Discovery and Optimization of Novel Pyrazolopyrimidines as Potent and Orally Bioavailable Allosteric HIV-1 Integrase Inhibitors

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Discovery and Optimization of Novel Pyrazolopyrimidines as Potent

and Orally Bioavailable Allosteric HIV‑1 Integrase Inhibitors

Guo Li,* Nicholas A. Meanwell, Mark R. Krystal, David R. Langley, B. Narasimhulu Naidu,

Prasanna Sivaprakasam, Hal Lewis, Kevin Kish, Javed A. Khan, Alicia Ng, George L. Trainor,

Christopher Cianci, Ira B. Dicker, Michael A. Walker, Zeyu Lin, Tricia Protack, Linda Discotto,

Susan Jenkins, Samuel W. Gerritz, and Annapurna Pendri *

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ABSTRACT: The standard of care for HIV-1 infection, highly active

antiretroviral therapy (HAART), combines two or more drugs from at

least two classes. Even with the success of HAART, new drugs with

novel mechanisms are needed to combat viral resistance, improve

adherence, and mitigate toxicities. Active site inhibitors of HIV-1

integrase are clinically validated for the treatment of HIV-1 infection.

Here we describe allosteric inhibitors of HIV-1 integrase that bind to the

LEDGF/p75 interaction site and disrupt the structure of the integrase

multimer that is required for the HIV-1 maturation. A series of

pyrazolopyrimidine-based inhibitors was developed with a vector in the

2-position that was optimized by structure-guided compound design.

This resulted in the discovery of pyrazolopyrimidine 3, which was

optimized at the 2- and 7-positions to aﬀord 26 and 29 as potent

allosteric inhibitors of HIV-1 integrase that exhibited low nanomolar

antiviral potency in cell culture and encouraging PK properties.

integrase strand transfer inhibitors (INSTIs) are clinically

useful therapeutics that include raltegravir, elvitegravir,

dolutegravir, and bictegravir (Figure 1).²These inhibitors

bind to the active site of the integrase enzyme, forming a

ternary complex with 3′ processed proviral DNA that impairs

strand transfer activity, the step that initiates the integration of

viral DNA into host DNA. In contrast, the allosteric integrase

inhibitors (ALLINIs) bind to a site located at the HIV-1

integrase dimer interface that is recognized by the host cell

transcription factor lens epithelium-derived growth factor

(LEDGF/p75).³⁻⁵The important role of LEDGF/p75 in

HIV-1 integration has been validated in vitro by site-directed

mutagenesis studies,^6,7RNAi knockdown experiments, and

cellular expression of the integrase binding domain (IBD) of

LEDGF that inhibits HIV-1 replication in cell culture.^8,9

Recent studies showed that the multifunctional ALLINIs not

only inhibit IN binding to its cofactor LEDGF/p75 but also

promote aberrant IN multimerization to block viral matura-

tion, leading to the production of structurally defective,

INTRODUCTION

■

Human immunodeﬁciency virus type 1 (HIV-1) is the

etiologic agent underlying acquired immunodeﬁciency syn-

drome (AIDS) and was ﬁrst recognized and diagnosed in the

early 1980s. After more than 30 years, HIV-1 infection remains

a signiﬁcant medical problem and is now a global epidemic that

infects 37 million people worldwide. At present, more than 40

drugs have been approved for the treatment of HIV-1/AIDS

and these are used to create combination regimens designed to

mitigate the emergence of resistance. These drugs belong

mainly to four classes: nucleoside and non-nucleoside

inhibitors of reverse transcriptase, integrase (IN) inhibitors,

and inhibitors of HIV-1 protease.¹In addition, the HIV-1 entry

inhibitors maraviroc (a CCR5 co-receptor antagonist),

enfuvirtide (a peptidic inhibitor of gp41 fusion), and

ibalizumab (a nonimmunosuppressive monoclonal antibody

that binds to the host cell receptor CD4) are available for later

lines of therapy. Key challenges have been the continued

emergence of resistance and the long-term toxicity associated

with approved anti-HIV-1 drugs. The identiﬁcation of

additional viral targets and the development of new classes

of antiviral compounds are essential in the continued ﬁght

against HIV/AIDS.

Special Issue: Women in Medicinal Chemistry

Received: October 10, 2019

HIV-1 integrase is the enzyme responsible for integrating

viral DNA into the host cell genome. Active site-targeted

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A

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Figure 1. Structures of Food and Drug Administration (FDA)-

approved HIV-1 active site-targeted integrase strand transfer

inhibitors.

Figure 2. Scaﬀold modiﬁcation and preliminary SAR exploration

associated with ALLINIs 2−6.

noninfectious virions.¹⁰⁻¹³The pleiotropic eﬀects of ALLINIs

suggest considerable promise as antiviral agents for the

development of HIV-1 therapeutics.³The potential of this

new class of inhibitor led us to explore compounds that bind to

the novel dimer interface pocket.

Structural modiﬁcation of the unsaturated 6,6-fused ring

system found in 1 to the 5,6-scaﬀold of 3 provided an

alternative core system that reduced the aromatic nature of the

fused heterocyclic ring while oﬀering topologically distinct

vectors for structural exploration and elaboration, particularly

at the C-2 position which bisects the vectors oﬀered by 1.

Indeed, the introduction of a phenyl substituent at the 2-

position of 3 aﬀorded 4 as a compound with improved potency

in the binding assay (K_i= 700 nM) which translated to

enhanced antiviral activity, WT EC₅₀= 620 nM, CC₅₀= 77

μM. Consistent with the emerging structure−activity relation-

ships (SARs) for ALLINI inhibitors, the carboxamide 5 and

alcohol 6 were considerably less potent than 4, with WT EC₅₀

values of 18 and >40 μM, respectively. As a consequence, 4

was adopted as a promising lead structure suitable for further

optimization.

The initial phase of study with 4 explored the eﬀect of

variation of the C-7 substituent, with the results compiled in

Table 1. Removal of the para-CH₃substituent from the 7-

phenyl moiety of 4 resulted in 7, which exhibited 5-fold

reduced potency in the binding assay while failing to

signiﬁcantly inhibit virus replication in cell culture. However,

the 4-OCH₃analogue 8 oﬀered improved potency in the

binding assay, although this compound was less active than 4

in the cell-based assay. Installation of CH₃or OCH₃

substituents at the meta- and ortho- positions of the C-7

phenyl ring, explored in 9-12, led to reduced activity in both

assays. However, the incorporation of a combination of para-

alkoxy and meta-alkyl substitution in the context of the

chromane ring¹⁴of 13 resulted in a 10-fold increase in potency

in the binding assay, although the magnitude did not fully

translate to antiviral activity in cell culture. Substitution of the

chromane ring led to the production of separable atropisomers

of which the major isomer 14 was more potent than the minor

isomer 15. The identity of the atropisomers was established by

single crystal X-ray structure determination which revealed 14

to be a mixture of two enantiomers, assigned as (S,S) and

(R,R) (Figure 3), while 15 was a mixture of the (S,R) and

(R,S) isomers (Figure 4). The separated diastereomers 14 and

15 showed no interconversion at temperatures ranging from 30

RESULTS AND DISCUSSION

■

The 2-(quinolin-3-yl)acetic acid derivative 1 was shown to

bind to the ALLINI site on HIV-1 integrase, presenting

rudimentary elements of a pharmacophore that were more fully

appreciated in the context of a cocrystal structure of the

compound bound to the integrase CCD dimer interface.^14,15

This structure revealed a well-deﬁned binding pocket that

could accommodate small molecules and indicated the

importance of both the tert-butoxyacetic acid moiety and the

quinoline 4-substituent as key recognition elements.¹⁴⁻¹⁷The

analysis of the structure focused attention on exploring

alternatives to the quinoline scaﬀold that would oﬀer

opportunities for unique substituent vector presentation and

which may lead to improved potency and enhanced drug-like

properties. The initial screening strategy relied upon evaluation

of test compounds in a binding assay in which displacement of

a tritiated version of 1 was monitored to provide K_ivalues.

Antiviral activity was assessed in a cell culture replication assay

using a fully replicating NL₄₋₃virus containing the Renilla

luciferase gene in place of nef (WT EC₅₀), with the cytotoxicity

(CC₅₀) of test compounds toward the host MT-2 cells

determined in parallel.

A computer-aided drug design (CADD) approach based on

careful analysis of the cocrystal structure allowed an in silico

assessment of the potential of a series of scaﬀolds that were

scored by a best ﬁt analysis for their complementarity to the

pocket formed by the integrase dimer. To aid in this analysis,

the biphenyl derivative 2 (Figure 2) was prepared as an

abbreviated model system for 1 and found to be poorly active

in the displacement assay with a K_ivalue of 67 μM, although

the compound exhibited weak but detectable antiviral activity

in cell culture (WT EC₅₀= 16 μM, CC₅₀= 127 μM)). Several

additional scaﬀolds were investigated for their potential to bind

to the pocket, and the pyrazolopyrimidine derivative 3

emerged as a compound with a K_ivalue of 2 μM in the

binding assay that inhibited WT virus in cell culture with an

EC₅₀value of 4.5 μM, a promising preliminary proﬁle.

B

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Table 1. Eﬀect of Substituent Variation at the 7-Position of Pyrazolopyrimidine 4 on Binding Potency and Antiviral Activity As

Explored with 7−19

a

b

K_iand EC₅₀values were the result of multiple determinations (n ≥ 2). K_iand EC₅₀values were obtained after single determinations (n = 1).

Figure 4. Single crystal X-ray structure of (S,R and R,S)-15.

Figure 3. Single crystal X-ray structure of (S,S and R,R)-14.

C

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Figure 5. (a) Snapshot of the cocrystal structure of (S,S)-14 bound to the HIV-1 integrase CCD dimer. Subunits 1 and 2 of the dimer are colored

cyan and green, respectively, and (S,S)-14 is colored orange. The catalytic domain of HIV-1 integrase that contains the DDE triad is highlighted in

purple patches for comparison to the LEDGF binding site where (S,S)-14 binds crystallographically. (b) Close-up view of the binding interactions

of (S,S)-14 in one of the two LEDGF binding pockets at the dimer interface. Hydrogen atoms were added in silico followed by a restrained

minimization of the X-ray structure. (c) F_o− F_celectron density map for (S,S)-14 and surrounding water molecules contoured at 3σ.

to 100 °C in NMR experiments conducted in DMSO-d₆, with

conﬁgurational stability attributed to the presence of the

adjacent bulky tert-butoxy moiety.

Both binding and antiviral activity were dependent on the

identity of the substituent at C-7, since the simple CH₃-

substituted analogue 16 was poorly active in both assays, as

was the substituted aniline 17. However, cyclic amine-based

substituents at C-7 were tolerated, as exempliﬁed by 18 and

19, with the latter compound exhibiting antiviral activity in cell

culture that was comparable to 4.

hydrogen-bonding interaction with the backbone amide NHs

of Glu170 and His171 of subunit 2 of the enzyme. In addition,

the side chain hydroxyl group of Thr174 of subunit 2 makes a

hydrogen-bonding interaction with one of the carboxylic acid

oxygen atoms. The tert-butoxy group occupies a hydrophobic

pocket created by Thr125, Gln95, Tyr99, and Thr174, while

the C7-chromane ring sculpts into another hydrophobic

pocket formed by Leu102, Ala129, Thr174, Met178, Trp132,

and Ala169. The interactions observed with respect to the

carboxylic acid, tert-butoxy, and chromane moieties are

consistent with what has been observed in the crystal

structures of other ALLINIs bound to HIV-1 integrase CCD

dimers.^18,19The pyrazolopyrimidine core of (S,S)-14 and the

2-phenyl substituent lie on the hydrophobic surface of the

subunit 1, and the molecule exhibits tight van der Waals

contact with Ala128 in the α3-helix(124−133) of subunit 1.

The dihedral angle between the core heterocycle and 2-phenyl

ring is ∼30°, which contrasts with the nearly coplanar

arrangement observed in the single crystal X-ray structure of

(S,S)-14 (Figure 3), reﬂecting the conformational mobility of

the substituent. An overlay of the previously solved ALLINI-

bound crystal structures of HIV-1 integrase CCD dimers

revealed an interesting slight shift in the α3-helix(124−133),

presumably to maximize the hydrophobic contacts between

Ala128 and the 2-phenyl ring of (S,S)-14. Two crystallographic

water molecules bridge the N4 nitrogen atom of the

pyrazolopyrimidine core of (S,S)-14 with protein residues.

An image of F_o− F_celectron density for (S,S)-14 and

surrounding water molecules contoured at 3σ is shown in

Figure 5c. Interestingly, a polyethylene glycol fragment was

found to occupy a site just above the ligand. Its positioning

corresponds to the positioning of the side chains of Tyr226,

Trp235, and Lys266 in the carboxyl terminal domain (CTD)

of the CCD−CTD interface discerned in the full length crystal

structure of HIV-1 integrase solved by Gupta and colleagues.²⁰

Manual docking of (S,S)-14 into this full length crystal

structure of HIV-1 integrase reveals a snug ﬁt of the 2-phenyl

group in an invagination created at the CTD−CCD interface.

Comparison of the cocrystal structures of the dimeric HIV-1

integrase CCD bound to the LEDGF IBD²¹(Figure 6a) and

(S,S)-14 (Figure 5b) revealed some interesting insights into

binding interactions. The carboxylic acid moiety of (S,S)-14

engages the backbone amide groups of HIV-1 integrase via two

hydrogen-bonding interactions, while the pyrazolopyrimidine

The (S,S)- and (R,R)-enantiomers of 14 were successfully

separated by chromatography using a chiral column, with the

(S,S)-enantiomer 14 found to be ∼100-fold more potent (K_i=

0.09 0.02 μM, WT EC₅₀= 0.11 0.06 μM) than the (R,R)-

enantiomer (K_i> 53 μM, WT EC₅₀= 13 μM). Similarly,

compound 15 was successfully resolved by chiral column

chromatography; however, both enantiomers were inactive in

the cell culture antiviral assay. A single crystal X-ray structure

of racemic 14 (Figure 3) revealed the presence of the S,S- and

R,R-enantiomers in one unit cell. In both conﬁgurations, the

methyl group on the phenyl and the tert-butoxy group on the

acid-containing side chain project in opposite directions, while

the C-2 phenyl and pyrazolopyrimidine rings are coplanar and

the plane of the chromane heterocycle is almost perpendicular

to the core (the dihedral angle between the two planes is

∼74°). In contrast to 14, the phenyl and the pyrazolopyr-

imidine elements of 15 do not approach coplanarity (Figure

4). However, the bicyclic chromane ring adopts a dihedral

angle relative to the plane of the pyrimidine of ∼66°. Thus, the

major diﬀerence between 14 and 15 is the conformation of the

distal phenyl ring, while the elements proximal to the

carboxylic acid moiety diﬀer only slightly in their disposition.

In order to obtain further insight into the binding

interactions between 14 and the enzyme, a cocrystal structure

of the racemic compound and the dimeric HIV-1 integrase

catalytic core domain (CCD) was determined (Figure 5). A

full view of the HIV-1 integrase CCD dimer cocrystallized with

two molecules of (S,S)-14 is shown in Figure 5a. The structure

demonstrated that the inhibitor occupied the same binding

pocket as the LEDGF IBD and that the binding is preferential

for the more potent (S,S)-enantiomer [the (R,R)-isomer was

not observed]. A magniﬁed view of one of the inhibitor

binding pockets at the dimer interface is shown in Figure 5b.

The carboxylic acid group of (S,S)-14 makes a bidentate

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Table 2. Eﬀect of Structural Modiﬁcation of the 2-Aryl

Moiety on the Antiviral Activity, PAMPA Permeability, and

Metabolic Stability in Human and Rat Liver Microsomes of

f

Pyrazolopyrimidines 20−24 Compared to 14

Figure 6. (a) Cocrystal structures of the dimeric HIV-1 integrase

CCD bound to the LEDGF IBD. (b) Molecular modeling of

substitution at the meta-position of the 2-phenyl group of (S,S)-14.

core packs into the hydrophobic pocket by relying primarily on

van der Waals interactions. The carboxylic acid moiety of

(S,S)-14 mimics Asp366 of LEDGF, while the chromane group

at C-7 mimics the hydrophobic side chain of Ile365. However,

(S,S)-14 does not take advantage of the interactions availed by

Val408 and Phe406 in LEDGF with the integrase protein.

Molecular modeling (Figure 6b) suggested that substituents at

the meta-position of the 2-phenyl ring of (S,S)-14 would

extend deeper into the LEDGF binding pocket in a fashion

that would more eﬀectively mimic the contacts made by

Val408 and Phe406 of the IBD of LEDGF. Consequently, this

was viewed as a useful avenue for additional structural

elaboration that may lead to improved antiviral activity and

the possibility of a higher genetic barrier to the emergence of

resistance.

This phase of the SAR survey is compiled in Table 2 and

revealed that the judicious introduction of substituents to the

2-phenyl ring does indeed lead to enhanced antiviral activity in

cell culture. The 4-CH₃substituent in 20 improved antiviral

potency by a modest 2-fold. That potency was only marginally

aﬀected by the introduction of the much larger 4-benzyloxy

moiety in 21. Further improvements were obtained with meta-

substitutions, as explored with 22−24, of which the 3-phenyl

derivative 24 oﬀered a 10-fold improvement over prototype

14, exhibiting a WT EC₅₀value of 15 nM, CC₅₀= 10 μM. The

biphenyl derivative 24 was 4-fold more potent than the

phenoxy homologue 23 and comparable to the 3-benzyloxy

analogue 22. In parallel with optimization of antiviral potency,

these inhibitors were proﬁled in a parallel artiﬁcial membrane

permeability assay (PAMPA) and evaluated for metabolic

stability in rat and human liver microsomal preparations.

Analogues 20−24 exhibited uniformly high permeability in a

parallel artiﬁcial membrane permeability assay (PAMPA) and

generally showed good metabolic stability in the microsomal

assays, suggestive of the potential to demonstrate bioavail-

ability following oral administration.

a

EC₅₀values were obtained by multiple determinations (n ≥ 2).

b

EC₅₀value was obtained after a single determination (n = 1).

c

PAMPA values were averaged from triplicate determinations (n = 3).

d

HLM/RLM denotes human and rat liver microsomes, respectively.

e

Metabolic stability % remaining values were averaged from duplicate

f

experiments (n = 2). All compounds were screened as a mixture of

the (S,S)- and (R,R)-isomers.

Table 3. Antiviral Activity of the Pyrazolopyrimidine

Inhibitors against the INSTI-Resistant G140S/Q148H HIV-

1 Strain (GQ EC₅₀) Compared to Raltegravir

a

EC₅₀values were obtained by multiple determinations (n ≥ 2).

EC₅₀value was obtained by single determination (n = 1).

A select set of pyrazolopyrimidines was evaluated for

antiviral eﬃcacy toward the double mutant G140S/Q148H

virus strain that confers resistance to some of the active site-

targeting INSTIs. GQ EC₅₀values are shown in Table 3 where

the GQ mutant inhibitory activity is compared to that toward

WT virus. (S,S and R,R)-14, (S,S)-14, and (S,S and R,R)-24

fully retained potency toward the resistant virus strain, whereas

the EC₅₀value for the INSTI raltegravir shifted by over 1000-

fold compared to its inhibitory eﬀect toward WT virus.

The beneﬁcial eﬀects of meta-substitutions of the C-2 phenyl

moiety in 22−24 prompted an examination of this structural

b

modiﬁcation in the context of the C-7 piperidine 19, with the

result that both the phenyl and benzyloxy derivatives 25 and

26, respectively, were more potent antiviral agents (Table 4).

Both 19 and 26 exhibited good PAMPA permeability and high

microsomal stability in both HLM and RLM.

Several of the compounds in Tables 2 and 4 combined

potent antiviral activity with good membrane permeability and

high microsomal stability, which suggested that they could be

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candidates for in vivo pharmacokinetic (PK) studies in the rat

to determine the potential for exposure following oral

administration. However, as a prelude to these studies,

compounds were evaluated in the antiviral cell culture assay

in the presence of human serum albumin (HSA EC₅₀) since

these data would provide a target trough concentration of drug

based on the assumption that in vivo activity would be

dependent on maintaining drug levels above the protein

binding-adjusted EC₉₀value. These data were obtained on the

more active (S,S)-isomers. The potency of (S,S)-24, the

prototype for the series presented in Table 5, was increased

compared to the racemic mixture with an EC₅₀value of 6 nM.

However, in the presence of 45 mg/mL of HSA, the

approximate concentration of this plasma protein in humans,

the EC₅₀value for (S,S)-24 was 110 nM, an 18-fold

attenuation. In an eﬀort to probe this aspect of the proﬁle of

the inhibitors, analogues 27−31 were prepared and the (S,S)-

enantiomers evaluated. As summarized in Table 5, analogues

(S,S)-27−31 exhibited excellent antiviral potency in the

absence of HSA, with WT EC₅₀values ranging from 3 to 6

nM, while all compounds demonstrated a reduced fold-shift in

the presence of HSA (5- to 6-fold) compared to (S,S)-24, with

the notable exception of the pyridine derivative (S,S)-30,

Table 4. Eﬀect of Structural Modiﬁcation of the 2-Aryl

Moiety on the Antiviral Activity, PAMPA Permeability, and

Metabolic Stability in Human and Rat Liver Microsomes of

C-7 Piperidine-Substituted Pyrazolopyrimidines 25 and 26

d

Compared to Prototype 19

a

EC₅₀values were obtained by multiple determinations (n ≥ 2).

b

PAMPA values were averaged from multiple replicates (n = 3).

c

Metabolic stability % remaining values were averaged from duplicate

d

experiments (n = 2). All compounds were screened as a mixture of

the S- and R-isomers.

Table 5. Antiviral Activity in the Absence and Presence of 45 mg/mL HSA, Fold Shift in Potency, Therapeutic Index, PAMPA

Permeability, and Metabolic Stability Associated with Pyrazolopyrimidine Derivatives (S,S)-24 and (S,S)-27−31

a

b

EC₅₀or CC₅₀values were obtained by multiple determinations (n ≥ 2). EC₅₀or CC₅₀value was obtained by single determination (n = 1).

c

d

PAMPA values were averaged from multiple replicates (n = 3). Metabolic stability % remaining values were averaged from duplicate experiments

(n = 2).

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a

Table 6. Rat PK Data for (S,S)-27−29

compd

C_maxSD (nM) t_maxSD (h) AUC_0−24hSD (nM·h) t_1/2SD (h) clearance SD (mL min⁻¹kg⁻¹

)

V_ssSD (L/kg) F (%)

(S,S)-27

(S,S)-28

(S,S)-29

1292 1062

1106 697

3

0

6300 2532

2991 958

4.1 0.1

7.1 0.3

28 5.7

0.5 0.1

1.8 1.8

0.7 0.0

32

59

44

2.3 1.3

2.7 3.8

3

2

1

0

803

0

4733

0

13

0

a

All compounds were dosed at 1 mpk (iv) and 5 mpk (po). All values were obtained from multiple determinations (n = 3) for both iv and po.

a

Scheme 1. Synthesis of Pyrazolopyrimidine 4

a

Reagents and conditions: (a) (i) Methyl acetoacetate, THF, heptane, piperidine, reﬂux; (ii) DDQ, CH₂Cl₂; (b) (i) DIBAL, THF, CH₂Cl₂; (ii)

PCC, CH₂Cl₂; (c) ZnI₂, TMSCN, CH₂Cl₂; (d) HCl (conc), 90 °C; (e) (i) SOCl₂, MeOH; (ii) HClO₄, tert-butyl acetate, CH₂Cl₂; (iii) LiOH, 1,4-

dioxane; (f) HClO₄, tert-butyl acetate, CH₂Cl₂.

which experienced a similar ∼20-fold shift. The therapeutic

index (TI) for this series of compounds is also captured in

Table 5 and was found to range from 1098 to 6360, with (S,S)-

29, one of the more potent inhibitors in the series, exhibiting a

TI value of 6360, which compares with a TI value of 66 783

recorded for BI-224436¹⁴when tested under these assay

conditions. The overall proﬁles of these compounds reﬂect a

combination of good membrane permeability and reasonable

metabolic stability, and those compounds with good HSA EC₅₀

values, (S,S)-27−29, were advanced into rat PK studies.

Rat PK studies monitored drug levels in plasma for 24 h

following oral administration of a 5 mg/kg dose. Results are

compiled in Table 6. All three compounds demonstrated low

to moderate clearance and low volumes of distribution. The

ortho-methyl (S,S)-27 and ortho-ﬂuoro (S,S)-29 derivatives

exhibited lower clearance than the ortho-methoxy analogue

(S,S)-28, although the latter demonstrated the highest oral

bioavailability of this short series of compounds. These results

indicate that the series oﬀers promising PK proﬁles.

CHEMISTRY

■

The initial synthetic route to pyrazolopyrimidine 4 originated

with the condensation of commercially available benzaldehyde

32, 3-aminopyrazole 33, and methyl acetoacetate in a three

component, one-pot reaction that was followed by oxidation

with DDQ to form the pyrazolopyrimidine core 34 (Scheme

1). The methyl ester of 34 was transformed into the aldehyde

35, which was directly converted to the cyanohydrin 36. tert-

Butyl ether formation using HClO₄and tert-butyl acetate

resulted in partial hydrolysis of the nitrile moiety of 36 to give

the carboxamide 5. Alternatively, the hydrolysis of 36 under

strongly acidic conditions provided the desired acid 6.

Esteriﬁcation of the acid 6 and subsequent treatment with

HClO₄and tert-butyl acetate in CH₂Cl₂installed the tert-butyl

ether followed by saponiﬁcation to yield the pyrazolopyr-

imidine acid 4.

The synthetic route to access compounds with structural

variation at the C-7 position is exempliﬁed in Scheme 2 and

utilized 41 as a common intermediate for C-7 modiﬁcation.

Reaction of aminopyrazole 33 with dimethyl 2-acetylsuccinate

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a

Scheme 2. Synthesis of Pyrazolopyrimidine 14, 15, 16, and 19

a

Reagents and conditions: (a) p-TsOH·H₂O, xylene, reﬂux; (b) POCl₃, reﬂux; (c) (i) KHMDS, THF, −78 °C; (ii) 2-(phenylsulfonyl)-3-

phenyloxaziridine (Davis reagent); (d) HClO₄, tert-butylacetate, CH₂Cl₂; (e) (i) 2-(8-ﬂuoro-5-methylchroman-6-yl)-4,4,5,5-tetramethyl-1,3,2-

dioxaborolane, Pd(PPh₃)₄, K₂CO₃, DMF, microwave, 130 °C; (ii) LiOH, 1,4-dioxane; (f) (i) Pd(PPh₃)₄, MeZnCl, THF; (ii) LiOH, 1,4-dioxane;

(g) (i) 4,4-dimethylpiperidine, HCl salt, DIEA, NMP, 50 °C; (ii) LiOH.

a

Scheme 3. Synthesis of Pyrazolopyrimidine (S,S)- and (R,R)-20 and -25

a

Reagents and conditions: (a) p-TsOH·H₂O, xylene, reﬂux; (b) POCl₃, reﬂux; (c) (i) KHMDS, THF, −78 °C; (ii) 2-(phenylsulfonyl)-3-

phenyloxaziridine (Davis reagent); (d) HClO₄, tert-butyl acetate, DCM; (e) (i) p-tolylboronic acid, Pd(PPh₃)₄, K₂CO₃(2M), DMF, microwave, 70

°C; (f) (i) 2-(8-ﬂuoro-5-methylchroman-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, K₃PO₄(2M), DMF, microwave, 130 °C; (ii)

LiOH 1,4-dioxane; (g) (i) 2-([1,1′-biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, K₂CO₃(2 M), DMF, microwave, 70 °C;

(ii) 4,4-dimethylpiperidine, HCl salt, DIEA, NMP, 50 °C; (iii) LiOH, 1,4-dioxane.

H

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a

Scheme 4. Chiral Synthesis of Pyrazolopyrimidine (S,S)-27

a

Reagents and conditions: (a) p-TsOH·H₂O, xylene, reﬂux; (b) POCl₃, reﬂux; (c) (i) KHMDS, THF, −78 °C; (ii) 2-(phenylsulfonyl)-3-

phenyloxaziridine (Davis reagent); (d) Dess−Martin periodinane, CH₂Cl₂; (e) (R)-1-methyl-3,3-diphenyltetrahydro-1H,3H-pyrrolo[1,2-

c][1,3,2]oxazaborole, catecholborane, toluene; (f) HClO₄, tert-butyl acetate, CH₂Cl₂; (g) 2-(8-ﬂuoro-5-methylchroman-6-yl)-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane, Pd(PPh₃)₄, K₃PO₄(2 M), DMF, microwave, 130 °C; (h) (i) o-tolylboronic acid, Pd(OAc)₂, S-Phos, K₃PO₄

(2M), DMF, microwave, 130 °C; (ii) LiOH, 1,4-dioxane.

(37) aﬀorded pyrazolopyrimidine 38, which was converted to

the chloride 39 using POCl₃. The benzylic hydroxyl group on

40 was introduced by deprotonation followed by treatment

with the Davis reagent²²to give 40 as a racemic mixture.

Alcohol 40 was treated with HClO₄and tert-butyl acetate in

CH₂Cl₂to aﬀord the tert-butyl ether 41, a versatile

intermediate. Subjecting 41 to a Suzuki−Miyaura coupling at

C-7 with arylboronic acids allowed the introduction of a

diverse set of aryl substituents. A Negishi coupling reaction was

used to introduce C-7 alkyl groups, while a nucleophilic

displacement reaction with diﬀerent amines or anilines resulted

in a series of C-7 amino-substituted analogs.

An alternative synthetic route was developed to introduce

modiﬁcations at C-2 and began with condensation of 5-bromo-

3-aminopyrazole (42) with 37 aﬀorded pyrazolopyrimidine 43,

which was converted to the chloride 44 using POCl₃. The

benzylic hydroxyl group on 45 was introduced by deprotona-

tion followed by treatment with Davis reagent to give 45 as a

racemic mixture. Alcohol 45 was then treated with HClO₄and

t-butyl acetate in CH₂Cl₂to procure the key intermediate 46

(Scheme 3). A selective Suzuki−Miyaura coupling at the C-2

position of 46 was realized by controlling the reaction

temperature and varying the base (70 °C, K₂CO₃). After

completion of the Suzuki−Miyaura coupling at the 2-position,

the 7-chloro was subjected to a second Suzuki−Miyaura

coupling that was conducted at 130 °C using K₃PO₄as the

base. Alternatively, the C-7 chlorine could be displaced by

amines. A ﬁnal hydrolysis aﬀorded acids 20 and 25.

was low and the separation was time-consuming. To alleviate

this problem, an eﬃcient chiral synthetic route was developed

to make the (S,S)-isomer of the biarylpyrazolopyrimidines

starting from aminopyrazole 48 (Scheme 4). The racemic

alcohol 51 was oxidized to the corresponding ketone using the

Dess−Martin reagent, setting the stage for an enantioselective

reduction using catecholborane in the presence of a chiral

oxazaborolidine,²³which resulted in the isolation of (S)-53

with 96% ee. Compound 53 was treated with HClO₄and tert-

butyl acetate to aﬀord the corresponding tert-butyl ether 54,

which was subjected to two consecutive Suzuki−Miyaura

coupling reactions. The phenyl-based chlorine is much less

reactive than the chlorine attached to the pyrimidine ring,

allowing for excellent selectivity and directing the ﬁrst Suzuki−

Miyaura coupling to C-7. The second Suzuki−Miyaura

coupling was achieved in the presence of Pd(OAc)₂using

the S-Phos ligand, and hydrolysis of 55 gave the targeted chiral

pyrazolopyrimidine (S,S)-27.

CONCLUSION

■

This manuscript describes the design, synthesis, and biological

activities of a series of novel pyrazolopyrimidines that act as

potent allosteric inhibitors of HIV-1 integrase and exhibit drug-

like properties. Separation, characterization, and screening of

four diastereomers (S,S)-14, (R,R)-14, (S,R)-15, and (R,S)-15

provided interesting SARs and demonstrated an impact of

conformational diﬀerences between atropisomers. A cocrystal

structure of (S,S)-14 with the dimeric HIV-1 integrase CCD

revealed that the inhibitor occupied the same binding pocket

as LEDGF. Molecular modeling based on the crystal structure

data predicted that substituents incorporated at the meta-

Although the active chiral compound (S,S)-14 and the less

active enantiomer (R,R)-14 could be separated by chiral

column chromatography, the yield of the active enantiomer

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H), 7.38−7.22 (m, 7 H), 5.22 (s, 1 H), 2.42 (s, 3 H), 0.93 (s, 9 H).

MS (C₁₉H₂₂O₃): m/z 321 (M + Na)⁺.

position of 2-phenyl group would extend deeper into the

ALLINI binding pocket, modiﬁcations postulated to lead to

increased binding aﬃnity and antiviral activity. This vector

proved to be a useful avenue for structural elaboration and

resulted in inhibitors with excellent antiviral activity in cell

culture. The most potent compound 29 exhibited a WT EC₅₀

value of 0.003 μM, while 26, which incorporates a dimethyl-

piperidine substituent at the 7- position, resulted in a WT EC₅₀

value of 0.06 μM. Both the vector on the 2-position and the

amino substituent at the 7-position of the core oﬀer unique

SARs for allosteric integrase inhibitors, unprecedented based

on prior patent publications²⁴and literature articles.^14,16,17

Three compounds, 27−29, that exhibited good overall proﬁles

in the in vitro antiviral and PK screening assays, including

minimal potency shifts in the presence of HSA, were advanced

into rat PK studies and showed promising PK proﬁles with

good oral bioavailability. Further studies based on this

chemotype led to allosteric HIV-1 IN inhibitors with excellent

cellular potency.²⁵

2-tert-Butoxy-2-(5-methyl-7-p-tolylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetic Acid, TFA Salt (3). Compound 3 was

prepared from 1H-pyrazol-3-amine in a manner similar to that

1

described for compound 4. H NMR (500 MHz, MeOD) δ (ppm):

8.03 (d, J = 2.4 Hz, 1H), 7.66−7.42 (m, 4 H), 6.63 (d, J = 2.4 Hz,

1H), 5.16 (s, 1 H), 2.70 (s, 3 H), 2.52 (s, 3 H), 0.98 (s, 9 H). MS

(C₂₀H₂₃N₃O₃): m/z 354 (M + H)⁺. HRMS calcd for C₂₀H₂₃N₃O₃

354.1812 (MH⁺); found 354.1819.

2-tert-Butoxy-2-(5-methyl-2-phenyl-7-p-tolylpyrazolo[1,5-

a]pyrimidin-6-yl)acetic Acid, TFA Salt (4). SOCl₂(0.0023 mL,

0.032 mmol) was added to a solution of 6 (6 mg, 0.016 mmol) in

MeOH (2 mL) and the reaction mixture stirred at 40 °C for 16 h.

The solvent was evaporated to give methyl 2-hydroxy-2-(5-methyl-2-

phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidin-6-yl)acetate. The crude

product was used directly for next step. MS (C₂₃H₂₁N₃O₃): m/z

388 (M + H)⁺. Perchloric acid (0.008 mL, 0.128 mmol) was added to

a solution of methyl 2-hydroxy-2-(5-methyl-2-phenyl-7-p-

tolylpyrazolo[1,5-a]pyrimidin-6-yl)acetate (6.20 mg, 0.016 mmol) in

tert-butyl acetate (0.3 mL) and the reaction mixture stirred for 2 h at

room temperature. The reaction mixture was quenched with H₂O and

diluted with EtOAc. The organic phase was washed with saturated

NaHCO₃and dried over Na₂SO₄. The solvent was evaporated to give

methyl 2-tert-butoxy-2-(5-methyl-2-phenyl-7-p-tolylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetate. The crude product was used directly for the

next step. MS (C₂₇H₂₉N₃O₃): m/z 444 (M + H)⁺. 1.5 N LiOH

aqueous solution (0.5 mL, 0.750 mmol) was added to a solution of

methyl 2-tert-butoxy-2-(5-methyl-2-phenyl-7-p-tolylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetate (7.10 mg, 0.016 mmol) in dioxane (0.5 mL)

and the mixture stirred at 50 °C for 2 h. The reaction mixture was

ﬁltered and the ﬁltrate was puriﬁed by preparative HPLC to aﬀord 4

as TFA salt (4 mg, 43.7% for 3 steps). ¹H NMR (400 MHz, MeOD) δ

(ppm): 7.91−7.82 (m, 2 H), 7.67 (dd, J = 7.8, 2.8 Hz, 2H), 7.49 (d, J

= 8.0 Hz, 2H), 7.43−7.28 (m, 3 H), 6.94 (s, 1 H), 5.19 (s, 1 H), 2.69

(s, 3 H), 2.53 (s, 3 H), 0.97 (s, 9 H). MS (C₂₆H₂₇N₃O₃): m/z 430 (M

+ H)⁺.

EXPERIMENTAL SECTION

■

Chemistry Information. Solvents and reagents were used directly

as obtained from commercial sources. Flash chromatography was

performed on silica gel 60 (0.040−0.063 particle size; EM Science

supply). ¹H NMR spectra were recorded on a Bruker DRX-500f

spectrometer at 500 MHz (or Bruker AV 400 MHz, Bruker DPX-

300B, or Varian Gemini 300 at 300 MHz as stated). The chemical

shifts are reported in ppm on the δ scale relative to δTMS = 0. The

following internal references were used for the residual protons in the

following solvents: CDCl₃(δ_H7.26), CD₃OD (δ_H3.30), acetic-d₄

(acetic acid-d₄) (δ_H11.6, 2.07), DMSOmix or DMSO-d₆CDCl₃(δ_H

2.50 and 8.25) (ratio 75%:25%), and DMSO-d₆(δ_H2.50). Standard

abbreviations were employed to describe the multiplicity patterns: s

(singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), m

(multiplet), b (broad), app (apparent). The coupling constants (J)

are reported in hertz. All liquid chromatography (LC) data were

recorded on a Shimadzu LC-10AS liquid chromatograph using a SPD-

10AV UV−vis detector with mass spectrometry (MS) data

determined using a Micromass Platform for LC in electrospray

mode. The purity of the ﬁnal compounds was determined by HPLC

as described above and is 95% or higher unless speciﬁed otherwise.

2-(tert-Butoxy)-2-(4′-methyl-[1,1′-biphenyl]-2-yl)acetic Acid

(2). Di-tert-butyl dicarbonate (4.453 g, 20.40 mmol) was added to a

mixture of methyl 2-(2-bromophenyl)-2-hydroxyacetate (2000 mg,

8.16 mmol), Mg(ClO₄)₂(273 mg, 1.224 mmol) in CH₂Cl₂(12 mL)

and stirred for 72 h at room temperature. The solvent was evaporated

and the residue puriﬁed by silica gel chromatography to provide

methyl 2-(2-bromophenyl)-2-(tert-butoxy)acetate (1000 g, 40.7%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.66 (d, 2H), 7.54 (d, 2H),

2-(tert-Butoxy)-2-(5-methyl-2-phenyl-7-(p-tolyl)pyrazolo-

[1,5-a]pyrimidin-6-yl)acetamide, TFA Salt (5). Perchloric acid

(0.008 mL, 0.128 mmol) was added to a solution of 36 (3 mg, 8.46

μmol) in tert-butyl acetate (0.3 mL) and the mixture stirred for 3 h at

room temperature. The reaction mixture was quenched with H₂O and

diluted with EtOAc. The solvent was evaporated and the residue was

puriﬁed by preparative HPLC to aﬀord 5 as TFA salt (2 mg, 52.4%).

¹H NMR (400 MHz, MeOD) δ (ppm): 7.87−7.85 (m, 2 H), 7.62−

7.58 (m, 2 H), 7.45−7.36 (m, 5 H), 6.93 (s, 1 H), 5.05 (s, 1 H), 2.66

(s, 3 H), 2.50 (s, 3 H), 1.40 (s, 9 H). MS (C₂₆H₂₈N₄O₂): m/z 429 (M

+ H)⁺.

2-Hydroxy-2-(5-methyl-2-phenyl-7-p-tolylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetic Acid (6). A solution of 36 (28 mg, 0.079

mmol) in conc HCl (400 μL, 4.87 mmol) was heated at 90 °C for 3 h.

The solvent was evaporated and the residue was puriﬁed by

preparative HPLC to aﬀord 6 (14 mg, 47.5%). ¹H NMR (400

MHz, MeOD) δ (ppm): 7.93−7.80 (m, 2 H), 7.57 (d, J = 8.3 Hz,

2H), 7.51−7.29 (m, 5 H), 6.95 (s, 1 H), 5.19 (s, 1 H), 2.67 (s, 3 H),

2.51 (s, 3 H). MS (C₂₂H₁₉N₃O₃): m/z 374 (M + H)⁺.

7.36−7.15 (m, 2 H), 6.93 (s, 1 H), 5.50 (s, 1 H), 3.70 (s, 3 H), 1.25

(s, 9 H). MS (C₁₃H₁₇BrO₃): m/z 323 (M + Na)⁺. A mixture of

methyl 2-(2-bromophenyl)-2-(tert-butoxy)acetate (30 mg, 0.100

mmol), p-tolylboronic acid (20.3 mg, 0.149 mmol), Pd(Ph₃P)₄

(17.3 mg, 0.015 mmol), 2 M K₃PO₄(200 μL) in DMF (2 mL)

was heated in a microwave reactor at 130 °C for 10 min. The reaction

mixture was ﬁltered and the ﬁltrate was puriﬁed by preparative HPLC

to give methyl 2-(tert-butoxy)-2-(4′-methyl-[1,1′-biphenyl]-2-yl)-

2-(tert-Butoxy)-2-(5-methyl-2,7-diphenylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetic Acid (7). The title compound was prepared

from methyl 2-tert-butoxy-2-(7-chloro-5-methyl-2-phenylpyrazolo-

[1,5-a]pyrimidin-6-yl)acetate in a manner similar to that described

1

for 14. H NMR (600 MHz, DMSO/CDCl₃) δ (ppm): 7.84 (d, J =

1

acetate (22 mg, 70.7%). H NMR (400 MHz, MeOD) δ (ppm):

7.6 Hz, 2H), 7.76−7.67 (m, 5 H), 7.42−7.34 (m, 3 H), 7.06 (s, 1 H),

4.97 (s, 1 H), 2.53 (s, 3 H), 0.91 (s, 9 H). MS (C₂₅H₂₅N₃O₃): m/z

416 (M + H)⁺.

2-(tert-Butoxy)-2-(7-(4-methoxyphenyl)-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid, TFA Salt (8).

The title compound was prepared from methyl 2-tert-butoxy-2-(7-

chloro-5-methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a

manner similar to that described for 14. ¹H NMR (600 MHz,

DMSO/CDCl₃) δ (ppm): 7.86 (d, J = 8.2 Hz, 2H), 7.75−7.65 (m, 2

7.58−7.56 (m, 1 H), 7.36−7.20 (m, 7 H), 5.24 (s, 1 H), 3.68 (s, 3 H),

2.42 (s, 3 H), 0.95 (s, 9 H). MS (C₂₀H₂₄O₃): m/z 335 (M + Na)⁺. 1

N LiOH aqueous solution (0.6 mL, 0.6 mmol was added to a solution

of methyl 2-(tert-butoxy)-2-(4′-methyl-[1,1′-biphenyl]-2-yl)acetate

(22 mg, 0.070 mmol) in dioxane (0.6 mL), and the mixture was

stirred at room temperature for 2 h. The reaction mixture was ﬁltered

and the ﬁltrate was puriﬁed by preparative HPLC to aﬀord 2 (15.7

mg, 74.0%). ¹H NMR (400 MHz, MeOD) δ (ppm): 7.60−7.58 (m, 1

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H), 7.43−7.35 (m, 3 H), 7.20 (d, J = 8.8 Hz, 2H), 7.03 (s, 1 H), 5.05

(s, 1 H), 3.92 (s, 3 H), 2.53 (s, 3 H), 0.92 (s, 9 H). MS

(C₂₆H₂₇N₃O₄): m/z 446 (M + H)⁺.

504 (M + H)⁺. (S,R and R,S)-15. ¹H NMR (300 MHz, DMSO-d₆) δ

(ppm): 13.26 (br s, 1H), 7.87 (d, J = 7.4 Hz, 2H), 7.49−7.37 (m,

3H), 7.20 (d, J = 11.2 Hz, 1H), 7.14 (s, 1H), 4.93 (s, 1H), 4.24−4.38

(m, 2H), 2.84−2.66 (m, 2H), 2.64 (s, 3H, s), 2.16−2.04 (m, 2H),

1.78 (s, 3H), 0.96 (s, 9H). MS (C₂₉H₃₀FN₃O₄): m/z 504 (M + H)⁺.

Single crystal structures for (S,S and R,R)-14 and (S,R and R,S)-15

have been deposited in the CCDC database under CCDC deposition

numbers 1862636 and 1862637, respectively.

2-tert-Butoxy-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-

methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid

(S,S)-14 and 2-tert-butoxy-2-(7-(8-ﬂuoro-5-methylchroman-6-

yl)-5-methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic

Acid (R,R)-14. The title compounds were synthesized using a two-

step method starting from the racemic ester precursor for compound

(S,S and R,R)-14. The racemic ester was separated into two

enantiomers using a chiral column to give (S,S)-methyl 2-tert-

butoxy-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate and (R,R)-methyl 2-tert-

butoxy-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate. Chiral separation meth-

od: Chiralpak AD-H preparative column, 20 mm × 250 mm, 5 μm.

Mobile phase: 15% MeOH in CO₂at 150 bar. Temp: 35 °C. Flow

rate: 45.0 mL/min for 14 min. UV was monitored at 254 nm.

Hydrolysis of these esters as described for compound 4 gave the title

compounds (S,S)-14 and (R,R)-14.

2-(tert-Butoxy)-2-(5-methyl-2-phenyl-7-(m-tolyl)pyrazolo-

[1,5-a]pyrimidin-6-yl)acetic Acid (9). The title compound was

prepared from methyl 2-tert-butoxy-2-(7-chloro-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a manner similar to

that described for 14. ¹H NMR (600 MHz, DMSO/CDCl₃) δ (ppm):

7.84 (d, J = 7.0 Hz, 2H), 7.55−7.47 (m, 4 H), 7.44−7.35 (m, 3 H),

7.05 (s, 1 H), 4.97 (s, 1 H), 2.53 (s, 3 H), 2.45 (s, 3 H), 0.92 (s, 9 H).

MS (C₂₆H₂₇N₃O₃): m/z 430 (M + H)⁺.

2-(tert-Butoxy)-2-(7-(3-methoxyphenyl)-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid (10). The title

compound was prepared from methyl 2-tert-butoxy-2-(7-chloro-5-

methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a manner

similar to that described for 14. ¹H NMR (600 MHz, DMSO/CDCl₃)

δ (ppm): 7.85 (d, J = 7.0 Hz, 2H), 7.59−7.56 (m, 1 H), 7.42−7.21

(m, 6 H), 7.05 (s, 1 H), 5.02 (s, 1 H), 3.84 (s, 3 H), 2.53 (s, 3 H),

0.93 (s, 9 H). MS (C₂₆H₂₇N₃O₄): m/z 446 (M + H)⁺.

2-(tert-Butoxy)-2-(5-methyl-2-phenyl-7-(o-tolyl)pyrazolo-

[1,5-a]pyrimidin-6-yl)acetic Acid (11). The title compound was

prepared from methyl 2-tert-butoxy-2-(7-chloro-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a manner similar to

that described for 14. ¹H NMR (600 MHz, DMSO/CDCl₃) δ (ppm):

7.79 (d, J = 7.6 Hz, 2H), 7.54−7.46 (m, 2 H), 7.41−7.33 (m, 5 H),

7.04 (s, 1 H), 4.81 (s, 1 H), 2.73 (s, 3 H), 2.53 (s, 3 H), 1.11 (s, 9 H).

MS (C₂₆H₂₇N₃O₃): m/z 430 (M + H)⁺.

2-(tert-Butoxy)-2-(7-(2-methoxyphenyl)-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid (12). The title

compound was prepared from methyl 2-tert-butoxy-2-(7-chloro-5-

methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a manner

similar to that described for 14. ¹H NMR (600 MHz, DMSO/CDCl₃)

δ (ppm): 7.82 (d, J = 7.6 Hz, 2H), 7.67−7.64 (m, 1 H), 7.52−7.18

(m, 6 H), 7.00 (s, 1 H), 4.94 (s, 1 H), 3.68 (s, 3 H), 2.53 (s, 3 H),

0.94 (s, 9 H). MS (C₂₆H₂₇N₃O₄): m/z 446 (M + H)⁺.

2-tert-Butoxy-2-(5,7-dimethyl-2-phenylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetic Acid, TFA Salt (16). A mixture of 41 (20

mg, 0.052 mmol), Pd(PPh₃)₄(20 mg, 0.017 mmol), methylzinc(II)

chloride (0.5 mL, 1.000 mmol), and THF (0.8 mL) was heated in a

microwave reactor at 130 °C for 15 min. The reaction mixture was

quenched with H₂O, extracted with EtOAc, and the combined organic

layer was dried over anhydrous Na₂SO₄, ﬁltered, and concentrated.

The residue was puriﬁed by preparative HPLC to give methyl 2-tert-

butoxy-2-(5,7-dimethyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)-

acetate as the TFA salt (15 mg, 60.4%). ¹H NMR (400 MHz, CDCl₃)

δ (ppm): 8.04−8.02 (m, 2 H), 7.56−7.38 (m, 3 H), 7.06 (s, 1 H),

5.41 (s, 1 H), 3.75 (s, 3 H), 3.10 (s, 3 H), 2.85 (s, 3 H), 1.28 (s, 9 H).

MS (C₂₁H₂₅N₃O₃): m/z 368 (M + H)⁺. An aqueous solution of 1.5 N

LiOH (0.5 mL, 0.750 mmol) was added to a solution of the TFA salt

of methyl 2-tert-butoxy-2-(5,7-dimethyl-2-phenylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetate (15 mg, 0.031 mmol) in dioxane (0.5 mL)

and the mixture stirred at room temperature for 4 h. The reaction

mixture was ﬁltered and the ﬁltrate was puriﬁed by preparative HPLC

2-(tert-Butoxy)-2-(7-(chroman-6-yl)-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid (13). The title

compound was prepared from methyl 2-tert-butoxy-2-(7-chloro-5-

methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a manner

similar to that described for 14. ¹H NMR (600 MHz, DMSO/CDCl₃)

δ (ppm): 7.96 (s, 1H), 7.87 (d, J = 7.0 Hz, 2H), 7.60−7.30 (m, 4 H),

7.02 (s, 1 H), 6.98 (d, J = 8.8 Hz, 1H), 5.07 (s, 1 H), 4.29 (br s, 2H),

2.84 (s, 2H), 2.61 (s, 3 H), 2.04 (br s, 2H), 0.93 (9 H, s). MS

(C₂₈H₂₉N₃O₄): m/z 472 (M + H)⁺.

1

to aﬀord the TFA salt of 16 (13 mg, 89%). H NMR (500 MHz,

CDCl₃) δ (ppm): 9.92 (br s, 1H), 8.06−7.89 (m, 2 H), 7.55−7.34

(m, 3 H), 7.07 (s, 1 H), 5.41 (s, 1 H), 3.10 (s, 3 H), 2.85 (s, 3 H),

1.27 (s, 9 H). MS (C₂₀H₂₃N₃O₃): m/z 354 (M + H)⁺.

2-(tert-Butoxy)-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-

methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid,

TFA Salt (S,S and R,R)-14 and (S,R and R,S)-15. A mixture of

41 (50 mg, 0.1 mmol), Pd(PPh₃)₄(17.27 mg, 0.015 mmol), 2-(8-

ﬂuoro-5-methylchroman-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

(58.2 mg, 0.199 mmol), and 2 M K₃PO₄solution (300 μL) in DMF

(4 mL) was heated in a microwave reactor at 130 °C for 15 min. The

reaction mixture was ﬁltered and the ﬁltrate puriﬁed by preparative

HPLC to aﬀord (S,S and R,R)-methyl 2-(tert-butoxy)-2-(7-(8-ﬂuoro-

5-methylchroman-6-yl)-5-methyl-2-phenylpyrazolo[1,5-a]pyrimidin-

6-yl)acetate (30 mg, 54.3%) and (S,R and R,S) isomer (1.6 mg, 2.9%)

as TFA salt. (S,S and R,R)-Methyl 2-(tert-butoxy)-2-(7-(8-ﬂuoro-5-

methylchroman-6-yl)-5-methyl-2-phenylpyrazolo[1,5-a]pyrimidin-6-

yl)acetate. ¹H NMR (400 MHz, CD₃OD) δ (ppm): 7.83 (dd, J = 8.2,

1.6 Hz, 2H), 7.48−7.27 (m, 3 H), 7.04−6.85 (m, 2 H), 5.08 (s, 1 H),

4.33−4.30 (m, 2 H), 3.68 (s, 3 H), 2.81 (t, J = 6.5 Hz, 2H), 2.75 (s, 3

H), 2.20−2.14 (m, 2 H), 1.87 (s, 3 H), 1.17 (s, 9 H). MS

(C₃₀H₃₂FN₃O₄): m/z 518 (M + H)⁺. (S,R and R,S)-Methyl 2-(tert-

butoxy)-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate. MS (C₃₀H₃₂FN₃O₄):

m/z 518 (M + H)⁺. Hydrolysis of these esters as described for 4

gave the title compounds (S,S and R,R)-14 and (S,R and R,S)-15. (S,S

and R,R)-14. ¹H NMR (500 MHz, CD₃OD) δ (ppm): 7.83 (d, J = 7.2

Hz, 2H), 7.42−7.34 (m, 3H), 6.96 (d, J = 10.99 Hz, 1H), 6.90 (s,

1H), 4.58 (s, 1H), 4.41−4.23 (m, 2H), 2.85−2.80 (m, 5H), 2.18−

2.14 (m, 2H), 1.97 (s, 3H), 1.17 (s, 9H). MS (C₂₉H₃₀FN₃O₄): m/z

2-(tert-Butoxy)-2-(5-methyl-2-phenyl-7-(p-tolylamino)-

pyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid (17). The title com-

pound was prepared from methyl 2-tert-butoxy-2-(7-chloro-5-methyl-

2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a manner similar to

1

that described for 19. H NMR (500 MHz, DMSO-d₆) δ (ppm):

7.84−7.83 (m, 2 H), 7.43−7.35 (m, 4H), 7.10 (d, J = 7.9 Hz, 2H),

6.92 (s, 1H), 6.87 (br s, 1H), 5.29 (s, 1H), 2.54 (s, 3H), 2.30 (s, 3H),

1.08 (s, 9H). MS (C₂₆H₂₈N₄O₃): m/z 445 (M + H)⁺.

2-(7-(Azepan-1-yl)-5-methyl-2-phenylpyrazolo[1,5-a]-

pyrimidin-6-yl)-2-(tert-butoxy)acetic Acid (18). The title com-

pound was prepared from methyl 2-tert-butoxy-2-(7-chloro-5-methyl-

2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate in a manner similar to

that described for 19. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.06

(d, J = 7.5 Hz, 2H), 7.54−7.48 (m, 2 H), 7.45−7.41 (m, 1 H), 7.06 (s,

1 H), 5.88 (s, 1 H), 2.55 (s, 3 H), 1.92−1.79 (m, 8 H), 1.22 (s, 9 H).

MS (C₂₅H₃₂N₄O₃): m/z 437 (M + H)⁺.

2-(tert-Butoxy)-2-(7-(4,4-dimethylpiperidin-1-yl)-5-methyl-

2-phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid (19). DIEA

(0.047 mL, 0.272 mmol) was added to a mixture of 41 (40 mg, 0.091

mmol) and 4,4-dimethylpiperidine, HCl salt (13.55 mg, 0.091 mmol)

in NMP (1 mL) and heated at 50°C for 2 h. 1 N LiOH (0.272 mL,

0.272 mmol) was added to the reaction mixture, and the contents

were heated at 50°C for 2 h. The reaction mixture was ﬁltered and the

ﬁltrate puriﬁed by preparative HPLC to aﬀord 19 as TFA salt (13.5

K

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mg, 30%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.05 (d, J = 7.8

Hz, 2H). 7.54−7.48 (m, 2 H), 7.45−7.41 (m, 1 H), 7.03 (s, 1 H),

5.77 (s, 1 H), 2.53 (s, 3 H), 1.65−1.52 (m, 4 H), 1.20 (s, 9 H), 1.12

(br s, 6H). MS (C₂₆H₃₄N₄O₃): m/z 451 (M + H)⁺.

(2S)-2-(2-([1,1′-Biphenyl]-3-yl)-7-(8-ﬂuoro-5-methylchro-

man-6-yl)-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-(tert-

butoxy)acetic Acid ((S,S)-24). The title compound was prepared

from (2S)-methyl 2-(tert-butoxy)-2-(2-(3-chlorophenyl)-7-(8-ﬂuoro-

5-methylchroman-6-yl)-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)-

2-tert-Butoxy-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-

methyl-2-p-tolylpyrazolo[1,5-a]pyrimidin-6-yl)acetic Acid,

TFA Salt (S,S and R,R)-20. A mixture of the TFA salt of 47 (8

mg, 0.016 mmol), Pd(PPh₃)₄(2.69 mg, 2.326 μmol), 2-(8-ﬂuoro-5-

methylchroman-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (9.06

mg, 0.031 mmol), 2 M K₃PO₄solution (50 μL), and DMF (0.7

mL) was heated in a microwave reactor at 130 °C for 15 min. The

reaction mixture was ﬁltered and the ﬁltrate puriﬁed by preparative

HPLC to aﬀord methyl 2-tert-butoxy-2-(7-(8-ﬂuoro-5-methylchro-

man-6-yl)-5-methyl-2-p-tolylpyrazolo[1,5-a]pyrimidin-6-yl)acetate as

1

acetate in a manner similar to that described for (S,S)-27. H NMR

(500 MHz, CDCl₃) δ (ppm): 8.05−7.98 (m, 1H), 7.81 (d, J = 7.6 Hz,

1H), 7.65−7.58 (m, 2H), 7.55 (d, J = 7.6 Hz, 1H), 7.46 (t, J = 7.5 Hz,

3H), 7.40−7.33 (m, 1H), 6.98−6.90 (m, 2H), 5.09 (s, 1H), 4.38−

4.27 (m, 2H), 2.82−2.66 (m, 5H), 2.15 (d, J = 6.1 Hz, 2H), 1.93 (s,

3H), 1.21 (s, 9H). MS (C₃₅H₃₄FN₃O₄): m/z 580 (M + H)⁺.

2-(2-([1,1′-Biphenyl]-3-yl)-7-(4,4-dimethylpiperidin-1-yl)-5-

methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-(tert-butoxy)acetic

Acid ((S and R)-25). A mixture of 46 (90 mg, 0.230 mmol),

Pd(PPh₃)₄(40 mg, 0.035 mmol), [1,1′-biphenyl]-3-ylboronic acid

(45.6 mg, 0.230 mmol), 2 M K₂CO₃solution (300 μL), and DMF (4

mL) was heated in a microwave reactor at 70 °C for 60 min. The

mixture was ﬁltered and the ﬁltrate puriﬁed by preparative HPLC to

aﬀord methyl 2-(2-([1,1′-biphenyl]-3-yl)-7-chloro-5-methylpyrazolo-

[1,5-a]pyrimidin-6-yl)-2-(tert-butoxy)acetate as the TFA salt (47 mg,

35.3%). MS (C₂₆H₂₆ClN₃O₃): m/z 464 (M + H)⁺. A mixture of

methyl 2-(2-([1,1′-biphenyl]-3-yl)-7-chloro-5-methylpyrazolo[1,5-a]-

pyrimidin-6-yl)-2-(tert-butoxy)acetate, TFA salt (25 mg, 0.043

mmol), 4,4-dimethylpiperidine HCl salt (16.13 mg, 0.108 mmol),

and DIEA (0.038 mL, 0.216 mmol) in CH₂Cl₂(3 mL) was stirred at

rt for 18 h. The solvent was evaporated, dioxane (1 mL) and NaOH

(1 mL, 1.0 mmol) added and the mixture heated at 50 °C for 1 h. The

mixture was ﬁltered and the ﬁltrate was puriﬁed by preparative HPLC

to aﬀord (S and R)-25 as the TFA salt (1.5 mg, 6.7%). ¹H NMR (500

MHz, DMSO-d₆) δ (ppm): 8.36 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H),

7.79−7.71 (m, 3H), 7.61 (t, J = 7.63 Hz, 1H), 7.52 (t, J = 7.27 Hz,

2H), 7.44−7.41 (m, 1H), 7.11 (s, 1H), 5.63 (br s, 1H), 3.48−3.26

(m, 4H), 2.54 (s, 3H), 1.67−1.51 (m, 4H), 1.18 (s, 9H), 1.12 (br s,

6H). MS (C₃₂H₃₈N₄O₃): m/z 527 (M + H)⁺.

2-(2-(3-(Benzyloxy)phenyl)-7-(4,4-dimethylpiperidin-1-yl)-

5-methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-(tert-butoxy)acetic

Acid ((S and R)-26). The title compound was prepared from methyl

2-(2-bromo-7-chloro-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-tert-

butoxyacetate in a manner similar to that described for (S and R)-25.

¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 7.72−7.67 (m, 1H), 7.63

(d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.2 Hz, 2H), 7.44−7.41 (m, 3H),

7.37−7.34 (m, 1H), 7.07 (dd, J = 8.2, 1.8 Hz, 1H), 7.03 (s, 1H), 5.71

(s, 1H), 5.21 (s, 2H), 3.36 (br s, 4H), 2.52 (s, 3H), 1.51−1.67 (m,

4H), 1.19 (s, 9H), 1.11 (br s, 6H). MS (C₃₃H₄₀N₄O₄): m/z 557 (M +

H)⁺.

(2S)-2-(tert-Butoxy)-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-

5-methyl-2-(2′-methyl-[1,1′-biphenyl]-3-yl)pyrazolo[1,5-a]-

pyrimidin-6-yl)acetic Acid ((S,S)-27). A mixture of dicyclohexyl-

(2′,6′-dimethoxy-[1,1′-biphenyl]-2-yl)phosphine (595 mg, 1.449

mmol), Pd(OAc)₂(163 mg, 0.725 mmol), o-tolylboronic acid (296

mg, 2.174 mmol), 2 M K₃PO₄solution (200 μL), and 55 (400 mg,

0.725 mmol) in DMF (1.5 mL) was heated in a microwave reactor at

130 °C for 30 min. The mixture was ﬁltered and the ﬁltrate puriﬁed

by silica gel chromatography to aﬀord (2S)-methyl 2-(tert-butoxy)-2-

(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-methyl-2-(2′-methyl-[1,1′-bi-

phenyl]-3-yl)pyrazolo[1,5-a]pyrimidin-6-yl)acetate (225.6 mg,

51.2%). ¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.86−7.76 (m,

2H), 7.42 (t, J = 7.8 Hz, 1H), 7.29−7.26 (m, 5H), 6.88−6.86 (m,

2H), 4.99 (s, 1H), 4.39−4.27 (m, 2H), 3.64 (s, 3H), 2.83−2.70 (m,

5H), 2.26 (s, 3H), 2.22−2.13 (m, 2H), 1.84 (s, 3H), 1.16 (s, 9H). MS

(C₃₇H₃₈FN₃O₄): m/z 608 (M + H)⁺. 1 N NaOH aqueous solution (9

mL, 9 mmol) was added to a solution of (2S)-methyl 2-(tert-butoxy)-

2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-methyl-2-(2′-methyl-[1,1′-

biphenyl]-3-yl)pyrazolo[1,5-a]pyrimidin-6-yl)acetate (1.28 g, 2.1

mmol) in dioxane (12 mL) and the mixture stirred at 50 °C for 2

h. The mixture was diluted with H₂O (50 mL), neutralized with

AcOH, and extracted with EtOAc (3 × 100 mL). The organic phase

was combined, dried over Na₂SO₄, concentrated and the crude

product puriﬁed by preparative HPLC to aﬀord (S,S)-27 (840 mg,

66%). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 7.86−7.75 (m, 2H),

7.50 (t, J = 7.7 Hz, 1H), 7.40−7.22 (m, 5H), 7.18 (s, 1H), 7.09 (d, J =

1

the TFA salt (5 mg, 49.9%). H NMR (400 MHz, CDCl₃) δ (ppm):

7.72 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 7.01 (s, 1 H), 6.89

(d, J = 10.5 Hz, 1H), 5.06 (s, 1 H), 4.40−4.33 (m, 2 H), 3.68 (s, 3

H), 2.86 (s, 3 H), 2.81−2.76 (m, 2 H), 2.38 (s, 3 H), 2.23−2.16 (m, 2

H), 1.86 (s, 3 H), 1.19 (s, 9 H). MS (C₃₁H₃₄FN₃O₄): m/z 532 (M +

H)⁺. A mixture of 1 N LiOH aqueous solution (0.5 mL, 0.5 mmol)

and methyl 2-tert-butoxy-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-

methyl-2-p-tolylpyrazolo[1,5-a]pyrimidin-6-yl)acetate, TFA salt (5

mg, 7.74 μmol) in dioxane (0.5 mL) was stirred at 50 °C for 1 h.

The mixture was ﬁltered and the ﬁltrate puriﬁed by preparative HPLC

1

to aﬀord (S,S and R,R)-20 (4 mg, 82%) as the TFA salt. H NMR

(500 MHz, MeOD) δ (ppm): 7.71 (d, J = 8.2 Hz, 2H), 7.21 (d, J =

7.9 Hz, 2H), 6.94 (d, J = 10.7 Hz, 1H), 6.88 (s, 1 H), 5.01 (s, 1 H),

4.40−4.24 (m, 2 H), 2.86−2.78 (m, 2 H), 2.76 (s, 3 H), 2.35 (s, 3 H),

2.22−2.10 (m, 2 H), 1.87 (s, 3 H), 1.17 (s, 9 H). MS

(C₃₀H₃₂FN₃O₄): m/z 518 (M + H)⁺.

2-(2-(4-(Benzyloxy)phenyl)-7-(8-ﬂuoro-5-methylchroman-6-

yl)-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-tert-butoxyace-

tic Acid, TFA Salt ((S,S and R,R)-21). The title compound was

prepared from methyl 2-(2-bromo-7-chloro-5-methylpyrazolo[1,5-

a]pyrimidin-6-yl)-2-tert-butoxyacetate in a manner similar to that

described for compound (S,S and R,R)-20. ¹H NMR (400 MHz,

CDCl₃) δ (ppm): 7.76 (d, J = 8.8 Hz, 2H), 7.47−7.32 (m, 5H),

7.04−6.95 (m, 3H), 6.91 (d, J = 10.5 Hz, 1H), 5.11 (d, J = 2.0 Hz,

3H), 4.44−4.25 (m, 2H), 2.92−2.59 (m, 5H), 2.28−2.10 (m, 2H),

1.89 (s, 3H), 1.22 (s, 9H). MS (C₃₆H₃₆FN₃O₅): m/z 610 (M + H)⁺.

2-(2-(3-(Benzyloxy)phenyl)-7-(8-ﬂuoro-5-methylchroman-6-

yl)-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-tert-butoxyace-

tic Acid, TFA Salt ((S,S and R,R)-22). The title compound was

prepared from methyl 2-(2-bromo-7-chloro-5-methylpyrazolo[1,5-

a]pyrimidin-6-yl)-2-tert-butoxyacetate in a manner similar to that

described for compound (S,S and R,R)-20. ¹H NMR (500 MHz,

CDCl₃) δ (ppm): 7.48−7.27 (m, 8H), 6.96 (dd, J = 8.2, 1.8 Hz, 1H),

6.93−6.88 (m, 2H), 5.08 (s, 3H), 4.46−4.12 (m, 2H), 2.92−2.73 (m,

5H), 2.26−2.08 (m, 2H), 1.91 (s, 3H), 1.20 (s, 9H). MS

(C₃₆H₃₆FN₃O₅): m/z 610 (M + H)⁺.

2-tert-Butoxy-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-5-

methyl-2-(3-phenoxyphenyl)pyrazolo[1,5-a]pyrimidin-6-yl)-

acetic Acid, TFA Salt ((S,S and R,R)-23). The title compound was

prepared from methyl 2-(2-bromo-7-chloro-5-methylpyrazolo[1,5-

a]pyrimidin-6-yl)-2-tert-butoxyacetate in a manner similar to that

described for (S,S and R,R)-20. ¹H NMR (400 MHz, CDCl₃) δ

(ppm): 7.62−7.49 (m, 2H), 7.41−7.31 (m, 3H), 7.17−6.95 (m, 5H),

6.90 (d, J = 10.8 Hz, 1H), 5.12 (s, 1H), 4.50−4.22 (m, 2H), 2.89−

2.66 (m, 5H), 2.17 (dd, J = 6.0, 4.0 Hz, 2H), 1.90 (s, 3H), 1.28−1.09

(m, 9H). MS (C₃₅H₃₄FN₃O₅): m/z 596 (M + H)⁺.

2-(2-([1,1′-Biphenyl]-3-yl)-7-(8-ﬂuoro-5-methylchroman-6-

yl)-5-methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-(tert-butoxy)-

acetic Acid, TFA Salt ((S,S and R,R)-24). The title compound was

prepared from methyl 2-(2-bromo-7-chloro-5-methylpyrazolo[1,5-

a]pyrimidin-6-yl)-2-tert-butoxyacetate in a manner similar to that

described for (S,S and R,R)-20. ¹H NMR (500 MHz, CDCl₃) δ

(ppm): 8.05−7.98 (m, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.65−7.58 (m,

2H), 7.55 (d, J = 7.6 Hz, 1H), 7.46 (t, J = 7.5 Hz, 3H), 7.40−7.33 (m,

1H), 6.98−6.90 (m, 2H), 5.09 (s, 1H), 4.38−4.27 (m, 2H), 2.82−

2.66 (m, 5H), 2.15 (d, J = 6.1 Hz, 2H), 1.93 (s, 3H), 1.21 (s, 9H). MS

(C₃₅H₃₄FN₃O₄): m/z 580 (M + H)⁺.

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1.0 Hz, 1H), 4.83 (s, 1H), 4.34−4.22 (m, 2H), 2.82−2.66 (m, 5H),

2.24 (s, 3H), 2.13−2.02 (m, 2H), 1.83 (s, 3H), 1.09 (s, 9H). MS

(C₃₆H₃₆FN₃O₄): m/z 594 (M + H)⁺. Anal. (C₃₆H₃₆FN₃O₄with

1.26% H₂O) Calcd: C, 71.91; H, 6.18; N, 6.99; F, 3.16. Found: C,

71.67; H, 6.25; N, 7.07; F, 2.79.

solution. The aqueous layer was extracted with CH₂Cl₂, and the

combined organic layer was dried over anhydrous Na₂SO₄, ﬁltered,

and concentrated. The residue was puriﬁed by silica gel chromatog-

raphy to give (5-methyl-2-phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidin-

6-yl)methanol (344 mg, 46.7%). ¹H NMR (500 MHz, CDCl₃) δ

(ppm): 1.61 (1 H, t, J = 5.0 Hz), 2.49 (3 H, s), 2.78 (3 H, s), 4.59 (2

H, d, J = 5.0 Hz), 6.88 (1 H, s), 7.29−7.42 (5 H, m), 7.56 (2 H, d, J =

8.2 Hz), 7.81−7.96 (2 H, m). MS (C₂₁H₁₉N₃O): m/z 330 (M + H)⁺.

To a stirred solution of (5-methyl-2-phenyl-7-p-tolylpyrazolo[1,5-

a]pyrimidin-6-yl)methanol (100 mg, 0.304 mmol) in CH₂Cl₂(8 mL)

was added PCC (98 mg, 0.455 mmol). The reaction mixture was

stirred at room temperature for 16 h. The solvent was evaporated and

the residue was puriﬁed by silica gel chromatography to give the title

compound 35 (82 mg, 83%). ¹H NMR (500 MHz, CDCl₃) δ (ppm):

9.80 (s, 1 H), 7.91 (dd, J = 8.1, 1.4 Hz, 2H), 7.60 (d, J = 7.9 Hz, 2H),

7.50−7.33 (m, 5 H), 6.98 (s, 1 H), 2.89 (s, 3 H), 2.52 (s, 3 H). MS

(C₂₁H₁₇N₃O): m/z 328 (M + H)⁺.

2-Hydroxy-2-(5-methyl-2-phenyl-7-p-tolylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetonitrile (36). To a solution of 35 (30 mg, 0.092

mmol) in CH₂Cl₂(2 mL) at 0 °C was added ZnI₂(14.63 mg, 0.046

mmol) followed by TMS-CN (0.049 mL, 0.367 mmol). The reaction

mixture was stirred at room temperature for 4 h and diluted with

CH₂Cl₂(100 mL), washed with H₂O, and dried over Na₂SO₄. The

solvent was evaporated and the residue was puriﬁed by preparative

HPLC to give the title compound 36 (15 mg, 46.2%) as a TFA salt.

¹H NMR (500 MHz, CDCl₃) δ (ppm): 7.76−7.63 (m, 2 H), 7.44−

7.30 (m, 4 H), 7.25−7.16 (m, 3 H), 6.85 (s, 1 H), 5.39 (s, 1 H), 2.89

(s, 3 H), 2.46 (s, 3 H). MS (C₂₂H₁₈N₄O): m/z 355 (M + H)⁺.

Methyl 2-(5-Methyl-7-oxo-2-phenyl-4,7-dihydropyrazolo-

[1,5-a]pyrimidin-6-yl)acetate (38). p-TsOH.H₂O (50 mg, 0.263

mmol) was added to a solution of 3-phenyl-1H-pyrazol-5-amine 33 (4

g, 25.1 mmol) and dimethyl 2-acetylsuccinate 37 (12 mL, 74.0 mmol)

in xylene (120 mL) and the mixture heated at reﬂux under a Dean−

Stark trap for 20 h. The solid was ﬁltered and washed with hexanes to

aﬀord 38 (6.4 g, 86%). ¹H NMR (400 MHz, MeOD) δ (ppm): 8.06−

7.87 (m, 2 H), 7.53−7.34 (m, 3 H), 6.46 (s, 1 H), 3.72 (s, 3 H), 3.66

(s, 2 H), 2.37 (s, 3 H). MS (C₁₆H₁₅N₃O₃): m/z 298 (M + H)⁺.

Methyl 2-(7-Chloro-5-methyl-2-phenylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetate (39). POCl₃(25 mL, 268 mmol) was

added to 38 (3 g, 10.09 mmol) and the mixture heated at reﬂux for 1

h. After cooling, the reaction mixture was added dropwise to ice−

water to aﬀord a brown precipitate. The solid was ﬁltered oﬀ, washed

with H₂O, and dissolved in EtOAc. The organic solution was washed

with saturated NaHCO₃and dried over Na₂SO₄. The solvent was

evaporated to give 39 (2.77 g, 84%). ¹H NMR (400 MHz, DMSO-d₆)

δ (ppm): 8.07 (d, J = 7.0 Hz, 2H), 7.58−7.43 (m, 3 H), 7.29 (s, 1 H),

4.04 (s, 2 H), 3.71 (s, 3 H), 2.58 (s, 3 H). MS (C₁₆H₁₄ClN₃O₂): m/z

316 (M + H)⁺.

Methyl 2-(7-Chloro-5-methyl-2-phenylpyrazolo[1,5-a]-

pyrimidin-6-yl)-2-hydroxyacetate (40). A solution of 39 (1 g,

3.17 mmol) in THF (24 mL) was added dropwise over 40 min to a

stirred solution of KHMDS (0.5 M in toluene, 9.50 mL, 4.75 mmol)

in THF (24 mL) maintained at −78 °C. The mixture was stirred at

−78 °C for 30 min before adding a solution of 3-phenyl-2-

(phenylsulfonyl)-1,2-oxaziridine (1.241 g, 4.75 mmol) in THF (24

mL) over 20 min. The reaction mixture was stirred for additional 30

min at −78 °C before being quenched by the addition of saturated

aqueous NH₄Cl solution (4 mL). The reaction mixture was allowed

to warm to room temperature, diluted with EtOAc (100 mL) and the

organic phase washed with H₂O and brine and dried over Na₂SO₄.

The solvent was evaporated and the residue puriﬁed by silica gel

chromatography to provide 40 (535 mg, 50.9%). ¹H NMR (500

MHz, CDCl₃) δ ppm: 8.02−8.00 (m, 2 H), 7.50−7.38 (m, 3 H), 6.94

(s, 1 H), 5.76 (s, 1 H), 5.29 (s, 1 H), 3.83 (s, 3 H), 2.62 (s, 3 H). MS

(C₁₆H₁₄ClN₃O₃): m/z 332 (M + H)⁺.

(2S)-2-(tert-Butoxy)-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-

2-(2′-methoxy-[1,1′-biphenyl]-3-yl)-5-methylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetic Acid ((S,S)-28). The title compound was

prepared from (2S)-methyl 2-(tert-butoxy)-2-(2-(3-chlorophenyl)-7-

(8-ﬂuoro-5-methylchroman-6-yl)-5-methylpyrazolo[1,5-a]pyrimidin-

1

6-yl)acetate in a manner similar to that described for (S,S)-27. H

NMR (500 MHz, CDCl₃) δ (ppm): 8.00−7.95 (m, 1H), 7.79 (d, J =

7.6 Hz, 1H), 7.51−7.47 (m, 1H), 7.45−7.39 (m, 1H), 7.37−7.31 (m,

2H), 7.08−6.97 (m, 2H), 6.93 (d, J = 10.7 Hz, 1H), 6.90 (s, 1H),

5.08 (s, 1H), 4.39−4.24 (m, 2H), 3.79 (s, 3H), 2.82−2.64 (m, 5H),

2.15 (d, J = 3.7 Hz, 2H), 1.92 (s, 3H), 1.21 (s, 9H). MS

(C₃₆H₃₆FN₃O₅): m/z 610 (M + H)⁺.

(2S)-2-(tert-Butoxy)-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-

2-(2′-ﬂuoro-[1,1′-biphenyl]-3-yl)-5-methylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetic Acid ((S,S)-29). The title compound was

prepared from (2S)-methyl 2-(tert-butoxy)-2-(2-(3-chlorophenyl)-7-

(8-ﬂuoro-5-methylchroman-6-yl)-5-methylpyrazolo[1,5-a]pyrimidin-

1

6-yl)acetate in a manner similar to that described for (S,S)-27. H

NMR (500 MHz, CDCl₃) δ (ppm): 7.95 (d, J = 1.2 Hz, 1H), 7.88−

7.75 (m, 1H), 7.57−7.40 (m, 3H), 7.37−7.29 (m, 1H), 7.24−7.12

(m, 2H), 7.01−6.82 (m, 2H), 5.06 (s, 1H), 4.36−4.24 (m, 2H),

2.80−2.63 (m, 5H), 2.13 (dd, J = 6.1, 3.1 Hz, 2H), 1.89 (s, 3H), 1.17

(s, 9H). MS (C₃₅H₃₃F₂N₃O₄): m/z 598 (M + H)⁺.

(2S)-2-(tert-Butoxy)-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-

5-methyl-2-(3-(pyridin-4-yl)phenyl)pyrazolo[1,5-a]pyrimidin-

6-yl)acetic Acid, TFA Salt ((S,S)-30). The title compound was

prepared from (2S)-methyl 2-(tert-butoxy)-2-(2-(3-chlorophenyl)-7-

(8-ﬂuoro-5-methylchroman-6-yl)-5-methylpyrazolo[1,5-a]pyrimidin-

1

6-yl)acetate in a manner similar to that described for (S,S)-27. H

NMR (500 MHz, CDCl₃) δ (ppm): 8.91 (d, J = 6.41 Hz, 2H), 8.13−

8.05 (m, 4H), 7.75−7.60 (m, 2H), 7.00 (s, 1H), 6.92 (d, J = 10.38

Hz, 1H), 5.09 (s, 1H), 4.33 (t, J = 5.04 Hz, 2H), 2.79−2.69 (m, 5H),

2.22−2.11 (m, 2H), 1.95 (s, 3H), 1.21 (s, 9H). MS (C₃₄H₃₃FN₄O₄):

m/z 581 (M + H)⁺. HRMS calcd for C₃₄H₃₃FN₄O₄(MH⁺) 581.2559,

found 581.2574.

(2S)-2-(tert-Butoxy)-2-(7-(8-ﬂuoro-5-methylchroman-6-yl)-

2-(3-(6-methoxypyridin-3-yl)phenyl)-5-methylpyrazolo[1,5-

a]pyrimidin-6-yl)acetic Acid ((S,S)-31). The title compound was

prepared from (2S)-methyl 2-(tert-butoxy)-2-(2-(3-chlorophenyl)-7-

(8-ﬂuoro-5-methylchroman-6-yl)-5-methylpyrazolo[1,5-a]pyrimidin-

1

6-yl)acetate in a manner similar to that described for (S,S)-27. H

NMR (500 MHz, DMSO-d₆) δ (ppm): 8.55 (d, J = 2.4 Hz, 1H),

8.13−8.05 (m, 2H), 7.79 (d, J = 7.9 Hz, 1H), 7.66 (d, J = 7.9 Hz,

1H), 7.51 (t, J = 7.6 Hz, 1H), 7.26 (s, 1H), 7.09 (d, J = 11.3 Hz, 1H),

6.95 (d, J = 8.5 Hz, 1H), 4.78 (s, 1H), 4.34−4.21 (m, 2H), 3.92 (s,

3H), 2.75−2.73 (m, 2H), 2.71 (s, 3H), 2.08 (t, J = 5.6 Hz, 2H), 1.83

(s, 3H), 1.09 (s, 9H). MS (C₃₅H₃₅FN₄O₅): m/z 611 (M + H)⁺.

Methyl 5-Methyl-2-phenyl-7-p-tolylpyrazolo[1,5-a]-

pyrimidine-6-carboxylate (34). To a stirred solution of 4-

methylbenzaldehyde 32 (1.2 g, 9.99 mmol), 3-phenyl-1H-pyrazol-5-

amine 33 (1.6 g, 9.99 mmol), and methyl 3-oxobutanoate (1.3 g,

10.99 mmol) in THF (80 mL) and heptane (20 mL) was added

piperidine (30 μL, 0.303 mmol). The reaction mixture was heated at

reﬂux for 20 h, the solvent was evaporated, and the crude material was

dissolved in CH₂Cl₂. DDQ (2.041 g, 8.99 mmol) was added, and the

mixture was stirred at room temperature for 1 h. The solvent was

evaporated and the residue puriﬁed by silica gel chromatography to

1

provide 34 (2.3 g, 64%). H NMR (500 MHz, CDCl₃) δ (ppm):

7.99−7.86 (m, 2H), 7.63 (d, J = 7.9 Hz, 2H), 7.47−7.31 (m, 5H),

6.93 (s, 1 H), 3.64 (s, 3 H), 2.65 (s, 3 H), 2.47 (s, 3 H). MS

(C₂₂H₁₉N₃O₂): m/z 358 (M + H)⁺.

Methyl 2-tert-Butoxy-2-(7-chloro-5-methyl-2-

phenylpyrazolo[1,5-a]pyrimidin-6-yl)acetate (41). CH₂Cl₂(2

mL) and HClO₄(0.027 mL, 0.452 mmol) were added sequentially to

a suspension of methyl 2-(7-chloro-5-methyl-2-phenylpyrazolo[1,5-

a]pyrimidin-6-yl)-2-hydroxyacetate 40 (100 mg, 0.301 mmol) in tert-

5-Methyl-2-phenyl-7-p-tolylpyrazolo[1,5-a]pyrimidine-6-

carbaldehyde (35). DIBAL-H (1 M in THF, 6.72 mL, 6.72 mmol)

was added dropwise to a stirred solution of 34 (800 mg, 2.238 mmol)

in CH₂Cl₂(50 mL). The reaction mixture was stirred at room

temperature for 1 h before being quenched with saturated NH₄Cl

M

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Article

butyl acetate (2 mL), and the mixture was stirred for 2 h at room

temperature. The reaction mixture was quenched with H₂O, diluted

with EtOAc and the organic phase washed with saturated NaHCO₃

and dried over Na₂SO₄. The solvent was evaporated and the residue

puriﬁed by silica gel chromatography to provide 41 (71 mg, 60.7%).

¹H NMR (500 MHz, CDCl₃) δ (ppm): 8.01 (d, J = 7.3 Hz, 2H),

7.79 (m, 1H), 7.51−7.27 (m, 2H), 6.62−6.35 (m, 1H), 3.72 (s, 3H),

3.67 (s, 2H), 2.39 (s, 3H). MS (C₁₆H₁₄ClN₃O₃): m/z 332 (M + H)⁺.

Methyl 2-(7-Chloro-2-(3-chlorophenyl)-5-methylpyrazolo-

[1,5-a]pyrimidin-6-yl)acetate (50). A mixture of 49 (12 g, 36.2

mmol) and POCl₃(50 mL) was heated at reﬂux for 2.5 h. After

cooling, the reaction mixture was added dropwise to ice−water to

precipitate a brown solid which was ﬁltered and washed with H₂O

before dissolving in EtOAc. The organic layer was washed with

saturated NaHCO₃, dried over Na₂SO₄and concentrated to give 50

(11.9 g, 94%). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.03 (t, J = 1.8

Hz, 1H), 7.94−7.82 (m, 1H), 7.48−7.34 (m, 2H), 6.94 (s, 1H), 3.93

(s, 2H), 3.78 (s, 3H), 2.63 (s, 3H). MS (C₁₆H₁₃Cl₂N₃O₂): m/z 350

(M + H)⁺.

7.52−7.34 (m, 3 H), 6.93 (s, 1 H), 5.66 (s, 1 H), 3.73 (s, 3 H), 1.27

(s, 9 H). MS (C₂₀H₂₂ClN₃O₃): m/z 388 (M + H)⁺.

Methyl 2-(2-Bromo-7-hydroxy-5-methylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetate (43). p-TsOH·H₂O (2 mg, 10.51 μmol)

was added to a solution of 42 (0.2 g, 1.235 mmol) and 37 (0.697 g,

3.70 mmol) in xylene (10 mL) and the mixture heated at reﬂux under

a Dean−Stark trap for 8 h. The solid was ﬁltered and washed with

hexanes to aﬀord 43 (0.201 g, 54.2%). ¹H NMR (400 MHz, MeOD)

δ (ppm): 6.20 (s, 1 H), 3.71 (s, 3 H), 3.65 (s, 2 H), 2.37 (s, 3 H). MS

(C₁₀H₁₀BrN₃O₃): m/z 300 (M + H)⁺.

Methyl 2-(7-Chloro-2-(3-chlorophenyl)-5-methylpyrazolo-

[1,5-a]pyrimidin-6-yl)-2-hydroxyacetate (51). KHMDS (44.5

mL, 22.27 mmol) was added dropwise over 30 min to a stirred

solution of 50 (7.8 g, 22.27 mmol) in THF (40 mL) maintained at

−78 °C. The mixture was stirred at −78 °C for 30 min before adding

a solution of 3-phenyl-2-(phenylsulfonyl)-1,2-oxaziridine (8.73 g, 33.4

mmol) in THF (50 mL) over 30 min. After stirring for an additional 2

h at −78 °C, the mixture was quenched with saturated NH₄Cl

aqueous solution (40 mL) and allowed to warm to room temperature

before being diluted with EtOAc (200 mL). The organic phase was

washed with H₂O and brine, dried over Na₂SO₄, and concentrated to

leave a residue that was puriﬁed by silica gel chromatography to

Methyl 2-(2-Bromo-7-chloro-5-methylpyrazolo[1,5-a]-

pyrimidin-6-yl)acetate (44). POCl₃(1 mL, 10.73 mmol) was

added to 43 (180 mg, 0.600 mmol) and the mixture heated at reﬂux

for 1 h. After cooling, the reaction mixture was added dropwise to

ice−water to aﬀord a brown solid precipitate. The solid was ﬁltered

1

oﬀ and washed with H₂O to give 44 (158 mg, 83%). H NMR (500

MHz, DMSO-d₆) δ (ppm): 6.99 (s, 1 H), 4.01 (s, 2 H), 3.69 (s, 3 H),

2.56 (s, 3 H). MS (C₁₀H₉BrClN₃O₂): m/z 318 (M + H)⁺.

Methyl 2-(2-Bromo-7-chloro-5-methylpyrazolo[1,5-a]-

pyrimidin-6-yl)-2-hydroxyacetate (45). A solution of 44 (300

mg, 0.942 mmol) in THF (6 mL) was added dropwise over 20 min to

a stirred solution of KHMDS (0.5 M in toluene, 2.83 mL, 1.413

mmol) in THF (6 mL) maintained at −78 °C. The mixture was

stirred at −78 °C for 30 min before adding a solution of 3-phenyl-2-

(phenylsulfonyl)-1,2-oxaziridine (369 mg, 1.413 mmol) in THF (6

mL) over 15 min. The mixture was stirred for an additional 60 min at

−78 °C before being quenched with saturated NH₄Cl aqueous

solution (4 mL). The mixture was allowed to warm to room

temperature, diluted with EtOAc (100 mL) and the organic phase

washed with H₂O and brine before being dried over Na₂SO₄. The

solvent was evaporated and the residue puriﬁed by silica gel

1

provide 51 (4.2 g, 51.5%). H NMR (500 MHz, CDCl₃) δ (ppm):

8.05−7.97 (m, 1H), 7.88 (dt, J = 6.7, 1.8 Hz, 1H), 7.44−7.38 (m,

2H), 6.93 (s, 1H), 5.76 (s, 1H), 3.84 (s, 3H), 2.62 (s, 3H). MS

(C₁₆H₁₃Cl₂N₃O₃): m/z 366 (M + H)⁺.

Methyl 2-(7-Chloro-2-(3-chlorophenyl)-5-methylpyrazolo-

[1,5-a]pyrimidin-6-yl)-2-oxoacetate (52). Dess−Martin period-

inane (6.37 g, 15.02 mmol) was added to a mixture of 51 (5.0 g, 13.65

mmol) in CH₂Cl₂(20 mL), and the mixture was stirred at room temp

for 1 h. The mixture was diluted with EtOAc (100 mL), the organic

layer was washed with saturated NaHCO₃solution (100 mL), dried

over Na₂SO₄, and concentrated. The residue was puriﬁed by silica gel

1

chromatography to aﬀord 52 (3.8 g, 76%). H NMR (500 MHz,

CDCl₃) δ (ppm): 8.10−7.98 (m, 1H), 7.89 (td, J = 4.4, 1.5 Hz, 1H),

7.51−7.36 (m, 2H), 7.00 (s, 1H), 4.00 (s, 3H), 2.62 (s, 3H). MS

(C₁₆H₁₁Cl₂N₃O₃): m/z 364 (M + H)⁺.

1

chromatography to provide 45 (85 mg, 27%). H NMR (400 MHz,

CDCl₃) δ (ppm): 6.71 (s, 1 H), 5.74 (s, 1 H), 3.84 (s, 3 H), 2.63 (s, 3

H). MS (C₁₀H₉BrClN₃O₃): m/z 334.

(S)-Methyl 2-(7-Chloro-2-(3-chlorophenyl)-5-

methylpyrazolo[1,5-a]pyrimidin-6-yl)-2-hydroxyacetate (53).

(R)-1-Methyl-3,3-diphenylhexahydropyrrolo[1,2-c][1,3,2]-

oxazaborole/toluene (1.797 mL of a 1.1 M solution, 1.977 mmol) was

added to a stirred solution of 52 (1.8 g, 4.94 mmol) in anhydrous

toluene (30 mL) and the mixture cooled to −40 °C (acetonitrile/dry

ice bath). A solution of catecholborane (50% by weight) in toluene

(1.695 mL, 6.92 mmol) was added over 30 min and the mixture

stirred at −45 to −35 °C for 2 h and at −25 °C to −15 °C for an

additional 1 h. Saturated Na₂CO₃solution (20 mL) was added to

quench the reaction, and the mixture was stirred vigorously for 30 min

and extracted with EtOAc. The organic layer was washed with

saturated Na₂CO₃solution, dried over Na₂SO₄, and concentrated to

leave a residue that was puriﬁed by silica gel chromatography (15−

50% EtOAc/hexane) to aﬀord 53 (1.5 g, 83%). ¹H NMR (500 MHz,

CDCl₃) δ (ppm): 8.05−7.97 (m, 1H), 7.88 (dt, J = 6.7, 1.8 Hz, 1H),

7.44−7.38 (m, 2H), 6.93 (s, 1H), 5.76 (s, 1H), 3.84 (s, 3H), 2.62 (s,

3H). MS (C₁₆H₁₃Cl₂N₃O₃): m/z 366 (M + H)⁺.

(S)-Methyl 2-(tert-Butoxy)-2-(7-chloro-2-(3-chlorophenyl)-5-

methylpyrazolo[1,5-a]pyrimidin-6-yl)acetate (54). tert-Butyl

acetate (20 mL) and HClO₄(0.282 mL, 3.28 mmol) were added

to a solution of 53 (1.0 g, 2.73 mmol) in CH₂Cl₂(100 mL), and the

mixture was stirred for 16 h at room temperature. The mixture was

quenched with H₂O, diluted with EtOAc and the organic phase

washed with saturated NaHCO₃and dried over Na₂SO₄. The solvent

was evaporated and the residue puriﬁed by silica gel chromatography

to provide 54 (520 mg, 50%). ¹H NMR (400 MHz, CDCl₃) δ (ppm):

8.07−8.00 (m, 1H), 7.89 (dt, J = 6.7, 1.9 Hz, 1H), 7.48−7.37 (m,

2H), 6.93 (s, 1H), 5.68 (s, 1H), 3.75 (s, 3H), 2.69 (s, 3H), 1.29 (s,

9H). MS (C₂₀H₂₁Cl₂N₃O₃): m/z 422 (M + H)⁺.

Methyl 2-(2-Bromo-7-chloro-5-methylpyrazolo[1,5-a]-

pyrimidin-6-yl)-2-tert-butoxyacetate (46). CH₂Cl₂(2 mL) and

HClO₄(0.022 mL, 0.359 mmol) were added to a suspension of 45

(80 mg, 0.239 mmol) in tert-butyl acetate (2 mL) and the mixture

stirred for 4 h at room temperature. The reaction mixture was

quenched with H₂O, diluted with EtOAc and the organic phase

washed with saturated NaHCO₃and dried over Na₂SO₄. The solvent

was evaporated and the residue puriﬁed by silica gel chromatography

to provide 46 (56 mg, 59.9%). ¹H NMR (500 MHz, MeOD) δ

(ppm): 6.75 (s, 1 H), 5.75 (s, 1 H), 3.74 (s, 3 H), 2.62 (s, 3 H), 1.27

(s, 9 H). MS (C₁₄H₁₇BrClN₃O₃): m/z 390.

Methyl 2-tert-Butoxy-2-(7-chloro-5-methyl-2-p-

tolylpyrazolo[1,5-a]pyrimidin-6-yl)acetate, TFA Salt (47). A

mixture of 46 (28 mg, 0.072 mmol), Pd(PPh₃)₄(12.42 mg, 10.75

μmol), p-tolylboronic acid (10.72 mg, 0.079 mmol), 2 M K₂CO₃

solution (100 μL), and DMF (3 mL), was heated in a microwave

reactor at 70 °C for 60 min. The mixture was ﬁltered and the ﬁltrate

puriﬁed by preparative HPLC to aﬀord 47 (16 mg, 43.3%) as the TFA

salt. ¹H NMR (500 MHz, MeOD) δ (ppm): 7.92 (d, J = 7.9 Hz, 2H),

7.30 (d, J = 7.9 Hz, 2H), 6.98 (s, 1 H), 5.79 (s, 1 H), 3.75 (s, 3 H),

2.62 (s, 3 H), 2.40 (s, 3 H), 1.29 (s, 9 H). MS (C₂₁H₂₄ClN₃O₃): m/z

402 (M + H)⁺.

Methyl 2-(2-(3-Chlorophenyl)-7-hydroxy-5-methylpyrazolo-

[1,5-a]pyrimidin-6-yl)acetate (49). p-TsOH·H₂O (100 mg, 0.526

mmol) was added to a solution of 48 (23 g, 119 mmol) and dimethyl

2-acetylsuccinate 37 (22.35 g, 119 mmol) in o-xylene (200 mL) and

the mixture heated at reﬂux under a Dean−Stark trap for 2 h. The

solid was ﬁltered and washed with hexanes to aﬀord 49 (39 g, 99%).

¹H NMR (500 MHz, MeOD) δ (ppm): 8.14−7.95 (m, 1H), 7.93−

N

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Article

(2S)-Methyl 2-(tert-Butoxy)-2-(2-(3-chlorophenyl)-7-(8-ﬂuo-

ro-5-methylchroman-6-yl)-5-methylpyrazolo[1,5-a]pyrimidin-

6-yl)acetate ((S,S)-55). A mixture of 54 (420 mg, 0.995 mmol),

Pd(PPh₃)₄(115 mg, 0.099 mmol), 2-(8-ﬂuoro-5-methylchroman-6-

yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (320 mg, 1.094 mmol),

2M K₂CO₃solution (400 μL) in DMF (4 mL) was heated in a

microwave reactor at 125 °C for 45 min. The mixture was ﬁltered and

the ﬁltrate puriﬁed by silica gel chromatography to aﬀord 55 (204 mg,

37.2%). The enantiomeric excess was determined by a chiral SFC

method: Chiralpak AD-H analytical column, 4.6 × 250 mm, 5 m;

Mobile Phase: 15% MeOH in CO₂. Temp: 35 °C. Flow rate: 2.0 mL/

min. for 10 min. UV monitored @ 266nm. Injection: 5 L of ∼2.0 mg/

mL solution in 50:50 MeOH:CHCl₃. The enantiomeric excess was

Compounds are titrated in 10 μL volumes in 25% DMSO over a ﬁnal

concentration range of 100 μM to 0.0512 nM (ten 5-fold dilutions).

Initially, HIV-1 integrase was incubated with PVT SPA beads in

1.25× cocktail (minus [³H]-1) for 1 h with rocking in a tube. [³H]-1

was added, vortexed, and then 40 μL was immediately added to 10 μL

of test compound in a white opaque 96-well plate (Corning 3600,

Corning, NY). The plate was rocked for 1 h at room temperature in

the dark. After sitting in the dark overnight, the plate was read on a

Microbeta plate reader (PerkinElmer, Waltham MA). Data were

imported into ToolSet (BMS, Wallingford, CT) and analyzed in

CurveMaster (BMS, Wallingford, CT) to calculate K_ivalues.

Cell Lines and Viruses. MT-2 and HEK 293T cells were

obtained from the NIH AIDS Research and Reference Reagent

Program. Cells were propagated in RPMI 1640 media supplemented

with 10% heat inactivated fetal bovine serum, 10 mM HEPES buﬀer,

pH 7.55, 2 mM ^L-glutamine, 100 units/mL penicillin G, 100 μg/mL

streptomycin. The proviral DNA clone of NL₄₋₃was obtained

through the NIH AIDS Research and Reference Reagent Program.

1

93.0%. H-NMR (500 MHz, CDCl3) δ (ppm): 7.83−7.78 (m, 1H),

7.69 (dt, J = 6.7, 1.8 Hz, 1H), 7.35−7.28 (m, 2H), 6.87 (d, J = 10.7

Hz, 1H), 6.84 (s, 1H), 5.00 (s, 1H), 4.48−4.26 (m, 2H), 3.64 (s, 3H),

2.85−2.67 (m, 5H), 2.30−2.13 (m, 2H), 1.84 (s, 3H), 1.16 (s, 9H).

MS (C₃₀H₃₁ClFN₃O₄): m/z 552 (M + H)⁺.

Crystallization, Data Collection, and Structure Reﬁnement

for the HIV-1 Integrase CCD:(S,S)-14 Complex. Crystals of

HIV(50-212)-C56S-W131E-F185K were prepared by the sitting drop

vapor diﬀusion method. The protein stock solution consisted of 12.5

mg/mL (0.691 mM based on the calculated MW of 18 098 Da) in 25

mM Hepes, pH 7.5, 500 mM NaCl, 1 mM EDTA, and 5 mM DTT.

The addition of 50 mM MgCl₂to the buﬀer prior to complexing

improved crystal screening hits; however, later magnesium was added

directly to the crystallization screen. The protein was complexed with

1.74 mM compound 14 from a 100 mM stock solution dissolved in

100% DMSO. The protein/ligand complex was incubated for 4 h at 4

°C and clariﬁed by centrifugation. Crystallization screens were

prepared using a SCREENMAKER 96+8 (Innovadyne, Santa Rosa,

CA, USA) on MRC 2 well sitting drop trays. Crystals grew from

reservoir solution conditions of 2.0 M (NH₄)₂SO₄and 100 mM citric

acid, pH 3.5. Drops were formed from 0.8 μL of the protein solution

and 0.8 μL of the reservoir solution (total initial volume of 1.6 μL),

mixed, and placed at 20 °C to equilibrate. Crystals appeared within 3

days and grew for an additional 7 days. Single crystals were removed

and transferred to a cryo-solution consisting of 1.75 M (NH₄)₂SO₄,

75 mM citric acid, pH 3.5, 10% (v/v) glycerol, and 10% (v/v) PEG

400. Crystals were ﬂash frozen in liquid N₂and stored prior to data

collection. Data for the HIV-1 integrase/(S,S)-14 complex were

collected at beamline 17ID at IMCA-CAT of the Advanced Photon

Source at Argonne National Laboratory. The wavelength used was 1.0

Å, and the detector was a DECTRIS PILATUS 6M. The diﬀraction

images were indexed, integrated, and scaled with XDS using

autoPROC (Global Phasing Ltd., Cambridge, England). The

structure was determined by molecular replacement using the

program PHASER²⁶with a previously reported structure of HIV

integrase (PDB entry 6NCJ) used as the starting model in the

reﬁnement. The structure was reﬁned with the program autoBUSTER

(Global Phasing Ltd., Cambridge, England) and was examined and

manually reﬁtted with the program COOT.²⁷Grade (Global Phasing

Ltd., Cambridge, England) was used to generate a CIF restraint

dictionary for (S,S)-14, and rhoﬁt (Global Phasing Ltd., Cambridge,

England) was used to place the ligand. Reﬁnement was completed

using Coot and autoBUSTER, and the statistics are included in

Supporting Information. Display graphics were produced with

For analysis of the potency of ALLINI compounds, an NL₄₋₃

-

derived replication competent virus, NLRepRluc/SacII (the unique

SacII site was engineered at the end of the integrase gene), was

generated from a proviral clone. In this clone, a section of the nef gene

from NL₄₋₃was replaced with the Renilla luciferase gene. This virus is

infectious and can undergo multiple cycles of replication in cell

culture, while the luciferase reporter provides a simple and easy

method for quantitating the extent of virus growth and consequently

the antiviral activity of test compounds. The NLRepRLuc/SacII virus

was prepared by transfection of HEK 293T cells using the

LipofectAMINE PLUS kit from Invitrogen (Carlsbad, CA) according

to the manufacturer, and the virus generated was titered in MT-2

cells, using luciferase enzyme activity as an end point.

Drug Susceptibility and Cytotoxicity Assays. Titrated virus

was used to infect MT-2 cells in the presence of serial dilutions of

ALLINI compounds. A 10 mM stock compound in 100% DMSO was

used to make the serial dilutions. Final DMSO concentration in each

well was 1%. After 3−4 days of incubation, cells were processed and

quantitated for virus growth by the amount of expressed luciferase.

Assay medium was RPMI 1640 supplemented with 10% heat

inactivated fetal bovine serum (FBS), 100 units/mL penicillin G/

100 μg/mL streptomycin, 10 mM HEPES buﬀer, pH 7.55, and 2 mM

L-glutamine. Luciferase was quantitated using Enduren from Promega

(Madison, WI). The 50% eﬀective concentration (EC₅₀) was

calculated by using the exponential form of the median eﬀect

equation where (Fa) = 1/[1+ (ED₅₀/drug conc)m].²⁸Cytotoxicity in

MT-2 cells was determined after 4 days incubation using XTT (2,3-

bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxani-

lide) to measure cell viability using an established protocol.²⁹

Evaluation of in Vitro Metabolism Rates in Liver Micro-

somes.³⁰The metabolic stability (Metstab) assay evaluates

cytochrome P450 (CYP)-mediated metabolism of test compounds

in vitro using human and rat microsomes after a 10 min incubation.

The incubation was automated on a Biomek FX automation

workstation (Beckman Coulter, Fullerton, CA, USA). Each

compound was incubated in duplicate in the respective species at a

concentration of 0.5 μM. Compounds were received as 3.5 mM

solutions in DMSO and were diluted with CH₃CN to 50 μM before

being added to the prewarmed (37 °C) microsomal suspension (1

mg/mL) prepared in 100 mM sodium phosphate, pH 7.4, and 6.6

mM MgCl₂. The reaction was initiated by adding 17 μL of prewarmed

5 mM NADPH in 100 mM sodium phosphate, pH 7.4, into 153 μL of

reaction mix. The concentration of DMSO in the incubation mixture

was 0.014%. Reaction components were mixed well, and 75 μL was

transferred into 150 μL of quench solution at 0 min time point (t0)

and again at the 10 min incubation time point (t10). Quenched

mixtures were centrifuged at 1500 rpm in an Allegra X-12 centrifuge

(Beckman Coulter) for 15 min, and 90 μL of the supernatant was

then transferred to a separate 96-well plate for analysis. Metabolism

rate was determined based on the parent compound disappearance

over time, as measured by LC−MS/MS.

̈

PyMOL version 1.4 (Schrodinger). The coordinates of the HIV-1

integrase CCD:(S,S)-14 complex have been deposited in the PDB

under PDB code 6UM8.

Virology Materials and Methods. Competitive Binding

Assay Method. Compounds were evaluated for their ability to

displace a tritiated LEDGF inhibitor ([³H]-1) from binding to HIV-1

integrase via a LEDGF inhibitor displacement assay (LIDIA). Relative

inhibitor avidity for HIV-1 integrase is expressed as a K_ivalue. The

ﬁnal concentrations of reagents in the 50 μL LIDIA assay are 26 mM

MOPS buﬀer, pH 7.0, 11 mM DTT, 100 mM NaCl, 20 mM MgCl₂,

0.05% NP-40, 5% PEG-8000, 27 μM [³H]-1, 13 μM puriﬁed HIV-1

integrase, and 2 mg/mL PVT SPA beads (polyvinyltoluene

scintillation proximity assay beads. PerkinElmer, Waltham, MA).

O

https://dx.doi.org/10.1021/acs.jmedchem.9b01681

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

PAMPA Evaluation. PAMPA assay was conducted in a sandwich

setup, consisting of the donor and acceptor chambers separated by an

artiﬁcial lipid membrane. Test compound (100 μM) was diluted with

donor buﬀer at pH 7.4 and placed in the donor side and allowed to

permeate to the acceptor side through the artiﬁcial membrane over a

4 h incubation. After incubation the “sandwich” plate was separated.

The donor and acceptor solutions, along with the t0 solution, were

then measured with a UV plate reader, and the permeability value was

calculated by pION software.

In Vivo Rat PK Studies. All animal studies were performed under

the approval of the Bristol-Myers Squibb Animal Care and Use

Committee and in accordance with the Association for Assessment

and Accreditation of Laboratory Animal Care. The pharmacokinetic

proﬁles of 27, 28, and 29 were determined in male Sprague-Dawley

rats. Animals were dosed iv by cannula implanted in an acceptable

vein with an infusion period of 10 min for rats. Animals were dosed

orally by gavage following an overnight fast. The vehicle for both iv

and po studies was 10% dimethylacetamide (DMAC)/90% PEG400.

Blood was collected via a venous port and centrifuged to obtain

plasma. Samples were treated with CH₃CN containing an internal

standard, centrifuged, and analyzed by LC−MS/MS. Pharmacokinetic

parameters were obtained by noncompartmental analysis of plasma

concentration versus time data (KINETICA software).

B. Narasimhulu Naidu − Department of Chemistry, Bristol-

Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States

Prasanna Sivaprakasam − Department of Computer-Aided

Drug Design & Molecular Analytics, Bristol-Myers Squibb

Research and Development, Princeton, New Jersey 08543-4000,

United States

Hal Lewis − Department of Molecular Structure and Design,

Bristol-Myers Squibb Research and Development, Princeton,

New Jersey 08543-4000, United States

Kevin Kish − Department of Molecular Structure and Design,

Bristol-Myers Squibb Research and Development, Princeton,

New Jersey 08543-4000, United States

Javed A. Khan − Department of Molecular Structure and Design,

Bristol-Myers Squibb Research and Development, Princeton,

New Jersey 08543-4000, United States

Alicia Ng − Department of Materials Science, Bristol-Myers

Squibb Research and Development, Wallingford, Connecticut

06492, United States

George L. Trainor − Department of Chemistry, Bristol-Myers

Squibb Research and Development, Princeton, New Jersey

08543-4000, United States

Christopher Cianci − Department of Virology Discovery

Biology, Bristol-Myers Squibb Research and Development,

Wallingford, Connecticut 06492, United States

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01681.

Ira B. Dicker − Department of Virology Discovery Biology,

Bristol-Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States

Michael A. Walker − Department of Chemistry, Bristol-Myers

Squibb Research and Development, Wallingford, Connecticut

06492, United States

Crystallography data for compounds (S,S and R,R)-14

and (S,R and R,S)-15; crystallization, data collection,

and structure reﬁnement results for the HIV-1 integrase

CCD:(S,S)-14 complex (PDF)

Zeyu Lin − Department of Virology Discovery Biology, Bristol-

Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States

Tricia Protack − Department of Virology Discovery Biology,

Bristol-Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States

Linda Discotto − Department of Virology Discovery Biology,

Bristol-Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States

Molecular formula strings and some data (XLSX)

Accession Codes

The coordinates of the HIV-1 integrase CCD:(S,S)-14

complex have been deposited in the PDB under PDB code

6UM8. Single crystal structures for (S,S and R,R)-14 and (S,R

and R,S)-15 have been deposited in the CCDC database under

CCDC deposition numbers 1862636 and 1862637, respec-

tively.

Susan Jenkins − Department of Pharmaceutical Candidate

Optimization, Bristol-Myers Squibb Research and Development,

Wallingford, Connecticut 06492, United States

Samuel W. Gerritz − Department of Early Discovery Chemistry,

Bristol-Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States

AUTHOR INFORMATION

■

Corresponding Authors

Guo Li − Department of Early Discovery Chemistry, Bristol-

Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States; Email: guo.li@bms.com

Annapurna Pendri − Department of Early Discovery Chemistry,

Bristol-Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States; orcid.org/0000-0002-

6070-5171; Email: annapurna.pendri@bms.com

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.9b01681

Author Contributions

The manuscript was written through contributions of all

authors.

Authors

Nicholas A. Meanwell − Department of Chemistry, Bristol-

Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States; orcid.org/0000-0002-

8857-1515

Mark R. Krystal − Department of Virology Discovery Biology,

Bristol-Myers Squibb Research and Development, Wallingford,

Connecticut 06492, United States

David R. Langley − Department of Computer-Aided Drug

Design & Molecular Analytics, Bristol-Myers Squibb Research

and Development, Princeton, New Jersey 08543-4000, United

States

Notes

The authors declare no competing ﬁnancial interest.

The authors are or were at the time these studies employees

and shareholders of Bristol-Myers Squibb.

ACKNOWLEDGMENTS

■

Robert Gentles and Stella Huang are acknowledged for helpful

discussions and NMR interpretation. The Bristol-Myers

Squibb Department of Discovery Synthesis and BBRC

Bangalore, India, are acknowledged for synthetic support. We

P

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(12) Desimmie, B. A.; Schrijvers, R.; Demeulemeester, J.;

Borrenberghs, D.; Weydert, C.; Thys, W.; Vets, S.; Van Remoortel,

B.; Hofkens, J.; De Rijck, J.; Hendrix, J.; Bannert, N.; Gijsbers, R.;

Christ, F.; Debyser, Z. LEDGINs inhibit late stage HIV-1 replication

by modulating integrase multimerization in the virions. Retrovirology

2013, 10, 57.

thank Wilson Shou, Amy Sarjeant, Xianmei Cai, Bhupinder

Sandhu, and Kathy Mosure for help and support.

ABBREVIATIONS USED

■

AIDS, acquired immunodeﬁciency syndrome; ALLINI, allos-

teric integrase inhibitor; AUC, area under the curve; CADD,

computer-aided drug design; CCD, catalytic core domain;

CD4, cluster of diﬀerentiation 4; CCR5, C−C chemokine

receptor 5; CTD, carboxyl terminal domain; DDQ, 2,3-

dichloro-5,6-dicyanobenzoquinone; DIBAL, diisobutylalumi-

num hydride; DTT, dithiothreitol; EDTA, ethylenediamine-

tetraacetic acid; FDA, U.S. Food and Drug Administration;

gp41, glycoprotein 41; HAART, highly active antiretroviral

therapy; HIV-1, human immunodeﬁciency virus type 1; HLM,

human liver microsome; HSA, human serum albumin; IBD,

integrase binding domain; IN, integrase; INSTI, integrase

strand transfer inhibitor; iv, intravenous; LEDGF, lens

epithelium-derived growth factor; PAMPA, parallel artiﬁcial

membrane permeability assay; PCC, pyridinium chlorochro-

mate; PK, pharmacokinetic; po, per os; RLM, rat liver

microsome; SAR, structure−activity relationship; TI, ther-

apeutic index; WT, wild-type

(13) Le Rouzic, E.; Bonnard, D.; Chasset, S.; Bruneau, J. M.;

Chevreuil, F.; Le Strat, F.; Nguyen, J.; Beauvoir, R.; Amadori, C.;

́

Brias, J.; Vomscheid, S.; Eiler, S.; Levy, N.; Delelis, O.; Deprez, E.;

Saïb, A.; Zamborlini, A.; Emiliani, S.; Ruff, M.; Ledoussal, B.; Moreau,

F.; Benarous, R. Dual inhibition of HIV-1 replication by integrase-

LEDGF allosteric inhibitors is predominant at the post-integration

stage. Retrovirology 2013, 10, 144.

(14) Fader, L. D.; Malenfant, E.; Parisien, M.; Carson, R.; Bilodeau,

F.; Landry, S.; Pesant, M.; Brochu, C.; Morin, S.; Chabot, C.; Halmos,

T.; Bousquet, Y.; Bailey, M. D.; Kawai, S. H.; Coulombe, R.; LaPlante,

S.; Jakalian, A.; Bhardwaj, P. K.; Wernic, D.; Schroeder, P.; Amad, M.;

Edwards, P.; Garneau, M.; Duan, J.; Cordingley, M.; Bethell, R.;

Mason, S. W.; Bos, M.; Bonneau, P.; Poupart, M.-A.; Faucher, A.-M.;

Simoneau, B.; Fenwick, C.; Yoakim, C.; Tsantrizos, Y. Discovery of BI

224436, a noncatalytic site integrase inhibitor (NCINI) of HIV-1.

ACS Med. Chem. Lett. 2014, 5, 422−427.

(15) Tsantrizos, Y. S.; Bailey, M. D.; Bilodeau, F.; Carson, R. J.;

Coulombe, R.; Fader, L.; Halmos, T.; Kawai, S.; Landry, S.; Laplante,

S.; Morin, S.; Parisien, M.; Poupart, M.-A.; Simoneau, B. Preparation

of 2-(tert-Butyloxy)-2-(2-methylquinolin-3-yl)acetic Acid Derivatives

as Inhibitors of Human Immunodeﬁciency Virus Replication. World

Patent WO2009/062285A1, 2009.

(16) Fader, L. D.; Carson, R.; Morin, S.; Bilodeau, F.; Chabot, C.;

Halmos, T.; Bailey, M. D.; Kawai, S. H.; Coulombe, R.; Laplante, S.;

Mekhssian, K.; Jakalian, A.; Garneau, M.; Duan, J.; Mason, S. W.;

Simoneau, B.; Fenwick, C.; Tsantrizos, Y.; Yoakim, C. Minimizing the

contribution of enterohepatic recirculation to clearance in rat for the

NCINI class of inhibitors of HIV. ACS Med. Chem. Lett. 2014, 5,

711−716.

(17) Fader, L. D.; Bailey, M. D.; Beaulieu, E.; Bilodeau, F.; Bonneau,

P.; Bousquet, Y.; Carson, R. J.; Chabot, C.; Coulombe, R.; Duan, J.;

Fenwick, C.; Garneau, M.; Halmos, T.; Jakalian, A.; James, C.; Kawai,

S. H.; Landry, S.; Laplante, S. R.; Mason, S. W.; Morin, S.; Rioux, N.;

Simoneau, B.; Surprenant, S.; Thavonekham, B.; Thibeault, C.; Trinh,

T.; Tsantrizos, Y.; Tsoung, J.; Yoakim, C.; Wernic, D. Aligning

potency and pharmacokinetic properties for pyridine-based NCINIs.

ACS Med. Chem. Lett. 2016, 7, 797−801.

(18) Tsiang, M.; Jones, G. S.; Niedziela-Majka, A.; Kan, E.; Lansdon,

E. B.; Huang, W.; Hung, M.; Samuel, D.; Novikov, N.; Xu, Y.;

Mitchell, M.; Guo, H.; Babaoglu, K.; Liu, X.; Geleziunas, R.;

Sakowicz, R. New class of HIV-1 integrase (IN) inhibitors with a

dual mode of action. J. Biol. Chem. 2012, 287, 21189−21203.

(19) Gupta, K.; Brady, T.; Dyer, B. M.; Malani, N.; Hwang, Y.; Male,

F.; Nolte, R. T.; Wang, L.; Velthuisen, E.; Jeffrey, J.; Van Duyne, G.

D.; Bushman, F. D. Allosteric inhibition of human immunodeficiency

virus integrase. J. Biol. Chem. 2014, 289, 20477−20488.

(20) Gupta, K.; Turkki, V.; Sherrill-Mix, S.; Hwang, Y.; Eilers, G.;

Taylor, L.; McDanal, C.; Wang, P.; Temelkoff, D.; Nolte, R. T.;

Velthuisen, E.; Jeffrey, J.; Van Duyne, G. D.; Bushman, F. D.

Structural basis for inhibitor-induced aggregation of HIV integrase.

PLoS Biol. 2016, 14, e1002584.

REFERENCES

■

(1) Gubernick, S. I.; Felix, N.; Lee, D.; Xu, J. J.; Hamad, B. The HIV

therapy market. Nat. Rev. Drug Discovery 2016, 15, 451.

(2) Demeulemeester, J.; De Maeyer, M.; Debyser, Z. HIV-1

integrase drug discovery comes of age. Top. Med. Chem. 2013, 15,

1−52.

(3) Christ, F.; Debyser, Z. The LEDGF/p75 integrase interaction, a

novel target for anti-HIV therapy. Virology 2013, 435, 102−109.

(4) Engelman, A. E. Multifaceted HIV integrase functionalities and

therapeutic strategies for their inhibition. J. Biol. Chem. 2019, 294,

15137−15157.

(5) Poeschla, E. M. Integrase, LEDGF/p75 and HIV replication.

Cell. Mol. Life Sci. 2008, 65, 1403−1424.

(6) Emiliani, S.; Mousnier, A.; Busschots, K.; Maroun, M.; Van

Maele, B.; Tempe, D.; Vandekerckhove, L.; Moisant, F.; Ben-Slama,

L.; Witvrouw, M.; Christ, F.; Rain, J.-C.; Dargemont, C.; Debyser, Z.;

Benarous, R. Integrase mutants defective for interaction with

LEDGF/p75 are impaired in chromosome tethering and HIV-1

replication. J. Biol. Chem. 2005, 280, 25517−25523.

(7) Busschots, K.; Voet, A.; De Maeyer, M.; Rain, J. C.; Emiliani, S.;

Benarous, R.; Desender, L.; Debyser, Z.; Christ, F. Identification of

the LEDGF/p75 binding site in HIV-1 integrase. J. Mol. Biol. 2007,

365, 1480−1492.

(8) De Rijck, J.; Vandekerckhove, L.; Gijsbers, R.; Hombrouck, A.;

Hendrix, J.; Vercammen, J.; Engelborghs, Y.; Christ, F.; Debyser, Z.

Overexpression of the lens epithelium-derived growth factor/p75

integrase binding domain inhibits human immunodeficiency virus

replication. J. Virol. 2006, 80, 11498−11509.

(9) Llano, M.; Saenz, D. T.; Meehan, A.; Wongthida, P.; Peretz, M.;

Walker, W. H.; Teo, W.; Poeschla, E. M. An essential role for

LEDGF/p75 in HIV integration. Science 2006, 314, 461−464.

(10) Jurado, K. A.; Wang, H.; Slaughter, A.; Feng, L.; Kessl, J. J.;

Koh, Y.; Wang, W.; Ballandras-Colas, A.; Patel, P. A.; Fuchs, J. R.;

Kvaratskhelia, M.; Engelman, A. Allosteric integrase inhibitor potency

is determined through the inhibition of HIV-1 particle maturation.

Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 8690−8695.

(11) Feng, L.; Sharma, A.; Slaughter, A.; Jena, N.; Koh, Y.; Shkriabai,

N.; Larue, R. C.; Patel, P. A.; Mitsuya, H.; Kessl, J. J.; Engelman, A.;

Fuchs, J. R.; Kvaratskhelia, M. The A128T resistance mutation reveals

aberrant protein multimerization as the primary mechanism of action

of allosteric HIV-1integrase inhibitors. J. Biol. Chem. 2013, 288,

15813−15820.

(21) Cherepanov, P.; Ambrosio, A. L. B.; Rahman, S.; Ellenberger,

T.; Engelman, A. Structural basis for the recognition between HIV-1

integrase and transcriptional coactivator p75. Proc. Natl. Acad. Sci. U.

S. A. 2005, 102, 17308−17313.

(22) Davis, F. A.; Vishwakarma, L. C.; Billmers, J. G.; Finn, J.

Synthesis of α -hydroxycarbonyl compounds (acyloins): direct

oxidation of enolates using 2-sulfonyloxaziridine. J. Org. Chem.

1984, 49, 3241−3243.

(23) Corey, E. J.; Link, J. O. A general, catalytic, and enantioselective

synthesis of α -amino acids. J. Am. Chem. Soc. 1992, 114, 1906−1908.

Q

https://dx.doi.org/10.1021/acs.jmedchem.9b01681

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(24) Carlens, G.; Chaltin, P.; Christ, F.; Debyser, Z.; Marchand, A.;

Marchand, D.; Voet, A.; De Maeyer, M. Novel Antiviral Compounds.

World Patent WO2011/076765A1, 2011.

(25) Peese, K. M.; Allard, C. W.; Connolly, T.; Johnson, B. L.; Li, C.;

Patel, M.; Sorensen, M. E.; Walker, M. A.; Meanwell, N. A.;

McAuliffe, B.; Minassian, B.; Krystal, M.; Parker, D. D.; Lewis, H. A.;

Kish, K.; Zhang, P.; Nolte, R. T.; Simmermacher, J.; Jenkins, S.;

Cianci, C.; Naidu, B. N. 5,6,7,8-Tetrahydro-1,6-naphthyridine

derivatives as potent HIV-1-integrase-allosteric-site inhibitors. J.

Med. Chem. 2019, 62, 1348−1361.

(26) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.

D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.

Crystallogr. 2007, 40, 658−674.

(27) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features

and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, 66, 486−501.

(28) Johnson, V. A.; Byington, R. T. Infectivity Assay. In Techniques

in HIV Research; Stockton Press: New York, NY, 1990.

(29) Weislow, O. S.; Kiser, R.; Fine, D. L.; Bader, J.; Shoemaker, R.

H.; Boyd, M. R. New soluble-formazan assay for HIV-1 cytopathic

effects: application to high-flux screening of synthetic and natural

products for AIDS-antiviral activity. J. Natl. Cancer Inst. 1989, 81,

577−586.

(30) Kieltyka, K.; Zhang, J.; Li, S.; Vath, M.; Baglieri, C.; Ferraro, C.;

Zvyaga, T. A.; Drexler, D. M.; Weller, H. N.; Shou, W. Z. A high-

throughput bioanalytical platform using automated infusion for

tandem mass spectrometric method optimization and its application

in a metabolic stability screen. Rapid Commun. Mass Spectrom. 2009,

23, 1579−1597.

R

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Article Doi

Discovery and Optimization of Novel Pyrazolopyrimidines as Potent and Orally Bioavailable Allosteric HIV-1 Integrase Inhibitors

DOI: 10.1021/acs.jmedchem.9b01681

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