X-ray Characterization and Structure-Based Optimization of Striatal-Enriched Protein Tyrosine Phosphatase Inhibitors

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX-XXX

X‑ray Characterization and Structure-Based Optimization of Striatal-

Enriched Protein Tyrosine Phosphatase Inhibitors

Michael R. Witten,^†Lisa Wissler,^‡Melanie Snow,^§,○Stefan Geschwindner,^‡Jon A. Read,^∥

Nicholas J. Brandon,^⊥Angus C. Nairn,^#Paul J. Lombroso,^#,∇Helena Kack,*^,‡

̈

and Jonathan A. Ellman*^,†

^†Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States

^‡Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Gothenburg, Sweden

^§Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Macclesﬁeld SK10 4TG, United

Kingdom

^∥Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge CB4 0WG, United

Kingdom

^⊥Neuroscience, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Cambridge, Massachusetts 02139, United

States

^#Department of Psychiatry, Yale University, New Haven, Connecticut 06520, United States

^∇Child Study Center, Yale University, New Haven, Connecticut 06520, United States

S

* Supporting Information

ABSTRACT: Excessive activity of striatal-enriched protein

tyrosine phosphatase (STEP) in the brain has been detected in

numerous neuropsychiatric disorders including Alzheimer’s

disease. Notably, knockdown of STEP in an Alzheimer mouse

model eﬀected an increase in the phosphorylation levels of

downstream STEP substrates and a signiﬁcant reversal in the

observed cognitive and memory deﬁcits. These data point to

the promising potential of STEP as a target for drug discovery

in Alzheimer’s treatment. We previously reported a substrate-

based approach to the development of low molecular weight STEP inhibitors with K_ivalues as low as 7.8 μM. Herein, we disclose

the ﬁrst X-ray crystal structures of inhibitors bound to STEP and the surprising ﬁnding that they occupy noncoincident binding

sites. Moreover, we utilize this structural information to optimize the inhibitor structure to achieve a K_iof 110 nM, with 15−60-

fold selectivity across a series of phosphatases.

methyl-^D-aspartate receptor (NMDAR)²³⁻²⁵and the GluA2

INTRODUCTION

■

subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepro-

Proper cognitive function requires robust synaptic connections

pionic acid receptor (AMPAR),²⁶which induces their internal-

possessing the ability to strengthen or weaken based upon

ization via endocytosis. Long-term potentiation (LTP),^27,28an

tightly regulated neuronal signaling.^1,2Aberrations in systems

increase in synaptic strength and plasticity that relies on ERK1/

within the central nervous system (CNS) that control synaptic

2²⁹and Pyk2,³⁰as well as transmembrane NMDAR³¹and

plasticity have been implicated in various cognitive and

AMPAR function,³²is one of the fundamental neuronal

neurological disorders such as Alzheimer’s disease (AD),^3,4

schizophrenia,⁵⁻⁷and fragile X syndrome.⁸⁻¹⁰Striatal-enriched

mechanisms underlying learning and memory. Dephosphor-

ylation of these kinases and transmembrane ion channels

protein tyrosine phosphatase (STEP, PTPN5) belongs to a

consequently results in an overall reduction in synaptic control

family of tyrosine-speciﬁc protein phosphatases (PTPs)

of LTP.

enriched in the CNS, particularly the striatum, hippocampus,

and cortex.¹¹⁻¹⁸Recent studies into STEP’s function have led

The potential of STEP as a therapeutic target in AD was

suggested through genetic reduction of STEP activity in a triple

to the conclusion that it opposes synaptic strengthening

transgenic AD mouse model.³³Knockout of STEP expression

through its actions on substrate proteins.

in these mice improved cognitive function up to the level of

STEP-mediated dephosphorylation of extracellular signal-

related kinases 1 and 2 (ERK1/2),¹⁹mitogen-activated protein

normal wild-type mice as determined by several memory and

kinase p38,²⁰proline-rich tyrosine kinase 2 (Pyk2),²¹and the

Src family tyrosine kinase Fyn leads to their inactivation.²²

Received: August 30, 2017

STEP also dephosphorylates the GluN2B subunit of the N-

A

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cognitive tests. Furthermore, mice null for STEP exhibited

restored levels of phospho-GluN2B in hippocampal synapto-

somal membranes as well as enhanced levels of phospho-

ERK1/2 and phospho-Fyn. Taken together, these ﬁndings

suggest that STEP represents a promising therapeutic target for

AD.

However, a signiﬁcant caveat toward the development of

STEP inhibitors is the widespread recognition that PTPs are

extremely challenging targets for the development of small

molecule inhibitors due to the highly positively charged nature

and the signiﬁcant structural homology of PTP active sites.³⁴

Indeed, no PTP inhibitors have yet been approved for clinical

use despite the clear physiological importance of these enzymes

and their complementary activity to the well-established kinase

drug targets.³⁵

Table 1. Thermodynamic Parameters for Compound

Interaction with STEP Phosphatase Domain Determined by

a

ITC at 30 °C

inhibitor

K_D(μM)

ΔH (kcal/mol)

−TΔS (kcal/mol)

1

2

3

70.4 4.7

27.2 0.6

31.4 0.8

−2.26 0.07

−7.71 0.06

−7.63 0.07

−2.12

2.77

a

The parameters were extracted from the complete protein titration by

the best ﬁt according to a single site binding model with ﬁxed

stoichiometry (n = 1), yielding the binding enthalpy (ΔH), entropy

(−TΔS) and the association constant (K_a), from which the

dissociation constant (K_D= 1/K_a) was calculated. The errors represent

the errors from the data ﬁtting.

energy. Combining this with information about the diﬀerent

chemical structures, one can postulate a major diﬀerence in the

binding mode of 1 in comparison with 2 and 3 although such

diﬀerences in thermodynamic proﬁles are not necessarily a

hallmark for alterations in the ligand interaction or binding

mode.⁴¹The multifactorial character of the enthalpy signal

together with solvation changes and conformational eﬀects

always call for an additional analysis using structural data, as we

discuss in the next section.

We have previously reported a series of low molecular weight

STEP inhibitors, developed using the substrate activity

screening (SAS) approach (Figure 1).³⁶We have also described

X-ray Characterization of Inhibitors Bound to STEP.

To enable structure-based design with the phosphonate

inhibitors 1, 2, and 3 as starting points and to better

understand the origin of the thermodynamic diﬀerences, crystal

structures were generated of these compounds in complex with

STEP (Figure 2). Data was collected from cryocooled crystals

Figure 1. Previously reported inhibitors shown to bind STEP with low

micromolar aﬃnity.

benzopentathiepin inhibitors of STEP.^37,38Since then, there

has only been one report of STEP inhibitors designed by in

silico virtual screening, with similar potencies to our own.³⁹

There has also been a cheminformatic study on drug

repurposing, but this report does not provide binding data.⁴⁰

Herein, we disclose thermodynamic characterization and X-

ray crystal structures of inhibitors 1, 2, and 3 bound to STEP,

the ﬁrst structures of STEP inhibitor complexes. Unexpectedly,

the inhibitors do not all reside in the same binding pocket on

the enzyme, and none of them fully occupy the active site.

Furthermore, we have utilized the structural information

obtained from these X-ray structures to develop our lead

inhibitor with a K_iof 110 nM, representing an improvement in

inhibition by nearly 2 orders of magnitude.

Figure 2. X-ray crystal structure of (A) inhibitor 1 bound to STEP in

orange, (B) inhibitor 2 bound to STEP in cyan, and (C) inhibitor 3

bound to STEP in skyblue. (D) A superposition of a substrate bound

STEP structure (PDB 1CJZ) and inhibitor 2 bound to STEP shows

that the phosphonic acid in 2 binds to a distinctly diﬀerent site from

that of the substrate.⁴²Putative hydrogen bonds are marked as dashed

lines. All panels are shown in the same orientation. The enzyme is

depicted as a surface model colored by electrostatics as calculated by

Pymol.⁴³

RESULTS AND DISCUSSION

■

Thermodynamic Characterization of Inhibitors Bound

to STEP. Isothermal titration calorimetry (ITC) was used to

conﬁrm direct binding of phosphonates 1, 2, and 3 to the STEP

phosphatase domain. Titration experiments of the STEP

phosphatase domain with the compounds indicate clear target

engagement with aﬃnities in the two-digit micromolar range

(Table 1). While the related compounds 2 and 3 representing

two epimers of the same scaﬀold show an undistinguishable and

enthalpy-driven thermodynamic signature speaking toward a

conserved and equivalent binding mode, compound 1 indicates

a signiﬁcantly weaker binding aﬃnity. This change can be

mainly attributed to the largely reduced enthalpic contribution

when comparing it with 2 and 3, which is only partially

compensated by an entropic contribution to the overall binding

soaked overnight in 4−5 mM solution of compound dissolved

in DMSO. The structures were subsequently solved by

molecular replacement using the previously reported STEP

apo structure (PDB 2BV5) as a search model.¹⁷Data

processing and reﬁnement statistics can be found in Supporting

Information, Table S1, and the resulting electron density for

the inhibitors in Supporting Information, Figure S1. The overall

structure of the phosphatase domain is very similar to the apo

B

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Figure 3. (A) Details of interactions made by inhibitor 1. (B) Details of interactions made by inhibitor 2. The WPD loop in STEP is colored purple

and the active site loop yellow.

structures, and no major conformational diﬀerences are noted

in the loops surrounding the catalytic site.

counterintuitive. Hydrophobic interactions are typically not

associated with enthalpy gains due to the hydrophobic eﬀect,⁴¹

which in this case speaks toward a more complex protein

solvation behavior both within these hydrophobic regions as

well as the close vicinity of the α-hydroxyphosphonic acid

pharmacophore. The co-crystal structures of 2 and 3 also

explain their near-identical inhibitory potency despite the

opposite stereochemistry of their benzhydryl alcohols. STEP

makes the same interactions with inhibitors 2 and 3 and does

not make speciﬁc contacts to the benzhydryl alcohols, which

are solvent exposed.

Inhibitor 1 binds in STEP’s active site groove, but

surprisingly, its negatively charged phosphonate headgroup is

nearly 8 Å distant from catalytic Cys472 (Figure 3A,

Supporting Information, Figure S2A). This result was

unexpected insofar as the α-hydroxyphosphonic acid pharma-

cophore is a nonhydrolyzable phosphate mimetic that might be

expected to bind at the same site as the corresponding

phosphate substrate.³⁶Instead, phosphonate 1 rests in a

separate but nearby positively charged pocket, which in the

apo structure is occupied by a sulfate ion.¹⁷In a previously

described structure of a STEP (C472S) mutant and the

substrate phosphotyrosine, the side chain of Arg478 interacts

with the phosphate group of the substrate.⁴²Here, Arg478

adopts a diﬀerent conformation in order to coordinate one of

the phosphonic acid oxygens in compound 1 (Supporting

Information, Figure S3). Even though the two sites do not

overlap, this shift may interfere with the integrity of the active

site and thereby reduce substrate aﬃnity. Furthermore the

backbone rings of phosphonate 1 curl into a hydrophobic

cleft,⁴⁴while the phenolic −OH of the central ring does not

make speciﬁc contact with the enzyme, rather it is solvent

exposed.

An overlay of the crystal structures of inhibitors 1 and 2

bound to STEP illustrates their unanticipated, noncoincident

binding (Figure 4). Although the two compounds reside in

STEP co-crystal structures were also obtained for inhibitors 2

and 3 (Figure 2B,C). The α-hydroxyphosphonic acid makes

several speciﬁc, direct hydrogen-bonding contacts to the same

positively charged cavity bound by inhibitor 1 and is distinct

from substrate binding. The nonoverlapping occupancy of

substrate phosphotyrosine and inhibitor 2 in STEP’s active site

is depicted in Figure 2D. The phosphonate oxygens in

inhibitors 2 and 3 bind to Gln520, Lys439, Trp435, and

Arg478, along with water molecules supported by Thr517,

Lys439, Gln520, and Gln516 (Figure 3B, Supporting

Information, Figure S2B). The α-hydroxy substituent of 2

and 3 is also directly involved in this hydrogen-bonding

network, which is consistent with the fact that the (S)-

conﬁguration of the alcohol stereocenter is necessary for strong

inhibition.³⁶

Surprisingly, except for the binding site of the α-

hydroxyphosphonic acid pharmacophore, inhibitors 2 and 3

interact with hydrophobic regions of the enzyme that are

completely distinct from that observed for inhibitor 1. The

unexpected diﬀerence in binding mode is in line with the large

diﬀerence in the thermodynamic proﬁle shown by ITC,

although the observed enthalpy gain appears at ﬁrst sight

Figure 4. Overlay of X-ray crystal structures of inhibitors 1 (orange)

and 2 (cyan) bound to STEP. The enzyme is depicted as a surface

model colored by electrostatics as calculated by Pymol.⁴³

separate shallow, hydrophobic clefts, the phosphonate head

groups both align into the same noncatalytic positively charged

pocket. This crystallographic understanding provided essential

inspiration for expansion of the inhibitor scaﬀold into more

potent structures (vide infra).

Structure-Based Optimization of STEP Inhibitors. We

set out to improve the binding aﬃnity of these phosphonate

inhibitors. To avoid stereochemical complications and for ease

of synthesis, we elected to begin optimization around the

diﬂuorophosphonic acid⁴⁵scaﬀold of inhibitor ( )-4 (Chart 1)

rather than the more complex structure of chiral hydrox-

yphosphonic acids 2 and 3. This highly homologous, previously

reported diﬂuorophosphonate has only slightly lower potency

than alcohols 2 and 3.³⁶The use of racemates streamlined

C

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a

atom (10), we observed a trivial increase in binding aﬃnity,

which was not pronounced enough to justify proceeding with

the heavier halogen.

Chart 1. Optimization of Distal Aromatic Ring

Next, we turned to the central aromatic ring. While the

hydrogen atoms at the 4-, 5-, and 6-positions all project into

solvent, it appeared that we could take advantage of a small

hydrophobic surface adjacent to the 2-position. Molecular

modeling suggested that ﬂuorine would be an ideal ﬁt for this

volume. Indeed, diﬂuorophosphonic acid 11 demonstrated 2-

fold better potency than inhibitor 9.

At this point, we sought to benchmark our improved

inhibitor 11 against α-hydroxyphosphonic acids that more

closely resembled the compounds observed in the co-crystal

structure (i.e., 2 and 3, vide supra). Restoration of the hydroxyl

group adjacent to the phosphorus atom resulted in a pair of

enantiomeric inhibitors ((R)-12 and (S)-12, Chart 3).

a

K_ivalues were measured in duplicate or quadruplicate (( )-13) using

a continuous in vitro inhibition assay by employing p-nitro-

phenylphosphate (pNPP) as a chromogenic substrate.⁴⁶Commercial

detergent, 0.01% v/v, was included in the assay buﬀer to preclude

incidental inhibition by nonproductive formation of micelles.⁴⁷⁻⁴⁹

Chart 3. Enantiopure Hydroxyphosphonate and Racemic

a

Fluorophosphonate Analogues Based on Inhibitor 11

synthetic considerations and is justiﬁed in that the benzhydryl

alcohol does not make speciﬁc contacts with the enzyme target

or aﬀect activity (see 2 and 3, vide supra).

We ﬁrst explored alterations to the distal aromatic ring. In

the crystal structure, we observed a small, hydrophobic cavity at

the ortho-position. Attempting to ﬁll this pocket with a

relatively small ﬂuorine-substituent (( )-5) resulted in a

negligible change in potency. However, the larger chlorine

substituent (( )-6) provided a small but measurable improve-

ment.

This substitution pattern was also tested in achiral

phosphonic acids lacking the benzhydryl hydroxyl group

(Chart 2). The trend observed in Chart 1 of decreasing K_i

with the presence of larger halogen substitution holds for this

series of inhibitors (7−9) as well, and all three are more potent

than the parent alcohols. Upon moving to a larger bromine

a

K_ivalues were measured in duplicate or quadruplicate (( )-13). K_i

values were measured in triplicate using a continuous in vitro

inhibition assay by employing p-nitrophenylphosphate (pNPP) as a

chromogenic substrate.⁴⁶Commercial detergent, 0.01% v/v, was

included in the assay buﬀer to preclude incidental inhibition by the

nonproductive formation of micelles.⁴⁷⁻⁴⁹

Chart 2. Removal of Benzhydryl Alcohol from Inhibitor

Scaﬀold

Consistent with the ﬁndings in our previous publication³⁶

and with the X-ray crystal structure of inhibitors 2 and 3, these

phosphonic acids exhibit chiral discrimination, with the (S)-

enantiomer possessing nearly 6-fold higher activity than the

(R)-enantiomer. Importantly, hydroxyphosphonic acid (S)-12

was not more potent than the simpler achiral inhibitor 11.

However, racemic monoﬂuorophosphonic acid ( )-13 was

measurably more active than compound 11.

a

We then returned to the overlay of the co-crystal structures

of inhibitors 1 and 2 bound to STEP (Figure 4B) for further

guidance. Speculating that top hits 11 and ( )-13 reside in the

same binding pocket as inhibitor 2, we hypothesized that we

could build into the space occupied by phosphonic acid 1 if we

grafted its ﬁrst aromatic ring onto our optimized scaﬀold.

Similar linking strategies have been reported by groups at

SmithKline Beecham in the context of cathepsin K

inhibitors.^50,51Molecular modeling indicated that this could

be successfully accomplished with a phenyl ketone, as in

phosphonic acid ( )-14 (Table 2). Indeed, this modiﬁcation

provided a 4-fold enhancement in potency over compound 11

and a 3-fold enhancement over monoﬂuorophosphonic acid

( )-13.

a

Saturation of the newly added ring resulted in a 2-fold drop

in activity (( )-15), and moving to the piperidine amide

(( )-16) resulted in a further 3-fold diminution in binding

aﬃnity. Nonetheless, given the ability of the ketone inhibitors

to potentially undergo a reverse phosphono-Claisen condensa-

K_ivalues were measured in duplicate or quadruplicate (( )-13) using

a continuous in vitro inhibition assay by employing p-nitro-

phenylphosphate (pNPP) as a chromogenic substrate.⁴⁶Commercial

detergent, 0.01% v/v, was included in the assay buﬀer to preclude

incidental inhibition by nonproductive formation of micelles.⁴⁷⁻⁴⁹

D

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hypothesis that (−)-22 makes direct and speciﬁc contacts

with the binding pocket.

Table 2. Ligands Based on Expanding Inhibitor ( )-13 into

Space Occupied by Inhibitor 1

a

Given the high structural homology among PTP active sites,

we tested (−)-22 against a series of PTPs and the dual-

speciﬁcity phosphatase MKP5 (Table 3). Notably, this

a

Table 3. Selectivity of (−)-22 against Several Phosphatases

phosphatase

K_i(μM)

selectivity

STEP

0.11 0.01

5.9 0.5

6.2 0.2

4.8 0.4

6.9 0.1

1.6 0.2

PTP1B

LMW-Ptp

TC-Ptp

MKP5

LAR

54

56

44

63

15

a

K_ivalues were measured in duplicate using a continuous in vitro

inhibition assay by employing p-nitrophenylphosphate (pNPP) as a

chromogenic substrate.⁴⁶Commercial detergent, 0.01% v/v, was

included in the assay buﬀer to preclude incidental inhibition by

nonproductive formation of micelles.⁴⁷⁻⁴⁹

sulfonamide exhibited greater than 40-fold selectivity against

all but one (LAR) other phosphatases assayed. Furthermore, in

preliminary studies for future in vivo evaluation, (−)-22 was

determined to have respectable aqueous solubility (11 2 μM,

in PBS at pH 7.4) and complete stability to rat plasma over 1 h

as well as excellent stability to rat liver microsomes (88% and

92% remaining after 1 h with and without NADPH).⁵²

CHEMICAL SYNTHESIS

The preparation of diﬂuorophosphonate inhibitors ( )-5−11

all employed either input 24 or 25 (Scheme 1). Zinc- and

■

Scheme 1. Synthesis of Inputs for Diﬂuorophosphonate

Inhibitors

a

K_ivalues were measured in duplicate, triplicate ((−)-22), or

quadruplicate (20) using a continuous in vitro inhibition assay by

employing p-nitrophenylphosphate (pNPP) as a chromogenic

substrate.⁴⁶Commercial detergent, 0.01% v/v, was included in the

assay buﬀer to preclude incidental inhibition by nonproductive

formation of micelles.⁴⁷⁻⁴⁹

tion, we were motivated to proceed with more stable amide-

based inhibitors. We found that we could greatly streamline our

syntheses by removal of the α-ﬂuorine substituent (( )-17)

with no appreciable diﬀerence in potency (see Chemical

Synthesis section for synthetic sequences). Switching from the

piperidine amide to the morpholine amide (( )-18) likely

improved solubility and moderately enhanced eﬃcacy. The

acyclic dimethyl amide (( )-19) was comparable in potency to

piperidine amide ( )-17, but secondary amide ( )-20 was

considerably weaker.

a

Reagents: (a) (i) Zn, DMA; (ii) CuBr, (iii) 1-bromo-4-iodobenzene,

DMA; (b) TMSI, CH₂Cl₂; (c) B₂pin₂, KOAc, Pd(dppf)Cl₂, dioxane.

A nearly 4-fold jump in activity accompanied the switch from

amide ( )-18 to sulfonamide ( )-21, a change that also

engendered improved solubility. Given the submicromolar

inhibition of STEP demonstrated by racemic inhibitor ( )-21,

we wished to assay the individual enantiomers of this

compound to test for chiral discrimination. We were unable

to resolve the enantiomers of ( )-21, and the protected

precursors of this inhibitor were not conﬁgurationally stable.

However, we were able to access both enantiomers of α-

ﬂuorinated analogue 22 (vide infra). The (+)-enantiomer was

over 35-fold less potent than the (−)-enantiomer, demonstrat-

ing substantial chiral discrimination and supporting the

CuBr-promoted coupling of diethyl (bromodiﬂuoromethyl)

phosphonate with 1-bromo-4-iodobenzene provided phos-

phonic acid diethyl ester 23.⁵³This material was then

deprotected with iodotrimethylsilane or borylated under

Miyaura conditions to provide intermediates 24 and 25,

respectively.

Inhibitors ( )-5 and ( )-6 were accessed through a three-

step process beginning with formation of the Grignard reagent

from 3-bromoiodobenzene by transmetalation with isopropyl-

magnesium iodide followed by addition to the appropriate

E

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a

benzaldehyde (26 or 27) to form alcohols ( )-28 and ( )-29

(Scheme 2a). While the synthesis of 4,5-dichloro-2-ﬂuoroben-

Scheme 3. Synthesis of Inhibitors 8−11

a

Scheme 2. Synthesis of Inhibitors ( )-5 and ( )-6

a

Reagents: (a) (i) iPrMgCl, THF, (ii) aldehyde, THF; (b) 25,

Pd(PPh₃)₄, K₂CO₃, toluene/EtOH/H₂O; (c) TMSI, CH₂Cl₂; (d) Fe,

EtOH, AcOH; (e) (i) HCl, NaNO₂, H₂O, (ii) KI, H₂O, CH₂Cl₂; (f)

(i) iPrMgCl, THF, (ii) DMF.

a

Reagents: (a) (i) iPrMgCl, THF, (ii) aldehyde, THF; (b) B₂pin₂,

KOAc, Pd(dppf)Cl₂, dioxane; (c) 24, Pd(PPh₃)₄, Na₂CO₃, DME/

EtOH/H₂O; (d) (i) iPrMgCl, THF, (ii) DMF.

a

Scheme 4. Synthesis of Inhibitors (R)-12 and (S)-12

zaldehyde 26 has been previously reported,⁵⁴we approached

2,4,5-trichlorobenzaldehyde 27 by transmetalation of the

corresponding 1,2,4-trichloro-5-iodobenzene with isopropyl-

magnesium iodide followed by addition to DMF (Scheme 2b).

Miyaura borylation of aryl bromides ( )-28 and ( )-29 gave

the corresponding pinacol boronates ( )-30 and ( )-31.

Subsequent Suzuki cross-coupling with phosphonic acid 24

aﬀorded ligands ( )-5 and ( )-6.

The synthesis of inhibitors 8−11 was accomplished through

a similar sequence (Scheme 3a). Transmetalation of aryl iodide

32 or 33⁵⁵followed by Grignard addition to the appropriate

benzaldehyde (26, 27, or 34) provided benzhydryl alcohols

( )-35−( )-38. Benzaldehyde 34 was prepared through a

three-step process beginning with the iron-mediated reduction

of 1-bromo-4,5-dichloro-2-nitrobenzene to the corresponding

aniline (43, Scheme 3b). Sandmeyer reaction of aniline 43

provided iodide 44, which was converted to benzaldehyde 34

through the same process as used above for aldehyde 27.

Suzuki cross-coupling of aryl iodides ( )-35−( )-38 with

boronic ester 25 gave phosphonate esters ( )-39−( )-42,

which were then treated with iodotrimethylsilane to concom-

itantly remove the ethyl ester protecting groups and the

benzhydryl hydroxyl group, to provide the desired phosphonic

acids 8−11.

a

Reagents: (a) (i) iPrMgCl, THF, (ii) aldehyde, THF; (b) Et₃SiH,

BF₃·Et₂O, CH₂Cl₂; (c) B₂pin₂, KOAc, Pd(dppf)Cl₂, dioxane; (d)

Pd(PPh₃)₄, Na₂CO₃, DME/EtOH/H₂O.

in the presence of boron triﬂuoride. Miyaura borylation to

aﬀord boronic ester 47 was followed by Suzuki cross coupling

with enantiomercially pure α-hydroxyphosphonic acids (R)-48

and (S)-48 to provide both enantiomers of inhibitor 12.

Monoﬂuorophosphonic acid ( )-13 was prepared in four

steps, starting with the α-ﬂuorination of diethyl (4-

bromobenzyl)phosphonate using NFSI (Scheme 5).⁵⁶The

resultant aryl bromide (( )-49) was treated with bis-

Enantiomerically pure inhibitors (R)-12 and (S)-12 were

synthesized by using a related set of reactions (Scheme 4).

First, transmetalation of 1,2,4-trichloro-5-iodobenzene and

addition to 3-bromo-2-ﬂuorobenzaldehyde generated alcohol

( )-45, which was reduced to aryl bromide 46 by triethylsilane

F

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a

which were subsequently cross-coupled with aryl iodide

( )-38. α-Fluorination of ketones 58 and 59 was accomplished

by treatment with a combination of NFSI and selectﬂuor.

Subsequent deprotection and hydrodehydroxylation of 60 and

61 provided inhibitors ( )-14 and ( )-15.

Scheme 5. Synthesis of Inhibitor ( )-13

Amide inhibitor ( )-16 was synthesized in ﬁve steps, starting

with an LDA-promoted amide formation between diethyl (4-

bromobenzyl)phosphonate and piperidine-1-carbonyl chloride

(62) to generate phosphonate ( )-63 (Scheme 7). Fluorina-

a

Scheme 7. Synthesis of Inhibitor ( )-16

a

Reagents: (a) (i) LiHMDS, THF, (ii) TMSCl, THF, (iii) NFSI,

THF; (b) B₂pin₂, KOAc, Pd(dppf)Cl₂, dioxane; (c) ( )-38,

Pd(PPh₃)₄, K₂CO₃, toluene/EtOH/H₂O; (d) TMSI, CH₂Cl₂.

a

Reagents: (a) LDA, THF; (b) LDA, NFSI, THF; (c) B₂pin₂, KOAc,

(pinacoloto)diboron under Miyaura conditions to provide

boronate ( )-50. Cross-coupling with the previously described

aryl iodide ( )-38 then generated a diastereomeric mixture of

benzhydryl alcohols 51. Treatment with iodotrimethylsilane

resulted in cleavage of the ethyl esters and loss of the

benzhydryl hydroxyl group to aﬀord inhibitor ( )-13.

We accessed β-ketophosphonates ( )-14 and ( )-15 by ﬁrst

performing an LDA-mediated phosphono-Claisen condensa-

tion between diethyl (4-bromobenzyl)phosphonate and the

appropriate commercially available ester (52 or 53, Scheme

6).⁵⁷This was followed by Miyaura borylation of aryl bromides

( )-54 and ( )-55 to aﬀord pinacol esters ( )-56 and ( )-57,

Pd(dppf)Cl₂, dioxane; (d) ( )-38, Pd(PPh₃)₄, K₂CO₃, toluene/

EtOH/H₂O; (e) TMSI, CH₂Cl₂.

tion with LDA and NFSI to provide amide ( )-64 was

followed by borylation under standard conditions. Suzuki cross-

coupling of boronate ( )-65 with aryl iodide ( )-38 aﬀorded a

diastereomeric mixture of amides 66, which was concomitantly

deprotected and hydrodehydroxylated with iodotrimethylsilane

to provide phosphonate ( )-16.

The synthesis of tertiary amide inhibitors ( )-17−( )-19

was carried out by ﬁrst reducing benzhydryl alcohol ( )-38 to

the diarylmethane 67 with triethylsilane and borylating diethyl

(4-bromobenzyl)phosphonate to boronate ester 68 (Scheme

8). These two components were subsequently cross-coupled to

aﬀord phosphonate 69, which was treated with LDA and the

appropriate carbamic chloride (62, 70, or 71). The resultant

amides ( )-72−( )-74 were treated without puriﬁcation with

iodotrimethylsilane to give inhibitors ( )-17−( )-19.

a

Scheme 6. Synthesis of Inhibitors ( )-14 and ( )-15

We approached secondary amide inhibitor ( )-20 in six

steps from commercially available methyl 2-(4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetate (Scheme

9). After Suzuki coupling with aryl iodide 67 in ethanol

solvent, we observed partial transesteriﬁcation of the methyl

ester to the ethyl ester, resulting in a trivial mixture of

intermediates 75a and 75b, which were hydrolyzed to the

corresponding acid (76). α-Bromination under Hell−Volhard−

Zelinsky conditions resulted in additional bromination at the

benzhydryl position, providing a diastereomeric mixture of

acids 77, which was treated with methylamine and CDI to

aﬀord amide 78. Arbuzov reaction in neat triethyl phosphite

provided phosphonate 79. Exposure to iodotrimethylsilane

resulted in removal of the ethyl ester protecting groups and

reduction of the benzhydryl bromide to give inhibitor ( )-20.

Inhibitor ( )-21 was produced starting with sulfonamide

formation between (4-bromophenyl)methanesulfonyl chloride

and morpholine to generate intermediate 80 (Scheme 10). This

material was borylated to give 81, which was then cross-coupled

a

Reagents: (a) LDA, THF; (b) B₂pin₂, KOAc, Pd(dppf)Cl₂, dioxane;

(c) ( )-38, Pd(PPh₃)₄, K₂CO₃, toluene/EtOH/H₂O; (d) NFSI,

selectﬂuor, CH₂Cl₂; (e) TMSI, CH₂Cl₂.

G

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a

Scheme 8. Synthesis of Inhibitors ( )-17−( )-19

Scheme 10. Synthesis of Inhibitor ( )-21

a

Reagents: (a) Na₂CO₃, MeCN; (b) B₂pin₂, KOAc, Pd(dppf)Cl₂,

dioxane; (c) 67, Pd(PPh₃)₄, K₂CO₃, toluene/EtOH/H₂O; (d) (i)

LDA, THF, (ii) diethyl chlorophosphate; (e) TMSI, CH₂Cl₂.

chlorophosphate⁵⁸gave phosphonate ( )-83, which was

deprotected with iodotrimethylsilane to aﬀord inhibitor

( )-21. The enantiomers of inhibitor ( )-21 did not separate

by preparative chiral chromatography and precursor ( )-83

racemized under separation conditions due to the highly acidic

nature of the α-hydrogen. To access enantiomerically pure

inhibitors, we prepared the α-ﬂuorinated sulfonamides (+)-22

and (−)-22.

a

Reagents: (a) Et₃SiH, TFA, CH₂Cl₂; (b) B₂pin₂, KOAc, Pd(dppf)Cl₂,

dioxane; (c) Pd(PPh₃)₄, K₂CO₃, toluene/EtOH/H₂O; (d) LDA,

THF; (e) TMSI, CH₂Cl₂.

a

Scheme 9. Synthesis of Inhibitor ( )-20

Accordingly, sulfonamide 82 was ﬂuorinated with NFSI, to

give ( )-84, which was converted to the racemic mixture of α-

ﬂuorophosphonates ( )-85 by deprotonation and addition of

diethyl chlorophosphate (Scheme 11).⁵⁸This racemic mixture

was resolved by chiral supercritical ﬂuid chromatography to

provide isolated enantiomers 85a and 85b.⁵⁹Finally, treatment

of the separated isomers with iodotrimethylsilane aﬀorded

enantiomeric inhibitors (+)-22 and (−)-22.

CONCLUSIONS

■

Despite the causative link between STEP overactivity and the

hallmark symptoms of AD, only limited progress has been

reported on the development of potent and selective inhibitors

of this PTP. In this article, we have presented co-crystal

structures of our previously reported inhibitors with STEP,

displaying a surprising example of noncoincident binding of

related compound structures. Furthermore, neither inhibitor

scaﬀold fully resides in the anticipated active site of the enzyme.

We were able to successfully use structural data from the

crystal structures in order to iteratively improve our inhibitor

design and expand the architecture of one compound to occupy

space ﬁlled by the other. Ultimately, we identiﬁed phosphonate

(−)-22 with a K_iof 110 nM. Importantly, this inhibitor

exhibited promising selectivity over other tyrosine and dual-

a

Reagents: (a) 67, Pd(PPh₃)₄, K₂CO₃, toluene/EtOH/H₂O; (b)

NaOH, MeOH, EtOH; (c) Br₂, PCl₃, PhCl; (d) MeNH₂, CDI,

CH₂Cl₂; (e) P(OEt)₃; (f) TMSI, CH₂Cl₂.

with aryl iodide 67 to provide sulfonamide 82. From here,

deprotonation with LDA followed by addition of diethyl

H

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a

NTA Superﬂow (Qiagen) packed in an XK26 column (GE

Healthcare) using an AKTA explorer (GE Healthcare). The column

Scheme 11. Synthesis of Inhibitors (+)-22 and (−)-22

̈

was then washed to A280 baseline with load buﬀer (50 mM Tris, pH

7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, 5% glycerol,

washed with 50 mM imidazole) and eluted with load buﬀer containing

250 mM imidazole. Samples of the fractions were analyzed by SDS-

PAGE and those containing the protein were pooled. Protein

concentration was determined by absorbance at 280 nm throughout.

The pool was concentrated (10 kDa MWCO centrifugal concen-

trators, Millipore) and loaded onto a pre-equilibrated (50 mM HEPES,

pH 7.5, 100 mM NaCl, 0.5 mM TCEP, 5% glycerol) High Load

Superdex 75 16/600 (GE Healthcare). SDS-PAGE was used to pool

fractions containing the phosphatase domain. The S75 pool was

diluted 4× into anion exchange buﬀer (50 mM HEPES, pH 7.5, 1 mM

TCEP, 5% glycerol) and loaded onto a pre-equilibrated 6 mL

Resource Q column (GE Healthcare). The column was washed to

A280 baseline and eluted with a 5 CV 10% elution buﬀer (anion

exchange buﬀer +1 M NaCl) step, followed by a 20 CV gradient of

20−50% elution buﬀer. Fractions were pooled based on SDS-PAGE.

The Q Pool was concentrated as before to 8 mL and loaded onto a Hi

Load Superdex 200 26/600 (GE Healthcare) pre-equilibrated with

storage buﬀer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM

TCEP). Fractions were pooled based on SDS-PAGE, aliquoted, and

snap-frozen in liquid nitrogen before storing at −80 °C.

Isothermal Titration Calorimetry (ITC). The ITC titration

experiments were carried out on a MicroCal ITC-200 system (Malvern

Instrument Ltd.) using protein that had been passed through a PD-10

column (GE-Healthcare) equilibrated with 20 mM Tris-HCl (pH 7.6),

150 mM NaCl, 1 mM TCEP, 0.05% (v/v) Tween 20, and 2% DMSO.

Because of the expectation of low aﬃnity and low heat signals,

titrations of 52 μM STEP phosphatase domain were conducted at 30

°C by injecting 30 × 1.2 μL aliquots at a concentration of 1.6 mM of

the respective compound with 90 s waiting time between subsequent

injections. The thermodynamic parameters of the binding were

extracted by analysis of the binding isotherms by applying a single site

binding model (setting the stoichiometry value n to 1 due to low c-

value titration) using the Microcal Origin version 7.0 software package,

yielding binding enthalpy (ΔH), entropy (ΔS), and association

constant (K_a), from which the dissociation constant K_Dwas calculated

(K_D= 1/K_a)

Crystallization and Structure Determination. Prior to

crystallization setup, 6His-TEV-STEP258−539 was washed with

fresh buﬀer (20 mM HEPES, pH 7.4, 0.1 M NaCl, 1 mM TCEP)

and reconcentrated to 4.9 mg/mL. Crystals were grown at 4 °C in

hanging drops using 2 μL of protein and 1 μL of well solution (26−

28% PEG3350, 0.1 M Bis-Tris, pH 5.5, 0.2 M lithium sulfate). Rodlike

crystals typically appeared after 1−2 days but continued to grow for

approximately 2 weeks. Structure complexes with compounds 1, 2, and

3 were obtained by soaking apo crystals in 28% PEG3350, 0.1 M Bis-

tris, pH 5.5, 1 mM TCEP and 4−5 mM of compound for 18−26 h.

Crystals were cryoprotected using 20% glycerol and frozen under

liquid nitrogen prior to data collection. X-ray diﬀraction data was

collected on a Rigaku A200 CDD detector mounted on a rotating

anode (FRE+, Rigaku). Data were integrated and processed using

autoPROC.⁶⁰The crystals belong to space group P2₁2₁2₁with one

molecule in the asymmetric unit. The structures were solved with

molecular replacement by using the Protein Data Bank (PDB) entry

2BV5 as search model.¹⁷Structural reﬁnement was carried out by

using autoBUSTER⁶¹and manual rebuilding was done using Coot.⁶²

Statistics from the integration, scaling, and reﬁnement are presented in

Supporting Information, Table S1. All structure illustrations were

prepared using the program Pymol.⁴³Crystallographic coordinates

have been deposited into the Protein Data Bank (accession codes: 1,

5OW1; 2, 5OVR; 3, 5OVX).

a

Reagents: (a) (i) NaHMDS, THF, (ii) NFSI, THF; (b) (i) LDA,

THF, (ii) diethyl chlorophosphate, (iii) preparative chiral SFC; (c)

TMSI, CH₂Cl_2.

speciﬁcity phosphatases. The determination of the co-crystal

structure of sulfonamide (−)-22 with STEP is a priority for

future eﬀorts to further enhance potency and selectivity and,

most importantly, to identify suitable replacements for the

polar and acidic phosphonic acid functionality as would likely

be necessary for target engagement within the CNS.

EXPERIMENTAL SECTION

■

General Methods. Flasks were ﬁtted with rubber septa. Reactions

were conducted under air unless noted. Stainless steel syringes were

used to transfer air- and moisture-sensitive liquids. Flash chromatog-

raphy was performed using silica gel 60 (230−400 mesh). Commercial

reagents were used as received with the following exceptions:

dichloromethane, 1,4-dioxane, and tetrahydrofuran were dried by

passing through columns of activated alumina. Diisopropylamine was

distilled from NaOH at 760 Torr. n-Butyllithium was titrated using N-

benzylbenzamide as an indicator. Chemical shifts for protons are

reported in parts per million downﬁeld from tetramethylsilane and are

referenced to residual protium in the NMR solvent (CHCl₃= δ 7.26).

Chemical shifts for carbon are reported in parts per million downﬁeld

from tetramethylsilane and are referenced to the carbon resonances of

the solvent (CDCl₃= δ 77.2). Data are represented as follows:

chemical shift (multiplicity (br = broad, s = singlet, d = doublet, t =

triplet, q = quartet, quin = quintet, m = multiplet), coupling constants

in Hertz (Hz), integration). The mass spectral data were obtained on

an Agilent 6550A quadrupole time-of-ﬂight LC/MS spectrometer

(ESI-TOF). Enzymatic assays were performed on a PerkinElmer

Envision 2100 plate reader. Synthesis, characterization, and enzymatic

assays of inhibitors 1, 2, 3, ( )-4, and 7 have been previously

reported.³⁶The purity of all new inhibitors (≥95%) has been

determined by HPLC analysis.

Protein Production for Isothermal Titration Calorimetry and

Crystallography. The STEP phosphatase domain (6His-TEV-

STEP258-539) DNA was synthesized and cloned by GeneArt Life

Technologies and expressed in Escherichia coli (BL21-Gold; DE3) at

18 °C with overnight expression. Frozen E. coli cell paste was

resuspended in lysis buﬀer (50 mM Tris HCl, pH 7.5, 500 mM NaCl,

0.5 mM TCEP, complete protease inhibitor tablets-EDTA free

(Roche), 2.5 units/mL Benzonase) using a polytron homogenizer.

Cells were lysed using one pass through a Basic Z (Constant Systems)

at 25K psi. The cell lysate was then clariﬁed by centrifugation at

25000g. The supernatant was loaded onto pre-equilibrated 10 mL Ni

General Procedure for Determination of Inhibitor K_i.

Reaction volumes of 100 μL were used in 96-well plates. An amount

of 65 μL of water was added to each well, followed by 5 μL of 20×

buﬀer (1.0 M imidazole, 1.0 M NaCl, 0.2% Triton-X 100, pH 7.0), 10

μL of 10× DTT (50 mM, 5 mM in assay), 5 μL of STEP phosphatase

(1 μM, 50 nM in assay), and 5 μL of the appropriate inhibitor dilution

I

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in DMSO (with 2-fold serial dilutions). The assay plate was incubated

for 5 min at 27 °C, at which point the reaction was started by addition

of 10 μL of a 10× pNPP substrate (5 mM, 500 μM in assay). The plate

was then immediately placed into a spectrophotometric plate reader,

and 20 min of kinetic data was obtained (405 nm, 27 °C). The initial

rate data collected were used for determination of K_ivalues. For K_i

determination, the kinetic values were obtained directly from

nonlinear regression of substrate−velocity curves in the presence of

various concentrations of inhibitor. Assays were run in at least

duplicate. Nonlinear regression analysis was accomplished in Prism

(GraphPad) using single site competitive inhibition. Dose−response

curves for each inhibitor as well as additional information are provided

in the Supporting Information.

Synthesis and Characterization of Inhibitors and Intermedi-

ates. ( )-((3′-((4,5-Dichloro-2-ﬂuorophenyl)(hydroxy)methyl)-[1,1′-

biphenyl]-4-yl)diﬂuoromethyl)phosphonic Acid (( )-5). Phosphonic

acid 24 (254 mg, 0.885 mmol, 1.0 equiv), boronic ester ( )-30 (514

mg, 1.29 mmol, 1.5 equiv), and Na₂CO₃·H₂O (642 mg, 5.18 mmol, 5.9

equiv) were dissolved in DME (3.9 mL), EtOH (0.97 mL), and H₂O

(0.97 mL). With stirring, N₂was bubbled through the biphasic mixture

for 1 h. Pd(PPh₃)₄(102 mg, 0.0879 mmol, 0.10 equiv) was then

added, and N₂was bubbled through the stirred solution for an

additional 20 min. The ﬂask was next equipped with a reﬂux

condenser, and the reaction mixture was stirred at 80 °C under an

atmosphere of N₂for 18 h. The solution was cooled to 23 °C and

concentrated. The residue was dissolved in minimal MeOH and

puriﬁed by reversed-phase gradient chromatography (5−100% MeCN

in H₂O with 0.1% TFA). Volatile components were removed, and the

resulting aqueous solution was lyophilized to aﬀord ( )-5 as a white

powder (99 mg, 23%). IR (ﬁlm) ν_max3308 (br), 1607, 1468, 1259,

1118, 1054, 921, 836, 794 cm⁻¹. ¹H NMR (400 MHz, CD₃OD) δ 7.55

(d, J = 7.0 Hz, 1H), 7.50−7.45 (m, 5H), 7.34 (dt, J = 7.6, 1.5 Hz, 1H),

7.22 (t, J = 7.7 Hz, 1H), 7.17−7.13 (m, 1H), 7.09 (d, J = 9.6 Hz, 1H),

5.86 (s, 1H). ¹⁹F NMR (376 MHz, CD₃OD) δ −110.9 (d, J = 113.8

Hz), −119.2. ³¹P NMR (162 MHz, CD₃OD) δ 4.90 (t, J = 113.6 Hz).

MS (ESI-TOF) calcd for C₂₀H₁₄Cl₂F₃O₄P [M − H]⁻474.9886, found

474.9888.

(Diﬂuoro(3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)methyl)-

phosphonic Acid (9). Reaction of phosphonate ( )-40 (171 mg,

0.311 mmol, 1.0 equiv) and iodotrimethylsilane (0.27 mL, 380 mg,

1.90 mmol, 6.1 equiv) in CH₂Cl₂(1.0 mL, 0.245 M) according to the

procedure for the preparation of 8 above aﬀorded 9 as a white solid

(134 mg, 90%). IR (ﬁlm) ν_max1605, 1461, 1353, 1260, 1131, 1067,

984, 927, 835, 798 cm⁻¹. ¹H NMR (400 MHz, CD₃OD) δ 7.69−7.64

(m, 4H), 7.57 (s, 1H), 7.51−7.46 (m, 2H), 7.40−7.35 (m, 2H), 7.17

(d, J = 7.8 Hz, 1H), 4.10 (s, 2H). ¹⁹F NMR (376 MHz, CD₃OD) δ

−110.9 (d, J = 113.6 Hz). ³¹P NMR (162 MHz, CD₃OD) δ 5.12. MS

(ESI-TOF) calcd for C₂₀H₁₄Cl₃F₂O₃P [M − H]⁻1:1 474.9641 and

476.9612, found 1:1 474.9637 and 476.9613.

((3′-(2-Bromo-4,5-dichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

diﬂuoromethyl)phosphonic Acid (10). Reaction of phosphonate

( )-41 (122 mg, 0.205 mmol, 1.0 equiv) and iodotrimethylsilane

(0.12 mL, 164 mg, 0.821 mmol, 4.0 equiv) in CH₂Cl₂(0.65 mL, 0.268

M) according to the procedure for the preparation of 8 above aﬀorded

10 as a white solid (62 mg, 58%). IR (ﬁlm) ν_max1609, 1458, 1347,

1254, 1132, 1053, 927, 836, 795 cm⁻¹. ¹H NMR (400 MHz, CD₃OD)

δ 7.68 (s, 1H), 7.63−7.58 (m, 4H), 7.46−7.41 (m, 2H), 7.34−7.30 (m,

2H), 7.11 (d, J = 7.6 Hz, 1H), 4.06 (s, 2H). ¹⁹F NMR (376 MHz,

CD₃OD) δ −110.7 (d, J = 113.3 Hz). ³¹P NMR (162 MHz, CD₃OD)

δ 4.81 (t, J = 113.2 Hz). MS (ESI-TOF) calcd for C₂₀H₁₄BrCl₂F₂O₃P

[M − H]⁻1:1 518.9136 and 520.9116, found 1:1 518.9135 and

520.9115.

(Diﬂuoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

methyl)phosphonic Acid (11). Reaction of phosphonate ( )-42 (142

mg, 0.249 mmol, 1.0 equiv) and iodotrimethylsilane (0.21 mL, 299

mg, 1.50 mmol, 6.0 equiv) in CH₂Cl₂(0.86 mL, 0.232 M) according

to the procedure for the preparation of 8 above aﬀorded 11 as a white

solid (90 mg, 73%). IR (ﬁlm) ν_max1459, 1259, 1205, 1132, 1068, 985,

928, 839, 798 cm⁻¹. ¹H NMR (400 MHz, CD₃OD) δ 7.42 (d, J = 8.2

Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.20 (s, 1H), 7.10 (td, J = 7.5, 2.0

Hz, 1H), 7.05 (s, 1H), 6.90 (t, J = 7.5 Hz, 1H), 6.86 (td, J = 7.7, 7.3,

2.0 Hz, 1H), 3.82 (s, 2H). ¹⁹F NMR (376 MHz, CD₃OD) δ −110.9

(d, J = 112.9 Hz), −123.7. ³¹P NMR (162 MHz, CD₃OD) δ 4.84 (t, J

= 105.4 Hz). MS (ESI-TOF) calcd for C₂₀H₁₃Cl₃F₃O₃P [M − H]⁻1:1

492.9547 and 494.9518, found 1:1 492.9548 and 494.9522.

( )-(Diﬂuoro(3′-(hydroxy(2,4,5-trichlorophenyl)methyl)-[1,1′-bi-

phenyl]-4-yl)methyl)phosphonic Acid (( )-6). Reaction of phos-

phonic acid 24 (297 mg, 1.03 mmol, 1.0 equiv), boronic ester ( )-31

(623 mg, 1.51 mmol, 1.5 equiv), Na₂CO₃·H₂O (757 mg, 6.10 mmol,

5.9 equiv), and Pd(PPh₃)₄(117 mg, 0.101 mmol, 0.10 equiv) in DME

(4.4 mL), EtOH (1.1 mL), and H₂O (1.1 mL) according to the

procedure for the preparation of ( )-5 above aﬀorded ( )-6 as a

white solid (131 mg, 26%). IR (ﬁlm) ν_max3296 (br), 1452, 1400, 1352,

(R)-((2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

(hydroxy)methyl)phosphonic Acid ((R)-12). (R)-((4-Bromophenyl)-

(hydroxy)methyl)phosphonic acid (R)-48 was synthesized according

to ref 36 with the following clariﬁcation. Please note that the use of ^L-

(−)-menthol provides tris-[(1R,2S,5R)-menth-2-yl]phosphite. After

reaction with 4-bromobenzaldehyde, the phosphonate with the (S)-

alcohol crystallizes ﬁrst at 23 °C, then the diastereomer with the (R)-

conﬁgured alcohol crystallizes at 0 °C.³⁶Reaction of (R)-((4-

bromophenyl)(hydroxy)methyl)phosphonic acid (R)-48 (96 mg,

0.360 mmol, 1.0 equiv), boronic ester 47 (167 mg, 0.401 mmol, 1.1

equiv), Na₂CO₃·H₂O (279 mg, 2.25 mmol, 6.2 equiv), and Pd(PPh₃)₄

(43.5 mg, 0.0376 mmol, 0.10 equiv) in DME (1.74 mL), EtOH (0.42

mL), and H₂O (0.42 mL) according to the procedure for the

preparation of ( )-5 above aﬀorded (R)-12 as a white solid (5.3 mg,

1

1257, 1171, 1130, 1030, 915, 835, 699 cm⁻¹. H NMR (400 MHz,

CD₃OD) δ 7.63 (s, 1H), 7.45−7.43 (m, 4H), 7.42 (t, J = 1.7 Hz, 1H),

7.30 (d, J = 4.8 Hz, 2H), 7.17 (t, J = 7.7 Hz, 1H), 7.09 (d, J = 7.7 Hz,

1H), 5.88 (s, 1H). ¹⁹F NMR (376 MHz, CD₃OD) δ −110.9 (d, J =

113.5 Hz). ³¹P NMR (162 MHz, CD₃OD) δ 4.85 (t, J = 112.4 Hz).

MS (ESI-TOF) calcd for C₂₀H₁₄Cl₃F₂O₄P [M − H]⁻1:1 490.9591

and 492.9561, found 1:1 490.9592 and 492.9564.

((3′-(4,5-Dichloro-2-ﬂuorobenzyl)-[1,1′-biphenyl]-4-yl)-

diﬂuoromethyl)phosphonic Acid (8). Phosphonate ( )-39 (218 mg,

0.410 mmol, 1.0 equiv) was dissolved in anhydrous CH₂Cl₂(1.3 mL,

0.248 M). To this mixture was added iodotrimethylsilane (0.35 mL,

492 mg, 2.46 mmol, 6.0 equiv), then the ﬂask was sealed under a

polyethylene stopper. The reaction mixture was stirred for 20 h at 23

°C, then concentrated. The residue was dissolved in minimal MeOH

and puriﬁed by reversed-phase gradient chromatography (5−100%

MeCN in H₂O with 0.1% TFA). Volatile components were removed,

and the resulting aqueous solution was lyophilized to aﬀord 8 as a

white powder (180 mg, 95%). IR (ﬁlm) ν_max3373 (br), 1652, 1478,

1

3%). IR (ﬁlm) ν_max3365 (br), 1674, 1457, 1135, 1070, 932 cm⁻¹. H

NMR (400 MHz, CD₃OD) δ 7.64 (s, 1H), 7.60 (d, J = 7.9 Hz, 2H),

7.48−7.45 (m, 2H), 7.39−7.34 (m, 2H), 7.18 (t, J = 7.6 Hz, 1H), 7.11

(td, J = 7.7, 1.7 Hz, 1H), 4.86−4.82 (m, 1H), 4.15 (s, 2H). ¹⁹F NMR

(376 MHz, CD₃OD) δ −124.0. ³¹P NMR (162 MHz, CD₃OD) δ 17.0

(br). MS (ESI-TOF) calcd for C₂₀H₁₅Cl₃FO₄P [M − H]⁻1:1

472.9685 and 474.9655, found 1:1 472.9683 and 474.9654.

(S)-((2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

(hydroxy)methyl)phosphonic Acid ((S)-12). Reaction of (S)-((4-

bromophenyl)(hydroxy)methyl)phosphonic acid (S)-48 (83 mg,

0.314 mmol, 1.0 equiv), boronic ester 47 (189 mg, 0.454 mmol, 1.4

equiv), Na₂CO₃·H₂O (258 mg, 2.08 mmol, 6.6 equiv), and Pd(PPh₃)₄

(38.6 mg, 0.0334 mmol, 0.11 equiv) in DME (1.60 mL), EtOH (0.40

mL), and H₂O (0.40 mL) according to the procedure for the

preparation of ( )-5 above aﬀorded (S)-12 as a white solid (8.6 mg,

6%). Spectroscopic results agree with data reported for the

enantiomeric phosphonic acid (R)-12.

1

1372, 1259, 1121, 1049, 926, 839, 783 cm⁻¹. H NMR (400 MHz,

CD₃OD) δ 7.51−7.44 (m, 4H), 7.31−7.28 (m, 2H), 7.19 (t, J = 7.4

Hz, 2H), 7.10 (d, J = 9.3 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 3.81 (s,

2H). ¹⁹F NMR (376 MHz, CD₃OD) δ −110.7 (d, J = 113.2 Hz),

−118.6. ³¹P NMR (162 MHz, CD₃OD) δ 4.93 (t, J = 105.7 Hz). MS

(ESI-TOF) calcd for C₂₀H₁₄Cl₂F₃O₃P [M − H]⁻458.9937, found

458.9937.

J

DOI: 10.1021/acs.jmedchem.7b01292

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(Fluoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

methyl)phosphonic Acid (( )-13). Reaction of phosphonate 51 (145

mg, 0.263 mmol, 1.0 equiv) and iodotrimethylsilane (0.22 mL, 309

mg, 1.54 mmol, 5.9 equiv) in CH₂Cl₂(1.0 mL, 0.216 M) according to

the procedure for the preparation of 8 above aﬀorded ( )-13 as a

white solid (85 mg, 68%). IR (ﬁlm) ν_max1457, 1408, 1354, 1205, 1167,

1134, 1070, 1014, 843, 799, 757 cm⁻¹. ¹H NMR (600 MHz, CD₃OD)

δ 7.55 (s, 1H), 7.52−7.47 (m, 4H), 7.34−7.28 (m, 2H), 7.13 (t, J = 7.6

Hz, 1H), 7.07 (t, J = 7.2 Hz, 1H), 5.65 (dd, J = 44.7, 8.3 Hz, 1H), 4.07

(s, 2H). ¹⁹F NMR (376 MHz, CD₃OD) δ −123.9, −201.1 (d, J = 83.2

Hz). ³¹P NMR (162 MHz, CD₃OD) δ 12.7 (d, J = 85.3). MS (ESI-

TOF) calcd for C₂₀H₁₄Cl₃F₂O₃P [M − H]⁻1:1 474.9641 and

476.9612, found 1:1 474.9641 and 476.9627.

7.06 (td, J = 7.2, 1.7 Hz, 1H), 4.61 (d, J = 23.0 Hz, 1H), 4.06 (s, 2H),

3.68−3.61 (m, 1H), 3.42−3.33 (m, 3H), 1.53−1.47 (m, 3H), 1.45−

1.34 (m, 2H), 1.07−0.99 (m, 1H). ¹⁹F NMR (376 MHz, CD₃OD) δ

−123.9. ³¹P NMR (162 MHz, CD₃OD) δ 18.7. MS (ESI-TOF) calcd

for C₂₆H₂₄Cl₃FNO₄P [M − H]⁻1:1 568.0420 and 570.0390, found

1:1 568.0419 and 570.0396.

( )-(1-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

2-morpholino-2-oxoethyl)phosphonic Acid (( )-18). Reaction of

phosphonate ( )-73 (56 mg crude, theoretical 0.0892 mmol, 1.0

equiv) and iodotrimethylsilane (0.07 mL, 98 mg, 0.492 mmol, ∼5.5

equiv) in CH₂Cl₂(0.38 mL, 0.198 M) according to the procedure for

the preparation of 8 above aﬀorded ( )-18 as a white solid (14 mg,

28% over 2 steps). IR (ﬁlm) ν_max2925, 2859, 1630, 1459, 1356, 1206,

1

1115, 1070, 1036, 939, 860, 820, 590 cm⁻¹. H NMR (600 MHz,

( )-(1-Fluoro-1-(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphen-

yl]-4-yl)-2-oxo-2-phenylethyl)phosphonic Acid (( )-14). Reaction of

phosphonate 60 (186 mg, 0.285 mmol, 1.0 equiv) and iodotrime-

thylsilane (0.24 mL, 337 mg, 1.69 mmol, 5.9 equiv) in CH₂Cl₂(1.2

mL, 0.198 M) according to the procedure for the preparation of 8

above aﬀorded ( )-14 as a white solid (46 mg, 28%). IR (ﬁlm) ν_max

CD₃OD) δ 7.63 (s, 1H), 7.59 (d, J = 6.5 Hz, 2H), 7.51 (d, J = 6.7 Hz,

2H), 7.39−7.35 (m, 2H), 7.19 (t, J = 7.6 Hz, 1H), 7.16−7.10 (m, 1H),

4.64 (d, J = 22.8 Hz, 1H), 4.14 (s, 2H), 3.68−3.59 (m, 5H), 3.58−3.53

(m, 1H), 3.46−3.41 (m, 1H), 3.34−3.30 (m, 1H). ¹⁹F NMR (376

MHz, CD₃OD) δ −124.0. ³¹P NMR (162 MHz, CD₃OD) δ 17.2. MS

(ESI-TOF) calcd for C₂₅H₂₂Cl₃FNO₅P [M − H]⁻1:1 570.0212 and

572.0183, found 1:1 570.0213 and 572.0189.

1

2361, 2339, 1684, 1653, 1457, 1259, 1205, 1069, 668 cm⁻¹. H NMR

(600 MHz, CD₃OD) δ 7.82 (d, J = 8.5 Hz 2H), 7.74 (dd, J = 8.5, 2.1

Hz, 2H), 7.60−7.56 (m, 3H), 7.51 (td, J = 7.5, 1.2 Hz, 1H), 7.39−7.33

(m, 4H), 7.16 (t, J = 7.6 Hz, 1H), 7.11 (t, J = 6.4 Hz, 1H), 4.11 (s,

2H). ¹⁹F NMR (376 MHz, CD₃OD) δ −123.9, −170.4 (d, J = 85.2

Hz). ³¹P NMR (162 MHz, CD₃OD) δ 9.2 (d, J = 85.3). MS (ESI-

TOF) calcd for C₂₇H₁₈Cl₃F₂O₄P [M − H]⁻1:1 578.9904 and

580.9874, found 1:1 578.9903 and 580.9880.

( )-(2-(Dimethylamino)-1-(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-

[1,1′-biphenyl]-4-yl)-2-oxoethyl)phosphonic Acid (( )-19). Reaction

of phosphonate ( )-74 (124 mg crude, theoretical 0.212 mmol, 1.0

equiv) and iodotrimethylsilane (0.18 mL, 253 mg, 1.27 mmol, ∼6.0

equiv) in CH₂Cl₂(0.90 mL, 0.196 M) according to the procedure for

the preparation of 8 above aﬀorded ( )-19 as a white solid (40 mg,

36% over 2 steps). IR (ﬁlm) ν_max1626, 1458, 1408, 1354, 1204, 1134,

( )-(2-Cyclohexyl-1-ﬂuoro-1-(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-

[1,1′-biphenyl]-4-yl)-2-oxoethyl)phosphonic Acid (( )-15). Reaction

of phosphonate 61 (36 mg, 0.0544 mmol, 1.0 equiv) and

iodotrimethylsilane (0.05 mL, 70.3 mg, 0.351 mmol, 6.5 equiv) in

CH₂Cl₂(0.31 mL, 0.151 M) according to the procedure for the

preparation of 8 above aﬀorded ( )-15 as a white solid (22 mg, 68%).

IR (ﬁlm) ν_max2931, 2854, 2361, 2337, 1708, 1457, 1205, 1151, 1070,

1020 cm⁻¹. ¹H NMR (600 MHz, CD₃OD) δ 7.75 (dd, J = 8.5, 2.1 Hz,

2H), 7.61 (s, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.38−7.33 (m, 2H), 7.17

(t, J = 7.6 Hz, 1H), 7.14−7.08 (m, 1H), 4.12 (s, 2H), 3.09 (tq, J =

11.2, 3.5 Hz, 1H), 1.83 (d, J = 13.1 Hz, 1H), 1.76 (dt, J = 12.9, 3.7 Hz,

1H), 1.68−1.61 (m, 3H), 1.43−1.34 (m, 1H), 1.34−1.22 (m, 2H),

1.16 (qt, J = 12.4, 3.6 Hz, 1H), 1.08 (qd, J = 13.9, 13.0, 3.8 Hz, 1H).

1

1070, 1003, 938, 800 cm⁻¹. H NMR (600 MHz, CD₃OD) δ 7.61 (s,

1H), 7.58 (d, J = 6.2 Hz, 2H), 7.48 (d, J = 6.7 Hz, 2H), 7.37−7.33 (m,

2H), 7.17 (t, J = 7.6 Hz, 1H), 7.11 (td, J = 7.3, 1.8 Hz, 1H), 4.63 (d, J

= 22.8 Hz, 1H), 4.12 (s, 2H), 3.04 (s, 3H), 2.95 (s, 3H). ¹⁹F NMR

(376 MHz, CD₃OD) δ −124.0. ³¹P NMR (162 MHz, CD₃OD) δ 18.0.

MS (ESI-TOF) calcd for C₂₃H₂₀Cl₃FNO₄P [M − H]⁻1:1 528.0107

and 530.0077, found 1:1 528.0106 and 530.0081.

( )-(1-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

2-(methylamino)-2-oxoethyl)phosphonic Acid (( )-20). Reaction of

phosphonate 79 (31 mg crude, maximum 0.0533 mmol, 1.0 equiv)

and iodotrimethylsilane (0.05 mL, 70.3 mg, 0.351 mmol, 6.6 equiv) in

CH₂Cl₂(0.24 mL, 0.184 M) according to the procedure for the

preparation of 8 above aﬀorded ( )-20 as a white solid (5.6 mg, 20%).

¹⁹F NMR (376 MHz, CD₃OD) δ −124.0, −177.8 (d, J = 81.6 Hz). ³¹

P

1

IR (ﬁlm) ν_max1644, 1459, 1410, 1354, 1204, 1135, 1070 cm⁻¹. H

NMR (162 MHz, CD₃OD) δ 8.5 (d, J = 82.1). MS (ESI-TOF) calcd

for C₂₇H₂₄Cl₃F₂O₄P [M − H]⁻1:1 585.0373 and 587.0344, found 1:1

585.0374 and 587.0351.

NMR (600 MHz, CD₃OD) δ 7.63 (s, 1H), 7.60−7.57 (m, 2H), 7.44

(d, J = 6.8 Hz, 2H), 7.37−7.33 (m, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.10

(ddd, J = 8.5, 6.9, 1.7 Hz, 1H), 4.13 (s, 2H), 3.99 (d, J = 21.6 Hz, 1H),

2.78 (s, 3H). ¹⁹F NMR (376 MHz, CD₃OD) δ −124.0. ³¹P NMR (162

MHz, CD₃OD) δ 14.2. MS (ESI-TOF) calcd for C₂₂H₁₈Cl₃FNO₄P [M

− H]⁻1:1 513.9950 and 515.9921, found 1:1 513.9945 and 515.9915.

( )-((2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

(morpholinosulfonyl)methyl)phosphonic Acid (( )-21). Reaction of

phosphonate ( )-83 (249 mg crude, maximum 0.374 mmol, 1.0

equiv) and iodotrimethylsilane (0.32 mL, 450 mg, 2.25 mmol, 6.0

equiv) in CH₂Cl₂(1.7 mL, 0.185 M) according to the procedure for

the preparation of 8 above aﬀorded ( )-21 as a white solid (60 mg,

18% over 2 steps). IR (ﬁlm) ν_max2362, 2336, 1457, 1344, 1262, 1154,

( )-(1-Fluoro-1-(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphen-

yl]-4-yl)-2-oxo-2-(piperidin-1-yl)ethyl)phosphonic Acid (( )-16). Re-

action of phosphonate 66 (maximum of 56.2 mg, 0.0850 mmol, 1.0

equiv) and iodotrimethylsilane (0.07 mL, 98.4 mg, 0.492 mmol, 5.8

equiv) in CH₂Cl₂(0.35 mL, 0.203 M) according to the procedure for

the preparation of 8 above aﬀorded ( )-16 as a white solid (17.6 mg,

35%). IR (ﬁlm) ν_max2940, 2858, 1629, 1456, 1404, 1354, 1256, 1205,

1163, 1069, 1022, 954, 915, 821, 794, 579 cm⁻¹. ¹H NMR (600 MHz,

CD₃OD) δ 7.62 (s, 1H), 7.60−7.58 (m, 4H), 7.38 (td, J = 7.6, 1.8 Hz,

1H), 7.36 (s, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.16−7.13 (m, 1H), 4.13

(s, 2H), 3.65−3.60 (m, 1H), 3.53−3.47 (m, 1H), 3.42−3.36 (m, 1H),

3.29−3.23 (m, 1H), 1.61−1.50 (m, 4H), 1.39−1.31 (m, 1H), 1.07−

0.99 (m, 1H). ¹⁹F NMR (376 MHz, CD₃OD) δ −123.9, −168.9 (d, J

= 91.9 Hz). ³¹P NMR (162 MHz, CD₃OD) δ 10.4 (d, J = 91.8). MS

(ESI-TOF) calcd for C₂₆H₂₃Cl₃F₂NO₄P [M − H]⁻1:1 586.0326 and

588.0296, found 1:1 586.0324 and 588.0298.

1

1114, 1070, 1018, 941, 870, 807, 736 cm⁻¹. H NMR (600 MHz,

CD₃OD) δ 7.77 (d, J = 7.8 Hz, 2H), 7.64 (s, 1H), 7.59 (d, J = 7.1 Hz,

2H), 7.41 (td, J = 7.5, 1.8 Hz, 1H), 7.38 (s, 1H), 7.21 (t, J = 7.6 Hz,

1H), 7.16 (ddd, J = 8.4, 7.1, 1.8 Hz, 1H), 5.00 (d, J = 21.1 Hz, 1H),

4.16 (s, 2H), 3.54 (ddd, J = 11.5, 6.4, 3.0 Hz, 2H), 3.46 (ddd, J = 11.4,

6.3, 3.0 Hz, 2H), 3.21−3.16 (m, 2H), 3.00−2.92 (m, 2H). ¹⁹F NMR

(376 MHz, CD₃OD) δ −123.9. ³¹P NMR (162 MHz, CD₃OD) δ 10.0.

MS (ESI-TOF) calcd for C₂₄H₂₂Cl₃FNO₆PS [M − H]⁻1:1 605.9882

and 607.9853, found 1:1 605.9892 and 907.9859.

(+)-(Fluoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-

yl)(morpholinosulfonyl)methyl)phosphonic Acid ((+)-22). Reaction

of phosphonate 85b (30 mg, 0.0439 mmol, 1.0 equiv) and

iodotrimethylsilane (0.04 mL, 56.3 mg, 0.281 mmol, 6.4 equiv) in

CH₂Cl₂(0.20 mL, 0.183 M) according to the procedure for the

preparation of 8 above aﬀorded (+)-22 as a white solid (25 mg, 90%).

( )-(1-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

2-oxo-2-(piperidin-1-yl)ethyl)phosphonic Acid (( )-17). Reaction of

phosphonate ( )-72 (131 mg assuming 100% yield in previous step,

0.209 mmol, 1.0 equiv) and iodotrimethylsilane (0.18 mL, 253 mg,

1.27 mmol, 6.0 equiv) in CH₂Cl₂(0.89 mL, 0.196 M) according to the

procedure for the preparation of 8 above aﬀorded ( )-17 as a white

solid (52 mg, 44% over 2 steps). IR (ﬁlm) ν_max2938, 2858, 1606,

1

1515, 1457, 1409, 1354, 1205, 1134, 1069, 1016, 938 cm⁻¹. H NMR

(600 MHz, CD₃OD) δ 7.54 (s, 1H), 7.51 (dd, J = 8.4, 2.3 Hz, 2H),

7.45 (d, J = 6.9 Hz, 2H), 7.32−7.27 (m, 2H), 7.11 (t, J = 7.6 Hz, 1H),

K

DOI: 10.1021/acs.jmedchem.7b01292

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

IR (ﬁlm) ν_max1645, 1458, 1349, 1204, 1161, 1134, 1114, 1071, 963,

68%). R_f= 0.30 (silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm) ν_max1399,

1

541 cm⁻¹. H NMR (400 MHz, CD₃OD) δ 7.93 (d, J = 8.2 Hz, 2H),

1361, 1328, 1272, 1144, 1125, 1091, 1045, 1018, 963, 857, 657, 574

1

7.64−7.58 (m, 3H), 7.41 (td, J = 7.5, 1.9 Hz, 1H), 7.36 (s, 1H), 7.20

(t, J = 7.6 Hz, 1H), 7.17−7.12 (m, 1H), 4.14 (s, 2H), 3.59−3.49 (m,

2H), 3.48−3.41 (m, 2H), 3.29−3.23 (m, 2H), 3.16−2.86 (m, 2H). ¹⁹F

NMR (376 MHz, CD₃OD) δ −124.8, −167.5 (d, J = 70.5 Hz). ³¹P

NMR (162 MHz, CD₃OD) δ 3.6 (d, J = 69.6 Hz). MS (ESI-TOF)

calcd for C₂₄H₂₁Cl₃F₂NO₆PS [M − H]⁻1:1 623.9788 and 625.9759,

cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.88 (d, J = 7.7 Hz, 2H), 7.61

(d, J = 7.4 Hz, 2H), 4.23−4.07 (m, 4H), 1.35 (s, 12H), 1.30 (t, J = 7.1

H, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −109.5 (d, J = 115.7 Hz). ³¹

P

NMR (162 MHz, CDCl₃) δ 6.24 (t, J = 115.6 Hz). MS (ESI-TOF)

calcd for C₁₇H₂₆BF₂O₅P [M + H⁺] 391.1652, found 391.1659.

2,4,5-Trichlorobenzaldehyde (27). 1,2,4-Trichloro-5-iodobenzene

(4.00 g, 13.0 mmol, 1.0 equiv) was dissolved in anhydrous THF (40

mL, 0.269 M), and the resultant solution was cooled to −78 °C in a

dry ice/acetone bath. Dropwise by syringe, under an atmosphere of

N₂, iPrMgCl (7.0 mL of a 2.0 M solution in THF, 14.0 mmol, 1.1

equiv) was added to the stirred reaction mixture. The mixture was

then stirred for 20 min at −78 °C. At this point, anhydrous DMF (1.5

mL, 1.42 g, 19.5 mmol, 1.5 equiv) was rapidly added to the solution

and the ﬂask was allowed to warm to 23 °C. The reaction mixture was

stirred under N₂for 1 h, then the reaction was quenched with satd aq

NH₄Cl (20 mL). The aqueous layer was extracted with EtOAc (2 × 40

mL), then the pooled organic phases were dried over Na₂SO₄, ﬁltered,

and concentrated to aﬀord 27 as a white powder, which required no

further puriﬁcation (2.59 g, 95%). R_f= 0.70 (silica gel, 4:1 hexanes/

EtOAc). IR (ﬁlm) ν_max1682, 1572, 1442, 1349, 1189, 1074, 931, 899

cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 7.98 (s, 1H), 7.59

(s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 187.6, 139.5, 136.0, 132.8,

132.2, 131.8, 130.6. MS (ESI-TOF) calcd for C₇H₃Cl₃O [M + H₃O⁺]

226.9428, found 226.9429.

20

found 1:1 623.9790 and 625.9767. [α]_D= +2.7 (c = 0.1, DMSO).

(−)-(Fluoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-

yl)(morpholinosulfonyl)methyl)phosphonic Acid ((−)-22). Reaction

of phosphonate 85a (34 mg, 0.0498 mmol, 1.0 equiv) and

iodotrimethylsilane (0.05 mL, 70.3 mg, 0.351 mmol, 7.1 equiv) in

CH₂Cl₂(0.23 mL, 0.178 M) according to 8 above aﬀorded (−)-22 as a

white solid (21 mg, 66%). Spectroscopic results agree with data

reported for phosphonic acid (+)-22. [α]_D²⁰= −2.5 (c = 0.1, DMSO).

Diethyl ((4-Bromophenyl)diﬂuoromethyl)phosphonate (23). Fol-

lowing a reported procedure,⁵³a ﬂame-dried round-bottom ﬂask was

charged with preactivated Zn dust (3.47 g, 53.0 mmol, 5.0 equiv) and

anhydrous DMA (11.2 mL), then heated to 55 °C under an

atmosphere of N₂. Dropwise over 30 min, with stirring under N₂,

diethyl (bromodiﬂuoromethyl)phosphonate (9.4 mL, 14.1 g, 52.9

mmol, 5.0 equiv) was added to the Zn suspension. The reaction

mixture was then stirred at 55 °C under N₂for an additional 30 min,

after which the reaction ﬂask was removed from heating and sonicated

for 3 h. Solid copper(I) bromide (7.56 g, 52.7 mmol, 4.9 equiv) was

added in one portion, and the reaction mixture was stirred at 23 °C

under N₂for 30 min. Next, 1-bromo-4-iodobenzene (3.02 g, 10.7

mmol, 1.0 equiv) in anhydrous DMA (6.0 mL) was added dropwise by

syringe under N₂. The reaction mixture was stirred under N₂at 23 °C

for 20 h, and then H₂O (15 mL) and Et₂O (15 mL) were added. The

suspension was ﬁltered through a pad of Celite, and the aqueous layer

was extracted with Et₂O (3 × 20 mL). The pooled organic layers were

dried over Na₂SO₄, ﬁltered, and concentrated. The crude product was

puriﬁed by ﬂash chromatography (silica gel, 19:1 to 4:1 hexanes/

EtOAc) to aﬀord 23 as a yellow oil (3.53 g, 97%). R_f= 0.41 (silica gel,

2:1 hexanes/EtOAc). IR (ﬁlm) ν_max1596, 1487, 1397, 1271, 1128,

( )-(3-Bromophenyl)(4,5-dichloro-2-ﬂuorophenyl)methanol

(( )-28). Following our previously reported procedure,³⁶1-bromo-3-

iodobenzene (0.25 mL, 559 mg, 1.97 mmol, 1.0 equiv) was dissolved

in anhydrous THF (6.0 mL), and the resultant mixture was cooled to 0

°C in an ice bath. With stirring, under an atmosphere of N₂, iPrMgCl

(0.99 mL of a 2.0 M solution in THF, 1.97 mmol, 1.0 equiv) was

added to the solution dropwise by syringe. The reaction mixture was

stirred under N₂at 0 °C for 20 min. Then 4,5-dichloro-2-

ﬂuorobenzaldehyde (26)⁵⁴(381 mg, 1.97 mmol, 1.0 equiv) in

minimal anhydrous THF (2.0 mL) was added dropwise by syringe.

The reaction mixture was allowed to slowly warm to 23 °C, and after 2

h, the reaction was quenched with satd aq NH₄Cl (5 mL) and Et₂O (5

mL). The aqueous layer was extracted with Et₂O (2 × 15 mL), and the

pooled organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated. The crude product was puriﬁed by ﬂash chromatography

(silica gel, 19:1 to 3:1 hexanes/EtOAc) to aﬀord ( )-28 as a colorless

oil (543 mg, 79%). R_f= 0.48 (silica gel, 4:1 hexanes/EtOAc). IR (ﬁlm)

ν_max3326 (br), 1604, 1572, 1467, 1374, 1187, 1118, 1036, 682 cm⁻¹.

¹H NMR (400 MHz, CDCl₃) δ 7.63 (d, J = 7.0 Hz, 1H), 7.53 (t, J =

1.9 Hz, 1H), 7.43 (ddd, J = 7.8, 2.0, 1.2 Hz, 1H), 7.29 (dq, J = 7.7, 1.0

Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 9.4 Hz, 1H), 6.01 (d, J =

2.9 Hz, 1H), 2.43 (d, J = 3.6 Hz, 1H). ¹⁹F NMR (376 MHz, CDCl₃) δ

−118.3. MS (ESI-TOF) calcd for C₁₃H₈BrCl₂FO [M − OH]⁺1:1

330.9087 and 332.9066, found 1:1 330.9084 and 332.9064.

( )-(3-Bromophenyl)(2,4,5-trichlorophenyl)methanol (( )-29).

Reaction of 1-bromo-3-iodobenzene (0.24 mL, 542 mg, 1.91 mmol,

1.0 equiv), iPrMgCl (0.95 mL of a 2.0 M solution in THF, 1.90 mmol,

1.0 equiv), and aldehyde 27 (401 mg, 1.91 mmol, 1.0 equiv) in

anhydrous THF (7.2 mL, 0.228 M) according to the procedure for the

preparation of ( )-28 above aﬀorded ( )-29 as a colorless oil (625

mg, 89%). R_f= 0.56 (silica gel, 4:1 hexanes/EtOAc). IR (ﬁlm) ν_max

3306 (br), 1571, 1455, 1353, 1182, 1072, 1034, 885, 794, 682 cm⁻¹.

¹H NMR (400 MHz, CDCl₃) δ 7.72−7.70 (m, 1H), 7.52−7.49 (m,

1

1099, 1009, 975, 937, 878, 821, 750, 569 cm⁻¹. H NMR (400 MHz,

CDCl₃) δ 7.52 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 7.9 Hz, 2H), 4.22−4.04

(m, 4H), 1.24 (td, J = 6.5, 0.8 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃)

δ −108.9 (d, J = 115.3 Hz). ³¹P NMR (162 MHz, CDCl₃) δ 5.67 (t, J

= 114.8 Hz). MS (ESI-TOF) calcd for C₁₁H₁₄BrF₂O₃P [M + H⁺] 1:1

343.0 and 345.0, found 1:1 342.9 and 345.0.

((4-Bromophenyl)diﬂuoromethyl)phosphonic Acid (24). Phospho-

nate 23 (643 mg, 1.87 mmol, 1.0 equiv) was dissolved in anhydrous

CH₂Cl₂(5.8 mL, 0.293 M). To this mixture was added

iodotrimethylsilane (0.59 mL, 828 mg, 4.14 mmol, 2.2 equiv), then

the ﬂask was sealed under a polyethylene stopper. The reaction

mixture was stirred for 20 h at 23 °C, then concentrated. The residue

was dissolved in minimal MeOH and puriﬁed by reversed-phase

gradient chromatography (5−100% MeCN in H₂O with 0.1% TFA).

Volatile components were removed, and the resulting aqueous

solution was lyophilized to aﬀord 24 as a white powder (358 mg,

67%). IR (ﬁlm) ν_max1595, 1487, 1398, 1254, 1126, 1051, 1014, 981,

920, 819, 560 cm⁻¹. ¹H NMR (400 MHz, CD₃OD) δ 7.42 (d, J = 8.2

Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H). ¹⁹F NMR (376 MHz, CD₃OD) δ

−111.3 (d, J = 111.8 Hz). ³¹P NMR (162 MHz, CD₃OD) δ 4.21 (t, J =

111.8 Hz). MS (ESI-TOF) calcd for C₇H₆BrF₂O₃P [M − H]⁻1:1

284.9133 and 286.9113, found 1:1 284.9135 and 286.9115.

Diethyl(diﬂuoro(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

phenyl)methyl)phosphonate (25). Phosphonate 23 (624 mg, 1.82

mmol, 1.0 equiv), bis(pinacoloto)diboron (572 mg, 2.25 mmol, 1.2

equiv), and KOAc (541 mg, 5.51 mmol, 3.0 equiv) were dissolved in

anhydrous 1,4-dioxane (8.2 mL, 0.222 M). With stirring, N₂was

bubbled through the reaction mixture for 20 min. Then, Pd(dppf)Cl₂·

CH₂Cl₂(148 mg, 0.182 mmol, 0.10 equiv) was added, and the ﬂask

was outﬁtted with a reﬂux condenser. The reaction mixture was stirred

under N₂at 65 °C for 18 h, then cooled to 23 °C and concentrated.

The crude product was puriﬁed by ﬂash chromatography (silica gel,

19:1 to 1:1 hexanes/EtOAc) to aﬀord 25 as a yellow oil (480 mg,

1H), 7.45−7.41 (m, 2H), 7.28−7.24 (m, 1H), 7.21 (t, J = 7.8 Hz, 1H),

6.03 (d, J = 3.0 Hz, 1H), 2.66 (br s, 1H). ¹³C NMR (100 MHz,

CDCl₃) δ 143.5, 140.5, 132.6, 132.0, 131.5, 131.0, 130.9, 130.4, 130.0,

129.4, 125.6, 122.9, 71.6. MS (ESI-TOF) calcd for C₁₃H₈BrCl₃O [M −

OH]⁺1:1 346.8791 and 348.8771, found 1:1 346.8792 and 348.8764.

( )-(4,5-Dichloro-2-ﬂuorophenyl)(3-(4,4,5,5-tetramethyl-1,3,2-di-

oxaborolan-2-yl)phenyl)methanol (( )-30). Benzyhydryl alcohol

( )-28 (217 mg, 0.619 mmol, 1.0 equiv), bis(pinacoloto)diboron

(185 mg, 0.727 mmol, 1.2 equiv), and KOAc (180 mg, 1.83 mmol, 3.0

equiv) were dissolved in anhydrous 1,4-dioxane (3.3 mL, 0.187 M).

With stirring, N₂was bubbled through the reaction mixture for 20 min.

L

DOI: 10.1021/acs.jmedchem.7b01292

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Then, Pd(dppf)Cl₂·CH₂Cl₂(51 mg, 0.062 mmol, 0.10 equiv) was

added and the ﬂask was outﬁtted with a reﬂux condenser. The reaction

mixture was stirred under N₂at 65 °C for 18 h, then cooled to 23 °C

and concentrated. The crude product was puriﬁed by ﬂash

chromatography (silica gel, 19:1 to 3:1 hexanes/EtOAc) to aﬀord

( )-30 as a colorless oil (237 mg, 97%). R_f= 0.65 (silica gel, 2:1

hexanes/EtOAc). IR (ﬁlm) ν_max3431 (br), 2979, 1467, 1431, 1359,

CDCl₃) δ 143.4, 140.4, 137.4, 135.9, 132.5, 131.9, 131.0, 130.8, 130.5,

129.3, 126.2, 94.7, 71.4. MS (ESI-TOF) calcd for C₁₃H₈Cl₃IO [M −

OH]⁺1:1 394.8653 and 396.8623, found 1:1 394.8650 and 396.8624.

( )-(2-Bromo-4,5-dichlorophenyl)(3-iodophenyl)methanol

(( )-37). Reaction of 1,3-diiodobenzene 32 (508 mg, 1.54 mmol, 1.5

equiv), iPrMgCl (0.77 mL of a 2.0 M solution in THF, 1.53 mmol, 1.5

equiv), and aldehyde 34 (260 mg, 1.02 mmol, 1.0 equiv) in anhydrous

THF (3.8 mL, 0.224 M) according to the procedure for the

preparation of ( )-35 above aﬀorded ( )-37 as a colorless oil

which foamed under vacuum (423 mg, 90%). R_f= 0.48 (silica gel, 4:1

hexanes/EtOAc). IR (ﬁlm) ν_max3301 (br), 1566, 1450, 1422, 1346,

1

1143, 854, 707 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.83 (t, J = 1.4

Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.68 (d, J = 7.0 Hz, 1H), 7.43 (dt, J

= 7.9, 1.6 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.14 (d, J = 9.4 Hz, 1H),

6.06 (d, J = 3.0 Hz, 1H), 2.35 (d, J = 3.5 Hz, 1H), 1.34 (s, 12H). ¹⁹F

NMR (376 MHz, CDCl₃) δ −117.9. MS (ESI-TOF) calcd for

C₁₉H₂₀BCl₂FO₃[M − OH]⁺379.0834, found 379.0834.

1

1178, 1133, 1031, 904, 788, 733, 681 cm⁻¹. H NMR (400 MHz,

CDCl₃) δ 7.72 (t, J = 1.8 Hz, 1H), 7.67−7.61 (m, 3H), 7.28 (d, J = 7.7

Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 5.95 (s, 1H), 2.91 (s, 1H). ¹³C NMR

(100 MHz, CDCl₃) δ 143.4, 142.0, 137.4, 136.0, 134.0, 132.8, 132.6,

130.5, 129.7, 126.3, 120.3, 94.7, 73.5. MS (ESI-TOF) calcd for

C₁₃H₈BrCl₂IO [M − OH]⁺1:1 438.8147 and 440.8127, found 1:1

438.8154 and 440.8132.

( )-(3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-

(2,4,5-trichlorophenyl)methanol (( )-31). Reaction of benzhydryl

alcohol ( )-29 (625 mg, 1.70 mmol, 1.0 equiv), bis(pinacoloto)-

diboron (523 mg, 2.06 mmol, 1.2 equiv), KOAc (496 mg, 5.06 mmol,

3.0 equiv), and Pd(dppf)Cl₂·CH₂Cl₂(141 mg, 0.172 mmol, 0.10

equiv) in 1,4-dioxane (9.0 mL, 0.189 M) according to the procedure

for the preparation of ( )-30 above aﬀorded ( )-31 as a white solid

(623 mg, 88%). R_f= 0.55 (silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm)

ν_max3433 (br), 1454, 1357, 1277, 1142, 850, 732, 707 cm⁻¹. ¹H NMR

(400 MHz, CDCl₃) δ 7.84 (s, 1H), 7.78 (s, 1H), 7.75 (dt, J = 6.8, 1.5

Hz, 1H), 7.43 (s, 1H), 7.39−7.32 (m, 2H), 6.10 (s, 1H), 2.32 (br s,

1H), 1.34 (s, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 141.1, 140.6,

135.0, 133.4, 132.2, 131.7, 131.1, 130.9, 129.9, 129.6, 128.3, 84.1, 72.5,

25.0. MS (ESI-TOF) calcd for C₁₉H₂₀BCl₃O₃[M + H⁺] 413.0644,

found 413.0643.

2-Bromo-4,5-dichlorobenzaldehyde (34). Reaction of aryl iodide

44 (579 mg, 1.65 mmol, 1.0 equiv), iPrMgCl (0.83 mL of a 2.0 M

solution in THF, 1.66 mmol, 1.0 equiv), and DMF (0.19 mL, 180 mg,

2.46 mmol, 1.5 equiv) in THF (4.6 mL, 0.293 M) according to the

procedure for the preparation of 27 above aﬀorded 34 as a white solid

(338 mg, 81%). Spectroscopic results agree with previously reported

data.⁶⁴

( )-(2-Fluoro-3-iodophenyl)(2,4,5-trichlorophenyl)methanol

(( )-38). Reaction of 2-ﬂuoro-1,3-diiodobenzene 33⁵⁵(2.00 g, 5.74

mmol, 1.2 equiv), iPrMgCl (2.9 mL of a 2.0 M solution in THF, 5.80

mmol, 1.2 equiv), and aldehyde 27 (1.01 g, 4.80 mmol, 1.0 equiv) in

anhydrous THF (15.0 mL, 0.268 M) according to the procedure for

the preparation of ( )-35 above aﬀorded ( )-38 as a white solid (1.97

g, 95%). R_f= 0.56 (silica gel, 4:1 hexanes/EtOAc). IR (ﬁlm) ν_max3288

1

(br), 1446, 1353, 1225, 1130, 1074, 1034, 906, 732 cm⁻¹. H NMR

(400 MHz, CDCl₃) δ 7.74−7.68 (m, 2H), 7.46 (s, 1H), 7.20 (td, J =

7.3, 6.8, 1.6 Hz, 1H), 6.90 (t, J = 7.8 Hz, 1H), 6.34 (s, 1H), 2.56 (s,

1H). ¹⁹F NMR (376 MHz, CDCl₃) δ −97.4. MS (ESI-TOF) calcd for

C₁₃H₇Cl₃FIO [M − OH]⁺1:1 412.8558 and 414.8529, found 1:1

412.8559 and 414.8539.

( )-Diethyl ((3′-((4,5-Dichloro-2-ﬂuorophenyl)(hydroxy)methyl)-

[1,1′-biphenyl]-4-yl)diﬂuoromethyl)phosphonate (( )-39). Benz-

hydryl alcohol ( )-35 (302 mg, 0.760 mmol, 1.0 equiv), boronic

ester 25 (366 mg, 0.938 mmol, 1.2 equiv), and K₂CO₃(1.06 g, 7.63

mmol, 10 equiv) were dissolved in toluene (24.2 mL), EtOH (8.3

mL), and H₂O (4.9 mL). With stirring, N₂was bubbled through the

biphasic mixture for 1 h. Pd(PPh₃)₄(37.3 mg, 0.0323 mmol, 0.04

equiv) was then added, and N₂was bubbled through the stirred

solution for an additional 20 min. The ﬂask was next equipped with a

reﬂux condenser, and the reaction mixture was stirred at 80 °C under

an atmosphere of N₂for 16 h. The solution was cooled to 23 °C and

concentrated. The crude residue was puriﬁed by ﬂash chromatography

(silica gel, 19:1 to 1:1 hexanes/EtOAc) to aﬀord ( )-39 as an orange

solid (218 mg, 54%). R_f= 0.17 (silica gel, 2:1 hexanes/EtOAc). IR

(ﬁlm) ν_max3380 (br), 1607, 1466, 1371, 1258, 1117, 1019, 947, 838,

( )-(4,5-Dichloro-2-ﬂuorophenyl)(3-iodophenyl)methanol

(( )-35). 1,3-Diiodobenzene 32 (966 mg, 2.93 mmol, 1.5 equiv) was

dissolved in anhydrous THF (7.0 mL), and the resultant mixture was

cooled to −20 °C. With stirring, under an atmosphere of N₂, iPrMgCl

(1.46 mL of a 2.0 M solution in THF, 2.93 mmol, 1.5 equiv) was

added to the solution dropwise by syringe. The reaction solution was

stirred under N₂at −20 °C for 20 min. Then 4,5-dichloro-2-

ﬂuorobenzaldehyde 26⁵⁴(377 mg, 1.95 mmol, 1.0 equiv) in minimal

anhydrous THF (2.5 mL) was added dropwise by syringe. The

reaction mixture was allowed to slowly warm to 23 °C, and after 2 h,

the reaction was quenched with satd aq NH₄Cl (10 mL) and Et₂O (10

mL). The aqueous layer was extracted with Et₂O (2 × 25 mL), and the

pooled organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated. The crude product was puriﬁed by ﬂash chromatography

(silica gel, 19:1 to 3:1 hexanes/EtOAc) to aﬀord ( )-35 as a colorless

oil (495 mg, 64%). R_f= 0.52 (silica gel, 4:1 hexanes/EtOAc). IR (ﬁlm)

1

794, 571 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 7.1 Hz,

1H), 7.68−7.60 (m, 5H), 7.52 (dt, J = 7.6, 1.6 Hz, 1H), 7.44 (t, J = 7.6

Hz, 1H), 7.38 (dt, J = 7.6, 1.4 Hz, 1H), 7.15 (d, J = 9.4 Hz, 1H), 6.11

(d, J = 3.2 Hz, 1H), 4.28−4.11 (m, 4H), 2.96 (d, J = 3.7 Hz, 1H), 1.32

(td, J = 7.1, 0.7 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −108.3 (d, J

= 117.2 Hz), −118.1. ³¹P NMR (162 MHz, CDCl₃) δ 6.21 (t, J =

116.9 Hz). MS (ESI-TOF) calcd for C₂₄H₂₂Cl₂F₃O₄P [M + H⁺]

533.0658, found 533.0660.

1

ν_max3324 (br), 1568, 1466, 1374, 1118, 1035 cm⁻¹. H NMR (400

MHz, CDCl₃) δ 7.73 (t, J = 1.8 Hz, 1H), 7.64 (s, 1H), 7.62 (s, 1H),

7.32 (d, J = 7.8 Hz, 1H), 7.16 (d, J = 9.4 Hz, 1H), 7.08 (t, J = 7.8 Hz,

1H), 5.98 (d, J = 3.6 Hz, 1H), 2.42 (d, J = 3.7 Hz, 1H). ¹³C NMR (100

MHz, CDCl₃) δ 157.6 (d, J = 249.9 Hz), 144.1, 137.5, 135.4, 135.3,

130.6, 128.8, 128.7, 125.7, 118.1, 117.8, 94.8, 68.8 (d, J = 2.3 Hz). ¹⁹F

NMR (376 MHz, CDCl₃) δ −118.1. MS (ESI-TOF) calcd for

C₁₃H₈Cl₂FIO [M − OH]⁺378.8948, found 378.8951.

( )-Diethyl (Diﬂuoro(3′-(hydroxy(2,4,5-trichlorophenyl)methyl)-

[1,1′-biphenyl]-4-yl)methyl)phosphonate (( )-40). Reaction of

benzhydryl alcohol ( )-36 (199 mg, 0.481 mmol, 1.0 equiv), boronic

ester 25 (225 mg, 0.577 mmol, 1.2 equiv), K₂CO₃(668 mg, 4.83

mmol, 10.0 equiv), and Pd(PPh₃)₄(19.5 mg, 0.0169 mmol, 0.04

equiv) in toluene (15.3 mL), EtOH (5.1 mL), and H₂O (3.1 mL)

according to the procedure for the preparation of ( )-39 above

aﬀorded ( )-40 as a yellow oil (190 mg, 72%). R_f= 0.22 (silica gel, 2:1

hexanes/EtOAc). IR (ﬁlm) ν_max3382 (br), 1452, 1259, 1129, 1020,

( )-(3-Iodophenyl)(2,4,5-trichlorophenyl)methanol (( )-36). Re-

action of 1,3-diiodobenzene 32 (945 mg, 2.86 mmol, 1.5 equiv),

iPrMgCl (1.43 mL of a 2.0 M solution in THF, 2.86 mmol, 1.5 equiv),

and aldehyde 27 (398 mg, 1.90 mmol, 1.0 equiv) in anhydrous THF

(7.0 mL, 0.225 M) according to the procedure for the preparation of

( )-35 above aﬀorded ( )-36 as a white solid (612 mg, 78%). R_f=

0.60 (silica gel, 4:1 hexanes/EtOAc). IR (ﬁlm) ν_max3294 (br), 1706,

1566, 1453, 1421, 1352, 1179, 1131, 1070, 1029, 904, 793, 731, 570

1

803, 576 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.83 (s, 1H), 7.68−

7.60 (m, 5H), 7.52 (dt, J = 7.7, 1.6 Hz, 1H), 7.45−7.40 (m, 2H), 7.36

(dt, J = 7.7, 1.5 Hz, 1H), 6.16 (s, 1H), 4.27−4.10 (m, 4H), 1.32 (t, J =

7.1 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −108.3 (d, J = 116.8

Hz). ³¹P NMR (162 MHz, CDCl₃) δ 6.19 (t, J = 117.0 Hz). MS (ESI-

TOF) calcd for C₂₄H₂₂Cl₃F₂O₄P [M + H⁺] 1:1 549.0362 and

551.0333, found 1:1 549.0362 and 551.0329.

1

cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.70−7.68 (m, 2H), 7.62 (dt, J

= 7.9, 1.4 Hz, 1H), 7.42 (s, 1H), 7.26 (d, J = 7.9 Hz, 1H), 7.05 (t, J =

7.8 Hz, 1H), 5.96 (s, 1H), 3.0 (br s, 1H). ¹³C NMR (100 MHz,

M

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( )-Diethyl ((3′-((2-Bromo-4,5-dichlorophenyl)(hydroxy)methyl)-

[1,1′-biphenyl]-4-yl)diﬂuoromethyl)phosphonate (( )-41). Reaction

of benzhydryl alcohol ( )-37 (129 mg, 0.281 mmol, 1.0 equiv),

boronic ester 25 (112 mg, 0.287 mmol, 1.0 equiv), K₂CO₃(389 mg,

2.82 mmol, 10.0 equiv), and Pd(PPh₃)₄(10.3 mg, 0.0089 mmol, 0.03

equiv) in toluene (8.9 mL), EtOH (3.0 mL), and H₂O (1.8 mL)

according to the procedure for the preparation of ( )-39 above

aﬀorded ( )-41 as a colorless oil (70 mg, 42%). R_f= 0.16 (silica gel,

2:1 hexanes/EtOAc). IR (ﬁlm) ν_max3374 (br), 1449, 1259, 1125,

( )-(3-Bromo-2-ﬂuorophenyl)(2,4,5-trichlorophenyl)methanol

(( )-45). 1,2,4-Trichloro-5-iodobenzene (458 mg, 1.49 mmol, 1.2

equiv) was dissolved in anhydrous THF (3.6 mL), and the resultant

mixture was cooled to −20 °C. With stirring, under an atmosphere of

N₂, iPrMgCl (0.73 mL of a 2.0 M solution in THF, 1.46 mmol, 1.2

equiv) was added to the solution dropwise by syringe. The reaction

was stirred under N₂at 0 °C for 20 min. Then 3-bromo-2-

ﬂuorobenzaldehyde (257 mg, 1.27 mmol, 1.0 equiv) in minimal

anhydrous THF (1.5 mL) was added dropwise by syringe. The

reaction mixture was allowed to slowly warm to 0 °C, and after 2 h, the

reaction was quenched with satd aq NH₄Cl (15 mL) and Et₂O (15

mL). The aqueous layer was extracted with Et₂O (2 × 25 mL), and the

pooled organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated. The crude product was puriﬁed by ﬂash chromatography

(silica gel, 19:1 to 3:1 hexanes/EtOAc) to aﬀord ( )-45 as a white

solid (479 mg, 98%). R_f= 0.53 (silica gel, 4:1 hexanes/EtOAc). IR

1

1043, 800 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.79 (s, 1H), 7.70−

7.62 (m, 6H), 7.54 (dt, J = 7.6, 1.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H),

7.38 (dt, J = 7.8, 1.5 Hz, 1H), 6.15 (d, J = 3.0 Hz, 1H), 4.29−4.11 (m,

4H), 2.63 (d, J = 3.6 Hz, 1H), 1.34 (t, J = 7.0 Hz, 6H). ¹⁹F NMR (376

MHz, CDCl₃) δ −108.3 (d, J = 116.6 Hz). ³¹P NMR (162 MHz,

CDCl₃) δ 6.25 (t, J = 116.5 Hz). MS (ESI-TOF) calcd for

C₂₄H₂₂BrCl₂F₂O₄P [M + H⁺] 1:1 592.9857 and 594.9836, found 1:1

592.9850 and 594.9826.

1

(ﬁlm) ν_max3289 (br), 1453, 1354, 1229, 1130, 1035 cm⁻¹. H NMR

(

)-Diethyl (Diﬂuoro(2′-ﬂuoro-3′-(hydroxy(2,4,5-

(400 MHz, CDCl₃) δ 7.69 (s, 1H), 7.50 (ddd, J = 8.1, 6.6, 1.7 Hz,

1H), 7.44 (s, 1H), 7.08−7.14 (m, 1H), 7.00 (td, J = 7.9, 1.0 Hz, 1H),

6.31 (d, J = 3.1 Hz, 1H), 3.02 (d, J = 4.0 Hz, 1H). ¹³C NMR (100

MHz, CDCl₃) δ 156.7 (d, J = 249.0 Hz), 139.4, 133.7, 132.8, 131.8,

131.1, 131.0, 130.0 (d, J = 14.4 Hz), 129.7 (d, J = 1.1 Hz), 127.6 (d, J

= 3.2 Hz), 125.4 (d, J = 4.5 Hz), 109.6 (d, J = 21.2 Hz), 66.5 (d, J =

3.3 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −111.2. MS (ESI-TOF)

calcd for C₁₃H₇BrCl₃FO [M − OH]⁺1:1 364.8697 and 366.8677,

found 1:1 364.8696 and 366.8667.

trichlorophenyl)methyl)-[1,1′-biphenyl]-4-yl)methyl)phosphonate

(( )-42). Reaction of benzhydryl alcohol ( )-38 (144 mg, 0.334

mmol, 1.0 equiv), boronic ester 25 (146 mg, 0.374 mmol, 1.0 equiv),

K₂CO₃(464 mg, 3.36 mmol, 10.1 equiv), and Pd(PPh₃)₄(26.7 mg,

0.0231 mmol, 0.07 equiv) in toluene (9.4 mL), EtOH (3.2 mL), and

H₂O (1.9 mL) according to the procedure for the preparation of

( )-39 above aﬀorded ( )-42 as an orange oil (142 mg, 75%). R_f=

0.19 (silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm) ν_max3374 (br), 1455,

1259, 1128, 1045, 1021 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s,

1H), 7.69−7.66 (m, 2H), 7.63−7.59 (m, 2H), 7.46 (s, 1H), 7.40 (td, J

= 7.4, 2.1 Hz, 1H), 7.28−7.24 (m, 1H), 7.21 (t, J = 7.6 Hz, 1H), 6.43

(d, J = 3.1 Hz, 1H), 4.27−4.12 (m, 4H), 3.09 (d, J = 3.8 Hz, 1H), 1.33

(t, J = 7.1 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −108.4 (d, J =

116.3 Hz), −123.0. ³¹P NMR (162 MHz, CDCl₃) δ 6.13 (t, J = 116.3

Hz). MS (ESI-TOF) calcd for C₂₄H₂₁Cl₃F₃O₄P [M + H⁺] 1:1

567.0268 and 569.0238, found 1:1 567.0265 and 569.0236.

1-(3-Bromo-2-ﬂuorobenzyl)-2,4,5-trichlorobenzene (46). Benz-

hydryl alcohol ( )-45 (789 mg, 2.05 mmol, 1.0 equiv) was dissolved

in anhydrous CH₂Cl₂(76 mL, 0.025 M) in a two-necked round-

bottom ﬂask equipped with a reﬂux condenser, and the resultant

mixture was cooled to 0 °C in an ice bath. By syringe, under an

atmosphere of N₂, triethylsilane (3.3 mL, 2.40 g, 20.7 mmol, 10.1

equiv) was added to the reaction mixture, followed by boron

triﬂuoride diethyl etherate (2.5 mL, 2.88 g, 20.2 mmol, 9.9 equiv).

The ﬂask was placed in an oil bath, and the solution was reﬂuxed at 40

°C for 18 h. The reaction mixture was then cooled to 23 °C, and the

reaction was quenched by the addition of satd aq NaHCO₃(25 mL).

The organic layer was dried over Na₂SO₄, ﬁltered, and concentrated.

The crude product was puriﬁed by ﬂash chromatography (silica gel,

19:1 to 4:1 hexanes/EtOAc) to aﬀord 46 as a colorless oil (620 mg,

82%). R_f= 0.73 (silica gel, 4:1 hexanes/EtOAc). IR (ﬁlm) ν_max1452,

2-Bromo-4,5-dichloroaniline (43). 1-Bromo-4,5-dichloro-2-nitro-

benzene (618 mg, 2.28 mmol, 1.0 equiv) was dissolved in a 1:1

mixture of EtOH and glacial AcOH (12.6 mL, 0.181 M) in a ﬂame-

dried round-bottom ﬂask equipped with a reﬂux condenser. Iron

powder (533 mg, 9.54 mmol, 4.2 equiv) was added to the solution,

and the reaction mixture was heated to 105 °C with stirring under an

atmosphere of N₂for 90 min. At this point, the mixture was cooled to

23 °C, diluted with H₂O (30 mL), and neutralized with solid Na₂CO₃.

The aqueous solution was extracted with CH₂Cl₂(3 × 20 mL), and

then the pooled organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated. The crude product was puriﬁed by ﬂash chromatography

(silica gel, 19:1 to 3:1 hexanes/EtOAc) to aﬀord 43 as a white solid

(485 mg, 88%). R_f= 0.50 (silica gel, 4:1 hexanes/EtOAc). IR (ﬁlm)

ν_max3439, 3348, 1610, 1472, 1376, 1271, 1243, 1138, 1047, 875, 850

cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 7.46 (s, 1H), 6.82 (s, 1H), 4.13

(br s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 143.8, 133.2, 132.1, 121.3,

116.2, 107.3. MS (ESI-TOF) calcd for C₆H₄BrCl₂N [M + H⁺] 1:1

239.8977 and 241.8956, found 1:1 239.8974 and 241.8952.

1-Bromo-4,5-dichloro-2-iodobenzene (44). Aniline 43 (485 mg,

2.01 mmol, 1.0 equiv) was dissolved in conc HCl (37% aq, 1.0 mL)

and H₂O (2.6 mL), and the resultant mixture was cooled to 0 °C in an

ice bath. Slowly, a solution of sodium nitrite (151 mg, 2.19 mmol, 1.1

equiv) in H₂O (2.2 mL) was poured into the aniline solution. This

reaction mixture was stirred under an atmosphere of N₂at 0 °C for 1

h. Slowly, a solution of potassium iodide (3.38 g, 20.4 mmol, 10.1

equiv) in ice-cold H₂O (4.4 mL) was added to the reaction mixture,

immediately followed by CH₂Cl₂(3.8 mL). The resultant biphasic

mixture was vigorously stirred under N₂at 23 °C for 18 h. The

reaction was then quenched by the addition of solid Na₂SO₃(82.4 mg,

0.654 mmol, 0.32 equiv), and resulting mixture was stirred for 15 min.

The aqueous layer was extracted with CH₂Cl₂(2 × 10 mL), and then

the pooled organic layers were washed with brine (10 mL). The

organic phase was dried over Na₂SO₄, ﬁltered, and concentrated. The

crude product was puriﬁed through a plug of silica gel, eluting with

hexanes, to aﬀord 44 as a white solid (569 mg, 80%). Spectroscopic

results agree with previously reported data.⁶³

1

1355, 1225, 1070, 786 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.49 (s,

1H), 7.46 (ddd, J = 8.1, 6.5, 1.8 Hz, 1H), 7.24 (s, 1H), 7.08−7.03 (m,

1H), 6.96 (t, J = 7.8 Hz, 1H), 4.07 (s, 2H). ¹³C NMR (100 MHz,

CDCl₃) δ 157.4 (d, J = 247.2 Hz), 136.9 (d, J = 1.0 Hz), 133.0, 132.4,

132.0 (d, J = 1.6 Hz), 131.7, 131.4, 131.0, 130.0 (d, J = 3.7 Hz), 127.0

(d, J = 16.5 Hz), 125.3 (d, J = 4.5 Hz), 109.6 (d, J = 21.4 Hz), 32.3 (d,

J = 3.0 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ − 110.3.

2-(2-Fluoro-3-(2,4,5-trichlorobenzyl)phenyl)-4,4,5,5-tetramethyl-

1,3,2-dioxaborolane (47). Reaction of aryl bromide 46 (400 mg, 1.09

mmol, 1.0 equiv), bis(pinacoloto)diboron (331 mg, 1.30 mmol, 1.2

equiv), KOAc (322 mg, 3.28 mmol, 3.0 equiv), and Pd(dppf)Cl₂·

CH₂Cl₂(89.6 mg, 0.110 mmol, 0.10 equiv) in 1,4-dioxane (6.3 mL,

0.172 M) according to the procedure for the preparation of ( )-30

above aﬀorded 47 as a colorless oil (387 mg, 86%). R_f= 0.63 (silica

gel, 4:1 hexanes/EtOAc). IR (ﬁlm) ν_max1450, 1363, 1280, 1125, 1071,

1

850 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.65 (ddd, J = 7.4, 5.6, 1.9

Hz, 1H), 7.47 (s, 1H), 7.24−7.18 (m, 2H), 7.09 (t, J = 7.4 Hz, 1H),

4.04 (s, 2H), 1.36 (s, 12H). ¹⁹F NMR (376 MHz, CDCl₃) δ −106.9.

( )-Diethyl ((4-Bromophenyl)ﬂuoromethyl)phosphonate

(( )-49). Following a reported procedure,⁵⁶nBuLi (2.4 mL of a 1.92

M solution in hexanes, 4.61 mmol, 3.0 equiv) was diluted in anhydrous

THF (13 mL), and the resultant solution was placed under an

atmosphere of N₂and cooled to −78 °C in a dry ice/acetone bath.

Dropwise by syringe, a solution of diethyl (4-bromobenzyl)-

phosphonate (0.35 mL, 480 mg, 1.56 mmol, 1.0 equiv) and

bis(trimethylsilyl)amine (1.1 mL, 851 mg, 5.28 mmol, 3.4 equiv) in

anhydrous THF (13 mL) was added to the reaction mixture. The

mixture was stirred under N₂at −78 °C for 10 min, then allowed to

warm to 23 °C for 1 h. Rapidly, chlorotrimethylsilane (0.22 mL, 188

N

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Journal of Medicinal Chemistry

Article

mg, 1.73 mmol, 1.1 equiv) in anhydrous THF (6.5 mL) was added to

the reaction mixture by syringe, and it was stirred under N₂at 23 °C

for 15 min, at which point the mixture was cooled to −90 °C in a

liquid nitrogen/hexanes bath. Once the reaction mixture had cooled,

NFSI (640 mg, 2.03 mmol, 1.3 equiv) in anhydrous THF (13 mL) was

added slowly by syringe under N₂. The reaction mixture was stirred at

−90 °C for 15 min then allowed to warm to 0 °C over the course of 1

h. The reaction was quenched at 0 °C by addition of 1 M aq LiOH,

and the resulting mixture was stirred for an additional 15 min. The

organic layer was washed with more 1 M aq LiOH (2 × 10 mL) then

poured into a mixture of 3 M aq HCl (15 mL), CH₂Cl₂(15 mL), and

ice (10 g). The resultant aqueous layer was extracted with CH₂Cl₂(2

× 15 mL), then the pooled organic layers were combined, dried over

Na₂SO₄, ﬁltered, and concentrated. The crude product was puriﬁed by

ﬂash chromatography (silica gel, 19:1 to 1:1 hexanes/EtOAc) to aﬀord

( )-49 as a colorless oil (465 mg, 92%). R_f= 0.18 (silica gel, 2:1

hexanes/EtOAc). IR (ﬁlm) ν_max1487, 1397, 1257, 1164, 1017, 971,

1.25 mmol, 1.0 equiv) and methyl benzoate 52 (0.16 mL, 174 mg, 1.28

mmol, 1.0 equiv) were dissolved in anhydrous THF (4.0 mL), and the

resultant solution was cooled to 0 °C in an ice bath. Dropwise by

syringe, under an atmosphere of N₂, the LDA solution was added to

the stirred reaction mixture, and stirring was continued for 15 min at 0

°C. The reaction was quenched with 5 M aq HCl to a pH of 4, then

diluted with EtOAc (10 mL). The aqueous layer was extracted with

EtOAc (2 × 10 mL), then the pooled organic layers were washed with

H₂O (5 mL) and brine (5 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated. The crude product was puriﬁed by

ﬂash chromatography (silica gel, 19:1 to 1:1 hexanes/EtOAc) to aﬀord

( )-54 as a colorless oil (455 mg, 89%). R_f= 0.08 (silica gel, 2:1

hexanes/EtOAc). IR (ﬁlm) ν_max1681, 1595, 1486, 1247, 1019, 963,

773 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 7.94−7.91 (m, 2H), 7.55−

7.50 (m, 1H), 7.47−7.38 (m, 6H), 5.30 (d, J = 22.5 Hz, 1H), 4.15−

3.98 (m, 4H), 1.22−1.16 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ

193.2 (d, J = 5.2 Hz), 136.4 (d, J = 5.0 Hz), 133.6, 131.9 (d, J = 2.7

Hz), 131.4 (d, J = 6.3 Hz), 130.7 (d, J = 9.1 Hz), 128.9, 128.7, 122.2

(d, J = 4.1 Hz), 63.3 (d, J = 6.8 Hz), 63.0 (d, J = 7.0 Hz), 53.6 (d, J =

137.1 Hz), 16.3 (d, J = 4.7 Hz), 16.2 (d, J = 4.7 Hz). ³¹P NMR (162

MHz, CDCl₃) δ 18.7. MS (ESI-TOF) calcd for C₁₈H₂₀BrO₄P [M +

H⁺] 1:1 411.0355 and 413.0335, found 1:1 411.0355 and 413.0337.

( )-Diethyl (1-(4-Bromophenyl)-2-cyclohexyl-2-oxoethyl)-

phosphonate (( )-55). Reaction of iPr₂NH (0.35 mL, 253 mg, 2.50

mmol, 2.0 equiv), nBuLi (1.3 mL of a 1.88 M solution in hexanes, 2.44

mmol, 2.0 equiv), diethyl (4-bromobenzyl)phosphonate (0.28 mL,

264 mg, 1.25 mmol, 1.0 equiv), and methyl cyclohexanecarboxylate 53

(0.18 mL, 179 mg, 1.26 mmol, 1.0 equiv) according to the procedure

for the preparation of ( )-54 above aﬀorded ( )-55 as a colorless oil

(184 mg, 35%). R_f= 0.15 (silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm)

ν_max2981, 2930, 2854, 1710, 1487, 1448, 1250, 1163, 1049, 1022, 962,

1

833 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.52 (d, J = 8.5 Hz, 2H),

7.34 (dd, J = 8.6, 1.9 Hz, 2H), 5.63 (dd, J = 44.7, 8.0 Hz, 1H), 4.20−

4.00 (m, 4H), 1.30−1.23 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ

132.1 (dd, J = 19.0, 1.6 Hz), 131.8 (d, J = 2.3 Hz), 128.4 (dd, J = 6.8,

5.6 Hz), 127.3 (dd, J = 10.7, 4.1 Hz), 88.9 (dd, J = 10.7, 4.1 Hz), 64.0

(d, J = 6.9 Hz), 63.6 (d, J = 6.9 Hz), 16.5 (d, J = 2.2 Hz), 16.4 (d, J =

2.3 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −201.5 (d, J = 83.7 Hz). ³¹

P

NMR (162 MHz, CDCl₃) δ 14.2 (d, J = 83.7 Hz). MS (ESI-TOF)

calcd for C₁₁H₁₅BrFO₃P [M + H⁺] 1:1 324.9999 and 326.9979, found

1:1 324.9999 and 326.9978.

( )-Diethyl (Fluoro(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)phenyl)methyl)phosphonate (( )-50). Reaction of phosphonate

( )-49 (368 mg, 1.13 mmol, 1.0 equiv), bis(pinacoloto)diboron (345

mg, 1.36 mmol, 1.2 equiv), KOAc (331 mg, 3.37 mmol, 3.0 equiv),

and Pd(dppf)Cl₂·CH₂Cl₂(96.2 mg, 0.118 mmol, 0.10 equiv) in 1,4-

dioxane (5.0 mL, 0.226 M) according to the procedure for the

preparation of ( )-30 above aﬀorded ( )-50 as a colorless oil (400

mg, 95%). R_f= 0.15 (silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm) ν_max

1

860, 760, 547 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.46 (d, J = 8.3

Hz, 2H), 7.33 (dd, J = 8.6, 2.4 Hz, 2H), 4.58 (d, J = 23.1 Hz, 1H),

4.12−3.97 (m, 4H), 2.58 (tt, J = 11.1, 3.4 Hz, 1H), 1.85 (d, J = 12.9

Hz, 1H), 1.80−1.69 (m, 3H), 1.64 (d, J = 10.9 Hz, 1H), 1.43−1.32

(m, 1H), 1.28−1.14 (m, 10H). ¹³C NMR (100 MHz, CDCl₃) δ 205.9

(d, J = 5.1 Hz), 131.9 (d, J = 2.6 Hz), 131.4 (d, J = 6.6 Hz), 130.4 (d, J

= 9.0 Hz), 122.2 (d, J = 3.9 Hz), 63.4 (d, J = 6.8 Hz), 63.1 (d, J = 7.1

Hz), 56.5 (d, J = 132.9 Hz), 52.1 (d, J = 5.4 Hz), 28.7, 28.2, 25.8, 25.7,

25.4, 16.5 (d, J = 3.0 Hz), 16.4 (d, J = 3.0 Hz). ³¹P NMR (162 MHz,

CDCl₃) δ 18.4. MS (ESI-TOF) calcd for C₁₈H₂₆BrO₄P [M + H⁺] 1:1

417.0825 and 419.0804, found 1:1 417.0824 and 419.0802.

1

1613, 1448, 1359, 1261, 1144, 1022, 963, 855 cm⁻¹. H NMR (400

MHz, CDCl₃) δ 7.82 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 6.3 Hz, 2H),

5.69 (dd, J = 44.9, 8.2 Hz, 1H), 4.21−3.96 (m, 4H), 1.33 (s, 12H),

1.27 (t, J = 7.0 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 135.9 (dd, J

= 18.3, 1.7 Hz), 134.9 (d, J = 2.3 Hz), 125.9 (dd, J = 6.9, 5.4 Hz),

124.8 (dd, J = 10.5, 4.0 Hz), 89.5 (dd, J = 184.5, 168.6 Hz), 84.1, 63.9

(d, J = 7.0 Hz), 63.5 (d, J = 6.8 Hz), 25.0, 16.52, 16.46. ¹⁹F NMR (376

MHz, CDCl₃) δ −202.5 (d, J = 83.4 Hz). ³¹P NMR (162 MHz,

CDCl₃) δ 14.7 (d, J = 83.2 Hz). MS (ESI-TOF) calcd for

C₁₇H₂₇BFO₅P [M + H⁺] 373.1746, found 373.1746.

( )-Diethyl (2-Oxo-2-phenyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-diox-

aborolan-2-yl)phenyl)ethyl)phosphonate (( )-56). Reaction of

phosphonate ( )-54 (426 mg, 1.04 mmol, 1.0 equiv), bis(pinacoloto)-

diboron (319 mg, 1.26 mmol, 1.2 equiv), KOAc (306 mg, 3.12 mmol,

3.0 equiv), and Pd(dppf)Cl₂·CH₂Cl₂(90.2 mg, 0.111 mmol, 0.11

equiv) in 1,4-dioxane (5.9 mL, 0.178 M) according to the procedure

for the preparation of ( )-30 above aﬀorded ( )-56 as a white solid

(421 mg, 88%). R_f= 0.08 (silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm)

Diethyl (Fluoro(2′-ﬂuoro-3′-(hydroxy(2,4,5-trichlorophenyl)-

methyl)-[1,1′-biphenyl]-4-yl)methyl)phosphonate (51). Reaction of

benzhydryl alcohol ( )-38 (134 mg, 0.311 mmol, 1.0 equiv), boronic

ester ( )-50 (137 mg, 0.369 mmol, 1.2 equiv), K₂CO₃(427 mg, 3.09

mmol, 9.9 equiv), and Pd(PPh₃)₄(22.2 mg, 0.0192 mmol, 0.06 equiv)

in toluene (8.7 mL), EtOH (3.0 mL), and H₂O (1.8 mL) according to

the procedure for the preparation of ( )-39 above aﬀorded 51 as a

yellow foam under vacuum (145 mg, 85%). R_f= 0.12 (silica gel, 2:1

hexanes/EtOAc). IR (ﬁlm) ν_max3337 (br), 1454, 1239, 1026, 803

1

ν_max1683, 1608, 1360, 1250, 1144, 1024, 963, 857, 774 cm⁻¹. H

NMR (400 MHz, CDCl₃) δ 7.93−7.90 (m, 2H), 7.77 (d, J = 7.7 Hz,

2H), 7.53−7.49 (m, 3H), 7.41−7.36 (m, 2H), 5.33 (d, J = 22.1 Hz,

1H), 4.18−3.99 (m, 4H), 1.31 (s, 12H), 1.26−1.19 (m, 6H). ¹³C

NMR (100 MHz, CDCl₃) δ 193.6 (d, J = 5.3 Hz), 136.6 (d, J = 5.8

Hz), 135.3 (d, J = 2.8 Hz), 134.7 (d, J = 9.2 Hz), 133.5, 129.2, 129.11,

129.05, 128.7, 84.0, 63.4 (d, J = 6.6 Hz), 63.1 (d, J = 7.1 Hz), 55.0 (d, J

= 138.2 Hz), 25.0, 16.5 (d, J = 4.1 Hz), 16.4 (d, J = 4.0 Hz). ³¹P NMR

(162 MHz, CDCl₃) δ 19.3. MS (ESI-TOF) calcd for C₂₄H₃₂BO₆P [M

+ H⁺] 459.2102, found 459.2100.

1

cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.79 (s, 1H), 7.58−7.52 (m,

4H), 7.46 (s, 1H), 7.39 (td, J = 7.3, 2.3 Hz, 1H), 7.24−7.17 (m, 2H),

6.43 (d, J = 3.0 Hz, 1H), 5.72 (dd, J = 44.7, 7.9 Hz, 1H), 4.22−4.03

(m, 4H), 3.09 (br s, 1H), 1.32−1.27 (m, 6H). ¹⁹F NMR (376 MHz,

CDCl₃) δ −123.1, −201.1 (d, J = 84.5 Hz). ³¹P NMR (162 MHz,

CDCl₃) δ 14.8 (d, J = 84.5 Hz). MS (ESI-TOF) calcd for

C₂₄H₂₂Cl₃F₂O₄P [M + H⁺] 1:1 549.0362 and 551.0333, found 1:1

549.0362 and 551.0335.

( )-Diethyl (2-Cyclohexyl-2-oxo-1-(4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)phenyl)ethyl)phosphonate (( )-57). Reaction of

phosphonate ( )-55 (184 mg, 0.440 mmol, 1.0 equiv), bis-

(pinacoloto)diboron (136 mg, 0.537 mmol, 1.2 equiv), KOAc (130

mg, 1.33 mmol, 3.0 equiv), and Pd(dppf)Cl₂·CH₂Cl₂(41.2 mg, 0.0505

mmol, 0.11 equiv) in 1,4-dioxane (2.5 mL, 0.176 M) according to the

procedure for the preparation of ( )-30 above aﬀorded ( )-57 as a

colorless oil (119 mg, 58%). R_f= 0.14 (silica gel, 2:1 hexanes/EtOAc).

IR (ﬁlm) ν_max2980, 2930, 2855, 1711, 1609, 1448, 1396, 1361, 1328,

(

)-Diethyl (1-(4-Bromophenyl)-2-oxo-2-phenylethyl)-

phosphonate (( )-54). Following a reported procedure,⁵⁷iPr₂NH

(0.35 mL, 253 mg, 2.50 mmol, 2.0 equiv) was diluted in anhydrous

THF (1.0 mL), and the resultant solution was cooled to −78 °C in dry

ice/acetone. Dropwise by syringe, with stirring under an atmosphere

of N₂, nBuLi (1.4 mL of a 1.80 M solution in hexanes, 2.52 mmol, 2.0

equiv) was added to the iPr₂NH solution in order to generate LDA.

Separately, diethyl (4-bromobenzyl)phosphonate (0.28 mL, 264 mg,

1

1253, 1144, 1091, 1024, 964 cm⁻¹. H NMR (400 MHz, CDCl₃) δ

O

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Journal of Medicinal Chemistry

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7.77 (d, J = 7.4 Hz, 2H), 7.46−7.42 (m, 2H), 4.62 (d, J = 22.5 Hz,

1H), 4.11−3.95 (m, 4H), 2.63−2.51 (m, 1H), 1.89−1.82 (m, 1H),

1.81−1.56 (m, 4H), 1.36−1.32 (m, 13H), 1.26−1.18 (m, 10H). ¹³C

NMR (100 MHz, CDCl₃) δ 206.2 (d, J = 5.4 Hz), 135.1 (d, J = 2.5

Hz), 134.3 (d, J = 8.7 Hz), 131.4 (d, J = 6.6 Hz), 129.1 (d, J = 6.4 Hz),

84.0, 63.3 (d, J = 6.7 Hz), 62.9 (d, J = 7.0 Hz), 57.5 (d, J = 133.5 Hz),

51.9 (d, J = 3.9 Hz), 28.6, 28.4, 25.8, 25.7, 25.5, 25.0, 16.5 (d, J = 2.2

Hz), 16.4 (d, J = 2.3 Hz). ³¹P NMR (162 MHz, CDCl₃) δ 18.9. MS

(ESI-TOF) calcd for C₂₄H₃₈BO₆P [M + H⁺] 465.2572, found

465.2571.

Diethyl (1-(2′-Fluoro-3′-(hydroxy(2,4,5-trichlorophenyl)methyl)-

[1,1′-biphenyl]-4-yl)-2-oxo-2-phenylethyl)phosphonate (58). Reac-

tion of benzhydryl alcohol ( )-38 (333 mg, 0.773 mmol, 1.0 equiv),

boronic ester ( )-56 (421 mg, 0.918 mmol, 1.2 equiv), K₂CO₃(1.06

g, 7.65 mmol, 9.9 equiv), and Pd(PPh₃)₄(89.7 mg, 0.0776 mmol, 0.10

equiv) in toluene (50 mL), EtOH (16 mL), and H₂O (10 mL)

according to the procedure for the preparation of ( )-39 above

aﬀorded 58 as a pale-yellow foam (341 mg, 69%). R_f= 0.08 (silica gel,

2:1 hexanes/EtOAc). IR (ﬁlm) ν_max3299 (br), 1682, 1452, 1239,

1048, 968, 728 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 7.97 (dd, J = 8.4,

1.3 Hz, 2H), 7.77 (s, 1H), 7.67−7.64 (m, 1H), 7.62−7.58 (m, 1H),

7.54−7.49 (m, 3H), 7.46−7.40 (m, 3H), 7.37 (td, J = 7.5, 2.1 Hz, 1H),

7.22 (ddd, J = 8.3, 6.5, 2.0 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.41 (s,

1H), 5.38 (d, J = 22.2 Hz, 1H), 4.20−4.01 (m, 4H), 3.12 (br s, 1H),

1.24−1.19 (m, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −123.0. ³¹P

NMR (162 MHz, CDCl₃) δ 19.4. MS (ESI-TOF) calcd for

C₃₁H₂₇Cl₃FO₅P [M + H⁺] 1:1 635.0718 and 637.0689, found 1:1

635.0726 and 637.0681.

selectﬂuor (18.6 mg, 0.0524 mmol, 0.30 equiv) in CH₂Cl₂(2.5 mL,

0.070 M) in the presence of 4 Å molecular sieves according to the

procedure for the preparation of 60 above aﬀorded 61 as a colorless oil

(36 mg, 31%). R_f= 0.46 (silica gel, 1:1 hexanes/EtOAc). IR (ﬁlm) ν_max

3367 (br), 2981, 2932, 2855, 1717, 1452, 1246, 1071, 1044, 1020

1

cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1H), 7.70 (dd, J = 8.6,

2.4 Hz, 2H), 7.58−7.50 (m, 2H), 7.46 (s, 1H), 7.40 (td, J = 7.4, 2.0

Hz, 1H), 7.25−7.17 (m, 2H), 6.43 (d, J = 3.8 Hz, 1H), 4.20−4.06 (m,

4H), 3.00 (ddt, J = 11.4, 7.7, 3.7 Hz, 1H), 2.92 (d, J = 4.2 Hz, 1H),

1.91 (d, J = 13.4 Hz, 1H), 1.79 (d, J = 12.9 Hz, 1H), 1.70−1.55 (m,

3H), 1.41 (dt, J = 15.4, 10.8 Hz, 1H), 1.32−1.23 (m, 10H). ¹⁹F NMR

(376 MHz, CDCl₃) δ −123.0, −177.9 (d, J = 86.0 Hz). ³¹P NMR (162

MHz, CDCl₃) δ 11.1 (d, J = 84.0 Hz). MS (ESI-TOF) calcd for

C₃₁H₃₂Cl₃F₂O₅P [M + H⁺] 1:1 659.1094 and 661.1064, found 1:1

659.1094 and 661.1076.

( )-Diethyl (1-(4-Bromophenyl)-2-oxo-2-(piperidin-1-yl)ethyl)-

phosphonate (( )-63). iPr₂NH (0.35 mL, 253 mg, 2.50 mmol, 2.0

equiv) was diluted in anhydrous THF (1.0 mL), and the resultant

solution was cooled to −78 °C in dry ice/acetone. Dropwise by

syringe, with stirring under an atmosphere of N₂, nBuLi (1.3 mL of a

1.92 M solution in hexanes, 2.50 mmol, 2.0 equiv) was added to the

iPr₂NH solution in order to generate LDA. Separately, diethyl (4-

bromobenzyl)phosphonate (0.28 mL, 264 mg, 1.25 mmol, 1.0 equiv)

and piperidine-1-carbonyl chloride 62 (0.16 mL, 189 mg, 1.28 mmol,

1.0 equiv) were dissolved in anhydrous THF (4.0 mL), and the

resultant solution was cooled to 0 °C in an ice bath. Dropwise by

syringe, under an atmosphere of N₂, the LDA solution was added to

the stirred reaction mixture, and stirring was continued for 15 min at 0

°C. The reaction was quenched with 5 M aq HCl to a pH of 4, and

then the resulting mixture was diluted with EtOAc (10 mL). The

aqueous layer was extracted with EtOAc (2 × 10 mL), then the pooled

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated. The

crude product was puriﬁed by ﬂash chromatography (silica gel, 99:1 to

4:1 CH₂Cl₂/MeOH) to aﬀord ( )-63 as a colorless oil (512 mg,

98%). R_f= 0.08 (silica gel, 1:1 hexanes/EtOAc). IR (ﬁlm) ν_max1635,

1486, 1438, 1249, 1216, 1053, 1011, 957, 852, 780, 730, 554 cm⁻¹. ¹H

NMR (400 MHz, CDCl₃) δ 7.42 (d, J = 8.5 Hz, 2H), 7.28 (dd, J = 8.6,

2.4 Hz, 2H), 4.42 (d, J = 22.8 Hz, 1H), 4.28−4.16 (m, 2H), 4.05−3.91

(m, 2H), 3.68 (ddd, J = 13.4, 6.3, 3.4 Hz, 1H), 3.40−3.31 (m, 1H),

3.29−3.23 (m, 2H), 1.55−1.36 (m, 5H), 1.23 (t, J = 7.1 Hz, 3H), 1.17

(t, J = 7.1 Hz, 3H) 1.08−0.98 (m, 1H). ¹³C NMR (100 MHz, CDCl₃)

δ 165.4 (d, J = 2.9 Hz), 131.8 (d, J = 2.8 Hz), 131.7 (d, J = 9.5 Hz),

131.0 (d, J = 5.9 Hz), 121.8 (d, J = 4.0 Hz), 63.6 (d, J = 6.5 Hz), 62.4

(d, J = 7.0 Hz), 49.9 (d, J = 145.0 Hz), 47.8. 43.3, 26.1, 25.4, 24.3, 16.5

(d, J = 6.1 Hz), 16.3 (d, J = 6.3 Hz). ³¹P NMR (162 MHz, CDCl₃) δ

19.5. MS (ESI-TOF) calcd for C₁₇H₂₅BrNO₄P [M + H⁺] 1:1 418.0777

and 420.0757, found 1:1 418.0780 and 420.0763.

Diethyl (2-Cyclohexyl-1-(2′-ﬂuoro-3′-(hydroxy(2,4,5-

trichlorophenyl)methyl)-[1,1′-biphenyl]-4-yl)-2-oxoethyl)-

phosphonate (59). Reaction of benzhydryl alcohol ( )-38 (111 mg,

0.258 mmol, 1.0 equiv), boronic ester ( )-57 (119 mg, 0.256 mmol,

1.0 equiv), K₂CO₃(308 mg, 2.23 mmol, 8.6 equiv), and Pd(PPh₃)₄

(30.0 mg, 0.0260 mmol, 0.10 equiv) in toluene (11.1 mL), EtOH (3.6

mL), and H₂O (2.2 mL) according to the procedure for the

preparation of ( )-39 above aﬀorded 59 as a colorless oil (112 mg,

68%). R_f= 0.08 (silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm) ν_max2981,

1

2931, 2855, 1710, 1451, 1241, 1020, 965, 908, 729, 542 cm⁻¹. H

NMR (400 MHz, CDCl₃) δ 7.49 (m, 3H), 7.46−7.42 (m, 2H), 7.37 (t,

J = 7.4, 1.9 Hz, 1H), 7.31 (dd, J = 8.6, 2.4 Hz, 1H), 7.23 (td, J = 7.2,

6.6, 1.9 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 6.41 (d, J = 3.3 Hz, 1H),

4.65 (d, J = 22.8 Hz, 1H), 4.14−3.96 (m, 4H), 3.62 (s, 1H), 2.63−2.53

(m, 1H), 1.92−1.59 (m, 5H), 1.44−1.31 (m, 1H), 1.27−1.16 (m,

10H). ¹⁹F NMR (376 MHz, CDCl₃) δ −123.1. ³¹P NMR (162 MHz,

CDCl₃) δ 19.1. MS (ESI-TOF) calcd for C₃₁H₃₃Cl₃FO₅P [M + H⁺]

1:1 641.1188 and 643.1158, found 1:1 641.1189 and 643.1166.

Diethyl (1-Fluoro-1-(2′-ﬂuoro-3′-(hydroxy(2,4,5-trichlorophenyl)-

methyl)-[1,1′-biphenyl]-4-yl)-2-oxo-2-phenylethyl)phosphonate

(60). Phosphonate 58 (341 mg, 0.537 mmol, 1.0 equiv), NFSI (513

mg, 1.63 mmol, 3.0 equiv), and selectﬂuor (47.7 mg, 0.135 mmol, 0.25

equiv) were dissolved in anhydrous CH₂Cl₂(7.5 mL, 0.072 M) in the

presence of 4 Å molecular sieves. The reaction mixture was stirred

under an atmosphere of N₂at 23 °C for 1 week, then concentrated.

The resultant residue was puriﬁed by ﬂash chromatography (silica gel,

1:1 CH₂Cl₂/Et₂O then 19:1 to 1:1 hexanes/EtOAc) to aﬀord 60 as a

foamy white solid (186 mg, 53%). R_f= 0.34 (silica gel, 1:1 hexanes/

EtOAc). IR (ﬁlm) ν_max3344 (br), 2982, 1684, 1450, 1258, 1070, 1020

( )-Diethyl (1-(4-Bromophenyl)-1-ﬂuoro-2-oxo-2-(piperidin-1-yl)-

ethyl)phosphonate (( )-64). iPr₂NH (0.23 mL, 166 mg, 1.64 mmol,

1.5 equiv) was diluted in anhydrous THF (1.0 mL), and the resultant

solution was cooled to −78 °C in dry ice/acetone. Dropwise by

syringe, with stirring under an atmosphere of N₂, nBuLi (0.86 mL of a

1.88 M solution in hexanes, 1.62 mmol, 1.5 equiv) was added to the

iPr₂NH solution in order to generate LDA. Separately, phosphonate

( )-63 (452 mg, 1.08 mmol, 1.0 equiv) and NFSI (1.02 mg, 3.24

mmol, 3.0 equiv) were dissolved in anhydrous THF (6.0 mL), and the

resultant solution was cooled to 0 °C in an ice bath. Dropwise by

syringe, under an atmosphere of N₂, the LDA solution was added to

the stirred reaction mixture, which was then allowed to warm to 23 °C.

After 24 h, the reaction was quenched with satd aq NH₄Cl (10 mL),

and the resulting mixture was diluted with EtOAc (20 mL). The

aqueous layer was extracted with EtOAc (2 × 20 mL), and the pooled

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated. The

crude product was puriﬁed by ﬂash chromatography (silica gel, 99:1 to

4:1 CH₂Cl₂/MeOH) to aﬀord ( )-64 as a pale-yellow oil (154 mg,

33%). R_f= 0.12 (silica gel, 1:1 hexanes/EtOAc). IR (ﬁlm) ν_max2935,

1624, 1484, 1444, 1396, 1261, 1163, 1063, 1022, 972, 790, 747, 648,

1

cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.86 (dt, J = 8.6, 1.7 Hz, 2H),

7.77 (s, 1H), 7.72 (dd, J = 8.6, 2.4 Hz, 2H), 7.67−7.57 (m, 2H), 7.52

(ddt, J = 7.1, 5.6, 1.5 Hz, 1H), 7.45 (s, 1H), 7.38 (t, J = 7.7 Hz, 3H),

7.24 (dd, J = 6.6, 1.8 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.42 (s, 1H),

4.29−4.13 (m, 4H), 3.11 (br s, 1H), 1.33−1.23 (m, 6H). ¹⁹F NMR

(376 MHz, CDCl₃) δ −123.0, −169.8 (d, J = 85.8 Hz). ³¹P NMR (162

MHz, CDCl₃) δ 11.3 (d, J = 85.9 Hz). MS (ESI-TOF) calcd for

C₃₁H₂₆Cl₃F₂O₅P [M + H⁺] 1:1 653.0624 and 655.0595, found 1:1

653.0623 and 655.0601.

Diethyl (2-Cyclohexyl-1-ﬂuoro-1-(2′-ﬂuoro-3′-(hydroxy(2,4,5-

trichlorophenyl)methyl)-[1,1′-biphenyl]-4-yl)-2-oxoethyl)-

phosphonate (61). Reaction of phosphonate 59 (112 mg, 0.175

mmol, 1.0 equiv), NFSI (165 mg, 0.524 mmol, 3.0 equiv), and

1

579, 552 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.53 (d, J = 8.4 Hz,

2H), 7.38 (dd, J = 8.8, 2.3 Hz, 2H), 4.29−4.16 (m, 4H), 3.65−3.56

(m, 1H), 3.54−3.45 (m, 1H), 3.35−3.25 (m, 1H), 3.18−3.10 (m, 1H),

P

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Article

1.61−1.47 (m, 4H), 1.42−1.33 (m, 1H), 1.26 (dt, J = 11.2, 7.1 Hz,

6H), 1.05−0.95 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 164.5 (dd, J

= 17.3, 4.5 Hz), 132.8 (dd, J = 19.4, 2.9 Hz), 131.7 (dd, J = 2.3, 1.3

Hz), 126.8 (dd, J = 7.7, 4.0 Hz), 123.6 (dd, J = 4.0, 1.5 Hz), 64.4 (d, J

= 6.8 Hz), 64.2 (d, J = 6.8 Hz), 46.5 (d, J = 11.3 Hz), 44.4, 25.8, 25.7,

24.4, 16.6 (d, J = 3.9 Hz), 16.5 (d, J = 4.0 Hz). ¹⁹F NMR (376 MHz,

CDCl₃) δ −167.7 (d, J = 90.8 Hz). ³¹P NMR (162 MHz, CDCl₃) δ

11.2 (d, J = 90.7 Hz). MS (ESI-TOF) calcd for C₁₇H₂₄BrFNO₄P [M +

H⁺] 1:1 436.0683 and 438.0663, found 1:1 436.0683 and 438.0668.

( )-Diethyl (1-Fluoro-2-oxo-2-(piperidin-1-yl)-1-(4-(4,4,5,5-tetra-

methyl-1,3,2-dioxaborolan-2-yl)phenyl)ethyl)phosphonate

(( )-65). Reaction of phosphonate ( )-64 (54 mg, 0.123 mmol, 1.0

equiv), bis(pinacoloto)diboron (39.1 mg, 0.154 mmol, 1.3 equiv),

KOAc (37.4 mg, 0.381 mmol, 3.1 equiv), and Pd(dppf)Cl₂·CH₂Cl₂

(13.6 mg, 0.0167 mmol, 0.14 equiv) in 1,4-dioxane (0.63 mL, 0.195

M) according to the procedure for the preparation of ( )-30 above

aﬀorded ( )-65 as a pale-yellow oil (46 mg, 76%). R_f= 0.04 (silica gel,

2:1 hexanes/EtOAc). IR (ﬁlm) ν_max2979, 2936, 1645, 1445, 1398,

Diethyl ((2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-

yl)methyl)phosphonate (69). Reaction of aryl iodide 67 (816 mg,

1.97 mmol, 1.0 equiv), boronic ester 68 (835 mg, 2.36 mmol, 1.2

equiv), K₂CO₃(2.72 g, 19.7 mmol, 10.0 equiv), and Pd(PPh₃)₄(227

mg, 0.197 mmol, 0.10 equiv) in toluene (119 mL), EtOH (38 mL),

and H₂O (23 mL) according to the procedure for the preparation of

( )-39 above aﬀorded 69 as a yellow oil (897 mg, 88%). R_f= 0.04

(silica gel, 2:1 hexanes/EtOAc). IR (ﬁlm) ν_max1458, 1247, 1204, 1134,

1

1053, 1024, 961, 856, 801, 760, 592, 543 cm⁻¹. H NMR (400 MHz,

CDCl₃) δ 7.52−7.47 (m, 3H), 7.38 (dd, J = 8.3, 2.5 Hz, 2H), 7.33 (dd,

J = 7.5, 1.9 Hz, 1H), 7.26 (s, 1H), 7.14 (t, J = 4.6 Hz, 1H), 7.08 (td, J =

7.2, 1.9 Hz, 1H), 4.10 (s, 2H), 4.05 (dqd, 4H), 3.20 (d, J = 21.7 Hz,

2H), 1.27 (t, J = 7.1 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −122.1.

³¹P NMR (162 MHz, CDCl₃) δ 26.1. MS (ESI-TOF) calcd for

C₂₄H₂₃Cl₃FO₃P [M + H⁺] 1:1 515.0507 and 517.0478, found 1:1

515.0507 and 517.0482.

( )-Diethyl (1-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphen-

yl]-4-yl)-2-oxo-2-(piperidin-1-yl)ethyl)phosphonate (( )-72).

iPr₂NH (0.12 mL, 86.6 mg, 0.856 mmol, 4.1 equiv) was diluted in

anhydrous THF (0.4 mL), and the resultant solution was cooled to

−78 °C in dry ice/acetone. Dropwise by syringe, with stirring under an

atmosphere of N₂, nBuLi (0.44 mL of a 1.88 M solution in hexanes,

0.827 mmol, 4.0 equiv) was added to the iPr₂NH solution in order to

generate LDA. Separately, phosphonate 69 (108 mg, 0.209 mmol, 1.0

equiv) and piperidine-1-carbonyl chloride 62 (0.04 mL, 47 mg, 0.320

mmol, 1.5 equiv) were dissolved in anhydrous THF (0.6 mL), and the

resultant solution was cooled to 0 °C in an ice bath. Dropwise by

syringe, under an atmosphere of N₂, half of the LDA solution (0.48

mL, 0.414 mmol, 2.0 equiv) was added to the stirred reaction mixture,

and stirring was continued for 15 min at 0 °C. The reaction was

quenched with 5 M aq HCl to a pH of 4, and the resulting mixture was

then diluted with EtOAc (10 mL). The aqueous layer was extracted

with EtOAc (2 × 10 mL), then the pooled organic layers were dried

over Na₂SO₄, ﬁltered, and concentrated. The crude ( )-72 product

(147 mg) was used in the next step without further puriﬁcation.

( )-Diethyl (1-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphen-

yl]-4-yl)-2-morpholino-2-oxoethyl)phosphonate (( )-73). Mixture

of iPr₂NH (0.12 mL, 86.6 mg, 0.856 mmol, 4.0 equiv) and nBuLi

(0.44 mL of a 1.92 M solution in hexanes, 0.845 mmol, 4.0 equiv) in

THF (0.4 mL) according to the procedure for the preparation of

( )-72 above generated LDA. Half of this LDA solution (0.48 mL,

0.423 mmol, 2.0 equiv) was then reacted with phosphonate 69 (109

mg, 0.212 mmol, 1.0 equiv) and morpholine-4-carbonyl chloride 70

(0.03 mL, 38 mg, 0.257 mmol, 1.2 equiv) according to the procedure

for the preparation of ( )-72 above to aﬀord crude ( )-73 (56 mg),

which was used in the next step without further puriﬁcation.

( )-Diethyl (2-(Dimethylamino)-1-(2′-ﬂuoro-3′-(2,4,5-trichloro-

benzyl)-[1,1′-biphenyl]-4-yl)-2-oxoethyl)phosphonate (( )-74). Mix-

ture of iPr₂NH (0.16 mL, 116 mg, 1.14 mmol, 4.2 equiv) and nBuLi

(0.56 mL of a 1.92 M solution in hexanes, 1.08 mmol, 4.0 equiv) in

THF (0.4 mL) according to the procedure for the preparation of

( )-72 above generated LDA. Half of this LDA solution (0.56 mL,

0.540 mmol, 2.0 equiv) was then reacted with phosphonate 69 (139

mg, 0.269 mmol, 1.0 equiv) and dimethylcarbamic chloride 71 (0.03

mL, 35 mg, 0.326 mmol, 1.2 equiv) according to the procedure for the

preparation of ( )-72 above to aﬀord crude ( )-74 (124 mg), which

was used in the next step without further puriﬁcation.

Methyl 2-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-

yl)acetate (75a) and Ethyl 2-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-

[1,1′-biphenyl]-4-yl)acetate (75b). Aryl iodide 67 (636 mg, 1.53

mmol, 1.0 equiv), methyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)phenyl)acetate (509 mg, 1.84 mmol, 1.2 equiv), and K₂CO₃(2.11

g, 15.3 mmol, 10 equiv) were dissolved in toluene (92 mL), EtOH (29

mL), and H₂O (18 mL). With stirring, N₂was bubbled through the

biphasic mixture for 1 h. Pd(PPh₃)₄(178 mg, 0.154 mmol, 0.10 equiv)

was then added, and N₂was bubbled through the stirred solution for

an additional 20 min. The ﬂask was next equipped with a reﬂux

condenser, and the reaction mixture was stirred at 80 °C under an

atmosphere of N₂for 20 h. The solution was cooled to 23 °C and

concentrated. The crude residue was puriﬁed by ﬂash chromatography

1

1362, 1264, 1145, 1065, 1024, 972, 580 cm⁻¹. H NMR (400 MHz,

CDCl₃) δ 7.83 (d, J = 7.7 Hz, 2H), 7.49 (dd, J = 8.3, 2.2 Hz, 2H),

4.30−4.14 (m, 4H), 3.70−3.57 (m, 1H), 3.54−3.40 (m, 1H), 3.35−

3.22 (m, 1H), 3.19−3.09 (m, 1H), 1.65−1.46 (m, 6H), 1.35 (s, 12H),

1.28−1.21 (m, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −168.4 (d, J =

91.9 Hz). ³¹P NMR (162 MHz, CDCl₃) δ 11.5 (d, J = 91.9 Hz).

Diethyl (1-Fluoro-1-(2′-ﬂuoro-3′-(hydroxy(2,4,5-trichlorophenyl)-

methyl)-[1,1′-biphenyl]-4-yl)-2-oxo-2-(piperidin-1-yl)ethyl)-

phosphonate (66). Reaction of benzhydryl alcohol ( )-38 (39.7 mg,

0.0920 mmol, 1.0 equiv), boronic ester ( )-65 (45.4 mg, 0.0939

mmol, 1.0 equiv), K₂CO₃(109 mg, 0.788 mmol, 8.6 equiv), and

Pd(PPh₃)₄(10.1 mg, 0.00874 mmol, 0.09 equiv) in toluene (5.3 mL),

EtOH (1.7 mL), and H₂O (1.0 mL) according to the procedure for the

preparation of ( )-39 above aﬀorded 66 as a yellow oil (55.7 mg,

1

92%). Even after chromatography, 66 provided a complex H NMR

spectrum, precluding rigorous characterization. The material was

identiﬁed by LCMS and brought forward in impure form to the

subsequent step. MS (ESI-TOF) calcd for C₃₀H₃₁Cl₃F₂NO₅P [M +

H⁺] 1:1 660.1046 and 662.1017, found 1:1 660.1047 and 662.1026.

1,2,4-Trichloro-5-(2-ﬂuoro-3-iodobenzyl)benzene (67). Benzhydr-

yl alcohol ( )-38 (1.97 g, 4.56 mmol, 1.0 equiv) was dissolved in 1:1

CH₂Cl₂/TFA (8.4 mL, 0.215 M) in a ﬂame-dried round-bottom ﬂask

equipped with a reﬂux condenser. To this reaction mixture, with

stirring under N₂, was added triethylsilane (2.2 mL, 1.60 mg, 13.8

mmol, 3.0 equiv) by syringe. The reaction mixture was then heated to

40 °C and stirred under N₂for 16 h. At this point, the solution was

concentrated and the crude product was puriﬁed by ﬂash

chromatography (silica gel, 19:1 to 4:1 hexanes/EtOAc) to aﬀord

67 as a colorless oil (1.14 g, 60%). R_f= 0.76 (silica gel, 4:1 hexanes/

EtOAc). IR (ﬁlm) ν_max1445, 1354, 1222, 1134, 1068, 928, 887, 867,

1

840, 805, 784, 753, 710, 677 cm⁻¹. H NMR (400 MHz, CDCl₃) δ

7.65 (ddd, J = 7.8, 5.9, 1.7 Hz, 1H), 7.49 (s, 1H), 7.23 (s, 1H), 7.07

(td, J = 7.2, 1.6 Hz, 1H), 6.84 (t, J = 7.7 Hz, 1H), 4.06 (s, 2H). ¹³C

NMR (100 MHz, CDCl₃) δ 159.9 (d, J = 245.3 Hz), 138.3 (d, J = 1.6

Hz), 136.9, 133.0, 132.0 (d, J = 1.5 Hz), 131.6, 131.4, 131.1 (d, J = 3.8

Hz), 130.9, 126.2 (d, J = 17.9 Hz), 126.0 (d, J = 4.2 Hz), 81.8 (d, J =

26.1 Hz), 32.5 (d, J = 2.7 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ −96.3.

Diethyl (4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-

phosphonate (68). Reaction of diethyl (4-bromobenzyl)phosphonate

(0.70 mL, 959 mg, 3.12 mmol, 1.0 equiv), bis(pinacoloto)diboron

(957 mg, 3.77 mmol, 1.2 equiv), KOAc (921 mg, 9.39 mmol, 3.0

equiv), and Pd(dppf)Cl₂·CH₂Cl₂(252 mg, 0.309 mmol, 0.10 equiv) in

1,4-dioxane (17.7 mL, 0.170 M) according to the procedure for the

preparation of ( )-30 above aﬀorded 68 as a yellow oil (1.05 g, 95%).

R_f= 0.18 (silica gel, 1:1 hexanes/EtOAc). IR (ﬁlm) ν_max2979, 1612,

1396, 1359, 1324, 1250, 1144, 1089, 1053, 1024, 960, 857, 659, 547

1

cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 7.0 Hz, 2H), 7.28

(dd, J = 8.0, 2.5 Hz, 2H), 4.03−3.93 (m, 4H), 3.15 (d, J = 21.9 Hz,

2H), 1.32 (s, 12H), 1.25−1.19 (m, 6H). ¹³C NMR (100 MHz, CDCl₃)

δ 135.1, 135.0, 129.3, 129.2, 83.9, 62.3 (d, J = 6.8 Hz), 34.2 (d, J =

137.4 Hz), 25.0, 16.5 (d, J = 6.0 Hz). ³¹P NMR (162 MHz, CDCl₃) δ

25.9.

Q

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(silica gel, 19:1 to 1:1 hexanes/EtOAc) to aﬀord an inconsequential,

separable mixture of 75a (115 mg, 17%) and 75b (177 mg, 26%) as

Diethyl (1-(3′-(Bromo(2,4,5-trichlorophenyl)methyl)-2′-ﬂuoro-

[1,1′-biphenyl]-4-yl)-2-(methylamino)-2-oxoethyl)phosphonate

(79). Amide 78 (32 mg, 0.0625 mmol, 1.0 equiv) was dissolved in

triethyl phosphite (0.04 mL, 38.8 mg, 0.233 mmol, 3.7 equiv) in a

sealed tube. The resultant solution was heated to 120 °C for 2 h then

cooled to 23 °C. The crude product was loaded directly onto a ﬂash

chromatography column (silica gel, 99:1 to 9:1 CH₂Cl₂/MeOH) to

elute 79 as a yellow oil (31 mg, 85%). Even after chromatography, 79

provided a complex ¹H NMR spectrum, precluding rigorous

characterization. The material was brought forward in impure form

to the subsequent step.

1

yellow oils. 75a: H NMR (500 MHz, CDCl₃) δ 7.52−7.49 (m, 3H),

7.38−7.31 (m, 3H), 7.26 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.09 (ddd, J

= 8.5, 7.0, 1.8 Hz, 1H), 4.11 (s, 2H), 3.72 (s, 3H), 3.68 (s, 2H). 75b:

¹H NMR (500 MHz, CDCl₃) δ 7.53−7.49 (m, 3H), 7.39−7.32 (m,

3H), 7.27 (s, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.13−7.06 (m, 1H), 4.18

(q, J = 7.1 Hz, 2H), 4.11 (s, 2H), 3.66 (s, 2H), 1.28 (t, J = 7.1 Hz,

3H).

2-(2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)acetic

Acid (76). Esters 75a (115 mg, 0.262 mmol) and 75b (177 mg, 0.391

mmol) were dissolved in 3:1 MeOH/EtOH (8.8 mL, 0.071 M) in a

ﬂame-dried round-bottom ﬂask outﬁtted with a reﬂux condenser. To

this mixture, with stirring under an atmosphere of N₂, was added 5 M

aq NaOH (0.39 mL, 1.95 mmol, 3.0 equiv). The reaction mixture was

heated at reﬂux (70 °C) for 3 h, then cooled to room temperature.

The solution was concentrated, and the residue was redissolved in

H₂O (10 mL) and EtOAc (10 mL). The aqueous layer was washed

with EtOAc (5 mL) then acidiﬁed to pH 2 by addition of 2 M aq HCl.

The acidifed aqueous solution was extracted with ethyl acetate (3 × 10

mL), and these last three organic extractions were combined, dried

over Na₂SO₄, ﬁltered, and concentrated to aﬀord spectroscopically

pure 76 (276 mg, > 99%) as a pale-yellow oil, which was used in the

subsequent step without further puriﬁcation. R_f= 0.11 (silica gel, 4:1

hexanes/EtOAc). ¹H NMR (400 MHz, CDCl₃) δ 7.55−7.49 (m, 3H),

7.40−7.31 (m, 3H), 7.27 (s, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.14−7.05

(m, 1H), 4.11 (s, 2H), 3.71 (s, 2H). ¹⁹F NMR (376 MHz, CDCl₃) δ

−122.1.

4-((4-Bromobenzyl)sulfonyl)morpholine (80). Na₂CO₃(395 mg,

3.73 mmol, 2.0 equiv) was suspended in MeCN (8.4 mL, 0.213 M),

then morpholine (0.32 mL, 319 mg, 3.66 mmol, 2.0 equiv) was added

to the reaction mixture. With stirring, solid (4-bromophenyl)-

methanesulfonyl chloride (501 mg, 1.86 mmol, 1.0 equiv) was

added, and the ﬂask was sealed with a septum under an atmosphere of

N₂. After stirring for 2 h, the reaction mixture was diluted with H₂O

(15 mL), and the aqueous solution was extracted with EtOAc (2 × 20

mL). The pooled organic phases were dried over Na₂SO₄, ﬁltered, and

concentrated to aﬀord 80 as a white solid, which required no further

puriﬁcation (585 mg, 98%). R_f= 0.10 (silica gel, 4:1 hexanes/EtOAc).

IR (ﬁlm) ν_max2660, 1488, 1454, 1342, 1330, 1260, 1155, 1112, 1071,

1

1014, 951, 843, 762, 729 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.53

(d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 4.16 (s, 2H), 3.67−3.62

(m, 4H), 3.18−3.10 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 132.4,

132.2, 127.7, 123.4, 66.8, 55.9, 46.3.

4-((4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-

sulfonyl)morpholine (81). Reaction of sulfonamide 80 (579 mg, 1.81

mmol, 1.0 equiv), bis(pinacoloto)diboron (557 mg, 2.19 mmol, 1.2

equiv), KOAc (534 mg, 5.44 mmol, 3.0 equiv), and Pd(dppf)Cl₂·

CH₂Cl₂(148 mg, 0.181 mmol, 0.10 equiv) in 1,4-dioxane (12.4 mL,

0.146 M) according to the procedure for the preparation of ( )-30

above aﬀorded 81 as a white solid (586 mg, 88%). R_f= 0.23 (silica gel,

2:1 hexanes/EtOAc). IR (ﬁlm) ν_max2978, 2916, 2859, 1612, 1453,

1402, 1362, 1342, 1323, 1296, 1259, 1150, 1111, 1092, 1072, 951, 861,

2-Bromo-2-(3′-(bromo(2,4,5-trichlorophenyl)methyl)-2′-ﬂuoro-

[1,1′-biphenyl]-4-yl)acetic Acid (77). Carboxylic acid 76 (276 mg,

0.653 mmol, 1.0 equiv) was dissolved in chlorobenzene (1.55 mL,

0.400 M) in a two-neck ﬂame-dried round-bottom ﬂask equipped with

a reﬂux condenser. This solution was sealed with septa under an

atmosphere of N₂. To the reaction mixture was added phosphorus

trichloride (0.02 mL, 32 mg, 0.229 mmol, 0.35 equiv) by syringe, then

it was heated to 105 °C. Once this temperature had been achieved,

bromine (0.06 mL, 187 mg, 1.17 mmol, 1.8 equiv) was added with

stirring, under N₂, by syringe. After 2 h at 105 °C, additional

phosphorus trichloride (0.02 mL, 32 mg, 0.229 mmol, 0.35 equiv) and

bromine (0.06 mL, 187 mg, 1.17 mmol, 1.8 equiv) were consecutively

added by syringe. The reaction mixture was stirred under N₂at 105 °C

for an additional 2 h before it was cooled to 23 °C. H₂O (0.5 mL) and

satd aq NaHSO₃(0.5 mL) were added, and the aqueous layer was

extracted with CH₂Cl₂(3 × 5 mL). The pooled organic layers were

dried over Na₂SO₄, ﬁltered, and concentrated. The crude product was

puriﬁed by ﬂash chromatography (silica gel, 19:1 to 1:1 hexanes/

EtOAc) to aﬀord 77 as a yellow oil (309 mg, 94%). R_f= 0.12 (silica

1

765, 659 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.81 (d, J = 8.0 Hz,

2H), 7.41 (d, J = 8.0 Hz, 2H), 4.24 (s, 2H), 3.61−3.56 (m, 4H), 3.10−

3.05 (m, 4H), 1.35 (s, 12H). ¹³C NMR (100 MHz, CDCl₃) δ 135.3,

132.4, 131.5, 130.1, 84.2, 66.8, 57.1, 46.3, 25.0. MS (ESI-TOF) calcd

for C₁₇H₂₆BNO₅S [M + Na⁺] 390.1517, found 390.1518.

4-(((2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-yl)-

methyl)sulfonyl)morpholine (82). Reaction of aryl iodide 67 (465 mg,

1.12 mmol, 1.0 equiv), boronic ester 81 (488 mg, 1.33 mmol, 1.2

equiv), K₂CO₃(1.55 g, 11.2 mmol, 10.0 equiv), and Pd(PPh₃)₄(131

mg, 0.114 mmol, 0.10 equiv) in toluene (50 mL), EtOH (17 mL), and

H₂O (9 mL) according to the procedure for the preparation of ( )-39

above aﬀorded 82 as a white solid (474 mg, 80%). R_f= 0.20 (silica gel,

2:1 hexanes/EtOAc). IR (ﬁlm) ν_max2858, 1456, 1410, 1343, 1317,

1258, 1153, 1112, 1070, 950, 735 cm⁻¹. ¹H NMR (400 MHz, CDCl₃)

δ 7.58 (dd, J = 8.3, 1.7 Hz, 2H), 7.52−7.47 (m, 3H), 7.35 (td, J = 7.4,

2.0 Hz, 1H), 7.27 (s, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.12 (td, J = 7.7,

7.2, 1.9 Hz, 1H), 4.27 (s, 2H), 4.11 (s, 2H), 3.69−3.62 (m, 4H), 3.20−

3.14 (m, 4H). ¹⁹F NMR (376 MHz, CDCl₃) δ −121.9.

( )-Diethyl ((2′-Fluoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-

4-yl)(morpholinosulfonyl)methyl)phosphonate (( )-83). Following

a reported procedure,⁵⁸iPr₂NH (0.09 mL, 65.0 mg, 0.642 mmol, 1.1

equiv) was diluted in anhydrous THF (1.1 mL), and the resultant

solution was cooled to 0 °C in an ice bath. Dropwise by syringe, with

stirring under an atmosphere of N₂, nBuLi (0.36 mL of a 1.88 M

solution in hexanes, 0.677 mmol, 1.2 equiv) was added to the iPr₂NH

solution in order to generate LDA. The LDA solution was then cooled

to −78 °C in dry ice/acetone and stirred under N₂for 15 min.

Dropwise by syringe, with stirring, under N₂, a solution of sulfonamide

82 (298 mg, 0.564 mmol, 1.0 equiv) in anhydrous THF (9.7 mL) was

added to the chilled LDA solution. After addition, the reaction mixture

was stirred under N₂at −78 °C for 10 min. Then, diethyl

chlorophosphate (0.13 mL, 155 mg, 0.900 mmol, 1.6 equiv) was

added by syringe, and the reaction mixture was stirred at −78 °C

under N₂for 1 h. The reaction mixture was warmed to 0 °C in an ice

1

gel, 4:1 hexanes/EtOAc). IR (ﬁlm) ν_max1720, 1452, 1071 cm⁻¹. H

NMR (500 MHz, CDCl₃) δ 7.75 (s, 1H), 7.64 (d, J = 8.2 Hz, 2H),

7.54 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.42 (td, J = 7.6, 1.8

Hz, 1H), 7.29−7.24 (m, 1H), 6.80 (s, 1H), 5.41 (s, 1H). ¹⁹F NMR

(376 MHz, CDCl₃) δ −120.3.

2-Bromo-2-(3′-(bromo(2,4,5-trichlorophenyl)methyl)-2′-ﬂuoro-

[1,1′-biphenyl]-4-yl)-N-methylacetamide (78). Carboxylic acid 77

(309 mg, 0.614 mmol, 1.0 equiv) was dissolved in anhydrous CH₂Cl₂

(5.8 mL, 0.100 M). To the reaction mixture was added carbonyl

diimidazole (112 mg, 0.688 mmol, 1.1 equiv), and the resultant

solution was stirred after sealing the ﬂask with a polyethylene stopper

for 30 min. Then, methylamine (0.34 mL of a 2.0 M solution in THF,

0.680 mmol, 1.1 equiv) was added by syringe, and the polyethylene

stopper was placed back on the ﬂask. The reaction mixture was stirred

at 23 °C for 24 h, then concentrated. The crude product was puriﬁed

by ﬂash chromatography (silica gel, 19:1 to 1:1 hexanes/EtOAc) to

aﬀord 78 as a white foam (104 mg, 33%). R_f= 0.23 (silica gel, 2:1

hexanes/EtOAc). IR (ﬁlm) ν_max1657, 1563, 1453, 1409, 1353, 1071,

1

908, 733 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.75 (s, 1H), 7.53−

7.49 (m, 5H), 7.41 (td, J = 7.5, 1.8 Hz, 1H), 7.28−7.22 (m, 1H), 6.79

(s, 1H), 6.74−6.69 (m, 1H), 5.48 (s, 1H), 2.93 (d, J = 4.9 Hz, 3H). ¹⁹F

NMR (376 MHz, CDCl₃) δ −120.3. MS (ESI-TOF) calcd for

C₂₂H₁₅Br₂Cl₃FNO [M + H⁺] 593.8622, found 593.8620.

R

DOI: 10.1021/acs.jmedchem.7b01292

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

bath and stirred for an additional hour before the reaction was

quenched with brine (10 mL). The aqueous layer was extracted with

CH₂Cl₂(3 × 15 mL) then EtOAc (10 mL). The combined organic

layers were washed with H₂O (10 mL), dried over Na₂SO₄, ﬁltered,

and concentrated. The crude product was ﬂash chromatographed

(silica gel, 99:1 to 9:1 CH₂Cl₂/MeOH) to aﬀord recovered 82 (120

mg, 40%) and ( )-83 as a yellow oil (249 mg), which provided a

complex ¹H NMR spectrum, precluding rigorous characterization. The

material was brought forward in impure form to the subsequent step.

( )-4-((Fluoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-

4-yl)methyl)sulfonyl)morpholine (( )-84). Sulfonamide 82 (474 mg,

0.896 mmol, 1.0 equiv) was dissolved in anhydrous THF (12 mL), and

the resultant solution was cooled to −78 °C in a dry ice/acetone bath.

Under an atmosphere of N₂, with stirring, sodium bis(trimethylsilyl)-

amide (0.49 mL of a 2.0 M solution in THF, 0.980 mmol, 1.1 equiv)

was added to the reaction mixture dropwise by syringe. The mixture

was stirred for 30 min at −78 °C. Then, a solution of NFSI (311 mg,

0.988 mmol, 1.1 equiv) in anhydrous THF (2.5 mL) was added to the

stirred solution, dropwise by syringe under N₂. The ﬂask was warmed

to 23 °C, and stirring under N₂was continued for 4 h, at which point

the reaction was quenched by the addition of satd aq NH₄Cl (15 mL).

The aqueous layer was extracted with EtOAc (3 × 20 mL), then the

pooled organic extracts were dried over Na₂SO₄, ﬁltered, and

concentrated. The crude product was puriﬁed by ﬂash chromatography

(silica gel, 19:1 to 2:1 hexanes/EtOAc) to aﬀord ( )-84 as a white

foam (221 mg, 45%). R_f= 0.36 (silica gel, 2:1 hexanes/EtOAc). IR

(ﬁlm) ν_max1459, 1407, 1353, 1300, 1261, 1204, 1162, 1114, 1070, 957,

yl)(morpholinosulfonyl)methyl)phosphonate (85a and 85b). Phos-

phonate ( )-85 was separated into enantiomerically pure isomers 85a

(ﬁrst peak) and 85b (second peak) with >99% chemical purity and

>99% ee using chiral preparatory supercritical ﬂuid chromatography.⁵⁹

The racemic compound was loaded as a 5 mg/mL solution in EtOH,

with an injection volume of 1.0 mL, onto a ChiralPak AD-H column

(2 cm × 25 cm), and eluted with 40% MeOH/CO₂at 100 bar, with a

ﬂow rate of 50 mL/min. Peaks were visualized using UV at 220 nm.

Analytical chromatograms were obtained by injecting compound

solutions onto a ChiralPak AD-H column (25 cm × 0.46 cm),

followed by elution with 40% MeOH/CO₂at a ﬂow rate of 3 mL/min.

Peaks were visualized using UV at 254 nm. See Supporting

Information for SFC traces.

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b01292.

Experimental procedures, SFC traces for 85a and 85b,

NMR spectra for all inhibitors, dose−response curves for

all inhibitors, and X-ray crystallographic details (PDF)

Molecular formula strings (CSV)

Accession Codes

The X-ray crystal structures have been deposited in the Protein

Data Bank (1, 5OW1; 2, 5OVR; 3, 5OVX). Authors will release

the atomic coordinates and experimental data upon article

publication.

1

910, 847, 736, 572 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.65−7.62

(m, 4H), 7.51 (s, 1H), 7.35 (td, J = 7.4, 2.0 Hz, 1H), 7.27 (s, 1H), 7.18

(t, J = 7.5 Hz, 1H), 7.13 (td, J = 7.2, 2.0 Hz, 1H), 6.16 (d, J = 46.0 Hz,

1H), 4.12 (s, 2H), 3.74−3.69 (m, 4H), 3.41−3.36 (m, 4H). ¹⁹F NMR

(376 MHz, CDCl₃) δ −121.8, −176.4. MS (ESI-TOF) calcd for

C₂₄H₂₀Cl₃F₂NO₃S [M + Na⁺] 1:1 568.0090 and 570.0060, found 1:1

568.0091 and 570.0070.

AUTHOR INFORMATION

Corresponding Authors

*For J.A.E.: phone, 203-432-3647; E-mail, jonathan.ellman@

yale.edu.

■

( )-Diethyl (Fluoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-bi-

phenyl]-4-yl)(morpholinosulfonyl)methyl)phosphonate (( )-85).

Following a reported procedure,⁵⁸iPr₂NH (0.07 mL, 51.0 mg, 0.500

mmol, 1.2 equiv) was diluted in anhydrous THF (1.0 mL), and the

resultant solution was cooled to 0 °C in an ice bath. Dropwise by

syringe, with stirring under an atmosphere of N₂, nBuLi (0.26 mL of a

1.90 M solution in hexanes, 0.494 mmol, 1.2 equiv) was added to the

iPr₂NH solution in order to generate LDA. The LDA solution was

then cooled to −78 °C in dry ice/acetone and stirred under N₂for 15

min. Dropwise by syringe, with stirring, under N₂, a solution of

sulfonamide ( )-84 (221 mg, 0.405 mmol, 1.0 equiv) in anhydrous

THF (4.0 mL) was added to the chilled LDA solution. After addition,

the reaction mixture was stirred under N₂at −78 °C for 10 min. Then,

diethyl chlorophosphate (0.09 mL, 108 mg, 0.623 mmol, 1.5 equiv)

was added by syringe, and the reaction mixture was stirred at −78 °C

under N₂for 1 h. The reaction mixture was warmed to 0 °C in an ice

bath and stirred for an additional 90 min before the reaction was

quenched with brine (10 mL). The aqueous layer was extracted with

CH₂Cl₂(3 × 15 mL) then EtOAc (10 mL). The combined organic

layers were dried over Na₂SO₄, ﬁltered, and concentrated. The crude

product was ﬂash chromatographed (silica gel, 1:1 CH₂Cl₂/EtOAc) to

aﬀord ( )-85 as a colorless oil (157 mg, 57%). R_f= 0.10 (silica gel, 2:1

hexanes/EtOAc). IR (ﬁlm) ν_max1458, 1359, 1262, 1165, 1134, 1115,

*For H.K.: phone, +46 31 7064060; E-mail, helena.kack@

astrazeneca.com.

ORCID

Stefan Geschwindner: 0000-0002-2154-8345

Helena Kack: 0000-0002-5456-3269

̈

Jonathan A. Ellman: 0000-0001-9320-5512

Present Address

^○For M.S.: Regulatory Project Management, Global Medicines

Development, AstraZeneca, Macclesﬁeld, Cheshire SK10 2NA,

United Kingdom.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by the NIH (R35GM122473 to J.A.E.

and F32GM114880 to M.R.W.). P.J.L. acknowledges support

from Yale Center for Clinical Investigation, grant no. 1097115.

We gratefully acknowledge Terence Wu (Yale) for mass

spectrometric assistance and Dr. Kate D. Otley (Yale) for

experimental assistance and helpful discussions. Dr. Rebecca F.

Wissner (Yale) is acknowledged for comments on this

manuscript.

1

1060, 1022, 964 cm⁻¹. H NMR (400 MHz, CDCl₃) δ 7.90 (dd, J =

8.7, 1.9 Hz, 2H), 7.65 (d, J = 7.6 Hz, 2H), 7.51 (s, 1H), 7.37 (td, J =

7.4, 1.9 Hz, 1H), 7.26 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.13 (td, J =

7.6, 7.2, 1.9 Hz, 1H), 4.44−4.31 (m, 2H), 4.22−3.97 (m, 6H), 3.63−

3.52 (m, 2H), 3.52−3.44 (m, 2H), 3.31−3.22 (m, 2H), 1.39 (td, J =

7.1, 0.8 Hz, 3H), 1.20 (td, J = 7.1, 0.8 Hz, 3H). ¹⁹F NMR (376 MHz,

CDCl₃) δ −121.7, −167.5 (d, J = 71.4 Hz). ³¹P NMR (162 MHz,

CDCl₃) δ 6.3 (d, J = 71.4 Hz). MS (ESI-TOF) calcd for

C₂₈H₂₉Cl₃F₂NO₆PS [M + Na⁺] 1:1 704.0379 and 706.0350, found

1:1 704.0379 and 706.0346.

ABBREVIATIONS USED

■

AcOH, acetic acid; AD, Alzheimer’s disease; AMPAR, α-amino-

3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; aq,

aqueous; CH₂Cl₂, dichloromethane; CNS, central nervous

system; CO₂, carbon dioxide; conc, concentrated; CV, column

volumes; DME, 1,2-dimethoxyethane; DMF, N,N-dimethylfor-

(+)-Diethyl (Fluoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-bi-

phenyl]-4-yl)(morpholinosulfonyl)methyl)phosphonate and (−)-Di-

ethyl (Fluoro(2′-ﬂuoro-3′-(2,4,5-trichlorobenzyl)-[1,1′-biphenyl]-4-

S

DOI: 10.1021/acs.jmedchem.7b01292

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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mamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol;

EDTA, ethylenediaminetetraacetic acid; ERK, extracellular

signal related kinases; Et₂O, diethyl ether; EtOAc, ethyl acetate;

EtOH, ethanol; H₂O, water; HCl, hydrochloric acid; iPr₂NH,

diisopropylamine; iPrMgCl, isopropylmagnesium chloride;

ITC, isothermal titration calorimetry; K₂CO₃, potassium

carbonate; KOAc, potassium acetate; LAR, leukocyte common

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MeOH, methanol; MKP5, mitogen-activated protein (MAP)

kinase phosphatase 5; MWCO, molecular weight cutoﬀ; N₂,

nitrogen; Na₂CO₃, sodium carbonate; Na₂CO₃·H₂O, sodium

carbonate monohydrate; Na₂SO₃, sodium sulﬁte; Na₂SO₄,

sodium sulfate; NaCl, sodium chloride; NaHCO₃, sodium

bicarbonate; NaHSO₃, sodium bisulﬁte; NaOH, sodium

hydroxide; n-BuLi, n-butyllithium; NFSI, N-ﬂuorobenzenesul-

fonimide; NH₄Cl, ammonium chloride; NMDAR, N-methyl-D-

aspartate receptor; PBS, phosphate-buﬀered saline; Pd(dppf)-

Cl₂·CH₂Cl₂, [1,1′-bis(diphenylphosphino)ferrocene]-

dichloropalladium(II) complex with dichloromethane; Pd-

(PPh₃)₄, tetrakis(triphenylphosphine)palladium(0); pNPP,

para-nitrophenylphosphate; PTP, protein tyrosine phospha-

tase; PTP1B, protein tyrosine phosphatase 1B; Pyk2, proline-

rich tyrosine kinase 2; SAS, substrate activity screening; satd,

saturated; STEP, striatal-enriched protein tyrosine phosphatase;

TCEP, tris(2-carboxyethyl)phosphine; TC-Ptp, T-cell protein

tyrosine phosphatase; TFA, triﬂuoroacetic acid; THF, tetrahy-

drofuran

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X-ray Characterization and Structure-Based Optimization of Striatal-Enriched Protein Tyrosine Phosphatase Inhibitors

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