Wei Yan

Lingtian Zhang

Fengping Lv

Marialuisa Moccia

Francesca Carlomagno

Christophe Landry

Massimo Santoro

Fabien Gosselet

Brendan Frett

Ph.D Hong-Yu Li

Sarah Skerratt
email: sarahskerratt1@gmail.com

Nicholas Mcconnell
email: nicholas.mcconnell@ucsf.edu

Discovery of pyrozolo-thienopyrimidinylamino-phenyl acetamides as type-II pan-tropomyosin receptor kinase (TRK) inhibitors: design, synthesis, and biological evaluation

Keywords: kinase inhibitor, TRKA, type-II, colorectal cancer kinase inhibitor, TRK, type-II, colorectal cancer

published or not. The documents may come

Introduction

The tropomyosin receptor kinase (TRK) family enzymes belong to the transmembrane receptor tyrosine kinases superfamily. The TRKs are primarily expressed in human neuronal tissues that regulate synaptic strength and plasticity in the mammalian nervous system. [START_REF] Lai | TrkB phosphorylation by Cdk5 is required for activity-dependent structural plasticity and spatial memory[END_REF][START_REF] Huang | Trk receptors: roles in neuronal signal transduction[END_REF] The TRK family consists of three subtypes, and each is activated by a specific ligand: TRKA and TRKB are activated by nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), respectively, while TRKC is primarily turned on by neurotrophin 3 (NT3). [START_REF] Lai | TrkB phosphorylation by Cdk5 is required for activity-dependent structural plasticity and spatial memory[END_REF][START_REF] Huang | Trk receptors: roles in neuronal signal transduction[END_REF][START_REF] Yan | Insights into current tropomyosin receptor kinase (TRK) inhibitors: development and clinical application[END_REF] Upon ligand binding, TRKs become dimerized and autophosphorylated thus trigger downstream signal transduction pathways including RAS/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT), and phospholipase C-γ (PLC-γ)/PKC pathways, which consequently modulate cell growth, proliferation, survival, and differentiation. [START_REF] Amatu | NTRK gene fusions as novel targets of cancer therapy across multiple tumour types[END_REF] Initially, the TRKA oncogene was discovered in colon cancer. [START_REF] Martin-Zanca | A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences[END_REF][START_REF] Martin-Zanca | Molecular and biochemical characterization of the human trk proto-oncogene[END_REF] So far, NTRK fusions are found to occur in 3.3% of lung cancers, 2.2% of colorectal cancers, 16.7% of thyroid cancers, 2.5% of glioblastomas, and 7.1% of pediatric gliomas. [START_REF] Yan | Insights into current tropomyosin receptor kinase (TRK) inhibitors: development and clinical application[END_REF] Within the TRK family, TRKA is the most commonly identified oncogene, which is found at a rate of approximately 7.4% across multiple tumor types (0.4% for TRKB and 3.4% for TRKA, respectively). [START_REF] Yan | Insights into current tropomyosin receptor kinase (TRK) inhibitors: development and clinical application[END_REF][START_REF] Vaishnavi | TRKing down an old oncogene in a new era of targeted therapy[END_REF] Moreover, it has been found that TRKA also mediates the stimulation of early tumor progression. [START_REF] Edel | An in vivo functional genetic screen reveals a role for the TRK-T3 oncogene in tumor progression[END_REF] Therefore, the blockade of TRKA signaling is considered as an attractive clinical approach for the treatment of cancers. To date, only two TRKA inhibitors were approved by U.S. food and drug administration. Larotrectinib was approved in November 2018 for solid tumors with NTRK gene fusions;[9] and entrectinib was approved in August 2019 for NTRK gene fusion-positive solid tumors or ROS1-positive non-small cell lung cancer.

[10] According to the classification of Shokat et al., [START_REF] Dar | The evolution of protein kinase inhibitors from antagonists to agonists of cellular signaling[END_REF] these two drugs both belong to type-I kinase inhibitor. Type-I inhibitors bind to the kinase only at the highly conserved adenosine triphosphate (ATP) binding site in the "DFG-motif in"

(active) conformation and are thus less selective or active. Distinct from type-I inhibitors, type-II inhibitors extend themselves into an allosteric hydrophobic pocket adjacent to the ATP binding site and bind to the kinase in the "DFG-motif out" (inactive) conformation, which makes type-II inhibitors more active; moreover, given that the allosteric pocket residues are much less conserved between kinases, type-II inhibitors are therefore more selective as well.

One concern in the development of TRK kinase inhibitors is the potential risk of central nervous system (CNS) adverse events, regarding to their critical roles in the CNS normal function. [START_REF] Mattson | Glutamate and neurotrophic factors in neuronal plasticity and disease[END_REF][START_REF] Noble | The lighter side of BDNF[END_REF][START_REF] Sanchez-Ortiz | TrkA gene ablation in basal forebrain results in dysfunction of the cholinergic circuitry[END_REF][START_REF] Nikoletopoulou | Neurotrophin receptors TrkA and TrkC cause neuronal death whereas TrkB does not[END_REF] Unwanted adverse events related to the drug exposure in the CNS had been reported. [START_REF] Wang | Trk kinase inhibitors as new treatments for cancer and pain[END_REF] Therefore, the development of peripherally restricted TRK inhibitors is a rational and feasible approach to minimize the potential CNS toxicity. [START_REF] Skerratt | The discovery of a potent, selective, and peripherally restricted pan-Trk inhibitor (PF-06273340) for the treatment of pain[END_REF] Because of their binding modes, type-II kinase inhibitors generally bear a larger molecular weight and are more polar than type-I inhibitors, and are thus considered to be less permeable through the blood-brain barrier (BBB). [START_REF] Obermeier | Development, maintenance and disruption of the blood-brain barrier[END_REF][START_REF] Daneman | The blood-brain barrier[END_REF] For example, the classical type-II BCR-ABL inhibitor imatinib (Gleevec®) exhibited excellent efficacy and safety profile and low CNS exposure. [START_REF] Takayama | Imatinib mesylate has limited activity against the central nervous system involvement of Philadelphia chromosome-positive acute lymphoblastic leukaemia due to poor penetration into cerebrospinal fluid[END_REF] In this context, the development of type-II TRKA inhibitors is highly desired. Currently, several type-II TRK inhibitors have been reported, but the majority of them are in the early discovery stage. [START_REF] Yan | Insights into current tropomyosin receptor kinase (TRK) inhibitors: development and clinical application[END_REF][START_REF] Subramanian | Lead identification and characterization of hTrkA type 2 inhibitors[END_REF][START_REF] Wang | Discovery of (E)-N-(4-methyl-5-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-yl)thiazol-2-yl)-2-(4methylpiperazin-1-yl)acetamide (IHMT-TRK-284) as a novel orally available Type II TRK kinase inhibitor capable of overcoming multiple resistant mutants[END_REF][START_REF] Cui | Design, synthesis and biological evaluation of 3-(imidazo[1,2-a]pyrazin-3-ylethynyl)-2-methylbenzamides as potent and selective pantropomyosin receptor kinase (TRK) inhibitors[END_REF] One of the most advanced type-II TRKA inhibitor CE-245677 was discontinued in Phase I clinical trials; [START_REF] Skerratt | The discovery of a potent, selective, and peripherally restricted pan-Trk inhibitor (PF-06273340) for the treatment of pain[END_REF] its successor PF-06273340 completed the Phase I clinical trial in 2014, but no further progress was reported ever since. [START_REF] Loudon | Demonstration of an anti-hyperalgesic effect of a novel pan-Trk inhibitor PF-06273340 in a battery of human evoked pain models[END_REF] Therefore, the exploitation of novel chemical scaffolds for type-II TRKA inhibitors is highly demanded.

As a part of our continuous effort on the development of kinase inhibitors, [START_REF] Yan | Discovery of 1,3-diaryl-pyridones as potent VEGFR-2 inhibitors: design, synthesis, and biological evaluation[END_REF][START_REF] Yan | Discovery of 3-(5'-substituted)-benzimidazole-5-(1-(3,5-dichloropyridin-4-yl)ethoxy)-1H-indazoles as potent fibroblast growth factor receptor inhibitors: design, synthesis, and biological evaluation[END_REF][START_REF] Frett | Fragment-based discovery of a dual pan-RET/VEGFR2 kinase inhibitor optimized for single-agent polypharmacology[END_REF][START_REF] Moccia | Bioisosteric discovery of NPA101.3, a second-generation RET/VEGFR2 inhibitor optimized for single-agent polypharmacology[END_REF][START_REF] Lakkaniga | Pyrrolo[2,3-d]pyrimidine derivatives as inhibitors of RET: Design, synthesis and biological evaluation[END_REF][START_REF] Lakkaniga | Discovery of SP-96, the first non-ATP-competitive Aurora Kinase B inhibitor, for reduced myelosuppression[END_REF] especially TRKA inhibitors, [START_REF] Frett | Identification of pyrazine-based TrkA inhibitors: design, synthesis, evaluation, and computational modeling studies[END_REF] we recently screened a small in-house kinase compounds library, and compounds 1b and 2b were found to exhibit moderate TRKA inhibitory activity. Although thienopyrimidines derivatives had been reported having a variety of biological activities including anti-parasitic activity [START_REF] Woodring | Evaluation of aromatic 6-substituted thienopyrimidines as scaffolds against parasites that cause trypanosomiasis, leishmaniasis, and malaria[END_REF]; Bertrand et al, disclosed the co-crystal structure of EX429 with TRKA and TRKB, [START_REF] Bertrand | The crystal structures of trkA and trkB suggest key regions for achieving selective inhibition[END_REF] but the medicinal chemistry effort towards the discovery of this compound remained unknown. Therefore, we hypothesized that our thienopyrimidine derivativescould serve as a starting point for the discovery and development of novel TRK pan-inhibitors. From the initial hits 1b and 2b, utilizing the structure-based drug design (SBDD) approach, a series of potent TRKA inhibitors was designed and synthesized. The representative lead 4c exhibited strong in vitro TRKA inhibition and anti-proliferative activity.

In this paper, we report the design, synthesis, and biological evaluation of these compounds.

Results and discussion

Structure and activity relationship (SAR)

We quickly synthesized a few analogues of compounds 1b and 2b to explore the SAR of these two closely related scaffolds. Unfortunately, all compounds 1a-1c and 2a-2c were only moderately or weakly active in enzymatic and cellular assays (Tables 1 &2). To understand the binding mode and further improve the potency, compounds 1b and 2b were docked to the TRKA protein (PDB ID: 6PL1). As shown in Figure 2A, compound 1b bound to the TRKA protein in a classical type-II manner: a π-π interaction was found between the phenyl ring and Phe589, two hydrogen bonds with Glu560 and DFG-motif residue Asp668 were formed from the amide moiety, and the allosteric DFG pocket was occupied with the 3-(tert-butyl)-1-methyl-1H-pyrazole moiety;

while in compound 2b, a hydrogen bond from the N1 of 4-amino-thieno [3,2-d]pyrimidine was formed with the hinge region residue Met592 (Figure 2B). Compounds 1a-1c features with the direct carbon-carbon connection between the phenyl ring and the thieno-pyrimidine core. Although the structural feature had been proved to be favored in a few kinase inhibitors (e.g. JAK inhibitor ruxolitinib and BTK inhibitor ibrutinib), [START_REF] Saha | The Exploration of Chirality for Improved Druggability within the Human Kinome[END_REF] based on the molecular docking result, we discovered that this structural type is too rigid to have a strong binding with the TRKA kinase. In compounds 2a-2c, the phenyl ring was bridged to the thieno-pyrimidine core through a nitrogen atom, which allows a flexible binding. As shown in the superimposed structures of 1b and 2b (Figure 2C), the spatial distance between the phenyl rings of 1b and 2b was only 1.8 Å. Therefore, we envisioned that a hybrid of these two scaffolds would be able to retain the original crucial interactions with the TRKA protein while maintaining a flexible binding for the optimal affinity. A series of compounds were then synthesized and evaluated. The SAR study showed that the nature of R 3 group in the allosteric pocket had significant impacts on the activity (Table 3). Similar to that observed in the compound 1 series, aliphatic group substituted derivatives (3a and 3b) were 2-40 fold less active than aromatic substituted derivatives (3c-f) in enzymatic assays, and the cellular potency was completely abolished. Among the aromatic substituted derivatives, the five-membered heterocyclic rings (3e-f) were more favored than substituted phenyls (3c-d). Moreover, the ethynyl derivative 3d was about 3-fold less active than its trifluoromethyl analogue 3c, probably due to a steric clash between the ethynyl fragment and the allosteric pocket residues. Compound 3e was docked with TRKA (Figure 3). As expected, the π-π interaction between the phenyl acetamide fragment and the enzyme was retained, and a hydrogen bond was found between the hinge region residue Met592 and the N1 atom of compound 3e. carbon green, oxygen red, nitrogen blue, sulfur yellow, and hydrogen white.

Although compounds in the series III displayed excellent enzymatic potency in the low micromolar range, but the cellular potency was relatively weak. We envisioned that the low potency in cells could be due to poor aqueous solubility. As it was observed in the binding study of compound 3e with TRKA kinase (Figure 3), the thiophenyl moiety of thieno-pyrimidine orients toward to the solvent front. We hypothesized that the introduction of a polar substituent group to the thiophenyl moiety could improve aqueous solubility while maintaining good ligand-protein interactions. Therefore, a methyl-pyrazole group was introduced to the 6-position of the thienopyrimidine moiety. To our delight, the resulting compounds 4a and 4b displayed approximately 10-fold increase in both enzymatic and cellular potencies than the parent compounds 3c and 3d (Table 3); strikingly, 4c demonstrated a 20-fold improvement in enzymatic potency and 100-fold improvement in cellular potency than the parent compound 3e.

Encouraged by these promising results, we proceeded to synthesize more derivatives to further explore SAR in the allosteric pocket. As shown in Table 3, the replacement of methyl-pyrazole in 4c with thiazole (compound 4d) resulted in 20-and 150-fold decreases in enzymatic and cellular potencies, respectively.

Compound 4e with morpholinoethyl attached to the pyrazole, exhibited a comparable enzymatic potency to 4c;

but was far less potent in the cellular assay. It can be explained with that the high polarity of compound 4e limits its cell membrane permeability. Several compounds bearing a phenyl ring (4f-h) showed comparable cellular potency with 4a and 4b, although compound 4h was less active in the enzymatic assay than other compounds (4f-g). When the terminal phenyl group was replaced with a benzylic group (4i), a 25-fold drop in potency was observed (vs 4a), indicating a potential steric clash with a surrounding residue; while a pyridine replacement (4j) resulted in a 70-fold decrease in enzymatic potency, probably due to an unfavored electrostatic interaction between pyridine and surrounding residues. Diaryl urea is an important pharmacophore in constructing type-II anticancer kinase inhibitors, exemplified by the success of sorafenib (Nexavar®). Therefore, compound 4k bearing a urea linker was prepared and evaluated, which demonstrated comparable activities in both enzymatic and cellular levels as its amide partner 4c. Superimposed structures of 4c and 4e. The atoms of 4e are colored as follows: carbon brown, oxygen red, nitrogen blue, sulfur yellow, and hydrogen white. Zoomed view on the morpholine moiety of 4e.

As illustrated in the proposed binding mode of 4c with the TRKA kinase (Figure 4A), all interactions observed between 3e and the TRKA enzyme were retained with compound 4c, with the terminal pyrazole group laying at the solvent front (Figure 4B). To rationalize the excellent enzymatic potency, compound 4e was docked with the TRKA protein as well. Compound 4e nicely superimposed with 4c; meanwhile, the morpholine moiety of 4e extended out of the allosteric hydrophobic pocket and was able to form a salt bridge with the carboxylic acid group of Glu560 or Asp668 (Figure 4C). This proposed binding mode explained its excellent enzymatic potency very well. The low potency could be explained with the low cell permeability of compound 4e.

To explore the SAR in the R 5 region, the methyl-pyrazole was replaced (Table 5). 4-(Methylsulfonyl)phenyl group was established as a bioisosteric group of methyl-pyrazole which can avoid the formation of de-methylation metabolite. [START_REF] Moccia | Bioisosteric discovery of NPA101.3, a second-generation RET/VEGFR2 inhibitor optimized for single-agent polypharmacology[END_REF] However, the resulting compounds 4l and 4m totally abolished the potency in the enzymatic assay. Although it is unexpected, the loss of the potency can be partially explained with potential steric clash between the bulky methylsulfonyl (4l) or trimethoxyl group (4m) with surrounding residues; because as observed in Figure 4B, the methyl-pyrazole of 4c was tightly surrounded by the solvent front residues. In line with the above speculation, a less hindered pyrrolidin-ethoxy-phenyl group was well tolerated and compound 4n displayed a comparable enzymatic and cellular potency to compound 4h. The tolerability of a solubilizing group from the pyrazole moiety is an important SAR discovery which would enable the further optimization of solubility and drug properties. In order to explore the ability of these new compounds to inhibit TRK-derived oncoproteins in cells, we analyzed their effects on the phosphorylation of TRKA using the human colorectal cancer derived cell line KM12 which endogenously harbours TPM3-TRKA rearrangement. As shown in Figure 5, compounds 4c and 4k inhibited the TPM3-TRKA autophosphorylation in a dose-dependent manner. Both compounds completely inhibited TPM3-TRKA autophosphorylation at the concentration of 100 nM; while 4k was able to significantly inhibit the TPM3-TRKA autophosphorylation on Y490 and Y674/Y675 at the concentration of 10 nM. These results suggested that both compounds 4c and 4k exhibited an effective blockage of the TRKA autophosphorylation with a relatively low concentration in cells, which is correlated with the anti-proliferative activity.

Selectivity profile

To assess the selectivity of our compounds, inhibitory activities of 4c and 4g were tested against a panel of kinases and NCI-60 human cancer cell lines, respectively. As shown in Table 6, 4c exhibited pan-TRK inhibitory activity and was almost equal potent against TRKA-C, but was much less active against other kinases.

Furthermore, as illustrated in Figure 6, 4g also exhibited excellent selectivity against TRKA-driven KM12 cells.

At the drug concentration of 10 µM, 4g demonstrated nearly 80% of growth inhibition for KM12, while only minimal inhibitory activity was observed for the remaining 59 cancer cell lines. These data indicated our compounds are selective against TRK oncogenes at both enzymatic and cellular levels. To evaluate the BBB permeability, entrectinib and 4c were subjected to the in vitro BBB transport assay.

As shown in Figure 7, the diffusion of entrectinib (permeability coefficient (Pe) = 1.02 x 10 -3 cm/min) across the BBB was observed in our human in vitro BBB model, which was in line with published data that entrectinib crossed the BBB in the mouse xenograft model. [START_REF] Menichincheri | Discovery of entrectinib: a new 3-aminoindazole as a potent anaplastic lymphoma kinase (ALK), c-ros oncogene 1 kinase (ROS1), and pan-tropomyosine receptor kinases (Pan-TRKs) inhibitor[END_REF] Compound 4c (Pe = 3.03 × 10 -3 cm/min) was more permeable than entrectinib, which was predicted from its lower molecular weight (500.6 Da vs 560.6 Da of entrectinib) and higher predicted LogP (5.15 vs 4.45 of entrectinib). We envisioned that by strategically increasing molecular weight and lowing LogP could lead to the discovery of derivatives having desirable cancer cell killing activity and low BBB permeability.

Chemistry

As shown in Scheme 1, the key intermediates acids 7, 11, 14, and 15 were synthesized through either a telescope coupling-hydrolysis reaction (7 and 14) or a successive substitution-hydrolysis reaction (11 and 15).

Final compounds 1, 3, and 4a-j were synthesized from the corresponding acids and amines using HATU as the acylation reagent in DMF at room temperature; amide 16 was synthesized from acid 15 following the same procedure, and then compound 16 was coupled with the corresponding boronic acids to give final compounds to give compound 22, but the self-cyclized product 23 was also isolated in 30% yield, which was not expected since the amino group was hindered by the bulky Ts groups. The Ts-protection group was removed by stirring to give compound 24 in H2SO4 at ambient temperature. Amine 24 was too hydrophilic to be extracted from water.

Therefore, the reaction mixture was concentrated after base quenching, and the resulting crude was used directly for next step without purification.

Experimental Section

Chemistry.

All starting materials and reagents were either obtained from commercial suppliers or prepared according to literature reported procedures. All purchased chemicals and solvents were used without further purification unless otherwise noted. Flash chromatography was performed using silica gel (300-400 mesh) on Teledyne ISCO CombiFlash. All reactions were monitored by thin-layer chromatography (TLC) or liquid chromatographymass spectrometry (LC-MS) (Thermo Surveyor HPLC System with Thermo Finnigan LCQ Deca). 1

N-(3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)-2-(4-(thieno[3,2-d]pyrimidin-4-yl)phenyl)acetamide (1b).

The title compound was synthesized from 7 and 3-(tert-butyl)-1-methyl-1H-pyrazol-5-amine following general procedure A. White solid (82%). 1 H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 9.27 (s, 1H), 8.57 (d, J = 5.5 Hz, 1H), 8.17 1.19 (s, 9H). 13

N-(3-ethynylphenyl)-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)amino)phenyl)acetamide (4b).

The title compound was synthesized from 14 and 3-ethynylaniline following general procedure A. Beige solid (73%). 1

N-(3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4yl)amino)phenyl)acetamide (4c).

The title compound was synthesized from 14 and 3-(tert-butyl)-1-methyl-1Hpyrazol-5-amine following general procedure A. White solid (58%). 1

N-(4-(tert-butyl)thiazol-2-yl)-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4yl)amino)phenyl)acetamide (4d

N-(3-(tert-butyl)-1-(2-morpholinoethyl)-1H-pyrazol-5-yl)-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2d]pyrimidin-4-yl)amino)phenyl)acetamide (4e).

The title compound was synthesized from 14 and 25 following general procedure A. Beige solid (55%). 1

N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4yl)amino)phenyl)acetamide (4f

N-(4-(tert-butyl)phenyl)-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)amino)phenyl)acetamide (4g).

The title compound was synthesized from 14 and 4-(tert-butyl)aniline following general procedure A.

White solid (73%). 1

N-(3-fluorobenzyl)-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)amino)phenyl)acetamide (4i).

The title compound was synthesized from 14 and (3-fluorophenyl)methanamine following general procedure A.

Yellow solid (58%).

Synthesis of compound 4k.

Step 1. 18 (0.3 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.5 mmol), and NaHCO3 (1.2 mmol) were dissolved in THF (2 mL) and H2O (2 mL). The solution was degassed for 5 min, then Pd(PPh3)4 (0.03 mmol) was added. The reaction mixture was irradiated under microwave at 140 °C for 0.5 h.

The consumption of starting materials was detected by LC-MS analysis. The reaction mixture was concentrated in vacuum. The resulting residue was purified by silica gel chromatography to get yellow solid (120 mg, yield 86%). 1

Synthesis of compound 24.

Step 1. 2-(5-amino-3-(tert-butyl)-1H-pyrazol-1-yl)ethanol [START_REF] Saha | The Exploration of Chirality for Improved Druggability within the Human Kinome[END_REF]

Enzymatic assay:

TRKA-C activity was measured via a mobility shift assay monitoring the separation of the phosphorylated product from substrate. In 96-well plates, compound stock solution (20 mM in DMSO) were diluted with kinase buffer (10 mM HEPES, 0.05% Triton X-100, 5% glycerol, 5 mM DTT, 10 mM MgCl2, 10 mM MnCl2 and 0.1 mg/mL BSA) in 12-point ½log dilutions (2 mM-6.32 nM). The TRKA enzyme (Life Technologies, CA, USA) was diluted to 2.35 µg/ml with the kinase buffer. Then, 4 µl of the TRKA solution and 1 µl of the diluted compound solution were transferred to a 384-well assay plate to be incubated for 1 hour. 1 µl of kinase buffer was added in the positive control instead of the compound solution and 5 µl of the kinase buffer were added to the negative control.

Cellular assay:

KM12 cells were cultured using RPMI-1640 (Gibco, CA, USA) growth medium containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, USA), 100 U/ml penicillin (Gibco, Carlsbad, USA) and 100 U/ml streptomycin (Gibco, CA, USA). The cells were cultured at 37°C in a humidified atmosphere with 5% CO2. To do the viability assay, cells were seeded in 96-well cell culture plates. After 24-hour incubation, cells were exposed to 7 concentrations of compounds ranging from 200 to 0.0002 µM for 72 hours. Meantime, DMSO were added to the controls. Resazurin staining viability assay (Biotium, CA, USA) was used to determine the cell viability at the end of compound treatment. Then, IC50 values were calculated from the dose-response curves which were generated in GraphPad Prism 8. Error was determined as the standard deviation between two independent IC50 measurements.

Western blot:

Cells were harvested in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1.5 mM MgCl2, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml) and clarified by centrifugation at 10,000 x g. Protein concentration was estimated with a modified Bradford assay (Bio-Rad, Munich, Germany). Antigens were revealed by an enhanced chemiluminescence detection kit (Pierce ECL, ThermoFisher Scientific, Waltham, MA, USA) [START_REF] Sanchez-Ortiz | TrkA gene ablation in basal forebrain results in dysfunction of the cholinergic circuitry[END_REF]. Phospho-TRKA (Santa Cruz, CA, USA). Anti phospho-tyrosine, clone 4G10 (05-321) antibody was from Merck Millipore (Billerica, MA, USA). Secondary antibodies coupled to horseradish peroxidase were purchased from Bio-Rad.

Molecular modeling:

Molecular modeling studies were performed using AutoDock Tools, AutoDock Vina, [START_REF] Menichincheri | Discovery of entrectinib: a new 3-aminoindazole as a potent anaplastic lymphoma kinase (ALK), c-ros oncogene 1 kinase (ROS1), and pan-tropomyosine receptor kinases (Pan-TRKs) inhibitor[END_REF]

In vitro human brain-like endothelial cells (BLEC) BBB model:

A human BBB model was then used in order to predict entrectinib and 4c passage. The human in vitro BBB model derived from hematopoietic stem cells was prepared as described previously. [START_REF] Cecchelli | A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells[END_REF] Infants' parents signed a consent form and cells were manipulated following the procedure validated by the local ethical committee and French ministry of Research. Briefly, CD34+ cells were isolated from human umbilical cord blood by ficoll density gradient and using the mini-MACS immunomagnetic separation system (Miltenyi Biotec, Bergisch Gladbach), according to the manufacturer's recommendations. These CD34 positive cells were subsequently were transferred onto 1% (w/v) gelatin coated 24-well plates (2 x 10 5 cells/well) and incubated in endothelial growth medium (EGM-2; Lonza, Gaithersburg, MD, USA) with 20% (v/v) fetal bovine serum (FBS; Invitrogen,Carlsbad, USA) and 50 ng/mL vascular endothelial growth factor (VEGF165; PrepoTech Inc., Rocky Hill, USA), at 5% CO2, 37°C. After 2-3 weeks they are differentiated into endothelial cells and endothelial markers are checked by western blot, FACs and quantitative PCR. [START_REF] Pedroso | Improved survival, vascular differentiation and wound healing potential of stem cells co-cultured with endothelial cells[END_REF] Then, CD34+derived endothelial cells were co-cultivated with bovine brain pericytes in Transwell™ Costar polycarbonate inserts, 0.4 μm pore size, coated with matrigel® (Corning) in a complete medium (ScienCell). Brain péricytes were isolated as previously described. [START_REF] Risau | Blood-brain barrier pericytes are the main source of gamma-glutamyltranspeptidase activity in brain capillaries[END_REF] After 6 days of co-culture, CD34+-derived endothelial cells have acquired BBB properties induced by brain pericytes as previously described. [START_REF] Heymans | Contribution of brain pericytes in blood-brain barrier formation and maintenance: a transcriptomic study of cocultured human endothelial cells derived from hematopoietic stem cells[END_REF] This model is then named Human brain like-endothelial cells (BLECs) and is routinely used to determine molecules toxicity and delivery into the CNS.

Endothelial permeability measurement

Endothelial permeability coefficients were determined as described by Dehouck et al. [START_REF] Dehouck | Drug transfer across the blood-brain barrier: correlation between in vitro and in vivo models[END_REF] The inserts, containing an endothelial cell monolayer or only coated with matrigel, were transferred into 12-well plates containing 1.5 mL of Ringer-HEPES (RH) bufferer saline (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2, 0.6 mM NaHCO3, 5 mM HEPES, pH 7.4) per well, thus constituting the abluminal compartment. Then, 0.5 mL of RH buffer saline containing 10 µM of the compound to be tested (entrectinib or 4c) was added to the upper (luminal) compartment, in place of the culture medium in contact with the BLECs. After different timepoints (30, 60, and 120 min), filters were transferred into a new well filled with RH buffer saline. All incubations were performed at 37 °C and stopped at 120 minutes.

To assess the toxicity of the molecules on the BLECs, Lucifer yellow (LY) (Sigma-Aldrich, L0259) that poorly crosses the BBB, was used as the monolayer integrity marker. Diffusion of 25 µM LY was tested alone (control)

or incubated with entrectinib or 4c, as described above. At 120 min, a 200 µL aliquot from a lower compartment at each time point and a 20 µL aliquot of the initial solution placed in the upper compartment were measured using a fluorimeter (Synergy H1; BioTek, Winooski, VT, USA) to calculate the LY concentration.

Concentrations of entrectinib or 4c were determined by LC-MS (Waters ACQUITY UPLC System with ACQUITY QDa mass spectrometer) in the solutions from the upper and lower compartments. The endothelial permeability coefficients (Pe) to LY, entrectinib and 4c, were calculated using the clearance principle to generate a concentration-independent parameter as described by Siflinger-Birnboim et al. [START_REF] Siflinger-Birnboim | Molecular sieving characteristics of the cultured endothelial monolayer[END_REF] First, the volumes cleared were plotted versus time (30 and 60 min) and the slopes were estimated by linear regression for both insert only coated with matrigel (PSf) and for insert coated with matrigel and seeded with cells (PSt). PSe was calculated according to the following formula: 1/PSe = 1/PSt-1/PSf where "S" represented the surface area of the porous membrane of the insert. PSe was divided by the membrane surface (1.12 cm²) to generate the endothelial Pe to the studied molecules expressed in cm/min.

To assess possible adsorption, aggregation and/or degradation of molecules during the transport experiments, their mass balances (recovery in %) were calculated at 120 min from the amount of compound recovered in both compartments at the end of the experiment divided by the total amount added in the donor compartment at the start of the experiment.

Figure 1 .

 1 Figure 1. Chemical structures of larotrectinib (Vitrakvi®) and entrectinib (Rozlytrek®).

6 a

 6 IC50 values are shown as the mean ± SD of two independent assays. b Inhibition percentage values are shown as the average of two independent assays.

Figure 2 .

 2 Figure 2. Proposed binding modes of 1b and 2b with TRKA. The protein structure of TRKA is shown as orange ribbon. The hinge region of TRKA is colored in violet, and the DFG-motif is colored in blue. Key residues are colored as follows: carbon grey, oxygen red, nitrogen blue, and hydrogen white. The hydrogen bonds are colored in black, and π-π interaction is colored in teal. (A) The atoms of 1b are colored as follows: carbon green, oxygen red, nitrogen blue, sulfur yellow, and hydrogen white. (B) The atoms of 2b are colored as follows: carbon brown, nitrogen blue, sulfur yellow, fluorine cyan, and hydrogen white. (C) Superimposed structures of 1b and 2b. Zoomed view on the phenyl rings.

Figure 3 .

 3 Figure 3. Proposed binding mode of compounds 3e with TRKA. The protein structure of TRKA is shown as orange ribbon. The hinge region of TRKA is colored in violet, and the DFG-motif is colored in blue. Key residues are colored as follows: carbon grey, oxygen red, nitrogen blue, and hydrogen white. The hydrogen bonds are colored in black, and pi-pi interaction is colored in teal. The atoms of 3e are colored as follows:

Figure 4 .

 4 Figure 4. Proposed binding mode of compounds 4c and 4e with TRKA. The protein structure of TRKA is shown as orange ribbon. The hinge region of TRKA is colored in violet, and the DFG-motif is colored in blue. Key residues are colored as follows: carbon grey, oxygen red, nitrogen blue, and hydrogen white. The hydrogen bonds are colored in black. (A) The atoms of 4c are colored as follows: carbon green, oxygen red, nitrogen blue, sulfur yellow, and hydrogen white. (B) The protein structure of TRKA is shown as an azure surface. (C)

11 aFigure 5 .

 115 Figure 5. Inhibition of TPM3-TRKA phosphorylation by 4c, and 4k in human colorectal carcinoma derived cell

Figure 6 .

 6 Figure 6. NCI-60 human cancer cell lines screen of compound 4g.

Figure 7 .

 7 Figure 7. Transport assessment of entrectinib and 4c across in vitro BBB model.

4l- n .

 n Compounds 12 and 17 were irradiated under microwave to produce 18, which underwent a successive coupling and de-protection procedure to generate amine 19. Urea compound 4k was prepared from aniline 19 and 3-(tert-butyl)-1-methyl-1H-pyrazol-5-amine in the presence of triphosgene. To synthesize intermediate 24, 20 was reacted with an excessive amount of TsCl to give compound 21; compound 21 was heated in morpholine

Scheme 1 .Scheme 2 .

 12 Scheme 1. Synthesis of final compounds. Reagents and conditions: (a) Pd(PPh3)4, NaHCO3, THF, H2O, microwave, 140 °C, 0.5 h; (b) LiOH, THF, H2O, microwave, 80 °C, 0.5 h; (c) amines, HATU, Et3N, DMF, R.T., overnight; (d) i PrOH, microwave, 100 °C, 0.5 h.

  (d, J = 7.0 Hz, 2H), 7.73 (d, J = 5.5 Hz, 1H), 7.61 (d, J = 7.2 Hz, 2H), 6.08 (s, 1H), 3.84 (s, 2H), 3.61 (s, 3H),

  4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)-N-(3-(trifluoromethyl)phenyl)acetamide (3c). The title compound was synthesized from 11 and 3-(trifluoromethyl)aniline following general procedure A. Beige solid (88%).

1 .

 1 H NMR (400 MHz, CDCl3) δ 8.56 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.52 -7.45 (m, 3H), 7.42 (d, J = 8.4 Hz, 2H), 6.64 (s, 1H), 3.94 (s, 3H), 1.55 (s, 9H). LC-MS (ESI) m/z: [M + H] + 423.2. The solid was dissolved in DCM (2 mL) and TFA (1 mL) was added. The mixture was stirred at ambient temperature li completion of the reaction was monitored by LC-MS analysis. The reaction mixture was concentrated in vacuum. The resulting residue was dissolved in DCM (100 mL) and washed with saturated NaHCO3 solution, water and brine twice. The organic phase was concentrated in vacuum to get 19 as a yellow solid (85 mg, 93%). 1 H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H), 7.70 (s, 1H), 7.63 (s, 1H), 7.30 (s, 1H), 7.22 (d, J = 8.6 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 3.93 (s, 3H). LC-MS (ESI) m/z: [M + H] + 323.Step 2. 3-(tert-butyl)-1-methyl-1H-pyrazol-5-amine (0.06 mmol) and triphogene (0.03 mmol) was dissolved in DCM (1 mL). A solution of Et3N in DCM (0.15 mmol Et3N in 1 mL DCM) was added slowly at 0 °C. The reaction mixture was warmed to ambient temperature and stirred for 2 h. The reaction mixture was added slowly into a solution of 19 (0.05 mmol) in DCM (1 mL) at ambient temperature. The resulting reaction mixture was stirred at ambient temperature for 5 h. The completion of the reaction was monitored by LC-MS analysis. The reaction mixture was poured into water and extracted with DCM. The organic phase was washed with water and brine twice, and concentrated in vacuum. The resulting residue was purified by silica gel chromatography to get 4k as a yellow solid (20 mg, 66%). 1 H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 7.78 (s, 1H), 7.70 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H),

  , 9H). LC-MS (ESI) m/z: [M + H] + 502.0. HPLC purity: 97.2%, tR = 7.90 min. HRMS (ESI) m/z: [M + H] + calcd for C25H28N9OS 502.2138; found 502.2128.General procedure C: Synthesis of compounds 4l-n.[START_REF] Wang | Trk kinase inhibitors as new treatments for cancer and pain[END_REF] (0.1 mmol), boronic acid (0.15 mmol), and NaHCO3 (0.3 mmol) were dissolved in THF (2mL) and H2O (2mL). The solution was degassed for 5 min, then Pd(PPh3)4 (0.005 mmol) was added. The reaction mixture was irradiated under microwave at 140 °C for 0.5 h. The consumption of starting materials was detected by LC-MS analysis. The reaction mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and brine twice, and concentrated in vacuum. The resulting residue was purified by silica gel chromatography to get the title compound.N-(3-chloro-4-fluorophenyl)-2-(4-((6-(4-(methylsulfonyl)phenyl)thieno[3,2-d]pyrimidin-4yl)amino)phenyl)acetamide (4l). The title compound was synthesized from 16 and (4-(methylsulfonyl)phenyl)boronic acid following general procedure C. Yellow solid (66%).1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.83 (s, 1H), 8.59 (s, 1H), 8.17 -8.10 (m, 3H), 8.06 (d, J = 8.5 Hz, 2H), 7.94 (dd, J = 6.8, 2.5 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.52 -7.47 (m, 1H), 7.40 -7.31 (m, 3H), 3.65 (s, 2H), 3.29 (s, 3H).

1 .

 1 General procedure D: Synthesis of acids 7 and 14. Chloride 5 or bromide 13(3.5 mmol), boronic ester (4.1 mmol), and NaHCO3 (14 .0 mmol) were dissolved in THF (5mL) and H2O (5mL). The solution was degassed for 5 min, then Pd(PPh3)4 (0.2 mmol) was added. The reaction mixture was irradiated under microwave at 140 °C for 0.5 h. The consumption of starting materials was detected by LC-MS analysis. LiOH (14.0 mmol) was added and the reaction mixture was irradiated under microwave at 100 °C for 0.5 h. The consumption of starting materials was detected by LC-MS analysis. The resulting mixture was adjusted to pH 3-4 with 1 N HCl aq solution. The resulting suspension was stirred for 5 h then filtered. The residue was dried under vacuum at 45 °C overnight to get the titled compound.2-(4-(thieno[3,2-d]pyrimidin-4-yl)phenyl)acetic acid[START_REF] Vaishnavi | TRKing down an old oncogene in a new era of targeted therapy[END_REF]. The title compound was synthesized from 5 and 6 following general procedure D. Grey solid (yield, 82%)1 H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.57 (d, J = 5.5 Hz, 1H), 8.14 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 5.5 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 3.73 (s, 2H). LC-MS (ESI) m/z: [M + H] + 271.0.2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)amino)phenyl)acetic acid (14).The title compound was synthesized from 13 and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole following general procedure D. Grey solid (yield, 77%) 1 H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H), 9.58 (s, 1H), 8.51 (s, 1H), 8.29 (s, 1H), 7.96 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.54 (s, 1H), 7.24 (d, J = 8.4 Hz, 2H), 3.91 (s, 3H), 3.55 (s, 2H). LC-MS (ESI) m/z: [M + H] + 366.Synthesis of compound 11. A mixture of 10 (4.0 mmol) and LiOH (16.0 mmol) in THF (5 mL) and H2O (5 mL) was irradiated under microwave at 100 °C for 0.5 h. The consumption of starting materials was detected by LC-MS analysis. The resulting mixture was adjusted to pH 3-4 with 1 N HCl solution. The resulting suspension was stirred for 5 h then filtered. The residue was dried under vacuum at 45 °C overnight to get the titled compound (980 mg, yield 86%). 1 H NMR (400 MHz, DMSO-d6) δ 12.31 (s, 1H), 9.68 (s, 1H), 8.56 (s, 1H), 8.22 (d, J = 5.2 Hz, 1H), 7.70 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 5.4 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 3.56 (s, 2H). LC-MS (ESI) m/z: [M + H] + 286.3.

3 [

 3 After the pre-incubation, 5 µl of the substrate mix containing 10 µM ATP and 3 µM 5-FAM tagged peptide (peptide substrate #22, 5-FAM-EEPLYWSFPAKKK-CONH2 [PerkinElmer, MA, USA]) in kinase buffer were added to the assay plate. Final running concentrations were as follows: ATP (5 μM), peptide (1.5 μM), compounds (0.2 mM-0.632 nM). Entrectinib was used as the running control. The plate was run on the Caliper EZ Reader II (PerkinElmer, MA, USA) with separation buffer (100 mM HEPES, 10 mM EDTA, 0.015% Brij-35, 0.1% CR-PerkinElmer, MA, USA]) until 10-20% conversion based on the positive control wells. The following separation conditions were utilized: upstream voltage -500V; downstream voltage, -1900V; chip pressure -0.8. Doseresponse curves were generated in GraphPad Prisim 8 and IC50 values were obtained from the curves. IC50 values were generated in duplicate and error was calculated from the standard deviation between two values.

  Tyr490) (#9141), and phospho-TRKA (Tyr674/675) (#4621) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-TRKA (C-14) (#sc-11) antibody was from Santa Cruz Biotechnology



  A series of novel thieno[3,2-d]pyrimidine type-II pan-TRK inhibitors were designed and synthesized. Representative compound 4c was discovered through structure-based drug design (SBDD) approach  Compound 4c exhibited an effective blockage of TRKA autophosphorylation. Compound 4g exhibited excellent selectivity for KM12 in the NCI-60 human cancer cell lines screen

Table 1 .

 1 Structure-activity relationship of series I compounds. a

	Compound	R 1	TRKA Inhibition % (@ 20 µM) b	KM12 cell IC50 (µM)
	1a		18.0	>200
	1b		31.4	25.2 ± 4.8
	1c		18.0	86.5 ± 11.2
	Entrectinib	--	IC50=0.0027 ± 0.0007 µM 0.014 ± 0.009

a IC50 values are shown as the mean ± SD of two independent assays.

b Inhibition percentage values are shown as the average of two independent assays.

Table 2 .

 2 Structure-activity relationship of series II compounds. a

			TRKA Inhibition	KM12 cell
	Compound	R 2	% (@ 20 µM) b	IC50 (µM)
	2a		18.0	102.6 ± 10.3

Table 3 .

 3 Structure-activity relationship of series III compounds. a

	C li c k t o B U Y N O W ! P D F -X C hange E d it o r w w w . t r a c k er-s of t w a r e . c o m		t P D r w w w .	a F -X C hange c k er-s of t w a r e E d it o r C li c k t o B U Y N O W ! . c o m
		TRKA	KM12 cell
	Compound	R 3
		IC50 (µM)	IC50 (µM)

a IC50 values are shown as the mean ± SD of two independent assays.

Table 4 .

 4 Structure-activity relationship of series IV compounds (part 1). a

	P D	F -X	W ! C hange E d it o r
			N O
			B U Y
			t o
	w w w . C li c k t r a
					TRKA	KM12 cell
			Compound	X	R 4
					IC50 (µM)	IC50 (µM)
			4a	CH2	0.14 ± 0.03	1.49 ± 0.24
			4b	CH2	0.54 ± 0.07	1.42 ± 0.30
			4c	CH2	0.075 ± 0.001	0.14 ± 0.02
			4d	CH2	1.95 ± 0.19	20.92 ± 5.99
			4e	CH2	0.026 ± 0.001	1.65 ± 0.27

c k er-s of t w a r e . c o m C li c k t o B U Y N O W ! P D F -X C hange E d it o r w w w . t r a c k er-s of t w a r e . c o m

	4f	CH2		0.092 ± 0.054	1.02 ± 0.16
	4g	CH2		0.094 ± 0.016	0.09 ± 0.01
	4h	CH2		1.58 ± 0.17	1.38 ± 0.20
	4i	CH2		3.61 ± 0.34	>200
	4j	CH2		9.70 ± 1.59	ND b
	4k	NH		0.058 ± 0.008	0.13 ± 0.04
	Entrectinib	--	--	0.0027 ± 0.0007	0.012 ± 0.00002

a IC50 values are shown as the mean ± SD of two independent assays. b Not determined.

Table 5 .

 5 Structure-activity relationship of series IV compounds (part 2). a

		TRKA	KM12 cell
	Compound	R 5
		IC50 (µM)	IC50 (µM)

Table 6 .

 6 Kinase selectivity profile of compound 4c. a

	Kinase	IC50 (µM)
	TRKA	0.075 ± 0.001
	TRKB	0.074 ± 0.007
	TRKC	0.055 ± 0.014
	Aurora A	0.893 ± 0.006
	Aurora B	1.99 ± 0.24
	CSF-1R	13.6 ± 0.3
	MAP4K4	41.5 ± 7.8
	EGFR	176.09 ± 18.21
	NEK2	>200

a IC50 values are shown as the mean ± SD of two independent assays.

  H NMR spectral data were recorded on Varian 400 NMR spectrometer, and13 C NMR was recorded on Varian Mercury

	101 NMR spectrometer at ambient temperature. Chemicals shifts (δ) were reported in parts per million, coupling
	constants (J) values were in hertz, and the splitting patterns were abbreviated as follows: s for singlet; d for
	doublet; t for triplet; q for quartet; and m for multiplet. All final compounds for biological evaluation were purified
	to ≥95% purity as determined by Agilent 1100 HPLC instrument (column: Phenomenex kinetex 150 × 4.6 mm, 5
	μm; solvent A: H2O/0.1% TFA; solvent B: MeOH/0.1% TFA; run time 12 min.; time/%B: 0.0/30, 6.0/100, 7.0/100,
	74 (s,
	1H), 3.71 -3.62 (m, 1H), 3.61 (s, 2H), 1.71 -1.49 (m, 4H), 1.47 -1.36 (m, 2H), 1.33 -1.17 (m, 2H). 13 C NMR
	(101 MHz, DMSO-d6) δ 168.86, 161.87, 158.81, 154.58, 140.37, 138.43, 134.71, 129.79, 128.07, 127.15,
	124.44, 66.82, 50.87, 42.09, 31.69, 26.63, 23.76, 19.57. LC-MS (ESI) m/z: [M + H] + 368.1. HPLC purity: 100%,
	tR (retention time) = 6.87 min.

7.01/30,12.0/30; flow rate: 0.8 mL/min.; wavelength: 254 nm).

General procedure A: Synthesis of compounds 1, 3, 4a-j, and 16. Acids such as 7, 11, 14, or 15 (0.14 mmol), amine (0.16 mmol), HATU (0.16 mmol), and Et3N (0.28 mmol) were dissolved in anhydrous DMF (1 mL). The reaction mixture was stirred at ambient temperature overnight. The completion of the reaction was monitored by LC-MS analysis. The reaction mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and brine twice, and concentrated in vacuum. The resulting residue was purified by silica gel chromatography to get the title compound.

N-((1R,2R)-2-hydroxycyclohexyl)-2-(4-(thieno[3,2-d]pyrimidin-4-yl)phenyl)acetamide (1a). The title compound

was synthesized from 7 and (1R,2R)-2-aminocyclohexanol hydrochloride following general procedure A. White solid (73%).

1

H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.56 (d, J = 5.5 Hz, 1H), 8.12 (d, J = 7.9 Hz, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 5.6 Hz, 1H), 7.54 (d, J = 8.0 Hz, 2H), 4.62 (d, J = 3.8 Hz, 1H), 3.

  C NMR (101 MHz, DMSO-d6) δ 168.44, 161.91, 158.72, 158.62, 154.60, 139.05, 138.50, 136.17, 135.12, 129.98 (2C), 128.26 (2C), 127.21, 124.46, 94.96, 41.91, 35.30, 31.77, 30.30 (3C). LC-MS (ESI) m/z: [M + H] + 406.3. HPLC purity: 100%, tR = 7.53 min.

	N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(thieno[3,2-d]pyrimidin-4-yl)phenyl)acetamide (1c). The title compound was
	synthesized from 7 and 5-(tert-butyl)isoxazol-3-amine following general procedure A. White solid (85%). 1 H
	NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 9.26 (d, J = 1.1 Hz, 1H), 8.57 (d, J = 5.6 Hz, 1H), 8.15 (d, J = 7.5
	Hz, 2H), 7.73 (d, J = 5.5 Hz, 1H), 7.59 (d, J = 7.8 Hz, 2H), 6.59 (s, 1H), 3.83 (s, 2H), 1.27 (s, 9H). 13 C NMR
	(101 MHz, DMSO-d6) δ 180.47, 168.94, 161.90, 158.71, 157.87, 154.59, 138.67, 138.50, 135.21, 130.00 (2C),
	128.27 (2C), 127.23, 124.45, 93.12, 32.47, 28.29 (3C). LC-MS (ESI) m/z: [M + H] + 393.3. HPLC purity:
	97.9%, tR = 8.02 min.
	The title
	compound was synthesized from 11 and (1R,2S)-2-aminocyclohexanol following general procedure A. Beige
	solid (70%). 1 H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.54 (s, 1H), 8.23 -8.18 (m, 1H), 7.65 (d, J = 8.4
	Hz, 2H), 7.62 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 5.4 Hz, 1H), 7.25 (d, J = 8.3 Hz, 2H), 4.59 (d, J = 3.9 Hz, 1H),
	3.71 (s, 1H), 3.63 (t, J = 8.6 Hz, 1H), 3.44 (s, 2H), 3.19 -3.14 (m, 1H), 1.70 -1.18 (m, 7H). 13 C NMR (101
	MHz, DMSO-d6) δ 169.47, 160.33, 155.27, 154.02, 137.18, 134.15, 132.14, 129.02 (2C), 124.35, 122.27 (2C),

N-((1S,2S)-2-hydroxycyclohexyl)-2-(4-(thieno

[3,2-d]

pyrimidin-4-ylamino)phenyl)acetamide (3a). The title compound was synthesized from 11 and (1R,2R)-2-aminocyclohexanol hydrochloride following general procedure A. Beige solid (67%).

1

H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.55 (d, J = 1.8 Hz, 1H), 8.21 -8.18 (m, 1H), 7.68 -7.64 (m, 2H), 7.62 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 5.4 Hz, 1H), 7.25 (d, J = 8.3 Hz, 2H), 4.61 -4.56 (m, 1H), 3.71 (s, 1H), 3.64 (s, 1H), 3.45 (s, 2H), 1.70 -1.47 (m, 4h), 1.46 -1.34 (m, 2H), 1.31 -1.16 (m, 2H).

13

C NMR (101 MHz, DMSO-d6) δ 169.48, 160.33, 155.27, 154.02, 137.18, 134.14, 132.13, 129.03 (2C), 124.34, 122.28 (2C), 115.28, 66.86, 50.77, 41.79, 39.52, 31.72, 26.64, 23.77, 19.56. LC-MS (ESI) m/z: [M + H] + 383.3. HPLC purity: 100%, tR = 5.26 min. N-((1R,2S)-2-hydroxycyclohexyl)-2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamide (3b). 115.27, 66.85, 50.76, 41.78, 31.72, 26.64, 23.76, 19.56. LC-MS (ESI) m/z: [M + H] + 383.4. HPLC purity: 100%, tR = 5.34 min.

  1 H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 9.69 (s, 1H), 8.56 (s, 1H), 8.21 (d, J = 5.4 Hz, 1H), 8.12 (s, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.55 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 5.4 Hz, 1H), 7.39 (d, J = 130.48, 129.17 (2C), 124.35, 122.31(2C), 115.35, 93.10, 41.98, 32.47, 28.31(3C). LC-MS (ESI) m/z: [M + H] + 407.1. HPLC purity: 99.2%, tR = 6.56 min.

	C li c k t o B U Y N O W ! P D F -X C hange E d it o r w w w . t r a c k er-s of t w a r e . c o m	t P D r w w w .	a F -X C hange c k er-s of t w a r e E d it o r C li c k t o B U Y N O W ! . c o m
	2-(4-((6-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-d]pyrimidin-4-yl)amino)phenyl)-N-(3-
	(trifluoromethyl)phenyl)acetamide (4a). The title compound was synthesized from 14 and 3-
	7.7 Hz, 1H), 7.34 (d, J = 8.2 Hz, 2H), 3.68 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 169.85, 160.32, 155.24, (trifluoromethyl)aniline following general procedure A. Beige solid (62%). 1 H NMR (400 MHz, DMSO-d6) δ
	153.99, 139.95, 137.66, 134.19, 130.76, 129.96, 129.44 (q, J = 32 Hz), 129.21 (2C), 124.09 (q, J = 272 Hz), 10.50 (s, 1H), 9.60 (s, 1H), 8.51 (s, 1H), 8.28 (s, 1H), 8.12 (s, 1H), 7.96 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.71
	124.34, 122.60, 122.31 (2C), 119.52 (q, J = 4 Hz), 115.37, 115.11 (q, J = 4 Hz), 42.78. LC-MS (ESI) m/z: (d, J = 8.3 Hz, 2H), 7.57 -7.52 (m, 2H), 7.39 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.3 Hz, 2H), 3.91 (s, 3H), 3.67 (s,
	[M + H] + 429.0. HPLC purity: tR = 6.72 min. 2H). 13 C NMR (101 MHz, DMSO-d6) δ 169.88, 161.21, 154.56, 154.29, 141.85, 139.96, 137.79, 136.90,
	N-(3-ethynylphenyl)-2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamide (3d). The title compound was 130.56, 129.97,129.44 (q, J = 32 Hz), 129.22 (2C), 129.18, 124.09 (q, J = 272 Hz), 122.61, 122.08 (2C),
	3.66 (s, 2H), 3.58 (s, 3H), 1.19 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 169.10, 160.37, 158.60, 155.24,
	154.01, 137.62, 136.27, 134.19, 130.85, 129.15 (2C), 124.36, 122.30 (2C), 115.36, 95.02, 41.64, 35.27, 31.78,
	30.32 (3C). LC-MS (ESI) m/z: [M + H] + 421.2. HPLC purity: 100%, tR = 6.15 min.
	N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamide (3f). The title compound
	was synthesized from 11 and 5-(tert-butyl)isoxazol-3-amine following general procedure A. Beige solid (69%).

synthesized from 11 and 3-ethynylaniline following general procedure A. Beige solid (83%).

1

H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 9.69 (s, 1H), 8.56 (s, 1H), 8.20 (d, J = 5.4 Hz, 1H), 7.82 (s, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 5.4 Hz, 1H), 7.37 -7.28 (m, 3H), 7.15 (d, J = 7.6 Hz, 1H), 4.16 (s, 1H), 3.65 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 169.51, 160.34, 155.24, 154.00, 139.41, 137.59, 134.17, 130.97, 129.18, 129.16 (2C), 126.43, 124.35, 122.32 (2C), 122.00, 121.94, 119.69, 115.35, 83.35, 80.51, 42.79. LC-MS (ESI) m/z: [M + H] + 385.1. HPLC purity: 99.2%, tR = 6.18 min.

N-(3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)-2-(4-(thieno[3,2-d]pyrimidin-4-ylamino)phenyl)acetamide (3e)

.

The title compound was synthesized from 11 and 3-(tert-butyl)-1-methyl-1H-pyrazol-5-amine following general procedure A. Beige solid (72%). 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.68 (s, 1H), 8.56 (s, 1H), 8.21 (d, J = 5.4 Hz, 1H), 7.72 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 5.4 Hz, 1H), 7.33 (d, J = 8.0 Hz, 2H), 6.06 (s, 1H), 1 H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 9.68 (s, 1H), 8.55 (s, 1H), 8.21 (dd, J = 5.4, 1.3 Hz, 1H), 7.70 (d, J = 7.4 Hz, 2H), 7.45 (dd, J = 5.4, 1.3 Hz, 1H), 7.30 (d, J = 7.7 Hz, 2H), 6.58 (s, 1H), 3.65 (s, 2H), 1.27 (s, 9H).

13 C NMR (101 MHz, DMSO-d6) δ 180.40, 169.54, 160.36, 157.94, 155.22, 153.99, 137.68, 134.19, 119.52 (q, J = 4 Hz), 118.22, 115.31, 115.11 (q, J = 4 Hz), 113.20, 54.89, 42.79. LC-MS (ESI) m/z: [M + H] + 509.2. HPLC purity: 99.4%, tR = 6.96 min. HRMS (ESI) m/z: [M + H] + calcd for C25H20F3N6OS 509.1371; found 509.1354.

  ). The title compound was synthesized from 14 and 4-(tert-butyl)thiazol-2-amine

	following general procedure A. Yellow solid (62%). 1 H NMR (400 MHz,DMSO-d6) δ 12.30 (s, 1H), 9.59 (s, 1H),
	8.51 (s, 1H), 8.28 (s, 1H), 7.96 (s, 1H), 7.69 (d, J = 8.3 Hz, 2H), 7.54 (s, 1H), 7.31 (d, J = 8.3 Hz, 2H), 3.91 (s,
	3H), 3.73 (s, 2H), 1.26 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 169.24, 161.21, 160.12, 157.15, 154.54,
	154.28, 141.85, 137.88, 136.89, 130.06, 129.25 (2C), 129.17, 122.11 (2C), 118.20, 115.29, 113.18, 104.60,
	41.16, 38.82, 34.10, 29.75 (3C). LC-MS (ESI) m/z: [M + H] + 504.1. HPLC purity: 95.7%, tR = 7.28 min. HRMS
	(ESI) m/z: [M + H] + calcd for C25H26N7OS2 504.1640; found 504.1635.

  C NMR (101 MHz, DMSO-d6) δ 169.49, 160.76, 154.91, 154.69, 152.99 (J = 243 Hz), 147.08, 141.19, 137.49, 137.32, 136.45 (J = 3 Hz), 130.99, 129.26 (2C), 128.09 (2C), 127.07 (2C), 122.88, 122.32 (2C), 120.45, 119.36 (J = 6 Hz), 119.09 (J = 18 Hz), 116.92 (J = 21 Hz), 115.80, 43.42, 42.68. LC-MS (ESI) m/z: [M + H] + 567.3. HPLC purity 95.0%, tR = 7.06 min.

	N-(3-chloro-4-fluorophenyl)-2-(4-((6-(3,4,5-trimethoxyphenyl)thieno[3,2-d]pyrimidin-4-
	yl)amino)phenyl)acetamide (4m). The title compound was synthesized from 16 and (3,4,5-
	trimethoxyphenyl)boronic acid following general procedure C. Beige solid (62%). 1 H NMR (400 MHz, DMSO-
	d6) δ 10.37 (s, 1H), 9.68 (s, 1H), 8.57 (s, 1H), 7.96 -7.92 (m, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.51 -7.46 (m,
	1H), 7.38 J = 9.1 Hz, 1H), 7.35 -7.30 (m, 2H), 7.14 (s, 2H), 3.90 (s, 6H), 3.73 (s, 3H), 3.64 (s, 2H). 13 C
	NMR (101 MHz, DMSO-d6) δ 169.52, 161.04, 154.60, 154.46, 153.40 (2C), 152.99 (J = 243 Hz), 149.59,
	138.76, 137.77, 136.45 (J = 3 Hz), 130.58, 129.21 (2C), 128.24, 121.88 (2C), 120.48, 120.45, 119.36 (J = 7
	Hz), 119.07 (J = 18 Hz), 116.92 (J = 21 Hz), 114.67, 103.81 (2C), 60.17, 56.09 (2C), 42.67. LC-MS (ESI) m/z:
	[M + H] + 579.1. HPLC purity 98.0%, tR = 7.49 min.
	N-(3-chloro-4-fluorophenyl)-2-(4-((6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)thieno[3,2-d]pyrimidin-4-
	yl)amino)phenyl)acetamide (4n). The title compound was synthesized from 16 and (4-(2-(pyrrolidin-1-
	yl)ethoxy)phenyl)boronic acid following general procedure C. Yellow solid (52%). 1 H NMR (400 MHz, DMSO-
	d6) δ 10.35 (s, 1H), 9.61 (s, 1H), 8.49 (s, 1H), 7.91 (dd, J = 6.9, 2.5 Hz, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.72 (s,
	1H), 7.67 (d, J = 8.4 Hz, 2H), 7.48 -7.42 (m, 1H), 7.33 (t, J = 9.1 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.04 (d, J =
	8.8 Hz, 2H), 4.10 (t, J = 5.8 Hz, 2H), 3.60 (s, 2H), 2.78 (t, J = 5.8 Hz, 2H), 2.48 -2.44 (m, 3H), 1.69 -1.61 (m,
	4H). 13 C NMR (101 MHz, DMSO-d6) δ 169.54, 161.34, 159.71, 154.66, 154.39, 153.01 (J = 243 Hz), 149.59,
	137.71, 136.47 (J = 3 Hz), 130.73, 129.21 (2C), 127.74 (2C), 125.13, 122.22 (2C), 120.47, 119.36 (J = 7 Hz),
	119.08 (J = 18 Hz), 118.92, 116.93 (J = 22 Hz), 115.29 (2C), 114.16, 66.96, 54.18, 53.98 (2C), 42.70, 23.15
	(2C). LC-MS (ESI) m/z: [M + H] + 602.2. HPLC purity 98.1%, tR = 6.34 min.

  mixture was refluxed overnight. The consumption of starting materials was detected by LC-MS analysis. Water (100 mL) was added into the reaction mixture. Separate the organic phase and concentrated in vacuum. The resulting residue was purified by silica gel chromatography to get 21 as a beige solid (8.8 g, yield 50%).1 H NMR (400 MHz, CDCl3) δ 7.77 (dd, J = 11.5, 8.3 Hz, 6H), 7.36 (d, J = 8.1 Hz, 4h), 7.30 (d, J = 8.1 Hz, 2H),

	C li c k t o B U Y N O W ! P D F -X C hange E d it o r w w w . t r a c k er-s of t w a r e . c o m	t P D r w w w .	a F -X C hange c k er-s of t w a r e E d it o r C li c k t o B U Y N O W ! . c o m
	5.66 (s, 1H), 4.35 (t, J = 6.4 Hz, 2H), 3.91 (t, J = 6.4 Hz, 2H), 2.49 (s, 6H), 2.42 (s, 3H). LC-MS (ESI) m/z:
	[M + H] + 646.2.
	Step 2. A mixture of 21 (3.0 mmol) in morpholine (30 mL) was heated to 120 °C overnight. The consumption of
	starting materials was detected by LC-MS analysis. Water (100 mL) and DCM (200 mL) was added into the
	reaction mixture. The organic phase was washed with water and brine twice, and concentrated in vacuum. The
	resulting residue was purified by silica gel chromatography to get 22 as colorless oil (0.8 g, yield 64%). 1 H
	NMR (400 MHz, CDCl3) δ 7.63 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H), 5.99 (s, 1H), 3.91 (t, J = 4.6 Hz,
	4h), 3.88 -3.83 (m, 2H), 2.68 -2.62 (m, 2H), 2.58 (s, 4h), 2.39 (s, 3H), 1.22 (s, 9H). LC-MS (ESI) m/z: 646.2
	[M + H] + . Also get 23 as colorless oil (0.36 g, yield 30%). 1 H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 7.9 Hz, 2H),
	7.26 (d, J = 8.1 Hz, 2H), 5.91 (s, 1H), 4.11 (t, J = 7.6 Hz, 2H), 3.97 (t, J = 7.5 Hz, 2H), 2.39 (s, 3H), 1.26 (s,
	9H). LC-MS (ESI) m/z: [M + H] + 320.1.
	Step 3. A mixture of 22 (0.9 mmol) in H2SO4 was stirred at ambient temperature overnight. The consumption of
	starting materials was detected by LC-MS analysis. The reaction mixture was poured into iced water and
	(27.3 mmol) and Et3N (81.8 mmol) was dissolved
	in DCM (100 mL). A solution of TsCl (81.8 mmol) in DCM (100 mL) was added dropwise. The resulting reaction

adjusted to pH 8-9 with 2N NaOHaq solution. The resulting mixture was concentrated in vacuum to get crude 24 as a white solid. LC-MS (ESI) m/z: [M + H] + 253.1.

  box for the ATP binding site, which includes the hinge region, DFG residues and the residues of the αC-helix facing the ATP pocket. to be docked were assigned torsions around rotatable bonds using AutoDock Tools and were docked into the grid by using Autodock Vina. The docking results were visualized and analyzed with Maestro 2018-2.

	and Maestro
	2018-2. TRKA crystal structure (PDB ID: 6PL1) was retrieved from RCSB Protein Data Bank. Using AutoDock
	Tools, the TRK model was prepared as follows: (i) remove all waters; (ii) add all hydrogen as 'Polar Only'; (iii)
	generate a grid

M.B (%)= Drug amount recovered in media at the end of the experiment x 100 Drug amount in the donor compartment at the onset of the experiment

Acknowledgments

The authors would like to thank Dr. Ryoichi Fujiwara, the director of UAMS Analytical and Bioanalytical Core (CORE B), for providing services for quantification of small molecules. This work was supported by NCI 1R01CA197178 (to HL) and the POR Campania FESR 2014-2020 "SATIN" grant (to MS and FC).

Supplementary Material -For Publication Online

Click here to access/download Supplementary Material -For Publication Online supporting information_20210108.pdf

Author Information

Corresponding Author *H. L.: e-mail, HLi2@uams.edu

Author contributions

The idea was conceived by WY, BF and HL. The synthesis was done by WY and FL. The biological assays were performed by LZ, MM, FC and CL. The article was written by WY and edited by all. † WY and LZ contributed equally.

Declaration of competing interest

The authors declare no competing financial interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: 1 H NMR, 3 C NMR and HPLC spectra of final compounds. Supplementary information for endothelial permeability measurement assay.


Capturing Ultrasmall EMT Zeolite from Template-Free Systems

Eng-Poh Ng, 1,2 Daniel Chateigner, 3 Thomas Bein, 4 Valentin Valtchev, 1 Svetlana Mintova 1 * Small differences between the lattice energies of different zeolites suggest that kinetic factors are of major importance in controlling zeolite nucleation. Thus, it is critical to control the nucleation kinetics in order to obtain a desired microporous material. Here, we demonstrate how careful investigation of the very early stages of zeolite crystallization in colloidal systems can provide access to important nanoscale zeolite phases while avoiding the use of expensive organic templates. We report the effective synthesis of ultrasmall (6-to 15-nanometer) crystals of the large-pore zeolite EMT from template-free colloidal precursors at low temperature (30°C) and very high yield.

Z eolites are metastable crystalline aluminosilicate molecular sieves with uniform pores of molecular dimensions that are widely applied in catalysis, separations, and adsorption (1)[START_REF] Breck | Zeolites and Molecular Sieves System[END_REF][START_REF] Choi | [END_REF](4). The EMT-type zeolite has one of the lowest framework densities for a microporous material [START_REF] Ch | Atlas of Zeolite Framework Types[END_REF] and is a hexagonal polytype of the cubic FAU-type zeolite that plays a very important role in catalysis, for example, in fluid catalytic cracking (FCC) of hydrocarbons [START_REF] Vaughan | Properties and Applications of Zeolites[END_REF]. Similar to the FAU-type material, the EMT framework topology has a three-dimensional large (12-membered ring) pore system. The cubic FAU polymorph features only one type of supercage (with a volume of 1.15 nm 3 ), but a different stacking of faujasite sheets creates two cages in the EMT zeolite: a hypocage (0.61 nm 3 ) and a hypercage (1.24 nm 3 )( 7). The EMT material shows interesting catalytic properties different from FAU as an FCC catalyst, but the very high price of the product so far precludes practical uses (8,9).

In addition, several EMT-FAU intergrown phases (CSZ-1, ECR-30, ZSM-20, ZSM-3) have also been reported (9)(10)(11)(12)(13)(14). The synthesis of pure EMT-type zeolite is possible by templating with the expensive 18-crown-6 ether and using tightly controlled synthesis parameters [START_REF] Dougnier | [END_REF]. Many studies have been carried out to reduce the consumption of the crown ether template, for instance, by recycling after the synthesis (15) or using the socalled "SINTEF" tumbling approach (16,17), steam-assisted crystallization (18), surfactants (19), or other organic and inorganic auxiliary additives (20)(21)(22). Although the cost of producing EMT has been reduced, all attempts toward a synthesis of EMT-type zeolite without an organic structure-directing agent (OSDA) have been un-successful thus far. Moreover, the 18-crown-6 ether template stimulates the crystallization of micrometer-sized EMT crystals, and no attempts for the preparation of nanosized crystals have been reported.

Certain nanosized molecular sieves have been obtained at moderate (60°to 130°C) and low temperatures (25°to 50°C) (23)(24)(25)(26). Low-temperature synthesis techniques for discrete zeolite nanocrystals from organic-free precursor systems are highly desired, as they would reduce cost and hazardous wastes, save energy, and possibly alter the properties of the materials (26,27). Here, we describe a template-free Na 2 O-Al 2 O 3 -SiO 2 -H 2 O precursor system as a foundation for the preparation of a nanosized EMT molecular sieve, where the ratios between different compounds, nucleation temperature and times, and type of heating have been adjusted to avoid phase transformations (e.g., to FAU and SOD) and to stabilize the EMTtype crystals at a small particle size. We report the synthesis of ultrasmall hexagonal EMT nanocrystals (diameter of 6 to 15 nm) at the low temperature of 30°C without using any organic template; that is, from Na-rich precursor suspensions. Strikingly, this synthesis strategy requires no organic Fig. 1. Ultrasmall EMT crystals with hexagonal morphology synthesized from template-free precursor suspension at 30°C for 36 hours. The individual crystals are schematically presented with asizeof10to15nmand a thickness of 2 to 3 nm. Scale bars, 10 nm. template. Moreover, the absence of an organic template implies that no high-temperature calcination step is required for opening up the pore system for the intended applications.

Typically, a high concentration of OSDA is needed to prepare nanosized zeolites to achieve a high degree of supersaturation by which the crystal size can be controlled and the resulting nanoparticles can be stabilized. We synthesized the ultrasmall (6-to 15-nm) and nanosized (50-to 70-nm) EMT crystals at near ambient conditions within a very short time; that is, 36 hours under conventional heating and 4 min under microwave irradiation, respectively (see supporting online material,fig. S1).Weusedthese"soft" conditions to avoid the formation of the more stable and denser phase hydroxysodalite (SOD) and the formation of a FAU-type phase observed in precursor suspensions with slight changes of the oxide ratios and water content (fig. S2).

The ultrasmall EMT crystals prepared at 30°C for 36 hours under conventional heating are shown in Fig. 1. Nearly 63% of the amorphous aluminosilicates were transformed into ultrasmall hexagonal EMT zeolite (Si/Al = 1.14). The particles were single crystals with a size of ~6 to 15 nm, and they contained channels in a highly ordered hexagonal arrangement. The x-ray diffraction (XRD) pattern of this sample (fully crystalline EMT-type zeolite) exhibited broadened Bragg peaks, which suggests the presence of very small crystallite sizes (Fig. 2D). The pattern was indexed using the hexagonal EMT structure in the P6 3 /mmc space group, with a reliability factor as low as weighted R w =1 . 5 % ,B r a g gR B = 1.12%, and experimental R exp = 0.62%, which gave rise to a goodness-of-fit of 6. In addition, zeolites X and Y (FAU-type structures) were considered in the modeling; however, they did not fit the first three peaks characteristic of the EMT zeolite, nor the anisotropic line broadening (fig. S3). The refined cell parameters, a = 1.7616(1) nm and c = 2.838(2) nm, correspond very well to the EMT-type structure [START_REF] Ch | Atlas of Zeolite Framework Types[END_REF]. Moreover, the refined shape of the particles was a hexagonal crystal with mean sizes of 10 nm along the [100] direction, 15 nm along [110], and 2.0 nm along [001]. The refined shape of the crystallites matched the size and shape of the EMT crystals measured with high-resolution transmission electron microscopy (HRTEM) (Figs. 1 and2D). Moreover, the fitting of this XRD pattern (sample synthesized at 30°C for 36 hours) with isotropic and platelike EMT crystals did not provide better fitting results (fig. S3 and table S1).

We investigated the entire process of nucleation and growth of the ultrasmall EMT crystals in the system subjected to conventional heating for a total period of 36 hours. HRTEM images of the solid particles extracted at different crystallization times revealed the presence of amorphous gelafter8to14hoursandfullycrystallineEMT particles after 36 hours of heating at 30°C (Fig. 3). We did not observe any difference between the TEM pictures of samples heated for 8 and 14 hours. Amorphous objects of about equal size with a diameter of 2 to 10 nm were seen in these samples (Fig. 3A); some of these particles had a size and morphology similar to the final EMT crystallites. The XRD pattern of the sample heated only for 8 hours did not exhibit any Bragg peaks, confirming the amorphous nature (Fig. 2A), whereas analysis of the diffraction pattern for the sample heated for 14 hours (Fig. 2B) resulted in nonsatisfactory fitting based on the use of one phase only (either amorphous or crystalline). Thus, we used a mixture of amorphous and nanocrystalline EMT-type zeolite for the fitting. On the basis of this refinement (Fig. 2B), the relative amount of the crystalline phase calculated is ~30%. More developed anisotropy of both unit-cell (c/a = 1.44) and mean crystallite shape ( 2 . 1×2 . 3×1 . 0n m 3 ) was calculated for this sample based on the XRD. Although the XRD pattern corresponds to an entirely amorphous sample, based on the Rietveld refinement, we concluded that the particles had anisotropic shapes with mostly developed hexagonal platelike morphology (fig. S4 andtable S2) (28). Moreover, the refined crystallite volume was comparable to the unit-cell volume, which is a signature of the starting of EMT growth (fig. S5). After 24 hours of heating, ultrasmall crystallites of zeolite EMT appeared in the amorphous matrix (Fig. 3B). The XRD pattern of this sample contains amorphous matter and low intense Bragg peaks (Fig. 2C). The size of the crystalline domains calculated based on XRD is 5.5 × 2.0 × 5.1 nm 3 (table S3). Moreover, the crystalline particles existing in the aluminosilicate system after 14 and 24 hours heating at 30°C already exhibited the hexagonal shape (insets in Fig. 2, B andC).

As the crystallization proceeded, the intensity of the Bragg peaks increased, but they were still broad because of the small crystalline domains. After 36 hours of heating, the amorphous particles were turned entirely into crystalline matter, as demonstrated with HRTEM and XRD (Figs. 2D and3C). In the fully crystalline sample, we detected well-formed hexagonal particles with crystalline fringes. A closer look at these nanocrystals (Fig. 1) revealed that the apparent size and the hexagonal arrangement of the micropores corre-spond to the EMT-type zeolite. Further evidence for the high crystallinity of the samples was pro-videdbyN 2 sorption and spectroscopic data (infrared and nuclear magnetic resonance spectroscopy) (figs. S6 to S9 and table S4). The nitrogen sorption measurement of the fully crystalline sample (36 hours) revealed a type I sorption isotherm; micropores of 7.3 Å were observed in addition to textural mesoporosity (pores of 2 to 50 nm) attributed to the random packing of the ultrasmall nanocrystals. The Brunauer-Emmett-Teller surface area for the ultrasmall and nanosized EMT materials was 578 and 562 m 2 /g, and the pore volume was 0.78 and 0.84 cm 3 /g, respectively (table S4).

We compared the ultrasmall EMT crystals synthesized under conventional heating (Fig. 4, A and B) with nanosized EMT crystals synthesized under microwave heating (Fig. 4, C andD) at 30°C. The HRTEM images of the sample prepared with microwave treatment revealed the presence of small EMT crystals with the characteristic hexagonal platelike morphology (Fig. 4C). The crystalline solids showed a uniform par-ticlesizeof50to70nm(Fig. 4Dandfigs.S1and S10). The selected-area electron diffraction (SAED) pattern of the nanosized EMT exhibits sixfold symmetry, which is indicative of the EMT structural features (Fig. 4F and table S5). The distance between two fringes based on the TEM measurement is 1.3 nm. The ABABAB stacking of the faujasite sheets is observed in the EMT nanocrystals; this stacking gives the hexagonal crystal shape and EMT topology (Fig. 4E). Unlike high silica micrometer-sized EMT zeolite, the nanosized EMT grow favorably in the a direction, rather than in the c direction, thus resulting in the thin hexagonal-plate form due to the limited crystal growth via the layer-by-layer mechanism along the c direction (29).

The homogeneity of the two samples, ultrasmall (6-to 15-nm) and nanosized (50-to -70 nm) EMT crystals, is illustrated at two different magnifications in Fig. 4, A to D. The hexagonal morphology of both samples is evident. Moreover, their colloidal stability was examined by measuring the zeta potential values, which are equal to -45 mV. This negative charge leads to electrostatic stabilization of the hexagonal nanoparticles.

Why has EMT never been observed in organic-free synthesis solutions? The reason may be surprisingly simple. When the same synthesis solutions as described above were heated at the same temperature for extended times or at higher temperatures, the nanoscale EMT materials converted into the well-known FAU and SOD structures (fig. S2). We propose that under appropriate conditions the EMT is the first kinetic, metastable product in this synthesis field, followed by conversion into the more stable cubic FAU and more dense SOD structures. This hypothesis is strongly supported by several reports on EMT/FAU intergrowths (9)(10)(11)(12)(13)(14). Indeed, we suggest that it may be possible to capture other important zeolite phases that occur as intergrowths by exploiting the very early stages of synthesis and thus avoiding the use of organic reagents that are commonly needed to stabilize the desired phases.

From an environmental perspective, the synthesis of EMT zeolite presented here is extremely attractive, as the nanocrystals can be easily synthesized at very high yield at near ambient temperature without using any organic templates, suggesting that scale-up of an energy-efficient synthesis would be easily feasible. These nanoscale EMT materials offer exciting opportunities for both fundamental study and potential industrial applications. The possible green mass production of EMT-type zeolite provides excellent opportunities for applications in catalysis, adsorption, and separations involving larger molecules and for designing thin films, membranes, or nanoscale devices.

Fig. 2 .

 2 Fig.2. XRD patterns representing the evolution of ultrasmall EMT crystals from template-free precursor suspensions at 30°C under conventional heating for (A)8hours,(B)14hours,(C)24hours,and (D)3 6h o u r s .V e r t i c a lt i c k sc o r r e s p o n dt ol i n ei ndexing of the EMT phase. The difference diagram between calculated and experimental points is shown at the bottom of each XRD pattern. (Insets) Crystallite sizes and shapes calculated based on the XRD data. The whole pattern was fitted using the combined analysis formalism[START_REF] Chateigner | Combined Analysis: Structure-Texture-Microstructure-Phase-Stresses-Reflectivity Determination by X-ray and Neutron Scattering[END_REF] implemented in the MAUD program[START_REF] Lutterotti | MAUD (Material Analysis Using Diffraction): A User Friendly JAVA Program for Rietveld Texture Analysis and More,i nTextures of Materials[END_REF], based on the Rietveld analysis approach. Fourier analysis was applied to deconvolute the instrumental-and sample-broadening parts from the measured XRD lines. The instrumental contribution to the line broadening was calibrated with an LaB 6 standard powder from the National Institute of Standards and Technology. The Popa formalism was then used to describe anisotropic crystallite sizes and shapes[START_REF] Popa | [END_REF], and an arbitrary texture correction model (31)wasusedtoaccountforthemoderate preferred orientations introduced in the EMT powder in a flat sample holder. a.u., arbitrary units.

Fig. 3 .

 3 Fig. 3. TEM images of (A) amorphous-shaped particles in the template-free precursor suspension after 8 to 14 hours, (B) birth of ultrasmall EMT nuclei after 24 hours, and (C) fully crystalline ultrasmall EMT after 36 hours of conventional heating at 30°C. Scale bars, 10 nm.

Fig. 4 .

 4 Fig. 4. TEM images of ultrasmall EMT crystals at different magnifications with scale bars of (A)10nmand(B) 500 nm, as well as nanosized EMT single crystals with scale bars of (C)2 0n ma n d (D)5 00n m .( E)Magnified TEM image and corresponding schematic diagram of the framework structure (ABABA stacking of faujasite sheets highlighted in purple and blue). (F) SAED pattern of a nanosized EMT single crystal, projected along [100].

Acknowledgments: We thank S. Schmidt (LMU) for collecting the TEM data and SRIF-ANR-06-NANO-007-01 Region and FEDER of Down Normandy, NIM (Nano-Initiative-Munich), for financial support.


Emmanouil Markoulakis
email: markoul@hmu.gr.

A grand unification charge flux manifold for the quarks

Using our previous published findings of the possible charge electromagnetic flux manifold of the electron we outline describe here how the electromagnetic and in generalization the electroweak force can be unified with the strong force of the quarks inside the nucleus of the atom showing that both are different formations (i.e. manifolds) of intrinsic charge electromagnetic flux. Their different electromagnetic charge flux manifolds are dictated by their fine structure constant, αEM~1/137 for the electromagnetic force interaction for electrons and αS=1 for the strong force interaction for quarks. In particular we show how the helical charge flux manifold of quarks opposite to the horn spheroid charge flux manifold of electrons leads to the increased strength αS/ αEM~137 strong force of the quarks and its peculiar property to increase when two bounded quarks are separated to longer distance from their rest state within the confinement of the nucleon.

Introduction

We have shown in our previous publication [START_REF] Markoulakis | A ½ spin fiber model for the electron[END_REF] that the intrinsic charge flux manifold of the electron is normal EM flux in a relativistic dynamic manifold described as spirals on a sphere forming a horn spheroid shape or joined dipole vortex loosely similar to the magnetic field of an electrical solenoid: In fig. 1 we see the possible EM flux dynamic manifold of the intrinsic charge of the free electron at rest freeze in time 3D imprint. The fine structure constant αEM~1/137 as a geometric proportionality constant of its charge manifold is also shown as well expressed as a magnetic flux distribution ratio of the flux on the sphere and the flux inside the horn tube characteristic of the manifold shown above.

Based on our above findings for the electron and therefore also electromagnetic force and given the fine structure constant value αS=1 (see fig. 1 above, in the case of the quark, diameter at equator equals diameter of horn tube) for the strong force, our extended model predicts [START_REF]STANDARD MODEL (ST) TABLE OF ELEMENTARY PARTICLES AMENDED WITH THEIR RESPECTIVE ENERGY MASS MANIFOLDS[END_REF] that the intrinsic fractional charge of a quark is essentially a helix shaped EM flux manifold as shown in fig. 2: The fractional charge of a quark is actually EM flux in a helix formation similar to the wire formation of an electrical solenoid or a mechanical spring. This spring-like intrinsic flux manifold of the quark explains also geometrically its fine structure constant αS=1 value, why the strong force interaction of the quarks is much stronger than the EM force of the electrons (i.e. approximately αS/ αEM~137 times stronger) and also why the strong force increases when we try to stretch out two bounded quarks from their rest instantaneous positions. The model also explains why isolated quarks cannot exist since as shown above their manifold is an open ended helix opposite to the free electron closed loop charge formation thus a fractional charge for the quark. Quarks can only exist in a bounded state with other quarks forming a closed loop of EM flux. Fractional charge value of the quarks shown in fig. 2 depending on the number of spirals per unit length and sign of charge -or + on intrinsic chirality of the quark.

Analogous to a mechanical spring and Hooke's law, the force between two bounded quarks inside the nucleus of an atom increases as we try to stretch them out to longer separation:

() F V r   (1)
Where, () 4 ()

3 s rc V r kr r     ħ ( 2
)
where V(r) the Yukawa potential, constant k relates to the field energy per unit length and is called string tension. For short relative short distances (less than the two quarks rest distance) this resembles the Coulomb law, while for large distances (equal or more than the two quarks rest distance) the kr factor dominates within the confinement of the nucleon. It is important to note that the coupling αs also varies according to the forced distance between the quarks opposite to the rest distance value which is always 1.

Phenomenology and proposed experiment

The strong and EM force unification as briefly described here as being two different manifolds of the same EM flux energy phenomena could be relative easily demonstrated qualitatively but also quantitatively by manipulating to a helix formation a given magnetic flux in space as we shown is possible in one of our previous publications [START_REF] Markoulakis | A synthetic macroscopic magnetic unipole[END_REF] emulating therefore macroscopically a quark and then comparing the magnetic torque generated on a ferromagnetic material by this helix flux field with that of a normal dipole magnetic flux field of equal flux density. If the result shows a much stronger force for the helix field then this will be a positive outcome for the proposed herein grand unification model. Also two helical field sources could be combined in series in space to demonstrate that when we try to separate the sources to a longer distance the required external force actually increases.

Conclusion

We have shown herein that strong force of quarks and EM force of electrons are actually two different geometry of intrinsic charge EM flux manifolds therefore providing the ground for the grand unification of the these apparent different phenomena.

Fig. 1

 1 Fig. 1 Electron intrinsic charge EM flux manifold and its fine structure constant geometry [1].

Fig. 2

 2 Fig. 2 Intrinsic charge EM flux manifolds of quarks and charged leptons [2].