Design, Synthesis and SAR in 2,4,7-Trisubstituted Pyrido[3,2-d]Pyrimidine Series as Novel PI3K/mTOR Inhibitors

This work describes the synthesis, enzymatic activities on PI3K and mTOR, in silico docking and cellular activities of various uncommon 2,4,7 trisubstituted pyrido[3,2-d]pyrimidines. The series synthesized offers a chemical diversity in C-7 whereas C-2 (3-hydroxyphenyl) and C-4 groups (morpholine) remain unchanged, in order to provide a better understanding of the molecular determinants of PI3K selectivity or dual activity on PI3K and mTOR. Some C-7 substituents were shown to improve the efficiency on kinases compared to the 2,4-di-substituted pyrimidopyrimidine derivatives used as references. Six novel derivatives possess IC50 values on PI3Kα between 3 and 10 nM. The compounds with the best efficiencies on PI3K and mTOR induced micromolar cytotoxicity on cancer cell lines possessing an overactivated PI3K pathway.


Introduction
The phosphatidylinositol 3-kinase (PI3K) pathway controls cell proliferation, growth, differentiation, protein synthesis, glucose metabolism, migration, and apoptosis [1,2]. Its activation is initiated by the binding of the corresponding ligands to tyrosine kinase receptors and G-protein coupled receptors (GPCRs). This results in phosphorylation of a regulatory subunit of PI3K (the first enzyme of the pathway) and the subsequent activation of p110, a catalytic subunit of PI3K. This activation leads to the production of phosphatidylinositol 3,4,5-triphosphate (PIP3), a lipid second messenger, at the plasma membrane. PIP3 levels are negatively regulated by phosphatase and tensin homologue (PTEN). PIP3 allows the recruitment of Akt at the membrane through its pleckstrin homology domain (PH). Akt is activated by phosphorylation at the plasma membrane [3][4][5].
Once activated, signaling through Akt propagates to a diverse array of substrates, including the mammalian target of rapamycin (mTOR), a key regulator of protein translation. mTOR is a serine/threonine protein kinase that interacts with several proteins, forming two distinct complexes named mTOR complex 1 (mTORC1) and 2 (mTORC2), which regulate different cellular processes including metabolism, growth, proliferation, and survival [6][7][8][9]. Moreover, activation of the pathway is not vertical, and it has been shown that mTOR exerts a negative feedback loop on PI3K: mTOR once activated will inhibit PI3K through IRS-1 (Insulin Receptor Substrate-1) [10,11]. Furthermore, it has been observed in several studies that external factors (growth factors, nutrient intake) can activate Akt and mTOR, thereby bypassing PI3K and thus restarting cancer genesis [12][13][14][15].
The PI3K pathway is one of the most commonly activated signaling pathways in diverse cancer types, resulting in an extended survival growth and angiogenesis of tumor cells [16][17][18]. In a retrospective analysis of 19 784 patients, Millis et al. identified aberrations in the PI3K/Akt/mTOR pathway in 38% of the solid tumors histologically analyzed; 30% correspond to PTEN loss and 13%, 6% and 1% to mutations in PI3KCA (PI3Kα), PTEN and Akt1, respectively [19].
For these reasons, the need to develop compounds that can inhibit the PI3K pathway has been a great motivation for research teams around the world. Currently developed PI3K pathway inhibitors can be divided into five classes: (i) mTOR inhibitors, (ii) pan-class I PI3K inhibitors, (iii) dual PI3K/mTOR inhibitors, (iv) isoform selective PI3K inhibitors and (v) Akt inhibitors [20][21][22][23][24]. Pharmaceutical companies and academic institutes accomplish significant efforts in the clinical development of PI3K pathway inhibitors for solid tumor treatment, including exploring effective combinations, predictive biomarkers, target patient populations, as well as underlying resistance mechanisms. To date, 126 clinical trials are currently ongoing using Akt inhibitors (including 10 phase III trials) and 140 clinical trials (including 14 phase III) are currently being conducted on mTOR inhibitors either as monotherapy or as part of a combination therapy for many cancer types. For instance, 235 clinical trials are under way on PI3K inhibitors (including 30 phase III) and concern mainly lymphoma and solid tumors.
Once activated, signaling through Akt propagates to a diverse array of substrates, including the mammalian target of rapamycin (mTOR), a key regulator of protein translation. mTOR is a serine/threonine protein kinase that interacts with several proteins, forming two distinct complexes named mTOR complex 1 (mTORC1) and 2 (mTORC2), which regulate different cellular processes including metabolism, growth, proliferation, and survival [6][7][8][9]. Moreover, activation of the pathway is not vertical, and it has been shown that mTOR exerts a negative feedback loop on PI3K: mTOR once activated will inhibit PI3K through IRS-1 (Insulin Receptor Substrate-1) [10,11]. Furthermore, it has been observed in several studies that external factors (growth factors, nutrient intake) can activate Akt and mTOR, thereby bypassing PI3K and thus restarting cancer genesis [12][13][14][15].
The PI3K pathway is one of the most commonly activated signaling pathways in diverse cancer types, resulting in an extended survival growth and angiogenesis of tumor cells [16][17][18]. In a retrospective analysis of 19′784 patients, Millis et al. identified aberrations in the PI3K/Akt/mTOR pathway in 38% of the solid tumors histologically analyzed; 30% correspond to PTEN loss and 13%, 6% and 1% to mutations in PI3KCA (PI3Kα), PTEN and Akt1, respectively [19].
For these reasons, the need to develop compounds that can inhibit the PI3K pathway has been a great motivation for research teams around the world. Currently developed PI3K pathway inhibitors can be divided into five classes: (i) mTOR inhibitors, (ii) panclass I PI3K inhibitors, (iii) dual PI3K/mTOR inhibitors, (iv) isoform selective PI3K inhibitors and (v) Akt inhibitors [20][21][22][23][24]. Pharmaceutical companies and academic institutes accomplish significant efforts in the clinical development of PI3K pathway inhibitors for solid tumor treatment, including exploring effective combinations, predictive biomarkers, target patient populations, as well as underlying resistance mechanisms. To date, 126 clinical trials are currently ongoing using Akt inhibitors (including 10 phase III trials) and 140 clinical trials (including 14 phase III) are currently being conducted on mTOR inhibitors either as monotherapy or as part of a combination therapy for many cancer types. For instance, 235 clinical trials are under way on PI3K inhibitors (including 30 phase III) and concern mainly lymphoma and solid tumors.
Currently, two drugs inhibiting the mTOR signaling pathway (Temserolimus and Everolimus) through the binding of FKBP-12 have been approved by the FDA (Food and Drug Administration) for cancer treatment (advanced renal cell carcinoma, tuberous sclerosis) [25,26]. Four PI3K inhibitors have also been FDA-approved ( Figure 1): the two benzopyrimidinones idelalisib and duvelisib (PI3K δ and γ-selective) for three types of blood cancer hematologic malignancy or chronic lymphocytic leukemia, respectively [27,28] as well as the tricyclic heterocyclic copanlisib (PI3K α and δ-selective) for follicular lymphoma [29]. Finally, the thiazole derivative, alpelisib, was approved for PIK3CA-mutated advanced breast cancer treatment [24,30].  The mTOR inhibitor effects as cancer monotherapy have been limited, perhaps because they only exhibit poor proapoptotic activity, being mainly cytostatic and because of the existence of a negative feedback loop on PI3K/Akt, resulting in enhanced PI3K/Akt upon mTOR inhibition. Additionally, selective PI3K and Akt inhibitors might not be sufficient to block the entire pathway because of the possible independent activation of mTOR. Therefore, dual PI3K/mTOR inhibitors with pan class I PI3K and mTOR inhibition combine multiple therapeutic efficiencies in a single molecule. They reduce the risk of drug resistance development and prevent PI3K/Akt reactivation due to the negative feedback loop. Due to the awareness of the importance of the retroactive loop, only early phases of clinical trials (25) are found on dual PI3K/mTOR inhibitors and one of them includes morpholinylpyrimidine buparlisib (BKM120 in phase III for metastatic breast cancer) [31].
In this context, we have previously generated a library of C-2,4 di-substituted pyridopyrimidines I, that were screened for their dual PI3K/mTOR inhibition. Some derivatives are active in the nanomolar range on both enzymatic targets and two of them are highly potent on cancer cells in the submicromolar range without any toxicity on healthy cells. In order to explore the molecular interactions in the active sites of the two kinases and to understand the structural elements leading to selectivity against PI3K or duality against both PI3K and mTOR in this novel chemical series, we focused our efforts on C-2,4,7 trisubstituted pyridopyrimidines of type II (Figure 2). This series will offer chemical diversity in C-7 whereas the 2-(3-hydroxyphenyl) and C-4-morpholine groups remained unchanged, because these two pharmacophoric substituents confer a dual inhibition potency on 1.
mTOR. Therefore, dual PI3K/mTOR inhibitors with pan class I PI3K and mTOR inhibition combine multiple therapeutic efficiencies in a single molecule. They reduce the risk of drug resistance development and prevent PI3K/Akt reactivation due to the negative feedback loop. Due to the awareness of the importance of the retroactive loop, only early phases of clinical trials (25) are found on dual PI3K/mTOR inhibitors and one of them includes morpholinylpyrimidine buparlisib (BKM120 in phase III for metastatic breast cancer) [31].
In this context, we have previously generated a library of C-2,4 di-substituted pyridopyrimidines I, that were screened for their dual PI3K/mTOR inhibition. Some derivatives are active in the nanomolar range on both enzymatic targets and two of them are highly potent on cancer cells in the submicromolar range without any toxicity on healthy cells. In order to explore the molecular interactions in the active sites of the two kinases and to understand the structural elements leading to selectivity against PI3K or duality against both PI3K and mTOR in this novel chemical series, we focused our efforts on C-2,4,7 tri-substituted pyridopyrimidines of type II (Figure 2). This series will offer chemical diversity in C-7 whereas the 2-(3-hydroxyphenyl) and C-4-morpholine groups remained unchanged, because these two pharmacophoric substituents confer a dual inhibition potency on 1. In this work, we first established the straightforward pathways able to provide various inventive trisubstituted pyridopyrimidines II from a unique synthetic trichlorinated skeleton 2. Molecular diversity exploration in C-7 position was highlighted by the introduction of vinyl, oximes, chlorine, as well as methyl and variously substituted methylene groups (alcohols, ethers, amines, triazoles and oxazoles) which could become, after SAR analysis, key elements in a better understanding of the drug kinase interactions. With this aim in mind, each final compound was evaluated on the one hand on PI3Kα and mTOR targets in vitro, and on a representative cancer cell line panel. The ability of each compound to inhibit PI3K in cells was checked on one of these cell lines by evaluating the amount of p-Akt by Western blot. In silico docking studies were used to support medicinal chemistry efforts and proved to be successful in explaining the SAR of inhibitors.

Synthesis
To explore the molecular diversity in C-7 position, derivative 4 was synthesized after two steps from the 2,4,7-trichloropyrido 3,2-d pyrimidine 2 [32]. Condensation of morpholine under SNAr first selectively occurred in C-4 position. Next a C-2 regio-specific arylation with the homemade 3-methoxymethoxyphenyltrifloroborate potassium salt or with 3-hydroxyphenyl boronic acid led, under microwave irradiation, to compounds 4 and 5 in satisfying yields (Scheme 1) [33,34]. The last palladium cross coupling reactions concerned In this work, we first established the straightforward pathways able to provide various inventive trisubstituted pyridopyrimidines II from a unique synthetic trichlorinated skeleton 2. Molecular diversity exploration in C-7 position was highlighted by the introduction of vinyl, oximes, chlorine, as well as methyl and variously substituted methylene groups (alcohols, ethers, amines, triazoles and oxazoles) which could become, after SAR analysis, key elements in a better understanding of the drug kinase interactions. With this aim in mind, each final compound was evaluated on the one hand on PI3Kα and mTOR targets in vitro, and on a representative cancer cell line panel. The ability of each compound to inhibit PI3K in cells was checked on one of these cell lines by evaluating the amount of p-Akt by Western blot. In silico docking studies were used to support medicinal chemistry efforts and proved to be successful in explaining the SAR of inhibitors.

Synthesis
To explore the molecular diversity in C-7 position, derivative 4 was synthesized after two steps from the 2,4,7-trichloropyrido 3,2-d pyrimidine 2 [32]. Condensation of morpholine under S N Ar first selectively occurred in C-4 position. Next a C-2 regio-specific arylation with the homemade 3-methoxymethoxyphenyltrifloroborate potassium salt or with 3-hydroxyphenyl boronic acid led, under microwave irradiation, to compounds 4 and 5 in satisfying yields (Scheme 1) [33,34]. The last palladium cross coupling reactions concerned the use of 5 in a C-7 methylation involving AlMe 3 to generate 6 in a 70% yield, and the use of derivative 4 in very efficient vinylation under microwave irradiation to give 7 [32]. To be able to introduce diversity on the C-7 position, we prepared the aldehyde 8 using Lemieux-Johnson oxidation conditions (NaIO 4 and OsO 4 ) from 4 in a near quantitative manner. Finally, the MOM protective group removal of 7 and 8 with HCl (4M in dioxane) led to 9 and 10 in excellent yields. the use of 5 in a C-7 methylation involving AlMe3 to generate 6 in a 70% yield, and the use of derivative 4 in very efficient vinylation under microwave irradiation to give 7 [32]. To be able to introduce diversity on the C-7 position, we prepared the aldehyde 8 using Lemieux-Johnson oxidation conditions (NaIO4 and OsO4) from 4 in a near quantitative manner. Finally, the MOM protective group removal of 7 and 8 with HCl (4M in dioxane) led to 9 and 10 in excellent yields. Reductive aminations were performed on 8 in the presence of cyclic primary or secondary amines and NaBH(OAc)3 or NaBH3CN as hydride sources, respectively (Scheme 2). The small C-7 methyleneaminoalkyl library was obtained with yields ranging between 40 and 91%. Noteworthy, each derivative was isolated as a chlorhydrate salt as the MOM protective groups were directly removed after the reductive amination by acidic hydrolysis. Scheme 2. Reagents and conditions: (a) primary amine, NaBH3CN, CH2Cl2, r.t., 12 h; (b) secondary amine, NaBH(OAc)3, CH2Cl2/DMF, acetic acid, r.t., 5 h; (c) HCl 4 M, dioxane, r.t., 6 h, for 11: 40%, for 12: 61%, for 13: 91%.
Classical aldehyde reduction of 8 gave access to primary alcohol 14 in a quantitative yield (Scheme 3). Williamson methylation and MOM deprotection led to ether 16 in satisfying yield. Additionally, primary alcohol was iodinated and next transformed in azido 18 whereas its MOM deprotection led to 19. Triazole moieties were generated via Huisgen 1,3-dipolar cycloaddition starting from 19 and using several commercially available or homemade terminal propargylic alkynes [35]. The copper source was adapted as a function of the reactivity and the attempted products 20-23 were synthesized in moderate to Reductive aminations were performed on 8 in the presence of cyclic primary or secondary amines and NaBH(OAc) 3 or NaBH 3 CN as hydride sources, respectively (Scheme 2). The small C-7 methyleneaminoalkyl library was obtained with yields ranging between 40 and 91%. Noteworthy, each derivative was isolated as a chlorhydrate salt as the MOM protective groups were directly removed after the reductive amination by acidic hydrolysis. Reductive aminations were performed on 8 in the presence of cy ondary amines and NaBH(OAc)3 or NaBH3CN as hydride sources, re 2). The small C-7 methyleneaminoalkyl library was obtained with yiel 40 and 91%. Noteworthy, each derivative was isolated as a chlorhydra protective groups were directly removed after the reductive aminatio ysis. Scheme 2. Reagents and conditions: (a) primary amine, NaBH3CN, CH2Cl2, r amine, NaBH(OAc)3, CH2Cl2/DMF, acetic acid, r.t., 5 h; (c) HCl 4 M, dioxane, r 12: 61%, for 13: 91%.
Classical aldehyde reduction of 8 gave access to primary alcohol 14 in a quantitative yield (Scheme 3). Williamson methylation and MOM deprotection led to ether 16 in satisfying yield. Additionally, primary alcohol was iodinated and next transformed in azido 18 whereas its MOM deprotection led to 19. Triazole moieties were generated via Huisgen 1,3-dipolar cycloaddition starting from 19 and using several commercially available or homemade terminal propargylic alkynes [35]. The copper source was adapted as a function of the reactivity and the attempted products 20-23 were synthesized in moderate to excellent yields. Treatment of alcohol 23 with DAST failed but the same fluorination method was successfully employed from protecting compound 22, leading to compound 24 in a good yield after phenol deprotection. Aldehyde 8 provided the cyanomethyl derivative 26 using a small excess of TosMIC in presence of t-BuOK, followed by a hydrolysis in acidic media. Next, Oximes 31 and 32 were next straightforwardly prepared using 10 with the adapted hydroxylamine. To finish, the side chain homologation of 6 was carried out using a Wittig/oxidative cleavage procedure and crude aldehyde furnished the oxime 28 in moderate yield. The presence of 28 gave us the opportunity to build the oxazolic derivative 30, which could be considered as a direct isoster of 21 (Scheme 4). Aldehyde 8 provided the cyanomethyl derivative 26 using a small excess of TosMIC in presence of t-BuOK, followed by a hydrolysis in acidic media. Next, Oximes 31 and 32 were next straightforwardly prepared using 10 with the adapted hydroxylamine. To finish, the side chain homologation of 6 was carried out using a Wittig/oxidative cleavage procedure and crude aldehyde furnished the oxime 28 in moderate yield. The presence of 28 gave us the opportunity to build the oxazolic derivative 30, which could be considered as a direct isoster of 21 (Scheme 4).

Kinase Assays
In vitro activities toward the PI3Kα isoform were measured using the final phenolic compounds and IC 50 values (Table 1) were compared to 1 which inhibits PI3Kα and mTOR with IC 50 = 19 and 37 nM, respectively. Considering the PI3K target, introduction of a C-7 substitution had clearly an influence on the observed kinase activity. A chlorine or methyl group led to derivatives 5 and 6 that are slightly more active than 1 (entries 1, 2). Comparatively, a hydroxymethyl group (15, entry 7) led to a significant 30-fold reduction in the inhibition level as if a donor/acceptor hydrogen (DAH) group in this position was not tolerated. The introduction of vinyl, methoxymethyl and cyanomethyl groups partially restored the activity of the corresponding derivatives (entries 3,8,14). Finally, the zwitterionic azido derivative 19 exhibited a spectacular IC 50 of 10 nM (entry 9). Even if they exist as an isomer mixture and could be considered as unstable in living cells, oximes were evaluated and as attempted the hydroxyloxime function of 31 was less tolerated than its more hydrophobic methylated analogue 32 (entries 16,17), which inhibited the enzyme with an excellent 3 nM IC 50 value.

Kinase Assays
In vitro activities toward the PI3Kα isoform were measured using the final phenolic compounds and IC50 values (Table 1) were compared to 1 which inhibits PI3Kα and mTOR with IC50 = 19 and 37 nM, respectively. Considering the PI3K target, introduction of a C-7 substitution had clearly an influence on the observed kinase activity. A chlorine or methyl group led to derivatives 5 and 6 that are slightly more active than 1 (entries 1, 2). Comparatively, a hydroxymethyl group (15, entry 7) led to a significant 30-fold reduction in the inhibition level as if a donor/acceptor hydrogen (DAH) group in this position was not tolerated. The introduction of vinyl, methoxymethyl and cyanomethyl groups partially restored the activity of the corresponding derivatives (entries 3,8,14). Finally, the zwitterionic azido derivative 19 exhibited a spectacular IC50 of 10 nM (entry 9). Even if they exist as an isomer mixture and could be considered as unstable in living cells, oximes were evaluated and as attempted the hydroxyloxime function of 31 was less tolerated than its more hydrophobic methylated analogue 32 (entries 16,17), which inhibited the enzyme with an excellent 3 nM IC50 value.

Kinase Assays
In vitro activities toward the PI3Kα isoform were measured using the fi compounds and IC50 values ( Table 1) were compared to 1 which inhibits PI3Kα with IC50 = 19 and 37 nM, respectively. Considering the PI3K target, introduc substitution had clearly an influence on the observed kinase activity. A chlori group led to derivatives 5 and 6 that are slightly more active than 1 (entries 1, atively, a hydroxymethyl group (15, entry 7) led to a significant 30-fold redu inhibition level as if a donor/acceptor hydrogen (DAH) group in this posit tolerated. The introduction of vinyl, methoxymethyl and cyanomethyl grou restored the activity of the corresponding derivatives (entries 3,8,14). Final terionic azido derivative 19 exhibited a spectacular IC50 of 10 nM (entry 9). exist as an isomer mixture and could be considered as unstable in living cells, evaluated and as attempted the hydroxyloxime function of 31 was less toler more hydrophobic methylated analogue 32 (entries 16,17), which inhibited with an excellent 3 nM IC50 value.

Kinase Assays
In vitro activities toward the PI3Kα isoform were measured using the final phenolic compounds and IC50 values ( Table 1) were compared to 1 which inhibits PI3Kα and mTOR with IC50 = 19 and 37 nM, respectively. Considering the PI3K target, introduction of a C-7 substitution had clearly an influence on the observed kinase activity. A chlorine or methyl group led to derivatives 5 and 6 that are slightly more active than 1 (entries 1, 2). Comparatively, a hydroxymethyl group (15, entry 7) led to a significant 30-fold reduction in the inhibition level as if a donor/acceptor hydrogen (DAH) group in this position was not tolerated. The introduction of vinyl, methoxymethyl and cyanomethyl groups partially restored the activity of the corresponding derivatives (entries 3,8,14). Finally, the zwitterionic azido derivative 19 exhibited a spectacular IC50 of 10 nM (entry 9). Even if they exist as an isomer mixture and could be considered as unstable in living cells, oximes were evaluated and as attempted the hydroxyloxime function of 31 was less tolerated than its more hydrophobic methylated analogue 32 (entries 16,17), which inhibited the enzyme with an excellent 3 nM IC50 value.

Kinase Assays
In vitro activities toward the PI3Kα isoform were measured using the fi compounds and IC50 values ( Table 1) were compared to 1 which inhibits PI3Kα with IC50 = 19 and 37 nM, respectively. Considering the PI3K target, introduc substitution had clearly an influence on the observed kinase activity. A chlori group led to derivatives 5 and 6 that are slightly more active than 1 (entries 1, atively, a hydroxymethyl group (15, entry 7) led to a significant 30-fold redu inhibition level as if a donor/acceptor hydrogen (DAH) group in this posit tolerated. The introduction of vinyl, methoxymethyl and cyanomethyl grou restored the activity of the corresponding derivatives (entries 3,8,14). Final terionic azido derivative 19 exhibited a spectacular IC50 of 10 nM (entry 9). exist as an isomer mixture and could be considered as unstable in living cells, evaluated and as attempted the hydroxyloxime function of 31 was less toler more hydrophobic methylated analogue 32 (entries 16,17), which inhibited with an excellent 3 nM IC50 value.

Kinase Assays
In vitro activities toward the PI3Kα isoform were measured using the final phenolic compounds and IC50 values ( Table 1) were compared to 1 which inhibits PI3Kα and mTOR with IC50 = 19 and 37 nM, respectively. Considering the PI3K target, introduction of a C-7 substitution had clearly an influence on the observed kinase activity. A chlorine or methyl group led to derivatives 5 and 6 that are slightly more active than 1 (entries 1, 2). Comparatively, a hydroxymethyl group (15, entry 7) led to a significant 30-fold reduction in the inhibition level as if a donor/acceptor hydrogen (DAH) group in this position was not tolerated. The introduction of vinyl, methoxymethyl and cyanomethyl groups partially restored the activity of the corresponding derivatives (entries 3,8,14). Finally, the zwitterionic azido derivative 19 exhibited a spectacular IC50 of 10 nM (entry 9). Even if they exist as an isomer mixture and could be considered as unstable in living cells, oximes were evaluated and as attempted the hydroxyloxime function of 31 was less tolerated than its more hydrophobic methylated analogue 32 (entries 16,17), which inhibited the enzyme with an excellent 3 nM IC50 value.  The improved activity for several compounds containing relatively rem such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could b of the stabilization of molecules with residues located in an aprotic but polar this idea in mind, we decided to increase the size of the C-7 appendix, introd and large amines on the C-7 sp 3 methylene. When amino residues were cyclop or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took plac micromolar range. The best result was obtained with the morpholine group a an IC50 = 43 nM. Since substituents containing several polyheteroatomic funct azides and oximes had shown a promising inhibitory potency, we next pe biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong ele hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared ence of the more hydrophobic dimethylamino or methoxymethyl residues (e of 20 and 21, which became one of the most active compounds against PI3K w C-7 substituent (IC50 = 10 nM). Finally, switching from a triazole to an oxazole bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays struct ities, we next examined the inhibitory activity on mTOR of molecules posses on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attem selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed grees of activity against both mTOR and PI3K (Table 2). The improved activity for several compounds containing relatively remote groups such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could be indicative of the stabilization of molecules with residues located in an aprotic but polar region. With this idea in mind, we decided to increase the size of the C-7 appendix, introducing small and large amines on the C-7 sp 3 methylene. When amino residues were cyclopropylamine or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took place in the submicromolar range. The best result was obtained with the morpholine group as 12 exhibits an IC50 = 43 nM. Since substituents containing several polyheteroatomic functions such as azides and oximes had shown a promising inhibitory potency, we next performed the biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong electrically rich hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared to the presence of the more hydrophobic dimethylamino or methoxymethyl residues (entries 10, 11) of 20 and 21, which became one of the most active compounds against PI3K with a stable C-7 substituent (IC50 = 10 nM). Finally, switching from a triazole to an oxazole ring did not bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays structural similarities, we next examined the inhibitory activity on mTOR of molecules possessing an IC50 on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attempted, all the selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed diverse degrees of activity against both mTOR and PI3K (Table 2). The improved activity for several compounds containing relatively rem such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could b of the stabilization of molecules with residues located in an aprotic but polar this idea in mind, we decided to increase the size of the C-7 appendix, introd and large amines on the C-7 sp 3 methylene. When amino residues were cyclop or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took plac micromolar range. The best result was obtained with the morpholine group a an IC50 = 43 nM. Since substituents containing several polyheteroatomic funct azides and oximes had shown a promising inhibitory potency, we next pe biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong ele hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared ence of the more hydrophobic dimethylamino or methoxymethyl residues (e of 20 and 21, which became one of the most active compounds against PI3K w C-7 substituent (IC50 = 10 nM). Finally, switching from a triazole to an oxazole bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays struct ities, we next examined the inhibitory activity on mTOR of molecules posse on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attem selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed grees of activity against both mTOR and PI3K (Table 2). The improved activity for several compounds containing relatively remote groups such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could be indicative of the stabilization of molecules with residues located in an aprotic but polar region. With this idea in mind, we decided to increase the size of the C-7 appendix, introducing small and large amines on the C-7 sp 3 methylene. When amino residues were cyclopropylamine or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took place in the submicromolar range. The best result was obtained with the morpholine group as 12 exhibits an IC50 = 43 nM. Since substituents containing several polyheteroatomic functions such as azides and oximes had shown a promising inhibitory potency, we next performed the biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong electrically rich hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared to the presence of the more hydrophobic dimethylamino or methoxymethyl residues (entries 10, 11) of 20 and 21, which became one of the most active compounds against PI3K with a stable C-7 substituent (IC50 = 10 nM). Finally, switching from a triazole to an oxazole ring did not bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays structural similarities, we next examined the inhibitory activity on mTOR of molecules possessing an IC50 on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attempted, all the selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed diverse degrees of activity against both mTOR and PI3K (Table 2). The improved activity for several compounds containing relatively rem such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could b of the stabilization of molecules with residues located in an aprotic but polar this idea in mind, we decided to increase the size of the C-7 appendix, introd and large amines on the C-7 sp 3 methylene. When amino residues were cyclop or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took plac micromolar range. The best result was obtained with the morpholine group a an IC50 = 43 nM. Since substituents containing several polyheteroatomic funct azides and oximes had shown a promising inhibitory potency, we next pe biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong ele hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared ence of the more hydrophobic dimethylamino or methoxymethyl residues (en of 20 and 21, which became one of the most active compounds against PI3K w C-7 substituent (IC50 = 10 nM). Finally, switching from a triazole to an oxazole bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays struct ities, we next examined the inhibitory activity on mTOR of molecules posses on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attem selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed grees of activity against both mTOR and PI3K (Table 2). The improved activity for several compounds containing relatively remote groups such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could be indicative of the stabilization of molecules with residues located in an aprotic but polar region. With this idea in mind, we decided to increase the size of the C-7 appendix, introducing small and large amines on the C-7 sp 3 methylene. When amino residues were cyclopropylamine or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took place in the submicromolar range. The best result was obtained with the morpholine group as 12 exhibits an IC50 = 43 nM. Since substituents containing several polyheteroatomic functions such as azides and oximes had shown a promising inhibitory potency, we next performed the biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong electrically rich hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared to the presence of the more hydrophobic dimethylamino or methoxymethyl residues (entries 10, 11) of 20 and 21, which became one of the most active compounds against PI3K with a stable C-7 substituent (IC50 = 10 nM). Finally, switching from a triazole to an oxazole ring did not bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays structural similarities, we next examined the inhibitory activity on mTOR of molecules possessing an IC50 on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attempted, all the selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed diverse degrees of activity against both mTOR and PI3K (Table 2). The improved activity for several compounds containing relatively rem such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could b of the stabilization of molecules with residues located in an aprotic but polar r this idea in mind, we decided to increase the size of the C-7 appendix, introd and large amines on the C-7 sp 3 methylene. When amino residues were cyclop or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took place micromolar range. The best result was obtained with the morpholine group a an IC50 = 43 nM. Since substituents containing several polyheteroatomic funct azides and oximes had shown a promising inhibitory potency, we next pe biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong ele hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared ence of the more hydrophobic dimethylamino or methoxymethyl residues (en of 20 and 21, which became one of the most active compounds against PI3K w C-7 substituent (IC50 = 10 nM). Finally, switching from a triazole to an oxazole bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays struct ities, we next examined the inhibitory activity on mTOR of molecules posses on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attem selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed grees of activity against both mTOR and PI3K (Table 2). The improved activity for several compounds containing relatively remote groups such as methoxyether, vinyl, azido and methyl oxime groups in C-7 could be indicative of the stabilization of molecules with residues located in an aprotic but polar region. With this idea in mind, we decided to increase the size of the C-7 appendix, introducing small and large amines on the C-7 sp 3 methylene. When amino residues were cyclopropylamine or N-methylpiperazine (entries 4, 6), enzymatic inhibition activity took place in the submicromolar range. The best result was obtained with the morpholine group as 12 exhibits an IC 50 = 43 nM. Since substituents containing several polyheteroatomic functions such as azides and oximes had shown a promising inhibitory potency, we next performed the biological evaluation of several azole derivatives.
Triazole substitutions confirmed the previous observation that strong electrically rich hydroxyl or fluorine groups are not well tolerated (entries 12, 13) compared to the presence of the more hydrophobic dimethylamino or methoxymethyl residues (entries 10, 11) of 20 and 21, which became one of the most active compounds against PI3K with a stable C-7 substituent (IC 50 = 10 nM). Finally, switching from a triazole to an oxazole ring did not bring additional inhibition potency (entry 15).
As mTOR belongs to the same kinase family as PI3K and displays structural similarities, we next examined the inhibitory activity on mTOR of molecules possessing an IC 50 on PI3K lower than that measured for 1 (i.e., IC 50 PI3K = 19 nM). As attempted, all the selected 2,4,7-trisubstituted pyrido[3,2-d]pyrimidine compounds displayed diverse degrees of activity against both mTOR and PI3K (Table 2).
It appears immediately that the introduction of a substituent in C-7 decreases the inhibition of mTOR. Nevertheless, the selected derivatives remain active against this kinase and offer IC 50 around 100 nM. Moreover, IC 50 measurements showed that chemical modifications based on 1 offered a quasi-selective PI3K inhibitor 32. Additionally, we increased the number of potent dual inhibitors such as 5, 19 and 21, which possess a similar selectivity index (SI) around 10 value. With these three compounds, our aim of improving the inhibition efficiency against PI3k with a C-7 substituent with a moderate impact on the duality of inhibition is achieved. Finally, the two dual derivatives 6 and 21 were chosen to measure their selectivity against the other PI3K isoforms. Values show a very important selectivity for PI3Kα versus other isoforms except for derivative 21 which led to a good additional activity on isoform β. Addition of the triazole ring in C-7 might therefore modify the interaction of the molecule with this isoform. bring additional inhibition potency (entry 15). As mTOR belongs to the same kinase family as PI3K and displays structural similarities, we next examined the inhibitory activity on mTOR of molecules possessing an IC50 on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attempted, all the selected 2,4,7-trisubstituted pyrido [3,2-d]pyrimidine compounds displayed diverse degrees of activity against both mTOR and PI3K (Table 2). bring additional inhibition potency (entry 15). As mTOR belongs to the same kinase family as PI3K and displays structural similarities, we next examined the inhibitory activity on mTOR of molecules possessing an IC50 on PI3K lower than that measured for 1 (i.e., IC50 PI3K = 19 nM). As attempted, all the selected 2,4,7-trisubstituted pyrido [3,2-d]pyrimidine compounds displayed diverse degrees of activity against both mTOR and PI3K (Table 2). It appears immediately that the introduction of a substituent in C-7 decreases the inhibition of mTOR. Nevertheless, the selected derivatives remain active against this kinase and offer IC50 around 100 nM. Moreover, IC50 measurements showed that chemical modifications based on 1 offered a quasi-selective PI3K inhibitor 32. Additionally, we increased the number of potent dual inhibitors such as 5, 19 and 21, which possess a similar selectivity index (SI) around 10 value. With these three compounds, our aim of improving the inhibition efficiency against PI3k with a C-7 substituent with a moderate impact on the duality of inhibition is achieved. Finally, the two dual derivatives 6 and 21 were chosen to measure their selectivity against the other PI3K isoforms. Values show a very important selectivity for PI3Kα versus other isoforms except for derivative 21 which led to a good additional activity on isoform β. Addition of the triazole ring in C-7 might therefore modify the interaction of the molecule with this isoform.

Docking Studies
To investigate the binding mode of the ligands and analyze the substituent effects on the inhibition and selectivity against PI3Kα and mTOR, we carried out docking studies. The structures of mTOR [36] and PI3Kα [37] were retrieved from the Protein Data Bank (PDB) [38]. Both structures interact with the pyridinylfuranopyrimidine inhibitor PI-103 that contains a morpholine group binding to the hinge through a hydrogen bond. The hydroxy moiety of the phenol is forming a hydrogen bond donor to the carboxylic acid group of Asp2195 side chain at the back of the inner pocket of PI3K. The PI3Kα and mTOR aligned structures were extracted from the MOE kinase-ligand complex library [39]. As stressed by Bryant et al. [40] or Wright et al. [41], the two targets share a high structure similarity but a low sequence identity.
All the compounds were docked into the active site of each studied protein, PI3Kα and mTOR. All the compounds exhibited a similar binding mode in which the pyridopyrimidine scaffold of all the docked compounds was well superimposed, creating similar interactions with each active site of the proteins. Only compound 6 has the flipped It appears immediately that the introduction of a substituent in C-7 decreases the inhibition of mTOR. Nevertheless, the selected derivatives remain active against this kinase and offer IC50 around 100 nM. Moreover, IC50 measurements showed that chemical modifications based on 1 offered a quasi-selective PI3K inhibitor 32. Additionally, we increased the number of potent dual inhibitors such as 5, 19 and 21, which possess a similar selectivity index (SI) around 10 value. With these three compounds, our aim of improving the inhibition efficiency against PI3k with a C-7 substituent with a moderate impact on the duality of inhibition is achieved. Finally, the two dual derivatives 6 and 21 were chosen to measure their selectivity against the other PI3K isoforms. Values show a very important selectivity for PI3Kα versus other isoforms except for derivative 21 which led to a good additional activity on isoform β. Addition of the triazole ring in C-7 might therefore modify the interaction of the molecule with this isoform.

Docking Studies
To investigate the binding mode of the ligands and analyze the substituent effects on the inhibition and selectivity against PI3Kα and mTOR, we carried out docking studies. The structures of mTOR [36] and PI3Kα [37] were retrieved from the Protein Data Bank (PDB) [38]. Both structures interact with the pyridinylfuranopyrimidine inhibitor PI-103 that contains a morpholine group binding to the hinge through a hydrogen bond. The hydroxy moiety of the phenol is forming a hydrogen bond donor to the carboxylic acid group of Asp2195 side chain at the back of the inner pocket of PI3K. The PI3Kα and mTOR aligned structures were extracted from the MOE kinase-ligand complex library [39]. As stressed by Bryant et al. [40] or Wright et al. [41], the two targets share a high structure similarity but a low sequence identity.
All the compounds were docked into the active site of each studied protein, PI3Kα and mTOR. All the compounds exhibited a similar binding mode in which the pyridopyrimidine scaffold of all the docked compounds was well superimposed, creating similar interactions with each active site of the proteins. Only compound 6 has the flipped It appears immediately that the introduction of a substituent in C-7 decreases the inhibition of mTOR. Nevertheless, the selected derivatives remain active against this kinase and offer IC50 around 100 nM. Moreover, IC50 measurements showed that chemical modifications based on 1 offered a quasi-selective PI3K inhibitor 32. Additionally, we increased the number of potent dual inhibitors such as 5, 19 and 21, which possess a similar selectivity index (SI) around 10 value. With these three compounds, our aim of improving the inhibition efficiency against PI3k with a C-7 substituent with a moderate impact on the duality of inhibition is achieved. Finally, the two dual derivatives 6 and 21 were chosen to measure their selectivity against the other PI3K isoforms. Values show a very important selectivity for PI3Kα versus other isoforms except for derivative 21 which led to a good additional activity on isoform β. Addition of the triazole ring in C-7 might therefore modify the interaction of the molecule with this isoform.

Docking Studies
To investigate the binding mode of the ligands and analyze the substituent effects on the inhibition and selectivity against PI3Kα and mTOR, we carried out docking studies. The structures of mTOR [36] and PI3Kα [37] were retrieved from the Protein Data Bank (PDB) [38]. Both structures interact with the pyridinylfuranopyrimidine inhibitor PI-103 that contains a morpholine group binding to the hinge through a hydrogen bond. The hydroxy moiety of the phenol is forming a hydrogen bond donor to the carboxylic acid group of Asp2195 side chain at the back of the inner pocket of PI3K. The PI3Kα and mTOR aligned structures were extracted from the MOE kinase-ligand complex library [39]. As stressed by Bryant et al. [40] or Wright et al. [41], the two targets share a high structure similarity but a low sequence identity.
All the compounds were docked into the active site of each studied protein, PI3Kα and mTOR. All the compounds exhibited a similar binding mode in which the pyridopyrimidine scaffold of all the docked compounds was well superimposed, creating similar interactions with each active site of the proteins. Only compound 6 has the flipped It appears immediately that the introduction of a substituent in C-7 decreases the inhibition of mTOR. Nevertheless, the selected derivatives remain active against this kinase and offer IC50 around 100 nM. Moreover, IC50 measurements showed that chemical modifications based on 1 offered a quasi-selective PI3K inhibitor 32. Additionally, we increased the number of potent dual inhibitors such as 5, 19 and 21, which possess a similar selectivity index (SI) around 10 value. With these three compounds, our aim of improving the inhibition efficiency against PI3k with a C-7 substituent with a moderate impact on the duality of inhibition is achieved. Finally, the two dual derivatives 6 and 21 were chosen to measure their selectivity against the other PI3K isoforms. Values show a very important selectivity for PI3Kα versus other isoforms except for derivative 21 which led to a good additional activity on isoform β. Addition of the triazole ring in C-7 might therefore modify the interaction of the molecule with this isoform.

Docking Studies
To investigate the binding mode of the ligands and analyze the substituent effects on the inhibition and selectivity against PI3Kα and mTOR, we carried out docking studies. The structures of mTOR [36] and PI3Kα [37] were retrieved from the Protein Data Bank (PDB) [38]. Both structures interact with the pyridinylfuranopyrimidine inhibitor PI-103 that contains a morpholine group binding to the hinge through a hydrogen bond. The hydroxy moiety of the phenol is forming a hydrogen bond donor to the carboxylic acid group of Asp2195 side chain at the back of the inner pocket of PI3K. The PI3Kα and mTOR aligned structures were extracted from the MOE kinase-ligand complex library [39]. As stressed by Bryant et al. [40] or Wright et al. [41], the two targets share a high structure similarity but a low sequence identity.
All the compounds were docked into the active site of each studied protein, PI3Kα and mTOR. All the compounds exhibited a similar binding mode in which the pyridopyrimidine scaffold of all the docked compounds was well superimposed, creating similar interactions with each active site of the proteins. Only compound 6 has the flipped

Docking Studies
To investigate the binding mode of the ligands and analyze the substituent effects on the inhibition and selectivity against PI3Kα and mTOR, we carried out docking studies. The structures of mTOR [36] and PI3Kα [37] were retrieved from the Protein Data Bank (PDB) [38]. Both structures interact with the pyridinylfuranopyrimidine inhibitor PI-103 that contains a morpholine group binding to the hinge through a hydrogen bond. The hydroxy moiety of the phenol is forming a hydrogen bond donor to the carboxylic acid group of Asp2195 side chain at the back of the inner pocket of PI3K. The PI3Kα and mTOR aligned structures were extracted from the MOE kinase-ligand complex library [39]. As stressed by Bryant et al. [40] or Wright et al. [41], the two targets share a high structure similarity but a low sequence identity.
All the compounds were docked into the active site of each studied protein, PI3Kα and mTOR. All the compounds exhibited a similar binding mode in which the pyridopyrimidine scaffold of all the docked compounds was well superimposed, creating similar interactions with each active site of the proteins. Only compound 6 has the flipped hydroxyphenyl ring in PI3Kα. As suggested before, the side chains at the C-7 position point towards the solvent area and more precisely to the glycine rich loop for PI3Kα (Figure 3a) and the activation loop for mTOR (Figure 3b).
Analysis of the intermolecular interactions between the compounds and residues of the active site clearly showed that the oxygen atom of the morpholine moiety forms a hydrogen bond with Val851 of PI3Kα and Val2240 of mTOR located in the hinge region. The pyridopyrimidine scaffold is positioned in the ATP active site and the hydroxy group of the phenol moiety at the C-2 position is forming a hydrogen bond donor with the carboxylic acid group of Asp810 and Asp2195 residues of PI3Kα and mTOR, respectively.
In order to understand the duality observed with several derivatives, we focused on the docking solutions of derivative 6. Compared to other compounds, the docking poses of 6 ( Table 2, entries 2) specifically show an additional interaction in mTOR such as the π-CH interaction between the pyridine ring and the Met2345 residue ( Figure 4).  Analysis of the intermolecular interactions between the compounds and residues of the active site clearly showed that the oxygen atom of the morpholine moiety forms a hydrogen bond with Val851 of PI3Kα and Val2240 of mTOR located in the hinge region. The pyridopyrimidine scaffold is positioned in the ATP active site and the hydroxy group of the phenol moiety at the C-2 position is forming a hydrogen bond donor with the carboxylic acid group of Asp810 and Asp2195 residues of PI3Kα and mTOR, respectively. In order to understand the duality observed with several derivatives, we focused on the docking solutions of derivative 6. Compared to other compounds, the docking poses of 6 ( Table 2, entries 2) specifically show an additional interaction in mTOR such as the π-CH interaction between the pyridine ring and the Met2345 residue (Figure 4).  While compounds 6, 19 and 32 present higher activity on PI3Kα versus mTOR, docking results show similar binding modes in both kinases ( Figure 5). Additional computational experiments, such as molecular dynamics simulations or desolvation thermodynamics by displacing water molecules in the active site are needed to understand this discrepancy.  Analysis of the intermolecular interactions between the compounds and residues of the active site clearly showed that the oxygen atom of the morpholine moiety forms a hydrogen bond with Val851 of PI3Kα and Val2240 of mTOR located in the hinge region. The pyridopyrimidine scaffold is positioned in the ATP active site and the hydroxy group of the phenol moiety at the C-2 position is forming a hydrogen bond donor with the carboxylic acid group of Asp810 and Asp2195 residues of PI3Kα and mTOR, respectively. In order to understand the duality observed with several derivatives, we focused on the docking solutions of derivative 6. Compared to other compounds, the docking poses of 6 ( Table 2, entries 2) specifically show an additional interaction in mTOR such as the π-CH interaction between the pyridine ring and the Met2345 residue ( Figure 4).  While compounds 6, 19 and 32 present higher activity on PI3Kα versus mTOR, docking results show similar binding modes in both kinases ( Figure 5). Additional computational experiments, such as molecular dynamics simulations or desolvation thermodynamics by displacing water molecules in the active site are needed to understand this discrepancy.

Cell Assays
The most active derivative 32 on PI3Kα was first tested on six different cell lines hepatocellular carcinoma Huh-7, colorectal adenocarcinoma Caco-2, mammary c noma MDA-MB231, spontaneously immortalized keratinocytes Hacat, and normal man fibroblast (Table 3).

Cell Assays
The most active derivative 32 on PI3Kα was first tested on six different cell lines, i.e., hepatocellular carcinoma Huh-7, colorectal adenocarcinoma Caco-2, mammary carcinoma MDA-MB231, spontaneously immortalized keratinocytes Hacat, and normal human fibroblast (Table 3). This compound exhibited the same activity profile and induced a cytotoxicity in the micromolar range for Huh-7, Caco-2, Hacat and normal human fibroblasts thereby attesting a good cellular penetration, while MDA-MB231 cells were less sensitive. This resistance might be due to the overactivation of the Ras/MAPK pathway in these cells which harbor activating mutation in Ras and Raf [42]. Indeed, PI3K inhibition might not compensate for this pathway overactivation.

Cell Assays
The most active derivative 32 on PI3Kα was first tested on six different cell lines, i.e., hepatocellular carcinoma Huh-7, colorectal adenocarcinoma Caco-2, mammary carcinoma MDA-MB231, spontaneously immortalized keratinocytes Hacat, and normal human fibroblast (Table 3). This compound exhibited the same activity profile and induced a cytotoxicity in the micromolar range for Huh-7, Caco-2, Hacat and normal human fibroblasts thereby attesting a good cellular penetration, while MDA-MB231 cells were less sensitive. This resistance might be due to the overactivation of the Ras/MAPK pathway in these cells which harbor activating mutation in Ras and Raf [42]. Indeed, PI3K inhibition might not compensate for this pathway overactivation.

Cell Assays
The most active derivative 32 on PI3Kα was first tested on six different cell lines, i.e., hepatocellular carcinoma Huh-7, colorectal adenocarcinoma Caco-2, mammary carcinoma MDA-MB231, spontaneously immortalized keratinocytes Hacat, and normal human fibroblast (Table 3). This compound exhibited the same activity profile and induced a cytotoxicity in the micromolar range for Huh-7, Caco-2, Hacat and normal human fibroblasts thereby attesting a good cellular penetration, while MDA-MB231 cells were less sensitive. This resistance might be due to the overactivation of the Ras/MAPK pathway in these cells which harbor activating mutation in Ras and Raf [42]. Indeed, PI3K inhibition might not compensate for this pathway overactivation.

Cell Assays
The most active derivative 32 on PI3Kα was first tested on six different cell lines, i.e., hepatocellular carcinoma Huh-7, colorectal adenocarcinoma Caco-2, mammary carcinoma MDA-MB231, spontaneously immortalized keratinocytes Hacat, and normal human fibroblast (Table 3). This compound exhibited the same activity profile and induced a cytotoxicity in the micromolar range for Huh-7, Caco-2, Hacat and normal human fibroblasts thereby attesting a good cellular penetration, while MDA-MB231 cells were less sensitive. This resistance might be due to the overactivation of the Ras/MAPK pathway in these cells which harbor activating mutation in Ras and Raf [42]. Indeed, PI3K inhibition might not compensate for this pathway overactivation. This compound exhibited the same activity profile and induced a cytotoxicity in the micromolar range for Huh-7, Caco-2, Hacat and normal human fibroblasts thereby attesting a good cellular penetration, while MDA-MB231 cells were less sensitive. This resistance might be due to the overactivation of the Ras/MAPK pathway in these cells which harbor activating mutation in Ras and Raf [42]. Indeed, PI3K inhibition might not compensate for this pathway overactivation.
Compounds 5, 19 and 21, which inhibit the two kinase of interest, were then tested on the same cell lines panel. This time, fibroblasts were less sensitive than Huh-7, Caco-2 and Hacat cell lines. This result suggests that these compounds have more effects on fast proliferating cells such as cancer and immortalized cells lines with the exception of MDA-MB231 cells. Furthermore, on Caco-2 cells, compound 21 appeared to be the more cytotoxic while compound 19 appeared to have an intermediate effect between compounds 21 and 5. To further understand the origins of these different cytotoxicities, Akt phosphorylation (on T308) was analyzed by Western blot after treatment of Caco-2 cells with 5, 19 and 21 compounds. Figure 6A,B shows that compounds 5, 19 and 21 differentially affect Akt phosphorylation in cells and that their degree of cytotoxicity on Caco-2 survival perfectly correlates with their ability to inhibit PI3K pathway. By analogy, the best cytotoxic values displayed by 21 on all the other cell lines, especially MDA-MB-231 might be explained by a stronger PI3K pathway inhibition-indeed 21 possesses an additional effect on the PI3Kβ isoform-rather than an impact of triazole on other cell target(s). lation (on T308) was analyzed by Western blot after treatment of Caco-2 cells with 5, 19 and 21 compounds. Figure 6A,B shows that compounds 5, 19 and 21 differentially affect Akt phosphorylation in cells and that their degree of cytotoxicity on Caco-2 survival perfectly correlates with their ability to inhibit PI3K pathway. By analogy, the best cytotoxic values displayed by 21 on all the other cell lines, especially MDA-MB-231 might be explained by a stronger PI3K pathway inhibition-indeed 21 possesses an additional effect on the PI3Kβ isoform-rather than an impact of triazole on other cell target(s).

Chemistry
General procedure A: To a solution of halogenated derivative (1.0 eq.), in 1.2-dimethoxyethane was added boronic acid (1.5 eq.). An aqueous solution (1 M) of potassium carbonate (3.0 eq.) was then injected and the mixture was degassed by argon bubbling for 15 min. Pd(PPh3)4 (0.05 eq.) was added and the mixture was heated to 150 °C for 1 h by microwave irradiation. The solvent was removed in vacuo. The crude product was purified by flash chromatography.
General procedure C: To a solution of MOM protected compound (1.0 eq.), in dioxane was added a solution of HCl in dioxane, 4.0 M (6.0 eq.). The mixture was stirred at room temperature until completion monitored by TLC. The solvent was removed by filtration and the product was washed with diethyl ether prior to drying the solid under reduced pressure.
General procedure D: To a solution of aldehyde in dry CH2Cl2/DMF, 6/1 (6 mL), was added the secondary amine. After cooling the reaction to 0 °C, NaBH(OAc)3 (5.0 eq.) was added. After 10 min of stirring, four drops of acetic acid were added to the mixture. The reaction was stirred at room temperature for 5 h before adding water (5 mL) and extracting the product. The combined organic layers were washed with a solution of saturated NaHCO3 (2 × 10 mL) and dried over MgSO4 and filtered. The solvent was removed under

Chemistry
General procedure A: To a solution of halogenated derivative (1.0 eq.), in 1.2-dimethoxyethane was added boronic acid (1.5 eq.). An aqueous solution (1 M) of potassium carbonate (3.0 eq.) was then injected and the mixture was degassed by argon bubbling for 15 min. Pd(PPh 3 ) 4 (0.05 eq.) was added and the mixture was heated to 150 • C for 1 h by microwave irradiation. The solvent was removed in vacuo. The crude product was purified by flash chromatography.
General procedure C: To a solution of MOM protected compound (1.0 eq.), in dioxane was added a solution of HCl in dioxane, 4.0 M (6.0 eq.). The mixture was stirred at room temperature until completion monitored by TLC. The solvent was removed by filtration and the product was washed with diethyl ether prior to drying the solid under reduced pressure.
General procedure D: To a solution of aldehyde in dry CH 2 Cl 2 /DMF, 6/1 (6 mL), was added the secondary amine. After cooling the reaction to 0 • C, NaBH(OAc) 3 (5.0 eq.) was added. After 10 min of stirring, four drops of acetic acid were added to the mixture. The reaction was stirred at room temperature for 5 h before adding water (5 mL) and extracting the product. The combined organic layers were washed with a solution of saturated NaHCO 3 (2 × 10 mL) and dried over MgSO 4 and filtered. The solvent was removed under reduced pressure. The crude product then underwent the reaction as described in general procedure C to afford the final compound. [21]. To a solution of 1 [20] (1.0 g, 6.13 mmol) in phosphorus oxychloride (10 mL) was added phosphorus pentachloride (7.65 g, 36.78 mmol, 6.0 eq.). The mixture was heated by microwave irradiation at 160 • C for 2 h. The crude product was dissolved in CH 2 Cl 2 (100 mL) and was then poured in ice. The mixture was stirred at room temperature for 6 h and then extracted. The combined organic layers were washed with water (2 × 20 mL) and dried over MgSO 4 and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 /petroleum ether, 5/5) to afford 2 as a yellow solid (0.934 g, 65%). 1

3-(7-((Cyclopropylamino)methyl)-4-morpholinylpyrido[3,2-d]pyrimidin-2-yl)phenol hydrochloride salt (11).
To a solution of 8 (70 mg, 0.184 mmol) in dry CH 2 Cl 2 (6 mL), was added a small amount of MgSO 4 and cyclopropylamine (12 µL, 0.184 mmol, 1.0 eq.). The reaction was stirred at room temperature for 12 h before filtering the MgSO 4 and removing the solvent in vacuo. The crude product was diluted in methanol (6 mL) and NaBH 3 CN (60 mg, 0.92 mmol, 5.0 eq.) was added to the mixture. Once the reaction stopped bubbling (15 min), the crude product was extracted. The combined organic layers were washed with a solution of saturated NaHCO 3 (2 × 10 mL) and dried over MgSO 4 and filtered. The crude product was then subjected to the general procedure C to afford 11 as a white solid (28 mg, 40%).  (14). To a solution of NaBH 4 (60 mg, 1.58 mmol, 2.0 eq.) in MeOH (12 mL), was added 8 (300 mg, 0.79 mmol). The mixture was stirred at room temperature for two hours. The solvent was then removed under reduced pressure, the crude product was diluted in CH 2 Cl 2 (30 mL), the organic layer was washed with brine (10 mL), dried over MgSO 4 (15). The reaction was carried out as described in general procedure C using 14 (

3-(7-((4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)-4-morpholinylpyrido[3,2-d] pyrimidin-2-yl)phenol (23).
To a solution of 19 (82 mg, 0.23 mmol) in MeCN (4 mL) were added CuI (4 mg, 0.01 mmol, 0.05 eq.), 2-propyn-1-ol (16 µL, 0.25 mmol, 1.1 eq.) and a few drops of triethylamine until the products were soluble in the solvent. The reaction was stirred at room temperature for 12 h, before removing the solvent in vacuo. The crude product was dissolved in CH 2 Cl 2 (30 mL) and the organic layer was washed with a saturated solution of NaHCO 3 (10 mL) and brine (10 mL). The organic layer was dried over MgSO 4 and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica gel (CH 2 Cl 2 /MeOH, 98/2) to afford 23 as a yellow solid (49 mg, 51%). R f (CH 2 (24). Derivative 18 (35 mg, 0.17 mmol) was subjected to the triazole formation using the same procedure as described for 23. The crude material was dissolved in dry CH 2 Cl 2 (4 mL) and the mixture was cooled to 0 • C under inert atmosphere. A solution of DAST (11 µL, 0.083 mmol, 1.1 eq) was added slowly. After stirring at 0 • C, 22 µL of DAST (0.34 mmol, 2.0 eq) was added again. The mixture was stirred at 0 • C for 1 h. Hydrolysis was performed with a saturated solution of NaHCO 3 (10 mL). The organic layer was extracted with CH 2 Cl 2 (3 × 10 mL). The organic layer was dried over MgSO 4 and filtered. The solvent was removed under reduced pressure. The crude product directly underwent the reaction described in general procedure C to afford 24 as a yellow solid (44 mg, 62%). Mp: 168-170 • C. IR (Diamond ATR, cm −1 ) ν: 3354, 3046, 1620, 1562, 1506, was injected in a plate followed by the addition of 2.5 µL of PI3Kα and ATP/PIP2 (5 µL) solutions. After incubating for an hour at room temperature with no agitation and with the plate sealed, the kinase reaction was stopped by adding the Detection Solution to yield final concentrations of 10 nM for the tracer, 2 nM for the antibody and 30 mM of EDTA (5 µL). All of these compounds were diluted in a Detection Buffer (PV3574, Invitrogen) and the plate was sealed and incubated for the optimal time of 30 min at room temperature without shaking before the reading step. Emission values at 615 nm (corresponding to the donor) and 665 nm (corresponding to the acceptor) were measured and the TR-FRET signal corresponding to the ratio Em 665/Em 615 was calculated.

3-(7-((4-(Fluoromethyl)-1H-1,2,3-triazol-1-yl)methyl)-4-morpholinylpyrido[3,2-d] pyrimidin-2-yl)phenol
The LANCE TM Ultra kit (Perkin Elmer) contains a synthetic peptide of 4E-BP1, a natural substrate of mTOR, linked to an acceptor fluorophore (ULight) and an antibody against the phosphorylated form of this peptide linked to a donor fluorophore (Eu). The amount of antibody bound to the peptide is directly related to its rate of phosphorylation. Therefore, the TR-FRET signal decreases with the rate of peptide phosphorylation and a higher concentration of mTOR inhibitor. The experimental process was the following: inhibitor solution (2.5 µL) was injected in a plate followed by the addition of 2.5 µL of mTOR, and ATP/peptide (2.5 µL) solutions. After incubating for two hours at room temperature with no agitation and with the plate sealed, the kinase reaction was stopped by adding 5 µL of a solution at 32 mM EDTA. After 5 min of plate agitation, the detection solution (CR97-100, Perkin-Elmer) containing the antibody (5 µL) was added in each well to yield a final concentration of 2 nM of the antibody. The plate was sealed and incubated for the optimal time of 1h at room temperature without shaking before the assay readout at 665 nm.
Kinase tests using capillary electrophoresis (CE): The CE system used was a 1600 Hewlett Packard 3D CE (Agilent, Waldbronn, Germany) equipped with a photodiode array detection system. Agilent software 3D-CE Chemstation (rev B.04.02) was used to pilot the CE system and for signal acquisition. CE analyses were performed in silica capillaries (66 cm total length, 57.5 cm effective length, 50 µm i.d.) purchased from Polymicro Technologies (Phoenix, AZ, USA). New capillaries were conditioned by 1.0 M NaOH for 30 min and water for 5 min. They were then coated by rinsing with cationic PDADMAC solution (0.2% w/v) [46,47]. The separation was conducted at −30 kV (reverse polarity) and at 30 • C. All rinse cycles were carried out at 4 bars. Between runs, the capillary was flushed for 1 min with water, 2 min with PDADMAC and 3 min with BGE.
All solutions were prepared with pure water, filtered through 0.45 µm PVDF Filter and stored at 4 • C. For PI3K and mTOR assays, the incubation buffer was HEPES/NaOH/MgCl 2 (25 mM/8.84 mM/5 mM). Its pH was 7.2 and ionic strength was about 60 mM. The BGE was tris/phosphate/MgCl 2 (117 mM/62.5 mM/5 mM), its pH was 7.2 and ionic strength was 180 mM. All buffers were prepared fresh each day. Their pH was measured with a MeterLab PHM201 Portable pH-Meter (Radiometer Analytical, Villeurbanne, France). Stock solutions of ATP (2 mM) and ADP (2 mM) were prepared in the incubation buffer and diluted to 50 µM. Inhibitor stock solutions were prepared by dissolving 1 mg in 1 mL of incubation buffer and DMSO (<4% v/v).
In all enzymatic assays, after incubation, the electric field (of −30 kV) was applied to separate the ADP formed during the enzymatic reaction from the other reactants. ADP was detected at 254 nm for quantification. No inhibitor plug was injected to determine the maximum activity of the enzyme; it was replaced by a plug of incubation buffer. Control blank assays were done without injecting the enzyme.
ATP concentrations were set in all assays to be identical to those used in conventional methods (assay kits) for comparison. For IC 50 determination, enzyme and substrate concentration remained constant whereas at least 11 concentrations of inhibitors were used around the reported IC 50 value in order to precisely determine IC 50 . Assays were performed in triplicate (n = 3). The dose-response curve for enzyme inhibition was carried out by plotting the enzyme activity (%) versus Log [Inhibitor]. GraphPad Prism ® (GraphPad