Synthesis and PI 3-Kinase Inhibition Activity of Some Novel 2,4,6-Trisubstituted 1,3,5-Triazines

A number of new trisubstituted triazine phosphatidylinositol 3-kinase (PI3K) inhibitors were prepared via a three-step procedure utilizing sequential nucleophilic aromatic substitution and cross-coupling reactions. All were screened as PI3K inhibitors relative to the well-characterized PI3K inhibitor, ZSTK474. The most active inhibitors prepared here were 2–4 times more potent than ZSTK474. A leucine linker was attached to the most active inhibitor since it would remain on any peptide-containing prodrug after cleavage by a prostate-specific antigen, and it did not prevent inhibition of protein kinase B (Akt) phosphorylation, and hence, the inhibition of PI3K by the modified inhibitor.


Introduction
Androgen ablation therapy remains the most successful systemic treatment for prostate cancer. In most cases, the disease initially responds, but it eventually recurs as advanced androgen-independent prostate cancer, for which no effective treatment is currently available. Prostate cancer cells that acquire androgen independence become resistant to chemotherapy and radiation therapy [1][2][3]. To further extend the survival of prostate cancer patients, it is necessary to understand the mechanisms that underlie the responses of prostate cancer cells to therapies.
Normal prostate epithelial cells undergo apoptosis after androgen levels decrease [4]. It was proposed that, in order to survive, androgen-independent prostate cancers activate androgen receptors (ARs) by very low levels of androgen, or transactivate ARs in the absence of androgen [5,6]. Several therapeutic strategies for complete androgen ablation were developed, including combinations of gonadotropin-releasing hormone analogs (e.g., leuprolide) that cause continuous stimulation of the pituitary gland, abiraterone acetate (an inhibitor of androgen biosynthesis), and enzalutamide (MDV3100), an AR antagonist that prevents the binding of androgens to AR, nuclear translocation, and chromatin binding of AR. However, even with complete blockage of AR signaling, disease eventually progresses [7,8]. Accumulating evidence suggests that advanced prostate cancer engages androgen-independent signal transduction pathways that inhibit apoptosis, and hence, "bypass" the requirement for AR activation [9,10].
Numerous publications emphasize the role of anti-apoptotic signaling via protein kinase cascades in the therapeutic resistance of advanced androgen-independent prostate cancer, among which the

Chemistry
Triazines offer synthetic advantages over pyrimidines in that the addition of substituents onto its core does not produce regioisomers that have to be separated. The synthesis and characterization of trisubstituted triazines and pyrimidines remains an active area of PI3K inhibition research [38][39][40][41][42][43][44][45][46][47]. We focused most of our synthetic efforts on trisubstituted triazines (Scheme 1; 1) which contain (1) a linker group terminating in a primary alcohol which can be used as a peptide linkage point, (2) a secondary amine (largely morpholine due to its known importance [47]), and (3) a hydrogen-bonding aromatic or heteroaromatic group.
The triazine and pyrimidine cores offered simplicity in synthesis coupled with good PI3K inhibition activity [37,[48][49][50][51][52][53][54][55][56][57][58][59]. The triazines (1, Scheme 1) present no regiochemical addition problems, and the trichlorotriazine (cyanuric chloride) starting material (5, Scheme 1) was less expensive than the 2,4,6-trichloropyrimidine; as such, we focused our efforts on the triazines. Modeling, as previously described [34,53,55,58,59], indicated that compounds containing the pyrimidine or triazine core which exhibit K i s < 1 µm and good water solubility and stability often contained a morpholine substituent, as well as an aromatic or heteroaromatic ring containing a hydrogen-bond donor. The third and final position on the pyrimidine/triazine core was left to be the attachment point for a functional group capable of serving as the link to the PSA cleavable peptide sequence (Mu-LEHSSKLQL). Our initial work with LY 294002 analogs [34] used linkers that terminated in OH or NH 2 groups; so, we chose those types of linkers again. In addition to this linker, we were also interested in having a functional group in one of the other two core substituents which could interact with amino acids upon binding in PI3Ks, i.e., an ATP mimic with chemical reactivity more like wortmannin. Wortmannin functions as an irreversible PI3K inhibitor via a 1,4 conjugate addition reaction (a reaction with a soft electrophile, a Michael reaction) which takes place between the Lys 833 NH 2 and the α,β-unsaturated ester which is contained within the fused furan and lactone rings [60][61][62].
we report our work on triazines.

Chemistry
Triazines offer synthetic advantages over pyrimidines in that the addition of substituents onto its core does not produce regioisomers that have to be separated. The synthesis and characterization of trisubstituted triazines and pyrimidines remains an active area of PI3K inhibition research [38][39][40][41][42][43][44][45][46][47]. We focused most of our synthetic efforts on trisubstituted triazines (Scheme 1; 1) which contain (1) a linker group terminating in a primary alcohol which can be used as a peptide linkage point, (2) a secondary amine (largely morpholine due to its known importance [47]), and (3) a hydrogen-bonding aromatic or heteroaromatic group. Given the limiting parameters of a trisubstituted nitrogen heterocycle core containing a linker, an aromatic hydrogen-bond donor, and a heteroatom-containing secondary amine, we could, in theory, commence synthetic work by putting on any one of these three groups. In practice, we envisioned the last halogen on the heterocyclic core being replaced by a cross-coupling reaction rather than nucleophilic substitution; so, we left this for our third step. This decision meant we could investigate synthetic routes that started by adding linkers first or secondary amines (Scheme 2) first. The triazine and pyrimidine cores offered simplicity in synthesis coupled with good PI3K inhibition activity [37,[48][49][50][51][52][53][54][55][56][57][58][59]. The triazines (1, Scheme 1) present no regiochemical addition problems, and the trichlorotriazine (cyanuric chloride) starting material (5, Scheme 1) was less expensive than the 2,4,6-trichloropyrimidine; as such, we focused our efforts on the triazines. Modeling, as previously described [34,53,55,58,59], indicated that compounds containing the pyrimidine or triazine core which exhibit Kis < 1 µm and good water solubility and stability often contained a morpholine substituent, as well as an aromatic or heteroaromatic ring containing a hydrogen-bond donor. The third and final position on the pyrimidine/triazine core was left to be the attachment point for a functional group capable of serving as the link to the PSA cleavable peptide sequence (Mu-LEHSSKLQL). Our initial work with LY 294002 analogs [34] used linkers that terminated in OH or NH2 groups; so, we chose those types of linkers again. In addition to this linker, we were also interested in having a functional group in one of the other two core substituents which could interact with amino acids upon binding in PI3Ks, i.e., an ATP mimic with chemical reactivity more like wortmannin. Wortmannin functions as an irreversible PI3K inhibitor via a 1,4 conjugate addition reaction (a reaction with a soft electrophile, a Michael reaction) which takes place between the Lys 833 NH2 and the α,β-unsaturated ester which is contained within the fused furan and lactone rings [60][61][62].
Given the limiting parameters of a trisubstituted nitrogen heterocycle core containing a linker, an aromatic hydrogen-bond donor, and a heteroatom-containing secondary amine, we could, in theory, commence synthetic work by putting on any one of these three groups. In practice, we envisioned the last halogen on the heterocyclic core being replaced by a cross-coupling reaction rather than nucleophilic substitution; so, we left this for our third step. This decision meant we could investigate synthetic routes that started by adding linkers first or secondary amines (Scheme 2) first.

First Addition Reaction
When we investigated the addition of aminoalcohols as the first step, we found the addition of 4-aminobenzylalcohol, 4-aminomethylbenzylalcohol, and 4-aminophenylethanol to all react sluggishly with cyanuric chloride at low temperatures (0 °C to −20 °C), and to give mixtures of monoand di-addition products at higher temperatures (≥25 °C). Furthermore, 6-aminohexanol reacted well with cyanuric chloride to provide monosubstituted triazine (7, Scheme 2) with a 64% yield. We found Venkatesan's procedure [63] of performing these reactions in acetone followed by the addition of ice Scheme 2. Nucleophilic aromatic substitution reactions of chlorotriazines.

First Addition Reaction
When we investigated the addition of aminoalcohols as the first step, we found the addition of 4-aminobenzylalcohol, 4-aminomethylbenzylalcohol, and 4-aminophenylethanol to all react sluggishly with cyanuric chloride at low temperatures (0 • C to −20 • C), and to give mixtures of mono-and di-addition products at higher temperatures (≥25 • C). Furthermore, 6-aminohexanol reacted well with cyanuric chloride to provide monosubstituted triazine (7, Scheme 2) with a 64% yield. We found Venkatesan's procedure [63] of performing these reactions in acetone followed by the addition of ice water to be often preferable to other methods, since the monosubstituted triazine products precipitate under those reactions conditions. Morpholine and ethyl isonipecotate also reacted well with with cyanuric chloride under these conditions, producing 9a and 9b (Scheme 2).

Second Addition Reaction
Compound 9a (Scheme 2) was then treated with a variety of aromatic primary amines, 9b (Scheme 2) was treated with 4-aminophenethylalcohol, and compound 7 (Scheme 2) was treated with morpholine. Primary amines were added to compounds 9a and 9b (Scheme 2) using K 2 CO 3 in DMF with good yield. The morpholine/aminohexanol di-addition product (10a, Scheme 2) could be prepared as described above, starting from 9 (Scheme 2), or prepared starting from compound 7 (Scheme 2) using Et 3 N in CH 3 CN with essentially equal ease. Compounds 10a, 10b, and 10e (Scheme 2) required chromatographic purification whereas 10c and 10d (Scheme 2) did not, which perhaps accounts for the higher isolated yields in those two cases.

Third Addition Reaction
A variety of aromatic substituents were then added to these disubstituted triazines (10a-d, Scheme 2) via cross-coupling reactions, and a number of cross-coupling reaction conditions were investigated (Scheme 3). In many cases, the polarity of the final products was as much of a challenge as the cross-coupling conditions in determining isolated yields of cross-coupled products (for instance, the organic solubility of 10d over 10b lead us to pursue 10d (Scheme 3)). Pd(PPh 3 ) 4 (10 mol %) was used initially as a catalyst in DMSO, THF, and DME (4:1 organic solvent, 2 M Na 2 CO 3 ). DME proved superior, and we observed no product when the 2 M Na 2 CO 3 was eliminated. Pd(dppf)Cl 2 , Pd(OAc) 2 /PPh 3 , Pd[(PtBu) 3 ] 2 , and Pd 2 dba 3 /PPh 3 were all investigated as catalysts, and the in situ-generated Pd(PPh 3 ) 4 from Pd 2 dba 3 provided the consistently best yields of cross-coupled product. PPh 3 proved superior as a ligand to SPhos, JohnPhos, and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene. Polystyrene-bound PPh 3 used with Pd 2 dba 3 was ineffective, but polystyrene-bound Pd(PPh 3 ) 4 (PS-Pd(PPh 3 ) 4 ) was used successfully with 13h (Scheme 3). In other cases, isolated yields of cross-coupled products were typically lower with PS-Pd(PPh 3 ) 4 , but its use eliminated the chromatographic separation of these polar cross-coupled products from the byproduct, OPPh 3 . Our final optimized cross-coupling conditions were to use 1.5 equivalents of boronic acid or ester in 4:1 DME:2 M Na 2 CO 3 . Pd 2 dba 3 (10 mol %) and PPh 3 (80 mol %) were added to a sealable microwave tube, which was degassed and sealed, and irradiated 10-50 min at 300 W with a 100 • C temperature cut-off. These cross-coupling conditions never produced products from 10e (Scheme 3) in greater than 20% isolated yield; as such, those compounds were not pursued further. water to be often preferable to other methods, since the monosubstituted triazine products precipitate under those reactions conditions. Morpholine and ethyl isonipecotate also reacted well with with cyanuric chloride under these conditions, producing 9a and 9b (Scheme 2).

Second Addition Reaction
Compound 9a (Scheme 2) was then treated with a variety of aromatic primary amines, 9b (Scheme 2) was treated with 4-aminophenethylalcohol, and compound 7 (Scheme 2) was treated with morpholine. Primary amines were added to compounds 9a and 9b (Scheme 2) using K2CO3 in DMF with good yield. The morpholine/aminohexanol di-addition product (10a, Scheme 2) could be prepared as described above, starting from 9 (Scheme 2), or prepared starting from compound 7 (Scheme 2) using Et3N in CH3CN with essentially equal ease. Compounds 10a, 10b, and 10e (Scheme 2) required chromatographic purification whereas 10c and 10d (Scheme 2) did not, which perhaps accounts for the higher isolated yields in those two cases.

Third Addition Reaction
A variety of aromatic substituents were then added to these disubstituted triazines (10a-d, Scheme 2) via cross-coupling reactions, and a number of cross-coupling reaction conditions were investigated (Scheme 3). In many cases, the polarity of the final products was as much of a challenge as the cross-coupling conditions in determining isolated yields of cross-coupled products (for instance, the organic solubility of 10d over 10b lead us to pursue 10d (Scheme 3)). Pd(PPh3)4 (10 mol %) was used initially as a catalyst in DMSO, THF, and DME (4:1 organic solvent, 2 M Na2CO3). DME proved superior, and we observed no product when the 2 M Na2CO3 was eliminated. Pd(dppf)Cl2, Pd(OAc)2/PPh3, Pd[(PtBu)3]2, and Pd2dba3/PPh3 were all investigated as catalysts, and the in situgenerated Pd(PPh3)4 from Pd2dba3 provided the consistently best yields of cross-coupled product. PPh3 proved superior as a ligand to SPhos, JohnPhos, and 9,9-dimethyl-4,5bis(diphenylphosphino)xanthene. Polystyrene-bound PPh3 used with Pd2dba3 was ineffective, but polystyrene-bound Pd(PPh3)4 (PS-Pd(PPh3)4) was used successfully with 13h (Scheme 3). In other cases, isolated yields of cross-coupled products were typically lower with PS-Pd(PPh3)4, but its use eliminated the chromatographic separation of these polar cross-coupled products from the byproduct, OPPh3. Our final optimized cross-coupling conditions were to use 1.5 equivalents of boronic acid or ester in 4:1 DME:2 M Na2CO3. Pd2dba3 (10 mol %) and PPh3 (80 mol %) were added to a sealable microwave tube, which was degassed and sealed, and irradiated 10-50 min at 300 W with a 100 °C temperature cut-off. These cross-coupling conditions never produced products from 10e (Scheme 3) in greater than 20% isolated yield; as such, those compounds were not pursued further.

Addition of Leucine to the Lead Compound (13h, Scheme 3)
All compounds 11-13 (Scheme 3) were screened as PI3K inhibitors, and compared directly to ZSTK 474 in this assay (see Supplementary Materials for Western blots). Trisubstituted triazine (13h, Scheme 2) was our most active compound as a PI3K inhibitor (see screening discussion below); therefore, we undertook its preparation where leucine was added to the primary OH in the linker group. In our earlier work [34], we showed that a PSA-cleavable peptide (Mu-LEHSSKLQ) added to produce prostate-specific prodrugs cleaved between L and Q; thus, any peptide-linked PI3K inhibitor contains a leucine residue. Two different routes to the desired compound were explored. In one case, Boc-protected leucine was added to the primary alcohol in the linker in disubstituted triazine (10d, Scheme 4), and then, this compound (14, Scheme 4) was subjected to cross-coupling conditions to produce 15 (Scheme 4), which was immediately deprotected. In an alternate route, Boc-protected leucine was added directly to 13h (Scheme 4) to produce 15 (Scheme 4), and then, the protecting group was removed to yield 16 (Scheme 4).

Addition of Leucine to the Lead Compound (13h, Scheme 3)
All compounds 11-13 (Scheme 3) were screened as PI3K inhibitors, and compared directly to ZSTK 474 in this assay (see Supplementary Materials for Western blots). Trisubstituted triazine (13h, Scheme 2) was our most active compound as a PI3K inhibitor (see screening discussion below); therefore, we undertook its preparation where leucine was added to the primary OH in the linker group. In our earlier work [34], we showed that a PSA-cleavable peptide (Mu-LEHSSKLQ) added to produce prostate-specific prodrugs cleaved between L and Q; thus, any peptide-linked PI3K inhibitor contains a leucine residue. Two different routes to the desired compound were explored. In one case, Boc-protected leucine was added to the primary alcohol in the linker in disubstituted triazine (10d, Scheme 4), and then, this compound (14, Scheme 4) was subjected to cross-coupling conditions to produce 15 (Scheme 4), which was immediately deprotected. In an alternate route, Boc-protected leucine was added directly to 13h (Scheme 4) to produce 15 (Scheme 4), and then, the protecting group was removed to yield 16 (Scheme 4).

Scheme 4.
Addition of leucine to lead compound, 13h.

Biological Activity
To assess the biological activity of the new PI3K inhibitor compounds, trisubstituted triazine (13h) and 16 (Scheme 4), the experiments were conducted in C4-2 prostate cancer cells that represent castration-resistant metastatic prostate cancer. C4-2 cells are characterized by constitutive activation of the PI3K pathway due to the loss of expression of the PI3 phosphatase, PTEN. Protein kinase B/Akt is among the best characterized downstream effectors of the PI3K pathway. Activation of PI3K leads to the accumulation of phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 binds to the pleckstrin homology (PH) domains of Akt and pyruvate dehydrogenase kinases (PDKs), and recruits Akt and PDKs to the plasma membrane. This, in turn, leads to the phosphorylation of Akt at T308 by PDK1, and to phosphorylation at S473 by the rapamycin-insensitive mTOR:Rictor:Gβ complex. Thus, the phosphorylation of Akt at S473 and T308 faithfully reflect activation of the PI3K pathway in most cell lines, and are routinely used to monitor PI3K activity [64].
The analysis of the phosphorylation of S473 Akt and of T308 in C4-2 cells was used in this study to assess PI3K inhibition by triazine derivatives. Figure 1 shows representative western blots that illustrate the inhibition of S473 Akt phosphorylation by 13h and by 16 (Scheme 4), which contains the leucine linker.
Quantitation of western blots using image J software showed that respective IC50 values for 13h and 16 (Scheme 4) were 6.3-and 3.6-fold, respectively, more potent compared to the wellcharacterized PI3K inhibitor, ZSTK474. Thus, the 50% inhibition of S473 Akt phosphorylation was observed at lower concentrations of 13h and 16 (Scheme 4) than the 50% inhibition by ZSTK474. The comparison of phosphorylated S473 Akt (pS473 Akt) at 1 µM of ZSTK474, and compounds 13h or 16 (Scheme 4), showed that the inhibition of S473 Akt phosphorylation by compound 13h (Scheme 4) at

Biological Activity
To assess the biological activity of the new PI3K inhibitor compounds, trisubstituted triazine (13h) and 16 (Scheme 4), the experiments were conducted in C4-2 prostate cancer cells that represent castration-resistant metastatic prostate cancer. C4-2 cells are characterized by constitutive activation of the PI3K pathway due to the loss of expression of the PI3 phosphatase, PTEN. Protein kinase B/Akt is among the best characterized downstream effectors of the PI3K pathway. Activation of PI3K leads to the accumulation of phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) in the plasma membrane. PIP 3 binds to the pleckstrin homology (PH) domains of Akt and pyruvate dehydrogenase kinases (PDKs), and recruits Akt and PDKs to the plasma membrane. This, in turn, leads to the phosphorylation of Akt at T308 by PDK1, and to phosphorylation at S473 by the rapamycin-insensitive mTOR:Rictor:Gβ complex. Thus, the phosphorylation of Akt at S473 and T308 faithfully reflect activation of the PI3K pathway in most cell lines, and are routinely used to monitor PI3K activity [64].
The analysis of the phosphorylation of S473 Akt and of T308 in C4-2 cells was used in this study to assess PI3K inhibition by triazine derivatives. Figure 1 shows representative western blots that illustrate the inhibition of S473 Akt phosphorylation by 13h and by 16 (Scheme 4), which contains the leucine linker.
Quantitation of western blots using image J software showed that respective IC 50 values for 13h and 16 (Scheme 4) were 6.3-and 3.6-fold, respectively, more potent compared to the well-characterized PI3K inhibitor, ZSTK474. Thus, the 50% inhibition of S473 Akt phosphorylation was observed at lower concentrations of 13h and 16 (Scheme 4) than the 50% inhibition by ZSTK474. The comparison of phosphorylated S473 Akt (pS473 Akt) at 1 µM of ZSTK474, and compounds 13h or 16 (Scheme 4), showed that the inhibition of S473 Akt phosphorylation by compound 13h (Scheme 4) at 1 µM was 3.74 times more potent than inhibition by ZSTK474 at the same concentration (p = 0.0015); whereas inhibition by compound 16 (Scheme 4) was 2.25-fold more potent compared to ZSTK474 (p = 0.038).

General Methods
Unless otherwise stated, all reactions were performed in a flame-dried round-bottom flask or a glass microwaveable tube. The flame-dried glassware was further cooled and maintained under an atmosphere of nitrogen or argon using standard Schlenk techniques before the start of the reaction. Reagents were purchased from common chemical suppliers, and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on 2.5 cm × 7.5 cm Silica G TLC Plates (200-µm thickness) from Sorbtech (Norcross, GA, USA). TLC plates were pre-coated with a fluorescent indicator, and after plate development, were examined under 254-nm UV light. Flash chromatography was performed using SiliaFlash P60 230-400 mesh silica gel from Silicycle. All 1 Hand 13 C-NMR spectra were recorded using Bruker Avance 300 MHz or 500 MHz multinuclear spectrometers at ambient temperature. Chemical shifts were reported in parts per million (δ) relative to tetramethylsilane (TMS) or to residual resonances of the deuterated solvents. Coupling constants (J values) were reported in Hertz (Hz), and spin multiplicities were indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), and m (multiplet).
When stated, samples were sent off for elemental analysis to Atlantic Microlab, Inc. (Norcross, GA, USA). Samples were submitted to either the University of North Carolina at Chapel Hill's Chemistry High Resolution Mass Spectrometry Facility or to Northwestern University's High Resolution Mass Spectrometry Facility for HRMS analysis. HRMS analysis was also performed within Wake Forest University's Chemistry Department using Thermo Scientific's LTQ HRMS

General Methods
Unless otherwise stated, all reactions were performed in a flame-dried round-bottom flask or a glass microwaveable tube. The flame-dried glassware was further cooled and maintained under an atmosphere of nitrogen or argon using standard Schlenk techniques before the start of the reaction. Reagents were purchased from common chemical suppliers, and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on 2.5 cm × 7.5 cm Silica G TLC Plates (200-µm thickness) from Sorbtech (Norcross, GA, USA). TLC plates were pre-coated with a fluorescent indicator, and after plate development, were examined under 254-nm UV light. Flash chromatography was performed using SiliaFlash P60 230-400 mesh silica gel from Silicycle. All 1 H-and 13 C-NMR spectra were recorded using Bruker Avance 300 MHz or 500 MHz multinuclear spectrometers at ambient temperature. Chemical shifts were reported in parts per million (δ) relative to tetramethylsilane (TMS) or to residual resonances of the deuterated solvents. Coupling constants (J values) were reported in Hertz (Hz), and spin multiplicities were indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), and m (multiplet).

Conclusions
In conclusion, we conducted the synthesis and pilot characterization of a new trisubstituted triazine PI3K inhibitor with increased potency at 1 M (2-4-fold) compared to the well-characterized PI3K inhibitor, ZSTK474. At concentrations lower than 1 M, our data cannot be unambiguously interpreted, and additional experiments are needed in the future. We also demonstrated that this new compound can be used to produce a latent prodrug selectively activated in prostate tumors that secretes the prostate-specific antigen (PSA) protease. Thus, attachment of a leucine linker did not prevent the inhibition of Akt phosphorylation, and hence, the inhibition of PI3K by the modified inhibitor. The leucine linker remains on the prodrug after cleavage by PSA of an inhibitor peptide, preventing penetration of the inactive prodrug into target cells. Future experiments will examine the toxicity profile of the new PI3K inhibitor, and will determine if prodrugs based on this compound would provide more potent inhibition of PI3K in prostate tumors that secrete PSA compared to currently used PI3K inhibitors.