Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

Article

pubs.acs.org/jmc

Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol

Binding Protein 4: Potential Treatment of Atrophic Age-Related

Macular Degeneration and Stargardt Disease

Christopher L. Cioﬃ,*^,†Boglarka Racz,^§Emily E. Freeman,^†Michael P. Conlon,^†Ping Chen,^†

Douglas G. Staﬀord,^†Daniel M. C. Schwarz,^†Lei Zhu,^‡Douglas B. Kitchen,^‡Keith D. Barnes,^†

Nicoleta Dobri,^§Enrique Michelotti,^#Charles L. Cywin,^○William H. Martin,^⊥Paul G. Pearson,^∥

Graham Johnson,^∥and Konstantin Petrukhin*^,§

^†Department of Medicinal Chemistry, and ^‡Computer Assisted Drug Discovery, AMRI, East Campus, 3 University Place, Rensselaer,

New York 12144, United States

^§Department of Ophthalmology, Columbia University Medical Center, New York, New York 10032, United States

^∥iCuraVision LLC, 31194 La Baya Drive, Suite 101, Westlake Village, California 91362, United States

^⊥WHM Consulting LLC, 111 Sterling City Road, Lyme, Connecticut 06371, United States

^#National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, United States

^○National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, United States

S

* Supporting Information

ABSTRACT: Antagonists of retinol-binding protein 4 (RBP4) impede ocular

uptake of serum all-trans retinol (1) and have been shown to reduce cytotoxic

bisretinoid formation in the retinal pigment epithelium (RPE), which is

associated with the pathogenesis of both dry age-related macular degeneration

(AMD) and Stargardt disease. Thus, these agents show promise as a potential

pharmacotherapy by which to stem further neurodegeneration and concomi-

tant vision loss associated with geographic atrophy of the macula. We previ-

ously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists,

represented by bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo analogue 4. We

describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a

suitable isostere for the anthranilic acid appendage of 4, which led to the

discovery of standout antagonist 33. Analogue 33 possesses exquisite in vitro

RBP4 binding aﬃnity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels in vivo in a

robust manner (>90%).

factor in autosomal recessive Stargardt disease, an untreatable

and inherited form of juvenile-onset macular degeneration

caused by genetic mutations in the Abca4 gene. Abca4 is a key

component of the visual retinoid cycle, and this critical gene

product loss is functionally recapitulated in the Abca4^−/−knock-

out mouse model.⁵The best studied bisretinoid component of

INTRODUCTION

■

Atrophic (dry) age-related macular degeneration (AMD) is a

chronic and slowly progressing neurodegenerative ocular

disorder that involves geographic atrophy of the central region

of the retina called the macula.¹Approximately 85% to 90%

of reported cases of macular degeneration are of the atrophic

form,¹and this disorder is the leading cause of vision loss for

individuals aged 60 years or older.²The dry AMD patient

population is estimated at 11 million individuals in the US

alone and is expected to double by 2020.³Currently, there is no

FDA-approved treatment available for patients suﬀering from

this debilitating disease.

Accumulation of lipofuscin, a granular substance comprising

cytotoxic bisretinoid ﬂuorophores, in the retinal pigment epithe-

lium (RPE) has been implicated as one of several pathogenic

factors contributing to photoreceptor cell degeneration in the

macula of dry AMD patients.⁴In addition, dramatic retinal accu-

mulation of lipofuscin is also believed to be the key etiological

lipofuscin is N-retinide-N-retinylidene ethanolamine (A2E),⁴ⁱ

a

naturally occurring byproduct of the visual cycle derived from

phosphatidylethanolamine and two molecules of all-trans retinal.

A2E induces several toxic eﬀects that may lead to the observed

abnormalities in the dry AMD RPE.^4h,6Therefore, it is postu-

lated that disruption or inhibition of de novo A2E formation in

the RPE may provide a pharmacological intervention point by

which to delay or retard the neurodegenerative processes associ-

ated with dry AMD and Stargardt disease.⁷Currently, there are

Received: March 26, 2015

A

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Figure 1. Retinol (1), fenretinide (2), A1120 (3), and 2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]pyrrole-2-

carboxamido)benzoic acid (4).

several agents in various stages of development that interfere

with the visual cycle under clinical investigation for the treatment

of dry AMD.^3,8We recently reported our initial eﬀorts in this

ﬁeld, which are based on the hypothesis that reduction of the

ocular uptake of serum all-trans retinol (retinol, vitamin A) (1)

(Figure 1) via reduction in circulating levels of serum retinol

binding protein 4 (RBP4) can lead to a reduced rate of bisretinoid

A2E accumulation in the retina.⁹

favorable RBP4 binding aﬃnity and provided several key

structural elements that allowed for rapid structure−activity

relationship (SAR) exploration. However, the compound

suﬀers from poor human liver microsomal (HLM) stability

(∼3% remaining after 30 min incubation), which was a major

liability that needed to be addressed. Thus, our goal was to

develop novel chemical matter with improved drug-like charac-

teristics relative to 3, and our eﬀorts led to the discovery of the

novel bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo RBP4

antagonist 4.⁹Analogue 4 possesses a favorable balance of suitable

in vitro RBP4 binding (scintillation proximity assay (SPA)) and

functional RBP4-TTR interaction antagonist (HTRF) activity

with signiﬁcantly improved HLM stability (100% remaining after a

30 min incubation). Furthermore, 4 possesses good pharmacoki-

netic (PK) and pharmacodynamic (PD) properties, leading to

robust and sustained lowering (>85%) of serum RBP4 levels in

both acute and chronic rodent oral dosing studies.⁹

We sought to build upon our reported SAR eﬀorts by further

exploring the anthranilic acid appendage of 3 and compound 4.

Speciﬁcally, we wished to investigate whether a pyrimidine-4-

carboxylic acid could provide a suitable isostere for the amide of

3 while still presenting the acid group as a favorable interaction.

Pyrimidine reduces the number of rotatable bonds and was

expected to lead to improved RBP4 binding aﬃnity (Figure 2).

Our overarching goal was to further enhance the in vitro and

in vivo RBP4 potency observed for 4 while maintaining its

excellent microsomal stability and other favorable drug-like

characteristics. Guided by our RBP4 computational docking

models,⁹we initially designed and synthesized illustrative

examples containing alternative cores that link together the

ortho-triﬂuoromethylphenyl moiety of 3 and 4 with the newly

proposed 6-methylpyrimidine-4-carboxylic acid fragment. We

subsequently explored the SAR of the pyrimidine-4-carboxylic

acid appended to the bicyclic [3.3.0]-octahydrocyclopenta[c]-

pyrrolo core featured in compound 4. Lastly, we also included

within our test set a focused series of bicyclic [3.3.0]-octa-

hydrocyclopenta[c]pyrrolo core analogues bearing a 6-methyl-

pyrimidine-4-carboxylic acid fragment in conjunction with variable

aryl head groups.

Retinol is transported systemically via a tertiary complex with

RBP4 and transthyretin (TTR).¹⁰Non-TTR complexed RBP4,

a low molecular weight (21 kDa) lipocalin serum protein,¹¹is

rapidly cleared from the bloodstream through glomerular

ﬁltration. RBP4-TTR complexation is retinol dependent as apo-

RBP4 interacts poorly with TTR.¹⁰It was suggested that lower

serum retinol levels achieved via inhibition of RBP4 would

modulate the visual cycle, which is fueled by a constant delivery

of retinol to the RPE by RBP4.¹²As a consequence of reduced

retinol supply to fuel the visual cycle, the rate of bisretinoid

formation in the RPE would be slowed or halted and the

concomitant progression of geographic atrophy within the dry

AMD retina would be retarded.^7aIndeed, this hypothesis is

supported by preclinical and clinical data obtained for the

retinoid-based RBP4 antagonist fenretinide (2). In preclinical

studies, fenretinide also lowered circulating plasma RBP4 levels

in vivo^7a,13and signiﬁcantly reduced the accumulation of lipo-

fuscin bisretinoids in the Abca4^−/−mouse model of enhanced

retinal lipofuscinogenesis.^7aIn extended clinical studies,

fenretinide lowered serum RBP4 levels and reduced the rate

of the geographic atrophy growth in a subset of the treated

AMD patients wherein an approximate 70% (or greater) reduc-

tion in circulating serum RBP4 levels was achieved.¹⁴

We have shown that chronic oral administration of the

nonretinoid RBP4 antagonist A1120 (3) (previously developed

by Amgen for the potential treatment of diabetes)¹⁵reduced

the accumulation of lipofuscin bisretinoids by approximately

50% in the Abca4^−/−mouse model of enhanced retinal lipo-

fuscinogenesis.¹⁶This reduction was correlated with a 75%

reduction in serum RBP4.¹⁶In our more recent report,⁹we

described medicinal chemistry eﬀorts conducted in the pursuit

of designing novel nonretinoid RBP4 antagonists that utilized

3 as a rational design starting point. Compound 3 exhibits

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Figure 2. Medicinal chemistry work plan for pyrimidine-4-carboxylic acid containing antagonists of RBP4.

a

Scheme 1

a

Reagents and conditions: (a) (i) n-BuLi, THF, −78 °C, 40 min; (ii) 1-benzylpiperidin-4-one, THF, −78 °C; (b) SOCl₂, 0 °C, 2 h; (c) (i)

HCO₂NH₄, 10% Pd/C, CH₃OH, reﬂux; (ii) 4.0 M HCl solution in 1,4-dioxane, CH₃CN, rt, 2 h; (d) methyl 2-chloro-6-methylpyrimidine-4-

carboxylate, i-Pr₂NEt Et₃N, DMF, 60 °C, 16 h; (e) (i) LiOH·H₂O, H₂O, THF, rt, 16 h; (ii) 2 N aq HCl.

methyl 2-chloro-6-methylpyrimidine-4-carboxylate gave ester

13, which underwent saponiﬁcation in the presence of LiOH·

H₂O to give upon acidiﬁcation the desired carboxylic acid 14.

The synthesis of 6-methyl-2-((3aR,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidine-4-

carboxylic acid (18) is depicted in Scheme 3. Palladium-catalyzed

amination of 1-bromo-2-(triﬂuoromethyl)benzene (5) with

(3aR,6aS)-tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-

carboxylate provided intermediate 15, which underwent HCl-

mediated Boc-deprotection to give amine hydrochloride 16.

Treatment of 16 with methyl 2-chloro-6-methylpyrimidine-4-

carboxylate gave the corresponding methyl ester 17, which

was hydrolyzed with LiOH·H₂O and then acidiﬁed to aﬀord

carboxylic acid 18 in good yield.

CHEMISTRY

■

The synthesis of 6-methyl-2-(4-(2-(triﬂuoromethyl)phenyl)-

piperidin-1-yl)pyrimidine-4-carboxylic acid (10) is presented in

Scheme 1.^9,17Addition of the lithium salt of 1-bromo-2-

(triﬂuoromethyl)benzene (5) to 1-benzylpiperidin-4-one gave

tertiary alcohol 6, which was readily dehydrated in the presence

of thionyl chloride to furnish tetrahydropyridine 7. Reduction

of the oleﬁn of 7 with concomitant N-benzyl deprotection

using ammonium formate and 10% Pd/C followed by exposure

to HCl provided piperidine hydrochloride salt 8. Treatment of

8 with methyl 2-chloro-6-methylpyrimidine-4-carboxylate in the

presence of i-Pr₂NEt in DMF at 60 °C aﬀorded methyl ester 9,

which underwent facile saponiﬁcation with LiOH·H₂O. Acid-

iﬁcation aﬀorded carboxylic acid 10 in good yield.

Construction of the [3.3.0]-octahydrocyclopenta[c]pyrrolo

series of analogues 32−36 was achieved following our previ-

ously reported route (Scheme 4).^9,18LiAlH₄reduction of com-

mercially available dione 19 and subsequent N-Boc protection

of the resulting secondary amine gave tetrahydroisoindole 20 in

good yield. NaIO₄and RuO₂·H₂O promoted oxidative cleavage

of 20 gave ring-opened diacid 21, which underwent Dieckman

The synthesis of 6-methyl-2-(4-(2-(triﬂuoromethyl)phenyl)-

piperazin-1-yl)pyrimidine-4-carboxylic acid (14) is highlighted

in Scheme 2. A palladium-catalyzed amination reaction between

1-bromo-2-(triﬂuoromethyl)benzene (5) and tert-butyl piper-

azine-1-carboxylate aﬀorded aryl piperazine 11, which under-

went smooth Boc-deprotection in the presence of HCl to

provide piperazine hydrochloride 12. Treatment of 12 with

C

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a

Scheme 2

a

Reagents and conditions: (a) tert-butyl piperazine-1-carboxylate, Pd(OAc)₂, BINAP, NaOt-Bu toluene, 110 °C, 16 h; (b) 2.0 M HCl solution in

Et₂O, CH₂Cl₂, rt, 3 h; (c) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr₂NEt, DMF, 60 °C, 16 h; (d) (i) LiOH·H₂O, H₂O, THF, rt, 16 h;

(ii) 2 N aq HCl.

a

Scheme 3

a

Reagents and conditions: (a) (3aR,6aS)-tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate, Pd(OAc)₂, BINAP, NaOt-Bu, toluene,

110 °C, 16 h; (b) 2.0 M HCl solution in Et₂O, CH₂Cl₂, rt, 3 h; (c) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr₂NEt, DMF, 60 °C, 16 h;

(d) (i) LiOH·H₂O, H₂O, THF, rt, 16 h; (ii) 2 N aq HCl.

a

Scheme 4

a

Reagents and conditions: (a) (i) LiAlH₄(1.0 M solution in THF), THF, 70 °C, 16 h; (ii) Boc₂O, CH₂Cl₂, rt, 16 h; (b) (i) NaIO₄, RuO₂·H₂O,

CH₃CN, CCl₄, H₂O, rt, 24 h; (c) Ac₂O, NaOAc, 120 °C, 3 h; (d) (i) LiHMDS (1.0 M solution in THF), THF, −78 °C, 30 min; (ii),

PhN(SO₂CF₃)₂, THF, −78 °C to rt, 3 h; (e) (2-(triﬂuoromethyl)phenyl)boronic acid, Pd(PPh₃)₄, 2 M Na₂CO₃, DME, 80 °C, 6 h; (f) H₂(40 psi),

10% Pd/C, CH₃OH, rt, 16 h; (g) 2.0 M HCl in Et₂O, CH₂Cl₂, 0 °C to rt, 24 h; (h) 2-chloro-6-substitutedpyrimidine-4-carboxylic acid methyl ester,

Et₃N, DMF, 60 °C, 16 h; (i) (i) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (ii) 2 N HCl.

D

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a

Scheme 5

a

Reagents and conditions: (a) TFA, CH₂Cl₂, rt, 3 h; (b) H₂(50 psi), 10% Pd/C, CH₃OH, rt, 6 h; (c) (i) 2-chloro-6-methylpyrimidine-4-carboxylic

acid, Et₃N, DMF, 60 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl.

a

Scheme 6

a

Reagents and conditions: (a) (i) 2,4-dichloro-5,6-dimethylpyrimidine, Pd(OAc)₂, Cs₂CO₃, JohnPhos, toluene, reﬂux, 16 h; (ii) Mo(CO)₆,

Pd(OAc)₂, BINAP, CH₃OH, CH₃CN, 80 °C, 16 h; (iii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 16 h; (b) (i) methyl 2-chloro-5-methylpyrimidine-4-

carboxylate, Et₃N, DMF, 60 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (c) (i) methyl 2-chloro-4-methylpyrimidine-5-carboxylate or

methyl 2-chloropyrimidine-5-carboxylate, i-Pr₂NEt, 70 °C (microwave irradiation), 1.5 h or Et₃N, DMF, 70 °C, 16 h; (ii) LiOH·H₂O, THF, H₂O, rt,

16 h or 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl.

condensation with concomitant decarboxylation in acetic anhy-

dride at 120 °C to give ketone 22. Ketone 22 was converted

to the corresponding racemic triﬂate ( )-23 in the presence

of 1,1,1-triﬂuoro-N-phenyl-N-((triﬂuoromethyl)sulfonyl)meth-

anesulfonamide. Intermediate ( )-23 underwent facile Suzuki

Pd-catalyzed cross-coupling with (2-(triﬂuoromethyl)phenyl)-

boronic acid conversion to give styrene ( )-24. Catalytic

reduction of ( )-24 was conducted under 40 psi H₂in the

presence of 10% Pd/C in CH₃OH and exclusively provided

endo-product 25. HCl-induced Boc-deprotection of 25 followed

by reaction with a corresponding 6-substituted 2-chloropyr-

imidine-4-carboxylic acid methyl ester aﬀorded analogues 27−

31. Methyl esters 27−31 were subsequently hydrolyzed and

acidiﬁed to the corresponding acids 32−36.

The preparation of exo-analogue 40 was achieved via our

previously reported route, which is shown in Scheme 5.^9,18

Reduction of des-Boc oleﬁn ( )-37 provided a mixture of endo-

and exo-isomers 38 and 39, which were isolated via reversed

phase column chromatography. Treatment of exo-amine 39

with methyl 2-chloro-5-methylpyrimidine-4-carboxylate fol-

lowed by saponiﬁcation with 2 N NaOH and subsequent

acidiﬁcation provided the desired exo-acid 40.

Pyrimidine analogues 41−44 were accessed via intermediate

26 (Scheme 6). Compound 41 was furnished via a palladium-

catalyzed amination of 2,4-dichloro-5,6-dimethylpyrimidine

with 26, followed by carbonylation with Mo(CO)₆and sub-

sequent saponiﬁcation of the resulting methyl ester. Treatment

of 26 with methyl 2-chloro-5-methylpyrimidine-4-carboxylate

E

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a

Scheme 7

a

Reagents and conditions: (a) (i) 2-chloro-6-methylpyrimidine-4-carbonitrile, i-Pr₂NEt, DMF, 60 °C, 16 h; (b) LiOH·H₂O, THF, H₂O, 70 °C, 18 h;

(c) NaN₃, NH₄Cl, DMF, 130 °C (microwave irradiation), 1 h.

followed by hydrolysis and acidiﬁcation gave acid 42. Analogues

43 and 44 were prepared via treatment of 26 with either methyl

2-chloro-4-methylpyrimidine-5-carboxylate or methyl 2-chlor-

opyrimidine-5-carboxylate followed by saponiﬁcation and

acidiﬁcation of the corresponding methyl esters.

Boc-deprotection aﬀorded the endo-amine hydrochloride inter-

mediates 60a−f. Treatment of these secondary amines with

2-methyl 2-chloro-6-methylpyrimidine-4-carboxylate followed

by hydrolysis and acidiﬁcation of the corresponding methyl

esters provided the desired acids 61−66.

The preparation of the carboxylic acid isosteres carboxamide

46 and tetrazole 47 are depicted in Scheme 7. Treatment of 26

with 2-chloro-6-methylpyrimidine-4-carbonitrile provided ni-

trile intermediate 45, which was hydrolyzed with LiOH·H₂O in

THF and H₂O at 70 °C to provide carboxamide 46. Nitrile 45

also underwent cycloaddition with NaN₃to furnish tetrazole 47

in good yield.

RESULTS AND DISCUSSION

■

Computational docking of an initial proposed set of 6-methyl-

pyrimidine-4-carboxylic acid ligands was conducted within our

model derived from the cocrystal structure of 3 bound to RBP4

(PDB code: 3fmz)¹⁵using standard precision (SP) mode. The

docked poses were then reﬁned using the extra precision (XP)

A series of diverse analogues (48−54) designed to further

explore pyrimidine carboxylic acid SAR is shown in Scheme 8.

The corresponding substituted chloro or bromo-substituted

picolinic, nicotinic, isonicotinic, or benzoic acid methyl esters

were aminated with amine 26 using a combination of either

Pd(OAc)₂or Pd₂(dba)₃and ligands XantPhos or JohnPhos in

the presence of Cs₂CO₃in reﬂuxing toluene or 1,4-dioxane.

The intermediary methyl esters were subsequently hydrolyzed

and acidiﬁed to give the corresponding acid analogues 48−54.

The syntheses of alternative heteroaromatic analogues 55−

57 are highlighted in Scheme 9. Methyl 6-chloropyrazine-2-

carboxylate, methyl 6-chloropyridazine-4-carboxylate, and

methyl 2-chloro-5-methylthiazole-4-carboxylate were independ-

ently reacted with amine 26 in the presence of either Et₃N or

i-Pr₂NEt in DMF at 60 °C followed by saponiﬁcation of

the resulting intermediary methyl esters with LiOH·H₂O

and acidiﬁcation to provide analogues 55, 56, and 57, respec-

tively.

mode of Glide (version 5.8, Schrodinger, LLC.). These docked

poses of the proposed ligands were minimized within the

binding site of the 3fmz model. All molecular mechanics (MM)

̈

minimization used Impact (version 5.8, Schrodinger, LLC).

̈

We modiﬁed the binding site by incorporating a structural

water molecule within the anthranilic acid binding region of

3fmz. Several reported RBP4 crystal structures show two water

molecules (W1 and W2) housed within the anthranilic acid

region of the RBP4 binding site, where they are engaged in

H-bond interactions with Arg121, Gln98, and Tyr90. However,

3fmz is devoid of these water molecules likely because the

region is surface exposed, and the solvent is likely quite mobile.

However, our docking and minimization procedures would be

less accurate if a solvent hole were left in this fairly polar

portion of the site. Therefore, to account for partially structured

water molecules, we added structural waters from other RBP4

crystal structures. When the structural water W2 of 1fel (RBP4

cocrystallized with fenretinide) was minimized within 3fmz,

strong H-bond interactions among it, compound 3, and Arg121

were observed (data not shown). This observation suggests that

structural water W2 could be playing a critical role in the bind-

ing of 3 to RBP4. Therefore, we have incorporated this struc-

tural water within our 3fmz docking model so as to ensure that

any key diﬀerences with regard to critical H-bond interactions

The synthesis of a series of analogues possessing varied aryl

head groups (61−66) is depicted in Scheme 10. Palladium-

catalyzed Suzuki cross-coupling of triﬂate ( )-23 with the

appropriately substituted aryl boronic acids provided the corre-

sponding styrene intermediates represented by ( )-58a−f in

good yield. Hydrogenation of the oleﬁns followed by HCl-promoted

F

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a

Scheme 8

a

Reagents and conditions: (a) (i) methyl 6-chloro-4-methylpicolinate, Pd(OAc)₂, XantPhos, Cs₂CO₃, toluene, 110 °C, 16 h; (ii) 2 N aq NaOH,

THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl; (b) (i) methyl 2-chloro-6-methylisonicotinate, Pd(OAc)₂, XantPhos, Cs₂CO₃, toluene, 110 °C, 16 h;

(ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl; (c) (i) methyl 6-chloro-3-methylpicolinate, Pd(OAc)₂, JohnPhos, Cs₂CO₃, toluene,

reﬂux, 14 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 72 h; (iii) 2 N aq HCl; (d) (i) methyl 5-bromonicotinate, Pd₂(dba)₃, XantPhos, Cs₂CO₃, 1,4-

dioxane, reﬂux, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 4 h; (iii) 2 N aq HCl; (e) methyl 3-bromobenzoate, Pd(OAc)₂, BINAP, NaOt-Bu,

toluene, 110 °C, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 16 h; (iii) 2 N aq HCl; (f) (i) methyl 2-chloro-6-methylnicotinate, Pd₂(dba)₃,

XantPhos, Cs₂CO₃, toluene, 100 °C, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, 50 °C, 30 min; (iii) 2 N aq HCl; (g) (i) methyl 2-chloronicotinate,

Pd(OAc)₂, XantPhos, Cs₂CO₃, 1,4-dioxane, 110 °C, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 16 h; (iii) 2 N aq HCl.

a

Scheme 9

a

Reagents and conditions: (a) (i) methyl 2-chloropyrazine-6-carboxylate or methyl 3-chloropyridazine-5-carboxylate or methyl 2-chloro-5-

methylthiazole-4-carboxylate, Et₃N or i-Pr₂NEt, DMF, 60 °C, 16 h or i-Pr₂NEt, DMSO, 110 °C, 24 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt,

30 min-16 h; (iii) 2 N aq HCl.

are observed for all newly proposed analogues and so that

ligand poses do not ﬁll a nonexistent hole.

from the backbone of Leu37, and the carboxylic acid forms a

salt bridge with Arg121 and two H-bonds with Tyr90 and

Gln98.^9,15These key interactions are potentially playing critical

roles in inducing and stabilizing the conformational changes

observed within the aforementioned RBP4 loops. To see if

the 6-methylpyrimidine-4-carboxylic acid moiety could provide

a suitable isosteric replacement for the anthranilic acid, we

initially conducted a 3fmz docking overlay of 3 with proposed

comparator 10. Indeed, analogue 10 presented a similar dock-

ing geometry relative to that of 3 (Figure 3); both compounds

are shown extending through the β-barrel core of the RBP4

As previously reported, the functional antagonist activity ob-

served for 3 is attributed to conformational changes it induces

in RBP4 loops β3-β4 and β5-β6.^9,15These changes block com-

plex formation with TTR, leading to reduced RBP4 plasma

levels as a result of increased glomerular excretion of the

uncomplexed protein. The anthranilic carboxylic acid fragment

of 3 is positioned at the opening of the RBP4 binding cavity

where it is engaged in several key binding interactions; the

carboxamido carbonyl oxygen atom of 3 accepts an H-bond

G

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a

Scheme 10

a

Reagents and conditions: (a) (i) substituted phenyl or pyridyl boronic acid, Pd(PPh₃)₄, 2.0 M Na₂CO₃, DME, 80 °C, 6 h; (b) H₂(40 psi), 10%

Pd/C, CH₃OH, rt, 16 h; (c) 2.0 M HCl in Et₂O, CH₂Cl₂, 0 °C to rt, 24 h; (d) (i) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, Et₃N, DMF,

60 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl.

Figure 3. (A) Overlay of minimized bound conformations of RBP4 antagonist 10 (orange) and 3 (green) within 3fmz. (B) Expanded view of the

anthranilic acid and 6-methylpyrimidine-4-carboxylic acid fragments of 3 and analogue 10. Both moieties are shown engaging in H-bond interactions

with Arg121, Gln98, and Tyr90. H-bond interactions to 10 are indicated with blue lines. Residues making contact with ligands are labeled and

illustrated in stick format. Molecular surface is colored by hydrophobicity (green) and hydrophilicity (purple) using contact preferences (MOE,

Chemical Computing Group, Inc., Montreal, CA).

binding cavity with their respective ortho-triﬂuoromethylphenyl

fragments projecting into the internal lipophilic β-ionone

pocket. The overlays also show the carboxylic acid moieties of

both compounds positioned within close proximity to one

another and engaged in the same key H-bond interactions

with Arg121, Gln98, and Tyr90. It should be noted that the

geometry of the pyrimidine moiety suggests the unexpected

replacement of the H-bond donor amide with the H-bond

acceptor pyrimidine. The geometry shown in Figure 3B likely

induces some very interesting protonation states among the

acid group, Tyr90, Arg121, the modeled water molecule, and

the pyrimidine, likely stabilized by Gln98. The movement of

Gln98 to this region appears to be a driving force for the block-

ing motion of loops β3-β4 and β5-β6. These conformations are

H

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signiﬁcantly diﬀerent (2.7 Å) from those in the fenretinide

antagonist structure 1fel. Buoyed by the favorable geometric

alignment observed in this docking experiment, we sought

to synthesize compound 10 and other similar analogues that

contain alternative cores linking together the ortho-triﬂuoro-

methylphenyl and 6-methylpyrimidine-4-carboxylic acid frag-

ments. Acknowledging our previous success with the bicyclic

[3.3.0]-octahydrocyclopenta[c]pyrrole core used to generate

the aforementioned RBP4 antagonist 4,⁹we ensured that

this core scaﬀold was included within our initial sample set of

analogues.

In Vitro Binding of Compounds to RBP4. Analogue

binding aﬃnities for RBP4 were measured using our previously

described scintillation proximity assay (SPA).¹⁶Nonlinear regression

analysis conducted following saturation binding of retinol to

RBP4 revealed a K_dof 62.5 nM in our assay conditions, which

is in line with the previously reported 70−190 nM range of

values.^10d,19For compound characterization, we measured the

competitive displacement of radiolabeled retinol added to the

reaction mix at 10 nM which roughly corresponds to the

generally recommended 1/10 of the K_dconcentration of

radioligand. The IC₅₀values were calculated from the 12-point

compound titrations using a four-parameter logistic equation.

Compounds with appreciable RBP4 binding potency (gen-

erally, with IC₅₀<150 nM) were further assessed for the ability

to antagonize the retinol-dependent RBP4-TTR interaction

using the HTRF assay.¹⁶

Assessment of Antagonistic Activity in the HTRF

RBP4-TTR Interaction Assay. The ability of an analogue to

disrupt the retinol-induced interaction of RBP4 with TTR was

examined using the previously described HTRF assay¹⁶that

probes retinol-dependent RBP4-TTR interaction. We used

bacterially expressed maltose binding protein (MBP)-tagged

RBP4 and commercially available TTR labeled directly with

Eu³⁺cryptate along with a d2-conjugated anti-MBP monoclonal

antibody in order to implement this assay. Retinol added to

the reaction mix at 1 μM concentration stimulates RBP4-TTR

interaction which brings europium in close proximity to the d2

dye with the following ﬂuorescence energy transfer (FRET) to

d2. The FRET signal, measured as a 668 nm emission, was nor-

malized using the 620 nm europium emission to compensate

for the pipetting and dispensing errors. Following a 12-point

dose titration (20 μM−0.1 nM), the IC₅₀values were calculated

using a standard four parameter logistic nonlinear regression

model equation. In order to monitor a correlation between the

binding aﬃnity of compounds for RBP4 and their potency as

antagonists of the retinol-dependent RBP4-TTR interaction, we

tracked a ratio of IC₅₀values in the HTRF and SPA assays.

Despite the diﬀerence in binding potency, for the majority of

analogues, this ratio tended to be within the 5−20 range.

Structure−Activity Relationships. An initial series of

analogues containing both the 6-methylpyrimidine-4-carboxylic

acid and corresponding methyl ester moieties were prepared

with the following core structures; piperidine (9 and 10), piper-

azine (13 and 14), bicyclic [3.3.0]-octahydropyrrolo[3,4-c]-

pyrrole (17 and 18), and bicyclic [3.3.0]-octahydrocyclopenta-

[c]pyrrole (28 and 33). The RBP4 SPA binding aﬃnity, HTRF

functional antagonist activity, and microsomal stability data for

these new analogues is shown in Table 1. Notably, the most

direct comparator of 3, analogue 10, exhibited similar in vitro

RBP4 binding aﬃnity and functional antagonist activity (RBP4

SPA IC₅₀= 35.3 nM; RBP4 HTRF IC₅₀= 188 nM), verifying

that the 6-methylpyrimidine-4-carboxylic acid fragment was

indeed a suitable isostere for anthranilic acid. Interestingly, the

pyrimidine-4-carboxylic acid of 10 also conferred the additional

beneﬁt of signiﬁcantly improving human liver microsomal

stability (68% remaining after 30 min incubation) relative to

that of 3. The corresponding methyl ester of 10 (9) was found

to be ∼3-fold more potent than 3, and the remaining methyl

esters 13, 17, and 28 also exhibited appreciable in vitro RBP4

potency in both assays. However, methyl esters 13 and 28

suﬀered from extensive degradation in the presence of human

(HLM) and rat (RLM) liver microsomes. Piperazine acid ana-

logue 14 was less potent than 3 (RBP4 SPA IC₅₀= 89.5 nM;

RBP4 HTRF IC₅₀= 511 nM), and it presented only a modest

improvement in HLM stability, whereas bicyclic [3.3.0]-

octahydropyrrolo[3,4-c]pyrrole 18 was slightly more potent

and exhibited better HLM stability. Incorporation of the

bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrole core provided

standout analogue 33, which exhibited signiﬁcantly improved

in vitro RBP4 potency (RBP4 SPA IC₅₀= 12.8 0.4 nM; RBP4

HTRF IC₅₀= 43.6

10.5 nM) and excellent microsomal

stability. Subsequent docking analysis of 33 revealed favorable

alignment within the RBP4 binding cavity (Figure 4). The

6-methylpyrimidine-4-carboxylic acid appendage of 33 is

engaged in key H-bond interactions as the bicyclic [3.3.0]-

octahydrocyclopenta[c]pyrrolo core extends through the β-

barrel channel and projects the ortho-triﬂuoromethylphenyl ring

slightly farther into the lipophilic hydrophobic β-ionone cavity

than 3. We postulate that the excellent RBP4 potency observed

for 33 potentially results from two possible factors (or a combi-

nation thereof): (1) more favorable van der Waals interactions

resulting from the deeper extension and optimal angle of

projection of the ortho-triﬂuoromethylphenyl ring into the lipo-

philic β-ionone cavity and/or (2) more fortiﬁed key H-bond

interactions due to increased rotational restriction of the

6-methylpyrimidine-4-carboxylic acid relative to the anthranilic

acid of 3 and 4 (two fewer rotatable bonds). In addition, the

exo-isomer 40 was also prepared and found to be devoid of

RBP4 activity (SPA IC₅₀> 3 μM, data not shown). The RBP4

activity diﬀerences observed between potent endo-isomer 33

and inactive exo-isomer 40 were consistent with our previously

reported ﬁndings for these geometric isomers of this scaﬀold

class.⁹The results obtained from this initial sample set of

probe compounds prompted us to further explore the SAR of

analogue 33.

The SAR of the 6-methylpyrimidine-4-carboxylic acid region

of analogue 33 is summarized in Table 2. Initial eﬀorts within

Table 2 focused on examining the impact on potency of

incorporating various chemotypes at the 6-position of the

pyrimidine-4-carboxylic acid ring. Limited exploration of this

region revealed that the size of the substituent was important

and that in vitro binding aﬃnity and functional antagonist activity

trends do not generally correlate. For example, removal of the

6-methyl group of analogue 33 to furnish 32 led to a ∼2-fold

diminishment in potency in both the SPA (RBP4 SPA IC₅₀

=

20.8 0.5 nM) and HTRF (RBP4 HTRF IC₅₀= 79.7 nM)

assays. However, when a triﬂuoromethyl group was introduced

to the 6-position (34), the SPA binding aﬃnity was recovered

(RBP4 SPA IC₅₀= 9.6 nM), yet HTRF potency remained

roughly 2-fold lower (RBP4 HTRF IC₅₀= 88.0 nM).

Introduction of the larger isopropyl group (36) led to a signi-

ﬁcant diminishment in potency relative to that of 33, which may

be indicative of a size/steric tolerability limit in that region of the

binding space. In addition, there was no diﬀerentiation between

substituents possessing electron-donating or electron-withdrawing

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Table 1. RBP4 SPA Binding Aﬃnity, RBP4 HTRF, and Liver Microsomal Stability Data for 6-Methylpyrimidine-4-carboxylic

Acid and Methyl Ester Analogues

microsomal stability (% remaining)

a

c

b

c

d

e

compd

RBP4 SPA IC₅₀(nM)

RBP4 HTRF IC₅₀(nM)

HLM

RLM

3

15.0 0.005

122 0.035

294

3

85

4

72.7

4.1

100

ND

68

75

9

132

ND

96

10

13

14

17

18

28

33

35.3

9.7

188

1402

0.0

18

0.0

63

89.5

3.9

511

74.0

ND

80

ND

76

57.4

6.3

140

100

0.2

94

9.8

93

12.8 0.4

43.6 10.5

a

b

IC₅₀values for the SPA assay obtained in the presence of a ﬁxed, 10 nM concentration of ³H-retinol. IC₅₀values for the HTRF assay obtained in

c

the presence of 1 μM concentration of retinol. For compounds tested multiple times (more than twice), the IC₅₀data are represented as the mean

standard deviation. For those compounds that were only tested twice, the IC₅₀data are shown as the mean of two independent experiments and

d

e

not as the mean standard deviation. HLM = human liver microsomes. RLM = rat liver microsomes. Compound concentration was 10 μM, and

incubation time with either human or rat microsomes was 30 min. ND = not determined.

character, as triﬂuoromethyl analogue 34 and methoxy analogue

35 exhibited similar binding aﬃnity. The incorporation of an

additional methyl group at the 5-position to give 5,6-dimethyl

analogue 41 resulted in a 5-fold decrease in potency, and

monosubstitution of a methyl group at the 5-position (42) led to

a further diminishment in potency. These data suggest that the

most potent analogues of this series require one substituent of

limited size to be positioned meta to the pyrimidine-4-carboxylic

acid and that substitution ortho to the carboxylic acid is dis-

favored. We also examined the impact of repositioning the

carboxylic acid of 33 from the 4- to the 5-position on the

pyrimidine ring. Interestingly, analogues 43 and 44 were tolerated

the unsubstituted nicotinic acid 51 was devoid of RBP4 activity.

Removal of all nitrogen atoms to furnish benzoic acid 52 provi-

ded an analogue with appreciable binding aﬃnity yet was very

weakly potent in the HTRF assay (RBP4 SPA IC₅₀= 83.2 nM;

RBP4 HTRF IC₅₀= 3956 nM). These data suggest that while

the methyl-substituted pyridine congeners of 33 (48 and 49)

exhibit good binding aﬃnity, both nitrogen atoms of pyrimidine

33 appear to be required for optimal functional antagonist

activity in the HRTF assay. In addition, the positioning of a

methyl group ortho to the carboxylic acid within this pyridine

series (50) had the same deleterious eﬀect on SPA binding

and HTRF potency that was observed for 5-methyl substituted

pyrimidine analogue 42. We next investigated the eﬀect of

repositioning the carboxylic acid adjacent to the bicyclic [3.3.0]-

octahydrocyclopenta[c]pyrrolo core, as docking of nicotinic acid

analogue 54 indicated favorable geometric alignment within the

RBP4 binding cavity (Figure 5). Indeed, analogues 53 and 54

were tolerated and exhibited comparable SPA binding aﬃnities

relative to one another (RBP4 SPA IC₅₀= 68.4 nM and 43.8 nM,

respectively); however, 54 was found to be 5-fold more potent

than 53 in the HTRF assay. Interestingly, the HTRF trend

observed between nicotinic acids 53 and 54 suggests that sub-

stitution on this heteroaromatic system in a manner analogous

to the 6-methyl group of pyrimidine 33 is not favorable with

regard to functional antagonist activity. Lastly, we also pursued

alternative heteroaromatic analogues pyrazine 55, pyridazine 56,

and thiazole 57. Of this set, pyridazine 56 and thiazole 57 were

found to possess good binding aﬃnity (RBP4 SPA IC₅₀= 23.0 nM

and 29.3 nM, respectively); however, both analogues were

and exhibited appreciable binding aﬃnity (RBP4 SPA IC₅₀

=

40.8 nM and 171 nM, respectively); however, both analogues

were signiﬁcantly less potent than 33 in the HTRF assay (RBP4

HTRF IC₅₀= 523 nM and 1864 nM, respectively). Lastly,

primary carboxamide 46 and tetrazole 47 served as suitable

carboxylic acid isosteres within this series as both compounds

displayed RBP4 binding aﬃnities comparable to that of 33.

However, 46 and 47 were found to be signiﬁcantly less potent in

the HTRF assay (RBP4 HTRF IC₅₀= 248 nM and 175 nM,

respectively).

In parallel, we evaluated replacing the pyrimidine-4-

carboxylic acid of 33 with picolinic, isonicotinic, nicotinic,

and benzoic acid moieties (Table 3). 4-Methyl picolinic acid 48

and 2-methyl isonicotinic acid 49 exhibited binding aﬃnities

within a 5-fold range of 33; however, they were both nearly

10-fold less potent in the HTRF assay (RBP4 HTRF IC₅₀

=

295 nM and 228 nM, respectively). In contrast, 3-methyl

picolinic acid 50 was signiﬁcantly less potent in both assays and

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Figure 4. (A) Overlay of minimized bound conformations of RBP4 antagonist 33 (magenta) and 3 (orange) within 3fmz. (B) Expanded view of the

anthranilic acid and 6-methylpyrimidine-4-carboxylic acid fragments of 3 (green) and analogue 33 (magenta). Both moieties are shown engaging in

H-bond interactions with Arg121, Gln98, and Tyr90. H-bond interactions to 33 are indicated with blue lines. Residues making contact with ligands

are labeled and illustrated in stick format. The molecular surface is colored by hydrophobicity (green) and hydrophilicity (purple) using contact

preferences (MOE, Chemical Computing Group, Inc., Montreal, CA).

signiﬁcantly less potent than benchmark 33 in the HTRF assay

(RBP4 HTRF IC₅₀= 437 nM and 495 nM, respectively).

We also probed the SAR associated with the aryl headgroup

region of compound 33 with a focused set of substituted

aromatic and heteroaromatic analogues that are shown in Table

4. 5-Fluoro-2-(triﬂuoromethyl)phenyl analogue 61 exhibited

permeable in the MDR1-MDCK permeability assay. The ob-

served CL_intvalues suggest very low predicted hepatic clearance,

and the % plasma protein binding (PPB) data indicate low

unbound fractions. Interestingly, unlike previously reported an-

thranilic acid analogue 4, which exhibited signiﬁcant CYP2C9

inhibitory activity (IC₅₀= 340 nM),⁹33 was found to be clean in

a standard CYP panel (all CYP IC₅₀values >34 μM). Additional

selectivity proﬁling revealed no signiﬁcant oﬀ-target activity at

the hERG channel or within a standard screening panel of ﬁfty-

ﬁve GPCRs, enzymes, ion channels, and transporters (data not

shown). Lastly, 33 did not induce human PXR activation (data

not shown).

In Vivo Activity: PK Characteristics of 33 in Rats. Single

dose PK studies conducted with Sprague−Dawley rats at

5.1 μmol/kg (2 mg/kg) IV and 18.2 μmol/kg (5 mg/kg) PO

doses showed very low plasma clearance (5.06 mL/min/kg)

and a very long half-life of 22 h (Table 6). The compound

was well absorbed and slowly eliminated from plasma after

oral administration with an observed C_maxof 62.2 μM and

corresponding T_maxat 2.67 h. Very high exposures were

observed (AUC_INFwas 3636 μM·h) and the estimated % F

exceeded 100%. The greater than 100% oral bioavailability

may be related to the very low plasma clearance of 33 and some

nonlinear pharmacokinetics over the 5.1 to 12.7 μmol/kg dose

range.

potency comparable to that of parent 33 (RBP4 SPA IC₅₀

=

46.3 1.1 nM; RBP4 HTRF IC₅₀= 33.4 1.8 nM). In

contrast, incorporation of a ﬂuorine ortho to the triﬂuoromethyl

group to give 3-ﬂuoro-2-(triﬂuoromethyl)phenyl analogue 62

was not well tolerated and led to a 20-fold loss in SPA bind-

ing potency (RBP4 SPA IC₅₀= 196 nM). Replacing the

ortho-triﬂuoromethyl group of 61 and 62 with chlorine to give

analogues 63 and 64 led to a precipitous drop in potency (SPA

IC₅₀= 532 198 nM and 137 nM; HTRF IC₅₀= 347 nM and

504 nM, respectively). Lastly, introduction of polarity by means

of pyridine analogues 65 and 66 yielded appreciable SPA

binding aﬃnity; however, the compounds were weakly potent

in the HRTF assay.

In light of the excellent RBP4 potency and robust

microsomal stability observed for analogue 33, the compound

was selected for further in vitro and in vivo evaluation. The

in vitro pharmacological proﬁle is presented in Table 5. Com-

pound 33 exhibited excellent kinetic solubility in phosphate

buﬀered saline (PBS) (pH 7.4) and was classiﬁed as highly

K

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Table 2. RBP4 SPA Binding Aﬃnity and RBP4 HTRF for Pyrimidine Carboxylic Acid Analogues

a

b

IC₅₀values for the SPA assay obtained in the presence of a ﬁxed, 10 nM concentration of ³H-retinol. IC₅₀values for the HTRF assay obtained in

c

the presence of 1 μM concentration of retinol. For compounds tested multiple times (more than twice), the IC₅₀data are represented as the mean

standard deviation. For those compounds that were only tested twice, the IC₅₀data are shown as the mean of two independent experiments and

not as the mean standard deviation.

We also conducted a subsequent 7-day, subchronic rodent

oral dosing PK−PD study with 33 (Figure 6). Sprague−Dawley

rats received either a single 18.2 μmol/kg (5 mg/kg) oral dose

or a once daily (QD) 18.2 μmol/kg oral dose of 33 over a

period of 7-days. Blood was collected from the single dose-

treated rats (Group 1) at predose, 15 and 30 min, and 1, 2, 4, 8,

L

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Table 3. RBP4 SPA Binding Aﬃnity and RBP4 HTRF for Pyridine and Other Heteroaromatic Carboxylic Acid Analogues

a

b

IC₅₀values for the SPA assay obtained in the presence of a ﬁxed, 10 nM concentration of ³H-retinol. IC₅₀values for the HTRF assay obtained in

c

the presence of 1 μM concentration of retinol. For compounds tested multiple times (more than twice), the IC₅₀data are represented as the mean

standard deviation. For those compounds that were only tested twice, the IC₅₀data are shown as the mean of two independent experiments and

not as the mean standard deviation. ND = not determined.

12, 24, 36, and 48 h post dose on Day 1. Blood was collected

from the 7-day QD dose-treated rats (Group 3) at predose

(Days 1−6; for RBP4 biomarker analysis only) and predose, 15

and 30 min, and 1, 2, 4, 8, 12, 24, 36, and 48 h post-last dose on

Day 7. A vehicle dose group (Group 2) was also included in the

study and blood was collected on Days 1 and 7 as a control

group for RBP4 biomarker analysis. The plasma exposure levels

of 33 were high in all samples of Groups 1 and 3. On Day 1,

compound 33 reached the peak concentration rapidly after

dose administration and declined slowly thereafter (Figure 6,

A). Mean plasma levels of 33 were approximately 20 μM at the

48 h time-point. As a result of the slow clearance of 33 from

M

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Figure 5. (A) Overlay of minimized bound conformations of RBP4 nicotinic acid antagonist 54 (yellow) and 3 (A1120, green) within 3fmz.

(B) Expanded view of the anthranilic acid and nicotinic acid fragments of 3 (green) and analogue 54 (yellow). Both moieties are shown engaging in

H-bond interactions with Arg121, Gln98, and Tyr90. H-bond interactions to 54 are indicated with blue lines. Residues making contact with ligands

are labeled and illustrated in stick format. The molecular surface is colored by hydrophobicity (green) and hydrophilicity (purple) using contact

preferences (MOE, Chemical Computing Group, Inc., Montreal, CA).

plasma, the mean predose plasma concentration on Day 7 was

148.1 58.2 μM (Figure 6B). After the administration of the

seventh daily dose, the mean plasma level increased to 178.9

59.4 μM at the 4 h time-point (Figure 6A). At 48 h after this

dose, the mean concentration of compound 33 remained high

at 97.1 48.6 μM. The PK parameters for the single and 7-day

doses were also determined. The observed C_maxvalues were

observed (Figure 7, A), while the 7-day QD oral administration

in rats at 18.2 μmol/kg induced an approximately 95% reduction

in plasma RBP4 (Figure 7, A). Comparison of the dynamics of

RBP4 lowering in the compound 33-treated animals with that of

the vehicle-treated rats over 7 days of QD dosing conﬁrmed the

magnitude of the compound eﬀect on the level of plasma RBP4

(Figure 7, B). The extent of the RBP4 lowering (Figure 7) shows

a clear correlation with plasma compound levels (Figure 6). With

regard to this eﬀect, even though the plasma protein binding of

33 is signiﬁcant (rat 99.4%, Table 5), the high circulating plasma

levels, long exposure, and very low clearance mean that the

projected plasma free drug concentration of 33 signiﬁcantly

exceeds that required to antagonize RBP4/TTR interaction

measured by either the SPA or HTRF binding assays (Table 4).

Antagonists of the RBP4-TTR interaction from diﬀerent

structural classes had previously established the correlation

among RBP4 lowering, serum retinol lowering, and bisretinoid

reduction in the Abca4^−/−mouse model of Stargardt disease.^7a,15

While we did not measure retinol lowering by 33 in our in vivo

studies, it seemed reasonable to expect that 33 would reduce

serum retinol and show the desired preclinical chronic eﬃcacy in

the Abca4^−/−mice given its pronounced RBP4-lowering activity

(Figure 7) and the established correlations between levels of

serum RBP4 and serum retinol.^7a,15,20This and additional data

describing the nonrodent pharmacology of compound 33 will be

published elsewhere.

66.5

23.6 μM with a T_maxof 2.75

2.17 h and 183.1

55.2 μM with a T_maxof 2.17 1.76 h on Days 1 and 7, respec-

tively. Exposure levels based on AUC_lastwere 1697 279 μM·h

on Day 1 and 6385 2501 μM·h on Day 7. The elimination

phase t_1/2values were calculated to be 31.9 17.7 h and 49.4

17.7 h for Day 1 and Day 7, respectively. Notably, plasma

collected before the administration of the ﬁnal dose on Day 7

contained approximately 150 μM of 33, which is consistent

with the observed long t_1/2and suggests that steady state levels

were achieved after 6 to 7 days of QD oral administration

(Figure 6, B).

In Vivo Activity: PK−PD Correlations of Analogue 33.

In order to establish the in vivo target engagement, provide

proof of in vivo activity, and document PK−PD correlations, we

studied the eﬀect of single and 7-day dosing of 33 on the level

of plasma RBP4 in rats. Aliquots of plasma samples collected

during the PK experiments were used to analyze plasma RBP4

concentrations. After a single 18.2 μmol/kg (5 mg/kg) oral

dose of 33, a maximum of 85% decrease in plasma RBP4 was

N

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Table 4. RBP4 SPA Binding Aﬃnity and RBP4 HTRF for Pyridine and Other Heteroaromatic Carboxylic Acid Analogues

a

b

IC₅₀values for the SPA assay obtained in the presence of a ﬁxed, 10 nM concentration of ³H-retinol. IC₅₀values for the HTRF assay obtained in

c

the presence of 1 μM concentration of retinol. For compounds tested multiple times (more than twice), the IC₅₀data are represented as the mean

standard deviation. For those compounds that were only tested twice, the IC₅₀data are shown as the mean of two independent experiments and

not as the mean standard deviation. ND = not determined.

Table 5. In Vitro ADMET Proﬁle for 33

MDCK-MDR1 permeability P_app

b

d

hepatic CL_int(μL/mL/min)

% PPB

(×10⁻⁶cm/s)

c

hERG

a

solubility

H

R

M

cyno CYP inhibition (IC₅₀) 2C9, 2C19, 2D6, 3A4

0.7 All >34 μM

(IC₅₀)

H

R

A-B

74.7

B-A

82.2

class

>100 μM

<0.7

>30 μM

99.9

99.4

high permeability

a

b

c

Kinetic solubility measured in PBS (pH 7.4). Hepatic intrinsic clearance; H = human; R = rat; M = mouse; cyno = cynomolgus monkey. Patch-

Xpress’ patch-clamp assay; compounds were tested (n = 3) in a ﬁve-point concentration−response on HE293 cells stably expressing the hERG

d

channel. % PPB = plasma protein binding.

exhibiting exquisite in vitro RBP4 potency (SPA IC₅₀= 12.8

0.4 nM and HTRF IC₅₀= 43.6 10.5 nM) and excellent in vitro

microsomal stability. Follow-up analogues of 33 that explored the

6-methylpyrimidine-4-carboxylic acid region of the scaﬀold were

subsequently pursued. Although these analogues displayed in-

triguing SAR trends, none of them proved to be better than 33 in

terms of in vitro RBP4 potency. Concurrent with this campaign,

SAR exploration of the aryl headgroup of 33 was also conducted.

Similar to the aforementioned 6-methylpyrimidine-4-carboxylic

acid SAR eﬀort, the aryl headgroup campaign did not yield a more

potent compound than 33.

CONCLUSIONS

■

We sought to investigate whether a 6-methylpyrimidine-4-car-

boxylic acid fragment could serve as a suitable isostere for the

anthranilic acid moiety of 3 and the previously reported bicyclic

[3.3.0]-octahydrocyclopenta[c]pyrrolo RBP4 antagonist 4

with the goal of further improving RBP4 potency and drug-

like characteristics. We initially investigated probe analogues

(10, 14, 18, and 33) that featured diverse cores in which the

ortho-triﬂuoromethylphenyl headgroup of 3 and 4 was retained.

Of this focused sample set of compounds, bicyclic [3.3.0]-

octahydrocyclopenta[c]pyrrolo 33 emerged as a standout analogue

O

DOI: 10.1021/acs.jmedchem.5b00423

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Journal of Medicinal Chemistry

Article

a

Table 6. In Vivo PK Data for Analogue 33 Following IV and PO Administration in Rats

b

c

d

e

f

g

i

route

dose (μmol/kg)

C₀(μM)

CL (mL/h/kg)

t_1/2(h)

V_ss(mL/kg)

AUC_last(μM·h)

AUC_INF(μM·h)

% F

IV

5.1

58.6 (1.8)

5.06 (0.49)

22.0 (3.0)

156 (9.0)

795 (48)

1017 (93)

NA

h

d

e

f

g

i

h

route

dose (μmol/kg)

C_max(μM)

T_max(h)

t_1/2(h)

V_ss(mL/kg)

AUC_last(μM·h)

AUC_INF(μM·h)

% F

PO

12.7

62.2 (4.3)

2.67 (1.15)

38.8 (17.3)

NA

2011 (326)

3636 (1588)

143 (62.4)

a

̈

Data are represented as the mean with standard deviation in parentheses (mean (SD)). Dosing groups consisted of three drug naıve adult male

Sprague−Dawley rats. IV administration: the test article was administered at the 5.1 μmol/kg (2 mg/kg) dose; test article vehicle = 3% DMA/45%

PEG300/12% EtOH/40% sterile H₂O; PO administration, test article was administered at the 12.7 μmol/kg (5 mg/kg) dose; vehicle = 2% Tween

80 in 0.9% saline. Bioavailability; F = (AUC_INFpo× dose_iv) ÷ AUC_INFiv× dose_po). Observed initial concentration of compound in plasma at time

zero. Total body clearance. Apparent half-life of the terminal phase of elimination of the compound from plasma. Volume of distribution at steady

state. Area under the plasma concentration versus time curve from 0 to the last time point that the compound was quantiﬁable in plasma. Area

under the plasma concentration versus time curve from 0 to inﬁnity. Maximum observed concentration of the compound in plasma. Time of

b

c

d

e

f

g

h

i

maximum observed concentration of the compound in plasma.

Figure 6. (A) Plasma exposure levels of analogue 33 in Sprague−Dawley rats following a single (Day 1) and 7-day (Day 7) QD oral dose

̈

administration at 18.2 μmol/kg (5 mg/kg). Data represented as the mean (SD). Each dosing group consisted of three drug naive adult male

Sprague−Dawley rats; the test article vehicle was 2% Tween 80 in 0.9% saline. The diﬀerence in plasma compound concentration between Day 1

and Day 7 samples was statistically signiﬁcant at all time points studied (P < 0.05; two-tailed unpaired Student’s t test). (B) Plasma concentration of

analogue 33 at predose time points during the 7-day QD oral dose administration in three rats from Group 3. Data represented as the mean (SD).

The predose compound concentration was statistically signiﬁcantly increased at Day 7 in comparison to the Day 2 predose level (P < 0.05; two-tailed

unpaired Student’s t test). (C) Pharmacokinetic parameters for 33 in Sprague−Dawley rats after a single dose (Day 1) and seven QD oral doses

(Day 7) at 18.2 μmol/kg. Data are represented as the mean with standard deviation shown in parentheses.

were used as received from vendors, and no attempts were made to

purify or dry these components further. Removal of solvents under

reduced pressure was accomplished with a Buchi rotary evaporator at

approximately 28 mmHg pressure using a Teﬂon-linked KNF vacuum

pump. Thin layer chromatography was performed using 1″ × 3″

AnalTech No. 02521 silica gel plates with ﬂuorescent indicator.

Visualization of TLC plates was made by observation with short wave

UV light (254 nm lamp), 10% phosphomolybdic acid in ethanol, or in

iodine vapors. Preparative thin layer chromatography was performed

using Analtech, 20 × 20 cm, 1000 μm preparative TLC plates. Flash

column chromatography was carried out using a Teledyne Isco

CombiFlash Companion Unit with RediSep Rf silica gel columns. If

needed, products were puriﬁed by reverse phase chromatography,

using a Teledyne Isco CombiFlash Companion Unit with RediSep

Gold C18 reverse phase column. Proton NMR spectra were obtained

Analogue 33 was further evaluated in vitro and in vivo and

was found to possess a favorable ADME proﬁle, no limiting oﬀ-

target in vitro pharmacology, and excellent PK characteristics.

In addition, the compound induced a robust and sustained

lowering (>90%) of serum RBP4 levels upon 7-day QD oral

dosing studies conducted in rats. Taken collectively, these data

suggest that compound 33 shows particular promise as a

potential oral treatment for dry AMD and Stargardt disease.

EXPERIMENTAL SECTION

■

General Chemistry. All reactions were performed under a dry

atmosphere of nitrogen unless otherwise speciﬁed. Indicated reaction

temperatures refer to the reaction bath, while room temperature (rt) is

noted as 25 °C. Commercial grade reagents and anhydrous solvents

P

DOI: 10.1021/acs.jmedchem.5b00423

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Journal of Medicinal Chemistry

Article

Figure 7. (A) Plasma RBP4 levels (μg/mL) in male Sprague−Dawley rats following single (Day 1, Group 1) and 7-day (Day 7, Group 3) 18.2 μmol/kg

(5 mg/kg) QD dose administration of analogue 33. Data represented as the mean (SD). (B) Plasma RBP4 levels (μg/mL) at predose time-points

during the 7-day 5 mg/kg QD oral dose administration of analogue 33 (Group 3) or test article vehicle (Group 2) in Sprague−Dawley rats. Data

represented as the mean (SD).

either on a 300 MHz Bruker Nuclear Magnetic Resonance Spectrometer

or on 500 MHz Bruker Nuclear Magnetic Resonance Spectrometer,

and chemical shifts (δ) are reported in parts per million (ppm). Cou-

pling constant (J) values are given in Hz, with the following spectral

pattern designations: s, singlet; d, doublet; t, triplet, q, quartet; dd,

doublet of doublets; m, multiplet; and br, broad. Tetramethylsilane

was used as an internal reference. Melting points are uncorrected and

were obtained using a MEL-TEMP electrothermal melting point

apparatus. Mass spectroscopic analyses were performed using positive

mode electron spray ionization (ESI) on a Varian ProStar LC-MS with

a 1200L quadrapole mass spectrometer. High pressure liquid chro-

matography (HPLC) purity analysis was performed using a Varian Pro

Star HPLC system with a binary solvent system A and B using gradient

elusion [A, H₂O with 0.05% triﬂuoroacetic acid (TFA); B, CH₃CN

with 0.05% TFA] and a ﬂow rate = 1 mL/min, with UV detection at

223 nm. All ﬁnal compounds were puriﬁed to ≥95% purity, and these

purity levels were measured by a Varian Pro Star HPLC system. The

following Varian Pro Star HPLC methods were used to establish

compound purity: (A) Phenomenex C18(2) column (3.0 × 250 mm);

mobile phase, A = H₂O with 0.05% TFA and B = CH₃CN with 0.05%

TFA; gradient, 0−90% B (0.0−20.0 min); UV detection at 254 nm.

(B) Phenomenex C18(2) column (3.0 × 250 mm); mobile phase,

A = H₂O with 0.05% TFA and B = CH₃CN with 0.05% TFA; gradient,

0−100% B (0.0−20 min); UV detection at 254 nm.

6-Methyl-2-(4-(2-(triﬂuoromethyl)phenyl)piperidin-1-yl)-

pyrimidine-4-carboxylic Acid (10). Step A. To a solution of 1-bromo-

2-(triﬂuoromethyl)benzene (5, 35.0 g, 156 mmol) in THF (350 mL)

cooled to −78 °C was slowly added a solution of n-BuLi (70.4 mL,

2.5 M in THF, 176 mmol) over a period of 15 min. The mixture

stirred at −78 °C for 40 min was allowed to warm to 0 °C and then

cooled back down to −78 °C. To this was added a solution of

1-benzylpiperidin-4-one (22.1 g, 117 mmol) in THF (80 mL) over a

period of 10 min. The resulting mixture continued to stir at −78 °C

for 2 h. The reaction was carefully quenched with aqueous, saturated

aq NH₄Cl solution (500 mL), and the mixture was extracted with

EtOAc (300 mL). The organic extract was washed with H₂O, brine,

dried over Na₂SO4, ﬁltered, and concentrated under reduced pressure.

The resulting residue was chromatographed over silica gel (Isco

CombiFlash Companion unit, 330 g RediSep column, 0−30% EtOAc

in hexanes) to give 1-benzyl-4-(2-(triﬂuoromethyl)phenyl)piperidin-4-

ol (6) as a light-yellow oil (29.2 g, 74%): ¹H NMR (500 MHz, CDCl₃)

δ 7.78 (d, J = 1.6 Hz, 1H), 7.59 (m, 1H), 7.47 (m, 1H), 7.36 (m, 5H),

7.31 (m, 2H), 3.58 (s, 2H), 2.80 (m, 2H), 2.55 (m, 2H), 2.27 (m, 2H),

1.88 (m, 2H); MS (ESI+) m/z 336 [M + H]⁺.

Step B. A 0 °C cooled solution of 1-benzyl-4-(2-(triﬂuoromethyl)-

phenyl)piperidin-4-ol (6, 29.2 g, 87.1 mmol) in SOCl₂(60 mL) was

stirred for 2 h and was then diluted with CH₂Cl₂(250 mL). The

mixture was carefully poured into a saturated aq NaHCO₃solution

(200 mL). The biphasic mixture was separated, and the aqueous layer

was further extracted with CH₂Cl₂(400 mL). The combined organic

layers were washed with brine, dried over Na₂SO₄, ﬁltered, and con-

centrated. The resulting residue was chromatographed over silica gel

(Isco CombiFlash Companion unit, 330 g RediSep column, 0−30%

EtOAc in hexanes) to give 1-benzyl-4-(2-(triﬂuoromethyl)phenyl)-

1

1,2,3,6-tetrahydropyridine (7) as a light-yellow oil (13.5 g, 49%): H

NMR (500 MHz, CDCl₃) δ 7.63 (d, J = 1.6 Hz, 1H), 7.48 (m, 1H),

7.39 (m, 5H), 7.28 (m, 2H), 5.56 (s, 1H), 0.68 (s, 2H), 3.14 (m, 2H),

2.70 (m, 2H), 2.39 (m, 2H); MS (ESI+) m/z 318 [M + H]⁺.

Step C. A mixture of 1-benzyl-4-(2-(triﬂuoromethyl)phenyl)-

1,2,3,6-tetrahydropyridine (7, 13.6 g, 42.5 mmol), 10% Pd/C (3.0 g),

and ammonium formate (26.8 g, 425 mmol) in CH₃OH (800 mL) was

heated at reﬂux for 2 h. The mixture was cooled to rt and was ﬁltered

over Celite. The ﬁltrate was concentrated, and the resulting residue was

chromatographed over silica gel (Isco CombiFlash Companion unit,

330 g RediSep column, 0−10% CH₃OH with 1% NH₄OH in CH₂Cl₂)

to give 4-(2-(triﬂuoromethyl)phenyl)piperidine as a colorless oil (2.0 g,

1

21%): H NMR (500 MHz, CDCl₃) δ 7.61 (d, J = 1.7 Hz, 1H), 7.52

(m, 2H), 7.29 (m, 1H), 3.21 (m, 2H), 3.07 (m, 1H), 2.80 (m, 2H), 2.33

(bs, 1H), 1.77 (m, 4H); MS (ESI+) m/z 230 [M + H]⁺. To a solution

of 4-(2-(triﬂuoromethyl)phenyl)piperidine (5.6 g, 24.5 mmol) in

CH₃CN (30 mL) was added a 4 M solution of HCl in 1,4-dioxane

(6.1 mL, 24.5 mmol) at rt. The mixture was stirred for 2 h and was then

concentrated under reduced pressure to give 4-(2-(triﬂuoromethyl)-

phenyl)piperidine hydrochloride (8) as a white solid (6.4 g, >99%): MS

(ESI+) m/z 230 [M + H]⁺.

Step D. A mixture of 4-(2-(triﬂuoromethyl)phenyl)piperidine

hydrochloride (8, 0.050 g, 0.19 mmol), methyl 2-chloro-6-methylpyr-

imidine-4-carboxylate (0.046 g, 0.21 mmol), and i-Pr₂NEt (0.04 mL,

0.23 mmol) in DMF (2 mL) was heated at 60 °C for 16 h. The

reaction was concentrated under reduced pressure, and the resulting

residue was chromatographed over silica gel (0% to 100% EtOAc in

hexanes) to give methyl 6-methyl-2-(4-(2-(triﬂuoromethyl)phenyl)-

piperidin-1-yl)pyrimidine-4-carboxylate (9) as an oﬀ-white solid

1

(0.049 g, 68%): mp 109−112 °C; H NMR (500 MHz, CDCl₃) δ

7.70−7.61 (m, 3H), 7.43 (m, 1H), 7.02 (s, 1H), 4.94 (m, 2H), 3.85 (s,

3H), 3.16 (m, 1H), 2.99 (m, 2H), 2.38 (s, 3H), 1.79−1.68 (m, 4H);

MS (ESI+) m/z 380 [M + H]⁺; HPLC >99% (AUC), (Method A),

t_R= 16.8 min.

Q

DOI: 10.1021/acs.jmedchem.5b00423

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Journal of Medicinal Chemistry

Article

Step E. A solution of methyl 6-methyl-2-(4-(2-(triﬂuoromethyl)-

phenyl)piperidin-1-yl)pyrimidine-4-carboxylate (9, 0.049 g, 0.13 mmol)

and LiOH·H₂O (0.030 g, 0.72 mmol) in THF (2 mL) and H₂O (2 mL)

was stirred at rt for 16 h. The mixture was acidiﬁed to pH 5 with 2 N aq

HCl and extracted with CH₂Cl₂(3 × 10 mL). The combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to give 6-methyl-2-(4-(2-

(triﬂuoromethyl)phenyl)piperidin-1-yl)pyrimidine-4-carboxylic acid

(10) as an oﬀ-white solid (0.043 g, 91%): mp 166−169 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 13.27 (bs, 1H), 7.70−7.61 (m, 3H),

7.43 (m, 1H), 6.99 (s, 1H), 4.98 (m, 2H), 3.13 (m, 1H), 2.98 (m, 2H),

2.37 (s, 3H), 1.75−1.72 (m, 4H); MS (ESI+) m/z 366 [M + H]⁺;

HPLC >99% (AUC), (Method A), t_R= 15.1 min.

6-Methyl-2-(4-(2-(triﬂuoromethyl)phenyl)piperazin-1-yl)-

pyrimidine-4-carboxylic Acid (14). Step A. To a mixture of tert-butyl

piperazine-1-carboxylate (1.0 g, 5.37 mmol), 1-bromo-2-(triﬂuoromethyl)-

benzene (5, 1.0 mL, 7.34 mmol), BINAP (0.135 g, 0.22 mmol), and

NaOt-Bu (0.670 g, 6.57 mmol) in toluene (20 mL) was added Pd(OAc)₂

(0.024 g, 0.11 mmol). The mixture was heated at 110 °C for 16 h then

allowed to cool to rt. The mixture was ﬁltered over Celite, and the ﬁlter

cake was washed with EtOAc. The ﬁltrate was concentrated under

reduced pressure, and the resulting residue was puriﬁed by ﬂash column

chromatography (Isco CombiFlash Rf unit, 40 g Gold RediSep column,

0%−100% EtOAc in hexanes) to give tert-butyl 4-(2-(triﬂuoromethyl)-

phenyl)piperazine-1-carboxylate (11) as an oﬀ-white solid (1.63 g, 92%):

¹H NMR (300 MHz, CDCl₃) δ 7.64 (m, 1H), 7.55 (m, 1H), 7.33 (m,

column chromatography (Isco CombiFlash Rf unit, 40 g Gold RediSep

column, 0%−100% EtOAc in hexanes) to give (3aR,6aS)-tert-butyl 5-(2-

(triﬂuoromethyl)phenyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carbox-

1

ylate (15) as an oﬀ-white solid (1.35 g, 81%): H NMR (300 MHz,

CDCl₃) δ 7.61 (m, 1H), 7.46 (m, 1H), 7.17 (m, 1H), 7.02 (m, 1H),

3.70 (m, 2H), 3.38−3.28 (m, 4H), 3.14 (m, 2H), 2.93 (m, 2H); MS

(ESI+) m/z 357 [M + H]⁺.

Step B. A solution of (3aR,6aS)-tert-butyl 5-(2-(triﬂuoromethyl)-

phenyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (15, 1.37 g,

3.84 mmol) and 2.0 M HCl in Et₂O (10 mL, 20 mmol) in CH₂Cl₂

(10 mL) was stirred at rt for 3 h. The mixture was concentrated under

reduced pressure to provide (3aR,6aS)-2-(2-(triﬂuoromethyl)phenyl)-

octahydropyrrolo[3,4-c]pyrrole hydrochloride (16) as an oﬀ-white

1

solid (0.968 g, 86%): H NMR (300 MHz, CDCl₃) δ 9.18 (bs, 1H),

10.08 (bs, 1H), 9.56 (bs, 1H), 7.64 (m, 1H), 7.53 (m, 1H), 7.43 (m,

1H), 7.21 (m, 1H), 3.72 (m, 2H), 3.12 (m, 8H); MS (ESI+) m/z 257

[M + H]⁺.

Step C. A mixture of (3aR,6aS)-2-(2-(triﬂuoromethyl)phenyl)-

octahydropyrrolo[3,4-c]pyrrole hydrochloride (16, 0.100 g, 0.34 mmol),

methyl 2-chloro-6-methylpyrimidine-4-carboxylate (0.070 g, 0.38 mmol),

and i-Pr₂NEt (0.1 mL, 0.46 mmol) in DMF (4 mL) was heated at 60 °C

for 16 h. The reaction was concentrated under reduced pressure, and

the resulting residue was chromatographed over silica gel (0% to 10%

CH₃OH in CH₂Cl₂) to give methyl 6-methyl-2-((3aR,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-

pyrimidine-4-carboxylate (17) as an oﬀ-white solid (0.039 g, 28%): ¹H

NMR (300 MHz, DMSO-d₆) δ 7.60−7.51 (m, 3H), 7.34 (m, 1H),

7.08 (s, 1H), 3.87 (m, 5H), 3.47−3.38 (m, 4H), 3.14−3.07 (m, 4H),

2.37 (s, 3H); MS (ESI+) m/z 407 [M + H]⁺; HPLC >99% (AUC),

(Method A), t_R= 19.1 min.

1H), 7.21 (m, 1H), 3.57 (m, 4H), 2.88 (m, 4H), 1.48 (s, 9H); MS (ESI+)

m/z 331 [M + H]⁺.

Step B. A solution of tert-butyl 4-(2-(triﬂuoromethyl)phenyl)-

piperazine-1-carboxylate (11, 1.63 g, 4.93 mmol) and 2.0 M HCl in

Et₂O (10 mL, 20 mmol) in CH₂Cl₂(10 mL) was stirred at rt for 3 h.

The mixture was concentrated under reduced pressure to provide

1-(2-(triﬂuoromethyl)phenyl)piperazine hydrochloride (12) as an

oﬀ-white solid (1.23 g, 94%): ¹H NMR (300 MHz, DMSO-d₆) δ

9.18 (bs, 1H), 7.74 (m, 2H), 7.55 (m, 1H), 7.38 (m, 1H), 3.36 (m,

4H), 3.07 (m, 4H); MS (ESI+) m/z 231 [M + H]⁺.

Step D. A mixture of 6-methyl-2-((3aR,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidine-4-carbox-

ylate (17, 0.031 g, 0.076 mmol) and LiOH·H₂O (0.031 g, 0.74 mmol)

in THF (2 mL) and H₂O (1 mL) was stirred at rt for 16 h. The

mixture was acidiﬁed to pH 5 with 2 N aq HCl and extracted with

CH₂Cl₂(3 × 10 mL). The combined organic extracts were washed

with brine, dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure to give 6-methyl-2-(4-(2-(triﬂuoromethyl)phenyl)-

piperazin-1-yl)pyrimidine-4-carboxylic acid (18) as an oﬀ-white solid

Step C. A mixture of 1-(2-(triﬂuoromethyl)phenyl)piperazine

hydrochloride (12, 0.100 g, 0.37 mmol), methyl 2-chloro-6-methyl-

pyrimidine-4-carboxylate (0.076 g, 0.41 mmol), and i-Pr₂NEt (0.15 mL,

0.86 mmol) in DMF (3 mL) was heated at 60 °C for 16 h. The reaction

was concentrated under reduced pressure, and the resulting residue was

chromatographed over silica gel (0% to 100% EtOAc in hexanes) to give

methyl 6-methyl-2-(4-(2-(triﬂuoromethyl)phenyl)piperazin-1-yl)-

pyrimidine-4-carboxylate (13) as an oﬀ-white solid (0.089 g, 63%):

1

(0.043 g, 91%): H NMR (300 MHz, DMSO-d₆) δ 13.53 (bs, 1H),

7.61−7.58 (m, 2H), 7.34 (m, 1H), 7.10 (m, 1H), 6.99 (s, 1H), 3.86

(m, 2H), 3.47−3.35 (m, 4H), 3.14 (m, 4H), 2.36 (s, 3H); MS (ESI+)

m/z 393 [M + H]⁺; HPLC 97.6% (AUC), (Method A), t_R= 16.8 min.

(3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)octahydrocyclopenta-

[c]pyrrole Hydrochloride (26). Step A (i). To a 0 °C cooled solution of

LiAlH₄in THF (1.0 M, 800 mL, 800 mmol) in THF (800 mL) was

carefully added (3aR,7aS)-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-

dione (19, 53.7 g, 0.35 mol) portion-wise. An exotherm of ∼5 °C

occurred upon each addition of 19. Upon complete addition, the mix-

ture was allowed to warm to rt followed by heating at 70 °C for 16 h.

The mixture was allowed to cool back to rt and then further cooled to

0 °C. The reaction was carefully quenched by slow addition of H₂O

(30 mL), 15% aq NaOH solution (30 mL), followed by another bolus

of H₂O (90 mL). The rate of quenching was done carefully so as to

maintain an internal temperature below 25 °C. The mixture was stirred

for 1 h and was ﬁltered through Celite. The aqueous ﬁltrate was

extracted with Et₂O (2 × 100 mL), and the organic extracts were

combined and concentrated under reduced pressure. The resulting

residue was puriﬁed using a Kugelrohr distillation apparatus to give

(3aR,7aS)-2,3,3a,4,7,7a-hexahydro-1H-isoindole as a clear, colorless oil

(19.45 g, 44%): ¹H NMR (500 MHz, CDCl₃) δ 5.29 (s, 2H), 3.88 (bs,

1H), 3.26 (m, 2H), 2.82 (m, 2H), 2.41−2.19 (m, 4H), 1.96 (m, 2H).

Step B. To a 0 °C cooled solution of (3aR,7aS)-2,3,3a,4,7,7a-

hexahydro-1H-isoindole (11.5 g, 93.5 mmol) in CH₂Cl₂(200 mL) was

added Boc₂O (24.5 g, 112 mmol) and the mixture stirred at rt for 16 h.

The mixture was washed with H₂O (100 mL), brine (100 mL), dried

over Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash column chromatography (Isco CombiFlash

Rf unit, 330 g RediSep column, 0% to 30% EtOAc in hexanes) to give

(3aR,7aS)-tert-butyl 3a,4,7,7a-tetrahydro-1H-isoindole-2(3H)-carboxylate

1

mp 103−106 °C; H NMR (300 MHz, DMSO-d₆) δ 7.71−7.57 (m,

3H), 7.39 (m, 1H), 7.06 (s, 1H), 3.90 (m, 4H), 3.85 (s, 3H), 2.94 (m,

4H), 2.39 (s, 3H); MS (ESI+) m/z 381 [M + H]⁺; HPLC 98.9%

(AUC), (Method A), t_R= 16.5 min.

Step D. A mixture of methyl 6-methyl-2-(4-(2-(triﬂuoromethyl)-

phenyl)piperazin-1-yl)pyrimidine-4-carboxylate (13, 0.080 g, 0.21 mmol)

and LiOH·H₂O (0.050 g, 1.19 mmol) in THF (2 mL) and H₂O (2 mL)

was stirred at rt for 16 h. The mixture was acidiﬁed to pH 5 with 2 N aq

HCl and extracted with CH₂Cl₂(3 × 10 mL). The combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and concen-

trated under reduced pressure to give 6-methyl-2-(4-(2-(triﬂuoromethyl)-

phenyl)piperazin-1-yl)pyrimidine-4-carboxylic acid (14) as an oﬀ-white

solid (0.043 g, 91%): ¹H NMR (300 MHz, DMSO-d₆) δ 13.53 (bs, 1H),

7.91−7.80 (m, 3H), 7.58 (m, 1H), 7.01 (s, 1H), 4.12 (m, 4H), 2.70 (m,

4H), 2.37 (s, 3H); MS (ESI+) m/z 367 [M + H]⁺; HPLC >99% (AUC),

(Method A), t_R= 14.8 min.

6-Methyl-2-((3aR,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

pyrrolo[3,4-c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic Acid (18). Step

A. To a mixture of (3aR,6aS)-tert-butyl hexahydropyrrolo[3,4-c]-

pyrrole-2(1H)-carboxylate (1.0 g, 4.70 mmol), 1-bromo-2-(triﬂuoro-

methyl)benzene (5, 1.0 mL, 7.34 mmol), BINAP (0.117 g, 0.19 mmol),

and NaOt-Bu (0.587 g, 6.11 mmol) in toluene (10 mL) was added

Pd(OAc)₂(0.021 g, 0.10 mmol). The mixture was heated at 110 °C for

16 h then allowed to cool to rt. The mixture was ﬁltered over Celite, and

the ﬁlter cake was washed with EtOAc. The ﬁltrate was concentrated

under reduced pressure, and the resulting residue was puriﬁed by ﬂash

R

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(20) as an oil (20.10 g, 49%): ¹H NMR (500 MHz, CDCl₃) δ 5.64 (s,

2H), 3.39 (m, 2H), 3.20 (m, 2H), 3.15 (m, 2H), 2.23−2.19 (m, 4H),

1.97 (m, 2H), 1.57 (s, 9H).

using a Parr Shaker apparatus at rt for 16 h. The mixture was ﬁltered

through Celite, and the ﬁltrate was concentrated under reduced pres-

sure. The resulting residue was puriﬁed by ﬂash column chromatog-

raphy (Isco CombiFlash Rf unit, 40 g RediSep column, 0%−30% EtOAc

in hexanes) to give (3aR,5r,6aS)-tert-butyl 5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (25) as a

clear, viscous oil (0.910 g, 85%): H NMR (500 MHz, CDCl₃) δ

7.69 (m, 1H), 7.51 (m, 2H), 7.25 (m, 1H), 3.49 (m, 5H), 2.75 (m, 2H),

2.92 (m, 2H), 1.52 (m, 2H), 1.48 (s, 9H).

Step C. To a 0 °C cooled mixture of (3aR,7aS)-tert-butyl 3a,4,7,7a-

tetrahydro-1H-isoindole-2(3H)-carboxylate (20, 66.78 g, 224 mmol)

in CH₃CN (600 mL), CCl₄(400 mL), and H₂O (800 mL) was added

NaIO₄(192.3 g, 899 mmol) followed by RuO₂·H₂O (1.19 g, 8.94 mmol).

The mixture was stirred at rt for 24 h with mechanical stirring and then

ﬁltered through Celite. The ﬁlter cake was washed with 10% CH₃OH

in CH₂Cl₂(200 mL), and the biphasic mother liquor was separated.

The aqueous phase was further extracted with CH₂Cl₂(3 × 150 mL),

and the combined organic extracts were washed with H₂O (100 mL),

brine (100 mL), dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The residue was ﬁltered through a plug of silica

gel using a CH₃OH/CH₂Cl₂eluent system (2%−10% CH₃OH in

CH₂Cl₂). The ﬁltrate was concentrated under reduced pressure to give

2,2′-((3S,4R)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl)diacetic acid

(21) as a solid (46.75 g, 72%): ¹H NMR (500 MHz, DMSO-d₆) δ 12.2

(s, 2H), 3.38 (m, 2H), 3.02 (m, 2H), 2.49 (m, 2H), 2.32 (m, 2H), 2.29

(m, 2H), 1.42 (s, 9H).

1

Step H. To a 0 °C cooled solution of (3aR,5r,6aS)-tert-butyl 5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrole-2(1H)-car-

boxylate (25, 7.94 g, 22.32 mmol) in CH₂Cl₂(60 mL) was added a 2.0

M HCl solution in Et₂O (60 mL), and the mixture was allowed to stir

at rt for 24 h. The mixture was diluted with Et₂O (200 mL), and the

precipitated product was ﬁltered to give (3aR,5r,6aS)-5-(2-(triﬂuo-

romethyl)phenyl)octahydrocyclopenta[c]pyrrole hydrochloride (26)

1

as a white solid (5.90 g, 91%): H NMR (500 MHz, CDCl₃) δ 10.17

(bs, 1H), 8.06 (m, 1H), 7.59 (m, 1H), 7.53 (m, 1H), 7.27 (m, 1H),

3.42 (m, 2H), 3.38 (m, 3H), 3.01 (m, 2H), 2.36 (m, 2H), 1.96 (m,

2H); MS (ESI+) m/z 256 [M + H]⁺.

Methyl 2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylate (27). Step A.

To a solution of (3aR,5R,6aS)-5-(2-(triﬂuoromethyl)phenyl)octa-

hydrocyclopenta[c]pyrrole hydrochloride (26, 0.050 g, 0.17 mmol)

and Et₃N (0.05 mL, 0.51 mmol) in DMF (10 mL) was added methyl

2-chloropyrimidine-4-carboxylate (0.029 g, 0.17 mmol), and the

resulting solution was stirred at 60 °C for 16 h. The reaction was

diluted with H₂O (200 mL) and extracted with EtOAc (3 × 100 mL).

The combined organic extracts were washed with H₂O (3 × 100 mL),

brine (100 mL), dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The resulting residue was chromatographed

over silica gel (0% to 30% EtOAc in hexanes) to give methyl 2-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)pyrimidine-4-carboxylate (27) as an oﬀ-white solid

(0.055 g, 77%): MS (ESI+) m/z 392 [M + H]⁺.

Methyl 6-Methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylate

(28). Compound 28 was prepared according to a similar procedure

described for the synthesis of 27. ¹H NMR (300 MHz, CDCl₃) δ 7.61

(m, 1H), 7.58 (m, 2H), 7.23 (m, 1H), 7.05 (s, 1H), 3.95 (s, 3H), 3.82

(m, 4H), 3.59 (m, 1H), 2.92 (m, 2H), 2.44 (s, 3H), 2.40 (m, 2H), 1.69

(m, 2H); MS (ESI+) m/z 406 [M + H]⁺; HPLC >99% purity

(Method C), t_R= 16.3 min.

Step D. To a suspension of 2,2′-((3S,4R)-1-(tert-butoxycarbonyl)-

pyrrolidine-3,4-diyl)diacetic acid (21, 6.97 g, 24.31 mmol) in Ac₂O

(50 mL) was added NaOAc (1.99 g, 24.31 mmol), and the mixture

was heated at 120 °C for 3 h. The mixture was allowed to cool to rt

and ﬁltered through Celite. The ﬁlter cake was washed with Et₂O (5 ×

50 mL), and the mother liquor was concentrated under reduced pres-

sure. The resulting residue was puriﬁed by ﬂash column chromatog-

raphy (Isco CombiFlash Rf unit, 120 g RediSep column, 0%−30%

EtOAc in hexanes) to give (3aR,6aS)-tert-butyl 5-oxohexahydro-

cyclopenta[c]pyrrole-2(1H)-carboxylate (22) as a white foam (2.17 g,

1

40%): H NMR (500 MHz, CDCl₃) δ 3.69 (m, 2H), 3.22 (m, 2H),

2.91 (m, 2H), 2.50 (m, 2H), 2.17 (m, 2H), 1.46 (s, 9H).

Step E. To a −78 °C cooled solution of (3aR,6aS)-tert-butyl 5-

oxohexahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (22, 22.35 g,

99.2 mmol) in THF (500 mL) was slowly added a solution of

LiHMDS in THF (1.0 M, 129 mL). The mixture continued to stir at

−78 °C for 30 min, then a solution of 1,1,1-triﬂuoro-N-phenyl-N-

((triﬂuoromethyl)sulfonyl)methanesulfonamide (49.65 g, 139 mmol)

in THF (150 mL) was slowly added. The mixture was stirred for an

additional 1 h at −78 °C and was then allowed to stir at rt for 2 h. The

mixture was concentrated under reduced pressure, and the residue

was puriﬁed by ﬂash column chromatography (Isco CombiFlash Rf

unit, 330 g RediSep column, 0%−50% EtOAc in hexanes) to give

( )-(3aS,6aS)-tert-butyl 5-(((triﬂuoromethyl)sulfonyl)oxy)-3,3a,6,6a-

tetrahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (( )-23) as a

clear, viscous oil (1.56 g, quantitative): ¹H NMR (500 MHz,

CDCl₃) δ 5.58 (s, 1H), 3.62 (m, 1H), 3.53 (m, 1H), 3.46 (m, 2H),

3.19 (m, 1H), 2.95 (m, 2H), 2.46 (m, 1H), 1.47 (s, 9H).

Methyl 6-(triﬂuoromethyl)-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-car-

boxylate (29). Compound 29 was prepared according to a similar

procedure described for the synthesis of 27. MS (ESI+) m/z 460

[M + H]⁺.

Methyl 6-methoxy-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylate

(30). Compound 30 was prepared according to a similar procedure

described for the synthesis of 27. MS (ESI+) m/z 422 [M + H]⁺.

Methyl 6-isopropyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylate

(31). Compound 31 was prepared according to a similar procedure

described for the synthesis of 27. MS (ESI+) m/z 434 [M + H]⁺.

2-((3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic Acid (32). Step

A: A solution of methyl 2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxy-

late (27, 0.050 g, 0.12 mmol) and 2 N aq NaOH (5 mL) in a 1:1

mixture of CH₃OH/THF (10 mL) stirred at rt for 16 h. The mixture

was neutralized at 0 °C with 2 N aq HCl and extracted with CH₂Cl₂

(3 × 10 mL). The combined organic extracts were washed with brine,

dried over Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

The resulting residue was chromatographed over silica gel (0% to 10%

CH₃OH in CH₂Cl₂) to give 2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carbox-

ylic acid (32) as an oﬀ-white solid (0.044 g, 92%): mp 183−187 °C;

¹H NMR (300 MHz, DMSO-d₆) δ 8.56 (s, 1H), 7.71 (m, 1H), 7.65

Step F. To an N₂degassed mixture of ( )-(3aS,6aS)-tert-butyl

5-(((triﬂuoromethyl)sulfonyl)oxy)-3,3a,6,6a-tetrahydrocyclopenta[c]-

pyrrole-2(1H)-carboxylate (( )-23, 14.79 g, 41.4 mmol), 2-triﬂuor-

omethylphenylboronic acid (19.70 g, 104 mmol), and a 2 M aqueous

solution of Na₂CO₃(250 mL) in DME (500 mL) was added

Pd(PPh₃)₄(4.80 g, 4.16 mmol). The mixture was heated at 80 °C for 6

h, then cooled to rt, and diluted with H₂O (500 mL). The aqueous

mixture was extracted with EtOAc (2 × 200 mL), and the combined

organic extracts were washed with H₂O (200 mL), brine (200 mL),

dried over Na₂SO₄, ﬁltered, and concentrated under reduced pres-

sure. The residue was puriﬁed by ﬂash column chromatography (Isco

CombiFlash Rf unit, 330 g RediSep column, 0%−10% EtOAc in

hexanes) to give ( )-(3aR,6aS)-tert-butyl 5-(2-(triﬂuoromethyl)-

phenyl)-3,3a,6,6a-tetrahydrocyclopenta[c]pyrrole-2(1H)-carboxylate

1

(( )-24 as a clear, viscous oil (13.70 g, 94%): H NMR (500 MHz,

CDCl₃) δ 7.65 (m, 1H), 7.47 (m, 2H), 7.25 (m, 1H), 5.58 (s, 1H),

3.85−3.42 (m, 4H), 3.23 (m, 1H), 2.98 (m, 2H), 2.49 (m, 1H), 1.47

(s, 9H).

Step G. A mixture of ( )-(3aR,6aS)-tert-butyl 5-(2-(triﬂuoromethyl)-

phenyl)-3,3a,6,6a-tetrahydrocyclopenta[c]pyrrole-2(1H)-carboxylate

(( )-24, 8.63 g, 24.41 mmol) and 10% Pd/C (1.57 g, wet, 10% w/w) in

CH₃OH (50 mL) was subjected to an atmosphere of H₂gas (40 psi)

(m, 2H), 7.39 (m, 1H), 7.06 (s, 1H), 3.73 (m, 2H), 3.64 (m, 2H), 3.43

S

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Journal of Medicinal Chemistry

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(m, 1H), 2.86 (m, 2H), 2.30 (m, 2H), 1.64 (m, 2H); MS (ESI+) m/z

378 [M + H]⁺; HPLC >99% purity (Method A), t_R= 10.5 min.

6-Methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic

Acid (33). Compound 33 was prepared according to a similar

2-chloropyrimidine-4-carboxylate (0.067 g, 0.39 mmol), and the

resulting solution was stirred at 60 °C for 16 h. The reaction was

diluted with H₂O (200 mL) and extracted with EtOAc (3 × 100 mL).

The combined organic extracts were washed with H₂O (3 × 100 mL),

brine (100 mL), dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The resulting residue was chromatographed over

silica gel (0% to 30% EtOAc in hexanes) to give methyl 6-methyl-2-

((3aR,5s,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)pyrimidine-4-carboxylate as an oﬀ-white solid (0.123 g,

78%): MS (ESI+) m/z 406 [M + H]⁺.

Step D. A solution of methyl 6-methyl-2-((3aR,5s,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

pyrimidine-4-carboxylate (0.100 g, 0.24 mmol) and 2 N aq NaOH

(5 mL) in a 1:1 mixture of CH₃OH/THF (10 mL) stirred at room

temperature for 16 h. The mixture was carefully at 0 °C with 2 N aq

HCl and extracted with CH₂Cl₂(3 × 10 mL). The combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and con-

centrated under reduced pressure. The resulting residue was chro-

matographed over silica gel (0% to 10% CH₃OH in CH₂Cl₂) to give

6-methyl-2-((3aR,5s,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic acid (40) as an

oﬀ-white solid (0.089 g, 93%): mp 175−178 °C; ¹H NMR (300 MHz,

CDCl₃) δ 7.68−7.45 (m, 3H), 7.29 (m, 1H), 7.15 (s, 1H), 4.00 (m,

2H), 3.75 (m, 1H), 3.46 (m, 2H), 3.17 (m, 2H), 2.48 (s, 3H), 2.17−

1.90 (m, 4H); MS (ESI+) m/z 392 [M + H]⁺; HPLC >99% purity

(Method A), t_R= 14.2 min.

5,6-Dimethyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic

Acid (41). Step A. To a mixture of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)octahydrocyclopenta[c]pyrrole hydrochloride (26, 0.400 g,

1.37 mmol), 2,4-dichloro-5,6-dimethylpyrimidine (0.242 g, 1.37 mmol),

JohnPhos (0.048 g, 0.137 mmol), and Cs₂CO₃(1.34 g, 4.11 mmol) in

toluene (10 mL) was added Pd(OAc)₂(0.015 g, 0.068 mmol). The

mixture was heated at reﬂux for 16 h then allowed to cool to rt. The

mixture was ﬁltered over Celite, and the ﬁlter cake was washed with

EtOAc. The ﬁltrate was concentrated under reduced pressure, and the

resulting residue was puriﬁed by ﬂash column chromatography (Isco

CombiFlash Rf unit, 40 g Gold RediSep column, 0%−15% EtOAc in

hexanes) to give (3aR,5r,6aS)-2-(4-chloro-5,6-dimethylpyrimidin-2-yl)-5-

(2-(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]pyrrole as a white

solid (0.271 g, 50%): MS (ESI+) m/z 396 [M + H]⁺.

Step B. To a mixture of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

octahydrocyclopenta[c]pyrrole hydrochloride (26, 0.200 g, 0.50 mmol),

Mo(CO)₆(0.160 g, 0.60 mmol), and BINAP (0.009 g, 0.05 mmol) in

CH₃OH (10 mL), and CH₃CN (10 mL) was added Pd(OAc)₂(0.005 g,

0.024 mmol). The mixture was heated at 80 °C for 16 h then allowed

to cool to rt. The mixture was ﬁltered over Celite, and the ﬁlter cake

was washed with EtOAc. The ﬁltrate was concentrated under reduced

pressure, and the resulting residue was puriﬁed by ﬂash column

chromatography (Isco CombiFlash Rf unit, 40 g Gold RediSep

column, 0%−15% EtOAc in hexanes) to give methyl 5,6-dimethyl-2-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)pyrimidine-4-carboxylate as a white solid (0.132 g,

63%): MS (ESI+) m/z 420 [M + H]⁺.

1

procedure described for the synthesis of 32. Mp 129−131 °C; H

NMR (300 MHz, DMSO-d₆) δ 7.73 (m, 1H), 7.64 (m, 2H), 7.36, (m,

1H), 6.51, (s, 1H), 3.90 (s, 3H), 3.76 (m, 2H), 3.72, (m, 2H), 2.88 (m,

2H), 2.22 (m, 2H), 1.67 (m, 2H); ¹³C NMR (300 MHz, DMSO-d₆) δ

169.02, 166.47, 160.56, 157.04, 142.84, 132.72, 128.29, 127.09, 126.71,

126.34, 125.28, 125.19, 122.80, 107.82, 52.08, 42.65, 42.28, 41.19,

23.94; MS (ESI+) m/z 408 [M + H]⁺; HRMS (ESI+) for

C₂₀H₂₁F₃N₃O₂[M + H]⁺calcd 392.1586, found 392.1570; HPLC

>99% purity (Method A), t_R= 14.3 min.

6-(Triﬂuoromethyl)-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic

Acid (34). Compound 34 was prepared according to a similar proce-

1

dure described for the synthesis of 32. Mp 155−156 °C; H NMR

(500 MHz, DMSO-d₆) δ 13.92 (bs, 1H), 7.57 (m, 1H), 7.65 (m, 1H),

7.59 (m, 1H), 7.40 (m, 1H), 7.33 (s, 1H), 3.78 (m, 2H), 3.68 (m, 2H),

3.30 (m, 1H), 2.93 (m, 2H), 2.29 (m, 2H), 1.64 (m, 2H); MS (ESI+)

m/z 446 [M + H]⁺; HPLC >99% purity (Method A), t_R= 12.9 min.

6-Methoxy-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic

Acid (35). Compound 35 was prepared according to a similar proce-

1

dure described for the synthesis of 32. Mp 171−174 °C; H NMR

(300 MHz, CDCl₃) δ 7.73 (m, 1H), 7.64 (m, 2H), 7.36, (m, 1H),

6.51, (s, 1H), 3.90 (s, 3H), 3.76 (m, 2H), 3.72, (m, 2H), 2.88 (m, 2H),

2.22 (m, 2H), 1.67 (m, 2H); MS (ESI+) m/z 408 [M + H]⁺; HPLC

>99% purity (Method A), t_R= 16.2 min.

6-Isopropyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic

Acid (36). Compound 36 was prepared according to a similar proce-

1

dure described for the synthesis of 32. H NMR (300 MHz, DMSO-

d₆) δ 13.21 (bs, 1H), 7.72 (m, 1H), 7.65, (m, 2H), 7.39 (m, 2H), 7.01,

(s, 1H), 3.75 (m, 2H), 3.71, (m, 2H), 3.42 (m, 1H), 2.90, (m, 3H),

2.49 (m, 2H), 1.66 (m, 2H), 1.23 (s, 6H); MS (ESI+) m/z 420 [M +

H]⁺; HPLC >99% purity (Method A), t_R= 13.9 min.

6-Methyl-2-((3aR,5s,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic

Acid (40). Step A. To a solution of ( )-(3aR,6aS)-tert-butyl 5-(2-

(triﬂuoromethyl)phenyl)-3,3a,6,6a-tetrahydrocyclopenta[c]pyrrole-

2(1H)-carboxylate (( )-24, 120 mg, 0.34 mmol) in CH₂Cl₂(3 mL)

was added TFA (3 mL) and the resulting solution stirred at rt for 3 h.

The residue was dissolved in CH₂Cl₂(25 mL) and washed with

saturated aq NaHCO₃solution (25 mL), brine (25 mL), dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure to provide

( )-(3aS,6aR)-5-(2-(triﬂuoromethyl)phenyl)-1,2,3,3a,4,6a-hexahydro-

cyclopenta[c]pyrrole (( )-37) as an oﬀ-white solid (0.146 mg,

1

> 99%): H NMR (300 MHz, CDCl₃) δ 9.21 (br s, 1H), 8.18 (br s,

1H), 7.67 (d, J = 7.8 Hz, 1H), 7.51−7.34 (m, 3H), 5.57 (s, 1H), 3.82

(br s, 1H), 3.62−3.54 (m, 2H), 3.39−3.09 (m, 4H), 2.62−2.56 (m, 1H).

Step B. To a solution of ( )-(3aS,6aR)-5-(2-(triﬂuoromethyl)-

phenyl)-1,2,3,3a,4,6a-hexahydrocyclopenta[c]pyrrole (( )-37, 0.680 g,

1.92 mmol) in CH₃OH (25 mL) was added Pd/C (10% w/w, Degussa

type E101 NE/W, 0.140 g). The mixture was subjected to an

atmosphere of H₂(50 psi) at rt for 6 h and was ﬁltered through Celite.

The ﬁltrated was concentrated under reduced pressure, and the result-

ing residue was puriﬁed by reversed phase column chromatography

(Isco C18 Reversed Phase Gold column, 10%−30% CH₃CN in H₂O

with 0.05% TFA). The resulting material was dissolved in CH₂Cl₂and

washed with saturated aqueous NaHCO₃, dried over Na₂SO₄, ﬁltered,

and concentrated under reduced pressure to give (3aR,5s,6aS)-5-(2-

(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]pyrrole (39) as a

Step C. A mixture of 5,6-dimethyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoro-

methyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-

carboxylate (0.130 g, 0.309 mmol) and LiOH·H₂O (0.038 g, 0.929 mmol)

in THF (4 mL), CH₃OH (4 mL), and H₂O (4 mL) stirred at rt for 16 h.

The mixture was acidiﬁed to pH 6 via the addition of 2 N aq HCl and

then extracted with CH₂Cl₂(3 × 50 mL). The combined organic extracts

were washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The resulting residue was puriﬁed by ﬂash

column chromatography (Isco CombiFlash Rf unit, 40 g RediSep column,

0%−8% CH₃OH in CH₂Cl₂) to give 5,6-dimethyl-2-((3aR,5r,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

pyrimidine-4-carboxylic acid (41) as a white solid (0.115 g, 92%): mp

140−142 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 7.72 (m, 3H), 7.32 (m,

1H), 3.62 (m, 4H), 2.89 (m, 2H), 2.43 (s, 3H), 2.23 (m, 2H), 2.09 (s,

3H), 1.66 (m, 2H); MS (ESI+) m/z 406 [M + H]⁺; HPLC >99%

(AUC), (Method A), t_R= 13.3 min.

1

white solid (0.070 g, 14%): H NMR (300 MHz, CDCl₃) δ 7.61 (d,

J = 7.8 1H), 7.50 (m, 2H), 7.30−7.24 (m, 1H), 3.54−3.42 (m, 1H),

3.32−3.26 (m, 2H), 2.81−2.68 (m, 2H), 2.51−2.46 (m, 2H), 1.84−

1.76 (m, 4H); MS (ESI+) m/z 256 [M + H]⁺.

Step C. To a solution of (3aR,5s,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)octahydrocyclopenta[c]pyrrole (39, 0.100 g, 0.39 mmol) and

Et₃N (0.16 mL, 1.17 mmol) in DMF (10 mL) was added methyl

T

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5-Methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic

Acid (42). Step A. To a solution of (3aR,5R,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)octahydrocyclopenta[c]pyrrole hydrochloride (26, 0.050 g,

0.17 mmol) and Et₃N (0.05 mL, 0.51 mmol) in DMF (10 mL) was

added methyl 2-chloro-5-methylpyrimidine-4-carboxylate (0.031 g,

0.17 mmol), and the resulting solution was stirred at 60 °C for 16 h.

The reaction was allowed to cool to rt and then diluted with H₂O

(200 mL). The aqueous mixture was extracted with EtOAc (3 × 100 mL),

and the combined organic extracts were washed with H₂O (3 × 100 mL),

brine (100 mL), dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The resulting residue was chromatographed over

silica gel (0% to 30% EtOAc in hexanes) to give methyl 5-methyl-2-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)pyrimidine-4-carboxylate as an oﬀ-white solid (0.015 g,

22%): MS (ESI+) m/z 406 [M + H]⁺.

16 h. The mixture was allowed to cool to rt and was diluted with H₂O

(20 mL). The aqueous mixture was extracted with EtOAc (3 ×

20 mL), and the combined organic extracts were washed with H₂O

(3 × 100 mL), brine (100 mL), dried over Na₂SO₄, ﬁltered, and con-

centrated under reduced pressure. The resulting residue was chro-

matographed over silica gel (0% to 30% EtOAc in hexanes) to give

methyl 2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-5-carboxylate as an oﬀ-white

solid (0.035 g, 52%): MS (ESI+) m/z 392 [M + H]⁺.

Step B. A solution of methyl 2-((3aR,5r,6aS)-5-(2-(triﬂuoro-

methyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-5-

carboxylate (0.030 g, 0.071 mmol) and 2 N aq NaOH (5 mL) in a 1:1

mixture of CH₃OH/THF (10 mL) stirred at room temperature for

16 h. The mixture was carefully neutralized at 0 °C with 2 N HCl and

extracted with CH₂Cl₂(3 × 10 mL). The combined organic extracts

were washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The resulting residue was chromatographed

over silica gel (0% to 10% CH₃OH in CH₂Cl₂) to 2-((3aR,5r,6aS)-5-

(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

pyrimidine-5-carboxylic acid (44) as an oﬀ-white solid (0.026 g, 91%):

mp 218−222 °C; ¹H NMR (500 MHz, CDCl₃) δ 8.90 (bs, 1H), 7.32−

7.51 (m, 3H), 7.21 (m, 3H), 3.41−3.91 (m, 5H), 2.89 (m, 2H), 2.32

(m, 2H), 1.64 (m, 2H); MS (ESI+) m/z 378 [M + H]⁺; HPLC >99%

(AUC), (Method A), t_R= 17.6 min.

6-Methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxamide

(46). Step A. A mixture of 5-(2-(triﬂuoromethyl)phenyl)octahy-

drocyclopenta[c]pyrrole hydrochloride (26, 0.068 g, 0.355 mmol),

2-chloro-6-methylpyrimidine-4-carbonitrile (0.054 g, 0.352 mmol),

and i-Pr₂NEt (0.14 mL, 1.05 mmol) in DMF (2 mL) was heated at

60 °C for 16 h. The mixture was allowed to cool to rt and then poured

into brine (50 mL). The aqueous mixture was extracted with EtOAc

(3 × 50 mL), and the combined organic extracts were washed with

saturated NaHCO₃(3 × 50 mL), dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was

chromatographed over silica gel (0−50% EtOAc in hexanes) to aﬀord

6-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carbonitrile (45) as an oﬀ-

white solid (0.072 g, 55%): ESI MS m/z 373 [M + H]⁺.

Step B. A mixture of 6-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoro-

methyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-

carbonitrile (45, 0.049 g, 0.133 mmol) LiOH·H₂O (0.030 g, 0.665

mmol) in THF (4 mL) and H₂O (2 mL) was heated at 70 °C for 18 h.

The mixture was allowed to cool to rt and was then acidiﬁed to pH 6

with 2 N aq HCl. The aqueous mixture was extracted with CH₂Cl₂

(3 × 50 mL), and the combined organics were dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. The residue was

chromatographed over silica gel (0−5% methanol in CH₂Cl₂) to

yield 6-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxamide

(46) as an oﬀ-white solid (0.041 g, 78%): mp 183−187 °C; ¹H NMR

(300 MHz, CDCl₃) δ 7.77 (s, 1H), 7.66−7.58 (m, 1H), 7.58−7.41

(m, 2H), 7.20 (s, 1H), 5.57 (s, 1H), 3.85−3.64 (m, 4H) 3.64−3.45

(m, 1H), 3.01−3.85 (m, 2H), 2.46 (s, 3H), 2.45−2.32 (m, 2H),

1.72−1.56 (m, 2H), aromatic CH obscured by residual peak; ESI MS

m/z: 254.1 [M + H]⁺; HPLC >99% (AUC), (Method B), t_R= 17.5

min.

Step B. A solution of methyl 5-methyl-2-((3aR,5r,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

pyrimidine-4-carboxylate (0.015 g, 0.04 mmol) and 2 N aq NaOH

(5 mL) in a 1:1 mixture of CH₃OH/THF (10 mL) stirred at rt for

16 h. The mixture was carefully neutralized at 0 °C with 2 N aq HCl

and extracted with CH₂Cl₂(3 × 10 mL). The combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and con-

centrated under reduced pressure. The resulting residue was

chromatographed over silica gel (0% to 10% CH₃OH in CH₂Cl₂) to

5-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboxylic acid (42) as an

1

oﬀ-white solid (0.013 g, 92%): H NMR (300 MHz, CD₃OD) δ 8.12

(s, 1H), 7.67−7.78 (m, 3H), 7.31 (m, 1H), 3.41−3.61 (m, 5H), 2.91

(m, 2H), 2.34 (m, 4H), 2.15 (s, 3H), 1.67 (m, 2H); MS (ESI+) m/z

392 [M + H]⁺; HPLC >99% (AUC), (Method A), t_R= 14.2 min.

4-Methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-5-carboxylic

Acid (43). Step A. A solution of (3aR,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)octahydrocyclopenta[c]pyrrole hydrochloride (26, 0.100 g,

0.340 mmol), ethyl 2-chloro-6-methylpyrimidine-5-carboxylate (76 mg,

0.38 mmol), and i-Pr₂NEt (0.15 mL, 0.86 mmol) in DMF (5 mL) was

heated at 70 °C for 90 min via microwave irradiation. The mixture was

allowed to cool to rt then concentrated under reduced pressure. The

resulting residue was chromatographed over silica gel (Isco CombiFlash

Companion unit, 12 g Redisep column, 0−100% EtOAc in hexanes) to

provide methyl 4-methyl-2-((3aR,6aS)-5-(2-(triﬂuoro-

methyl)phenyl)hexahydrocyclopenta[c]pyrrole-2(1H)-yl)pyrimidine-5-

carboxylate as a colorless semisolid (99 mg, 69%): ¹H NMR (300 MHz,

CDCl₃) δ 8.87 (s, 1H), 7.61 (dd, J = 7.8 Hz, 1H), 7.50−7.48 (m, 2H),

4.36−4.29 (m, 2H) 3.88−3.77 (m, 4H), 3.59−3.56 (m, 1H), 3.06−2.94

(m, 2H), 2.71 (s, 3H), 2.44−2.35 (m, 2H), 1.69−1.56 (m, 3H), 1.39−

1.35 (m, 3H); MS (ESI+) m/z 420 [M + H]⁺.

Step B. A mixture of methyl 4-methyl-2-((3aR,6aS)-5-(2-(triﬂuo-

romethyl)phenyl)hexahydrocyclopenta[c]pyrrole-2(1H)-yl)-

pyrimidine-5-carboxylate (0.099 g, 0.241 mmol) and LiOH·H₂O

(0.100 g, 2.38 mmol) in THF (4 mL) and H₂O (2 mL) stirred at rt for

18 h and was then heated at 40 °C for 7 h. The mixture was allowed to

cool to rt and was diluted with H₂O (4 mL) and acidiﬁed with 2 N aq

HCl to pH 6. The mixture was extracted with EtOAc (3 × 10 mL),

and the combined extracts were dried over Na₂SO₄, ﬁltered, and con-

centrated under reduced pressure to provide 4-methyl-2-((3aR,6aS)-5-

(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

pyrimidine-5-carboxylic acid (43) as a white solid (0.020 g, 21%): mp

(3aR,5r,6aS)-2-(4-Methyl-6-(1H-tetrazol-5-yl)pyrimidin-2-yl)-5-(2-

(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]pyrrole (47). Step

A. A mixture of 6-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-4-carboni-

trile (45, 0.093 g, 0.250 mmol), NaN₃(0.195 g, 3.0 mmol), and

NH₄Cl (0.160 g, 3.0 mmol) in DMF (5 mL) was heated at 130 °C in a

sealed tube via microwave irradiation for 1 h. The mixture was allowed

to cool to rt and then diluted with H₂O (50 mL). The aqueous

mixture was extracted with EtOAc (3 × 50 mL), and the combined

organic extracts were washed with H₂O (3 × 50 mL) and brine. The

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The resulting residue was puriﬁed by reversed phase

column chromatography (Isco RediSep 12 g Gold C18 reverse phase

1

82−85 °C; H NMR (300 MHz, DMSO-d₆) δ 12.57 (br s, 1H), 8.73

(s, 1H), 7.73−7.61 (m, 3H), 7.41−7.38 (m, 1H), 3.82−3.62 (m, 4H),

2.86 (br s, 2H), 2.58 (s, 3H), 2.26 (br s, 2H), 1.63 (br s, 2H); MS

(ESI+) m/z 392 [M + H]⁺; HPLC 98.5% purity (Method H), t_R=

17.4 min.

2-((3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrimidine-5-carboxylic Acid (44).

Step A. A mixture of (3aR,5R,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

octahydrocyclopenta[c]pyrrole hydrochloride (26, 0.050 g, 0.172 mmol)

methyl 2-chloropyrimidine-5-carboxylate (0.029 g, 0.17 mmol), and

Et₃N (0.05 mL, 0.51 mmol) in DMF (5 mL) was heated at 70 °C for

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DOI: 10.1021/acs.jmedchem.5b00423

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column, 0%−100% CH₃CN in H₂O) to provide (3aR,5r,6aS)-2-(4-

methyl-6-(1H-tetrazol-5-yl)pyrimidin-2-yl)-5-(2-(triﬂuoromethyl)-

phenyl)octahydrocyclopenta[c]pyrrole (47) as a white solid (0.074 g,

71%): mp 150−158 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 7.71−7.59

(m, 3H), 7.41 (m, 1H), 7.21 (s, 1H), 3.85−3.65 (m, 4H), 3.49 (m,

1H), 2.98 (m, 2H), 2.42 (s, 3H), 2.32 (m, 2H), 1.62 (m, 2H); MS

(ESI+) m/z 416 [M + H]⁺; HPLC 98.3% (AUC), (Method A), t_R=

18.0 min.

4-Methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)picolinic Acid (48). Step A.

A mixture of (3aR,5R,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydro-

cyclopenta[c]pyrrole hydrochloride (26, 0.158 g, 0.54 mmol), methyl

2-chloro-4-methylnicotinate (0.100 g, 0.54 mmol), Pd(OAc)₂(0.011 g,

0.05 mmol), Xantphos (0.011 g), and Cs₂CO₃(0.050 g, 0.15 mmol) in

toluene (10 mL) was heated at 110 °C for 16 h. The reaction was

allowed to cool to rt then diluted with H₂O (200 mL) and extracted

with EtOAc (3 × 100 mL). The combined organic extracts were washed

with H₂O (3 × 100 mL), brine (100 mL), dried over Na₂SO₄, ﬁltered,

and concentrated under reduced pressure. The resulting residue was

chromatographed over silica gel (0% to 30% EtOAc in hexanes) to give

methyl 4-methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)picolinate as an oﬀ-white solid

(0.129 g, 59%).

Step B. A solution of methyl 4-methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuo-

romethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)picolinate

(0.129 g, 0.07 mmol) and 2 N aq NaOH (10 mL) in a 1:1 mixture of

CH₃OH/THF (20 mL) stirred at room temperature for 16 h. The

mixture was carefully neutralized at 0 °C with 2 N aq HCl and

extracted with CH₂Cl₂(3 × 10 mL). The combined organic extracts

were washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The resulting residue was chromatographed

over silica gel (0% to 10% CH₃OH in CH₂Cl₂) to give 4-methyl-6-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)picolinic acid (48) as an oﬀ-white solid (0.106 g,

88%): mp 152−155 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 12.51 (bs,

1H), 7.71 (m, 3H), 7.61 (m, 1H), 7.14 (s, 1H), 6.62 (s, 1H), 3.55 (m,

4H), 3.34 (m, 4H), 2.88 (m, 2H), 2.29 (m, 5H), 1.62 (m, 2H); MS

(ESI+) m/z 391 [M + H]⁺; HPLC >99% (AUC), (Method A), t_R=

13.4 min.

2-Methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)isonicotinic Acid (49). Step

A. A mixture of (3aR,5R,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydro-

cyclopenta[c]pyrrole hydrochloride (26, 0.160 g, 0.55 mmol), methyl

2-chloro-6-methylisonicotinate (0.101 g, 0.55 mmol), Pd(OAc)₂

(0.011 g, 0.05 mmol), Xantphos (0.011 g), and Cs₂CO₃(0.050 g,

0.15 mmol) in toluene (10 mL) was stirred at 110 °C for 16 h. The

reaction was allowed to cool to rt and diluted with H₂O (200 mL) and

extracted with EtOAc (3 × 100 mL). The combined organic extracts

were washed with H₂O (3 × 100 mL), brine (100 mL), dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The result-

ing residue was chromatographed over silica gel (0% to 30% EtOAc in

hexanes) to give methyl 2-methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuoro-

methyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)isonicotinate

as an oﬀ-white solid (0.140 g, 63%).

3-Methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)picolinic Acid (50). Step A.

To a mixture of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydro-

cyclopenta[c]pyrrole hydrochloride (26, 0.400 g, 1.37 mmol),

6-chloro-3-methylpicolinic acid (0.254 g, 1.37 mmol), JohnPhos

(0.048 g, 0.137 mmol), and Cs₂CO₃(1.34 g, 4.11 mmol) in toluene

(7 mL) was added Pd(OAc)₂(0.015 g, 0.068 mmol). The mixture was

heated at reﬂux for 14 h then allowed to cool to rt. The mixture was

ﬁltered over Celite, and the ﬁlter cake was washed with EtOAc. The

ﬁltrate was concentrated under reduced pressure, and the resulting

residue was puriﬁed by ﬂash column chromatography (Isco CombiFlash

Rf unit, 40 g Gold RediSep column, 0%−15% EtOAc in hexanes) to

give methyl 3-methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)picolinate as a white solid

(0.366 g, 66%): MS (ESI+) m/z 405 [M + H]⁺.

Step B. A mixture of methyl 3-methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuo-

romethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)picolinate

(0.366 g, 0.905 mmol) and LiOH·H₂O (0.114 g, 2.71 mmol) in THF

(4 mL), CH₃OH (4 mL), and H₂O (4 mL) was stirred at rt for 72 h.

The mixture was acidiﬁed to pH 6 via the addition of 2 N aq HCl and

then extracted with CH₂Cl₂(3 × 50 mL). The combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and con-

centrated under reduced pressure. The resulting residue was puriﬁed

by ﬂash column chromatography (Isco CombiFlash Rf unit, 40 g

RediSep column, 0%−8% CH₃OH in CH₂Cl₂) to give 3-methyl-6-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)picolinic acid (50) as a white solid (0.337 g, 95%):

mp 148−150 °C; ¹H NMR (500 MHz, CDCl₃) δ 11.97 (bs, 1H), 7.67

(s, 1H), 7.59−7.50 (m, 3H), 7.27 (m, 1H), 6.65 (s, 1H), 3.70−3.44

(m, 5H), 2.94 (m, 2H), 2.61 (s, 3H), 1.70 (m, 2H), 1.50 (m, 2H); MS

(ESI+) m/z 391 [M + H]⁺; HPLC >99% (AUC), (Method A), t_R=

11.8 min.

5-((3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)nicotinic Acid (51). Step A. To a

mixture of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydro-

cyclopenta[c]pyrrole hydrochloride (26, 0.153 g, 0.524 mmol),

methyl 5-bromonicotinate (0.103 g, 0.477 mmol), XantPhos (0.083 g,

0.140 mmol), and Cs₂CO₃(0.465 g, 1.43 mmol) in 1,4-dioxane (8 mL)

was added Pd₂(dba)₃(0.043 g, 0.047 mmol). The mixture was heated at

reﬂux for 16 h, then allowed to cool to rt. The mixture was concentrated

under reduced pressure, and the resulting residue was puriﬁed by ﬂash

column chromatography (Isco CombiFlash Rf unit, 40 g Gold RediSep

column, 0%−25% CH₃OH in CH₂Cl₂) to give methyl 5-((3aR,5r,6aS)-

5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

nicotinate as a yellow ﬁlm (0.077 g, 41%): ¹H NMR (500 MHz, CDCl₃)

δ 8.59 (s, 1H), 7.62 (s, 1H), 7.53 (m, 1H), 7.48 (m, 3H), 7.29 (m, 1H),

3.94 (s, 3H), 3.52 (m, 1H), 3.43 (m, 4H), 2.98 (m, 2H), 2.45 (m, 2H),

1.67 (m, 2H); MS (ESI+) m/z 391 [M + H]⁺.

Step B. A mixture of methyl 5-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)nicotinate (0.076 g,

0.194 mmol) and LiOH·H₂O (0.081 g, 1.94 mmol) in THF (4 mL),

CH₃OH (2 mL), and H₂O (1 mL) was stirred at rt for 4 h. The mixture

was acidiﬁed to pH 2 via the addition of 2 N aq HCl and then diluted

with H₂O (40 mL). The aqueous mixture was extracted with CH₂Cl₂

(3 × 50 mL), and the combined organic extracts were washed with

brine, dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure to provide 5-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)nicotinic acid (51) as a yellow

Step B. A solution of methyl 2-methyl-6-((3aR,5r,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

isonicotinate (0.140 g, 0.08 mmol) and 2 N aq NaOH (10 mL) in a

1:1 mixture of CH₃OH/THF (20 mL) was stirred at room tempera-

ture for 16 h. The mixture was carefully neutralized at 0 °C with 2 N

aq HCl and extracted with CH₂Cl₂(3 × 10 mL). The combined

organic extracts were washed with brine, dried over Na₂SO₄, ﬁltered,

and concentrated under reduced pressure. The resulting residue was

chromatographed over silica gel (0% to 10% CH₃OH in CH₂Cl₂) to

give 2-methyl-6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)isonicotinic acid (49) as an

oﬀ-white solid (0.124 g, 86%): mp 121−124 °C; ¹H NMR (500 MHz,

DMSO-d₆) δ 12.81 (bs, 1H), 7.77 (s, 1H), 7.66 (m, 3H), 7.40 (m,

1H), 6.60 (s, 1H), 3.52 (m, 4H), 3.45 (m, 1H), 2.80 (m, 2H), 2.36 (s,

3H), 2.24 (m, 2H), 1.60 (m, 2H); MS (ESI+) m/z 391 [M + H]⁺;

HPLC >99% (AUC), (Method A), t_R= 10.1 min.

1

solid (0.063 g, 88%): mp 302−304 °C; H NMR (500 MHz, DMSO-

d₆) δ 13.17 (bs, 1H), 8.39 (s, 1H), 8.23 (s, 1H), 7.72−7.59 (m, 3H),

7.38 (m, 2H), 3.44 (m, 5H), 2.94 (m, 2H), 2.30 (m, 2H), 1.65 (m, 2H);

MS (ESI+) m/z 377 [M + H]⁺; HPLC >99% (AUC), (Method B), t_R=

7.8 min.

3-((3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)benzoic Acid (52). Step A. To a mixture

of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]-

pyrrole hydrochloride (26, 0.250 g, 0.86 mmol), NaOt-Bu (0.180 g,

0.89 mmol), and BINAP (0.105 g, 0.17 mmol) in toluene (10 mL) was

added Pd(OAc)₂(0.020 g, 0.089 mmol). The mixture was heated at

110 °C for 16 h, then allowed to cool to rt. The mixture was ﬁltered

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over Celite, and the ﬁlter cake was washed with EtOAc. The ﬁltrate

was concentrated under reduced pressure, and the resulting residue

was puriﬁed by ﬂash column chromatography (Isco CombiFlash

Rf unit, 40 g Gold RediSep column, 0%−15% EtOAc in hexanes) to

give methyl 3-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)benzoate as a white solid (0.132 g,

63%): MS (ESI+) m/z 390 [M + H]⁺.

CH₃OH/THF (20 mL) was stirred at room temperature for 16 h. The

mixture was carefully neutralized at 0 °C with 2 N HCl and extracted

with CH₂Cl₂(3 × 10 mL). The combined organic extracts were

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The resulting residue was chromato-

graphed over silica gel (0% to 10% CH₃OH in CH₂Cl₂) to give

2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta-

[c]pyrrol-2(1H)-yl)nicotinic acid (54) as an oﬀ-white solid (0.057 g,

Step B. A mixture of methyl 3-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)benzoate (0.396 g,

0.95 mmol) and LiOH·H₂O (0.400 g, 9.5 mmol) in THF (4 mL),

CH₃OH (2 mL), and H₂O (1 mL) stirred at rt for 16 h. The mixture

was acidiﬁed to pH 2 via the addition of 2 N aq HCl and then diluted

with H₂O (40 mL). The aqueous mixture was extracted with CH₂Cl₂

(3 × 50 mL), and the combined organic extracts were washed with

brine, dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure to provide 3-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)benzoic acid (52) as a yellow

1

85%): mp 158−160 °C; H NMR (500 MHz, DMSO-d₆) δ 8.20 (s,

1H), 7.79 (m, 1H), 7.63 (m, 3H), 7.39 (m, 1H), 6.71 (s, 1H), 3.53 (m,

4H), 3.48 (m, 1H), 2.80 (m, 2H), 2.24 (m, 2H), 1.61 (m, 2H); MS

(ESI+) m/z 377 [M + H]⁺; HPLC >99% (AUC), (Method A), t_R=

10.4 min.

6-((3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyrazine-2-carboxylic Acid (55). Step

A. A mixture of 5-(2-(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]-

pyrrole hydrochloride (26, 0.057 g, 0.29 mmol), methyl 2-chloro-

pyrazine-6-carboxylate (0.051 g, 0.29 mmol), and i-Pr₂NEt (0.14 mL,

1.09 mmol) in DMF (10 mL) was heated at 60 °C for 16 h. The

mixture was allowed to cool to rt and poured into brine (50 mL).

The aqueous mixture was extracted with EtOAc (3 × 50 mL), and the

combined organic extracts were washed with a saturated NaHCO₃

(3 × 50 mL), dried over Na₂SO₄, and concentrated under reduced

pressure. The resulting residue was chromatographed over silica gel

(0−50% EtOAc in hexanes) to aﬀord methyl 6-((3aR,5r,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

pyrazine-2-carboxylate (66 mg, 57%): ¹H NMR (300 MHz, CDCl₃) δ

8.53 (s, 1H), 8.10 (s, 1H), 7.66−7.56 (m, 1H), 7.56−7.42 (m, 2H),

3.99 (s, 3H), 3.81−3.48 (m, 5H) 3.07−2.92 (m, 2H), 2.51−2.34 (m,

2H), 1.75−1.61 (m, 2H), aromatic CH obscured by a residual peak;

ESI MS m/z 392 [M + H]⁺.

1

solid (0.026 g, 18%): H NMR (300 MHz, DMSO-d₆) δ 12.72 (bs,

1H), 7.72−7.63 (m, 3H), 7.41 (m, 1H), 7.30−7.20 (m, 3H), 6.89 (m,

1H), 3.40 (m, 5H), 2.91 (m, 2H), 2.31 (m, 2H), 1.64 (m, 2H); MS

(ESI+) m/z 376 [M + H]⁺; HPLC 98.8% (AUC), (Method B), t_R=

18.6 min.

6-Methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)nicotinic Acid (53). Step A.

To a mixture of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydro-

cyclopenta[c]pyrrole hydrochloride (26, 0.250 g, 0.857 mmol), methyl

2-chloro-6-methylnicotinate (0.146 g, 0.787 mmol), XantPhos (0.135 g,

0.233 mmol), and Cs₂CO₃(0.647 g, 1.98 mmol) in toluene (25 mL)

was added Pd₂(dba)₃(0.071 g, 0.077 mmol). The mixture was heated

at 100 °C for 16 h, then allowed to cool to rt. The mixture was

concentrated under reduced pressure, and the resulting residue was

puriﬁed by ﬂash column chromatography (Isco CombiFlash Rf unit, 24 g

Gold RediSep column, 0%−25% CH₃OH in CH₂Cl₂) to give methyl

6-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)nicotinate as a crude yellow ﬁlm (0.036 g).

Step B. A mixture of methyl 6-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuo-

romethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)nicotinate

(0.036 g, 0.089 mmol) and LiOH·H₂O (0.037 g, 0.89 mmol) in THF

(4 mL), CH₃OH (4 mL), and H₂O (1 mL) stirred at 50 °C for 30

min. The mixture was acidiﬁed to pH 2 via the addition of 2 N aq HCl

and then diluted with H₂O (40 mL). The aqueous mixture was

extracted with CH₂Cl₂(3 × 20 mL), and the combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was

puriﬁed by reversed phase column chromatography (Isco RediSep 12 g

Gold C18 reverse phase column, 0%−100% CH₃CN in H₂O) to

provide 6-methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)nicotinic acid (53) as a

white solid (0.007 g, 20% over two steps): mp 156−157 °C; ¹H

NMR (500 MHz, DMSO-d₆) δ 13.28 (bs, 1H), 7.71−7.59 (m, 3H),

7.38 (m, 1H), 6.87 (s, 1H), 6.74 (s, 1H), 3.55 (m, 4H), 3.41 (m, 1H),

2.89 (m, 2H), 2.35 (s, 3H), 2.27 (m, 2H), 1.65 (m, 2H); MS (ESI+)

m/z 391 [M + H]⁺; HPLC 98.6% (AUC), (Method A), t_R= 8.4 min.

2-((3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)nicotinic Acid (54). Step A. A mixture of

(3aR,5R,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]-

pyrrole hydrochloride (26, 0.160 g, 0.55 mmol), methyl

2-chloronicotinate (0.071 g, 0.41 mmol), Pd(OAc)₂(0.011 g, 0.05 mmol),

Xantphos (0.011 g), and Cs₂CO₃(0.050 g, 0.15 mmol) in toluene

(10 mL) was stirred at 110 °C for 16 h. The reaction was diluted with

H₂O (200 mL) and extracted with EtOAc (3 × 100 mL). The

combined organic extracts were washed with H₂O (3 × 100 mL),

brine (100 mL), dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The resulting residue was chromatographed

over silica gel (0% to 30% EtOAc in hexanes) to give methyl 2-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)nicotinate as an oﬀ-white solid (0.07 g, 33%).

Step B. A solution of 2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)nicotinate (0.70 g,

0.18 mmol) and 2 N aq NaOH (10 mL) in a 1:1 mixture of

Step B. A mixture of methyl 6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrazine-2-carboxy-

late (66.0 mg, 0.169 mmol) and LiOH·H₂O (0.027 g, 0.64 mmol) in

THF (4 mL), CH₃OH (4 mL), and H₂O (1 mL) was stirred at rt for

30 min. The mixture was acidiﬁed to pH 6 using 2 N aq HCl and

extracted with CH₂Cl₂(3 × 20 mL), and the combined organic

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was

puriﬁed by reversed phase column chromatography (Isco RediSep 12 g

Gold C18 reverse phase column, 0%−100% CH₃CN in H₂O) to

provide 2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)nicotinic acid (55) as a white solid

1

(0.015 g, 24%). mp 182−185 °C; H NMR: (300 MHz, CDCl₃) δ

8.60 (s, 1H), 8.29−8.00 (br s, 1H), 7.74−7.55 (m, 1H), 7.55−7.39 (m,

2H), 3.93−3.40 (m, 5H), 2.79−2.54 (m, 2H) 2.50−2.22 (m, 2H),

1.80−1.44 (m, 2H); ESI MS m/z: 378 [M + H]⁺; HPLC 98.7%

(AUC), (Method B), t_R= 14.0 min.

6-((3aR,5r,6aS)-5-(2-(Triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)pyridazine-4-carboxylic Acid (56). Step

A. A mixture of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)octahydro-

cyclopenta[c]pyrrole hydrochloride (26, 0.500 g, 1.71 mmol), methyl

3-chloropyridazine-5-carboxylate (0.295 g, 1.71 mmol), and Et₃N

(0.71 mL, 5.15 mmol) in DMF (10 mL) was heated at 60 °C for 16 h.

The mixture was then allowed to cool to rt and diluted with H₂O

(50 mL). The aqueous mixture was extracted with EtOAc (3 × 50 mL),

and the combined organic extracts were washed with H₂O (3 × 50 mL)

and brine. The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was puriﬁed

by ﬂash column chromatography (Isco CombiFlash Rf unit, 40 g

RediSep column, 0%−50% EtOAc in hexanes) to give methyl 6-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)pyridazine-4-carboxylate as an oil (0.069 g, 10%): MS

(ESI+) m/z 392 [M + H]⁺.

Step B. A mixture of methyl 6-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyridazine-4-carboxy-

late (0.060 g, 0.153 mmol) and LiOH·H₂O (0.020 g, 0.477 mmol) in

THF (5 mL) and H₂O (5 mL) was stirred at rt for 16 h. The mixture

was acidiﬁed to pH 6 via the addition of 2 N aq HCl and then

extracted with CH₂Cl₂(3 × 20 mL). The combined organic extracts

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were washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure to provide 6-((3aR,5r,6aS)-5-(2-(triﬂuoro-

methyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyridazine-4-

Step C. To a 0 °C cooled solution of (3aR,5r,6aS)-tert-butyl 5-(5-

ﬂuoro-2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrole-

2(1H)-carboxylate (4.50 g, 12.1 mmol) in CH₂Cl₂(60 mL) was added

a 2.0 M HCl solution in Et₂O (100 mL), and the mixture was allowed

to stir at rt for 24 h. The mixture was diluted with Et₂O (200 mL), and

the precipitated product was ﬁltered to give (3aR,5r,6aS)-5-(5-ﬂuoro-2-

(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]pyrrole hydrochloride

(60a) as a white solid (3.45 g, 92%): MS (ESI+) m/z 274 [M + H]⁺.

1

carboxylic acid (56) as a light-yellow solid (0.054 g, 95%): H NMR

(300 MHz, DMSO-d₆) δ 13.92 (bs, 1H), 8.82 (s, 1H), 7.75−7.62 (m,

3H), 7.38 (m, 1H), 7.22, (s, 1H), 3.72−3.66 (m, 4H), 3.44 (m, 1H),

2.94 (m, 2H), 2.29 (m, 2H), 1.67 (m, 2H); MS (ESI+) m/z 378 [M +

H]⁺; HPLC 97.4% (AUC), (Method A), t_R= 12.8 min.

Step D. To

a solution of (3aR,5r,6aS)-5-(5-ﬂuoro-2-

5-Methyl-2-((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)thiazole-4-carboxylic Acid

(57). Step A. To a solution of (3aR,5r,6aS)-5-(2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrole-2(1H)-carbonyl chloride

(0.300 g, 1.03 mmol) in DMSO (8 mL) and ethyl 2-chloro-5-methyl-

thiazole-4-carboxylate (0.211 g, 1.03 mmol) was added i-Pr₂NEt (0.40 g,

3.09 mmol). The resulting solution was heated at 110 °C for 24 h. The

reaction was allowed to cooled to rt and diluted with H₂O (30 mL) and

extracted with EtOAc (3 × 30 mL). The combined organic extracts

were washed with brine (2 × 30 mL), dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was chro-

matographed over silica gel (Isco CombiFlash Rf unit, 12 g Redisep

column, 0−30% EtOAc in hexanes) to give ethyl 5-methyl-2-

((3aR,5r,6aS)-5-(2-(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]-

pyrrol-2(1H)-yl)thiazole-4-carboxylate as an oﬀ-white solid (0.042 g,

9%): ¹H NMR (300 MHz, DMSO-d₆) δ 7.76−7.70 (m, 1H), 7.68−7.54

(m, 2H), 7.42−7.35 (m, 1H), 4.22 (q, J = 6.9 Hz, 2H), 3.55−3.42 (m,

2H), 3.42−3.35 (m, 2H), 2.97−2.83 (m, J = 2H), 2.55 (s, 3H), 2.32−

2.18 (m, 3H), 1.74−1.53 (m, 2H), 1.27 (t, J = 6.9 Hz, 3H); MS (ESI+)

m/z 425 [M + H]⁺.

(triﬂuoromethyl)phenyl)octahydrocyclopenta[c]pyrrole hydrochloride

(1.0 g, 3.23 mmol) and Et₃N (1.43 mL, 10.29 mmol) in DMF (50 mL)

was added a methyl 2-chloropyrimidine-4-carboxylate (0.641 g, 3.43

mmol), and the resulting solution was stirred at 60 °C for 16 h. The

reaction was diluted with H₂O (200 mL) and extracted with EtOAc

(3 × 100 mL). The combined organic extracts were washed with H₂O

(3 × 100 mL) and brine (100 mL), dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was chro-

matographed over silica gel (0% to 30% EtOAc in hexanes) to give

methyl 2-((3aR,5r,6aS)-5-(5-ﬂuoro-2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-4-carboxy-

late as an oﬀ-white solid (1.14 g, 84%): MS (ESI+) m/z 424 [M + H]⁺.

Step E. A solution of methyl 2-((3aR,5r,6aS)-5-(5-ﬂuoro-2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-6-

methylpyrimidine-4-carboxylate (1.0 g, 2.36 mmol) and 2 N aq NaOH

(20 mL) in a 1:1 mixture of CH₃OH/THF (40 mL) was stirred at

room temperature for 16 h. The mixture was carefully neutralized at

0 °C with 2 N aq HCl and extracted with CH₂Cl₂(3 × 10 mL). The

combined organic extracts were washed with brine, dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. The resulting

residue was chromatographed over silica gel (0% to 10% CH₃OH in

CH₂Cl₂) to give 2-((3aR,5r,6aS)-5-(5-ﬂuoro-2-(triﬂuoromethyl)-

phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-

Step B. A mixture of ethyl 5-methyl-2-((3aR,5r,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

thiazole-4-carboxylate (0.040 g, 0.094 mmol) and LiOH·H₂O (0.039 g,

0.94 mmol) in THF (2 mL), CH₃OH (1 mL), and H₂O (0.5 mL) was

stirred at rt for 16 h. The mixture was then acidiﬁed to pH 5 with 2 N

aq HCl and diluted with H₂O (50 mL). The resulting solids were

collected by ﬁltration to provide 5-methyl-2-((3aR,5r,6aS)-5-(2-

(triﬂuoromethyl)phenyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-

thiazole-4-carboxylic acid (57) as a white solid (0.030 g, 81%): mp

1

4-carboxylic acid (61) as an oﬀ-white solid (0.831 g, 86%): H NMR

(500 MHz, DMSO-d₆) δ 7.74 (m, 1H), 7.54 (m, 1H), 7.24 (m, 1H),

6.63 (s, 1H), 3.63 (m, 4H), 3.58 (m, 1H), 2.82 (m, 2H), 2.36 (m, 2H),

2.27 (s, 3H), 1.61 (m, 2H); MS (ESI+) m/z 410 [M + H]⁺; HPLC

>99% (AUC) (Method B), t_R= 10.8 min.

1

2-((3aR,5r,6aS)-5-(3-Fluoro-2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-4-car-

boxylic Acid (62). Compound 62 was prepared according to a similar

166−170 °C; H NMR (500 MHz, DMSO-d₆) δ 12.41 (br s, 1H),

7.72 (d, J = 8.0 Hz, 1H), 7.67−7.58 (m, 2H), 7.42−7.36 (m, 1H),

3.51−3.45 (m, 2H), 3.42−3.32 (m, 3H), 2.95−2.85 (m, 2H), 2.53 (s,

3H), 2.30−2.22 (m, 2H), 1.66−1.57 (m, 2H); MS (ESI+) m/z 397

[M + H]⁺; HPLC >99% (AUC), (Method B), t_R= 14.5 min.

2-((3aR,5r,6aS)-5-(5-Fluoro-2-(triﬂuoromethyl)phenyl)-

hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-4-car-

boxylic Acid (61). Step A. To a N₂degassed mixture of (3aS,6aS)-tert-

butyl 5-(((triﬂuoromethyl)sulfonyl)oxy)-3,3a,6,6a-tetrahydro-

cyclopenta[c]pyrrole-2(1H)-carboxylate (( )-23, 5.0 g, 14.0 mmol),

(5-ﬂuoro-2-(triﬂuoromethyl)phenyl)boronic acid (2.91 g, 14.0 mmol),

and aqueous 2 M Na₂CO₃(100 mL) in DME (200 mL) was added

Pd(PPh₃)₄(0.500 g, 1.4 mmol). The mixture was heated at 80 °C for

6 h, then allowed to cool to rt and diluted with H₂O (500 mL). The

aqueous mixture was extracted with EtOAc (2 × 200 mL), and the

combined organic extracts were washed with H₂O (200 mL) and brine

(200 mL), dried over Na₂SO₄, ﬁltered, and concentrated under re-

duced pressure. The residue was puriﬁed by ﬂash column chromato-

graphy (Isco CombiFlash Rf unit, 330 g Redisep column, 0% to 10%

EtOAc in hexanes) to give (3aR,6aS)-tert-butyl 5-(5-ﬂuoro-2-

(triﬂuoromethyl)phenyl)-3,3a,6,6a-tetrahydrocyclopenta[c]pyrrole-

2(1H)-carboxylate (58a) as a clear, viscous oil (4.88 g, 94%).

Step B. A mixture of 5-(5-ﬂuoro-2-(triﬂuoromethyl)phenyl)-

3,3a,6,6a-tetrahydrocyclopenta[c]pyrrole-2(1H)-carboxylate (4.8 g,

12.9 mmol) and 10% Pd/C (1.57 g, wet, 10% w/w) in CH₃OH

(50 mL) was subjected to an atmosphere of H₂gas (40 psi) using a Parr

Shaker apparatus for 16 h at rt. The mixture was ﬁltered through Celite,

and the ﬁltrate was concentrated under reduced pressure. The resulting

residue was puriﬁed by ﬂash column chromatography (Isco CombiFlash Rf

unit, 40 g Redisep column, 0% to 30% EtOAc in hexanes) to give

(3aR,5r,6aS)-tert-butyl 5-(5-ﬂuoro-2-(triﬂuoromethyl)phenyl)hexahydro-

cyclopenta[c]pyrrole-2(1H)-carboxylate (59a) as a clear, viscous oil

(4.5 g, 95%).

1

procedure described for the synthesis of 61. H NMR (300 MHz,

DMSO-d₆) δ 7.52−7.44 (m, 1H), 7.02−6.99 (d, J = 8.4 Hz, 1H),

6.89−6.84 (m, 2H), 3.80−3.73 (m, 2H), 3.46−3.42 (m, 4H), 3.14−

3.04 (m, 4H); ESI MS m/z 411 [M + H]⁺; HPLC >99.0% purity

(Method B), t_R= 16.8 min.

2-((3aR,5r,6aS)-5-(2-Chloro-5-ﬂuorophenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-4-carboxylic Acid

(63). Compound 63 was prepared according to a similar procedure

described for the synthesis of 61. Mp 183−185 °C; ¹H NMR (500 MHz,

DMSO-d₆) δ 13.22 (br s, 1H). 7.44 (dd, J = 9.0, 5.5 Hz, 1H), 7.34 (dd,

J = 10.5, 3.0 Hz, 1H), 7.10−7.05 (m, 1H), 6.99 (s, 1H), 3.70−3.64 (m,

2H), 3.62−3.57 (m, 2H), 3.50−3.41 (m, 1H), 2.92−2.81 (m, 2H), 2.36

(s, 3H), 2.34−2.25 (m, 2H), 1.57−1.47 (m, 2H); MS (ESI+) m/z 376

[M + H]⁺; HPLC >99% purity (Method B), t_R= 12.7 min.

2-((3aR,5r,6aS)-5-(2-Chloro-3-ﬂuorophenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-4-carboxylic Acid

(64). Compound 64 was prepared according to a similar procedure

1

described for the synthesis of 61. H NMR (300 MHz, DMSO-d₆) δ

7.35−7.28 (m, 2H), 7.28−7.19 (m, 1H), 6.68 (s, 1H), 3.62−3.54 (m,

4H), 3.53−3.41 (m, 1H), 2.91−2.76 (m, 2H), 2.40−2.26 (m, 2H),

2.33 (s, 3H), 1.56−1.41 (m, 2H); MS (ESI+) m/z 376 [M + H]⁺;

HPLC >99% purity (Method B), t_R= 10.9 min.

2-((3aR,5r,6aS)-5-(2-Chloro-3-ﬂuorophenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-4-carboxylic Acid

(65). Compound 65 was prepared according to a similar procedure

described for the synthesis of 61.Mp 176−180 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 13.31 (br s, 1H), 8.99 (s, 1H), 8.65 (d, J = 5.1 Hz, 1H),

7.65 (d, J = 5.1 Hz, 1H), 6.99 (s, 1H), 3.75−3.62 (m, 4H), 3.41−3.35

(m, 1H), 2.91−2.90 (m, 2H), 2.37 (s, 3H), 2.32−2.27 (m, 2H),

1.80−1.74 (m, 2H); MS (ESI+) m/z 393 [M + H]⁺; HPLC 98.3%

purity (Method H), t_R= 13.8 min.

X

DOI: 10.1021/acs.jmedchem.5b00423

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2-((3aR,5r,6aS)-5-(2-Chloro-3-ﬂuorophenyl)hexahydro-

cyclopenta[c]pyrrol-2(1H)-yl)-6-methylpyrimidine-4-carboxylic Acid

(66). Compound 66 was prepared according to a similar procedure

described for the synthesis of 61. Mp 161−165 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 13.31 (br s, 1H), 8.55−8.53 (m, 1H), 8.22

(d, J = 8.1 Hz, 1H), 7.67−7.62 (m, 1H), 7.00 (s, 1H), 3.71−3.61 (m,

4H), 3.44−3.38 (m, 1H), 2.91−2.89 (m, 2H), 2.37 (s, 3H), 2.31−2.23

(m, 2H), 1.67−1.57 (m, 2H); MS (ESI+) m/z 393 [M + H]⁺; HPLC

>99.0% purity (Method B), t_R= 14.7 min.

sodium hydroxide; SOCl₂, thionyl chloride; Pd(PPh₃)₄,

tetrakis(triphenylphosphine)palladium(0); PhN(SO₂CF₃)₂,

1,1,1-triﬂuoro-N-phenyl-N-((triﬂuoromethyl)sulfonyl)-

methanesulfonamide; LiAlH₄, lithium aluminum hydride; Gly,

glycine; Tyr, tyrosine; Arg, arginine; Gln, glutamine; Leu,

leucine; Phe, phenylalanine; CYP, cytochrome P450; CYP2C9,

cytochrome P450 2C9; CYP2C19, cytochrome P450 2C19;

CYP2D6, cytochrome P450 2D6; CYP3A4, cytochrome P450

3A4; GPCR, G-protein coupled receptor; hERG, human ether-

ASSOCIATED CONTENT

̀

a-go-go-related gene; PK, pharmacokinetics; PD, pharmacody-

■

namics; iv, intravenous; po, oral; qd, once daily; Cl, clearance;

V_ss, volume of distribution at steady state; AUC, area under the

curve; % F, % bioavailability; SAR, structure−activity relation-

ship; SPR, structure−property relationship; ADME, absorption,

distribution, metabolism, elimination; PPB, plasma protein

binding; THF, tetrahydrofuran; DME, dimethoxyethane;

CH₂Cl₂, dichloromethane; CH₃CN, acetonitrile; aq, aqueous

S

* Supporting Information

1

In vivo protocols and H NMR spectral data. The Supporting

Information is available free of charge on the ACS Publications

website at DOI: 10.1021/acs.jmedchem.5b00423.

AUTHOR INFORMATION

Corresponding Authors

*(C.L.C.) Phone: 518-512-2955. E-mail: christopher.cio ﬃ @

amriglobal.com.

■

REFERENCES

■

*(K.P.) Phone: 212-305-9040. E-mail: kep4@cumc.columbia.

edu.

(1) Petrukhin, K. New therapeutic targets in atrophic age-related

macular degeneration. Expert Opin. Ther. Targets 2007, 11 (5), 625−39.

(2) Hubschman, J. P.; Reddy, S.; Schwartz, S. D. Age-related macular

degeneration: current treatments. Clin. Ophthalmol. 2009, 3, 155−66.

(3) Macular Degeneration Facts and Statistics. http://www.

brightfocus.org/macular/about/understanding/facts.html (accessed

June 10, 2015).

Notes

The authors declare the following competing ﬁnancial interest(s):

C.L.C., N.D., E.E.F., M.P.C., P.C., L.Z., G.J., and K.P. are

inventors on the patent applications for compounds disclosed in

this paper that are assigned to The Trustees of Columbia

University in the City of New York and licensed to iCura Vision.

K.P., G.J., and P.G.P. are co-founders of iCura Vision.

(4) (a) Young, R. W. Pathophysiology of age-related macular

degeneration. Surv. Ophthalmol. 1987, 31 (5), 291−306. (b) Dorey, C.

K.; Wu, G.; Ebenstein, D.; Garsd, A.; Weiter, J. J. Cell loss in the aging

retina. Relationship to lipofuscin accumulation and macular degener-

ation. Invest. Ophthalmol. Visual Sci. 1989, 30 (8), 1691−9. (c) Holz, F.

G.; Bellman, C.; Staudt, S.; Schutt, F.; Volcker, H. E. Fundus

autofluorescence and development of geographic atrophy in age-

related macular degeneration. Invest. Ophthalmol. Visual Sci. 2001, 42

(5), 1051−6. (d) Holz, F. G.; Bellmann, C.; Margaritidis, M.; Schutt,

F.; Otto, T. P.; Volcker, H. E. Patterns of increased in vivo fundus

autofluorescence in the junctional zone of geographic atrophy of the

retinal pigment epithelium associated with age-related macular

degeneration. Graefe's Arch. Clin. Exp. Ophthalmol. 1999, 237 (2),

145−52. (e) Holz, F. G.; Bindewald-Wittich, A.; Fleckenstein, M.;

Dreyhaupt, J.; Scholl, H. P.; Schmitz-Valckenberg, S. Progression of

geographic atrophy and impact of fundus autofluorescence patterns in

age-related macular degeneration. Am. J. Ophthalmol. 2007, 143 (3),

463−72. (f) Schmitz-Valckenberg, S.; Fleckenstein, M.; Scholl, H. P.;

Holz, F. G. Fundus autofluorescence and progression of age-related

macular degeneration. Surv. Ophthalmol. 2009, 54 (1), 96−117.

(g) Finnemann, S. C.; Leung, L. W.; Rodriguez-Boulan, E. The

lipofuscin component A2E selectively inhibits phagolysosomal

degradation of photoreceptor phospholipid by the retinal pigment

epithelium. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (6), 3842−7.

(h) Suter, M.; Reme, C.; Grimm, C.; Wenzel, A.; Jaattela, M.; Esser, P.;

Kociok, N.; Leist, M.; Richter, C. Age-related macular degeneration.

The lipofusion component N-retinyl-N-retinylidene ethanolamine

detaches proapoptotic proteins from mitochondria and induces

apoptosis in mammalian retinal pigment epithelial cells. J. Biol.

Chem. 2000, 275 (50), 39625−30. (i) Sparrow, J. R.; Fishkin, N.;

Zhou, J.; Cai, B.; Jang, Y. P.; Krane, S.; Itagaki, Y.; Nakanishi, K. A2E, a

byproduct of the visual cycle. Vision Res. 2003, 43 (28), 2983−90.

(j) Sparrow, J. R.; Gregory-Roberts, E.; Yamamoto, K.; Blonska, A.;

Ghosh, S. K.; Ueda, K.; Zhou, J. The bisretinoids of retinal pigment

epithelium. Prog. Retinal Eye Res. 2012, 31 (2), 121−35. (k) Delori, F.

C. RPE Lipofuscin in Ageing and Age-Related Macular Degeneration.

In Retinal Pigment Epithelium and Macular Diseases (Documenta

Ophthalmologica); Coscas, G.; Piccolino, F. C., Eds.; Kluwer Academic

Publishers: Dordrecht, The Netherlands, 1995; Vol. 62, pp 37−45.

(5) Weng, J.; Mata, N. L.; Azarian, S. M.; Tzekov, R. T.; Birch, D. G.;

Travis, G. H. Insights into the function of Rim protein in

ACKNOWLEDGMENTS

■

This study was supported by NIH Grants U01 NS074476 (to

K.P.), P30 EY019007 (Core Support for Vision Research), and

unrestricted funds from Research to Prevent Blindness (New

York, NY) to the Department of Ophthalmology, Columbia

University. We thank The Burch Family Foundation, the Mary

Jaharis-John Catsimatidis Scholarship Fund, the Kaplen

Foundation, and the Eye Surgery Fund for gifts supporting

this study. This project has also been funded in whole or in part

with Federal funds from the National Institute of Neurological

Disorders and Stroke, the National Eye Institute, the NIH

Blueprint Neurotherapeutics Network, Blueprint for Neuro-

science Research Program, National Institutes of Health,

Department of Health and Human Services, under Contract

No. HHSN271201100013C.

ABBREVIATIONS USED

■

AMD, age-related macular degeneration; A2E, N-retinide-N-

retinylidene ethanolamine; SPA, scintillation proximity assay;

HTRF, homogeneous time-resolved ﬂuorescence assay; HLM,

human liver microsomes; RLM, rat liver microsomes; n-BuLi, n-

butyl lithium; THF, tetrahydrofuran; DMF, N,N-dimethylfor-

mamide; DME, dimethoxyethane; Et₂O, diethyl ether; EtOAc,

ethyl acetate; CH₃OH, methyl alcohol; EtOH, ethyl alcohol;

Boc₂O, di-tert-butyl dicarbonate; TFA, triﬂuoroacetic acid;

Pd(OAc)₂, palladium(II) acetate; Pd₂(dba)₃, tris-

(dibenzylideneacetone) dipalladium(0); BINAP, 2,2′-bis-

(diphenylphosphino)-1,1′-binaphthalene; XantPhos, 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene; JohnPhos, (2-

biphenyl)di-tert-butylphosphine; Ac₂O, acetic anhydride;

NaOAc, sodium acetate; LiHMDS, lithium bis(trimethylsilyl)-

amide; i-Pr₂NEt, N,N-diisopropylethylamine; Et₃N, triethyl-

amine; LiOH·H₂O, lithium hydroxide monohydrate; NaOH,

Y

DOI: 10.1021/acs.jmedchem.5b00423

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

photoreceptors and etiology of Stargardt’s disease from the phenotype

in abcr knockout mice. Cell (Cambridge, MA, U. S.) 1999, 98 (1),

13−23.

structure of human retinol-binding protein (RBP) with its carrier

protein transthyretin reveals an interaction with the carboxy terminus

of RBP. Biochemistry 1999, 38 (9), 2647−53. (c) Noy, N.; Slosberg, E.;

Scarlata, S. Interactions of retinol with binding proteins: studies with

retinol-binding protein and with transthyretin. Biochemistry 1992, 31

(45), 11118−24. (d) Noy, N.; Xu, Z. J. Interactions of retinol with

binding proteins: implications for the mechanism of uptake by cells.

Biochemistry 1990, 29 (16), 3878−83. (e) Monaco, H. L. The

transthyretin-retinol-binding protein complex. Biochim. Biophys. Acta,

Protein Struct. Mol. Enzymol. 2000, 1482 (1−2), 65−72. (f) Monaco,

H. L. Three-dimensional structure of the transthyretin-retinol-binding

protein complex. Clin. Chem. Lab. Med. 2002, 40 (12), 1229−36.

(g) Monaco, H. L.; Rizzi, M.; Coda, A. Structure of a complex of two

plasma proteins: transthyretin and retinol-binding protein. Science

1995, 268 (5213), 1039−41.

(6) (a) Sparrow, J. R.; Cai, B. Blue light-induced apoptosis of A2E-

containing RPE: involvement of caspase-3 and protection by Bcl-2.

Invest. Ophthalmol. Visual Sci. 2001, 42 (6), 1356−62. (b) Bergmann,

M.; Schutt, F.; Holz, F. G.; Kopitz, J. Inhibition of the ATP-driven

proton pump in RPE lysosomes by the major lipofuscin fluorophore

A2-E may contribute to the pathogenesis of age-related macular

degeneration. FASEB J. 2004, 18 (3), 562−4. (c) Sparrow, J. R.;

Parish, C. A.; Hashimoto, M.; Nakanishi, K. A2E, a lipofuscin

fluorophore, in human retinal pigmented epithelial cells in culture.

Invest. Ophthalmol. Visual Sci. 1999, 40 (12), 2988−95. (d) De, S.;

Sakmar, T. P. Interaction of A2E with model membranes. Implications

to the pathogenesis of age-related macular degeneration. J. Gen.

Physiol. 2002, 120 (2), 147−57. (e) Vives-Bauza, C.; Anand, M.;

Shirazi, A. K.; Magrane, J.; Gao, J.; Vollmer-Snarr, H. R.; Manfredi, G.;

Finnemann, S. C. The age lipid A2E and mitochondrial dysfunction

synergistically impair phagocytosis by retinal pigment epithelial cells. J.

Biol. Chem. 2008, 283 (36), 24770−80. (f) Zhou, J.; Jang, Y. P.; Kim,

S. R.; Sparrow, J. R. Complement activation by photooxidation

products of A2E, a lipofuscin constituent of the retinal pigment

epithelium. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (44), 16182−7.

(g) Radu, R. A.; Hu, J.; Yuan, Q.; Welch, D. L.; Makshanoff, J.; Lloyd,

M.; McMullen, S.; Travis, G. H.; Bok, D. Complement system

dysregulation and inflammation in the retinal pigment epithelium of a

mouse model for Stargardt macular degeneration. J. Biol. Chem. 2011,

286 (21), 18593−601. (h) Ben-Shabat, S.; Parish, C. A.; Vollmer, H.

R.; Itagaki, Y.; Fishkin, N.; Nakanishi, K.; Sparrow, J. R. Biosynthetic

studies of A2E, a major fluorophore of retinal pigment epithelial

lipofuscin. J. Biol. Chem. 2002, 277 (9), 7183−90. (i) Rozanowska, M.;

Jarvis-Evans, J.; Korytowski, W.; Boulton, M. E.; Burke, J. M.; Sarna, T.

Blue light-induced reactivity of retinal age pigment. In vitro generation

of oxygen-reactive species. J. Biol. Chem. 1995, 270 (32), 18825−30.

(j) Sparrow, J. R.; Zhou, J.; Ben-Shabat, S.; Vollmer, H.; Itagaki, Y.;

Nakanishi, K. Involvement of oxidative mechanisms in blue-light-

induced damage to A2E-laden RPE. Invest. Ophthalmol. Visual Sci.

2002, 43 (4), 1222−7. (k) Dontsov, A. E.; Sakina, N. L.; Golubkov, A.

M.; Ostrovsky, M. A. Light-induced release of A2E photooxidation

toxic products from lipofuscin granules of human retinal pigment

epithelium. Dokl. Biochem. Biophys. 2009, 425, 98−101.

(11) Schlehuber, S.; Skerra, A. Lipocalins in drug discovery: from

natural ligand-binding proteins to ″anticalins″. Drug Discovery Today

2005, 10 (1), 23−33.

(12) Wolf, G. Multiple functions of vitamin A. Physiol. Rev. 1984, 64

(3), 873−937.

(13) (a) Berni, R.; Formelli, F. In vitro interaction of fenretinide with

plasma retinol-binding protein and its functional consequences. FEBS

Lett. 1992, 308 (1), 43−5. (b) Adams, W. R.; Smith, J. E.; Green, M.

H. Effects of N-(4-hydroxyphenyl)retinamide on vitamin A metabo-

lism in rats. Exp. Biol. Med. 1995, 208 (2), 178−85.

(14) Mata, N. L.; Lichter, J. B.; Vogel, R.; Han, Y.; Bui, T. V.;

Singerman, L. J. Investigation of Oral Fenretinide for Treatment of

Geographic Atrophy in Age-Related Macular Degeneration. Retina

2013, 33 (3), 498−507.

(15) Motani, A.; Wang, Z.; Conn, M.; Siegler, K.; Zhang, Y.; Liu, Q.;

Johnstone, S.; Xu, H.; Thibault, S.; Wang, Y.; Fan, P.; Connors, R.; Le,

H.; Xu, G.; Walker, N.; Shan, B.; Coward, P. Identification and

characterization of a non-retinoid ligand for retinol-binding protein 4

which lowers serum retinol-binding protein 4 levels in vivo. J. Biol.

Chem. 2009, 284 (12), 7673−80.

(16) Dobri, N.; Qin, Q.; Kong, J.; Yamamoto, K.; Liu, Z.; Moiseyev,

G.; Ma, J.-X.; Allikmets, R.; Sparrow, J. R.; Petrukhin, K. A1120, a

Nonretinoid RBP4 Antagonist, Inhibits Formation of Cytotoxic

Bisretinoids in the Animal Model of Enhanced Retinal Lipofuscino-

genesis. Invest. Ophthalmol. Visual Sci. 2013, 54 (1), 85−95.

(17) Swanson, D. M.; Dubin, A. E.; Shah, C.; Nasser, N.; Chang, L.;

Dax, S. L.; Jetter, M.; Breitenbucher, J. G.; Liu, C.; Mazur, C.; Lord, B.;

Gonzales, L.; Hoey, K.; Rizzolio, M.; Bogenstaetter, M.; Codd, E. E.;

Lee, D. H.; Zhang, S. P.; Chaplan, S. R.; Carruthers, N. I. Identification

and biological evaluation of 4-(3-trifluoromethylpyridin-2-yl)-

piperazine-1-carboxylic acid (5-trifluoromethylpyridin-2-yl)amide, a

high affinity TRPV1 (VR1) vanilloid receptor antagonist. J. Med.

Chem. 2005, 48 (6), 1857−72.

(18) (a) Dart, M. J.; Searle, X. B.; Tietje, K. R.; Toupence, R. B.

Azabicyclic Compounds Are Central Nervous System Active Agents.

PCT Int. Appl. WO2004016604 A2. Google Patents: 2004. (b) Lauffer,

D.; Li, P.; Waal, N.; Mcginty, K.; Tang, Q.; Ronkin, S.; Farmer, L.;

Shannon, D.; Jacobs, D. C-Met Protein Kinase Inhibitors. PCT Int.

Appl. WO2009045992 A2. Google Patents: 2009. (c) Guillemont, J. E.

(7) (a) Radu, R. A.; Han, Y.; Bui, T. V.; Nusinowitz, S.; Bok, D.;

Lichter, J.; Widder, K.; Travis, G. H.; Mata, N. L. Reductions in serum

vitamin A arrest accumulation of toxic retinal fluorophores: a potential

therapy for treatment of lipofuscin-based retinal diseases. Invest.

Ophthalmol. Visual Sci. 2005, 46 (12), 4393−401. (b) Radu, R. A.;

Mata, N. L.; Nusinowitz, S.; Liu, X.; Sieving, P. A.; Travis, G. H.

Treatment with isotretinoin inhibits lipofuscin accumulation in a

mouse model of recessive Stargardt’s macular degeneration. Proc. Natl.

Acad. Sci. U. S. A. 2003, 100 (8), 4742−7. (c) Maeda, A.; Maeda, T.;

Golczak, M.; Imanishi, Y.; Leahy, P.; Kubota, R.; Palczewski, K. Effects

of potent inhibitors of the retinoid cycle on visual function and

photoreceptor protection from light damage in mice. Mol. Pharmacol.

2006, 70 (4), 1220−9. (d) Palczewski, K. Retinoids for treatment of

retinal diseases. Trends Pharmacol. Sci. 2010, 31 (6), 284−95.

(8) Petrukhin, K. Pharmacological inhibition of lipofuscin accumu-

lation in the retina as a therapeutic strategy for dry AMD treatment.

Drug Discovery Today: Ther. Strategies 2013, 10 (1), e11−e20.

(9) Cioffi, C. L.; Dobri, N.; Freeman, E. E.; Conlon, M. P.; Chen, P.;

Stafford, D. G.; Schwarz, D. M. C.; Golden, K. C.; Zhu, L.; Kitchen, D.

B.; Barnes, K. D.; Racz, B.; Qin, Q.; Michelotti, E.; Cywin, C. L.;

Martin, W. H.; Pearson, P. G.; Johnson, G.; Petrukhin, K. Design,

Synthesis, and Evaluation of Nonretinoid Retinol Binding Protein 4

Antagonists for the Potential Treatment of Atrophic Age-Related

Macular Degeneration and Stargardt Disease. J. Med. Chem. 2014, 57

(18), 7731−7757.

G.; Lanco̧ is, D. F. A.; Motte, M. M. S.; Koul, A.; Balemans, W. M. A.;

Arnoult, E. P. A. Antibacterial Cyclopenta[c]pyrrole Substituted 3,4-

Dihydro-1H-[1,8]naphthyridinones. PCT Int. Appl. WO2013021054

A1. Google Patents: 2013.

(19) Cogan, U.; Kopelman, M.; Mokady, S.; Shinitzky, M. Binding

affinities of retinol and related compounds to retinol binding proteins.

Eur. J. Biochem. 1976, 65 (1), 71−8.

(20) Gamble, M. V.; Ramakrishnan, R.; Palafox, N. A.; Briand, K.;

Berglund, L.; Blaner, W. S. Retinol binding protein as a surrogate

measure for serum retinol: studies in vitamin A-deficient children from

the Republic of the Marshall Islands. Am. J. Clin. Nutr. 2001, 73 (3),

594−601.

(10) (a) Kanai, M.; Raz, A.; Goodman, D. S. Retinol-binding protein:

the transport protein for vitamin A in human plasma. J. Clin. Invest.

1968, 47 (9), 2025−44. (b) Naylor, H. M.; Newcomer, M. E. The

Z

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Article Doi

Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

DOI: 10.1021/acs.jmedchem.5b00423

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