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Introduction of pyrrolidineoxy or piperidineamino group at the 4-position of quinazoline leading to novel quinazoline-based phosphoinositide 3-kinase delta (PI3Kδ) inhibitors

Abstract Phosphoinositide 3-kinase Delta (PI3Kδ) plays a key role in B-cell signal transduction and inhibition of PI3Kδ was confirmed to have clinical benefit in certain types of activation of B-cell malignancies. Herein, we reported a novel series of 4-pyrrolidineoxy or 4-piperidineamino substituted quinazolines, showing potent PI3Kδ inhibitory activities. Among these compounds, 12d, 14b and 14c demonstrated higher potency against PI3Kδ with the half maximal inhibitory concentration (IC50) values of 4.5, 3.0, and 3.9 nM, respectively, which were comparable to idelalisib (IC50 = 2.7 nM). The further PI3K isoforms selectivity evaluation showed that compounds 12d, 14b and 14c have excellent PI3Kδ selectivity over PI3Kα, PI3Kβ, and PI3Kγ. Moreover, compounds 12d, 14b and 14c also displayed different anti-proliferative profiles against a panel of four human B cell lines including Ramos, Raji, RPMI-8226, and SU-DHL-6. The molecular docking simulation indicated several key hydrogen bonding interactions were formed. This study suggests the introduction of pyrrolidineoxy or piperidineamino groups into the 4-position of quinazoline leads to new potent and selective PI3Kδ inhibitors.


Introduction
Phosphoinositide 3-kinases (PI3Ks) play a pivotal role in multiple cellular functions including cell growth, development, migration, angiogenesis, and survival 1 . Upon stimulation of cytokine signaling, PI3Ks function as an intracellular secondary messenger transforming phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3) via phosphorylation catalysation, thereafter activation of the downstream signal transducer (Akt, mTOR) and subsequent activator of transcription 2 . There are four PI3K isoforms validated, including PI3Ka, PI3Kb, PI3Kc, and PI3Kd. PI3Ka and PI3Kb are ubiquitously expressed in multiple cells whereas PI3Kd and PI3Kc are identified as predominant expression in hematopoietic cells 3 . In particular, PI3Kd is found responsible for the B-cell receptor (BCR) signaling downstream transduction and constitutive studies show activation of BCR signaling pathway is a hallmark of B-cell malignancies such as chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), and indolent non-Hodgkin's lymphoma (iNHL) 4 . Therefore, PI3Kd is thought as suitable target for the potential treatment of certain B-cell malignancies, as well as immunologic disorders (due to its specific role in controlling immune cell function) 5 . Notably, small molecules selective PI3Kd inhibitor idelalisib (Compound 1) has been recently approved by Food and Drug Administration (FDA) for treatment of CLL, FL, and SLL, which solidify the therapeutic concept of PI3Kd inhibitor (Figure 1) 6,7 .
Despite the first-in-class approved, potent and oral selective PI3Kd inhibitor, idelalisib was tagged with black-box warning and demonstrated struggling with severe adverse events in the later clinical validation 8 . Therefore, there is an urgent need to develop second generation PI3Kd inhibitor with lower toxicity and fewer side effects. Duvelisib (Compound 2), another potent PI3Kd inhibitor, shared chemical similarity to idelalisib, however, this was recently terminated in the phase III clinical trials due to underneath efficiency. Many other analogues derived from the chemical structure of idelalisib were recently reported and showed strong PI3Kd efficacy and selectivity, for instances Compounds 3 (PI3Kd: half maximal inhibitory concentration (IC 50 ) ¼ 2.2 nM), 4 (PI3Kd: IC 50 ¼ 1.0 nM), and 5 (PI3Kd: IC 50 ¼ 4.6 nM) [9][10][11] . Nevertheless, our drug discovery efforts are engaged into the development of PI3Kd inhibitors with novel and distinct chemotypes. Recently, we reported a new series of potent PI3Kd inhibitors, chemically featured by a quinazoline scaffold and a 6-benzamide moiety such as Compound 7 (PI3Kd: IC 50 ¼ 17 nM) 12 , derived from the Novartis's patented Compound 6 (PI3Kd: IC 50 ¼ 9 nM) with potent PI3Kd inhibition and selectivity [13][14][15] . A subsequent structural modification was carried out and a series of 4-anilinequinazolines was synthesised, exemplified by Compound 8 (PI3Kd: IC 50 ¼ 9.3 nM) showing improved PI3Kd inhibition 16 . Later, further structural investigation by replacing the 4-aniline with a 4-pyrrolidineamino moiety led to a series of potent and selective PI3Kd inhibitors, such as Compound 9 (PI3Kd: IC 50 ¼ 2.7 nM), showing equivalence to idelalisib in our examination (Figure 1

PI3Kd inhibitory activity for the title compounds
All the newly synthesised compounds were assessed for their PI3Kd inhibitory activities and idelalisib was employed as the positive control. The 4-pyrrolidineoxy substituted quinazoline analogs were firstly examined and the results are shown in Table 1   inhibitory activity, which was approximately equivalent to control drug idelalisib (IC 50 ¼ 2.7 nM; Table 1).
Subsequently, the 4-piperidineamino substituted quinazoline analogues were evaluated and the data are shown in Table 2. The initial (S)-4-(1-Boc-piperidin-3-yl)amino Compound 14a showed weak PI3Kd inhibitory activity with an inhibitory ratio of 51% at the concentration of 100 nM. However, replacement of tert-butoxy group with diverse cyclic aliphatic substituents afforded highly improved PI3Kd inhibitory activity. Analogue of Compound 14b bearing a cyclopropyl group gave an IC 50 value of 3 nM, and analogue of Compound 14c tailed with a cyclobutyl group showed almost comparable potency, with an IC 50 value of 3.9 nM, whereas analogues of Compound 14d with a cyclopentyl terminal and Compound 14e containing a tetrahydro-2H-pyran-4-yl tail showed a little less potent PI3Kd inhibition than that of Compound 14b, with IC 50 values of 8.7 and 5.2 nM, respectively. Again, an attempt to shift the cyclic group to non-cyclic alkyl group such as tert-butyl (Compound 14f: 54%) resulted in PI3Kd inhibition largely reduced. Otherwise, an exploration of changing the (S)-4-(piperidin-3-yl)amino side chain into 4-(piperidin-4-yl)amino group was also conducted, and three analogues were synthesised. However, unfortunately, Compound 16a bearing a Boc group (Compound 16a: 72%) and Compound 16b with a cyclopropyl group (Compound 16 b: 70%) showed moderate PI3Kd inhibition, while Compound 16c incorporated with tetrahydro-2H-pyran-4-yl group(Compound 16c: 48%) produced unsatisfactorily weak potency. This suggested the spatial orientation of the tailed acyl substituents was critical for PI3Kd inhibition, which was consistent to the structure-activity relationship of our previously reported 6aryl substituted 4-anilinequinazoline series. In this preliminary PI3Kd inhibition evaluation, three compounds 12d, 14b, and 14c showed IC 50 values beneath 5 nM, being approximately comparable to idelalisib, which were picked out for further evaluation.

Isoform selectivity of the new PI3Kd inhibitors
Based on the above preliminary PI3Kd inhibitory activity results, Compounds 12d, 14b, and 14c were subsequently evaluated for their selectivity among PI3Ka, PI3Kb, and PI3Kc. As shown in Table 3, all three compounds 12d, 14b, and 14c showed much lower potency against other three PI3K isoforms than that of PI3Kd, although they displayed moderate PI3Ka inhibition. Compound 12d with an IC 50 value of 4.5 nM against PI3Kd demonstrated 11-fold, 131-fold, and 103-fold selectivity over PI3Ka, PI3Kb and PI3Kc, respectively, whereas Compounds 14 b and 14c displayed the similar PI3Kd selectivity which were 12-and 15-fold over PI3Ka, 105and 96-fold over PI3Kb, and 34-and 31-fold over PI3Kc, respectively. Moreover, it was noted that selectivity of compound 12d against the PI3Kb and PI3Kc isoforms was much higher than idelalisib, although the poor selectivity against PI3Ka was observed (Table 3).

In vitro anti-proliferative assays of the new PI3Kd inhibitors
Furthermore, Compounds 12d, 14b, and 14c were tested for their anti-proliferative activities against four human B cell lines including Ramos, Raji, RPMI-8226, and SU-DHL-6with idelalisib and SAHA as reference compounds. As shown in Table 4, Compound 12d exhibited most potent anti-proliferation against RPMI-8226 (IC 50 ¼ 44 nM) among these four cell lines, whereas Compound 14b showed significantly potent anti-proliferative activity against Ramos, Raji, and SU-DHL-6, but moderate anti-proliferation against RPMI-8226 and Compound 14c also showed strong anti-proliferativity against SU-DHL-6 with an IC 50 value of 1.49 nM. It was found that the reference PI3Kd inhibitor idelalisib displayed markedly anti-proliferative activity against SU-DHL-6, whereas another reference drug SAHA (vorinostat) afforded significantly anti-proliferation against Ramos, Raji, and RPMI-8226. In a word, three Compounds 12d, 14b, and 14c as well as idelalisib were observed showing different anti-proliferative profiles in the four human B cell lines (Table 4).

Molecular modeling study
To further understand the potent PI3Kd inhibition, molecular docking simulations of Compounds 12d, 14b, and 14c within human PI3Kd enzyme were performed. As shown in Figure 3, the docked pose of each Compound (12d, 14b and 14c) ma es the similarly favorable interactions with the PI3Kd binding pocket of structure 2WXP as expected, namely, three key hydrogen bonds with the hinge residue, the quinazoline scaffold with Val828, the methoxypyridyl moiety with Lys779, as well as the carbonyl group with Asn836. Moreove r, it was observed that, although, the oxygen of the tetrahydro-2H-pyran-4-yl group in Compound 20a formed an additional hydrogen bond with Asp753, it seemed to show little contribution for improving the inhibitory activity in this case (Figure 3).
profiles against a panel of four human B cell lines. The molecular docking study indicated several key hydrogen bonding interactions formations, which may explain their higher PI3Kd. This study suggests the introduction of pyrrolidineoxy or piperidineamino groups into the 4-position of quinazoline leads to new potent and selective PI3Kd inhibitors Disclosure statement