Fungal-Selective Resorcylate Aminopyrazole Hsp90 Inhibitors: Optimization of Whole-Cell Anticryptococcal Activity and Insights into the Structural Origins of Cryptococcal Selectivity

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Fungal-Selective Resorcylate Aminopyrazole Hsp90 Inhibitors:

Optimization of Whole-Cell Anticryptococcal Activity and Insights

into the Structural Origins of Cryptococcal Selectivity

Paul T. Marcyk, Emmanuelle V. LeBlanc, Douglas A. Kuntz, Alice Xue, Francisco Ortiz, Richard Trilles,

Stephen Bengtson, Tristan M. G. Kenney, David S. Huang, Nicole Robbins, Noelle S. Williams,

́

Damian J. Krysan, Gilbert G. Prive,* Luke Whitesell,* Leah E. Cowen,* and Lauren E. Brown *

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ABSTRACT: The essential eukaryotic chaperone Hsp90 regulates the form and function of diverse client proteins, many of which

govern thermotolerance, virulence, and drug resistance in fungal species. However, use of Hsp90 inhibitors as antifungal therapeutics

has been precluded by human host toxicities and suppression of immune responses. We recently described resorcylate

aminopyrazoles (RAPs) as the ﬁrst class of Hsp90 inhibitors capable of discriminating between fungal (Cryptococcus neoformans,

Candida albicans) and human isoforms of Hsp90 in biochemical assays. Here, we report an iterative structure−property optimization

toward RAPs capable of inhibiting C. neoformans growth in culture. In addition, we report the ﬁrst X-ray crystal structures of C.

neoformans Hsp90 nucleotide binding domain (NBD), as the apoprotein and in complexes with the non-species-selective Hsp90

inhibitor NVP-AUY922 and three RAPs revealing unique ligand-induced conformational rearrangements, which reaﬃrm the

hypothesis that intrinsic diﬀerences in protein ﬂexibility can confer selective inhibition of fungal versus human Hsp90 isoforms.

both act via distinct mechanisms to deplete ergosterol, the major

component of fungal membranes.^9,10Resistance to each class

has emerged, particularly for the azoles, with fungal infections

becoming increasingly diﬃcult to cure. A third class,

antimetabolite pyrimidines, are only used in combination with

other antifungals due to rapid emergence of resistance.¹¹The

fourth antifungal class, the cell wall-targeting echinocandins,

have limited activity against Cryptococcus and no CNS

penetration.¹²⁻¹⁵Given the limited array of antifungal drug

classes and targets and the dramatically increasing incidence of

INTRODUCTION

■

Invasive fungal diseases impose a major economic and public

health burden worldwide, killing over 1.5 million each year.¹⁻³

Approximately 90% of deaths due to fungal infection are caused

by Candida, Aspergillus, and Cryptococcus species.⁴Of these,

Cryptococcus is now recognized as one of the most signiﬁcant

fungal threats to human health, with incidence increasing in both

immunocompromised and immunocompetent hosts.^1,5Recent

data from the WHO place the annual global burden of

cryptococcal meningitis, the major clinical manifestation of the

disease, in excess of 223 000 cases annually, causing more than

150 000 attributable mortalities and approximately 30% of all

AIDS-associated deaths.^6,7Cryptococcal meningitis has a 100%

mortality rate if left untreated, and mortality rates remain high at

30% in resource-rich and 70% in resource-poor contexts.⁸Of the

meager four drug classes approved for treatment of systemic

fungal infections, only three are eﬀective against Cryptococcus.

Azoles and polyenes, which can each be used as single agents,

Received: October 20, 2020

Published: January 14, 2021

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Figure 1. Generic structure of resorcylate aminopyrazoles (RAPs, left) and summary of activity and selectivity proﬁle for previously reported ﬁrst-

generation cryptococcal-selective RAP series of type 1, which are N-methylated at R¹and bear a monosubstituted phenyl ring at R²to increase fungal

selectivity.

resistance to current agents, the discovery of new chemotypes

that act in ways that are mechanistically distinct from the

available armamentarium is desperately needed.

nonmacrocyclic resorcylate drug candidates such as AT13387

(onalespib),⁴³⁻⁴⁶NVP-AUY922 (luminespib),⁴⁷⁻⁵²and STA-

9090 (ganetespib),⁵³⁻⁶¹we more recently disclosed the design,

synthesis, and characterization of resorcylate aminopyrazoles

(RAPs). RAPs are the ﬁrst class of inhibitors capable of

selectively inhibiting fungal isoforms of Hsp90 (speciﬁcally, C.

neoformans and C. albicans) over their human orthologues

Hsp90α/β, TRAP1, and Grp94.⁶²Our development of

increasingly potent and fungal-selective RAPs, as well as

structure−activity relationships (SARs) for 112 early members

of the series against C. neoformans and C. albicans culminated in

our ﬁrst generation of fungal-selective lead RAPs (1, Figure 1),

which were all N-methylated at R¹and C-arylated at R².

Interestingly, species-speciﬁc divergences in potency and fungal

selectivity (FS) occurred with ortho/para positioning of R²aryl-

ring substituents, while activity and selectivity generally

converged with meta-substituents. However, despite this

interesting biochemical activity proﬁle, whole-cell permeance

of RAPs was an unsolved problem; of the 94 compounds from

this study that showed potent cryptococcal Hsp90 binding

(EC₅₀≤ 1 μM in fungal lysate), only six had whole-cell minimal

inhibitory concentrations (MICs) ≤ 25 μM and only one of

these six ﬁt the desired proﬁle of high biochemical potency and

high fungal selectivity.

In fungi, the cell wall presents an inherent impediment to

passive small-molecule permeability that is not present in

mammalian cells and other microorganisms, which lack this

strong protective barrier.⁶³As a key virulence factor, C.

neoformans also elaborates a dense polysaccharide capsule,

which in addition to the plasma membrane and semipermeable

ﬁbrillary network of the cell wall contributes to limiting the

accumulation of passively permeable compounds within this

organism.⁶⁴⁻⁶⁶In C. neoformans as in other fungi, a network of

eﬄux pumps also prevents accumulation of many xenobiotics.⁶⁷

In this report, we thus designate “permeant” molecules as having

the combined features of active or passive cell penetration and

evasion of eﬄux in C. neoformans so as to accumulate within the

fungus and engage the target.⁶⁷

Targeting core hubs of stress response circuitry is a powerful,

yet unexploited strategy to cripple fungal pathogens. A prime

example is the eukaryotic molecular chaperone Hsp90.¹⁶⁻¹⁹

Hsp90 is essential in all fungi, and in addition to enabling

survival, Hsp90 client proteins are also critical to virulence and

drug resistance mechanisms.²⁰Although profound compromise

of Hsp90 expression or function is lethal to fungi, more modest

compromise via chemical inhibitors or genetic reduction

prevents and reverses resistance to antifungals in culture.²¹⁻²³

In Cryptococcus neoformans, recent studies have implicated

Hsp90 in its thermotolerance, capsule assembly, and sensitivity

to antifungals, strongly inﬂuencing its virulence in a nematode

model.^24,25Beyond an intrinsic potential for broad-spectrum

single-agent antifungal activity, the unique eﬀects on fungal

biology inherent to Hsp90 inhibitors also render them strong

candidates for the development of combination treatment

regimens that would actively impede mechanisms of drug

resistance.

Due in large part to its prominent role in supporting the

function of numerous oncoproteins, Hsp90 has been studied

extensively as an anticancer target.²⁶Unfortunately, all Hsp90

inhibitors currently being developed as anticancer drugs exert

mammalian host toxicities, especially suppression of innate and

acquired immune mechanisms, that preclude their use as

antifungals.²⁷In fact, although numerous drug candidates have

progressed into clinical trials over the past decade, none have

been approved due to limited anticancer eﬃcacy at their

maximally tolerated doses.²⁸One approach to reducing toxicity

and improving therapeutic index for cancer and other

indications has been the pursuit of paralog-selective inhibitors

across the four Hsp90 family members in humans: Hsp90α,

Hsp90β, Trap1, and Grp94.^29,30The resorcylate scaﬀold, one of

several privileged Hsp90-inhibitory chemotypes, has been

modiﬁed to confer human paralog selectivity with applications

in oncology and glaucoma.³¹⁻³⁸In addition, selective purine

mimetics, such as TAS-116³⁹and modiﬁed analogues of

BIIB021 selectively targeting Trap1,³⁰have been described.

Paralog selectivity has also been reported for modiﬁed

benzamides resembling SNX-2112.^40,41

Among the six cryptococcal-permeant analogues in our ﬁrst-

generation RAP library, we identiﬁed unique structural features

that appeared to distinguish these compounds from non-

permeant near-neighbors. Here, we now report signiﬁcant

achievements toward improving whole-cell anticryptococcal

activity via a rational structure−property approach. In addition,

we describe the ﬁrst X-ray structures of the C. neoformans Hsp90

nucleotide binding domain (NBD), crystallized as the

apoprotein and in co-complexes with three fungal-selective

RAPs. Together, these new developments deﬁne the binding

We previously described eﬀorts to develop Hsp90 inhibitors

that are selective for fungal isoforms, initially templated from the

resorcylic acid macrolactone natural products radicicol and

monocillin.⁴²Building on successes in achieving Candida

albicans selectivity with semisynthetic radicicol and monocillin

oximes, as well as the strong preclinical track record of

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mode of this novel Hsp90 inhibitor class, providing insights into

the most plausible chemostructural origins of their observed

fungal selectivity. Equally important from the therapeutic

perspective, they advance our understanding of the structure−

activity and structure−property relationships critical to

conferring selective, whole-cell anticryptococcal activity.

densation of 3a−ab with a variety of N-alkyl-hydrazines

incorporated the R¹alkyl group of the 5-aminopyrazole building

blocks (4a−bb).

The resorcinol core was prepared by the formylation of 3,5-

dimethoxybromobenzene (5) and subsequent demethylation

with BBr₃(Scheme 2). Bis-benzyl protection of 6 gave stable

common intermediate (7), which could be elaborated with

various amide groups. Pinnick oxidation of the aldehyde (7),

followed by hexaﬂuorophosphate azabenzotriazole tetramethyl

uronium (HATU)-mediated amide coupling with isoindoline

hydrochloride or substituted ﬂuoroisoindoline derivatives, gave

resorcylate amide bromides (8a−c). The synthesis was

completed by the Buchwald−Hartwig cross-coupling of aryl

bromides 8a−c with 5-aminopyrazole (4a−bb) followed by

global deprotection with palladium on carbon to give the

targeted RAP compounds (9a−bi).

RESULTS AND DISCUSSION

■

Rational Optimization of RAPs toward the Combined

Features of Potent, Fungal-Selective Hsp90 Inhibition

and Improved Whole-Cell Activity. To arrive at rationally

designed, second-generation RAPs with the combined qualities

of fungal accumulation and fungal selectivity, we sought to

merge the structural features found to confer biochemical

selectivity for fungal Hsp90 with nascent features, which

appeared to track with permeance. To enable eﬃcient

elaboration of new second-generation analogues, the RAP

scaﬀold (Figure 1) was synthesized in a convergent manner

using an alternate protecting group strategy from our published

ﬁrst-generation set,⁶²allowing for modular variation of the

aminopyrazole and amide portion of the molecule. By

synthesizing bespoke 5-aminopyrazoles, various N-alkyl groups

at R¹as well as diﬀerential substitution patterns on the R²

aromatic ring could be incorporated (Scheme 1). Starting from

To determine target binding aﬃnity, we used a ﬂuorescence

polarization (FP)-based competitive binding assay.^42,62Use of

whole-cell lysates enables assessment of binding to Hsp90 while

complexed with relevant cochaperones and, in the case of

human lysate, to the entire repertoire of Hsp90 family members

expressed in human cells. Given these factors, FP in lysate is

highly relevant to selectivity in the whole-cell context but can

only provide relative quantiﬁcation of binding aﬃnity because

the absolute concentration of the protein targets is unknown. In

comparison, FP using puriﬁed NBD proteins allows for

measurement of assay-independent ligand dissociation con-

stants.⁶⁸In our published ﬁrst-generation set, the most potent

and selective inhibitors were meta-methoxy-substituted RAPs

10−12 (Figure 2A), which disappointingly all failed to inhibit

cryptococcal growth at concentrations of up to 25 μM. In

addition to ﬁve nonselective whole-cell-active RAPs (13−17,

Figure 2B), the only RAP exhibiting the combined features of

>5-fold cryptococcal selectivity plus a cryptococcal MIC ≤ 25

μM was the R²ortho-tolyl-substituted compound 18. This

compound was one of only three examples of an RAP bearing an

ortho-substituted R²aryl ring and was the single instance among

these three with an isoindoline amide. Notably, despite a roughly

equal proportion of isoindoline, pyridopyrrolidine, and

pyrazolopyrrolidine amides within the early SAR set (Figure

a

Scheme 1. Preparation of Building Block Aminopyrazoles 4

a

Conditions: (a) MeCN, n-BuLi, tetrahydrofuran (THF), −78 °C to

room temperature (rt); (b) R¹NH-NH₂, MeOH, 120 °C, microwave

or R¹NH-NH₂·HCl, NEt₃, MeOH, 120 °C, microwave.

commercially available mono- and disubstituted methyl

benzoate derivatives (2a−ab), acylation of deprotonated

acetonitrile aﬀorded α-cyanoketone products 3a−ab. Con-

a

Scheme 2. Convergent Synthesis of RAPs 9a−bi from Resorcylate Amides 8a−c and Aminopyrazoles 4a−bb

a

Conditions: (a) POCl₃, DMF, 0 °C to 100 °C; (b) BBr₃, CH₂Cl₂, 0 °C to rt; (c) BnBr, K₂CO₃, MeCN, reﬂux; (d) NaOCl₂, NaH₂PO₄·H₂O, 2-

methyl-2-butene, THF/t-BuOH/H₂O; (e) isoindoline·HCl (for 8a), 4-ﬂuoroisoindoline·HCl (for 8b), or 5-ﬂuoroisoindoline·HCl (for 8c), HATU,

Et₃N, THF/CH₂Cl₂; (f) Pd(OAc)₂(10 mol %), Xantphos (20 mol %), Cs₂CO₃, toluene, 130 °C or Pd₂(dba)₃(4 mol %), Xantphos (10 mol %),

NaOPh, dioxane, 170 °C, microwave; (g) Pd/C (cat.), H₂, MeOH or Pd(OH)₂/C (cat.), H₂, EtOAc.

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Figure 2. (A) RAPs with meta-substituted R²aryl rings exhibiting high fungal selectivity (FS) for cryptococcal Hsp90 over human isoforms. (B)

Among 94 RAPs with fungal target aﬃnity (H99 EC₅₀) ≤ 1 μM, only six demonstrate whole-cell antifungal activity (MIC ≤ 25 μM). Values for

compounds with either fold-selectivity >5 or MIC₈₀≤ 12.5 μM are highlighted in blue. Values for compounds with both fold-selectivity >5 and MIC₈₀

≤ 12.5 μM are highlighted in red.

1), whole-cell activity was limited solely to isoindoline or

ﬂuoroisoindolines at this site.

isoindoline or ﬂuoroisoindoline amide. Analogues 9d and 9e

were prepared as examples of such features in combination with

the ortho-tolyl group at R². The introduction of an N-tert-butyl

group (9d) at R¹was suﬃcient to retain both fungal selectivity

(11.1-fold) and whole-cell activity (MIC 12.5 μM) that was

comparable to the N-Me parent 18 (14.1-fold and MIC 10 μM,

respectively). Isoindoline ﬂuorination of 18, however, was

detrimental to both whole-cell activity and selectivity (com-

pound 9e).

Based on these observations, we postulated that lipophilicity

at the amide may be a key driver for permeance, and opted to

explore other ortho-substituents in combination with isoindo-

line (Table 1, entries 1−3) to discern whether compound 18

represented an outlier or was suggestive of a potential structural

trend. Gratifyingly, improved whole-cell activity was also

observed in the biochemically potent ortho-ﬂuorinated (9a),

ortho-chlorinated (9b), and ortho-triﬂuoromethylated (9c)

analogues. However, this improvement was oﬀset by a

disappointing drop in fungal selectivity for all three compounds.

Given the comparatively higher selectivity observed with the

ortho-methylated parent compound, we next designed analogues

in which this substituent was retained, and instead paired with

other potentially permeance-enhancing features. Revisiting the

cohort of permeant compounds in Figure 2B, another structural

commonality that we deemed unique to these compounds

compared to the larger set was the presence of relatively large,

lipophilic groups at both R¹and R², again in the presence of an

In our prior study,⁶²similar biochemical potencies and fungal

selectivities were observed among compounds bearing a meta-

tolyl or meta-methoxyphenyl group at R². In contrast, at the

ortho site, we found that new compound 9f, the methoxy

analogue of 18, showed a precipitous loss of selectivity and no

measurable whole-cell activity, despite high binding aﬃnity for

Hsp90. Compounds 9g−9l were next prepared to again test the

strategy of increased bulk and lipophilicity at R¹to improve

permeance; for these analogues, whole-cell activities were again

improved, but gains in fungal selectivity were only modest.

Interestingly, converting the methoxy to triﬂuoromethoxy

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Table 1. Early Exploration of the Impact of (1) Ortho-Substituents at the R²Aryl Ring and (2) Increased Aliphatic Bulk at R¹on

c

Biochemical Potency and Selectivity in Binding Cryptococcal Hsp90, and in Growth Inhibition of C. neoformans in Culture

a

EC₅₀values were determined by ﬂuorescence polarization (FP)-based equilibrium competition assay performed in 384-well format using whole-

b

cell lysates prepared from C. neoformans and serial compound dilutions. All determinations were performed in duplicate. To calculate fold-

selectivity, the EC₅₀values determined by FP using human HepG2 cell lysate were divided by the EC₅₀value determined using C. neoformans lysate.

The resulting ratio was then normalized by the ratio of values determined for the Hsp90 inhibitor geldanamycin using lysate of each cell type.

Results for key selective compounds were conﬁrmed by repeat assay in lysates, as well as by measuring ligand dissociation constants (K_i) using

c

puriﬁed NBDs in FP assays (see Supporting Tables 1 and 2; Figures S1 −S3). Concentration of compound resulting in >80% inhibition of fungal

cell growth compared to vehicle control. Values for compounds with either fold-selectivity >5 or MIC₈₀≤ 12.5 μM are highlighted in blue. Values

for compounds with both fold-selectivity >5 and MIC₈₀≤ 12.5 μM are highlighted in red.

delivered mixed results, with the N-methylated compound 9n

exhibiting no selectivity and submicromolar whole-cell activity,

while the N-tert-butyl compound 9o showed modest 6.8-fold

selectivity in combination with an impressive MIC of 1.8 μM.

With initial conﬁrmation that the whole-cell activity of R²-

arylated RAPs could be improved via increased bulk and

lipophilicity at R¹, we next sought to deploy this strategy on R²

meta-substituted scaﬀolds, which had trended toward the

highest cryptococcal Hsp90 selectivity in our previous study.

Compounds 9p, 9q, and 9r were each prepared as N-tert-butyl

analogues of earlier N-methylated inhibitors, which had

exhibited fungal selectivities ranging from low (e.g., meta-

triﬂuoromethyl) to the highest observed in the study at 12- or

33-fold (meta-methyl or meta-methoxy). Unfortunately,

although this modiﬁcation was again successful in achieving

suﬃcient permeance for whole-cell activity, biochemical

potencies dropped to the low micromolar range and a

concomitant drop in selectivity was also observed. Similar

trends were also observed with meta-triﬂuoromethoxylated

compounds 9s and 9t. In contrast, improved selectivity and

potency were observed with meta-ﬂuorination at R²(com-

pounds 9u and 9v) with measurable, albeit high MIC values

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Table 2. Eﬀorts to Improve Whole-Cell Anticryptococcal Activities and Selectivities for Inhibitors N-Methylated at R¹, via a

c

Paring of Ortho/Meta-Substituents on the R²Aryl Ring

a

EC₅₀values were determined by FP-based equilibrium competition assay performed in a 384-well format using whole-cell lysates prepared from C.

b

neoformans and serial compound dilutions. All determinations were performed in duplicate. To calculate fold-selectivity, the EC₅₀value

determined in human HepG2 cell lysate was divided by the EC₅₀value determined in C. neoformans lysate. The resulting ratio was then normalized

by the ratio of values determined for the Hsp90 inhibitor geldanamycin using lysate of each cell type. Results for key selective compounds were

conﬁrmed by repeat assay in lysates, as well as by measuring ligand dissociation constants (K_i) using puriﬁed NBDs in FP assays (see Supporting

c

Tables 1 and 2). Concentration of compound resulting in >80% inhibition of fungal cell growth compared to vehicle control. Values for

compounds with either fold-selectivity >5 or MIC₈₀≤ 12.5 μM are highlighted in blue. Values for compounds with both fold-selectivity >5 and

MIC₈₀≤ 12.5 μM are highlighted in red.

against whole cells. Replacement of the ﬂuorine with chlorine in

these analogues (9w and 9x), plus an N-cyclohexyl variant (9y)

reduced biochemical potencies from nanomolar to low micro-

molar range. Again, with these chlorinated analogues, there was

an apparent trade-oﬀ between fungal selectivity >5-fold and

whole-cell activity <25 μM, with no compounds espousing both

features.

In sum, this initial cohort of second-generation RAPs

conﬁrmed our prior observations: while selectivity and

permeance were most often orthogonal qualities, the combina-

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Table 3. Strategic Merging of Bulky Aliphatic Moieties at R¹and Optimal Disubstituted Aromatics at R²Result in Advanced Leads

c

with Signiﬁcantly Improved Activity Proﬁles

a

EC₅₀values were determined by FP-based equilibrium competition assay performed in 384-well format using whole-cell lysates prepared from C.

b

neoformans and serial compound dilutions. All determinations were performed in duplicate. To calculate fold-selectivity, the EC₅₀value

determined in human HepG2 cell lysate was divided by the EC₅₀value determined in C. neoformans lysate. The resulting ratio was then normalized

to the ratio of values determined for the nonselective inhibitor geldanamycin using lysate of each cell type. Results for key selective compounds

were conﬁrmed by repeat assay in lysates, as well as by measuring ligand dissociation constants (K_i) using puriﬁed NBDs in FP assays (see

c

Supporting Tables 1 and 2). Concentration of compound resulting in >80% inhibition of fungal cell growth compared to vehicle control. Values

for compounds with either fold-selectivity >5 or MIC₈₀≤ 12.5 μM are highlighted in blue. Values for compounds with both fold-selectivity >5 and

MIC₈₀≤ 12.5 μM are highlighted in red.

tion of key structural features could in some cases (e.g.,

compounds 9d, 9u, 9v) provide an inroad to optimized

inhibitors.

μM or lower). Of note, the meta-chlorination present in

analogues 9ak and 9ao again appeared to dampen biochemical

potency, as was observed in 9w−9y (Table 1). This reduced

cryptococcal potency in turn lowered fungal selectivity in

comparison to other near-neighbor analogues. In contrast, meta-

ﬂuorination appeared to consistently yield biochemical

potencies below 100 nM, fungal selectivities greater than 15-

fold, and whole-cell activity at or below MIC 12.5 μM when

paired with either a suitable ortho-substituent (9ac−9ae, 9am,

9an) or a bulky aliphatic group at R¹(9v, Table 1). Compound

9as, bearing two ortho-ﬂuorine substituents in a 2,6 arrange-

ment, exhibited moderate potency and whole-cell activity, but a

complete loss of selectivity. Finally, ortho substitution (9at,

9au) was also suﬃcient to achieve permeance in combination

with additional para-substituents, but consistent with our

previously published SAR, these para-substituted analogues

were not found to confer any cryptococcal selectivity.

With two successful permeance-enhancing strategies identi-

ﬁed, we next examined the combination of both approaches

(Table 3). Gratifyingly, merging our optimal disubstitution

patterns at R²with a variety of bulky aliphatic moieties at R¹

(tert-butyl, isopropyl, cyclopentyl, and cyclohexyl) aﬀorded

inhibitors 9av−9bi with almost universally improved activity

proﬁles. Notably, all compounds in this series had measurable

MICs at or below 25 μM, with several compounds achieving the

best cryptococcal whole-cell growth inhibition observed for

fungal-selective RAPs to date at 3.13 μM (9bb, 9bd, 9bf, and

9bi). Among this series, the strongest whole-cell potencies

appeared to track to the N-tert-butyl and N-cyclohexyl

Although the trend was not consistent across all analogues, we

did note that in several instances, conversion of the R¹N-methyl

to N-tert-butyl dampened fungal selectivity in addition to

improving whole-cell activity. In response, we next pursued an

alternative method of pairing the apparent permeance-inducing

ortho-substituent with a selectivity-inducing meta-substituent,

on scaﬀolds that retained the R¹N-methylation (Table 2). Here,

we observed improved fungal selectivities with the 2,3-dimethyl

series 9z−9ab, but this R²group generally did not render the

inhibitors permeant. Interestingly, this set also exhibited an

extreme sensitivity to positioning of the isoindoline ﬂuorine

substituent with respect to biochemical potency. In contrast, we

had signiﬁcant success with R²-ﬂuorinated analogues. Com-

pounds 9ac−9ae, with an R²2,5-substitution pattern embody-

ing one of two possible hybrids of monosubstituted compounds

18 and 9u, showed dramatically improved potencies and

selectivities, in concert with good whole-cell MICs ranging

from 6.25 to 14.1 μM. Similarly, improved activity proﬁles were

observed among 2-ﬂuoro-3-methyl analogues 9af−9ah. Inspired

by these promising results, we next examined an array of

disubstituted aryl rings at R², paired with isoindoline amide A

(compounds 9ai−9au). While selectivity and permeance

sometimes diverged, we identiﬁed a number of ortho/meta

pairings (again in both 2,3- and 2,5-relationships), which

conferred high selectivity, and in select cases (9ak, 9am, 9an,

and 9ar) good concomitant whole-cell activity (MIC values 12.5

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Table 4. Mammalian Cytotoxicity and Microsomal Stability Assessments for 24 Select RAPs, Including 22 RAPs with Biochemical

a

FS >5 and C. neoformans MIC ≤ 12.5 μM

a

All whole-cell testing results are representative of two independent experiments, each performed with technical triplicates. Compounds with NIH

3T3 therapeutic index >2 are highlighted in green. Compounds with intermediate microsomal stability (CL_int∼ 20−70 μL/min/mg)⁶⁹are

highlighted in orange.

Figure 3. Scatter plots of the top 22 most fungal-selective, whole-cell-active RAPs showing that (A) biochemical potency (H99 lysate pEC50, x-axis)

and whole-cell antifungal activity (C. neoformans MIC, μM, y-axis) are poorly correlated. Similarly, no correlation is seen between fungal selectivity and

therapeutic index (TI, mammalian IC₅₀÷ C. neoformans MIC) in (B) NIH 3T3 cells or (C) HepG2 cells. In (A−C), scatter points are colored based on

relative H99 selectivity in cellular lysates. (D) Structures of key highlighted compounds.

substitutions at R¹, with the corresponding N-isopropyl (9ba,

9be) and N-cyclopentyl (9bc, 9bh) congeners espousing

slightly higher MICs at 6.25 μM. Interestingly, while

substitution of the isoindoline amide of 9bf for 4-ﬂuoroisoindo-

line (compound 9bg) depressed potency to increase the lysate

EC₅₀to the low micromolar range, this compound still exhibited

selectivity and whole-cell activity comparable to near-neighbor

analogues with lysate EC₅₀’s in the 100−400 nM range.

With a larger cohort of whole-cell-active, fungal-selective

compounds in hand, we next sought to further evaluate these

compounds for potential use in animals. In our prior study, poor

metabolic stability was identiﬁed as a signiﬁcant liability among

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several of our most promising ﬁrst-generation RAPs, with many

surprisingly, we observed a near-inverse relationship between

cellular TI and lysate fold-selectivity; the most selective

compound in mouse ﬁbroblasts (9o) was among the least

selective in lysates, whereas the RAP exhibiting the highest lysate

selectivity (9ad) was among the least selective in NIH 3T3 cells

(Figure 3B,D). In HepG2 cells, which due to their oncogenic

nature are known to be hypersensitive to Hsp90 inhibition, none

of the compounds exhibited cytotoxicity less than the minimum

concentration required to suppress C. neoformans growth (all

TIs < 1, Figure 3C,D). From these data, we conclude that

although progress has been made, achieving adequate fungal

permeance remains a challenge, a well-recognized hurdle for

many small-molecule xenobiotics.⁷⁰

Structural Insights into Fungal Selectivity of RAPs. In

addition to pursuing a property-driven optimization of our RAP

series, we performed structural studies to better understand the

binding mode of these inhibitors and the basis of their

selectivity. We determined the crystal structures (Supporting

Tables 5 and 6) of the C. neoformans NBD domain in the apo

state, in complex with the nonselective Hsp90 inhibitor NVP-

AUY922 (luminespib)⁷¹and in complex with the fungal-

selective RAPs 10, 18 (Figure 2), and 19 (Figure 4).⁶²These

structures provide the ﬁrst insights into the mode of RAP

binding and also reveal a plausible structural rationale for their

fungal selectivity.

compounds showing very high microsomal metabolism (T_1/2

10 min), and only one RAP with intermediate (CL_int∼ 20−70,

_1/220−60 min) metabolic stability. In this study, we examined

<

T

a wider array of RAPs from our second-generation set, including

all of the most promising compounds summarized in Table 4, to

better understand the metabolic liabilities present and attempt

rational modiﬁcations to minimize them. Starting from ﬁrst-

generation RAP 18 (T_1/2= 6 min), we found that a number of

structural modiﬁcations conferred a net stabilizing eﬀect, with

microsomal half-lives for nearly half of the compounds (11 of

24) extended to >20 min. Such modiﬁcations include

conversion of the R¹methyl to tert-butyl (9ax) and cyclohexyl

(9az), ﬂuorination of the isoindoline at the 4- (9ad) and 5-

position (9ae), and conversion of the R²ortho-methyl

substituent to ﬂuorine (9am) and chlorine (9an). However,

matched-pair analysis for other members of the cohort (not

shown) showed that these same types of single structural

modiﬁcations impart divergent eﬀects on microsomal stability,

depending on the structure of the parent RAP. While it is

diﬃcult to discern clear structure−property relationships or

pinpoint exact sites of metabolism from these data, we postulate

that the observed trends are consistent with either metabolism

that is largely occurring at the isoindoline with extreme

sensitivity to CYP450 binding eﬀects imparted by the entire

aminopyrazole subunit, or more likely that several potential

metabolic soft spots exist on the RAP scaﬀold, and that access to

these potential hot spots by CYP450 enzymes is strongly

governed by each site’s positioning relative to other substituents

exerting distal eﬀects on CYP450 binding. While the extension

of half-lives from the low to intermediate range represents a key

advancement, there is clearly room for further optimization of

metabolic stability.

Multiple copies of the protein were present in the asymmetric

units of the crystals in all cases (three in apo, six in complex with

Next, we examined the downstream translatability of potency

and selectivity in lysates to the context of whole cells. Strikingly,

a plot of biochemical vs whole-cell potency (Figure 3A) clearly

shows that these values are noncorrelative. The lack of

correlation suggests that although barriers to cryptococcal

permeance have been lowered with this series, as-yet-undeﬁned

mechanisms still reduce compound accumulation in Crypto-

coccus and, as a result, dampen whole-cell potency to levels well

below what would be expected based on biochemical target

engagement. This point is perhaps best exempliﬁed by direct

comparison of compounds 9bg and 9ac (Figure 3A); despite a

>30-fold diﬀerence in biochemical potency in cryptococcal H99

lysate (1.15 μM vs 34 nM, respectively), both compounds were

equally eﬀective in H99 whole cells (MIC 6.25 μM).

To investigate whether permeance limitations in Cryptococcus

extended to mammalian cells, we proceeded to evaluate the

cytotoxicity of our most promising compounds, again mainly

focused on those with FS >5 and MIC ≤ 12.5 μM, in mammalian

systems (Table 4). These RAPs were tested in nine-point dose

response for cytotoxic activity against human liver cancer

(HepG2) and mouse ﬁbroblast (NIH 3T3) cell lines (Table 4,

Supporting Tables 3 and 4; Figures S4 and S5). The disparity in

these cell lines between whole-cell activity and biochemical

potency for our RAPs was not as great as in Cryptococcus. As a

result, we found that high fungal target selectivity in lysates did

not translate to a useful therapeutic index (TI = mammalian IC₅₀

÷ C. neoformans MIC₈₀) in most cases. Using the noncancerous

cell line NIH 3T3, known to be less sensitive to Hsp90 inhibition

than cancer cells such as HepG2,⁴³only a handful of compounds

showed a modest therapeutic window (TI 1.5−3.1). Somewhat

Figure 4. Chemical structures of ligands including RAP 19;⁶²

previously reported C. albicans-selective inhibitor CMLD013075;⁴²

clinical inhibitors NVP-AUY922,⁷¹AT13387, and SNX-2112; and

purine inhibitor PU3.⁷²

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Figure 5. Overlay of human, C. albicans, and C. neoformans Hsp90 NBD structures, viewed from the “back” side of the domain. In this view, the ligand-

binding pocket and lid helices α4−α6 are behind the protein, away from the viewer, and are deemphasized to accentuate the changes in the β-sheet and

α1. (A) Superposition of Cα traces from 21 structures that share a similar core. Traces are colored black, with β-strands in blue with blue labels. The lid

is in light gray behind strands β5-β4-β7. Bound ligands in the nucleotide binding pocket are shown in pale orange behind strands β4−β7. The

structures include the human protein in the apo state (PDB ID 1YER and 1YES, 2 chains) and in complex with radicicol (4EGK, 1 chain), SNX-2112

(4NH7, 2 chains), AUY922 (2VCI, 1 chain), AT13387 (2XJX, 1 chain), and PU3 (1UY6, 1 chain); C. albicans apo (6CJI, 1 chain) and in complex with

ADP (6CJJ, 1 chain), radicicol (6CJL, 2 chains), and SNX-2112 (6CJR, 1 chain); and C. neoformans apo (7K9R, 3 chains) and in complex with

AUY922 (7K9S, 6 chains). (B) Same as (A), but with the addition of structures from C. neoformans with compounds 10 (7K9W, 2 chains), 18 (7K9V, 2

chains), and 19 (7K9U, 2 chains), shown in red. This set shows consistent changes in β5, β6, and α1 and a disordering of strand β1 (not modeled in the

crystal structures). (C) Same as (A), but with the addition of chains from C. albicans in complex with AUY922 (6CJS, 1 chain) and CMLD013075 (20)

(6CJP, 2 chain), shown in magenta. Changes in the core structure are seen in β6, β1, and α1. (D) Individual structures from the box in (B) showing C.

neoformans in complex with AUY922 (7K9S, left) and 18 (7K9V, right).

AUY922, and two with each of 10, 18, and 19), providing

independent views of the protein in diﬀerent states (Supporting

Figure S6). Excluding the ﬂexible lid domain that spans residues

86−124 and includes helices α4−α6, the core backbone

structure of the C. neoformans NBD in the apo state and in

complex with AUY922 aligns well with the previously

determined human and C. albicans structures (Figure 5A).

The conservation of this core, which includes the entire β sheet

and α1, across three distantly related species in both apo and

ligand-bound states demonstrates that this core is rigid and not

easily deformed. In the more ﬂexible lid domain, two of the three

C. neoformans apo chains resemble the “closed” (catalytically

active) state of the human structure (PDB: 1YER⁷³), as deﬁned

by the inward positioning of the catalytic loop between helices

α4 and α5, while the third chain adopts the “open” state, similar

to that seen in the human structure (1YES⁷³). In the apo and

AUY922 structures, the lid adopts a range of conformational

states and the catalytic loop between helices α4 and α5 ranges in

length from two to eight residues. The lid regions in the six RAP

structures, however, are similar to each other but diﬀer from the

previously described structures. Notably, helices α4 and α5 are

fused into a single continuous helix from residues 86−110 that

curves around the nucleotide binding site (Supporting Figure

S6). The structure of this lid region is similar in all six RAP

complex structures; however, there are minor diﬀerences in the

conformation of the fused helix, including a bulge⁷⁴at residue

L93 in three of six cases. In addition, and in contrast to the C.

neoformans apo and AUY922 structures, the six RAP structures

show consistent changes in the conserved core of the domain.

Notably, strand β1 could not be modeled due to disorder, while

α1 and the following loop connecting to α2 are shifted relative to

the canonical structure (Figure 5B). Similar N-terminal changes

involving β1 and α1 have been observed in structures of C.

albicans with AUY922 and the C. albicans-selective Hsp90

inhibitor CMLD013075 (20, Figure 4), although the speciﬁc

details diﬀer (Figure 5C).⁴²

Overall, ﬁve regions of the NBDs make contact with the

ligands: residues from α2; the loop that precedes α4; the α4−α5

segment of the lid; helix α7; and residues on the inward-facing

strands of β4, β5, and β7 of the main sheet (Supporting Figure

S7). With the exception of the lid, there is little structural

variability in the backbone of these elements, and most of the

side chains adopt similar rotamers irrespective of the species or

bound ligand. As previously described, the vast majority of the

Hsp90 residues in direct contact with ligands are identical

between the three species, with the exception of positions S38/

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A41/S52 on α2 and M173/L176/V186 on β7 (C. neoformans/

C. albicans/human residue numbering). These side chains

occupy similar volumes in all structures and are roughly isosteric,

but they do make contributions to the surface of the pocket that

accepts the resorcinol ring, among others. These positions may

modulate ligand-binding aﬃnity and thus may contribute to

species speciﬁcity.

A comparison of the C. neoformans complex with 10 and the

human complex with AT13387 shows excellent overlap between

the common resorcinol and isoindoline rings (Figure 6A). In

originally designed to mimic the CMLD013075 oxime, the

aminopyrazole instead sits in an orthogonal position, with a π-

stacking interaction between the R²aryl ring and Hsp90 F124.

The R²aryl rings in the RAP series introduce an important

feature that distinguishes these compounds from previously

reported inhibitors.⁶²A comparison of the structures of the

human and two fungal NBDs in complex with AUY922 reveal

highly similar structures, with the exception of moderate shifts in

the α4-α5 lid region (Figure 7A). However, in all six RAP

structures, the lid is displaced outward in response to the R²

rings (Figure 7B). Hydrophobic residues L93, F124, and W148

are in close contact in all of the apo and AUY922 complexes

(Figure 7C,D) but are displaced in the RAP structures to

accommodate the aryl rings (Figure 7E,F). The aromatic

residues F124 and W148 in the NBD are on the relatively well-

ﬁxed α7 and β5 strand elements, while L93 is on the more

malleable lid and is more easily displaced to accept the aryl ring.

The disruption of this packing and outward displacement of L93

promotes the adoption of an α-helical conformation in the loop

between α4 and α5, resulting in the formation of a continuous

helix that spans the N-terminus of α4 to the C-terminus of α5

(residues 86−110) (Supporting Figure S6). Equivalent L93/

F124/W148 packing is observed in the majority of apo and

complex NBD structures from human and C. albicans, and

similar disruptions of this core have been observed by the

indazole ring of the nonselective inhibitor SNX-2112 in

human⁴¹and C. albicans,⁴²and the methoxy-substituted aryl

ring of PU3 in human (Figure 6B).^72,75Notably, the RAP R²aryl

ring penetrates more deeply into this site and engages F124 in

π−π stacking interaction (Figures 6 and 7), potentially oﬀsetting

the energetic cost of repacking the hydrophobic core.

Figure 6. Comparison with previously reported complexes. (A)

Overlay of the human Hsp90/AT13387 complex (2XJX, cyan carbon

atoms) with the C. neoformans/10 complex (7K9W, orange carbon

atoms). The surface of the fungal protein is shown, and the AT13387

piperazine ring is omitted for clarity. (B) Staggered view of aligned

ligands from four complexes: the two chains from the C. neoformans/10

crystal structure (7K9W), human/SNX-2112 (4NH7), and human/

PU3 (1UY6). The gray oval represents the deep hydrophobic pocket of

Hsp90, indicated in the left part of (A).

comparison to other liganded Hsp90 structures in the PDB, the

RAP aminopyrazole ring is positioned in an induced pocket that

is similar to, but slightly deeper than, the pocket induced by the

indazole ring of nonselective inhibitor SNX-2112.^41,42While

The C. neoformans structures reveal two packing arrange-

ments of the RAP R²aryl groups (Figure 6B and Supporting

Figure S6). In four of the complexes (chain A of 10, chains A and

Figure 7. Ligand-driven reorganization of the C. neoformans binding pocket. (A) Overlay of the human (light blue, 2VCI), C. albicans (red, 6CJS), and

C. neoformans (orange, 7K9S) complexes with AUY922. (B) Overlay of the C. neoformans NBD in complex with AUY422 (orange, 7K9S) and RAP

compounds 10 (7K9W), 18 (7K9V), and 19 (7K9U) (dark blue). (C−F) Residues L93, F125, and W128 from helices α4, α7, and strand β5,

respectively, are in packing contact in the apo (6CJI, C) and AUY922 (7K9S, D) complexes, but are disrupted by the R²aryl ring of the RAPs (7K9W,

E/F). The two chains in the co-crystals of the C. neoformans NBD with 10 reveal diﬀerent rotamers of the meta-methoxy aryl ring, shown in (E) and

(F).

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Figure 8. RAP binding disrupts the β-sheet. (A) Overlay of the C. neoformans and human structures from Figure 5 were divided into two sets. Set A

includes all of the eight human structures [1YER, 1YES, 4EGK, 4NH7 (two chains), 2VCI, 2XJX, and 1UY6] and the nine C. neoformans apo and

AUY922 structures [7K9R (three chains) and 7K9S (six chains)] and is shown in black Cα trace. Set B includes the six fungal RAP complexes [7K9U

(two chains), 7K9V (two chains), and 7K9W (two chains)] and is shown in red Cα trace. Selected side chains from set A are shown in stick

representation with van der Waals surface and fungal/human numbering. (B) The same backbone traces are represented, but with the side chains from

set B as well as the RAP inhibitor 10 in space ﬁlling representation. Note that strand β1 is present only in the structures from set A.

Figure 9. Scatter plots of cryptococcal selectivity, biochemical potency, and whole-cell activity for Table 4 RAPs, binned by the aminopyrazole

substructure class as deﬁned by the R¹and R²substitution patterns. The y-axis depicts cryptococcal fold-selectivity on a logarithmic scale. Points are

colored by H99 lysate EC₅₀on a logarithmic scale, on a gradient ranging from cyan (10 μM) to red (1 nM). Point symbols indicate whole-cell activity

class, with circles corresponding to compounds with little to no whole-cell activity (H99 MIC ≥ 25 μM) and stars indicating whole-cell-active

compounds with an H99 MIC < 25 μM.

B of 18, and chain A of 19), the aryl ring is in the position shown

in Figure 7E, while in the other two (chain B of 10 and 19), the

conformer is as in Figure 7F. Thus, the pocket can adjust to two

rotamers of the ligand aryl ring relative to the aminopyrazole

ring. These structures, in combination with the SNX-2112 and

AT13387 structures, demonstrate the plasticity of this induced

binding pocket in both fungal and human Hsp90s. While we

expect that the RAPs can induce similar rearrangements in the

human protein, the weaker K_d’s strongly suggest that the

energetics of repacking diﬀers between the two species.

Although there is no obvious trend to show how the aryl ring

substituents may favor diﬀerent binding states, these structures

clearly show that ring substitution is a viable strategy to

modulate the binding aﬃnity within this series of inhibitors.

The insertion of RAP aryl rings not only displaces and

remodels the α4-α5 lid but also has smaller but signiﬁcant eﬀects

on β5 due to backbone shifts at residue W148 (Figures 7E,F and

8). This has a domino eﬀect that aﬀects neighboring strand β6

by displacing F156, ultimately resulting in the loss of the N-

terminal β1 strand of the sheet (Figure 8). In turn, the disruption

of strand β1 results in the shift in α1 since the N-terminus of this

helix is no longer anchored to the protein (Figures 5 and 8).

Each of the three major ligand-induced conformational

changes seen in the C. neoformans RAP complexes (disruption

of the L93/F125/W128 core, fusion of the lid helices, and

disruption of the N-terminus) have been observed in other NBD

complexes, but not in the same structures (Supporting Table 7).

Given the current data, it is challenging to propose a single

unifying model to explain how these changes are correlated to

each other and to ligand binding. We propose, however, that the

fusion of the lid helices is not directly correlated with the other

observed changes and is most likely driven by direct interactions

with the ligands. Support for this model can be found in the

structures with AUY922, which are known for all three species

under consideration. The binding of AUY922 to the C. albicans

NBD results in a fused lid helix and disrupted N-terminus, but

does not disrupt the core, while none of these changes are

present in the human AUY922 complex. In addition, a fused lid

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Figure 10. (A) Two predominant structural classes among RAPs with optimal combined biochemical potency/fungal selectivity/whole-cell activity

proﬁle, each with slightly divergent activity proﬁles. (B) Representative structures and activities of top inhibitors 9ac, 9ad, 9ba, and 9bb from these

subclasses.

helix is seen in two of the six AUY922 complexes with the C.

neoformans NBDs, but all have an intact L93/F125/W128 core

and an ordered N-terminus. The second pattern that emerges

from the set of complexes is that the disruption of the N-terminal

region co-occurs with the disruption of the W148/F156 stack

between strands β5 and β6. This stack is disrupted in some, but

not all, of the ligand complexes that disrupt the L93/F125/

W128 core. These include the six C. neoformans/RAP structures

presented here and the C. albicans complexes with AUY922 and

CMLD013075.⁴²Complexes with SNX-2112 (human, C.

albicans) and PU3 (human) are able to disrupt the core packing,

but do not reach deeply enough into the induced pocket to aﬀect

the position of W148, and consequently, these ligands do not

disrupt the β1/α1 N-terminus. It is notable that N-terminal

remodeling has been observed in approximately half of the

available C. neoformans and C. albicans structures, but never in a

human structure. This may simply reﬂect the selection of

structures that are currently available, or it may be because the

fungal NBDs are more prone to structural rearrangements in

regions outside of the lid. Overall, these results reﬂect the

complex interplay between the ligands and the proteins and

support the idea that Hsp90 inhibitors can be designed with

species-speciﬁc properties.

Summary of Structure−Activity Relationships and

Insights from Modeling. With X-ray crystallographic

structures providing an improved understanding of the binding

modes of R¹-methylated, R²monosubstituted RAPs, we next

attempted to further rationalize the observed structure−activity

and structure−property relationships across diﬀerent RAP

structural subtypes. Figure 9 shows a scatter plot summarizing

the cryptococcal fold-selectivity (y-axis), biochemical potency

(color), and whole-cell activity (symbol) of the cohort of

inhibitors from Table 4, binned on the x-axis based on their

patterns of substitution at R¹(methyl vs bulky aliphatic

substituents) as well as at the R²phenyl ring (2-substituted, 3-

substituted, 2,3-disubstituted, and 2,5-disubstituted). From this

plot, we can glean generalized SAR trends through head-to-head

comparisons of diﬀerent cohorts. For example, in comparing

groups A−B and E−F, it is clear that ortho-monosubstituted

compounds in group A generally have very good (low nM)

biochemical potencies but are limited in their fungal selectivity.

In contrast, meta-monosubstitutions (groups B and F) generally

lead to suppression of biochemical potency, in some cases to the

micromolar range. Interestingly, for the meta-monosubstituted

subset B, selectivity does not appear to track as cleanly with

biochemical activity compared to the ortho-monosubstituted

subset A. Also apparent is the general overall improvement in

biochemical potency and selectivity that is achieved when

incorporating a second substituent at R²(groups C/D/G/H as

compared to groups A/B/E/F). It is also notable that group to

group, these improvements in selectivity do sometimes come at

a cost: for example, the most potent and selective N-methylated,

2,3-disubstituted inhibitors (group D) tend to have poor whole-

cell activity, and highly selective inhibitors bearing bulky R¹

substituents are generally less potent than their N-methylated

counterparts (cf. groups G/H and groups C/D).

In judging these structural subclasses by their full complement

of relevant parameters, two clear “optimal” themes emerge

(Figure 10A): ﬁrst, for compounds that are N-methylated at R¹,

overall whole-cell activity is best among R²-disubstituted

compounds where the substituents have a 2,5 relationship

(group C); in contrast, compounds bearing bulky aliphatic

groups at R¹generally exhibit improved potency and selectivity

when R²is 2,3-disubstituted (group H). Interestingly, these two

orthogonal subsets also diverge somewhat in the nature of their

“optimal” properties; while group C is exemplary in biochemical

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Figure 11. Results of computational modeling using RAP-liganded X-ray crystal structures. (A) X-ray crystal structure (7K9V) with compound 18

bound at Chain A. (B, C) Comparative views of two rotamers of compound 9an docked into structure 7K9V Chain A. Docking scores are −15.3 and

−14.5 kcal/mol, respectively. (D, E) Comparative views of two rotamers of compound 9ba docked into structure 7K9V Chain A. Docking scores are

−14.7 and −14.5 kcal/mol, respectively. (F, G) Comparative views of two rotamers of compound 9bc docked into structure 7K9V Chain A. Docking

scores are −14.2 and −14.4 kcal/mol, respectively.

potency and selectivity, group H is stronger in terms of whole-

cell anticryptococcal activity; this feature is even more

pronounced when considering the relative whole-cell potencies,

which for simplicity are not represented here. In Figure 10B, we

provide representative leading members of each of these two

classes, as starting points for future optimizations of potency;

selectivity; whole-cell activity; and additional absorption,

distribution, metabolism, and excretion (ADME) parameters

including metabolic stability.

rotamers that were observed for compound 10 in the

independent chains of the 7K9W structure, underscoring the

ﬂexibility of this pocket to accommodate disubstituted R²aryl

rings in multiple orientations. Select docking poses of

compounds 9an, 9ba, and 9bc, which illustrate this phenom-

enon, are depicted in Figure 11B−G.

Out of the set of RAPs docked, only the R¹-cyclohexyl-

substituted compounds (9j, 9az, 9bd, and 9bi) failed to produce

any poses in any of the six chains under the applied constraints,

which we ascribe to the entropic and steric factors described

above. For the remaining 20 compounds, very high docking

scores were obtained across compounds with widely varying

biochemical potencies and selectivities; as such, it is diﬃcult to

fully rationalize the divergent activities on the basis of this

structural modeling. However, this limited computational study

does conﬁrm an overall compatibility of the R²-disubstituted

RAPs with the RAP-induced binding pocket. In addition, the

accommodation of multiple R²substituents and in multiple

orientations suggests that the potency gains observed for

disubstituted compounds may in part arise due to additional

beneﬁcial van der Waals interactions and an improved

occupancy of the deep hydrophobic cavity in the R²-induced

pocket.^78,79What is not clear from this modeling, however, is

whether these added R¹/R²substituents may induce further

changes to the protein structure, nor do they explain what role

the size and nature of the R¹/R²groups play in other important

improvements such as whole-cell permeance and reduced

mammalian cytotoxicity. Given the number of unexpected

ligand-induced conformations that have been observed in fungal

Hsp90 structures to date, further crystallographic work is needed

to fully understand the binding modes of the optimized R²-

disubstituted compounds described herein.

We next attempted to model these various structural

subclasses into our existing X-ray structures to rationalize

structure−activity trends observed among the R²-disubstituted

inhibitors. In doing so, it is important to consider that due to the

inherent plasticity of the Hsp90 NBD and the number and scope

of remodeling events which have so far been documented across

diﬀerent species and in co-complex with diﬀerent ligand

structural subclasses, any eﬀort to model other RAP structural

subclasses into these rigid, RAP-induced receptors is of only

limited and speculative utility. Nonetheless, in each RAP-

liganded X-ray structure, the R¹-methyl substituent is projected

outward and solvent-exposed; we posit that expansion of this

group to a larger lipophilic moiety is likely to create entropic

penalties due to bulk water disruption, which may in part explain

the general overall trend of lowered biochemical EC₅₀’s as the

size and lipophilicity of this substituent increases. Given the

proximity of this solvent-exposed group to a polar region of the

binding pocket periphery, it is also possible that incompatible

sterics and polarities also compound this issue. In addition, since

the overall structural disruptions of the NBD appear to be

induced by the R²aryl ring, we postulate that the added steric

demands imposed by a second substituent on this ring may be

the source of further improvements to selectivity. While we are

reluctant to deﬁnitively propose binding modes for the R²-

disubstituted RAPs, we have performed computational (Glide)

docking^76,77of the 24 RAPs from Table 4 to explore whether the

pockets induced by compounds 10, 18, and 19 are large enough

to accommodate larger R¹and R²substituents in similar binding

poses without any further protein structure remodeling (Figure

11 and Supporting Table 8). Glide docking with positioning

restricted to the reference RAP ligand core produced excellently

scored poses (−14.3 to −15.5 kcal/mol), for 20 out of the 24

compounds into at least one of the six independent chains with a

core atom root-mean-square deviation (RMSD) to the native

ligand of <0.1 Å. Notably, each compound also produced at least

one high-scored docking pose for each of the two types of R²

CONCLUSIONS

■

Continuing in our pursuit of fungal-selective, resorcylate

inhibitors of the protein-folding chaperone Hsp90, and building

on early leads from our prior study of resorcylate amino-

pyrazoles (RAPs), we have used a rational structure−property

approach to improve the whole-cell fungal permeance of a lead

series which exhibits high selectivity for cryptococcal Hsp90.

Toward this goal, we have achieved synthesis of 22 new fungal-

selective RAPs that eﬀectively inhibit growth of C. neoformans

whole cells at low-to-sub-micromolar concentrations. Further

pharmacological and biological characterization of these RAPs

showed that despite moderate improvements in both micro-

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9af 459.2-340.1; 9am 463.1-344.1; 9ar 529.1-410.1; 9av 497.2-322.0;

9ax 501.2-326.0; 9az 527.2-326.0; 9ba 487.2-326.1; 9bb 501.2-326.0;

9bc 513.2-326.0; 9bd 527.2-326.1; 9be 491.2-330.1; 9bf 505.2-330.0;

9bg 523.2-330.1; 9bi 531.2-330.1. An Agilent (Santa Clara, CA) C18

XDB column (5 μm, 50 mm × 4.6 mm) was used for chromatography

for compounds detected in negative mode under the following

conditions: Buﬀer A: dH₂O + 0.1% formic acid, Buﬀer B: methanol +

0.1% formic acid, 0−1.0 min 5% B, 1.0−2 min gradient to 100% B, 2−

3.5 min 100% B, 3.5−3.6 min gradient to 5% B, 3.6−4.5 5% B.

Tolbutamide (transition 269.1-169.9) from Sigma (St. Louis, MO) was

used as an internal standard (IS). Conditions for compounds evaluated

in positive mode were nearly identical, except the gradient conditions:

0−1.0 min 5% B, 1.0−1.5 min gradient to 100% B, 1.5−3.0 min 100% B,

3.0−3.2 min gradient to 5% B, 3.2−4.5 5% B, and N-benzylbenzamide

(Sigma) was used as the internal standard (transition 212.1−91.1).

Solvents (methanol and water) and formic acid were all of LC-MS/MS

grade (“Optima”) and purchased from Fisher Scientiﬁc (Waltham,

MA).

Mouse Liver Microsome Stability. Female ICR/CD-1 mouse

microsome fractions were purchased from BioIVT (Westbury, NY),

and the protocol provided with the microsomes for assessment of

compound stability was followed as detailed herein. All chemical

reagents aside from the aforementioned solvents were purchased from

Sigma (St. Louis, MO). Microsome protein (0.5 mg/mL) was added to

a glass screw cap tube; then, 50 mM Tris, pH 7.5 solution, containing

the compound of interest in DMSO (ﬁnal DMSO concentration, 0.1%)

was added on ice. The ﬁnal concentration of compound after addition

of all reagents was 2 μM. A nicotinamide adenine dinucleotide

phosphate (NADPH)-regenerating system (1.7 mg/mL NADP, 7.8

mg/mL glucose-6-phosphate, 6 U/mL glucose-6-phosphate dehydro-

genase in 2% w/v NaHCO₃/10 mm MgCl₂) was added for analysis of

Phase I metabolism after both the microsome/compound mixture and

regenerating system were warmed to 37 °C for 5 min. The tube was

then placed in a 37 °C shaking water bath. At varying time points after

addition of the NADPH regenerating system, the reaction was stopped

by the addition of 0.5 mL of methanol containing an internal standard

(IS) and formic acid such that the ﬁnal concentration of tolbutamide IS

was 50 ng/mL and that of N-benzylbenzamide IS was 100 ng/mL, and

acid was 0.1%. Time 0 samples were quenched with methanol prior to

addition of compound. The samples were incubated 10 min at room

temperature and then spun at 16 100g for 5 min in a microcentrifuge at

4 °C. The supernatant was analyzed by LC-MS/MS. The method

described in McNaney et al.⁶⁹was used with modiﬁcation for

determination of metabolic stability half-life by substrate depletion. A

“% remaining” value was used to assess the metabolic stability of a

compound over time. The LC-MS/MS peak area of the incubated

sample at each time point was divided by the LC-MS/MS peak area of

the time 0 (T0) sample and multiplied by 100. The natural log (LN) of

the % remaining of compound was then plotted versus time (in

minutes) and a linear regression curve plotted going through y-

intercept at LN(100). The metabolism of some compounds failed to

show linear kinetics at a later time point, so those time points were

excluded. The half-life (T_1/2) was calculated as T_1/2= −0.693/slope. To

determine intrinsic clearance,⁸²⁻⁸⁵which allows for comparison of data

obtained using diﬀerent volumes and protein concentrations, the

following calculations were employed:

somal stability and whole-cell anticryptococcal activity, further

optimization of fungal permeance is still needed to achieve an

adequate therapeutic window for use in infection-relevant

contexts. We have also solved the ﬁrst X-ray crystal structures of

C. neoformans Hsp90 in various states, including structures in

complex with three fungal-selective RAPs. Conformational

rearrangements induced by RAPs, while unique within the

direct binding site, create downstream conformational reorgan-

izations that are consistent with other changes induced by a

resorcylate compound that selectively binds C. albicans Hsp90.

Further studies to parlay these foundational ﬁndings into

improved fungal-selective Hsp90 inhibitors for use as chemical

biological probes and eventually therapeutics are ongoing.

EXPERIMENTAL SECTION

■

Fungal Strains and Culture Conditions. The strain used in this

study was C. neoformans H99.⁸⁰Archive of this strain was maintained at

−80 °C in 25% glycerol. Active cultures were maintained on solid (2%

agar) yeast extract peptone (YPD, 1% yeast extract, 2% bacopeptone,

and 2% glucose) at 4 °C for no more than 1 month.

Antifungal Sensitivity Testing. Minimum inhibitory concen-

trations (MICs) were determined in ﬂat-bottom, 96-well-plate format

using RPMI medium (Gibco SKU no. 318000-089, 3.5% MOPS, 2%

glucose, pH 7.0). A modiﬁed broth microdilution protocol was used as

previously described,^23,81except that a relative viable cell number was

monitored by standard dye-reduction assay after a 3 h incubation with

resazurin at 37 °C. All compounds were formulated in dimethyl

sulfoxide (DMSO, Sigma-Aldrich Co.). Each compound was tested in

duplicate in at least two independent experiments.

Mammalian Cell Culture and Cytotoxicity Testing. The cell

lines NIH 3T3 (CRL-1658) and HepG2 (HB-8065) were purchased

from the American Type Culture Collection (ATCC). The cells were

maintained in RPMI medium supplemented with 10% fetal bovine

serum at 37 °C under 5% CO₂. Experiments were performed using cells

within 10 passages post recovery from low-passage stocks maintained in

liquid nitrogen and conﬁrmed negative for mycoplasma contamination

by PCR-based assay. The cells were plated in a 384-well format at a

density of 2000 per well (HepG2) or 2500 per well (NIH 3T3) in

RPMI supplemented with 10% fetal bovine serum (FBS). Following

overnight adherence, twofold dilutions of test inhibitors were added to

wells and plates were incubated for 72 h. Subsequent determination of

relative viable cell number was performed using standard resazurin dye-

reduction assay with incubation at 37 °C for 3 h and measurement in a

Tecan Spark microplate reader using SparkControl software (version

2.2). Nonlinear four-parameter curve ﬁtting of raw dose−response data

was performed in GraphPad Prism 7 to determine IC₅₀values. Results

reported are representative of two independent experiments, each

performed with technical triplicate.

FP Assays. FP assays performed with whole-cell lysates and puriﬁed

C. neoformans Hsp90 NBD were performed as previously described.⁶²

In calculating fold-selectivity for compounds, a normalization factor of

0.84 was applied based on the EC₅₀values determined in human and

fungal cell lysate for geldanamycin, the same Hsp90 inhibitor used to

generate the FP probe itself.

NBD Expression and Puriﬁcation. Recombinant Hsp90 NBDs

volume of incubation (μL)

V(μL/mg) =

were expressed and puriﬁed as previously described.^42,62

protein in the incubation (mg)

Analytical LC-MS/MS Conditions. Compound levels for in vitro

metabolic stability assays were monitored by LC-MS/MS using an AB

Sciex (Framingham, MA) 4000 QTRAP mass spectrometer coupled to

a Shimadzu (Columbia, MD) Prominence LC. The parent ion and the

two most prominent daughter ions were followed to conﬁrm

compound identity, although only the most abundant daughter was

used for quantitation. Analytes were detected with the mass

spectrometer in negative multiple reaction monitoring (MRM) mode

for the following compounds: 9i 541.3-404.0; 9j 541.2-404.0; 9n 477.1-

358.0; 9o 551.3-432.0; 9ac 457.1-337.9; 9ad 475.2-338.1; 9ae 475.1-

337.9; 9ah 475.2-338.0; 9ak 477.1-357.9. The following compounds

were detected in positive MRM mode: 9d 483.2-308.0; 9v 487.2-312.0;

Vx0.693

t_1/2

intrinsic clearance (CL_int)(μL/min /mg protein) =

Statistical Methods. For FP experiments in support of SAR

studies, GraphPad Prism 5.0 was used to perform curve ﬁtting and

calculate the concentrations of compounds resulting in 50% reduction

in maximal polarization signal (EC₅₀). All curve ﬁts demonstrated a

correlation coeﬃcient (R²) > 0.95. The number of independent

experiments performed and the number of technical replicates in each

experiment are provided in the legends of ﬁgures and tables

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characterizing the biochemical and biological activities of compounds.

In calculating the error of selectivity determinations, the fractional error

of measurements in each species was summed to yield a composite error

for the derived ratio.

Protein Crystallization and Structure Determination. Ali-

quots of puriﬁed C. neoformans NBD (residues 1−211 or 1−215) were

crystallized either alone (apo) or in the presence of AUY922 or RAPs

10, 18, and 19. Crystals were obtained by mixing one part of protein

solution at 10−15 mg/mL (20 mM Hepes, pH 7.5, 500 mM NaCl, 8%

glycerol, 10% DMSO with or without 2 mM ligand) with one part of

reservoir solution. Crystals were obtained by sitting-drop vapor

diﬀusion at 21 °C. The best diﬀracting crystals were produced with a

reservoir containing 20−25% PEG6K, 0.1 M Hepes, pH 7, and 1 M

LiCl₂.

Crystals were cryo-protected by passage through paratone before

ﬂash-cooling in liquid nitrogen. Diﬀraction data were collected at the

APS or NSLS II synchrotrons, as indicated in Supplementary Tables 5

and 6. Data were processed and integrated with XDS⁸⁶and Aimless,⁸⁷

and structures were determined by molecular replacement using C.

albicans Hsp90 NBD (PDB ID 6CJI) as a search model for the apo

structure. The apo structure was then used to solve the RAP complexes

by molecular replacement. Model building was done with Coot,⁸⁸and

the structures were reﬁned with Phenix.⁸⁹Software used in this project

was curated by SBGrid.⁹⁰

Computational Docking. Molecular docking of the 24 com-

pounds listed in Table 4 was performed using six separate docking grids

(7K9U Chain A, 7K9U Chain B, 7K9V Chain A, 7K9V Chain B, 7K9W

Chain A, and 7K9W Chain B) using the Glide docking program

under a nitrogen atmosphere unless otherwise noted. Analytical

UPLC−MS experiments were performed using a Waters Acquity

(ultraperformance liquid chromatography) with a binary solvent

manager, an SQ mass spectrometer, a Waters 2996 photodiode array

(PDA) detector, and an evaporative light-scattering detector (ELSD).

All microwave experiments were performed on a CEM Discover

microwave reactor, using a sealed 10 or 35 mL vessel with temperatures

monitored by an external sensor. All compounds tested in biological

assays were determined to be >95% pure by UPLC−MS-ELSD analysis.

α-Cyanoketone Synthesis. α-Cyanoketones 3a, 3c, 3e, 3g, 3h, 3i,

3k, and 3l were obtained from commercial sources.

General Procedure A. To a ﬂame-dried nitrogen-ﬂushed round-

bottom ﬂask equipped with a magnetic stir bar were added anhydrous

THF (4 mL) and nBuLi solution (3 mmol, 1.2 equiv). The solution was

cooled to −78 °C in a dry ice/acetone bath. To the reaction ﬂask was

added a solution of MeCN (0.26 mL, 5 mmol, 2 equiv, 2 M in THF)

dropwise over 2 min. The mixture was stirred for 1 h at −78 °C. A

solution of methyl ester 2 (2.5 mmol, 2 M in THF) was prepared and

added to the reaction ﬂask dropwise over 2 min. The reaction was

stirred at −78 °C for 30 min and then at room temperature for 2 h. The

reaction was quenched with 1 M HCl (30 mL), extracted with EtOAc

(3 × 50 mL), washed with brine, dried with Na₂SO₄, and concentrated

under reduced pressure. Puriﬁcation by silica ﬂash chromatography

(2−40% EtOAc in hexanes) aﬀorded the desired α-cyanoketone

product 3.

3-Oxo-3-(2-(triﬂuoromethyl)phenyl)propanenitrile (3b). 3b was

prepared from methyl 3-(triﬂuoromethyl)benzoate (0.78 mL, 5 mmol)

according to General Procedure A. 3b (799 mg, 3.75 mmol, 74% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.82−7.75 (m, 1H), 7.73−7.64 (m,

2H), 7.54−7.48 (m, 1H), 3.95 (s, 2H).

̈

(Schrodinger Maestro Version 12.4.079). Proteins were treated using

̈

Schrodinger’s Protein Preparation Wizard with retention of waters,

optimization of hydrogen bonds, and restrained minimization

converging heavy atoms to RMSD 0.3 Å. Protein structures were

kept rigid during docking experiments. Docking grids were prepared

using a receptor-based box centered at the native ligand for each chain.

Ligands were prepared using Schrodinger’s LigPrep and kept at a

neutral ionization state. Docking was run at Standard precision with

ﬂexible ligands, with the docking restricted to a reference position

(identiﬁed using Maximum Common Substructure) deﬁned by the 5-

((1H-pyrazol-5-yl)amino)benzene-1,3-diol core substructure from the

native ligand of each chain. The output was set to report up to ﬁve poses

per ligand, and Epik state penalties were included in the calculation of

docking score. All of the output docking poses fell into one of the two

possible R²aryl-ring rotamers as originally observed for compound 10

in the 7K9W X-ray structure. Where two R²aryl-ring rotamers were

present, the highest-scored poses for each of the two rotamers were kept

for analysis; otherwise, only the highest-scored pose for each compound

into each chain was kept. Tabular results for docking each compound

into each chain are reported in Supporting Table 8.

3-Oxo-3-(2-(triﬂuoromethoxy)phenyl)propanenitrile (3e). 3e was

prepared from methyl 2-(triﬂuoromethoxy)benzoate (1.10 g, 5 mmol)

according to General Procedure A. 3e (1.08 g, 4.73 mmol, 94% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.88 (dd, J = 7.9, 1.8 Hz, 1H), 7.70−

7.63 (m, 1H), 7.49−7.41 (m, 1H), 7.41−7.33 (m, 1H), 4.05 (s, 2H).

3-Oxo-3-(3-(triﬂuoromethoxy)phenyl)propanenitrile (3i). 3i was

prepared from methyl 3-(triﬂuoromethoxy)benzoate (1.10 g, 5 mmol)

according to General Procedure A. 3i (979 mg, 4.27 mmol, 85% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.86−7.81 (m, 1H), 7.79−7.75 (m,

1H), 7.62−7.55 (m, 1H), 7.53−7.48 (m, 1H), 4.13 (s, 2H).

3-(2,3-Dimethylphenyl)-3-oxopropanenitrile (3l). 3l was prepared

from methyl 2,3-dimethylbenzoate (985 mg, 6 mmol) according to

1

General Procedure A. 3l (647 mg, 3.74 mmol, 62% yield). H NMR

(400 MHz, CDCl₃) δ 7.37 (d, J = 7.7 Hz, 2H), 7.21 (t, J = 7.7 Hz, 1H),

3.99 (s, 2H), 2.40 (s, 3H), 2.34 (s, 3H).

3-(5-Fluoro-2-methylphenyl)-3-oxopropanenitrile (3m). 3m was

prepared from methyl 5-ﬂuoro-2-methyl-benzoate (1.01 g, 5 mmol)

according to General Procedure A. 3m (706 mg, 3.98 mmol, 66%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.34−7.27 (m, 2H), 7.20 (td, J =

8.1, 2.6 Hz, 1H), 4.02 (s, 2H), 2.52 (s, 3H).

1

Chemistry Methods. General Methods. H NMR spectra were

recorded at 400 or 500 MHz at ambient temperature. ¹³C NMR spectra

were recorded at 101 or 126 MHz at ambient temperature. ¹⁹F NMR

spectra were recorded at 470 MHz at ambient temperature. Chemical

shifts are reported in parts per million. Data for ¹H NMR are reported as

follows: chemical shift, multiplicity (app = apparent, br = broad, s =

singlet, d = doublet, t = triplet, q = quartet, sxt = sextet, hept = heptet, m

= multiplet,), coupling constants, and integration. All ¹³C NMR spectra

were recorded with complete proton decoupling. Analytical thin-layer

chromatography was performed using 0.25 mm silica gel 60-F plates.

Silica ﬂash chromatography was performed using prepacked columns

(SI-HC, puriFlash or Premium Universal, Yamazen) on either an

Interchim puriFlash450 or Yamazen Smart Flash EPCLC W-Prep2XY

system. All mass-guided preparative high-performance liquid chroma-

tography (HPLC) experiments were performed using an acetonitrile/

water gradient (mobile phase modiﬁed with 0.01% formic acid) on a

Waters FractionLynx system equipped with a 600 HPLC pump, a

micromass ZQ quadrupole, Waters 996 diode array, and Sedere Sedex

75 ELS detectors, using an XBridge Prep C18 5 μM OBD 19 mm

diameter column of either 100 or 250 mm length. Isolated yields refer to

chromatographically and spectroscopically pure compounds, unless

otherwise stated. All reactions were carried out in oven-dried glassware

3-(2-Fluoro-3-methylphenyl)-3-oxopropanenitrile (3n). 3n was

prepared from methyl 2-ﬂuoro-3-methyl-benzoate (1.01 g, 6 mmol)

according to General Procedure A. 3n (1020 mg, 5.76 mmol, 95%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.76 (t, J = 7.3 Hz, 1H), 7.51−

7.43 (m, 1H), 7.22−7.13 (m, 1H), 4.12−4.05 (m, 2H), 2.34 (s, 3H).

3-(2,3-Diﬂuorophenyl)-3-oxopropanenitrile (3o). 3o was prepared

from methyl 2,3-diﬂuorobenzoate (1.03 g, 6 mmol) according to

1

General Procedure A. 3o (694 mg, 3.83 mmol, 63% yield). H NMR

(400 MHz, CDCl₃) δ 7.76−7.68 (m, 1H), 7.53−7.43 (m, 1H), 7.30−

7.21 (m, 1H), 4.11 (d, J = 2.4 Hz, 2H).

3-(2-Methyl-3-(triﬂuoromethyl)phenyl)-3-oxopropanenitrile (3p).

3p was prepared from methyl 2-methyl-3-(triﬂuoromethyl)benzoate

(501 mg, 2.3 mmol) according to General Procedure A. 3p (449 mg,

1.98 mmol, 85% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.84 (d, J = 7.9

Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 4.00 (s, 2H),

2.57 (s, 3H).

3-(3-Chloro-2-ﬂuorophenyl)-3-oxopropanenitrile (3q). 3q was

prepared from methyl 3-chloro-2-ﬂuoro-benzoate (471 mg, 2.5

mmol) according to General Procedure A. 3q (443 mg, 2.24 mmol,

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89% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.89−7.81 (m, 1H), 7.73−

7.64 (m, 1H), 7.30−7.19 (m, 1H), 4.11 (s, 2H).

5-Aminopyrazole Synthesis. 5-Aminopyrazoles 4a, 4f, and 4s

were obtained from commercial sources.

General Procedure B. To a microwave vial equipped with a

magnetic stir bar were added α-cyanoketone 3 (1 equiv), methyl

hydrazine (1.1 equiv), and anhydrous methanol (1 M). The vial was

capped and heated in a microwave reactor maintaining 120 °C for 90

min. The vial was cooled to room temperature and the reaction was

concentrated under reduced pressure. The crude residue was puriﬁed

by silica ﬂash chromatography (5-60% EtOAc in CH₂Cl₂) to aﬀord the

desired 5-aminopyrazole.

General Procedure C. To a microwave vial equipped with a

magnetic stir bar were added α-cyanoketone 3 (1 equiv), N-alkyl-

hydrazine hydrochloride (1.1 equiv), triethylamine (1.5 equiv), and

anhydrous methanol (1 M). The vial was capped and heated in a

microwave reactor maintaining 120 °C for 90 min. The vial was cooled

to room temperature and the reaction was concentrated under reduced

pressure. The crude residue was puriﬁed by silica ﬂash chromatography

(5−50% EtOAc in hexanes) to aﬀord the desired 5-aminopyrazole.

3-(2-Chlorophenyl)-1-methyl-1H-pyrazol-5-amine (4b). 4b was

prepared from 2-chlorobenzoylacetonitrile 3a (898 mg, 5 mmol, 1

equiv) according to General Procedure B. 4b (508 mg, 2.45 mmol, 48%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.76 (dd, J = 7.8, 1.8 Hz, 1H),

7.41 (dd, J = 7.8, 1.5 Hz, 1H), 7.32−7.18 (m, 2H), 6.11 (s, 1H), 3.75 (s,

3H), 3.56 (s, 2H).

3-(2-Fluoro-3-(triﬂuoromethoxy)phenyl)-3-oxopropanenitrile

(3r). To a 20 mL scintillation vial were added 2-ﬂuoro-3-

(triﬂuoromethoxy)benzoic acid (504 mg, 2.25 mmol), concentrated

sulfuric acid (0.2 mL), and methanol (6 mL, 0.38 M). The vial was

sealed, and the reaction was heated at 75 °C for 18 h. The reaction was

cooled to room temperature, diluted with ethyl acetate (50 mL),

washed with saturated sodium bicarbonate solution (3 × 50 mL),

washed with brine (50 mL), dried with Na₂SO₄, and concentrated

under reduced pressure. The crude methyl ester was carried on without

further puriﬁcation and treated according to General Procedure A. 3r

(445 mg, 1.80 mmol, 80% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.96−

7.88 (m, 1H), 7.66−7.58 (m, 1H), 7.39−7.31 (m, 1H), 4.12 (d, J = 2.7

Hz, 2H).

3-(2,5-Diﬂuorophenyl)-3-oxopropanenitrile (3s). 3s was prepared

from methyl 2,5-diﬂuorobenzoate (516 mg, 3 mmol) according to

1

General Procedure A. 3s (248 mg, 1.37 mmol, 45% yield). H NMR

(400 MHz, CDCl₃) δ 7.72−7.60 (m, 1H), 7.40−7.28 (m, 1H), 7.25−

7.14 (m, 1H), 4.10 (d, J = 2.7 Hz, 2H).

3-(2-Chloro-5-ﬂuorophenyl)-3-oxopropanenitrile (3t). 3t was

prepared from methyl 2-chloro-5-ﬂuoro-benzoate (471 mg, 2.5

mmol) according to General Procedure A. 3t (441 mg, 2.23 mmol,

89% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.53−7.45 (m, 1H), 7.42−

7.36 (m, 1H), 7.31−7.22 (m, 1H), 4.19 (s, 2H).

1-Methyl-3-(2-(triﬂuoromethyl)phenyl)-1H-pyrazol-5-amine (4c).

4c was prepared from 3b (710 mg, 3.33 mmol, 1 equiv) according to

General Procedure B. 4c (501 mg, 2.1 mmol, 62% yield). ¹H NMR (400

MHz, CDCl₃) δ 7.74−7.60 (m, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.42 (t, J =

7.7 Hz, 1H), 5.85−5.77 (m, 1H), 3.77−3.72 (m, 3H), 3.69−3.51 (m,

2H).

3-(5-Chloro-2-ﬂuorophenyl)-3-oxopropanenitrile (3u). 3u was

prepared from methyl 5-chloro-2-ﬂuoro-benzoate (471 mg, 2.5

mmol) according to General Procedure A. 3u (445 mg, 2.25 mmol,

1

90% yield). H NMR (400 MHz, CDCl₃) δ 7.94 (m, 1H), 7.59 (m,

1H), 7.18 (m, 1H), 4.08 (d, J = 2.6 Hz, 2H).

1-(tert-Butyl)-3-(o-tolyl)-1H-pyrazol-5-amine (4d). 4d was pre-

pared from o-toluoylacetonitrile 3c (239 mg, 1.5 mmol, 1 equiv) and

tert-butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1 equiv)

according to General Procedure C. 4d (221 mg, 0.96 mmol, 64% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.56−7.51 (m, 1H), 7.37−7.30 (m,

1H), 7.23−7.12 (m, 2H), 5.77 (s, 1H), 3.56 (s, 2H), 2.51 (s, 3H), 1.69

(s, 9H).

3-(2-Fluoro-5-methylphenyl)-3-oxopropanenitrile (3v). 3v was

prepared from methyl 2-ﬂuoro-5-methyl-benzoate (505 mg, 3 mmol)

according to General Procedure A. 3v (323 mg, 1.82 mmol, 60% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.74 (dd, J = 7.1, 2.4 Hz, 1H), 7.45−

7.34 (m, 1H), 7.08 (dd, J = 11.3, 8.4 Hz, 1H), 4.08 (dd, J = 2.5, 0.6 Hz,

2H), 2.38 (s, 3H).

3-(2-Fluoro-5-(triﬂuoromethyl)phenyl)-3-oxopropanenitrile (3w).

3w was prepared from methyl 2-ﬂuoro-5-(triﬂuoromethyl)benzoate

(511 mg, 2.3 mmol) according to General Procedure A. 3w (489 mg,

2.11 mmol, 91% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.29−8.21 (m,

1H), 7.94−7.86 (m, 1H), 7.42−7.33 (m, 1H), 4.14−4.10 (m, 2H).

3-(2-Fluoro-5-(triﬂuoromethoxy)phenyl)-3-oxopropanenitrile

(3x). To a 20 mL scintillation vial were added 2-ﬂuoro-5-

(triﬂuoromethoxy)benzoic acid (504 mg, 2.25 mmol), concentrated

sulfuric acid (0.2 mL), and methanol (6 mL, 0.38 M). The vial was

sealed, and the reaction was heated at 75 °C for 18 h. The reaction was

cooled to room temperature, diluted with ethyl acetate (50 mL),

washed with saturated sodium bicarbonate solution (3 × 50 mL),

washed with brine (50 mL), dried with Na₂SO₄, and concentrated

under reduced pressure. The crude methyl ester was carried on without

further puriﬁcation and treated according to General Procedure A. 3x

(414 mg, 1.68 mmol, 74% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.83

(dd, J = 5.9, 2.9 Hz, 1H), 7.53−7.46 (m, 1H), 7.31−7.25 (m, 1H),

4.13−4.09 (m, 2H).

1-Methyl-3-(o-tolyl)-1H-pyrazol-5-amine (4e). 4e was prepared

from o-toluoylacetonitrile 3c (239 mg, 1.5 mmol, 1 equiv) according to

1

General Procedure B. 4e (135 mg, 0.72 mmol, 47% yield). H NMR

(400 MHz, CDCl₃) δ 7.55−7.46 (m, 1H), 7.23−7.17 (m, 3H), 5.73 (s,

1H), 3.74 (s, 3H), 3.51 (s, 2H), 2.46 (s, 3H).

1-Isopropyl-3-(2-methoxyphenyl)-1H-pyrazol-5-amine (4g). 4g

was prepared from 2-methoxybenzoylacetonitrile 3d (175 mg, 1

mmol, 1 equiv) and isopropylhydrazine hydrochloride (122 mg, 1.1

mmol, 1.1 equiv) according to General Procedure C. 4g (191 mg, 0.83

mmol, 82% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.00−7.86 (m, 1H),

7.26−7.24 (m, 1H), 7.08−6.87 (m, 2H), 6.13 (s, 1H), 4.50−4.32 (m,

1H), 3.88 (s, 3H), 3.45 (s, 2H), 1.52 (d, J = 6.7 Hz, 6H).

1-Cyclohexyl-3-(2-methoxyphenyl)-1H-pyrazol-5-amine (4h). 4h

was prepared from 2-methoxybenzoylacetonitrile 3d (175 mg, 1 mmol,

1 equiv) and cyclohexylhydrazine hydrochloride (166 mg, 1.1 mmol,

1.1 equiv) according to General Procedure C. 4h (231 mg, 0.85 mmol,

84% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.99−7.84 (m, 1H), 7.26−

7.18 (m, 1H), 7.08−6.89 (m, 2H), 6.12 (s, 1H), 4.06−3.91 (m, 1H),

3.88 (s, 3H), 3.45 (s, 2H), 2.12−1.86 (m, 6H), 1.79−1.67 (m, 1H),

1.49−1.20 (m, 3H).

1-Cyclopentyl-3-(2-methoxyphenyl)-1H-pyrazol-5-amine (4i). 4i

was prepared from 2-methoxybenzoylacetonitrile 3d (175 mg, 1.1

mmol, 1 equiv) and cyclopentylhydrazine hydrochloride (150 mg, 1.1

mmol, 1.1 equiv) according to General Procedure C. 4i (204 mg, 0.79

mmol, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.01−7.80 (m, 1H),

7.27−7.20 (m, 2H), 7.05−6.84 (m, 2H), 6.13 (s, 1H), 4.64−4.30 (m,

1H), 3.88 (s, 3H), 3.47 (s, 2H), 2.24−2.11 (m, 2H), 2.11−1.89 (m,

4H), 1.74−1.61 (m, 2H).

1-Isobutyl-3-(2-methoxyphenyl)-1H-pyrazol-5-amine (4j). 4j was

prepared from 2-methoxybenzoylacetonitrile 3d (175 mg, 1 mmol, 1

equiv) and isobutylhydrazine hydrochloride (137 mg, 1.1 mmol, 1.1

equiv) according to General Procedure C. 4j (206 mg, 0.84 mmol, 84%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.93−7.85 (m, 1H), 7.30−7.22

3-(2,6-Diﬂuorophenyl)-3-oxopropanenitrile (3y). 3y was prepared

from methyl 2,6-diﬂuorobenzoate (258 mg, 1.5 mmol) according to

1

General Procedure A. 3y (169 mg, 0.93 mmol, 62% yield). H NMR

(400 MHz, CDCl₃) δ 7.60−7.50 (m, 1H), 7.09−6.99 (m, 2H), 4.02−

3.94 (m, 2H).

3-(4-Chloro-2-ﬂuorophenyl)-3-oxopropanenitrile (3z). 3z was

prepared from methyl 4-chloro-2-ﬂuoro-benzoate (1.13 g, 6 mmol)

according to General Procedure A. 3z (1.08 g, 5.46 mmol, 91% yield).

¹H NMR (400 MHz, CDCl₃) δ 8.00−7.87 (m, 1H), 7.34−7.28 (m,

1H), 7.28−7.20 (m, 1H), 4.10−4.03 (m, 2H).

3-(2-Chloro-4-ﬂuorophenyl)-3-oxopropanenitrile (3aa). 3aa was

prepared from methyl 2-chloro-4-ﬂuoro-benzoate (1.13 g, 6 mmol)

according to General Procedure A. 3aa (1.11 g, 5.62 mmol, 93% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.79−7.70 (m, 1H), 7.25−7.19 (m,

1H), 7.16−7.06 (m, 1H), 4.15 (s, 2H).

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(m, 1H), 7.05−6.90 (m, 2H), 6.12 (s, 1H), 3.88 (s, 3H), 3.80 (d, J = 7.4

Hz, 2H), 3.44 (s, 2H), 2.36−2.19 (m, 1H), 0.97 (d, J = 6.6 Hz, 6H).

1-(tert-Butyl)-3-(2-methoxyphenyl)-1H-pyrazol-5-amine (4k). 4k

was prepared from 2-methoxybenzoylacetonitrile 3d (168 mg, 0.96

mmol, 1 equiv) and tert-butylhydrazine hydrochloride (131 mg, 1.05

mmol, 1.1 equiv) according to General Procedure C. 4k (193 mg, 0.79

mmol, 81% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.01−7.92 (m, 1H),

7.25−7.19 (m, 1H), 7.05−6.95 (m, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.18

(s, 1H), 3.88 (s, 3H), 3.56 (s, 2H), 1.69 (s, 9H).

7.64−7.58 (m, 1H), 7.31−7.24 (m, 1H), 7.24−7.17 (m, 1H), 5.88 (s,

1H), 3.61 (s, 2H), 1.69 (s, 9H).

3-(3-Chlorophenyl)-1-cyclohexyl-1H-pyrazol-5-amine (4w). 4w

was prepared from 3-chlorobenzoylacetonitrile 3k (269 mg, 1.5

mmol, 1 equiv) and cyclohexylhydrazine hydrochloride (226 mg, 1.5

mmol, 1 equiv) according to General Procedure C. 4w (328 mg, 1.19

mmol, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.72 (t, J = 2.0 Hz,

1H), 7.61−7.54 (m, 1H), 7.26−7.20 (m, 1H), 7.20−7.15 (m, 1H), 5.81

(s, 1H), 3.95−3.81 (m, 1H), 3.52 (s, 2H), 2.00−1.82 (m, 6H), 1.70 (d,

J = 12.0 Hz, 1H), 1.50−1.24 (m, 3H).

3-(2,3-Dimethylphenyl)-1-methyl-1H-pyrazol-5-amine (4x). 4x

was prepared from 3l (350 mg, 2 mmol, 1 equiv) according to General

Procedure B. 4x (197 mg, 0.98 mmol, 48% yield). ¹H NMR (400 MHz,

CDCl₃) δ 7.32−7.26 (m, 2H), 7.13−7.08 (m, 2H), 5.66 (s, 1H), 3.73

(s, 3H), 3.51 (s, 2H), 2.33 (s, 3H), 2.31 (s, 3H).

1-Methyl-3-(2-(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-amine

(4l). 4l was prepared from 3e (458 mg, 2 mmol, 1 equiv) according to

1

General Procedure B. 4l (348 mg, 1.35 mmol, 67% yield). H NMR

(400 MHz, CDCl₃) δ 8.03−7.96 (m, 1H), 7.33−7.27 (m, 3H), 6.03 (s,

1H), 3.74 (s, 3H), 3.54 (s, 2H).

1-(tert-Butyl)-3-(2-(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-

amine (4m). 4m was prepared from 3e (343 mg, 1.5 mmol, 1 equiv)

and tert-butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1 equiv)

according to General Procedure C. 4m (303 mg, 1.01 mmol, 67%

yield). ¹H NMR (400 MHz, CDCl₃) δ 8.10−8.00 (m, 1H), 7.37−7.22

(m, 4H), 6.06 (s, 1H), 3.56 (s, 2H), 1.74−1.65 (m, 9H).

1-(tert-Butyl)-3-(3-methoxyphenyl)-1H-pyrazol-5-amine (4n). 4n

was prepared from 3-methoxybenzoylacetonitrile 3f (219 mg, 1.25

mmol, 1 equiv) and tert-butylhydrazine hydrochloride (171 mg, 1.38

mmol, 1.1 equiv) according to General Procedure C. 4n (288 mg, 1.17

mmol, 93% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.35−7.29 (m, 2H),

7.28−7.21 (m, 1H), 6.84−6.76 (m, 1H), 5.89 (s, 1H), 3.85 (s, 3H),

3.59 (s, 2H), 1.75−1.59 (m, 9H).

3-(5-Fluoro-2-methylphenyl)-1-methyl-1H-pyrazol-5-amine (4y).

4y was prepared from 3m (298 mg, 1.68 mmol, 1 equiv) according to

1

General Procedure B. 4y (194 mg, 0.95 mmol, 56% yield). H NMR

(400 MHz, CDCl₃) δ 7.32−7.22 (m, 1H), 7.18−7.10 (m, 1H), 6.93−

6.84 (m, 1H), 5.72 (s, 1H), 3.72 (s, 3H), 3.54 (s, 2H), 2.41 (s, 3H).

3-(2-Fluoro-3-methylphenyl)-1-methyl-1H-pyrazol-5-amine (4z).

4z was prepared from 3n (461 mg, 2.6 mmol, 1 equiv) according to

1

General Procedure B. 4z (299 mg, 1.46 mmol, 55% yield). H NMR

(400 MHz, CDCl₃) δ 7.77−7.68 (m, 1H), 7.13−6.97 (m, 2H), 6.07−

5.93 (m, 1H), 3.74 (s, 3H), 3.56 (s, 2H), 2.33−2.28 (m, 3H).

3-(2,3-Diﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine (4aa). 4aa

was prepared from 3o (221 mg, 1.22 mmol, 1 equiv) according to

General Procedure B. 4aa (142 mg, 0.68 mmol, 55% yield). ¹H NMR

(400 MHz, CDCl₃) δ 7.74−7.62 (m, 1H), 7.11−6.99 (m, 2H), 6.05 (d,

J = 4.0 Hz, 1H), 3.75 (s, 3H), 3.55 (s, 2H).

1-Methyl-3-(2-methyl-3-(triﬂuoromethyl)phenyl)-1H-pyrazol-5-

amine (4ab). 4ab was prepared from 3p (284 mg, 1.25 mmol, 1 equiv)

according to General Procedure B. 4ab (138 mg, 0.54 mmol, 43%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.65−7.57 (m, 2H), 7.32−7.27

(m, 1H), 5.68 (s, 1H), 3.79−3.75 (m, 3H), 3.66 (s, 2H), 2.51 (d, J = 1.8

Hz, 3H).

3-(3-Chloro-2-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine

(4ac). 4ac was prepared from 3q (247 mg, 1.25 mmol, 1 equiv)

according to General Procedure B. 4ac (180 mg, 0.80 mmol, 63%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.86 (t, J = 7.4 Hz, 1H), 7.30 (t, J

= 7.3 Hz, 1H), 7.08 (m, 1H), 6.05 (d, J = 4.2 Hz, 1H), 3.76 (s, 3H),

3.71−3.49 (m, 2H).

3-(2-Fluoro-3-(triﬂuoromethoxy)phenyl)-1-methyl-1H-pyrazol-5-

amine (4ad). 4ad was prepared from 3r (247 mg, 1 mmol, 1 equiv)

according to General Procedure B. 4ad (202 mg, 0.73 mmol, 73%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.92−7.85 (m, 1H), 7.25−7.09

(m, 2H), 6.09−5.99 (m, 1H), 3.74 (s, 3H), 3.60 (s, 2H).

3-(2,5-Diﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine (4ae). 4ae

was prepared from 3s (248 mg, 1.37 mmol, 1 equiv) according to

General Procedure B. 4ae (193 mg, 0.92 mmol, 67% yield). ¹H NMR

(400 MHz, CDCl₃) δ 7.68−7.59 (m, 1H), 7.12−6.98 (m, 1H), 6.98−

6.82 (m, 1H), 6.04 (d, J = 4.1 Hz, 1H), 3.74 (s, 3H), 3.54 (s, 2H).

3-(2-Chloro-5-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine (4af).

4af was prepared from 3t (247 mg, 1.25 mmol, 1 equiv) according to

General Procedure B. 4af (159 mg, 0.71 mmol, 56% yield). ¹H NMR

(400 MHz, CDCl₃) δ 7.57−7.49 (m, 1H), 7.41−7.31 (m, 1H), 6.98−

6.85 (m, 1H), 6.15 (s, 1H), 3.74 (s, 3H), 3.69−3.43 (m, 2H).

3-(5-Chloro-2-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine

(4ag). 4ag was prepared from 3u (247 mg, 1.25 mmol, 1 equiv)

according to General Procedure B. 4ag (183 mg, 0.81 mmol, 64%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.98−7.88 (m, 1H), 7.20−7.13

(m, 1H), 7.08−6.93 (m, 1H), 6.03 (d, J = 4.1 Hz, 1H), 3.74 (s, 3H),

3.53 (s, 2H).

1-(tert-Butyl)-3-(m-tolyl)-1H-pyrazol-5-amine (4o). 4o was pre-

pared from m-toluoylacetonitrile 3g (199 mg, 1.25 mmol, 1 equiv) and

tert-butylhydrazine hydrochloride (171 mg, 1.38 mmol, 1.1 equiv)

according to General Procedure C. 4o (276 mg, 1.20 mmol, 96% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.62−7.57 (m, 1H), 7.56−7.48 (m,

1H), 7.23 (t, J = 7.6 Hz, 1H), 7.10−7.01 (m, 1H), 5.89 (s, 1H), 3.58 (s,

2H), 2.37 (s, 3H), 1.69 (s, 9H).

1-(tert-Butyl)-3-(3-(triﬂuoromethyl)phenyl)-1H-pyrazol-5-amine

(4p). 4p was prepared from 3-(triﬂuoromethyl)benzoylacetonitrile 3h

(266 mg, 1.25 mmol, 1 equiv) and tert-butylhydrazine hydrochloride

(171 mg, 1.38 mmol, 1.1 equiv) according to General Procedure C. 4p

(255 mg, 0.90 mmol, 71% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.04−

7.95 (m, 1H), 7.93−7.85 (m, 1H), 7.52−7.39 (m, 2H), 5.93 (s, 1H),

3.63 (s, 2H), 1.70 (s, 9H).

1-Methyl-3-(3-(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-amine

(4q). 4q was prepared from 3i (344 mg, 1.5 mmol, 1 equiv) according to

1

General Procedure B. 4q (246 mg, 0.96 mmol, 63% yield). H NMR

(400 MHz, CDCl₃) δ 7.70−7.60 (m, 1H), 7.60−7.55 (m, 1H), 7.37 (t, J

= 8.0 Hz, 1H), 7.15−7.06 (m, 1H), 5.86 (s, 1H), 3.72 (s, 3H), 3.57 (s,

2H).

1-(tert-Butyl)-3-(3-(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-

amine (4r). 4r was prepared from 3i (344 mg, 1.65 mmol, 1 equiv) and

tert-butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1 equiv)

according to General Procedure C. 4r (248 mg, 0.83 mmol, 55% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.74−7.63 (m, 1H), 7.62−7.57 (m,

1H), 7.35 (t, J = 8.0 Hz, 1H), 7.12−7.04 (m, 1H), 5.89 (s, 1H), 3.60 (s,

2H), 1.69 (s, 9H).

1-(tert-Butyl)-3-(3-ﬂuorophenyl)-1H-pyrazol-5-amine (4t). 4t was

prepared from 3-ﬂuorobenzoylacetonitrile 3j (245 mg, 1.5 mmol, 1

equiv) and tert-butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1

equiv) according to General Procedure C. 4t (222 mg, 0.95 mmol, 63%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.54−7.40 (m, 2H), 7.35−7.25

(m, 1H), 6.98−6.88 (m, 1H), 5.89 (s, 1H), 3.61 (s, 2H), 1.69 (s, 9H).

3-(3-Chlorophenyl)-1-methyl-1H-pyrazol-5-amine (4u). 4u was

prepared from 3-chlorobenzoylacetonitrile 3k (898 mg, 5 mmol, 1

equiv) according to General Procedure B. 4u (614 mg, 2.96 mmol, 59%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.72 (s, 1H), 7.61 (d, J = 7.6 Hz,

1H), 7.33−7.21 (m, 2H), 5.86 (s, 1H), 3.75 (s, 3H), 3.60 (s, 2H).

1-(tert-Butyl)-3-(3-chlorophenyl)-1H-pyrazol-5-amine (4v). 4v

was prepared from 3-chorobenzoylacetonitrile 3k (224 mg, 1.25

mmol, 1 equiv) and tert-butylhydrazine hydrochloride (171 mg, 1.38

mmol, 1.1 equiv) according to General Procedure C. 4v (232 mg, 0.93

mmol, 74% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.77−7.73 (m, 1H),

3-(2-Fluoro-5-methylphenyl)-1-methyl-1H-pyrazol-5-amine

(4ah). 4ah was prepared from 3v (323 mg, 1.82 mmol, 1 equiv)

according to General Procedure B. 4ah (271 mg, 1.32 mmol, 72%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.81−7.64 (m, 1H), 7.14−6.80

(m, 2H), 6.13−5.91 (m, 1H), 3.81−3.66 (m, 3H), 3.66−3.46 (m, 2H),

2.33 (s, 3H).

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3-(2-Fluoro-5-(triﬂuoromethyl)phenyl)-1-methyl-1H-pyrazol-5-

amine (4ai). 4ai was prepared from 3w (289 mg, 1.25 mmol, 1 equiv)

according to General Procedure B. 4ai (109 mg, 0.42 mmol, 33% yield).

¹H NMR (400 MHz, CDCl₃) δ 8.33−8.24 (m, 1H), 7.54−7.43 (m,

1-(tert-Butyl)-3-(2-ﬂuoro-3-methylphenyl)-1H-pyrazol-5-amine

(4au). 4au was prepared from 3n (266 mg, 1.5 mmol, 1 equiv) and tert-

butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1 equiv)

according to General Procedure C. 4au (238 mg, 0.96 mmol, 64%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.86−7.78 (m, 1H), 7.11−6.94

(m, 2H), 6.10−5.97 (m, 1H), 3.55 (s, 2H), 2.33−2.25 (m, 3H), 1.69 (s,

9H).

1-Cyclopentyl-3-(2-ﬂuoro-3-methylphenyl)-1H-pyrazol-5-amine

(4av). 4av was prepared from 3n (177 mg, 1 mmol, 1 equiv) and

cyclopentylhydrazine hydrochloride (150 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4av (208 mg, 0.80 mmol, 80%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.91−7.79 (m, 1H), 7.20−6.99

(m, 2H), 6.09−6.02 (m, 1H), 4.74−4.49 (m, 1H), 2.37−2.23 (m, 3H),

2.24−1.88 (m, 6H), 1.80−1.56 (m, 2H).

1-Cyclohexyl-3-(2-ﬂuoro-3-methylphenyl)-1H-pyrazol-5-amine

(4aw). 4aw was prepared from 3n (266 mg, 1.5 mmol, 1 equiv) and

cyclohexylhydrazine hydrochloride (226 mg, 1 mmol, 1 equiv)

according to General Procedure C. 4aw (322 mg, 1.18 mmol, 78%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.82−7.72 (m, 1H), 7.10−6.97

(m, 2H), 6.02 (d, J = 4.2 Hz, 1H), 4.02−3.86 (m, 1H), 3.47 (s, 2H),

2.30 (d, J = 2.4 Hz, 3H), 2.07−1.83 (m, 6H), 1.78−1.68 (m, 1H),

1.49−1.22 (m, 3H).

1H), 7.23−7.13 (m, 1H), 6.10−6.00 (m, 1H), 3.78 (s, 3H), 3.69 (s,

2H).

3-(2-Fluoro-5-(triﬂuoromethoxy)phenyl)-1-methyl-1H-pyrazol-5-

amine (4aj). 4aj was prepared from 3x (247 mg, 1 mmol, 1 equiv)

according to General Procedure B. 4aj (163 mg, 0.59 mmol, 59% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.86−7.81 (m, 1H), 7.11−7.05 (m,

2H), 6.04 (dd, J = 4.1, 0.7 Hz, 1H), 3.75 (s, 3H), 3.59 (s, 2H).

3-(2,6-Diﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine (4ak). 4ak

was prepared from 3y (169 mg, 0.93 mmol, 1 equiv) according to

1

General Procedure B. 4ak (48 mg, 0.23 mmol, 24% yield). H NMR

(400 MHz, CDCl₃) δ 7.26−7.15 (m, 1H), 6.94 (t, J = 8.3 Hz, 2H), 5.90

(d, J = 1.9 Hz, 1H), 3.77 (s, 3H), 3.53 (s, 2H).

3-(4-Chloro-2-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine (4al).

4al was prepared from 3z (296 mg, 1.5 mmol, 1 equiv) according to

General Procedure B. 4al (172 mg, 0.76 mmol, 50% yield). ¹H NMR

(400 MHz, CDCl₃) δ 7.90 (t, J = 8.3 Hz, 1H), 7.17−7.08 (m, 2H), 6.01

(d, J = 4.1 Hz, 1H), 3.75 (s, 3H), 3.70−3.51 (m, 2H).

3-(2-Chloro-4-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-amine

(4am). 4am was prepared from 3aa (296 mg, 1.5 mmol, 1 equiv)

according to General Procedure B. 4am (135 mg, 0.60 mmol, 39%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.80−7.69 (m, 1H), 7.21−7.09

(m, 1H), 7.09−6.95 (m, 1H), 6.06 (s, 1H), 3.74 (s, 3H), 3.60 (s, 2H).

1-(tert-Butyl)-3-(2,3-dimethylphenyl)-1H-pyrazol-5-amine (4ao).

4ao was prepared from 3l (260 mg, 1.5 mmol, 1 equiv) and tert-

butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1 equiv)

according to General Procedure C. 4ao (122 mg, 0.50 mmol, 33%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.33−7.27 (m, 1H), 7.12−7.03

(m, 2H), 5.69 (s, 1H), 3.60 (s, 2H), 2.37 (s, 3H), 2.30 (s, 3H), 1.69 (s,

9H).

3-(5-Fluoro-2-methylphenyl)-1-isopropyl-1H-pyrazol-5-amine

(4ap). 4ap was prepared from 3m (177 mg, 1 mmol, 1 equiv) and

isopropylhydrazine hydrochloride (82 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4ap (93 mg, 0.40 mmol, 40% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.30−7.23 (m, 1H), 7.23−7.08 (m,

3-(2,3-Diﬂuorophenyl)-1-isopropyl-1H-pyrazol-5-amine (4ax).

4ax was prepared from 3o (181 mg, 1 mmol, 1 equiv) and

isopropylhydrazine hydrochloride (82 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4ax (128 mg, 0.54 mmol, 54%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.84−7.74 (m, 1H), 7.14−6.99

(m, 2H), 6.08−6.02 (m, 1H), 4.52−4.37 (m, 1H), 3.76−3.55 (m, 2H),

1.52 (d, J = 6.4 Hz, 6H).

1-(tert-Butyl)-3-(2,3-diﬂuorophenyl)-1H-pyrazol-5-amine (4ay).

4ay was prepared from 3o (272 mg, 1.5 mmol, 1 equiv) and tert-

butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1 equiv)

according to General Procedure C. 4ay (244 mg, 0.97 mmol, 64%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.83−7.71 (m, 1H), 7.13−6.96

(m, 2H), 6.12−6.04 (m, 1H), 3.78−3.51 (m, 2H), 1.69 (s, 9H).

1-Cyclopentyl-3-(2,3-diﬂuorophenyl)-1H-pyrazol-5-amine (4az).

4az was prepared from 3o (181 mg, 1 mmol, 1 equiv) and

cyclopentylhydrazine hydrochloride (150 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4az (193 mg, 0.73 mmol, 73%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.82−7.73 (m, 1H), 7.11−6.98

(m, 2H), 6.05 (d, J = 4.1 Hz, 1H), 4.55 (p, J = 7.4 Hz, 1H), 3.85−3.53

(m, 2H), 2.27−1.91 (m, 6H), 1.78−1.59 (m, 2H).

1-Cyclohexyl-3-(2,3-diﬂuorophenyl)-1H-pyrazol-5-amine (4bb).

4bb was prepared from 3o (181 mg, 1.5 mmol, 1 equiv) and

cyclohexylhydrazine hydrochloride (166 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4bb (237 mg, 0.85 mmol, 85%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.92−7.83 (m, 1H), 7.10 (t, J =

6.1 Hz, 2H), 6.10 (d, J = 3.8 Hz, 1H), 4.24−4.04 (m, 1H), 2.22−2.05

(m, 1H), 2.03−1.86 (m, 5H), 1.74−1.66 (m, 1H), 1.51−1.26 (m, 3H).

Recorcylate Amide Bromide Synthesis. 2-Bromo-4,6-dime-

thoxybenzaldehyde (S1). To a 250 mL round-bottom ﬂask equipped

with a magnetic stir bar were added 3,5-dimethoxybromobenzene (10.0

g, 46.1 mmol) and DMF (23 mL, 2 M). The mixture was cooled to 0 °C,

and POCl₃(12.8 mL, 138 mmol, 3 equiv) was added dropwise over 5

min. The reaction was warmed to room temperature and then heated to

90 °C for 6 h. The reaction was cooled to room temperature and poured

into ice water (200 mL). The reaction was quenched with a slow

addition of KOH (55 g) to reach pH 14. The slurry was warmed to

room temperature and stirred for 16 h. The aqueous phase was

extracted with Et₂O (3 × 200 mL), and the combined organic extracts

were washed with water (3 × 100 mL) and brine (150 mL), dried with

Na₂SO₄, and concentrated under reduced pressure. No further

puriﬁcation was required aﬀording aldehyde S1 as a brown solid

(10.4 g, 92% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.31 (s, 1H), 6.78

(d, J = 2.2 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H), 3.95−3.80 (m, 6H).

UPLC/MS [M + H] = 245.359, t_R= 1.41 min.

1H), 6.92−6.83 (m, 1H), 5.72 (s, 1H), 4.51−4.32 (m, 1H), 3.65 (s,

2H), 2.43 (s, 3H), 1.51 (d, J = 6.6 Hz, 6H).

1-(tert-Butyl)-3-(5-ﬂuoro-2-methylphenyl)-1H-pyrazol-5-amine

(4aq). 4aq was prepared from 3m (266 mg, 1.5 mmol, 1 equiv) and tert-

butylhydrazine hydrochloride (206 mg, 1.65 mmol, 1.1 equiv)

according to General Procedure C. 4aq (118 mg, 0.48 mmol, 31%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.32−7.27 (m, 1H), 7.17−7.10

(m, 1H), 6.89−6.79 (m, 1H), 5.78 (s, 1H), 3.58 (s, 2H), 2.46 (s, 3H),

1.68 (s, 9H).

1-Cyclopentyl-3-(5-ﬂuoro-2-methylphenyl)-1H-pyrazol-5-amine

(4ar). 4ar was prepared from 3m (177 mg, 1 mmol, 1 equiv) and

cyclopentylhydrazine hydrochloride (150 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4ar (202 mg, 0.78 mmol, 78%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.34−7.24 (m, 1H), 7.18−7.10

(m, 1H), 6.88 (m, 1H), 5.75 (s, 1H), 4.67−4.53 (m, 1H), 2.43 (s, 3H),

2.23−1.87 (m, 6H), 1.67 (m, 2H).

1-Cyclohexyl-3-(5-ﬂuoro-2-methylphenyl)-1H-pyrazol-5-amine

(4as). 4as was prepared from 3m (177 mg, 1 mmol, 1 equiv) and

cyclohexylhydrazine hydrochloride (166 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4as (185 mg, 0.68 mmol, 68%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.29−7.21 (m, 1H), 7.18−7.08

(m, 1H), 6.93−6.83 (m, 1H), 5.75−5.70 (m, 1H), 4.08−3.92 (m, 1H),

3.77 (s, 2H), 2.42 (s, 3H), 2.04−1.85 (m, 7H), 1.71 (d, J = 12.6 Hz,

1H), 1.50−1.29 (m, 2H).

3-(2-Fluoro-3-methylphenyl)-1-isopropyl-1H-pyrazol-5-amine

(4at). 4at was prepared from 3n (177 mg, 1 mmol, 1 equiv) and

isopropylhydrazine hydrochloride (82 mg, 1.1 mmol, 1.1 equiv)

according to General Procedure C. 4at (149 mg, 0.64 mmol, 64%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.83 (td, J = 7.4, 2.0 Hz, 1H),

7.13−6.99 (m, 2H), 6.03 (d, J = 4.1 Hz, 1H), 4.46 (hept, J = 6.6 Hz,

1H), 3.73 (s, 1H), 2.30 (d, J = 2.3 Hz, 3H), 1.52 (d, J = 6.6 Hz, 6H).

2-Bromo-4,6-dihydroxybenzaldehyde (6). To a ﬂamed-dried 250

mL round-bottom ﬂask equipped with a magnetic stir bar was added 2-

bromo-4,6-dimethoxybenzaldehyde S1 (10 g, 40.8 mmol). The ﬂask

was ﬁtted with a rubber septum, evacuated, and backﬁlled with N₂.

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Anhydrous CH₂Cl₂(150 mL, 0.27 M) was added, and the mixture was

cooled to 0 °C. In a separate round-bottom ﬂask, a solution of BBr₃

(11.6 mL, 122 mmol, 3 equiv) in anhydrous CH₂Cl₂(30 mL, 4 M) was

prepared. The BBr₃solution was added dropwise via a cannula over 15

min. The reaction was slowly warmed to room temperature as the ice

bath melted and stirred for 18 h. The reaction was poured into ice water

(300 mL), extracted with EtOAc (3 × 200 mL), washed with brine (300

mL), dried with Na₂SO₄, and concentrated under reduced pressure.

The crude product was puriﬁed by silica ﬂash chromatography (5−50%

acetone in hexanes) aﬀording aldehyde 6 (6.61 g, 75% yield) as a light

purple solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.19 (s, 1H), 9.97 (s,

1H), 6.71 (d, J = 2.1 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H). UPLC/MS [M +

H] = 216.956, t_R= 1.29 min.

2,4-Bis(benzyloxy)-6-bromobenzaldehyde (7). To a 200 mL

round-bottom ﬂask equipped with a magnetic stir bar were added 2-

bromo-4,6-dihydroxybenzaldehyde 6 (3.33 g, 15.3 mmol), K₂CO₃(5.3

g, 38.3 mmol, 2.5 equiv), benzyl bromide (4.6 mL, 38.3 mmol, 2.5

equiv), and MeCN (45 mL, 0.33 M). The ﬂask was ﬁtted with a reﬂux

condenser, and the reaction was heated at reﬂux in a 90 °C oil bath for

16 h. The reaction was cooled to room temperature, and the salts were

removed by vacuum ﬁltration and washed with EtOAc (100 mL). The

ﬁltrate was concentrated under reduced pressure and puriﬁed by silica

ﬂash chromatography (2−40% EtOAc in hexanes) aﬀording aldehyde 7

(5.0 g, 82% yield) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ

10.39 (s, 1H), 7.49−7.29 (m, 10H), 6.89 (d, J = 2.1 Hz, 1H), 6.57 (d, J

= 2.2 Hz, 1H), 5.13 (s, 2H), 5.07 (s, 2H); ¹³C NMR (101 MHz,

CDCl₃) δ 188.86, 163.47, 162.92, 135.66, 135.43, 128.94, 128.87,

128.71, 128.40, 127.76, 127.18, 126.64, 117.63, 113.07, 100.33, 70.96,

70.76. UPLC/MS [M + H] = 397.337, t_R= 2.30 min.

General Procedure D. To a 100 mL round-bottom ﬂask equipped

with a magnetic stir bar were added 2,4-bis(benzyloxy)-6-bromoben-

zaldehyde 7 (1.59 g, 4 mmol), t-BuOH (12 mL), and THF (10 mL). In

a separate ﬂask, sodium chlorite (1.09 g, 12 mmol, 3 equiv) and sodium

monobasic phosphate monohydrate (4.14 g, 30 mmol, 7.5 equiv) were

dissolved in H₂O (10 mL). The aqueous solution was added to the

reaction portion-wise over 2 min, turning the reaction bright yellow. 2-

Methyl-2-butene (5.1 mL, 48 mmol, 12 equiv) was added, and the

reaction was stirred for 30 min until the yellow color dissipated and the

reaction turned colorless again. The reaction was quenched with 3 M

HCl (30 mL) and extracted with EtOAc (3 × 50 mL). The combined

organic extracts were washed with saturated NH₄Cl solution (2 × 50

mL), washed with brine (50 mL), dried with Na₂SO₄, and concentrated

under reduced pressure. The resulting benzoic acid was immediately

used without puriﬁcation.

In a 100 mL round-bottom ﬂask equipped with a magnetic stir bar,

the crude benzoic acid was dissolved in CH₂Cl₂(12 mL) and THF (12

mL). Amine (4.40 mmol, 1.1 equiv), N,N-diisopropylethylamine

(DIPEA, 1.74 mL, 10 mmol, 2.5 equiv), and HATU (1.83 g, 4.80 mmol,

1.2 equiv) were added. The reaction was stirred at room temperature

for 3 h. The reaction was quenched with saturated NaHCO₃solution

(30 mL), extracted with CH₂Cl₂(3 × 50 mL), washed with brine (75

mL), dried with Na₂SO₄, and concentrated under reduced pressure.

The crude product was puriﬁed by silica ﬂash chromatography (5−60%

EtOAc in hexane) to aﬀord the desired amide 8.

6.54 (m, 1H), 5.09−4.99 (m, 6H), 4.67−4.40 (m, 2H). UPLC/MS [M

+ H] = 532.195, t_R= 2.09 min.

(2,4-Bis(benzyloxy)-6-bromophenyl)(5-ﬂuoroisoindolin-2-yl)-

methanone (8c). 8c was prepared according to General Procedure D

from 5-ﬂuoroisoindoline hydrochloride (382 mg, 2.2 mmol). 8c (917

mg, 1.72 mmol) was obtained in 86% yield.

¹H NMR (400 MHz, CDCl₃) δ 7.55−7.32 (m, 5H), 7.33−7.19 (m,

6H), 7.13−6.92 (m, 2H), 6.87−6.78 (m, 1H), 6.58 (dd, J = 2.2, 1.2 Hz,

1H), 5.09−4.90 (m, 6H), 4.60−4.38 (m, 2H). UPLC/MS [M + H] =

532.438, t_R= 2.08 min.

RAP Synthesis. General Procedure E. E1a: To a ﬂame-dried 2D

vial equipped with a magnetic stir bar was added resorcylate amide

bromide 8a−c (0.15 mmol, 1 equiv), aminopyrazole 4a−bb (0.165

mmol, 1.1 equiv), Pd(OAc)₂(15 μmol, 0.1 equiv), xantphos (30 μmol,

0.2 equiv), and Cs₂CO₃(0.3 mmol, 2 equiv). The vial was ﬁtted with a

rubber septum, evacuated, backﬁlled with N₂, and anhydrous toluene

(0.6 mL, 0.25 M) was added. The septum was replaced with a cap, and

the vial was sealed with PTFE tape. The reaction was heated at 130 °C

for 16 h in a heating block. The reaction was cooled to room

temperature and ﬁltered through silica plug eluting with EtOAc (12

mL). The ﬁltrate was concentrated and used without further

puriﬁcation.

E1b: To a ﬂame-dried microwave vial equipped with a magnetic stir

bar were added resorcylate amide bromide 8a−c (0.15 mmol, 1 equiv),

aminopyrazole 4a−bb (0.165 mmol, 1.1 equiv), Pd₂(dba)₃(6 μmol,

0.04 equiv), xanthphos (15 μmol, 0.1 equiv), and sodium phenoxide

(0.225 mmol, 1.5 equiv). The vial was ﬁtted with a rubber septum,

evacuated, backﬁlled with N₂, and anhydrous 1,4-dioxane (0.1 M

reaction concentration) was added. The septum was replaced with a

microwave cap, and the reaction was heated in a microwave holding at

170 °C for 2 h. The reaction was cooled to room temperature and

ﬁltered through a silica plug eluting with EtOAc (8× reaction volume).

The ﬁltrate was concentrated and used without further puriﬁcation.

E2a: In a 2D vial equipped with a magnetic stir bar were added

benzyl-protected RAP (1 equiv), MeOH (2 mL), and Pd/C (20% wt).

The vial was ﬁtted with a rubber septum and a hydrogen balloon.

Hydrogen was bubbled through the solution for 10 min; then, the

reaction was stirred under hydrogen atmosphere for 16 h or until full

conversion was observed by UPLC/MS. The reaction was ﬁltered

through celite plug eluting with MeOH (6 mL) and EtOAc (6 mL) and

concentrated. The crude material was puriﬁed by mass-guided

preparative HPLC.

E2b: In a 2D vial equipped with a magnetic stir bar was added benzyl-

protected RAP (1 equiv), EtOAc (2 mL), and Pd(OH)₂/C (20% wt).

The vial was ﬁtted with a rubber septum and a hydrogen balloon.

Hydrogen was bubbled through the solution for 10 min; then, the

reaction was stirred under hydrogen atmosphere for 16 h or until full

conversion was observed by UPLC/MS. The reaction was ﬁltered

through celite plug eluting with MeOH (6 mL) and EtOAc (6 mL) and

concentrated. The crude material was puriﬁed by mass-guided

preparative HPLC.

(2-((3-(2-Fluorophenyl)-1-methyl-1H-pyrazol-5-yl)amino)-4,6-

dihydroxyphenyl)(isoindolin-2-yl)methanone (9a). Amide bromide

8a (45 mg, 0.087 mmol, 1 equiv) was coupled to 5-amino-3-(2-

ﬂuorophenyl)-1-methylpyrazole 4a (18.4 mg, 0.096 mmol, 1.1 equiv)

according to General Procedure E1a. The resulting product was

deprotected according to General Procedure E2a. 9a (16.7 mg, 0.037

mmol) was obtained in a 43% yield over two steps. ¹H NMR (500 MHz,

DMSO-d₆) δ 9.64 (s, 1H), 9.31 (s, 1H), 7.89 (dt, J = 1.90, 7.8 Hz, 1H),

7.29−7.35 (m, 3H), 7.24−7.29 (m, 2H), 7.22−7.24 (m, 1H), 7.21 (s,

2H), 7.20 (s, 1H), 6.36 (d, J = 3.9 Hz, 1H), 5.89 (d, J = 2.0 Hz, 1H),

5.62 (d, J = 2.0 Hz, 1H), 4.63−4.86 (m, 4H), 3.63 (s, 3H); ¹³C NMR

(126 MHz, DMSO-d₆) δ 166.5, 160.3, 159.4, 158.3, 155.7, 143.9, 142.8,

141.6, 141.6, 129.2, 129.1, 127.6, 127.4, 124.6, 123.0, 121.3, 121.2,

116.3, 116.1, 104.5, 99.8, 99.7, 94.6, 93.0, 40.6, 35.2.

(2,4-Bis(benzyloxy)-6-bromophenyl)(isoindolin-2-yl)methanone

(8a). 8a was prepared according to General Procedure D from

isoindoline hydrochloride (685 mg, 4.40 mmol, 1.1 equiv). 8a (1.67 g,

1

3.24 mmol) was obtained in 81% yield as a colorless solid. H NMR

(400 MHz, CDCl₃) δ 7.48−7.22 (m, 13H), 7.15 (d, J = 7.3 Hz, 1H),

6.85 (d, J = 2.1 Hz, 1H), 6.59 (d, J = 2.1 Hz, 1H), 5.16−5.05 (m, 2H),

5.02 (d, J = 7.1 Hz, 4H), 4.68−4.45 (m, 2H); ¹³C NMR (101 MHz,

CDCl₃) δ 165.90, 160.46, 156.51, 136.54, 136.47, 136.16, 136.03,

128.86, 128.70, 128.48, 128.12, 127.86, 127.71, 127.62, 127.04, 123.27,

122.74, 122.19, 120.39, 110.63, 100.80, 70.75, 70.63, 53.32, 51.89.

UPLC/MS [M + H] = 514.231, t_R= 2.10 min.

(2,4-Bis(benzyloxy)-6-bromophenyl)(4-ﬂuoroisoindolin-2-yl)-

methanone (8b). 8b was prepared according to General Procedure D

from 4-ﬂuoroisoindoline (290 mg, 2.11 mmol). 8b (585 mg, 1.10

mmol) was obtained in 57% yield. H NMR (400 MHz, CDCl₃) δ

7.47−7.21 (m, 11H), 7.15−6.87 (m, 2H), 6.86−6.80 (m, 1H), 6.68−

(2-((3-(2-Chlorophenyl)-1-methyl-1H-pyrazol-5-yl)amino)-4,6-

dihydroxyphenyl)(isoindolin-2-yl)methanone (9b). Amide bromide

8a (51 mg, 0.1 mmol, 1 equiv) was coupled to 4b (23 mg, 0.11 mmol,

1.1 equiv) according to General Procedure E1a. The resulting product

was deprotected according to General Procedure E2a. 9b (3.4 mg,

1

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0.007 mmol) was obtained in 7% yield over two steps. ¹H NMR (500

MHz, DMSO-d₆): δ 9.64 (s, 1H), 9.32 (s, 1H), 7.78−7.72 (m, 1H),

7.50−7.45 (m, 1H), 7.38−7.29 (m, 4H), 7.29−7.25 (m, 2H), 7.21 (s,

1H), 6.48 (s, 1H), 5.90 (d, J = 2.1 Hz, 1H), 5.65 (d, J = 2.1 Hz, 1H),

4.82−4.69 (m, 4H), 3.62 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.34, 159.23, 155.52, 145.71, 143.74, 140.82, 132.16, 130.72, 130.20,

129.88, 128.88, 127.23, 127.14, 122.86, 104.28, 100.19, 94.50, 92.87,

35.04. UPLC/MS [M + H] = 461.337, t_R= 2.36 min.

(2,4-Dihydroxy-6-((1-methyl-3-(2-(triﬂuoromethyl)phenyl)-1H-

pyrazol-5-yl)amino)phenyl)(isoindolin-2-yl)methanone (9c). Amide

bromide 8a (77 mg, 0.15 mmol, 1 equiv) was coupled to 4c (40 mg,

0.165 mmol, 1.1 equiv) according to General Procedure E1a. The

resulting product was deprotected according to General Procedure E2a.

9c (13 mg, 0.026 mmol) was obtained in 17% yield over two steps. ¹H

NMR (500 MHz, DMSO-d₆): δ 9.63−9.59 (m, 1H), 9.32 (s, 1H), 7.78

(dd, J = 8.0, 1.3 Hz, 1H), 7.68−7.63 (m, 1H), 7.63−7.60 (m, 1H),

7.58−7.53 (m, 1H), 7.41−7.25 (m, 4H), 7.23 (s, 1H), 6.18 (s, 1H),

5.91 (d, J = 2.1 Hz, 1H), 5.64 (d, J = 2.1 Hz, 1H), 4.75 (s, 4H), 3.61 (s,

3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.32, 159.22, 155.48,

146.45, 143.68, 140.85, 137.21, 133.27, 132.20, 131.56, 128.06, 127.25,

126.09, 125.26, 122.88, 104.34, 99.67, 94.51, 92.87, 35.02. UPLC/MS

[M + H] = 495.601, t_R= 1.49 min.

(2-((1-(tert-Butyl)-3-(o-tolyl)-1H-pyrazol-5-yl)amino)-4,6-

dihydroxyphenyl)(isoindolin-2-yl)methanone (9d). Amide bromide

8a (62 mg, 0.120 mmol, 1 equiv) was coupled to 4d (30 mg, 0.132

mmol, 1.1 equiv) according to General Procedure E1b. The resulting

product was deprotected according to General Procedure E2a. 9d (18.5

mg, 0.038 mmol) was obtained in 32% yield over two steps. ¹H NMR

(500 MHz, DMSO-d₆): δ 9.69 (s, 1H), 9.29 (s, 1H), 7.58−7.51 (m,

1H), 7.38−7.32 (m, 2H), 7.32−7.26 (m, 2H), 7.24−7.16 (m, 3H), 6.98

(s, 1H), 6.37 (s, 1H), 5.85 (d, J = 2.1 Hz, 1H), 5.70 (d, J = 2.1 Hz, 1H),

4.81 (s, 4H), 2.48 (s, 3H), 1.55 (s, 9H); ¹³C NMR (126 MHz, DMSO-

d₆): δ 167.36, 159.88, 156.00, 147.57, 145.54, 139.92, 137.13, 135.37,

133.63, 131.35, 128.69, 127.73, 127.45, 126.14, 123.31, 103.48, 103.20,

94.24, 92.56, 59.70, 29.89, 21.90 UPLC/MS [M + H] = 483.575, t_R=

1.81 min.

(m, 3H), 7.17−7.21 (m, 3H), 6.90 (br t, J = 7.50 Hz, 1H), 6.85 (d, J =

8.31 Hz, 1H), 6.72 (s, 1H), 6.49 (s, 1H), 5.97 (d, J = 0.98 Hz, 1H), 5.76

(d, J = 1.96 Hz, 1H), 4.94−5.09 (m, 2H), 4.81−4.93 (m, 2H), 3.75 (s,

3H), 3.66 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 168.8, 160.5, 156.8,

156.4, 147.1, 145.3, 140.3, 136.7, 129.0, 128.3, 127.7, 122.9, 122.3,

120.9, 111.4, 103.1, 101.6, 95.6, 93.8, 77.5, 55.5, 53.0, 40.9, 35.1.

UPLC/MS [M + H] = 457.409, t_R= 1.56 min.

(2,4-Dihydroxy-6-((1-isopropyl-3-(2-methoxyphenyl)-1H-pyra-

zol-5-yl)amino)phenyl)(isoindolin-2-yl)methanone (9g). Amide bro-

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4g (38 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1a. The resulting

product was deprotected according to General Procedure E2a. 9g (22

mg, 0.046 mmol) was obtained in 37% yield over two steps. ¹H NMR

(500 MHz, DMSO-d₆): δ 9.58 (s, 1H), 9.25 (s, 1H), 7.91 (m, 1H),

7.42−7.21 (m, 5H), 7.12−6.94 (m, 3H), 6.46 (s, 1H), 5.84 (d, J = 2.1

Hz, 1H), 5.54 (d, J = 2.1 Hz, 1H), 4.77 (m, 4H), 4.50−4.37 (p, J = 6.6

Hz, 1H), 3.85−3.78 (m, 3H), 1.35 (d, J = 6.6 Hz, 6H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.62, 159.35, 156.32, 155.64, 145.20, 145.08,

139.03, 136.90, 128.51, 127.40, 127.25, 123.03, 122.49, 120.49, 111.89,

103.84, 102.14, 94.11, 92.40, 55.49, 47.87, 22.45. UPLC/MS [M + H]

= 485.231, t_R= 1.52 min.

(2-((1-Cyclohexyl-3-(2-methoxyphenyl)-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9h). Amide bro-

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4h (45 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1a. The resulting

product was deprotected according to General Procedure E2a. 9h (20

mg, 0.039 mmol) was obtained in 26% yield over two steps. ¹H NMR

(500 MHz, DMSO-d₆): δ 9.66 (s, 1H), 9.33 (s, 1H), 8.01−7.96 (m,

1H), 7.43 (t, J = 4.4 Hz, 2H), 7.40−7.31 (m, 3H), 7.17−7.09 (m, 2H),

7.09−7.02 (m, 1H), 6.56 (s, 1H), 5.94 (d, J = 2.1 Hz, 1H), 5.65 (d, J =

2.1 Hz, 1H), 4.86 (s, 4H), 4.15−4.05 (m, 1H), 3.89 (s, 3H), 1.99−1.81

(m, 6H), 1.69−1.65 (m, 1H), 1.36−1.19 (m, 3H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.49, 159.16, 156.15, 155.46, 144.91, 144.86,

138.97, 136.70, 128.33, 127.25, 127.07, 122.84, 122.30, 120.32, 111.72,

103.76, 102.07, 94.01, 92.29, 55.32, 55.25, 32.34, 25.12, 24.91. UPLC/

MS [M + H] = 526.363, t_R= 3.06 min.

(2-((1-Cyclohexyl-3-(2-methoxyphenyl)-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(4-ﬂuoroisoindolin-2-yl)methanone (9i).

Amide bromide 8b (48 mg, 0.090 mmol, 1 equiv) was coupled to 4h

(27 mg, 0.099 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9i (19 mg, 0.035 mmol) was obtained in 39% yield over two steps.

¹H NMR (500 MHz, DMSO-d₆): δ 9.59 (s, 1H), 9.26 (s, 1H), 7.93−

7.85 (m, 1H), 7.37−7.30 (m, 1H), 7.28−7.22 (m, 1H), 7.19 (d, J = 7.6

Hz, 1H), 7.10 (t, J = 8.8 Hz, 1H), 7.07−7.00 (m, 2H), 7.00−6.93 (m,

1H), 6.46 (s, 1H), 5.85 (d, J = 2.1 Hz, 1H), 5.57 (d, J = 2.1 Hz, 1H),

4.92−4.68 (m, 4H), 4.07−3.97 (m, 1H), 3.79 (s, 3H), 1.91−1.70 (m,

7H), 1.61−1.57 (m, 1H), 1.28−1.14 (m, 3H), ¹³C NMR (126 MHz,

DMSO-d₆): δ 166.43, 159.25, 156.11, 155.52, 144.96, 144.87, 138.97,

128.29, 127.01, 122.24, 120.29, 113.63 (d, J = 19.4 Hz), 111.67, 103.45,

102.04, 93.99, 92.49, 79.16, 55.27, 32.34, 25.11, 24.91. UPLC/MS [M

+ H] = 543.598, t_R= 1.79 min.

(2-((1-Cyclohexyl-3-(2-methoxyphenyl)-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(5-ﬂuoroisoindolin-2-yl)methanone (9j).

Amide bromide 8c (48 mg, 0.90 mmol, 1 equiv) was coupled to 4h

(27 mg, 0.099 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9j (19 mg, 0.034 mmol) was obtained in 38% yield over two steps.

¹H NMR (500 MHz, DMSO-d₆): δ 9.57 (s, 1H), 9.24 (s, 1H), 7.91−

7.86 (m, 1H), 7.43−7.32 (m, 1H), 7.27−7.22 (m, 1H), 7.22−7.18 (m,

1H), 7.11−7.06 (m, 1H), 7.04−7.01 (m, 2H), 6.45 (s, 1H), 5.84 (d, J =

2.1 Hz, 1H), 5.57 (d, J = 2.1 Hz, 1H), 4.73 (s, 6H), 3.80 (s, 3H), 1.93−

1.71 (m, 6H), 1.66−1.55 (m, 1H), 1.31−1.13 (m, 3H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.44, 159.20, 156.12, 155.48, 144.91, 144.88,

139.00, 128.30, 127.05, 124.54, 122.27, 120.30, 114.22, 111.68, 110.06,

103.62, 102.01, 94.00, 92.44, 79.17, 55.30, 48.58, 32.35, 25.12. UPLC/

MS [M + H] = 543.322, t_R= 1.79 min.

(2,4-Dihydroxy-6-((1-methyl-3-(o-tolyl)-1H-pyrazol-5-yl)amino)-

phenyl)(5-ﬂuoroisoindolin-2-yl)methanone (9e). Compound 9e was

obtained according to the route and procedures described in ref 62. (2-

Bromo-4,6-bis(methoxymethoxy)phenyl)(5-ﬂuoroisoindolin-2-yl)-

methanone (78 mg, 0.177 mmol, 1 equiv), 2-methyl-5-(o-tolyl)pyrazol-

3-amine (4e) (22 mg, 0.195 mmol, 1.1 equiv), Pd₂(dba)₃(8.1 mg, 5

mol %), xantphos (10.3 mg, 10 mol %), sodium phenoxide (31 mg,

0.266 mmol, 1.5 equiv), and anhydrous dioxane (2 mL) were heated to

120 °C in a 2 dram vial under an atmosphere of nitrogen. The reaction

was cooled to room temperature, diluted with ethyl acetate, and passed

through a prewetted 3 mL hydromatrix cartridge eluting with ethyl

acetate. The eluents were dried with sodium sulfate, ﬁltered, and

condensed by rotary evaporation. Puriﬁcation on a short silica column

with a gradient of 25% to 40% acetone in hexanes aﬀorded a yellow

gum, which was then dissolved in methanol (5 mL) and 2 M aqueous

HCl (0.53 mL). The reaction was heated to 50 °C overnight, cooled to

room temperature, and the solvent was removed by rotary evaporation.

The crude residue was puriﬁed by mass-guided preparative HPLC to

1

aﬀord 9e (24.2 mg, 28% yield over two steps). H NMR (500 MHz,

CDCl₃) δ 7.41 (d, J = 7.34 Hz, 1H), 7.06−7.17 (m, 4H), 6.92 (dt, J =

1.96, 8.56 Hz, 1H), 6.86 (br d, J = 8.31 Hz, 1H), 6.81 (s, 1H), 6.10 (s,

1H), 5.97 (d, J = 0.98 Hz, 1H), 5.78 (d, J = 1.47 Hz, 1H), 4.91 (br s,

2H), 4.72−4.86 (m, 2H), 3.64 (s, 3H), 2.33 (s, 3H); ¹³C NMR (126

MHz, CDCl₃) δ 168.8, 161.8, 160.5, 156.4, 150.4, 144.8, 140.6, 136.0,

132.9, 132.0, 130.9, 129.1, 128.0, 126.0, 124.2, 124.1, 115.1, 114.9,

110.2, 110.0, 100.0, 95.9, 94.0, 52.9, 52.4, 40.9, 21.2. UPLC/MS [M +

H] = 459.393, t_R= 1.70 min.

(2,4-Dihydroxy-6-((3-(2-methoxyphenyl)-1-methyl-1H-pyrazol-5-

yl)amino)phenyl)(isoindolin-2-yl)methanone (9f). Compound 9f was

obtained from (2-bromo-4,6-bis(methoxymethoxy)phenyl)-

(isoindolin-2-yl)methanone⁶²and 5-(2-methoxyphenyl)-2-methyl-

pyrazol-3-amine (4f) according to the same procedures described

above for 9e. 9f (17.8 mg) was obtained in 35% yield over two steps. ¹H

NMR (500 MHz, CDCl₃) δ 7.76 (dd, J = 1.47, 7.83 Hz, 1H), 7.21−7.25

(2-((1-Cyclopentyl-3-(2-methoxyphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9k).

Amide bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4i

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(42 mg, 0.165 mmol, 1.1 equiv) according to General Procedure E1a.

The resulting product was deprotected according to General Procedure

E2a. 9k (30.5 mg, 0.060 mmol) was obtained in 40% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.59 (s, 1H), 7.92−7.87 (m,

1H), 7.39−7.32 (m, 2H), 7.30−7.22 (m, 3H), 7.10−7.01 (m, 2H),

7.01−6.93 (m, 1H), 6.47 (s, 1H), 5.86 (d, J = 2.1 Hz, 1H), 5.55 (d, J =

2.1 Hz, 1H), 4.77 (s, 4H), 4.58 (p, J = 7.3 Hz, 1H), 3.81 (s, 3H), 2.04−

1.78 (m, 6H), 1.67−1.47 (m, 2H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.52, 159.21, 156.18, 155.48, 145.00, 144.83, 139.60, 136.76, 128.38,

127.26, 127.10, 122.86, 122.30, 120.37, 111.75, 103.72, 101.94, 94.04,

92.39, 56.91, 55.34, 32.25, 24.16. UPLC/MS [M + H] = 511.295, t_R=

1.74 min.

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4n (40 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1b. The resulting

product was deprotected according to General Procedure E2a. 9p (32.3

mg, 0.065 mmol) was obtained in 43% yield over two steps. ¹H NMR

(500 MHz, DMSO-d₆): δ 9.70 (s, 1H), 9.28 (s, 1H), 7.38−7.32 (m,

3H), 7.32−7.25 (m, 4H), 6.97 (s, 1H), 6.86−6.80 (m, 1H), 6.58 (d, J =

0.6 Hz, 1H), 5.86 (d, J = 2.1 Hz, 1H), 5.67 (d, J = 2.1 Hz, 1H), 4.99−

4.66 (m, 4H), 3.78 (s, 3H), 1.56 (s, 9H); ¹³C NMR (126 MHz, DMSO-

d₆): δ 166.90, 159.49, 159.42, 155.54, 146.34, 145.08, 140.43, 136.66,

135.22, 129.55, 127.28, 122.86, 117.14, 112.72, 110.05, 103.03, 100.57,

93.82, 92.24, 59.36, 55.02, 29.41 UPLC/MS [M + H] = 499.255, t_R=

1.66 min.

(2,4-Dihydroxy-6-((1-isobutyl-3-(2-methoxyphenyl)-1H-pyrazol-

5-yl)amino)phenyl)(isoindolin-2-yl)methanone (9l). Amide bromide

8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4j (40 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1a. The resulting

product was deprotected according to General Procedure E2a. 9l (20.7

mg, 0.0415 mmol) was obtained in 28% yield over two steps. ¹H NMR

(500 MHz, DMSO-d₆): δ 9.63 (s, 1H), 9.29 (s, 1H), 7.90−7.84 (m,

1H), 7.34 (s, 2H), 7.31−7.24 (m, 3H), 7.08 (s, 1H), 7.07−7.04 (m,

1H), 6.98−6.94 (m, 1H), 6.50 (s, 1H), 5.87 (d, J = 2.1 Hz, 1H), 5.67 (d,

J = 2.1 Hz, 1H), 4.77 (s, 4H), 3.82 (s, 3H), 3.72 (d, J = 7.3 Hz, 2H),

2.14−2.03 (m, 1H), 0.78 (d, J = 6.7 Hz, 6H); ¹³C NMR (126 MHz,

DMSO-d₆): δ 167.03, 159.72, 156.66, 155.95, 145.55, 144.68, 140.56,

137.11, 128.92, 127.72, 127.56, 123.31, 122.57, 120.83, 112.24, 104.24,

101.43, 94.67, 92.89, 55.82, 55.00, 29.14, 20.25. UPLC/MS [M + H] =

499.343, t_R= 1.74 min.

(2-((1-(tert-Butyl)-3-(2-methoxyphenyl)-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9m). Amide bro-

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4k (40 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1a. The resulting

product was deprotected according to General Procedure E2a. 9m

(12.4 mg, 0.025 mmol) was obtained in 12.5% yield over two steps.

¹H NMR (500 MHz, DMSO-d₆): δ 9.67 (s, 1H), 9.26 (s, 1H), 7.94−

7.89 (m, 1H), 7.41−7.33 (m, 2H), 7.31−7.24 (m, 3H), 7.08−7.02 (m,

1H), 6.97 (s, 1H), 6.94−6.91 (m, 1H), 6.54 (d, J = 0.7 Hz, 1H), 5.84 (d,

J = 2.1 Hz, 1H), 5.62 (d, J = 2.1 Hz, 1H), 4.81 (s, 4H), 3.81 (s, 3H), 1.55

(s, 9H); ¹³C NMR (126 MHz, DMSO-d₆): δ 167.35, 159.83, 156.68,

155.97, 145.81, 143.75, 139.66, 137.12, 128.81, 127.73, 127.46, 123.31,

122.73, 120.82, 112.21, 105.28, 103.41, 94.12, 92.51, 59.69, 55.81,

29.91. UPLC/MS [M + H] = 499.343, t_R= 1.62 min.

(2-((1-(tert-butyl)-3-(m-tolyl)-1H-pyrazol-5-yl)amino)-4,6-

dihydroxyphenyl)(isoindolin-2-yl)methanone (9q). Amide bromide

8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4o (38 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1b. The resulting

product was deprotected according to General Procedure E2a. 9q (34.7

mg, 0.072 mmol) was obtained in 48% yield over two steps. ¹H NMR

(500 MHz, DMSO-d₆): δ 9.70 (s, 1H), 9.28 (s, 1H), 7.61−7.57 (m,

1H), 7.57−7.52 (m, 1H), 7.40−7.32 (m, 2H), 7.32−7.21 (m, 3H),

7.11−7.04 (m, 1H), 7.00−6.96 (m, 1H), 6.53 (s, 1H), 5.86 (d, J = 2.1

Hz, 1H), 5.69 (d, J = 2.1 Hz, 1H), 5.06−4.62 (m, 4H), 2.32 (s, 3H),

1.56 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.92, 159.43,

155.55, 146.55, 145.06, 140.42, 137.50, 136.67, 133.72, 128.37, 127.82,

127.29, 125.19, 122.86, 121.91, 103.06, 100.22, 93.84, 92.26, 59.31,

29.44, 21.10. UPLC/MS [M + H] = 483.247, t_R= 1.85 min.

(2-((1-(tert-Butyl)-3-(3-(triﬂuoromethyl)phenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9r).

Amide bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4p

(46.7 mg, 0.165 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9r (49.8 mg, 0.093 mmol) was obtained in 62% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.72 (s, 1H), 9.29 (s, 1H),

8.10−8.02 (m, 2H), 7.64−7.57 (m, 2H), 7.41−7.31 (m, 2H), 7.31−

7.24 (m, 2H), 7.03 (s, 1H), 6.75 (s, 1H), 5.88 (d, J = 2.1 Hz, 1H), 5.70

(d, J = 2.1 Hz, 1H), 5.04−4.59 (m, 4H), 1.58 (s, 9H); ¹³C NMR (126

MHz, DMSO-d₆): δ 167.33, 159.89, 156.01, 145.47, 145.35, 141.49,

137.11, 130.13, 129.91 (q, J = 30.8 Hz), 128.95, 127.73, 125.82,

124.14−123.94 (m), 123.65, 123.30, 121.23−121.03 (m), 103.64,

101.20, 94.43, 92.85, 60.13, 29.80. UPLC/MS [M + H] = 537.270, t_R=

1.94 min.

(2,4-Dihydroxy-6-((1-methyl-3-(2-(triﬂuoromethoxy)phenyl)-1H-

pyrazol-5-yl)amino)phenyl)(isoindolin-2-yl)methanone (9n). Amide

bromide 8a (77 mg, 0.15 mmol, 1 equiv) was coupled to 4l (42 mg,

0.165 mmol, 1.1 equiv) according to General Procedure E1b. The

resulting product was deprotected according to General Procedure E2a.

9n (36 mg, 0.70 mmol) was obtained in 47% yield over two steps. ¹H

NMR (500 MHz, DMSO-d₆): δ 9.63 (s, 1H), 9.29 (s, 1H), 8.00−7.92

(m, 1H), 7.44−7.36 (m, 3H), 7.32 (s, 2H), 7.28−7.23 (m, 3H), 6.35 (s,

1H), 5.92 (d, J = 2.1 Hz, 1H), 5.62 (d, J = 2.1 Hz, 1H), 4.75 (s, 4H),

3.63 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.34, 159.27,

155.52, 145.02, 143.73, 143.28, 141.43, 136.73, 128.92, 128.62, 127.64,

127.25, 126.95, 122.85, 121.53, 121.22, 119.18, 104.39, 99.57, 94.56,

92.88, 35.17. UPLC/MS [M + H] = 511.162, t_R= 1.49 min.

(2-((1-(tert-Butyl)-3-(2-(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-

yl)amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9o).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4m

(41 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9o (14.7 mg, 0.027 mmol) was obtained in 22% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.69 (s, 1H), 9.26 (s, 1H),

8.02−7.97 (m, 1H), 7.45−7.37 (m, 3H), 7.37−7.31 (m, 2H), 7.32−

7.26 (m, 2H), 7.02 (s, 1H), 6.44 (s, 1H), 5.86 (d, J = 2.1 Hz, 1H), 5.62

(d, J = 2.1 Hz, 1H), 4.80 (s, 4H), 1.56 (s, 9H); ¹³C NMR (126 MHz,

DMSO-d₆): δ 166.78, 159.40, 155.50, 144.95, 141.74, 140.21, 136.60,

128.80, 128.69, 127.62, 127.27, 127.04, 122.83, 121.57, 121.28, 119.24,

103.26, 103.19, 93.90, 92.08, 59.77, 29.32.UPLC/MS [M + H] =

553.609, t_R= 1.87 min.

(2,4-Dihydroxy-6-((1-methyl-3-(3-(triﬂuoromethoxy)phenyl)-1H-

pyrazol-5-yl)amino)phenyl)(isoindolin-2-yl)methanone (9s). Amide

bromide 8a (77 mg, 0.15 mmol, 1 equiv) was coupled to 4q (42 mg,

0.165 mmol, 1.1 equiv) according to General Procedure E1b. The

resulting product was deprotected according to General Procedure E2a.

9s (43 mg, 0.084 mmol) was obtained in 56% yield over two steps. ¹H

NMR (500 MHz, DMSO-d₆): δ 9.64 (s, 1H), 9.30 (s, 1H), 7.76−7.70

(m, 1H), 7.69−7.64 (m, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.34−7.29 (m,

2H), 7.29−7.23 (m, 3H), 7.22 (s, 1H), 6.58 (s, 1H), 5.91 (d, J = 2.1 Hz,

1H), 5.66 (d, J = 2.1 Hz, 1H), 4.92−4.51 (m, 4H), 3.63 (s, 3H); ¹³C

NMR (126 MHz, DMSO-d₆): δ 166.40, 159.27, 155.55, 148.90−

148.76 (m), 146.67, 142.02, 136.68, 135.92, 130.55, 127.24, 123.63,

122.84, 121.15, 119.45, 119.11, 116.59, 104.27, 97.13, 94.52, 93.09,

35.10. UPLC/MS [M + H] = 511.162, t_R= 1.62 min.

(2-((1-(tert-Butyl)-3-(3-(triﬂuoromethoxy)phenyl)-1H-pyrazol-5-

yl)amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9t).

Amide bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4r

(49 mg, 0.165 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9t (39.4 mg, 0.071 mmol) was obtained in 47% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.71 (s, 1H), 9.28 (s, 1H),

7.82−7.77 (m, 1H), 7.73−7.68 (m, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.40−

7.32 (m, 2H), 7.32−7.21 (m, 3H), 7.02 (s, 1H), 6.69 (s, 1H), 5.87 (d, J

= 2.1 Hz, 1H), 5.68 (d, J = 2.1 Hz, 1H), 5.05−4.58 (m, 4H), 1.57 (s,

9H); ¹³C NMR (126 MHz, DMSO-d₆): δ 167.33, 159.89, 156.01,

149.38−149.25 (m), 145.44, 145.40, 141.38, 137.11, 136.64, 131.01,

127.73, 124.08, 121.61, 119.79, 119.57, 117.16, 103.60, 101.30, 94.40,

92.80, 60.10, 29.79. UPLC/MS [M + H] = 553.278, t_R= 544.246 min.

(2-((1-(tert-Butyl)-3-(3-methoxyphenyl)-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9p). Amide bro-

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(2-((3-(3-Fluorophenyl)-1-methyl-1H-pyrazol-5-yl)amino)-4,6-

dihydroxyphenyl)(isoindolin-2-yl)methanone (9u). Amide bromide

8a (45 mg, 0.087 mmol, 1 equiv) was coupled to 2-methyl-5-(3-

ﬂuorophenyl)pyrazol-3-amine 4s (18 mg, 0.096 mmol, 1.1 equiv)

according to General Procedure E1a. The resulting product was

deprotected according to General Procedure E2a. 9u (24.7 mg, 0.056

mmol) was obtained in 64% yield over two steps. ¹H NMR (400 MHz,

methanol-d₄) δ 7.45−7.39 (m, 1H), 7.38−7.26 (m, 2H), 7.26−7.21 (m,

4H), 7.01−6.89 (m, 1H), 6.38 (s, 1H), 5.96 (d, J = 2.1 Hz, 1H), 5.79 (d,

J = 2.1 Hz, 1H), 4.95−4.72 (m, 4H), 3.71 (s, 3H); (500 MHz, DMSO-

d₆): δ 9.63 (s, 1H), 9.29 (s, 1H), 7.59−7.53 (m, 1H), 7.50−7.46 (m,

1H), 7.41−7.35 (m, 1H), 7.35−7.29 (m, 2H), 7.30−7.25 (m, 2H), 7.19

(s, 1H), 7.10−7.03 (m, 1H), 6.54 (s, 1H), 5.89 (d, J = 2.1 Hz, 1H), 5.64

(d, J = 2.1 Hz, 1H), 4.84−4.68 (m, 4H), 3.61 (s, 3H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.83, 162.99 (d, J = 242.2 Hz), 159.70, 155.99,

147.48 (d, J = 2.4 Hz), 144.29, 142.26, 136.54 (d, J = 8.2 Hz), 130.93 (d,

J = 8.5 Hz), 127.69, 123.30, 121.06, 114.28 (d, J = 21.7 Hz), 111.51 (d, J

= 22.4 Hz), 104.64, 97.54, 94.90, 93.42, 35.51. UPLC/MS [M + H] =

445.408, t_R= 2.31 min.

(2-((1-(tert-Butyl)-3-(3-ﬂuorophenyl)-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9v). Amide bro-

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4t (38.5 mg,

0.165 mmol, 1.1 equiv) according to General Procedure E1b. The

resulting product was deprotected according to General Procedure E2a.

9v (32.8 mg, 0.067 mmol) was obtained in 45% yield over two steps. ¹H

NMR (500 MHz, DMSO-d₆): δ 9.71 (s, 1H), 9.28 (s, 1H), 7.64−7.59

(m, 1H), 7.59−7.51 (m, 1H), 7.44−7.38 (m, 1H), 7.37−7.32 (m, 2H),

7.32−7.25 (m, 2H), 7.11−7.04 (m, 1H), 7.00 (s, 1H), 6.65 (s, 1H),

5.87 (d, J = 2.1 Hz, 1H), 5.67 (d, J = 2.1 Hz, 1H), 4.97−4.71 (m, 4H),

1.56 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.88, 162.58 (d, J =

242.3 Hz), 159.43, 155.56, 145.35 (d, J = 2.6 Hz), 144.99, 140.75,

136.67, 136.31 (d, J = 8.1 Hz), 130.50 (d, J = 8.6 Hz), 127.29, 122.86,

120.65 (d, J = 2.7 Hz), 113.75 (d, J = 21.0 Hz), 111.06 (d, J = 22.4 Hz),

103.11, 100.80, 93.91, 92.30, 59.58, 29.37. UPLC/MS [M + H] =

487.501, t_R= 1.82 min.

1H), 7.71−7.65 (m, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.34−7.25 (m, 5H),

7.10 (s, 1H), 6.55 (s, 1H), 5.86 (d, J = 2.1 Hz, 1H), 5.61 (d, J = 2.1 Hz,

1H), 4.75 (s, 4H), 4.07−3.98 (m, 1H), 1.90−1.70 (m, 6H), 1.61−1.55

(m, 1H), 1.26−1.13 (m, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.42, 159.16, 155.49, 146.69, 144.42, 140.58, 136.65, 135.92, 133.37,

130.38, 127.24, 126.83, 124.07, 123.18, 122.82, 104.02, 97.97, 94.27,

92.75, 55.44, 40.43, 32.24, 24.85. UPLC/MS [M + H] = 529.319, t_R=

1.90 min.

(2-((3-(2,3-Dimethylphenyl)-1-methyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9z). Amide bro-

mide 8a (77 mg, 0.15 mmol, 1 equiv) was coupled to 4x (33.2 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1a. The resulting

product was deprotected according to General Procedure E2a. 9z (5.5

1

mg, 0.012 mmol) was obtained in 8% yield over two steps. H NMR

(500 MHz, DMSO-d₆): 9.60 (s, 1H), 9.30 (s, 1H), 7.34 (s, 2H), 7.31−

7.26 (m, 2H), 7.24−7.18 (m, 1H), 7.17 (s, 1H), 7.13−7.09 (m, 1H),

7.06 (t, J = 7.5 Hz, 1H), 6.13 (s, 1H), 5.88 (d, J = 2.1 Hz, 1H), 5.66 (d, J

= 2.1 Hz, 1H), 4.82−4.70 (m, 4H), 3.60 (s, 3H), 2.30 (s, 3H), 2.26 (s,

3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.40, 159.24, 155.50,

149.40, 143.88, 140.45, 136.76, 133.87, 133.85, 128.76, 127.25, 126.92,

125.06, 122.87, 104.09, 99.74, 94.34, 92.74, 34.85, 20.36, 16.59. UPLC/

MS [M + H] = 455.303, t_R= 1.52 min.

(2-((3-(2,3-Dimethylphenyl)-1-methyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(4-ﬂuoroisoindolin-2-yl)methanone (9aa).

Amide bromide 8b (66.6 mg, 0.125 mmol, 1 equiv) was coupled to

4x (27.7 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9aa (17.5 mg, 0.037 mmol) was obtained in 30% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.66 (s, 1H), 9.34 (s,

1H), 7.40−7.30 (m, 1H), 7.25−7.14 (m, 3H), 7.15−7.08 (m, 2H), 7.05

(t, J = 7.5 Hz, 1H), 6.14 (s, 1H), 5.89 (d, J = 2.1 Hz, 1H), 5.68 (d, J = 2.1

Hz, 1H), 4.83−4.70 (m, 4H), 3.17 (s, 3H), 2.30 (s, 3H), 2.26 (s, 3H);

¹³C NMR (126 MHz, DMSO-d₆): δ 166.42, 159.39, 157.13 (d, J =

241.2 Hz), 155.58, 149.41, 144.01, 140.43, 136.76, 133.84 (d, J = 4.3

Hz), 129.93, 128.77, 126.89, 125.06, 123.23, 119.19, 113.69 (d, J = 19.2

Hz), 103.74, 99.74, 94.34, 92.89, 34.86, 20.37, 16.59. UPLC/MS [M +

H] = 473.751, t_R= 1.54min.

(2-((3-(3-Chlorophenyl)-1-methyl-1H-pyrazol-5-yl)amino)-4,6-

dihydroxyphenyl)(isoindolin-2-yl)methanone (9w). Amide bromide

8a (129 mg, 0.25 mmol, 1 equiv) was coupled to 4u (57 mg, 0.275

mmol, 1.1 equiv) according to General Procedure E1b. The resulting

product was deprotected according to General Procedure E2b. 9w

(20.4 mg, 0.044 mmol) was obtained in 18% yield over two steps. ¹H

NMR (500 MHz, DMSO-d₆): δ 9.43 (s, 1H), 9.07 (s, 1H), 7.49 (t, J =

1.9 Hz, 1H), 7.45−7.39 (m, 1H), 7.11 (t, J = 7.9 Hz, 1H), 7.09−7.00

(m, 5H), 6.95 (s, 1H), 6.30 (s, 1H), 5.65 (d, J = 2.1 Hz, 1H), 5.40 (d, J =

2.1 Hz, 1H), 4.65−4.33 (m, 4H), 3.36 (s, 3H); ¹³C NMR (126 MHz,

DMSO-d₆): δ 166.38, 159.25, 155.56, 146.72, 143.76, 141.87, 136.95,

135.67, 133.40, 130.40, 127.23, 126.92, 124.13, 123.14, 122.83, 104.20,

96.93, 94.49, 93.01, 35.06. UPLC/MS [M + H] = 461.175, t_R= 1.54

min.

(2-((3-(2,3-Dimethylphenyl)-1-methyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(5-ﬂuoroisoindolin-2-yl)methanone (9ab).

Amide bromide 8c (53.2 mg, 0.1 mmol, 1 equiv) was coupled to 4x

(22.1 mg, 0.11 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9ab (8.2 mg, 0.017 mmol) was obtained in 17% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.60 (s, 1H), 9.30 (s, 1H),

7.38−7.34 (m, 1H), 7.24−7.18 (m, 2H), 7.17 (s, 1H), 7.13−7.03 (m,

3H), 6.12 (s, 1H), 5.88 (d, J = 2.1 Hz, 1H), 5.66 (d, J = 2.0 Hz, 1H),

5.05−4.42 (m, 4H), 3.60 (s, 3H), 2.29 (s, 3H), 2.26 (s, 3H); ¹³C NMR

(126 MHz, DMSO-d₆): δ 166.35, 159.28, 155.51, 149.38, 143.93,

140.44, 138.77, 136.74, 133.83 (d, J = 2.8 Hz), 128.75, 126.89, 125.04,

110.01 (d, J = 24.7 Hz), 103.93, 99.74, 94.32, 92.81, 34.85, 20.35, 16.58.

UPLC/MS [M + H] = 473.670, t_R= 1.53 min.

(2-((3-(5-Fluoro-2-methylphenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ac).

Amide bromide 8a (77 mg, 0.15 mmol, 1 equiv) was coupled to 4y

(34 mg, 0.165 mmol, 1.1 equiv) according to General Procedure E1a.

The resulting product was deprotected according to General Procedure

E2a. 9ac (9 mg, 0.020 mmol) was obtained in 13% yield over two steps.

¹H NMR (500 MHz, DMSO-d₆): δ 9.60 (s, 1H), 9.29 (s, 1H), 7.36−

7.22 (m, 6H), 7.20 (s, 1H), 7.01 (td, J = 8.4, 2.9 Hz, 1H), 6.34 (s, 1H),

5.89 (d, J = 2.0 Hz, 1H), 5.66 (d, J = 2.0 Hz, 1H), 4.78−4.70 (m, 4H),

3.61 (s, 3H), 2.41 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 160.37,

154.39 (d, J = 240.2 Hz), 153.23, 149.51, 141.62, 137.82, 134.93,

130.69, 128.85 (d, J = 7.4 Hz), 126.51 (d, J = 7.8 Hz), 124.91 (d, J = 2.8

Hz), 121.23, 116.84, 108.17 (d, J = 21.8 Hz), 107.62 (d, J = 21.1 Hz),

98.17, 93.52, 88.41, 86.87, 29.00, 14.55. UPLC/MS [M + H] =

459.452, t_R= 1.43 min.

(2-((3-(5-Fluoro-2-methylphenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(4-ﬂuoroisoindolin-2-yl)methanone

(9ad). Amide bromide 8b (66.6 mg, 0.125 mmol, 1 equiv) was coupled

to 4y (28.2 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

(2-((1-(tert-Butyl)-3-(3-chlorophenyl)-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9x). Amide bro-

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4v (41 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1b. The resulting

product was deprotected according to General Procedure E2b. 9x (6.4

1

mg, 0.013 mmol) was obtained in 9% yield over two steps. H NMR

(500 MHz, DMSO-d₆): δ 9.70 (s, 1H), 9.27 (s, 1H), 7.79 (t, J = 1.9 Hz,

1H), 7.77−7.69 (m, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.37−7.33 (m, 2H),

7.33−7.27 (m, 3H), 7.00 (s, 1H), 6.66 (s, 1H), 5.85 (d, J = 2.1 Hz, 1H),

5.67 (d, J = 2.1 Hz, 1H), 4.93−4.69 (m, 4H), 1.56 (s, 9H); ¹³C NMR

(126 MHz, DMSO-d₆): δ 166.84, 159.40, 155.53, 145.03, 144.90,

140.81, 136.31, 135.90, 133.40, 130.43, 127.27, 126.88, 124.08, 123.20,

122.85, 103.11, 100.71, 93.90, 92.29, 59.59, 29.35. UPLC/MS [M + H]

= 503.236, t_R= 1.86 min.

(2-((3-(3-Chlorophenyl)-1-cyclohexyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9y). Amide bro-

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4w (46 mg, 0.165

mmol, 1.1 equiv) according to General Procedure E1b. The resulting

product was deprotected according to General Procedure E2b. 9y (85

mg, 0.016 mmol) was obtained in 11% yield over two steps. ¹H NMR

(500 MHz, DMSO-d₆): δ 9.59 (s, 1H), 9.25 (s, 1H), 7.76 (t, J = 1.9 Hz,

1161

https://dx.doi.org/10.1021/acs.jmedchem.0c01777

J. Med. Chem. 2021, 64, 1139−1169

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

E1b. The resulting product was deprotected according to General

Procedure E2a. 9ad (27 mg, 0.056 mmol) was obtained in 45% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.66 (s, 1H), 9.34 (s,

1H), 7.36−7.30 (m, 1H), 7.30−7.26 (m, 1H), 7.26−7.21 (m, 2H),

7.20−7.15 (m, 1H), 7.08 (t, J = 8.8 Hz, 1H), 7.00 (td, J = 8.4, 2.9 Hz,

1H), 6.34 (s, 1H), 5.90 (d, J = 2.1 Hz, 1H), 5.68 (d, J = 2.1 Hz, 1H),

4.87−4.71 (m, 4H), 3.62 (s, 3H), 2.40 (s, 3H); ¹³C NMR (126 MHz,

DMSO-d₆): δ 166.39, 160.39 (d, J = 240.5 Hz), 159.37, 147.62 (d, J =

2.3 Hz), 143.98, 140.95, 134.80 (d, J = 7.9 Hz), 132.51 (d, J = 8.0 Hz),

130.88 (d, J = 2.9 Hz), 129.85, 119.14, 114.12 (d, J = 22.1 Hz), 113.71

(d, J = 7.1 Hz), 113.55 (d, J = 8.8 Hz), 103.85, 99.51, 94.43, 93.12, 89.17

(d, J = 104.5 Hz), 35.02, 20.57. UPLC/MS [M + H] = 477.489, t_R=

1.53 min.

2.24 (d, J = 2.1 Hz, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.30,

160.78, 159.24, 156.66 (d, J = 244.7 Hz), 155.55, 143.88, 142.11 (d, J =

181.7 Hz), 130.16, 125.03−124.87 (m), 124.75 (d, J = 7.8 Hz), 124.41,

123.85 (d, J = 4.3 Hz), 120.74 (d, J = 12.4 Hz), 114.34 (d, J = 24.9 Hz),

109.81 (d, J = 25.6 Hz), 104.12, 99.50 (d, J = 10.4 Hz), 94.48, 93.18,

35.05, 14.26 (d, J = 4.8 Hz). UPLC/MS [M + H] = 477.445, t_R= 1.51

min.

(2-((3-(2,3-Diﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ai). Amide bro-

mide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4aa (34.5 mg,

0.165 mmol, 1.1 equiv) according to General Procedure E1a. The

resulting product was deprotected according to General Procedure E2a.

9ai (5.7 mg, 0.012 mmol) was obtained in 8% yield over two steps. ¹H

NMR (500 MHz, DMSO-d₆): δ 9.61 (s, 1H), 9.30 (s, 1H), 7.70−7.63

(m, 1H), 7.35−7.28 (m, 3H), 7.27−7.22 (m, 3H), 7.22−7.16 (m, 1H),

6.37 (d, J = 3.7 Hz, 1H), 5.90 (d, J = 2.0 Hz, 1H), 5.65 (d, J = 2.1 Hz,

1H), 4.79−4.66 (m, 4H), 3.64 (s, 3H); ¹³C NMR (126 MHz, DMSO-

d₆): δ 166.28, 159.19, 155.53, 143.71, 141.77, 136.38, 132.64, 127.16,

124.43, 122.90, 122.78, 104.41, 94.60, 93.20, 35.16. UPLC/MS [M +

H] = 463.241, t_R= 1.45 min.

(2-((3-(5-Fluoro-2-methylphenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(5-ﬂuoroisoindolin-2-yl)methanone

(9ae). Amide bromide 8c (66.6 mg, 0.125 mmol, 1 equiv) was coupled

to 4y (28.2 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9ae (19 mg, 0.40 mmol) was obtained in 32% yield over

1

two steps. H NMR (500 MHz, DMSO-d₆): δ 9.61 (s, 1H), 9.30 (s,

1H), 7.37−7.31 (m, 1H), 7.29−7.25 (m, 1H), 7.25−7.22 (m, 1H),

7.22−7.16 (m, 2H), 7.12−7.04 (m, 1H), 7.04−6.98 (m, 1H), 6.32 (s,

1H), 5.89 (d, J = 2.1 Hz, 1H), 5.66 (d, J = 2.1 Hz, 1H), 4.80−4.65 (m,

4H), 3.62 (s, 3H), 2.40 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.32, 160.35 (d, J = 240.5 Hz), 159.26, 155.54, 147.60 (d, J = 2.4 Hz),

143.90, 140.96, 134.80 (d, J = 7.7 Hz), 132.49 (d, J = 8.1 Hz), 130.87 (d,

J = 2.9 Hz), 124.50 (d, J = 8.8 Hz), 114.22, 114.05, 113.67, 113.51,

109.96 (d, J = 23.2 Hz), 104.03, 99.51, 94.41, 93.04, 35.01, 20.56.

UPLC/MS [M + H] = 477.489, t_R= 1.52 min.

(2-((3-(2-Fluoro-3-methylphenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9af).

Amide bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4z

(33.8 mg, 0.165 mmol, equiv) according to General Procedure E1a.

The resulting product was deprotected according to General Procedure

E2a. 9af (13.7 mg, 0.030 mmol) was obtained in 20% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.61 (s, 1H), 9.29 (s, 1H),

7.74−7.67 (m, 1H), 7.35−7.29 (m, 2H), 7.29−7.23 (m, 2H), 7.21 (s,

1H), 7.20−7.15 (m, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.35 (d, J = 4.1 Hz,

1H), 5.89 (d, J = 2.1 Hz, 1H), 5.64 (d, J = 2.0 Hz, 1H), 4.81−4.67 (m,

4H), 3.62 (s, 3H), 2.24 (d, J = 2.1 Hz, 3H); ¹³C NMR (126 MHz,

DMSO-d₆): δ 166.33, 159.21, 157.64 (d, J = 246.4 Hz), 155.51, 143.78,

142.88, 141.33, 130.20, 127.19, 125.07−124.96 (m), 124.79 (d, J = 17.8

Hz), 123.88, 122.81, 120.73, 104.26, 99.46, 94.47, 35.04, 14.29 (d, J =

5.0 Hz). UPLC/MS [M + H] = 459.496, t_R= 1.49 min.

(2-((3-(2-Fluoro-3-methylphenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(4-ﬂuoroisoindolin-2-yl)methanone

(9ag). Amide bromide 8b (66.6 mg, 0.125 mmol, 1 equiv) was coupled

to 4z (28.2 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9ag (39 mg, 0.082 mmol) was obtained in 66% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.68−9.64 (m, 1H),

9.34 (s, 1H), 7.73−7.66 (m, 1H), 7.35−7.27 (m, 1H), 7.25 (s, 1H),

7.20−7.13 (m, 2H), 7.10−7.03 (m, 2H), 6.33 (d, J = 4.1 Hz, 1H), 5.91

(d, J = 2.1 Hz, 1H), 5.68 (d, J = 2.0 Hz, 1H), 4.87−4.67 (m, 4H), 3.63

(s, 3H), 2.23 (d, J = 2.1 Hz, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.36, 159.34, 157.65 (d, J = 247.1 Hz), 155.61, 144.25, 142.85,

141.38, 130.21, 124.92 (d, J = 4.1 Hz), 124.76, 123.87 (d, J = 3.6 Hz),

120.73 (d, J = 12.5 Hz), 119.08, 113.59 (d, J = 19.1 Hz), 103.95, 99.49

(d, J = 10.2 Hz), 94.50, 93.29, 79.16, 35.05, 14.28 (d, J = 4.8 Hz).

UPLC/MS [M + H] = 477.489, t_R= 1.44 min.

(2-((3-(2-Fluoro-3-methylphenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(5-ﬂuoroisoindolin-2-yl)methanone

(9ah). Amide bromide 8c (53.2 mg, 0.1 mmol, 1 equiv) was coupled to

4z (22.6 mg, 0.11 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9ah (18 mg, 0.038 mmol) was obtained in 38% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H), 9.26 (s,

0H), 7.73−7.66 (m, 1H), 7.35−7.29 (m, 1H), 7.22 (s, 1H), 7.20−7.12

(m, 2H), 7.11−7.02 (m, 2H), 6.32 (d, J = 4.1 Hz, 1H), 5.89 (d, J = 2.1

Hz, 1H), 5.66 (d, J = 2.1 Hz, 1H), 4.84−4.56 (m, 4H), 3.63 (s, 3H),

(2,4-Dihydroxy-6-((1-methyl-3-(2-methyl-3-(triﬂuoromethyl)-

phenyl)-1H-pyrazol-5-yl)amino)phenyl)(isoindolin-2-yl)methanone

(9aj). Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4ab (35 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9aj (3.4 mg, 0.007 mmol) was obtained in 6% yield over

1

two steps. H NMR (500 MHz, DMSO-d₆): δ 9.60 (s, 1H), 9.30 (s,

1H), 7.66 (t, J = 7.8 Hz, 2H), 7.38 (t, J = 7.8 Hz, 1H), 7.34−7.31 (m,

3H), 7.31−7.25 (m, 2H), 7.23 (s, 1H), 6.27 (s, 1H), 5.89 (d, J = 2.1 Hz,

1H), 5.68 (d, J = 2.0 Hz, 1H), 4.83−4.69 (m, 4H), 3.62 (s, 3H), 2.49 (s,

3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.34, 159.22, 155.52,

147.64, 143.75, 140.96, 136.17, 133.85, 133.27, 127.23, 125.93, 122.84,

104.25, 100.08, 94.47, 93.01, 34.99, 16.38; ¹⁹F NMR (470 MHz,

DMSO-d₆): δ −59.28. UPLC/MS [M + H] = 509.514, t_R= 1.56 min.

(2-((3-(3-Chloro-2-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ak).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4ac

(31 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2b. 9ak (18 mg, 0.037 mmol) was obtained in 30% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H), 9.31 (s, 1H),

7.87−7.80 (m, 1H), 7.51−7.44 (m, 1H), 7.33−7.27 (m, 2H), 7.27−

7.19 (m, 4H), 6.38 (d, J = 4.0 Hz, 1H), 5.90 (d, J = 2.1 Hz, 1H), 5.66 (d,

J = 2.1 Hz, 1H), 4.87−4.60 (m, 4H), 3.64 (s, 3H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.29, 159.20, 155.55, 154.13 (d, J = 250.7 Hz),

143.69, 141.84−141.68 (m), 136.63, 129.05, 127.16, 126.21 (d, J = 3.6

Hz), 125.29 (d, J = 4.3 Hz), 122.86, 122.77, 120.45 (d, J = 18.0 Hz),

104.41, 99.55 (d, J = 9.8 Hz), 94.61, 93.22, 35.18; ¹⁹F NMR (470 MHz,

DMSO-d₆) δ −116.80 to −124.29 (m). UPLC/MS [M + H] = 479.219,

t_R= 1.46 min.

(2-((3-(2-Fluoro-3-(triﬂuoromethoxy)phenyl)-1-methyl-1H-pyra-

zol-5-yl)amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone

(9al). Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4ad (38 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9al (29 mg, 0.056 mmol) was obtained in 45% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.63 (s, 1H), 9.32 (s,

1H), 7.93−7.86 (m, 1H), 7.49−7.41 (m, 1H), 7.34−7.20 (m, 6H), 6.40

(d, J = 3.7 Hz, 1H), 5.91 (d, J = 2.1 Hz, 1H), 5.67 (d, J = 2.1 Hz, 1H),

4.81−4.64 (m, 4H), 3.65 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.32, 159.23, 155.57, 150.83 (d, J = 254.4 Hz), 143.69, 141.93 (d, J =

1.9 Hz), 141.50, 136.65, 136.23−136.00 (m), 127.18, 126.41 (d, J = 3.3

Hz), 125.00 (d, J = 4.5 Hz), 123.32 (d, J = 9.5 Hz), 122.79, 122.19,

121.12, 119.07, 104.49, 99.50 (d, J = 9.2 Hz), 94.68, 93.28, 35.22; ¹⁹F

NMR (470 MHz, DMSO-d₆) δ −57.82 (d, J = 4.9 Hz), −134.22 to

−134.33 (m). UPLC/MS [M + H] = 529.562, t_R= 1.54 min.

(2-((3-(2,5-Diﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9am). Amide

bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to 4ae (34.5

mg, 0.165 mmol, 1.1 equiv) according to General Procedure E1a. The

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resulting product was deprotected according to General Procedure E2a.

9am (14.6 mg, 0.032 mmol) was obtained in 21% yield over two steps.

¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H), 9.30 (s, 1H), 7.62−

156.06, 144.19, 142.61 (d, J = 2.3 Hz), 141.56, 137.13, 127.62, 126.55−

125.77 (m), 125.39, 124.86−124.44 (m), 123.21 (d, J = 3.0 Hz), 122.48

(d, J = 13.2 Hz), 118.08 (d, J = 23.5 Hz), 105.03, 100.17 (d, J = 10.3

Hz), 95.20, 93.91, 49.04, 35.69. UPLC/MS [M + H] = 513.212, t_R=

1.63 min.

7.56 (m, 1H), 7.34−7.23 (m, 6H), 7.19−7.11 (m, 1H), 6.38 (d, J = 4.0

Hz, 1H), 5.90 (d, J = 2.1 Hz, 1H), 5.63 (d, J = 2.1 Hz, 1H), 4.84−4.60

(m, 4H), 3.64 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.28,

159.19, 157.26, 155.53, 154.23 (d, J = 243.9 Hz), 143.70, 141.85 (d, J =

2.4 Hz), 141.70, 127.19, 122.80, 118.04−117.56 (m), 115.52−114.98

(m), 113.10−112.60 (m), 104.44, 99.60 (d, J = 10.1 Hz), 94.61, 93.15,

35.17 (d, J = 241.7 Hz); ¹⁹F NMR (470 MHz, DMSO-d₆) δ −118.66 to

−118.78 (m), −121.71 to −121.84 (m) UPLC/MS [M + H] =

463.241, t_R= 1.46 min.

(2-((3-(2-Chloro-5-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9an).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4af

(31 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2b. 9an (18 mg, 0.038 mmol) was obtained in 30% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H), 9.32 (s, 1H),

7.56−7.48 (m, 2H), 7.33−7.29 (m, 2H), 7.28−7.22 (m, 3H), 7.22−

7.15 (m, 1H), 6.56 (s, 1H), 5.91 (d, J = 2.1 Hz, 1H), 5.66 (d, J = 2.0 Hz,

1H), 4.80−4.69 (m, 4H), 3.64 (s, 3H); ¹³C NMR (126 MHz, DMSO-

d₆): δ 166.30, 160.66 (d, J = 243.7 Hz), 159.21, 155.54, 144.63 (d, J =

1.9 Hz), 143.70, 141.25, 136.60, 133.85 (d, J = 8.5 Hz), 132.11 (d, J =

8.7 Hz), 127.21, 125.85 (d, J = 3.0 Hz), 122.82, 115.74 (d, J = 22.7 Hz),

115.64 (d, J = 22.1 Hz), 104.43, 100.19, 94.61, 93.12, 35.15; ¹⁹F NMR

(470 MHz, DMSO-d₆) δ −115.27 to −115.40 (m). UPLC/MS [M +

H] = 479.259, t_R= 1.53 min.

(2-((3-(2-Fluoro-5-(triﬂuoromethoxy)phenyl)-1-methyl-1H-pyra-

zol-5-yl)amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone

(9ar). Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4aj (38 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9ar (33 mg, 0.063 mmol) was obtained in 50% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.64 (d, J = 1.3 Hz,

1H), 9.32 (s, 1H), 7.82−7.76 (m, 1H), 7.40−7.19 (m, 7H), 6.40 (d, J =

4.1 Hz, 1H), 5.91 (d, J = 2.1 Hz, 1H), 5.65 (d, J = 2.0 Hz, 1H), 4.84−

4.62 (m, 4H), 3.65 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.31,

159.22, 157.27 (d, J = 248.5 Hz), 155.58, 144.54−144.35 (m), 143.74,

142.08 (d, J = 2.3 Hz), 141.25 (d, J = 2.2 Hz), 136.71, 127.18, 122.78,

122.65, 121.59 (d, J = 8.6 Hz), 121.09, 119.21 (d, J = 5.1 Hz), 119.05,

118.12 (d, J = 24.9 Hz), 104.53, 99.71 (d, J = 10.5 Hz), 94.70, 93.34,

35.23; ¹⁹F NMR (470 MHz, DMSO-d₆) δ −57.20, −117.44 to −117.53

(m). UPLC/MS [M + H] = 529.529, t_R= 1.66 min.

(2-((3-(2,6-Diﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9as). Amide bro-

mide 8a (45 mg, 0.087 mmol, 1 equiv) was coupled to 4ak (20 mg,

0.096 mmol, 1.1 equiv) according to General Procedure E1a. The

resulting product was deprotected according to General Procedure E2a.

9as (20.6 mg, 0.045 mmol) was obtained in 52% yield over two steps.

¹H NMR (500 MHz, DMSO-d₆): δ 9.63 (s, 1H), 9.33 (s, 1H), 7.44−

7.36 (m, 1H), 7.32 (s, 2H), 7.30−7.24 (m, 2H), 7.24−7.17 (m, 1H),

7.14 (t, J = 8.3 Hz, 2H), 6.27−6.23 (m, 1H), 5.89 (d, J = 2.1 Hz, 1H),

5.61 (d, J = 2.1 Hz, 1H), 4.87−4.56 (m, 4H), 3.62 (s, 3H); ¹³C NMR

(126 MHz, DMSO-d₆): δ 166.35, 159.73 (dd, J = 249.7, 7.3 Hz),

159.25, 155.53, 143.72, 140.91, 137.83, 129.40, 127.26, 112.02 (d, J =

25.9 Hz), 112.02 (d, J = 15.0 Hz), 111.43, 104.32, 101.85−100.61 (m),

94.52, 92.84, 35.13; ¹⁹F NMR (470 MHz, DMSO-d₆) δ −112.05 to

−112.16 (m). UPLC/MS [M + H] = 463.225, t_R= 1.36 min.

(2-((3-(4-Chloro-2-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9at).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4al

(31 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2b. 9at (7 mg, 0.014 mmol) was obtained in 11% yield over two steps.

¹H NMR (500 MHz, DMSO-d₆): δ 9.61 (s, 1H), 9.30 (s, 1H), 7.89 (t, J

= 8.5 Hz, 1H), 7.46−7.40 (m, 1H), 7.32−7.21 (m, 6H), 6.34 (d, J = 4.0

Hz, 1H), 5.89 (d, J = 2.1 Hz, 1H), 5.63 (d, J = 2.1 Hz, 1H), 4.87−4.52

(m, 4H), 3.63 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.29,

159.20, 157.79 (d, J = 250.8 Hz), 155.53, 143.72, 141.72 (t, J = 2.5 Hz),

136.72, 132.15 (d, J = 10.5 Hz), 128.50 (d, J = 4.6 Hz), 127.18, 124.84

(d, J = 3.2 Hz), 122.80, 120.19 (d, J = 11.8 Hz), 116.65 (d, J = 26.1 Hz),

104.39, 99.42 (d, J = 9.7 Hz), 94.58, 93.11, 35.14; ¹⁹F NMR (470 MHz,

DMSO-d₆) δ −113.56 to −113.67 (m). UPLC/MS [M + H] = 479.259,

t_R= 1.58 min.

(2-((3-(2-Chloro-4-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9au).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4am (31 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2b. 9au (18 mg, 0.037 mmol) was obtained in 30% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H), 9.31 (s,

1H), 7.80−7.74 (m, 1H), 7.45 (dd, J = 8.9, 2.7 Hz, 1H), 7.32 (s, 2H),

7.29−7.20 (m, 4H), 6.45 (s, 1H), 5.90 (d, J = 2.1 Hz, 1H), 5.66 (d, J =

2.0 Hz, 1H), 4.85−4.64 (m, 4H), 3.62 (s, 3H); ¹³C NMR (126 MHz,

DMSO-d₆): δ 166.33, 160.98 (d, J = 247.9 Hz), 159.22, 155.53, 144.92,

143.74, 140.96, 136.58, 131.46 (d, J = 10.5 Hz), 131.29 (d, J = 8.7 Hz),

128.94 (d, J = 3.4 Hz), 127.22, 122.84, 117.17 (d, J = 24.9 Hz), 114.56

(d, J = 21.1 Hz), 104.33, 99.96, 94.54, 93.00, 35.04; ¹⁹F NMR (470

MHz, DMSO-d₆) δ −113.06 to −113.20 (m). UPLC/MS [M + H] =

479.219, t_R= 1.52 min.

(2-((3-(5-Chloro-2-ﬂuorophenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ao).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4ag (31 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2b. 9ao (18 mg, 0.037 mmol) was obtained in 30% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.63 (s, 1H), 9.32 (s,

1H), 7.89−7.82 (m, 1H), 7.39−7.33 (m, 1H), 7.33−7.21 (m, 6H), 6.38

(d, J = 4.0 Hz, 1H), 5.91 (d, J = 2.1 Hz, 1H), 5.65 (d, J = 2.1 Hz, 1H),

4.96−4.53 (m, 4H), 3.64 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.30, 159.21, 157.66 (d, J = 248.4 Hz), 155.55, 143.69, 141.92 (d, J =

2.3 Hz), 141.36 (d, J = 2.1 Hz), 136.61, 128.57−128.40 (m), 127.19,

126.41 (d, J = 4.3 Hz), 122.87 (d, J = 13.3 Hz), 122.79, 118.19 (d, J =

24.2 Hz), 104.49, 99.62 (d, J = 9.9 Hz), 94.67, 93.25, 35.20; ¹⁹F NMR

(470 MHz, DMSO-d₆) δ −118.55 to −118.64 (m). UPLC/MS [M +

H] = 479.219, t_R= 1.57 min.

(2-((3-(2-Fluoro-5-methylphenyl)-1-methyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ap).

Amide bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to

4ah (33.9 mg, 0.165 mmol, 1.1 equiv) according to General Procedure

E1a. The resulting product was deprotected according to General

Procedure E2a. 9ap (12.5 mg, 0.027 mmol) was obtained in 18% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.60 (s, 1H), 9.29 (s,

1H), 7.72−7.66 (m, 1H), 7.37−7.30 (m, 2H), 7.30−7.23 (m, 2H), 7.19

(s, 1H), 7.14−7.05 (m, 2H), 6.33 (d, J = 3.9 Hz, 1H), 5.89 (d, J = 2.1

Hz, 1H), 5.63 (d, J = 2.1 Hz, 1H), 4.82−4.67 (m, 4H), 3.62 (s, 3H),

2.30 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.32, 159.20,

156.44 (d, J = 244.4 Hz), 155.49, 143.78, 142.78, 141.37 (d, J = 2.1 Hz),

133.37 (d, J = 3.2 Hz), 129.35, 127.55 (d, J = 4.1 Hz), 127.21, 120.56 (d,

J = 11.9 Hz), 115.75 (d, J = 22.3 Hz), 104.31, 99.55, 94.48, 92.90, 35.03,

20.22. UPLC/MS [M + H] = 459.256, t_R= 1.52 min.

(2-((3-(2-Fluoro-5-(triﬂuoromethyl)phenyl)-1-methyl-1H-pyra-

zol-5-yl)amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone

(9aq). Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4ai (36 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9aq (36 mg, 0.071 mmol) was obtained in 57% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.66 (s, 1H), 9.34 (s,

1H), 8.21−8.15 (m, 1H), 7.72−7.65 (m, 1H), 7.49−7.41 (m, 1H), 7.28

(q, J = 3.2 Hz, 3H), 7.25−7.17 (m, 2H), 6.42 (d, J = 4.1 Hz, 1H), 5.93

(d, J = 2.1 Hz, 1H), 5.68 (s, 1H), 4.72 (s, 4H), 3.67 (s, 3H); ¹³C NMR

(126 MHz, DMSO-d₆): δ 166.78, 161.24 (d, J = 254.5 Hz), 159.68,

(2-((1-(tert-Butyl)-3-(2,3-dimethylphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9av).

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Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4ao

(33 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9av (31 mg, 0.063 mmol) was obtained in 50% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.68 (s, 1H), 9.30 (s, 1H),

7.37−7.32 (m, 2H), 7.32−7.25 (m, 3H), 7.16−7.05 (m, 2H), 6.99 (s,

1H), 6.24 (s, 1H), 5.86 (d, J = 2.1 Hz, 1H), 5.72 (d, J = 2.1 Hz, 1H),

4.89−4.75 (m, 4H), 2.34 (s, 3H), 2.27 (s, 3H), 1.55 (s, 9H); ¹³C NMR

(126 MHz, DMSO-d₆): δ 166.92, 159.44, 147.76, 145.08, 139.31,

136.86, 136.70, 133.91, 133.87, 128.68, 127.27, 126.89, 125.09, 122.85,

103.17, 103.03, 93.79, 92.11, 79.17, 59.17, 29.48, 20.43, 16.60. UPLC/

MS [M + H] = 497.600, t_R= 1.80 min.

(2-((3-(5-Fluoro-2-methylphenyl)-1-isopropyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9aw).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4ap (32 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9aw (40 mg, 0.083 mmol) was obtained in 66% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.63 (s, 1H), 9.30 (s,

1H), 7.38−7.29 (m, 3H), 7.30−7.22 (m, 3H), 7.13 (s, 1H), 7.05−6.97

(m, 1H), 6.35 (s, 1H), 5.88 (d, J = 2.1 Hz, 1H), 5.63 (d, J = 2.1 Hz, 1H),

4.87−4.69 (m, 4H), 4.51−4.40 (m, J = 6.9 Hz, 1H), 2.44 (s, 3H), 1.35

(d, J = 6.5 Hz, 6H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.48, 160.43

(d, J = 240.5 Hz), 159.26, 155.56, 147.71 (d, J = 2.4 Hz), 144.58,

139.50, 136.72, 135.06 (d, J = 7.9 Hz), 132.59 (d, J = 8.0 Hz), 130.93 (d,

J = 2.9 Hz), 127.25, 122.85, 114.15 (d, J = 22.0 Hz), 113.51 (d, J = 20.7

Hz), 103.90, 100.20, 94.21, 92.57, 47.90, 22.20, 20.66; ¹⁹F NMR (470

MHz, DMSO-d₆) δ −117.99 to −118.09 (m). UPLC/MS [M + H] =

487.545, t_R= 1.70 min.

(2-((1-(tert-Butyl)-3-(5-ﬂuoro-2-methylphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ax).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4aq (34 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9ax (35.5 mg, 0.067 mmol) was obtained in 54% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.70 (s, 1H), 9.29 (s,

1H), 7.40−7.32 (m, 3H), 7.32−7.22 (m, 3H), 7.05−6.97 (m, 2H), 6.47

(s, 1H), 5.86 (d, J = 2.1 Hz, 1H), 5.70 (d, J = 2.1 Hz, 1H), 4.95−4.62

(m, 4H), 3.35 (s, 3H), 2.46 (s, 3H), 1.56 (s, 9H); ¹³C NMR (126 MHz,

DMSO-d₆): δ 166.90, 160.47 (d, J = 240.3 Hz), 159.45, 155.56, 146.05

(d, J = 2.4 Hz), 145.01, 139.78, 136.68, 134.88 (d, J = 7.7 Hz), 132.67

(d, J = 7.9 Hz), 130.90 (d, J = 2.9 Hz), 127.28, 122.85, 114.10 (d, J =

22.0 Hz), 113.48 (d, J = 20.8 Hz), 103.08, 102.94, 93.86, 92.18, 59.47,

29.38, 20.82; ¹⁹F NMR (470 MHz, DMSO-d₆) δ −117.90 to −118.08

(m). UPLC/MS [M + H] = 501.569, t_R= 1.87 min.

(2-((1-Cyclopentyl-3-(5-ﬂuoro-2-methylphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ay).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4ar

(36 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9ay (31 mg, 0.060 mmol) was obtained in 50% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.64 (s, 1H), 9.31 (s, 1H),

7.39−7.30 (m, 3H), 7.30−7.22 (m, 3H), 7.15 (s, 1H), 7.04−6.97 (m,

1H), 6.36 (s, 1H), 5.88 (d, J = 2.1 Hz, 1H), 5.63 (d, J = 2.1 Hz, 1H),

4.87−4.69 (m, 4H), 4.61 (p, J = 7.1 Hz, 1H), 2.44 (s, 3H), 1.92 (s, 4H),

1.87−1.76 (m, 2H), 1.59−1.50 (m, 2H); ¹³C NMR (126 MHz, DMSO-

d₆): δ 166.50, 160.44 (d, J = 240.3 Hz), 159.25, 155.56, 147.70 (d, J =

2.3 Hz), 144.43, 140.18, 136.71, 134.96 (d, J = 7.7 Hz), 132.62 (d, J =

8.0 Hz), 130.92 (d, J = 2.8 Hz), 127.26, 122.84, 114.17 (d, J = 21.9 Hz),

113.51 (d, J = 20.7 Hz), 103.93, 100.05, 94.28, 92.72, 57.03, 32.28,

24.18, 20.72; ¹⁹F NMR (470 MHz, DMSO-d₆) δ −117.94 to −118.06

(m). UPLC/MS [M + H] = 513.564, t_R= 1.83 min.

2.9 Hz, 1H), 6.36 (s, 1H), 5.87 (d, J = 2.1 Hz, 1H), 5.64 (d, J = 2.1 Hz,

1H), 4.87−4.70 (m, 4H), 4.07−4.00 (m, 1H), 2.43 (s, 3H), 1.90−1.70

(m, 6H), 1.64−1.55 (m, 1H), 1.25−1.12 (m, 3H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.47, 160.42 (d, J = 240.3 Hz), 159.20, 155.52,

147.59 (d, J = 2.1 Hz), 144.50, 139.62, 136.65, 135.02 (d, J = 7.7 Hz),

132.57 (d, J = 7.9 Hz), 130.91 (d, J = 2.9 Hz), 127.25, 122.82, 114.13 (d,

J = 22.0 Hz), 113.47 (d, J = 20.6 Hz), 103.99, 100.25, 94.25, 92.65,

55.33, 32.27, 25.05, 24.89, 20.69; ¹⁹F NMR (470 MHz, DMSO-d₆) δ

−118.01 to −118.16 (m). UPLC/MS [M + H] = 527.578, t_R= 1.90

min.

(2-((3-(2-Fluoro-3-methylphenyl)-1-isopropyl-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9ba).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4at

(32 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9ba (38 mg, 0.078 mmol) was obtained in 65% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H), 9.29 (s, 1H),

7.79−7.71 (m, 1H), 7.36−7.30 (m, 2H), 7.30−7.23 (m, 2H), 7.21−

7.15 (m, 1H), 7.14 (s, 1H), 7.09 (t, J = 7.6 Hz, 1H), 6.34 (d, J = 4.1 Hz,

1H), 5.87 (d, J = 2.1 Hz, 1H), 5.61 (d, J = 2.1 Hz, 1H), 4.85−4.68 (m,

4H), 4.52−4.40 (m, 1H), 2.25 (d, J = 2.1 Hz, 3H), 1.36 (d, J = 6.2 Hz,

6H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.46, 159.25, 157.71 (d, J =

247.1 Hz), 155.56, 144.57, 142.99, 139.93 (d, J = 2.2 Hz), 136.72,

130.21 (d, J = 5.1 Hz), 127.26, 125.17 (d, J = 3.8 Hz), 124.86 (d, J =

17.6 Hz), 123.91 (d, J = 3.8 Hz), 122.87, 121.09 (d, J = 12.4 Hz),

104.00, 100.52 (d, J = 10.1 Hz), 94.26, 92.62, 48.02, 22.23, 14.35 (d, J =

4.8 Hz); ¹⁹F NMR (470 MHz, DMSO-d₆) δ −120.64 to −121.59 (m).

UPLC/MS [M + H] = 487.545, t_R= 1.70 min.

(2-((1-(tert-Butyl)-3-(2-ﬂuoro-3-methylphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9bb).

Amide bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to

4au (41 mg, 0.165 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9bb (41.8 mg, 0.084 mmol) was obtained in 56% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.70 (s, 1H), 9.29 (s,

1H), 7.80−7.73 (m, 1H), 7.38−7.32 (m, 2H), 7.31−7.25 (m, 2H),

7.22−7.15 (m, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.02 (s, 1H), 6.43 (d, J =

4.2 Hz, 1H), 5.87 (d, J = 2.1 Hz, 1H), 5.68 (d, J = 2.1 Hz, 1H), 4.86−

4.71 (m, 4H), 2.26 (d, J = 2.1 Hz, 3H), 1.57 (s, 9H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.87, 159.42, 157.76 (d, J = 246.9 Hz), 155.56,

144.96, 141.26, 140.21 (d, J = 2.3 Hz), 136.68, 130.19 (d, J = 4.8 Hz),

127.27, 125.09 (d, J = 3.8 Hz), 124.84 (d, J = 17.5 Hz), 123.93 (d, J =

3.8 Hz), 122.85, 121.03 (d, J = 12.6 Hz), 103.29 (d, J = 10.1 Hz),

103.18, 93.94, 92.22, 59.59, 29.37, 14.32 (d, J = 4.7 Hz); ¹⁹F NMR (470

MHz, DMSO-d₆) δ −121.11 to −121.25 (m). UPLC/MS [M + H] =

501.437, t_R= 1.88 min.

(2-((1-Cyclopentyl-3-(2-ﬂuoro-3-methylphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9bc).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4av

(36 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9bc (43 mg, 0.085 mmol) was obtained in 68% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.63 (s, 1H), 9.30 (s, 1H),

7.77−7.70 (m, 1H), 7.36−7.30 (m, 2H), 7.26 (dd, J = 5.6, 3.2 Hz, 2H),

7.21−7.15 (m, 2H), 7.09 (t, J = 7.6 Hz, 1H), 6.35 (d, J = 4.1 Hz, 1H),

5.88 (d, J = 2.1 Hz, 1H), 5.62 (d, J = 2.1 Hz, 1H), 4.76 (s, 4H), 4.61 (p, J

= 7.3 Hz, 1H), 2.25 (d, J = 2.1 Hz, 3H), 1.95−1.89 (m, 4H), 1.87−1.76

(m, 2H), 1.58−1.52 (m, 2H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.50, 159.25, 157.72 (d, J = 247.0 Hz), 155.56, 144.44, 142.94,

140.62 (d, J = 2.3 Hz), 136.72, 130.23 (d, J = 5.0 Hz), 127.27, 125.17 (d,

J = 3.9 Hz), 124.87 (d, J = 17.5 Hz), 123.94 (d, J = 3.8 Hz), 122.86,

121.06 (d, J = 12.4 Hz), 104.03, 100.43 (d, J = 10.0 Hz), 94.34, 92.75,

57.15, 32.22, 24.15, 14.35 (d, J = 4.7 Hz); ¹⁹F NMR (470 MHz, DMSO-

d₆) δ −119.76 to −122.97 (m). UPLC/MS [M + H] = 513.564, t_R=

1.80 min.

(2-((1-Cyclohexyl-3-(5-ﬂuoro-2-methylphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9az).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4as

(38 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9az (35 mg, 0.066 mmol) was obtained in 53% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.60 (s, 1H), 9.28 (s, 1H),

7.36−7.29 (m, 3H), 7.30−7.21 (m, 3H), 7.11 (s, 1H), 7.00 (td, J = 8.4,

(2-((1-Cyclohexyl-3-(2-ﬂuoro-3-methylphenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9bd).

Amide bromide 8a (77 mg, 0.150 mmol, 1 equiv) was coupled to

4aw (45 mg, 0.165 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

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Procedure E2a. 9bd (50.7 mg, 0.096 mmol) was obtained in 64% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.60 (s, 1H), 9.28 (s,

1H), 7.77−7.70 (m, 1H), 7.35−7.30 (m, 2H), 7.29−7.22 (m, 2H),

7.20−7.13 (m, 1H), 7.13−7.05 (m, 2H), 6.35 (d, J = 4.1 Hz, 1H), 5.87

(d, J = 2.1 Hz, 1H), 5.62 (d, J = 2.0 Hz, 1H), 4.84−4.64 (m, 4H), 4.10−

4.00 (m, 1H), 2.24 (d, J = 2.0 Hz, 3H), 1.79 (d, J = 28.9 Hz, 6H), 1.61−

1.57 (m, 1H), 1.26−1.11 (m, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ

166.50, 159.23, 157.71 (d, J = 247.1 Hz), 155.55, 144.52, 142.90,

140.04 (d, J = 2.1 Hz), 136.68, 130.19 (d, J = 4.8 Hz), 127.27, 125.15 (d,

J = 3.8 Hz), 124.86 (d, J = 17.5 Hz), 123.90 (d, J = 3.8 Hz), 122.85,

121.09 (d, J = 12.4 Hz), 104.08, 100.60 (d, J = 10.1 Hz), 94.32, 92.70,

55.49, 32.30, 25.12, 24.93, 14.35 (d, J = 4.7 Hz); ¹⁹F NMR (470 MHz,

DMSO-d₆) δ −120.94 to −121.05 (m). UPLC/MS [M + H] = 527.589,

t_R=1.90 min.

(2-((3-(2,3-Diﬂuorophenyl)-1-isopropyl-1H-pyrazol-5-yl)amino)-

4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9be). Amide bro-

mide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4ax (33 mg,

0.138 mmol, 1.1 equiv) according to General Procedure E1b. The

resulting product was deprotected according to General Procedure E2a.

9be (33 mg, 0.067 mmol) was obtained in 56% yield over two steps. ¹H

NMR (500 MHz, DMSO-d₆): δ 9.65 (s, 1H), 9.31 (s, 1H), 7.75−7.68

(m, 1H), 7.41−7.09 (m, 7H), 6.37 (d, J = 3.7 Hz, 1H), 5.89 (d, J = 2.1

Hz, 1H), 5.61 (d, J = 2.0 Hz, 1H), 4.76−4.71 (m, 4H), 4.53−4.43 (m,

1H), 1.38−1.33 (m, 6H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.40,

159.22, 155.62, 150.30 (d, J = 244.6 Hz), 147.06 (d, J = 248.9 Hz),

144.44, 140.40, 127.20, 124.63, 123.59, 122.80, 122.50, 115.63 (d, J =

16.6 Hz), 104.14, 100.40 (d, J = 8.7 Hz), 94.41, 92.85, 48.16, 22.16.

UPLC/MS [M + H] = 491.531, t_R= 1.59 min.

(m, 2H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.45, 159.24, 155.58,

150.22 (d, J = 248.2 Hz), 147.18 (d, J = 248.2 Hz), 144.33, 141.78,

141.07, 136.74, 127.22, 124.68, 123.55 (d, J = 8.6 Hz), 122.81, 122.52,

115.65 (d, J = 16.8 Hz), 104.18, 100.35 (d, J = 8.9 Hz), 94.49, 92.99,

57.27, 32.23, 24.14. UPLC/MS [M + H] = 517.533, t_R= 1.75 min.

(2-((1-Cyclohexyl-3-(2,3-diﬂuorophenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9bi).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to

4bb (38 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9bi (46 mg, 0.087 mmol) was obtained in 73% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.64 (s, 1H), 9.30 (s,

1H), 7.74−7.66 (m, 1H), 7.34−7.09 (m, 7H), 6.37 (d, J = 3.8 Hz, 1H),

5.89 (d, J = 2.1 Hz, 1H), 5.63 (d, J = 2.1 Hz, 1H), 4.76−4.72 (m, 4H),

4.13−4.03 (m, 1H), 1.91−1.70 (m, 6H), 1.62−1.58 (m, 1H), 1.21−

1.15 (m, 3H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.87, 159.64,

156.03, 151.83, 146.30, 142.17, 140.97, 137.05, 127.65, 125.04, 123.99

(d, J = 8.6 Hz), 123.22, 122.93, 116.04 (d, J = 16.8 Hz), 104.68, 100.90

(d, J = 9.4 Hz), 94.92, 93.43, 56.01, 40.87, 25.51, 25.33. UPLC/MS [M

+ H] = 531.547, T_R= 1.85 min.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01777.

Supporting Tables 1−8, Supporting Figures S1−S7

(PDF)

(2-((1-(tert-Butyl)-3-(2,3-diﬂuorophenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9bf).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4ay

(34.6 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9bf (33.4 mg, 0.066 mmol) was obtained in 53% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.74 (s, 1H), 9.32 (s, 1H),

7.77−7.70 (m, 1H), 7.37−7.18 (m, 6H), 7.05 (s, 1H), 6.48 (d, J = 3.8

Hz, 1H), 5.88 (d, J = 2.1 Hz, 1H), 5.67 (d, J = 2.1 Hz, 1H), 4.82−4.79

(m, 4H), 1.57 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆): δ 166.86,

159.44, 155.62, 150.40 (dd, J = 244.2, 12.7 Hz), 147.11 (dd, J = 249.6,

13.8 Hz), 144.89, 140.68 (d, J = 1.9 Hz), 140.21 (d, J = 3.4 Hz), 136.68,

127.30, 124.87−124.61 (m), 123.55 (d, J = 8.3 Hz), 122.87, 122.52 (t, J

= 3.0 Hz), 115.71 (d, J = 17.1 Hz), 103.35, 103.29 (d, J = 1.9 Hz), 94.06,

92.34, 59.91, 29.34; ¹⁹F NMR (470 MHz, DMSO-d₆) δ −138.74 to

−139.72 (m), −142.92 to −143.90 (m). UPLC/MS [M + H] =

505.538, t_R= 1.83 min.

(2-((1-(tert-Butyl)-3-(2,3-diﬂuorophenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(4-ﬂuoroisoindolin-2-yl)methanone

(9bg). Amide bromide 8b (67 mg, 0.125 mmol, 1 equiv) was coupled to

4ay (35 mg, 0.138 mmol, 1.1 equiv) according to General Procedure

E1b. The resulting product was deprotected according to General

Procedure E2a. 9bg (34 mg, 0.066 mmol) was obtained in 53% yield

over two steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.77 (s, 1H), 9.33 (s,

1H), 7.77−7.70 (m, 1H), 7.38−7.27 (m, 2H), 7.26−7.17 (m, 2H),

7.14−7.04 (m, 2H), 6.47 (d, J = 3.7 Hz, 1H), 5.88 (d, J = 2.1 Hz, 1H),

5.67 (d, J = 2.1 Hz, 1H), 4.82 (m, 4H), 1.58 (s, 9H); ¹³C NMR (126

MHz, DMSO-d₆): δ 166.80, 159.53, 155.66, 149.70 (d, J = 423.9 Hz),

144.98, 140.66, 140.18, 129.92, 124.69, 123.51 (d, J = 8.5 Hz), 122.46,

119.14, 115.66 (d, J = 17.2 Hz), 114.03, 113.68 (d, J = 19.1 Hz), 103.38,

102.94, 94.01, 92.45, 59.92, 29.32. UPLC/MS [M + H] = 523.576, t_R=

1.86 min.

Conﬁrmation of lysate EC_50s(Table 1); conﬁrmation of

fungal selectivity using puriﬁed NBDs (Table 2); HepG2

cytotoxicity dose−response data and statistics (Table 3);

NIH 3T3 cytotoxicity dose−response data and statistics

(Table 4); crystallographic data for apoprotein and NVP-

AUY922-liganded structures (Table 5); crystallographic

data for RAP-liganded structures (Table 6); features of

selected Hsp90 NBD structures (Table 7); summary of

computational docking results for the compounds in

Table 4 (Table 8); correlation between lysate and puriﬁed

NBD potencies in C. neoformans (Figure S1); correlation

between lysate and puriﬁed NBD potencies in human

(Figure S2); correlation between fold-selectivities calcu-

lated using lysates and puriﬁed NBDs (Figure S3);

HepG2 cytotoxicity dose−response curves (Figure S4);

NIH 3T3 cytotoxicity dose−response curves (Figure S5);

ligand variability in the C. neoformans RAP complexes

(Figure S6); and contact sites between ligands and

structural elements of HSP90 (Figure S7) (PDF)

Molecular formula strings and associated biological data

(CSV)

Coordinate ﬁles used to generate Figure 11B (PDB)

Coordinate ﬁles used to generate Figure 11C (PDB)

Coordinate ﬁles used to generate Figure 11D (PDB)

Coordinate ﬁles used to generate Figure 11E (PDB)

Coordinate ﬁles used to generate Figure 11F (PDB)

Coordinate ﬁles used to generate Figure 11G (PDB)

(2-((1-Cyclopentyl-3-(2,3-diﬂuorophenyl)-1H-pyrazol-5-yl)-

amino)-4,6-dihydroxyphenyl)(isoindolin-2-yl)methanone (9bh).

Amide bromide 8a (64 mg, 0.125 mmol, 1 equiv) was coupled to 4az

(36 mg, 0.138 mmol, 1.1 equiv) according to General Procedure E1b.

The resulting product was deprotected according to General Procedure

E2a. 9bh (44 mg, 0.085 mmol) was obtained in 68% yield over two

steps. ¹H NMR (500 MHz, DMSO-d₆): δ 9.74 (s, 1H), 9.40 (s, 1H),

7.81 (t, J = 7.2 Hz, 1H), 7.46−7.25 (m, 7H), 6.48 (d, J = 3.7 Hz, 1H),

6.00 (d, J = 2.1 Hz, 1H), 5.72 (d, J = 2.0 Hz, 1H), 4.87−4.83 (m, 4H),

4.73 (p, J = 7.3 Hz, 1H), 2.04 (s, 4H), 1.96−1.88 (m, 2H), 1.70−1.61

Accession Codes

Coordinates have been deposited in the PDB with the following

accession numbers: apo (7K9R), NVP-AUY922 (7K9S),

compound 19 (7K9U), compound 18 (7K9V), and compound

10 (7K9W). The authors will release coordinates and

experimental data upon article publication.

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Funding

NIH R01AI120958

Notes

AUTHOR INFORMATION

Corresponding Authors

■

́

Gilbert G. Prive − Princess Margaret Cancer Centre, Toronto,

The authors declare the following competing ﬁnancial

interest(s): L.E.C. is a cofounder, Chief Scientiﬁc Oﬃcer, and

shareholder of Bright Angel Therapeutics, a platform company

for the development of novel antifungal therapeutics. L.E.C. is a

consultant for Boragen, a small molecule development company

focused on leveraging the unique chemical properties of boron

chemistry for crop protection and animal health. L.W. is a co-

founder and shareholder of Bright Angel Therapeutics. L.E.B.,

D.S.H., P.T.M., L.E.C. and L.W. are named as inventors on a

patent application pertaining to ﬁndings reported here.

Ontario M5G 1L7, Canada; Department of Medical

Biophysics and Department of Biochemistry, University of

Toronto, Toronto, Ontario M5G 1L7, Canada;

Email: Gil.Prive@uhnresearch.ca

Luke Whitesell − Department of Molecular Genetics, University

of Toronto, Toronto, Ontario M5G 1M1, Canada;

Email: luke.whitesell@utoronto.ca

Leah E. Cowen − Department of Molecular Genetics, University

of Toronto, Toronto, Ontario M5G 1M1, Canada;

orcid.org/0000-0001-5797-0110; Email: leah.cowen@

utoronto.ca

Lauren E. Brown − Department of Chemistry and Center for

Molecular Discovery (BU-CMD), Boston University, Boston,

Massachusetts 02215, United States; orcid.org/0000-

0001-9489-484X; Email: brownle@bu.edu

ACKNOWLEDGMENTS

■

This work was supported by a grant from the National Institutes

of Health (Grant R01AI120958 to L.E.C., L.E.B., and D.J.K.).

L.E.C. holds a Canada Research Chair in Microbial Genomics &

Infectious Disease and is Co-Director of the CIFAR Program,

Fungal Kingdom: Threats & Opportunities. G.G.P. was

supported by grants from NSERC and the Samuel Waxman

Cancer Research Foundation.

Authors

Paul T. Marcyk − Department of Chemistry and Center for

Molecular Discovery (BU-CMD), Boston University, Boston,

Massachusetts 02215, United States; orcid.org/0000-

0001-5414-0293

Emmanuelle V. LeBlanc − Department of Molecular Genetics,

University of Toronto, Toronto, Ontario M5G 1M1, Canada;

orcid.org/0000-0003-4278-0984

ABBREVIATIONS USED

■

Hsp90, heat shock protein 90; Trap1, TNF receptor-associated

protein 1; Grp94, 94 kDa glucose-regulated protein; NBD,

nucleotide binding domain; ATP, adenosine triphosphate; Cl_int,

intrinsic clearance; DIPEA, N,N-diisopropylethylamine; ELSD,

evaporative light-scattering detector; FBS, fetal bovine serum;

FP, ﬂuorescence polarization; FS, fungal selectivity; HATU,

hexaﬂuorophosphate azabenzotriazole tetramethyl uronium; IS,

internal standard; MRM, multiple reaction monitoring; TI,

therapeutic index; UPLC, ultraperformance liquid chromatog-

raphy; WHO, World Health Organization; YPD, yeast extract

peptone dextrose

Douglas A. Kuntz − Princess Margaret Cancer Centre, Toronto,

Ontario M5G 1L7, Canada

Alice Xue − Department of Molecular Genetics, University of

Toronto, Toronto, Ontario M5G 1M1, Canada

Francisco Ortiz − Department of Biochemistry, UT

Southwestern Medical Center, Dallas, Texas 75390-9038,

United States

Richard Trilles − Department of Chemistry and Center for

Molecular Discovery (BU-CMD), Boston University, Boston,

Massachusetts 02215, United States

Stephen Bengtson − Department of Chemistry and Center for

Molecular Discovery (BU-CMD), Boston University, Boston,

Massachusetts 02215, United States; orcid.org/0000-

0002-9383-5159

Tristan M. G. Kenney − Department of Medical Biophysics,

University of Toronto, Toronto, Ontario M5G 1L7, Canada;

orcid.org/0000-0001-8242-4349

David S. Huang − Department of Chemistry and Center for

Molecular Discovery (BU-CMD), Boston University, Boston,

Massachusetts 02215, United States; orcid.org/0000-

0003-0256-5307

Nicole Robbins − Department of Molecular Genetics,

University of Toronto, Toronto, Ontario M5G 1M1, Canada

Noelle S. Williams − Department of Biochemistry, UT

Southwestern Medical Center, Dallas, Texas 75390-9038,

United States

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S.; Ngoma, J.; Gondwe-Chunda, L.; Perfect, C.; Shroufi, A.; Andrieux-

Meyer, I.; Chan, A.; Schutz, C.; Hosseinipour, M.; Van der Horst, C.;

Klausner, J. D.; Boulware, D. R.; Heyderman, R.; Lalloo, D.; Day, J.;

Jarvis, J. N.; Rodrigues, M.; Jaffar, S.; Denning, D.; Migone, C.;

Complete contact information is available at:

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The manuscript was written through contributions of all

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Fungal-Selective Resorcylate Aminopyrazole Hsp90 Inhibitors: Optimization of Whole-Cell Anticryptococcal Activity and Insights into the Structural Origins of Cryptococcal Selectivity

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