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. 2021 Nov 2;12(11):1794–1801. doi: 10.1021/acsmedchemlett.1c00412

Macrocyclization as a Source of Desired Polypharmacology. Discovery of Triple PI3K/mTOR/PIM Inhibitors

Sonia Martínez-González †, Rosa M Alvarez †, José I Martín †, Ana Belén García †, Concepción Riesco-Fagundo †, Carmen Varela †, Antonio Rodríguez Hergueta †, Esther González Cantalapiedra †, M I Albarrán †, Elena Gómez-Casero †, Antonio Cebriá †, Enara Aguirre †, Nuria Ajenjo †, David Cebrián †, Bruno Di Geronimo †, Darren Cunningham ‡, Michael O’Neill ‡, Harish P G Dave §, Carmen Blanco-Aparicio †, Joaquín Pastor †,*
PMCID: PMC8591745  PMID: 34795869

Abstract

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The PI3K/AKT/mTOR and PIM kinase pathways contribute to the development of several hallmarks of cancer. Cotargeting of these pathways has exhibited promising synergistic therapeutic effects in liquid and solid tumor types. To identify molecules with combined activities, we cross-screened our collection of PI3K/(±mTOR) macrocycles (MCXs) and identified the MCX thieno[3,2-d]pyrimidine derivative 2 as a moderate dual PI3K/PIM-1 inhibitor. We report the medicinal chemistry exploration and biological characterization of a series of thieno[3,2-d]pyrimidine MCXs, which led to the discovery of IBL-302 (31), a potent, selective, and orally bioavailable triple PI3K/mTOR/PIM inhibitor. IBL-302, currently in late preclinical development (AUM302), has recently demonstrated efficacy in neuroblastoma and breast cancer xenografts. Additionally, during the course of our experiments, we observed that macrocyclization was essential to obtain the desired multitarget profile. As a matter of example, the open precursors 35–37 were inactive against PIM whereas MCX 28 displayed low nanomolar activity.

Keywords: Polypharmacology, PI3K(±mTOR)/PIM inhibitors, anticancer therapies, macrocycles

The PI3K/AKT/mTOR signaling pathway is implicated in several cellular functions including cell growth, proliferation, differentiation, motility, survival, angiogenesis, and intracellular trafficking. Activation of this pathway contributes to the development of tumors.1 Targeting the PI3K/AKT/mTOR pathway is an important therapeutic approach to treat cancer. Several inhibitors have reached clinical trials, and the United States Food and Drug Administration has approved five of them: alpelisib (PI3Kα inhibitor), idelalisib (PI3Kδ), copanlisib (PI3Kα,δ), duvelisib (PI3Kδ,γ), and umbralisib (PI3Kδ, CK-Iε).2,3

The Proviral Insertion site in Moloney murine leukemia virus (PIM) protein family plays important roles in cell proliferation, apoptosis, cell cycle regulation, survival, and migration.4−6 Overexpression or dysfunction of PIM kinases has been observed in many types of hematological malignancies and solid tumors. Several PIM inhibitors have been reported,7 and among them the pan-PIM inhibitors PIM447, INCB053914, and TP-3654 are currently in clinical trials.8−10

The PI3K/AKT/mTOR and PIM pathways have overlapping functions regulating different cellular processes implicated in tumorigenesis, through the convergence in several substrates such as BAD, c-MYC, PRAS40, and 4E-BP1 among others, so influencing each other.11 Moreover, PIM kinases have been implicated in mechanisms of resistance to PI3K inhibitors.12 Our group authored one of the first published reports demonstrating that the combination of a PI3K inhibitor (GDC-0941) with a PIM inhibitor (ETP-45299) was strongly synergistic and antiproliferative in MV4:11 (AML) cells, indicating that the simultaneous inhibition of PIM and PI3K kinases could have a clinical benefit.13 Since then, several studies have supported the combination of inhibitors of the PI3K/AKT/mTOR and PIM pathways in different liquid and solid tumor types.14−18 Therefore, the presence of this polypharmacology in a single compound could result in better therapeutic effects and potentially lower off-target toxicity compared with the corresponding combination of individual drugs.19,20 To the best of our knowledge, none of the advanced molecules in clinical phases present the desired cross activities against PI3K(±mTOR)/PIM. Only the nonselective research tool PI3K inhibitor LY294002 has been reported as a very weak inhibitor of PIM-1 (IC50 = 4 μM).21

Over the past few years, our group has designed and developed a family of macrocycles (MCXs) inspired by omipalisib (GSK2126458, 1, Table 1), a well-known pan-PI3K and mTOR inhibitor.22 Our MCXs contain key pharmacophoric elements to achieve PI3K(±mTOR) inhibition, such as a N acceptor in the explored bicyclic scaffolds and the N-(2-methoxy-pyridin-3-yl)-benzenesulfonamide motif, both present in omipalisib. This collection of MCXs was prepared by substitution of the bicyclic scaffolds with aryl, heteroaryl, and heterocycloalkyl moieties, which were connected via amide linkers to the aryl ring of the methoxypyridinylbenzenesulfonamide fragment. The cross-screening of these molecules against PIM-1 identified MCX thieno[3,2-d]pyrimidine derivative 2 (unpublished previously, Table 1) as a prototype of dual PI3K/PIM inhibitor (IC50s: PI3K 16.8 nM; PIM-1 279 nM; mTOR > 10 μM). Conversely, MCXs with other scaffolds retained a selective PI3K(±mTOR) profile, without PIM-1 activity as omipalisib.23 Here, we report the medicinal chemistry exploration around MCX 2 which led to the discovery of IBL-302 (31), a potent, selective, and orally bioavailable triple PI3K/mTOR/PIM inhibitor (Table 1). The synthetic route used to prepare the described compounds is outlined in Scheme 1.

Table 1. Biological Activity of GSK2126458 (1) and MCXsa.

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a

The values are the average of three independent experiments expressed in nM, except for those marked with an asterisk (*) that represent duplicate experiments. IC50 was determined for PI3K-α (ADP-Glo), mTOR (LanthaScreen), and PIM-1 (ADP Hunter Plus assay).

Scheme 1. Synthesis of Macrocycles 2, 16–34.

Scheme 1

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Reagents and conditions: (a) LDA, THF, −78 °C, 1–2 h then I2, 2 h. (b) (5-Amino-6-methoxypyridin-3-yl)boronic acid pinacol ester, Na2CO3 2M, PdCl2(PPh3)2, 1,4-dioxane, reflux, 1–30 h, 70–90% yield. (c) Heteroaryl boronic acid/boronate ester, Na2CO3 2 M or saturated aqueous solution, or K2CO3, PdCl2(PPh3)2 or Pd(dppf)Cl2, 1,4-dioxane or DME, 85 °C-120 °C, 1–24 h, 50–80% yield. (d) N-Heterocycles, TEA, nBuOH, 100–120 °C, > 85% yield. (e) 3-(Chlorosulfonyl)benzyl derivative, pyridine, 0 °C to rt. (f). Lithium hydroxide monohydrate or KOH, 1,4-dioxane/water or MeOH/water, rt, >85% yield. (g) DCM/TFA, 0 °C to rt, quantitative yield. (h) HATU, HOAt in DMF, rt. Then slow addition of a solution of 14-15 in DMF and DIPEA, rt, 10–24 h, 4–52% yield.

Commercial available precursors 3 and 4 were transformed into iodine derivatives 5 and 6. Regioselective reaction with (5-amino-6-methoxypyridin-3-yl)boronic acid pinacol ester under Suzuki Pd-catalyzed conditions provided 7 and 8, respectively. Next, Suzuki reaction with corresponding boronic esters yielded 9a–f, 10a–c and nucleophilic aromatic substitution with N-heterocycles gave 10m–o. Then, sulfonamide formation reaction with carboxylic acid/ester phenyl sulfonyl chlorides rendered 12 and 13 in good yields, after basic ester hydrolysis. Compounds 12f–h were synthesized from 7 by doing first the sulfonamide reaction to get 11 and then introducing the 4-position heteroaryl moiety via Pd-catalyzed coupling. Boc deprotection with trifluoroacetic acid provided 14 and 15. Finally, macrocyclization was done via intramolecular lactam bridge formation between amino and carboxylic acid moieties under high dilution conditions to produce MCXs 2 and 16–34. The synthesized macrocyclic compounds were then evaluated against PI3Kα, mTOR, and PIM-1 (Table 1).

We followed several criteria for the design of the initial structure–activity relationship (SAR) exploration of MCX 2 (compounds 2–24). First, we kept the thieno[3,2-d]pyrimidine scaffold unchanged due to the importance of the central core for achieving PIM-1 activity (Table S1). Next, we selected several A-rings which rendered potent PI3K(±mTOR) inhibition with other scaffolds, such as phenyl, 4-pyridyl, 3-pyridyl, and pyrazolyl, with the latter two providing MCXs with good pharmacokinetics.23 Furthermore, we included other phenyl isosteres based on thiophene ring and other 5-membered ring heterocycles such as furane and thiazole. Finally, the methoxypyridinylbenzenesulfonamide fragment and the amide linkage were maintained along the exploration due also to their known contribution to PI3K(±mTOR) inhibition and synthetic feasibility reasons.23

We replaced then the phenyl moiety of 2 with heteroaryls (16–23). This modification led to noticeable changes in the combined activity, including an outstanding improvement in PIM-1 inhibition for certain compounds. The 2-(5-aminomethyl)thiophenyl MCX 16 led to a more than 20-fold increase in PIM-1 potency vs the phenyl analogue 2 (IC50 = 13 nM and 279 nM respectively), and maintained similar PI3K activity. Disappointingly, the corresponding furanyl analogue 20 did not improve the PIM-1 activity of MCX 2 and was demonstrated to be a weak PI3K inhibitor (IC50 = 381 nM). The replacement by thiazole (23) was also quite detrimental for PI3K and PIM-1 activities (IC50 = 123 nM and >10 μM, respectively). 2-(4-Aminomethyl)thiophenyl MCX 21 showed an improvement in PIM-1 activity (IC50 = 13 nM), and it was 2-fold less potent than 2 against PI3K. By contrast, 3-(5-aminomethy)thiophenyl 22 was an almost 2-fold better PIM-1 inhibitor (IC50 = 154 nM) than 2 and maintained the PI3K activity. The replacement of the phenyl ring of 2 with a pyrazol moiety (18) produced a negligible effect on PIM-1 (IC50 = 254 nM) but afforded 3-fold weaker PI3K inhibition (IC50 = 58 nM). Other replacements by 6-membered aromatic heterocycles such as 3-pyridyl (17) led to 6-fold better potency for PIM-1 (IC50 = 46 nM) and decreased PI3K inhibition (IC50 = 75 nM). The corresponding 4-pyridyl analogue (19) proved to be a worse PI3K/PIM-1 dual inhibitor (IC50 = 50 nM and 649 nM respectively). Regarding mTOR, none of the analogues of MCX 2 displayed significant inhibition, showing IC50 values greater than 10 μM with the exception of MCX 16 (IC50 = 5.7 μM).

Next, compound 16, with nanomolar dual PI3K/PIM-1 activity and signs of mTOR inhibition, was selected for further exploration (24–28). The presence of a methyl group at the 2-thiophenyl ring (24) was slightly detrimental for the PIM-1 activity (IC50 = 44 nM) but gave better PI3K inhibition (7 nM) when compared with MCX 16. Substitutions in the para-position of the phenyl ring (MeO–, 25; F–, 26; and Cl–, 27) were allowed and resulted in ∼3-fold improvement in PI3K potency (∼6 nM all) while maintaining PIM-1 activity in the halo-derivatives (IC50 = 15 nM and 11 nM) but suffered a drop in MCX 25 (IC50 = 66 nM). MCX 26 improved the mTOR activity over 16, but it was still a micromolar inhibitor.

We also investigated the presence of a methyl group at the 7-position of the thienopyrimidine scaffold (MCX 28). This modification produced a remarkable improvement of the combined PI3K/PIM-1 activity (IC50 = 0.2 nM and 18 nM) and a significant increase of mTOR inhibition (IC50 = 138 nM). The observed boost in PI3K/mTOR activity was likely due to the accommodation of the methyl group of 28 into a small hydrophobic pocket available in the hinge region of these kinases. The filling of this pocket has been used to gain activity and overall kinase selectivity for PI3K inhibitors.24−27

Based on the interesting biochemical profile of 28, we explored other MCXs keeping the 7-methyl thienopyrimidine core (29–34). We also incorporated C-4 heterocycloalkyl rings, which could act as nonaromatic bioisosteres. We observed a significant improvement in PI3K activity in MCXs 29–31, compared to the nonmethyl analogues 17, 18, and 26. The impact of the methyl fragment was outstanding in the case of the 3-pyrazolyl MCX 30 and the F-derivative MCX 31, yielding subnanomolar PI3K inhibition (IC50 = 0.4 nM, and 0.1 nM respectively). The mTOR activity of the methylated MCXs followed a similar trend, transforming the inactive unsubstituted MCXs 17 and 18 into submicromolar inhibitors 29 and 30 (IC50 ∼ 200 nM). Notably, MCXs 31 reached a potent IC50 value of 74 nM. Regarding PIM-1 activity, the effect of the methyl-thienopyrimidine scaffold was less important and only minor changes were observed when keeping the IC50 values in the same range as the corresponding unmethylated pairs.

The exploration of MCXs with nonaromatic heterocycles, such as morpholine (S-enantiomer) and pyrrolidine (R- and S-enantiomers), gave potent PI3K and PIM-1 activity, demonstrating the feasibility of this bioisosteric replacement in these molecules. However, the dual PI3K/PIM activity of MCXs 32–34 was slightly below that of the thienyl analogue 28. Furthermore, the mTOR activity was weak for the morpholine MCX and negligible for the pyrrolydine enantiomers.

In general, the SAR interpretation for MCXs is often complex and less straightforward than that for “open small molecules”. The coupling of bond rotations and intramolecular interactions could enable the transmission of three-dimensional information from one side of a MCX to the other. In other words, making relatively small structural modifications to a MCX can result in local conformational changes that propagate along the ring to affect distal structural features.28 The combination of the C4-substituent and the methylation of the scaffold in our MCX could lead to different preferred conformations for each, which would affect their key interactions and activity against PI3K/mTOR/PIM.

The conformational sampling of MCXs and the selection of candidate conformers for molecular modeling studies is not trivial and straightforward even for available software packages.29 Based on these precedents, we did not attempt to interpret in detail the complete SAR, as it could be quite speculative in the absence of protein–MCX complex structures. Nevertheless, we carried out tentative molecular docking studies of MCXs 2, 16, and 28 in PIM-1 (PDB ID 4A7C). All MCXs adopted similar binding modes, accommodating the thienopyrimidine scaffold in the hinge area of PIM-1. The methoxypyridinylbenzenesulfonamide fragment displayed key electrostatic and H-bond interactions with Lys67. Of note, MCXs 16 and 28 established extra bidentate H-bond interactions between the amide linker with the backbone N–H of Ser46 and the backbone C=O of Glu171. MCX 2 only showed a single H-bond with the backbone N–H of Ser46. The presence of the thienyl moiety in MCXs 16 and 28 favored a conformation where the amide group of the linker was well positioned to produce the double interaction (Figure S1). Perhaps the PIM-1 activity of the explored MCXs could be rationalized using similar arguments. The Induced Fit Docking (IFD) scores of the proposed models were similar: 2, −10.05 kcal mol–1; 16, −10.67 kcal mol–1; and 28, −11.04 kcal mol–1. The IFD scoring function ranked the three MCXs with similar binding affinity in their models, but its performance was limited to address the differences in activity of ∼1 log unit observed between MCX 2 and the thienyl analogues 16–28. This was not surprising since accurate prediction of binding affinity remains a challenging task in the field of structure-based virtual screening.30

We were intrigued by the contribution of the macrocyclization step to the observed PI3K(±mTOR)/PIM activity of the potent thieno[3,2-d]pyrimidine MCXs. We selected MCX 28 as a case study and synthesized and tested the open precursors 35–37 (Figure 1). We hypothesized that the PI3K(±mTOR) activity of 28 was brought about primarily by the open precursors, which share key pharmacophoric features with GSK2126458 (1), and not due to their macrocyclization. As expected, the macrocyclization was not significant for the PI3K activity of 28 since 35–37 were already low nanomolar PI3K inhibitors. The mTOR activity was also modulated by the ring closing step in functionalized precursors 35 and 36. Conversely, the contribution of macrocyclization to the PIM-1 activity of MCX 28 was outstanding and unexpected. The open precursors 35–37 were inactive, whereas 28 displayed a potent nanomolar PIM-1 inhibition. The IFD models of MCX 28 and open analogues 35–37 in PIM-1 revealed similar binding modes for all compounds except for 35. The IFD score of MCX 28 (−11.04 kcal mol–1) was much higher than IFD scores of the open analogues (35 = −7.19 kcal mol–1, 36 = −8.00 kcal mol–1, 37 = −6.45 kcal mol–1), supporting the experimental data. The IFD scoring function was able to discern among highly active and inactive compounds.

Figure 1.

Figure 1

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Open precursors and biological activity in comparison with MCX 28.

To the best of our knowledge, it is the first reported case in which a macrocyclization strategy has achieved a desired polypharmacological profile in a MCX, starting from an “open precursor” without said combined activity.31 It is likely that the results of 28 could be extrapolated to all MCXs with potent PIM-1 activity.

The SAR studies identified compounds with dual PI3K/PIM activity and most notably the triple PI3K/mTOR/PIM-1 inhibitors (28 and 31). These MCXs were tested against PI3K and PIM isoforms (Table 2). Both MCXs demonstrated a strong pan-PI3K profile, already observed for PI3K inhibitors of this class,23 displaying subnanomolar Ki values. They also behaved as pan-PIM inhibitors with IC50 values in the nanomolar range. The pan-PIM profile has been postulated as the preferred one for a PIM inhibitor since it would avoid compensatory overexpression of PIM-2,3 isoforms when only PIM-1 is inhibited.32

Table 2. PI3K Isoform Ki and PIM Isoform IC50 Dataa.

Nr PI3Kα PI3Kβ PI3Kδ PI3Kγ PIM-1 PIM-2 PIM-3
28 0.25 0.37 0.08 0.44 18.2 13.3 4.39
31 0.27 0.31 0.13 0.17 22.8 8.04 5.75
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a

The values are the average of two independent experiments in nM. Ki for PI3K isoforms was determined with a HTRF technology assay. IC50 for PIM-1 was determined via ADP Hunter Plus assay.

Next, we evaluated the cellular activity of 28 and 31 in comparison with GSK2126458 (1) in the MV4:11 cell line, which has activated PI3K/mTOR and PIM pathways. Inhibition of phospho-AKT on Ser473 and phospho-BAD on Ser112 was measured as downstream targets.33,34 MCXs 28 and 31 displayed potent modulation of both pathways with EC50 values in the low nanomolar range for inhibition of p-AKT (EC50 < 1.0 nM both; assay limit) and p-BAD (EC50 = 34 nM and 60 nM respectively). As expected, GSK2126458 (1) was able to inhibit the phosphorylation of AKT (EC50 < 10 nM) but not p-BAD (EC50 > 10 μM).

Both MCXs displayed excellent in vitro metabolic stability (>98%) in human liver microsomes. However, in mouse and rat microsomes, 28 (90%, 87% respectively) was more stable than 31 (63%, 65%). The inhibition of CYPs-450 was negligible, above 50 μM for both with the exception of CYP-2C9 where MCX 28 gave micromolar activity (IC50 = 4.61 μM), which is far from its nanomolar PI3K/mTOR/PIM activity. Furthermore, we did not detect hERG binding below 30 μM for both inhibitors. Preliminary pharmacokinetic studies were performed in BALB-C mice after I.V. and P.O. administration (Table 3). The plasma clearance was low and almost identical (0.06 L/h/kg), which represented ∼1% of the hepatic blood flow for mice, and their volume of distribution was low to moderate (Vd ∼ 0.3 L/kg). Both MCXs were orally bioavailable with absorbed fractions of 35% and 124%, respectively, at the doses tested.

Table 3. Pharmacokinetic Profile of MCXs 28 and 31a.

route parameter 28 31
I.V. dose 1 0.7
  AUClast 16 596 11 176
  T1/2 3.66 2.96
  Cl 0.060 0.063
  Vd 0.31 0.26
P.O. dose 10 5
  Cmax 2929 9370
  Tmax 0.25 5
  T1/2 23.4 14.6
  AUClast 58 235 99 055
  F% 35.08 124
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a

PK studies. The data were adjusted to a noncompartmental model using Winnolin software. Units: Dose (mg/kg); area under the curve AUC (h.ng/mL); half-life T1/2 (h); clearance Cl (L/h/kg); volume of distribution Vd (L/kg); maximun concentration Cmax (ng/mL); time of maximum concentration Tmax (h).

Despite the good overall profile of both MCXs, compound 31 was prioritized because of to its more balanced triple PI3K/mTOR/PIM inhibition and pharmacokinetics. The kinase selectivity of MCX 31 was assessed in a high-throughput competition binding assay against a panel of 468 kinases at 1 μM (KINOMEscan assay platform).35 The selectivity of 31 was outstanding with a selectivity score S (35) of 0.052 (Figure S2, Table S2). 31 exhibited strong inhibition of the signal produced by untreated controls for their expected targets of PI3K isoforms and mutants, and mTOR and PIM isoforms. The only major off-target detected and confirmed was TTK (Kd 0.9 nM).

SAR studies identified that MCX 28 and 31 had a potent pan-PI3K and pan-PIM profile. These compounds were subsequently renamed as IBL-301 and IBL-302 (Inflection Biosciences), respectively, and later as AUM301 and AUM302 (AUM Biosciences), respectively. MCX 28 (IBL-301/AUM301) decreased PIM-1, c-Myc, pBAD, p4EBP1 (Thr37/46), and peIF4B (S406) protein levels in vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in NSCLC tumor tissue explants.36 Subsequently, MCX 31 (IBL-302/AUM302) was taken up as the lead candidate for further development. It has shown efficacy and tolerability, for instance, in a neuroblastoma xenograft and PDX model, leading to prolonged survival of treated mice as single agent, and in combination with low doses of cisplatin.37 Furthermore, in HER2/+PI3KCA mutated breast cancer cell line derived xenografts, it has also shown single-agent efficacy.38 Currently, AUM302 is in late preclinical development and undergoing IND-enabling studies.

In summary, we have discovered and characterized a series of novel orally bioavailable, cell active, and selective triple PI3K/mTOR/PIM inhibitors, exemplified by the advanced candidate MCX 31 (IBL-302/AUM302), currently in late preclinical development. To the best of our knowledge, this is the first reported case of MCXs which enable a pursued polypharmacology not available in their non-MCX precursors. This strategy could be applied by medicinal chemists to other bioactive compounds to enrich their target profile and efficacy.

Acknowledgments

We thank Genoveva Mateos and Manuel Urbano for compound handling and support of databases and Elena Hernández-Encinas who reanalyzed PK data. We are thankful for the financial support of Ministry of Science and Innovation (MICINN) of Spain (ADE08/90038).

Glossary

Abbreviations

MCX

macrocycle

AML

acute myeloid leukemia

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00412.

  • Details of synthetic procedures, docking studies, NMR spectra, and selectivity and biological assays (PDF)

Author Present Address

# Medicinal Chemistry. Diseases of the developing world, GSK. C/Severo Ochoa, 2, E-28760, Spain

Author Present Address

Lilly S.A., Av. de la Industria, 30, Alcobendas, E-28108, Spain

Author Present Address

∇ Pharmacology unit. GSK. C/Severo Ochoa, 2, E-28760, Spain

Author Present Address

∥ Bristol Myers Squibb, C/Quintanadueñas 6, E-28050, Spain

Author Present Address

⊥ Tumour Bank Unit, CNIO

Author Contributions

The manuscript was written through contributions of all authors./All authors have given approval to the final version of the manuscript.

The Ministry of Science and Innovation (MICINN) of Spain supported this research through the project ADE08/90038.

The authors declare no competing financial interest.

Special Issue

Published as part of the ACS Medicinal Chemistry Letters virtual special issue “Medicinal Chemistry in Portugal and Spain: A Strong Iberian Alliance”.

Supplementary Material

ml1c00412_si_001.pdf (1.6MB, pdf)

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