzymatic level have been found. The synthetized Abl T315 inhibitors allowed to asses the proposed binding mode and gave a consistent SAR. Based on the obtained results it is clear that the 8-aminoquinoline-based inhibitors are lead compounds which can be further developed in an optimization process to gain activity and better selectivity in order to be able to enter in vivo studies.