Design, Synthesis, and Characterization of an Orally Active Dual-Specific ULK1/2 Autophagy Inhibitor that Synergizes with the PARP Inhibitor Olaparib for the Treatment of Triple-Negative Breast Cancer

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Design, Synthesis, and Characterization of an Orally Active Dual-

Speciﬁc ULK1/2 Autophagy Inhibitor that Synergizes with the PARP

Inhibitor Olaparib for the Treatment of Triple-Negative Breast

Cancer

Huiyu Ren, Nicole A. Bakas, Mitchell Vamos, Apirat Chaikuad, Allison S. Limpert, Carina D. Wimer,

Sonja N. Brun, Lester J. Lambert, Lutz Tautz, Maria Celeridad, Douglas J. She ﬄ er, Stefan Knapp,

Reuben J. Shaw, and Nicholas D. P. Cosford*

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sı Supporting Information

ABSTRACT: Inhibition of autophagy, the major cellular recycling pathway in

mammalian cells, is a promising strategy for the treatment of triple-negative

breast cancer (TNBC). We previously reported SBI-0206965, a small molecule

inhibitor of unc-51-like autophagy activating kinase 1 (ULK1), which is a key

regulator of autophagy initiation. Herein, we describe the design, synthesis, and

characterization of new dual inhibitors of ULK1 and ULK2 (ULK1/2). One

inhibitor, SBP-7455 (compound 26), displayed improved binding aﬃnity for

ULK1/2 compared with SBI-0206965, potently inhibited ULK1/2 enzymatic

activity in vitro and in cells, reduced the viability of TNBC cells and had oral

bioavailability in mice. SBP-7455 inhibited starvation-induced autophagic ﬂux in

TNBC cells that were dependent on autophagy for survival and displayed synergistic cytotoxicity with the poly (ADP-ribose)

polymerase (PARP) inhibitor olaparib against TNBC cells. These data suggest that combining ULK1/2 and PARP inhibition may

have clinical utility for the treatment of TNBC.

INTRODUCTION

suppresses autophagy by directly phosphorylating and inhibiting

ULK1/2 function (Figure S1).

■

Macroautophagy (hereafter referred to as autophagy) is a highly

conserved pathway in eukaryotic cells that mediates the

coordinated lysosome-dependent disassembly and recycling of

cellular components. By degrading damaged organelles,

misfolded or toxic proteins, and pathogens, autophagy maintains

ULK1/2 are the only serine/threonine (S/T) kinases in the

core autophagy pathway, and their involvement at the ﬁrst

committed step in the autophagy cascade makes them attractive

druggable targets for inhibiting autophagy. ULK1/2 play crucial

roles in autophagy by phosphorylating the other three proteins

in the ULK1/2 preinitiation complex (ATG13, ATG101, and

1

−3

cellular homoeostasis and promotes cell survival under stress.

Autophagic ﬂux is maintained at a basal level in all cells, but it can

be further increased by various stressors, such as elevated levels

of reactive oxygen species (ROS), deprivation of nutrients, and

9

−11

FIP200)

and proteins in the downstream Beclin1 initiation

complex: Beclin1 (encoded by the gene BECN1), VPS34,

4

12

exposure to chemotherapeutic agents.

ATG9, and ATG16L1.

Autophagy is a multistep pathway initiated through the ULK

preinitiation complex, which consists of unc-51-like autophagy

activating kinase 1 (ULK1) or its homologue ULK2, autophagy

related protein 13 (ATG13), ATG101 and focal adhesion kinase

As a major cellular recycling process, autophagy plays

important roles in numerous diseases, including cancer.

Autophagy promotes tumor growth in several ways: by

providing nutrients and small molecule building blocks to

13,14

5

,6

(FAK) family-interacting protein (FIP200) (Figure S1).

support the elevated metabolic demands of tumor cells,

Autophagy is stimulated under stress conditions by activation

of AMP-activated protein kinase (AMPK) and inhibited under

nonstressed conditions by the mechanistic target of rapamycin

15−18

regulating oxidative stress,

and contributing to the

7

,8

Received: May 22, 2020

(

mTOR). AMPK activates autophagy by two mechanisms:

ﬁrst, by inhibiting mTOR activity via phosphorylation of the

mTOR complex components raptor and tuberous sclerosis

complex 2 (TSC2); and second, by directly phosphorylating

ULK1/2 at multiple sites N-terminal to the mTOR-phosphor-

ylation site, triggering ULK1/2 activation. Conversely, mTOR

©

XXXX American Chemical Society

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A

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development of drug resistance. ⁹⁻²³Some tumor types require

a higher basal level of autophagy than normal cells, which

suggests that autophagy inhibition may be a feasible therapeutic

1

24

approach for certain cancers. One example is triple-negative

breast cancer (TNBC), which accounts for about 15−20% of all

24

breast cancer cases. TNBC cells do not express clinically

signiﬁcant levels of estrogen receptors, progesterone receptors,

or of the human epidermal growth factor receptor 2 (HER2),

thus rendering them insensitive to endocrine or anti-HER2-

25,26

targeted treatments.

Both basic research and clinical studies

support the hypothesis that autophagy inhibition is a promising

27

therapeutic strategy for TNBC. However, the only drugs

available for clinical studies are the weak autophagy inhibitors

28,29

chloroquine (CQ) and hydroxychloroquine (HCQ),

which

are not autophagy-speciﬁc and have poor toxicity proﬁles

resulting in serious adverse side eﬀects. Thus, we considered that

small molecule ULK1/2 inhibitors that block autophagy

30

initiation may hold promise for the treatment of TNBC.

We previously reported SBI-0206965 (compound 1 in Table

, Figure S2), a ﬁrst-generation chemical probe that inhibits

1

ULK1 enzymatic activity in vitro and in cells, as demonstrated by

inhibition of phosphorylation of the downstream substrates

11

VPS34 and Beclin1. While SBI-0206965 has proven to be a

31−33

valuable autophagy probe,

we were keen to improve its on-

target potency and drug-like properties, including oral

bioavailability. Herein, we describe new dual ULK1/2 inhibitors

that resulted from a combination of a structure-based and

rational design. We demonstrate that the optimized autophagy

inhibitor SBP-7455 (26) selectively blocks autophagic ﬂux in

human TNBC cell lines and exhibits favorable PK and PD

properties in vivo. In addition, we show that compound 26 acts

synergistically with poly(ADP-ribose)polymerase (PARP)

inhibitors to kill TNBC cells, suggesting that combination

therapy with ULK1/2 inhibitors may have utility for the

treatment of drug-resistant TNBC.

Figure 1. Crystal structure of the kinase domain of ULK2 bound to

chemical probe SBI-0206965 (1). (A) Crystal structure of SBI-0206965

bound to the interlobe ATP site in the kinase domain of ULK2 at 2.7 Å

(PDB ID: 6YID). (B) SBI-0206965 forms hydrogen bonds to Cys88 in

the hinge region. (C) Detailed interactions with hinge residue atoms

and Lys39 are indicated. (D) In silico docking of compound 3 to the

crystal structure of ULK2.

of the DFG loop. The trimethoxyphenyl ring was located along

the hinge toward the solvent-exposed region of the binding

pocket. The bromine of the pyrimidine scaﬀold ﬁlled a malleable

“

back pocket” cavity proximal to the gatekeeper residue Met85.

RESULTS AND DISCUSSION

■

The trimethoxyphenyl and pyrimidinyl rings of SBI-0206965

adopted nearly coplanar geometries, while the N-methylbenza-

mide ring was rotated ∼6° out of plane. Overall, SBI-0206965

adopted a Type I binding mode that targeted the ULK1/2-active

SAR Studies. To obtain a structure-based pharmacophore

for improving the ULK1/2 potency of SBI-0206965 (Figure

S2), we obtained crystals of the ULK2 kinase domain (1-277)

34

35−37

bound to SBI-0206965. The kinase domains of ULK1 and

ULK2 share high sequence identity (78%) with near-complete

structural correspondence of residues in the interlobe region.

We recently reported that several small molecule kinase

inhibitors exhibit comparable potencies against recombinant

conformation.

The currently reported structures of inhibitors bound to

ULK1 or ULK2 suggest the presence of common speciﬁcity-

determining features that belie the observed binding pocket

plasticity. In all compounds with high potency, the cyclopropyl

and triﬂuoromethyl groups, or the halogen atoms located at the

5-position of the pyrimidine are placed into the back pocket of

ULK1/2. This is evident in the structures of MRT67307 or

MRT68921 bound to ULK2 (PDB ID: 6QAU and 6QAV,

respectively). ULK1/2 accepts diverse Type I sca ﬀ olds; the

reported pyrazolylquinazoline (Shokat compound, Figure S2)

even binds to ULK1 in a ﬂipped orientation (PDB ID:

4WNO). However, the structure of SBI-0206965 bound to

ULK2 revealed multiple adjacent sites for expanded interaction,

providing the opportunity to construct second-generation

autophagy inhibitors using structure-based design.

Optimization and Characterization of ULK Inhibitors.

Using the hinge-binding pyrimidine scaﬀold of SBI-0206965 as

the basis for next-generation ULK inhibitors, we conducted a

focused biochemical screen on a small number of 2,4,5-

trisubsitituted pyrimidine compounds and identiﬁed the

benzo[d][1,3]dioxole 2 and the related N -cyclopropyl

derivative 3 as promising candidates for further optimization

34

ULK1 and ULK2. By utilizing a similar strategy, crystals of the

T150D ULK2 kinase domain bound to SBI-0206965 were

obtained at a resolution of 2.7 Å (Figure 1A−C and Table S1).

The T150D mutant of ULK2 was used to mimic the active form

of the kinase domain because it more readily forms high-quality

crystals than the wild-type (WT) protein. The crystallized form

of ULK2 bound to SBI-0206965 adopted face-to-face

interactions with a symmetrical protomer counterpart in

which the activation loop and C helix of the two protomers

34

38

3

4

interdigitated.

Our results show that SBI-0206965 bound to the ATP

binding site of ULK2 adopts an orientation that formed a

speciﬁc bidentate hydrogen bond between the trisubstituted

pyrimidine scaﬀold and backbone residue Cys88 in the hinge

region. The compact binding mode of SBI-0206965 conformed

to the dimensions of the ATP binding site with a high degree of

competitive occupancy. The amide substituent of the N-

methylbenzamide ring extended toward the catalytic residues

4

B

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a 19 kDa luciferase domain and cell-permeable bivalent

ﬂuorescent energy transfer (FRET) probe that emits a signal.

A kinase inhibitor that is a cell penetrant competes with the

a

Scheme 1. SAR Strategy Around ULK1/2 Fragments

42

ﬂuorescent probe and reduces the signal. This assay was

compatible with a high-throughput workﬂow and oﬀered a

reliable metric to compare the cellular potencies of com-

43

pounds. In addition, protein thermal shift (PTS) assays were

employed to conﬁrm the biochemical potency and determine

the relative ability of compounds to stabilize ULK1 or ULK2, as

44

measured by a shift in protein-melting temperatures (Tm).

The co-crystal structure of SBI-0206965 bound to ULK2,

coupled with our in silico modeling data and previous structural

34

studies around ULK1 and ULK2, suggested that improved

potency could be elicited by systematic evaluation of aryl-

2

substituents at the R position (Table 1). Analogues were

synthesized in a modular approach by two sequential

substitutions on the pyrimidine scaﬀold (Scheme 1B). Thus,

2

,4-dichloro-5-(triﬂuoromethyl)pyrimidine was reacted with

a

(

A) Compounds 2 and 3 identiﬁed in a screen for ULK1 inhibition,

various alkyl amines under basic conditions to aﬀord key

intermediate A. Compound A was then reacted with a panel of

aryl amines in acetic acid utilizing microwave irradiation to

provide the target inhibitors (B) with diverse electronic and

polar characteristics. The analogues were tested in dose−

response mode in the ADP-Glo kinase assay (Figure S3), and

4

with IC₅₀values (ADP-Glo assay) and (B) a synthetic scheme for N -

cyclopropyl-5-(triﬂuoromethyl)pyrimidine ULK inhibitors.

(Scheme 1A). Because compound 3 possessed a 5-triﬂuor-

omethyl to insert in the hydrophobic back pocket and

convenient 2,4-diamine substituents to explore space in and

around the active site, we used the crystal structure of ULK2 to

model the interactions of 3 within the ligand binding site (Figure

the data for compounds with IC values better than 50 nM are

shown in Table 1.

50

Since our docking studies revealed that the aniline moiety

2

(

R ) is directed toward solvent-exposed space, it is perhaps

1

D). Flexible ligand docking with compound 3 was performed

39

unsurprising that a range of aryl substituents were tolerated. For

example, systematic expansion of the dioxole ring to the dioxine

using the ICM docking algorithm. The data obtained from

these docking studies supported a binding mode in which

nitrogen in the 1-position of the pyrimidine and the amino group

in the 2-position of the pyrimidine ring form hydrogen-bonding

interactions with the backbone atoms of Cys88 in the hinge

region. These data are in agreement with the co-crystal structure

of ULK2 bound to SBI-0206965, which showed the

corresponding 2-aminopyrimidine moiety of SBI-0206965 in a

similar position, undergoing similar interactions. The docking

data indicated that the triﬂuoromethyl group in the 5-position of

the pyrimidine ring in 3 occupied the hydrophobic back pocket

formed by the side chains of Val23, Ala37, Lys39, Val69, Met85,

and Leu138, thus providing additional favorable interactions

compared with the bromo group at the corresponding position

in SBI-0206965. The docking data also suggested that the

cyclopropylamine moiety in the 4-position of the pyrimidine

ring of 3 formed hydrophobic interactions with the sidechains of

Val15 and Val23 and with backbone atoms of Gly16 within the

β1 and β2 sheets, respectively. Finally, the benzo[d][1,3]dioxole

moiety was found to undergo hydrophobic interactions with the

side chains of Val15, Tyr87, and Leu138 and with backbone

atoms of Asp89, Gly90, and Gly91. Notably, the benzo[d]-

5

or dioxepine 6 improved the IC compared with 3 (21 nM) to

50

14 and 7 nM, respectively, in the ADP-Glo assay (Table 1). On

the other hand, the diﬂuorodioxole derivative 4 (42 nM) was

less potent than 3. The series of derivatives in which the dioxole

moiety was replaced with a 5-membered heteroaromatic ring (7,

8, 9, 10, 11, and 15) showed subtle variations in potency. Thus,

the indole (8), pyrrolopyridine (11), and benzoxazole (15) were

approximately equipotent with 3, while the benzimidazole (7)

and benzotriazole (10) derivatives were about 4-fold more

potent (IC50 values 5 and 6 nM, respectively). We also tested

1

analogues of 11 in which the cyclopropyl moiety (R ) was

replaced with a cyclobutyl (12) or a methyl (13) group, resulting

in no signiﬁcant improvement in potency. Interestingly, in the

series of 6,6-fused ring analogues (14, 16−19), all except the

pyrido[1,4]oxazine derivative (14) were more potent than 3.

Notably, the tetralone derivative (19) was the most potent

analogue with an IC50 value of 3 nM in the ADP-Glo assay.

Finally, in the series of six-membered ring aryl and heteroaryl

derivatives (20−26), the potencies ranged from 13 to 50 nM in

the ADP-Glo assay, with 21, 24, and 26 being the most potent.

To verify the binding aﬃnities and benchmark, the relative

stabilization of protein compared with SBI-0206965, ﬁfteen of

the most potent analogues were tested in the PTS assay against

the kinase domain of ULK1 (Table S2 ). The largest thermal shift

observed was ΔTm = 9.5 °C for compound 26, compared with

ΔTm = 8.3 °C for SBI-0206965. Interestingly, the PTS data

followed a similar trend to the ADP-Glo data, with the more

potent compounds in the ADP-Glo assay generally exhibiting a

larger ΔTm in the PTS assay (Table S2 ).

[

1,3]dioxole moiety was not involved in any speciﬁc hydrogen

bonding interactions, suggesting that it could be replaced with

other moieties that might add additional polar interactions or

could be truncated to improve ligand eﬃciency.

To fully characterize new ULK1/2 inhibitors and facilitate

expansion of the SAR, we established a versatile testing platform.

34,40

We used an in vitro end-point ADP-Glo kinase assay

as the

primary assay to measure the biochemical inhibitory potency of

compounds (Table 1 and Figure S3 ). To determine on-target

kinase engagement in live cells, we employed a bioluminescence

energy transfer NanoBRET assay that we optimized for ULK1

We next utilized the ULK1 NanoBRET intracellular kinase

assay to quantify the target engagement of compounds in live

cells, marking the ﬁ rst report of a NanoBRET assay for ULK1

(Table 1 and Figure S4). The IC₅₀values, shown in Table 1,

41

(

Table 1 and Figure S4 ). This quantitative technique relies on

a proximity-based energy transfer between a protein tagged with

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a

Table 1. Structures of ULK1/2 Inhibitors and IC₅₀Values Measured by ADP-Glo and NanoBRET Assays

a

(

*)Compounds 1 and 26 are SBI-0206965 and SBP-7455, respectively. ADP-Glo and NanoBRET assay results are presented as the means ± SD

of three independent experiments performed in triplicate (ADP-Glo) or duplicate (NanoBRET).

D

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indicate the capacity of the inhibitors to both enter the cell and

bind to full-length ULK1 kinase at intracellular physiological

levels of ATP. Compared with the ADP-Glo kinase assay, the

IC₅₀values for compounds were right-shifted in the ULK1

NanoBRET cellular assay. The results in the NanoBRET assay

proved to be critical to the selection of compounds for further

characterization. Thus, our ﬁrst-generation chemical probe SBI-

tional permeability of the same subset of compounds in Caco-2

cells (Table S5). All the compounds tested, with the exception of

11 and 19, which also have limited solubility in PBS (Table S4),

had high levels of permeability (Papp >10 × 10 cm/s), and the

majority had limited potential as a PgP substrate (eﬄux ratio <

2).

Given these membrane permeability data, the right-shifts

observed in the NanoBRET assays relative to the ADP-Glo

assays are more likely due to competition from cellular levels of

ATP and not due to diﬀerences in membrane permeability.

Based on the data shown in Tables 1 and 2 and Tables S4 and S5,

compounds 7, 15, 16, 21, 23, and 26 were selected for further

characterization.

−

6

0

7

2

206965 is active in this assay, with a benchmark IC value of

50

85 nM. Remarkably, only ﬁve of the new inhibitors (7, 16, 21,

3, and 26) exhibited IC values in the sub-micromolar range,

50

with the potencies of the other analogues ranging from 1.0 to

>

10 μM (Table 1).

The most potent ULK1 inhibitors, as well as SBI-0206965,

were next tested for their ability to inhibit ULK2 in vitro and in

cells. Thus, ULK2 biochemical inhibition (using the ADP-Glo

assay) and cellular target engagement (using the NanoBRET

assay) were measured for compounds 1, 7, 15, 16, 21, 23, and 26

Evaluation of Downstream Target Engagement by

ULK1/2 Inhibitors in Mammalian Cells. We previously

reported a method for quantifying the phosphorylation of

VPS34 at Ser249 when ULK1 and VPS34 are overexpressed in

(Table 2). As shown in Table 2, in general, the compounds were

11

HEK293T cells. We have established a similar assay to detect

the phosphorylation of Beclin1 at Ser15. We therefore

performed Western blot analysis to determine whether the

new ULK1/2 inhibitors inhibit phosphorylation of Beclin1 or

Table 2. Inhibitory Activities of Selected Compounds against

ULK2

45

VPS34 (Figure S1 and Figure 2).

compound

1

7

ULK2 ADP-Glo IC₅₀(nM) ULK2 NanoBRET IC₅₀(nM)

a

For these experiments, Flag-tagged Beclin1 or VPS34 and

Myc-tagged ULK1 were overexpressed in HEK293T cells, and

the cells were incubated with DMSO or ULK1/2 inhibitors (10

μM, 1 h). Cell lysates were then subjected to Western blot

analysis with antibodies to Ser15-phosphorylated and total

Beclin1 or Ser249-phosphorylated and total VPS34. Impor-

tantly, the ULK1 speciﬁcity of the inhibitors was veriﬁed by

examining cells overexpressing a Myc-tagged kinase-inactive

(KI) mutant in place of the WT enzyme. As expected, control

(DMSO-treated) cells overexpressing WT ULK1 showed a

dramatic increase in Beclin1 phosphorylation compared with

untransfected cells or ULK1-KI-expressing cells (Figure 2A).

Treatment with SBI-0206965 reduced Beclin1 phosphorylation

by ∼30% compared with control (DMSO), and a similar level of

inhibition was observed for compounds 7, 15, 16, 21, and 23.

The most pronounced eﬀect was displayed by compound 26,

which inhibited Beclin1 phosphorylation by ∼60%. We

observed the same pattern of activity for our compounds

when phosphorylation of VPS34 Ser249 by ULK1 was measured

2448 ± 298

720 ± 60

4578 ± 234

703 ± 84

1

2

5

6

1

3

6

1193 ± 88

714 ± 99

695 ± 244

1329 ± 94

476 ± 21

4256 ± 513

1647 ± 200

1885 ± 141

1687 ± 75

1158 ± 116

a

Compounds 1 and 26 are SBI-0206965 and SBP-7455, respectively.

Mean ± SD, representative of three independent experiments

performed in triplicate (ADP-Glo) or in duplicate (NanoBRET).

much less potent against ULK2 compared with ULK1 in both

assays, except compound 7, which was similarly potent against

ULK1 and ULK2 in the NanoBRET assay, with IC values 585

50

and 703 nM, respectively. As demonstrated with ULK1,

compound IC₅₀values were generally right-shifted in the

ULK2 NanoBRET assay relative to the ULK2 ADP-Glo assay.

Notably, however, compound 26 was signiﬁcantly more potent

than SBI-0206965 in both ULK2 assays (Table 2).

The right-shifts in potency for the NanoBRET versus ADP-

Glo assays for ULK1 and ULK2 could be due to a combination

of eﬀects, including membrane permeability, ATP competition,

inﬂuence of the activation state and cellular protein interactions.

These diﬀerences are due to the cellular nature of the

NanoBRET assay, which requires compounds to cross the

cytoplasmic membrane to interact with the target and to then

compete with higher levels of ATP present in the NanoBRET

assays, which are performed under cellular levels of ATP as

(

Figure 2B). All of the ULK inhibitors greatly reduced VPS34

phosphorylation (by ∼70−80%), with compound 26 again

having the greatest eﬀect (∼90% inhibition) (Figure 2B). These

data conﬁrm that our ULK1/2 inhibitors eﬀectively engage their

intended targets in the cellular environment and reveal the

superior potency of 26, which was selected for further evaluation

and characterization.

PK Analysis of Compound 26. As noted previously, SBI-

206965 has a limited value as an in vivo probe due to poor

0

systemic exposure following ip dosing and no measurable oral

bioavailability in mice. To determine whether compound 26 had

improved PK properties compared with SBI-0206965, a single

dose of 26 (30 mg/kg) was orally administered to mice and its

plasma concentration was measured over the following 24 h

using LC−MS/MS. This analysis showed that the time to peak

plasma concentration (Tmax) for 26 was approximately 1 h and

the maximum plasma concentration (Cmax) was 990 nM, which

was about 3-fold higher than the IC50 value for 26 (328 nM)

against ULK1 in the NanoBRET assay (Figure 3A). The plasma

concentration of compound 26 remained above the ULK1 IC₅₀

for almost 4 h after oral dosing. These data indicate that 26 has

opposed to K levels of ATP utilized in the ADP-Glo assays.

m

Since we were keen to identify ULK1/2 inhibitors with

improved drug-like properties, we next evaluated aqueous

solubility, membrane permeability, and whether compounds

could act as a substrate for drug transporters such as P-

glycoprotein (PgP). We selected a subset of compounds and

determined kinetic solubility in three media: PBS pH 7.4 buﬀer,

fasted simulated intestinal ﬂuid (FaSSIF), or fasted simulated

gastric ﬂuid (FaSSGF). The results, shown in Table S4,

indicated that several compounds had reasonable solubility

(

>5 μM) in PBS, increased solubility in FaSSIF, and greatly

increased solubility in FaSSGF. We also assessed the bidirec-

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Figure 2. ULK1/2 inhibitors block phosphorylation of downstream autophagy target proteins. Western blot analysis (upper panels) and quantiﬁcation

lower panels) of Beclin1 and VPS34 phosphorylation. HEK293T cells were transfected with Myc-tagged kinase inactive (KI) or wildtype (WT)

(

ULK1 and WT Flag-tagged (A) Beclin1 or (B) VPS34 for 24 h. Cells were then treated with DMSO or the indicated ULK1/2 inhibitors at 10 μM for 1

h, lysed, and subjected to Western blotting with the indicated antibodies. Blots were quantiﬁed by densitometry. Data are expressed as the ratio of

pBeclin1/Beclin1 or pVPS34/VPS34 normalized to DMSO-treated samples. Mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs

SBI-0206965 by two-sided unpaired test.

improved PK properties compared with SBI-0206965, which

will enable analysis of its PD properties in vivo.

ATG7, or BECN1 and pharmacologically with the lysosomo-

tropic autophagy inhibitor CQ. Interestingly, the TNBC cells

were dependent on autophagy for survival even in complete

media and thus in the absence of stressors known to stimulate

autophagy. To examine whether this was true for other core

autophagy genes, we performed shRNA-mediated KD of ULK1

or BECN1 in the human TNBC cell lines MDA-MB-468, MDA-

MB-231, and BT549. Western blot analysis conﬁrmed that the

respective target proteins were eﬀectively and speciﬁcally

silenced compared with control cells expressing scramble

shRNA (Figure S5). Analysis of cell survival and proliferation

revealed that ULK1 or BECN1 KD markedly reduced the

proliferation of all three cell lines over 8 days, with near-

complete abrogation of growth in ULK1-KD and BECN1-KD

MDA-MB-468 cells and in ULK1-KD BT549 cells (Figure 4).

These data demonstrate that all three TNBC cells require

autophagy initiation for survival and growth, with the most

pronounced eﬀects observed for MDA-MB-468 and BT549

cells.

In Vivo Target Engagement of SBP-7455. As noted

above, ATG13 resides with ULK1 in the autophagy preinitiation

complex in cells. When autophagy is initiated, it is phosphory-

lated by ULK1 at Ser318. ATG13 is therefore a reliable PD

biomarker for ULK1 activity. To evaluate the PD proﬁle and in

vivo target engagement, mice were dosed with compound 26

(

10 mg/kg) or vehicle by oral gavage, and liver samples were

collected after 2 h. The eﬀects of compound 26 on ULK1 and

ATG13 were assessed in the liver lysates. Western blot analysis

revealed robust inhibition of pATG13 (Ser318), as well as

downregulation of total ATG13 and ULK1 levels by compound

26 (Figure 3B). Based on the PK and PD proﬁles, our data show

that compound 26 is an eﬃcacious tool that can be used to

modulate autophagy in vivo.

TNBC Cells Require ULK1 Activity and Autophagy

Initiation for Survival and Proliferation. A growing body of

evidence supports a role for autophagy in the survival and

proliferation of TNBC cells, suggesting that autophagy

inhibition may be an eﬀective strategy for the treatment of

ULK1/2 Inhibitors Reduce TNBC Cell Viability. Because

our KD results showed that MDA-MB-468 cells were the most

dependent on autophagy initiation, we next examined the eﬀects

of our ULK1/2 inhibitors in this cell line. MDA-MB-468 cells

were incubated for 72 h with varying concentrations of SBI-

0206965, compound 26 (SBP-7455), or CQ as a positive

control. As shown in Figure 5A, MDA-MB-468 cells were

2

6,28,46,47

TNBC.

For example, Maycotte and co-workers used

short hairpin RNAs (shRNAs) to target the core autophagy

genes ATG5, ATG7, and BECN1 in a panel of breast tumor

2

4

cells. Their data showed that TNBC cell lines were sensitive to

autophagy inhibition by gene knockdown (KD) of ATG5,

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Figure 5. ULK1/2 inhibitors are cytotoxic as single agents against

TNBC cells. (A) MDA-MB-468 cells were treated with the indicated

concentrations of each ULK1/2 inhibitor or chloroquine (CQ) and

incubated for 72 h. Cell viability was then measured using CellTiter-

Glo. Compound treatment started at 30 μM for the ULK1/2 inhibitors

and 90 μM for CQ with serial dilutions of 1:3. IC50 values were

determined using GraphPad Prism 8. The dose−response curves are

representative of three independent experiments performed in

quadruplicate; mean ± SD. (B) Compound 26 (SBP-7455) promotes

apoptosis in TNBC cells under nutrient-deprived conditions. MDA-

MB-468 cells were treated with DMSO or compound 26 (10 μM) in

normal growth media (RPMI with 10% FBS, upper panel) or starvation

media (EBSS, lower panel) for 18 h. The cells were then collected, and

apoptosis was analyzed with PE-Annexin V/7-AAD staining by ﬂow

cytometry. Numbers indicate the percentage of cells in each gate.

Shown is a representative of three independent experiments with

similar results.

Figure 3. PK and PD analyses of compound 26 (SBP-7455). (A) Mice

were dosed orally with compound 26 (30 mg/kg in 5% DMSO, 10%

Tween 80, and 85% H O). Plasma samples were taken at the indicated

2

time points, and the concentration of compound was determined by

LC−MS/MS analysis. The data were analyzed using PKSolver software.

Mean ± SD of N = 3. Dotted line indicates the IC of compound 26 in

5

0

the ULK1 NanoBRET assay. (B) Mice were dosed with compound 26

10 mg/kg) or vehicle (5% DMSO, 10% Tween 80, and 85% H O) via

(

2

oral gavage. Liver samples were harvested and lysed 2 h post dosing, and

in vivo target engagement was assessed by Western blot. N = 3. All the

lanes were on the same gel; the lanes between vehicle and SBP-7455

were cropped out.

Figure 4. Ablation of ULK1 or BECN1 by shRNA-mediated knockdown reduces the viability of TNBC cells. MDA-MB-468 (A), BT549 (B), and

MDA-MB-231 (C) cells transduced with scramble, ULK1-targeting, or BECN1-targeting shRNAs and selected in puromycin for 3 days before analysis.

Cell viability was measured with the Promega CellTiter-Glo assay on the indicated days. Mean ± SD of three independent experiments, each

performed in quadruplicate. Data are normalized to the viability of scramble shRNA control cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs scramble

control by two-sided unpaired t-test.

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relatively insensitive to CQ (IC = 10.6 μM) compared with 26

50

(

IC = 0.3 μM) or SBI-0206965 (IC = 2.1 μM). These data

50 50

are consistent with the results of the in vitro (ADP-Glo) and

cellular (NanoBRET) ULK1/2 kinase assays, which revealed

more potent inhibition by compound 26 compared with SBI-

0

206965. Mechanistically, the ULK1/2 inhibitors regulate

autophagy initiation, whereas the pleiotropic drug CQ inhibits

end-stage lysosomal degradation in the autophagy pathway.

Accordingly, our data suggest that TNBC cell survival and

growth may be more sensitive to inhibition early in the

autophagy pathway as opposed to downstream.

Given the role of autophagy as a survival mechanism for

cancer cells in response to nutrient deprivation, we hypothesized

that our new autophagy inhibitor, compound 26, would increase

apoptosis in starved cells compared with cells provided with full

nutrients. To investigate this, wild-type MDA-MB-468 cells

were treated with compound 26 in normal media (RPMI with

1

0% FBS) or low-glucose, amino acid-free Earle’s balanced salt

solution (EBSS) for 18 h. Apoptosis was measured with Annexin

V and 7-AAD using ﬂow cytometry, and the results are shown in

Figure 5B. In normal media, the same percentages of cells

(6.8%) were in late-stage apoptosis regardless of whether they

were treated with DMSO or compound 26 (Figure 5B, panels i

and ii). Under starvation conditions, however (Figure 5B, panels

iii and iv), a much larger percentage of cells (18.4%) treated with

compound 26 were in late-stage apoptosis compared with

DMSO-treated cells (7.6%). Furthermore, a higher percentages

of starved cells (42.9%) underwent early stage apoptosis

compared with cells in normal media (28.9%) when treated

with compound 26 (Figure 5B, panels ii and iv). The eﬀects of

compound 26 were much more pronounced than the

percentages of cells (5.6 and 5.7%) in early stage apoptosis for

the DMSO controls (Figure 5B, panels i and iii). Taken together,

these data are consistent with enhanced cell killing by

compound 26 under starved conditions that activate autophagy.

ULK1/2 Inhibitors Block Autophagic Flux in TNBC

Cells. Autophagic ﬂux is the cellular process, whereby

autophagosomes fuse with autolysosomes and subsequently

degrade their cargo. To conﬁrm that autophagic ﬂux, and

therefore autophagy, is inhibited by compound 26, we employed

a ﬂow cytometry-based assay using MDA-MB-468 cells

expressing a tandem-labeled mCherry-EGFP-LC3 chimeric

reporter protein to count the number of autophagosomes and

4

8

autolysosomes. GFP ﬂuorescence, but not mCherry ﬂuo-

rescence, is quenched in the acidic (pH ≤ 5) environment of

lysosomes; therefore, inhibition of autophagic ﬂux is reﬂected by

a decrease in the cellular ratio of mCherry to GFP ﬂuorescence

Figure 6. ULK1/2 inhibitors reduce autophagic ﬂux. (A) MDA-MB-

468 cells expressing mCherry-EGFP-LC3 fusion protein were used to

monitor autophagic ﬂux, as measured by the ratio of mCherry:GFP by

ﬂow cytometry. (B) Cells were incubated in normal growth media

(Figure 6A). In the absence of an autophagy stimulus, the MDA-

MB-468 cells exhibited low (∼55%) or intermediate (∼40%)

basal levels of autophagy with only ∼5% of the cell population in

high ﬂux (Figure 6B and Figure S6). After incubation (18 h) in

EBSS to starve the cells and induce autophagy, the percentage of

cells with high autophagic ﬂux increased to >90%. Treatment of

starved cells with 10 μM SBI-0206965 or compound 26

signiﬁcantly reduced the mCherry:GFP ratio, indicating

inhibition of the starvation-induced increase in autophagic ﬂ ux

(

control), with EBSS (starved) or EBSS plus the ULK1/2 inhibitors

(10 μM) for 18 h before autophagic ﬂux was assessed. Three

independent experiments were performed; mean ± SD. *P < 0.001

by two-sided unpaired t-test of the high autophagic ﬂux population. (C)

Ultrastructural analysis of cells in (B) by TEM to monitor

autophagosome accumulation. Red arrows indicate autophagosomes.

(i) Representative untreated control cells, (ii) EBSS (starved) cells, (iii)

cells treated with SBI-0206965 (10 μM), and (iv) cells treated with

compound 26 (10 μM). Images are representative of 15−25 cells for

each set of conditions analyzed by TEM.

(

by ∼10% and ∼ 60%, respectively; Figure 6B and Figure S6).

Autophagosomes in the cells were visualized by transmission

electron microscopy (TEM) (Figure 6C). The amount of

autophagosomes under diﬀerent conditions was consistent with

the data from the autophagic ﬂux assay, with the highest number

of autophagosomes identiﬁed in starved cells (Figure 6C, ii).

Again, similar to the autophagic ﬂux assay, compound 26

(Figure 6C, iv) decreased the number of autophagosomes to a

greater extent than SBI-0206965 (Figure 6C, iii). These data

show that the compounds eﬀectively inhibited autophagy in

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TNBC cells, with compound 26, a more potent ULK1/2

inhibitor than SBI-0206965, being the most eﬀective.

SBP-7455 Exhibits Synergistic Cytotoxicity with Poly

ADP-ribose) Polymerase (PARP) Inhibitors against

(

TNBC Cells. PARPs are a family of proteins that maintain

genome stability and are responsible for DNA-damage repair in

4

9

cells. Since DNA damage can induce cancer cell death, PARP

inhibition, which leads to the accumulation of single-stranded

50,51

breaks, is a viable strategy to treat cancer.

Several PARP

inhibitors, including olaparib, niraparib, rucaparib, and

talazoparib, have received regulatory approval for the treatment

5

0,51

of cancer.

However, the emergence of resistance has led to

interest in combination therapies that can resensitize tumor cells

52

to the eﬀects of PARP inhibitors. Recent evidence has

emerged that PARP inhibitors activate autophagy in cancer cells,

and that this may lead to resistance. For example, inhibition of

autophagy with CQ was found to enhance the eﬀects of

niraparib in xenograft models of hepatocellular carcinoma, while

autophagy inhibition either genetically (ATG5 KD) or with CQ

increased the cytotoxicity of talazoparib in models of pediatric

53,54

chronic myelogenous leukemia (CML).

Because PARP

inhibitors have been approved for the treatment of TNBC, we

investigated whether combining compound 26 with PARP

inhibitors would increase cytotoxicity against TNBC cells.

Activity of the PARP inhibitors olaparib, niraparib, and

rucaparib was ﬁrst tested against MDA-MB-468 cells to

determine their potencies as single agents (Figure S7 ). Based

on these data, we selected olaparib (IC₅₀= 10.1 μM) and

niraparib (IC = 7.6 μM) for further studies. We tested di ﬀerent

5

0

concentrations of compound 26 with niraparib (Figure S9 ) and

olaparib (Figure 7 and Figure S8 ) to measure the combined

eﬀect of inhibiting both ULK1/2 and PARP. MDA-MB-468

cells were treated with compound 26 (0−15 μM) and either

niraparib or olaparib (0−15 μM) in a dose-reponse matrix, and

the synergistic eﬀect was analyzed with Combeneﬁt using the

5

Loewe model. We observed strong antiproliferative synergy

between 26 and both olaparib (Figure 7 and Figure S8) and

niraparib (Figure S9 ), suggesting that autophagy inhibition

enhances the cytotoxic eﬀects of PARP inhibitors toward TNBC

cells. Speciﬁcally, signiﬁcant synergy was observed with

concentrations of 26 at 0.021−15 μM and olaparib at 3.8−15

μM (Figure 7), and with 26 at 0.19−1.7 μM and niraparib at

Figure 7. Compound 26 (SBP-7455) and the PARP inhibitor olaparib

synergize to kill MDA-MB-468 cells. (A) MDA-MB-468 cells were

treated with 26 (0.25 nM−15 μM) and olaparib (0.47−15 μM) for 72

h, and cell viability was assessed (CellTiter-Glo assay). A synergy graph

was generated using the Combeneﬁt Loewe model. Experiments were

repeated three times (n = 4) with similar results. (B) Normalized cell

viability after incubation with DMSO (vehicle control), olaparib (7.5

μM), compound 26 (0.19 μM), or a combination of both compounds.

Mean ± SD. *P < 0.025, **P < 0.01, ***P < 0.001 P vs by two-sided

unpaired t-test.

3

.8−7.5 μM (Figure S9). Of note, the synergistic eﬀect was

greater with olaparib than with niraparib, with the maximum

synergy scores of 34 and 26, respectively. These data support the

hypothesis that an ULK1/2 inhibitor in combination with a

PARP inhibitor may have therapeutic utility for the treatment of

TNBC.

autophagic ﬂux levels in untreated MDA-MB-468 cells in Figure

8 compared with Figure 6B can be attributed to the

measurement of basal autophagy at diﬀerent time points,

speciﬁcally 18 h in Figure 6B versus 48 h in Figure 8.

SBP-7455 Reverses Olaparib-Induced Upregulation of

Autophagic Flux in TNBC Cells. To investigate the

mechanism by which compound 26 potentiates the cytotoxic

eﬀects of PARP inhibitors, we treated MDA-MB-468 cells

expressing mCherry-EGFP-LC3 with olaparib (30 μM) and/or

CONCLUSIONS

■

We utilized a combination of rational- and structure-based

design, based on a high-resolution structure of our ﬁrst-

generation probe SBI-0206965 bound to ULK2, to create a

series of novel small molecule ULK1/2 inhibitors. The best

compounds potently inhibited ULK1 and ULK2 in vitro and in

cells, as measured by ADP-Glo, PTS, and Nano-BRET assays.

SBP-7455 (compound 26) was identiﬁed as a highly eﬀective

inhibitor of phosphorylation of the ULK1 downstream

substrates Beclin1 and Vps34 and exhibited promising PK and

PD properties when dosed orally in mice. Importantly,

inhibition of autophagic ﬂux by shRNA-mediated KD of

2

6 (10 μM) for 48 h and then quantiﬁed autophagic ﬂux.

Olaparib alone was a strong inducer of autophagy, as

demonstrated by an approximately 30% increase in autophagic

ﬂux compared with control cells (Figure 8 and Figure S10).

However, concomitant treatment with compound 26 signiﬁ-

cantly suppressed the olaparib-induced increase in autophagic

ﬂux to a level similar to that observed in untreated control cells

Figure 8 and Figure 10). These data suggest that compound 26

potentiates olaparib activity, at least in part, by abrogating the

ability of olaparib to induce autophagy. Of note, the higher

(

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EXPERIMENTAL SECTION

■

General Chemistry Information. All reactions were performed

with oven-dried glassware under a nitrogen atmosphere with magnetic

stirring. All solvents and chemicals were purchased from commercial

sources and used without further puriﬁcation unless otherwise stated.

Reactions conducted under microwave irradiation were performed in a

CEM Discover microwave reactor using 10 mL reaction vessels.

Reaction progress was monitored by reverse-phase HPLC and/or thin-

layer chromatography (TLC). Chromatographic puriﬁcation was

carried out using prepacked silica or C18 cartridges (from RediSep

and Luknova) and eluted using an ISCO companion system. Reverse-

phase puriﬁcations were conducted using water and acetonitrile doped

with 0.1% formic acid. Purity and characterization of compounds were

established by a combination of LC−MS and NMR. HPLC−MS

analyses were performed on a Shimadzu 2010EV LC−MS using the

following conditions: a Kromasil C18 column (reverse phase, 4.6 mm _

50 mm), a linear gradient from 10% acetonitrile and 90% water to 95%

acetonitrile and 5% water over 4.5 min, a ﬂow rate of 1 mL/min, and UV

photodiode array detection from 200 to 300 nm. High-resolution ESI-

TOF mass spectra were acquired from the Scripps Research Institute

Center for Metabolomics and Mass Spectrometry (La Jolla, California).

Figure 8. Compound 26 (SBP-7455) inhibits the increase in

autophagic ﬂux induced by the PARP inhibitor olaparib. MDA-MB-

4

68 cells expressing mCherry-EGFP-LC3 were treated with DMSO

control, compound 26 (10 μM), olaparib (30 μM), or a combination of

both for 48 h. Two independent experiments were performed; mean ±

SD. *P < 0.05, **P < 0.01 for the high autophagic ﬂux population by

two-sided unpaired t-test.

1

13

Proton ( H) and carbon ( C) NMR spectra were obtained on a

JEOL400 spectrometer at 400 and 101 MHz, respectively. Chemical

shifts are reported in δ (ppm) and were internally referenced to

1

deuterated solvent signals. The data for H NMRs are reported in terms

ULK1 was replicated with the optimized ULK1/2 inhibitor

SBP-7455 in MDA-MB-468 TNBC cells.

of chemical shift (δ ppm), multiplicity, coupling constant (Hz), and

1

3

proton integration. The data for C NMR are reported in terms of

chemical shift (δ ppm) and coupling constant (Hz). The purity of ﬁnal

products submitted for biological testing was determined by analytical

HPLC−MS to be ≥95%. For NMR spectrum and LCMS traces of

compounds 7, 15, 16, 21, 23, and 26, see the Supporting Information .

TNBC therapy is particularly challenging due to its resistance

to antihormonal and HER2 targeted therapies. PARP inhibitors

are eﬀective in a subset (∼20%) of TNBC patients that harbor

56

BRCA mutations; however, ﬁnding eﬀective ways to sensitize

WT BRCA TNBC to anti-cancer therapies is an area of ongoing

research. Our results suggest that autophagy inhibition may be

an attractive option. In the present study, we found that the

autophagy inhibitor SBP-7455 was cytotoxic as a single agent

against TNBC cells. In a clinical setting, it is likely that

autophagy inhibition will be most eﬀectively deployed as an

adjunct to existing therapies. Therefore, we also showed that

SBP-7455 and the PARP inhibitor olaparib synergistically kill

MDA-MB-468 TNBC cells. Thus, in these cells, PARP

inhibition combined with autophagy inhibition is more eﬀective

than either tumor cell killing mechanism alone.

2

-Chloro-N-cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-

amine. A solution of 2,4-dichloro-5-(triﬂuoromethyl)pyrimidine (1.00

g, 4.61 mmol), cyclopropylamine (0.32 mL, 4.61 mmol), and N,N-

diisopropylethylamine (0.80 mL, 4.61 mmol) in acetonitrile (15 mL)

was microwaved at 70 °C for 10 min in a 38 mL pressure vessel. The

reaction mixture was then concentrated in vacuo and puriﬁed by

automated reverse-phase chromatography (water-acetonitrile eluent).

Tan solid (0.349 g, 32% yield). LC−MS (ESI) calcd for C

H

ClF

6

N

8

3 3

+

1

[

M + H] : 238.04; found 238.30. H NMR (400 MHz, DMSO-d ): δ

8

0

.39 (s, 1H), 7.93 (d, J = 3.6 Hz, 1H, 1H), 2.88 (dq, J = 7.2, 3.6 Hz, 1H),

.79−0.72 (m, 2H), 0.70−0.64 (m, 2H). C NMR (101 MHz, DMSO-

1

3

d ): δ 162.66, 159.73, 155.42, 123.34 (q, J = 271 Hz), 105.31 (q, J = 32

6

Hz), 24.72, 6.30.

Our results from the present study are in alignment with data

from other groups on overcoming the protective eﬀects of

autophagy in advanced cancers. For example, recent ﬁndings in

pancreatic ductal adenocarcinoma (PDAC) suggest that

inhibition of RAF-MEK-ERK signaling activates a compensatory

General Method 1. A solution of commercially purchased 2-

chloro-N-cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (1 equiv)

and the appropriate aniline (1 equiv) in acetic acid (2 mL) was

microwaved at 110 °C for 10 min and then concentrated in vacuo. The

product was puriﬁed by automated reverse-phase chromatography

(water-acetonitrile eluent).

57,58

pro-autophagic LKB1-AMPK-ULK1 axis.

Thus, a growing

2

N -(Benzo[d][1,3]dioxol-5-yl)-5-(triﬂuoromethyl)-

pyrimidine-2,4-diamine (2). A solution of 2,4-dichloro-5-

body of preclinical and clinical evidence suggests that autophagy

activation is a common mechanism of drug resistance in many

advanced cancers; therefore, combination therapies that block

autophagy, such as those described here, could have wide

(triﬂuoromethyl)pyrimidine (85 mg, 0.392 mmol), benzo[d][1,3]-

dioxol-5-amine (54 mg, 0.394 mmol), and N,N-diisopropylethylamine

(0.068 mL) in acetonitrile (3 mL) was microwaved at 100 °C for 10

min. Ammonia (0.22 mL of a 7 M solution in MeOH) and N,N-

diisopropylethylamine (0.07 mL) were added, and the reaction mixture

was microwaved at 100 °C for 10 min, then concentrated in vacuo.

Product was puriﬁed by automated reverse-phase chromatography.

Brown solid (15 mg, 13%). LC−MS (ESI) calcd for C H F N O [M

59

therapeutic applications. We focused our eﬀorts on the

development of inhibitors of ULK1/2 as initiating kinases in the

autophagy signaling pathway. It is likely that autophagy

inhibitors acting at later steps in the pathway would have

distinct toxicity proﬁles compared with inhibitors of autophagy

initiation. Blockade of lysosomal degradation leads to the

accumulation of unprocessed autophagosomes, whereas block-

ade of the upstream stages causes a buildup of the autophagy

1

2

10

3

4

2

+

1

+

H] : 299.08, found 299.28. H NMR (400 MHz, DMSO-d ): δ 9.39

6

(s, 1H), 8.15 (s, 1H), 7.54 (s, 2H), 7.11 (d, J = 10.5 Hz, 1H), 6.76 (d, J =

1

3

8.4 Hz, 1H), 5.93 (s, 2H). C NMR (101 MHz, DMSO-d

59.8, 155.1, 147.1, 142.2, 134.8, 125.3 (q, J = 269 Hz), 112.5, 107.9,

102.3, 100.9, 96.5 (q, J = 31 Hz).

): δ 161.2,

6

1

30

cargo; namely, protein aggregates and damaged organelles.

2

4

N -(Benzo[d][1,3]dioxol-5-yl)-N -cyclopropyl-5-(tri-

ﬂuoromethyl)pyrimidine-2,4-diamine (3). The title compound

was prepared according to General Method 1 using 2-chloro-N-

cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (60 mg, 0.253

mmol) and benzo[d][1,3]dioxol-5-amine (35 mg, 0.253 mmol). Purple

Chemical probes, such as the one described here, that act at

diﬀerent stages in the autophagy pathway could serve as tools to

assess the relative importance of each component of the

autophagic machinery from a therapeutic perspective.

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solid (56 mg, 66% yield). LC−MS (ESI) calcd for C H F N O [M +

(triﬂuoromethyl)pyrimidin-4-amine (70 mg, 0.295 mmol) and 1H-

1

5

14

3

4

2

+

1

H] : 339.11, found 339.00. H NMR (400 MHz, DMSO-d ): δ 9.61 (s,

indol-5-amine (39 mg, 0.295 mmol). Black solid (72 mg, 73% yield).

6

+

1

H), 8.16 (s, 1H), 7.82 (br. s, 1H), 7.18 (dd, J = 8.5, 2.2 Hz, 1H), 7.15

LC−MS (ESI) calcd for C H F N [M + H] : 334.13, found 334.15.

1

6

15

3

5

1

(br. s, 1H), 6.83 (d, J = 8.6 Hz, 1H), 5.95 (s, 2H), 2.79 (dq, J = 6.2, 3.4

H NMR (400 MHz, DMSO-d ): δ 10.92 (s, 1H), 9.47 (s, 1H), 8.25 (s,

6

1

3

Hz, 1H), 0.78 (td, J = 7.2, 4.9 Hz, 2H), 0.67 (dt, J = 7.3, 4.6 Hz, 2H). C

1H), 8.15 (s, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.33−7.24 (m, 2H), 7.02 (s,

1H), 6.32 (t, J = 2.6 Hz, 1H), 2.87 (s, 1H), 0.85−0.76 (m, 2H), 0.74−

NMR (101 MHz, DMSO-d ): δ 160.6, 159.4, 154.4, 146.5, 142.3,

6

1

3

1

34.3, 124.96 (q, J = 269 Hz), 111.8, 107.8, 102.3, 100.7, 24.5, 6.7.

N -Cyclopropyl-N -(2,2-diﬂuorobenzo[d][1,3]dioxol-5-yl)-5-

0.64 (m, 2H). C NMR (101 MHz, DMSO-d ): δ 166.2, 161.0, 159.4,

6

4

2

154.5, 154.4, 132.1, 127.5, 125.6, 125.2 (q, J = 269 Hz), 115.5, 110.8,

+

(triﬂuoromethyl)pyrimidine-2,4-diamine (4). The title compound

1

00.9, 24.5, 6.7. HRMS (ESI-TOF) calcd for C H F N [M + H] :

1

6

15

3

5

was prepared according to General Method 1 using 2-chloro-N-

cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (50 mg, 0.210

mmol) and 2,2-diﬂuorobenzo[d][1,3]dioxol-5-amine (36 mg, 0.210

mmol). The crude product was puriﬁed by automated normal phase

chromatography on silica gel (DCM eluent). White solid (35 mg, 44%

3

34.1280; found 334.1267.

N -Cyclopropyl-N -(1H-indazol-5-yl)-5-(triﬂuoro-methyl)-

4

2

pyrimidine-2,4-diamine (9). The title compound was prepared

according to General Method 1 using 2-chloro-N-cyclopropyl-5-

(triﬂuoromethyl)pyrimidin-4-amine (80 mg, 0.337 mmol) and 1H-

indazol-5-amine (45 mg, 0.337 mmol). The product was isolated by

precipitation and ﬁltration with acetone. Pink solid (70 mg, 62% yield).

+

yield). LC−MS (ESI) calcd for C H F N O [M + H] : 375.09,

1

5

12

5

4

2

1

found 375.20. H NMR (400 MHz, DMSO-d ): δ 9.93 (s, 1H), 8.30

6

+

(

br. s, 1H), 8.21 (s, 1H), 7.46 (dd, J = 8.8, 2.2 Hz, 1H), 7.30 (d, J = 8.8

Hz, 1H), 2.81 (tq, J = 6.7, 3.1 Hz, 1H), 0.84−0.75 (m, 2H), 0.72−0.66

m, 2H). ¹³C NMR (101 MHz, DMSO-d ): δ 160.5, 159.4, 154.6,

LC−MS (ESI) calcd for C H F N [M + H] : 335.13, found 335.15.

1

5

14

3

6

1

H NMR (400 MHz, DMSO-d ): δ 11.00 (s, 1H), 8.07 (s, 1H), 7.57 (s,

6

(

2H), 2.97−2.88 (m, 1H), 0.89−0.82 (m, 2H), 0.78 (dt, J = 7.8, 3.9 Hz,

2H). C NMR (101 MHz, DMSO-d ): δ 172.1, 158.9, 152.6, 145.3,

137.5, 133.5, 133.0, 129.9, 122.8, 122.7 (d, J = 270 Hz), 121.4, 110.6,

25.5, 6.6. HRMS (ESI-TOF) calcd for C H F N [M + H] :

6

1

3

1

54.5, 142.6, 137.3, 137.2, 131.4 (t, J = 252 Hz), 124.8 (q, J = 269 Hz)

6

14.2, 109.7, 101.6, 24.5, 6.7.

4

2

+

N -Cyclopropyl-N -(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-

1

5

14

3

6

(triﬂuoromethyl)pyrimidine-2,4-diamine (5). A solution of 2,4-

335.1232; found 335.1219.

dichloro-5-(triﬂuoromethyl)pyrimidine (85 mg, 0.392 mmol), cyclo-

propanamine (22 mg, 0.385 mmol), and N,N-diisopropylethylamine

2

4

N -(1H-Benzo[d][1,2,3]triazol-6-yl)-N -cyclopropyl-5-

(triﬂuoromethyl)pyrimidine-2,4-diamine (10). The title com-

pound was prepared according to General Method 1 using 2-chloro-N-

cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (90 mg, 0.379

mmol) and 1H-benzo [d][l,2,3]triazol-5-amine (51 mg, 0.379

mmol). The product was isolated by precipitation and ﬁltration with

acetone. White solid (97 mg, 76% yield). LC−MS (ESI) calcd for

C H F N [M + H] : 336.12, found 336.20. H NMR (400 MHz,

DMSO-d ): δ 11.27 (br. s, 1H), 8.63 (s, 1H), 8.46 (s, 1H), 7.91 (d, J =

8.8 Hz, 1H), 7.50 (dd, J = 9.0, 1.9 Hz, 1H), 2.93 (dq, J = 7.2, 3.4 Hz,

1H), 0.95 (td, J = 7.2, 4.9 Hz, 2H), 0.75 (dt, J = 7.0, 4.8 Hz, 2H).

NMR (101 MHz, DMSO-d ): δ 159.1, 154.0, 147.0, 138.0, 136.8,

136.0, 119.4, 116.9, 123.0 (q, J = 270 Hz), 102.8, 98.8 (q, J = 34 Hz),

25.5, 6.7. HRMS (ESI-TOF) calcd for C H F N [M + H] :

336.1185; found 336.1186.

N -Cyclopropyl-N -(1H-pyrrolo[2,3-b]pyridin-5-yl)-5-

(triﬂuoromethyl)pyrimidine-2,4-diamine (11). A solution of 2,4-

dichloro-5-(triﬂuoromethyl)pyrimidine (85 mg, 0.392 mmol), cyclo-

propanamine (22 mg, 0.385 mmol), and N,N-diisopropylethylamine

(0.07 mL) in acetonitrile (3 mL) was microwaved at 100 °C for 10 min.

The mixture was concentrated in vacuo and processed according to

General Method 1 with the addition of 1H-pyrrolo[2,3-b]pyridin-5-

amine (52 mg, 0.391 mmol) in acetic acid (2 mL). The mixture was

microwaved at 120 °C for 10 min. Brown solid (13 mg, 10%). LC−MS

(0.068 mL) in acetonitrile (3 mL) was microwaved at 100 °C for 10

min, then concentrated in vacuo. 2,3-Dihydrobenzo[b][1,4]dioxin-6-

amine (59 mg, 0.390 mmol) was added, and the reaction mixture was

processed according to General Method 1. The mixture was

microwaved at 120 °C for 10 min. White solid (15 mg, 11%). LC−

+

1

+

1

MS (ESI) calcd for C H F N O [M + H] : 353.12, found 353.31. H

1

6

16

3

4

2

14 13 3 7

NMR (400 MHz, DMSO-d ): δ 11.98 (s, 1H), 9.53 (s, 1H), 8.15 (s,

6

1

0

1

H), 7.79 (s, 1H), 7.18 (dd, J = 8.8, 2.6 Hz, 1H), 6.75 (d, J = 8.7 Hz,

1

3

H), 4.23−4.15 (m, 4H), 2.79 (br. s, 1H), 0.82 (dt, J = 6.8, 3.4 Hz, 2H),

C

1

3

.70−0.65 (m, 2H). C NMR (101 MHz, DMSO-d ): δ 160.6, 159.5,

6

54.5, 154.4, 142.9, 138.3, 134.1, 125.0 (q, J = 269 Hz), 116.5, 112.5,

+

08.4, 64.3, 64.0, 24.6, 6.7. HRMS (ESI-TOF) calcd for C H F N O

1

6

16

3

4

2

14 13

3

7

+

[

M + H] : 353.1225; found 353.1216.

4

2

4

2

N -Cyclopropyl-N -(3,4-dihydro-2H-benzo[b][1,4] dioxepin-

7

-yl)-5-(triﬂuoromethyl)pyrimidine-2,4-diamine (6). The title

compound was prepared according to General Method 1 using 2-

chloro-N-cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (80 mg,

0.337 mmol) and 3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-amine (56

mg, 0.337 mmol). White solid (103 mg, 84% yield). LC−MS (ESI)

+

1

calcd for C H F N O [M + H] : 367.14, found 367.30. H NMR

17

18

3

4

2

(

400 MHz, DMSO-d ): δ 9.61 (s, 1H), 8.17 (s, 1H), 7.77 (br. s, 1H),

6

7

4

2

7

1

.36 (dd, J = 8.8, 2.6 Hz, 1H), 7.16 (br. s, 1H), 6.87 (d, J = 8.6 Hz, 1H),

.07 (t, J = 5.3 Hz, 2H), 4.03 (t, J = 5.3 Hz, 2H), 2.83−2.76 (m, 1H),

.05 (q, J = 6.0, 5.4z Hz, 1H), 0.82 (dt, J = 7.1, 3.4 Hz, 2H), 0.67 (td, J =

+

1

(ESI) calcd for C H F N [M + H] : 335.12, found 335.25. H NMR

(400 MHz, DMSO-d ): δ 11.48 (s, 1H), 9.67 (s, 1H), 8.61 (s, 1H), 8.51

(s, 1H), 8.19 (s, 1H), 7.42−7.38 (m, 1H), 7.15−7.07 (m, 1H), 6.37

(dd, J = 3.5, 1.8 Hz, 1H), 2.84 (br. s, 1H), 0.79 (td, J = 7.1, 4.7 Hz, 2H),

0.72−0.65 (m, 2H). C NMR (101 MHz, DMSO-d ): δ 161.2, 159.5,

154.6, 154.6, 144.8, 136.8, 130.0, 126.5, 125.1 (q, J = 269 Hz), 119.0,

99.6, 24.5, 6.7.

1

5

14

3

6

.0, 3.5 Hz, 2H). ¹C NMR (101 MHz, DMSO-d ): δ 160.6, 159.4,

3

6

54.5, 154.4, 150.9, 145.9, 135.9, 124.9 (q, J = 270 Hz), 121.1, 114.2,

1

3

12.6, 70.6, 32.1, 24.6, 6.7. HRMS (ESI-TOF) calcd for C H F N O

1

7

18

3

4

2

6

+

[

M + H] : 367.1382; found 367.1368.

2

4

N -(1H-Benzo[d]imidazol-6-yl)-N -cyclopropyl-5-

4

2

(triﬂuoromethyl)pyrimidine-2,4-diamine (7). The title compound

N -Cyclobutyl-N -(1H-pyrrolo[2,3-b]pyridin-5-yl)-5-

(triﬂuoromethyl)pyrimidine-2,4-diamine (12). A solution of 2,4-

dichloro-5-(triﬂuoromethyl)pyrimidine (120 mg, 0.553 mmol), cyclo-

butanamine (49 mg, 0.689 mmol), and N,N-diisopropylethylamine

(0.12 mL) in acetonitrile (3 mL) was microwaved at 100 °C for 10 min.

1H-Pyrrolo[2,3-b]pyridin-5-amine (92 mg, 0.691 mmol) and acetic

acid (3 mL) were added, and the reaction mixture was processed

according to General Method 1 and microwaved at 100 °C for 20 min.

Tan solid (50 mg, 42% yield). LC−MS (ESI) calcd for C H F N [M

was prepared according to General Method 1 using 2-chloro-N-

cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (65 mg, 0.274

mmol) and 1H-benzo[d]imidazol-6-amine (36 mg, 0.274 mmol).

The mixture was microwaved at 130 °C for 20 min. White solid (41 mg,

+

4

5% yield) LC−MS (ESI) calcd for C H F N [M + H] : 335.13;

1

5

13

3

6

1

found 335.00. H NMR (400 MHz, DMSO-d ): δ 9.70 (s, 1H), 8.49

6

(

br. s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 7.48 (s, 2H), 7.13 (br. s, 1H),

2

.89 (dq, J = 6.9, 3.3 Hz, 1H), 0.93−0.80 (m, 2H), 0.73−0.64 (m, 2H).

1

6

16

3

6

13

+

1

C NMR (101 MHz, DMSO-d ): δ 160.8, 159.5, 154.5, 154.4, 141.6,

+ H] : 349.14, found 349.25. H NMR (400 MHz, DMSO-d ): δ 11.52

6

1

36.7, 135.2, 125.0 (q, J = 269 Hz), 115.8, 115.3, 104.5, 24.6, 6.8.

(s, 1H), 9.54 (br. s, 1H), 8.42 (s, 1H), 8.31 (s, 1H), 8.18 (s, 1H), 7.42

+

HRMS (ESI-TOF) calcd for C H F N [M + H] : 335.1232; found

(t, J = 3.0 Hz, 1H), 6.94 (d, J = 6.9 Hz, 1H), 6.38 (dd, J = 3.3, 1.9 Hz,

1

5

14

3

6

1

3

335.1223.

1H), 4.60 (br. s, 1H), 2.29−2.08 (m, 4H), 1.74−1.53 (m, 2H).

C

4

2

N -Cyclopropyl-N -(1H-indol-5-yl)-5-(triﬂuoromethyl)-

NMR (101 MHz, DMSO-d ): δ 161.3, 157.2, 154.9, 154.8, 145.0,

6

pyrimidine-2,4-diamine (8). The title compound was prepared

according to General Method 1 using 2-chloro-N-cyclopropyl-5-

137.3, 129.6, 126.6, 125.0 (q, J = 269 Hz), 119.8, 119.0, 99.5, 45.9, 29.9,

14.8.

K

https://dx.doi.org/10.1021/acs.jmedchem.0c00873

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4

2

+

N -Methyl-N -(1H-pyrrolo[2,3-b]pyridin-5-yl)-5-

triﬂuoromethyl)pyrimidine-2,4-diamine (13). To a solution of

2

(ESI-TOF) calcd for C H F N [M + H] : 346.1280; found

17 15 3 5

(

346.1284.

4

2

,4-dichloro-5-(triﬂuoromethyl)pyrimidine (400 mg, 1.844 mmol) in

N -Cyclopropyl-N -(quinolin-3-yl)-5-(triﬂuoromethyl)-

pyrimidine-2,4-diamine (17). The title compound was prepared

according to General Method 1 using 2-chloro-N-cyclopropyl-5-

(triﬁuoromethyl)pyrimidin-4-amine (60 mg, 0.253 mmol) and

quinolin-3-amine (36 mg, 0.253 mmol). The mixture was microwaved

at 120 °C for 20 min. The product was isolated by precipitation with

acetone and further puriﬁed by automated reverse-phase chromatog-

raphy of the ﬁltrate. White solid (8 mg, 9% yield). LC−MS (ESI) calcd

dichloromethane and tert-butanol (10 mL, 1:1) at −10 °C under

nitrogen was added zinc(II) chloride (502 mg, 3.69 mmol) and stirred

at −10 to 0 °C. After 1 h, 1H-pyrrolo[2,3-b]pyridin-5-amine (245 mg,

0

.844 mmol) and triethylamine (0.28 mL, 2.03 mmol) were added. The

reaction mixture was warmed to room temperature. The dichloro-

methane was removed in vacuo, and the solid precipitate was ﬁltered

and washed with water. The crude product (620 mg) was isolated as a

0:30 mix of isomers, with N-(4-chloro-5-(triﬂuoromethyl)pyrimidin-

-yl)-lH-pyrrolo[2,3-b]pyridin-5-amine as the major isomer. LC−MS

ESI) calcd for C H ClF N [M + H] : 314.04, found 313.80.

A solution of N-(4-chloro-5-(triﬂuoromethyl)pyrimidin-2-yl)-lH-

pyrrolo[2,3-b]pyridin-5-amine (100 mg, 0.319 mmol, containing 30%

of the regioisomer) and methanamine (0.19 mL, 0.96 mmol) in DMF

2 mL) was microwaved at 100 °C for 20 min and concentrated in

+

1

7

for C17

H F N [M + H] : 346.13 found 345.80. H NMR (400 MHz,

15 3 5

DMSO-d ): δ 10.20 (s, 1H), 9.12 (d, J = 2.7 Hz, 1H), 9.06 (br. s, 1H),

.29 (s, 1H), 7.93 (dd, J = 8.5, 1.5 Hz, 1H), 7.79 (dd, J = 7.8, 1.7 Hz,

H), 7.62−7.49 (m, 2H), 7.35 (br. s, 1H), 2.96 (tq, J = 7.1, 3.7 Hz, 1H),

.88 (td, J = 7.0, 4.8 Hz, 2H), 0.74 (dt, J = 6.9, 4.9 Hz, 2H). C NMR

101 MHz, DMSO-d ): δ 160.9, 159.5, 154.6, 145.3, 143.4, 134.1,

28.6, 128.1, 127.3, 127.0, 127.0, 124.7 (q, J = 270 Hz), 120.7, 24.6, 6.8.

HRMS (ESI-TOF) calcd for C H F N [M + H] : 346.1280; found

2

(

6

+

8

1

0

12

8

3

5

1

3

(

6

1

(

+

vacuo. The product was puriﬁed by automated reverse-phase

chromatography. Tan solid (20 mg, 20% yield). LC−MS (ESI) calcd

17 15

3

5

3

46.1281.

4

2

+

1

N -Cyclopropyl-N -(quinoxalin-6-yl)-5-(triﬂuoromethyl)-

for C H F N [M + H] : 309.11, found 308.95. H NMR (400 MHz,

1

3

12

3

6

pyrimidine-2,4-diamine (18). The title compound was prepared

according to General Method 1 using 2-chloro-N-cyclopropyl-5-

(triﬂuoromethyl)pyrimidin-4-amine (60 mg, 0.253 mmol) and

quinoxalin-6-amine (37 mg, 0.253 mmol). The mixture was micro-

waved at 120 °C for 20 min. White solid (17 mg, 19% yield). LC−MS

DMSO-d ): δ 11.47 (s, 1H), 9.51 (br. s, 1H), 8.43 (s, 1H), 8.31 (s, 1H),

.14 (s, 1H), 7.43−7.37 (m, 1H), 7.07 (br. s, 1H), 6.38 (dd, J = 5.0, 2.0

Hz, 1H), 2.91 (d, J = 4.2 Hz, 3H). C NMR (101 MHz, DMSO-d ): δ

6

8

13

6

1

61.6, 158.6, 154.5, 154.4, 145.0, 137.4, 129.8, 126.7, 125.3 (q, J = 269

Hz), 119.2, 99.8, 28.1.

+

1

4

2

(ESI) calcd for C16

H F N [M + H] : 347.13, found 347.10. H NMR

14 3 6

400 MHz, DMSO-d ): δ 10.31 (s, 1H), 9.15 (s, 1H), 8.83 (d, J = 1.8

Hz, 1H), 8.74 (d, J = 1.9 Hz, 1H), 8.31 (s, 1H), 8.09 (dd, J = 9.2, 2.3 Hz,

H), 7.99 (d, J = 9.1 Hz, 1H), 7.45 (s, 1H), 2.91 (dq, J = 7.1, 3.2 Hz,

H), 1.03 (dt, J = 7.2, 3.6 Hz, 2H), 0.74 (td, J = 7.1, 3.6 Hz, 2H).

NMR (101 MHz, DMSO-d ): δ 160.7, 159.6, 154.7, 154.6, 145.8,

143.7, 143.1, 141.6, 138.6, 129.0, 124.7 (q, J = 273 Hz), 124.5, 114.5,

N -Cyclopropyl-N -(4-ethyl-3,4-dihydro-2H-pyrido[3,2-b]-

1,4]oxazin-7-yl)-5-(triﬂuoromethyl)pyrimidine-2,4-diamine

14). The title compound was prepared according to General Method 1

(

6

[

(

1

using 2-chloro-N-cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine

120 mg, 0.505 mmol) and 4-ethyl-3,4-dihydro-2H-pyrido[3,2-b]-

1,4]oxazin-7-amine (91 mg, 0.505 mmol). Pink solid (76 mg, 40%

yield). LC−MS (ESI) calcd for C H F N O [M + H] : 381.17, found

81.33. H NMR (400 MHz, DMSO-d ): δ 9.45 (s, 1H), 8.13 (s, 1H),

.99 (s, 1H), 4.18 (t, J = 4.5 Hz, 2H), 3.53 (q, J = 7.0 Hz, 2H), 3.37 (t, J

4.4 Hz, 2H), 2.82−2.69 (m, 1H), 1.05 (t, J = 7.0 Hz, 3H), 0.77 (dt, J =

.9, 3.3 Hz, 2H), 0.65 (m, 2H). C NMR (101 MHz, DMSO-d ): δ

60.8, 159.4, 154.5, 142.5, 138.6, 130.5, 127.7, 125.0 (q, J = 269 Hz),

1

3

C

(

[

6

+

1

7

20

3

6

+

1

24.8, 6.8. HRMS (ESI-TOF) calcd for C H F N [M + H] :

3

16 14

3

6

3

47.1232; found 347.1216.

6

7

-((4-(Cyclopropylamino)-5-(triﬂuoromethyl)pyrimidin-2-

=

1

3

yl)amino)-3,4-dihydronaphthalen-1(2H)-one (19). The title

compound was prepared according to General Method 1 using 2-

chloro-N-cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (60 mg,

0.253 mmol) and 6-amino-3,4-dihydro-naphthalen-1(2H)-one (41 mg,

6

1

6

14.4, 64.3, 44.4, 41.6, 24.4, 11.4, 6.7.

2

4

N - ( B e n z o [ d ] o x a z o l - 6 - y l ) - N - c y c l o p r o p y l - 5 -

0

.253 mmol). The mixture was microwaved at 110 °C for 20 min and

then concentrated in vacuo. Ethyl acetate-dichloromethane was added,

and the precipitate ﬁltered to obtain the desired product. Tan solid (61

(

triﬂuoromethyl)pyrimidine-2,4-diamine (15). The title com-

pound was prepared according to General Method 1 using 2-chloro-N-

cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine (65 mg, 0.274

mmol) and benzo[d]oxazol-6-amine (37 mg, 0.274 mmol). The

mixture was microwaved at 130 °C for 20 min. Brown solid (4 mg, 4%

yield). LC−MS (ESI) calcd for C H F N O [M + H] : 336.11, found

35.95. H NMR (400 MHz, DMSO-d ): δ 10.02 (s, 1H), 8.79 (s, 1H),

+

mg, 67% yield). LC−MS (ESI) calcd for C H F N O [M + H] :

1

8

18

3

4

1

3

63.15, found 363.15. H NMR (400 MHz, DMSO-d ): δ 11.10 (s,

6

+

1H), 8.47 (s, 1H), 8.27 (br. s, 1H), 7.92 (br. s, 1H), 7.85 (d, J = 8.7 Hz,

1H), 7.71 (dd, J = 8.7, 2.2 Hz, 1H), 2.95 (tq, J = 7.2, 3.6 Hz, 1H), 2.90

t, J = 6.0 Hz, 2H), 2.54 (t, J = 6.4 Hz, 2H), 2.02 (p, J = 6.3 Hz, 2H),

.92−0.80 (m, 2H), 0.84−0.72 (m, 2H). C NMR (101 MHz, DMSO-

d ): δ 196.2, 172.1, 159.2, 155.1, 148.5, 145.9, 142.8, 127.6, 127.4, 123.2

q, J = 270 Hz), 118.4, 117.8, 99.01 (q, J = 34 Hz), 38.0, 29.8, 25.3, 22.9,

.1. HRMS (ESI-TOF) calcd for C H F N O [M + H] : 363.1433;

1

5

13

3

5

1

3

8

6

(

0

.58 (s, 1H), 8.23 (s, 1H), 7.77−7.55 (m, 2H), 7.28 (s, 1H), 2.87 (dq, J

13

=

6.9, 3.4 Hz, 1H), 0.85 (dt, J = 6.8, 3.3 Hz, 2H), 0.76−0.66 (m, 2H).

13

6

C NMR (101 MHz, DMSO-d ): δ 160.7, 159.5, 154.6, 154.5, 153.3,

6

(

7

1

49.9, 138.6, 134.0, 124.8 (q, J = 269 Hz), 119.4, 116.8, 101.3, 24.6, 6.7.

HRMS (ESI-TOF) calcd for C H F N O [M + H] : 336.1072; found

+

18 18 3 4

1

5

13

3

5

found 363.1439.

3

36.1064.

4

2

N -Cyclopropyl-N -(pyridazin-4-yl)-5-(triﬂuoromethyl)-

pyrimidine-2,4-diamine (20). Glassware and the stir bar were ﬂame

dried, and nitrogen was bubbled through reagents and solvents prior to

use. 2-Chloro-N-cyclopropyl-5-(triﬂuoromethyl)pyrimidin-4-amine

70 mg, 0.295 mmol), pyridazin-4-amine (28 mg, 0.295 mmol), 9,9-

dimethyl-(9H-xanthene-4,5-diyl)bis(diphenyl)phosphane (17 mg,

.029 mmol), diacetoxypalladium (3.3 mg, 0.015 mmol), and cesium

carbonate (120 mg, 0.589 mmol) were mixed in 1,4-dioxane (1 mL).

The mixture was microwaved at 140 °C for 20 min, ﬁltered through

celite with methanol, and concentrated in vacuo. The crude product

was puriﬁed by automated reverse-phase chromatography. White solid

5 mg, 6% yield). LC−MS (ESI) calcd for C H F N [M + H] :

97.11, found 296.75. H NMR (400 MHz, DMSO-d ): δ 10.39 (s,

H), 9.51 (d, J = 2.1 Hz, 1H), 8.96 (d, J = 5.9 Hz, 1H), 8.35−8.28 (m,

2H), 7.54 (d, J = 2.4 Hz, 1H), 2.87 (dq, J = 7.2, 3.7 Hz, 1H), 0.86 (td, J =

7.0, 4.8 Hz, 2H), 0.70 (dt, J = 7.2, 4.6 Hz, 2H).

N -Cyclopropyl-N -(5-methoxypyridin-3-yl)-5-

(triﬂuoromethyl)pyrimidine-2,4-diamine (21). Glassware and the

4

2

N -Cyclopropyl-N -(quinolin-6-yl)-5-(triﬂuoromethyl)-

pyrimidine-2,4-diamine (16). The title compound was prepared

according to General Method b using 2-chloro-N-cyclopropyl-5-

(

triﬂuoromethyl)pyrimidin-4-amine (60 mg, 0.253 mmol), copper

1.6 mg, 0.025 mmol) and quinolin-6-amine (36 mg, 0.253 mmol). The

reaction mixture was microwaved at 120 °C for 10 min. The product

was isolated by precipitation with acetone and further puriﬁed by

automated reverse-phase chromatography of the ﬁltrate. White solid (7

mg, 8% yield). LC−MS (ESI) calcd for C H F N [M + H] : 346.13

found 345.95. H NMR (400 MHz, DMSO-d6): δ 10.09 (s, 1H), 8.76

d, J = 2.4 Hz, 1H), 8.72 (dd, J = 4.1, 1.7 Hz, 1H), 8.27 (s, 1H), 8.13

dd, J = 8.3, 1.6 Hz, 1H), 8.04 (dd, J = 9.1, 2.4 Hz, 1H), 7.93 (d, J = 9.1

Hz, 1H), 7.43 (dd, J = 8.3, 4.2 Hz, 1H), 7.29 (s, 1H), 2.99 (dq, J = 7.1,

.4 Hz, 1H), 0.88 (td, J = 7.1, 4.8 Hz, 2H), 0.78−0.69 (m, 2H).

NMR (101 MHz, DMSO-d6): δ 160.8, 159.5, 154.6 (q, J = 5 Hz),

(

0

+

1

7

15

3

5

1

+

(

2

1

2

12

3

6

1

6

1

3

C

4

2

1

48.2, 144.2, 138.3, 135.1, 129.0, 128.53, 124.8 (q, J = 270 Hz), 124.1,

21.7, 113.9, 40.2, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 24.6, 6.8. HRMS

L

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

+

stir bar were ﬂame dried, and nitrogen was bubbled through reagents

and solvents prior to use. 2-Chloro-N-cyclopropyl-5-(triﬂuoromethyl)-

pyrimidin-4-amine (65 mg, 0.274 mmol), 5-methoxypyridin-3-amine

mg, 16% yield). LC−MS (ESI) calcd for C H F N O [M + H] :

2

23

3

5

2

1

446.18, found 446.35. H NMR (400 MHz, DMSO-d ): δ 10.25 (s,

6

1H), 8.36 (s, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 8.7 Hz, 2H),

7.47 (d, J = 9.1 Hz, 1H), 7.07 (d, J = 8.7 Hz, 2H), 2.76 (dq, J = 7.1, 3.3

Hz, 1H), 2.45−2.35 (m, 1H), 1.82−1.68 (m, 1H), 1.10−0.82 (m, 4H),

0.82−0.73 (m, 4H), 0.70−0.47 (m, 4H).

N - C y c l o p r o p y l - N - ( 3 , 4 - d i m e t h o x y p h e n y l ) - 5 -

(triﬂuoromethyl)pyrimidine-2,4-diamine (26, SBP-7455). A

solution of 4-dichloro-5-(triﬂuoromethyl)pyrimidine (503 mg, 2.32

mmol) and zinc chloride (379 mg, 2.78 mmol) in 1,2-dichloroethane

and tert-butanol (1:1, 20 mL) was set to stir at 0 °C. After 30 min, a

solution of 3,4-dimethoxyaniline (355 mg, 2.32 mmol) and triethyl-

amine (0.40 mL, 2.78 mmol) in 1,2-dichloroethane and tert-butanol

(

34 mg, 0.274 mmol), (9,9-dimethyl-9H-xanthene-4,5-diyl)bis-

diphenylphosphane) (16 mg, 0.027 mmol), diacetoxypalladium (3.1

mg, 0.014 mmol), and cesium carbonate (134 mg, 0.410 mmol) were

mixed in 1,4-dioxane (1 mL). The mixture was microwaved at 140 °C

for 20 min, ﬁltered through celite with methanol, and concentrated in

vacuo. The crude product was puriﬁed by automated reverse-phase

chromatography. White solid (9 mg, 10% yield). LC−MS (ESI) calcd

4

2

+

1

for C H F N O [M + H] : 326.13, found 325.90. H NMR (400

14

15

3

5

MHz, DMSO-d ): δ 9.85 (s, 1H), 8.56 (d, J = 2.1 Hz, 1H), 8.20 (s, 1H),

6

8

.06 (t, J = 2.3 Hz, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.25 (br. s, 1H), 3.76

(

1:1, 2 mL) was added dropwise to the reaction mixture. The reaction

(

s, 3H), 2.82 (dq, J = 7.2, 3.4 Hz, 1H), 0.76 (td, J = 7.2, 4.7 Hz, 2H),

.65 (dt, J = 7.1, 4.8 Hz, 2H). C NMR (101 MHz, DMSO-d ): δ

1

3

mixture was stirred at room temperature for 4 h then concentrated in

vacuo. The crude product was triturated with methanol, ﬁltered, and

dried to yield 4-chloro-N-(3,4-dimethoxyphenyl)-5-(triﬂuoromethyl)-

0

6

1

60.8, 159.4, 155.3, 154.7, 154.6, 137.7, 133.7, 129.2, 124.7 (q, J = 270

Hz), 111.4, 55.5, 24.6, 6.6.

pyrimidin-2-amine. White solid (702 mg, 91% yield) used crude in the

2

-((4-(Cyclopropylamino)-5-(triﬂuoromethyl)pyrimidin-2-

+

yl)amino)phenol (22). The title compound was prepared according

to General Method 1 using 2-chloro-N-cyclopropyl-5-

next step. LC−MS (ESI) calcd for C13

H

12ClF

3

N

6

O

3

2

[M + H] : 334.06

): δ 10.49 (s, 1H), 8.74

(s, 1H), 7.35 (d, J = 2.6 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 6.93 (d, J =

8.7 Hz, 1H), 3.74 (s, 3H), 3.74 (s, 3H). C NMR (101 MHz, DMSO-

): δ 160.6, 158.0, 148.6, 145.5, 131.6, 123.1 (q, J = 270 Hz), 112.9,

1

found 334.00. H NMR (400 MHz, DMSO-d

(

triﬂuoromethyl)pyrimidin-4-amine (60 mg, 0.253 mmol) and 2-

aminophenol (28 mg, 0.253 mmol). The mixture was microwaved at

10 °C for 20 min and then concentrated in vacuo. Ethyl acetate was

1

3

1

d

6

added, and the precipitate ﬁltered to obtain the desired product. Brown

112.0, 106.1, 55.7, 55.5.

solid (57 mg, 73% yield). LC−MS (ESI) calcd for C H F N O [M +

A solution of 4-chloro-N-(3,4-dimethoxyphenyl)-5-

(triﬂuoromethyl)pyrimidin-2-amine (500 mg, 1.50 mmol), cyclo-

propanamine (125 μL, 1.80 mmol) and N,N-diisopropyl ethylamine

1

4

14

3

4

+

1

H] : 31z1.11, found 311.15. H NMR (400 MHz, DMSO-d ): δ 10.07

6

(

7

s, 1H), 8.26 (s, 1H), 8.17 (d, J = 11.6 Hz, 2H), 7.20 (s, 1H), 6.90−6.84

(

1

0.31 mL, 1.80 mmol) in DMF (2 mL) was microwaved at 120 °C for

0 min. The crude product was concentrated in vacuo and puriﬁed by

automated reverse-phase chromatography. White solid (291 mg, 55%

m, 2H), 6.84−6.74 (m, 1H), 2.84 (dq, J = 7.0, 3.5 Hz, 1H), 0.77 (td, J =

1

3

.1, 4.6 Hz, 2H), 0.73−0.62 (m, 2H). C NMR (101 MHz, DMSO-d ):

6

δ 160.6, 159.5, 154.6, 154.5, 146.8, 127.6, 124.8 (q, J = 270 Hz), 123.0,

20.6, 119.1, 115.2, 24.5, 6.6.

+

yield). LC−MS (ESI) calcd for C H F N O [M + H] : 355.14 found

3

1

16 18

3

4

2

1

2

4

55.50. H NMR (400 MHz, DMSO-d ): δ 9.46 (s, 1H), 8.16 (s, 1H),

N -(4-Aminophenyl)-N -cyclopropyl-5-(triﬂuoro-methyl)-

pyrimidine-2,4-diamine (23). A solution of 2-chloro-N-cyclopropyl-

-(triﬂuoromethyl)pyrimidin-4-amine (60 mg, 0.253 mmol) and

6

7.55 (br. s, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.05 (br. s, 1H), 6.88 (d, J =

8.8 Hz, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 2.91 (dq, J = 6.9, 3.3 Hz, 1H),

5

1

3

benzene-1,4-diamine (55 mg, 0.505 mmol) in butan-l-ol (1 mL) was

microwaved at 120 °C for 20 min and concentrated in vacuo. Acetone

was added, and the mixture was ﬁltered. The ﬁltrate was concentrated

to give the desired product. Brown solid (92 mg, 98% yield). LC−MS

0.77 (td, J = 7.2, 4.9 Hz, 2H), 0.67 (dt, J = 7.3, 4.4 Hz, 2H). C NMR

(101 MHz, DMSO-d ): δ 160.8, 159.4, 154.5, 154.4, 148.5, 144.2,

133.8, 125.0 (q, J = 269 Hz), 112.2, 111.4, 105.2, 55.8, 55.6, 24.5, 6.6.

6

+

HRMS (ESI-TOF) calcd for C16

found 355.1388.

H F N O [M + H] : 355.1382;

18 3 4 2

+

1

(

ESI) calcd for C H F N [M + H] : 310.13, found 310.00. H NMR

14 15 3 5

(

6

1

400 MHz, DMSO-d ): δ 9.24 (s, 1H), 8.09 (s, 1H), 7.49 (br. s, 1H),

Crystallization and Structure Determination. ULK2 kinase

6

34

.95 (s, 1H), 6.49 (d, J = 8.5 Hz, 2H), 4.75 (br. s, 2H), 2.85−2.77 (m,

domain was puriﬁed and crystallized as previously described. Data

1

3

H), 0.78−0.68 (m, 2H), 0.67−0.61 (m, 2H). C NMR (101 MHz,

collection was performed at an SLS X06SA, and data were processed

6

0,61

DMSO-d ): δ 160.8, 159.4, 154.4 (q, J = 4.6 Hz), 143.8, 129.4, 125.2 (q,

and scaled with XDS and aimless, respectively.

Molecular

6

J = 269 Hz), 121.1, 113.8, 24.4, 6.6.

replacement was performed using a Phaser and the published ULK2

62

structure (PDB ID: 6QAV). The structure was rebuilt in COOT and

N-(4-((4-(Cyclopropylamino)-5-(triﬂuoromethyl)pyrimidin-

63,64

2

-yl)amino)phenyl)cyclopropanecarbox-amide (24). A solution

reﬁned using REFMAC.

are summarized in Table S1 .

Data collection and reﬁnement statistics

2

4

of N -(4-aminophenyl)-N -cyclopropyl-5-(triﬂuoromethyl)-

pyrimidine-2,4-diamine (20 mg, 0.065 mmol), cyclopropanecarbonyl

chloride (6.2 μL, 0.068 mmol), and triethylamine (0.012 mL, 0.084

mmol) in DMF (1 mL) was microwaved at 60 °C for 10 min and

concentrated in vacuo. The crude product was puriﬁed by automated

reverse phase chromatography. White solid (3 mg, 12% yield). LC−MS

In Silico Docking. Fully continuously ﬂexible small-molecule ligand

docking to the ULK2 protein, represented by grid interaction

i

potentials, was performed using the ICM docking algorithm [ ] as

implemented in the ICM-Pro program (v3.8, Molsoft, LLC.). The

coordinates of the three-dimensional structure of ULK2 (PDB ID:

6YID) reported here were converted into an ICM object, charges were

assigned, orientations of sidechain amides were corrected, and

hydrogen atoms added and their positions optimized by energy

minimization using the MMFF force ﬁeld. The docking receptor site

was deﬁned as an 8 Å radius around the SBI-0206965 ligand. An energy-

minimized three-dimensional molecular model of the compound was

generated and docking performed using the implemented routine in

ICM.

+

1

(

ESI) calcd for C H F N O [M + H] : 378.16, found 378.00. H

18 19 3 5

NMR (400 MHz, DMSO-d ): δ 10.06 (s, 1H), 9.61 (br. s, 1H), 8.17 (s,

6

1

2

0

1

2

H), 7.80 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.9 Hz, 2H), 7.11 (br. s, 1H),

.84 (td, J = 7.2, 3.6 Hz, 1H), 1.80−1.69 (m, 1H), 0.84−0.72 (m, 6H),

1

3

.71−0.60 (m, 2H). C NMR (101 MHz, DMSO-d ): δ 171.1, 160.7,

6

59.4, 154.5, 154.5, 135.5, 133.7, 124.97 (q, J = 269 Hz), 119.6, 119.3,

4.5, 14.0, 6.9, 6.6. HRMS (ESI-TOF) calcd for C H F N O [M +

1

8

19

3

5

+

H] : 378.1542; found 378.1530.

N-(Cyclopropanecarbonyl)-N-(4-((4-(cyclopropyl amino)-5-

ULK1/2 ADP-Glo Assay. The kinase reaction was performed in 5

μL total volume containing 25 μM ATP (Sigma-Aldrich #A7699). For

the ULK1 ADP-Glo assay, the reaction employed 2 μg/mL

recombinant human ULK1 protein (1-649, SignalChem #U01-11G)

and 80 μg/mL myelin basic protein (MBP, Sigma-Aldrich #M1891).

The ULK2 assay included 4 μg/mL recombinant human ULK2 protein

(1-478, SignalChem #U02-11G) and 80 μg/mL MBP. IC values were

determined in 16-dose assay mode with threefold serial dilutions

starting at 30 μM compound, performed in triplicate. Staurosporine, a

nonselective protein kinase inhibitor, was used as a positive control.

(

triﬂuoromethyl)pyrimidin-2-yl)amino)phenyl)cyclopropane-

2

4

carboxamide (25). A solution of N -(4-aminophenyl)-N -cyclo-

propyl-5-(triﬂuoromethyl)pyrimidine-2,4-diamine (43 mg, 0.139

mmol), cyclopropanecarbonyl chloride (16 mg, 0.153 mmol) and N-

ethyl-N-isopropyl propan-2-amine (0.03 mL, 0.167 mmol) in DMF (1

mL) was microwaved at 60 °C for 10 min. Additional cyclo-

propanecarbonyl chloride (16 mg, 0.153 mmol) was added and

microwaved at 60 °C for an additional 10 min. The crude product was

puriﬁed by automated reverse-phase chromatography. White solid (10

5

0

M

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

ULK1/2 NanoBRET Assay. Human embryonic kidney cells

HEK293T) were transfected with a NanoLuc-ULK1 Fusion Vector

Promega #NV2211) or a NanoLuc-ULK2 Fusion Vector (Promega

NV2221) using the jetPRIME transfection reagent (Polyplus

samples ﬂash frozen in liquid nitrogen then homogenized on ice in CST

lysis buﬀer. The protein lysates were equilibrated for protein levels

using a BCA protein assay kit (Pierce) and resolved on 8% gels. The

lysates were immunoblotted with pATG13 S318 (Rockland #600-401-

C49S), ATG13 (CST #13273), ULK1 (CST #8054), and β-actin

(Sigma A5441) antibodies to evaluate on-target inhibition in the livers.

Lentiviral Preparation and Viral Infection of Cells. Lentiviruses

containing shRNA-encoding pLKO.1 vectors were prepared by the

Sanford Burnham Prebys Viral Vector Core Facility following a

(

#

Transfection #114-15) for 24 h. Cells were trypsinized and resuspended

in Opti-MEM I (1X) Reduced Serum Medium (Gibco, #11058−021)

3

and plated at approximately 7 × 10 cells/34 μL/well total volume in

nonbinding surface 384 well plates (Corning #3574). Complete

NanoBRET 20× Tracer K-5 reagent, prepared according to the

manufacturer’s directions, was added at 2 μL/well, and the plate was

mixed on an orbital shaker for 15 s at 700 rpm. Compounds were

serially diluted at 10× the ﬁnal concentration in an assay medium

65

previously described protocol. The ULK1 and BECN1 target

sequences are listed in Table S3. For the viral infection, MDA-MB-

5

468 cells were plated at 1 × 10 /well of 6-well plates and incubated for

(

Opti-MEM I, Reduced Serum Medium) and added to the cells at 4

μL/well, and the assay plate was mixed for 15 s at 700 rpm. The assay

24 h. The viruses were mixed with 8 μg/mL Polybrene (Santa Cruz

Biotechnology #sc-134220) and added to the cells. Three days later,

infected cells were selected by the addition of 1 μg/mL puromycin

(Gibco #A11138-03) for an additional 3 days, harvested by

trypsinization, and then dispensed into 96-well plates for experiments.

Apoptosis Assay. MDA-MB-468 cells were treated with

compound 26 or DMSO for 18 h after which cells the were trypsinized

and washed twice with cold PBS. The cells were then stained using a PE

Annexin V Apoptosis Detection Kit I (BD Pharmingen #559763).

Speciﬁcally, the cells were resuspended in 1× binding buﬀer at a

plate was incubated for 2 h in a 37 °C incubator with 5% CO and then

2

equilibrated to rt for 15 min. 3X Complete Substrate plus Inhibitor

Solution was prepared according to the manufacturer’s directions, with

Extracellular NanoLuc Inhibitor (60 μM), and added to the cells at a

ﬁnal working concentration of 20 μM/well. The assay plate and

incubated at rt for 2−3 min, and the signals at the donor (450 nm) and

acceptor (610 nm) emission wavelengths were measured using a Spark

multimode microplate reader (Tecan).

6

Thermal Shift Assay. Recombinant kinase domains of ULK1 and

concentration of 1 × 10 cells/mL, and 100 μL of the solution was

ULK2 (2 μM) were mixed with each ULK1/2 inhibitor (10 μM), and

transferred to a 5 mL culture tube. PE Annexin V (5 μL) and 7-AAD (5

μL) were added to the tube with cells. The mix was incubated for 15

min at room temperature in the dark after gentle vortexing. Next, 1×

binding buﬀer (400 μL) was added to each tube, and ﬂow cytometry to

detect PE-Annexin V and 7-AAD was performed within 1 h using a

LSRFortessa 14-color (BD Biosciences) to analyze apoptosis. Events of

34

the assay was performed as previously described.

pBeclin1 Assay. HEK293T cells were incubated in 6-well dishes at

5

6

× 10 cells/well in normal growth media (DMEM containing 10%

fetal bovine serum [FBS] and 1% penicilin/streptomycin) for 24 h and

then transfected with WT or KI Myc-tagged ULK1 plus WT Flag-

tagged Beclin1 using a jetPRIME transfection reagent (Polyplus

Transfection #114-15). At 24 h posttransfection, the cells were

incubated in fresh media containing DMSO or ULK1/2 inhibitor (10

μM) for 1 h. The cells were lysed in RIPA buﬀer (Sigma #R0278)

containing protease inhibitors (Roche #5892970001) and phosphatase

inhibitors (Roche #4906837001). The lysates were immunoblotted

with a Beclin1 (pSer15) antibody (Abbiotec #254515) or glycer-

aldehyde 3-phosphate dehydrogenase (GAPDH; 14C10 Cell Signaling

Technology [CST] #2118S) and then stripped with Restore Western

Blot Stripping Buﬀer (Thermo Scientiﬁc #21059) and incubated with

Beclin1 antibody (CST #3738S) to probe total Beclin1. Densitometry

analysis was performed with Image Lab 5.2.1 (Bio-Rad).

1

0,000 were collected for each sample.

mCherry-GFP-LC3 FACS Assay. MDA-MB-468 cells stably

expressing the mCherry-GFP-LC3 fusion protein were plated in 6-

5

well plates at a density of 6 × 10 cells/well in growth media DMEM/

F12 + GlutaMAX-1 (Gibco) supplemented with 10% FBS (Gibco) and

1

× Anti-Anti (Gibco) and allowed to grow overnight. Cells were

treated, trypsinized, and then resuspended in PBS, and analytical

cytometry was performed in the Sanford Burnham Prebys ﬂow

cytometry core using an LSRFortessa 14-color (BD Biosciences) with

a 488 nm and 610 nM laser. For ﬂow cytometry, WT MDA-MB-468

cells were used as the unstained control, and MDA-MB-468 cells

expressing either mCherry or GFP were used for compensation. Events

of 10,000 were collected for each sample.

5

pVPS34 Assay. HEK293T cells (6 × 10 ) cells were incubated for

2

4 h as described for pBeclin1 and then transfected with WT or KI Myc-

TEM Imaging of Autophagy Vacuoles. Cells were seeded on 10

cm acrylic culture dishes (Corning) until 70% conﬂuence, ﬁxed in a

cold solution containing 4% formaldehyde (EMS), 2.5% glutaraldehyde

tagged ULK1 plus WT Flag-tagged Vps34 (WT Vps34) using a

standard Lipofectamine 2000 transfection protocol (Invitrogen

11668500). At 24 h posttransfection, the cells were incubated in

fresh media containing DMSO or ULK1/2 inhibitor (10 μM) for 1 h,

and the cells were lysed with cell lysis buﬀer (CST #9803). The cell

lysates were immunoblotted with anti-Myc tag (CST #2278), anti-

FLAG tag polyclonal (Sigma #F7425), and anti-pVPS34 (CST

13857) antibodies. Bands were quantiﬁed as described for pBeclin1.

PK and PD Studies. All animal care and handling was in accordance

with the NIH guidelines for the Care and Use of Laboratory Animals,

and the procedures were approved by the Sanford Burnham Prebys

Medical Discovery Institute Institutional Animal Care and Use

Committee. Female C57BL/6J mice (8 weeks of age) were purchased

from the Jackson Laboratory, housed in pathogen-free conditions on a

#

(

EMS), 100 mM cacocylate buﬀer (Sigma) for 1 min using a microwave

oven (at 150 watts, Ted Pella), and additional 40 min at 4 °C. The

samples were washed in the same buﬀer (3 × 15 min), post-ﬁxed with

1

% osmium tetroxide (EMS) and 1.2% potassium ferrocyanide in

cacodylate buﬀer (100 mM) for 40 min, washed with buﬀer (3 × 15

min), stained with 1% uranyl acetate (EMS) for 40 min, and washed

with Milliq water (3 × 15 min). The cells were scraped oﬀ the plates,

transferred to 1.5 mL eppendorfs, and pelleted for 15 min at 15,000 rpm

#

(

Eppendorf Centrifuge model 5415R). The cells were dehydrated in an

acetone series until 100% (3×) and embedded in an Epon resin.

Ultrathin sections of 50 nm were obtained with a diamond knife

(

Diatome), collected on 300 mesh copper grids, and post-stained with

1

2 h light/dark cycle, and given free access to food and water. Mice were

orally administered compound 26 (30 mg/kg) formulated in vehicle

5% DMSO, 10% Tween-80, and 85% distilled and deionized H 0). At

UranyLess (EMS) for 5 min. Sections were observed in a Zeiss Libra

1

20 operated at 80 kV, and high-resolution images (4K × 4K) obtained

(

2

with a CCD camera Gatan US4000.

the indicated times of postdosing, blood samples were collected retro-

orbitally and plasma was separated by centrifugation. Plasma samples

were extracted with acetonitrile: water (4:1) with 0.1% formic acid-

containing indomethacin as an internal standard. Samples were

centrifuged, and supernatants were diluted with acetonitrile: water

and analyzed by LC−MS/MS on a Shimadzu Nexera X2 HPLC

coupled to an AB Sciex 6500 QTRAP. For the PD studies, mice were

orally administered compound 26 (10 mg/kg) in a vehicle (5% DMSO,

Statistics. The 50% inhibitory concentrations (IC₅₀values) were

determined by analyzing the log of the concentration-response curves

by nonlinear regression analysis using Prism 8 (GraphPad).

ASSOCIATED CONTENT

■

*

sı Supporting Information

1

0% Tween-80, and 85% distilled and deionized H2O) or vehicle alone.

The mice were sacriﬁced 2 h post-dose by cervical dislocation, and liver

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00873.

N

https://dx.doi.org/10.1021/acs.jmedchem.0c00873

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Sonja N. Brun − Molecular and Cell Biology Laboratory, The

Overview of the autophagy pathway, structures of ULK1/

Salk Institute for Biological Studies, San Diego, California

2

inhibitors: SBI-0206965 and Shokat compound, data

92037, United States

collection and reﬁnement statistics for the ULK2

structure, representative dose−response curves of com-

pounds in the ADP-Glo assay, representative dose−

response curves of compounds in the NanoBRET assay,

protein thermal shift data of selected compounds, shRNA

target sequences for knockdown studies, Western blot

analysis of shRNA-mediated ULK1 and BECN1 knock-

down in three TNBC cell lines, data showing the

downregulation of starvation-induced autophagic ﬂux in

MDA-MB-468 cells by ULK inhibitors, data showing that

PARP inhibitors kill MDA-MB-468 cells with low

potency, data showing that SBP-7455 and olaparib

synergize to inhibit the viability of MDA-MB-468 cells,

data showing that SBP-7455 and niraparib synergize to

inhibit the viability of MDA-MB-468 cells, data showing

that induction of autophagic ﬂux in MDA-MB-468 cells

by olaparib is inhibited by concomitant treatment with

SBP-7455, kinetic solubility of selected compounds,

bidirectional permeability of selected compounds in

Caco-2 cells, NMR and LC−MS spectra of selected

compounds, and molecular formula strings (PDF).

Lester J. Lambert − Cancer Molecules & Structures Program,

NCI-Designated Cancer Center, Sanford Burnham Prebys

Medical Discovery Institute, La Jolla, California 92037, United

States

Lutz Tautz − Cancer Molecules & Structures Program, NCI-

Designated Cancer Center, Sanford Burnham Prebys Medical

Discovery Institute, La Jolla, California 92037, United States

Maria Celeridad − Cancer Molecules & Structures Program,

NCI-Designated Cancer Center, Sanford Burnham Prebys

Medical Discovery Institute, La Jolla, California 92037, United

States

Douglas J. Sheﬄer − Cancer Molecules & Structures Program,

NCI-Designated Cancer Center, Sanford Burnham Prebys

Medical Discovery Institute, La Jolla, California 92037, United

States

Stefan Knapp − Structural Genomics Consortium, Buchmann

Institute for Molecular Life Sciences and Institute of

Pharmaceutical Chemistry, Goethe-University Frankfurt,

Frankfurt 60438, Germany; orcid.org/0000-0001-5995-

6494

Reuben J. Shaw − Molecular and Cell Biology Laboratory, The

Salk Institute for Biological Studies, San Diego, California

Accession Codes

92037, United States

X-ray crystal structure of SBI-0206965 in complex with ULK2,

PDB ID: 6YID. Authors will release the atomic coordinates and

experimental data upon article publication.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c00873

Author Contributions

AUTHOR INFORMATION

■

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Corresponding Author

Nicholas D. P. Cosford − Cancer Molecules & Structures

Program, NCI-Designated Cancer Center, Sanford Burnham

Prebys Medical Discovery Institute, La Jolla, California 92037,

United States; orcid.org/0000-0001-7228-4709;

Email: ncosford@sbpdiscovery.org

Funding

This work was supported by the Epstein Family Foundation, a

Sanford Burnham Prebys National Cancer Institute Cancer

Center Support Grant P30 CA030199, a Larry L. Hillblom

Foundation Grant 2019-A-005-NET, and a 2019 Pancreatic

Cancer Action Network Translational Research Grant 19-65-

COSF to NDPC. NAB was supported by the National Cancer

Institute of the National Institutes of Health under award

number T32CA211036. S.K. and A.C. are grateful for support

from the SGC, a registered charity (no. 1097737) that receives

funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,

Canada Foundation for Innovation, Eshelman Institute for

Innovation, Genome Canada through Ontario Genomics

Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Dia-

mond, Innovative Medicines Initiative 2 Joint Undertaking

Authors

Huiyu Ren − Cancer Molecules & Structures Program, NCI-

Designated Cancer Center, Sanford Burnham Prebys Medical

Discovery Institute, La Jolla, California 92037, United States

Nicole A. Bakas − Cancer Molecules & Structures Program,

NCI-Designated Cancer Center, Sanford Burnham Prebys

Medical Discovery Institute, La Jolla, California 92037, United

States

Mitchell Vamos − Cancer Molecules & Structures Program,

NCI-Designated Cancer Center, Sanford Burnham Prebys

Medical Discovery Institute, La Jolla, California 92037, United

States

(

EUbOPEN grant 875510), Janssen, Merck KGaA (a.k.a. EMD

in Canada and US), Merck & Co (aka MSD outside Canada and

US), Pﬁzer, Sao Paulo Research Foundation-FAPESP, Takeda,

Apirat Chaikuad − Structural Genomics Consortium,

Buchmann Institute for Molecular Life Sciences and Institute of

Pharmaceutical Chemistry, Goethe-University Frankfurt,

Frankfurt 60438, Germany; orcid.org/0000-0003-1120-

̃

and Wellcome. S.K. and A.C. are also grateful for funding by the

DKTK and FCI cancer network as well as the DFG funded

SFB1177 (selective autophagy). The content is solely the

responsibility of the authors and does not necessarily represent

the oﬃcial views of the National Institutes of Health.

2

209

Allison S. Limpert − Cancer Molecules & Structures Program,

NCI-Designated Cancer Center, Sanford Burnham Prebys

Medical Discovery Institute, La Jolla, California 92037, United

States

Notes

The authors declare no competing ﬁnancial interest.

Carina D. Wimer − Cancer Molecules & Structures Program,

NCI-Designated Cancer Center, Sanford Burnham Prebys

Medical Discovery Institute, La Jolla, California 92037, United

States

ACKNOWLEDGMENTS

We thank Dr. Andrew Thorburn (U.C. Denver) for providing

the MDA-MB-468 cells expressing mCherry-GFP-LC3, Dr. Do-

■

O

https://dx.doi.org/10.1021/acs.jmedchem.0c00873

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Hyung Kim (University of Minnesota) for sharing the ULK1

shRNA construct, the Viral Vector Core Facility at Sanford

Burnham Prebys Medical Discovery Institute (SBP) for

generating lentiviruses, the Flow Cytometry Core at SBP for

Sigl, V.; Aumayr, K.; Schmauss, G.; Fellner, N.; Handschuh, S.;

Glosmann, M.; Pasierbek, P.; Schlederer, M.; Resch, G. P.; Ma, Y.;

Yang, H.; Popper, H.; Kenner, L.; Kroemer, G.; Penninger, J. M. A dual

role for autophagy in a murine model of lung cancer. Nat. Commun.

2014, 5, 3056.

̈

5

help generating the data in Figure B, and the Waitt Advanced

(16) Rosenfeldt, M. T.; O’Prey, J.; Morton, J. P.; Nixon, C.; MacKay,

Biophotonics Core at the Salk Institute for Biological Studies for

taking the TEM images. The authors thank the staﬀ at Swiss

Light Source for their assistance with data collection, which was

also supported by the funding from the European Union’s

Horizon 2020 research and innovation program under grant

agreement number 730872, project CALIPSOplus.

G.; Mrowinska, A.; Au, A.; Rai, T. S.; Zheng, L.; Ridgway, R.; Adams, P.

D.; Anderson, K. I.; Gottlieb, E.; Sansom, O. W.; Ryan, K. M. p53 status

determines the role of autophagy in pancreatic tumour development.

Nature 2013, 504 , 296.

(

17) White, E. The role for autophagy in cancer. J. Clin. Invest. 2015,

25, 42−46.

18) Guo, J. Y.; Xia, B.; White, E. Autophagy-mediated tumor

promotion. Cell 2013, 155, 1216−1219.

19) O ’Donovan, T. R.; O ’Sullivan, G. C.; McKenna, S. L. Induction

1

(

ABBREVIATIONS USED

■

(

ULK, Unc-51-like autophagy activating kinase; TNBC, triple-

negative breast cancer; PARP, poly (ADP-ribose) polymerase;

SAR, structure−activity relationship; PTS, protein thermal shift;

GFP, green ﬂuorescent protein; LC3, MAP1LC3B; TEM,

transmission electron microscope

of autophagy by drug-resistant esophageal cancer cells promotes their

survival and recovery following treatment with chemotherapeutics.

Autophagy 2011, 7, 509−524.

(20) Chen, S.; Rehman, S. K.; Zhang, W.; Wen, A.; Yao, L.; Zhang, J.

Autophagy is a therapeutic target in anticancer drug resistance. Biochim.

Biophys. Acta, (BBA)-Rev. Cancer 2010, 1806, 220−229.

REFERENCES

(21) Sui, X.; Chen, R.; Wang, Z.; Huang, Z.; Kong, N.; Zhang, M.;

■

(

1) Bento, C. F.; Renna, M.; Ghislat, G.; Puri, C.; Ashkenazi, A.;

Vicinanza, M.; Menzies, F. M.; Rubinsztein, D. C. Mammalian

Han, W.; Lou, F.; Yang, J.; Zhang, Q.; Wang, X.; He, C.; Pan, H.

Autophagy and chemotherapy resistance: a promising therapeutic

target for cancer treatment. Cell death dis. 2013, 4, No. e838.

(22) Holohan, C.; Van Schaeybroeck, S.; Longley, D. B.; Johnston, P.

G. Cancer drug resistance: an evolving paradigm. Nat. Rev. Cancer

2013, 13, 714.

(23) Limpert, A. S.; Lambert, L. J.; Bakas, N. A.; Bata, N.; Brun, S. N.;

Shaw, R. J.; Cosford, N. D. P. Autophagy in cancer: regulation by small

molecules. Trends Pharmacol. Sci. 2018, 39, 1021−1032.

autophagy: how does it work? Annu. Rev. Biochem. 2016 , 85 , 685 −713.

(2) Kimmelman, A. C. The dynamic nature of autophagy in cancer.

Genes Dev. 2011, 25, 1999−2010.

3) Yang, Z.; Klionsky, D. J. Eaten alive: a history of macroautophagy.

Nat. Cell Biol. 2010, 12, 814.

4) Amaravadi, R. K.; Yu, D.; Lum, J. J.; Bui, T.; Christophorou, M. A.;

(

Evan, G. I.; Thomas-Tikhonenko, A.; Thompson, C. B. Autophagy

inhibition enhances therapy-induced apoptosis in a Myc-induced

model of lymphoma. J. Clin. Invest. 2007, 117, 326−336.

(24) Maycotte, P.; Gearheart, C. M.; Barnard, R.; Aryal, S.; Levy, J. M.

M.; Fosmire, S. P.; Hansen, R. J.; Morgan, M. J.; Porter, C. C.;

Gustafson, D. L.; Thorburn, A. STAT3-mediated autophagy depend-

ence identifies subtypes of breast cancer where autophagy inhibition

can be efficacious. Cancer Res. 2014, 74, 2579−2590.

(5) Hosokawa, N.; Sasaki, T.; Iemura, S.-i.; Natsume, T.; Hara, T.;

Mizushima, N. Atg101, a novel mammalian autophagy protein

interacting with Atg13. Autophagy 2009, 5, 973−979.

(6) Ganley, I. G.; Lam, D. H.; Wang, J.; Ding, X.; Chen, S.; Jiang, X.

(25) Yao, H.; He, G.; Yan, S.; Chen, C.; Song, L.; Rosol, T. J.; Deng, X.

ULK1 · ATG13 FIP200 complex mediates mTOR signaling and is

Triple-negative breast cancer: is there a treatment on the horizon?

Oncotarget 2017, 8, 1913.

essential for autophagy. J. Biol. Chem. 2009 , 284 , 12297 −12305.

(7) Laplante, M.; Sabatini, D. M. mTOR signaling in growth control

(

26) Prat, A.; Perou, C. M. Deconstructing the molecular portraits of

breast cancer. Mol. Oncol. 2011, 5, 5−23.

27) O’Reilly, E. A.; Gubbins, L.; Sharma, S.; Tully, R.; Guang, M. H.

and disease. Cell 2012, 149, 274−293.

8) Galluzzi, L.; Pietrocola, F.; Levine, B.; Kroemer, G. Metabolic

control of autophagy. Cell 2014, 159, 1263−1276.

9) Papinski, D.; Schuschnig, M.; Reiter, W.; Wilhelm, L.; Barnes, C.

(

Z.; Weiner-Gorzel, K.; McCaffrey, J.; Harrison, M.; Furlong, F.; Kell,

M.; McCann, A. The fate of chemoresistance in triple negative breast

cancer (TNBC). BBA clin. 2015, 3, 257−275.

(

A.; Maiolica, A.; Hansmann, I.; Pfaffenwimmer, T.; Kijanska, M.;

Stoffel, I.; Lee, S. S.; Brezovich, A.; Lou, J. H.; Turk, B. E.; Aebersold, R.;

Ammerer, G.; Peter, M.; Kraft, C. Early steps in autophagy depend on

direct phosphorylation of Atg9 by the Atg1 kinase. Mol. Cell 2014, 53,

(28) Chittaranjan, S.; Bortnik, S.; Dragowska, W. H.; Xu, J.;

Abeysundara, N.; Leung, A.; Go, N. E.; DeVorkin, L.; Weppler, S. A.;

Gelmon, K.; Yapp, D. T.; Bally, M. B.; Gorski, S. M. Autophagy

inhibition augments the anticancer effects of epirubicin treatment in

anthracycline-sensitive and-resistant triple-negative breast cancer. Clin.

Cancer Res. 2014, 20, 3159−3173.

4

(

71−483.

10) Papinski, D.; Kraft, C. Regulation of autophagy by signaling

through the Atg1/ULK1 complex. J. Mol. Biol. 2016, 428, 1725−1741.

11) Egan, D. F.; Chun, M. G. H.; Vamos, M.; Zou, H.; Rong, J.;

(

(29) Vogel, R. I.; Coughlin, K.; Scotti, A.; Iizuka, Y.; Anchoori, R.;

Miller, C. J.; Lou, H. J.; Raveendra-Panickar, D.; Yang, C.-C.; Sheffler,

D. J.; Teriete, P.; Asara, J. M.; Turk, B. E.; Cosford, N. D. P.; Shaw, R. J.

Small molecule inhibition of the autophagy kinase ULK1 and

identification of ULK1 substrates. Mol. Cell 2015, 59, 285−297.

Roden, R. B. S.; Marastoni, M.; Bazzaro, M. Simultaneous inhibition of

deubiquitinating enzymes (DUBs) and autophagy synergistically kills

breast cancer cells. Oncotarget 2015, 6, 4159.

(30) Amaravadi, R. K.; Kimmelman, A. C.; Debnath, J. Targeting

(12) Alsaadi, R. M.; Losier, T. T.; Tian, W.; Jackson, A.; Guo, Z.;

Autophagy in Cancer: Recent Advances and Future Directions. Cancer

discovery 2019, 9, 1167−1181.

Rubinsztein, D. C.; Russell, R. C. ULK1-mediated phosphorylation of

ATG16L1 promotes xenophagy, but destabilizes the ATG16L1

Crohn ’s mutant. EMBO Rep. 2019, 20, No. e46885.

(31) Hwang, D. Y.; Eom, J.-I.; Jang, J. E.; Jeung, H.-K.; Chung, H.;

Kim, J. S.; Cheong, J.-W.; Min, Y. H. ULK1 inhibition as a targeted

therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia. J.

Exp. Clin. Cancer Res. 2020, 39, 1 −14.

(32) Qiu, L.; Zhou, G.; Cao, S. Targeted inhibition of ULK1 enhances

daunorubicin sensitivity in acute myeloid leukemia. Life Sci. 2020, 243,

117234.

(

13) Saha, S.; Panigrahi, D. P.; Patil, S.; Bhutia, S. K. Autophagy in

health and disease: A comprehensive review. Biomed. Pharmacother.

018, 104, 485−495.

14) Galluzzi, L.; Bravo-San Pedro, J. M.; Levine, B.; Green, D. R.;

2

(

Kroemer, G. Pharmacological modulation of autophagy: therapeutic

potential and persisting obstacles. Nat. Rev. Drug Discovery 2017, 16,

4

(

87.

15) Rao, S.; Tortola, L.; Perlot, T.; Wirnsberger, G.; Novatchkova,

M.; Nitsch, R.; Sykacek, P.; Frank, L.; Schramek, D.; Komnenovic, V.;

(33) Tang, F.; Hu, P.; Yang, Z.; Xue, C.; Gong, J.; Sun, S.; Shi, L.;

Zhang, S.; Li, Z.; Yang, C.; Zhang, C.; Xie, C. SBI0206965, a novel

inhibitor of Ulk1, suppresses non-small cell lung cancer cell growth by

P

https://dx.doi.org/10.1021/acs.jmedchem.0c00873

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

modulating both autophagy and apoptosis pathways. Oncol. Rep. 2017,

(50) Krishnakumar, R.; Kraus, W. L. The PARP side of the nucleus:

molecular actions, physiological outcomes, and clinical targets. Mol. Cell

2010, 39, 8−24.

3

(

7, 3449−3458.

34) Chaikuad, A.; Koschade, S. E.; Stolz, A.; Zivkovic, K.; Pohl, C.;

Shaid, S.; Ren, H.; Lambert, L. J.; Cosford, N. D. P.; Brandts, C. H.;

Knapp, S. Conservation of structure, function and inhibitor binding in

UNC-51-like kinase 1 and 2 (ULK1/2). Biochem. J. 2019, 476, 875−

(51) Rouleau, M.; Patel, A.; Hendzel, M. J.; Kaufmann, S. H.; Poirier,

G. G. PARP inhibition: PARP1 and beyond. Nat. Rev. Cancer 2010, 10,

2

(

93−301.

52) Drea

therapy. Crit. Rev. Oncol. Hematol. 2016, 108, 73−85.

53) Zai, W.; Chen, W.; Han, Y.; Wu, Z.; Fan, J.; Zhang, X.; Luan, J.;

́

n, A.; Lord, C. J.; Ashworth, A. PARP inhibitor combination

8

(

87.

35) Roskoski, R., Jr. Classification of small molecule protein kinase

(

inhibitors based upon the structures of their drug-enzyme complexes.

Tang, S.; Jin, X.; Fu, X.; Gao, H.; Ju, D.; Liu, H. Targeting PARP and

autophagy evoked synergistic lethality in hepatocellular carcinoma.

Carcinogenesis 2019, 41, 345−357.

Pharmacol. Res. 2016, 103, 26−48.

(36) de Oliveira, P. S. L.; Ferraz, F. A. N.; Pena, D. A.; Pramio, D. T.;

Morais, F. A.; Schechtman, D. Revisiting protein kinase −substrate

interactions: Toward therapeutic development. Sci. Signaling 2016, 9,

re3−re3.

(54) Liu, Y.; Song, H.; Song, H.; Feng, X.; Zhou, C.; Huo, Z. Targeting

autophagy potentiates the anti-tumor effect of PARP inhibitor in

pediatric chronic myeloid leukemia. AMB Express 2019, 9, 108.

(37) Dar, A. C.; Shokat, K. M. The evolution of protein kinase

(55) Di Veroli, G. Y.; Fornari, C.; Wang, D.; Mollard, S.; Bramhall, J.

inhibitors from antagonists to agonists of cellular signaling. Annu. Rev.

Biochem. 2011, 80, 769−795.

L.; Richards, F. M.; Jodrell, D. I. Combenefit: an interactive platform for

the analysis and visualization of drug combinations. Bioinformatics

(38) Lazarus, M. B.; Novotny, C. J.; Shokat, K. M. Structure of the

2

(

016, 32, 2866−2868.

human autophagy initiating kinase ULK1 in complex with potent

56) Papadimitriou, M.; Mountzios, G.; Papadimitriou, C. A. The role

inhibitors. ACS Chem. Biol. 2015, 10, 257−261.

of PARP inhibition in triple-negative breast cancer: unraveling the wide

(

39) Abagyan, R.; Totrov, M.; Kuznetsov, D. ICM a new method for

spectrum of synthetic lethality. Cancer Treat. Rev. 2018, 67, 34−44.

protein modeling and design: applications to docking and structure

(57) Kinsey, C. G.; Camolotto, S. A.; Boespflug, A. M.; Guillen, K. P.;

prediction from the distorted native conformation. J. Comput. Chem.

Foth, M.; Truong, A.; Schuman, S. S.; Shea, J. E.; Seipp, M. T.; Yap, J.

T.; Burrell, L. D.; Lum, D. H.; Whisenant, J. R.; Gilcrease, G. W., III.;

Cavalieri, C. C.; Rehbein, K. M.; Cutler, S. L.; Affolter, K. E.; Welm, A.

L.; Welm, B. E.; Scaife, C. L.; Snyder, E. L.; McMahon, M.; Tomar, G.;

Papke, B.; Hobbs, G. A.; Yan, L.; Hayes, T. K.; Diehl, J. N.; Goode, G.

D.; Chaika, N. V.; Wang, Y.; Zhang, G.-F.; Witkiewicz, A. K.; Knudsen,

E. S.; Petricoin, E. F., III; Singh, P. K.; Macdonald, J. M.; Tran, N. L.;

Lyssiotis, C. A.; Ying, H.; Kimmelman, A. C.; Cox, A. D.; Der, C. J.

Protective autophagy elicited by RAF → MEK → ERK inhibition

suggests a treatment strategy for RAS-driven cancers. Nat. Med. 2019,

1

(

994, 15, 488−506.

40) Zegzouti, H.; Zdanovskaia, M.; Hsiao, K.; Goueli, S. A. ADP-Glo:

A Bioluminescent and homogeneous ADP monitoring assay for kinases.

Assay Drug Dev. Technol. 2009, 7, 560−572.

(41) Robers, M. B.; Dart, M. L.; Woodroofe, C. C.; Zimprich, C. A.;

Kirkland, T. A.; Machleidt, T.; Kupcho, K. R.; Levin, S.; Hartnett, J. R.;

Zimmerman, K.; Zimmerman, K.; Niles, A. L.; Ohana, R. F.; Daniels, D.

L.; Slater, M.; Wood, M. G.; Cong, M.; Cheng, Y.-Q.; Wood, K. V.

Target engagement and drug residence time can be observed in living

cells with BRET. Nat. Commun. 2015, 6, 1−10.

2

(

5, 620.

58) Bryant, K. L.; Stalnecker, C. A.; Zeitouni, D.; Klomp, J. E.; Peng,

(42) Vasta, J. D.; Corona, C. R.; Wilkinson, J.; Zimprich, C. A.;

S.; Tikunov, A. P.; Gunda, V.; Pierobon, M.; Waters, A. M.; George, S.

D.; Tomar, G.; Papke, B.; Hobbs, G. A.; Yan, L.; Hayes, T. K.; Diehl, J.

N.; Goode, G. D.; Chaika, N. V.; Wang, Y.; Zhang, G.-F.; Witkiewicz, A.

K.; Knudsen, E. S.; Petricoin, E. F., III; Singh, P. K.; Macdonald, J. M.;

Tran, N. L.; Lyssiotis, C. A.; Ying, H.; Kimmelman, A. C.; Cox, A. D.;

De, C. J. Combination of ERK and autophagy inhibition as a treatment

approach for pancreatic cancer. Nat. Med. 2019 , 25 , 628 −640.

Hartnett, J. R.; Ingold, M. R.; Zimmerman, K.; Machleidt, T.; Kirkland,

T. A.; Huwiler, K. G.; Ohana, R. F.; Slater, M.; Otto, P.; Cong, M.;

Wells, C. I.; Berger, B.-T.; Hanke, T.; Ding, C. G. K.; Drewry, D. H.;

Huber, K. V. M.; Willson, T. M.; Knapp, S.; Muller, S.; Meisenheimer,

̈

P. L.; Fan, F.; Wood, K. V.; Robers, M. B. Quantitative, wide-spectrum

kinase profiling in live cells for assessing the effect of cellular ATP on

target engagement. Cell Chem. Biol. 2018, 25, 206−214.e11.

(

59) Rebecca, V. W.; Amaravadi, R. K. Emerging strategies to

effectively target autophagy in cancer. Oncogene 2016, 35, 1−11.

60) Kabsch, W. Xds. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,

6, 125−132.

61) Evans, P. R.; Murshudov, G. N. How good are my data and what

is the resolution? Acta Crystallogr., Sect. D: Biol. Crystallogr. 2013, 69,

(43) Robers, M. B.; Friedman-Ohana, R.; Huber, K. V. M.; Kilpatrick,

L.; Vasta, J. D.; Berger, B.-T.; Chaudhry, C.; Hill, S.; Mu

̈

ller, S.; Knapp,

(

6

(

S.; Wood, K. V. Quantifying target occupancy of small molecules within

living cells. Annu. Rev. Biochem. 2020, 89, 557−581.

(44) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential

scanning fluorimetry to detect ligand interactions that promote protein

stability. Nat. Protoc. 2007, 2, 2212.

1

204−1214.

(62) McCoy, A. J. Acknowledging errors: advanced molecular

replacement with Phaser. In Protein Crystallography; Springer: Humana

Press: New York, NY, 2017; pp. 421 −453.

(45) Russell, R. C.; Tian, Y.; Yuan, H.; Park, H. W.; Chang, Y.-Y.; Kim,

J.; Kim, H.; Neufeld, T. P.; Dillin, A.; Guan, K.-L. ULK1 induces

autophagy by phosphorylating Beclin-1 and activating VPS34 lipid

kinase. Nat. Cell Biol. 2013, 15, 741.

(63) Emsley, P. Tools for ligand validation in Coot. Acta Crystallogr.,

Sect. D: Struct. Biol. 2017, 73, 203−210.

(46) Lefort, S.; Joffre, C.; Kieffer, Y.; Givel, A.-M.; Bourachot, B.;

(64) Skubak, P.; Murshudov, G. N.; Pannu, N. S. Direct incorporation

́

Zago, G.; Bieche, I.; Dubois, T.; Meseure, D.; Vincent-Salomon, A.;

Camonis, J.; Mechta-Grigoriou, F. Inhibition of autophagy as a new

means of improving chemotherapy efficiency in high-LC3B triple-

negative breast cancers. Autophagy 2014, 10, 2122−2142.

of experimental phase information in model refinement. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2196−2201.

(

65) Cunningham, T. J.; Michael, S. Y.; McKeithan, W. L.; Spiering,

S.; Carrette, F.; Huang, C.-T.; Bushway, P. J.; Tierney, M.; Albini, S.;

Giacca, M.; Mano, M.; Puri, P. L.; Sacco, A.; Ruiz-Lozano, P.; Riou, J.-

F.; Umbhauer, M.; Duester, G.; Mercola, M.; Colas, A. R. Id genes are

essential for early heart formation. Genes Dev. 2017, 31, 1325−1338.

(47) Liang, D. H.; Choi, D. S.; Ensor, J. E.; Kaipparettu, B. A.; Bass, B.

L.; Chang, J. C. The autophagy inhibitor chloroquine targets cancer

stem cells in triple negative breast cancer by inducing mitochondrial

damage and impairing DNA break repair. Cancer Lett. 2016, 376, 249−

2

(

58.

48) Gump, J. M.; Thorburn, A. Sorting cells for basal and induced

autophagic flux by quantitative ratiometric flow cytometry. Autophagy

014, 10, 1327 −1334.

49) Bai, P. Biology of poly (ADP-ribose) polymerases: the factotums

of cell maintenance. Mol. Cell 2015, 58, 947−958.

2

(

Q

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Design, Synthesis, and Characterization of an Orally Active Dual-Specific ULK1/2 Autophagy Inhibitor that Synergizes with the PARP Inhibitor Olaparib for the Treatment of Triple-Negative Breast Cancer

DOI: 10.1021/acs.jmedchem.0c00873

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