Discovery and SAR of Novel Disubstituted Quinazolines as Dual PI3Kalpha/mTOR Inhibitors Targeting Breast Cancer

PI3Kalpha/mTOR Inhibitors Targeting Breast Cancer

Letter

Discovery and SAR of Novel Disubstituted Quinazolines as Dual

Aisha A. K. Al-Ashmawy,* Khaled M. Elokely, Oscar Perez-Leal, Mario Rico, John Gordon,

George Mateo, Abdelsattar M. Omar, Magid Abou-Gharbia, and Wayne E. Childers, Jr.*

Cite This: https://dx.doi.org/10.1021/acsmedchemlett.0c00289

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ABSTRACT: The dual PI3Kα/ m TOR inhibitors represent a

promising molecularly targeted therapy for cancer. Here, we

documented the discovery of new 2,4-disubstituted quinazoline

analogs as potent dual PI3Kα/sm TOR inhibitors. Our structure

based chemistry endeavor yielded six excellent compounds 9e, 9f,

g, 9k, 9m, and 9o with single/double digit nanomolar IC values

against both enzymes and acceptable aqueous solubility and

stability to oxidative metabolism. One of those analogs, 9m,

possessed a sulfonamide substituent, which has not been described for this chemical scaﬀold before. The short direct synthetic

routes, structure−activity relationship, in vitro 2D cell culture viability assays against normal ﬁbroblasts and 3 breast cancer cell lines,

and in vitro 3D culture viability assay against MCF7 cells for this series are described.

KEYWORDS: 4-Morpholinoquinazolines, PI3Kα, mammalian target of rapamycin, mTOR. dual inhibitors, breast cancer cell lines

he human PI3K/Akt/mTOR signaling pathway is

implicated in vital processes such as cell proliferation,

upstream regulator receptor tyrosine kinases (RTKs). Regard-

less of the mechanistic cause, the end result is tumorigenesis

and angiogenesis.

survival, and metabolism. This central pathway receives growth

signals and then propagates them through a series of

phosphorylations and dephosphorylations of downstream

Oncology drug discovery for these cancers targets key nodes

in this pathway to avoid the feedback of downstream eﬀectors

7−9

eﬀectors. Phosphatidylinositol-3-kinases (PI3Ks) are lipid

and to gain expected synergistic suppression.

Upregulation

of the PI3K pathway is a major mechanism of resistance to

kinases which catalyze the phosphorylation of

phosphatidylinisotol(4,5)-biphosphate (PIP2) to

phosphatidylinositol(3,4,5)-triphosphate (PIP3). This secon-

dary messenger is the master key in signal transfer through the

activation of protein kinase B (Akt) and other downregulators

such as mammalian target of rapamycin (mTOR). According

to structural features and substrate speciﬁcity, PI3Ks are

divided into four classes, I (subclassiﬁed as IA and IB), II, III,

and IV. Class IV enzymes are also deﬁned as phosphoinositide-

many molecularly targeted cancer therapeutics. Therefore,

the design of small molecule ATP competitive dual PI3Kα/

mTOR inhibitors is an appealing approach which has been

underserved to date as there is only one approved drug on the

market. There are, however, some advanced candidates in

diﬀerent phases of development (Figure 1). Pﬁzer is currently

performing Phase II clinical trials on gedatosilib (PKI-587, 1),

a potent selective triazine derivative, for the treatment of breast

cancer and acute myeloid leukemia. Sanoﬁ-Aventis is

examining voxtalisib (XL-765, 2) in phase I studies for the

-kinase related protein kinases (PIKKs), and they share a

conserved catalytic domain sequence to that of PI3Ks. PI3Kα

12,13

oral treatment of lymphoma and solid tumors.

PI-103 (3)

is an example of Class IA, while mTOR, a serine-threonine

was the ﬁrst quinazoline analog reported to display PI3Kα

kinase, belongs to Class IV.

inhibitory activity. However, it did not enter clinical trials

Dysfunction of the PI3K/Akt/mTOR signaling pathway has

been implicated in many human cancers such as breast,

because of its rapid in vivo metabolism. A trisubstituted

quinazoline derivative (4), exhibited a potent dual PI3Kα/

mTOR inhibition with good antiproliferative activity against

ovarian, prostate, colorectal, and glioblastoma. Breast cancer

is the most common cause of death among women.

Approximately 12% of women may acquire breast cancer

over the period of their lifetime (www.breastcancer.org).

Generally, the pathological activation of this pathway is

thought to be due to ampliﬁcation of proto-oncogenes (e.g.,

PI3KCA, Akt), silencing of tumor suppressor genes (e.g.,

phosphatase and tensin homologue deleted on chromosome

ten (PTENS):PIP3 phosphatase), and/or overexpression of

Received: May 28, 2020

Accepted: October 6, 2020

XXXX American Chemical Society

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

Letter

Figure 1. Quinazolines as dual PI3Kα/mTOR inhibitors.

the U937 cell line. And in 2019, the FDA approved the ﬁrst

α-subtype speciﬁc PI3K inhibitor from Novartis, alpelisib

The syntheses of these compounds were accomplished

following the 2-step direct synthetic route shown in Scheme 1.

(

Piqray, 5), for use in combination with endocrine therapy in

postmenopausal women with PI3Kα-mediated advanced breast

Scheme 1. Synthesis of 4-Morpholinoquinazoline

Derivatives C2 Substituted by Aryl, Heteroaryl, and

Substituted Aromatic Amines

cancer.

In 2017, we reported a series of pyridopyrimidine-based

ATP-competitive dual PI3Kα/mTOR inhibitors. Our dis-

ubstituted analogs demonstrated potent inhibitory activity

against both enzymes with selectivity toward PI3Kα over

mTOR. Compound A (Figure 2 ) was exempliﬁed with PI3K α

Figure 2. Initial strategy for designing 2-substituted-4-morpholino-

quinazolines.

Reagents and conditions: (i) morpholine, triethyl amine, dry THF,

stir 3 h, rt; (ii) (Het)ArB(OH) or pinacolester, K CO , Pd(PPh ) ,

3 4

DME, 150 °C, 20 min microwave; (iii) aromatic amines, NaI, DMA,

^I^C⁵0 ⁼⁰^.⁵ⁿ^M^,^m^T^O^R^I^C50 = 104 nM, and excellent

antiproliferative activity against MCF7 with IC₅₀= 170 nM.

Docking studies with compound A and the crystal structures of

PI3Kα and mTOR revealed structural features of the C2

substituents that impacted on binding to both enzymes and

imparted selectivity for PI3Kα over mTOR. In this paper we

report our eﬀorts to extrapolate those structural revelations to

the quinazoline scaﬀold.

Our ﬁrst strategy was to simplify the structure by replacing

the diaryl urea moiety with substituted aryl, heteroaryl, or

substituted aromatic amines as depicted in Figure 2. We

wished to explore whether these substituents would mimic the

diaryl urea moiety in both the binding aﬃnity and inhibitory

activity toward the two enzymes.

160 °C, 15 min microwave.

Nucleophilic displacement of the 4-chloro group on the

commercially available 2,4-dichloroquinazoline with morpho-

line under dry basic conditions gave intermediate 6. Under

microwave irradiation, this intermediate was coupled with

either boronic acids or pinacol esters under Suzuki−Miyaura

conditions to form compounds 7a −e or treated with

substituted anilines to yield 8a−g (Supporting Information).

The HotSpot radioisotope ﬁlter binding assay was used to

assess the in vitro enzymatic inhibitory activity for the novel

quinazoline derivatives at 10 μM concentration in the presence

of 10 μM ATP and using PI-103 as reference (Table S2).

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

binding pockets as represented in Figure 3 .

Letter

Inhibitory activity against human PI3Kα was assessed by

measuring the conversion of phosphatidylinisotol(4,5)-biphos-

phate to phosphatidylinositol(3,4,5)-triphosphate. The ability

to inhibit human mTOR was assessed by measuring the

phosphorylation of factor 4E-binding protein1 (4EBP1). In

this series, all the compounds showed a moderate inhibitory

activity at this concenteration. Consequently, the best three

compounds 7d, 8d, and 8e were subjected to a concentration−

response study to determine IC values against both enzymes.

The syntheses of these substituted ureas were achieved

through the in situ conversion of 7e to the isocyanate by

reaction with triphosgene under dry conditions followed by

treatment with the appropriate amine/aniline to give 9a−u as

presented in Scheme 2.

Scheme 2. Synthesis of 4-Morpholinoquinazoline−Urea

Derivatives Substituted by Alkyl (9a,b), Substituted Phenyl

(9c−l), and Substituted Benzenesulfonamides (9m−u)

Results are shown in Table 1 together with in vitro ADME

Table 1. Kinase Inhibitory Activity, Solubility, and Mouse

Liver Microsomal Stability of 7d, 8d, and 8e

Of the 21 synthesized analogs, 16 aﬀorded ≥90% inhibition

at 10 μM concentration against both PI3Kα and mTOR in the

Hotspot radioisotope ﬁlter binding assay (Table S2). IC₅₀

values were determined for these 16 analogs (Table 2). The

type and size of the substituent directly inﬂuenced the

potencies for both enzymes. The newly synthesized derivatives

possessed low to excellent aqueous solubility in 2% DMSO/

phosphate buﬀered solution. Concerning the mouse micro-

somal metabolic stability, these new analogs demonstrated a

range from moderate (e.g., t₁_/₂= 23.5 min for 9s) to excellent

(

many t₁_/₂value > 60 min) stability. Results of 9a−u are

shown in Table 2.

screening data measuring the maximum kinetic aqueous

solubility in 2% DMSO/phosphate-buﬀered saline (pH =

Initial SAR studies revealed that the replacement of the

phenyl urea ring with a short alkyl side chain such as methyl 9a

or ethylalcohol 9b caused an increase in selectivity for mTOR

over PI3Kα by 2-fold to 3-fold. Substitution of the alkyl group

with phenyl (9c) enhanced potency for PI3Kα and reduced

potency for mTOR, giving an analog that was 4-fold selective

for PI3Kα. Interestingly, incorporation of heteroatoms in that

phenyl ring (9d) eliminated PI3Kα selectivity (IC for PI3Kα

.4) and metabolic stability in mouse liver microsomes,

expressed in t₁_/₂(min).

Unfortunately, the strategy of simplifying the structure

yielded weak dual PI3Kα/mTOR inhibitors with IC values in

the micromolar range. Representative data are shown in Table

, and data for 7a−e and 8a−g are provided in Table S1.

Maximum kinetic aqueous solubility for these analogs, in

general, was better than that seen for PI-103 and t₁_/₂values in

mouse liver microsomes ranged from 2.1 min to >60 min, with

many analogs demonstrating superior stability to that seen with

PI-103 (t₁_/₂= 7.1 min). The results obtained in the kinase

inhibition assays highlighted the critical role of the phenylurea

moiety in this series of derivatives for potent dual enzyme

inhibition. Therefore, we reverted to our second optimization

strategy in which we kept the urea moiety intact and

substituted it with alkyl groups and aryl rings containing

hydrogen bond donating or accepting functional groups to

explore the structural requirements of the PI3Kα and mTOR

and mTOR = 114 nM and 115 nM, respectively).

In order to probe the size and electronic nature of the

binding pockets around the urea moiety, we prepared a series

of analogs with hydrogen bonding substituents on the phenyl

ring. These included hydroxymethyl in the para (9e) and meta

(9f) positions as well as the para-hydroxyethyl derivative (9g).

An increase in both PI3Kα and mTOR inhibitory potency was

realized with all three derivatives compared to the unsub-

stituted analog 9c, and the para-substitution pattern appeared

to be better tolerated. All three analogs displayed selectivity for

PI3Kα over mTOR, especially 9g which was 40-fold selective.

These results suggested the presence of a hydrogen bond

acceptor near the para-position of the phenyl ring and the

possibility that the binding pocket on PI3Kα may be somewhat

larger/longer than the pocket on mTOR.

Substituting structurally similarly sized groups with oxygen-

containing hydrogen bond acceptor moieties in the para

position (9h, 4-acetyl; 9i, 4-propanyl; 9j, 4-carboethoxy)

caused a loss in potency for both enzymes. However, a

signiﬁcant increase in potency against PI3Kα was realized

when the strongly hydrogen bond accepting amide groups

(-NHCO-, -CONH-) were inserted in the para position of the

phenylurea ring (9k and 9l, PI3Kα IC = 0.7 nM and 2.5 nM,

Figure 3. Second strategy for designing 4-morpholino-quinazoline

urea derivatives.

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Letter

Table 2. Kinase Inhibitory Activity, Aqueous Solubility, and Mouse Liver Microsomal Stability of 9a−u

^†Percent inhibition at a concentration of 10 μM.

respectively). Both compounds maintained reasonable potency

against mTOR (IC₅₀= 114 nM and 59.1 nM, respectively).

However, the order of the atoms within the amide substituent

had opposite eﬀects, with the switch from the acetylanilino

structure in 9k to the carboxamide structure in 9l resulting in a

loss of potency against PI3Kα and an increase in potency

against mTOR. Indeed, compound 9k was the most potent

PI3Kα inhibitor of all the analogs prepared for this study and

displayed the greatest selectivity for PI3Kα over mTOR (154-

fold) compared to other qunazolines within this series.

To further probe the PI3Kα and mTOR binding sites for

this series, we substituted the para-amide motif with

sulfonamide moieties. Compared to the corresponding

carboxamide derivative 9l, the unsubstituted sulfonamide

derivative 9m retained similar inhibitory potency for PI3Kα

but lost some potency against mTOR. Contrary to the amides

role in mTOR aﬃnity as the corresponding phenyl

sulfonamide 9s and the electron-poor 2-ﬂuorophenyl and

2,4-diphenyl derivatives 9t and 9u, respectively, also lost

potency against mTOR activity compared to the heteroaryl

analogs 9q and 9r. It should be noted that to our knowledge,

this is the ﬁrst time that the sulfonamide substituent has been

examined as part of the SAR of this class of PI3Kα/mTOR

inhibitor.

Most of the urea analogs prepared for this study possessed

low to moderate maximum kinetic aqueous solubility in 2%

DMSO/phosphate buﬀered saline, although those solubilities

were comparable to that seen with the standard PI-103. The

exception to this trend was the alkyl analogs 9a and 9b, for

which 192 μM and 200 μM solutions, respectively, were

obtained. With the exception of methylurea derivative 9a, all of

the prepared analogs demonstrated superior stability to

oxidative metabolism in mouse liver microsomes compared

to PI-103. These data were not inﬂuenced by solubility

limitations since the concentration of test compounds used in

the microsomal stability assays was 1 μM.

l and 9k, when the order of the atoms for the sulfonamide

group in 9m was reversed to give 9n, a loss in both PI3Kα and

mTOR inhibitory potency was seen. Further derivatization of

the sulfonamide motif present in 9m resulted in several analogs

that lost PI3Kα potency but retained inhibitory potency for

mTOR. These included the guanidinylsulfonamide 9o, the

thiazolyl analog 9p, the pyridinyl derivative 9q, and especially

the pyrimidinyl compound 9r, which was the most potent

mTOR inhibitor of the compounds prepared for this study

with an mTOR IC₅₀value = 37.4 nM. Either hydrogen

bonding or the electronic nature of the ring appeared to play a

To better understand our results, we performed docking

studies of compounds 9k (PI3Kα-selective) and 9r (mTOR-

selective) with human PI3Kα and mTOR using the published

cocrystal structures of these compounds with human PI3Kα

(PDB 4L23) and mTOR (PDB 4JT6) bound to PI-103.

Binding modes were predicted using Glide SP. Hydrogen bond

constraints were employed, followed by generalized energy

ACS Medicinal Chemistry Letters

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Letter

Figure 4. Docking of PI3Kα-selective inhibitor 9k and mTOR-selective inhibitor 9r with the crystal structures of human PI3Kα and mTOR

showing hydrogen bonds and surface maps. (A) 9k docked to PI3Kα with four critical hydrogen bonds shown; (B) 9k docked to mTOR with three

critical hydrogen bonds shown; (C) 9r docked to pI3Kα with 4 critical hydrogen bonds shown; (D) 9r docked to mTOR with 5 critical hydrogen

bonds shown.

minimizations, to endure that the morpholino groups of 9k

and 9r were oriented similarly to the morpholino moiety of PI-

PI3Kα to accommodate larger residues as predicted from the

SAR results.

03 in the binding pockets. The results are presented in Figure

The docking models do suggest reasons for the opposite

selectivity seen with 9k and 9r. For 9k (Figure 4A), two

additional hydrogen bonding interactions are predicted (the

acetimide -NH with Asp806 and the urea carbonyl oxygen with

Lys802), while 9k appears to form only one additional

hydrogen bond with mTOR (the acetimide -NH with

Gln2194) (Figure 4B). This could explain the selectivity for

PI3Kα over mTOR seen for 9k. Alternatively, the mTOR-

selective analog 9r appears to make 5 hydrogen bonding

interactions with mTOR (the three described above for 9k

plus two additional interactions between the urea carbonyl

oxygen and Glu2190 and the sulfonamide -NH and Asp2192

(Figure 4D) as opposed to 4 hydrogen bonds formed with

In agreement with results on our and other analogous

pyrido-pyrimidines, our models indicated that both 9k and 9r

retained the essential hydrogen bond between the morpholino

oxygen and Val851 in the hinge region of PI3Kα and Val2240

in the hinge region of mTOR. The hydrogen bonds predicted

between the urea N−H groups and the aspartates in the

hydrophobic binding pockets (Asp810 for PI3Kα; Asp2357 for

mTOR) were also retained. One end of the binding pocket on

PI3Kα appears to be solvent-exposed, and a ridge of amino

acid residues is present at the distal end of the binding pocket

on mTOR that may make it more diﬃcult for mTOR than for

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Letter

PI3Kα (the three described above for 9k plus an interaction

between the sulfonamide -NH and Asp805) (Figure 4C). This

discrepancy may explain the selectivity for mTOR seen with

in normal human dermal ﬁbroblasts. Results are provided in

Table 3 as percent of viable cells.

r, To conﬁrm that our compounds act as dual PI3Kα/mTOR

inhibitors, we treated MCF7 cells with 9k at concentrations of

2.5 and 125 nM for 4 h and then analyzed cell lysates for

Table 3. Percent of Cell Viability of 3 Breast Cancer Cell

Lines and Normal Skin Cells in the Presence of 10 μM

Compounds as Tested by CellTiter-Glo 2D

downstream targets of the two enzymes by Western blot. The

results are shown in Figure 5. Compound 9k dose-dependently

inhibited the production of the PI3Kα- and mTOR-associated

signaling components phospho-4EBP, phospho-P70SK6, and

phospho-AKT(473).

The most prevalent eﬀect on cancer cell viability by our

compounds was seen in the estrogen-sensitive, HER2-negative

MCF7 cell line. Four analogs, 9f, 9h, 9k, and 9m, also

demonstrated reasonable cytotoxic activity against the triple

negative cell line MDA-MD-468. Like the standard agent PI-

03, none of our quinazoline derivatives showed appreciable

cytotoxic activity in the claudin-low triple negative cell line

MA-MDB-231 that could not be attributed to overt cellular

toxicity. MDA-MB-231 cells are known to be less susceptible

to chemotherapy. Several of the quinazoline analogs (9b−f,

l−m, 9o−p) demonstrated a therapeutic index for MCF7

cells over normal cells that was superior to that seen with PI-

03, and two derivatives (9f, 9m) also demonstrated a

therapeutic index for MDA-MB-468 cells over normal cells.

These result are in contrast to the results obtained with the

previously reported pyrido-pyrimidines, where only one

analog possessed a therapeutic index for MCF7 over normal

ﬁbroblasts and none of the compounds demonstrated a

therapeutic index for cytotoxicity in the triple negative cell

lines over ﬁbroblasts.

Figure 5. Western blot analysis of PI3Kα and mTOR targets.

Phosphorylation changes of phospho-4EBP, phospho-P70S6, and

phospho-AKT(473) in a dose-dependent treatment of MCF7 cells for

h with compound 9k.

In vitro antitumor activity was initially evaluated by testing

the eﬀects of the more potent dual PI3Kα/mTOR inhibitors

on the cellular viability using a luminescent CellTiter-Glo assay

Promega, Madison, WI). Preliminary screening was carried

out in 2D cell culture using a single 10 μM concentration of

selected analogs against three breast cancer cell lines (MDA-

MB-468, MDA-MB-231, and MCF7). An approximation of

therapeutic index was determined by screening the compounds

Compounds that displayed >50% cytotoxicity in MCF7 cells

(shaded in red in Table 3) and >80% viability in dermal

ﬁbroblasts (shaded in green in Table 3) were examined for

their cytotoxic eﬀects on MCF7 cells in 3D cell culture using

CellTiter-Glo 3D assay (Promega, Madison, WI). 3D cancer

cell culture is believed to more accurately reﬂect in vivo tumor

cell physiology and morphology as well as tumor architec-

(

ture. Dose response curves (Figure S1) were generated for

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Letter

the most promising analogs (9b−g, 9l−m, 9o−p). We also

examined compound 9k as the most potent PI3Kα inhibitor

and compound 9g as the most potent combined PI3Kα/

mTOR inhibitor. IC₅₀values are given in Table 4.

Table 4. IC₅₀Values for 9b−g, 9k, 9l−m, and 9o−p against

MCF7 Cells in 3D Cell Culture (CellTiterGlo 3)

Cpd.

IC₅₀(μM)

0.7

1.2

1.6

0.2

0.3

Cpd.

IC₅₀(μM)

0.2

0.3

0.2

0.3

1.8

All of the analogs tested displayed potent cytotoxic activity

against MCF7 cells in 3D culture, with IC₅₀values ranging

from 0.2 μM to 1.8 μM. In general, IC₅₀values were in

agreement with the percent cytotoxic activity seen in the 2D

assay (Table 3). The exceptions to this were 9c, 9d, and 9p

which demonstrated IC₅₀values that were higher than those

obtained with the other analogs. The reasons for this are not

clear at this point. These compounds displayed somewhat

higher IC₅₀values against mTOR activity compared to other

analogs. However, other compounds with similar mTOR

inhibitory potency (e.g., 9f) were found to be potent cytotoxic

agents in the 3D cell culture model. One contributing factor

may be solubility limitations. The compounds with higher than

^e^x^p^e^c^t^e^d^I^C⁵0 values in the 3D culture model seemed to hit a

ceiling eﬀect (∼30% viability) at the 7.5 μM concentration,

with no further eﬀect seen at 15 μM. Permeability in the 3D

system could also have played a role. While the logP values for

all tested analogs are within a range predicted to impart

Figure 6. Kinome tree visualization of top predicted upstream serine/

threonine kinases (STKs) where kinase statistics values, encoded in

branch color and node color, indicate the overall change of the

peptide set that represents the kinase, with value <0 indicating lower

kinase activity in compound 9k (1 μM) treated MCF7 cells relative to

DMSO control. Kinase scores, encoded in node size, are used for

ranking kinases based on their signiﬁcance and speciﬁcity in terms of

the set of peptides used for the corresponding kinase.

24,25

reasonable cellular permeability (2.0−4.6),

the logP values

Removal of the urea moiety within the scaﬀold yielded weak

dual inhibitors. On the other hand, combining the urea group

with diﬀerent hydrogen bonding substituents resulted in

potent inhibitors that displayed a range of selectivity for

PI3Kα and mTOR. Substitution of the phenylurea ring with

short alkyl chains or 6-membered hetreoaryl/aryl sulfonamides

aﬀorded analogs with selectivity toward mTOR over PI3Kα.

Alternatively, substituting the phenylurea ring with the

acetimide moiety provided 9k, a potent inhibitor of PI3Kα

(IC₅₀= 0.7 nM) with 154-fold selectivity for PI3Kα over

mTOR. The selectivity seen within this series could be

explained by diﬀerences in the number of hydrogen bonding

interactions made with the two enzymes as predicted by

docking studies performed with the X-ray crystal structures of

human PI3Kα and mTOR. Nine derivatives demonstrated

potent cytotoxic activity against MCF7 cells in 2D culture and

a good therapeutic index as assessed in normal dermal

ﬁbroblasts. Two of these analogs also showed cytotoxic activity

against the triple negative breast cancer cell line MDB-MA-

468. Most of these analogs also displayed potent cytotoxic IC₅₀

values against MCF7 cells grown in 3D cell culture, with

potency in this assay aligning with potency against PI3Kα and

logP values predicted to impart good cellular permeability. In

addition, we identiﬁed a new substituent motif that imparted

potent PI3Kα and mTOR inhibitory potency to the

morpholino-quinazoline scaﬀold, namely the sulfonamide, as

exempliﬁed by compound 9m. This substituent has not been

reported previously for such compounds. Oﬀ-target kinase

inhibitory activity for a representative compound (9k)

included STKs within the RAS, ErbB, and MAPK signaling

pathways. All of these STK inhibitory activities could be

of 9c (2.03), 9d (3.94), and 9p (4.37) fall near the outer limits

of that range.

Since PI3Kα and mTOR both possess serine/threonine

kinase (STK) activity, we assessed the selectivity of compound

k within the STK class of kinases in MCF7 cells using

Pamgene’s proprietary Pamchip microarray-based peptide

activity proﬁle (Pamgene International, B.V., s’Hertogenbosch,

The Netherlands). The PamChip arrays generate relative

phosphorylation intensity data of peptides containing known

substrate recognition sites of STKs in the presence compound

k and DMSO control. Using a diﬀerential upstream kinase

analysis of bait peptides, we predicted top STKs (n = 20)

whose activity was signiﬁcantly inhibited by compound 9k at

the 1 μM concentration (1,300-fold higher than the IC₅₀for

PI3Kα inhibition; 10-fold higher than the IC₅₀for mTOR

inhibition) in comparison to DMSO control as shown in

Figure 6. The STK proﬁle conﬁrmed the PI3Kα and mTOR

inhibitory activity of 9k by demonstrating a decrease in levels

of phospho-P70S6K and phospho-NAUK1 (downstream

targets of mTOR) as well as phospho-AKTs (downstream

targets of PI3Kα). In addition, the STK proﬁle demonstrated

the inhibitory eﬀect of compound 9k on kinases activity

associated with the RAS, ErbB, and MAPK signaling pathways.

A list of eﬀected kinases and downstream phosphorylated

peptides is provided in the Supporting Information .

In conclusion, straightforward synthetic routes were used to

synthesize novel disubstituted quinazolines as potent dual

PI3Kα/mTOR inhibitors that demonstrated superior ther-

apeutic indices for cytotoxic activity against breast cancer cell

lines compared to earlier pyrido-pyrimidine compounds.

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

The Supporting Information is available free of charge at

Letter

contributing to the potent cytoxicity seen with 9k in MCF7

cell cultures. Based on pharmacological and in vitro ADME

properties, compounds 9e, 9f, 9g, 9k, 9m, and 9o will be

advanced to in vivo studies which will be the subject of future

reports.

Author Contributions

The main idea, all of the chemistry work, and writing the

manuscript was done by Aisha Al-Ashmawy. Docking study

was carried by KE. OP and MR carried out the in vitro

antitumor activity and Western blot. JG carried out in vitro

ADME assays. GM carried out the HRMS. AO initiated the

kinome screening assay. WC and MA helped in the editing and

writing of the manuscript. All authors have given approval to

the ﬁnal version of the manuscript.

ASSOCIATED CONTENT

■

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00289.

Funding

Funding was provided by The Egyptian Ministry of Higher

Education/Central Department of missions (CDM) and

United States Agency for International Development

(USAID) and by Deanship of Scientiﬁc Research (DSR) at

King Abdulaziz University, Jeddah under grant no. FP-61-42

for funding the kinome screening assay.

Synthetic procedures for compounds 2, 6, 7a−e, 8a−g,

and 9a−u, biological methods, in vitro ADME

procedures, and molecular modeling methods (PDF)

AUTHOR INFORMATION

■

Notes

Corresponding Authors

The authors declare no competing ﬁnancial interest.

Aisha A. K. Al-Ashmawy − Moulder Center for Drug Discovery

Research, Temple University, School of Pharmacy, Philadelphia,

Pennsylvania 19140, United States; Department of Therapeutic

Chemistry, Pharmaceutical and Drug Industries Research

Division, National Research Center, Cairo 12622, Egypt;

orcid.org/0000-0002-9219-5436 ;

Phone: +201013231824; Email: Aisha_pharmacy@

yahoo.com

Wayne E. Childers, Jr. − Moulder Center for Drug Discovery

Research, Temple University, School of Pharmacy, Philadelphia,

Pennsylvania 19140, United States ; Phone: 215-707-1079;

Email: wayne.childers@temple.edu

ACKNOWLEDGMENTS

■

The authors extend their appreciation for the Egyptian

Ministry of Higher Education for sponsoring Aisha Al-

Ashmawy as visiting scholar in addition to the United States

Agency for International Development (USAID) for sponsor-

ing Aisha Al-Ashmawy as postdoctoral fellow (GSP-P-75) for

completion of the work. The authors acknowledge, with

thanks, the DSR for technical and ﬁnancial support. The

authors thank Reaction Biology Corporation (Malvern, PA)

for running in vitro kinase assays. The authors also thank

Pamgene International B.V., especially Tushar Tomar and Dirk

Pijnenburg, for providing the kinome screening data.

Authors

Khaled M. Elokely − Moulder Center for Drug Discovery

Research, Temple University, School of Pharmacy, Philadelphia,

Pennsylvania 19140, United States; Department of

Pharmaceutical Chemistry, Tanta University, Tanta 31527,

Egypt; Division of Pharmaceutical Sciences, Arnold and Marie

Schwartz College of Pharmacy and Health Sciences, Long Island

University, Brooklyn, New York 11201, United States

Oscar Perez-Leal − Moulder Center for Drug Discovery

Research, Temple University, School of Pharmacy, Philadelphia,

Pennsylvania 19140, United States

Mario Rico − Moulder Center for Drug Discovery Research,

Temple University, School of Pharmacy, Philadelphia,

Pennsylvania 19140, United States

John Gordon − Moulder Center for Drug Discovery Research,

Temple University, School of Pharmacy, Philadelphia,

Pennsylvania 19140, United States

ABBREVIATIONS

■

PI3Ks, phosphatidylinositol-3-kinases; PIP2,

phosphatidylinisotol(4 , 5)- biph osphate; PIP3,

phosphatidylinositol(3,4,5)-triphosphate; Akt, protein kinase

B; mTOR, mammalian target of rapamycin; PIKKs,

phosphoinositide-3-kinase related protein kinases; PI3KCA,

phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic sub-

unit alpha; PTENS, phosphatase and tensin homologue

deleted on chromosome ten; RTKs, receptor tyrosine kinases;

4EBP1, factor 4E-binding protein1; DME, dimethoxyethane;

DMA, dimethylacetamide; DIPEA, N,N-diisopropylethyl-

amine; ADME, absorption, distribution metabolism, and

excretion; PDB, Protein Data Bank; STK, serine/threonine

kinase.

George Mateo − Moulder Center for Drug Discovery Research,

Temple University, School of Pharmacy, Philadelphia,

Pennsylvania 19140, United States

Abdelsattar M. Omar − Department of Pharmaceutical

Chemistry, Faculty of Pharmacy, King Abdulaziz University,

Jeddah 21589, Saudi Arabia; Department of Pharmaceutical

Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo

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