Synthesis and PI 3-kinase inhibition activity of some novel 2,4,6-trisubstituted 1,3,5-triazines

Article abstract of DOI:10.3390/molecules23071628

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.

Full text of DOI:10.3390/molecules23071628

molecules
Article
Synthesis and PI 3-Kinase Inhibition Activity of
Some Novel 2,4,6-Trisubstituted 1,3,5-Triazines
Ronald A. Nelson Jr. 1, Taylor Schronce 1, Yue Huang 2, Alanoud Albugami 3, George Kulik 2,3,
*
and Mark E. Welker 1,
*
1
Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA;
ranelson@wakehealth.edu (R.A.N.J.); taylorschronce@alumni.wfu.edu (T.S.)
Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest School of Medicine,
Medical Center Blvd., Winston-Salem, NC 27157, USA; yhuang@wakehealth.edu
Life Sciences program, College of Science, Alfaisal University, Riyadh 11533, Saudi Arabia;
aalbugumi@alfaisal.edu
2
3
*
Correspondence: gkulik@wakehealth.edu (G.K.); welker@wfu.edu (M.E.W.); Tel.: +1-336-713-7650 (G.K.);
+1-336-702-1953 (M.E.W.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 14 June 2018; Accepted: 2 July 2018; Published: 4 July 2018
Abstract: 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.
Keywords: triazine synthesis; PI3K inhibitor; prostate cancer
1. 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 [1–3]. 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 [
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 [ ]. Several
4]. It was
5,6
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
Molecules 2018, 23, 1628; doi:10.3390/molecules23071628
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phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway gained prominence [10
Continuous activation of PI3K/Akt signaling allows prostate tumors to inhibit apoptosis, maintaining
continued proliferation of malignant cells in a low-androgen environment [15 17]. Mutations leading to
–14].
–
activation of the PI3K/Akt pathway are reported in almost all advanced metastatic prostate cancer [18].
Loss of the phosphatase and tensin homolog (PTEN) phosphatase is the most common mechanism
of constitutive activation of PI3K signaling in cancer cells [19], which was documented in 15–30% of
primary, and over 60% of androgen-independent metastatic cancers [20–24].
Knocking out PTEN in the prostates of mice can trigger the development of prostate cancer [25].
Constitutive activation leads to “addiction” of cancer cells to the PI3K pathway; conversely,
an inhibition of the PI3K pathway sensitizes prostate cancer cells to apoptosis [14,26,27]. Understanding
PI3K/Akt/ mammalian target of rapamycin (mTOR) signaling pathways, and the development of
clinically useful inhibitors remain an active area of cancer research [28–33].
In 2012 [34], we reported that prodrugs containing PI3K inhibitors could be activated via peptide
cleavage by prostate-specific antigen (PSA). Cheng and co-workers also reported the generation
of prostate-selective PI3K inhibitors [35]. We already showed the specific inhibition of PI3K in
PSA-secreting prostate cancer cells by a Mu-LEHSSKLQL-LY294002 (prodrug-LY294002) [34]. However,
LY294002 inhibits PI3K at relatively high concentrations (25 µM) and is rapidly metabolized, so while
its ease of synthesis made it a good choice for proof of concept, these pharmacological properties make
it a poor candidate for in vivo studies.
We were confident that PSA would cleave the Mu-LEHSSKLQL peptide after glutamine (Q),
thus leaving leucine (L) attached to the PI3K inhibitor [36]. Computer modeling of the interactions
between PI3K inhibitors and the ATP-binding cleft of PI3K were used to identify positions at which
the PSA substrate peptide could be attached, so as to prevent the linker plus leucine from diminishing
PI3K inhibition. Following our review of PI3K inhibitor literature [37], we were most interested in
preparing trisubstituted pyrimidines and triazines as the next test of this prodrug concept, and here,
we report our work on triazines.
2. Results and Discussion
2.1. 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
–47].
We focused most of our synthetic efforts on trisubstituted triazines (Scheme 1; ) which contain (1)
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
and the trichlorotriazine (cyanuric chloride) starting material (
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 m and good water solubility and stability often contained a morpholine substituent,
,48
–59]. The triazines (
1, Scheme 1) present no regiochemical addition problems,
5, Scheme 1) was less expensive than
,
,
,
,
µ
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

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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
is contained within the fused furan and lactone rings [60–62].
α,β-unsaturated ester which
Scheme 1. Retrosynthesis of trisubstituted triazines.
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.
Scheme 2. Nucleophilic aromatic substitution reactions of chlorotriazines.
2.2. 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
di-addition products at higher temperatures (
−
20 C), and to give mixtures of mono- and
◦
≥
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

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under those reactions conditions. Morpholine and ethyl isonipecotate also reacted well with with
cyanuric chloride under these conditions, producing 9a and 9b (Scheme 2).
2.3. 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.
2.4. 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 situ-generated 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-4,5-bis(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.
Scheme 3. Cross coupling reactions of disubstituted chlorotriazines.

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2.5. Addition of Leucine to the Lead Compound (13h, Scheme 3)
All compounds 11
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);
–13 (Scheme 3) were screened as PI3K inhibitors, and compared directly
(
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.
2.6. 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:G
β
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 well-characterized
PI3K inhibitor, ZSTK474. Thus, the 50% inhibition of S473 Akt phosphorylation was observed at lower

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concentrations of 13h and 16 (Scheme 4) than the 50% inhibition by ZSTK474. The comparison of
phosphorylated S473 Akt (pS473 Akt) at 1 M of ZSTK474, and compounds 13h or 16 (Scheme 4),
showed that the inhibition of S473 Akt phosphorylation by compound 13h (Scheme 4) at 1 M was
µ
µ
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).
Figure 1. Representative western blots of S473 protein kinase B (Akt) phosphorylation in C4-2 prostate
cancer cells. (
A
) C4-2 cells were treated with ZSTK474 or compound 13h (Scheme 4; 10, 1, or 0.1
µM
each). ( ) C4-2 cells were treated with ZSTK474 or compound 16 (Scheme 4; 10, 1, or 0.1
B
µM each).
Equal loading was controlled by probing western blot for beta-actin.
3. Materials and Methods
3.1. Compound Synthesis
3.1.1. 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- m thickness) from Sorbtech (Norcross, GA, USA). TLC plates were pre-coated
µ
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 1H- and 13C-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

Molecules 2018, 23, 1628
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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 Orbitrap XL
(Waltham, MA, USA). LC–MS analyses were performed on samples using the direct injection method
for an Agilent 1100 SL meterspectro (Santa Clara, CA, USA). Lastly, TLC–MS analyses were performed
using Advion (Ithaca, NY, USA) Expression-LCMS and Advion (Ithaca, NY, USA) Plate Express
TLC-MS spectrometers.
3.1.2. 6-((4,6-Dichloro-1,3,5-triazin-2-yl)amino)hexan-1-ol (7, Scheme 2)
In a 500-mL round-bottom flask with a stir bar, cyanuric chloride (
was dissolved in acetonitrile (10 mL) and placed under nitrogen in an ice bath. Then, 6-amino-1-hexanol
, Scheme 2; 1.880 g, 16.1 mmol) was dissolved in acetonitrile (5 mL), and N,N-diisopropylethylamine
5, Scheme 2; 1.840 g, 10.0 mmol)
(6
(7.18 mL, 40.0 mmol) was added. The mixture was added dropwise to the dissolved cyanuric
chloride. After the dropwise addition, the ice bath was removed, and the reaction was allowed
to stir for approximately one hour. The solvent was removed by rotary evaporation and high vacuum.
The product was purified via column chromatography (silica, 1:1 ethyl acetate and heptanes) to
give 6-((4,6-dichloro-1,3,5-triazin-2-yl)amino)hexan-1-ol (7, Scheme 2; 1.697 g, 6.4 mmol, 64%) Rf 0.40
1
(1:1 ethyl acetate in heptane) as a yellow waxy substance. H-NMR (300 MHz, CDCl3)
δ
6.03 (br d,
168.7,
1H), 3.66 (t, J = 6 Hz, 2H), 3.47 (q, J = 6 Hz, 2H) 1.63–1.20 (m, 9H). 13C-NMR (75 MHz, CDCl3)
δ
167.5, 163.6, 60.5, 39.2, 30.2, 26.7, 24.1, 23.
3.1.3. 4-(4,6-Dichloro-1,3,5-triazin-2-yl)morpholine (9a, Scheme 2)
Cyanuric chloride (
5
, Scheme 2; 4.979 g, 27.0 mmol) was dissolved in acetone (50 mL) and cooled to
◦
−
20 C. Morpholine (
8
, Scheme 2; 0.7 eq., 1.654 g, 19.0 mmol) and triethylamine (0.7 eq., 1.922 g,
19.0 mmol) were mixed in an addition funnel, and added dropwise to the cooled reaction flask.
The reaction stirred for 15 min before being quenched with cold water (500 mL). The solid material was
allowed to settle in the flask before being filtered through a Buchner funnel, and was washed with cold
methanol. The solid white material was dried overnight using a high-vacuum pump, and then, further
purified using column chromatography (silica) with 20% ethyl acetate in hexanes as the eluent. White
1
product (9a, Scheme 2) [65] was collected (2.903 g, 12.4 mmol, 65%). H-NMR (300 MHz, CDCl3)
δ 3.89
(dd, J = 5.6, 4.0 Hz, 4H), 3.80–3.64 (m, 4H). 13C-NMR (75 MHz, CDCl3) δ 170.38, 164.04, 66.36, 44.45.
3.1.4. Ethyl 1-(4,6-dichloro-1,3,5-triazin-2-yl)piperidine-4-carboxylate (9b, Scheme 2)
Cyanuric chloride (
78 C. In a separate container, ethyl piperidine-4-carboxylate (
5
, Scheme 2; 1.840 g, 10.0 mmol) was added to a round-bottom flask and cooled
◦
to
−
8, Scheme 2; 1 eq., 1.570 g, 10.0 mmol)
and acetone (25 mL) were combined. This mixture was added dropwise to the cooled round-bottom
flask using a syringe pump at a rate of 25 mL per hour. The reaction was then concentrated using
rotary evaporation, before being purified using column chromatography (silica) with 1:1 ethyl acetate
in hexanes as the eluent. Product (9b, Scheme 2) was obtained as a white solid (1.434 g, 4.7 mmol, 47%).
1H-NMR (300 MHz, CDCl3)
11.0, 3.2 Hz, 2H), 2.63 (tt, J = 10.4, 4.1 Hz, 1H), 2.03 (dq, J = 12.6, 3.8 Hz, 2H), 1.76 (m, 2H), 1.27
(t, J = 7.1 Hz, 3H). 13C-NMR (101 MHz, CDCl3)
173.71, 170.31, 163.81, 60.81, 43.47, 40.55, 27.73, 14.20.
δ 4.54 (dt, J = 13.6, 4.0 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.24 (ddd, J = 13.9,
δ
3.1.5. 6-((4-Chloro-6-morpholino-1,3,5-triazin-2-yl)amino)hexan-1-ol (10a, Scheme 2)
Additionally, 6-((4,6-dichloro-1,3,5-triazin-2-yl)amino)hexan-1-ol ( , Scheme 2; 1.000 g, 4.0 mmol)
7
was dissolved in acetonitrile (25 mL). Triethylamine (2.09 mL, 15.0 mmol) and morpholine (0.342 mL,
4.0 mmol) were added dropwise to the reaction mixture while stirring. The reaction was allowed to stir
under nitrogen for approximately 48 h. The solvent was removed by rotary evaporation, and the crude
product was purified via column chromatography (silica, 1:1 ethyl acetate and dichloromethane) to
give 6-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)hexan-1-ol (10a, Scheme 2; 0.557 g, 1.76 mmol,

Molecules 2018, 23, 1628
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47%): Rf 0.57 (100% ethyl acetate) as a white powder. 1H-NMR (300 MHz, CDCl3)
δ
5.42 (s, 1H),
4.78–4.65 (br s, 1H), 3.87–3.59 (m, 10H), 3.38 (q, J = 6.6 Hz, 2H), 1.67–1.49 (m, 4H), 1.40 (m, 4H).
13C-NMR (75 MHz, CDCl3)
δ 168.9, 165.53, 164.22, 66.63, 62.77, 43.86, 40.84, 32.55, 29.17, 26.60, 25.43.
ESI MS calculated for C13H22N5O2Cl 315.8; found 316.2. HRMS [M + H]+ calculated for C13H23N5O2Cl
316.1540; found 316.1526.
3.1.6. (4-(((4-Chloro-6-morpholino-1,3,5-triazin-2-yl)amino)methyl)phenyl)methanol (10b, Scheme 2)
Furthermore, 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (9a, Scheme 2; 0.588 g, 2.5 mmol),
(4-(aminomethyl)phenyl)methanol (1 eq., 0.343 g, 2.5 mmol), and K2CO3 (1 eq., 0.343 g, 2.5 mmol)
were combined in a flask. The solid material was dissolved in DMF (10 mL), and was stirred for 72 h at
room temperature (heated if necessary on the last day). Brine (100 mL) was added to the flask and the
product was precipitated. The solid was filtered and dried overnight under high vacuum. The product
was purified using column chromatography (silica) with 50% ethyl acetate in hexanes as the eluent.
1
White product (10b, Scheme 2) was produced (0.428 g, 1.28 mmol, 51%). H-NMR (300 MHz, CDCl3)
δ
7.29 (m, 4H), 5.92 (s, 1H), 4.69 (s, 2H), 4.57 (d, J = 6.1 Hz, 2H), 3.79 (m, 4H), 3.69 (t, J = 4.6 Hz, 4H),
1.88 (br s, 1H). 13C-NMR (101 MHz, DMSO-d6)
δ 169.35, 168.74, 166.01, 165.66, 164.46, 164.32, 141.59,
137.98, 137.93, 127.65, 127.30, 126.91, 126.87, 66.16, 63.15, 44.00, 43.95. Most 13C-NMR resonances show
evidence of two conformers due to hindered rotation around the C–NHR bond.
3.1.7. Methyl 4-(((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)methyl)benzoate (10c, Scheme 2)
Moreover, 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (9a, Scheme 2; 0.825 g, 3.5 mmol),
potassium carbonate (1 eq., 0.500 g, 3.5 mmol), and anhydrous DMF (20 mL) were combined and
stirred at room temperature. A solution of methyl 4-(aminomethyl)benzoate (1 eq., 0.580 g, 3.5 mmol)
in anhydrous DMF (5 mL) was added dropwise, and was then stirred for 72 h at room temperature.
The reaction was poured into water (500 mL), and the precipitate was filtered using a Buchner funnel,
washed with 20 mL cold methanol, and dried overnight. White product (10c, Scheme 2) was obtained
(1.082 g, 2.98 mmol, 85%). Elemental analysis calculated for C16H18N5O3Cl: C, 52.82 (found 52.58);
1
H, 4.99 (found 5.01). H-NMR (300 MHz, CDCl3)
δ
8.01 (d, J = 8 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 4.66
165.09, 143.39, 129.92,
(d, J = 6.1 Hz, 2H), 3.93 (s, 3H), 3.81–3.70 (m, 9H). 13C-NMR (75 MHz, CDCl3)
δ
127.47, 127.06, 76.72, 66.59, 66.42, 52.14, 44.63, 44.07. HRMS [M + H]+ calculated for C16H19N5O3Cl:
364.1176; observed 364.1171.
3.1.8. 2-(4-((4-Chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (10d, Scheme 2)
Subsequently, 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (9a, Scheme 2; 3.393 g, 14.4 mmol),
4-aminophenethyl alcohol (1 eq., 1.980 g, 14.4 mmol), and potassium carbonate (1 eq., 1.980 g,
14.4 mmol) were combined. The solid material was dissolved in anhydrous DMF (90 mL), and was
stirred for 72 h at room temperature (monitored via TLC). The reaction contents were added into a
separatory funnel with 1:1 brine and ethyl acetate, and were extracted twice (2
layer was dried with sodium sulfate, filtered, and concentrated using rotary evaporation and high
×
100 mL). The organic
vacuum (no further purification needed). White product (10d, Scheme 2) was obtained (3.730 g,
11.1 mmol, 77%). Elemental analysis calculated for C15H18N5O2Cl: C, 53.65 (found 53.45); H, 5.40
1
(found 5.44). H-NMR (300 MHz, DMSO-d6)
δ 10.05 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.16 (d, J = 8.1 Hz,
2H), 4.60 (s, 1H), 3.71–3.53 (m, 10H), 2.68 (t, J = 7.1 Hz, 2H), 2.45 (s, 1H). 13C-NMR (75 MHz, DMSO-d6)
136.42, 134.34, 128.94, 120.08, 65.67, 62.16, 43.66, 38.40. HRMS [M + H]+ calculated for C15H19ClN5O2,
δ
336.1222; observed 336.1227.
3.1.9. Ethyl 1-(4-chloro-6-((4-(2-hydroxyethyl)phenyl)amino)-1,3,5-triazin-2-yl)piperidine-4-
carboxylate (10e, Scheme 2)
Ethyl 1-(4,6-dichloro-1,3,5-triazin-2-yl)piperidine-4-carboxylate (9b, Scheme 2; 0.687 g, 2.3 mmol),
4-aminophenethyl alcohol (1 eq., 0.309 g, 2.3 mmol), K2CO3 (1 eq., 0.311 g, 2.3 mmol), and DMF (8 mL)

Molecules 2018, 23, 1628
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were combined in a round-bottom flask and stirred for 72 h at room temperature. Brine (100 mL) was
added and extracted with ethyl acetate (2 50 mL). The organic layer was washed with additional
brine solution (3 50 mL). The organic layer was dried with sodium sulfate, filtered, and then,
×
×
concentrated via rotary evaporation. Purification involved using column chromatography (silica) with
1:1 ethyl acetate in hexanes as the eluent. Product was recovered as a white solid (0.457 g, 1.13 mmol,
1
50%). H-NMR (300 MHz, CDCl3)
δ 7.45 (d, J = 8 Hz, 2H), 7.21 (d, J = 8 Hz, 2H), 7.07 (s, 1H), 4.56 (br t,
2H), 4.16 (q, J = 7 Hz, 2H), 3.86 (m, 2H), 3.14 (m, 2H), 2.85 (t, J = 6.5 Hz, 2H), 2.60 (m, 1H), 2.00 (m, 2H),
1.73 (m, 2H) 1.27 (t, J = 7 Hz, 3H). 13C-NMR (75 MHz, CDCl3)
δ
174.17, 169.25, 164.37, 163.79, 136.28,
134.09, 129.52, 120.58, 63.62, 60.67, 43.14, 41.02, 38.57, 27.96, 27.80, 14.22 -. Elemental analysis calculated
for C19H24N5O3Cl, C, 56.22 (found 55.97); H, 5.96 (found 5.96).
3.1.10. N-(3-(4-((6-hydroxyhexyl)amino)-6-morpholino-1,3,5-triazin-2-yl)phenyl)acetamide (11c,
Scheme 3)
Firstly, 6-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)hexan-1-ol (10a, Scheme 3; 0.063 g,
0.2 mmol) and N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide (0.166 g, 0.4 mmol)
were combined with DME (4 mL) and 2 M Na2CO3 (1 mL, 2.0 mmol). The reaction mixture was
degassed with nitrogen, and PPh3 (0.021 g, 0.08 mmol) and Pd2(dba)3 (0.037 g, 0.04 mmol) were
◦
quickly added. The reaction mixture was placed in the CEM Discover under 300
µ
W and 100 C
conditions, and was set to hold for 30 minutes. After the completion time of 50 minutes, the reaction
mixture was greenish black. The reaction was allowed to cool in an ice bath, was quenched with
water (10 mL), and was extracted with ethyl acetate (3
×
10 mL). The organic layer was dried using
NaSO4, and was filtered and condensed by rotary evaporation. The product was purified with column
chromatography on silica gel with a 1:1 ethyl acetate and heptane solvent mobile phase to give
N-(3-(4-((6-hydroxyhexyl)amino)-6-morpholino-1,3,5-triazin-2-yl)phenyl)acetamide (11c, Scheme 3;
0.057g, 0.14 mmol, 69%): Rf 0.04 (1:1 ethyl acetate in heptane) as a light-brown solid. 1H-NMR
(300 MHz, DMSO-d6)
δ 10.04 (s, 1H), 8.42 (s, 1H), 8.04–7.74 (m, 2H), 7.50–7.29 (m, 2H), 4.32 (t, J = 5.1 Hz,
1H), 3.66 (d, J = 4.9 Hz, 6H), 3.44–3.22 (m, 6H), 2.06 (s, J = 1.6 Hz, 3H), 1.36–1.27 (m, 8H). 13C-NMR
(75 MHz, DMSO-d6)
δ 169.00, 168.20, 165.59, 164.43, 139.12, 137.43, 128.28, 122.44, 121.72, 118.58,
65.88, 60.58, 43.07, 32.43, 29.24, 28.75, 26.22, 25.14, 23.87. HRMS [M + H]+ calculated for C21H30N6O3:
415.2379 Found: 415.2450.
3.1.11. Ethyl 4-(((4-(2-aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)amino)methyl)benzoate
(12, Scheme 3)
Methyl 4-(((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)methyl)benzoate (10c, Scheme 3;
0.447g, 1.23 mmol), 2-aminopyrimidine-5-boronic acid pinacol ester (1.5 eq., 0.403 g, 1.83 mmol),
and a 34:1 DME (17 mL):2 M Na2CO3 (500 µL) solution were added into a microwaveable tube.
The mixture was degassed for 1 min before adding Pd(PPh3)4 (10 mol %). The microwave tube was
heated to 100 ◦C for 24 h before cooling to room temperature. The microwave tube was chilled on ice
1
before filtering the solid product (12, Scheme 3; 0.218 g, 0.52 mmol, 42%). H-NMR (300 MHz, CDCl3)
δ
9.17 (s, 2H), 8.03 (d, J = 6.0 Hz, 2H), 7.43 (d, J = 6.0 Hz, 2H), 5.36 (s, 2H), 4.72 (br s, 1H), 3.93 (s, 3H),
3.87–3.75 (m, 8H).
3.1.12. 2-(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol
(13a, Scheme 3)
Additionally, 2-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol
(10d,
Scheme 3; 0.200 g, 0.49 mmol), 2-aminopyrimidine-5-boronic acid pinacol ester (1.5 eq., 0.164 g,
0.74 mmol), and a 4:1 DME (8 mL) 2 M Na2CO3 (2 mL) solution were added to a microwaveable tube.
The solution was degassed before adding Pd2(dba)3 (10 mol %, 0.048 g, 0.049 mmol) and PPh3 (80 mol
%, 0.096 g, 0.38 mmol). The microwave tube was sealed and heated to 90 ◦C for 50 min in a microwave
reactor. The microwave tube was cooled to room temperature, and was decanted into a separatory

Molecules 2018, 23, 1628
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funnel containing 1:1 ether (10 mL) and water (10 mL). The aqueous phase was removed before adding
6 M HCl (10 mL) to the organic phase. The ether phase was removed, and 3 M NaOH (10 mL or enough
until all HCl was quenched) was added to the aqueous phase. The product was extracted using fresh
ether (10 mL), dried with sodium sulfate, filtered, and then concentrated via rotary evaporation to
1
produce a white powder (0.089 g, 0.23 mmol, 46%). H-NMR (300 MHz, DMSO-d6)
δ
9.57 (br s, 1H),
9.06 (s, 1H), 8.26–8.12 (m, 1H), 7.60 (d, J = 8 Hz, 2H), 7.28 (s, 1H), 7.16 (d, J = 8 Hz, 2H), 6.55 (m, 1H),
4.60 (br s, 1H), 3.82–3.57 (m, 8H), 3.68 (t, J = 7 Hz, 2H), 2.66 (t, J = 8 Hz, 2H).
3.1.13. 2-(4-((4-(4-Aminophenyl)-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (13b,
Scheme 3)
Furthermore, 2-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (10d
,
Scheme 3; 0.068 g, 0.20 mmol), 4-aminophenyl boronic acid pinacol ester (1.5 eq., 0.066 g, 0.30 mmol),
and a 4:1 DME (4mL):2 M Na2CO3 (1 mL) solution were added to a microwaveable tube. The mixture
was degassed before adding Pd2(dba)3 (10 mol %, 0.020 g, 0.020 mmol) and PPh3 (80 mol %, 0.040 g,
◦
0.16 mmol). The sealed microwave tube was heated to 90 C for 50 min in a microwave reactor.
The microwave tube was cooled to room temperature and 5 mL of brine solution was added before
extracting with ethyl acetate (3
×
5 mL). The organic layer was dried with sodium sulfate, filtered,
and concentrated via rotary evaporation. The material was purified using column chromatography
(silica) with 100% ethyl acetate as the eluent. Product was obtained a solid (0.024 g, 0.062 mmol, 31%).
1H-NMR (300 MHz, CDCl3)
δ 8.24 (d, J = 6 Hz, 2H), 7.58 (d, J = 6 Hz, 2H), 7.23 (d, J = 6 Hz, 2H), 6.72
(d, J = 6 Hz, 2H), 3.95–3.63 (m, 8H), 3.87 (t, J = 6.5 Hz, 2H), 2.86 (t, J = 6.5 Hz, 2H).
3.1.14. Tert-butyl(5-(4-((4-(2-hydroxyethyl)phenyl)amino)-6-morpholino-1,3,5-triazin-2-
yl)pyrimidin-2-yl)carbamate (13c, Scheme 3)
Moreover, 2-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (10d, Scheme 3;
0.068 g, 0.20 mmol), tert-butyl N-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl]carbamate
(1.5 eq., 0.096 g, 0.30 mmol), and a 4:1 DME (4 mL):2 M Na2CO3 (1 mL) solution were added to a
microwaveable tube. The mixture was degassed before adding Pd2dba3 (10 mol %, 0.020 g, 0.020 mmol)
and PPh3 (80 mol %, 0.040 g, 0.16 mmol). The sealed microwave tube was heated to 90 ◦C for 50 min
in a microwave reactor. The microwave tube was cooled to room temperature and the contents filtered
through a pad of celite, and then, rinsed with ethyl acetate (~10 mL). The organic layer was dried with
sodium sulfate, filtered, and concentrated via rotary evaporation. The material was then purified using
column chromatography (silica) with 100% ethyl acetate as the eluent. White product was recovered
as a solid (0.042 g, 0.084 mmol, 42%). Elemental analysis calculated for C24H30N8O4, C, 58.29 (found
1
57.46); H, 6.11 (found 6.09). H-NMR (300 MHz, CDCl3)
δ 9.46 (s, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.25
(d, J = 8 Hz, 2H), 7.04 (br s, 1H), 3.88 (s, 9H), 3.83–3.60 (m, 8H), 3.78 (t, J = 6.5 Hz, 2H), 2.87 (t, J = 6.5
Hz, 2H), 1.56 (s, 9H). HRMS [M + H]+ calculated for C24H31N8O4, 495.2467; observed 495.2463.
3.1.15. 2-(4-((4-(2-Methoxypyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol
(13d, Scheme 3)
Subsequently, 2-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (10d
Scheme 3; 0.068 g, 0.20 mmol), 2-methoxypyrimidine-5-boronic acid (1.5 eq., 0.046 g, 0.30 mmol),
and a 4:1 DME (4 mL):2 M Na2CO3 (1 mL) solution were added to a microwaveable tube. The mixture
was degassed before adding Pd2(dba)3 (10 mol %, 0.020 g, 0.020 mmol) and PPh3 (80 mol %, 0.040
g, 0.16 mmol). The sealed microwave tube was heated to 90 ◦C for 60 min in a microwave reactor.
The reaction mixture was cooled to room temperature then filtered through a pad of celite and rinsed
with ethyl acetate (~10 mL). The solution was concentrated via rotary evaporation and impurities
,
removed through trituration with chloroform (3
solid product (0.059 g, 0.14 mmol, 72%). H-NMR (300 MHz, CDCl3)
×
1 mL) and acetonitrile (3
×
1 mL) to yield a white
1
δ
9.40 (s, 2H), 7.59 (d, J = 6 Hz,

Molecules 2018, 23, 1628
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2H), 7.23 (d, J = 6 Hz, 2H), 7.01 (s, 1H), 4.07 (s, 3H), 3.88 (t, J = 6.6 Hz, 2H), 3.90–3.63 (m, 8H), 2.87
(t, J = 6.5 Hz, 3H). HRMS [M + H]+ calculated for C20H24N7O3, 410.1940; observed 410.1941.
3.1.16.
2-(4-((4-Morpholino-6-(6-(trifluoromethyl)pyridin-3-yl)-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol
(13f, Scheme 3)
Then, 2-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (10d, Scheme 3;
0.068 g, 0.20 mmol), 2-trifluoromethylpyridine-5-boronic acid (2 eq., 0.076 g, 0.40 mmol), and a 4:1 DME
(4 mL):2 M Na2CO3 (1 mL) solution were added to a microwaveable tube. The mixture was degassed
before adding Pd2dba3 (20 mol %, 0.037 g, 0.04 mmol) and PPh3 (40 mol %, 0.020 g, 0.08 mmol).
The sealed microwave tube was heated to 100 ◦C for 20 min in a CEM Discovery microwave reactor
(300
µW). The reaction was cooled to room temperature before adding water (5 mL) and extracting with
ethyl acetate (4
×
5 mL). The organic layer was dried with sodium sulfate, filtered, and concentrated
via rotary evaporation. Product was purified using column chromatography (silica) with 1:1 ethyl
acetate in hexanes as the eluent, which was gradually changed to 100% ethyl acetate. A cream-colored
1
product was recovered (0.071 g, 0.16 mmol, 80%). H-NMR (300 MHz, DMSO-d6)
δ 9.83 (br s, 1H),
9.58 (s, 1H), 8.85 (d, J = 6 Hz, 1H), 8.05 (d, J = 6 Hz, 1H), 7.66 (d, J = 6 Hz, 2H), 7.19 (d, J = 6 Hz, 2H),
4.63 (t, J = 6 Hz, 2H), 3.81–3.66 (m, 8H), 2.71 (t, J = 6 Hz, 2H). 13C-NMR (75 MHz, DMSO-d6)
δ
167.11,
164.29, 163.80, 149.44, 137.44, 137.10, 135.28, 133.77, 128.87, 120.60, 120.12, 65.86, 62.22, 43.43, 38.83,
38.43. HRMS [M + H]+ calculated for C21H22N6O2F3, 447.1756; observed 447.1743.
3.1.17. N-(4-(4-((4-(2-hydroxyethyl)phenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)phenyl)acetamide
(13h, Scheme 3)
Finally, 2-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (10d, Scheme 3;
0.068 g, 0.20 mmol), 4-acetylaminophenyl boronic acid pinacol ester (2 eq., 0.104 g, 0.40 mmol), and a 4:1
DME (4 mL):2 M Na2CO3 (1 mL) solution were added to a microwaveable tube. The mixture degassed
before adding PS–(PPh3)4–Pd (10 mol %, 0.182 g, 0.03 mmol). The sealed microwave tube was heated
to 100 ◦C for 30 min in a CEM Discovery microwave reactor. The reaction was cooled before adding
brine (5 mL) and extracting with ethyl acetate (3
×
5 mL). Saturated sodium bicarbonate solution
(5 mL) was added to the aqueous layer and extracted with fresh ethyl acetate (5 mL). The organic
layers were dried with magnesium sulfate, filtered, and concentrated via rotary evaporation. Product
was purified using column chromatography (silica) with 5% ethanol in dichloromethane (DCM) as
the eluent; gradually changed to 10% ethanol in DCM. Product was recovered as a solid (0.076 g,
1
0.17 mmol, 87%). H-NMR (300 MHz, DMSO-d6)
δ 10.18 (s, 1H), 9.55 (s, 1H), 8.29 (d, J = 6 Hz, 2H),
7.76–7.60 (m, 4H), 7.17 (d, J = 6 Hz, 2H), 4.60 (t, J = 6 Hz, 1H), 3.71–3.56 (m, 8H), 2.68 (t, J = 6 Hz,
2H), 2.08 (s, 3H). 13C-NMR (75 MHz, DMSO-d6)
δ 169.27, 168.61, 164.57, 164.05, 142.39, 137.58, 133.22,
130.93, 128.84, 128.76, 119.84, 118.18, 65.94, 62.26, 43.37, 24.10. HRMS [M + H]+ calculated C23H27N6O3,
435.2144; observed 435.2136.
3.1.18. 4-((4-Chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenethyl (tert-butoxycarbonyl)leucinate
(14, Scheme 4)
To begin, 2-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-ol (10d, Scheme 4;
0.068 g, 0.20 mmol), Boc-protected L-leucine (2 eq., 0.093 g, 0.40 mmol), dicyclohexylcarbodiimide
(DCC; 2 eq., 0.083 g, 0.40 mmol), and dimethylaminopyridine (DMAP) (1 eq., 0.024 g, 0.20 mmol) were
added to a round-bottom flask, and then, DCM (5 mL) was added before stirring overnight at room
temperature (monitored via TLC analysis). The reaction was diluted with fresh DCM (15 mL) and
washed with cold 1 M HCl (10 mL), saturated sodium bicarbonate solution (5 mL), and then, brine
solution (5 mL). The organic layer was dried with sodium sulfate, filtered, and then, concentrated
via rotary evaporation. The solid was purified by column chromatography using 1:1 ethyl acetate in
hexanes are the eluent. Product (14, Scheme 4) was recovered as a solid (0.045 g, 0.082 mmol, 41%).

Molecules 2018, 23, 1628
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1H-NMR (400 MHz, CDCl3)
δ
10.01 (s, 0H), 9.34 (d, J = 7.4 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.49–7.41
(m, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.20 (dd, J = 7.7, 5.6 Hz, 1H), 5.33 (s, 0H), 4.94–4.77 (m, 1H), 4.40–4.24
(m, 1H), 3.91 (t, J = 4.7 Hz, 1H), 3.87–3.76 (m, 1H), 3.79 (s, 1H), 3.74 (d, J = 4.8 Hz, 2H), 3.51 (s, 1H), 3.43
(s, 2H), 2.94 (td, J = 7.0, 3.7 Hz, 1H), 1.90 (dt, J = 12.4, 3.8 Hz, 2H), 1.74–1.59 (m, 2H), 1.48–1.34 (m, 9H),
1.38–1.23 (m, 1H), 1.22–1.05 (m, 2H), 0.98–0.86 (m, 6H).
3.1.19. 4-((4-(4-Acetamidophenyl)-6-morpholino-1,3,5-triazin-2-yl)amino)phenethyl
(tert-butoxycarbonyl)leucinate (15, Scheme 4)
Furthermore, 4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)amino)phenethyl (tert-butoxycarbonyl)
leucinate (14, Scheme 4; 0.055 g, 0.10 mmol), 4-acetylaminophenyl boronic acid pinacol ester (2 eq.,
0.052 g, 0.20 mmol), and a 4:1 DME (4 mL):2 M Na2CO3 (1 mL) solution were added to a microwaveable
tube. The mixture was degassed for 15 minutes before adding PS–(PPh3)4–Pd (10 mol %, 0.182 g,
◦
0.03 mmol). The sealed microwave tube was heated to 100 C for 30 min in a CEM Discovery
microwave reactor. The reaction was cooled before adding brine (5 mL) and extracting with ethyl
acetate (3 × 5 mL). Saturated sodium bicarbonate solution (5 mL) was added to the aqueous layer and
extracted with fresh ethyl acetate (5 mL). Organic layers were combined and dried with sodium sulfate,
filtered, and concentrated via rotary evaporation. Product was purified using column chromatography
with 1:1 ethyl acetate in hexanes as the eluent, which was gradually changed to 100% ethyl acetate.
Product (15, Scheme 4) was recovered (0.049 g, 0.075 mmol, 75%; identical by MS comparison to 15
(Scheme 4) described below), and was immediately deprotected in a second reaction.
3.1.20. 4-((4-(4-Acetamidophenyl)-6-morpholino-1,3,5-triazin-2-yl)amino)phenethyl
(tert-butoxycarbonyl)leucinate (15, Scheme 4)
In acetonitrile (2 mL), Boc-leucine (0.011 g, 0.049 mmol), N,N0-dicyclohexylcarbodiimide
(0.013 g, 0.065 mmol), and 4-dimethylaminopyridine (one crystal) were added. To this mixture,
N-(4-(4-((4-(2-hydroxyethyl)phenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)phenyl)acetamide (13h
,
Scheme 4; 0.014 g, 0.032 mmol) in acetonitrile (2 mL) was added and stirred for 48 h at room temperature.
After 48 h, an additional measure of Boc-leucine (0.011 g, 0.049 mmol), N,N0-dicyclohexylcarbodiimide
(0.013 g, 0.065 mmol), and 4-dimethylaminopyridine (one crystal) were added and stirred for an
additional 24 h at room temperature. After 24 h, the reaction was concentrated into a solid powder via
rotary evaporation, and then, purified using column chromatography (1:1 ethyl acetate in hexanes).
Product (15, Scheme 4) was recovered as a solid (0.009 g, 0.014 mmol, 43%). ESI MS M+ calculated for
C33H45N7O6 635.7; found 635.3 (100), 592.2 (10), 460.2 (10), 306.3 (34), 266.2 (22), 225.2 (10).
3.1.21. 4-((4-(4-Acetamidophenyl)-6-morpholino-1,3,5-triazin-2-yl)amino)phenethyl leucinate (16,
Scheme 4)
Additionally, 4-((4-(4-acetamidophenyl)-6-morpholino-1,3,5-triazin-2-yl) amino) phenethyl (tert-
butoxycarbonyl) leucinate (15, Scheme 4; 0.046 g, 0.071 mmol), para-toluenesulfonic acid (2 eq., 0.027 g,
0.14 mmol), and acetonitrile (5 mL) were added to a round-bottom flask and stirred for 48 h at room
temperature. On the second day, another equivalent of para-toluenesulfonic acid (0.014 g, 0.071 mmol)
was added. The reaction stirred for another 24 h before concentrating via rotary evaporation. The contents
were then dissolved in cold water (3 mL) and ethyl acetate (5 mL). The aqueous layer was removed, and
saturated sodium bicarbonate (3 mL) was added to the organic layer. This aqueous layer was extracted
three additional times with ethyl acetate (3 × 5mL). Organic layers were combined, dried with sodium
sulfate, filtered, and then, concentrated via rotary evaporation. Column chromatography (silica) was
performed using 1:1 ethyl acetate in hexanes as the eluent, which was gradually changed to 100% ethyl
acetate, then 10% ethanol in ethyl acetate. Product (16, Scheme 4) was recovered as a solid (0.005 g,
0.009 mmol, 13%; ~90% pure) and all this material was used for screening. ESI MS M+ calculated for
C29H37N7O4 547.6; found 547.8 (100), 527.9 (15), 434.8 (30), 414.9 (50), 301.9 (25), 283.9 (10).

Molecules 2018, 23, 1628
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3.2. Western Blot Analysis on Synthesized PI3K Inhibitors
General Methods
Media were removed and cells were washed gently twice using cold phosphate-buffered
saline (PBS) on ice. Lysates were generated using cold 20 mM HEPES, 150 mM NaCl, 1 mM
EDTA, 0.5% Na + deoxycholate, 1% Nonidet P-40, 1 mM DTT pH 7.4 buffer-containing protease
inhibitors (10
and 1 mM PMSF), and phosphatase inhibitors (1 mM NaVO4, 50 mM
p-nitrophenylphosphate, 40 mM NaF, and 1 g/mL microcystin). All reagents were purchased from
µg/mL aprotinin, 10
µg/mL leupeptin, 10
µg/mL pepstatin, 1 mM benzamidine,
β-glycerophosphate, 40 mM
µ
Sigma-Aldrich (St. Louis, MO, USA). Cell lysates were clarified by centrifugation at 15,000 rpm for
15 min at 4 ◦C, and the supernatant was collected and the protein content measured by a Bradford
assay (Bio-Rad Laboratories, Hercules, CA, USA) per the manufacturer’s directions. Proteins were
separated on SDS-PAGE on 12% gels, and were transferred onto a 0.45-µm nitrocellulose membrane
(PerkinElmer, Waltham, MA, USA). Western blotting was performed with the Odyssey CLx Infrared
Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) per the manufacturer’s instructions. Rabbit
polyclonal phosphorylated Akt (Ser473; D9E) XP, phosphorylated Akt (Thr308; D25E6) XP, and anti-Akt
were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-β-Actin
was purchased from Sigma-Aldrich. Secondary goat anti-mouse IRDye 680 and goat-anti rabbit
IRDye 800 were both purchased from Li-Cor Biosciences. Protein bands were quantified using ImageJ
software (National Institutes of Health, USA). Rabbit mAb #4060, Ref: 08/2015, Lot:19 (Cell Signaling).
ZSTK474 is an ATP-competitive inhibitor of class I PI3K isoforms with an IC50 value of 37 nM in a
cell-free assay. We used this PI3K inhibitor as a control when testing our synthesized compounds.
4. 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.
Supplementary Materials: Supplementary materials include spectral data for new compounds, and can be
accessed at.
Author Contributions: R.A.N.J. performed most of the synthetic work described here, while T.S. performed the
remainder, and Y.H. performed most of the biological evaluation work described here. A.A. and G.K. performed
some of the biological evaluation. R.A.N.J., M.W., and G.K. co-authored this manuscript.
Funding: This research was funded by R21CA182248 Grant from NCI, and SRG Grant 407071502154 from
Alfaisal University.
Acknowledgments: The authors acknowledge support from the Wake Forest University Collaborative Research
Fund and a Pilot Project Grant from the Comprehensive Cancer Center at Wake Forest Baptist Medical Center.
Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
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