with an oxygen which would not only change the electronics of the bicyclic ring but would also have different hydrogen bonding capabilities compared to Series I. Compounds 181-185 will evaluate the effect of replacing the pyrrolo[3,2-d]pyrimidine scaffold in 161 with a furo[3,2-d]pyrimidine scaffold and the importance of hydrogen bond donor vs acceptor at the 5-position.
Figure 29. Series VI
Substituted 7-benzyl pyrrolo[2,3-d]pyrimidines with general structure 186 (Figure 30) were reported as antiangiogenic, antimetastatic and antitumor agents.170 Substituents on
the benzyl group and the location of the benzyl group dictate RTK inhibitory activity in pyrrolo[2,3-d]pyrimidines. Hence, having determined the antitubulin effects of 161, it
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Figure 30. Series VII
was of interest to engineer RTK inhibitory activity without loss of antitubulin activity by incorporating the 7-benzyl group onto the pyrrolo[3,2-d]pyrimidine scaffold in 161. The results of this hybrid design afforded compound 187. Compounds 188 and 189 were designed to evaluate the importance of the 4'-OCH3 and the 4-NCH3 moieties,
respectively. Compound 190 was designed by incorporating a 2'-OCH3 group to explore
the effect of substituents at this position.
The general RTK pharmacophore model consists of an Adenine region, a Sugar binding pocket and a Phosphate binding region which binds the adenine ring, the sugar moiety and the triphosphate moiety of ATP respectively. Additionally, there are two hydrophobic regions I and II, neither of which are used by ATP for binding. The pyrrolo[3,2-d]pyrimidine ring of the designed compounds could bind to the Adenine
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Binding mode 1
Binding mode 2
In both binding modes 187 makes a hydrogen bond interaction with the backbone of Cysteine in the hinge region.
Figure 31. Predicted binding modes of 187 in VEGFR2
region similar to heterocyclic scaffold inhibitors such as pyrimidines, quinazolines and pyrrolo[2,3-d]pyrimidines.171 The 7-benzyl group was could access the Sugar binding
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pocket as shown in mode 1 [Figure 31]. Alternatively, the pyrrolo[3,2-d]pyrimidine compounds could adopt mode 2 in which the compounds are rotated around the 2-CH3-
C2 bond. In this mode the 7-benzyl group and the 4-anilino groups could occupy Hydrophobic region I and Sugar binding pockets respectively. These compounds could also adopt binding mode 3 in which the molecule is rotated by 60o (from mode 2). In this mode, the 7-benzyl group could orient towards the Phosphate binding pocket and the 4- anilino groups could occupy Hydrophobic region II. Docking studies performed on the X-ray crystal structure of VEGFR2 [pdb: IYWN]172 suggested the possibility of several binding modes. Representative low energy binding modes are shown in Figure 31.
Biological evaluation indicated that a single agent 187 that was designed to have both cytotoxic and antiangiogenic effects did indeed display these activities. Compound
187 has an inhibitory potency comparable with the clinically used sunitinib and clinically
evaluated semaxinib against VEGFR2. The antiangiogenic effect was probably mediated via VEGFR2 inhibition. The cytotoxic effect was mediated by tubulin inhibition and was independent of overexpression of Pgp and βIII-tubulin. The compound caused cellular microtubule depolymerization, arrested cells in the G2/M phase and triggered apoptotic
cell death.
Compound 187, showed a 3-digit nanomolar GI50 in all the NCI 60 tumor cell lines,
showing moderate cytotoxic activity (as desired) against tumor cells. The antiangiogenic component of 187 is not active in these cell culture assays. This perhaps is an indication of low toxicity to normal cells.
In vivo, compound 187 reduced tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 and U251 glioma models] and in a 4T1 triple negative
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breast orthotopic allograft model. In these in vivo models, the activity of 187 was superior to those of clinically used agents temozolomide (U251), docetaxel and sunitinib (MDA- MB-435 and 4T1) without overt toxicity to the animals.
Design of single agents with combination chemotherapy and multiple RTK inhibitory potential
The complexity of the angiogenic pathways implies that disrupting only a single aspect of angiogenesis may not result in significant clinical success. Multiple RTKs are co-activated in tumors and redundant inputs drive and maintain downstream signaling, thereby limiting the efficacy of therapies targeting single RTKs.19, 173 Resistance to anti- VEGF treatment is associated with increased PDGFR expression within the tumor, increased recruitment of pericytes to tumor vasculature, and increases in other proangiogenic factors.174 Preclinical models suggest that PDGF-mediated recruitment of pericytes may contribute to resistance to VEGF blockade. EGFR inhibition can lead to VEGFR2 up-regulation which subsequently promotes tumor growth signaling independent of EGFR and thus contributes to the resistance of EGFR inhibitors.175, 176
The effect of EGFR inhibition can also be partially overcome by activation of PDGFR signaling and the subsequent transactivation of HER-3 signaling to promote alternate tumor growth signaling.20, 175 Hence, targeting multiple RTKs maximizes the proportion
of angiogenic signalling that is effectively targeted.173, 175 Moreover, high intratumoral heterogeneity has been reported with different subpopulations producing distinct growth factors. 177-179 Targeting a single RTK could be ineffective due to subpopulations of cells that are either not affected by the drug mechanism and possess or acquire a greater drug
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resistance.180 Hence it was of interest to explore the effect of different structural changes on activity against the RTKs VEGFR2, PDGFRβ and EGFR in addition to having cytotoxic antitubulin effects with the goal of identifying single agents with antitubulin and multiple RTK inhibitory potential.
Figure 32. Series VIII