Evaluation of the Structure-Activity Relationship of Microtubule-Targeting 1,2,4-Triazolo[1,5- a]pyrimidines Identifies New Candidates for Neurodegenerative Tauopathies

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Evaluation of the Structure−Activity Relationship of Microtubule-

Targeting 1,2,4-Triazolo[1,5‑a]pyrimidines Identiﬁes New

Candidates for Neurodegenerative Tauopathies

Killian Oukolo ﬀ , Goodwell Nzou, Carmine Varricchio, Bobby Lucero, Thibault Alle, Jane Kovalevich,

Ludovica Monti, Anne-Sophie Cornec, Yuemang Yao, Michael J. James, John Q. Trojanowski,

Virginia M.-Y. Lee, Amos B. Smith, III, Andrea Brancale, Kurt R. Brunden,* and Carlo Ballatore*

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ABSTRACT: Studies in tau and Aβ plaque transgenic mouse

models demonstrated that brain-penetrant microtubule (MT)-

stabilizing compounds, including the 1,2,4-triazolo[1,5-a]-

pyrimidines, hold promise as candidate treatments for Alzheimer’s

disease and related neurodegenerative tauopathies. Triazolopyr-

imidines have already been investigated as anticancer agents;

however, the antimitotic activity of these compounds does not

always correlate with stabilization of MTs in cells. Indeed, previous

studies from our laboratories identiﬁed a critical role for the

fragment linked at C6 in determining whether triazolopyrimidines

promote MT stabilization or, conversely, disrupt MT integrity in

cells. To further elucidate the structure−activity relationship

(SAR) and to identify potentially improved MT-stabilizing candidates for neurodegenerative disease, a comprehensive set of 68

triazolopyrimidine congeners bearing structural modiﬁcations at C6 and/or C7 was designed, synthesized, and evaluated. These

studies expand upon prior understanding of triazolopyrimidine SAR and enabled the identiﬁcation of novel analogues that, relative to

the existing lead, exhibit improved physicochemical properties, MT-stabilizing activity, and pharmacokinetics.

endowed with generally more favorable drug-like absorption,

distribution, metabolism, and excretion-pharmacokinetics

(ADME-PK) properties than MT-stabilizing natural products⁹

have also shown promising results both in vitro and in

vivo.¹⁰⁻¹²Although the MT binding site of the triazolopyr-

imidines is known to be diﬀerent than that of taxol, epothilone

D, and other taxane site binders,¹³evaluation of a selected

prototype (3, Figure 1) in a mouse model of tauopathy

revealed that this compound could exert similar beneﬁcial

eﬀects to those previously observed with 1, indicating that

triazolopyrimidines hold promise as candidate treatments for

neurodegenerative tauopathies.¹²More recently, 3 has also

been shown to decrease axonal dystrophy, with a resulting

reduction of Aβ peptide and Aβ plaque deposition in a Tg

mouse model of senile plaque formation.¹⁴

INTRODUCTION

■

In Alzheimer’s disease (AD) and related neurodegenerative

tauopathies,¹the hyperphosphorylation of the microtubule

(MT)-associated protein tau promotes its dissociation from

MTs and eventual deposition into insoluble aggregates, which

are commonly referred to as neuroﬁbrillary tangles (NFTs)

and neuropil threads (NTs). Reduced tau binding to MTs is

believed to trigger alterations in the normal structure and

dynamics of MTs in the axons of neurons that ultimately cause

or contribute to impaired axonal transport and resulting axonal

dystrophy.²Given the evidence of neuronal MT abnormalities

in AD patients and in tau and Aβ plaque transgenic (Tg)

mouse models,³CNS-active MT-stabilizing agents have been

proposed as molecules that may provide therapeutic beneﬁts in

AD and related tauopathies.⁴Multiple preclinical studies⁵⁻⁸in

tau Tg mice have shown that relatively low and infrequent

doses of CNS-active MT-stabilizing natural products, such as

epothilone D (1, Figure 1) and dictyostatin (2, Figure 1), can

lead to signiﬁcant improvements in several endpoints including

an increase in MT density, a reduction in axonal dystrophy,

and a signiﬁcant lowering of both tau pathology and neuron

loss.⁵⁻⁷More recently, brain-penetrant MT-stabilizing 1,2,4-

triazolo[1,5-a]pyrimidines that are structurally simpler and are

Received: September 28, 2020

Published: January 7, 2021

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activity of these compounds may not always correlate with

stabilization of MTs in cells. Indeed, evaluation of

representative examples of triazolopyrimidines and related

phenylpyrimidines in cell-based assays of MT stabilization

revealed that varying the scaﬀold (i.e., triazolopyrimidine or

phenylpyrimidine) or substitution pattern results in molecules

that can either promote MT stabilization or, conversely,

disrupt MT integrity.¹¹For example, an evaluation of a

selected set of triazolopyrimidines and phenylpyrimidines for

their ability to produce elevations in known cellular markers of

stable MTs,¹⁹such as acetylated and de-tyrosinated α-tubulin

(AcTub and GluTub, respectively), revealed that typically the

phenylpyrimidines, such as 4, produce an unusual bell-shaped

concentration−response curve in these assays. In addition,

these compounds cause a concentration-dependent decrease in

total cellular α- and β-tubulin resulting from proteasome-

mediated degradation, with a visible reduction of MT

networks. Interestingly, although similar observations were

made for some triazolopyrimidines, including 5 and its brain-

penetrant¹⁰congener 6 (Figure 1), replacement of the alkoxide

side-chain of these molecules with a ﬂuorine atom, as in 7

(Figure 1) or 3, had a dramatic impact on the MT-stabilizing

properties of the compounds as demonstrated by the fact that

the latter molecules caused the desired linear concentration-

dependent increase in MT stabilization and MT mass in cells

over the tested concentrations without decreased cellular

tubulin levels or changing MT morphology.¹¹These ﬁndings,

combined with the observation that the amine fragment linked

at C7 of lead compound, 3, is likely to be the primary site of

metabolism,¹²led to a more systematic exploration of the SAR

of triazolopyrimidines modiﬁed at C6 and C7. The results

from these studies led to the identiﬁcation of selected

congeners that, relative to the existing lead, 3, exhibit improved

physicochemical properties, MT-stabilizing activity, and

pharmacokinetics.

Figure 1. Structure of MT-stabilizing natural products, epothilone D

and dictyostatin, and selected examples of MT-binding triazolopyr-

imidines and phenylpyrimidines.

MT-active triazolopyrimidines and structurally related

phenylpyrimidines (e.g., 4) have already been evaluated as

candidate treatments for cancer chemotherapy.^15,16This eﬀort,

which culminated in the identiﬁcation of cevipabulin (5) as a

clinical candidate,^17,18included a comprehensive assessment of

the structure−activity relationship (SAR) in cytotoxicity assays

that used rapidly dividing cancer cell-lines.¹⁵More recent

studies from our laboratories suggested that the antimitotic

a

Scheme 1

a

Reagents and reaction conditions: (a) For 76−85, 87−89, 91: Ar-X, CuBr, NaH, 1,4-dioxane, 60−100 °C, 12 h, 19−74%; For 86: Ar-CH₂Br,

NaH, DMF, 0 °C, 1 h, 90%; For 90 and 92: Ar-F, K₂CO₃, DMF, 60 °C, 4 h, 84−86%; (b) N-tributylamine, 170−180 °C, 2−6 h; (c) phosphorus

oxychloride, 110−130 °C, 6−16 h, 46−58% over two steps; (d) appropriate amine, DMF, rt−90 °C, 1−18 h, 12−50%; (e) Fe, NH₄Cl, H₂O/

MeOH (40/50), 80 °C, 2 h, 69%.

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a

Scheme 2

a

Reagents and reaction conditions: (a) For 24: NaOMe, THF, 0 to 60 °C, 14 h, 71%; For 25, 31: ROH or RSH, NaH, DMSO, 60 °C, 3 h, 81−

95%; for 28: CH₃SNa, DMSO, 60 °C, 3 h, quant; (b) m-CPBA (1 equiv), CH₂Cl₂, 20 °C, 2 h, 32% of 26, 52% of 27, 70% of 29; (c) m-CPBA (3

equiv), CH₂Cl₂, 20 °C, 3 h, 71%; (d) TEMPO, BAIB, CH₂Cl₂/H₂O (90/70), 20 °C, 2 h, 59%; (e) LiOH, MeOH/H₂O (1/1), 20 °C, 16 h, 39%.

of the ﬂuorinated phenyl ring (24−33, Scheme 2). In these

cases, the installation of the appropriate alcohol or thiol side-

chain was conducted via S_NAr followed by HPLC puriﬁcation

of the ﬁnal product. Sulfoxide (26, 29) and sulfone (27, 30)

derivatives were obtained from the corresponding thioethers

(i.e., 25, and 28, respectively) upon treatment with meta-

chloroperbenzoic acid (m-CPBA) (Scheme 2).

Triazolopyrimidine dichloride 102 was used to access a

series of congeners modiﬁed at position C7 (i.e., 34−61,

Scheme 3). In these cases, chemoselective displacement of the

chloro substituent at C7 was accomplished in moderate to

good yields by treating 102 with the appropriate amine. The

synthesis of C7 alkyl derivative 62 (Scheme 4) was initially

attempted adopting a known procedure²¹by reacting

dichloride 102 with the methyl ester of 4,4,4-triﬂuoro-3-

methylbutanoic acid in the presence of LiHMDS to obtain

110, followed by a Krapcho decarboxylation.²²The main

product isolated from this synthesis was the tetracyclic

compound 111 (Scheme 4 ) whose structure was conﬁrmed

CHEMISTRY

■

As part of these studies, a total of 68 compounds (7−75,

Schemes 1-5) have been synthesized and tested, including

eight previously described triazolopyrimidines (i.e., 7,¹⁵11,¹⁵

25,¹⁵31−33,²⁰34,¹²and 54¹³); 23 compounds exempliﬁed in

the patent literature (i.e., 10, 12, 14, 16, 18, 24, 28−30, 35, 41,

45−48, 50, 51, 52, 57, 60, 61, 64, 65); and 37 structurally

novel congeners. In all cases, the triazolopyrimidine ring was

accessed via cyclocondensation reaction between the appro-

priate diethylmalonate (76−92, Scheme 1) and 1H-1,2,4-

triazol-5-amine. Next, treatment with phosphorous oxychloride

provided the corresponding 5,7-dichloro triazolopyrimidines

(93−109, Scheme 1). Chemoselective amination at C7 with

(S)-1,1,1-triﬂuoropropan-2-amine furnished triazolopyrimi-

dines 7−22 (Scheme 1). Reduction of the para nitro derivative

22 provided the corresponding aniline derivative 23 (Scheme

1).

Triazolopyrimidine 7 was then used to access additional

derivatives bearing diﬀerent substitutions at the para position

Figure 2. Eﬀect of ﬂuorination of the aryl group at C6 on MT-stabilizing activity of Class I triazolopyrimidines. Black squares indicate the average

activity of test compounds at 1 and 10 μM, normalized to the activity of 100 nM of 5 (positive control).

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alkylation reaction at C7 was conducted using the tert-butyl

ester of 4,4,4-triﬂuoro-3-methylbutanoic acid to obtain

intermediate 112, which was then subjected to TFA-mediated

cleavage of the ester moiety and in situ decarboxylation

(Scheme 4).

Scheme 3

Finally, the synthesis of compounds 63−75 (Scheme 5) was

conducted by reacting the appropriate triazolopyrimidine

dichlorides (i.e., 95, 97, 106, or 107) with amine fragments

(Scheme 5). In selected cases (i.e., 67 and 68), puriﬁcation via

silica gel column chromatography aﬀorded individual atro-

poisomers, the structures of which could be determined via

single crystal X-ray structure determination of 68 (Scheme 5).

RESULTS

■

All test compounds were evaluated in a previously described¹¹

cell-based assay of MT-stabilization, in which compound-

dependent changes in tubulin polymerization and total tubulin

levels were determined after 4 h of incubation of QBI293 cells

with 1 and 10 μM compound via quantiﬁcation of acetylated

α-tubulin (AcTub) and total α-tubulin (α-Tub) in cell lysates

by ELISA. Although the changes in AcTub levels of

compound-treated cells relative to DMSO-treated cells provide

a valid assessment of the MT-stabilizing activity of test

compounds, day-to-day diﬀerences in the responsiveness of

cells to MT-stabilizing treatment can result in occasional

variability in the fold-change in AcTub levels. Thus, to account

for this variability and ultimately enable a ranking of diﬀerent

triazolopyrimidines based on MT-stabilizing activity, com-

pound-dependent changes in AcTub levels relative to DMSO-

treated cells (i.e., negative control) were also normalized to the

corresponding changes caused by 100 nM of cevipabulin (5),

which was routinely used as positive control.¹⁰

Previous evaluations of the MT-stabilizing activity of

selected examples of triazolopyrimidines in this assay suggested

that the nature of the substituent in the para position of the

ﬂuorinated phenyl can elicit one of two distinct cellular

responses: one characterized by a linear concentration-

dependent increase of markers of stable MTs (i.e., acetylated-

and detyrosinated-α-tubulin) with no eﬀect on total tubulin

levels at tested concentrations, which is typically observed with

triazolopyrimidines such as 3 and 7, and a second response

caused by compounds bearing the alkoxy side-chain (e.g., 5 and

6) that is characterized by a proteasome-dependent degrada-

tion of tubulin that is often associated with the appearance of a

bell-shaped concentration−response relationship in the AcTub

assay.¹¹To facilitate the presentation and discussion of the

SAR results, we will refer here to these two distinct cellular

responses as “Class I” and “Class II”, respectively, and

triazolopyrimidines 7 and 6 are used as deﬁning examples of

these classes.

As the replacement of the alkoxy side-chain of 5 or 6 (Class

II) with a ﬂuorine atom (as in 7, Class I) was found to produce

a profound impact on the MT-stabilizing activity of the

triazolopyrimidine compounds,¹¹our investigations of SARs

began by synthesizing and testing selected analogues (18−33,

Table 1) that could help identify speciﬁc stereoelectronic

characteristics of the substituent in the para position of the

ﬂuorinated phenyl ring that may be necessary/suﬃcient to

impart Class I activity. Evaluation of these compounds in the

QBI293 assay of MT-stabilization revealed a possible general

trend, indicating that active congeners bearing electron-

donating groups at the para position generally elicit a

signiﬁcant (i.e., >15%) loss of α-Tub levels when tested at

a

Reagents and reaction conditions: (a) appropriate amine, base,

DMF, 20−90 °C, 1−18 h, 12−50%.

by X-ray crystallography. The desired compound 62 was

ultimately obtained employing a similar approach in which

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a

Scheme 4

a

Reagents and reaction conditions: (a) appropriate ester of 4,4,4-triﬂuoro-3-methylbutanoic acid, HMDS, n-BuLi, THF, −78 °C, 2.5 h, 70% for

110, 25% for 112; (b) TFA, CH₂Cl₂, 20 °C, 16 h, 68%; (c) LiCl, DMSO, 130 °C (microwave irradiation), 3 h, 76%.

Figure 3. Eﬀect of both branching of aliphatic acyclic amines and homologation of endo- and exo-cyclic amines on MT-stabilizing activity of Class I

triazolopyrimidines. Test compounds are ordered along the X-axis based on the number of carbon atoms in the amine fragment. Black squares

indicate the average activity of test compounds at 1 and 10 μM, normalized to the activity of 100 nM of 5 (positive control). Compounds that did

not produce a statistically signiﬁcant elevation in AcTub at either 1 or 10 μM are not plotted and marked as inactive.

either 1 and/or 10 μM concentration, which is characteristic of

Class II compounds. For example, both para hydroxyl- (33)

and methoxy- (24) derivatives as well as thioalkyl compounds,

25 and 28, were all found to produce >20% reduction in

cellular α-Tub. In contrast, active analogues bearing electron-

withdrawing groups, such as cyano (20) and nitro (22), were

found to increase AcTub levels without producing a signiﬁcant

reduction in α-Tub levels (i.e., Class I phenotype). However, in

cases such as 21, where the chloro substituent produces

opposite mesomeric (+M) and inductive (−I) eﬀects, a

comparatively more moderate but still signiﬁcant loss of α-Tub

(19%) was observed when the compound was tested at a 10

μM concentration. These results indicate that the electronic

properties of the substituent in para may play an important

role in determining the phenotypic response. However,

diﬀerences in the shape/volume and/or the geometry of

hydrogen bonding of the substituent can modulate signiﬁcantly

the MT-stabilizing activity, independent of the phenotype.

This is best represented by comparing selected pairs of

compounds, such as nitrile (20) and sulfone (30), or alkyl-

sulfone (27) and the corresponding alkoxy compound (6).

Docking studies (vide infra) indicate that the larger volume of

the sulfone moiety is likely to aﬀect negatively the interaction

of the triazolopyrimidine with the binding site.

To investigate the eﬀect of the degree and pattern of

ﬂuorination of the phenyl ring at C6, compounds 8−16 were

evaluated in comparison with a Class I control compound, 7.

The results of these studies, summarized in Table 1 and Figure

2, show that all compounds within this series maintain MT-

stabilizing activity that is qualitatively similar to 7 (i.e., all

compounds elicit Class I phenotype); however, depending on

the number and position of the ﬂuorine atoms, these

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triazolopyrimidine congeners can diﬀer signiﬁcantly in terms of

potency. In general, the Class I MT-stabilizing activity appears

to increase with the number of ﬂuorine atoms in the ring, with

a preferred arrangement within the series being 2,4,6-

triﬂuorophenyl (Figure 2). Among derivatives bearing a

mono-ﬂuorinated phenyl fragment at C6, the ortho ﬂuoro

derivative (10) appeared to be more active than the

corresponding para substituted compound (9) at both 1 and

10 μM concentrations. Similarly, among triazolopyrimidines

bearing a di-ﬂuorinated phenyl at C6 (11−15), the 2,6-

diﬂuoro compound (12) was found to be the most active.

Finally, homologation of the 2,4,6-triﬂuorophenyl of 7 to the

corresponding 2,4,6-triﬂuorobenzyl congener (17) led to a

drastic reduction of MT-stabilizing activity.

Scheme 5

Comparative evaluation of Table 2 compounds (3, 34−62)

and 7 in the cell-based MT-stabilization assay helped deﬁne

several SAR elements of the fragment linked at the C7 position

of the triazolopyrimidine ring. Truncation of this fragment as

in 34 and 38 led to a dramatic loss of MT-stabilizing activity.

Replacement of the nitrogen atom at C7 of control compound,

7, with an oxygen atom (42) revealed that the ether linkage is

not permitted as it resulted in a drastic reduction of activity in

the cellular assay. Conversely, replacement of the nitrogen

atom at C7 with a methylene, as in 62, showed that the alkyl

linkage allows for retention of signiﬁcant activity in the AcTub

assay at both 1 and 10 μM. However, this compound was

found to cause a signiﬁcant reduction of α-Tub (i.e., ∼22%)

when tested at 10 μM. These observations demonstrate that

the nature of the fragment linked at C7 can play an important

role in determining both potency and cellular phenotype

elicited by MT-active triazolopyrimidines and that the

presence of a substituted nitrogen at this position may be a

necessary condition for Class I activity.

Further investigation of the SAR of triazolopyrimidines

featuring a substituted nitrogen at C7 included a comparison

between selected aliphatic and aromatic amines as well as

amides. Comparison of lead compound 3 with amide

derivative, 39, indicates that aliphatic amines may be preferred

over amides. Similarly, comparison of aniline derivative, 40,

with the corresponding cyclohexyl amine congener, 58, shows

that whereas the former is essentially devoid of activity at both

1 and 10 μM, the aliphatic derivative, 58, is signiﬁcantly active

at 10 μM without decreasing α-Tub levels. These results

suggest that aliphatic amines at C7 may be generally preferred

and, as a result, further exploration of the SAR focused on

identifying the key requisites of the aliphatic amine fragment,

which included an evaluation of linear, branched, and cyclic

amines of diﬀerent sizes. Comparison of the in vitro MT-

stabilizing activity of lead compound, 3, with congeners

featuring amine substituents at C7 that are either substructures

(i.e., 35−37) or that are incrementally larger fragments than

the C7 substituent of 3 (e.g., 60) clearly suggests that the MT-

stabilizing activity of triazolopyrimidines may increase with the

size and branching of the aliphatic fragment. As highlighted in

Figure 3, the extreme examples within this series are the linear

amine derivative, 36, which is devoid of MT-stabilizing activity

in the QBI293 assay, and the t-butyl derivative, 60, which

exhibits relatively potent activity as revealed by the marked

increases in AcTub levels at both 1 and 10 μM concentrations.

A similar trend also emerges from the comparison of series of

homologues featuring diﬀerent endo- (i.e., 49−54) or exo-

cyclic (i.e., 55−58) amines. Within each of these series,

relatively bulky fragments led to marked elevations of AcTub

a

Reagents and reaction conditions: (a) appropriate amine, DMF, rt, 1

h, 56−86%.

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Table 1. MT-Stabilizing Activity of Triazolopyrimidines Modiﬁed at C6

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Table 1. continued

a

Fold-changes in AcTub and total α-Tub levels in QBI293 cells after 4 h of incubation with test compounds at either 1 or 10 μM. Reported values

for AcTub and α-Tub represent the fold-change over control (DMSO)-treated cells (*p < 0.05 and **p < 0.01 by one-way ANOVA); numbers in

b

parentheses represent the fold-change of AcTub over positive control-treated cells (i.e., 100 nM of 5). Class I compounds are those producing a

concentration-dependent increase in AcTub levels and that do not cause >15% reduction in α-Tub at either concentration; Class II compounds are

those that cause >15% decrease in α-Tub at either 1 or 10 μM compound concentration. NA = not applicable as the test compound does not cause

signiﬁcant changes in AcTub or total α-Tub when tested at a 1 or a 10 μM concentration.

levels. However, comparison of the average MT-stabilizing

activity at 1 and 10 μM, relative to the positive control,

suggested that the optimal ring size in the endo- and exo-cyclic

amine series may be diﬀerent. Whereas in the exocyclic amine

series, the ﬁve-membered ring (57) seemed optimal, in the

case of endocyclic amines, the presence of a seven-membered

ring system (52) or a fused bicyclic system (54) produced the

highest activity (Figure 3). Finally, an evaluation of the eﬀect

of ﬂuorination and chiral conﬁguration of the branched amine

fragment at C7 was also conducted. Comparison of 7, which

features a ﬂuorinated isopropyl amine at C7, with correspond-

ing analogues 43, 44, and 35 that exhibit either 2, 1, or no

ﬂuorine substituents in the amine fragment indicates the

general trend linking the degree of ﬂuorination of the amine

fragment with potency. Similarly, ﬂuorination of 45 resulted in

a comparatively more potent congener (46). With respect to

the chiral conﬁguration, in the majority of cases examined, the

chirality of the amine was found to inﬂuence the potency of the

compound with one enantiomer being typically more active

than the other (cf., 3 and 45; 7 and 48; 60 and 61). In one

case, however, the chiral conﬁguration of the amine substituent

of 46 (R) and 47 (S) was also found to impact signiﬁcantly the

cellular phenotype. Whereas the R-enantiomer was found to be

relatively potent in the AcTub QBI293 assay without evidence

of a reduction in α-Tub levels, the opposite enantiomer (S)

produced a marked reduction in cellular α-Tub of approx-

imately ∼39% when tested at 10 μM, suggesting that 47 is

likely to negatively impact MT integrity. Notwithstanding this

notable exception, all other MT-active triazolopyrimidines

shown in Table 2 that feature an amine fragment at C7 were

found to be MT-stabilizing without producing losses in α-Tub.

Having identiﬁed the main characteristics of both the C6

and C7 fragments that may be required to obtain Class I MT-

stabilizing triazolopyrimidines, a focused series of derivatives

was designed by combining the most promising C6 and C7

fragments. In particular, as the C6 fragment SAR studies

highlighted the importance of a ﬂuorine atom in the ortho

position of the phenyl ring, whereas the para position may be

left unsubstituted or occupied by an electron-withdrawing

group, such as a ﬂuorine, a nitro, or a nitrile, we chose, in

addition to the 2,4,6-triﬂuorophenyl fragment, the correspond-

ing 2-ﬂuoro-, 2,6-diﬂuoro-, 2-ﬂuoro-4-cyano-, and 2,6-diﬂuoro-

4-cyano-phenyl C6 fragments. Similarly, among the amine

fragments at C7, all instances matched by molecular pair

analysis that were found to produce comparable or better

activity than 3 (i.e., 7, 52−54 and 60) were selected. By

systematically combining the preferred set of C6 and C7

fragments, a panel of 25 compounds could be designed that

based on SAR ﬁndings would be expected to exhibit relatively

potent Class I MT-stabilizing activity. As summarized in Table

3, several examples (13) from this set were synthesized and

tested. In all cases, these compounds were conﬁrmed to be

active Class I triazolopyrimidines. Nine analogues exhibited

comparable (i.e., 66, 67, 73, and 74) or improved (i.e., 64, 69

and 70−72) in vitro potency compared to lead compound 3,

with 72 being particularly eﬀective in the AcTub assay when

tested at either 10 or 1 μM. Evaluation of individual

atropoisomers, 67 and 68, indicated that the former may be

more potent than the latter. Among derivatives bearing

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Table 2. MT-Stabilizing Activity of Triazolopyrimidines Modiﬁed at C7

a

Fold-changes in AcTub and α-Tub levels in QBI293 cells after 4 h of incubation with test compounds at either 1 or 10 μM. Reported values for

AcTub and α-Tub represent the fold-change over control (DMSO)-treated cells (*p < 0.05 and **p < 0.01 by one-way ANOVA); numbers in

b

parentheses represent the fold-change of AcTub over positive control-treated cells (i.e., 100 nM of 5). Class I compounds are those producing a

concentration-dependent increase in AcTub levels and that do not cause >15% reduction in α-Tub at either concentration; class II compounds are

those that cause >15% decrease in α-Tub at either 1 or 10 μM compound concentration. NA = not applicable as the test compound does not cause

c

signiﬁcant changes in AcTub or total α-Tub when tested at a 1 or a 10 μM concentration. Test compound was tested at 10 and 30 μM.

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a

Table 3. MT-Stabilizing Activity of Triazolopyrimidines Modiﬁed at C7 and/or C6

a

Fold-changes in AcTub and α-Tub levels in QBI293 cells after 4 h of incubation with test compounds at either 1 or 10 μM. Reported values for

AcTub and α-Tub represent the fold-change over control (DMSO)-treated cells (*p < 0.05 and **p < 0.01 by one-way ANOVA); numbers in

parentheses represent the fold-change of AcTub over positive control-treated cells (i.e., 100 nM of 5).

ﬂuorinated cyanobenzene fragments at C6, evaluation of a

representative example (69) in primary neurons conﬁrmed

that compounds of this type increase AcTub levels and prevent

the characteristic loss of neuronal MTs that is observed after

incubation with the phosphatase inhibitor, okadaic acid (OA)

(Figure 4). Although the cellular data do not allow for

resolution of a detailed MT structure, the AcTub staining

clearly shows that incubation with 69 protects the MT network

from OA-induced collapse (Figure 4).

Interestingly, an assessment of lipophilicity of several

compounds (Figure 5), as determined by experimental

determinations of the distribution coeﬃcient between n-

octanol and water at pH 7.4 (i.e., log D_7.4values), revealed

that the presence of a nitrile moiety in the para position of the

phenyl ring at C6 produces a signiﬁcant lowering of log D_7.4

values that becomes more pronounced in 2,6-diﬂuorophenyl

congeners, such as 66, 69, and 72.

Importantly, an evaluation of brain-to-plasma (B/P) ratios

of selected examples 1 h after administration to mice

conﬁrmed that the nitrile derivatives 66, 69, and 72 are

readily brain-penetrant with B/P values >0.6 (Table 4). This

result was conﬁrmed by a 16 h brain and plasma PK study with

69 (Figure 6A). Moreover, a comparison of plasma PK of lead

compound 3 and 69 indicates a greater metabolic stability of

the nitrile-containing congener (Figure 6B).

Computational Studies. To investigate the nature of the

interaction of Class I triazolopyrimidines with tubulin and to

develop an in silico model that may ultimately be predictive of

MT-stabilizing activity, we conducted docking studies with

both active and inactive triazolopyrimidine congeners using the

X-ray cocrystal structure of a tubulin preparation in complex

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Figure 4. To conﬁrm that the nitrile-containing triazolopyrimidine derivatives identiﬁed in these studies can stabilize neuronal MTs under

conditions of tau loss-of-function, we examined the ability of representative compound, 69, to prevent the MT collapse that occurs from reduced

binding of hyperphosphorylated tau to axonal MTs after treatment of neuron cultures with the phosphatase inhibitor, OA. (A) Primary rat cortical

neurons treated with 1 μM reference compound 3 or 69 in the absence of OA (−OA) show increased axonal acetyl-tubulin staining relative to

those receiving vehicle only. Upon treatment with OA (+OA) in the absence of a compound (Vehicle), there is a dramatic reduction in axonal

AcTub staining with fragmentation of MTs and neuronal processes (also see ref 11). Co-addition of 3 or 69 (1 μM) with OA results in

normalization of AcTub staining and axonal processes. (B) ELISA determination of AcTub levels in homogenates from primary mouse cortical

neurons treated with 1 or 10 μM of 69, or vehicle, in the presence of OA. The higher concentration of 69 resulted in AcTub levels comparable to

those in neurons without OA treatment.

Figure 5. Comparison of selected compounds based on experimental log D_7.4values (triangles) and MT-stabilizing activity (squares) expressed as

the average activity in the AcTub assay at 1 and 10 μM normalized to positive control (i.e., 100 nM 5). log D_7.4values were determined via the

shake ﬂask method (experiments run by Analiza, Inc.).

with Class I triazolopyrimidine 54^13,23(PDB: 5NJH; Figure

7A). The X-ray studies highlighted two key π−π interactions

between 54 and tubulin: one involving the pyrimidine core and

Tyr224 of α-Tub and a second between the triﬂuorophenyl

group and Tyr210 (α-Tub).¹³Both of these interactions are

likely essential for a correct orientation of the triazolopyr-

imidine derivative within the binding pocket and substituents

that interfere with these critical interactions are likely to result

in reduced binding and biological activity. Indeed, docking of

inactive derivative, 30, within the triazolopyrimidine binding

site suggests that the relatively large volume of the sulfone

moiety disrupts the critical π−π stacking interaction with the

two tyrosine residues, leading to a diﬀerent binding pose

(Figure 7B) and, ultimately, a dramatic loss in MT-stabilizing

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C6, an additional H-bond interaction with the backbone of

Glu207 (α-Tub) is also observed, which, in some cases (e.g.,

72), may contribute to better ΔG of binding (Figure 7D) and

MT-stabilizing activity (see Figure 5).

Table 4. Ratio between Brain and Plasma Compound

Concentration 1 h after Administration (i.p. Injection) of

the Test Compound

cpd #

i.p. dose (mg/kg)

B/P

The binding modes of the diﬀerent compounds were further

evaluated by calculating the free energy of binding via a

Molecular Mechanics Generalized Born Surface Area

(MMGBSA) approach.²⁴Interestingly, by plotting the

computed MMGBSA free energetics of the Class I ligands in

complex with tubulin against the MT-stabilizing activity

(expressed as the average of the activity at 1 and 10 μM

compound concentrations in the QBI293 AcTub assay, relative

to the positive control), a moderately high Pearson correlation

was found [r = −0.70, P-value (two-tailed) < 0.0001, Figure 8].

A 3D-QSAR study was also performed through a “ﬁeld

points” template, obtained from the cocrystallized compound.

These ﬁeld points deﬁne the shape and electrostatic and

hydrophobic properties of the molecules and their spatial

distribution.²⁵For this study, 52 active and inactive Class I

compounds were randomly partitioned to a training set (47)

and a test set (5), while the experimental in vitro activity was

computed using the normalized activity, expressed as the log of

the average of the activity at 1 and 10 μM in the AcTub assay

relative to the positive control (5) and deﬁned as a dependent

variable. In this case, the model shows a comparatively better

correlation with a Pearson r = 0.90 (Figure 9A), which was

cross-validated by leave-one-out (LOO) technique, q²= 0.62.

Interestingly, the 3D-QSAR model highlighted the following

steric and electrostatic contributions to the predicted activity

(Figure 9B): a favorable electrostatic contribution (green) near

the para position of the phenyl ring at C6 and strong steric

contributions of the amine fragment at C7, as indicated by the

large size of the dark teal ﬁeld point (Figure 9B). Importantly,

the combination of the binding free energies and the QSAR

models (Figure 10) appeared to accurately place all of the most

active MT-stabilizing compounds identiﬁed in this study

within a well-deﬁned area of lower ΔG of binding and higher

QSAR predicted activity. Thus, the combinations of these

models may provide a valuable tool to guide future design of

novel potent MT-stabilizing triazolopyrimidines.

3

66

60

69

52

72

5

2.5

5

2.5

5

2.5

2.7

0.7

2.1

0.7

1.4

0.7

a

Cassette dosing of three compounds (66, 69, and 72).

Figure 6. Brain and plasma pharmacokinetics of 69 after 5 mg/kg i.p.

dosing to CD1 mice (A) and comparison of plasma pharmacokinetics

of 69 and 3.¹²

DISCUSSION

■

CNS-active MT-stabilizing compounds have been proposed as

promising therapeutic candidates to treat AD and related

neurodegenerative tauopathies.²⁶In this context, the essential

prerequisites for compound selection and advancement are the

ability to both cross the blood−brain barrier and normalize the

dynamics of MTs in the axons of neurons, such that eﬃcient

axonal transport can be maintained or restored. MT-stabilizing

triazolopyrimidines have a number of potentially attractive

features as MT-stabilizing compounds for CNS indications,⁹

including synthetic tractability, a generally good brain

penetration, and oral bioavailability. Selected members from

this class of compounds have already been the focus of

mechanism-of-action¹³and SAR studies that were based

largely on cell-free experiments and cytotoxicity studies in

cancer cell-lines.^15,16,27Although clearly informative, such

studies did not provide particular insight into the eﬀects that

triazolopyrimidines can have on cellular MTs. Studies from our

laboratories in which the activity of MT-active triazolopyr-

imidines was evaluated by monitoring compound-dependent

changes in cellular levels of tubulin as well as changes in levels

of known markers of MT-stabilization, such as acetylated- and

Figure 7. Diﬀerent views of the cocrystal structure (PBD: 5NJH) of

54 bound within a tubulin preparation (A) and the docked structures

of 30 (B), 3 (C), and 72 (D) within the triazolopyrimidine binding

site.

activity. In the case of active Class I compounds, including lead

compound 3 (Figure 7C), docking studies generally identify

very similar binding poses and sets of interactions as seen in

the cocrystal structure of 54. However, for derivatives bearing a

nitro or a nitrile at the para position of the ﬂuorinated ring at

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Figure 8. MMGBSA scores for the Class I compounds vs MT-stabilizing activity in QBI293 cells expressed as the log of the average of the activity

avg AcTub

1&10 μM

at 1 and 10 μM in the AcTub assay, relative to the positive control, logj

z.

AcTub_{0.1 μM}of 5

Figure 9. (A) QSAR predicted vs experimental MT-stabilizing activity in QBI293 cells for training and test sets. In both cases, MT-stabilizing

avg AcTub

i

y

1&10μM

j

z

activity is expressed as the log of the average of the activity at 1 and 10 μM in the AcTub assay, relative to the positive control, logj

z.

j

z

AcTub_0.1μMof 5

k

{

(B) Visual representation of the ﬁeld and steric contributions to predicted activity for compound 72.

detyrosinated-α-tubulin, showed that diﬀerent congeners can

generally elicit one of two cellular responses discriminated by

whether (Class II) or not (Class I) these compounds reduce

tubulin levels.¹¹Furthermore, although one selected prototype

from Class I triazolopyrimidines, 3, has proven useful in

obtaining validation of this class in mouse models of AD,

further characterization of the structural determinants that may

be most important to impart the desired MT-stabilizing eﬀect

in cells is a necessary step that could facilitate the identiﬁcation

of candidates for clinical development.

important for potency as well as ADME-PK properties,^11,12our

exploration of the SAR of triazolopyrimidines focused

primarily on identifying speciﬁc features (e.g., electrostatic,

hydrophobic, shape) of the C6 and C7 fragments that may be

preferred for Class I MT-stabilizing triazolopyrimidines. Data

from the cell-based MT-stabilization assays revealed diﬀerent

structural and property features that appear to have a relatively

high impact on the biological activity. In particular, a

comparison of active congeners of either class suggests that

the electrostatic characteristics of the substituent in the para

position of the phenyl ring at C6 play an important role in

determining the cellular phenotype, while the degree and

pattern of ﬂuorination can modulate potency (Figure 2).

Indeed, an analysis of in vitro biological activity versus the

Hammett σ values of the diﬀerent substituents in para suggests

Given that our previous studies showed that the substituent

in the para position of the phenyl ring (C6 fragment) of

triazolopyrimidines can play an important role in determining

the particular cellular response (i.e., Class I or II) elicited by

these compounds, whereas the C7 fragment may be especially

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Figure 10. 3D summary plot of Class I triazolopyrimidines showing the experimental MT-stabilizing activity in the QBI293 assay, the MMGBSA

score value (ΔG bind), and the QSAR predicted activity. The experimental MT-stabilizing activity of test compounds is plotted both via color

avg AcTub

1&10μM

coding and as the log of the average of the activity at 1 and 10 μM in the AcTub assay, relative to the positive control, logj

z, see the

AcTub_0.1μMof 5

Experimental Section for further details.

Figure 12. Activity atlas analysis revealing key features of Class I

triazolopyrimidines that are necessary for MT-stabilizing activity: (a)

red and cyan colors show positive and negative ﬁeld regions,

respectively; (b) brown beige regions show hydrophobic interaction

sites required for activity; (c) activity cliﬀs analysis revealing a

favorable hydrophobic region (green) and a favorable negative

electrostatic region (cyan).

Figure 11. Eﬀect of the stereoelectronic properties of the substituent

in para. Active Class I and II compounds are ranked based on the

Hammett σ_pvalues.²⁸

that the desired Class I MT-stabilizing activity generally

requires the presence of an electron-poor phenyl ring. Diﬀerent

ﬂuorinated phenyl fragments can be tolerated, especially those

bearing either one or two ﬂuorine atoms in the ortho positions,

and a para position that is either unsubstituted or substituted

with an additional ﬂuorine or other electron-withdrawing

groups, such as nitro and nitrile.

a general trend, whereby with the sole exception of the para-

chloro derivative, 21, positive σ values (e.g., ﬂuorine, nitro, or

nitrile) are typically conducive to Class I activity, whereas σ

values < 0 (e.g., hydroxyl or methoxy groups) appear to be

associated with the Class II phenotype (Figure 11). Thus, the

SAR data of the C6 fragment of triazolopyrimidines indicate

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With respect to the SAR of the fragment linked at C7, the

data presented in Table 2 revealed a number of structural and

property requirements for optimal MT-stabilizing activity. In

particular, in addition to aliphatic amines being typically

preferred (e.g., over ethers or aromatic amines), a general trend

emerged linking MT-stabilizing activity in cells with the

lipophilic character and size of the C7 fragment (Figure 3). An

interesting observation made during these studies was the fact

that in speciﬁc instances, the chirality of the aliphatic amine

fragment can play a role in determining the cellular phenotype

elicited by the triazolopyrimidine. Although the conﬁguration

of the chiral center of the amine fragment of 3 or 7 appears to

impact the potency of these compounds in the AcTub assay

without aﬀecting α-Tub levels (cf., 7 with 48, and 3 with 45),

in selected cases, such as compounds 46 and 47, the inversion

of chiral conﬁguration was found to cause a shift in the cellular

phenotype, with 46 being a Class I compound while its

enantiomer, 47, clearly produced a marked reduction (∼40%)

in α-Tub (Class II). Interestingly, docking studies with 46 and

47 did not reveal signiﬁcant diﬀerences in binding modes that

could help explain the diﬀerent cellular phenotypes elicited by

the two enantiomers. In addition, a side-by-side comparison of

the cytotoxicity of enantiomers 46 (Class I) and 47 (Class II)

in rapidly dividing QBI293 or HeLa cells did not reveal

signiﬁcant diﬀerences in IC₅₀values (Supporting Information),

suggesting that both Class I and II triazolopyrimidines exhibit

comparable antimitotic eﬀects. These observations indicate

that neither docking nor IC₅₀values in cell cytotoxicity assays

should be used to predict whether a triazolopyrimidine would

stabilize or disrupt the MT network. Given that such a

distinction is an essential aspect driving the prioritization of

candidates for neurodegenerative tauopathies, an evaluation of

compounds in a cellular assay that assesses compound eﬀect on

α/β-tubulin levels as well as increases in markers of MT

stabilization is ultimately a necessary step. Although such

assays have some limitations, such as a relatively low-

throughput and possible day-to-day variability that can be

minimized by normalizing the activity of test compounds to a

positive control, the SAR data generated in this study led to the

identiﬁcation of several characteristics that are important for

Class I MT-stabilizing triazolopyrimidines. Indeed, a qual-

itative assessment of the combined C6 and C7 SAR data of all

Class I triazolopyrimidines tested in this study, which was

conducted using Activity atlas analysis,²⁹identiﬁed distinct

regions/characteristics that are shared by the vast majority of

Class I congeners (Figure 12A,B); these include a positive

electrostatic region in the proximity of the amine fragment at

C7 (indicated in red, Figure 12A), a negative electrostatic

region in the proximity of the para position of the aryl group at

C6 (cyan, Figure 12A), and two hydrophobic interaction

regions near the ﬂuorinated phenyl at C6 and the aliphatic

amine fragment at C7 (yellow, Figure 12B). Furthermore, an

activity cliﬀ analysis (Figure 12C), which highlights structural

diﬀerences of similar compounds that produce a dispropor-

tionally high impact on the biological activity (i.e., activity cliﬀ

regions),³⁰revealed that an increase of negative electrostatic

ﬁeld at the para position of the phenyl ring (cyan), as

associated with substituents such as nitro and nitrile, as well as

the presence of a favorable hydrophobic region in C7 (green),

produce a drastic increase in MT-stabilizing activity.

Collectively, these ﬁndings, combined with the development

of relatively predictive models that are based on calculated

binding free energies and QSAR, provide valuable insights that

could facilitate the design of Class I triazolopyrimidines.

Importantly, by combining the preferred C6 and C7

fragments found in the course of these SAR studies, several

new Class I triazolopyrimidine analogues have been identiﬁed

(Table 3), including multiple examples (64, 69, and 70−72)

that exhibit both improved in vitro activity as well as reduced

lipophilic character compared to lead compound 3. In primary

cortical neurons, 69 was found to protect the MT-network

against OA-induced collapse in a manner comparable to 3

(Figure 4), suggesting that these new congeners, like 3, may be

beneﬁcial in normalizing MT-deﬁcits in tauopathy neurons.

Interestingly, although the eﬀect of the nitrile moiety in

lowering lipophilicity is well documented,³¹a direct compar-

ison of experimentally determined log D_7.4values of matched

paired compounds bearing a nitrile moiety in the para position

of the ring at C6 (cf., 66 with 67/68; 69 with 70; and 72 with

73) demonstrates that the ﬂuorination state of the aromatic

ring plays an equally important role as evidenced by the

signiﬁcantly lower log D_7.4values observed when both ortho

positions are ﬂuorinated (Figure 5). Of notice, in spite of the

reduced lipophilic character of 66, 69, and 72 compared to the

corresponding 2,4,6-triﬂuorophenyl congeners (3, 60, and 52),

these compounds retained excellent brain penetration (Table

4). Furthermore, evaluation of brain/plasma PK of 69 after i.p.

injection revealed that this compound exhibits greater

metabolic stability (i.e., reduced clearance and increased

plasma half-life) compared to existing lead (3). Thus, taken

together, our results show that an appropriate combination of

relatively bulky/lipophilic aliphatic amines at C7 with polar

ﬂuorinated cyanobenzene fragments at C6 results in Class I

congeners that exhibit both improved MT-stabilizing activity in

cells as well as improved lipophilic eﬃciency and metabolic

stability.

CONCLUSIONS

■

Brain-penetrant, MT-stabilizing triazolopyrimidines have pro-

duced promising results in diﬀerent mouse models of AD,

suggesting that compounds from this class may be considered

as candidate therapeutics for neurodegenerative tauopathies.

However, depending on the substitution pattern, this type of

compounds can elicit very diﬀerent cellular responses by either

promoting MT stabilization or, conversely, disrupting MT

integrity in cells. The studies presented here deﬁne the most

important stereoelectronic characteristics of the C6 and C7

fragments of MT-active triazolopyrimidines that can lead to

relatively potent MT-stabilizing activity in cells without

reduction in cellular tubulin levels. These results, which

provide additional insight into the SAR of MT-stabilizing

triazolopyrimidines, enabled both the development of

predictive models as well as the design of congeners exhibiting

improved properties and in vitro biological activity. Com-

pounds of this type hold promise as candidate treatments for

neurodegenerative tauopathies.

EXPERIMENTAL SECTION

■

Materials and Methods. All solvents were of reagent grade. All

reagents were purchased from Aldrich or Acros and used as received.

Thin-layer chromatography (TLC) was performed with 0.25 mm E.

Merck precoated silica gel plates. Silica gel column chromatography

was performed with silica gel 60 (particle size 0.040−0.062 mm)

supplied by Silicycle and Sorbent Technologies. TLC spots were

detected by viewing under a UV light. Melting points (mp) were

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acquired on a Mel-Temp II (model: 1001) and are uncorrected.

Infrared (IR) spectra were recorded on a Bruker, model Alpha

spectrometer (part number 1003271/03). Proton (¹H) and carbon

(¹³C) NMR spectra were recorded on a 500 MHz Bruker AMX-500

spectrometer or 600 MHz Bruker AVANCE III spectrometer.

Fluorine (¹⁹F) NMR spectra were recorded on a 500 MHz Bruker

AVANCE II spectrometer. Chemical shifts were reported relative to

puriﬁcation via silica gel ﬂash chromatography or by reverse-phase

HPLC yielded the desired product.

General Procedure D (Addition of the Side Chain). According

to reported procedures,^15,16to a suspension of NaH (4.0 equiv) in a

2:1 mixture of DMSO and tetrahydrofuran (THF) (0.35 M) was

added the appropriate aminoalcohol (4.0 equiv), and the mixture was

heated to 60 °C for 1 h. The resulting solution was treated with a

solution of triﬂuoroarene (1.0 equiv) in a 1:1 mixture DMSO and

THF (0.5 M). The reaction mixture was stirred at 60 °C for 3 h and

monitored by LCMS. If the starting material remained after 3 h,

additional aminoalcohol (4.0 equiv) and NaH (4.0 equiv) were

added, sequentially, and the reaction mixture was heated for 16 h.

Following complete consumption of the starting material, the reaction

mixture was cooled to rt and diluted with H₂O and EtOAc. The

organic layer was washed with H₂O and brine, and the combined

aqueous layers were extracted with EtOAc (×3). The combined

organic layers were dried over MgSO₄, ﬁltered, and concentrated.

Puriﬁcation by reverse-phase HPLC provided the desired product.

(S)-5-Chloro-6-phenyl-N-(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (8). General procedure C was

followed using 5,7-dichloro-6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine

(0.500 g, 1.88 mmol) (93) and (S)-2-amino-1,1,1-triﬂuoropropane

hydrochloride (0.840 g, 5.64 mmol). Puriﬁcation via preparative

reverse-phase chromatography provided the title compound as a white

powder (0.066 g, 0.19 mmol, 10%).¹H NMR (500 MHz, CDCl₃): δ

8.40 (s, 1H), 7.62−7.52 (m, 3H), 7.42−7.36 (m, 1H), 7.35−7.28 (m,

1H), 5.68 (s, 1H), 4.63 (s, 1H), 1.29 (d, J = 6.8 Hz, 3H) ppm; ¹³C

NMR (126 MHz, CDCl₃): δ 157.17, 155.44, 145.72, 131.67, 131.51,

130.54, 130.09, 129.96, 129.82, 124.90 (q, J = 282.4 Hz), 105.26,

50.67 (q, J = 31.5 Hz), 15.02 (q, J = 1.7 Hz) ppm; IR (ﬁlm) ν: 3435,

1619, 1577, 1491, 1460, 1384, 1366, 1338, 1264, 1248, 1179, 1145,

1085, 1024, 955, 906, 767, 742, 702, 652, 615 cm⁻¹; HRMS (ES⁺):

calcd for C₁₄H₁₂ClF₃N₅[M + H]⁺, 342.0728; found, 342.0726.

(S)-5-Chloro-6-(4-ﬂuorophenyl)-N-(1,1,1-triﬂuoropropan-2-

yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (9). General proce-

dure C was followed using 5,7-dichloro-4-ﬂuorophenyl-[1,2,4]-

triazolo[1,5-a]pyrimidine (0.300 g, 1.04 mmol) (94) and (S)-2-

amino-1,1,1-triﬂuoropropane hydrochloride (0.465 g, 3.11 mmol).

Puriﬁcation via preparative reverse-phase chromatography provided

the title compound as a white powder (0.126 g, 0.35 mmol, 34%). ¹H

NMR (500 MHz, CDCl₃): δ 8.39 (s, 1H), 7.42−7.35 (m, 1H), 7.36−

7.24 (m, 3H), 5.65 (s, 1H), 4.77 (s, 1H), 1.34 (d, J = 6.8 Hz, 3H)

ppm; ¹³C NMR (126 MHz, CDCl₃): δ 163.57 (d, J = 251.3 Hz),

157.29, 155.56, 153.93, 145.86, 133.63 (d, J = 8.4 Hz), 132.61 (d, J =

8.4 Hz), 127.57 (d, J = 3.7 Hz), 124.94 (q, J = 282.4 Hz), 117.34 (d, J

= 22.0 Hz), 117.16 (d, J = 22.0 Hz), 104.31, 50.83 (q, J = 31.6 Hz),

15.04 (d, J = 2.1 Hz) ppm; IR (ﬁlm) ν: 3421, 1619, 1574, 1504, 1461,

1366, 1337, 1247, 1180, 1146, 1096, 1024, 957, 841, 766, 738, 610

cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₁₀ClF₄N₅Na [M + Na]⁺,

382.0459; found, 382.0464.

1

solvents. Data for H NMR spectra are reported as follows: chemical

shift [ppm, referenced to protium; s = singlet, d = doublet, t = triplet,

q = quartet, quint = quintet, dd = doublet of doublets, td = triplet of

doublets, ddd = doublet of doublet of doublets, bs = broad singlet, m

= multiplet, coupling constant (Hz), and integration]. High-resolution

mass spectra were measured using an Agilent 6230 time-of-ﬂight mass

spectrometer with a Jet stream electrospray ionization source. Single-

crystal X-ray structure determinations were performed using a Bruker

MicroStar with an APEX II detector, double-bounce micro-focus

optics, and a Cu rotating anode source. Analytical reverse-phase

(Sunﬁre C18; 4.6 mm × 50 mm, 5 mL) HPLC was performed with a

Gilson HPLC equipped with UV and a mass detector. All samples

were analyzed employing a linear gradient from 10 to 90% of ACN in

H₂O over 8 min and ﬂow rate of 1 mL/min. Preparative reverse-phase

HPLC puriﬁcations were performed on a Gilson instrument

employing Waters SunFire preparative C₁₈OBD columns (5 μm 19

mm × 50 mm or 19 mm × 100 mm). Puriﬁcations were carried out

employing a linear gradient from 10 to 90% of ACN in H₂O for 15

min with a ﬂow rate of 20 mL/min. All ﬁnal compounds were found

to be >95% pure by HPLC.

General Procedure A (Synthesis of Diethylmalonate

Derivatives). To a suspension of NaH (60 wt % in mineral oil)

(1.00 equiv) in anhydrous 1,4-dioxane (previously degassed with N₂)

at 60 °C under N₂was slowly added diethyl malonate (3.00 equiv)

and the resulting mixture was stirred for 10 min. CuBr (1.20 equiv)

and aryl bromide derivate were then added and the reaction mixture

(1.75 mol/L of aryl bromide derivate) was heated to reﬂux overnight.

The reaction mixture was then cooled to rt, quenched with HCl 12 N

(1.40 equiv), ﬁltered, and washed with H₂O. The ﬁltrate was extracted

with EtOAc (×3). The combined organic extracts were washed with

brine, dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure. Puriﬁcation via silica gel column chromatography or

preparative reverse-phase HPLC provided the desired diethylmalo-

nate derivatives.

General Procedure B (Synthesis of Dichloro-triazolopyr-

imidine Derivatives). A mixture of the appropriate diethylmalonate

derivate (1.00 equiv), 3-amino-1,2,4-triazole (1.05 equiv), and

tributylamine (1.05 equiv) in a sealed tube was stirred at 170 °C

for 2 h. After cooling to 130 °C, the reaction mixture was diluted with

toluene to reach approximately a concentration of 1.00 M of the

diethylmalonate derivative. The reaction was then cooled to 50 °C

and an aqueous solution of NaOH (50 wt %) (0.160 mL/mmol of

diethylmalonate derivate) was added. The resulting mixture was

stirred at 0 °C for 10 min and then ﬁltered to obtain the

triazolopyrimidine disodic salt intermediate as a solid, which was

washed with cold toluene, dried, and used directly without further

puriﬁcation. This disodic derivate (1.00 equiv) and POCl₃(17.80

equiv) were mixed in a sealed tube and heated to 130 °C for 6 h. The

reaction was then quenched with H₂O and extracted with EtOAc

(×2). The combined organic extracts were washed with H₂O (×5),

then brine, dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. The resulting dichloro-triazolopyrimidine was used

directly without further puriﬁcation.

General Procedure C (Addition of the Amine). According to a

reported procedure,¹⁵to a solution of 5,7-dichloro-6-(2,4,6-triﬂuor-

ophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (1.0 equiv) in N,N-dime-

thylformamide (DMF) (0.1 M) at rt was added the appropriate amine

(1.5−3.0 equiv either as HCl salt or free base) followed by i-Pr₂NEt

(3.0 equiv) or Et₃N (3.0 equiv), if necessary. The reaction mixture

was stirred for 0.5−16 h and then diluted with H₂O. The organic layer

was washed with a 1 N HCl (×2), the aqueous phase was extracted

with EtOAc (×3), and the combined organic layers were washed with

brine (×2), dried over MgSO₄, ﬁltered, and concentrated. Finally,

5-Chloro-6-(2-ﬂuorophenyl)-N-(-1,1,1-triﬂuoropropan-2-yl)-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (10). General procedure

C was followed using 5,7-dichloro-6-(2-ﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.300 g, 1.04 mmol) (95) and (S)-2-amino-1,1,1-

triﬂuoropropane hydrochloride (0.465 g, 3.11 mmol). Puriﬁcation via

silica gel column chromatography (0−30% EtOAc in hexanes)

provided the title compound as a white powder (0.060 g, 0.17 mmol,

16%). ¹H NMR (500 MHz, CDCl₃): mixture of diastereomers δ 8.39

(s, 1H), 7.61−7.52 (m, 1H), 7.40−7.27 (m, 3H), 6.08−5.49 (m, 1H),

4.95−4.22 (m, 1H), 1.34 (d, J = 6.8 Hz, 3H) ppm; ¹³C NMR (126

MHz, CDCl₃): δ 161.79, 161.59, 159.80, 159.61, 157.71, 157.50,

155.50, 155.43, 154.20, 153.83, 145.83, 145.41, 133.41, 133.39,

132.79, 132.72, 132.66, 132.35, 132.34, 128.18, 128.00, 125.93,

125.76, 125.50, 125.47, 125.44, 125.41, 123.69, 123.51, 121.45,

121.27, 119.42, 119.30, 119.18, 117.21, 117.04, 116.70, 116.53, 99.20,

98.22, 51.26, 51.00, 50.75, 50.49, 50.24, 15.18, 15.16, 15.07, 15.05

ppm; IR (ﬁlm) ν: 3435, 1618, 1554, 1491, 1460, 1368, 1248, 1179,

1144, 1094, 1025, 960, 759, 651, 616 cm⁻¹; HRMS (ES⁺): calcd for

C₁₄H₁₀ClF₄N₅Na [M + Na]⁺, 382.0459; found, 382.0441.

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(S)-5-Chloro-6-(2,6-diﬂuorophenyl)-N-(1,1,1-triﬂuoropro-

pan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (12). General

procedure C was followed using 5,7-dichloro-6-(2,6-diﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (0.280 g, 0.93 mmol) (97) and (S)-

2-amino-1,1,1-triﬂuoropropane hydrochloride (0.417 g, 2.79 mmol).

Puriﬁcation via preparative reverse-phase chromatography provided

155.78, 155.73, 155.72, 155.66, 152.56, 152.50, 152.43, 152.38,

152.33, 152.20, 152.09, 151.96, 150.51, 150.46, 150.38, 150.35,

150.17, 150.06, 149.94, 145.95, 145.91, 145.82, 145.78, 145.74,

128.38, 128.34, 128.30, 127.36, 127.31, 127.28, 126.04, 125.99,

123.80, 123.75, 121.58, 121.55, 121.18, 121.05, 120.19, 120.17,

120.06, 120.05, 119.34, 119.29, 119.24, 119.19, 119.16, 119.02,

103.47, 103.35, 51.42, 51.35, 51.16, 51.10, 50.91, 50.84, 50.65, 50.59,

15.08 ppm; IR (ﬁlm) ν: 3436, 2923, 2853, 1632, 1463, 1383, 1270,

1147 cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₁₀ClF₅N₅[M + H]⁺,

378.0539; found, 378.0550.

1

the title compound as a white powder (0.024 g, 0.06 mmol, 7%). H

NMR (500 MHz, CDCl₃): δ 8.33 (s, 1H), 7.54−7.45 (m, 1H), 7.10−

7.00 (m, 2H), 6.19−6.10 (m, 1H), 4.76 (s, 1H), 1.39 (d, J = 6.9 Hz,

3H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 161.12 (dd, J = 252.3, 5.5

Hz), 160.82 (dd, J = 250.9, 5.3 Hz), 157.72, 155.26, 154.16, 145.89,

133.01 (t, J = 10.0 Hz), 124.59 (dd, J = 564.1, 282.1 Hz), 112.43 (dd,

J = 21.5, 3.4 Hz), 112.07 (dd, J = 21.5, 3.6 Hz), 108.90 (t, J = 20.2

Hz), 92.39, 50.86 (q, J = 32.3 Hz), 14.98 ppm. IR (ﬁlm) ν: 3227,

1630, 1602, 1460, 1383, 1266, 1239, 1195, 1126, 1003, 900, 809, 765,

652, 622 cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₁₀ClF₅N₅[M + H]⁺,

378.0539; found, 378.0556.

(S)-5-Chloro-6-(3,5-diﬂuorophenyl)-N-(1,1,1-triﬂuoropro-

pan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (13). General

procedure C was followed using 5,7-dichloro-6-(3,5-diﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (0.350 g, 1.16 mmol) (98) and (S)-

2-amino-1,1,1-triﬂuoropropane hydrochloride (0.521 g, 3.49 mmol).

Puriﬁcation via silica gel column chromatography (0−30% EtOAc in

hexanes) provided the title compound as a white powder (0.148 g,

0.39 mmol, 34%). ¹H NMR (500 MHz, CDCl₃): δ 8.40 (s, 1H), 7.03

(dt, J = 8.6, 2.3 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 7.5 Hz,

1H), 5.64 (s, 1H), 4.82 (s, 1H), 1.38 (d, J = 6.8 Hz, 3H) ppm; ¹³C

NMR (126 MHz, CDCl₃): δ 163.72 (dd, J = 253.2, 13.0 Hz), 163.66

(dd, J = 251.7, 11.7 Hz), 156.45, 155.71, 153.96, 145.57, 134.65 (t, J

= 10.0 Hz), 124.85 (q, J = 282.4 Hz), 115.03 (dd, J = 21.9, 3.7 Hz),

114.04 (dd, J = 21.9, 3.7 Hz), 105.99 (t, J = 24.8 Hz), 103.18, 51.07

(q, J = 31.6 Hz), 15.02 (d, J = 1.9 Hz) ppm; IR (ﬁlm) ν: 3435, 1620,

1590, 1521, 1493, 1460, 1434, 1384, 1362, 1333, 1264, 1192, 1159,

1125, 1081, 1028, 987, 956, 906, 855, 763, 670, 653, 628 cm⁻¹;

HRMS (ES⁻): calcd for C₁₄H₈ClF₅N₅[M − H]⁻, 376.0394; found,

376.0376.

(S)-5-Chloro-6-(2,3,4-triﬂuorophenyl)-N-(-1,1,1-triﬂuoropro-

pan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (16). General

procedure C was followed using 5,7-dichloro-6-(2,3,4-triﬂuorophen-

yl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.300 g, 0.94 mmol) (101) and

(S)-2-amino-1,1,1-triﬂuoropropane hydrochloride (0.422 g, 2.82

mmol). Puriﬁcation via preparative reverse-phase chromatography

provided the title compound as a white powder (0.050 g, 0.13 mmol,

13%). ¹H NMR (500 MHz, CDCl₃): mixture of diastereomers δ 8.37

(s, 1H), 7.24−7.05 (m, 2H), 6.05−5.61 (m, 1H), 5.20−4.47 (m, 1H),

1.42 (d, J = 6.3 Hz, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃): mixture

of diastereomers δ 157.56, 157.24, 155.75, 155.67, 154.40, 153.95,

153.77, 153.74, 153.69, 153.67, 153.62, 153.59, 151.74, 151.71,

151.66, 151.63, 151.58, 151.56, 151.39, 151.36, 151.31, 151.28,

151.06, 151.03, 150.98, 150.95, 149.37, 149.34, 149.29, 149.26,

149.05, 149.02, 148.97, 148.94, 146.17, 145.67, 142.19, 142.07,

141.95, 141.86, 141.74, 141.62, 140.16, 140.04, 139.91, 139.83,

139.70, 139.58, 128.20, 127.95, 127.23, 127.20, 127.18, 127.17,

127.14, 126.15, 126.14, 126.11, 126.10, 126.09, 126.08, 126.05,

125.95, 125.70, 123.71, 123.46, 121.47, 121.22, 117.07, 117.04,

116.96, 116.93, 116.91, 116.84, 116.80, 113.96, 113.92, 113.91,

113.88, 113.81, 113.78, 113.77, 113.74, 97.55, 96.47, 51.59, 51.34,

51.31, 51.08, 51.05, 50.83, 50.80, 50.55, 15.21, 15.19, 15.07, 15.06

ppm. IR (ﬁlm) ν: 3388, 3338, 1619, 1553, 1509, 1486, 1463, 1367,

1352, 1251, 1181, 1143, 1090, 1041, 1025, 987, 957, 906, 878, 808,

766, 738, 698, 680, 653, 619 cm⁻¹; HRMS (ES⁺): calcd for

C₁₄H₉ClF₆N₅[M + H]⁺, 396.0445; found, 396.0464.

(S)-5-Chloro-6-(2,5-diﬂuorophenyl)-N-(-1,1,1-triﬂuoropro-

pan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (14). General

procedure C was followed using 5,7-dichloro-6-(2,5-diﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (0.400 g, 1.33 mmol) (99) and (S)-

2-amino-1,1,1-triﬂuoropropane hydrochloride (0.596 g, 3.99 mmol).

Puriﬁcation via silica gel column chromatography (0−30% EtOAc in

hexanes) provided the title compound as a white powder (0.020 g,

0.05 mmol, 4%). ¹H NMR (500 MHz, CDCl₃): mixture of

diastereomers δ 8.40 (s, 1H), 7.32−7.24 (m, 2H), 7.15−7.02 (m,

1H), 6.07−5.46 (m, 1H), 4.71 (d, J = 156.9 Hz, 1H), 1.39 (d, J = 6.8

Hz, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃): mixture of

diastereomers δ 159.86, 159.84, 157.90, 157.88, 157.71, 157.68,

157.28, 157.15, 155.95, 155.93, 155.76, 155.73, 155.64, 155.58,

154.21, 153.94, 145.78, 145.47, 128.12, 127.98, 125.88, 125.74,

123.64, 123.49, 121.39, 121.25, 120.68, 120.62, 120.56, 120.54,

120.50, 120.47, 120.41, 120.35, 119.99, 119.97, 119.80, 119.78,

119.54, 119.51, 119.47, 119.44, 119.35, 119.32, 119.28, 119.25,

118.91, 118.89, 118.72, 118.70, 118.52, 118.45, 118.33, 118.26,

118.00, 117.93, 117.81, 117.74, 98.19, 97.30, 51.46, 51.25, 51.21,

50.99, 50.95, 50.74, 50.70, 50.49, 15.19, 15.18, 15.09, 15.08 ppm; IR

(ﬁlm) ν: 3441, 1622, 1494, 1461, 1424, 1383, 1251, 1182, 1145, 1092,

1025, 979, 877, 821, 775 cm⁻¹; HRMS (ES⁺): calcd for

C₁₄H₁₀ClF₅N₅[M + H]⁺, 378.0539; found, 378.0509.

(S)-5-Chloro-6-(3,4-diﬂuorophenyl)-N-(1,1,1-triﬂuoropro-

pan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (15). General

procedure C was followed using 5,7-dichloro-6-(3,4-diﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (0.150 g, 0.50 mmol) (100) and (S)-

2-amino-1,1,1-triﬂuoropropane hydrochloride (0.223 g, 1.49 mmol).

Puriﬁcation via preparative reverse-phase chromatography provided

the title compound as a white powder (0.007 g, 0.02 mmol, 4%).¹H

NMR (500 MHz, CDCl₃): mixture of diastereomers δ 8.42 (s, 1H),

7.45−7.34 (m, 1H), 7.22−7.05 (m, 2H), 5.71−5.47 (m, 1H), 5.02−

4.63 (m, 1H), 1.37 (d, J = 6.9 Hz, 3H) ppm; ¹³C NMR (126 MHz,

CDCl₃): mixture of diastereomers δ 157.04, 157.01, 156.89, 156.88,

(S)-5-Chloro-6-(2,4,6-triﬂuorobenzyl)-N-(1,1,1-triﬂuoropro-

pan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (17). General

procedure C was followed using 5,7-dichloro-6-(2,4,6-triﬂuoroben-

zyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.300 g, 0.90 mmol) (103) and

(S)-2-amino-1,1,1-triﬂuoropropane hydrochloride (0.404 g, 2.70

mmol). Puriﬁcation via silica gel column chromatography (0−25%

EtOAc in hexanes) provided the title compound as a white powder

(0.085 g, 0.21 mmol, 23%). ¹H NMR (500 MHz, CDCl₃): δ 8.32 (s,

1H), 6.73 (t, J = 8.3 Hz, 2H), 6.32−6.12 (m, 1H), 5.23 (d, J = 10.2

Hz, 1H), 4.27−4.08 (m, 2H), 1.50 (d, J = 6.9 Hz, 3H) ppm; ¹³C

NMR (126 MHz, CDCl₃): δ 162.21 (dt, J = 251.1, 16.0 Hz), 161.52

(dd, J = 247.9, 10.9 Hz), 161.41 (dd, J = 248.0, 10.8 Hz), 157.36,

155.42, 154.39, 146.49, 125.14 (q, J = 282.1 Hz), 108.38 (td, J = 19.0,

4.8 Hz), 101.31−100.77 (m), 100.73, 51.82 (q, J = 31.5 Hz), 21.06,

15.30 (d, J = 2.2 Hz) ppm; IR (ﬁlm) ν: 3427, 3276, 3143, 3001, 2926,

2855, 1621, 1552, 1496, 1464, 1443, 1378, 1337, 1274, 1244, 1182,

1145, 1118, 1091, 1035, 999, 959, 910, 842, 804, 762, 734, 680, 648,

613 cm⁻¹; HRMS (ES⁺): calcd for C₁₅H₁₁ClF₆N₅[M + H]⁺,

410.0602; found, 410.0608.

(S)-5-Chloro-6-(2,6-diﬂuoro-4-methylphenyl)-N-(1,1,1-tri-

ﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(18). General procedure C was followed using 5,7-dichloro-6-(2,6-

diﬂuoro-4-methylphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.050 g,

0.16 mmol) (104) and (S)-1,1,1-triﬂuoropropan-2-amine (0.038 g,

0.33 mmol). Puriﬁcation via silica gel column chromatography (0−

25% EtOAc in hexanes) provided the title compound as a white

powder (0.028 g, 0.071 mmol, 65%). ¹H NMR (600 MHz, CDCl₃): δ

8.36 (s, 1H), 6.91 (d, J = 9.0 Hz, 2H), 6.00−5.94 (m, 1H), 4.73 (s,

1H), 2.45 (s, 3H), 1.39 (d, J = 7.0 Hz, 3H) ppm; ¹³C NMR (150

MHz, CDCl₃): δ 160.75 (dd, J = 251.4, 7.0 Hz), 160.50 (dd, J =

249.9, 7.3 Hz), 158.07, 155.34, 154.11, 145.83, 144.86 (t, J = 10.2

Hz), 124.59 (q, J = 282.1 Hz), 112.93 (ddd, J = 65.8, 21.3, 4.0 Hz),

105.67 (t, J = 20.9 Hz), 92.50, 22.72−20.28 (m), 15.19 ppm; IR

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(ﬁlm) ν: 1618, 1559, 1143, 840 cm⁻¹; HRMS (ES⁺): calcd for

C₁₅H₁₂N₅ClF₅[M + H]⁺, 392.0696; found, 392.0693.

(23). To a solution of (S)-5-chloro-6-(2,6-diﬂuoro-4-nitrophenyl)-N-

(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(0.025 g, 0.059 mmol) (22) in 0.100 mL of H₂O/MeOH (1:1) were

added iron powder (0.017 g, 0.30 mmol) and ammonium chloride

(0.013 g, 0.24 mmol). The mixture was stirred at 80 °C for 2 h and

then ﬁltered and rinsed with hot methanol. The ﬁltrate was

evaporated under reduced pressure, and the resulting mixture was

puriﬁed by HPLC to aﬀord the product as a white solid (0.016 g, 0.04

mmol, 69%). ¹H NMR (600 MHz, CDCl₃): δ 8.37 (s, 1H), 6.37 (t, J

= 9.8 Hz, 2H), 5.93 (s, 1H), 4.86 (s, 1H), 4.28 (s, 2H), 2.17 (s, 1H),

1.41−1.39 (m, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 161.86

(ddd, J = 246.8, 32.1, 8.7 Hz), 158.99, 155.32, 154.09, 151.02 (t, J =

13.9 Hz), 146.15, 130.31, 128.98, 124.72 (q, J = 281.8 Hz), 98.19

(ddd, J = 63.6, 25.1, 3.2 Hz), 97.13 (t, J = 21.1 Hz), 92.99, 50.71 (q, J

= 32.4 Hz), 31.08, 15.35 ppm; IR (ﬁlm) ν: 3341, 1651, 1614, 1587,

1346 cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₁₁ClF₅N₆[M + H]⁺,

393.0648; found, 393.0646.

(S)-5-Chloro-6-(2,6-diﬂuoro-4-methoxyphenyl)-N-(1,1,1-tri-

ﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(24). To a solution of (S)-5-chloro-6-(2,4,6-triﬂuorophenyl)-N-

(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(0.020 g, 0.05 mmol) (7) in 0.3 mL of THF was added 0.04 mL of a

30% sodium methoxide solution in methanol at 0 °C. The reaction

mixture was heated for 2 h at 60 °C in a microwave reactor and then

cooled to rt and diluted with aqueous ammonium chloride solution.

The aqueous phase was extracted with EtOAc (×3). The combined

organic layers were washed with brine (×2), dried (MgSO₄), ﬁltered,

and concentrated. Puriﬁcation by reverse-phase HPLC provided the

title compound as a white solid (0.015 g, 0.036 mmol, 71% yield). ¹H

NMR (500 MHz; CDCl₃): δ 8.45 (s, 1H), 6.67−6.65 (m, 2H), 5.94−

5.93 (m, 1H), 4.77−4.74 (m, 1H), 3.90 (s, 3H), 1.41 (d, J = 6.8 Hz,

3H) ppm; ¹³C NMR (126 MHz; CDCl₃): δ 163.48 (t, J = 13.9 Hz),

161.80 (dd, J = 249.9, 9.0 Hz), 161.54 (dd, J = 248.4, 8.9 Hz), 159.55

(q, J = 40.7 Hz), 158.79, 155.09, 146.15, 124.63 (q, J = 282.0 Hz),

100.62 (t, J = 21.1 Hz), 98.93 (td, J = 26.8, 3.0 Hz), 92.71, 56.30,

50.87 (q, J = 32.1 Hz), 15.32 ppm; IR (ﬁlm) ν: 3340, 2953, 2924,

2848, 1642, 1618, 1579, 1553, 1147 cm⁻¹; HRMS (ES⁺): calcd for

C₁₅H₁₂ClF₅N₅O [M + H]⁺, 408.0651; found, 408.0652.

(S)-5-Chloro-6-(2,6-diﬂuoro-4-(triﬂuoromethyl)phenyl)-N-

(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-

amine (19). General procedure C was followed using 5,7-dichloro-6-

(2,6-diﬂuoro-4-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.350 g, 1.16 mmol) (105) and (S)-2-amino-1,1,1-

triﬂuoropropane hydrochloride (0.389 g, 2.60 mmol). Puriﬁcation via

silica gel column chromatography (0−30% EtOAc in hexanes)

provided the title compound as a white powder (0.240 g, 0.54 mmol,

62%). ¹H NMR (500 MHz, CDCl₃): δ 8.42 (s, 1H), 7.42 (d, J = 7.3

Hz, 2H), 5.87 (d, J = 10.5 Hz, 1H), 4.74 (s, 1H), 1.44 (d, J = 6.9 Hz,

3H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 161.32 (dd, J = 255.5, 5.5

Hz), 160.96 (dd, J = 253.8, 5.6 Hz), 157.25, 155.74, 154.36, 145.83,

135.62 (qt, J = 35.0, 9.8 Hz), 124.53 (q, J = 280.6 Hz), 122.33 (qt, J =

273.4, 2.9 Hz), 112.99 (t, J = 20.1 Hz), 110.42 (dp, J = 24.9, 3.7 Hz),

109.93 (dp, J = 25.0, 3.8 Hz), 91.17, 51.21 (q, J = 32.2 Hz), 15.31 (d,

J = 1.9 Hz) ppm; IR (ﬁlm) ν: 3208, 2925, 2855, 1620, 1555, 1461,

1436, 1369, 1249, 1179, 1143, 1111, 1090, 1037, 963, 909, 896, 868,

819, 767, 734, 680, 652, 612 cm⁻¹; HRMS (ES⁺): calcd for

C₁₅H₉ClF₈N₅[M + H]⁺, 446.0413; found, 446.0438.

(S)-4-(5-Chloro-7-((1,1,1-triﬂuoropropan-2-yl)amino)-

[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuorobenzonitrile

(20). General procedure C was followed using 4-(5,7-dichloro-

[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuorobenzonitrile (0.030

g, 0.09 mmol) (106) and (S)-1,1,1-triﬂuoropropan-2-amine (0.022

g, 0.19 mmol). Puriﬁcation via silica gel column chromatography (0−

40% EtOAc in hexanes) provided the title compound as a white

1

powder (0.020 g, 0.05 mmol, 54%). H NMR (600 MHz, CDCl₃): δ

8.41 (s, 1H), 7.44 (t, J = 7.1 Hz, 2H), 6.00 (d, J = 10.7 Hz, 1H), 4.77

(s, 1H), 1.45 (d, J = 6.9 Hz, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃):

δ 161.28 (dd, J = 256.4, 5.9 Hz), 160.87 (dd, J = 254.7, 6.0 Hz),

156.84, 155.68, 154.32, 124.46 (q, J = 282.1 Hz), 116.58 (t, J = 11.8

Hz), 116.47 (ddd, J = 67.6, 25.4, 4.1 Hz), 115.79 (d, J = 3.5 Hz),

114.93 (t, J = 20.0 Hz), 90.61, 51.27 (q, J = 32.2 Hz), 29.83, 15.14

ppm; ¹⁹F NMR (469 MHz): δ −78.31, −102.98, −105.00 ppm; IR

(KBr): 2923, 2239, 1617, 1556, 1141 cm⁻¹; HRMS (ES⁺): calcd for

C₁₅H₉N₆ClF₅[M + H]⁺, 403.0492; found, 403.0486.

(S)-5-Chloro-6-(4-chloro-2,6-diﬂuorophenyl)-N-(1,1,1-tri-

ﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(21). General procedure C was followed using 5,7-dichloro-6-(4-

chloro-2,6-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.050 g,

0.15 mmol) (108) and (S)-1,1,1-triﬂuoropropan-2-amine (0.035 g,

0.31 mmol). Puriﬁcation via silica gel column chromatography (0−

40% EtOAc in hexanes) provided the title compound as a white

powder (0.035 g, 0.085 mmol, 57%). ¹H NMR (600 MHz, CDCl₃): δ

8.40 (s, 1H), 7.18−7.16 (m, 2H), 5.93−5.88 (m, 1H), 4.75 (s, 1H),

1.43 (d, J = 6.9 Hz, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ

160.84 (ddd, J = 253.4, 48.9, 9.1 Hz), 157.67, 155.57, 154.21, 145.82,

138.62 (t, J = 14.0 Hz), 124.53 (q, J = 280.5 Hz), 114.47−113.17

(m), 51.00 (q, J = 32.9 Hz), 15.27 ppm. IR (ﬁlm) ν: 3000, 1571,

1517, 1142 cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₉Cl₂F₅N₅[M + H]⁺,

412.0150; found, 412.0146.

(S)-5-Chloro-6-(4-((3-(dimethylamino)propyl)sulﬁnyl)-2,6-

diﬂuorophenyl)-N-((S)-1,1,1-triﬂuoropropan-2-yl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (26) and (S)-5-Chloro-6-(4-

((3-(dimethylamino)propyl)sulfonyl)-2,6-diﬂuorophenyl)-N-

(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-

amine (27). To

a solution of (S)-5-chloro-6-(4-((3-

(dimethylamino)propyl)thio)-2,6-diﬂuorophenyl)-N-(1,1,1-triﬂuoro-

propan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (0.050 g, 0.100

mmol) (25) in CH₂Cl₂(1.00 mL) was added m-CPBA (70%, 0.025 g,

0.100 mmol). After 2 h, the reaction mixture was concentrated under

reduced pressure and puriﬁed by reverse-phase HPLC to obtain 26

(0.017 g, 0.03 mmol, 32%) and 27 (0.027 g, 0.05 mmol, 52%). (26):

¹H NMR (600 MHz, CDCl₃): δ 8.37 (s, 1H), 8.19 (s, 1H), 7.02 (d, J

= 8.0 Hz, 2H), 6.69 (s, 1H), 5.41 (s, 1H), 3.76−3.67 (m, 2H), 3.40

(d, J = 10.2 Hz, 7H), 3.22−3.15 (m, 2H), 2.44−2.37 (m, 2H), 1.43

(d, J = 6.9 Hz, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 166.95,

161.56 (dd, J = 253.3, 6.8 Hz), 160.98 (dd, J = 252.6, 6.6 Hz), 157.65,

155.36, 154.85, 146.84, 125.91 (q), 112.00−110.04 (m), 107.56−

105.57 (m), 92.69, 68.14, 57.73 (d, J = 11.8 Hz), 51.16 (q, J = 31.8

Hz), 29.62 (d, J = 2.0 Hz), 22.63 (d, J = 1.7 Hz), 14.82 ppm; IR

(ﬁlm) ν: 3053, 1589, 1535, 1178 cm⁻¹; HRMS (ES⁺): calcd for

(S)-5-Chloro-6-(2,6-diﬂuoro-4-nitrophenyl)-N-(1,1,1-triﬂuor-

opropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (22).

General procedure C was followed using 5,7-dichloro-6-(2,6-

diﬂuoro-4-nitrophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.070 g,

0.21 mmol) (109) and (S)-1,1,1-triﬂuoropropan-2-amine (0.048 g,

0.42 mmol). Puriﬁcation via silica gel column chromatography (0−

40% EtOAc in hexanes) provided the title compound as a white

1

C₁₉H₂₁ClF₅N₆OS [M + H]⁺, 511.1101; found, 511.1100. (27): H

1

powder (0.075 g, 0.18 mmol, 88%). H NMR (600 MHz, CDCl₃): δ

NMR (600 MHz, CDCl₃): mixture of diastereomers δ 8.38 (s, 1H),

8.11 (s, 1H), 7.50 (dd, J = 16.6, 7.4 Hz, 1H), 7.17 (d, J = 7.0 Hz,

0.5H), 5.88 (s, 0.5H), 5.59 (s, 0.5H), 3.86−3.78 (m, 1H), 3.77−3.70

(m, 0.5H), 3.69−3.62 (m, 1H), 3.36 (dd, J = 25.9, 3.6 Hz, 6H), 3.40−

3.27 (m, 0.5H), 2.99−2.90 (m, 1H), 2.51−2.40 (m, 1H), 2.36−2.26

(m, 1H), 1.42 (dd, J = 6.9, 2.9 Hz, 3H) ppm; ¹³C NMR (150 MHz,

CDCl₃): δ 167.52, 163.09−161.10 (m), 161.44 (ddd, J = 256.9, 21.4,

4.9 Hz), 157.00, 156.86, 155.45−155.33 (m), 155.12, 129.28−119.96

(m), 112.17 (td, J = 20.5, 4.6 Hz), 109.28−107.40 (m), 92.88, 92.30,

67.84, 67.54, 58.59, 58.51, 57.34, 56.93, 52.19, 51.77, 51.29 (td, J =

8.41 (s, 1H), 8.00−7.98 (m, 2H), 5.99 (d, J = 10.6 Hz, 1H), 4.78 (s,

1H), 1.46 (d, J = 6.8 Hz, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ

161.15 (dd, J = 257.2, 5.8 Hz), 160.73 (dd, J = 255.3, 6.1 Hz), 156.78,

155.75, 154.37, 150.43 (t, J = 10.5 Hz), 145.80, 124.47 (q, J = 282.2

Hz), 116.00 (t, J = 20.5 Hz), 108.53 (ddd, J = 61.2, 27.3, 4.0 Hz),

90.62, 51.38 (q, J = 32.1 Hz), 15.20 (d, J = 1.5 Hz) ppm; IR (ﬁlm) ν:

1618, 1530, 1489, 1143, 1043 cm⁻¹; HRMS (ES⁺): calcd for

C₁₄H₉ClF₅N₆O₂[M + H]⁺, 423.0390; found, 423.0384.

(S)-6-(4-Amino-2,6-diﬂuorophenyl)-5-chloro-N-(1,1,1-tri-

ﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

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31.8, 17.1 Hz), 15.61, 15.10, 14.68, 14.46 (d, J = 1.7 Hz) ppm; IR

(ﬁlm) ν: 3285, 1589, 1535, 1142 cm⁻¹; HRMS (ES⁺): calcd for

C₁₉H₂₁ClF₅N₆O₂S [M + H]⁺, 527.1050; found, 527.1045.

1124 cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₁₂ClF₃N₅[M + H]⁺,

342.0733; found, 342.0733.

5-Chloro-N-propyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (36). General procedure C was followed

using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.030 g, 0.09 mmol) (102) and propan-1-amine (0.012

g, 0.20 mmol). Puriﬁcation via silica gel column chromatography (0−

40% EtOAc in hexanes) provided the title compound as a white

(S)-5-Chloro-6-(2,6-diﬂuoro-4-(methylthio)phenyl)-N-(1,1,1-

triﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(28). To a suspension of sodium methanethiolate (0.331 mL, 0.71

mmol, 4 equiv) in DMSO (2 mL) at rt was added (S)-5-chloro-6-

(2,4,6-triﬂuorophenyl)-N-(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (0.070 g, 0.18 mmol, 1 equiv) (7) and the

mixture was heated for 3 h at 60 °C before it was quenched at rt with

a saturated NH₄Cl solution. The mixture was extracted with EtOAc

and the combined organic layers were washed 5 times with H₂O,

dried, and concentrated under reduced pressure to furnish the title

compound as a white solid (0.070 g, 0.17 mmol, 93%). ¹H NMR (600

MHz, CDCl₃): δ 8.38 (s, 1H), 6.94−6.92 (m, 2H), 5.92 (br s, 1H),

4.79 (br s, 1H), 2.56 (s, 3H), 1.41 (d, J = 6.0 Hz, 3H) ppm; ¹³C NMR

(150 MHz, CDCl₃): δ 161.79 (dd, J = 40.77, 6.0 Hz), 160.11 (dd, J =

39.3, 7.6 Hz), 158.15, 155.51, 154.22, 146.81 (t, J = 10.6 Hz), 145.95,

124.63 (q, J = 282.4 Hz), 109.06 (dd, J = 24.2, 3.0 Hz), 108.69 (dd, J

= 24.9, 3.0 Hz), 104.03 (t, J = 9.0 Hz), 92.28, 50.81 (q, J = 18.1 Hz),

15.31, 15.10 ppm; HRMS (ES⁺): calcd for C₁₄H₈N₄ClF₆O [M + H]⁺,

397.0285; found, 397.0279.

1

powder (0.020 g, 0.06 mmol, 62%). H NMR (600 MHz, CDCl₃): δ

8.33 (s, 1H), 6.84−6.82 (m, 2H), 6.48 (t, J = 5.9 Hz, 1H), 2.98 (q, J =

6.7 Hz, 2H), 1.57 (h, J = 7.3 Hz, 2H), 0.85 (t, J = 7.4 Hz, 4H) ppm;

¹³C NMR (150 MHz, CDCl₃): δ 163.93 (dt, J = 253.7, 15.4 Hz),

161.77 (ddd, J = 250.6, 14.9, 8.5 Hz), 158.16, 155.07, 153.65, 146.36,

107.18 (td, J = 20.8, 4.9 Hz), 101.12−100.62 (m), 88.91, 45.31,

29.83, 23.33, 11.04 ppm; IR (ﬁlm) ν: 2923, 1573, 1439, 1122 cm⁻¹;

HRMS (ES⁺): calcd for C₁₄H₁₂N₅ClF₃[M + H]⁺, 342.0728; found,

342.0725.

5-Chloro-N-iso-butyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (37). General procedure C was

followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.030 g, 0.09 mmol) (102) and 2-methylpropan-

1-amine (0.014 g, 0.20 mmol). Puriﬁcation via silica gel column

chromatography (0−40% EtOAc in hexanes) provided the title

5-Chloro-6-(2,6-diﬂuoro-4-(methylsulﬁnyl)phenyl)-N-((S)-

1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-

amine (29). To a solution of (S)-5-chloro-6-(2,6-diﬂuoro-4-

(methylthio)phenyl)-N-(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (0.029 g, 0.07 mmol, 1 equiv) (28) in

CH₂Cl₂(0.7 mL) at rt was added m-CPBA (0.012 g, 0.07 mmol, 1

equiv). After 1 h, the reaction was quenched with H₂O and extracted

twice with EtOAc. The combined organic fractions were washed with

a sat. aq. Na₂S₂O₃solution, dried, and concentrated under vacuum.

The crude yellow material was puriﬁed by reverse-phase HPLC (C18

column, H₂O/ACN 90/10 to 10/90, 15 min ramp) and lyophilized to

furnish the title compound (0.021 g, 0.05 mmol, 70%) as a white

1

compound as a white powder (0.027 g, 0.08 mmol, 81%). H NMR

(600 MHz, CDCl₃): δ 8.33 (s, 1H), 6.88−6.81 (m, 2H), 6.56 (t, J =

6.1 Hz, 1H), 2.84 (t, J = 6.5 Hz, 2H), 1.77−1.70 (m, 1H), 0.84 (d, J =

6.7 Hz, 8H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 164.05 (dt, J =

253.8, 15.1 Hz), 161.72 (ddd, J = 250.4, 14.9, 8.4 Hz), 158.13, 155.05,

153.63, 146.42, 107.31 (td, J = 20.8, 4.8 Hz), 102.26−98.14 (m),

88.95, 50.83, 29.05, 19.79 ppm; IR (ﬁlm) ν: 2961, 2924, 1637, 1577,

1434 cm⁻¹; HRMS (ES⁺): calcd for C₁₅H₁₄N₅ClF₃[M + H]⁺,

356.0884; found, 356.0887.

5-Chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidin-7-ol (38). This compound has been obtained as a side

product from the attempt at synthetizing 5-chloro-N-isobutyl-N-

(2,2,2-triﬂuoroethyl)-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidin-7-amine. General procedure C was followed using 5,7-

dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine

(0.064 g, 0.20 mmol) (102), 2-methyl-N-(2,2,2-triﬂuoroethyl)-

propan-1-amine hydrochloride (0.115 g, 0.60 mmol), and i-Pr₂NEt

(0.139 mL, 0.80 mmol). Puriﬁcation via preparative reverse-phase

1

solid. H NMR (600 MHz, CDCl₃): mixture of diastereomers δ 8.43

(s, 1H), 8.08 (s, 1H), 7.98 (d, J = 6.0 Hz, 1H), 7.58 (d, J = 6.0 Hz,

1.6H), 7.51 (d, J = 6.0 Hz, 0.4H), 7.42 (d, J = 9.0 Hz, 1H), 7.38 (d, J

= 6.0 Hz, 0.4H), 7.33 (d, J = 6.0 Hz, 0.6H), 5.93 (br s, 0.5H), 5.87 (br

s, 0.3H), 4.75 (br s, 0.4H), 4.62 (br s, 0.5H), 2.88 (s, 3H), 1.44−1.42

(m, 3H) ppm; HRMS (ES⁺): calcd for C₁₄H₈N₄ClF₆O [M + H]⁺,

440.0366; found, 440.0365.

(S)-5-Chloro-6-(2,6-diﬂuoro-4-(methylsulfonyl)phenyl)-N-

(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-

amine (30). To a solution of (S)-5-chloro-6-(2,6-diﬂuoro-4-

(methylthio)phenyl)-N-(1,1,1-triﬂuoropropan-2-yl)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (0.017 g, 0.04 mmol, 1 equiv) (28) in

CH2Cl2 (0.7 mL) at rt was added 3-chlorobenzoperoxoic acid (0.021

g, 0.12 mmol, 3 equiv). After 1 h, the reaction was quenched with

H₂O and extracted twice with EtOAc. The combined organic

fractions were washed with a sat. aq Na₂S₂O₃solution, dried, and

concentrated under vacuum. Puriﬁcation by reverse-phase HPLC

(C18 column, H₂O/ACN 90/10 to 10/90, 15 min ramp) provided

1

HPLC provided the title compound (0.023 g, 0.08 mmol, 38%). H

NMR (500 MHz; MeOD): δ 8.74 (s, 1H), 6.97 (dd, J = 8.7, 7.8 Hz,

2H) ppm; ¹³C NMR (126 MHz; MeOD): δ 164.78 (dt, J = 250.7,

16.4 Hz), 162.77 (ddd, J = 248.8, 15.2, 9.5 Hz), 159.40, 156.62,

108.75 (td, J = 21.2, 4.8 Hz), 102.66, 101.52−101.06 (m) ppm; IR

(ﬁlm) ν: 3446, 2789, 2684, 2549, 1683, 1667, 1655, 1635, 1616, 1600,

1558, 1532 cm⁻¹.

N-(5-Chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidin-7-yl)isobutyramide (39). To a solution of 5-chloro-6-

(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(0.035 g, 0.12 mmol, 1.00 equiv) (102) in anhydrous CH₂Cl₂(0.450

mL) was added Et₃N (0.022 mL, 0.15 mmol, 1.30 equiv) and DMAP

(0.001 g, 0.01 mmol, 0.10 equiv) under N₂and the resulting solution

was stirred at 0 °C. iso-Butyryl chloride (0.015 mL, 0.14 mmol, 1.20

equiv) was slowly added and the reaction was stirred at 0 °C for 30

min and rt for 18 h. Then, Et₃N (0.022 mL, 0.15 mmol, 1.30 equiv)

and iso-butyryl chloride (0.015 mL, 0.14 mmol, 1.20 equiv) were

added and the reaction was stirred at rt for 2 h. The reaction mixture

was then concentrated under reduced pressure. Puriﬁcation via

preparative reverse-phase HPLC provided the title compound (0.023

1

the title compound (0.013 g, 0.03 mmol, 71%) as a white solid. H

NMR (600 MHz, CDCl₃): δ 8.43 (s, 1H), 7.73 (d, J = 6.0 Hz, 2H),

5.82 (d, J = 6.0 Hz, 1H), 4.78 (br s, 1H), 3.21 (s, 3H), 1.45 (d, J = 6.0

Hz, 3H) ppm; HRMS (ES⁺): calcd for C₁₄5H₁₂N₄5ClF₅O₂S [M +

H]⁺, 456.0315; found, 456.0311.

5-Chloro-N-isopropyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (35). Following general proce-

dure C using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.064 g, 0.20 mmol) (102), propan-2-amine

(0.036 g, 0.60 mmol), i-Pr₂NEt (0.078 g, 0.60 mmol), and reverse-

phase HPLC puriﬁcation aﬀorded the title compound as a white solid

1

g, 0.06 mmol, 53%). H NMR (500 MHz, CDCl₃): δ 8.72 (s, 1H),

1

8.47 (s, 1H), 6.81 (t, J = 8.1 Hz, 2H), 2.66 (hept, J = 6.9 Hz, 1H),

1.12 (d, J = 6.9 Hz, 6H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ

173.47, 163.85 (dt, J = 252.8, 15.4 Hz), 160.51 (dd, J = 251.6, 9.0

Hz), 160.39 (dd, J = 251.7, 8.9 Hz), 158.48, 156.09, 153.73, 141.44,

107.40 (td, J = 19.8, 4.9 Hz), 104.48, 100.85 (td, J = 26.1, 3.4 Hz),

36.73, 19.10 ppm; IR (ﬁlm) ν: 3415, 2974, 1732, 1618, 1515, 1487,

1440, 1384, 1263, 1230, 1153, 1126, 1040, 997, 842, 738, 669 cm⁻¹;

(0.030 g, 0.09 mmol, 44% yield). H NMR (500 MHz; CDCl₃): δ

8.46 (s, 1H), 6.90−6.85 (m, 2H), 6.32 (d, J = 8.7 Hz, 1H), 3.62−3.60

(m, 1H), 1.19 (d, J = 6.4 Hz, 6H) ppm; ¹³C NMR (126 MHz;

CDCl₃): δ 164.27 (dt, J = 254.4, 15.2 Hz), 161.64 (dd, J = 251.0, 8.4

Hz), 161.53 (dd, J = 251.0, 8.4 Hz), 159.04, 153.77, 145.89, 106.92

(td, J = 20.7, 4.9 Hz), 101.46−101.02 (m), 90.00, 46.34, 23.82 ppm;

IR (ﬁlm) ν: 3344, 2982, 2936, 2873, 1615, 1579, 1264, 1208, 1171,

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HRMS (ES⁺): calcd for C₁₅H₁₁ClF₃N₅NaO [M + Na]⁺, 392.0502;

found, 392.0488.

J = 8.2 Hz, 1H), 5.75 (td, J = 55.4, 1.6 Hz, 1H), 4.32 (br s, 1H), 1.31

(d, J = 6.8 Hz, 3H) ppm; ¹³C NMR (151 MHz, CDCl₃): δ 164.46 (dt,

J = 254.9, 15.1 Hz), 161.5 (ddd, J = 250.66, 8.4, 5.4 Hz), 161.41 (ddd,

J = 252.17, 8.3, 5.3 Hz), 157.94, 155.46, 154.22, 114.59 (t, J = 246.2

Hz), 105.90 (td, J = 20.5, 4.8 Hz), 101.54 (dtd, J = 29.9, 25.9, 4.1

Hz), 90.88, 51.09 (t, J = 24.16 Hz), 42.38 (t, J = 552.1 Hz), 29.82,

14.61 (t, J = 3.8 Hz) ppm; IR (ﬁlm) ν: 1613, 1578, 1552, 1527, 1492,

1461, 1440, 1360, 1263, 1124, 1208, 1171, 1134, 1122, 1100, 1083

cm⁻¹; HRMS (ES⁺): calcd for C₁₅H₁₀ClF₅N₄[M + H]⁺, 378.0539;

found, 378.0537.

5-Chloro-N-phenyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (40). To a solution of 5,7-dichloro-6-

(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.053 g,

0.17 mmol) (102) and aniline (0.015 mL, 0.17 mmol) in DMF

(1.67 mL) was added K₂CO₃(0.046 g, 0.33 mmol) and the resulting

mixture was stirred for 24 h at 30 °C. The reaction mixture was then

diluted with H₂O. The organic layer was washed with a 1 N HCl

(×2), the aqueous phase was extracted with EtOAc (×3), and the

combined organic layers were washed with brine (×2), dried over

MgSO₄, ﬁltered, and concentrated. Puriﬁcation via silica gel column

chromatography (0−40% EtOAc in hexanes) provided the title

5-Chloro-N-(1-ﬂuoropropan-2-yl)-6-(2,4,6-triﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (44). General procedure

C was followed using 5,7-dichloro-6-(2,5-diﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidine (0.073 g, 0.23 mmol) (102), 1-ﬂuoropro-

pan-2-amine hydrochloride (0.053 g, 0.47 mmol), and Et₃N (0.130

mL, 0.91 mmol). Puriﬁcation via silica gel column chromatography

(0−30% EtOAc in hexanes) provided the title compound as a white

1

compound as a white powder (0.049 g, 0.13 mmol, 78%). H NMR

(500 MHz, CDCl₃): δ 8.43 (s, 1H), 8.24 (s, 1H), 7.16−7.11 (m, 3H),

7.10−7.00 (m, 2H), 6.36 (t, J = 8.2 Hz, 2H) ppm; ¹³C NMR (126

MHz, CDCl₃): δ 164.69 (dt, J = 255.2, 15.1 Hz), 161.57 (ddd, J =

253.8, 15.0, 8.2 Hz), 161.32 (ddd, J = 251.3, 14.9, 8.2 Hz), 158.29,

155.56, 153.98, 146.56, 124.53 (q, J = 283.5 Hz), 101.86 (td, J = 25.8,

4.1 Hz), 101.36 (td, J = 25.2, 2.4 Hz), 90.99, 58.99 (q, J = 29.4 Hz),

28.34, 19.81, 16.74 ppm; IR (ﬁlm) ν: 3423, 1638, 1613, 1563, 1491,

1440, 1335, 1261, 1197, 1122, 1036, 998, 940, 840, 766, 694, 668

cm⁻¹; HRMS (ES⁺): calcd for C₁₇H₁₀ClF₃N₅[M + H]⁺, 376.0571;

found, 376.0571.

1

powder (0.059 g, 0.16 mmol, 72%). H NMR (600 MHz, CDCl₃): δ

8.35 (s, 1H), 6.93−6.83 (m, 2H), 6.26 (d, J = 8.1 Hz, 1H), 4.37 (ddd,

J = 66, 9.6, 3.5 Hz, 1H), 4.29 (ddd, J = 66, 9.6, 4.3 Hz, 1H), 4.01 (br

s, 1H), 1.26 (d, J = 6.8 Hz, 3H) ppm; ¹³C NMR (151 MHz, CDCl₃):

δ 164.29 (dt, J = 254.6, 15.2 Hz), 161.53 (ddd, J = 250.7, 14.9, 8.3,

Hz), 161.52 (ddd, J = 249.9, 14.9, 8.3, Hz), 157.98, 155.28, 154.08,

146.19, 106.53 (td, J = 20.7, 4.7 Hz), 101.39 (tdd, J = 25.9, 9.1, 4.1

Hz), 90.03, 85.42 (d, J = 175.2 Hz), 49.79 (d, J = 19.6 Hz), 17.59 (d,

J = 5.3 Hz), 14.32 ppm; IR (ﬁlm) ν: 1639, 1611, 1594, 1580, 1492,

1468, 1440, 1358, 1239, 1207, 1171 cm⁻¹; HRMS (ES⁺): calcd for

C₁₅H₁₁ClF₄N₄[M + H]⁺, 360.0634; found, 360.0636.

(S)-5-Chloro-N-(3-methylbutan-2-yl)-6-(2,4,6-triﬂuorophen-

yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (45). General proce-

dure C was followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (0.119 g, 0.37 mmol) (102) and (S)-

3-methylbutan-2-amine (0.090 mL, 0.78 mmol). Puriﬁcation via silica

gel column chromatography (0−15% EtOAc in hexanes) provided the

title compound as a white powder (0.099 g, 0.27 mmol, 72%). The

spectroscopic properties of the compound were identical to those

reported for its enantiomer, 3.¹¹

N-Benzyl-5-chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (41). Following general procedure C

using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.064 g, 0.20 mmol) (102) and benzylamine (0.064 g,

0.60 mmol), reverse-phase HPLC puriﬁcation aﬀorded the title

1

compound as a yellow solid (0.024 g, 0.06 mmol, 31% yield). H

NMR (500 MHz; CDCl₃): δ 8.45 (s, 1H), 7.33−7.31 (m, 3H), 7.08−

7.06 (m, J = 6.5, 2.7 Hz, 2H), 6.89−6.88 (m, 1H), 6.89−6.86 (m,

1H), 6.72 (dd, J = 8.5, 6.8 Hz, 2H), 4.33 (d, J = 5.9 Hz, 2H) ppm; ¹³C

NMR (126 MHz; CDCl₃): δ 164.32 (dt, J = 254.1, 15.1 Hz), 164.01,

161.77 (ddd, J = 250.8, 14.9, 8.2 Hz), 159.63 (q, J = 40.8 Hz), 159.26,

154.03, 146.40, 135.30, 129.28, 128.75, 126.77, 106.56 (td, J = 20.7,

4.8 Hz), 101.15−100.70 (m), 90.43, 47.76 ppm; IR (ﬁlm) ν: 3352,

3268, 3113, 3067, 3033, 2928, 1642, 1617, 1595, 1575, 1495, 1441,

1124, 1036 cm⁻¹; HRMS (ES⁺): calcd for C₁₈H₁₂ClF₃N₅[M + H]⁺,

390.0733; found, 390.0730.

(R)-5-Chloro-N-(1,1,1-triﬂuoro-3-methylbutan-2-yl)-6-(2,4,6-

triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (46).

General procedure C was followed using 5,7-dichloro-6-(2,4,6-

triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.123 g, 0.38

mmol) (102) and (R)-1,1,1-triﬂuoro-3-methyl-2-butylamine (0.152

mL, 1.15 mmol). Puriﬁcation via silica gel column chromatography

(0−15% EtOAc in hexanes) provided the title compound as a white

5-Chloro-6-(2,4,6-triﬂuorophenyl)-7-((1,1,1-triﬂuoropro-

pan-2-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine (42). To sodium

hydride (60%, 0.015 g, 0.38 mmol) suspended in THF (3 mL) was

added 1,1,1-triﬂuoropropan-2-ol (0.034 mL, 0.38 mmol) and the

resulting mixture was stirred for 2 h while heating to reﬂux. 5,7-

Dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine

(0.100 g, 0.31 mmol) (102) was then added at rt. The reaction

mixture was stirred for 20 min before the reaction was quenched with

H₂O (5 mL) and extracted with EtOAc (×3). The combined organic

layers were dried over MgSO₄, ﬁltered, and concentrated under

reduced pressure. Puriﬁcation via silica gel column chromatography

(0−40% EtOAc in hexanes) provided the title compound (0.046 g,

1

powder (0.056 g, 0.13 mmol, 35%). H NMR (500 MHz, CDCl₃): δ

8.41 (s, 1H), 7.01−6.77 (m, 2H), 6.28 (s, 1H), 3.96 (s, 1H), 2.26−

2.09 (m, 1H), 1.03 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H) ppm;

¹³C NMR (126 MHz, CDCl₃): δ 164.69 (dt, J = 255.2, 15.1 Hz),

161.57 (ddd, J = 253.8, 15.0, 8.2 Hz), 161.32 (ddd, J = 251.3, 14.9,

8.2 Hz), 158.29, 155.56, 153.98, 146.56, 124.53 (q, J = 283.5 Hz),

101.86 (td, J = 25.8, 4.1 Hz), 101.36 (td, J = 25.2, 2.4 Hz), 90.99,

58.99 (q, J = 29.4 Hz), 28.34, 19.81, 16.74 ppm; IR (ﬁlm) ν: 3339,

2972, 2928, 1618, 1562, 1493, 1442, 1364, 1264, 1170, 1126, 1090,

1039, 999, 937, 911, 846, 768, 734, 705, 653 cm⁻¹; HRMS (ES⁺):

calcd for C₁₆H₁₂ClF₆N₅Na [M + Na]⁺, 446.0583; found, 446.0598.

(S)-5-Chloro-N-(1,1,1-triﬂuoro-3-methylbutan-2-yl)-6-(2,4,6-

triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (47).

General procedure C was followed using 5,7-dichloro-6-(2,4,6-

triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.123 g, 0.38

mmol) (102) and (S)-1,1,1-triﬂuoro-3-methyl-2-butylamine (0.152

mL, 1.15 mmol). Puriﬁcation via silica gel column chromatography

(0−15% EtOAc in hexanes) provided the title compound as a white

1

0.11 mmol, 37%). H NMR (600 MHz, CDCl₃): δ 8.51 (s, 1H),

6.89−6.77 (m, 2H), 6.21−6.12 (m, 1H), 1.63 (d, J = 6.6 Hz, 4H)

ppm; ¹³C NMR (150 MHz, CDCl₃): δ 164.14 (dt, J = 253.1, 15.2

Hz), 161.12 (ddd, J = 252.7, 15.1, 8.7 Hz), 160.86 (ddd, J = 251.3,

15.0, 8.6 Hz), 158.43, 156.71, 155.59 (d, J = 43.8 Hz), 151.92, 123.23

(q, J = 281.1 Hz), 104.10 (td, J = 20.5, 4.9 Hz), 102.40−98.42 (m),

76.02 (q, J = 32.7, 31.8 Hz), 14.07, 12.80 ppm; IR (ﬁlm) ν: 2925,

2854, 1606, 1517, 1493 cm⁻¹; HRMS (ES⁺): calcd for

C₁₄H₈N₄ClF₆O [M + H]⁺, 397.0285; found, 397.0279.

5-Chloro-N-(1,1-diﬂuoropropan-2-yl)-6-(2,4,6-triﬂuoro-

phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (43). General

procedure C was followed using 5,7-dichloro-6-(2,5-diﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (0.053 g, 0.17 mmol) (102), 1,1-

diﬂuoropropan-2-amine hydrochloride (0.045 g, 0.34 mmol), and

Et₃N (0.093 mL, 0.67 mmol). Puriﬁcation via silica gel column

chromatography (0−30% EtOAc in hexanes) provided the title

1

powder (0.039 g, 0.09 mmol, 24%). H NMR (500 MHz, CDCl₃): δ

8.41 (s, 1H), 6.99−6.84 (m, 2H), 6.29 (s, 1H), 3.98 (s, 1H), 2.24−

2.10 (m, J = 6.7 Hz, 1H), 1.03 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz,

3H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 164.63 (dt, J = 255.2,

15.0 Hz), 161.50 (ddd, J = 253.8, 14.9, 8.2 Hz), 161.25 (ddd, J =

251.3, 14.9, 8.2 Hz), 158.23, 155.49, 153.90, 146.50, 124.47 (q, J =

283.5 Hz), 101.80 (td, J = 25.8, 4.2 Hz), 101.30 (td, J = 27.1, 26.1, 3.7

Hz), 90.92, 58.93 (q, J = 29.4 Hz), 28.28, 19.75, 16.68 ppm; IR (ﬁlm)

1

compound as a white powder (0.035 g, 0.09 mmol, 56%). H NMR

(500 MHz, CDCl₃): δ 8.37 (s, 1H), 6.89 (t, J = 7.9 Hz, 2H), 6.00 (d,

1092

https://dx.doi.org/10.1021/acs.jmedchem.0c01605

J. Med. Chem. 2021, 64, 1073−1102

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

ν: 3339, 2972, 2930, 1618, 1562, 1493, 1459, 1442, 1364, 1264, 1170,

1126, 1090, 1066, 1038, 999, 938, 846, 768, 738, 704, 628 cm⁻¹;

HRMS (ES⁺): calcd for C₁₆H₁₃ClF₆N₅[M + H]⁺, 424.0758; found,

424.0762.

1H), 6.85 (t, J = 8.1 Hz, 2H), 3.45−3.39 (m, 4H), 1.76−1.67 (m,

4H), 1.65−1.58 (m, 4H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ

163.64 (dt, J = 253.0, 15.4 Hz), 161.10 (dd, J = 250.2, 8.8 Hz), 160.98

(dd, J = 250.3, 8.8 Hz), 158.21, 155.45, 155.08, 152.31, 109.08 (td, J

= 20.4, 5.0 Hz), 102.17−100.48 (m), 98.50, 53.70, 28.28, 28.23 ppm;

IR (ﬁlm) ν: 3436, 2932, 2858, 1637, 1593, 1525, 1492, 1443, 1357,

1284, 1259, 1231, 1200, 1171, 1152, 1125, 1100, 1035, 998, 968, 931,

842, 776, 760, 732, 656, 636, 614 cm⁻¹; HRMS (ES⁺): calcd for

C₁₇H₁₆ClF₃N₅[M + H]⁺, 382.1041; found, 382.1031.

7-(Azocan-1-yl)-5-chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidine (53). General procedure C was followed

using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.191 g, 0.60 mmol) (102) and azocane (0.142 g, 1.26

mmol). Puriﬁcation via silica gel column chromatography (0−20%

EtOAc in hexanes) provided the title compound as a white powder

(0.177 g, 0.45 mmol, 75%).

(R)-5-Chloro-6-(2,4,6-triﬂuorophenyl)-N-(1,1,1-triﬂuoropro-

pan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (48). General

procedure A was followed using (R)-1,1,1-triﬂuoropropan-2-amine

hydrochloride (0.090 g, 0.60 mmol), i-Pr₂NEt (0.078 g, 0.60 mmol),

and 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.064 g, 0.20 mmol) (102). Puriﬁcation via reverse-

phase HPLC aﬀorded the title compound as a white solid (0.017 g,

0.04 mmol, 21% yield). ¹H NMR (500 MHz; CDCl₃): δ 8.39 (s, 1H),

6.91 (dd, J = 8.4, 7.1 Hz, 2H), 5.92 (d, J = 10.3 Hz, 1H), 4.73 (s, 1H),

1.43 (d, J = 6.9 Hz, 3H) ppm; ¹³C NMR (126 MHz; CDCl₃): δ

164.64 (dt, J = 255.3, 15.1 Hz), 161.65 (ddd, J = 253.4, 15.1, 8.2 Hz),

161.32 (ddd, J = 251.7, 14.9, 8.2 Hz), 157.97, 155.60, 154.26, 145.97,

124.57 (q, J = 282.0 Hz), 105.51 (td, J = 20.7, 4.8 Hz), 101.61 (dtd, J

= 52.6, 26.1, 4.0 Hz), 91.50, 51.02 (q, J = 32.2 Hz), 15.30 ppm; IR

(ﬁlm) ν: 3335, 3205, 2924, 2852, 1621, 1558, 1272, 1251, 1175, 1145,

1120 cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₉ClF₆N₅[M + H]⁺,

396.0451; found, 396.0451.

7-(Azetidin-1-yl)-5-chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidine (49). General procedure C was followed

using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.064 g, 0.20 mmol) (102) and azetidine (0.034 g,

0.60 mmol). Puriﬁcation via reverse-phase HPLC aﬀorded the title

compound as a white solid (0.005 g, 0.02 mmol, 8% yield). ¹H NMR

(500 MHz; CDCl₃): δ 8.34 (s, 1H), 6.84−6.79 (m, 2H), 4.49−4.45

(m, 4H), 2.37 (quintet, J = 7.9 Hz, 2H) ppm; ¹³C NMR (126 MHz;

CDCl₃): δ 164.10 (dt, J = 253.9, 15.2 Hz), 162.35 (dd, J = 249.8, 8.2

Hz), 162.23 (dd, J = 249.7, 8.4 Hz), 158.12, 155.06, 155.04, 154.45,

146.35, 106.96 (td, J = 21.0, 4.6 Hz), 100.80−100.36 (m), 89.00,

56.73, 29.86, 17.36 ppm; IR (ﬁlm) ν: 2957, 2924, 2852, 1594, 1558,

1455, 1255, 1167, 1120, 1041 cm⁻¹; HRMS (ES⁺): calcd for

C₁₄H₉ClF₃N₅[M + H]⁺, 340.0577; found, 340.0573.

¹H NMR (500 MHz, CDCl₃): δ 8.32 (s, 1H), 6.81 (t, J = 7.9 Hz,

2H), 3.49−3.38 (m, 4H), 1.78−1.15 (m, 10H) ppm; ¹³C NMR (126

MHz, CDCl₃): δ 163.63 (dt, J = 253.5, 15.2 Hz), 160.93 (dd, J =

250.6, 8.9 Hz), 160.81 (dd, J = 250.5, 8.7 Hz), 158.19, 155.50, 154.73,

151.18, 108.91 (td, J = 20.2, 4.8 Hz), 102.27−100.10 (m), 98.25,

51.91, 27.67, 26.77, 24.36 ppm; IR (ﬁlm) ν: 3450, 2927, 2860, 1637,

1593, 1529, 1494, 1440, 1375, 1356, 1266, 1230, 1212, 1171, 1151,

1124, 1091, 1035, 998, 967, 914, 846, 761, 735, 656, 636, 612 cm⁻¹;

HRMS (ES⁺): calcd for C₁₈H₁₈ClF₃N₅[M + H]⁺, 396.1197; found,

396.1201.

5-Chloro-N-cyclopropyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (55). General procedure C was

followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.064 g, 0.20 mmol) (102), cyclopropanamine

(0.057 g, 0.60 mmol), and i-Pr₂NEt (0.078 g, 0.60 mmol).

Puriﬁcation via reverse-phase HPLC aﬀorded the title compound as

1

a beige solid (0.021 g, 0.06 mmol, 30% yield). H NMR (500 MHz;

CDCl₃): δ 8.34 (s, 1H), 6.84−6.80 (m, 3H), 2.36−2.33 (m, 1H),

0.68−0.65 (m, 2H), 0.48−0.44 (m, 2H) ppm; ¹³C NMR (126 MHz;

CDCl₃): δ 163.93 (dt, J = 252.6, 13.5 Hz), 162.00 (dd, J = 249.8, 8.5

Hz), 161.88 (dd, J = 250.1, 9.0 Hz), 158.41, 155.18, 153.53, 147.23,

147.17, 108.06 (td, J = 20.8, 5.0 Hz), 100.71−100.27 (m), 89.74,

89.70, 25.70, 25.56, 9.00, 8.96 ppm; IR (ﬁlm) ν: 3361, 3260, 3104,

2919, 1597, 1576, 1494, 1441, 1264, 1123, 1032 cm⁻¹; HRMS (ES⁺):

calcd for C₁₄H₁₉ClF₃N₅[M + H]⁺, 340.0577; found, 340.0574.

5-Chloro-N-cyclobutyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (56). General procedure C was

followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.030 g, 0.09 mmol) (102) and cyclobutanamine

(0.014 g, 0.20 mmol). Puriﬁcation via silica gel column chromatog-

raphy (0−40% EtOAc in hexanes) provided the title compound as a

white powder (0.025 g, 0.07 mmol, 75%). ¹H NMR (600 MHz,

CDCl₃): δ 8.35 (s, 1H), 6.85−6.81 (m, 2H), 6.61 (d, J = 7.5 Hz, 1H),

3.71 (q, J = 7.7 Hz, 1H), 2.08−1.94 (m, 4H), 1.80−1.73 (m, 1H),

1.60−1.51 (m, 1H), 1.24 (s, 1H) ppm; ¹³C NMR (150 MHz,

CDCl₃): δ 164.03 (dt, J = 253.8, 15.4 Hz), 161.71 (ddd, J = 250.5,

15.1, 8.4 Hz), 158.22, 155.04, 145.33, 120.10, 102.32−99.71 (m),

89.07, 48.80, 31.96, 14.62 ppm; IR (ﬁlm) 3285, 2838, 2178, 1571

cm⁻¹; HRMS (ES⁺): calcd for C₁₅H₁₂ClF₃N₅[M + H]⁺, 354.0728;

found, 354.0726.

5-Chloro-N-cyclopentyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (57). General procedure C was

followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.030 g, 0.09 mmol) (102) and cyclopentanamine

(0.017 g, 0.20 mmol). Puriﬁcation via silica gel column chromatog-

raphy (0−40% EtOAc in hexanes) provided the title compound as a

white powder (0.027 g, 0.07 mmol, 78%). ¹H NMR (600 MHz,

CDCl₃): δ 8.31 (s, 1H), 6.84−6.81 (m, 2H), 6.42 (d, J = 8.3 Hz, 1H),

3.63 (s, 1H), 1.82−1.59 (m, 4H), 1.62−1.38 (m, 4H) ppm. ¹³C NMR

(150 MHz, CDCl₃): δ 163.99 (dt, J = 253.7, 15.2 Hz), 161.66 (ddd, J

= 250.5, 15.0, 8.4 Hz), 158.19, 154.93, 145.81, 101.30−100.53 (m),

88.98, 55.11, 34.64, 23.92 ppm; IR (ﬁlm) ν: 3195, 2838, 1678, 1553

cm⁻¹; HRMS (ES⁺): calcd for C₁₆H₁₄N₅ClF₃[M + H]⁺, 368.0884;

found, 368.0881.

5-Chloro-7-(pyrrolidin-1-yl)-6-(2,4,6-triﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (50). General procedure C was

followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.064 g, 0.20 mmol) (102) and pyrrolidine (0.043

g, 0.60 mmol). Puriﬁcation by reverse-phase HPLC aﬀorded the title

1

compound as a yellow solid (0.009 g, 0.03 mmol, 14% yield). H

NMR (500 MHz; CDCl₃): δ 8.37 (s, 1H), 6.81 (dd, J = 8.4, 6.9 Hz,

2H), 3.71 (t, J = 6.5 Hz, 4H), 1.91 (dt, J = 6.2, 3.3 Hz, 4H) ppm; ¹³C

NMR (126 MHz; CDCl₃): δ 163.80 (dt, J = 252.3, 14.5 Hz), 161.66

(dd, J = 248.1, 7.1 Hz), 161.55 (dd, J = 249.6, 8.3 Hz), 158.54,

155.74, 154.26, 148.57, 109.66 (td, J = 20.7, 4.9 Hz), 100.94−100.50

(m), 92.85, 53.27, 25.94 ppm; IR (ﬁlm) ν: 3444, 1636, 1594, 1537,

1455, 1333, 1253, 1123 cm⁻¹. HRMS (ES⁺): calcd for C₁₅H₁₂ClF₃N₅

+

[M + H] : 354.0733; found, 354.0723.

5-Chloro-7-(piperidin-1-yl)-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidine (51). General procedure C was followed

using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.064 g, 0.20 mmol) (102) and piperidine (0.051 g,

0.60 mmol). Puriﬁcation via reverse-phase HPLC aﬀorded the title

1

compound as a yellow solid (0.012 g, 0.03 mmol, 17% yield). H

NMR (500 MHz; CDCl₃): δ 8.40 (s, 1H), 6.86 (dd, J = 8.4, 7.3 Hz,

2H), 3.27 (br s, 4H), 1.64 (br s, 6H) ppm; ¹³C NMR (126 MHz;

CDCl₃): δ 163.71 (dt, J = 252.8, 15.4 Hz), 161.19 (dd, J = 250.3, 8.7

Hz), 161.08 (dd, J = 250.1, 8.8 Hz), 158.24, 155.29, 151.15, 108.64

(td, J = 20.2, 4.8 Hz), 101.30−100.86 (m), 98.10, 51.13, 25.98, 23.71

ppm; IR (ﬁlm) ν: 2945, 2929, 2857, 1595, 1558, 1538, 1506, 1446,

1361, 1121, 1035 cm⁻¹; HRMS (ES⁺): calcd for C₁₆H₁₄ClF₃N₅[M +

H]⁺, 368.0890; found, 368.0877.

7-(Azepan-1-yl)-5-chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidine (52). General procedure C was followed

using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (0.197 g, 0.62 mmol) (102) and azepane (0.184 g, 1.85

mmol). Puriﬁcation via silica gel column chromatography (0−15%

EtOAc in hexanes) provided the title compound as a white powder

1

(0.057 g, 0.15 mmol, 24%). H NMR (500 MHz, CDCl₃): δ 8.36 (s,

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5-Chloro-N-cyclohexyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (58). General procedure C was

followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.100 g, 0.31 mmol) (102) and cyclohexanamine

(0.094 g, 0.94 mmol). Puriﬁcation via silica gel column chromatog-

raphy (0−40% EtOAc in hexanes) provided the title compound as a

white powder (0.119 g, 0.31 mmol, 99%). ¹H NMR (500 MHz,

CDCl₃): δ 8.33 (s, 1H), 6.92−6.81 (m, 2H), 6.37 (d, J = 8.7 Hz, 1H),

3.14−2.98 (m, 1H), 1.86−1.77 (m, 2H), 1.74−1.64 (m, 2H), 1.53

(dt, J = 13.2, 3.8 Hz, 1H), 1.30−1.18 (m, 2H), 1.12 (tdd, J = 12.9, 9.8,

6.1 Hz, 1H), 1.01−0.85 (m, 2H) ppm; ¹³C NMR (126 MHz,

CDCl₃): δ 164.08 (dt, J = 253.8, 15.1 Hz), 161.76 (dd, J = 250.7, 8.5

Hz), 161.65 (dd, J = 250.7, 8.4 Hz), 158.12, 155.00, 153.74, 145.59,

107.50 (td, J = 20.7, 4.8 Hz), 102.70−97.55 (m), 88.82, 52.94, 34.06,

24.92, 24.75 ppm; IR (ﬁlm) ν: 3341, 3103, 2934, 2856, 2239, 1636,

1610, 1578, 1492, 1439, 1367, 1341, 1313, 1263, 1207, 1159, 1125,

1036, 998, 976, 957, 930, 844, 766, 733, 655, 627 cm⁻¹; HRMS

(ES⁺): calcd for C₁₇H₁₆ClF₃N₅[M + H]⁺, 382.1041; found, 382.1054.

5-Chloro-N-neopentyl-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (59). General procedure C was

followed using 5,7-dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.050 g, 0.16 mmol) (102) and 2,2-dimethylpro-

pan-1-amine (0.039 mL, 0.33 mmol). Puriﬁcation via silica gel column

chromatography (0−30% EtOAc in hexanes) provided the title

Puriﬁcation via preparative reverse-phase HPLC (C18 column, 20

mL/min, 10 min ramp, 10 to 90% ACN/H₂O + 0.1% formic acid, tr =

8.5 min) provided the title compound (0.014 g, 0.04 mmol, 68%) as a

1

tan solid. H NMR (600 MHz, CDCl₃): δ 8.56 (s, 1H), 6.92−6.88

(m, 2H), 3.34 (dd, J = 13.9, 6.2 Hz, 1H), 3.22 (m, 1H), 3.05 (dd, J =

13.9, 8.4 Hz, 1H), 1.03 (d, J = 7.0 Hz, 4H) ppm; ¹³C NMR (151

MHz, CDCl₃): δ 164.45 (dt, 256.7, 18.1 Hz), 160.86 (ddd, 252.2,

15.1, 9.1 Hz), 160.61 (ddd, 250.7, 15.1, 9.06 Hz), 157.19, 157.02,

154.03, 148.77, 127.25 (q, 280.11 Hz) 112.29, 106.39 (td, 21.14, 4.9

Hz), 101.48 (dtd, J = 30.1, 25.9, 4.1 Hz), 35.37 (q, J = 27.6 Hz),

30.65, 12.88 ppm; IR (ﬁlm) ν: 1639, 1609, 1599, 1516, 1495, 1442,

1278, 1265, 1207, 1186, 1172, 1146, 1122, 1039, 999 cm⁻¹; HRMS

(ES⁺): calcd for C₁₅H₉ClF₆N₄[M + H]⁺, 395.0493; found, 395.0486.

(S)-4-(5-Chloro-7-((1,1,1-triﬂuoropropan-2-yl)amino)-

[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuorobenzonitrile (63).

General procedure C was followed using 4-(5,7-dichloro-[1,2,4]-

triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuorobenzonitrile (0.040 g, 0.13

mmol) (107) and (S)-1,1,1-triﬂuoropropan-2-amine (0.031 g, 0.27

mmol). Puriﬁcation by reverse-phase HPLC provided the title

1

compound as a white powder (0.031 g, 0.07 mmol, 54%). H NMR

(600 MHz, CDCl₃): mixture of diastereomers δ 8.41 (s, 1H), 7.68

(ddd, J = 7.8, 3.8, 1.5 Hz, 1H), 7.61 (ddd, J = 8.4, 3.8, 1.5 Hz, 1H),

7.57 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.3 Hz, 1H), 5.94 (d, J = 11.0 Hz,

1H), 5.57 (d, J = 10.8 Hz, 1H), 4.97 (s, 1H), 4.50 (s, 1H), 1.40 (d, J =

6.9 Hz, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 161.29 (d, J =

52.0 Hz), 159.61 (d, J = 50.4 Hz), 156.65 (d, J = 24.9 Hz), 155.72 (d,

J = 11.0 Hz), 154.20 (d, J = 57.5 Hz), 145.65 (d, J = 62.8 Hz), 135.00

(d, J = 2.0 Hz), 133.84 (d, J = 2.2 Hz), 129.21 (d, J = 4.2 Hz), 124.97

(dd, J = 16.2, 13.1 Hz), 124.67 (qd, J = 282.3, 34.7 Hz), 120.62 (dd, J

= 74.3, 25.2 Hz), 116.67 (d, J = 2.8 Hz), 116.33 (dd, J = 9.3, 5.2 Hz),

97.66, 96.61, 51.26 (dq, J = 37.9, 31.8 Hz), 15.13, 14.99 ppm; HRMS

(ES⁺): calcd for C₁₅H₁₀ClF₄N₆[M + H]⁺, 385.0586; found, 385.0589.

(R)-5-Chloro-6-(2,6-diﬂuorophenyl)-N-(3-methylbutan-2-

yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (64). General proce-

dure C was followed using 5,7-dichloro-6-(3,5-diﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (0.048 g, 0.16 mmol) (97) and

(R)-3-methylbutan-2-amine (0.028 g, 0.32 mmol). Puriﬁcation via

silica gel column chromatography (0−30% EtOAc in hexanes)

provided the title compound as a white powder (0.049 g, 0.14 mmol,

1

compound as a white powder (0.033 g, 0.09 mmol, 57%). H NMR

(500 MHz, CDCl₃): δ 8.33 (s, 1H), 6.85 (dd, J = 8.0, 7.2 Hz, 2H),

6.45 (s, 1H), 2.80 (d, J = 5.7 Hz, 2H), 0.88 (s, 9H) ppm; ¹³C NMR

(126 MHz, CDCl₃): δ 164.63 (dt, J = 255.2, 15.0 Hz), 161.50 (ddd, J

= 253.8, 14.9, 8.2 Hz), 161.25 (ddd, J = 251.3, 14.9, 8.2 Hz), 158.23,

155.49, 153.90, 146.50, 124.47 (q, J = 283.5 Hz), 101.80 (td, J = 25.8,

4.2 Hz), 101.30 (td, J = 27.1, 26.1, 3.7 Hz), 90.92, 58.93 (q, J = 29.4

Hz), 28.28, 19.75, 16.68 ppm; IR (ﬁlm) ν: 2959, 1636, 1611, 1573,

1495, 1438, 1358, 1263, 1250, 1124, 1036, 998, 843, 766, 533 cm⁻¹;

HRMS (ES⁺): calcd for C₁₆H₁₅ClF₃N₅[M + H]⁺, 370.1041; found,

370.1039.

(R)-5-Chloro-N-(3,3-dimethylbutan-2-yl)-6-(2,4,6-triﬂuoro-

phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (60). General

procedure C was followed using 5,7-dichloro-6-(2,4,6-triﬂuorophen-

yl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.150 g, 0.47 mmol) (102) and

(R)-3,3-dimethylbutan-2-amine (0.132 mL, 0.99 mmol). Puriﬁcation

via silica gel column chromatography (0−30% EtOAc in hexanes)

provided the title compound as a white powder (0.157 g, 0.86 mmol,

87%). The spectroscopic properties of the compound were identical

to those reported for 61.

1

87%). H NMR (600 MHz, CDCl₃): δ 8.32 (s, 1H), 7.54−7.49 (m,

1H), 7.09−7.06 (m, 2H), 6.34 (br s, 1H), 3.12 (br s, 1H), 1.61 (oct, J

= 6 Hz, 1H), 1.03 (d, J = 6 Hz, 3H), 0.77 (d, J = 6 Hz, 3H), 0.74 (d, J

= 6 Hz, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 160.77 (dt, J =

250.66, 5.3 Hz), 158.04, 154.89 (d, J = 4.5 Hz), 153.65, 145.94,

132.38−132.22 (m), 110.93 (t, J = 21.1 Hz), 112.08−111.81 (m),

89.78, 54.76, 33.65, 33.63, 18.21 (q, 3 Hz), 18.00 (q, J = 7.6 Hz),

17.80 (q, J = 3 Hz) ppm; HRMS (ES⁺): calcd for C₁₈H₁₇ClFN₆[M +

H]⁺, 352.1135; found, 352.1135.

(S)-5-Chloro-N-(3,3-dimethylbutan-2-yl)-6-(2,4,6-triﬂuoro-

phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (61). General

procedure C was followed using 5,7-dichloro-6-(2,4,6-triﬂuorophen-

yl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.100 g, 0.31 mmol) (102) and

(S)-3,3-dimethylbutan-2-amine (0.088 mL, 0.66 mmol). Puriﬁcation

via silica gel column chromatography (0−30% EtOAc in hexanes)

provided the title compound as a white powder (0.094 g, 0.24 mmol,

(R)-5-Chloro-6-(2-ﬂuorophenyl)-N-(3-methylbutan-2-yl)-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (65). General procedure

C was followed using 5,7-dichloro-6-(2-ﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (0.046 g, 0.16 mmol) (95) and (R)-3-methylbutan-

2-amine (0.028 g, 0.32 mmol). Puriﬁcation via silica gel column

chromatography (0−30% EtOAc in hexanes) provided the title

compound as a white powder (0.017 g, 0.051 mmol, 31%). ¹H NMR

(600 MHz, CDCl₃): mixture of diastereomers δ 8.33 (s, 1H), 7.52 (q,

J = 6 Hz, 1H), 7.36−7.29 (m, 2H), 7.24 (t, J = 6 Hz, 1H), 6.19 (br s,

1H), 3.15 (br s, 1H), 1.02 (d, J = 6 Hz, 1.5H), 0.96 (d, J = 6 Hz,

1.5H), 0.77 (t, J = 6 Hz, 3H), 0.74 (d, J = 6 Hz, 1.5H), 0.72 (d, J = 6

Hz, 1.5H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 160.83 (dd, J =

250.66, 9 Hz), 158.26 (d, J = 12.1 Hz), 153.91, 152.70, 146.02,

133.61, 133.61, 132.98, 132.06 (t, J = 8.3 Hz), 124.94 (d, J = 4.5 Hz),

124.89 (d, J = 3.0 Hz), 120.96 (d, J = 25.7 Hz), 120.85 (dd, J = 21.9,

1.6 Hz), 97.14, 96.99, 54.75, 54.59, 33.77, 33.47, 18.28, 17.98, 17.81,

17.73, 17.62 ppm; HRMS (ES⁺): calcd for C₁₈H₁₇ClFN₆[M + H]⁺,

334.1229; found, 334.1227.

1

78%). H NMR (500 MHz, CDCl₃): δ 8.31 (s, 1H), 6.92−6.74 (m,

2H), 6.44 (s, 1H), 3.08 (s, 1H), 1.00 (d, J = 6.7 Hz, 4H), 0.82 (s,

11H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 164.07 (dt, J = 254.0,

15.2 Hz), 161.52 (ddd, J = 250.8, 14.8, 8.4 Hz), 161.27 (ddd, J =

250.6, 14.9, 8.4 Hz), 158.13, 154.94, 153.61, 146.25, 107.54 (td, J =

20.6, 5.1 Hz), 101.51−100.58 (m), 88.60, 58.07, 34.72, 25.78, 16.58

ppm; IR (ﬁlm) ν: 1608, 1578, 1490, 1438, 1354, 1264, 1252, 1124,

999, 767, 623, 540 cm⁻¹; HRMS (ES⁺): calcd for C₁₇H₁₇ClF₃N₅[M +

H]⁺, 384.1197; found, 384.1198.

5-Chloro-7-(3,3,3-triﬂuoro-2-methylpropyl)-6-(2,4,6-tri-

ﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (62). To a sol-

ution of tert-butyl 2-(5-chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)-4,4,4-triﬂuoro-3-methylbutanoate

(0.026 g, 0.05 mmol, 1 equiv) (112) in CH₂Cl₂(2 mL) at rt was

added 2,2,2-triﬂuoroacetic acid (0.125 mL, 1.60 mmol, 30 equiv). The

mixture was stirred for 16 h at rt and then diluted with CH₂Cl₂and

quenched with a saturated NaHCO₃solution. The aqueous layer was

then extracted with CH₂Cl₂and the combined organic layers were

dried over MgSO₄, ﬁltered, and concentrated under reduced pressure.

(R)-4-(5-Chloro-7-((3-methylbutan-2-yl)amino)-[1,2,4]-

triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuorobenzonitrile (66).

General procedure C was followed using 4-(5,7-dichloro-[1,2,4]-

triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuorobenzonitrile (0.030 g, 0.09

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mmol) (106) and (R)-3-methylbutan-2-amine (0.017 g, 0.19 mmol).

Puriﬁcation by reverse-phase HPLC provided the title compound as a

white powder (0.028 g, 0.07 mmol, 81%). ¹H NMR (600 MHz,

DMSO-d₆): δ 8.62 (s, 1H), 8.11 (t, J = 7.8 Hz, 2H), 7.88 (s, 1H),

1.78 (dq, J = 13.9, 6.8 Hz, 1H), 1.10 (d, J = 6.6 Hz, 3H), 0.74 (dd, J =

6.8, 2.9 Hz, 6H) ppm; ¹³C NMR (151 MHz, DMSO-d₆): δ 160.71

(dd, J = 249.2, 6.8 Hz), 160.38 (dd, J = 249.5, 6.3 Hz), 155.02,

146.81, 117.00 (ddd, J = 26.3, 12.6, 3.9 Hz), 116.48 (d, J = 3.5 Hz),

114.83 (t, J = 12.8 Hz) ppm; ¹⁹F NMR (469 MHz): δ −103.93 (d, J =

9.9 Hz), −104.42 (d, J = 9.9 Hz) ppm; IR (ﬁlm) ν: 2966, 2220, 1690,

1562, 1422, 1204 cm⁻¹; HRMS (ES⁺): calcd for C₁₇H₁₆ClF₂N₆[M +

H]⁺, 377.1088; found, 377.1089.

(d, J = 2.2 Hz) ppm; HRMS (ES⁺): calcd for C₁₈H₁₉ClFN₆[M + H]⁺,

373.1338; found, 373.1338.

4-(5-Chloro-7-((1R,3s,5S)-3-methoxy-8-azabicyclo[3.2.1]-

octan-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuoro-

benzonitrile (71). General procedure C was followed using 4-(5,7-

dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuorobenzonitrile

(0.100 g, 0.31 mmol) (106), (1R,3R,5S)-3-methoxy-8-azabicyclo-

[3.2.1]octane hydrochloride (0.082 g, 0.46 mmol) and Et₃N (0.094 g,

0.92 mmol). Puriﬁcation by reverse-phase HPLC provided the title

1

compound as a white powder (0.112 g, 0.26 mmol, 85%). H NMR

(600 MHz, CDCl₃): δ 8.28 (s, 1H), 7.39 (d, J = 5.9 Hz, 2H), 4.57 (s,

2H), 3.45 (s, 1H), 3.23 (s, 3H), 2.13 (d, J = 7.4 Hz, 2H), 2.00 (d, J =

14.9 Hz, 2H), 1.92 (d, J = 14.7 Hz, 2H), 1.80−1.74 (m, 2H) ppm;

¹³C NMR (150 MHz, CDCl₃): δ 161.66 (d, J = 6.6 Hz), 159.98 (d, J

= 6.6 Hz), 156.99, 155.05, 146.39, 118.79, 116.34, 116.30, 116.18,

116.15, 116.11, 115.16 (t, J = 11.8 Hz), 92.08, 73.33, 62.34, 59.30,

56.53, 36.34, 27.98 ppm; HRMS (ES⁺): calcd for C₂₀H₁₈ClF₂N₆O [M

+ H]⁺, 431.1188; found, 431.1193.

4-(7-(Azepan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-

6-yl)-3,5-diﬂuorobenzonitrile (72). General procedure C was

followed using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-

3,5-diﬂuorobenzonitrile (0.020 g, 0.06 mmol) (106) and azepane

(0.013 g, 0.13 mmol). Puriﬁcation by reverse-phase HPLC provided

the title compound as a white powder (0.015 g, 0.04 mmol, 63%). ¹H

NMR (600 MHz, CDCl₃): δ 8.38 (s, 1H), 7.40 (d, J = 6.0 Hz, 2H),

3.45−3.39 (m, 4H), 1.78−1.70 (m, 4H), 1.64−1.60 (m, 4H) ppm;

¹³C NMR (150 MHz, CDCl₃): δ 160.74 (dd, J = 253.4, 6.6 Hz),

157.14, 155.26, 152.10, 118.61 (t, J = 19.7 Hz), 116.30−115.97 (m),

115.12 (t, J = 11.9 Hz), 97.32, 53.99, 51.22, 28.27, 28.17, 27.28, 27.07

ppm; ¹⁹F NMR (469 MHz): δ −105.22 ppm; IR (ﬁlm) ν: 2928, 2857,

2238, 1589, 1515, 1422 cm⁻¹; HRMS (ES⁺): calcd for C₁₈H₁₆ClF₂N₆

[M + H]⁺, 389.1088; found, 389.1085.

(R)-4-(5-Chloro-7-((3-methylbutan-2-yl)amino)-[1,2,4]-

triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuorobenzonitrile (67) and

(68). General procedure C was followed using 4-(5,7-dichloro-

[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuorobenzonitrile (0.048 g,

0.15 mmol) (107) and (R)-3-methylbutan-2-amine (0.037 g, 0.42

mmol). Puriﬁcation via silica gel column chromatography provided

atropoisomer 67 as a white solid (0.029 g, 0.08 mmol, 39%) and

atropoisomer 68 (0.023 g, 0.06 mmol, 30%). Recrystallization of 68

from CH2Cl2 followed by single crystal diﬀraction (mp 143.7−145.2)

permitted the structural assignment of the two atropoisomers (see

Supporting Information). 68 ¹H NMR (600 MHz, CDCl₃): δ 8.35 (s,

1H), 7.63 (dd, J = 12.0, 3.0 Hz, 1H), 7.56 (dd, J = 12.0, 3.0 Hz, 1H),

7.53 (t, J = 9.0 Hz, 1H), 6.29 (d, J = 12.0 Hz, 1H), 3.00 (br s, 1H),

1.64 (oct, J = 6.0 Hz, 1H), 0.99 (d, J = 6 Hz, 3H), 0.80 (d, J = 6 Hz,

3H), 0.77 (d, J = 6 Hz, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ

161.37, 159.70, 156.91, 155.15, 153.15, 145.67, 134.39, 128.71,

128.69, 127.13 (d, J = 16.6 Hz), 120.02 (d, J = 25.7 Hz), 116.98,

115.50 (d, J = 9.1 Hz), 94.53, 55.04, 33.48, 18.16, 17.80, 17.14 ppm.

1

67 H NMR (600 MHz, CDCl₃): δ 8.35 (s, 1H), 7.63 (dd, J = 12.0,

3.0 Hz, 1H), 7.56 (dd, J = 12.0, 3.0 Hz, 1H), 7.52 (t, J = 9.0 Hz, 1H),

6.30 (br s, 1H), 3.05 (br s, 1H), 1.61 (oct, J = 6.0 Hz, 1H), 1.05 (d, J

= 6 Hz, 3H), 0.79 (d, J = 6 Hz, 3H), 0.75 (d, J = 6 Hz, 3H) ppm; ¹³C

NMR (150 MHz, CDCl₃): δ 161.40, 159.73, 156.90, 155.19, 153.57,

145.67, 135.08, 134.40 (d, J = 3.0 Hz), 128.55 (d, J = 4.5 Hz), 127.03

(d, J = 15.1 Hz), 120.22 (d, J = 25.7 Hz), 117.01, 115.45 (d, J = 9.1

Hz), 94.37, 54.85, 33.80, 18.20, 17.90, 17.82 ppm; IR (ﬁlm) ν: 2963,

2220, 1608, 1570, 1260, 1156 cm⁻¹; HRMS (ES⁺): calcd for

C₁₈H₁₇ClFN₆[M + H]⁺, 359.1182; found, 359.1184.

4-(7-(Azepan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-

6-yl)-3-ﬂuorobenzonitrile (73). General procedure C was followed

using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuoro-

benzonitrile (0.040 g, 0.13 mmol) (107) and azepane (0.027 g,

0.27 mmol). Puriﬁcation by reverse-phase HPLC provided the title

1

compound as a white powder (0.027 g, 0.07 mmol, 56%). H NMR

(600 MHz, CDCl₃): δ 8.34 (s, 1H), 7.61 (dd, J = 7.9, 1.6 Hz, 1H),

7.53 (dd, J = 8.7, 1.6 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 3.38−3.33 (m,

4H), 1.71 (dq, J = 8.0, 4.2 Hz, 4H), 1.60 (p, J = 3.0 Hz, 4H) ppm; ¹³C

NMR (150 MHz, CDCl₃): δ 160.85, 159.18, 156.93, 155.27, 155.18,

151.61, 134.44 (d, J = 2.8 Hz), 128.60 (d, J = 3.9 Hz), 128.48, 120.11,

119.94, 117.09 (d, J = 2.8 Hz), 114.68 (d, J = 9.2 Hz), 103.59, 54.05,

28.27, 28.03 ppm; IR (ﬁlm) ν: 2926, 2235, 1589, 1519, 1446 cm⁻¹;

HRMS (ES⁺): calcd for C₁₈H₁₇ClFN₆[M + H]⁺, 371.1182; found,

371.1180.

(R)-4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]-

triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuorobenzonitrile (69).

General procedure C was followed using 4-(5,7-dichloro-[1,2,4]-

triazolo[1,5-a]pyrimidin-6-yl)-3,5-diﬂuorobenzonitrile (0.020 g, 0.06

mmol) (106) and (R)-3,3-dimethylbutan-2-amine (0.013 g, 0.13

mmol). Puriﬁcation by reverse-phase HPLC provided the title

1

compound as a white powder (0.018 g, 0.05 mmol, 75%). H NMR

(600 MHz, CDCl₃): δ 8.36 (s, 1H), 7.45−7.41 (m, 2H), 6.52 (d, J =

10.8 Hz, 1H), 2.94 (s, 1H), 1.02 (d, J = 6.7 Hz, 3H), 0.84 (s, 9H)

ppm; ¹³C NMR (150 MHz, CDCl₃): δ 161.33 (dd, J = 253.8, 6.2 Hz),

161.05 (dd, J = 253.8, 6.2 Hz), 157.12, 155.22, 146.03, 116.22 (d, J =

4.0 Hz), 116.13−115.95 (m), 115.95−115.82 (m), 58.57, 34.93,

25.86, 16.56 ppm; ¹⁹F NMR (469 MHz): δ −103.70 (d, J = 4.7 Hz),

−104.31 (d, J = 4.7 Hz) ppm; HRMS (ES⁺): calcd for C₁₈H₁₈ClF₂N₆

[M + H]⁺, 391.1244; found, 391.1241.

4-(7-(Azocan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-

6-yl)-3,5-diﬂuorobenzonitrile (74). General procedure C was

followed using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-

3,5-diﬂuorobenzonitrile (0.054 g, 0.17 mmol) (106) and azocane

(0.019 g, 0.17 mmol). Puriﬁcation via silica gel column chromatog-

raphy provided the title compound as a white solid (0.031 g, 0.08

mmol, 46%). ¹H NMR (600 MHz, CDCl₃): δ 8.29 (s, 1H), 7.30 (d, J

= 6.0 Hz, 2H), 3.36 (t, J = 5.7 Hz, 5H), 1.62 (t, J = 5.8 Hz, 5H), 1.52

(t, J = 5.5 Hz, 6H), 1.44 (d, J = 6.0 Hz, 2H) ppm; ¹³C NMR (151

MHz, CDCl₃): δ 160.68 (dd, J = 253.6, 6.5 Hz), 157.23, 155.73,

155.12, 151.06, 118.55 (t, J = 19.8 Hz), 116.38, 116.34, 116.23,

116.19, 115.23 (t, J = 11.8 Hz), 97.29, 52.24, 27.72, 26.80, 24.44

ppm; HRMS (ES⁺): calcd for C₁₈H₁₇ClFN₆[M + H]⁺, 403.1244;

found, 403.1240.

4-(7-(Azocan-1-yl)-5-chloro-[1,2,4]triazolo[1,5-a]pyrimidin-

6-yl)-3-ﬂuorobenzonitrile (75). General procedure C was followed

using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuoro-

benzonitrile (0.048 g, 0.15 mmol) (107) and azocane (0.035 g,

0.30 mmol). Puriﬁcation via silica gel column chromatography

provided the title compound as a white solid (0.032 g, 0.08 mmol,

53%). ¹H NMR (600 MHz, CDCl₃): mixture of diastereomers δ 8.59

(s, 0.4H), 8.42 (s, 0.3H), 7.80−7.77 (m, 0.6H), 7.30 (d, J = 6 Hz,

0.3H), 7.16 (d, J = 12 Hz, 0.4H), 3.41 (t, J = 6 Hz, 1.2H), 3.32 (br s,

(R)-4-(5-Chloro-7-((3,3-dimethylbutan-2-yl)amino)-[1,2,4]-

triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuorobenzonitrile (70). Gener-

al procedure C was followed using 4-(5,7-dichloro-[1,2,4]triazolo[1,5-

a]pyrimidin-6-yl)-3-ﬂuorobenzonitrile (0.040 g, 0.13 mmol) (107)

and (R)-3,3-dimethylbutan-2-amine (0.028 g, 0.27 mmol). Puriﬁca-

tion by reverse-phase HPLC provided the title compound as a white

1

powder (0.027 g, 0.07 mmol, 56%). H NMR (600 MHz, CDCl₃):

mixture of diastereomers δ 8.41−8.34 (m, 1H), 7.67−7.61 (m, 1H),

7.60−7.55 (m, 1H), 7.55−7.49 (m, 1H), 6.37 (s, 1H), 2.94−2.89 (m,

1H), 1.01 (d, J = 6.7 Hz, 1H), 0.94 (d, J = 6.7 Hz, 2H), 0.82 (s, 5H),

0.81 (s, 4H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 161.71−159.28

(m), 156.76, 156.67, 145.96, 135.28 (d, J = 2.4 Hz), 133.96 (d, J = 2.3

Hz), 128.65 (d, J = 4.1 Hz), 128.43 (d, J = 4.1 Hz), 119.96 (dd, J =

37.6, 25.4 Hz), 116.86 (d, J = 2.8 Hz), 115.42 (dd, J = 16.7, 9.3 Hz),

94.27, 94.14, 58.27, 58.07, 35.01, 34.82, 25.77 (d, J = 1.9 Hz), 16.42

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1.8H), 3.04 (br s, 1.8H), 1.70−1.41 (m, 10H) ppm; HRMS (ES⁺):

calcd for C₁₈H₁₇ClFN₆[M + H]⁺, 385.1338; found, 385.1332.

Diethyl 2-Phenylmalonate (76). General procedure A was

followed using diethyl malonate (6.122 g, 38.20 mmol) and

bromobenzene (2.002 g, 12.70 mmol). Puriﬁcation via silica gel

column chromatography (0−8% EtOAc in hexanes) provided the title

and 1-bromo-3,5-diﬂuorobenzene (2.000 g, 10.30 mmol). Puriﬁcation

via silica gel column chromatography (0−5% EtOAc in hexanes)

provided the title compound as a colorless oil (1.462 g, 5.36 mmol,

52%). ¹H NMR (500 MHz, CDCl₃): δ 6.99−6.93 (m, 2H), 6.76 (tt, J

= 8.9, 2.4 Hz, 1H), 4.57 (s, 1H), 4.28−4.13 (m, 4H), 1.25 (t, J = 7.1

Hz, 6H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 167.20, 162.86 (dd, J

= 248.7, 12.9 Hz), 136.08 (t, J = 9.9 Hz), 112.63 (dd, J = 20.7, 6.0

Hz), 103.87 (t, J = 25.2 Hz), 62.26, 57.35 (t, J = 1.9 Hz), 14.00 ppm;

IR (ﬁlm) ν: 3450, 2986, 1736, 1626, 1601, 1465, 1369, 1305, 1234,

1154, 1121, 1037, 995, 867, 738, 675 cm⁻¹; HRMS (ES⁺): calcd for

C₁₃H₁₄F₂NaO₄[M + Na]⁺, 295.0758; found, 295.0745.

1

compound as a colorless oil (0.833 g, 3.53 mmol, 28%). H NMR

(500 MHz, CDCl₃): δ 7.50−7.31 (m, 5H), 4.61 (s, 1H), 4.45−4.01

(m, 4H), 1.26 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, CDCl₃): δ

168.30, 132.94, 129.40, 128.73, 128.33, 61.94, 58.12, 14.16 ppm; IR

(ﬁlm) ν: 3450, 1731, 1638, 1455, 1368, 1305, 1217, 1147, 1029, 700

cm⁻¹; HRMS (ES⁺): calcd for C₁₃H₁₆NaO₄[M + Na]⁺, 259.0946;

found, 259.0952.

Diethyl 2-(2,5-Diﬂuorophenyl)malonate (82). General proce-

dure A was followed using diethyl malonate (5.000 g, 31.10 mmol)

and 2-bromo-1,4-diﬂuorobenzene (2.000 g, 10.30 mmol). Puriﬁcation

via silica gel column chromatography (0−10% EtOAc in hexanes)

provided the title compound as a colorless oil (1.767 g, 6.46 mmol,

Diethyl 2-(4-Fluorophenyl)malonate (77). General procedure

A was followed using diethyl malonate (5.491 g, 34.30 mmol) and 1-

bromo-4-ﬂuorobenzene (2.000 g, 11.40 mmol). Puriﬁcation via silica

gel column chromatography (8/8/84 EtOAc/CH₂Cl₂/hexanes)

provided the title compound as a colorless oil (0.818 g, 3.22 mmol,

1

62%). H NMR (500 MHz, CDCl₃): δ 7.25−7.19 (m, 1H), 7.06−

6.95 (m, 2H), 4.94 (s, 1H), 4.29−4.17 (m, 4H), 1.26 (t, J = 7.1 Hz,

6H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 167.10, 158.54 (dd, J =

242.3, 2.5 Hz), 156.62 (dd, J = 243.9, 2.5 Hz), 121.84 (dd, J = 17.0,

8.4 Hz), 117.20 (dd, J = 25.5, 3.2 Hz), 116.59 (dd, J = 15.0, 8.8 Hz),

116.39 (dd, J = 16.3, 8.6 Hz), 62.29, 50.35 (d, J = 2.7 Hz), 14.02

ppm; IR (ﬁlm) ν: 3452, 2986, 1737, 1631, 1499, 1369, 1303, 1226,

1154, 1031, 877, 819, 739 cm⁻¹; HRMS (ES⁺): calcd for

C₁₃H₁₄F₂NaO₄[M + Na]⁺, 295.0758; found, 295.0755.

1

28%). H NMR (500 MHz, CDCl₃): δ 7.41−7.36 (m, 2H), 7.07−

7.01 (m, 2H), 4.59 (s, 1H), 4.27−4.14 (m, 4H), 1.25 (t, J = 7.1 Hz,

6H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 168.10, 162.72 (d, J =

246.9 Hz), 131.11 (d, J = 8.3 Hz), 128.70 (d, J = 3.4 Hz), 115.59 (d, J

= 21.6 Hz), 61.99, 57.18, 14.07 ppm; IR (ﬁlm) ν: 3451, 2985, 2940,

1733, 1607, 1467, 1447, 1369, 1302, 1223, 1157, 1096, 1031, 841,

806, 753 cm⁻¹; HRMS (ES⁺): calcd for C₁₃H₁₅FNaO₄[M + Na]⁺,

277.0852; found, 277.0861.

Diethyl 2-(3,4-Diﬂuorophenyl)malonate (83). General proce-

dure A was followed using diethyl malonate (5.002 g, 31.10 mmol)

and 4-bromo-1,2-diﬂuorobenzene (2.001 g, 10.30 mmol). Puriﬁcation

via preparative reverse-phase HPLC provided the title compound as

an orange oil (0.530 g, 1.95 mmol, 19%). ¹H NMR (500 MHz,

CDCl₃): δ 7.33−7.28 (m, 1H), 7.18−7.07 (m, 2H), 4.56 (d, J = 2.9

Hz, 1H), 4.28−4.17 (m, 4H), 1.27 (td, J = 7.3, 2.2 Hz, 6H) ppm; ¹³C

NMR (126 MHz, CDCl₃): δ 167.67, 150.35 (dd, J = 272.8, 12.7 Hz),

150.35 (dd, J = 224.7, 12.6 Hz), 129.67 (dd, J = 6.2, 4.0 Hz), 125.77

(dd, J = 6.5, 3.7 Hz), 118.63 (d, J = 18.4 Hz), 117.34 (d, J = 17.4 Hz),

62.24, 57.00, 14.05 ppm; IR (ﬁlm) ν: 3450, 2986, 1734, 1613, 1520,

1441, 1369, 1287, 1154, 1032, 773 cm⁻¹; HRMS (ES⁺): calcd for

C₁₃H₁₅F₂O₄[M + H]⁺, 273.0938; found, 273.0941.

Diethyl 2-(2-Fluorophenyl)malonate (78). General procedure

A was followed using diethyl malonate (5.491 g, 34.30 mmol) and 1-

bromo-2-ﬂuorobenzene (2.000 g, 11.40 mmol). Puriﬁcation via silica

gel column chromatography (0−8% EtOAc in hexanes) provided the

title compound as a colorless oil (1.411 g, 5.55 mmol, 49%). ¹H NMR

(500 MHz, CDCl₃): δ 7.47 (td, J = 7.6, 1.8 Hz, 1H), 7.34−7.28 (m,

1H), 7.16 (t, J = 7.6 Hz, 1H), 7.07 (t, J = 9.1 Hz, 1H), 4.98 (s, 1H),

4.23 (p, J = 7.0 Hz, 4H), 1.27 (t, J = 7.1 Hz, 6H) ppm; ¹³C NMR

(126 MHz, CDCl₃): δ 167.66, 160.59 (d, J = 247.6 Hz), 130.54 (d, J

= 3.0 Hz), 130.04 (d, J = 8.3 Hz), 124.34 (d, J = 3.7 Hz), 120.51 (d, J

= 14.5 Hz), 115.51 (d, J = 22.2 Hz), 62.09, 50.61 (d, J = 3.3 Hz),

14.07 ppm; IR (ﬁlm) ν: 3458, 2984, 2940, 2907, 1737, 1617, 1589,

1494, 1458, 1369, 1303, 1232, 1149, 1094, 1030, 952, 860, 815, 756,

697 cm⁻¹; HRMS (ES⁺): calcd for C₁₃H₁₅FNaO₄[M + Na]⁺,

277.0852; found, 277.0858.

Diethyl 2-(2,4-Diﬂuorophenyl)malonate (79). General proce-

dure A was followed using diethyl malonate (5.000 g, 31.10 mmol)

and 1-bromo-2,4-diﬂuorobenzene (2.000 g, 10.30 mmol). Puriﬁcation

via silica gel column chromatography (0−5% EtOAc in hexanes)

provided the title compound as a colorless oil (1.907 g, 6.98 mmol,

68%). ¹H NMR (500 MHz, CDCl₃): δ 7.47 (td, J = 8.5, 6.3 Hz, 1H),

6.89 (td, J = 7.4, 3.8 Hz, 1H), 6.82 (td, J = 8.5, 2.5 Hz, 1H), 4.91 (s,

1H), 4.29−4.15 (m, 4H), 1.25 (t, J = 7.1 Hz, 6H) ppm; ¹³C NMR

(126 MHz, CDCl₃): δ 167.46, 162.83 (dd, J = 249.9, 12.2 Hz),

160.68 (dd, J = 250.0, 12.1 Hz), 131.58 (dd, J = 9.8, 4.5 Hz), 116.52

(dd, J = 14.6, 4.0 Hz), 111.64 (dd, J = 21.4, 3.7 Hz), 103.88 (t, J =

25.8 Hz), 62.17, 49.97 (d, J = 2.7 Hz), 14.01 ppm; IR (ﬁlm) ν: 3449,

1737, 1624, 1508, 1291, 1220, 1144, 1031, 970, 851 cm⁻¹; HRMS

(ES⁺): calcd for C₁₃H₁₅F₂O₄[M + H]⁺, 273.0938; found, 273.0946.

Diethyl 2-(2,6-Diﬂuorophenyl)malonate (80). General proce-

dure A was followed using diethyl malonate (4.981 g, 31.10 mmol)

and 2-bromo-1,3-diﬂuorobenzene (2.000 g, 10.30 mmol). Puriﬁcation

via preparative reverse-phase HPLC provided the title compound as a

colorless oil (2.082 g, 7.64 mmol, 74%). ¹H NMR (500 MHz,

CDCl₃): δ 7.34−7.27 (m, 1H), 6.93 (t, J = 8.1 Hz, 2H), 4.97 (s, 1H),

4.26 (q, J = 7.1 Hz, 4H), 1.27 (t, J = 7.1 Hz, 6H) ppm; ¹³C NMR

(126 MHz, CDCl₃): δ 167.05, 161.29 (dd, J = 250.6, 7.1 Hz), 130.27

(t, J = 11.9 Hz), 111.75 (t, J = 25.6 Hz), 111.65 (dd, J = 20.6, 4.6 Hz),

62.38, 47.32, 14.05 ppm; IR (ﬁlm) ν: 3460, 2985, 1746, 1628, 1595,

1471, 1370, 1303, 1274, 1236, 1157, 1096, 1035, 1003, 788, 698

cm⁻¹; HRMS (ES⁺): calcd for C₁₃H₁₄F₂NaO₄[M + Na]⁺, 295.0758;

found, 295.0757.

Diethyl 2-(2,3,4-Triﬂuorophenyl)malonate (84). General

procedure A was followed using diethyl malonate (4.560 g, 28.44

mmol) and 1-bromo-2,3,4-triﬂuorobenzene (2.000 g, 9.48 mmol).

Puriﬁcation via silica gel column chromatography (0−8% EtOAc in

hexanes) provided the title compound as a colorless oil (1.621 g, 5.58

1

mmol, 59%). H NMR (500 MHz, CDCl₃): δ 7.28−7.19 (m, 1H),

7.00 (tdd, J = 9.2, 7.0, 2.1 Hz, 1H), 4.90 (s, 1H), 4.29−4.19 (m, 4H),

1.28 (t, J = 7.1 Hz, 6H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ

166.98, 151.11 (ddd, J = 251.2, 9.8, 3.2 Hz), 149.78 (ddd, J = 251.2,

10.7, 3.4 Hz), 139.90 (dt, J = 251.5, 15.5 Hz), 124.30 (dt, J = 7.9, 3.9

Hz), 118.08 (dd, J = 11.8, 4.0 Hz), 112.31 (dd, J = 17.6, 3.9 Hz),

62.40, 50.06, 14.01 ppm; IR (ﬁlm) ν: 3405, 2986, 1738, 1617, 1513,

1490, 1370, 1305, 1243, 1151, 1033, 992, 865, 810, 672 cm⁻¹; HRMS

(ES⁺): calcd for C₁₃H₁₃F₃NaO₄[M + Na]⁺, 313.0664; found,

313.0663.

Diethyl 2-(2,4,6-Triﬂuorophenyl)malonate (85). General

procedure A was followed using diethyl malonate (8.408 g, 52.5

mmol) and 2-bromo-1,3,5-triﬂuorobenzene (3.692 g, 17.5 mmol).

Puriﬁcation via silica gel column chromatography (0−8% EtOAc in

hexanes) provided the title compound as a white powder (2.542 g,

1

8.75 mmol, 50% yield). H NMR (500 MHz; CDCl₃): δ 6.74−6.68

(m, 2H), 4.89 (s, 1H), 4.29−4.23 (m, 4H), 1.28 (t, J = 7.1 Hz, 6H)

ppm; ¹³C NMR (126 MHz, CDCl₃): δ 166.68, 162.78 (dt, J = 250.7,

15.1 Hz), 161.5 (dd, J = 250.7, 15.1 Hz), 161.44 (dd, J = 252.2, 15.1

Hz), 107.25 (dt, J = 18.1, 4.5 Hz), 100.81−100.43 (m), 62.41, 46.95,

14.04 ppm; LCMS: [M + H]⁺, 291.

Diethyl 2-(2,4,6-Triﬂuorobenzyl)malonate (86). To a suspen-

sion of NaH (60 wt % in mineral oil) (0.462 g, 11.56 mmol, 1.30

equiv) in anhydrous DMF (8.5 mL) at 0 °C under N₂was slowly

added diethyl malonate (1.712 g, 10.67 mmol, 1.20 equiv). After

addition, the reaction was stirred at 0 °C for 10 min. Then, 2-

(bromomethyl)-1,3,5-triﬂuorobenzene (2.001 g, 8.89 mmol, 1.00

Diethyl 2-(3,5-Diﬂuorophenyl)malonate (81). General proce-

dure A was followed using diethyl malonate (5.000 g, 31.10 mmol)

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equiv) in solution in anhydrous DMF (8.5 mL) was slowly added and

the reaction was stirred at 0 °C for 1 h. Then, the reaction was

quenched with a saturated solution of NH₄Cl and H₂O (85 mL) was

added. The mixture was extracted with EtOAc (×3). The combined

organic extracts were washed with brine, then dried over MgSO₄,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation via

silica gel column chromatography (0−5% EtOAc in hexanes)

provided the title compound as a colorless oil (2.43 g, 7.99 mmol,

pressure. Puriﬁcation via silica gel column chromatography (0−30%

EtOAc in hexanes) provided the title compound as a colorless oil

(6.734 g, 24.10 mmol, 84%). ¹H NMR (600 MHz, CDCl₃): δ 7.64 (t,

J = 7.6 Hz, 1H), 7.47 (dd, J = 8.0, 1.6 Hz, 1H), 7.38 (dd, J = 9.2, 1.6

Hz, 1H), 4.98 (s, 1H), 4.28−4.18 (m, 4H), 1.26 (t, J = 7.2 Hz, 6H)

ppm; ¹³C NMR (150 MHz, CDCl₃): δ 166.55, 160.04 (d, J = 252.0

Hz), 131.98 (d, J = 3.5 Hz), 128.26 (d, J = 4.0 Hz), 126.21 (d, J =

14.4 Hz), 119.23 (d, J = 26.0 Hz), 117.28 (d, J = 2.9 Hz), 113.71 (d, J

= 9.8 Hz), 62.55, 50.44 (d, J = 2.9 Hz), 14.00 ppm; IR (ﬁlm) ν: 2984,

2236, 1733, 1219 cm⁻¹; LCMS: [M + H]⁺, 280.

1

90%). H NMR (500 MHz, CDCl₃): δ 7.28−7.19 (m, 1H), 7.04−

6.95 (m, 1H), 4.90 (s, 1H), 4.29−4.19 (m, 4H), 1.28 (t, J = 7.1 Hz,

6H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 168.33, 161.72 (dd, J =

248.9, 11.3 Hz), 161.70 (dt, J = 248.9, 16.0, 15.5 Hz), 161.60 (dd, J =

249.0, 11.2 Hz), 109.78 (td, J = 20.1, 4.7 Hz), 100.35−99.84 (m),

61.63, 51.10, 21.45 (t, J = 2.3 Hz), 13.90 ppm; IR (ﬁlm) ν: 3437,

1733, 1639, 1499, 1444, 1371, 1304, 1239, 1153, 1048, 838, 617

cm⁻¹; HRMS (ES⁺): calcd for C₁₄H₁₅F₃NaO₄[M + Na]⁺, 327.0820;

found, 327.0818.

Diethyl 2-(4-Chloro-2,6-diﬂuorophenyl)malonate (91). Gen-

eral procedure A was followed using diethyl malonate (6.390 g, 39.57

mmol) and 2-bromo-5-chloro-1,3-diﬂuorobenzene (3.000 g, 13.19

mmol). Puriﬁcation via silica gel column chromatography (0−8%

EtOAc in hexanes) provided the title compound as a colorless oil

1

(1.424 g, 4.63 mmol, 35%). H NMR (600 MHz, CDCl₃): δ 6.96−

6.93 (m, 2H), 4.89 (s, 1H), 4.24 (q, J = 7.2 Hz, 4H), 1.28−1.24 (m,

6H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 166.40, 161.05 (dd, J =

252.7, 8.8 Hz), 135.40 (t, J = 13.4 Hz), 113.18−112.42 (m), 109.85

(t, J = 18.8 Hz), 62.4, 47.07, 14.01 ppm; IR (ﬁlm) ν: 2838, 1678,

1642, 1249 cm⁻¹; LCMS: [M + H]⁺, 308.

Diethyl 2-(2,6-Diﬂuoro-4-methylphenyl)malonate (87). Gen-

eral procedure A was followed using diethyl malonate (4.180 g, 26.10

mmol) and 2-bromo-1,3-diﬂuoro-5-methylbenzene (1.800 g, 8.69

mmol). Puriﬁcation via silica gel column chromatography (0−8%

EtOAc in hexanes) provided the title compound as a colorless oil

Diethyl 2-(2,6-Diﬂuoro-4-nitrophenyl)malonate (92). A

mixture of 1,2,3-triﬂuoro-5-nitrobenzene (1.000 g, 5.65 mmol, 1

equiv), potassium carbonate (1.562 g, 11.29 mmol, 2.00 equiv), and

diethyl malonate (0.914 g, 5.71 mmol, 1.01 equiv) in DMF (6.37 mL)

under N₂was stirred at 65 °C until the starting material was

consumed as indicated by TLC. The reaction mixture was cooled to

rt, washed with 1 N HCl (50 mL), and extracted with EtOAc (×3).

The organic layers were combined, washed with satd. aq NaCl, dried

over Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

Puriﬁcation via silica gel column chromatography (0−30% EtOAc in

hexanes) provided the title compound as a white solid (1.536 g, 4.85

1

(2.005 g, 6.99 mmol, 80%). H NMR (600 MHz, CDCl₃): δ 6.67−

6.65 (m, 2H), 4.83 (d, J = 3.0 Hz, 1H), 4.20−4.14 (m, 4H), 2.27 (d, J

= 3.2 Hz, 3H), 1.22−1.17 (m, 6H) ppm; ¹³C NMR (150 MHz,

CDCl₃): δ 166.89, 160.75 (dd, J = 249.1, 8.1 Hz), 141.32, 115.89−

110.83 (m), 107.42 (t, J = 18.8 Hz), 61.99, 46.98, 21.23 (d, J = 1.9

Hz), 13.87 ppm; IR (ﬁlm) ν: 3000, 1678, 1106, 1017 cm⁻¹. LCMS:

[M + H]⁺, 287.

Diethyl 2-(2,6-Diﬂuoro-4-(triﬂuoromethyl)phenyl)malonate

(88). General procedure A was followed using diethyl malonate

(2.160 g, 13.50 mmol) and 1,2,3-triﬂuoro-5-(triﬂuoromethyl)benzene

(0.900 g, 4.50 mmol). Puriﬁcation via silica gel column chromatog-

raphy (0−5% EtOAc in hexanes) provided the title compound as a

colorless oil (0.620 g, 1.82 mmol, 40%). ¹H NMR (500 MHz,

CDCl₃): δ 7.23 (d, J = 7.3 Hz, 2H), 4.99 (s, 1H), 4.27 (dt, J = 7.4, 6.5

Hz, 4H), 1.29 (t, J = 7.1 Hz, 6H) ppm; ¹³C NMR (126 MHz,

CDCl₃): δ 166.12, 161.21 (dd, J = 252.9, 7.4 Hz), 132.89 (qt, J =

34.8, 10.3 Hz), 122.63 (qt, J = 272.6, 3.3 Hz), 114.85 (t, J = 18.5 Hz),

109.41 (ddt, J = 26.3, 7.4, 3.8 Hz), 62.69, 47.34, 14.07 ppm; IR (ﬁlm)

ν: 3083, 2987, 1755, 1061, 1442, 1365, 1306, 1240, 1177, 1138, 1080,

1031, 910, 867, 719, 702 cm⁻¹; HRMS (ES⁺): calcd for

C₁₄H₁₃F₅NaO₄[M + Na]⁺, 363.0632; found, 363.0631.

Diethyl 2-(4-Cyano-2,6-diﬂuorophenyl)malonate (89). A

mixture of 3,4,5-triﬂuorobenzonitrile (1.000 g, 6.37 mmol, 1 equiv),

potassium carbonate (1.760 g, 12.70 mmol, 2.00 equiv), and diethyl

malonate (1.031 g, 6.43 mmol, 1.01 equiv) in DMF (6.37 mL) under

N₂was stirred at 65 °C until the starting material was consumed as

determined by TLC. The reaction mixture was cooled to rt, washed

with 1 M HCl (50 mL), and extracted with EtOAc (×3). The organic

layers were combined, washed with satd. aq NaCl, dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation via

silica gel column chromatography (0−30% EtOAc in hexanes)

provided the title compound as a white solid (1.408 g, 4.69 mmol,

73%). ¹H NMR (600 MHz, CDCl₃): δ 7.30 (d, J = 7.0 Hz, 2H), 5.01

(s, 1H), 4.31−4.24 (m, 4H), 1.29 (t, J = 7.4 Hz, 6H) ppm; ¹³C NMR

(150 MHz, CDCl₃): δ 165.54, 161.02 (dd, J = 254.0, 7.8 Hz), 116.78

(t, J = 18.5 Hz), 116.20 (t, J = 3.4 Hz), 115.94−115.48 (m), 113.88

(t, J = 12.4 Hz), 62.60, 47.24, 13.87 ppm; IR (ﬁlm) ν: 2856, 2178,

1695 cm⁻¹; LCMS: [M + H]⁺, 298.

1

mmol, 86%). H NMR (600 MHz, CDCl₃): δ 7.84 (d, J = 7.2 Hz,

2H), 5.00 (s, 1H), 4.27 (q, J = 7.1 Hz, 4H), 1.28 (t, J = 7.2 Hz, 6H)

ppm; ¹³C NMR (150 MHz, CDCl₃): δ 165.55, 160.93 (dd, J = 254.8,

7.7 Hz), 148.60 (t, J = 11.2 Hz), 118.05 (t, J = 18.7 Hz), 108.15−

107.61 (m), 62.84, 47.49, 14.04 ppm.

5,7-Dichloro-6-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (93).

General procedure B was followed using diethyl 2-phenylmalonate

(0.780 g, 3.30 mmol) (76) and 3-amino-1,2,4-triazole (0.292 g, 3.47

mmol). Then, the intermediate sodium 6-phenyl-[1,2,4]triazolo[1,5-

a]pyrimidine-5,7-bis(olate) (0.740 g, 2.71 mmol) and POCl₃(7.410

g, 48.46 mmol) were used to obtain the title compound as a brown

1

solid (0.527 g, 1.99 mmol, 73%). H NMR (500 MHz, CDCl₃): δ

8.56 (s, 1H), 7.58−7.51 (m, 3H), 7.38−7.34 (m, 2H) ppm; ¹³C NMR

(126 MHz, CDCl₃): δ 156.93, 156.65, 153.35, 139.90, 131.72, 130.07,

129.95, 129.16, 124.01 ppm; IR (ﬁlm) ν: 3437, 1637, 1458, 1383,

1267, 1205, 1180, 867, 802, 764, 740, 698, 652 cm⁻¹; HRMS (ES⁺):

calcd for C₁₁H₇Cl₂N₄[M + H]⁺, 265.0042; found, 265.0043.

5,7-Dichloro-6-(4-ﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (94). General procedure B was followed using diethyl 2-

(4-ﬂuorophenyl)malonate (0.700 g, 2.75 mmol) (77) and 3-amino-

1,2,4-triazole (0.242 g, 2.89 mmol). Then, the intermediate sodium 6-

(4-ﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-bis(olate)

(0.760 g, 2.61 mmol) and POCl₃(7.150 g, 46.45 mmol) were used to

obtain the title compound as a brown solid (0.481 g, 1.70 mmol,

67%). ¹H NMR (500 MHz, CDCl₃): δ 8.53 (s, 1H), 7.36 (dd, J = 8.4,

5.2 Hz, 2H), 7.29−7.20 (m, 2H) ppm; ¹³C NMR (126 MHz,

CDCl₃): δ 163.47 (d, J = 250.9 Hz), 156.95, 156.53, 153.29, 140.05,

132.18 (d, J = 8.6 Hz), 127.59 (d, J = 3.7 Hz), 123.03, 116.43 (d, J =

21.9 Hz) ppm; IR (ﬁlm) ν: 3421, 1600, 1529, 1504, 1460, 1330,

1267, 1229, 1203, 1183, 1160, 1089, 1024, 899, 837, 790 cm⁻¹;

HRMS (ES⁺): calcd for C₁₁H₆Cl₂FN₄[M + H]⁺, 282.9948; found,

282.9945.

Diethyl 2-(4-Cyano-2-ﬂuorophenyl)malonate (90). To a dry

ﬂask, under nitrogen, was added 3,4-diﬂuorobenzonitrile (4.000 g,

28.81 mmol, 1 equiv), K₂CO₃(7.954 g, 57.51 mmol, 2.00 equiv),

diethyl malonate (4.660 g, 29.10 mmol, 1.01 equiv), and anhydrous

DMF (24 mL) and it was heated at 65 °C until the starting material

was consumed based on TLC. The reaction mixture was cooled to rt

and added to a separatory funnel containing 50 mL of 1 N HCl. The

mixture was extracted with EtOAc (×3), and the combined organic

layers were washed with H₂O and satd. aq NaCl. The organic layer

was dried over anh. Na₂SO₄, ﬁltered, and concentrated under reduced

5,7-Dichloro-6-(2-ﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (95). General procedure B was followed using diethyl 2-

(2-ﬂuorophenyl)malonate (0.700 g, 2.75 mmol) (78) and 3-amino-

1,2,4-triazole (0.242 g, 2.89 mmol). Then, the intermediate sodium 6-

(2-ﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-bis(olate)

(0.760 g, 2.61 mmol) and POCl₃(7.150 g, 46.45 mmol) were used to

1097

https://dx.doi.org/10.1021/acs.jmedchem.0c01605

J. Med. Chem. 2021, 64, 1073−1102

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

obtain the title compound as a brown solid (0.572 g, 2.02 mmol,

77%). ¹H NMR (500 MHz, CDCl₃): δ 8.54 (t, J = 3.3 Hz, 1H), 7.59−

7.51 (m, 1H), 7.38−7.29 (m, 2H), 7.28−7.20 (m, 1H) ppm; ¹³C

NMR (126 MHz, CDCl₃): δ 159.79 (d, J = 249.6 Hz), 156.94,

156.57, 153.53, 140.57, 132.48 (d, J = 8.2 Hz), 131.78 (d, J = 1.8 Hz),

124.85 (d, J = 3.7 Hz), 119.39 (d, J = 15.9 Hz), 118.45, 116.38 (d, J =

21.1 Hz) ppm; IR (ﬁlm) ν: 3436, 1598, 1492, 1463, 1425, 1366,

1330, 1263, 1221, 1196, 1131, 1099, 1033, 900, 822, 792, 755, 652

cm⁻¹; HRMS (ES⁺): calcd for C₁₁H₅Cl₂FN₄Na [M + Na]⁺, 304.9768;

found, 304.9772.

cm⁻¹; HRMS (ES⁺): calcd for C₁₁H₅Cl₂F₂N₄[M + H]⁺, 300.9854;

found, 300.9857.

5,7-Dichloro-6-(3,4-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (100). General procedure B was followed using diethyl

2-(3,4-diﬂuorophenyl)malonate (0.456 g, 1.67 mmol) (83) and 3-

amino-1,2,4-triazole (0.147 g, 1.73 mmol). Then, the intermediate

sodium 6-(3,4-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-

bis(olate) (0.400 g, 1.30 mmol) and POCl₃(2.982 g, 23.11 mmol)

were used to obtain the title compound as a brown solid (0.183 g,

1

0.61 mmol, 40%). H NMR (500 MHz, CDCl₃): δ 8.57 (s, 1H),

7.41−7.32 (m, 1H), 7.26−7.21 (m, 1H), 7.16−7.11 (m, 1H) ppm;

5,7-Dichloro-6-(2,4-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (96). General procedure B was followed using diethyl 2-

(2,4-diﬂuorophenyl)malonate (0.800 g, 2.94 mmol) (79) and 3-

amino-1,2,4-triazole (0.260 g, 3.09 mmol). Then, the intermediate

sodium 6-(2,4-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-

bis(olate) (0.800 g, 2.60 mmol) and POCl₃(7.080 g, 46.23 mmol)

were used to obtain the title compound as a brown solid (0.455 g,

¹³C NMR (126 MHz, CDCl₃): δ 157.20, 156.17, 153.39, 151.41 (dd,

J = 252.7, 11.8 Hz), 150.60 (dd, J = 251.2, 12.8 Hz), 140.27, 128.21

(dd, J = 6.6, 4.6 Hz), 126.97 (dd, J = 6.7, 3.9 Hz), 122.03, 119.74 (d, J

= 18.4 Hz), 118.49 (d, J = 17.9 Hz) ppm; IR (ﬁlm) ν: 3377, 2918,

1606, 1460, 1383, 1269, 1180, 1120, 861, 793, 652 cm⁻¹; HRMS

(ES⁺): calcd for C₁₁H₅Cl₂F₂N₄[M + H]⁺, 300.9854; found, 300.9859.

5,7-Dichloro-6-(2,3,4-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (101). General procedure B was followed using diethyl

2-(2,3,4-triﬂuorophenyl)malonate (0.800 g, 2.75 mmol) (84) and 3-

amino-1,2,4-triazole (0.242 g, 2.89 mmol). Then, the intermediate

sodium 6-(2,3,4-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-

5,7-bis(olate) (0.740 g, 2.67 mmol) and POCl₃(6.151 g, 40.33

mmol) were used to obtain the title compound as a brown solid

(0.367 g, 1.15 mmol, 43%). ¹H NMR (500 MHz, CDCl₃): δ 8.59 (s,

1H), 7.24−7.16 (m, 1H), 7.16−7.09 (m, 1H) ppm; ¹³C NMR (126

MHz, CDCl₃): δ 157.39, 156.24, 153.75, 152.71 (ddd, J = 254.7, 9.8,

3.0 Hz), 149.54 (ddd, J = 253.6, 10.9, 3.6 Hz), 141.10, 140.64 (dt, J =

254.7, 15.1 Hz), 125.78 (ddd, J = 8.0, 4.4, 1.7 Hz), 117.08 (dd, J =

12.9, 4.0 Hz), 116.70 (d, J = 2.3 Hz), 113.42 (dd, J = 18.0, 3.9 Hz)

ppm; IR (ﬁlm) ν: 3387, 1601, 1485, 1458, 1383, 1343, 1274, 12010,

1180, 1053, 1024, 963, 915, 859, 820, 790, 761, 689, 650 cm⁻¹;

HRMS (ES⁺): calcd for C₁₁H₄Cl₂F₃N₄[M + H]⁺, 318.9760; found,

319.9764.

1

1.51 mmol, 58%). H NMR (500 MHz, CDCl₃): δ 8.50 (s, 1H),

7.39−7.32 (m, 1H), 7.06 (td, J = 8.3, 2.5 Hz, 1H), 6.99 (td, J = 9.1,

2.5 Hz, 1H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 164.23 (dd, J =

253.6, 11.8 Hz), 160.18 (dd, J = 252.2, 12.3 Hz), 156.94, 156.47,

153.47, 140.79, 132.91 (dd, J = 10.1, 3.4 Hz), 117.55, 115.54 (dd, J =

16.1, 4.1 Hz), 112.47 (dd, J = 21.9, 3.7 Hz), 104.96 (t, J = 25.4 Hz)

ppm; IR (ﬁlm) ν: 3451, 1600, 1503, 1464, 1424, 1368, 1335, 1267,

1196, 1143, 1128, 1092, 1022, 969, 890, 851, 796, 765, 735, 652, 631

cm⁻¹; HRMS (ES⁺): calcd for C₁₁H₅Cl₂F₂N₄[M + H]⁺, 300.9854;

found, 300.9850.

5,7-Dichloro-6-(2,6-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (97). General procedure B was followed using diethyl 2-

(2,6-diﬂuorophenyl)malonate (0.800 g, 2.94 mmol) (80) and 3-

amino-1,2,4-triazole (0.260 g, 3.09 mmol). Then, the intermediate

sodium 6-(2,6-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-

bis(olate) (0.600 g, 1.90 mmol) and POCl₃(5.310 g, 34.70 mmol)

were used to obtain the title compound as a brown solid (0.290 g,

5,7-Dichloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (102). General procedure B was followed using diethyl

2-(2,4,6-triﬂuorophenyl)malonate (3.66 g, 12.58 mmol) (85) and 3-

amino-1,2,4-triazole (1.07 g, 12.83 mmol). Then, the intermediate

sodium 6-(2,3,4-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-

5,7-bis(olate) (2.000 g, 6.29 mmol) and POCl₃(17.20 g, 112.0

mmol) were used to obtain the title compound as a brown solid (1.00

1

0.96 mmol, 49%). H NMR (500 MHz, CDCl₃): δ 8.59 (d, J = 1.4

Hz, 1H), 7.61−7.52 (m, 1H), 7.11 (t, J = 7.9 Hz, 2H) ppm; ¹³C NMR

(126 MHz, CDCl₃): δ 160.23 (dd, J = 252.0, 5.7 Hz), 157.17, 156.65,

153.87, 141.38, 133.06 (t, J = 10.2 Hz), 113.14, 112.16 (dd, J = 20.4,

4.2 Hz), 109.07 (t, J = 19.9 Hz) ppm; IR (ﬁlm) ν: 3427, 1629, 1602,

1460, 1384, 1266, 1194, 1003, 899, 806, 765, 652 cm⁻¹; HRMS

(ES⁺): calcd for C₁₁H₅Cl₂F₂N₄[M + H]⁺, 300.9854; found, 300.9866.

5,7-Dichloro-6-(3,5-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (98). General procedure B was followed using diethyl 2-

(3,5-diﬂuorophenyl)malonate (0.700 g, 2.57 mmol) (81) and 3-

amino-1,2,4-triazole (0.227 g, 2.70 mmol). Then, the intermediate

sodium 6-(3,5-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-

bis(olate) (0.700 g, 2.27 mmol) and POCl₃(6.200 g, 40.51 mmol)

were used to obtain the title compound as a brown solid (0.422 g,

1.40 mmol, 62%). ¹H NMR (500 MHz, CDCl₃): δ 8.60 (s, 1H), 7.02

(tt, J = 8.8, 2.4 Hz, 1H), 6.97−6.89 (m, 2H) ppm; ¹³C NMR (126

MHz, CDCl₃): δ 163.29 (dd, J = 251.5, 12.6 Hz), 157.31, 155.74,

153.47, 140.16, 134.33 (t, J = 10.3 Hz), 121.84, 113.71 (dd, J = 19.6,

6.9 Hz), 105.89 (t, J = 25.0 Hz) ppm; IR (ﬁlm) ν: 3434, 1628, 1595,

1460, 1435, 1121, 987, 833 cm⁻¹; HRMS (ES⁺): calcd for

C₁₁H₅Cl₂F₂N₄[M + H]⁺, 300.9854; found, 300.9871.

1

g, 3.14 mmol, 50%). H NMR (500 MHz, CDCl₃): δ 8.62 (s, 1H),

6.91 (t, J = 6.0 Hz, 2H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ

164.60 (dt, J = 253.7, 16.6 Hz), 160.77 (dd, J = 252.9, 9.1 Hz), 160.67

(dd, J = 252.9, 9.1 Hz), 157.36, 156.69, 141.67, 112.36, 105.67 (dt, J

= 19.6, 4.5 Hz), 101.63−101.26 (m) ppm.

5,7-Dichloro-6-(2,4,6-triﬂuorobenzyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (103). General procedure B was followed using diethyl

2-(2,4,6-triﬂuorobenzyl)malonate (0.800 g, 2.63 mmol) (86) and 3-

amino-1,2,4-triazole (0.232 g, 2.76 mmol). Then, the intermediate

sodium 6-(2,4,6-triﬂuorobenzyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-

bis(olate) (0.700 g, 2.06 mmol) and POCl₃(5.610 g, 36.60 mmol)

were used to obtain the title compound as a brown solid (0.355 g,

1.07 mmol, 52%). ¹H NMR (500 MHz, CDCl₃): δ 8.40 (s, 1H), 6.59

(t, J = 8.5 Hz, 2H), 4.27 (s, 2H) ppm; ¹³C NMR (126 MHz, CDCl₃):

δ 161.93 (dt, J = 250.5, 16.0 Hz), 161.44 (dd, J = 249.9, 10.6 Hz),

161.33 (dd, J = 249.8, 10.7 Hz), 156.68, 156.48, 152.67, 140.34,

118.92, 108.12 (td, J = 18.5, 4.8 Hz), 102.62−96.83 (m), 23.49 (t, J =

2.4 Hz) ppm; IR (ﬁlm) ν: 3408, 1633, 1471, 1266, 1118, 1038 cm⁻¹;

HRMS (ES⁺): calcd for C₁₂H₆Cl₂F₃N₄[M + H]⁺, 332.9916; found,

332.9906.

5,7-Dichloro-6-(2,5-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (99). General procedure B was followed using diethyl 2-

(2,5-diﬂuorophenyl)malonate (0.800 g, 2.94 mmol) (82) and 3-

amino-1,2,4-triazole (0.259 g, 3.09 mmol). Then, the intermediate

sodium 6-(2,5-diﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-

bis(olate) (0.800 g, 2.60 mmol) and POCl₃(7.080 g, 46.23 mmol)

were used to obtain the title compound as a brown solid (0.443 g,

5,7-Dichloro-6-(2,6-diﬂuoro-4-methylphenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidine (104). General procedure B was

followed using diethyl 2-(2,6-diﬂuoro-4-methylphenyl)malonate

(0.800 g, 2.79 mmol) (87) and 3-amino-1,2,4-triazole (0.247 g,

2.93 mmol). Then, the intermediate sodium 6-(2,6-diﬂuoro-4-

methylphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-bis(olate) (0.500

g, 1.55 mmol) and POCl₃(4.240 g, 27.60 mmol) were used to obtain

1

1.47 mmol, 57%). H NMR (500 MHz, CDCl₃): δ 8.58 (s, 1H),

7.28−7.23 (m, 2H), 7.11−7.07 (m, 1H) ppm; ¹³C NMR (126 MHz,

CDCl₃): δ 158.60 (dd, J = 245.1, 2.5 Hz), 157.24, 156.21, 156.03 (dd,

J = 245.9, 2.7 Hz), 153.68, 140.80, 120.50 (dd, J = 18.7, 8.8 Hz),

119.24 (dd, J = 23.9, 8.4 Hz), 118.42 (dd, J = 25.1, 2.2 Hz), 117.80

(dd, J = 24.2, 8.7 Hz), 117.43 ppm; IR (ﬁlm) ν: 3421, 1642, 1492,

1458, 1432, 1383, 1253, 1203, 1092, 1025, 858, 822, 775, 699, 651

1

the title compound as a brown solid (0.334 g, 1.06 mmol, 68%). H

NMR (600 MHz, CDCl₃): δ 8.59 (s, 1H), 6.92 (d, J = 8.5 Hz, 2H),

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2.46 (s, 3H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ 159.92 (dd, J =

251.1, 6.6 Hz), 157.07, 157.01, 153.85, 144.81 (d, J = 19.7 Hz),

141.44, 113.44, 112.74 (dd, J = 20.7, 3.5 Hz), 106.01 (d, J = 39.9 Hz),

21.88 (d, J = 1.9 Hz) ppm; IR (ﬁlm) ν: 3071, 1606, 1571, 1356 cm⁻¹;

LCMS: [M + H]⁺, 317.

150.51 (d, J = 10.5 Hz), 141.48, 116.12−115.19 (m), 111.63,

108.69−108.36 (m) ppm; IR (ﬁlm) ν: 2916, 2848, 1698, 1601, 1532

cm⁻¹; LCMS: [M + H]⁺, 347.

Methyl 2-(5-Chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-yl)-4,4,4-triﬂuoro-3-methylbutanoate

(110). To a solution of HMDS (0.101 g, 0.63 mmol, 1 equiv) in THF

(0.632 mL) at −78 °C was added n-butyllithium (0.369 mL, 0.63

mmol, 1 equiv) and the mixture was stirred at 0 °C for 1 h. Then,

methyl 4,4,4-triﬂuoro-3-methylbutanoate (0.117 g, 0.69 mmol, 1.1

equiv) was added dropwise at −78 °C and the reaction mixture was

stirred for 1 h. Then, a solution of 5,7-dichloro-6-(2,4,6-

triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.200 g, 0.63

mmol, 1 equiv) (102) in THF (0.500 mL) was added at −78 °C

and the resulting mixture was stirred for 2.5 h prior to quenching of

the reaction with 1 M HCl. The aqueous layer was then extracted

using EtOAc (×2) and washed with sat. aq NaCl, dried, and

concentrated under reduced pressure. Puriﬁcation via silica gel

chromatography using hexanes/EtOAc (5−20%) provided the

5,7-Dichloro-6-(2,6-diﬂuoro-4-(triﬂuoromethyl)phenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine (105). General procedure B was

followed using diethyl 2-(2,6-diﬂuoro-4-(triﬂuoromethyl)phenyl)-

malonate (0.600 g, 1.76 mmol) (88) and 3-amino-1,2,4-triazole

(0.156 g, 1.85 mmol). Then, the intermediate sodium 6-(2,6-diﬂuoro-

4-(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidine-5,7-bis-

(olate) (0.600 g, 1.60 mmol) and POCl₃(4.350 g, 28.41 mmol) were

used to obtain the title compound as a brown solid (0.392 g, 1.07

mmol, 66%). ¹H NMR (500 MHz, CDCl₃): δ 8.62 (s, 1H), 7.47 (d, J

= 7.2 Hz, 2H) ppm; ¹³C NMR (126 MHz, CDCl₃): δ 160.16 (dd, J =

254.8, 5.8 Hz), 157.22, 155.50, 153.81, 141.35, 135.19 (qt, J = 35.3,

34.6, 9.5 Hz), 122.14 (qt, J = 273.3, 3.2 Hz), 112.72 (t, J = 19.7 Hz),

111.49, 110.18−109.25 (m) ppm; IR (ﬁlm) ν: 3435, 1633, 1603,

1466, 1437, 1364, 1266, 1250, 1179, 1137, 1038, 916, 868, 801, 766,

705, 653 cm⁻¹; HRMS (ES⁻) calcd for C₁₂H₃Cl₂F₄N₄[M − F]⁻,

348.9676; found, 348.9662.

4-(5,7-Dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-di-

ﬂuorobenzonitrile (106). General procedure B was followed using

diethyl 2-(4-cyano-2,6-diﬂuorophenyl)malonate (0.500 g, 1.68 mmol)

(89) and 3-amino-1,2,4-triazole (0.148 g, 1.77 mmol). Then, the

intermediate sodium 6-(4-cyano-2,6-diﬂuorophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine-5,7-bis(olate) (0.400 g, 1.20 mmol) and POCl₃

(3.280 g, 21.41 mmol) were used to obtain the title compound as a

brown solid (0.210 g, 1.20 mmol, 53%). ¹H NMR (600 MHz,

CDCl₃): δ 8.65 (s, 1H), 7.47 (d, J = 6.2 Hz, 2H) ppm; ¹³C NMR

(150 MHz, CDCl₃): δ 161.21 (d, J = 6.4 Hz), 159.51 (d, J = 6.2 Hz),

157.66, 155.57, 116.85, 116.62−116.30 (m), 115.86 (t, J = 3.5 Hz),

114.64 (t, J = 19.6 Hz) ppm; IR (ﬁlm) ν: 3087, 2195, 1571, 1338,

1195 cm⁻¹; LCMS: [M + H]⁺, 327.

1

product (0.200 g, 0.44 mmol, 70%) as a white solid. H NMR (600

MHz, CDCl₃): mixture of diastereomers δ 8.53 (d, J = 6.6 Hz, 1H),

6.94−6.86 (m, 2H), 4.04 (d, J = 7.4 Hz, 1H), 3.92−3.85 (m, 1H),

3.62 (d, J = 10.4 Hz, 3H), 1.38 (d, J = 6.6 Hz, 1H), 0.80 (d, J = 7.4

Hz, 2H) ppm; HRMS (ES⁺): calcd for C₁₇H₁₂ClF₆N₄O₂[M + H]⁺,

453.0547; found, 453.0542.

7,9-Diﬂuoro-10-(3,3,3-triﬂuoro-2-methylpropyl)benzofuro-

[2,3-d][1,2,4]triazolo[1,5-a]pyrimidine (111). Methyl 2-(5-

chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-

4,4,4-triﬂuoro-3-methylbutanoate (0.020 g, 0.04 mmol, 1 equiv)

(110), LiCl (0.002 g, 0.04 mmol, 1 equiv), and DMSO (0.100 mL)

were stirred at 130 °C for 3 h (microwave irradiation). The reaction

mixture was then diluted in water and extracted with EtOAc (×3) and

the combined organic layers were washed with brine (×2), dried over

MgSO₄, ﬁltered, and concentrated. Puriﬁcation by reverse-phase

HPLC provided the title compound as a white solid (0.012 g, 0.03

mmol, 76%). X-ray quality crystals were obtained by slow evaporation

from a CH₂Cl₂/pentane solution: mp (CH₂Cl₂/pentane) 103.4−

4-(5,7-Dichloro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3-ﬂuo-

robenzonitrile (107). General procedure B was followed using

diethyl 2-(4-cyano-2,6-diﬂuorophenyl)malonate (0.500 g, 1.68 mmol)

(90) and 3-amino-1,2,4-triazole (0.148 g, 1.77 mmol). Then, the

intermediate sodium 6-(4-cyano-2-ﬂuorophenyl)-[1,2,4]triazolo[1,5-

a]pyrimidine-5,7-bis(olate) (3.000 g, 9.52 mmol) and POCl₃(26.000

g, 169.00 mmol) were used to obtain the title compound as a brown

1

107.4. H NMR (600 MHz, CDCl₃): δ 8.55 (s, 1H), 8.01 (s, 1H),

7.01 (ddd, J = 11.0, 9.3, 2.2 Hz, 1H), 6.86−6.79 (m, 3H), 4.11−4.05

(m, 2H), 3.88−3.78 (m, 2H), 3.30 (p, J = 7.3 Hz, 1H), 3.20 (dd, J =

13.9, 5.8 Hz, 2H), 3.05 (dt, J = 15.1, 7.6 Hz, 2H), 2.79 (dd, J = 14.0,

8.7 Hz, 2H), 1.20 (d, J = 7.0 Hz, 3H), 1.02 (d, J = 7.0 Hz, 3H) ppm;

¹³C NMR (150 MHz, CDCl₃): δ 165.15−164.48 (m), 163.01 (d, J =

1

solid (1.970 g, 9.52 mmol, 67%). H NMR (600 MHz, CDCl₃): δ

8.61 (s, 1H), 7.68 (dd, J = 7.9, 1.5 Hz, 1H), 7.61 (dd, J = 8.6, 1.5 Hz,

1H), 7.54 (t, J = 7.4 Hz, 1H) ppm; ¹³C NMR (150 MHz, CDCl₃): δ

160.47, 158.79, 157.48, 155.52, 153.79, 140.73, 133.32, 128.86 (d, J =

4.6 Hz), 124.68 (d, J = 15.7 Hz), 120.42 (d, J = 24.9 Hz), 116.76 (d, J

= 2.9 Hz), 116.69, 116.36 (d, J = 9.3 Hz) ppm.

15.1 Hz), 162.26−160.00 (m), 158.41, 156.38, 152.47, 150.91,

145.88, 143.80, 127.47 (dd, J = 279.5, 28.1 Hz), 108.69, 106.96,

106.21−104.64 (m), 102.08−100.00 (m), 97.85 (dd, J = 27.4, 4.6

Hz), 36.01 (q, J = 28.3, 27.6 Hz), 30.84, 29.86 ppm; HRMS (ES⁺):

calcd for C₁₅H₉F₅N₄O [M + H]⁺, 357.0769; found, 357.0768.

tert-Butyl-2-(5-chloro-6-(2,4,6-triﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)-4,4,4-triﬂuoro-3-methylbuta-

noate (112). To a solution of HMDS (0.054 g, 0.33 mmol, 1.6

equiv) in THF (3 mL) at −78 °C was added n-butyllithium (0.180

mL, 0.33 mmol, 1.6 equiv) and the mixture was stirred at 0 °C for 30

min. Then, tert-butyl 4,4,4-triﬂuoro-3-methylbutanoate (0.066 g, 0.31

mmol, 1.5 equiv) was added dropwise at −78 °C and the mixture was

stirred at this temperature for 1 h. Finally, a solution of 5,7-dichloro-6-

(2,4,6-triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (0.066 g,

0.21 mmol, 1 equiv) (102) in THF (0.500 mL) was added at −78

°C. After stirring for 2.5 h at this temperature, the reaction was

quenched with a 1 M HCl solution. The aqueous layer was then

extracted twice with EtOAc and the combined organic layers were

washed with brine, dried, and concentrated under reduced pressure.

Puriﬁcation via silica gel chromatography using a hexanes/EtOAc

gradient (95/05 to 80/20) provided tert-butyl 2-(5-chloro-6-(2,4,6-

triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-4,4,4-triﬂuoro-

5,7-Dichloro-6-(4-chloro-2,6-diﬂuorophenyl)-[1,2,4]-

triazolo[1,5-a]pyrimidine (108). General procedure B was

followed using diethyl 2-(4-chloro-2,6-diﬂuorophenyl)malonate (1.2

g, 3.91 mmol) (91) and 3-amino-1,2,4-triazole (0.350 g, 4.10 mmol).

Then, the intermediate sodium 6-(4-chloro-2,6-diﬂuorophenyl)-

[1,2,4]triazolo[1,5-a]pyrimidine-5,7-bis(olate) (1.000 g, 2.92 mmol)

and POCl₃(7.970 g, 52.00 mmol) were used to obtain the title

1

compound as a brown solid (0.710 g, 2.12 mmol, 73%). H NMR

(600 MHz, CDCl₃): δ 8.62 (s, 1H), 7.18 (d, J = 7.1 Hz, 2H) ppm;

¹³C NMR (150 MHz, CDCl₃): δ 161.29−159.09 (m), 157.36, 156.45,

153.95, 141.55, 138.63 (t, J = 13.0 Hz), 113.78−113.35 (m), 112.29,

108.08 (t, J = 20.0 Hz) ppm; IR (ﬁlm) ν: 3106, 1642, 1427, 1391,

1071 cm⁻¹; LCMS: [M + H]⁺, 336.

5,7-Dichloro-6-(2,6-diﬂuoro-4-nitrophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine (109). General procedure B was followed using

diethyl 2-(2,6-diﬂuoro-4-nitrophenyl)malonate (0.500 g, 1.58 mmol)

(92) and 3-amino-1,2,4-triazole (0.139 g, 1.65 mmol). Then, the

intermediate sodium 6-(2,6-diﬂuoro-4-nitrophenyl)-[1,2,4]triazolo-

[1,5-a]pyrimidine-5,7-bis(olate) (0.150 g, 0.43 mmol) and POCl₃

(1.160 g, 7.56 mmol) were used to obtain the title compound as a

brown solid (0.100 g, 0.29 mmol, 68%). ¹H NMR (600 MHz,

CDCl₃): δ 8.64 (s, 1H), 8.04 (d, J = 6.8 Hz, 2H) ppm; ¹³C NMR

(150 MHz, CDCl₃): δ 160.22 (dd), 157.70, 156.90, 155.39, 154.08,

1

3-methylbutanoate (0.026 g, 0.05 mmol, 25%) as a white solid. H

NMR (600 MHz, CDCl₃): mixture of diastereomers δ 8.56 (s, 0.6H),

8.55 (s, 0.4H), 6.91−6.87 (m, 2H), 4.14−4.04 (m, 0.4H), 3.95−3.83

(m, 1.2H), 3.72 (d, 0.4H), 1.38 (d, J = 6.7 Hz, 1.3H), 1.27 (s, 3.5H),

1.25 (s, 6.8H), 0.80 (d, J = 7.0 Hz, 2H) ppm; IR (ﬁlm) ν: 2922, 2852,

1598, 1736, 1493, 1460, 1441, 1371, 1271, 1252, 1201, 1182, 1173,

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1142, 1040 cm⁻¹; HRMS (ES⁺): calcd for C₂₀H₁₈ClF₆N₄O₂[M +

H]⁺, 495.1017; found, 495.1010.

Computational Studies. The X-ray crystal structure of the

tubulin in complex with a triazolopyrimidine was downloaded from

compound (1 μM), with methods as described.¹¹Finally, the cultures

were prepared for acetyl-tubulin staining as previously discussed.¹¹

Determination of Plasma and Brain Drug Concentrations.

All animal protocols were approved by the University of Pennsylvania

Institutional Animal Care and Use Committee (IACUC). Test

compounds were administered to groups of three 2−3-month-old

CD-1 mice (Charles River). For standard single time-point brain and

plasma determinations, the mice were injected i.p. with a single dose

of 5 mg/kg compound dissolved in DMSO. In some instances, the

mice were cassette-dosed via i.p. administration with 2−3 compounds

concurrently, each at 2.5 mg/kg. One hour following compound

administration, the mice were euthanized following an IACUC-

approved protocol. For multiple time-point studies, groups of 3 CD-1

mice injected i.p. with compound at 5 mg/kg were sacriﬁced at

varying times after compound administration. Whole-brain hemi-

spheres were homogenized in 10 mM ammonium acetate, pH 5.7

(50%, w/v), using a hand-held sonic homogenizer. Plasma was

obtained from blood collected in 0.5 M EDTA solution and

centrifuged for 10 min at 4500g at 4 °C. Compound levels with

plasma and brain homogenates were determined essentially as

previously described^10,11,37either in-house or at a contract laboratory

(Inotiv, Inc.).

̈

the Protein Data Bank and completed with MAESTRO (Schrodinger

Release 2019-3),³²waters, and other co-crystallized molecules were

removed, except for the ligand and GDP. Predicting protonation

states of protein residues were calculated considering a temperature of

300 K and a pH of 7, while the molecules were prepared considering

the ionization states at pH 7 2. A 12 Å docking grid (inner-box 10

Å and outer-box 20 Å) was prepared using as centroid the

cocrystallized ligand. The docking studies were performed using

Glide SP precision keeping the default parameters and setting, and it

was combined with “molecular mechanics generalized Born surface

area” (MMGBSA), implemented in the Prime module from Maestro,

to re-score the three output docking poses of each compound. Only

the best MMGBSA score and pose for each compound were plotted

in the graph and correlated with the normalized activity, expressed as

the log of the average of the activity at 1 and 10 μM in the AcTub

assay, relative to the positive control (5).

The 3D-QSAR ﬁeld-based and activity atlas models were

constructed using Forge software (Cresset Inc., Cambridgeshire,

UK).³³First, the molecular structures of each compound were

subjected to a ﬁeld-based alignment to the cocrystallized compound,

which was used as the reference structure. This method is based on

the 3D-shape and electrostatic potential similarity calculated by the

alignment and superposition between the reference compound and

the compounds in the database according to their electrostatic

distribution and volume occupied. Field point-based descriptors were

used for building the 3D-QSAR model after the alignment of 52 Class

I compounds with the known activity. The activity was computed

using the log of the average of the activity at 1 and 10 μM in the

AcTub assay and deﬁned as the dependent variable. The derived

QSAR model was assessed by the LOO technique to optimize the

activity-prediction model. Activity atlas was used to acquire qualitative

information of the ﬁeld and steric contributions signiﬁcant for the

activity.

Molecular Operating Environment (MOE) 2019.10 was used to

visualize the structures and acquire the images,³⁴while GraphPad

software³⁵and DataWarrior³⁶were used for the statistical analysis.

QBI293 Cell and Neuronal Acetyl-Tubulin and α-Tub

Determinations. Compound-induced changes in acetylated-tubulin

and α-Tub in QBI293 cells or primary mouse neurons were as

previously described.^5,11Brieﬂy, QBI293 cells were maintained in

Dulbecco’s modiﬁed Eagle’s medium containing 10% fetal bovine

serum, 2 mM ^L-glutamine (Mediatech), 50 units/mL penicillin, and

50 μg/mL streptomycin. For compound testing, cells were dissociated

with trypsin/EDTA and plated at a density of 6 × 10⁵cells/well in six-

well plates. After overnight incubation, the medium was aspirated and

fresh medium containing a vehicle or a test compound was added.

After 4 h of incubation, whole-cell extracts were prepared from the

QBI293 cells as described.^5,11The supernatant fraction from each

sample was collected and analyzed for protein content by BCA

assay.¹¹The acetyl-tubulin and α-Tub enzyme-linked immunosorbent

assays (ELISA) were performed on the supernatant fraction as

previously described.¹¹The amount of acetyl- and α-Tub protein in

each sample was extrapolated using standard curves generated from

serial dilutions with acetyl- or α-Tub preparations of known

concentrations. Acetyl-tubulin levels were determined in a similar

fashion in mouse cortical neuron cultures plated at 6 × 10⁵cells/well

in six-well plates, essentially as previously described for rat cortical

neurons.¹¹After 10 days of growth, the neurons were treated for 8 h

with 15 nM OA in the presence of the test compound (1 or 10 μM)

or vehicle (0.25% DMSO), and homogenates were prepared for

determination of acetyl-tubulin levels as previously described.¹¹

Acetyl-Tubulin Staining of Rat Cortical Neuron Cultures

Treated with Test Compounds in the Absence or Presence of

OA. Rat cortical neurons were grown in 24-well plates on coverslips

for 10 days as previously described.¹¹The cultures were then treated

for 8 h with or without 15 nM OA in the presence or absence of test

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01605.

■

sı

NMR spectra of test compounds; X-ray crystal structures

of compounds 68 (CCDC 1995770) and 111 (CCDC

2003391); the authors will release the atomic

coordinates and experimental data upon article pub-

lication (PDF)

SMILES string structures and the full data (CSV)

Chemical ﬁle format of compound 3 (PDB)

Chemical ﬁle format of compound 30 (PDB)

Chemical ﬁle format of compound 54 (PDB)

Chemical ﬁle format of compound 72 (PDB)

AUTHOR INFORMATION

Corresponding Authors

■

Kurt R. Brunden − Center for Neurodegenerative Disease

Research, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, United

States; Email: kbrunden@upenn.edu

Carlo Ballatore − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California, San Diego,

La Jolla, California 92093, United States; orcid.org/

0000-0002-2718-3850; Email: cballatore@health.ucsd.edu

Authors

Killian Oukoloﬀ − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California, San Diego,

La Jolla, California 92093, United States

Goodwell Nzou − Center for Neurodegenerative Disease

Research, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, United

States

Carmine Varricchio − Cardiﬀ School of Pharmacy and

Pharmaceutical Sciences, Cardiﬀ University, Cardiﬀ

CF103NB, U.K.; orcid.org/0000-0002-1673-4768

Bobby Lucero − Department of Chemistry & Biochemistry,

University of California San Diego, La Jolla, California

92093, United States

Thibault Alle − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California, San Diego,

1100

https://dx.doi.org/10.1021/acs.jmedchem.0c01605

J. Med. Chem. 2021, 64, 1073−1102

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pubs.acs.org/jmc

Article

(4) Lee, V. M. Y.; Daughenbaugh, R.; Trojanowski, J. Q.

Microtubule stabilizing drugs for the treatment of Alzheimers-disease.

Neurobiol. Aging 1994, 15, 87−89.

La Jolla, California 92093, United States; orcid.org/

0000-0001-8459-6443

Jane Kovalevich − Center for Neurodegenerative Disease

Research, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, United

States

Ludovica Monti − Skaggs School of Pharmacy and

Pharmaceutical Sciences, University of California, San Diego,

La Jolla, California 92093, United States

Anne-Sophie Cornec − Department of Chemistry, School of

Arts and Sciences, University of Pennsylvania, Philadelphia,

Pennsylvania 19104-6323, United States

Yuemang Yao − Center for Neurodegenerative Disease

Research, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, United

States

Michael J. James − Center for Neurodegenerative Disease

Research, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, United

States

John Q. Trojanowski − Center for Neurodegenerative Disease

Research, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, United

States

Virginia M.-Y. Lee − Center for Neurodegenerative Disease

Research, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, Pennsylvania 19104, United

States

(5) Brunden, K. R.; Zhang, B.; Carroll, J.; Yao, Y.; Potuzak, J. S.;

Hogan, A.-M. L.; Iba, M.; James, M. J.; Xie, S. X.; Ballatore, C.; Smith,

A. B., III; Lee, V. M. Y.; Trojanowski, J. Q. Epothilone D improves

microtubule density, axonal integrity, and cognition in a transgenic

mouse model of tauopathy. J. Neurosci. 2010, 30, 13861−13866.

(6) Makani, V.; Zhang, B.; Han, H.; Yao, Y.; Lassalas, P.; Lou, K.;

Paterson, I.; Lee, V. M. Y.; Trojanowski, J. Q.; Ballatore, C.; Smith, A.

B., III; Brunden, K. R. Evaluation of the brain-penetrant microtubule-

stabilizing agent, dictyostatin, in the PS19 tau transgenic mouse

model of tauopathy. Acta Neuropathol. Commun. 2016, 4, 106.

(7) Zhang, B.; Carroll, J.; Trojanowski, J. Q.; Yao, Y.; Iba, M.;

Potuzak, J. S.; Hogan, A.-M. L.; Xie, S. X.; Ballatore, C.; Smith, A. B.,

III; Lee, V. M. Y.; Brunden, K. R. The microtubule-stabilizing agent,

epothilone D, reduces axonal dysfunction, neurotoxicity, cognitive

deficits, and Alzheimer-like pathology in an interventional study with

aged tau transgenic mice. J. Neurosci. 2012, 32, 3601−3611.

(8) Barten, D. M.; Fanara, P.; Andorfer, C.; Hoque, N.; Wong, P. Y.

A.; Husted, K. H.; Cadelina, G. W.; DeCarr, L. B.; Yang, L.; Liu, V.;

Fessler, C.; Protassio, J.; Riff, T.; Turner, H.; Janus, C. G.;

Sankaranarayanan, S.; Polson, C.; Meredith, J. E.; Gray, G.; Hanna,

A.; Olson, R. E.; Kim, S.-H.; Vite, G. D.; Lee, F. Y.; Albright, C. F.

Hyperdynamic microtubules, cognitive deficits, and pathology are

improved in tau transgenic mice with low doses of the microtubule-

stabilizing agent BMS-241027. J. Neurosci. 2012, 32, 7137−7145.

(9) Ballatore, C.; Brunden, K. R.; Trojanowski, J. Q.; Lee, V. M.-Y.;

Smith, A. B., III. Non-naturally occurring small molecule microtubule-

stabilizing agents: a potential tactic for CNS-directed therapies. ACS

Chem. Neurosci. 2017, 8, 5−7.

Amos B. Smith, III − Department of Chemistry, School of Arts

and Sciences, University of Pennsylvania, Philadelphia,

Pennsylvania 19104-6323, United States; orcid.org/

0000-0002-1712-8567

Andrea Brancale − Cardiﬀ School of Pharmacy and

Pharmaceutical Sciences, Cardiﬀ University, Cardiﬀ

CF103NB, U.K.; orcid.org/0000-0002-9728-3419

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01605

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Financial support for this work has been provided by the NIH/

NIA (AG061173, AG044332).

́

(13) Saez-Calvo, G.; Sharma, A.; Balaguer, F. d. A.; Barasoain, I.;

ABBREVIATIONS

́

Rodríguez-Salarichs, J.; Olieric, N.; Munoz-Hernandez, H.; Berbís, M.

̃

■

́

A.; Wendeborn, S.; Penalva, M. A.; Matesanz, R.; Canales, A.; Prota,

̃

AcTub, acetylated α-tubulin; m-CPBA, meta-chloroperbenzoic

acid; MMGBSA,, molecular mechanics generalized Born

surface-area; MT, microtubule; NFTs, neuroﬁbrillary tangles;

NTs, neuropil threads; Tg, transgenic

A. E.; Jímenez-Barbero, J.; Andreu, J. M.; Lamberth, C.; Steinmetz, M.

O.; Díaz, J. F. Triazolopyrimidines are microtubule-stabilizing agents

that bind the vinca inhibitor site of tubulin. Cell Chem. Biol. 2017, 24,

737−750.

(14) Yao, Y.; Nzou, G.; Alle, T.; Tsering, W.; Maimaiti, S.;

Trojanowski, J. Q.; Lee, V. M. Y.; Ballatore, C.; Brunden, K. R.

Correction of microtubule defects within A β plaque-associated

dystrophic axons results in lowered A β release and plaque deposition.

Alzheimer’s Dementia 2020, 16, 1345−1357.

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Article Doi

Evaluation of the Structure-Activity Relationship of Microtubule-Targeting 1,2,4-Triazolo[1,5- a]pyrimidines Identifies New Candidates for Neurodegenerative Tauopathies

DOI: 10.1021/acs.jmedchem.0c01605

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