Identification of a New Heterocyclic Scaffold for Inhibitors of the Polo-Box Domain of Polo-like Kinase 1

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Identiﬁcation of a New Heterocyclic Scaﬀold for Inhibitors of the

Polo-Box Domain of Polo-like Kinase 1

Celeste N. Alverez, Jung-Eun Park, Kiran S. Toti, Yangliu Xia, Kristopher W. Krausz, Ganesha Rai,

Jeong K. Bang, Frank J. Gonzalez, Kenneth A. Jacobson,* and Kyung S. Lee *

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ABSTRACT: As a mitotic-speciﬁc target widely deregulated in various human cancers, polo-like kinase 1 (Plk1) has been

extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in

preclinical and clinical studies, their eﬃcacies are limited by dose-limiting cytotoxicity, mainly from oﬀ-target cross reactivity. The C-

terminal noncatalytic polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein−protein

interaction inhibitors. Here, we identiﬁed a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaﬀold that eﬃciently

inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure−activity relationship studies led to multiple inhibitors having

≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-speciﬁc phosphopeptide, PLHSpT (K_d∼ 450 nM).

In addition, S-methyl prodrugs eﬀectively inhibited mitotic progression and cell proliferation and their metabolic stability was

determined. These data describe a novel class of small-molecule inhibitors that oﬀer a promising avenue for future drug discovery

against Plk1-addicted cancers.

Plk1 contains an N-terminal kinase domain (KD) for ATP-

dependent catalysis and is characterized by the presence of the

C-terminal noncatalytic, but functionally essential, polo-box

domain (PBD).^11,12The PBD plays a key role in mediating

Plk1 functions by targeting its N-terminal catalytic activity to

distinct subcellular structures, such as centrosomes, kineto-

chores, and midbody, through speciﬁc protein−protein

interactions (PPIs).^12,13For more than a decade, extensive

eﬀorts were made to develop Plk1 inhibitors targeting the KD,

resulting in several advanced Plk1 ATP-competitive inhibitors

(Chart 1), such as volasertib/BI6727 1,¹⁴BI2536 2,¹⁵

GSK461364 3,¹⁶NMS-P937 4,¹⁷and TAK-960 5,¹⁸that

INTRODUCTION

■

Members of the polo subfamily of Ser/Thr protein kinases

[collectively, polo-like kinases (Plks)] play a key role in

regulating various aspects of the cell cycle and cell

proliferation.¹Among them, Plk1 is critically required for

proper mitotic progression, whereas other members play

distinct roles during interphase progression and exhibit little

functional overlap with other Plk family members.^1,2In

accordance with its importance in promoting mitosis, Plk1 is

largely upregulated in a broad range of human cancers and its

level of overexpression appears to correlate with aggressiveness

and poor prognosis for a wide spectrum of human cancers.^3,4

Notably, various cancer cells, but not their isogenic normal

cells, are addicted to high Plk1 levels and consequently require

Plk1 overexpression for their viability.⁵⁻⁷As the reversal of

addicted protein functions in cancer cells has proven to be an

attractive strategy to selectively kill cancer cells,⁸⁻¹⁰Plk1 is

considered a discriminating target for anticancer therapy.

Received: September 23, 2020

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Chart 1. Structures of Inhibitors of the KD (1−5) and the PBD (6) of Plk1

have been examined against various cancers.²However, they all

showed limited eﬃcacy with more-than-acceptable dose-

limiting toxicity in various preclinical or clinical trials. As

dose-limiting toxicity arises mainly from nonspeciﬁc activity of

these inhibitors,¹⁹improving the Plk1 speciﬁcity is likely the

major concern that needs to be addressed to achieve better

clinical outcomes. In fact, one of the common problems

associated with the currently available Plk1 ATP-competitive

inhibitors appears to be their low degree of selectivity against

other kinases,^20,21including two closely related Plk2 and Plk3

with potential tumor suppressor roles.^22,23

Unlike the KD inhibitors aimed at annihilating all Plk1

catalytic activity-dependent processes, inhibiting the Plk1 PBD

is expected to be less drastic because it could selectively

mitigate a subset of essential Plk1 functions requiring PBD-

dependent interactions.^7,24In addition, given a high aﬃnity

(low nM K_dvalues) interaction between Plk1 PBD and its

binding target sequence,²⁵the eﬀect of PBD inhibition is

expected to be less severe than that of KD inhibition. As Plk1 is

also required for the viability of normal cells, a moderate level

of inhibition could serve as a fail-safe mechanism that helps

prevent undesirable normal cell killing.

the KD inhibitors.^20,21Intriguingly, the blockade of Plk1 PBD-

dependent PPIs by short peptides or peptide mimetics is

suﬃcient for eﬀectively abrogating the proper mitotic

progression, thus imposing potent mitotic arrest and apoptotic

cell death in cancer cells but less in normal cells.^7,25,26These

ﬁndings suggest that PBD is an attractive target amenable for

in vitro PPI- and in vivo cell-based assays required to carry out

optimization and preclinical studies for potential leads.

Over the years, various Plk1 PBD inhibitors were generated

by either derivatizing the well-characterized minimal phospho-

peptide, PLHSpT 6a,²⁷or isolating small molecules interfering

with Plk1 PBD-dependent PPI.^20,21However, although

peptide-derived inhibitors provide valuable insights into the

binding interface of Plk1 PBD-dependent interactions, whether

they can serve as a lead for developing anti-PBD agents is

disputable, largely because of their poor permeability and

limited bioavailability.²¹In addition, although multiple small

molecule inhibitors targeting the Plk1 PBD were reported,

most exhibit a low level (IC₅₀of 50−1000 μM) of anti-Plk1

activity in cell-based assays,²⁸⁻³⁵while others are not

suﬃciently characterized at the cellular level.³⁶⁻⁴¹

Here, we report the identiﬁcation and exploration of

triazoloquinazolinone-based small-molecule compounds that

exhibit speciﬁc anti-Plk1 PBD activity in both in vitro

biochemical and cell-based assays. These compounds are

anticipated not to be chemically reactive as are many of the

current probes of Plk1 PBD that contain electrophilic

groups.^42,43Their low molecular weight (MW) (generally

At the structural level, Plk1 PBD represents a well-

characterized PPI domain that interacts with its short

phosphopeptide targets with a high aﬃnity.¹¹The unique

nature of PBD, which requires strict structural elements for

binding, holds the promise of generating speciﬁc inhibitors

capable of overcoming nonspeciﬁc toxicities associated with

B

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∼300−400 Da) along with moderate hydrophobicity and

activity of uncharged analogues predict facile availability in

intracellular compartments. Notably, the two heterocyclic

moieties of the inhibitors (i.e., quinazoline and triazole) are

frequently found in various FDA-approved drugs, suggesting

the potential druggability of this inhibitor class. We propose

the triazoloquinazolinone-based compounds described here as

attractive compounds that may lead to the development of a

new class of anti-PBD therapeutics against various Plk1-

addicted cancers.

Table 1. Inhibitory Activity of Triazoloquinazolinones

Modiﬁed in Zones 5 and 6 at the Plk1 PBD

a b

,

RESULTS

■

Screening Small Molecules to Target Plk1 PBD. A

small chemical library of ∼400 drug-like molecules in the

Molecular Recognition Section, National Institute of Diabetes

and Digestive and Kidney Diseases (NIDDK), National

Institutes of Health (NIH), was screened for the ability to

bind to the PBD of Plk1. The principle screening assay

consisted of an ELISA-based Plk1 PBD inhibition assay that

utilizes the speciﬁc interaction between the full-length human

Plk1 and a speciﬁc phospho-Thr (pT)-containing peptide

(Biotin-Ahx-C-ETFDPPLHSpTAI-NH₂) derived from a ki-

netochore-localizing Plk1-binding protein, PBIP1.^27,44When

necessary, secondary ﬂuorescence polarization (FP) assays

were carried out using a 5-carboxyﬂuorescein-labeled peptide

(FITC-Ahx-DPPLHSpTAI-NH₂)²⁶to conﬁrm the anti-Plk1

PBD activity from the primary ELISA-based assay and

eliminate false positives.

a

(IC₅₀values are n = 3 unless noted in parentheses) and microsomal

b

half-life, PAMPA assays, and aqueous solubility. ND, not

determined.

Among four hits identiﬁed was triazoloquinazolinone 7

(Chart 1), which inhibited PBD binding in the ELISA assay

with an IC₅₀of 4.38 μM. When compared to the previously

characterized phosphopeptide, PLHSpT 6a (IC₅₀of 14.74

μM), showing a K_dof ∼450 nM,²⁷the aﬃnity of the lead

compound 7 is anticipated to be at least threefold higher than

that of peptide 6a (Table 1). Compound 7 was previously

reported as a weak hit in a structure-based in silico screen to

identify ligands of the adenosine receptors.⁴⁵From this hit, a

family of congeners was synthesized by substitutions

introduced on all of the moieties of the lead compound 7

(Figure 1). Various substituents were introduced as R¹groups,

and aza analogues of the phenyl ring were also introduced.

Many alkyl, heteroalkyl, and arylalkyl modiﬁcations of the R²

group (phenylethyl in compound 7) were included. Some of

the analogues were obtained from commercial sources and

2-aminobenzoic acid with an isothiocyanate. However, because

of the limited availability of isothiocyanates, the preferred route

for preparation of the triazoloquinazolinones is shown in

Scheme 1. With the core triazoloquinazolinone formed,

additional analogues could be generated with a terminal

amine (e.g., 13, Scheme 2). Amides of this amino congener

(e.g., 14) were selected as a primary focus, and (1-cyano-2-

ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-

carbenium hexaﬂuorophosphate (COMU) was found to be the

best coupling agent and therefore used to synthesize all

additional analogues.

Structure−Activity Relationship. The structure−activity

relationship (SAR) of the 1-thioxo-2,4-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-5(1H)-one scaﬀold as PBD inhibitors was

explored (compounds 15−138, Tables 1−9). All compounds

were evaluated for their eﬃcacy against the full-length human

Plk1 in an ELISA assay (representative data shown in Figure 2)

and for their in vitro physiochemical properties (half-life,

permeability, and solubility). Because of the lack of structural

information for the binding of 7 to the PBD domain of Plk1,

early SAR studies began by exploring the 2,4-dihydro-3H-

1,2,4-triazole-3-thione moiety to determine empirically the

requirements for binding. Despite the prevalence of sulfur

atoms in small-molecule drug discovery, we began by replacing

and modifying the thiourea portion of the molecule. First, we

replaced the sulfur with a range of additional atoms including

oxygen in 15. We found that any replacement of the sulfur

resulted in complete loss of activity, indicating that a free 1-

thioxo group is essential for activity (Table 1). To determine

whether the thiocarbonyl was required, sulfur was methylated

and product 19 tested, but this methyl thioether was also

inactive. In addition, many alkyl groups were tried, and all were

found to be inactive (see Table S1, Supporting Information).

This showed that unmodiﬁed thiourea was required for

activity.

1

their identity conﬁrmed by H NMR and mass spectrometry.

Chemical Synthesis. The synthesis of analogues of 7

generally began with either the corresponding isatoic

anhydride (Scheme 1) or anthranilic acid (Supporting

Information). An isatoic anhydride (e.g., 10) was heated in

the presence of the desired amine to give the corresponding

intermediate 2-aminobenzamide (structure not shown). This

intermediate was heated in the presence of carbon disulﬁde

and potassium hydroxide to give the cyclized 2-thioxo-2,3-

dihydroquinazolin-4(1H)-one derivative (e.g., 11), which was

isolated by precipitation with treatment of water, followed by

ﬁltration. The corresponding 3-thione-1,2,4-triazole (e.g., 12)

was then typically formed in a one-pot procedure by ﬁrst

stirring with hydrazine in reﬂuxing ethanol to form the

hydrazine intermediate. This intermediate, after cooling, was

treated with pyridine and carbon disulﬁde. The desired

triazoloquinazolinone 12 was formed after heating the reaction

again to 80 °C. The 2-thioxo-2,3-dihydroquinazolin-4(1H)-

one intermediate could also be formed by reacting the desired

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Figure 1. Delineation of zones of SAR focus in the triazoloquinazolinone series.

Scheme 1. General Synthesis of 7-Fluoro Analogues

Scheme 2. Synthesis of Zone 4 Amides

Table 2. Inhibitory Activity of Early Triazoloquinazolinones

Modiﬁed in Zones 3 and 4 at the Plk1 PBD (IC₅₀Values Are

n = 3 Unless Noted in Parentheses) and Microsomal Half-

Life, PAMPA Assays, and Aqueous Solubility

We also explored S-methyl and S-acetyl derivatives

(compounds 139−144, Table 9) as potential prodrugs of the

corresponding active 1-thioxo derivatives. By decreasing the

polarity of the molecules, we expected to achieve better

intracellular levels of the active species, with the expectation

that the masking moiety on the S could be labile prior to

reaching the site of action.

We then explored the SAR of the side chain, R², by

systematically modifying the length and composition of the

linker as well as the end group of the side chain. First, we

simpliﬁed the side chain by removing the terminal phenyl ring

and maintaining the simpler alkyl chain of various lengths

(Table 2, 20−34). All alkyl chain lengths tested, from the

shorter ethyl group through the pentyl group, which maintain a

linear length similar to that of 7, showed improved potency

relative to the aryl-containing 7. This suggests a potential size

restriction in the area of the pocket where the side chain binds.

To further explore this, branching was introduced into the

alkyl side chain with the 3-methylbutane of 25 and the

isopropyl group of 26. However, these bulkier alkyl groups,

relative to their linear counterparts 23 and 20, respectively,

maintained potency in the range of 1−2 μM, suggesting that

the improvement in potency was not strictly due to removal of

the bulkier phenyl group.

Next, we tried to evaluate if incorporation of heteroatoms on

the alkyl group (R²) was tolerated (Table 2, 27−29). The CF₃

group of 27 also had the added beneﬁt of blocking potential

cytochrome P450 (CYP)-mediated metabolism of the side

chain. Though there was no observable change in the

metabolic stability of 27 relative to 21, 27 did maintain a

potency relative to that of 21. Incorporation of oxygen to give

ether side chains also improved the activity of 28 and 29

relative to 7 and maintained the activity relative to the

hydrocarbon chains. We also incorporated amide groups into

a

Racemic. ND, not determined.

the side chain to see if there were additional H-bonding

interactions that could be established with the PBD (Table 2,

30−32). All three amide-containing analogues showed a slight

improvement over 7 (IC₅₀= 2.92−3.90 μM vs IC₅₀= 4.38 μM,

respectively) but reduced activity relative to the alkyl chain-

D

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1.85 μM vs 4.38 μM, respectively). However, when the 4-

ﬂuorobenzyl group was present, it failed to show signiﬁcant

improvement over 7 (IC₅₀= 3.54 μM vs 4.38 μM,

respectively). This indicates that, unless directly connected

to the core, phenyl groups are not well tolerated when a linker

is present.

Table 3. Inhibitory Activity of Zone 5-Modiﬁed

Triazoloquinazolinone Derivatives at the Plk1 PBD (IC₅₀

Values Are n = 3 Unless Noted in Parentheses) and

Microsomal Half-Life, PAMPA Assays, and Aqueous

a

Solubility

Once an initial SAR was analyzed for the 2,4-dihydro-3H-

1,2,4-triazole-3-thione and the side chain, we chose to revisit

the triazole and determined whether alkylation of N-2 could be

tolerated. Four analogues (35−38) included a tertiary amine

with various small alkyl groups (Table 3). All four N-

substituted triazole analogues showed good activity, partic-

ularly the three analogues where R²was the longer propyl

group (IC₅₀= 1.63−1.83 μM). The ketone-containing

analogue 39 lacking a basic nitrogen was inactive (IC₅₀> 50

μM).

We then explored the fused phenyl ring of the core to

determine whether substitution would be tolerated in this

region. First, we began with halogenation at the 7-position

(Table 4, 40−42). As the size of the halogen increased, the

aﬃnity decreased (F, IC₅₀= 12.9 μM; Br, IC₅₀= 14.7 μM; I,

IC₅₀= 30.7 μM). The larger, hydrophobic 7-methyl group

(43) showed a potency similar to that of the 7-F analogue.

However, these analogues were still less potent overall than the

unsubstituted 7. We then wanted to determine if larger

substituents would be tolerated if a heteroatom was introduced

to make additional interactions. The 7-acetamide 44, 7-

dimethylamine 45, and 7-(1-morpholino) 46 were prepared

(Table 4). All three of these analogues with H-bonding

capabilities were approximately twofold more potent than 7

(IC₅₀= 1.54−2.77 μM vs IC₅₀= 4.38 μM, respectively). Next,

we explored the substitution pattern around the ring. To do so,

the mono-ﬂuoro analogues were prepared including 7-F (40),

8-F (41), and 9-F (42). They were shown to increase in

potency as the ﬂuorine moved around the ring from the 7- to

8- to 9-position (IC₅₀= 12.9, 8.29, and 2.58 μM, respectively).

Finally, we explored the replacement of the phenyl ring with

a

ND, not determined

containing analogues (IC₅₀= 2.92−3.90 μM vs IC₅₀= 1.03−

1.97 μM, respectively). This reduction in potency relative to

the alkyl chain-containing analogues could be due to the

branching of the fragment attached at the nitrogen, although

branching appeared to be tolerated for analogues 25 and 26. In

addition, the combination of the branching along with the

carbonyl of the amide resulted in an apparently less favorable

orientation of the side chain and reduced potency. The ﬁnal

side chains tested for these early SARs studied included

shorter-chain aryl groups (Table 2, 33 and 34). For analogue

33, the 2,3-dimethylphenyl group was attached directly to the

core and resulted in an improved activity relative to 7 (IC₅₀

=

Table 4. Inhibitory Activity of Triazoloquinazolinones Modiﬁed in Zones 1 and 2 at the Plk1 PBD (IC₅₀Values Are n = 3

a

Unless Noted in Parentheses) and Microsomal Half-Life, PAMPA Assays, and Aqueous Solubility

cmpd

R¹=

IC₅₀(μM, ELISA)

t_1/2(min, RLM)

PAMPA (1 × 10⁻⁶cm/s)

solub. (μg/mL)

7

H

4.38 0.41 (6)

12.92 1.85

14.74 0.81

30.71 1.31

11.29 0.92

2.19 0.10

2.77 0.33

1.54 0.21

8.29 0.59

2.58 0.12

6.16 0.44

27.78 4.07

7.55 0.44

ND

>30

13.3

>30

22.7

>30

15.8

>30

ND

77.0

162

1025

ND

253

356

1.2

91.0

141

16.8

202

51.8

ND

<1

2.2

<1

3.4

<1

3.4

26.9

17.3

4.9

40

41

42

43

44

45

46

47

48

49

50

51

7-F

7-Br

7-I

7-Me

7-NHAc

7-N(CH₃)₂

7-(1-morpholino)

8-F

9-F

H (6-aza)

H (8-aza)

H (9-aza)

a

ND, not determined.

E

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Table 5. Inhibitory Activity of Triazoloquinazolinones Modiﬁed in Combined Zones 1, 2, 3, and 4 at the Plk1 PBD (IC₅₀

a

Values Are n = 3 Unless Noted in Parentheses) and Microsomal Half-Life, PAMPA Assays, and Aqueous Solubility

a

ND, not determined.

pyridine. The 6-aza (49), 8-aza (50), and 9-aza (51) analogues

were prepared; however, these heteroaryl derivatives were

found to be less potent than 7 (>2−6-fold).

The next stage of our SAR exploration involved combining

features from each of the regions explored so far (Table 5).

First, we incorporated substitutions on the phenyl ring and

modiﬁed the side chain. On the 7-ﬂuoro core, we shortened

(52) and extended (53), the alkyl phenyl side chain. Both the

longer propyl and shorter methyl homologues showed similar

activity that was slightly less potent than that of the parent 7

respectively). The ether-containing analogue 56 was equi-

potent to its most similar 29. The two amide-containing side

chain analogues 57 and 58 with the 7-chloro and 7-methyl

cores, respectively, were about twofold more potent than the

unsubstituted 31 (IC₅₀= 1.49 μM, IC₅₀= 2.01 μM, and IC₅₀

=

3.90 μM, respectively).

After the promising results shown in Table 4 for the 7-

acetamide 44, three additional amides were prepared this time

in the 8-position to determine the tolerability of a larger group

in the 8-position. The 8-isopropyl amide with the propyl chain

(59, IC₅₀= 3.01 μM) was shown to be less potent than the

unsubstituted 21 (IC₅₀= 1.03 μM) but more potent than

either the 8-ﬂuoro (47, IC₅₀= 8.29 μM) or the 8-aza (50, IC₅₀

= 27.8 μM). Compounds 60 and 61 have an 8-cyclopentyl

amide substitution with alkyl side chains. However, when

comparing 60 and 25, both of which have a 3-methylbutane

side chain, the substitution in the 8-position was not beneﬁcial

as 60 was found to be sixfold less potent than 25. We had

already discovered that modifying the thiourea portion of the

with the ethyl linker (propyl, IC₅₀= 6.63 μM; methyl, IC₅₀

=

6.42 μM; ethyl, IC₅₀= 4.38 μM). We then revisited the alkyl

chains with the 7-chloro core (Table 5, 54−57). Compound

54 with the propyl side chain showed a small decrease in

potency from the unsubstituted 21 (IC₅₀= 1.77 μM and IC₅₀

=

1.03 μM, respectively). For compound 55 with the 3,3,3-

triﬂuoropropane side chain, the 7-chloro core resulted in a

nearly twofold loss of potency compared to its unsubstituted

counterpart, 27 (IC₅₀= 2.14 μM and IC₅₀= 1.25 μM,

F

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Table 6. Inhibitory Activity at the Plk1 PBD of Triazoloquinazolinones Modiﬁed in Zones 3 and 4 with a Preferred 7-Fluoro

Substitution, Except for 69 with 9-Fluoro (IC₅₀Values Are n = 3 Unless Noted in Parentheses) and Microsomal Half-Life,

PAMPA Assays, and Aqueous Solubility

a

b

c

d

Racemic. Relative stereochemistry, trans. TFA salt. Free base. ND, not determined.

2,4-dihydro-3H-1,2,4-triazole-3-thione was not tolerated; how-

ever, we wanted to determine whether the triazole ring itself

was required. To test this, we prepared the thiourea, 62, which

maintained the thiocarbonyl as well as the NH of the 2,4-

dihydro-3H-1,2,4-triazole-3-thione. Similar to most other

modiﬁcations of the 2,4-dihydro-3H-1,2,4-triazole-3-thione,

the thiourea of 62 was not tolerated and also resulted in a

highly metabolically labile analogue (IC₅₀> 50 μM, t_1/2RLM =

2.2 min).

Based on the observed results thus far, it became clear that

we could make the most signiﬁcant positive modiﬁcations to

the chemotype in the side chain. Therefore, we made a large

number of analogues on the 7-ﬂuoro core and varied the side

chain to expand our SAR. We began with linear alkyl groups,

which we had previously found to lead to low micromolar

aﬃnity. The shorter ethyl group (63) led to a small reduction

in aﬃnity compared to the unsubstituted 20 (IC₅₀= 1.98 μM

and IC₅₀= 1.49 μM, respectively), while the propyl analogue

(64) was found to be equipotent to the unsubstituted

equivalent (21). Previously the 3,3,3-triﬂuoropropyl group

led to one of the most potent analogues. Therefore, we

combined this group with the 7-F and found that the 7-F

analogue 65 was at least as potent as the corresponding

unsubstituted 27. We then chose to introduce additional H-

bonding groups that might increase interactions within the

binding site. To this end, hydroxy (66), amino (67−76), and

phosphorus (77, 78) derivatives were synthesized and tested.

The 1-propanol group, which is similar in length to the butyl

analogue (23), maintained good potency at 1.44 μM. For the

primary amines, both ethyl and propyl chains were tested, and

the three-carbon linker was found to be twice as potent as the

two-carbon linker. Also, as the 9-ﬂuoro substitution was

previously shown to be the most potent of the mono-ﬂuoro

substituents, the 9-F analogue with a propylamine chain (69)

was prepared but found to be less potent than the 7-F

derivative 68. This is unexpected as it is opposite to the trend

seen for the phenethyl series. When relatively simple dimethyl

amine analogues 70 and 71 were prepared, 70 was shown to be

G

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Table 7. continued

Table 7. Inhibitory Activity of Amide-Containing

Triazoloquinazolinones Modiﬁed in Zones 3 and 4 with

Preferred 7-Fluorine at the Plk1 PBD (IC₅₀Values Are n = 3

Unless Noted in Parentheses) and Microsomal Half-Life,

PAMPA Assays, and Aqueous Solubility

a

b

Relative stereochemistry, trans. Racemic. ND, not determined.

Table 8. Inhibitory Activity of 4 Phenylacetic Acid Amide

Triazoloquinazolinone Derivatives Modiﬁed in Zones 3 and

4 with Preferred 7-Fluorine at the Plk1 PBD (IC₅₀Values

Are n = 3 Unless Noted in Parentheses) and Microsomal

a

Half-Life, PAMPA Assays, and Aqueous Solubility

a

ND, not determined.

slightly more potent than the equivalent primary amine 67,

while the branched 1-dimethylaminoprop-2-yl analogue (71)

was slightly less potent. In the case of amide (72−74) and

sulfonamide (75 and 76) derivatives, regardless of the linker

length or the type of derivative, all analogues essentially

performed similarly (IC₅₀= ∼2.0 μM) with the benzamide 74

showing the most potent aﬃnity of the ﬁve. The phosphorus-

containing analogues, phosphonic acid 77 and phosphonate

diester 78, both showed signiﬁcantly weaker aﬃnity, with the

phosphonic acid being essentially inactive. In addition to these

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position is the most favorable for aﬃnity. However, in the case

of the pyridines, all three N positions were well tolerated. This

could be due to the chloro group extending into the pocket,

creating steric clashes, which were absent for the pyridines.

Nonaromatic heterocycles were also tested to determine

whether they would be more beneﬁcial than the aromatic

heterocycles. Azetidine 86, pyrrolidine 87, and piperidine 88

were all prepared to determine if there was a size preference for

the ring, and a clear preference was observed for the six-

membered piperidine (IC₅₀= 5.67 μM, IC₅₀= 5.67 μM, and

IC₅₀= 1.51 μM, respectively). With this information, we then

explored whether additional heteroatoms in the 4-position

would be tolerated. The 4,4-diﬂuoropiperidine 89 and the N-

Table 9. Inhibitory Activity of Prodrug

Triazoloquinazolinones and Their Corresponding Parent

Drugs at the Plk1 PBD (IC₅₀Values Are n = 3 Unless Noted

in Parentheses)

cmpd

R¹=

R⁵=

IC₅₀(μM)

a

21

64

H

7-F

9-F

9-Cl

9-OMe

H

7-F

H

1.03 0.08 (9)

1.16 0.06 (12)

2.06 0.07 (4)

2.49 0.02

2.43 0.01

1.33 0.08

>50

a

morpholino 90 were equipotent to 88, that is, all near IC₅₀

=

a

1.5 μM. However, when a second nitrogen was incorporated

into the ring in analogues 91 and 92, a discrepancy was

observed in the SAR. Based on the chloro-substituted phenyl

rings, both 91 and 92 were expected to be potent; however,

only 92 showed an aﬃnity similar to the other six-membered

ring heterocycles. Because the methyl group is much smaller

than the Boc group, the diﬀerence in aﬃnity is not due to the

size of the group. The more likely source of the diﬀerence is

the basicity of the nitrogen in the 4-position, given that the Boc

group signiﬁcantly decreases the pK_aof the nitrogen relative to

91. Finally, we also prepared the carbocyclic version of 89 to

determine if the heteroatom in the ring was required at all.

Compound 93 maintained the diﬂuoro substitution, which

could potentially generate H-bonding interactions. However,

there were no other heteroatoms with which to form H-bonds

or salt bridges, and as a result, the potency showed a small

(approximately twofold) decrease, indicating that though the

nitrogen is not required, it is beneﬁcial.

Expanding upon the SAR established above, we continued to

explore the side chain region by focusing on the propyl amide

motif, given that this serves as a good handle for further

modiﬁcation to probe the binding pocket (Table 7) and that

compound 74 with the benzamide was one of the more potent

analogues thus far. First, we prepared analogues 94 and 95

with polyethylene glycol (PEG) linkers to extend the primary

amine further into the pocket. While both compounds

maintained single-digit micromolar aﬃnity, the Boc-protected

95 was more potent than the free amine 94 (IC₅₀= 3.22 μM

and IC₅₀= 5.36 μM, respectively), which suggests a preference

for a less basic group and that the size of the terminal group is

unimportant. To determine whether this was related to a

preference for uncharged species or unprotonated species, the

L-Glu derivatives were prepared (96 and 97). While the fully

deprotected 96 was shown to be >twofold less potent than 94,

the t-butyl ester-protected 97 was <twofold less potent. This

does support the conclusion that, in the region where these

highly extended compounds reside, an uncharged species is

139

140

141

142

143

144

145

146

147

a

H

Ac

Me

9-F

9-Cl

9-OMe

a

Shown as the enol tautomers, while this series is shown in the

thiocarbonyl form elsewhere. ND, not determined.

heteroatom-containing analogues, several cyclopropane deriv-

atives were prepared (79 and 80). Both 79 and 80, which diﬀer

in the location of the cyclopropane in the chain, showed good

potency of approximately 1.0 μM, indicating that although

heteroatoms are well tolerated in the side chain, they are not

required.

Next, we explored the phenethyl side chain (Table 6), ﬁrst,

by substituting the phenyl moiety with chloro groups (81 and

82). While the 4-chloro analogue maintained an aﬃnity similar

to that of 7 (82, IC₅₀= 5.71 μM) and was over twofold more

potent than 40, the 2-chloro analogue was less potent than

either 7 or 40 (81, IC₅₀= 14.4 μM). This indicates that ortho

substitution is not well tolerated, which supports the earlier

conclusion that for the phenethyl derivatives the binding

pocket around the phenyl group is restrictive, and substituents

that make it bulkier led to negative interactions. Concluding

that the space around the phenyl ring was sterically limited, we

next chose to replace it with a pyridine to determine if H-

bonding opportunities are available without increasing the

steric bulk. All three pyridine analogues were synthesized, and

all were found to be ≥12-fold more potent than the phenyl-

containing 40. The trend for the potency (4-pyridine ≈ 2-

pyridine > 3-pyridine, best to worst) diﬀers from that observed

for the chloro substitution, which clearly indicated that the 4-

Figure 2. Representative inhibition curves in the ELISA assay. A previously characterized phosphopeptide, PLHSpT 6a (K_dof ∼450 nM²⁷), is

included for comparison. The curve numbers correspond to compound numbers.

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Figure 3. Comparative FP-based assays showing that compound 79 potently inhibits the PBD of Plk1 but not Plk2 and Plk3 with an IC₅₀of 0.47

μM. Under the same experimental conditions, PLHSpT 6a shows an IC₅₀of 22 μM, whereas its control nonphosphorylated form, PLHST 6b,

exhibits no detectable activity. Bars, mean standard deviation (s.d.).

preferred. We then chose to look at increasingly shorter chains

to determine their tolerability in the binding pocket. The PEG₃

chain analogues (98 and 99) showed the same preference for

the less basic, Boc-protected amine that was observed for the

PEG₄chain. However, when the chain was shorted further to

the hexylamine, the primary amine and the corresponding Boc-

protected amine were equipotent, indicating that the

preference for uncharged species begins at greater distances

from the main binding site for the chemotype. The Boc-^D-Glu-

OtBu (102) and Boc-^L-Glu-OtBu (103) derivatives showed a

preference for the natural amino acid derivative (>twofold

more potent than 102). Compound 103 was then deprotected,

giving either the Boc deprotected ^L-Glu-O^tBu 104 or the fully

deprotected 105. These analogues show that a negatively

charged species is not well tolerated (105 IC₅₀= 12.7 μM).

The succinic acid derivative, 106, still exhibited an intolerance

for negatively charged species as well, indicating that the two

charges likely present in 105 are not beneﬁcial either.

Moreover, the sulfonamide 107, which is thought to maintain

many of the same potential interactions without being

negatively charged, was shown to be the most potent of

these extended linear analogues with an IC₅₀= 2.18 μM.

After determining that the shorter chain molecules were

preferred, we focused on smaller, cyclic analogues. Beginning

with carbocyclic groups, the cyclopentyl amide 108 showed a

relatively good potency (IC₅₀= 1.47 μM), but the sterically

much bulkier trans-4-(t-butyl)cyclohexane amide 109 was

inactive. Consistent with the ﬁnding that a highly bulky group

was not tolerated, the 4-Boc-aminocyclohexane amide 110 was

found to be moderately potent (IC₅₀= 2.38 μM). However,

once again, when the Boc group was removed (111), the

potency decreased slightly, suggesting the preference for

uncharged species. A series of piperidine analogues (112−

114) were prepared to further assess this. In agreement with

the data above, all three piperidines, despite the location of the

nitrogen in the ring, were signiﬁcantly less active and nearly

inactive (IC₅₀= 27.7−44.0 μM). Notably, 4-tetrahydropyran

115 was found to show good potency (IC₅₀= 1.79 μM),

indicating that the protonation of the amine, which likely

occurs at physiological pH, is the feature not tolerated rather

than the presence of a heteroatom in the ring. Three additional

analogues with a methylene linker between the amide carbonyl

and the heterocycle were also prepared (116−118). All three

analogues contained a basic nitrogen in the ring and all three

showed moderate aﬃnity, reinforcing the preference for

neutral compounds in this side chain region. Finally, a series

of aromatic analogues of diﬀering lengths were prepared

(119−121). All three analogues, despite the presence of

heteroatoms or the overall length of the side chain, showed

good aﬃnity (IC₅₀= 1.23−1.78 μM).

Given that compound 120 showed the best aﬃnity of these

amide analogues, we decided to explore substitution around

the phenylacetamide portion (Table 8). Beginning with simple

halogen-containing analogues, compounds 122−125 were

prepared. Though not all positions were tested for each

halogen, a general trend did emerge. When comparing 122 and

123, the position of the halogen was found not to have a

signiﬁcant impact on the potency (meta IC₅₀= 1.42 μM and

para IC₅₀= 1.35 μM). Also, the halogen size had no eﬀect on

the aﬃnity of the analogue as seen by all four analogues

showing essentially the same potency, indicating that the size

of the group does not have a large impact on binding. Next, we

looked at substituents that would modulate the electronic

character of the ring (126−134). For electron-donating

groups, methyl (126), hydroxy (127), and methoxy (128−

130) analogues were prepared. Overall, electron-donating

groups were well tolerated, maintaining a similar aﬃnity

relative to 120. Similar to the trend observed for the halogens,

the location of these groups did not have a large impact on the

potency of the compound either. The electron-withdrawing

groups, triﬂuoromethyl (131−132) and cyano (133−134),

showed similar results. They were also well tolerated in all

positions, but the 3-CF₃derivative (132) was the least potent

but only slightly (<twofold) less potent than all other

substituted phenylacetamide analogues. The Boc-protected 4-

amino analogue was also twofold less potent than the parent

120. Finally, in the case of a series of pyridine analogues

prepared (136−138), all three analogues were less potent than

the phenyl analogue 120 and 2-pyridine was the weakest at

IC₅₀= 5.34 μM.

Plk1 PBD Speciﬁcity. To determine whether the above

modiﬁed compounds show Plk1 PBD-binding speciﬁcity, we

carried out FP-based inhibition assays using ﬂuorescein

isothiocyanate (FITC)-labeled peptides that speciﬁcally bind

to each of the PBDs from Plk1, Plk2, and Plk3.²⁶Under these

experimental settings, PLHSpT 6, but not its respective

nonphosphorylated peptide, speciﬁcally inhibited Plk1 PBD

with an IC₅₀of 22 μM (Figure 3). Under the same conditions,

representative, potent compound 79 inhibited Plk1 PBD with

an IC₅₀of 0.47 μM and failed to exhibit any detectable level of

inhibition against the PBDs from Plk2 and Plk3. An

approximately 40-fold increased potency for compound 79

over PLHSpT 6 is largely in good agreement with the data

obtained with ELISA-based assays described above. Similar to

this observation, expanded FP assays showed that several

additional compounds tested showed approximately the same

degree of Plk1 PBD speciﬁcity (Figure S3, Supporting

Information). The all-or-none selectivity for Plk1 PBD is

signiﬁcant, given that the well-characterized Plk1 KD inhibitor

BI2536 exhibits Plk1 selectivity of only around fourfold.⁴⁹

These data collectively suggest that, while the exact binding

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Figure 4. Antiproliferative eﬀect of 143 in HeLa cells. (A) Asynchronously growing HeLa cells were treated with 100 μM of compound 143 for 12

h, subjected to phase-contrast micrography (cells with a rounded morphology are marked with barbed arrows) and stained with DAPI. Cells

exhibiting mitotic and apoptotic DNA morphologies (arrows in DAPI) (bottom left) and abnormally misaligned or lagging chromosomes (bottom

right) were quantiﬁed from three independent experiments (n > 515 cells/sample/experiment). Bars, mean s.d., ***, P < 0.001 (unpaired two-

tailed t-test). (B) Cell numbers at the indicated time points were quantiﬁed from three independent experiments (n = an average of 2547 cells/

experiment for DMSO and 1673 cells/experiment for 143). Bars, mean

s.d. (C,D) Cells in (A) were immunostained with the indicated

antibodies to reveal delocalized Plk1 signals from centrosomes (marker: Cep63) and kinetochores (marker: CREST) (C) and abnormal spindle

morphologies (marker: α-tubulin) (D). Asterisks, centrosome-localized Plk1; brackets, kinetochore-associated Plk1. Arrowheads in (C), delocalized

Plk1 aggregates in the cytosol. Quantiﬁcation was carried out from three independent experiments [≥20 (DMSO) or 32 (143) centrosomes/

sample/experiment and ≥42 (DMSO) or 64 (143) kinetochores (∼4 kinetochores per cell)/sample/experiment for (C); ≥106 (DMSO) or 102

(143) cells/sample/experiment for (D)]. Bars, means s.d., ***, P < 0.001 (unpaired two-tailed t-test).

mode of these compounds is yet to be determined, they may at

least partially block the canonical S-pT/pS-P/X-binding region

of Plk1 PBD described previously.^11,27

Prodrug and Cellular Eﬃcacy. Having conﬁrmed that

many of the compounds generated through SAR exhibited

submicromolar IC₅₀values with a superb speciﬁcity for Plk1

PBD, we then examined their activities in whole-cell studies.

However, likely due to limited intracellular bioavailability

arising from low membrane permeability, we failed to observe

mitotic arrest, the phenotype that can be expected by

interfering with the function of Plk1 PBD.^25,26We reasoned

that alkylating the thiocarbonyl could improve the cellular

uptake of the compounds. In addition, it would directly block

oxidative metabolism of the thiourea as well as prevent

potential oxidation of the phenyl ring via steric factors,

consequently reducing clearance and allowing for greater

exposure of the drug at a site of action. Therefore, we selected

a small alkyl group or small alkyl ester as a starting point as

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they should be relatively readily cleaved within the body and

could allow us to gauge the level of stability necessary for the

prodrug to be beneﬁcial. Accordingly, compounds 142−147

were prepared as thioester or S-methyl prodrugs and examined

in the ELISA assay (Table 9) and in metabolic studies (see

below). The corresponding parent drugs were unsubstituted

21 for 142 and 143, 7-F analogue 64 for 144, 9-F analogue

139 for 145, 9-Cl analogue 140 for 146, and 9-OMe analogue

141 for 147. Not surprisingly, these prodrugs, except the S-acyl

group-containing 142, were inactive with IC₅₀> 50 μM (Table

9). Compound 142 appeared to rapidly become active by

releasing the active parental compound 21 because of the

reactivity of the S-acyl group toward electron-rich moieties

(see below and Figure S4, Supporting Information).

In HeLa cells, compounds 143, 144, and 145 were

compared for their ability to induce mitotic arrest and an

antiproliferation eﬀect, which are characteristics of Plk1 PBD

inhibition.^25,27Results showed that treatment of cells with 100

μM of either compound 143 or 145 eﬀectively induced

rounded cells with apoptotic or aberrant chromosome

morphologies (Figure 4A,B), indicative of a sustained mitotic

arrest. Consequently, both compounds inhibited cell prolifer-

ation, decreasing the total cell population nearly by 50% 3 days

after treatment. Considering that blocking the function of Plk1

PBD-dependent PPI is destined to be less drastic than that of

Plk1 KD^20,21and that several purported inhibitors reported to

date show a low level (IC₅₀of 50−1000 μM) of cellular

activities,²⁸⁻³⁵the activity of 143 or 145 is a modest but clear

improvement. Treatment with compound 144 also induced

mitotic arrest but at a somewhat reduced level, whereas control

DMSO failed to induce any detectable phenotype under the

same conditions. As expected, if the arrest was caused by

interfering with the PBD-dependent Plk1 function, compound

143 signiﬁcantly inhibited Plk1 localization to centrosomes

(approximately 20% inhibition) and kinetochores (approx-

imately 50% inhibition), frequently yielding cytosolic aggre-

gates of delocalized Plk1 as observed previously⁴⁴(Figure 4C).

A weaker blocking of Plk1 localization to the centrosome is

likely due to the presence of PBD-independent Plk1-binding

targets at this structure. Consistent with impaired Plk1

localization, spindle bipolarity was signiﬁcantly compromised

in 143-treated cells (Figure 4D). Under the same conditions,

the parent drugs, that is, 21, 64, and 139, lacked a detectable

level of cellular response. These ﬁndings suggest that, although

the prodrugs themselves failed to inhibit Plk1 PBD (Table 9)

because of the absence of a free 1-thioxy group, the S-methyl

thioethers had a more pronounced response, presumably by

promoting cell membrane permeability while regenerating the

parent drug intracellularly.

incubation with CYP using mouse liver microsomes (MLMs).

By inspection of the triazoloquinazolinone structures, they

display a potential for CYP-mediated hydroxylation on the

main quinazolinone aromatic ring as a preferred target. To our

surprise, not only were the expected hydroxylated metabolites

undetectable, but no other metabolites were produced in the

CYP incubation system for most of the tested compounds. To

further understand the metabolic pathways, similar MLM

incubations of the selected compound set were conducted with

uridine 5′-diphospho-glucuronic acid (UDPGA) as a cofactor

to allow glucuronidation reactions. As shown in Figure S1, a

new peak with 176 Da increase of the corresponding parent,

which is a characteristic gain for a glucuronide, appeared for

each of the tested compounds. It should be noted that for

some of these compounds, two peaks with the MWs

corresponding to one and two glucuronide additions were

apparent, but for the remainder, only one peak appeared with

the fragmentation pattern and MW representing a glucuronide.

To characterize the glucuronidated site, the MS/MS analysis

was performed to generate a fragment pattern for each of the

glucuronides. Upon entering the source, however, the parent

ions of the glucuronides fragmented into the daughter ions,

which are generated from the parent ion, rather than giving a

characteristic fragment to indicate the glucuronidation site

(Figure S2). Examining the structure of these compounds, the

thiocarbonyl S and the triazole ring secondary amine are two

sites that could potentially be attacked by uridine 5′-

diphospho-glucuronosyltransferase (UGT). Thus, it is highly

likely that one of the two glucuronides was generated from the

S-glucuronidation and the other from N-glucuronidation.

Remarkably, a considerable amount of parent ion remained

for the earlier-eluted glucuronides, while the parent ions of the

later-eluted glucuronides almost completely fragmented. This

is indicative of an N-glucuronide for the former and an S-

glucuronide for the latter, given that the C−N bond linking the

parent and the glucuronic acid in the former is much more

stable than the corresponding C−S bond in the latter. In

addition, only the S-glucuronide could be generated for the

amide- or pyridine-containing compounds as the parent ions of

their glucuronides were also completely fragmented. Thus, it is

reasonable to hypothesize that the hydrogen-bonding capa-

bilities of the amide- or pyridine-containing compounds may

account for their altered glucuronidation proﬁle where they

interacted with UGT in a pattern from which N-glucuronida-

tion was not favored. These results demonstrated that

noncirculating metabolites other than the circulating products

are expected to appear in vivo when these triazoloquinazoli-

nones are administrated to mice.

Stability of Selected Compounds and Their Prodrugs

in Mice. Despite the in vitro data suggesting that most

analogues were stable in RLMs up to 30 min (Tables 1−8),

further assays were required to fully understand the stability in

both in vitro and in vivo systems. Therefore, for selected

compounds, additional microsome-based stability assays were

performed to determine the major metabolites and class of

enzymes responsible for the modiﬁcations. Several molecules

including early hit-compounds (23, 46) and compounds with

submicromolar aﬃnity (i.e., 65, 79, 83, and 134) were

subjected to in vitro MLM assay. LC−MS/MS analysis

revealed that glucuronidation is the major metabolite. Note

that, in vitro glucuronidation resulted in two products, S- and

N-glucuronides in varying amounts, which resulted in two

glucuronidation peaks in LC-traces for some compounds

Analysis of Pharmacokinetics. To obtain metabolic

stability information, the half-life of the selected compounds

was determined in rat liver microsomes (RLMs) containing the

NADPH-generating system, which permits CYP-mediated

metabolic reactions.⁴⁶Parallel artiﬁcial membrane permeability

assay (PAMPA) permeability was measured using a high-

throughput protocol, as reported.⁴⁷Aqueous solubility was

determined by a published procedure.⁴⁸Most compounds

tested showed a half-life of >30 min, indicating that they could

be relatively stable in the CYP system. To further elucidate

their metabolic pathway and to prioritize compounds for in

vivo testing, several were selected as representative compounds

from each group with diﬀerent substitution patterns to

characterize the metabolites that may be generated during

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Figure 5. Summary of SAR studies on a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaﬀold for inhibition of Plk1 PBD.

(Figures S1 and S2, Supporting Information). To mitigate this

issue, the S-acetyl thioester prodrug 142 was made and found

to be active but also toxic and rapidly cleaved by liver

microsomes. The instability and toxicity of this compound was

expected, given the reactivity of the S-acyl group toward

electron-rich moieties. To conﬁrm this hypothesis, a model

reaction on 142 with ammonium acetate at pH 7.4 was

performed, which resulted in eﬃcient acyl transfer releasing the

parent drug 21 (Figure S4, Supporting Information). There-

fore, to enhance the stability of prodrugs, our attention was

turned to S-methyl thioether analogues (143−145), which

exhibited anti-Plk1 PBD activity in HeLa cells (Figure 4). Our

analyses with two of these analogues (143 and 144) revealed

that although they were stable in human, mouse, and rat

cytosol with t_1/2values up to 120 min, unlike their parental 21

and 64, they exhibited much shorter t_1/2values when reacted

with the microsomes from the corresponding three species

(Table S2). These ﬁndings suggest that the membrane-bound

enzymes in the microsomes are mainly responsible for the

metabolism of these prodrugs rather than the soluble cytosolic

enzymes.

The CYP-mediated reactions are the most likely metabolic

pathways these prodrugs would undergo given the nature of

the NADPH-containing microsome-based incubation system.

To further identify the metabolites generated in the microsome

reaction, 143 was incubated with MLMs for 1 h, and the

resulting mixture processed and subjected to LC−MS/MS

analysis (Figure S5A, Supporting Information). As expected,

CYP catalysis generated a hydroxylated product, which was

further shown by MS/MS fragmentography to be hydroxylated

on the main aromatic ring of the quinazolinone (Figure S5B,

Supporting Information). In addition, an S-demethylated

metabolite, also mediated by CYP, was found, for which the

structure was determined by comparing the fragmentation

pattern with that of the parent drug, 21. As demonstrated by

the in vitro results of MLM-treated nonprodrugs (Figures S1

and S2, Supporting Information), a glucuronidation could also

be expected for the demethylated product. To detect a

subsequent glucuronidation, 143 was incubated in a mixed

system containing both NADPH and UDPGA, the cofactor for

UDP-glucuronosyltransferase. After MS analysis, glucuronida-

tion of the demethylated metabolite was evidenced by the

appearance of a new peak having a 176 Da increase compared

to the demethylated product (Figure S5A, top, Supporting

Information).

Next, to achieve a comprehensive understanding of the

metabolic stability of the prodrugs, we performed the in vivo

experiment by injecting 143 in mice [20 mg/kg, intra-

peritoneal (IP) injection] under a protocol approved by the

National Cancer Institute Animal Care and Use Committee.

During the whole period of the experiment, no signs of toxicity

was observed under these conditions. Serum was collected at

15, 30, 60, 120, and 240 min after injection. In close agreement

with in vitro results in Figure S5, both the hydroxylated and

demethylated products were detected in the serum ≥15 min

postinjection (Figure S6, Supporting Information). While both

metabolites decreased time-dependently, the demethylated

product markedly increased in the serum ≥240 min post-

injection (Figure S6, Supporting Information). A trace amount

of the subsequent glucuronide of the demethylated metabolite

was also seen in the serum as it could be promptly excreted

from the liver to the bile, once generated, and can then

undergo enterohepatic circulation through which the demethy-

lated product is reabsorbed to the circulation. This could also

serve as an explanation for the increased demethylated

metabolite at the last time point. Compound 145 yielded

similar metabolites with a somewhat improved t_1/2value of

51.26 min (Table S3 and Figure S6, Supporting Information).

These results illustrated that the prodrugs could release the

corresponding parent drugs enzymatically, and the released

parent drugs levels could be longer lasting because of an

enterohepatic circulation. As the glucuronides are considered

pharmacologically inactive, whether the hydroxylated metab-

olite could also interact with Plk1 to exert an inhibitory eﬀect

requires further investigation.

DISCUSSION

■

Antimitotic drugs, such as taxanes and vinca alkaloids, that are

directed at inhibiting the dynamic function of microtubules

(MTs), have proven to be eﬀective in the treatment of

cancer.⁵⁰However, given the signiﬁcant side eﬀects of these

conventional anti-MT agents, targeting mitosis-speciﬁc and

cancer cell-addicted Plk1 has been considered an attractive

strategy for generating a cancer cell-selective therapeutic

agent.^3,20Indeed, a large body of studies show that several

Plk1 ATP-competitive inhibitors developed over the years

exhibit signiﬁcant activities against hematological malignan-

cies⁵¹⁻⁵⁵and several advanced solid tumors,⁵⁶⁻⁶⁰although

their less-than-acceptable speciﬁcities and dose-limiting tox-

icities have hampered further clinical applications.

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PBD has emerged as an alternative target for developing a

new class of Plk1 inhibitors that can potentially overcome the

hurdles facing ATP-competitive inhibitors. As PBD inhibitors

interfere only with the PBD-dependent Plk1 functions, they are

anticipated to incur mitotic stress suﬃcient to induce cell death

in cancer cells but not in normal cells.⁷Completely disrupting

Plk1 function would be detrimental even for normal cell

proliferation. While a high level of target speciﬁcity is one of

the inherent advantages that PPI inhibitors can achieve, the

primary challenge has been developing small (<500 Da)

molecules that still exhibit high aﬃnity to the shallow and often

nondescript interface of a target PPI.^61,62Because of this

diﬃculty, most of the purported PBD inhibitors reported to

date exhibit a poor (IC₅₀of 50−1000 μM) anti-Plk1 activity in

cell-based assays.²⁸⁻³⁵In addition, many of these compounds

appear to exhibit cross-reacting alkylating activities^42,43that

could impose dose-limiting nonspeciﬁc cytotoxicity.

SAR Summary. The overall conclusions of our SAR studies

are summarized in Figure 5. Our SAR studies identiﬁed six

analogues which showed submicromolar aﬃnity (65, 79, 83,

85, 129, and 134), which is at least an order of magnitude

more potent than the previously characterized Plk1 PBD-

speciﬁc phosphopeptide, PLHSpT 6a (K_d∼450 nM). All the

six compounds appeared to share two distinct features: (1) 7-F

substitution in zone 1 and (2) an unsubstituted triazole (zone

5). These are distinct because in our initial analogues exploring

zone 1, there was no obvious preference for the 7-substitution.

Then, when we combined early zone 1 and zone 3/4

modiﬁcation unsubstituted triazoles, the ﬂuorine was not

obviously the preferred group. The inactivity of thiourea-

containing analogue 62 and preference for an intact triazole

indicated that a π-stacking interaction rather than H-bonding

of the free NH is the important interaction. In addition, ﬁve of

the six analogues contained zone 4 groups that could form H-

bonding interactions, either CF₃(65), pyridine (83, 85), or

amides and an additional H-bond acceptor (HBA) (129, 134).

All ﬁve of these analogues contain HBA in positions that would

all be in the same general region of the binding pocket.

Compounds 129 and 134 have methoxy and cyano groups that

might interact in adjacent regions of the pocket. The only

submicromolar analogue that did not have additional H-

bonding capabilities, that is, the methyl cyclopropane zone 3/4

analogue (79), might still establish stabilizing hydrophobic

interactions.

allow for ﬂexibility in the binding pose of molecules with

various zone 1 substitutions. However, for the best analogues,

the 7-position with a small H-bonding group is preferred;

larger groups are tolerated but only with additional HBA

groups. Zone 2 did not tolerate modiﬁcation; the phenyl ring

was optimal. Zones 3 and 4 proved to be the most generally

amenable to modiﬁcation. When zone 4 was cyclic, the linker

length was best unmodiﬁed; however, when zone 4 was acyclic

(an alkyl chain, a heteroatom, or another functional group),

the entire chain could contain two or three carbons. This linker

could also be sterically restricted with a trans cyclopropane, but

this conclusion may not apply to all analogues diﬀering in zone

4. In zone 4, removal and replacement of the phenyl group

with unsubstituted heteroaryl groups was well tolerated, as was

replacement with amides, especially for phenylacetyl deriva-

tives. However, beyond the amide, hydrophilic or negatively

charged moieties were disfavored. This extreme diﬀerence in

aﬃnity between phosphorus derivatives 77 and 78 and the

other heteroatom-containing analogues could be due to the

much larger size of these groups or the highly negatively

charged phosphonic acid 77 at physiological pH.

All three nitrogen atoms of the triazole played an important

role in binding, and alkylation of the triazole NH but not the S

was accommodated. The tertiary amine substituents in 35−38

likely reside in a region that was formerly not accessed by the

scaﬀold and therefore can establish new interactions, allowing

for the improvement in aﬃnity. The reason for the inactivity of

N-substituted triazole analogue 39 is undetermined tertiary

amines can form salt bridges, which ketones cannot form, and

this could account for the potency diﬀerences. The diﬀerence

could be due to either electrostatic or steric interactions (the

acetophenone group in 39 is both larger and less ﬂexible than

the tertiary amines 35−38). Given that both tertiary amines

and carbonyl oxygens can form H-bonding interactions, H-

bonding may not account for the signiﬁcant aﬃnity diﬀerence.

The results provided here suggest that a 1-thioxo-2,4-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaﬀold

oﬀers promising structural and chemical features that can be

exploited for anti-Plk1 PBD drug discovery. While the triazole

moiety is one of the main components of antifungal drugs

widely used for the prophylaxis and treatment of invasive

fungal disease,^63,64the quinazoline moiety is frequently found

in several FDA-approved EGFR or HER2 inhibitors, such as

geﬁtinib, erlotinib, lapatinib, afatinib, and vandetanib.⁶⁵As

these approved drugs have been extensively tested in humans

for safety, available information from their prior clinical trials

could facilitate further development of the above-described

triazoloquinazolinone-based Plk1 PBD inhibitors. Drugs

containing the conjoined triazoloquinazolinone backbone

have not yet been reported in the drug depository (Drugbank;

https://www.drugbank.ca/), indicating the uniqueness of the

current triazoloquinazolinone leads. Remarkably, unlike

previously reported several Plk1 PBD inhibitors with

unacceptable alkylating activities,^42,43the identiﬁed triazolo-

quinazolinone chemotype does not bear any hallmark

structural features that could be a reactive liability. In addition,

triazoloquinazolinone-based compounds generally remain inert

in multiple inhibitor screenings⁶⁶⁻⁶⁹and speciﬁc modiﬁca-

tions, such as a methyl group at N-3 of the quinazolinone lead,

are required to induce biological eﬃcacy.^70,71These chemical

properties suggest a lower likelihood of oﬀ-target eﬀects when

dosed. In fact, potential oﬀ-target interactions for the initial hit,

7, and three other high-aﬃnity compounds (21, 68, and 127)

Absorption distribution metabolism excretion (ADME) data

were compared for the six most potent analogues (65, 79, 83,

85, 129, and 134). Five of the six analogues had half-lives of

>30 min in RLMs, and the sixth (129) had a fairly good (20.1

min) half-life (Tables 6 and 8). Notably, the PAMPA

permeability at pH 7.4 was favorable for six compounds (65,

83, 129, and 134) with moderate to good permeability (100 to

>200 × 10⁻⁶cm/s). However, only three analogues (65, 79,

and 129) showed moderate to good solubility (10 to >60 μg/

mL). Only compound 129 displayed both good permeability

and solubility (P = 311 × 10⁻⁶cm/s, S >65 μg/mL) (Table 8).

Despite its relatively shorter half-life than other analogues, 129

may represent the best balance of PLK1 PBD aﬃnity and

ADME properties.

Some regions of the scaﬀold tolerated broad structural

modiﬁcation, while a few did not. In zone 1, interdependence

on the groups in zones 3 and 4 deﬁned the preferred

substitution. This suggests that the core (zones 1, 2, and the

quinazolinone) binding region might have been unﬁlled to

N

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performed on an Agilent 1200 LC−MS system (Agilent Technolo-

gies) using a 3-min gradient of 4−100% acetonitrile (containing

0.025% triﬂuoroacetic acid) in water (containing 0.05% triﬂuoroacetic

acid) was used with an 8-min run time at a ﬂow rate of 1 mL/min.

The purity of compounds newly synthesized was demonstrated on an

Agilent 1200 LC−MS system (Agilent Technologies) using a 7-min

linear gradient of 4−100% acetonitrile (containing 0.025% triﬂuoro-

acetic acid) in water (containing 0.05% triﬂuoroacetic acid) followed

by a 4.5 min run time at a ﬂow rate of 1 mL/min and a Phenomenex

Luna C18 column (3 μm, 3 × 75 mm) at 50 °C. The purity of the

purchased compounds was determined using an Agilent ZORBAX

Eclipse XDB C18 column (5 mm, 4.6 × 250 mm) with a linear

gradient of 5−95% acetonitrile in water (containing 10 mM

triethylammonium acetate) for 20 min at a ﬂow rate of 1.0 mL/

were not detected at 10 μM, when they were screened at 45

receptors and channels by the National Institute of Mental

Health Psychoactive Drug Screening Program (https://pdsp.

unc.edu/pdspweb/) using standard radioligand binding and

functional assay methods.⁷²The only hits among these

compounds at 10 μM causing >50% binding inhibition were

for the primary amine 68 at M₃(100%), M₄(73%), and M₅

(84%) muscarinic acetylcholine receptors. Thus, there was no

pharmacological promiscuity observed for these four repre-

sentative analogues.

The low MWs (300−450 Da) of several promising

triazoloquinazolinone-based inhibitors described here oﬀer

considerable room for optimizations. Our extensive SAR

analyses revealed distinct patterns (Figure 3) that could

serve as a roadmap for further development. Notably, the

ELISA-based IC₅₀values for several compounds (65, 79, 83,

85, 129, and 134) reached <1 μM. As the previously

characterized Plk1 PBD-binding PLHSpT 6a (K_dof ∼450

nM)²⁷shows an IC₅₀of 14.74 μM under the same conditions,

these compounds are expected to have at least an order-of-

magnitude-higher aﬃnity than 6a. Moreover, the superb Plk1

PBD speciﬁcity of triazoloquinazolinone-derived inhibitors

(Figure 4) along with the cellular eﬃcacy of multiple prodrugs

(143, 144, and 145) (Figure 5) provides clear proof-of-

principle evidence that intervene in the Plk1 PBD-mediated

PPI is feasible with small drug-like compounds amenable for

potential clinical applications.

1

min. H and ¹³C NMR spectra were recorded on either a Varian 400

(100) MHz spectrometer or a Bruker 400 MHz spectrometer.

Chemical shifts are given in ppm (δ), calibrated to the residual solvent

signals and frequency calibrated internally by solvent for ¹⁹F NMR

(BrukerTopspin/MestReNova 10.0.2 or 14.1.0). High-resolution

mass spectrometry was recorded on either an Agilent 6210 time-of-

ﬂight LCMS system or a Waters Micromass spectrometer equipped

with a standard electrospray ionization (ESI) and modular Lock-

SprayTM interface. The purity of all the tested compounds (including

both newly synthesized and purchased, active compounds) were

demonstrated to be >95% at 254 nm, except commercially procured

compound 58, which was 93.5% pure. The synthesis of compounds

7−64 is described in the Supporting information.

CONCLUSIONS AND PERSPECTIVE

■

In this study, we used a 1-thioxo-2,4-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-5(1H)-one scaﬀold to synthesize S-methyl

prodrugs that eﬀectively inhibit PBD-dependent Plk1 function

in cultured cells. Further development of these inhibitors may

yield a new class of Plk1 PBD inhibitors that could oﬀer superb

speciﬁcity with versatile applicability either as a single agent or

as a part of combination chemotherapy. Combination

therapeutic regimens with various chemotherapeutic agents,

including cisplatin, cytarabine, methotrexate, and doxorubicin,

have already shown an improved antitumor activity with Plk1’s

ATP-competitive inhibitors in numerous studies.^{52,54,73−77}In

addition, as the most advanced ATP-competitive inhibitor 1¹⁴

was doomed in phase III clinical trials because of its less-than-

acceptable tolerability, combination treatment strategy with

this inhibitor may allow to achieve the synergistic anti-Plk1

eﬃcacy without causing the dose-limiting toxicity that

narrowed their therapeutic windows. As a promising avenue

for anti-Plk1 drug discovery, PBD inhibitors may prove to be

eﬀective in overcoming the hurdles facing current anti-Plk1

therapy and improving clinical outcomes.

General Procedure A. To a solution of appropriate 2-aminobenzoic

acid (1 equiv) and isothiocyanate (1.2 equiv) in EtOH (0.37 M) was

added triethylamine (1.2 equiv). The reaction was stirred at 80 °C for

1−2 h. The reaction was cooled to room temperature (RT) and

diluted with water. The solid (intermediate A) was ﬁltered and dried

under vacuum and used without further puriﬁcation.

General Procedure B. A solution of isatoic anhydride (1 equiv) in

acetonitrile (0.67 M) was added to the amine (1.5 equiv). In the case

of amine salts, triethylamine (1.5 equiv) was added to a solution of

the amine in acetonitrile (0.67 M), the salt was removed by ﬁltration,

and the free based amine solution was added to the reaction. The

reaction was heated at 50 °C for 30 min. The reaction was cooled to

RT and carbon disulﬁde (7 equiv) was added cold. The reaction was

then heated to 120 °C for 45 min. The reaction was diluted with

Et₂O, ﬁltered, rinsed with Et₂O, and dried. The solid (intermediate A)

was dried under vacuum and used without further puriﬁcation.

EXPERIMENTAL SECTION

■

Chemical Synthesis. General Methods for Chemistry. All

reactions were carried out under the nitrogen atmosphere using

anhydrous solvents. All moisture-sensitive reactions were also

performed with oven-dried glassware. Chemical reagents and

anhydrous solvents were obtained from commercial sources and

used as-is. Room temperature or rt refers to 25 2 °C. Preparative

puriﬁcation was performed on a Waters semipreparative HPLC

equipment. The column used was a Phenomenex Luna C18 (5 μm, 30

× 75 mm) at a ﬂow rate of 45 mL/min. The mobile phase consisted

of acetonitrile and water (each containing 0.1% triﬂuoroacetic acid).

A gradient of 10−50% acetonitrile over 8 min was used during the

puriﬁcation. Fraction collection was triggered by UV detection (220

nm). Initial analytical analysis during compound synthesis was

General Procedure C. A solution of isatoic anhydride (1 equiv) in

acetonitrile (0.67 M) was added to the amine (1.5 equiv). In the case

of amine salts, triethylamine (1.5 equiv) was added to a solution of

O

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the amine in acetonitrile (0.67 M), the salt was removed by ﬁltration,

and the free based amine solution was added to the reaction. The

reaction was heated at 60 °C for 3 h. The reaction was concentrated

and the residue dissolved in EtOH (0.67 M), and aq KOH (1.2

equiv) was added, followed by carbon disulﬁde (2 equiv). The

reaction was then heated to 120 °C for 2 h. The reaction was cooled

to RT, diluted with water, and washed with Et₂O. The solid

(intermediate A) was dried under vacuum and used without further

puriﬁcation.

After stirring the reaction mixture for 18 h at RT, cold 1 N aq HCl

was added with vigorous stirring. The precipitate was collected by

ﬁltration, washed with cold water and hexanes, and dried to aﬀord a

practically pure product (60 mg, 50%). Route 2: To a solution of 2-

amino-N-phenethylbenzamide (100 mg, 0.416 mmol) in anhydrous

ethanol (2 mL) at RT were added carbon disulﬁde (75 μL, 1.25

mmol) and solid KOH (32 mg, 0.916 mmol) sequentially. The

reaction mixture was stirred at 80 °C for 18 h at RT, cooled, and was

added to a cold 1 N aq HCl with vigorous stirring. The precipitate

was collected by ﬁltration, washed with cold water and hexanes, and

dried to aﬀord a practically pure product (70 mg, 60%). Note: The

product was puriﬁed further by silica-gel column chromatography

using 0−2% methanol in dichloromethane as an eluent. The puriﬁed

product gave better yield in the next step. ¹H NMR (400 MHz,

CDCl₃): δ 9.99 (s, 1H), 8.25−8.10 (m, 1H), 7.69 (ddd, J = 1.5, 7.3,

8.4 Hz, 1H), 7.45−7.40 (m, 2H), 7.38−7.32 (m, 2H), 7.27 (d, J = 5.6

Hz, 1H), 7.13 (dt, J = 0.7, 8.2 Hz, 1H), 4.82−4.65 (m, 2H), 3.19−

3.07 (m, 2H); HRMS: (M + H) for C₁₆H₁₄N₂OS calcd, 283.0905;

found, 283.0909.

General Procedure D. To a solution of intermediate A (1 equiv) in

EtOH (0.25−0.35 M) was added hydrazine (7 equiv). The reaction

was then heated to 80 °C for 4 h. The reaction was then cooled to RT

and pyridine (10 equiv) and carbon disulﬁde (10 equiv) were added.

The reaction was heated to 80 °C for 1−2 h. The reaction was poured

into cold water, and the product was ﬁltered and washed with water

or puriﬁed by HPLC.

2-Hydrazineyl-3-phenethylquinazolin-4(3H)-one: To a suspension

of 3-phenethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (200 mg,

0.708 mmol) in anhydrous ethanol (5 mL) was added anhydrous

hydrazine (0.33 mL, 10.62 mmol) and heated at 85 °C for 18 h. The

volatiles were evaporated under high vacuum, followed by

coevaporation with toluene (2×), giving 2-hydrazineyl-3-phenethyl-

quinazolin-4(3H)-one as a yellow solid and was used as such without

further puriﬁcation (200 mg, quantitative). ¹H NMR (400 MHz,

CDCl₃): δ 8.16 (dd, J = 1.6, 7.9 Hz, 1H), 7.68−7.56 (m, 1H), 7.45−

7.14 (m, 5H), 4.21 (dd, J = 6.7, 8.4 Hz, 2H), 3.09−2.99 (m, 1H);

HRMS: (M + H) for C₁₆H₁₆N₄O calcd, 281.1402; found, 281.1405.

4-Phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one: 2-Hydrazineyl-3-phenethylquinazolin-4(3H)-one (200

mg, 0.713 mmol) was dissolved in anhydrous ethanol (10 mL). To

the solution was added carbon disulﬁde (0.13 mL, 2.14 mmol),

followed by solid KOH (120 mg, 2.14 mmol), and it was heated to 80

°C for 18 h. The reaction mixture was cooled and poured into cold 1

N aq HCl solution. The precipitates were collected and puriﬁed by

silica-gel column chromatography using 0−2% methanol in dichloro-

methane as an eluent to aﬀord a pure product as a white solid (200

General Procedure E. To a solution of intermediate A (1 equiv) in

EtOH (0.35 M) was added water (2.8 equiv), followed by hydrazine

(7 equiv). The reaction was then heated to 80 °C for 4 h. The

reaction was then poured into cold water and concentrated. The

crude material was puriﬁed on a Teledyne ISCO CombiFlash System

by (dry-loading) (EtOAc/DCM: 0−3%) to give intermediate B.

General Procedure F. To a solution of carboxylic acid (1.5 equiv)

in DMF (0.2 M) was added COMU (1.5 equiv) and the reaction

stirred at RT for 30−45 min. Then, the amine (1 equiv) was added,

followed by DIPEA (2.2 equiv). The reaction was then stirred for 18 h

or 2.5 d at RT. The crude material was puriﬁed by HPLC.

4-Phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (7). Note: Compound 7 previously appeared

as an in silico hit in a screen for A_2Aadenosine receptor antagonists

{compound 30 in PMC2865168}.

2-Amino-N-phenethylbenzamide: A mixture of methylanthranilate

(0.856 mL, 6.615 mmol) and phenethylamine (1.3 mL, 10 mmol) was

stirred in a round-bottom ﬂask at 190 °C for 5 h. The product was

puriﬁed by silica-gel column chromatography (0.44 g, 28%). ¹H NMR

(400 MHz, CDCl₃): δ 7.40−7.14 (m, 6H), 6.68 (dd, J = 1.1, 8.1 Hz,

1H), 6.62 (ddd, J = 1.2, 7.2, 8.1 Hz, 1H), 6.06 (s, 1H), 5.49 (s, 2H),

3.70 (td, J = 5.8, 6.8 Hz, 2H), 2.94 (t, J = 6.9 Hz, 2H); HRMS: (M +

H) for C₁₅H₁₆N₂O calcd 241.1341; found, 241.1339.

1

mg, 87%). H NMR (400 MHz, DMSO-d₆): δ 14.10 (s, 1H), 10.23

(dd, J = 1.1, 8.6 Hz, 1H), 8.22 (dd, J = 1.6, 7.9 Hz, 1H), 7.91 (ddd, J

= 1.7, 7.3, 8.7 Hz, 1H), 7.62 (td, J = 1.1, 7.6 Hz, 1H), 7.38−7.15 (m,

4H), 4.32−4.15 (m, 2H), 3.08−2.92 (m, 2H); HRMS: (M + H) for

C₁₇H₁₄N₄OS, calcd 323.0967; found, 323.0966.

4-Phenethyl-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazoline-1,5-

dione (15). 2-Hydrazineyl-3-phenethylquinazolin-4(3H)-one (35 mg,

0.117 mmol) was suspended in anhydrous toluene (2 mL), and to it

was added carbonyl diimidazole (22 mg, 0.135 mmol) and heated to

reﬂux (115 °C) for 1.5 h. The volatiles evaporated under vacuum and

the residue was treated with 1 N aq HCl and 5% isopropanol-

dichloromethane. The organic layer was separated, dried over

Na₂SO₄, evaporated, and the residue was puriﬁed by silica-gel column

chromatography to aﬀord 7b as a white solid (13 mg, 34%). ¹H NMR

(400 MHz, DMSO-d₆): δ 12.07 (s, 1H), 8.65 (dd, J = 4.6, 9.1 Hz,

1H), 7.84 (dd, J = 3.0, 8.6 Hz, 1H), 7.75 (td, J = 3.1, 8.7 Hz, 1H),

7.35−7.19 (m, 4H), 4.26−4.05 (m, 2H), 3.08−2.92 (m, 2H); ¹⁹F

NMR (376 MHz, DMSO-d₆): δ −113.33 (td, J = 4.5, 8.1 Hz);

HRMS: (M + H) for C₁₇H₁₃N₄O₂F calcd, 325.1101; found, 325.1107.

3-Phenethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one: Route 1:

To a solution of 2-amino-N-phenethylbenzamide (100 mg, 0.416

mmol) in anhydrous DMF (2 mL) at RT was added carbon disulﬁde

(72 μL, 1.248 mmol) and DBU (136 μL, 0.916 mmol) sequentially.

P

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1-Amino-7-ﬂuoro-4-phenethyl-[1,2,4]triazolo[4,3-a]quinazolin-

5(4H)-one (16). 6-Fluoro-3-phenethyl-2-thioxo-2,3-dihydroquinazo-

lin-4(1H)-one was synthesized according to General Procedure A. 6-

Fluoro-2-hydrazineyl-3-phenethylquinazolin-4(3H)-one was synthe-

sized according to General Procedure E. Then, to a solution of 6-

ﬂuoro-2-hydrazinyl-3-phenethylquinazolin-4(3H)-one (0.1 g, 0.335

mmol) and cyanogen bromide (0.036 g, 0.335 mmol) in ethanol

(16.8 mL) was added sodium hydroxide (0.067 g, 1.675 mmol) and

the reaction was stirred at RT for 3 h. The reaction was neutralized

with sat. NaHCO₃and the solid ﬁltered. The solid was redissolved

and puriﬁed by HPLC to give 1-amino-7-ﬂuoro-4-phenethyl-[1,2,4]-

Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H); HRMS (ES-API) m/z: calcd for

C₁₁H₁₀N₄NaOS (M + Na), 269.0468; found, 269.0476.

4-Propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one (21). 4-Propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one was purchased from Enamine (Monmouth Jct.,

NJ).

4-Allyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one (22). 4-Allyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one was purchased from Enamine (Monmouth

Jct., NJ).

4-Butyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one (23). 4-Butyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one was purchased from Enamine (Monmouth Jct.,

NJ).

4-Pentyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one (24). 4-Pentyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one was purchased Enamine (Monmouth Jct., NJ).

4-Isopentyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (25). 4-Isopentyl-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one was purchased from Enamine

(Monmouth Jct., NJ).

1

triazolo[4,3-a]quinazolin-5(4H)-one, 2 TFA: H NMR (400 MHz,

DMSO-d₆): δ 8.29 (dd, J = 9.2, 4.2 Hz, 1H), 7.92 (dd, J = 8.5, 3.1 Hz,

1H), 7.82 (td, J = 8.6, 3.1 Hz, 1H), 7.34−7.24 (m, 4H), 7.22 (d, J =

7.0 Hz, 1H), 6.90 (s, 2H), 4.29 (dd, J = 9.5, 6.5 Hz, 2H), 3.06−2.98

(m, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −114.22; HRMS (ES-

API) m/z: calcd for C₁₇H₁₅FN₅O (M + H), 324.1255; found,

324.1264.

7-Fluoro-1-methyl-4-phenethyl-[1,2,4]triazolo[4,3-a]quinazolin-

5(4H)-one (17). 6-Fluoro-3-phenethyl-2-thioxo-2,3-dihydroquinazo-

lin-4(1H)-one was synthesized according to General Procedure A. 6-

Fluoro-2-hydrazineyl-3-phenethylquinazolin-4(3H)-one was synthe-

sized according to General Procedure E. Then, 6-ﬂuoro-2-hydrazinyl-

3-phenethylquinazolin-4(3H)-one (0.1 g, 0.335 mmol) was stirred in

Ac₂O (16.8 mL) at 100 °C for 5 h. The reaction was concentrated,

ﬁltered, and washed with water. The solid was dried and crystallized

from EtOH to give 7-ﬂuoro-1-methyl-4-phenethyl-[1,2,4]triazolo[4,3-

a]quinazolin-5(4H)-one (0.0453 g, 42%): ¹H NMR (400 MHz,

DMSO-d₆): δ 8.11 (dd, J = 9.2, 4.2 Hz, 1H), 7.95 (dd, J = 8.5, 3.1 Hz,

1H), 7.80 (ddd, J = 9.2, 8.0, 3.1 Hz, 1H), 7.29 (td, J = 9.0, 8.4, 6.1 Hz,

4H), 7.21 (td, J = 6.5, 2.2 Hz, 1H), 4.42−4.33 (m, 2H), 3.08−2.99

(m, 2H), 2.84 (s, 3H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −114.29

(td, J = 8.2, 4.0 Hz).

7-Fluoro-4-phenethyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one

(18). 6-Fluoro-3-phenethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-

one was synthesized according to General Procedure A. 6-Fluoro-2-

hydrazineyl-3-phenethylquinazolin-4(3H)-one was synthesized ac-

cording to General Procedure E. Then, 6-ﬂuoro-2-hydrazinyl-3-

phenethylquinazolin-4(3H)-one (0.1 g, 0.335 mmol) was stirred in

formic acid (16.8 mL) at 100 °C for 5 h. The reaction was

concentrated, redissolved, and puriﬁed by HPLC to give 7-ﬂuoro-4-

phenethyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one, TFA: ¹H

NMR (400 MHz, DMSO-d₆): δ 9.50 (s, 1H), 8.27 (dd, J = 8.8, 4.3

Hz, 1H), 7.89 (ddd, J = 11.0, 7.1, 3.9 Hz, 2H), 7.34−7.25 (m, 4H),

7.25−7.17 (m, 1H), 4.37 (dd, J = 9.2, 6.8 Hz, 2H), 3.09−3.01 (m,

2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.78 (td, J = 8.3, 4.1

Hz); HRMS (ES-API) m/z: calcd for C₁₇H₁₄FN₄O (M + H),

309.1146; found, 309.1155.

4-Isopropyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (26). 4-Isopropyl-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one was purchased from ChemDiv

(San Diego, CA).

1-Thioxo-4-(3,3,3-triﬂuoropropyl)-2,4-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-5(1H)-one (27). 2-Thioxo-3-(3,3,3-triﬂuoroprop-

yl)-2,3-dihydroquinazolin-4(1H)-one was synthesized according to

General Procedure B. Then, 1-thioxo-4-(3,3,3-triﬂuoropropyl)-2,4-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, TFA, was syn-

1

thesized according to General Procedure D: H NMR (400 MHz,

DMSO-d₆): δ 10.19 (d, J = 8.5 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H),

7.90 (t, J = 8.1 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 4.25 (t, J = 7.3 Hz,

2H), 2.72 (dt, J = 11.5, 7.4 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-

d₆): δ −64.31 (t, J = 11.3 Hz).

4-(3-Methoxypropyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (28). 4-(3-Methoxypropyl)-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was purchased from

Enamine (Monmouth Jct., NJ).

4-(3-Ethoxypropyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (29). 4-(3-Ethoxypropyl)-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was purchased from

Enamine (Monmouth Jct., NJ).

N-(sec-Butyl)-3-(5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propenamide (30). N-(sec-Butyl)-3-(5-oxo-1-

thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propenamide was purchased from ChemDiv (San Diego, CA).

N-Isopropyl-3-(5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propenamide (31). N-Isopropyl-3-(5-oxo-1-thi-

oxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propenamide was purchased from ChemDiv (San Diego, CA).

N-Isobutyl-3-(5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propenamide (32). N-Isobutyl-3-(5-oxo-1-thio-

xo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propenamide was purchased from ChemDiv (San Diego, CA).

4-(2,3-Dimethylphenyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-5(1H)-one (33). 4-(2,3-Dimethylphenyl)-1-thioxo-2,4-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was purchased

from Enamine (Monmouth Jct., NJ).

4-(4-Fluorobenzyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (34). 4-(4-Fluorobenzyl)-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was purchased from

ChemDiv (San Diego, CA).

2-((Cyclopropyl(methyl)amino)methyl)-4-propyl-1-thioxo-2,4-di-

hydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (35). 2-

((Cyclopropyl(methyl)amino)methyl)-4-propyl-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was purchased from

Enamine (Monmouth Jct., NJ).

1-(Methylthio)-4-phenethyl-[1,2,4]triazolo[4,3-a]quinazolin-

5(4H)-one (19). 3-Phenethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-

one was synthesized according to General Procedure A. Then, 4-

phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one was synthesized according to General Procedure D. Then,

1-(methylthio)-4-phenethyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-

one was synthesized as follows: to a solution of 4-phenethyl-1-thioxo-

2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (0.092 g,

0.223 mmol) in DMF (1.113 mL) was added K₂CO₃(0.037 g,

0.267 mmol) and MeI (0.017 mL, 0.267 mmol). The reaction was

quenched with methanol and concentrated. The reaction was stirred

for 3 h and quenched with methanol. The crude mixture was

concentrated and puriﬁed by HPLC to give 1-(methylthio)-4-

phenethyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one, TFA.

4-Ethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one (20). 3-Ethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one

was synthesized according to General Procedure A. Then, 4-ethyl-1-

thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, TFA,

1

was synthesized according to General Procedure D: H NMR (400

4-Methyl-2-((methyl(propyl)amino)methyl)-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (36). 4-Methyl-2-

((methyl(propyl)amino)methyl)-1-thioxo-2,4-dihydro-[1,2,4]-

MHz, DMSO-d₆): δ 10.20 (d, J = 8.5 Hz, 1H), 8.20 (d, J = 7.8 Hz,

1H), 7.87 (t, J = 8.0 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 4.05 (q, J = 7.1

Q

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Article

triazolo[4,3-a]quinazolin-5(1H)-one was purchased from Enamine

(Monmouth Jct., NJ).

2-((Methyl(propyl)amino)methyl)-4-propyl-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (37). 2-((Methyl-

(propyl)amino)methyl)-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one was purchased from Enamine

(Monmouth Jct., NJ).

2-(((Cyclopropylmethyl) (ethyl)amino)methyl)-4-propyl-1-thio-

xo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (38). 2-

(((Cyclopropylmethyl) (ethyl)amino)methyl)-4-propyl-1-thioxo-2,4-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was purchased

from Enamine (Monmouth Jct., NJ).

hydroquinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 7-bromo-4-phenethyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was synthesized accord-

1

ing to General Procedure D: H NMR (400 MHz, DMSO-d₆): δ

10.17 (d, J = 9.1 Hz, 1H), 8.25 (d, J = 2.4 Hz, 1H), 8.11 (dd, J = 9.0,

2.5 Hz, 1H), 7.34−7.17 (m, 5H), 4.25−4.17 (m, 2H), 3.02−2.94 (m,

2H); HRMS (ES-API) m/z: calcd for C₁₇H₁₄FN₄OS (M + H),

403.0046; found, 403.0046.

7-Iodo-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (42). 6-Iodo-3-phenethyl-2-thioxo-2,3-dihy-

droquinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 7-iodo-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one was synthesized according to

General Procedure D: ¹H NMR (400 MHz, DMSO-d₆): δ 10.00 (d, J

= 8.8 Hz, 1H), 8.41 (d, J = 2.1 Hz, 1H), 8.24 (dd, J = 8.9, 2.2 Hz,

1H), 7.32−7.18 (m, 5H), 4.24−4.16 (m, 2H), 3.02−2.93 (m, 2H).

7-Methyl-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-5(1H)-one (43). 6-Methyl-3-phenethyl-2-thioxo-2,3-di-

hydroquinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 7-methyl-4-phenethyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was synthesized accord-

2-(2-Oxo-2-phenylethyl)-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (39). 2-(2-Oxo-2-phenylethyl)-

4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one was purchased from ChemDiv (San Diego, CA).

7-Fluoro-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-5(1H)-one (40). 2-Amino-5-ﬂuoro-N-phenethylbenza-

mide: To a solution of 2-amino-5-ﬂuorobenzoic acid (250 mg, 1.61

mmol), phenethylamine (0.243 mL, 1.93 mmol), dimethylaminopyr-

idine (100 mg, 0.81 mmol), and triethylamine (0.45 mL, 3.22 mmol)

in anhydrous dichloromethane (5 mL) was added EDC·HCl (370 mg,

1.93 mmol) and stirred at RT for 18 h. Saturated aq NaHCO₃

solution and dichloromethane were added and stirred. The organic

layer was separated, dried over Na₂SO₄, and evaporated under

reduced pressure. The residue obtained was puriﬁed by silica-gel

chromatography to aﬀord the desired product as a white solid (200

1

ing to General Procedure D: H NMR (400 MHz, DMSO-d₆): δ

10.06 (d, J = 8.6 Hz, 1H), 8.02−7.97 (m, 1Hl), 7.71 (dd, J = 8.8, 2.2

Hz, 1H), 7.33−7.16 (m, 5H), 4.26−4.18 (m, 2H), 3.03−2.94 (m,

2H), 2.44 (s, 3H).

N-(5-Oxo-4-phenethyl-1-thioxo-1,2,4,5-tetrahydro-[1,2,4]-

triazolo[4,3-a]quinazolin-7-yl)acetamide (44). N-(4-Oxo-3-phe-

nethyl-2-thioxo-1,2,3,4-tetrahydroquinazolin-6-yl)acetamide was syn-

thesized according to General Procedure A. Then, N-(5-oxo-4-

phenethyl-1-thioxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]-

quinazolin-7-yl)acetamide was synthesized according to General

1

mg, 48%). H NMR (400 MHz, CDCl₃): δ 7.36 (t, J = 7.3 Hz, 2H),

7.27 (q, J = 6.7, 7.6 Hz, 3H), 6.96 (td, J = 2.9, 8.4 Hz, 1H), 6.89 (dd, J

= 2.8, 9.2 Hz, 1H), 6.64 (dd, J = 4.6, 9.0 Hz, 1H), 6.01 (s, 1H), 5.24

(s, 2H), 3.69 (q, J = 6.6 Hz, 2H), 2.94 (t, J = 6.9 Hz, 2H); ¹⁹F NMR

(376 MHz, CDCl₃): δ −111.91; HRMS: (M + H) for C₁₆H₁₅N₂OF

calcd, 259.1247; found, 259.1247.

1

Procedure D: H NMR (400 MHz, DMSO-d₆): δ 10.37 (s, 1H),

10.09 (d, J = 9.1 Hz, 1H), 8.51 (d, J = 2.6 Hz, 1H), 7.96 (dd, J = 9.1,

2.7 Hz, 1H), 7.26 (ddd, J = 19.6, 13.5, 7.3 Hz, 5H), 4.22 (dd, J = 9.4,

6.5 Hz, 2H), 2.99 (t, J = 7.9 Hz, 2H), 2.48 (t, J = 3.0 Hz, 2H), 2.08 (s,

3H); HRMS (ES-API) m/z: calcd for C₁₉H₁₈N₅O₂S (M + H),

380.1176; found, 380.1174.

7-(Dimethylamino)-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (45). 6-(Dimethylamino)-3-

phenethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure A. Then, 7-(dimethylamino)-4-

phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one, TFA, was synthesized according to General Procedure D:

¹H NMR (400 MHz, DMSO-d₆): δ 13.92 (s, 1H), 9.98 (d, J = 9.3 Hz,

6-Fluoro-3-phenethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one:

To a solution of 2-amino-5-ﬂuoro-N-phenethylbenzamide (100 mg,

0.387 mmol) in anhydrous DMF (2 mL) at RT was added carbon

disulﬁde (70 μL, 1.16 mmol) and DBU (127 μL, 0.851 mmol)

sequentially. After stirring the reaction mixture for 18 h at RT, cold 1

N aq HCl was added with vigorous stirring. The precipitate was

collected by ﬁltration, washed with cold water and hexanes, and dried

to give a white solid (110 mg, 95%). ¹H NMR (400 MHz, CDCl₃): δ

9.94 (s, 1H), 7.82 (dd, J = 2.9, 8.1 Hz, 1H), 7.40 (d, J = 7.7 Hz, 3H),

7.35 (t, J = 7.3 Hz, 1H), 7.27 (d, J = 4.9 Hz, 2H), 7.19−7.03 (m, 1H),

4.74 (dd, J = 6.4, 10.2 Hz, 2H), 3.11 (t, J = 8.3 Hz, 2H); ¹⁹F NMR

(376 MHz, CDCl₃): δ −114.59; HRMS: (M + H) for C₁₆H₁₃N₂OSF

calcd, 301.0811; found, 301.0813.

6-Fluoro-2-hydrazineyl-3-phenethylquinazolin-4(3H)-one: Follow-

ing the procedure mentioned for the synthesis of 2-hydrazineyl-3-

phenethylquinazolin-4(3H)-one, 6-ﬂuoro-3-phenethyl-2-thioxo-2,3-

dihydroquinazolin-4(1H)-one (110 mg, 0.366 mmol) gave product

6-ﬂuoro-2-hydrazineyl-3-phenethylquinazolin-4(3H)-one as a white

solid (80 mg, 73%). ¹H NMR (400 MHz, CDCl₃): δ 7.81 (dt, J = 1.9,

8.5 Hz, 1H), 7.41−7.20 (m, 8H), 4.20 (t, J = 7.4 Hz, 2H), 3.04 (t, J =

7.4 Hz, 2H); ¹⁹F NMR (376 MHz, CDCl₃): δ −117.99 (td, J = 4.8,

8.3 Hz); HRMS: (M + H) for C₁₆H₁₅N₄OF calcd, 299.1308; found,

299.1307.

1H), 7.33 (d, J = 3.1 Hz, 1H), 7.32−7.16 (m, 6H), 4.26−4.18 (m,

2H), 2.99 (s, 6H), 3.00−2.94 (m, 2H); HRMS (ES-API) m/z: calcd

for C₁₉H₂₀N₅OS (M + H), 366.1383; found, 366.1388; purity

(HPLC) 94.83%.

7-Morpholino-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-5(1H)-one (46). 6-Morpholino-3-phenethyl-2-thio-

xo-2,3-dihydroquinazolin-4(1H)-one was synthesized according to

General Procedure A. Then, 7-morpholino-4-phenethyl-1-thioxo-2,4-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, TFA, was syn-

1

thesized according to General Procedure D: H NMR (400 MHz,

DMSO-d₆): δ 13.97 (s, 1H), 10.02 (d, J = 9.3 Hz, 1H), 7.57 (d, J =

3.0 Hz, 1H), 7.53 (dd, J = 9.3, 3.0 Hz, 1H), 7.31−7.17 (m, 5H),

4.26−4.18 (m, 2H), 3.76 (t, J = 4.8 Hz, 4H), 3.22 (dd, J = 6.1, 3.6 Hz,

4H), 3.02−2.94 (m, 2H); HRMS (ES-API) m/z: calcd for

C₂₁H₂₂N₅O₂S (M + H), 408.1489; found, 408.1490.

7-Fluoro-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one: Following the procedure described for the

synthesis of 4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one, 6-ﬂuoro-2-hydrazineyl-3-phenethylquinazolin-

4(3H)-one (50 mg, 0.168 mmol) gave product 7-ﬂuoro-4-phenethyl-

1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one as a

8-Fluoro-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-5(1H)-one (47). 7-Fluoro-3-phenethyl-2-thioxo-2,3-di-

hydroquinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 8-ﬂuoro-4-phenethyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, TFA, was synthesized

1

white solid (20 mg, 35%). H NMR (400 MHz, DMSO-d₆): δ 14.16

(s, 1H), 10.31 (dd, J = 4.7, 9.3 Hz, 1H), 7.93 (dd, J = 3.1, 8.6 Hz,

1H), 7.83 (td, J = 3.1, 8.6 Hz, 1H), 7.27 (ddt, J = 7.3, 14.2, 19.9 Hz,

4H), 4.34−4.15 (m, 2H), 3.09−2.93 (m, 2H); ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −112.99 (td, J = 4.7, 8.3 Hz); HRMS: (M + H) for

C₁₇H₁₃N₄OSF calcd, 341.0872; found, 341.0871.

1

according to General Procedure D: H NMR (400 MHz, DMSO-

d₆): δ 10.11 (dd, J = 11.4, 2.5 Hz, 1H), 8.27 (dd, J = 8.8, 6.3 Hz, 1H),

7.48 (td, J = 8.4, 2.6 Hz, 1H), 7.26 (ddt, J = 13.0, 11.5, 7.1 Hz, 5H),

4.25−4.16 (m, 2H), 3.02−2.94 (m, 2H); ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −101.36 (dt, J = 11.9, 7.1 Hz); HRMS (ES-API) m/z:

calcd for C₁₇H₁₄FN₄OS (M + H), 341.0867; found, 341.0882.

7-Bromo-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-5(1H)-one (41). 6-Bromo-3-phenethyl-2-thioxo-2,3-di-

R

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9-Fluoro-4-phenethyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-5(1H)-one (48). 8-Fluoro-3-phenethyl-2-thioxo-2,3-di-

hydroquinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 9-ﬂuoro-4-phenethyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, TFA, was synthesized

5(1H)-one, TFA, was synthesized according to General Procedure D:

¹H NMR (400 MHz, DMSO-d₆): δ 10.23 (d, J = 9.1 Hz, 1H), 8.14

(d, J = 2.5 Hz, 1H), 8.00 (dd, J = 9.1, 2.5 Hz, 1H), 4.25 (t, J = 7.3 Hz,

2H), 2.72 (dt, J = 17.7, 9.2 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-

d₆): δ −64.33 (t, J = 11.3 Hz); HRMS (ES-API) m/z: calcd for

C₁₂H₉ClF₃N₄OS (M + H), 349.0132; found, 349.0141; purity

(HPLC) 91.99%.

7-Chloro-4-(3-isopropoxypropyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (56). 7-Chloro-4-(3-isopropox-

ypropyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one was purchased from ChemDiv (San Diego, CA).

3-(7-Chloro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)-N-isopropylpropanamide (57). 3-(7-Chloro-5-

oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

N-isopropylpropanamide one was purchased from ChemDiv (San

Diego, CA).

N-Isopropyl-3-(7-methyl-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propanamide (58). N-Isopropyl-

3-(7-methyl-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propanamide one was purchased from ChemDiv

(San Diego, CA). Purity (HPLC) 93.49%.

1

according to General Procedure D: H NMR (400 MHz, DMSO-

d₆): δ 13.88 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.83−7.73 (m, 1H),

7.62 (td, J = 8.0, 4.0 Hz, 1H), 7.25 (ddd, J = 18.6, 13.0, 7.3 Hz, 5H),

4.16 (dd, J = 9.2, 6.7 Hz, 2H), 3.02−2.94 (m, 2H); ¹⁹F NMR (376

MHz, DMSO-d₆): δ −93.38 (dd, J = 11.6, 4.1 Hz); HRMS (ES-API)

m/z: calcd for C₁₇H₁₄FN₄OS (M + H), 341.0867; found, 341.0859.

4-Phenethyl-1-thioxo-2,4-dihydropyrido[2,3-e][1,2,4]triazolo-

[4,3-a]pyrimidin-5(1H)-one (49). 3-Phenethyl-2-thioxo-2,3-

dihydropyrido[3,2-d]pyrimidin-4(1H)-one was synthesized according

to General Procedure A. Then, 4-phenethyl-1-thioxo-2,4-

dihydropyrido[2,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one,

TFA, was synthesized according to General Procedure D.

4-Phenethyl-1-thioxo-2,4-dihydropyrido[4,3-e][1,2,4]triazolo-

[4,3-a]pyrimidin-5(1H)-one (50). 3-Phenethyl-2-thioxo-2,3-

dihydropyrido[3,4-d]pyrimidin-4(1H)-one was synthesized according

to General Procedure A. Then, 4-phenethyl-1-thioxo-2,4-

dihydropyrido[4,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one,

N-Isopropyl-5-oxo-4-propyl-1-thioxo-1,2,4,5-tetrahydro-[1,2,4]-

triazolo[4,3-a]quinazoline-8-carboxamide (59). N-Isopropyl-5-oxo-

4-propyl-1-thioxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]-

quinazoline-8-carboxamide one was purchased from ChemDiv (San

Diego, CA).

1

TFA, was synthesized according to General Procedure D: H NMR

(400 MHz, DMSO-d₆): δ 11.36 (s, 1H), 8.82 (d, J = 5.0 Hz, 1H),

8.06 (d, J = 5.0 Hz, 1H), 7.33−7.22 (m, 4H), 7.20 (t, J = 7.1 Hz, 1H),

4.24−4.17 (m, 2H), 2.97 (t, J = 8.0 Hz, 2H).

N-Cyclopentyl-4-isopentyl-5-oxo-1-thioxo-1,2,4,5-tetrahydro-

[1,2,4]triazolo[4,3-a]quinazoline-8-carboxamide (60). N-Cyclopen-

tyl-4-isopentyl-5-oxo-1-thioxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-

a]quinazoline-8-carboxamideone was purchased from ChemDiv (San

Diego, CA).

N-Cyclopentyl-4-methyl-5-oxo-1-thioxo-1,2,4,5-tetrahydro-

[1,2,4]triazolo[4,3-a]quinazoline-8-carboxamide (61). N-Cyclopen-

tyl-4-methyl-5-oxo-1-thioxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]-

quinazoline-8-carboxamide one was purchased from ChemDiv (San

Diego, CA).

6-Phenethyl-9-thioxo-8,9-dihydropyrido[3,2-e][1,2,4]triazolo-

[4,3-a]pyrimidin-5(6H)-one (51). 3-Phenethyl-2-thioxo-2,3-

dihydropyrido[2,3-d]pyrimidin-4(1H)-one was synthesized according

to General Procedure A. Then, 6-phenethyl-9-thioxo-8,9-

dihydropyrido[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(6H)-one,

1

TFA, was synthesized according to General Procedure D: H NMR

(400 MHz, DMSO-d₆): δ 13.84 (s, 1H), 8.83 (dd, J = 4.7, 1.9 Hz,

1H), 8.52 (dd, J = 7.8, 1.9 Hz, 1H), 7.63 (dd, J = 7.8, 4.8 Hz, 1H),

7.24 (ddt, J = 20.7, 14.0, 7.3 Hz, 5H), 4.23−4.14 (m, 2H), 2.97 (dd, J

= 9.4, 6.4 Hz, 2H).

6-Fluoro-N-methyl-4-oxo-3-phenethyl-3,4-dihydroquinazoline-

1(2H)-carbothioamide (62). 6-Fluoro-3-phenethyl-2-thioxo-2,3-dihy-

droquinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 6-ﬂuoro-3-phenethyl-2,3-dihydroquinazolin-

4(1H)-one was synthesized as follows: to a solution of 6-ﬂuoro-3-

phenethyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (0.2 g, 0.666

mmol) and nickel(II)chloride (0.604 g, 4.66 mmol) in MeOH (5

mL) was added sodium borohydride (0.529 g, 13.98 mmol) carefully.

The reaction was stirred at RT for 30 min. The reaction was then

ﬁltered through Celite and the solid washed with MeOH. The ﬁltrate

was taken up in EtOAc and washed with water. The organics were

dried over MgSO₄, ﬁltered, and concentrated. The product was

recrystallized from EtOH.

4-Benzyl-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (52). 3-Benzyl-6-ﬂuoro-2-thioxo-2,3-dihydro-

quinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 4-benzyl-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one was synthesized according to

General Procedure D and puriﬁed by pouring into ice water and drops

of acetic acid were added. The solid was ﬁltered and recrystallized

from dioxane to give 4-benzyl-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (0.0226 g, 25%): ¹H NMR (400

MHz, DMSO-d₆): δ 10.30 (dd, J = 9.4, 4.7 Hz, 1H), 7.95 (dd, J = 8.6,

3.1 Hz, 1H), 7.82 (ddd, J = 9.3, 7.9, 3.1 Hz, 1H), 7.43−7.36 (m, 2H),

7.30 (dd, J = 8.1, 6.2 Hz, 2H), 7.29−7.21 (m, 1H), 5.22 (s, 2H); ¹⁹F

NMR (376 MHz, DMSO-d₆): δ −113.06 (td, J = 8.4, 4.9 Hz); HRMS

(ES-API) m/z: calcd for C₁₆H₁₂FN₄OS (M + H), 327.0710; found,

327.0716.

6-Fluoro-N-methyl-4-oxo-3-phenethyl-3,4-dihydroquinazoline-

1(2H)-carbothioamide, TFA, was synthesized as follows: to a solution

of 6-ﬂuoro-3-phenethyl-2,3-dihydroquinazolin-4(1H)-one (0.05 g,

0.185 mmol) in DCM (0.462 mL) was added 1,1′-thiocarbonyldii-

midazole (0.049 g, 0.277 mmol) and the reaction was stirred for 2 h at

RT. Methylamine hydrochloride (0.125 g, 1.850 mmol) and

triethylamine (0.284 mL, 2.035 mmol) were added, and the reaction

was stirred for 24 h. The reaction was concentrated under a stream of

7-Fluoro-4-(3-phenylpropyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (53). 6-Fluoro-3-(3-phenyl-

propyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure A. Then, 7-ﬂuoro-4-(3-phenyl-

propyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

1

one, TFA, was synthesized according to General Procedure D: H

1

N₂and puriﬁed by HPLC: H NMR (400 MHz, DMSO-d₆): δ 8.42

NMR (400 MHz, DMSO-d₆): δ 10.27 (dd, J = 9.3, 4.6 Hz, 1H), 7.90

(dd, J = 8.6, 3.1 Hz, 1H), 7.79 (td, J = 8.6, 3.1 Hz, 1H), 7.25−7.14

(m, 4H), 7.10 (t, J = 7.0 Hz, 1H), 4.07 (t, J = 7.2 Hz, 2H), 2.67 (t, J =

7.7 Hz, 2H), 2.02 (p, J = 7.4 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-

d₆): δ −113.19 (td, J = 8.4, 4.9 Hz).

7-Chloro-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (54). 7-Chloro-4-propyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one one was purchased from

ChemDiv (San Diego, CA).

7-Chloro-1-thioxo-4-(3,3,3-triﬂuoropropyl)-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (55). 6-Chloro-2-thioxo-3-

(3,3,3-triﬂuoropropyl)-2,3-dihydroquinazolin-4(1H)-one was synthe-

sized according to General Procedure B. Then, 7-chloro-1-thioxo-4-

(3,3,3-triﬂuoropropyl)-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

(s, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 6.3 Hz, 2H), 7.19 (dq, J

= 14.0, 7.2 Hz, 4H), 5.43 (s, 2H), 3.66 (t, J = 7.3 Hz, 2H), 2.93 (d, J =

4.2 Hz, 3H), 2.83 (t, J = 7.4 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-

d₆): δ −115.99; HRMS (ES-API) m/z: calcd for C₁₈H₁₈F₃N₃NaOS

(M + Na), 366.1047; found, 366.1052.

4-Ethyl-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (63). 3-Ethyl-6-ﬂuoro-2-thioxo-2,3-dihydroqui-

nazolin-4(1H)-one was synthesized according to General Procedure

A. Then, 4-ethyl-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one was synthesized according to General

1

Procedure D: H NMR (400 MHz, DMSO-d₆): δ 10.28 (dd, J =

9.3, 4.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.79 (ddd, J = 9.3,

8.0, 3.1 Hz, 1H), 4.06 (q, J = 7.1 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H);

S

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.15 (td, J = 8.5, 4.8 Hz);

HRMS (ES-API) m/z: calcd for C₁₁H₁₀FN₄OS (M + H), 265.0554;

found, 265.0557; purity (HPLC) 91.89%.

7-Fluoro-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (64). 7-Fluoro-4-propyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was purchased from

ChemDiv (San Diego, CA).

4-(3-Aminopropyl)-9-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-5(1H)-one (69). tert-Butyl (3-(8-ﬂuoro-4-oxo-2-

thioxo-1,4-dihydroquinazolin-3(2H)-yl)propyl)carbamate was synthe-

sized according to General Procedure A. Then, tert-butyl (3-(9-ﬂuoro-

5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

yl)propyl)carbamate was synthesized according to General Procedure

D. Then, 4-(3-aminopropyl)-9-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one was synthesized as follows: tert-

butyl (3-(9-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)carbamate (0.170 g, 0.432 mmol) was

dissolved in HCl in dioxane (1.512 mL, 6.05 mmol) and the solution

stirred at RT for 5 h. The reaction was quenched with triethylamine

(0.843 mL, 6.05 mmol), ﬁltered, and concentrated. The product was

puriﬁed by ISCO (EtOAc/hexanes, 0−100%) to give 4-(3-amino-

propyl)-9-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one, di-TFA salt: ¹⁹F NMR (376 MHz, DMSO-d₆):

δ −93.31 (dd, J = 11.6, 4.2 Hz).

7-Fluoro-1-thioxo-4-(3,3,3-triﬂuoropropyl)-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (65). 6-Fluoro-2-thioxo-3-

(3,3,3-triﬂuoropropyl)-2,3-dihydroquinazolin-4(1H)-one was synthe-

sized according to General Procedure B. Then, 7-ﬂuoro-1-thioxo-4-

(3,3,3-triﬂuoropropyl)-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one, TFA salt, was synthesized according to General

1

Procedure D: H NMR (400 MHz, DMSO-d₆): δ 10.27 (d, J = 6.5

Hz, 1H), 7.93 (dd, J = 8.6, 3.0 Hz, 1H), 7.82 (td, J = 9.3, 8.7, 3.1 Hz,

1H), 4.26 (t, J = 7.2 Hz, 2H), 2.72 (dtd, J = 18.6, 11.3, 7.7 Hz, 2H);

¹⁹F NMR (376 MHz, DMSO-d₆): δ −64.33 (t, J = 11.3 Hz), −112.81;

4-(2-(Dimethylamino)ethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (70). 3-(2-

(Dimethylamino)ethyl)-6-ﬂuoro-2-thioxo-2,3-dihydroquinazolin-

4(1H)-one was synthesized according to General Procedure B. Then,

4-(2-(dimethylamino)ethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one, di-TFA salt, was synthesized

according to General Procedure D: ¹H NMR (400 MHz, DMSO-d₆):

δ 10.30 (dd, J = 9.3, 4.6 Hz, 1H), 9.10 (s, 1H), 7.95 (dd, J = 8.5, 3.1

Hz, 1H), 7.86 (td, J = 8.5, 3.1 Hz, 1H), 4.40 (t, J = 5.6 Hz, 2H),

3.50−3.43 (m, 2H), 2.85 (s, 6H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−112.62 (q, J = 7.5 Hz); HRMS (ES-API) m/z: calcd for

C₁₃H₁₅FN₅OS (M + H), 308.0976; found, 308.0989.

4-(1-(Dimethylamino)propan-2-yl)-7-ﬂuoro-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (71). 3-(1-

(Dimethylamino)propan-2-yl)-6-ﬂuoro-2-thioxo-2,3-dihydroquinazo-

lin-4(1H)-one was synthesized according to General Procedure B.

Then, 4-(1-(dimethylamino)propan-2-yl)-7-ﬂuoro-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, di-TFA salt, was

synthesized according to General Procedure D: ¹H NMR (400

MHz, DMSO-d₆): δ 10.34 (dd, J = 9.3, 4.6 Hz, 1H), 9.24 (s, 1H),

7.94 (dd, J = 8.6, 3.1 Hz, 1H), 7.85 (td, J = 9.2, 8.7, 3.1 Hz, 1H), 4.05

(t, J = 12.3 Hz, 1H), 3.35 (s, 2H), 2.80 (d, J = 17.7 Hz, 6H), 1.52 (d, J

= 6.8 Hz, 3H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −112.68 (q, J =

7.8 Hz); HRMS (ES-API) m/z: calcd for C₁₄H₁₇FN₅OS (M + H),

322.1132; found, 322.1139.

N-(2-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)ethyl)acetamide (72). N-(2-(6-Fluoro-4-oxo-2-

thioxo-1,4-dihydroquinazolin-3(2H)-yl)ethyl)acetamide was synthe-

sized according to General Procedure B. Then, N-(2-(7-ﬂuoro-5-oxo-

1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

ethyl)acetamide, TFA salt, was synthesized according to General

Procedure D: ¹H NMR (400 MHz, DMSO-d₆): δ 10.30 (dd, J = 9.3,

4.6 Hz, 1H), 7.92 (dd, J = 8.6, 3.0 Hz, 1H), 7.88−7.76 (m, 2H), 4.08

(t, J = 5.8 Hz, 2H), 3.39 (q, J = 6.1 Hz, 2H), 1.64 (s, 3H); ¹⁹F NMR

(376 MHz, DMSO-d₆): δ −113.00 (q, J = 7.6, 6.7 Hz); HRMS (ES-

API) m/z: calcd for C₁₃H₁₃FN₅O₂S (M + H), 322.0769; found,

322.0780.

HRMS (ES-API) m/z: calcd for C₁₂H₈F₄N₄NaOS (M + Na),

355.0247; found, 355.0245.

7-Fluoro-4-(3-hydroxypropyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (66). 6-Fluoro-3-(3-hydroxy-

propyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 7-ﬂuoro-4-(3-hydroxyprop-

yl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one,

1

TFA salt, was synthesized according to General Procedure D: H

NMR (400 MHz, DMSO-d₆): δ 10.28 (dd, J = 9.4, 4.6 Hz, 1H), 7.90

(dd, J = 8.6, 3.1 Hz, 1H), 7.78 (td, J = 8.5, 3.0 Hz, 1H), 4.48 (t, J =

5.1 Hz, 1H), 4.08 (t, J = 7.4 Hz, 2H), 3.47 (q, J = 5.8 Hz, 2H), 1.89−

1.77 (m, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.18 (q, J =

7.4 Hz); HRMS (ES-API) m/z: calcd for C₁₂H₁₂FN₄O₂S (M + H),

294.0660; found, 294.0664.

4-(2-Aminoethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-5(1H)-one (67). tert-Butyl (2-(6-ﬂuoro-4-oxo-2-

thioxo-1,4-dihydroquinazolin-3(2H)-yl)ethyl)carbamate was synthe-

sized according to General Procedure C. Then, tert-butyl (2-(7-ﬂuoro-

5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

yl)ethyl)carbamate was synthesized according to General Procedure

D. Then, 4-(2-aminoethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one, di-TFA salt, was synthesized

by treating tert-butyl (2-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)ethyl)carbamate (0.112 g, 0.295

mmol) in DCM (1.230 mL) and MeOH (0.246 mL) with TFA

(0.989 mL, 12.84 mmol). The reaction was stirred at RT for 3 h, then

blown down, redissolved, and puriﬁed by HPLC: ¹H NMR (400

MHz, DMSO-d₆): δ 10.31 (dd, J = 9.3, 4.6 Hz, 1H), 7.95 (dd, J = 8.5,

3.1 Hz, 1H), 7.85 (ddd, J = 9.2, 7.6, 3.2 Hz, 3H), 4.32 (t, J = 5.8 Hz,

2H), 3.20 (t, J = 6.0 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−112.75 (td, J = 8.3, 4.7 Hz); HRMS (ES-API) m/z: calcd for

C₁₁H₁₀FN₅NaOS (M + Na), 302.0482; found, 302.0493.

4-(3-Aminopropyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-5(1H)-one (68). tert-Butyl (3-(6-ﬂuoro-4-oxo-2-

thioxo-1,4-dihydroquinazolin-3(2H)-yl)propyl)carbamate was synthe-

sized according to General Procedure C. Then, tert-butyl (3-(7-ﬂuoro-

5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

yl)propyl)carbamate was synthesized according to General Procedure

D. Then, 4-(3-aminopropyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one, di-TFA salt, was synthesized

by treating a solution of tert-butyl (3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)carbamate

(0.056 g, 0.142 mmol) in DCM (0.593 mL), MeOH (0.119 mL) was

added to TFA (0.477 mL, 6.19 mmol), and the reaction stirred at RT

for 3 h. The reaction was then concentrated, and the product was

puriﬁed by ISCO (DCM/MeOH +3.3% NH₄OH, 0−100%): ¹H

NMR (400 MHz, DMSO-d₆): δ 10.30 (dd, J = 9.4, 4.7 Hz, 1H), 7.92

(dd, J = 8.5, 3.1 Hz, 1H), 7.83 (ddd, J = 9.3, 7.9, 3.1 Hz, 1H), 7.65 (s,

3H), 4.10 (t, J = 6.8 Hz, 2H), 2.89 (q, J = 8.4, 7.3 Hz, 2H), 2.00 (p, J

= 7.0 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.06 (td, J =

8.3, 4.8 Hz); HRMS (ES-API) m/z: calcd for C₁₂H₁₃FN₅OS (M +

H), 294.0819; found, 294.0825.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)acetamide (73). tert-Butyl (3-(6-ﬂuoro-

4-oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)propyl)carbamate

was synthesized according to General Procedure C.

3-(3-Aminopropyl)-6-ﬂuoro-2-thioxo-2,3-dihydroquinazolin-

4(1H)-one: TFA (1.476 mL, 19.15 mmol) was added to a solution of

crude tert-butyl (3-(6-ﬂuoro-4-oxo-2-thioxo-1,4-dihydroquinazolin-

3(2H)-yl)propyl)carbamate (0.1556 g, 0.440 mmol) in DCM

(2.201 mL), and the reaction was stirred at RT for 2 h. The reaction

was then blown down, the residue dissolved in MeOH, gravity ﬁltered

through a PL-HCO₃SPE cartridge, and concentrated. The crude

product was used without further puriﬁcation.

N-(3-(6-Fluoro-4-oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)-

propyl)acetamide: to a solution of 3-(3-aminopropyl)-6-ﬂuoro-2-

thioxo-2,3-dihydroquinazolin-4(1H)-one (0.05 g, 0.197 mmol) in

pyridine (0.6 mL) was added acetyl chloride (0.021 mL, 0.296 mmol)

T

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Article

and the reaction stirred at 0 °C for 2 h. The reaction was then cooled

to RT and poured into ice water and ﬁltered, and the solid was

washed with water and Et₂O and dried. The crude product was used

without further puriﬁcation.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)acetamide, TFA salt, was synthesized

according to General Procedure D: ¹H NMR (400 MHz, DMSO-d₆):

δ 10.29 (dd, J = 9.4, 4.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H),

7.86−7.75 (m, 2H), 4.02 (dd, J = 8.2, 6.3 Hz, 2H), 3.09 (q, J = 6.6

Hz, 2H), 1.82 (p, J = 7.2 Hz, 2H), 1.76 (s, 3H); ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −113.17 (td, J = 8.2, 4.5 Hz); HRMS (ES-API) m/z:

calcd for C₁₇H₁₈F₃N₄OS (M + H), 336.0925; found, 336.0929; purity

(HPLC) 90.11%.

phosphonate was synthesized according to General Procedure B.

Then, diethyl (2-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-4(5H)-yl)ethyl)phosphonate, TFA salt, was synthe-

sized according to General Procedure D: ¹H NMR (400 MHz,

DMSO-d₆): δ 10.32 (d, J = 7.3 Hz, 1H), 7.92 (dd, J = 8.5, 3.1 Hz,

1H), 7.80 (ddd, J = 9.1, 7.9, 3.1 Hz, 1H), 4.25−4.15 (m, 2H), 4.00

(dqd, J = 8.1, 7.0, 3.5 Hz, 4H), 3.26 (s, 1H), 2.28−2.15 (m, 2H), 1.22

(t, J = 7.0 Hz, 6H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.07; ³¹

P

NMR (162 MHz, DMSO-d₆): δ 26.69; HRMS (ES-API) m/z: calcd

for C₁₅H₁₉FN₄O₄PS (M + H), 401.0843; found, 401.0856.

4-(Cyclopropylmethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (79). 3-(Cyclopropylmethyl)-6-

ﬂuoro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 4-(cyclopropylmethyl)-7-

ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

one, TFA salt, was synthesized according to General Procedure D: ¹H

NMR (400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 7.92

(dd, J = 8.6, 3.0 Hz, 1H), 7.80 (ddd, J = 9.3, 7.9, 3.1 Hz, 1H), 3.92 (d,

J = 7.1 Hz, 2H), 1.27 (s, 1H), 0.49−0.37 (m, 4H); ¹⁹F NMR (376

MHz, DMSO-d₆): δ −113.02 (td, J = 8.3, 4.7 Hz); HRMS (ES-API)

m/z: calcd for C₁₃H₁₂FN₄OS (M + H), 291.0710; found, 291.0720.

7-Fluoro-4-(trans-2-methylcyclopropyl)-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (80). 6-Fluoro-3-(trans-2-

methylcyclopropyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was

synthesized according to General Procedure B. Then, 7-ﬂuoro-4-

(trans-2-methylcyclopropyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-5(1H)-one, TFA salt, was synthesized according to

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)benzamide (74). N-(3-(7-Fluoro-5-oxo-

1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propyl)benzamide, TFA salt, was synthesized according to General

1

Procedure F: H NMR (400 MHz, DMSO-d₆): δ 10.31 (dd, J = 9.3,

4.7 Hz, 1H), 8.46 (t, J = 5.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H),

7.85−7.78 (m, 3H), 7.54−7.49 (m, 1H), 7.48−7.42 (m, 2H), 4.11

(dd, J = 8.1, 6.6 Hz, 2H), 3.36 (q, J = 6.6 Hz, 2H), 1.99 (p, J = 6.9 Hz,

2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.15 (td, J = 8.2, 4.6

Hz); HRMS (ES-API) m/z: calcd for C₁₉H₁₇FN₅O₂S (M + H),

398.1082; found, 398.1063.

N-(2-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)ethyl)methanesulfonamide (75). N-(2-(6-Fluo-

ro-4-oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)ethyl)-

methanesulfonamide was synthesized according to General Procedure

C. Then, N-(2-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-4(5H)-yl)ethyl)methanesulfonamide, TFA salt, was

synthesized according to General Procedure D: ¹H NMR (400 MHz,

DMSO-d₆): δ 10.30 (dd, J = 9.3, 4.7 Hz, 1H), 7.93 (dd, J = 8.6, 3.1

Hz, 1H), 7.81 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 7.17 (t, J = 6.4 Hz, 1H),

4.18 (t, J = 6.2 Hz, 2H), 3.36 (q, J = 6.2 Hz, 2H), 2.88 (s, 3H); ¹⁹F

NMR (376 MHz, DMSO-d₆): δ −112.99 (td, J = 8.3, 4.7 Hz); HRMS

(ES-API) m/z: calcd for C₁₂H₁₃FN₅O₃S₂(M + H), 358.0438; found,

358.0441.

1

General Procedure D: H NMR (400 MHz, DMSO-d₆): δ 13.99 (s,

1H), 10.26 (dd, J = 9.3, 4.6 Hz, 1H), 7.86 (dd, J = 8.8, 3.1 Hz, 1H),

7.76 (ddd, J = 10.7, 8.2, 3.0 Hz, 1H), 2.56 (dt, J = 7.3, 3.5 Hz, 1H),

1.27−1.19 (m, 1H), 1.15 (d, J = 6.0 Hz, 3H), 1.07 (dt, J = 9.6, 4.8 Hz,

1H), 0.90 (q, J = 6.6 Hz, 1H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−113.28 (q, J = 7.3, 6.8 Hz); HRMS (ES-API) m/z: calcd for

C₁₃H₁₂FN₄OS (M + H), 291.0710; found, 291.0717.

4-(2-Chlorophenethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (81). 3-(2-Chlorophenethyl)-6-

ﬂuoro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 4-(2-chlorophenethyl)-7-

ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

one, TFA salt, was synthesized according to General Procedure D: ¹H

NMR (400 MHz, DMSO-d₆): δ 10.28 (s, 1H), 7.88 (dd, J = 8.6, 2.9

Hz, 1H), 7.80 (t, J = 8.7 Hz, 1H), 7.39 (dd, J = 5.6, 3.7 Hz, 1H),

7.36−7.29 (m, 1H), 7.23 (dd, J = 5.9, 3.5 Hz, 2H), 4.27 (t, J = 7.5 Hz,

2H), 3.13 (t, J = 7.3 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−112.98; HRMS (ES-API) m/z: calcd for C₁₇H₁₃ClFN₄OS (M + H),

375.0477; found, 375.0481.

4-(4-Chlorophenethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (82). 3-(4-Chlorophenethyl)-6-

ﬂuoro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 4-(4-chlorophenethyl)-7-

ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

one, TFA salt, was synthesized according to General Procedure D: ¹H

NMR (400 MHz, DMSO-d₆): δ 10.28 (dd, J = 9.4, 4.6 Hz, 1H), 7.89

(dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 7.9, 3.1 Hz, 1H), 7.32 (d,

J = 8.5 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 4.21 (t, J = 7.7 Hz, 2H),

2.98 (t, J = 7.6 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.00

(td, J = 8.4, 5.0 Hz); HRMS (ES-API) m/z: calcd for C₁₇H_13ClFN₄OS

(M + H), 375.0477; found, 375.0480.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)methanesulfonamide (76). N-(3-(7-Flu-

oro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

4(5H)-yl)propyl)methanesulfonamide was synthesized by treating 4-

(3-aminopropyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (0.05 g, 0.170 mmol) in DCM (0.852 mL) at

RT with methanesulfonyl chloride (0.015 mL, 0.188 mmol) and

triethylamine (0.029 mL, 0.205 mmol). The reaction was stirred at

1

RT for 2 h, concentrated, and puriﬁed by HPLC: H NMR (400

MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 7.92 (dd, J = 8.6,

3.1 Hz, 1H), 7.81 (ddd, J = 9.3, 8.0, 3.2 Hz, 1H), 7.00 (t, J = 5.9 Hz,

1H), 4.07 (t, J = 7.1 Hz, 2H), 3.02 (q, J = 6.7 Hz, 2H), 2.86 (s, 3H),

1.89 (p, J = 7.1 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−113.18 (td, J = 8.3, 5.0 Hz); HRMS (ES-API) m/z: calcd for

C₁₃H₁₅FN₅O₃S₂(M + H), 372.0595; found, 372.0601; purity

(HPLC) 94.02%.

(2-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)ethyl)phosphonic Acid (77). (2-(7-Fluoro-5-

oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

ethyl)phosphonic acid was synthesized by treating a solution of

diethyl (2-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)ethyl)phosphonate (0.111 g, 0.277 mmol) in

DCM (4.62 mL) at 0 °C with bromotrimethylsilane (0.216 mL, 1.663

mmol) dropwise (fast). The reaction was then warmed to RT, and the

solution was stirred for 8 h. The reaction was concentrated,

7-Fluoro-4-(2-(pyridin-2-yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (83). 6-Fluoro-3-(2-(pyridin-2-

yl)ethyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 7-ﬂuoro-4-(2-(pyridin-2-

yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one, TFA salt, was synthesized according to General

1

redissolved, and puriﬁed by HPLC: H NMR (400 MHz, DMSO-

d₆): δ 10.27 (dd, J = 9.3, 4.6 Hz, 1H), 7.90 (dd, J = 8.7, 3.1 Hz, 1H),

7.79 (td, J = 8.5, 3.1 Hz, 1H), 4.24−4.14 (m, 2H), 2.06−1.93 (m,

2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.10 (q, J = 7.5 Hz);

³¹P NMR (162 MHz, DMSO-d₆): δ 20.70.

Diethyl (2-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-4(5H)-yl)ethyl)phosphonate (78). Diethyl (2-(6-

ﬂuoro-4-oxo-2-thioxo-1,4-dihydroquinazolin-3(2H)-yl)ethyl)-

1

Procedure D: H NMR (400 MHz, DMSO-d₆): δ 10.29 (dd, J =

9.4, 4.6 Hz, 1H), 8.46 (d, J = 5.0 Hz, 1H), 7.88 (dd, J = 8.6, 3.0 Hz,

1H), 7.80 (td, J = 8.6, 3.1 Hz, 1H), 7.76−7.73 (m, 1H), 7.36 (d, J =

7.7 Hz, 1H), 7.29−7.24 (m, 1H), 4.38 (t, J = 7.6 Hz, 2H), 3.20−3.11

(m, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −112.97 to −113.04

U

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(m); HRMS (ES-API) m/z: calcd for C₁₆H₁₃FN₅OS (M + H),

342.0819; found, 342.0818; purity (HPLC) 94.92%.

Then, 4-(2-(4,4-diﬂuoropiperidin-1-yl)ethyl)-7-ﬂuoro-1-thioxo-2,4-di-

hydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, di-TFA salt, was

synthesized according to General Procedure D: ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −95.47 (d, J = 213.5 Hz), −100.80 (d, J = 228.4 Hz),

−112.68; HRMS (ES-API) m/z: calcd for C₁₆H₁₆F₃N₅NaOS (M +

Na), 406.0920; found, 406.0937.

7-Fluoro-4-(2-(pyridin-3-yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (84). 6-Fluoro-3-(2-(pyridin-3-

yl)ethyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 7-ﬂuoro-4-(2-(pyridin-3-

yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

7-Fluoro-4-(2-morpholinoethyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (90). 6-Fluoro-3-(2-morpholi-

noethyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 7-ﬂuoro-4-(2-morpholi-

noethyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one, di-TFA salt, was synthesized according to General

1

5(1H)-one was synthesized according to General Procedure D: H

NMR (400 MHz, DMSO-d₆): δ 10.30 (dd, J = 9.3, 4.7 Hz, 1H), 8.56

(d, J = 2.2 Hz, 1H), 8.50 (dd, J = 4.9, 1.6 Hz, 1H), 7.92−7.85 (m,

2H), 7.82 (td, J = 8.6, 3.1 Hz, 1H), 7.46 (dd, J = 7.9, 5.0 Hz, 1H),

4.29 (t, J = 7.2 Hz, 2H), 3.08 (t, J = 7.2 Hz, 2H); ¹⁹F NMR (376

MHz, DMSO-d₆): δ −113.00 (td, J = 8.2, 4.6 Hz); HRMS (ES-API)

m/z: calcd for C₁₆H₁₃FN₅OS (M + H), 342.0819; found, 342.0811.

7-Fluoro-4-(2-(pyridin-4-yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (85). 6-Fluoro-3-(2-(pyridin-4-

yl)ethyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthesized

according to General Procedure B. Then, 7-ﬂuoro-4-(2-(pyridin-4-

yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one, TFA salt, was synthesized according to General

1

Procedure D: H NMR (400 MHz, DMSO-d₆): δ 10.31 (dd, J =

9.4, 4.5 Hz, 1H), 9.48 (s, 1H), 7.95 (dd, J = 8.6, 3.1 Hz, 1H), 7.86 (t,

J = 8.9 Hz, 1H), 4.41 (s, 2H), 3.98 (s, 2H), 3.57 (dd, J = 25.5, 15.2

Hz, 2H), 3.35 (s, 4H), 3.18 (s, 2H); ¹⁹F NMR (376 MHz, DMSO-

d₆): δ −112.55; HRMS (ES-API) m/z: calcd for C₁₅H₁₇FN₅O₂S (M +

H), 350.1082; found, 350.1091.

7-Fluoro-4-(2-(4-methylpiperazin-1-yl)ethyl)-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (91). 6-Fluoro-3-(2-

(4-methylpiperazin-1-yl)ethyl)-2-thioxo-2,3-dihydroquinazolin-

4(1H)-one was synthesized according to General Procedure B. Then,

7-ﬂuoro-4-(2-(4-methylpiperazin-1-yl)ethyl)-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, di-TFA salt, was synthe-

sized according to General Procedure D: HRMS (ES-API) m/z: calcd

for C₁₇H₁₈F₃N₄OS (M + H), 363.1398; found, 363.1384.

1

Procedure D: H NMR (400 MHz, DMSO-d₆): δ 10.30 (dd, J =

9.3, 4.7 Hz, 1H), 8.65−8.59 (m, 2H), 7.91−7.77 (m, 2H), 7.66 (d, J =

5.6 Hz, 2H), 4.34 (t, J = 7.1 Hz, 2H), 3.17 (t, J = 7.1 Hz, 2H); ¹⁹F

NMR (376 MHz, DMSO-d₆): δ −112.98 (td, J = 8.2, 4.7 Hz); HRMS

(ES-API) m/z: calcd for C₁₆H₁₃FN₅OS (M + H), 342.0819; found,

342.0825.

tert-Butyl 3-(2-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)ethyl)azetidine-1-carboxylate

(86). tert-Butyl 3-(2-(6-ﬂuoro-4-oxo-2-thioxo-1,4-dihydroquinazolin-

3(2H)-yl)ethyl)azetidine-1-carboxylate was synthesized according to

General Procedure B. Then, tert-butyl 3-(2-(7-ﬂuoro-5-oxo-1-thioxo-

1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)ethyl)-

azetidine-1-carboxylate, TFA salt, was synthesized according to

General Procedure D: ¹H NMR (400 MHz, DMSO-d₆): δ 10.28

(dd, J = 9.3, 4.6 Hz, 1H), 7.90 (dd, J = 8.6, 3.1 Hz, 1H), 7.79 (ddd, J

= 9.3, 7.9, 3.1 Hz, 1H), 3.98 (t, J = 6.8 Hz, 2H), 3.84 (s, 2H), 3.47 (s,

2H), 2.59−2.49 (m, 1H), 1.95 (q, J = 7.1 Hz, 2H), 1.33 (s, 8H); ¹⁹F

NMR (376 MHz, DMSO-d₆): δ −113.14 (q, J = 7.5 Hz); HRMS (ES-

API) m/z: calcd for C₁₉H₂₂FN₅NaO₃S (M + Na), 442.1320; found,

442.1332.

7-Fluoro-4-(2-(1-methylpyrrolidin-2-yl)ethyl)-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (87). 6-Fluoro-3-(2-

(1-methylpyrrolidin-2-yl)ethyl)-2-thioxo-2,3-dihydroquinazolin-

4(1H)-one was synthesized according to General Procedure B. Then,

7-ﬂuoro-4-(2-(1-methylpyrrolidin-2-yl)ethyl)-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, di-TFA salt, was synthe-

sized according to General Procedure D: ¹H NMR (400 MHz,

DMSO-d₆): δ 10.30 (dd, J = 9.3, 4.6 Hz, 1H), 9.43 (s, 1H), 7.92 (dd,

J = 8.5, 3.1 Hz, 1H), 7.83 (td, J = 8.5, 3.1 Hz, 1H), 4.11 (dp, J = 28.3,

7.4 Hz, 2H), 3.51 (d, J = 11.9 Hz, 1H), 3.01 (d, J = 10.1 Hz, 1H),

2.73 (s, 3H), 2.65 (d, J = 10.2 Hz, 1H), 2.43−2.25 (m, 1H), 2.04−

1.79 (m, 3H), 1.72 (dq, J = 16.0, 8.5 Hz, 1H); ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −112.96 (dt, J = 12.5, 6.1 Hz); HRMS (ES-API) m/z:

calcd for C₁₆H₁₈FN₅NaOS (M + Na), 370.1108; found, 370.1121.

7-Fluoro-4-(2-(piperidin-1-yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one (88). 6-Fluoro-3-(2-(piperidin-

1-yl)ethyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one was synthe-

sized according to General Procedure B. Then, 7-ﬂuoro-4-(2-

(piperidin-1-yl)ethyl)-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one, di-TFA salt, was synthesized according to

General Procedure D: ¹H NMR (400 MHz, DMSO-d₆): δ 10.31 (dd,

J = 9.3, 4.6 Hz, 1H), 8.78 (s, 1H), 7.96 (dd, J = 8.5, 3.0 Hz, 1H), 7.86

(td, J = 8.6, 3.0 Hz, 1H), 4.41 (t, J = 6.1 Hz, 2H), 3.61 (s, 2H), 3.44

(s, 2H), 2.96 (s, 3H), 1.89−1.28 (m, 5H); ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −112.56 (q, J = 7.4 Hz); HRMS (ES-API) m/z: calcd

for C₁₆H₁₉FN₅OS (M + H), 348.1289; found, 348.1305.

tert-Butyl 4-(2-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)ethyl)piperazine-1-carboxylate

(92). tert-Butyl 4-(2-(6-ﬂuoro-4-oxo-2-thioxo-1,4-dihydroquinazolin-

3(2H)-yl)ethyl)piperazine-1-carboxylate was synthesized according to

General Procedure B. Then, tert-butyl 4-(2-(7-ﬂuoro-5-oxo-1-thioxo-

1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)ethyl)-

piperazine-1-carboxylate, di-TFA salt, was synthesized according to

General Procedure D: HRMS (ES-API) m/z: calcd for

C₂₀H₂₆FN₆O₃S (M + H), 449.1766; found, 449.1786.

4-(2-(4,4-Diﬂuorocyclohexyl)ethyl)-7-ﬂuoro-1-thioxo-2,4-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (93). 3-(2-(4,4-

Diﬂuorocyclohexyl)ethyl)-6-ﬂuoro-2-thioxo-2,3-dihydroquinazolin-

4(1H)-one was synthesized according to General Procedure B. Then,

4-(2-(4,4-diﬂuorocyclohexyl)ethyl)-7-ﬂuoro-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one, TFA salt, was synthe-

sized according to General Procedure D: ¹H NMR (400 MHz,

DMSO-d₆): δ 10.28 (dd, J = 9.4, 4.6 Hz, 1H), 7.91 (dd, J = 8.7, 3.0

Hz, 1H), 7.84−7.74 (m, 1H), 4.05 (t, J = 7.4 Hz, 2H), 1.96 (d, J =

10.9 Hz, 2H), 1.88−1.58 (m, 6H), 1.46 (s, 1H), 1.16 (q, J = 12.3 Hz,

2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −89.54 (d, J = 231.6 Hz),

−99.44 (d, J = 231.4 Hz), −113.14 (q, J = 7.1, 6.2 Hz); HRMS (ES-

API) m/z: calcd for C₁₇H₁₈F₃N₄OS (M + H), 383.1148; found,

383.1166.

1-Amino-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-3,6,9,12-tetraoxapenta-

decan-15-amide (94). To a solution of 95 (0.025 g, 0.039 mmol) in

DCM (0.195 mL) was added TFA (0.066 mL, 0.858 mmol) and the

reaction stirred at RT overnight. The reaction was then blown down

and redissolved. The crude material was puriﬁed by RP-ISCO (H₂O/

MeCN + 0.2% NH₄OH, 10−100%) to give 1-amino-N-(3-(7-ﬂuoro-

5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

yl)propyl)-3,6,9,12-tetraoxapentadecan-15-amide, di-TFA salt: ¹H

NMR (400 MHz, CD₂Cl₂): δ 13.38 (s, 1H), 10.25 (dd, J = 9.3, 4.6

Hz, 1H), 8.04 (s, 3H), 7.95 (dd, J = 8.3, 3.0 Hz, 1H), 7.45 (ddd, J =

9.4, 7.5, 3.1 Hz, 1H), 7.40 (s, 1H), 5.53 (s, 1H), 4.21 (t, J = 6.7 Hz,

2H), 3.89−3.55 (m, 16H), 3.27 (d, J = 22.8 Hz, 4H), 2.57 (s, 2H),

2.01 (s, 2H); ¹⁹F NMR (376 MHz, CD₂Cl₂): δ −112.54 (td, J = 7.9,

4.4 Hz).

tert-Butyl (19-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)-15-oxo-3,6,9,12-tetraoxa-16-

azanonadecyl)carbamate (95). tert-Butyl (19-(7-ﬂuoro-5-oxo-1-

thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-15-

oxo-3,6,9,12-tetraoxa-16-azanonadecyl)carbamate, TFA salt, was

synthesized according to General Procedure F.

4-(2-(4,4-Diﬂuoropiperidin-1-yl)ethyl)-7-ﬂuoro-1-thioxo-2,4-di-

hydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (89). 3-(2-(4,4-

Diﬂuoropiperidin-1-yl)ethyl)-6-ﬂuoro-2-thioxo-2,3-dihydroquinazo-

lin-4(1H)-one was synthesized according to General Procedure B.

V

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(S)-22-Amino-1-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)-5,21-dioxo-8,11,14,17-tetraoxa-

4,20-diazapentacosan-25-oic Acid (96) and tert-Butyl (S)-22-

Amino-1-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)-5,21-dioxo-8,11,14,17-tetraoxa-4,20-diaza-

pentacosan-25-oate (97). tert-Butyl (S)-22-((tert-butoxycarbonyl)-

amino)-1-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)-5,21-dioxo-8,11,14,17-tetraoxa-4,20-diazapenta-

cosan-25-oate: To a solution of 95 (0.04 g, 0.074 mmol) in DMF

(0.190 mL) was added a solution of (S)-5-(tert-butoxy)-2-((tert-

butoxycarbonyl)amino)-5-oxopentanoic acid (Boc-^L-Glu-γ-t-Bu-ester,

0.067 g, 0.222 mmol) and COMU (0.063 g, 0.148 mmol) followed by

DIPEA (0.013 mL, 0.074 mmol). The reaction was then stirred

overnight at RT. The reaction was quenched with water and

concentrated. The product was puriﬁed by RP-ISCO (H₂O/MeCN +

3.3% NH₄OH, 10−75%) to give the protected amino acid

intermediate (0.0245 g, 40%): MS (ES-API) found (M + H), 826.2

and (M + Na), 848.1.

7.91 (dd, J = 8.5, 3.1 Hz, 1H), 7.80 (ddd, J = 9.1, 7.8, 2.8 Hz, 2H),

6.73 (t, J = 5.6 Hz, 1H), 4.02 (dd, J = 8.3, 6.3 Hz, 2H), 3.10 (q, J =

6.6 Hz, 2H), 2.86 (q, J = 6.6 Hz, 2H), 2.00 (t, J = 7.4 Hz, 2H), 1.82

(p, J = 7.0 Hz, 2H), 1.44 (p, J = 7.4 Hz, 2H), 1.37−1.27 (m, 11H),

1.24−1.11 (m, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.19;

HRMS (ES-API) m/z: calcd for C₂₃H₃₁FN₆NaO₄S (M + Na),

529.2004; found, 529.1994.

tert-Butyl (R)-4-((tert-butoxycarbonyl)amino)-5-((3-(7-ﬂuoro-5-

oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

yl)propyl)amino)-5-oxopentanoate (102). tert-Butyl (R)-4-((tert-

butoxycarbonyl)amino)-5-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-5-oxopen-

tanoate, TFA salt, was synthesized according to General Procedure F:

¹H NMR (400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H),

7.90 (dd, J = 8.6, 3.1 Hz, 1H), 7.85−7.76 (m, 2H), 6.82 (d, J = 8.2

Hz, 1H), 4.03 (t, J = 7.2 Hz, 2H), 3.90−3.80 (m, 1H), 3.12 (dp, J =

20.2, 6.6 Hz, 2H), 2.18 (t, J = 7.7 Hz, 2H), 1.83 (dt, J = 13.7, 6.5 Hz,

3H), 1.66 (dt, J = 14.3, 7.6 Hz, 1H), 1.36 (s, 9H), 1.35 (s, 9H); ¹⁹F

NMR (376 MHz, DMSO-d₆): δ −113.21 (t, J = 6.8 Hz); HRMS (ES-

API) m/z: calcd for C₂₆H₃₅FN₆NaO₆S (M + Na), 601.2215; found,

601.2214.

tert-Butyl (S)-4-((tert-butoxycarbonyl)amino)-5-((3-(7-ﬂuoro-5-

oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

yl)propyl)amino)-5-oxopentanoate (103). tert-Butyl (S)-4-((tert-

butoxycarbonyl)amino)-5-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-5-oxopen-

tanoate, TFA salt, was synthesized according to General Procedure F:

¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.15 (d, J = 11.0 Hz); HRMS

Compounds 96 and 97 were then prepared according to the

following method. TFA (0.022 mL, 0.291 mmol) and triisopropylsi-

lane (5.95 μL, 0.029 mmol) were added to a solution of the protected

amino acid intermediate (0.012 g, 0.015 mmol) in DCM (0.145 mL).

The reaction was stirred at RT for 2 h and concentrated. The mixture

of products was puriﬁed by HPLC to give 96 (2 TFA) and 97 (2

TFA): 96: HRMS (ES-API) m/z: calcd for C₂₈H₄₁FN₇O₉S (M + H),

670.2665; found, 670.2672. 97: ¹H NMR (400 MHz, dmso): δ 10.29

(dd, J = 9.3, 4.7 Hz, 1H), 8.47 (t, J = 5.7 Hz, 1H), 8.07 (s, 3H), 7.91

(dd, J = 8.6, 3.1 Hz, 1H), 7.86 (s, 1H), 7.83−7.76 (m, 1H), 4.02 (t, J

= 7.3 Hz, 2H), 3.72 (s, 1H), 3.56 (t, J = 6.5 Hz, 2H), 3.46−3.39 (m,

9H), 3.26−3.15 (m, 2H), 3.11 (q, J = 6.7 Hz, 2H), 2.27 (t, J = 7.2 Hz,

3H), 1.85 (dt, J = 23.5, 7.2 Hz, 3H), 1.37 (s, 9H); ¹⁹F NMR (376

MHz, dmso): δ −113.13 (dd, J = 8.2, 4.8 Hz); HRMS (ES-API) m/z:

calcd for C₃₂H₄₉FN₇O₉S (M + H), 726.3291; found, 726.3284.

3-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)-N-(3-(7-ﬂuoro-5-oxo-1-

thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propyl)propenamide (98). To a solution of 99 (0.1496 g, 0.251

mmol) in HCl in dioxane (0.627 mL, 2.507 mmol) was added

triisopropylsilane (0.079 g, 0.501 mmol), and the solution was stirred

at RT for 2 h and then concentrated. The product was puriﬁed by

HPLC to give 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(3-(7-ﬂuo-

ro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

4(5H)-yl)propyl)propenamide, di-TFA salt: HRMS (ES-API) m/z:

calcd for C₂₁H₃₀FN₆O₅S (M + H), 497.1977; found, 497.1973.

tert-Butyl (16-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)-12-oxo-3,6,9-trioxa-13-

azahexadecyl)carbamate (99). tert-Butyl (16-(7-ﬂuoro-5-oxo-1-

thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-12-

oxo-3,6,9-trioxa-13-azahexadecyl)carbamate, TFA salt, was synthe-

sized according to General Procedure F: HRMS (ES-API) m/z: calcd

for C₂₆H₃₇FN₆NaO₇S (M + Na), 619.2321; found, 619.2330.

6-Amino-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)hexanamide (100). Trii-

sopropylsilane (0.031 g, 0.193 mmol) was added to a solution of 101

(0.049 g, 0.097 mmol) in HCl in dioxane (0.242 mL, 0.967 mmol),

and the solution was stirred at RT for 2 h. The reaction was

concentrated and puriﬁed by HPLC to give 6-amino-N-(3-(7-ﬂuoro-

5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

(ES-API) m/z: calcd for C₂₆H₃₅FN₆NaO₆S (M + Na), 601.2215;

found, 601.2216.

tert-Butyl (S)-4-Amino-5-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihy-

dro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-5-oxo-

pentanoate (104) and (S)-4-Amino-5-((3-(7-ﬂuoro-5-oxo-1-thioxo-

1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-

amino)-5-oxopentanoic Acid (105). TFA (0.041 mL, 0.532 mmol)

and triisopropylsilane (10.90 μL, 0.053 mmol) were added to a

solution of 103 (0.0154 g, 0.027 mmol) in DCM (0.266 mL). The

reaction was stirred at RT for 2 h and concentrated. The products

were puriﬁed by HPLC to give tert-butyl (S)-4-amino-5-((3-(7-ﬂuoro-

5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-

yl)propyl)amino)-5-oxopentanoate, di-TFA salt, and (S)-4-amino-5-

((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)amino)-5-oxopentanoic acid; 104: ¹H

NMR (400 MHz, DMSO-d₆): δ 10.30 (dd, J = 9.3, 4.8 Hz, 1H),

8.25 (s, 3H), 8.01 (t, J = 5.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H),

7.82 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 4.07−3.99 (m, 2H), 3.92 (t, J =

6.3 Hz, 1H), 3.13 (q, J = 6.7 Hz, 2H), 2.22 (ddt, J = 22.8, 15.3, 7.4

Hz, 2H), 1.95 (q, J = 7.4 Hz, 2H), 1.85 (p, J = 7.1 Hz, 2H), 1.45 (s,

9H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.12 (dt, J = 7.8, 4.1

Hz); HRMS (ES-API) m/z: calcd for C₂₁H₂₈FN₆O₄S (M + H),

479.1871; found, 479.1866. 105: ¹H NMR (400 MHz, DMSO-d₆): δ

10.31 (dd, J = 9.3, 4.7 Hz, 1H), 8.26 (s, 3H), 8.02 (t, J = 5.7 Hz, 1H),

7.92 (dd, J = 8.5, 3.1 Hz, 1H), 7.83 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H),

4.05 (dd, J = 8.1, 6.4 Hz, 2H), 3.93 (t, J = 6.6 Hz, 1H), 3.15 (q, J =

6.7 Hz, 2H), 2.54 (s, 0H), 2.24 (ddt, J = 23.0, 15.4, 7.5 Hz, 2H), 1.97

(q, J = 7.4 Hz, 2H), 1.86 (p, J = 7.2 Hz, 2H), 1.46 (s, 9H); ¹⁹F NMR

(376 MHz, DMSO-d₆): δ −113.10 (td, J = 8.2, 4.8 Hz); HRMS (ES-

API) m/z: calcd for C₁₇H₂₀FN₆O₄S (M + H), 423.1245; found,

423.1232.

1

yl)propyl)hexanamide, di-TFA salt: H NMR (400 MHz, dmso): δ

10.29 (dd, J = 9.3, 4.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.81

(td, J = 7.9, 2.9 Hz, 2H), 7.61 (s, 3H), 4.02 (t, J = 7.4 Hz, 2H), 3.11

(q, J = 6.6 Hz, 2H), 2.75 (h, J = 6.0 Hz, 2H), 2.03 (t, J = 7.3 Hz, 2H),

1.82 (p, J = 7.2 Hz, 2H), 1.55−1.41 (m, 4H), 1.31−1.19 (m, 2H); ¹⁹F

NMR (376 MHz, dmso): δ −113.13 (td, J = 8.3, 4.8 Hz); HRMS

(ES-API) m/z: calcd for C₁₈H₂₃FN₆NaO₂S (M + Na), 430.1506;

found, 430.1512.

4-((3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propyl)amino)-4-oxobutanoic Acid (106). 4-

((3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)amino)-4-oxobutanoic acid, TFA salt,

1

was synthesized according to General Procedure F: H NMR (400

MHz, DMSO-d₆): δ 10.34 (s, 1H), 7.92 (dd, J = 8.6, 3.1 Hz, 1H),

7.86 (t, J = 5.6 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 4.05 (t, J

= 7.3 Hz, 2H), 3.12 (q, J = 6.7 Hz, 2H), 2.45−2.37 (m, 2H), 2.32−

2.24 (m, 3H), 1.84 (p, J = 7.1 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-

d₆): δ −113.28; HRMS (ES-API) m/z: calcd for C₁₆H₁₇FN₅O₄S (M +

H), 394.0980; found, 394.0979.

tert-Butyl (6-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-6-oxohexyl)-

carbamate (101). tert-Butyl (6-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-6-

oxohexyl)carbamate was synthesized according to General Procedure

F: ¹H NMR (400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H),

W

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-4-sulfamoylbutanamide (107). N-(3-

(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-4-sulfamoylbutanamide, TFA salt, was

ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

4(5H)-yl)propyl)piperidine-2-carboxamide was synthesized as fol-

lows: tert-butyl 2-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)carbamoyl)piperidine-1-

carboxylate (0.172 g, 0.341 mmol) was dissolved in HCl in dioxane

(0.852 mL, 3.41 mmol) and the solution stirred at RT for 2 h. The

reaction was concentrated and puriﬁed by HPLC (H₂O + 0.1%

NH₄OH/MeCN, 10−50%, 8 min, Waters XBridge Prep C18 OBD, 5

μm, 30 × 75 mm) to give N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)piperidine-2-car-

boxamide: ¹H NMR (400 MHz, dmso): δ 10.46 (dd, J = 9.3, 4.8 Hz,

1H), 8.28−8.22 (m, 1H), 7.87 (dd, J = 8.6, 3.2 Hz, 1H), 7.77 (td, J =

8.7, 3.1 Hz, 1H), 4.07 (t, J = 7.2 Hz, 2H), 3.49 (s, 1H), 3.22−3.06 (m,

3H), 2.79 (t, J = 11.8 Hz, 1H), 2.00−1.93 (m, 1H), 1.91−1.82 (m,

2H), 1.76−1.71 (m, 1H), 1.61 (d, J = 12.4 Hz, 1H), 1.49−1.37 (m,

4H); HRMS (ES-API) m/z: calcd for C₁₈H₂₂FN₆O₂S (M + H),

405.1503; found, 405.1517.

1

synthesized according to General Procedure F: H NMR (400 MHz,

DMSO-d₆): δ 10.31 (dd, J = 9.3, 4.7 Hz, 1H), 7.93 (dd, J = 8.6, 3.1

Hz, 1H), 7.87 (t, J = 5.6 Hz, 1H), 7.85−7.78 (m, 1H), 6.75 (s, 2H),

4.05 (dd, J = 8.1, 6.3 Hz, 2H), 3.13 (q, J = 6.6 Hz, 2H), 3.00−2.92

(m, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.94−1.81 (m, 4H); ¹⁹F NMR (376

MHz, DMSO-d₆): δ −113.14; HRMS (ES-API) m/z: calcd for

C₁₆H₂₀FN₆O₄S₂(M + H), 443.0966; found, 443.0977.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)cyclopentanecarboxamide (108). N-(3-

(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)cyclopentanecarboxamide, TFA salt, was

1

synthesized according to General Procedure F: H NMR (400 MHz,

DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1

Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 7.75 (t, J = 5.6 Hz, 1H),

4.02 (dd, J = 8.2, 6.5 Hz, 2H), 3.10 (q, J = 6.6 Hz, 2H), 1.82 (p, J =

7.0 Hz, 2H), 1.73−1.63 (m, 2H), 1.63−1.51 (m, 4H), 1.50−1.40 (m,

2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.19 (td, J = 8.2, 4.6

Hz); HRMS (ES-API) m/z: calcd for C₁₈H₂₁FN₅O₂S (M + H),

390.1395; found, 390.1387.

trans-4-(tert-Butyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)cyclohexane-1-car-

boxamide (109). trans-4-(tert-Butyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-

1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-

cyclohexane-1-carboxamide, TFA salt, was synthesized according to

General Procedure F: ¹H NMR (400 MHz, DMSO-d₆): δ 10.29 (dd, J

= 9.3, 4.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3,

8.0, 3.1 Hz, 1H), 7.67 (t, J = 5.7 Hz, 1H), 4.01 (dd, J = 8.4, 6.4 Hz,

2H), 3.09 (q, J = 6.5 Hz, 2H), 1.93 (td, J = 12.2, 2.9 Hz, 1H), 1.81 (p,

J = 6.9 Hz, 2H), 1.73 (d, J = 9.8 Hz, 4H), 1.26 (q, J = 12.2 Hz, 2H),

0.97−0.87 (m, 3H), 0.80 (s, 9H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−113.21 (td, J = 8.3, 4.6 Hz); HRMS (ES-API) m/z: calcd for

C₂₃H₃₁FN₅O₂S (M + H), 460.2177; found, 460.2177.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)piperidine-3-carboxamide (113). tert-

Butyl 2-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propyl)carbamoyl)piperidine-1-carboxylate

was synthesized according to General Procedure F. Then, N-(3-(7-

ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

4(5H)-yl)propyl)piperidine-3-carboxamide was synthesized as fol-

lows: tert-butyl 3-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)carbamoyl)piperidine-1-

carboxylate (0.172 g, 0.341 mmol) was dissolved in HCl in dioxane

(0.852 mL, 3.41 mmol) and the solution stirred at RT for 2 h. The

reaction was concentrated and puriﬁed by HPLC (H₂O + 0.1%

NH₄OH/MeCN, 10−50%, 8 min, Waters XBridge Prep C18 OBD, 5

μm, 30 × 75 mm) to give N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)piperidine-3-car-

1

boxamide: H NMR (400 MHz, dmso): δ 10.43 (s, 1H), 8.14 (t, J =

5.8 Hz, 1H), 7.87 (dd, J = 8.6, 3.2 Hz, 1H), 7.82−7.73 (m, 1H), 4.05

(t, J = 7.5 Hz, 2H), 3.11 (dq, J = 19.6, 7.8, 6.2 Hz, 5H), 2.96−2.90

(m, 1H), 2.89−2.80 (m, 2H), 1.84 (p, J = 7.1 Hz, 4H), 1.74 (app s,

1H), 1.56 (t, J = 9.7 Hz, 2H); HRMS (ES-API) m/z: calcd for

C₁₈H₂₂FN₆O₂S (M + H), 405.1503; found, 405.1491.

tert-Butyl (4-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)carbamoyl)cyclohexyl)-

carbamate (110). tert-Butyl (4-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-

carbamoyl)cyclohexyl)carbamate, di-TFA salt was synthesized

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)piperidine-4-carboxamide (114). tert-

Butyl 4-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propyl)carbamoyl)piperidine-1-carboxylate

was synthesized according to General Procedure F. Then, N-(3-(7-

ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

4(5H)-yl)propyl)piperidine-4-carboxamide was synthesized as fol-

lows: tert-butyl 4-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)carbamoyl)piperidine-1-

carboxylate (0.172 g, 0.341 mmol) was dissolved in HCl in dioxane

(0.852 mL, 3.41 mmol) and the solution stirred at RT for 2 h. The

reaction was concentrated and puriﬁed by HPLC (H₂O + 0.1%

NH₄OH/MeCN, 10−50%, 8 min, Waters XBridge Prep C18 OBD, 5

μm, 30 × 75 mm) to give N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)piperidine-4-car-

1

according to General Procedure F: H NMR (400 MHz, DMSO-

d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H),

7.80 (ddd, J = 9.4, 8.0, 3.2 Hz, 1H), 7.67 (t, J = 5.7 Hz, 1H), 6.65 (d, J

= 6.5 Hz, 1H), 4.06−3.98 (m, 2H), 3.43 (s, 1H), 3.11 (q, J = 6.5 Hz,

2H), 2.10 (dq, J = 12.2, 3.8 Hz, 1H), 1.82 (p, J = 7.7, 7.3 Hz, 2H),

1.77−1.68 (m, 1H), 1.64−1.53 (m, 2H), 1.47−1.37 (m, 5H), 1.36 (s,

9H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.17 (td, J = 8.3, 4.8

Hz); HRMS (ES-API) m/z: calcd for C₂₄H₃₁FN₆NaO₄S (M + Na),

541.2004; found, 541.2011.

4-Amino-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)cyclohexane-1-carboxa-

mide (111). tert-Butyl (4-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)carbamoyl)-

cyclohexyl)carbamate (0.0282 g, 0.054 mmol) was dissolved in HCl

in dioxane (0.136 mL, 0.544 mmol) and the solution stirred at RT for

2 h. The reaction was concentrated and puriﬁed by HPLC to give 4-

amino-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propyl)cyclohexane-1-carboxamide, di-TFA

1

boxamide: H NMR (400 MHz, dmso): δ 10.34 (s, 1H), 7.96 (t, J =

5.5 Hz, 1H), 7.91−7.87 (m, 1H), 7.83−7.76 (m, 1H), 4.02 (t, J = 7.4

Hz, 2H), 3.12 (dd, J = 12.4, 6.1 Hz, 2H), 2.87 (t, J = 12.2 Hz, 2H),

2.41−2.32 (m, 1H), 1.81 (d, J = 12.4 Hz, 5H), 1.69 (d, J = 13.1 Hz,

2H); HRMS (ES-API) m/z: calcd for C₁₈H₂₂FN₆O₂S (M + H),

405.1503; found, 405.1511; purity (HPLC) 93.09%.

1

salt: H NMR (400 MHz, DMSO-d₆): δ 10.31 (dd, J = 9.3, 4.7 Hz,

1H), 7.93 (dd, J = 8.5, 3.1 Hz, 1H), 7.88−7.76 (m, 2H), 7.71 (s, 3H),

4.04 (dd, J = 8.3, 6.4 Hz, 2H), 3.14 (q, J = 6.6 Hz, 2H), 2.33−2.23

(m, 0H), 1.91−1.78 (m, 5H), 1.72−1.63 (m, 6H), 1.58−1.47 (m,

1H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.09 (td, J = 8.4, 4.8

Hz); HRMS (ES-API) m/z: calcd for C₁₉H₂₄FN₆O₂S (M + H),

419.1660; found, 419.1642.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)piperidine-2-carboxamide (112). tert-

Butyl 2-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propyl)carbamoyl)piperidine-1-carboxylate

was synthesized according to General Procedure F. Then, N-(3-(7-

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)tetrahydro-2H-pyran-4-carboxamide

(115). N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propyl)tetrahydro-2H-pyran-4-carboxamide,

TFA salt, was synthesized according to General Procedure F: HRMS

(ES-API) m/z: calcd for C₁₈H₂₁FN₅O₃S (M + H), 406.1344; found,

406.1355.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(piperidin-4-yl)acetamide (116). tert-

Butyl 4-(2-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-2-oxoethyl)piperidine-1-

X

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

carboxylate was synthesized according to General Procedure F. Then,

N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(piperidin-4-yl)acetamide was synthe-

sized as follows: tert-butyl 4-(2-((3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-2-

oxoethyl)piperidine-1-carboxylate (0.0209 g, 0.040 mmol) was

dissolved in HCl in dioxane (0.101 mL, 0.403 mmol) and the

solution stirred at RT for 2 h. The reaction was concentrated and

puriﬁed by HPLC to give N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-2-(piperidin-4-yl)-

(t, J = 7.2 Hz, 2H), 3.32−3.23 (m, 2H), 3.10 (q, J = 6.7 Hz, 2H), 2.25

(s, 3H), 2.19 (dd, J = 7.9, 7.0 Hz, 2H), 2.13 (s, 3H), 1.81 (p, J = 7.0

Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.14 (dd, J = 8.3,

5.0 Hz); HRMS (ES-API) m/z: calcd for C₂₀H₂₂FN₆O₃S (M + H),

445.1453; found, 445.1475.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(3-ﬂuorophenyl)acetamide (122). N-

(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(3-ﬂuorophenyl)acetamide, TFA salt,

1

was synthesized according to General Procedure F: H NMR (400

1

acetamide, TFA: H NMR (400 MHz, DMSO-d₆): δ 10.31 (dd, J =

MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.08 (t, J = 5.6

Hz, 1H), 7.90 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1

Hz, 1H), 7.31 (td, J = 8.1, 6.5 Hz, 1H), 7.08−6.99 (m, 3H), 4.03 (t, J

= 7.3 Hz, 2H), 3.40 (s, 2H), 3.13 (q, J = 6.6 Hz, 2H), 1.84 (p, J = 7.1

Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.19 (q, J = 7.7, 7.1

Hz), −113.86 to −113.98 (m); HRMS (ES-API) m/z: calcd for

C₂₀H₁₈F₂N₅O₂S (M + H), 430.1144; found, 430.1153.

9.3, 4.7 Hz, 1H), 8.43 (s, 1H), 8.12 (s, 1H), 7.96−7.89 (m, 2H), 7.83

(ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 4.08−4.00 (m, 2H), 3.24 (d, J = 13.1

Hz, 2H), 3.13 (q, J = 6.6 Hz, 2H), 2.86 (dd, J = 21.5, 10.8 Hz, 3H),

2.02 (d, J = 7.0 Hz, 2H), 1.97−1.89 (m, 1H), 1.84 (p, J = 7.0 Hz,

2H), 1.77 (d, J = 14.0 Hz, 2H), 1.35−1.22 (m, 2H); ¹⁹F NMR (376

MHz, DMSO-d₆): δ −113.06 to −113.17 (m).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(4-methylpiperazin-1-yl)acetamide

(117). N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-

a]quinazolin-4(5H)-yl)propyl)-2-(4-methylpiperazin-1-yl)acetamide,

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(4-ﬂuorophenyl)acetamide (123). N-

(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(4-ﬂuorophenyl)acetamide, TFA salt,

1

di-TFA salt, was synthesized according to General Procedure F: H

was synthesized according to General Procedure F: H NMR (400

NMR (400 MHz, DMSO-d₆): δ 10.30 (dd, J = 9.3, 4.7 Hz, 1H), 9.49

(s, 1H), 7.97 (t, J = 5.5 Hz, 1H), 7.93 (dd, J = 8.5, 3.1 Hz, 1H), 7.82

(ddd, J = 9.3, 8.0, 3.2 Hz, 1H), 4.02 (t, J = 7.2 Hz, 2H), 3.40 (d, J =

10.2 Hz, 1H), 3.21−3.11 (m, 2H), 3.08 (s, 4H), 2.99 (d, J = 13.1 Hz,

3H), 2.78 (s, 3H), 1.84 (p, J = 6.8 Hz, 2H); ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −113.07 (td, J = 8.2, 4.6 Hz); HRMS (ES-API) m/z:

calcd for C₁₉H₂₅FN₇O₂S (M + H), 434.1769; found, 434.1763.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-morpholinoacetamide (118). N-(3-

(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-morpholinoacetamide, di-TFA salt,

MHz, DMSO-d₆): δ 10.29 (dd, J = 9.4, 4.7 Hz, 1H), 8.05 (t, J = 5.7

Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1

Hz, 1H), 7.30−7.22 (m, 2H), 7.14−7.05 (m, 2H), 4.02 (t, J = 7.3 Hz,

2H), 3.36 (s, 2H), 3.12 (q, J = 6.6 Hz, 2H), 1.84 (p, J = 7.1 Hz, 2H);

¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.18 (td, J = 8.3, 4.8 Hz),

−116.95 (ddd, J = 14.6, 9.1, 5.5 Hz); HRMS (ES-API) m/z: calcd for

C₂₀H₁₈F₂N₅O₂S (M + H), 430.1144; found, 430.1141; purity

(HPLC) 93.64%.

2-(3-Chlorophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide (124).

2-(3-Chlorophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide, TFA

1

was synthesized according to General Procedure F: H NMR (400

MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 10.18 (s, 1H),

8.55 (s, 1H), 7.92 (dd, J = 8.6, 3.1 Hz, 1H), 7.82 (ddd, J = 9.3, 8.0, 3.1

Hz, 1H), 4.05 (t, J = 7.1 Hz, 2H), 3.97−3.67 (m, 8H), 3.22 (q, J = 6.6

Hz, 2H), 1.89 (p, J = 7.1 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆):

δ −113.07 (td, J = 8.3, 4.6 Hz); HRMS (ES-API) m/z: calcd for

C₁₈H₂₂FN₆O₃S (M + H), 421.1453; found, 421.1449.

1

salt, was synthesized according to General Procedure F: H NMR

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.09 (t, J =

5.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1

Hz, 1H), 7.33−7.24 (m, 3H), 7.19 (dt, J = 7.5, 1.5 Hz, 1H), 4.03 (t, J

= 7.2 Hz, 2H), 3.39 (s, 2H), 3.27 (s, 1H), 3.12 (q, J = 6.7 Hz, 2H),

1.85 (p, J = 7.2 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−113.19 (d, J = 10.0 Hz); HRMS (ES-API) m/z: calcd for

C₂₀H₁₈ClFN₅O₂S (M + H), 446.0848; found, 446.0867; purity

(HPLC) 94.66%.

4-((1H-Imidazole-1-yl)methyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-

1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-

benzamide (119). 4-((1H-Imidazole-1-yl)methyl)-N-(3-(7-ﬂuoro-5-

oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propyl)benzamide, TFA salt, was synthesized according to General

2-(4-Bromophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide (125).

2-(4-Bromophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide, TFA

1

Procedure F: H NMR (400 MHz, DMSO-d₆): δ 10.30 (dd, J = 9.3,

4.7 Hz, 1H), 9.13 (t, J = 1.5 Hz, 1H), 8.52 (t, J = 5.7 Hz, 1H), 7.91

(dd, J = 8.5, 3.1 Hz, 1H), 7.86−7.77 (m, 3H), 7.75 (t, J = 1.7 Hz,

1H), 7.64 (t, J = 1.6 Hz, 1H), 7.57−7.45 (m, 2H), 5.46 (s, 2H), 4.11

(dd, J = 8.3, 6.3 Hz, 2H), 3.37−3.33 (m, 2H), 1.99 (p, J = 7.0 Hz,

2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.10 (td, J = 8.3, 4.7

Hz); HRMS (ES-API) m/z: calcd for C₂₃H₂₁FN₇O₂S (M + H),

478.1456; found, 478.1454.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-phenylacetamide (120). N-(3-(7-Flu-

oro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

4(5H)-yl)propyl)-2-phenylacetamide was synthesized according to

General Procedure F: ¹H NMR (400 MHz, DMSO-d₆): δ 10.31 (dd, J

= 9.3, 4.7 Hz, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.92 (dd, J = 8.6, 3.1 Hz,

1H), 7.81 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 7.33−7.15 (m, 5H), 4.04

(dd, J = 8.2, 6.4 Hz, 2H), 3.38 (s, 2H), 3.14 (q, J = 6.7 Hz, 2H), 1.86

(p, J = 7.2 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.17 (td,

J = 8.3, 4.8 Hz); HRMS (ES-API) m/z: calcd for C₁₉H₁₇FN₅O₂S (M

+ H), 398.1082; found, 398.1063.

1

salt, was synthesized according to General Procedure F: H NMR

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.6 Hz, 1H), 8.08 (t, J =

5.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.2, 7.9, 3.1

Hz, 1H), 7.50−7.42 (m, 2H), 7.22−7.15 (m, 2H), 4.02 (t, J = 7.3 Hz,

2H), 3.36 (s, 2H), 3.12 (q, J = 6.6 Hz, 2H), 1.84 (p, J = 7.0 Hz, 2H);

¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.19; purity (HPLC) 88.26%.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(p-tolyl)acetamide (126). N-(3-(7-

Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

4(5H)-yl)propyl)-2-(p-tolyl)acetamide, TFA salt, was synthesized

according to General Procedure F: ¹H NMR (400 MHz, DMSO-d₆):

δ 10.29 (dd, J = 9.4, 4.6 Hz, 1H), 7.99 (t, J = 5.8 Hz, 1H), 7.91 (dd, J

= 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 10.9, 8.0, 3.1 Hz, 1H), 7.09 (q, J =

8.0 Hz, 4H), 4.02 (t, J = 7.3 Hz, 2H), 3.11 (q, J = 6.6 Hz, 2H), 2.24

(s, 3H), 1.83 (p, J = 7.2 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−113.20; purity (HPLC) 89.66%.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(4-hydroxyphenyl)acetamide (127).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(4-hydroxyphenyl)acetamide, TFA

salt, was synthesized according to General Procedure F: H NMR

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 9.16 (s,

1H), 7.94−7.87 (m, 2H), 7.80 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 7.04−

3-(3,5-Dimethylisoxazol-4-yl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-

propenamide (121). 3-(3,5-Dimethylisoxazol-4-yl)-N-(3-(7-ﬂuoro-5-

oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propyl)propenamide, TFA salt, was synthesized according to General

Procedure F: H NMR (400 MHz, DMSO-d₆): δ 10.31 (dd, J = 9.3,

4.7 Hz, 1H), 7.92 (dd, J = 8.6, 3.0 Hz, 1H), 7.85−7.78 (m, 2H), 4.01

1

Y

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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6.98 (m, 2H), 6.69−6.60 (m, 2H), 4.06−3.98 (m, 2H), 3.23 (s, 2H),

3.10 (q, J = 6.6 Hz, 2H), 1.83 (p, J = 7.2 Hz, 2H); ¹⁹F NMR (376

MHz, DMSO-d₆): δ −113.19; purity (HPLC) 90.60%.

δ −113.18 (td, J = 8.4, 4.8 Hz); HRMS (ES-API) m/z: calcd for

C₂₁H₁₈FN₆O₂S (M + H) 437.1190; found, 437.1194.

2-(4-Cyanophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide (134).

2-(4-Cyanophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide, TFA

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(2-methoxyphenyl)acetamide (128).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(2-methoxyphenyl)acetamide, TFA

1

salt, was synthesized according to General Procedure F: H NMR

1

salt, was synthesized according to General Procedure F: H NMR

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.16 (t, J =

5.6 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1

Hz, 1H), 7.77−7.73 (m, 2H), 7.47−7.40 (m, 2H), 4.02 (t, J = 7.3 Hz,

2H), 3.50 (s, 2H), 3.18−3.09 (m, 2H), 1.84 (p, J = 7.1 Hz, 2H); ¹⁹F

NMR (376 MHz, DMSO-d₆): δ −113.17.

tert-Butyl (4-(2-((3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)amino)-2-oxoethyl)-

phenyl)carbamate (135). tert-Butyl (4-(2-((3-(7-ﬂuoro-5-oxo-1-

thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)-

propyl)amino)-2-oxoethyl)phenyl)carbamate, TFA salt, was synthe-

sized according to General Procedure F: ¹H NMR (400 MHz,

DMSO-d₆): δ 10.30 (dd, J = 9.3, 4.7 Hz, 1H), 9.23 (s, 1H), 7.98 (t, J

= 5.7 Hz, 1H), 7.92 (dd, J = 8.6, 3.1 Hz, 1H), 7.81 (ddd, J = 9.3, 8.0,

3.1 Hz, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.15−7.05 (m, 2H), 4.03 (dd, J

= 8.1, 6.5 Hz, 2H), 3.30 (s, 2H), 3.12 (q, J = 6.7 Hz, 2H), 1.84 (p, J =

7.1 Hz, 2H), 1.46 (s, 9H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−113.16 (td, J = 8.2, 4.8 Hz).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(pyridine-2-yl)acetamide (136). N-

(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(pyridine-2-yl)acetamide was synthe-

sized according to General Procedure F: ¹H NMR (400 MHz, dmso):

δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.48−8.42 (m, 1H), 8.13 (t, J = 5.8

Hz, 1H), 7.91 (dd, J = 8.6, 3.2 Hz, 1H), 7.80 (td, J = 8.6, 3.2 Hz, 1H),

7.71 (td, J = 7.8, 1.8 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.22 (dd, J =

7.5, 5.0 Hz, 1H), 4.04 (t, J = 7.2 Hz, 2H), 3.57 (s, 2H), 3.14 (q, J =

6.7 Hz, 2H), 2.52 (s, 1H), 1.85 (p, J = 7.2 Hz, 2H); ¹⁹F NMR (376

MHz, dmso): δ −113.14 to −113.23 (m).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(pyridine-3-yl)acetamide (137). N-

(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(pyridine-3-yl)acetamide was synthe-

sized according to General Procedure F: ¹H NMR (400 MHz,

DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.44−8.37 (m, 2H),

8.14 (t, J = 5.7 Hz, 1H), 7.90 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J =

9.3, 8.0, 3.1 Hz, 1H), 7.64 (dt, J = 7.8, 2.0 Hz, 1H), 7.31 (dd, J = 7.8,

4.8 Hz, 1H), 4.03 (dd, J = 8.3, 6.3 Hz, 2H), 3.42 (s, 2H), 3.13 (q, J =

6.5 Hz, 2H), 1.85 (p, J = 7.1 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-

d₆): δ −113.20 (d, J = 9.2 Hz).

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 7.91 (dd, J

= 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.2, 7.8, 3.0 Hz, 2H), 7.22−7.16

(m, 1H), 7.13 (dd, J = 7.5, 1.7 Hz, 1H), 6.92 (dd, J = 8.2, 1.1 Hz,

1H), 6.86 (td, J = 7.4, 1.1 Hz, 1H), 4.03 (t, J = 7.3 Hz, 2H), 3.74 (s,

3H), 3.34 (s, 2H), 3.13 (q, J = 6.6 Hz, 2H), 1.84 (p, J = 7.0 Hz, 2H);

¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.18 (td, J = 8.3, 4.7 Hz);

HRMS (ES-API) m/z: calcd for C₂₁H₂₁FN₅O₃S (M + H), 442.1344;

found, 442.1360.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(3-methoxyphenyl)acetamide (129).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(3-methoxyphenyl)acetamide, TFA

1

salt, was synthesized according to General Procedure F: H NMR

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.6 Hz, 1H), 8.01 (t, J =

5.7 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1

Hz, 1H), 7.22−7.13 (m, 1H), 6.82−6.73 (m, 3H), 4.03 (t, J = 7.2 Hz,

2H), 3.71 (s, 3H), 3.33 (s, 2H), 3.12 (q, J = 6.7 Hz, 2H), 1.84 (p, J =

7.2 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.19.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(4-methoxyphenyl)acetamide (130).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(4-methoxyphenyl)acetamide, TFA

1

salt, was synthesized according to General Procedure F: H NMR

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 7.96 (t, J =

5.8 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1

Hz, 1H), 7.17−7.09 (m, 2H), 6.87−6.79 (m, 2H), 4.02 (t, J = 7.3 Hz,

2H), 3.69 (s, 3H), 3.29 (s, 2H), 3.11 (q, J = 6.6 Hz, 2H), 1.83 (p, J =

7.1 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −113.19 (td, J =

8.2, 4.7 Hz).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(2-(triﬂuoromethyl)phenyl)-

acetamide (131). N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-2-(2-

(triﬂuoromethyl)phenyl)acetamide, TFA salt, was synthesized accord-

ing to General Procedure F: ¹H NMR (400 MHz, DMSO-d₆): δ

10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.05 (t, J = 5.7 Hz, 1H), 7.91 (dd, J =

8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.2, 7.9, 3.1 Hz, 1H), 7.65 (d, J = 7.7

Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 7.8 Hz, 2H), 4.04 (t, J =

7.3 Hz, 2H), 3.61 (s, 2H), 3.15 (q, J = 6.7 Hz, 2H), 1.85 (p, J = 7.1

Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ −58.53, −113.20 (td, J

= 8.3, 4.8 Hz).

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(3-(triﬂuoromethyl)phenyl)-

acetamide (132). N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)-2-(3-

(triﬂuoromethyl)phenyl)acetamide, TFA salt, was synthesized accord-

ing to General Procedure F: ¹H NMR (400 MHz, DMSO-d₆): δ

10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.14 (t, J = 5.6 Hz, 1H), 7.90 (dd, J =

8.6, 3.1 Hz, 1H), 7.80 (ddd, J = 9.3, 8.0, 3.1 Hz, 1H), 7.61−7.49 (m,

4H), 4.03 (dd, J = 8.1, 6.3 Hz, 2H), 3.50 (s, 2H), 3.13 (q, J = 6.8 Hz,

2H), 1.85 (p, J = 7.1 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−61.01, −113.20 (td, J = 8.2, 4.7 Hz); HRMS (ES-API) m/z: calcd

for C₂₁H₁₈F₄N₅O₂S (M + H), 480.1112; found, 480.1126.

N-(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(pyridine-4-yl)acetamide (138). N-

(3-(7-Fluoro-5-oxo-1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-4(5H)-yl)propyl)-2-(pyridine-4-yl)acetamide was synthe-

sized according to General Procedure F: ¹H NMR (400 MHz, dmso):

δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.51−8.44 (m, 2H), 8.23−8.16 (m,

1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.80 (td, J = 8.6, 3.2 Hz, 1H),

7.28 (s, 2H), 4.03 (t, J = 7.3 Hz, 2H), 3.44 (d, J = 4.1 Hz, 2H), 3.14

(q, J = 6.6 Hz, 2H), 1.85 (p, J = 7.2 Hz, 2H); ¹⁹F NMR (376 MHz,

dmso): δ −113.17 (td, J = 8.3, 4.7 Hz).

9-Fluoro-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (139). 8-Fluoro-3-propyl-2-thioxo-2,3-dihydro-

quinazolin-4(1H)-one, TFA salt, was synthesized according to

General Procedure A. Then, 9-ﬂuoro-4-propyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was synthesized accord-

1

2-(2-Cyanophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide (133).

2-(2-Cyanophenyl)-N-(3-(7-ﬂuoro-5-oxo-1-thioxo-1,2-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-4(5H)-yl)propyl)acetamide, TFA

ing to General Procedure D: H NMR (400 MHz, DMSO-d₆): δ

13.83 (s, 1H), 8.02 (dd, J = 7.9, 1.5 Hz, 1H), 7.77 (ddd, J = 11.5, 8.3,

1.5 Hz, 1H), 7.61 (td, J = 8.0, 4.1 Hz, 1H), 3.97−3.89 (m, 2H), 1.70

(sext, J = 7.5 Hz, 2H), 0.89 (t, J = 7.5 Hz, 3H); ¹⁹F NMR (376 MHz,

DMSO-d₆): δ −93.30 (dd, J = 11.5, 4.2 Hz).

9-Chloro-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (140). 8-Chloro-3-propyl-2-thioxo-2,3-dihy-

droquinazolin-4(1H)-one, TFA salt, was synthesized according to

General Procedure A. Then, 9-chloro-4-propyl-1-thioxo-2,4-dihydro-

[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one was synthesized accord-

1

salt, was synthesized according to General Procedure F: H NMR

(400 MHz, DMSO-d₆): δ 10.29 (dd, J = 9.3, 4.7 Hz, 1H), 8.20 (t, J =

5.6 Hz, 1H), 7.91 (dd, J = 8.6, 3.1 Hz, 1H), 7.85−7.73 (m, 2H), 7.63

(td, J = 7.7, 1.4 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.42 (td, J = 7.6,

1.2 Hz, 1H), 4.05 (t, J = 7.2 Hz, 2H), 3.65 (s, 2H), 3.16 (q, J = 6.7

Hz, 2H), 1.87 (p, J = 7.2 Hz, 2H); ¹⁹F NMR (376 MHz, DMSO-d₆):

Z

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1

ing to General Procedure D: H NMR (400 MHz, DMSO-d₆): δ

(0.027 mL, 0.426 mmol) were added to a solution of 9-methoxy-4-

propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

one (0.103 g, 0.355 mmol) in DMF (1.774 mL). The reaction was

stirred for 3 h and quenched with methanol. The crude mixture was

concentrated and puriﬁed by ISCO (EtOAc/hexanes, 0−90%) to give

9-methoxy-1-(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]quinazolin-

5(4H)-one.

13.78 (s, 1H), 8.10 (dd, J = 7.7, 1.5 Hz, 1H), 7.91 (dd, J = 8.0, 1.5 Hz,

1H), 7.60 (t, J = 7.9 Hz, 1H), 3.94−3.86 (m, 2H), 1.70 (sext, J = 7.5

Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H).

9-Methoxy-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one (141). 8-Methoxy-3-propyl-2-thioxo-2,3-dihy-

droquinazolin-4(1H)-one was synthesized according to General

Procedure A. Then, 9-methoxy-4-propyl-1-thioxo-2,4-dihydro-[1,2,4]-

triazolo[4,3-a]quinazolin-5(1H)-one was synthesized according to

Biochemical Assays. ELISA-Based PBD-Binding Inhibition

Assay. The assay was performed essentially as described previously.

Brieﬂy, a biotinylated PBIP1 p-T78 peptide (i.e., Biotin-Ahx-C-

ETFDPPLHSpTAI-NH₂) was ﬁrst diluted with 1× coating solution

(SeraCare, Gaithersburg, MD) to the ﬁnal concentration of 0.3 μM,

and then 100 μL of the resulting solution was immobilized onto a 96-

well streptavidin-coated plate (Nalgene Nunc, Rochester, NY). The

wells were washed once with PBS + 0.05% Tween 20 (PBST) and

incubated with 200 μL of PBS plus 1% BSA (blocking buﬀer) for 1 h

to prevent nonspeciﬁc binding. HEK293A lysates expressing HA-

EGFP-Plk1 were prepared in a TBSN [20 mM Tris−HCl (pH 8.0),

150 mM NaCl, 0.5% NP-40, 5 mM EGTA, 2 mM MgCl₂, 1.5 mM

EDTA, and protease inhibitor cocktail (Roche)] + 20% DMSO buﬀer

(∼20 μg total lysates in 100 μL buﬀer), mixed with the indicated

amount of the competitors (p-T78 peptide and its derivative

compounds) for 30 min, provided onto the biotinylated peptide-

coated ELISA wells, and then incubated with constant rocking for 1 h

at 25 °C. Following the incubation, ELISA plates were washed ﬁve

times with PBST. To detect bound HA-EGFP-Plk1, the plates were

probed for 2 h with 100 μL/well of anti-HA antibody at a

concentration of 0.5 μg/mL in the blocking buﬀer and then washed

ﬁve times. The plates were further probed for 1 h with 100 μL/well of

HRP-conjugated secondary antibody (GE Healthcare) at a 1:1000

dilution in the blocking buﬀer. The plates were washed ﬁve times with

PBST and incubated with 100 μL/well of 3,3′,5,5′-tetramethylbenzi-

dine solution (Sigma-Aldrich, St. Louis, MO) as a substrate until a

desired absorbance was reached. The reactions were stopped by the

addition of 100 μL/well of stop solution (Cell Signaling Technology,

Danvers, MA). The optical density was measured at 450 nm by using

a PerkinElmer Enspire Multimode Plate reader (PerkinElmer, Inc.,

Boston, MA). Data obtained from more than three independent

experiments were analyzed by GraphPad (San Diego, CA) Prism

software version 7.

PBD FP Binding Assays for Plk1 Speciﬁcity. FP assays were carried

out essentially as described previously.²⁶Brieﬂy, an appropriate 5-

carboxyﬂuorescein-labeled PBD-binding peptide for Plk1, Plk2, or

Plk3 was incubated at a ﬁnal concentration of 5 nM with various

concentrations of the bacterially expressed and puriﬁed PBD of Plk1,

Plk2 or Plk3, respectively, in a binding buﬀer [10 mM Tris (pH 8.0),

1 mM EDTA, 50 mM NaCl, and 0.01% Nonidet P-40]. Samples were

analyzed 30 min after mixing all components in a 384-well format

using the Molecular Devices (San Jose, CA). To eliminate the

possibility that the apparent anti-Plk1 PBD activity is not the result of

the compounds’ ability to inhibit the ligand peptide used for the Plk1

FP assay above (i.e., FITC-Ahx-DPPLHSpTAI-NH₂), a second Plk1

PBD-binding peptide (FITC-Ahx-GPMQSpTPLNG-NH₂)²⁵was

used to conﬁrm the activity (Figure S3B, Supporting Information).

SpectraMax Paradigm multimode microplate detection platform. All

experiments were performed in triplicate for each sample. The

obtained data were plotted using GraphPad Prism software version 7.

ADME Measurements. PAMPA permeability was measured using a

high-throughput protocol, as previously reported.⁴⁷Aqueous

solubility was determined by a published procedure.⁴⁸Microsomal

metabolic stability assays were performed as reported previously.⁴⁶

Cell-Based Assays. Culture, Immunostaining, and Confocal

Microscopy. HeLa cells were cultured as recommended by the

American Type Culture Collection (ATCC). Asynchronously

growing cells were treated with control DMSO or the indicated

concentration of 143. To reveal chromosome morphologies, cells

were treated with DAPI, ﬁxed, and subjected to confocal microscopy.

Immunostaining was carried out using anti-Plk1 (Santa Cruz

Biotechnology, Santa Cruz, CA), anti-Cep63 (MilliporeSigma), anti-

CREST (Antibodies Incorp, Davis, CA), and anti-α-tubulin (Sigma)

1

General Procedure D: H NMR (400 MHz, dmso): δ 13.67 (s, 1H),

7.71 (dd, J = 7.3, 1.7 Hz, 1H), 7.62−7.50 (m, 2H), 3.95−3.87 (m,

5H), 1.68 (sext, J = 7.5 Hz, 2H), 0.87 (t, J = 7.5 Hz, 3H).

S-(5-Oxo-4-propyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

1-yl) Ethanethioate (142). 3-Propyl-2-thioxo-2,3-dihydroquinazolin-

4(1H)-one was synthesized according to General Procedure A. Then,

4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one, TFA salt, was synthesized according to General

Procedure D. Then, S-(5-oxo-4-propyl-4,5-dihydro-[1,2,4]triazolo-

[4,3-a]quinazolin-1-yl) ethanethioate was synthesized by the follow-

ing method. Triethylamine (0.054 mL, 0.384 mmol) and acetic

anhydride (0.022 mL, 0.230 mmol) were added to a solution of 4-

propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

one (0.05 g, 0.192 mmol) in acetonitrile (0.549 mL). The reaction

was stirred for 2 h and quenched with water. The crude mixture was

concentrated and puriﬁed by HPLC to give S-(5-oxo-4-propyl-4,5-

dihydro-[1,2,4]triazolo[4,3-a]quinazolin-1-yl) ethanethioate, TFA:

¹H NMR (400 MHz, DMSO-d₆): δ 10.32 (dd, J = 8.6, 1.0 Hz,

1H), 8.24 (dd, J = 7.9, 1.6 Hz, 1H), 7.90 (ddd, J = 8.8, 7.3, 1.7 Hz,

1H), 7.64 (td, J = 7.6, 1.1 Hz, 1H), 4.06−4.00 (m, 2H), 2.65 (s, 3H),

1.77 (sext, J = 7.4 Hz, 2H), 0.93 (t, J = 7.5 Hz, 3H).

1-(Methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-

one (143). 4-Propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]-

quinazolin-5(1H)-one was synthesized as described above. Then, 1-

(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one

was synthesized by the following method. K₂CO₃(0.032 g, 0.230

mmol) and MeI (0.012 mL, 0.192 mmol) were added to a solution of

4-propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-

5(1H)-one (0.05 g, 0.192 mmol) in DMF (0.549 mL). The reaction

was stirred for 2 h and quenched with methanol. The crude mixture

was concentrated and puriﬁed by HPLC (conditions) to give 1-

(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one,

TFA.

7-Fluoro-1-(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]-

quinazolin-5(4H)-one (144). To a solution of 7-ﬂuoro-4-propyl-1-

thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one (0.05

g, 0.180 mmol) in DMF (0.513 mL) was added K₂CO₃(0.030 g,

0.216 mmol) and MeI (0.011 mL, 0.180 mmol). The reaction was

stirred for 2 h and quenched with methanol. The crude mixture was

concentrated and puriﬁed by HPLC (conditions) to give 7-ﬂuoro-1-

(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one,

TFA.

9-Fluoro-1-(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]-

quinazolin-5(4H)-one (145). K₂CO₃(0.027 g, 0.199 mmol) and MeI

(10.36 μL, 0.166 mmol) were added to a solution of 9-ﬂuoro-4-

propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

one (0.0461 g, 0.166 mmol) in DMF (0.473 mL). The reaction was

stirred for 3 h and quenched with methanol. The crude mixture was

concentrated and puriﬁed by HPLC to give 9-ﬂuoro-1-(methylthio)-

4-propyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one, TFA.

9-Chloro-1-(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]-

quinazolin-5(4H)-one (146). K₂CO₃(0.066 g, 0.480 mmol) and MeI

(0.030 mL, 0.480 mmol) were added to a solution of 9-chloro-4-

propyl-1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-

one (0.1179 g, 0.400 mmol) in DMF (2.000 mL). The reaction was

stirred for 3 h and quenched with methanol. The crude mixture was

concentrated and puriﬁed by ISCO (EtOAc/hexanes, 0−75%) to give

9-chloro-1-(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]quinazolin-

5(4H)-one.

9-Methoxy-1-(methylthio)-4-propyl-[1,2,4]triazolo[4,3-a]-

quinazolin-5(4H)-one (147). K₂CO₃(0.059 g, 0.426 mmol) and MeI

AA

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

antibodies. Confocal images were acquired using a Zeiss LSM780

equipped with a plan-apochromat 63× (N.A. 1.4) oil-immersion

objective lens, 34-channel GaAsP spectral detector (Carl Zeiss

Microscopy, LLC.), and 8-bit, 0.5 μm z-steps.

Image Quantiﬁcation and Statistical Analysis. To quantify

ﬂuorescence signal intensities, images were acquired under the same

settings and the images obtained after the maximum-intensity

projection of z-stacks were analyzed using the Zeiss ZEN v2.1

software (Carl Zeiss Microscopy, LLC). All the quantiﬁcations were

performed with randomly chosen ﬁelds from at least three

Extracted ion chromatograms of selected compounds

and their glucuronides in MLM incubations with

UDPGA after 60 min; MS/MS spectrum of selected

compounds and their glucuronides; comparative FP-

based assays showing the ability of additional

triazoloquinazolinone-derived compounds (20, 27, 89,

and 90) to speciﬁcally inhibit Plk1 PBD; acyl-transfer

from 142; extracted ion chromatograms of 143 and its

metabolites in MLM incubations with NADPH and

UDPGA after 60 min, MS/MS spectrum of 143, its

hydroxylated metabolite, and its demethylated metabo-

lite, and proposed metabolic pathways of 143 in mice;

extracted ion chromatograms of 143 and its metabolites

from MS after 20 mg/kg IP injection of C57BL/6 mice

analyzed 4 h post injection and extracted ion chromato-

grams of 145 and its metabolites from MS after 20 mg/

kg IP injection of C57BL/6 mice analyzed 4 h post

injection; inactive S-alkylated compounds; multispecies

microsome and cytosol stability of selected compounds;

and in vitro and in vivo stability of selected prodrugs

(PDF)

independent experiments. All values are given as mean

s. d. P

values were calculated by an unpaired t-test from the mean data of

each group in GraphPad Prism (***P < 0.001). The statistical details

including the deﬁnitions and value of n (e.g., number of experimental

replicates, cells, etc.) and standard deviations are provided in the

ﬁgure legends.

Metabolism Study. Animals. Male C57BL/6 mice from Charles

River Laboratories (Wilmington, MA) were housed in the National

Cancer Institute animal facility that is a pathogen-free environment

controlled for temperature, light (25 °C, 12 h light/dark cycle), and

humidity (45−65%) with free access to food and water. The National

Cancer Institute Animal Care and Use Committee approved all

animal experiments conducted in this study.

In Vivo Mouse Pharmacokinetics Study. 6−8-week-old male

C57BL/6 mice were selected for metabolite proﬁling. In brief, 200

μL/20 g mice of WA (20 mg/kg dissolved in 5% DMSO and 5%

Tween 80-contained saline) was administered intraperitoneally to

mice. Control mice were treated with saline containing 5% DMSO

and 5% Tween 80 alone. Each group consisted of three mice. Blood

samples were collected by retro-orbital bleeding at 4 h postdose. After

centrifugation for 10 min at 14,000g, serum was obtained and

prepared by mixing 20 μL serum with 180 μL 50% aqueous

acetonitrile. After centrifugation at 14,000g for 20 min, a 5 μL aliquot

of the supernatants was injected into a Waters UPLC-ESI-QTOFMS

system (Waters Corporation, Milford, MA).

In Vitro MLM Assay. The incubation system (200 μL) contained

100 mM phosphate buﬀer solution (pH = 7.4), 1.0 mg·mL⁻¹MLMs,

5 mM MgCl₂, and 20 μM test compound. The mixture was

preincubated at 37 °C for 3 min and initiated with 2 mM NADPH or

2 mM UDPGA. The reaction was stopped by an aliquot of 200 μL of

ice-cold acetonitrile after 60 min. After centrifugation at 14,000g for

10 min, a 5 μL aliquot of the supernatant was injected into a UHPLC-

ESIQTOFMS.

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Authors

Kenneth A. Jacobson − Molecular Recognition Section,

Laboratory of Bioorganic Chemistry, National Institute of

Diabetes and Digestive and Kidney Diseases, National

Institutes of Health, Bethesda, Maryland 20892, United

States; orcid.org/0000-0001-8104-1493; Phone: 301-

496-9024; Email: kennethj@niddk.nih.gov

Kyung S. Lee − Chemistry Section, Laboratory of Metabolism,

National Cancer Institute, National Institutes of Health,

Bethesda, Maryland 20892, United States; orcid.org/

0000-0001-8326-7162; Phone: 240-760-7276;

Email: kyunglee@mail.nih.gov

Authors

UHPLC-ESI-QTOFMS and UHPLC-ESI-TQMS Analysis. Metabolite

proﬁling and identiﬁcation were performed on an Acquity UPLC/

Synapt HDMS Q-TOF MS system (Waters Corp., Milford, MA) with

an ESI source. Separation was achieved on an Acquity C18 BEH

UPLC column (1.7 mm, 2.1 × 50 mm; Waters Corp.). The mobile

phase consisted of water containing 0.1% formic acid (A) and

acetonitrile containing 0.1% formic acid (B). The initial condition of

2% B was held for 0.5 min, with the following linear gradient

employed: 20% B at 4 min, 95% B at 8 min, to 99% B at 9 min, held

for 1 min for column ﬂushing, then returning to initial conditions for

2 min for column equilibration before the next injection. The ﬂow

rate of the mobile phase was set at 0.4 mL/min. Column temperature

was maintained at 40 °C throughout the run. Data were collected in

positive ion mode, which was operated in full-scan mode at 50−950

m/z. Nitrogen was used as both cone gas (50 L/h) and desolvation

gas (950 L/h). Source temperature and desolvation temperature were

set at 150 and 400 °C, respectively. The capillary voltage and cone

voltage were 3000 and 30 V, respectively. The structures of

metabolites of interest were elucidated by tandem MS fragmentog-

raphy with collision energies ramping from 15 to 40 eV, employing

the same chromatographic conditions described above.

Celeste N. Alverez − Chemistry Section, Laboratory of

Metabolism, National Cancer Institute, National Institutes of

Health, Bethesda, Maryland 20892, United States; Division

of Preclinical Innovation, National Center for Advancing

Translational Sciences, National Institutes of Health,

Rockville, Maryland 20850, United States

Jung-Eun Park − Chemistry Section, Laboratory of

Metabolism, National Cancer Institute, National Institutes of

Health, Bethesda, Maryland 20892, United States

Kiran S. Toti − Molecular Recognition Section, Laboratory of

Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States; orcid.org/

0000-0002-4528-4858

Yangliu Xia − Chemistry Section, Laboratory of Metabolism,

National Cancer Institute, National Institutes of Health,

Bethesda, Maryland 20892, United States

Kristopher W. Krausz − Chemistry Section, Laboratory of

Metabolism, National Cancer Institute, National Institutes of

Health, Bethesda, Maryland 20892, United States

Ganesha Rai − Division of Preclinical Innovation, National

Center for Advancing Translational Sciences, National

Institutes of Health, Rockville, Maryland 20850, United

States; orcid.org/0000-0001-9763-9641

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01669.

AB

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(3) Strebhardt, K. Multifaceted Polo-like Kinases: Drug Targets and

Antitargets for Cancer Therapy. Nat. Rev. Drug Discovery 2010, 9,

643−660.

Jeong K. Bang − Division of Magnetic Resonance, Korea Basic

Science Institute, Cheongju 28119, Republic of Korea;

orcid.org/0000-0001-5916-0912

Frank J. Gonzalez − Chemistry Section, Laboratory of

Metabolism, National Cancer Institute, National Institutes of

Health, Bethesda, Maryland 20892, United States

́

(4) de Carcer, G. The Mitotic Cancer Target Polo-like Kinase 1:

Oncogene or Tumor Suppressor? Genes 2019, 10, 208−221.

(5) Luo, J.; Emanuele, M. J.; Li, D.; Creighton, C. J.; Schlabach, M.

R.; Westbrook, T. F.; Wong, K.-K.; Elledge, S. J. A Genome-Wide

RNAi Screen Identifies Multiple Synthetic Lethal Interactions with

the Ras Oncogene. Cell 2009, 137, 835−848.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01669

(6) Sur, S.; Pagliarini, R.; Bunz, F.; Rago, C.; Diaz, L. A.; Kinzler, K.

W.; Vogelstein, B.; Papadopoulos, N. A Panel of Isogenic Human

Cancer Cells Suggests a Therapeutic Approach for Cancers with

Inactivated p53. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 3964−3969.

(7) Park, J.-E.; Kim, T.-S.; Kim, B. Y.; Lee, K. S. Selective Blockade

of Cancer Cell Proliferation and Anchorage-Independent Growth by

Plk1 Activity −Dependent Suicidal Inhibition of its Polo-Box Domain.

Cell Cycle 2015, 14, 3624−3634.

Author Contributions

C.N.A. and J.-E.P. contributed equally to this work. C.N.A. and

K.S.T. carried out the chemical synthesis under the supervision

of K.A.J.; J.-E.P. performed ELISA, FP, and cell-based assays

under the guidance of K.S.L.; Y.X. and K.W.K. executed mouse

PK analyses; G.R. and J.K.B. assisted in the synthetic chemistry

and provided in vitro assay reagents, respectively. C.N.A., J.-

E.P., Y.X., K.S.T., F.J.G., K.A.J., and K.S.L. designed the

experiments and wrote the paper.

(8) Weinstein, I. B. Addiction to Oncogenes − The Achilles Heal of

Cancer. Science 2002, 297, 63−64.

(9) McMurray, H. R.; Sampson, E. R.; Compitello, G.; Kinsey, C.;

Newman, L.; Smith, B.; Chen, S.-R.; Klebanov, L.; Salzman, P.;

Yakovlev, A.; Land, H. Synergistic Response to Oncogenic Mutations

Defines Gene Class Critical to Cancer Phenotype. Nature 2008, 453,

1112−1116.

(10) Luo, J.; Solimini, N. L.; Elledge, S. J. Principles of Cancer

Therapy: Oncogene and Non-Oncogene Addiction. Cell 2009, 136,

823−837.

(11) Elia, A. E. H.; Rellos, P.; Haire, L. F.; Chao, J. W.; Ivins, F. J.;

Hoepker, K.; Mohammad, D.; Cantley, L. C.; Smerdon, S. J.; Yaffe, M.

B. The Molecular Basis for Phosphodependent Substrate Targeting

and Regulation of Plks by the Polo-Box Domain. Cell 2003, 115, 83−

95.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Robert O’Connor and Stewart R. Durell for

structural assistance and Juan Marugan and Anton Simeonov

for helpful advice. This research was supported by the

Intramural Research Program of the National Institutes of

Health NCI FLEX (K.S.L.), NIDDK ZIADK31117 (K.A.J.),

and NCI ZIA BC005562 (F.J.G.).

(12) Lee, K. S.; Grenfell, T. Z.; Yarm, F. R.; Erikson, R. L. Mutation

of the Polo-Box Disrupts Localization and Mitotic Functions of the

Mammalian Polo Kinase Plk. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,

9301−9306.

(13) Seong, Y.-S.; Kamijo, K.; Lee, J.-S.; Fernandez, E.; Kuriyama,

R.; Miki, T.; Lee, K. S. A Spindle Checkpoint Arrest and a Cytokinesis

Failure by the Dominant-Negative Polo-Box Domain of Plk1 in U-2

OS Cells. J. Biol. Chem. 2002, 277, 32282−32293.

ABBREVIATIONS

■

ADME, absorption distribution metabolism excretion; ATP,

adenosine triphosphate; Boc, tert-butyloxycarbonyl; COMU,

(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-

morpholino-carbenium hexaﬂuorophosphate; CYP, cyto-

chrome P450; DCM, dichloromethane; DIPEA, N,N-diisopro-

pylethylamine; DMF, N,N-dimethylformamide; DMSO, dime-

thylsulfoxide; EGFR, epidermal growth factor receptor; ELISA,

enzyme-linked immunosorbent assay; FDA, Food and Drug

Administration; FITC, ﬂuorescein-5-isothiocyanate; FP, ﬂuo-

rescence polarization assay; HBA, H-bond acceptor; HER2,

human epidermal growth factor receptor 2; HPLC, high-

performance liquid chromatography; HRMS, high-resolution

mass spectrometry; IP, intraperitoneal; KD, kinase domain;

LCMS, liquid chromatography−mass spectrometry; MLM,

mouse liver microsome; MS/MS, tandem mass spectrometry;

MT, microtubule; NAPDH, reduced nicotinamide adenine

dinucleotide phosphate; PAMPA, parallel artiﬁcial membrane

permeability assay; PBD, polo-box domain; PBIP1, centro-

meric protein U; PEG, polyethylene glycol; PPI, protein−

protein interaction; pyr, pyridine; RLM, rat liver microsome;

RT, room temperature; SAR, structure−activity relationship;

TFA, triﬂuoroacetic acid; UDPGA, uridine 5′-diphosphoglu-

curonic acid; UGT, uridine diphosphate glucuronosyltransfer-

ase

(14) Rudolph, D.; Steegmaier, M.; Hoffmann, M.; Grauert, M.;

Baum, A.; Quant, J.; Haslinger, C.; Garin-Chesa, P.; Adolf, G. R. BI

6727, a Polo-like Kinase Inhibitor with Improved Pharmacokinetic

Profile and Broad Antitumor Activity. Clin. Cancer Res. 2009, 15,

3094−3102.

́

(15) Steegmaier, M.; Hoffmann, M.; Baum, A.; Lenart, P.;

̌

́

Petronczki, M.; Krssak, M.; Gurtler, U.; Garin-Chesa, P.; Lieb, S.;

̈

Quant, J.; Grauert, M.; Adolf, G. R.; Kraut, N.; Peters, J.-M.; Rettig,

W. J. BI 2536, a Potent and Selective Inhibitor of Polo-like Kinase 1,

Inhibits Tumor Growth in Vivo. Curr. Biol. 2007, 17, 316−322.

(16) Russo, M. A.; Kang, K. S.; Di Cristofano, A. The PLK1

Inhibitor GSK461364A is Effective in Poorly Differentiated and

Anaplastic Thyroid Carcinoma Cells, Independent of the Nature of

Their Driver Mutations. Thyroid 2013, 23, 1284−1293.

(17) Beria, I.; Bossi, R. T.; Brasca, M. G.; Caruso, M.; Ceccarelli, W.;

Fachin, G.; Fasolini, M.; Forte, B.; Fiorentini, F.; Pesenti, E.; Pezzetta,

D.; Posteri, H.; Scolaro, A.; Depaolini, S. R.; Valsasina, B. NMS-P937,

a 4,5-dihydro-1H-Pyrazolo[4,3-h]Quinazoline Derivative as Potent

and Selective Polo-like Kinase 1 Inhibitor. Bioorg. Med. Chem. Lett.

2011, 21, 2969−2974.

(18) Nie, Z.; Feher, V.; Natala, S.; McBride, C.; Kiryanov, A.; Jones,

B.; Lam, B.; Liu, Y.; Kaldor, S.; Stafford, J.; Hikami, K.; Uchiyama, N.;

Kawamoto, T.; Hikichi, Y.; Matsumoto, S.-i.; Amano, N.; Zhang, L.;

Hosfield, D.; Skene, R.; Zou, H.; Cao, X.; Ichikawa, T. Discovery of

TAK-960: An Orally Available Small Molecule Inhibitor of Polo-like

Kinase 1 (PLK1). Bioorg. Med. Chem. Lett. 2013, 23, 3662−3666.

(19) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.;

Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.;

Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.;

REFERENCES

■

(1) Zitouni, S.; Nabais, C.; Jana, S. C.; Guerrero, A.; Bettencourt-

Dias, M. Polo-like Kinases: Structural Variations Lead to Multiple

Functions. Nat. Rev. Mol. Cell Biol. 2014, 15, 433−452.

(2) Lee, K. S.; Burke, T. R., Jr.; Park, J.-E.; Bang, J. K.; Lee, E. Recent

Advances and New Strategies in Targeting Plk1 for Anticancer

Therapy. Trends Pharmacol. Sci. 2015, 36, 858−877.

AC

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard,

S.; Wodicka, L. M.; Zarrinkar, P. P. A Quantitative Analysis of Kinase

Inhibitor Selectivity. Nat. Biotechnol. 2008, 26, 127−132.

(20) Lee, K. S.; Burke, T. R., Jr.; Park, J.-E.; Bang, J. K.; Lee, E.

Recent Advances and New Strategies in Targeting Plk1 for Anticancer

Therapy. Trends Pharmacol. Sci. 2015, 36, 858−877.

by the Inhibition of Plk1 Polo Box Domain-Dependent Recognition.

J. Biol. Chem. 2009, 284, 2344−2353.

(35) Normandin, K.; Lavallee, J. F.; Futter, M.; Beautrait, A.;

Duchaine, J.; Guiral, S.; Marinier, A.; Archambault, V. Identification

of Polo-like Kinase 1 Interaction Inhibitors Using a Novel Cell-Based

Assay. Sci. Rep. 2016, 6, 37581.

(21) Park, J.-E.; Hymel, D.; Burke, T. R., Jr.; Lee, K. S. Current

Progress and Future Perspectives in the Development of Anti-Polo-

like Kinase 1 Therapeutic Agents. F1000Research 2017, 6, 1024.

(22) Syed, N.; Smith, P.; Sullivan, A.; Spender, L. C.; Dyer, M.;

Karran, L.; O’Nions, J.; Allday, M.; Hoffmann, I.; Crawford, D.;

Griffin, B.; Farrell, P. J.; Crook, T. Transcriptional Silencing of Polo-

like Kinase 2 (SNK/PLK2) is a Frequent Event in B-cell

Malignancies. Blood 2006, 107, 250−256.

(36) Scharow, A.; Knappe, D.; Reindl, W.; Hoffmann, R.; Berg, T.

Development of Bifunctional Inhibitors of Polo-like Kinase 1 with

Low-Nanomolar Activities Against the Polo-Box Domain. Chem-

BioChem 2015, 17, 759−767.

(37) Chen, Y.; Zhang, J.; Li, D.; Jiang, J.; Wang, Y.; Si, S.

Identification of a Novel Polo-like Kinase 1 Inhibitor that Specifically

Blocks the Functions of Polo-Box Domain. Oncotarget 2017, 8, 1234−

1246.

(23) Yang, Y.; Bai, J.; Shen, R.; Brown, S. A. N.; Komissarova, E.;

Huang, Y.; Jiang, N.; Alberts, G. F.; Costa, M.; Lu, L.; Winkles, J. A.;

Dai, W. Polo-like Kinase 3 Functions as a Tumor Suppressor and is a

Negative Regulator of Hypoxia-Inducible Factor-1 Alpha Under

Hypoxic Conditions. Cancer Res. 2008, 68, 4077−4085.

(38) Kim, T. G.; Lee, J. H.; Lee, M. Y.; Kim, K.-U.; Lee, J. H.; Park,

C. H.; Lee, B. H.; Oh, K.-S. Development of a High-Throughput

Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1

Based on Time-Resolved Fluorescence Energy Transfer. Biol. Pharm.

Bull. 2017, 40, 1454−1462.

́

(24) Hanisch, A.; Wehner, A.; Nigg, E. A.; Sillje, H. H. W. Different

(39) Chen, Y.; Li, Z.; Liu, Y.; Lin, T.; Sun, H.; Yang, D.; Jiang, C.

Identification of Novel and Selective Non-Peptide Inhibitors

Targeting the Polo-Box Domain of Polo-Like Kinase 1. Bioorg.

Chem. 2018, 81, 278−288.

Plk1 Functions Show Distinct Dependencies on Polo-Box Domain-

Mediated Targeting. Mol. Biol. Cell 2006, 17, 448−459.

̈

(25) Liu, F.; Park, J.-E.; Qian, W.-J.; Lim, D.; Graber, M.; Berg, T.;

Yaffe, M. B.; Lee, K. S.; Burke, T. R., Jr. Serendipitous Alkylation of a

Plk1 Ligand Uncovers a New Binding Channel. Nat. Chem. Biol. 2011,

7, 595−601.

̈

(40) Abdelfatah, S.; Berg, A.; Bockers, M.; Efferth, T. A Selective

Inhibitor of the Polo-Box Domain of Polo-like Kinase 1 Identified by

Virtual Screening. J. Adv. Res. 2019, 16, 145−156.

(26) Qian, W.-J.; Park, J.-E.; Lim, D.; Lai, C. C.; Kelley, J. A.; Park,

S.-Y.; Lee, K. W.; Yaffe, M. B.; Lee, K. S.; Burke, T. R., Jr. Mono-

anionic Phosphopeptides Produced by Unexpected Histidine

Alkylation Exhibit High Plk1 Polo-Box Domain-Binding Affinities

and Enhanced Antiproliferative Effects in HeLa Cells. Biopolymers

2014, 102, 444−455.

(41) Zhou, Y.; Yan, F.; Huo, X.; Niu, M.-M. Discovery of a Potent

PLK1-PBD Small-Molecule Inhibitor as an Anticancer Drug

Candidate Through Structure-Based Design. Molecules 2019, 24,

4351−4360.

(42) Lee, K. S.; Idle, J. R. Pinning Down the Polo-Box Domain.

Chem. Biol. 2008, 15, 415−416.

(43) Archambault, V.; Normandin, K. Several Inhibitors of the Plk1

Polo-Box Domain Turn Out to Be Non-Specific Protein Alkylators.

Cell Cycle 2017, 16, 1220−1224.

(44) Kang, Y. H.; Park, J.-E.; Yu, L.-R.; Soung, N.-K.; Yun, S.-M.;

Bang, J. K.; Seong, Y.-S.; Yu, H.; Garfield, S.; Veenstra, T. D.; Lee, K.

S. Self-regulated Plk1 Recruitment to Kinetochores by the Plk1-PBIP1

Interaction Is Critical for Proper Chromosome Segregation. Mol. Cell

2006, 24, 409−422.

(45) Carlsson, J.; Yoo, L.; Gao, Z.-G.; Irwin, J. J.; Shoichet, B. K.;

Jacobson, K. A. Structure-Based Discovery of A_2AAdenosine Receptor

Ligands. J. Med. Chem. 2010, 53, 3748−3755.

(46) Urban, D. J.; Martinez, N. J.; Davis, M. I.; Brimacombe, K. R.;

Cheff, D. M.; Lee, T. D.; Henderson, M. J.; Titus, S. A.; Pragani, R.;

Rohde, J. M.; Liu, L.; Fang, Y.; Karavadhi, S.; Shah, P.; Lee, O. W.;

Wang, A.; McIver, A.; Zheng, H.; Wang, X.; Xu, X.; Jadhav, A.;

Simeonov, A.; Shen, M.; Boxer, M. B.; Hall, M. D. Assessing

Inhibitors of Mutant Isocitrate Dehydrogenase Using a Suite of Pre-

Clinical Discovery Assays. Sci. Rep. 2017, 7, 12758.

(27) Yun, S.-M.; Moulaei, T.; Lim, D.; Bang, J. K.; Park, J.-E.;

Shenoy, S. R.; Liu, F.; Kang, Y. H.; Liao, C.; Soung, N.-K.; Lee, S.;

Yoon, D.-Y.; Lim, Y.; Lee, D.-H.; Otaka, A.; Appella, E.; McMahon, J.

B.; Nicklaus, M. C.; Burke, T. R., Jr.; Yaffe, M. B.; Wlodawer, A.; Lee,

K. S. Structural and Functional Analyses of Minimal Phosphopeptides

Targeting the Polo-Box Domain of Polo-Like Kinase 1. Nat. Struct.

Mol. Biol. 2009, 16, 876−882.

(28) Huggins, D. J.; Hardwick, B. S.; Sharma, P.; Emery, A.; Laraia,

L.; Zhang, F.; Narvaez, A. J.; Roberts-Thomson, M.; Crooks, A. T.;

Boyle, R. G.; Boyce, R.; Walker, D. W.; Mateu, N.; McKenzie, G. J.;

Spring, D. R.; Venkitaraman, A. R. Development of a Novel Cell-

Permeable Protein-Protein Interaction Inhibitor for the Polo-box

Domain of Polo-like Kinase 1. ACS Omega 2020, 5, 822−831.

(29) Sharma, P.; Mahen, R.; Rossmann, M.; Stokes, J. E.; Hardwick,

B.; Huggins, D. J.; Emery, A.; Kunciw, D. L.; Hyvonen, M.; Spring, D.

R.; McKenzie, G. J.; Venkitaraman, A. R. A Cryptic Hydrophobic

Pocket in the Polo-Box Domain of the Polo-like Kinase PLK1

Regulates Substrate Recognition and Mitotic Chromosome Segrega-

tion. Sci. Rep. 2019, 9, 15930.

(47) Sun, H.; Nguyen, K.; Kerns, E.; Yan, Z.; Yu, K. R.; Shah, P.;

Jadhav, A.; Xu, X. Highly Predictive and Interpretable Models for

PAMPA Permeability. Bioorg. Med. Chem. 2017, 25, 1266−1276.

(48) Sun, H.; Shah, P.; Nguyen, K.; Yu, K. R.; Kerns, E.; Kabir, M.;

Wang, Y.; Xu, X. Predictive Models of Aqueous Solubility of Organic

Compounds Built on a Large Dataset of High Integrity. Bioorg. Med.

Chem. 2019, 27, 3110−3114.

(30) Rubner, S.; Scharow, A.; Schubert, S.; Berg, T. Selective

Degradation of Polo-like Kinase 1 by a Hydrophobically Tagged

Inhibitor of the Polo-Box Domain. Angew. Chem., Int. Ed. 2018, 57,

17043−17047.

(31) Abdelfatah, S.; Berg, A.; Huang, Q.; Yang, L. J.; Hamdoun, S.;

Klinger, A.; Greten, H. J.; Fleischer, E.; Berg, T.; Wong, V. K. W.;

Efferth, T. MCC1019, a Selective Inhibitor of the Polo-Box Domain

of Polo-like Kinase 1 as Novel, Potent Anticancer Candidate. Acta

Pharm. Sin. B 2019, 9, 1021−1034.

́

(49) Lenart, P.; Petronczki, M.; Steegmaier, M.; Di Fiore, B.; Lipp, J.

J.; Hoffmann, M.; Rettig, W. J.; Kraut, N.; Peters, J.-M. The Small-

Molecule Inhibitor BI 2536 Reveals Novel Insights Into Mitotic Roles

of Polo-Like Kinase 1. Curr. Biol. 2007, 17, 304−315.

̈

(32) Reindl, W.; Yuan, J.; Kramer, A.; Strebhardt, K.; Berg, T. A

Pan-Specific Inhibitor of the Polo-Box Domains of Polo-like Kinases

(50) Jackson, J. R.; Patrick, D. R.; Dar, M. M.; Huang, P. S. Targeted

Anti-Mitotic Therapies: Can We Improve on Tubulin Agents? Nat.

Rev. Cancer 2007, 7, 107−117.

Arrests Cancer Cells in Mitosis. ChemBioChem 2009, 10, 1145−1148.

̈

(33) Reindl, W.; Yuan, J.; Kramer, A.; Strebhardt, K.; Berg, T.

Inhibition of Polo-Like Kinase 1 by Blocking Polo-Box Domain-

Dependent Protein-Protein Interactions. Chem. Biol. 2008, 15, 459−

466.

(51) Munch, C.; Dragoi, D.; Frey, A.-V.; Thurig, K.; Lubbert, M.;

̈ ̈

̈

Wasch, R.; Bogatyreva, L.; Hauschke, D.; Lassmann, S.; Werner, M.;

May, A. M. Therapeutic Polo-Like Kinase 1 Inhibition Results in

Mitotic Arrest and Subsequent Cell Death of Blasts in the Bone

(34) Watanabe, N.; Sekine, T.; Takagi, M.; Iwasaki, J.-i.; Imamoto,

N.; Kawasaki, H.; Osada, H. Deficiency in Chromosome Congression

AD

https://dx.doi.org/10.1021/acs.jmedchem.0c01669

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Identification of a New Heterocyclic Scaffold for Inhibitors of the Polo-Box Domain of Polo-like Kinase 1

DOI: 10.1021/acs.jmedchem.0c01669

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Full text of DOI:10.1021/acs.jmedchem.0c01669

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