Scholarly article on tert-butyl (S)-(3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)(2,4-dimethoxybenzyl)carbamate 1396257-04-7 from Journal of Medicinal Chemistry p. 8110

Article

pubs.acs.org/jmc

Discovery and Preclinical Pharmacology of a Selective ATP-

Competitive Akt Inhibitor (GDC-0068) for the Treatment of Human

Tumors

James F. Blake,*^,†Rui Xu,^†Josef R. Bencsik,^†Dengming Xiao,^†Nicholas C. Kallan,^†Stephen Schlachter,^†

Ian S. Mitchell,^†Keith L. Spencer,^†Anna L. Banka,^†Eli M. Wallace,^†Susan L. Gloor,^†

Matthew Martinson,^†Richard D. Woessner,^†Guy P.A. Vigers,^†Barbara J. Brandhuber,^†Jun Liang,^‡

Brian S. Saﬁna,^‡Jun Li,^‡Birong Zhang,^‡Christine Chabot,^‡Steven Do,^‡Leslie Lee,^‡Jason Oeh,^‡

Deepak Sampath,^‡Brian B. Lee,^‡Kui Lin,^‡Bianca M. Liederer,^‡and Nicholas J. Skelton^‡

^†Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301, United States

^‡Genentech Inc., 1 DNA Way, South San Francisco, California 94080-4990, United States

S

* Supporting Information

ABSTRACT: The discovery and optimization of a series of

6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are

ATP-competitive, selective inhibitors of protein kinase B/Akt

is reported. The initial design and optimization was guided by

the use of X-ray structures of inhibitors in complex with Akt1

and the closely related protein kinase A. The resulting

compounds demonstrate potent inhibition of all three Akt

isoforms in biochemical assays and poor inhibition of other

members of the cAMP-dependent protein kinase/protein

kinase G/protein kinase C extended family and block the

phosphorylation of multiple downstream targets of Akt in

human cancer cell lines. Biological studies with one such

compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic eﬀects on

downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase−Akt−

mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of

cancer.

competitive allosteric compounds. Several advanced Akt

inhibitors, representing both classes of compounds, were or are

being tested in clinical trials for the treatment of human cancers.⁵

Herein, we report on the discovery and preclinical character-

ization of 28 (GDC-0068), a highly selective pan-Akt inhibitor

that targets the ATP-binding cleft.

INTRODUCTION

■

Protein kinase B (PKB)/Akt is a serine−threonine kinase, a

downstream target for phosphatidylinositol 3-kinase (PI3K),

which comprises three closely related isoforms (Akt1, Akt2, and

Akt3). Akt functions as a pivotal node in the PI3K−Akt−mTOR

pathway; once activated, Akt can control key cellular processes

by phosphorylating substrates involved in apoptosis, tran-

scription, cell cycle progression, and translation.¹Akt activity is

frequently elevated in cancer due to ampliﬁcation and/or gain-of-

function mutations of upstream receptor tyrosine kinases and/or

PI3K, as well as loss of PTEN function, a negative regulator of

Akt.²Constitutive activation or overexpression of Akt isoforms

has been identiﬁed in a wide variety of human tumors, including

breast, prostate, ovarian carcinoma, and melanoma.²shRNA

knockdown of Akt in PTEN-null tumor xenograft models

demonstrates antitumor eﬀects with maximum eﬃcacy achieved

by inhibiting all three isoforms.³Combined, these factors

contribute to the attractiveness of inhibiting Akt activity as a

novel therapeutic approach to cancer treatment.⁴

As detailed in previous work,^6,7compounds exempliﬁed by 1

and 2 (Figure 1) demonstrated potent inhibition of Akt in

biochemical assays (Akt1 enzyme inhibition, IC₅₀, of 3 and 1 nM

for 1 and 2, respectively), reduced phosphorylation of Akt

substrates in cellular assays (e.g., reduction of p-PRAS40 levels in

LNCaP cells with IC₅₀values of 160 and 137 nM for 1 and 2,

respectively), and down-regulation of Akt signaling in xenograft

models of human cancer in nude mice. While the lack of kinase

selectivity and resulting tolerability issues hindered the develop-

ment of 1, compound 2 displayed a signiﬁcantly improved

selectivity proﬁle versus kinases such as PKA (PKA enzyme

inhibition IC₅₀/Akt1 enzyme inhibition IC₅₀= 35), ROCK1, and

Strategies for targeting Akt have included both ATP-

Received: July 12, 2012

competitive, active-site-directed inhibitors, and non-ATP-

Published: August 30, 2012

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followed by cyclization upon heating with formamide and

ammonium formate to deliver pyrimidones 6a−c. Phorphorous

oxychloride-mediated chlorination followed by SnAr introduc-

tion of the Boc-protected piperazine linker delivered 7a−c.

Oleﬁn 7c was converted to alcohol 7d by ozonolysis, followed by

reductive workup with NaBH₄and chiral supercritical ﬂuid

chromatography (SFC) separation of the resulting racemic

alcohol. 7d was then further functionalized by treatment with n-

perﬂuorobutanesulfonyl ﬂuoride (PBSF) and HF−Et₃N¹⁰to

aﬀord 7e. Removal of the Boc group in 7a−e provided 8a−e as

dihydrochloride salts.

Figure 1. Advanced proof-of-concept pan-Akt inhibitors.

C7-hydroxylated and -ﬂuorinated cyclopenta[d]pyrimidine

cores were synthesized as illustrated in Scheme 2. Introduction of

the C7-hydroxyl functionality was facilitated by N-oxide

formation of 7a followed by N-oxide acylation and concomitant

rearrangement upon heating to give acetate 9 in a 3:2 cis−trans

orientation. Hydrolysis of the acetate and subsequent Swern

oxidation led to ketone 10. Asymmetric transfer hydrogenation¹¹

using a ruthenium catalyst, RuCl(p-cymene)[(R,R)-TsDPEN],

gave the desired (R)-alcohol 12a in diastereomeric excess

ranging from 94% to 98%. To obtain diastereomerically pure

material, the alcohol was converted to the p-nitrophenyl ester,

which enabled separation by either column chromatography or

recrystallization. Hydrolysis of the p-nitrophenyl ester followed

by acid-mediated N-Boc deprotection revealed the key amine

intermediate 12b in excellent diastereomeric excess (>99% by

HPLC) as the dihydrochloride salt. The (S)-alcohol intermediate

13b was prepared in a similar manner except for using the S,S

ruthenium catalyst in the asymmetric hydrogenation step.

Ketone 10, (R)-alcohol 12a, and (S)-alcohol 13a were treated

with DAST¹²to give the corresponding ﬂuorinated products,

which were deprotected under acidic conditions to yield the

diﬂuoro core 11b, cis-ﬂuoro core 14b, and trans-ﬂuoro core 15b.

In Scheme 3, a convenient method for the preparation of

optically active β-phenylalanine amino acids is described. The

Evans auxiliary (R)-4-benzyloxazolidin-2-one (16) was coupled

with 2-(4-chlorophenyl)acetyl chloride to give oxazolidin-2-one

(17). Treatment of 17 with TiCl₄at −78 °C followed by

Mannich reaction of the resulting titanium enolate with N-

acyliminium ions generated in situ from N-(alkoxymethyl)-

other related AGC family members and was well tolerated while

showing signiﬁcant antitumor activity in PC3-NCI prostate

cancer xenograft models.⁷The present work builds on our

experiences with 1 and 2 and utilizes the dihydrocyclopentapyr-

imidine core (exempliﬁed by 3) as a platform to further explore

the selective inhibition of Akt. With suitable substitution around

this core, potent Akt-selective compounds have been identiﬁed

that exhibit druglike properties and are suitable candidates for

clinical evaluation.

CHEMISTRY

■

The general synthetic routes used to prepare 6,7-dihydro-5H-

cyclopenta[d]pyrimidine derivatives are outlined in the following

schemes.

As shown in Scheme 1, preparation of the 5(R)-methyl-

substituted cyclopenta[d]pyrimidine core requires rapid access

to the β-keto ester intermediate 5a. We decided to capitalize on

the commercially available natural product chiral pool.

Speciﬁcally, we searched for a starting material that could be

readily manipulated into the desired β-keto ester, while allowing

us to incorporate the desired methyl stereochemistry. Thus,

commercially available (+)-pulegone (4) was sequentially

reacted with bromine and sodium ethoxide to smoothly undergo

ring contraction via a Favorskii rearrangement. Subsequent

ozonolysis and reduction with zinc dust in acetic acid gave the

desired chiral keto ester 5a in excellent yield. Construction of the

pyrimidine ring was carried out by initial conversion of β-keto

esters 5a, 5b,⁸and 5c⁹to enamines by ammonium acetate

a

Scheme 1. Synthesis of 5-Substituted 6,7-Dihydro-5H-cyclopenta[d]pyrimidine Cores

a

Reagents and conditions: (a) (i) NaHCO₃, Et₂O, Br₂, 0 °C; (ii) 21% NaOEt, EtOH, 0 °C to rt, 12 h; (iii) semicarbazide hydrochloride, NaOAc,

H₂O−EtOH (2:1), reﬂux to rt, 12 h (64%); (b) (i) ozone, EtOAc, −78 °C; (ii) zinc dust, acetic acid, 0 °C, 2 h (94%); (c) NH₄OAc, MeOH, 12 h;

(d) NH₄CO₂, formamide, 150 °C, 24 h; (e) POCl₃, DCE, reﬂux, 6 h; (f) tert-butyl piperazine-1-carboxylate, DIEA, 1-BuOH, reﬂux, 16 h; (g) (i)

ozone, DCM, −78 °C, 15 min; (ii) EtSMe, rt, 1 h; (iii) NaBH₄, MeOH, 0 °C, 2 h (55%); (iv) chiral SFC separation; (h) PBSF, HF−Et₃N, (80%);

(i) 4 N HCl in dioxane, DCM, 12 h.

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a

Scheme 2. Synthesis of C7-Hydroxylated and -Fluorinated Cyclopenta[d]pyrimidine Cores

a

Reagents and conditions: (a) m-CPBA, NaHCO₃, CHCl₃, 0 °C to rt, 2 h (100%); (b) acetic anhydride, 100 °C, 2 h (89%); (c) LiOH, THF−H₂O

(5:1), 16 h (99%); (d) oxalyl chloride, DMSO, Et₃N, DCM, −78 °C to rt, 12 h (71%); (e) (i) RuCl(p-cymene)[(R,R)-TsDPEN] (for 12a) or

RuCl(p-cymene)[(S,S)-TsDPEN] (for 13a), formic acid, Et₃N, DCM, 12 h (ii) 4-nitrobenzoyl chloride, Et₃N, DCM, 0 °C to rt, 4 h; (iii) LiOH,

THF−H₂O (2:1), 0 °C to rt, 1 h; (f) DAST, DCM, −20 °C, 1 h; (g) 4 N HCl in dioxane, DCM, 12 h.

a

Scheme 3. Stereoselective Synthesis of β-Phenylalanine Amino Acids

a

Reagents and conditions: (a) n-BuLi, THF, −78 to −20 °C, 2-(4-chlorophenyl)acetyl chloride, 12 h (79%); (b) 1 M TiCl₄, DCM, −78 °C, DIEA,

rt, 1.5 h (72%); (c) DDQ, DCM, H₂O, 19 h (100%); (d) LiOH−H₂O, THF−H₂O (3:1), 0 °C, H₂O₂, 12 h.

carbamates 18 aﬀorded 19a−g in good yield and diastereoiso-

meric ratio (for example, 14:1 diastereomeric excess (de) for

19a). The diastereomers were easily separated by silica gel

column chromatography. Basic hydrolysis of the chiral auxiliary

provided the desired β-amino acids in excellent enantiopurity. N-

Unsubstituted amino acid 20h was synthesized by sequential

cleavage of the p-methoxybenzyl ether (PMB) group and the

chiral auxiliary in 19f. In a similar manner, preparation of amino

acids 23i−k with cyclic amines was accomplished by stereo-

selective coupling of the Evans imide 17 with α-methoxy

heterocycles 21i−k followed by basic hydrolysis of the

oxazolidinone auxiliary.¹³

With the requisite cores and β-phenylalanine amino acids in

hand, preparation of the desired compounds was achieved by

amide coupling, followed by Boc deprotection under acidic

conditions, as exempliﬁed in Scheme 4. The primary and

secondary amines were further elaborated by reductive

amination with Na(OAc)₃BH and aldehydes or ketones to

aﬀord the corresponding substituted amines.

Several analogues with CH₂CF₃- and t-Bu-substituted amines

or cyclic tertiary amines were synthesized by an alternative route

as outlined in Scheme 5. Alkylation of the primary amine 55a

with triﬂuoroethyl triﬂate followed by saponiﬁcation of the

methyl ester with KO(TMS) aﬀorded 58a as a potassium salt.

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a

Scheme 4. Amide Coupling and Further Elaboration of Amines

a

Reagents and conditions: (a) HBTU, Hunig’s base, DCM, 1 h; (b) 4 N HCl, DCM, rt; (c) aldehyde or ketone, Na(OAc) BH, Hunig’s base, DCE,

̈

3

rt, 16 h.

a

Scheme 5. Alternative Synthesis of Substituted Amines

a

Reagents and conditions: (a) CF CH OTf, Hunig’s base, THF−DMF (1:1), rt, 20 h, (93%); (b) KO(TMS), THF, rt, 18 h; (c) paraformaldehyde,

̈

3

2

10% NaOMe, DMSO, rt, 12 h; (d) MsCl, TEA, DCM, 0 °C to rt, 12 h; (e) HNR′R″, THF, 0 °C, 12 h; (f) (i) HBTU, Hunig’s base, DMF, 18 h; (ii)

chiral separation.

̈

Alternatively, phenyl acetate esters 56a,b were converted to the

acrylates 57a,b as previously reported.^6,7Michael addition of

amines followed by hydrolysis of the methyl ester provided 58b−

e as racemates, which were then coupled with the C7-

hydroxylated core 12b. Finally, all diastereomeric pairs were

resolved by chiral stationary-phase HPLC or chiral SFC to obtain

desired compounds 37, 39, 40, 51, and 52. The stereochemical

assignments of compounds 37, 39, 40, 51, and 52 were based on

the results of the Akt1 enzyme inhibition assay. All

diastereomeric pairs were tested, and the active diastereomer

was then assigned the stereochemistry shown in Scheme 5 on the

basis of the activity of highly similar compounds prepared

enantioselectively.

RESULTS AND DISCUSSION

■

Analysis of a series of X-ray structures of the pyrrolopyrimidine

and dihydrothienopyrimidine hinge-binding cores bound to

Akt1 and PKA suggested that increased steric bulk near the

gatekeeper residue tended to improve the selectivity proﬁle

relative to PKA and ROCK1/2. This was principally due to

diﬀerences between Akt1 and PKA that include Thr211 (Akt1)

to Val (PKA), Met281 (Akt1) to Leu, and Ala230 (Akt1) to Val,

which lead to a narrower and less polar cavity in PKA, which

should be less forgiving of larger hinge-binding functionality (cf.

Figure 2, ref 7). The saturated ring and larger sulfur atom of

dihydrothienopyrimidine 2 confer 35-fold selectivity for Akt1

versus PKA, while pyrrolopyrimidine 1 is only 2-fold selective.

The increased polarity of the Akt1 active site conferred by

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reasonable animal pharmacokinetics (PK).^6,7Thus, these

features were maintained during our initial exploration of the

hinge-binding motif.

From our previous eﬀorts, methyl substitution at the 5- and 6-

positions of the pyrrolopyrimidine core yielded a modest

increase in selectivity over PKA.⁶Coupled with the selectivity

data of the dihydrothienopyrimidine core, we targeted the

dihydrocyclopentapyrimidine core as it aﬀorded a better

platform to explore additional substitution around the saturated

ring. The ﬁrst of these analogues prepared (Figure 1, 3)

maintained the overall substitution pattern of 2 and proved to be

potent, with Akt1 IC₅₀= 6 nM, Akt2 IC₅₀= 12 nM, Akt3 IC₅₀= 5

nM, LNCaP cell p-PRAS40 IC₅₀= 287 nM, and 6-fold selectivity

over PKA (PKA IC₅₀= 33 nM). Screening of 3 against a broad

panel of 225 kinases found the compound displayed potent

inhibition against only 5 additional kinases (>90% inhibition at 1

μM versus PRKG1α, PRKG1β, p70S6K, MSK1, and MSK2) and

moderate potency against 14 kinases (>50% inhibition at 1 μM)

and thus appeared to be reasonably selective.¹⁵Permeability of 3

was high, as measured in a Caco-2 assay: 13.2 × 10⁻⁶cm/s (apical

to basolateral, A to B) and 19.2 × 10⁻⁶cm/s for the reverse

direction (basolateral to apical, B to A). The compound also

displayed a high predicted free fraction with 51% human plasma

protein binding. Unfortunately, 3 possessed a rapid in vitro

clearance in human hepatocytes (15 mL/min/kg). Given the

excellent selectivity, potency, and permeability properties of 3, it

represented a good starting point for further optimization eﬀorts.

From inspection of the X-ray structure of a related

dihydrothienopyrimidine inhibitor bound to Akt1, we reasoned

that, in the absence of signiﬁcant changes in the enzyme

structure, only relatively small substituents would be tolerated at

the 5- and 7-positions of the analogous dihydrocyclopentapyr-

imidine core (Figure 2). Moreover, due to the proximity of

protein atoms, there is more space available above the plane of

the bicycle in the 5-position and below the plane of the bicycle in

the 7-position. The environment above the plane of the bicycle is

Figure 2. X-ray crystal structure of Akt1 in complex with a

dihydrothienopyrimidine inhibitor (compound 26 from ref 7, PDB

code 3OW4) at 2.6 Å resolution showing the molecular surface of the

pocket in the vicinity of the hinge (hydrogens added for clarity).

Thr211 could be exploited with complementary polar atoms on

the inhibitor; for example, the dihydrofuranyl derivative of 2

showed >25-fold selectivity for Akt1 over PKA while maintaining

druglike properties.⁷Inhibition by a series of spirochromane

compounds has also revealed that PKA inhibition is much more

dependent on the nature of the hinge-binding motif than Akt

inhibition,¹⁴again pointing to utility of this region of the Akt

active site for generating selectivity. On the basis of these

observations and a desire to further improve the selectivity

proﬁle of 2, we decided to pursue additional changes to the

hinge-binding motif designed to take advantage of these key

insights. In previous analyses of the structure−activity relation-

ship (SAR) for the amino amide portion of pyrrolopyrimidine

and dihydrothienopyrimidine compounds, 4-chlorophenyl with

an isopropylamine substituent aﬀorded excellent potency, with

Table 1. Development of the Dihydrocyclopentapyrimidine Core SAR

a

Akt1 inhibition, IC₅₀,

nM

Akt2 inhibition, IC₅₀,

nM

Akt3 inhibition, IC₅₀,

nM

Akt p-PRAS40 LNCaP IC₅₀,

nM

PKA inhibition, IC₅₀,

nM

compd

R¹

CH₃

R²

3

25

26

33

27

28

29

30

31

32

H

6

2

12

4

5

3

287 18

ND

33

9

dimethyl

vinyl

>2000

2

3

1

6

2

1

5

0

2

176 31

123 32

846 202

157 30

740 53

152 31

901 272

8548 550

10

3

CH₂F

CH₂OH

CH₃

35 12

81 40

356 53

18 10

83 26

957 332

3100 705

1552 455

(R)-OH

(S)-OH

(R)-F

(S)-F

5

7

8

9

CH₃

68 38

249 11

73 27

CH₃

4

2

14

3

10

23

2

5

17

4

CH₃

12

4

35 13

541 179

>10000

CH₃

diF

1169 487

4177 879

4160 1466

a

Values are means of three or more experiments, and the standard deviation is given. ND = not determined.

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largely lipophilic (side chains of Ala177, Val164, Phe225, and

Met227), while below the plane it is more hydrophilic (side

chains of Thr211 and Thr291) and includes a water molecule

coordinated by the backbone and side chain of Glu228.

Interestingly, no water molecules are seen proximal to this site

in 64 publicly available crystal structures of PKA (multiple

inhibitor classes bound and with the majority of the crystals

diﬀracting to better than 2.5 Å) except in cases where Val123 of

PKA has been mutated to alanine (equivalent to Ala230 in Akt1),

presumably because the increased hydrophobic bulk of valine

makes this site unfavorable to water. We note that, in an aligned

set of 470 human kinase sequences, only 5% have an alanine

corresponding to Ala230 of Akt1, only 11% have a threonine

corresponding to Thr211, and only Akt has both. Thus,

hydrophilic ligand substituents in the vicinity of Thr211 and

the water molecule would be expected to improve selectivity over

not only PKA, but also most other kinases.

Figure 3. X-ray structure of 28 bound to Akt1, solved at 2.0 Å resolution.

Hydrogen atoms added for clarity (PDB code 4EKL).¹⁶

Polarity at the 5-position of the dihydrocyclopentapyrimidine

core (compound 27) did not improve the selectivity and resulted

in a loss of potency. Increasing the size of the C5 substituent with

hydrophobic groups (26, 33) tended to increase the potency

slightly, but aﬀorded no increase in selectivity over PKA. Note

that disubstitution at C5 (25) resulted in a signiﬁcant decrease in

potency, which is consistent with the narrow nature of both the

Akt and PKA active sites. One particularly interesting ﬁnding that

emerged from substitution on the core is the proﬁle of the cis

conﬁguration (Table 1, compounds 29 and 31) relative to the

corresponding trans analogues. From this comparison, the cis

substitution for both the ﬂuoro and hydroxy substituents

produces an equivalent proﬁle, with high potency against Akt1

and much lower potency against PKA. However, for the trans

conﬁguration (compounds 28 and 30), the proﬁle diverges

dramatically with over a 182-fold separation in PKA activities

between the two analogues. The (R)-F substituent of 30 is small

enough to ﬁt within the cavity near the hinge of PKA; however,

this is clearly not the case for the equivalent hydroxy substitution

as both the size and polarity of the substituent lead to a signiﬁcant

loss of PKA activity. Interestingly, even though both the R and S

conﬁgurations of both the ﬂuoro and hydroxy groups at C7 are

potent against Akt, the diﬂuoro analogue (compound 32) was

poorly tolerated. The C7-(R)-hydroxyl combined with the C5-

(R)-methyl substitution (Table 1, compound 28) gave ca. 620-

fold selectivity versus PKA and aﬀorded good cell potency.

Testing against a broad panel of 230 kinases, 28 only inhibited 3

kinases by >70% at 1 μM concentration (PRKG1α, PRKG1β,

and p70S6K, with subsequent IC₅₀values determined to be 98,

69, and 860 nM, respectively).¹⁵From our perspective, the

dihydrocyclopentapyrimidinol core had the desired pan-AKT

potency proﬁle and achieved the level of selectivity we felt would

ensure a wide safety margin on the basis of our prior eﬀorts.

The binding mode of the 6,7-dihydro-5H-cyclopenta[d]-

pyrimidine core was conﬁrmed by crystallography: the crystal

structure of 28 shows that the pyrimidine ring interacts via a

hydrogen bond to the amide NH of Ala230 (the N−N distance is

2.97 Å), illustrated in Figure 3. The hydroxyl donates a hydrogen

bond to the backbone carbonyl of Glu228 (the O−O distance is

2.66 Å). The isopropylamine side chain interacts in the carbonyl-

rich region with the carboxylate side chains of Glu234 (2.96 Å)

and Glu278 (2.75 Å). The 4-chlorophenyl group occupies a small

hydrophobic pocket under the P-loop that is formed when

Phe161 is displaced toward the C-helix. As noted above, one of

the key diﬀerences we targeted between Akt and PKA, or

ROCK1, is the presence of Ala230 (Akt1) in the hinge. The small

side chain of Ala230 in Akt1 creates a pocket that enables

substitution of the dihydrocyclopentapyrimidine core with

various groups that aﬀord a high degree of selectivity.

In the previous dihydrothieno- and dihydrofuropyrimidine

series, metabolism studies identiﬁed that amine dealkylation was

the major metabolic reaction.⁷On the basis of similar metabolite

identiﬁcation studies, N-dealkylation and oxidation of the core

hydroxyl substituent to form the corresponding ketone were the

primary routes of metabolism of 28 in vitro. The stability,

assessed by predicted in vitro clearance in human hepatocytes for

28, was 8 mL/min/kg. Given the nature of the amine

metabolism, we sought to synthesize analogues with varying

degrees of polarity, size, and basicity in an eﬀort to reduce the

oxidative loss of the isopropyl group, illustrated in Table 2.

As we observed in the dihydrothienopyrimidine series of

compounds,⁷the secondary amines tended to possess better cell

potency relative to the primary amines (e.g., 34 vs 35), likely due

to reduced permeability of the latter. The stability in human

hepatocytes for 35 decreased signiﬁcantly (predicted clearance of

11 mL/min/kg). Interestingly, inhibition of PKA does not

change for the primary amines, giving rise to a reduced selectivity

vs Akt1 for these compounds. Akt1 inhibition is tolerant of many

small aliphatic amine substitutions (35−38), while predicted

clearance ranged from 9 to 14 mL/min/kg in human hepatocytes

for these compounds. Similarly, the tertiary amine analogue of 28

led to only a slight degradation of potency and selectivity (42);

however, the predicted clearance increased to 11 mL/min/kg.

Larger cyclic aliphatic substitutions of the amine are also well

tolerated, with the enzyme potency, cell-based activity, and PKA

selectivity all within ca. 5-fold of those of 28 (41, 44, 45, and 46).

Constraining the amine also had little eﬀect on inhibition of Akt

(47 and 48), although in the case of the pyrrolidine analogue

(43) there was a degradation in PKA selectivity. Compound 43

did demonstrate improved stability (clearance in human

hepatocytes, 3 mL/min/kg). Likewise, polar additions to the

amine chain (39) produced lower clearance compounds (3 mL/

min/kg); however, this came at the expense of PKA selectivity.

The expected binding mode of these compounds has the amine

situated at the lip of the ATP pocket such that these larger

substitutions can project into the solvent, thus providing a

potential mechanism to alter the physicochemical properties of

the inhibitors without signiﬁcantly compromising the potency.

However, the basicity of the amine is an important driver of

potency; reducing the pK_abelow 6.5 led to an 8-fold drop in cell

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Table 2. Development of the Amine SAR

a

b

Values are means of three or more experiments, and the standard deviation is given. ND = not determined. pK_avalues were calculated using a

custom pK_amodel implemented in the MoKa software, version 1.1, from Molecular Discovery Ltd.¹⁷

potency (36), while reducing the pK_abelow 3.0 led to a 180-fold

decrease in enzyme activity (37). Although some of the

analogues in Table 2 did show modest improvements in potency,

none had dramatic improvements in metabolic stability while

retaining cellular potency and selectivity.

with the underside of the P-loop of Akt1 (Table 3). Replacing the

4-Cl with a 4-CF₃group improves the potency in the context of

the primary amine (comparing 49 and 34), although selectivity

over PKA is still only moderate. Addition of a 3-F group to the

secondary amines gives analogues with similar potency and

selectivity over PKA (compare 50 to 28 or 54 to 44). Combining

the 3-F with the 4-CF₃substitution led to a 3−4-fold

Finally, we explored the eﬀect of amine substitution in

conjunction with aromatic ring changes to probe the contacts

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Table 3. Development of the Cyclopentapyrimidinol Core SAR

a

Values are means of three or more experiments, and the standard deviation is given.

improvement in enzyme and cellular potency; however, this

substitution pattern reduces the selectivity ratio of PKA/Akt1 to

be ca. 35-fold or less (e.g., comparing 53 to 35 or 52 to 39).

While compounds 52 and 53 did show improved cellular

potency, the predicted stability (clearance of 7 and 13 mL/min/

kg in human hepatocytes, respectively) decreased, presumably

due to the increased lipophilicity of the 3-F-4-CF₃substitution.

From our exploration of the structure−activity relationship

around the dihydrocyclopentapyrimidinol core, 28 remained one

of the most selective compounds we have discovered, while

maintaining good potency against all three Akt isoforms and high

potency in cell-based assays. None of the analogues present in

Tables 2 and 3 oﬀered any signiﬁcant improvement of in vitro

stability and selectivity over 28, so 28 was advanced into

additional proﬁling. As with the corresponding dihydrocyclo-

pentapyrimidine (3), 28 is also predicted to have a high free-drug

fraction in plasma for both human and preclinical species with

39% human plasma protein binding and 44% measured in

monkey and 56% in mouse. Consistent with the plasma protein

binding results, the solubility of 28 was very high, being greater

than 10 mg/mL at three pH values (1.2, 6.5, and 7.4). 28 also

possessed low to moderate predicted in vitro clearance in human,

monkey, and mouse hepatocytes (8, 27, and 8 mL/min/kg,

respectively).

nM. We extended this analysis to other cell lines, including PC3,

MCF7-neo/HER2, and BT474M1. In all three lines, 28 was able

to inhibit overall viability with IC₅₀values in the range of 1−4

μM. More detailed analysis demonstrated that 28 induces a dose-

dependent block of the cell-cycle progression at the G1 phase

and a dose- and time-dependent increase in apoptosis and

necrosis in MCF7-neo/HER2 and BT474M1 cells (data not

shown; a more complete discussion of these studies will be

presented elsewhere). All four of the cell lines tested have

elevated levels of basal Akt signaling due to loss of PTEN (PC3

and LNCaP), mutation of PI3Kα (MCF7-neo/HER2), or

overexpression of Her2 (MCF7-neo/HER2 and BT474M1).

Thus, the inhibition of signaling and reduced viability suggest

that 28 will be useful in controlling human cancers in which

PI3K/Akt signaling is overly active. 28 was able to inhibit

phosphorylation of PRAS40 in all four of these cells lines with

IC₅₀values comparable to that observed in LNCaP cells (ca. 200

nM, data not shown).

PK studies of 28 in nu/nu mice were performed to support

pharmacodynamic and eﬃcacy studies. The animals were given a

single per os (po) dose of 28 at 12.5 and 50 mg/kg (0.5%

methylcellulose with 0.2% polysorbate 80 (MCT) dose

solutions). Systemic exposure increased in a more than dose-

proportional fashion with increasing dose, leading to good

exposures. Plasma concentrations were at or above 200 nM for

the 12.5 and 50 mg/kg dose groups, respectively, within 1 h of

drug administration. With the 50 mg/kg po dose, plasma

concentrations were approximately 7.4 μM at 1 h postdose and

0.5 μM at 9 h postdose; the concentration of 28 was above 200

nM for approximately 9 h (this concentration is higher than the

cellular IC₅₀for p-PRAS40 knockdown in LNCaP cells; see

Table 1). PK studies performed in rat and monkey also gave

In keeping with observations with other ATP-competitive Akt

inhibitors,¹⁸⁻²¹there is an increase of phosphorylated Akt levels

when cells are treated with 28.²²However, the data in Table 1

clearly show that 28 potently inhibits Akt signaling in LNCaP

cells (which have a high basal pAkt level due to loss of PTEN),

indicating that the increased level of phosphorylated Akt is not

functionally active in these cells.²²Additionally, 28 has a potent

antiproliferative eﬀect on this cell line with an IC₅₀of 95 16

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acceptable oral exposures (data not shown), suggesting that

reasonable oral exposures in humans can be achieved.

xenografts. Daily doses ranged from 25 to 100 mg/kg. Even the

lowest dose resulted in statistically signiﬁcant tumor growth

inhibition (49%, p < 0.009) when administered qd for 11 days

(Figure 5); hence, the minimum eﬃcacious dose was determined

A pharmacodynamic (PD) and PK study was performed in

nu/nu mice bearing subcutaneous PC3 prostate tumors to

correlate plasma drug levels of 28 with PD changes in the tumors.

Following administration of a single po dose, plasma and tumor

samples were collected from the animals between 1 and 24 h for

PK and PD analysis, respectively. As described above, robust Akt

pathway inhibition with 28 was determined in vitro on the basis

of the suppression of p-PRAS40. Therefore, this PD marker

relative to the total protein level was also evaluated in vivo.

Within 3 h of drug administration, there was a dose-dependent

decrease in the ratio of p-PRAS40 to tPRAS40 compared with

vehicle controls, with a >95% reduction achieved at 100 mg/kg

(Figure 4A). At 8 h postdose, plasma levels of 28 of >2.6 μM

Figure 5. Eﬀect of qd and bid oral dosing of 28 on PC3 prostate tumors

(mean tumor volume in cubic millimeters SEM). Dose levels are

expressed as free-base equivalents prepared in vehicle (0.5%

methylcellulose/0.2% Tween-80).

to be 25 mg/kg. The maximum tumor growth inhibition was

obtained with qd dosing of 28 for 11 days at 100 mg/kg (79%, p <

0.0001). In addition, half-maximal doses of 28 (50 mg/kg) given

orally bid resulted in a nearly equivalent tumor growth inhibition

of 81% when compared with qd dosing of 100 mg/kg (Figure 5,

not statistically diﬀerent). Thus, 28 is eﬃcacious against human

PC3 prostate cancer xenografts in vivo when dosed orally either

qd or bid. Overall body weight loss, including the vehicle control

group, was observed in all groups tested due to the cachexic

nature of this model.²³Doses of 0−100 mg/kg 28 qd caused less

than 10% mean body weight loss, whereas doses of 150 mg/kg qd

caused ≥20% loss of original body weight after eight doses, and

the mice had to be euthanized before the completion of the study

(data not shown).

CONCLUSIONS

■

Figure 4. PK/PD of 28 in PC3 prostate tumors. Tumor ratios of p-

PRAS40 to total PRAS40 (tPRAS40) were determined in female nude

mice bearing PC3 prostate tumor xenografts (average of ﬁve animals

SEM). Plasma concentrations of 28 were also measured. Samples were

collected 3 h (A) or 8 h (B) following administration of 12.5, 25, and 100

mg/kg doses (free base equivalents formulated in 0.5% methylcellulose/

0.2% Tween-80). The inhibition (%) of p-PRAS40/tPRAS40 is based

on comparison to the vehicle control and stated in parentheses. Average

drug levels SEM (μM) of 28 were determined by analysis of plasma

from ﬁve animals. Two asterisks indicate p < 0.001, determined by

Student’s t test to ﬁnd diﬀerences in biomarker eﬀects for dosing groups

vs the vehicle control.

We have described the discovery of 28, and related compounds,

for the treatment of human tumors. The novel ATP-competitive,

selective Akt inhibitors were optimized via structure-based

design to target unique features of the Akt ATP binding cleft,

resulting in exquisitely selective and potent inhibitors. In the

speciﬁc case of 28, this strategy led to good selectivity in a 230-

enzyme kinase panel. Extensive in vitro proﬁling has shown that

human cancer cell lines in which the PI3K/Akt pathway is

upregulated are sensitive to inhibition by 28; in spite of an

increase in pAkt levels, downstream signaling in this pathway is

inhibited by 28, providing a mechanistic explanation of the

antiproliferative and antisurvival eﬀects. The in vitro eﬀects are

recapitulated in the in vivo models, wherein we see good oral

exposures, a signiﬁcant inhibition of Akt signaling following a

single dose of 28, and a robust inhibition of tumor growth

following repeated qd or bid oral dosing in a mouse xenograft

model of PI3K/Akt-driven cancer. Given all of these data, 28 is

currently being investigated in human clinic trials for the

treatment of cancers driven by aberrant PI3K/Akt signaling.

were maintained with 100 mg/kg, and this correlated with an

87% inhibition of p-PRAS40/tPRAS40 (Figure 4B). Further-

more, pS6RP is also signiﬁcantly reduced under these conditions

(data not shown). These data demonstrate that 28 is able to

signiﬁcantly inhibit the Akt pathway in PC3 prostate tumors for

at least 8 h postdose at 100 mg/kg.

Multiple doses of 28 were also administered daily (qd) or

twice daily (bid) in nude mice bearing PC3 prostate cancer

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Franklin Lakes, NJ) at a density of 1500 cells/well and incubated

overnight to 1.5 days at 37 °C and 5% CO₂. Serial dilutions of inhibitor

were added to the cells, which were then incubated for another 96 h. Cell

viability was determined by measuring the cellular ATP levels as

described in the manufacturer’s protocol (CellTiter-Glo Luminescent

Cell Viability Assay Kit, catalog no. G7573, Promega, Madison, WI).

Dose−response curves were generated using the four-parameter logistic

model, and 50% inhibitory concentration (IC₅₀) values were determined

from these curve ﬁts.

In Vivo Eﬃcacy and PK/PD. For in vivo tumor xenograft studies,

female nu/nu (nude) mice were inoculated subcutaneously in the right

hind ﬂank with PC3 cells suspended in Hank’s balanced salt solution

(HBSS). When tumors reached a mean volume of 150 mm³, the animals

were size matched and distributed into treatment groups consisting of

10 animals/group. Tumor volume was calculated as follows: tumor size

(mm³) = (longer measurement × (shorter measurement)²) × 0.5.

Following data analysis, p values were determined using Dunnett’s t test

with JMP statistical software, version 7.0 (SAS Institute). Mouse body

weights were recorded twice weekly using an Adventura Pro AV812

scale (Ohaus Corp.). Mice were promptly euthanized when the tumor

volume exceeded 2000 mm³or if body weight loss was ≥20% of the

starting weight per IACUC protocol guidelines.

For PK/PD studies, blood and tumor samples were collected at 1, 3,

8, and 24 h after a single dose of 28 from PC3 tumor bearing mice. Blood

samples (approximately 800 μL) were collected from each animal at the

scheduled sample collection time by terminal cardiac puncture into

tubes containing K₂EDTA as an anticoagulant and centrifuged at 1500−

2000g to isolate plasma. The concentration of 28 in each plasma sample

was determined by a nonvalidated LC/MS/MS assay in the DMPK

Bioanalytical Department at Genentech. The assay lower limit of

quantitation (LLOQ) was 0.005 μM. Tumor samples were dissociated

in Tris lysis buﬀer containing 150 mM NaCl, 20 mM Tris (pH 7.5), 1

mM EDTA, 1 mM EGTA, and 1% Triton X-100 (Meso Scale Discovery;

Gaithersburg, MD). Protein concentrations were determined using the

BCA Protein Assay Kit (Pierce, Rockford, IL). The Invitrogen

(Camarillo, CA) human enzyme-linked immunosorbent assay

(ELISA) kits were used to determine the levels of total PRAS40 and

PRAS40 phosphorylated at Thr246 (p-PRAS40). The assay quantiﬁes

protein levels on the basis of measurements of absorbance. The colored

product is directly proportional to the concentration of p-PRAS40 and

tPRAS40 present in the specimen. The Meso Scale Discovery Multi-

Spot Biomarker Detection System (Meso Scale Discovery) was used to

determine the levels of total S6RP and S6RP phosphorylated at Ser235/

236 (pS6RP). These assays quantify protein levels on the basis of

measurements of electrochemiluminescence intensity. Levels of

phosphorylated protein were normalized to total protein levels in 28-

treated tumors and compared to the vehicle control.

Chemistry. All reaction reagents and solvents (anhydrous grade)

were purchased and used without further puriﬁcation. ¹H NMR spectra

were recorded on a Varian INOVA 400 instrument. Chemical shifts are

reported in parts per million relative to an internal standard of TMS in

CDCl₃or DMSO-d₆. HPLC analysis was conducted according to

methods A−E, with the retention time (t_R) expressed in minutes at UV

detection of 254 nM. Chromatography was performed on a Varian

Prostar with a YMC ODS-C18-AQ column (4.6 × 50 mm, 3 μm) at 40

°C with a ﬂow rate of 2.0 mL/min. Mobile phase A was 10 mM NH₄OAc

in water with 1% isopropyl alcohol. Mobile phase B was 10 mM

NH₄OAc, 1% H₂O, and isopropyl alcohol in acetonitrile. HPLC method

A: The gradient was 5% B to 95% B in 5 min. HPLC method B: The

gradient was 0% B to 95% B in 5 min. HPLC method C:

Chromatography was performed on an Agilent HPLC instrument

with a Zorbax SB C18 column (4.6 × 50 mm, 3 μm) with a ﬂow rate of

2.0 mL/min. Mobile phase A was 0.1% TFA in water, and mobile phase

B was 0.075% TFA in acetonitrile. The gradient was 5% B to 95% B in 9

min. HPLC method D: Chromatography was performed on an Agilent

6140 HPLC instrument with a Zorbax SB C18 column (2.1 × 30 mm,

1.8 μm) at 40 °C with a ﬂow rate of 0.4 mL/min. Mobile phase A was

0.05% TFA in water, and mobile phase B was 0.05% TFA in acetonitrile.

The gradient was 3% B to 95% B in 8.5 min. HPLC method E:

Chromatography was performed on an Agilent 6140 HPLC instrument

EXPERIMENTAL SECTION

■

Enzymatic Assays. The assay for the determination of Akt1/2/3

and PKA kinase activity employs the IMAP ﬂuorescence polarization

(FP) phosphorylation detection reagent (IMAP Screening Express Kit,

catalog no. R8073, Molecular Devices, Sunnyvale, CA) to detect

ﬂuorescently labeled peptide substrates that have been phosphorylated

by the respective kinases. The Akt enzymes employed in these studies

consisted of recombinant baculovirus expressed, amino-terminal,

polyhistidine-tagged, full-length, wild-type human forms (GenBank

accession numbers M63167, NP_001617, and NP_005456) and were

obtained from Millipore (Akt1, catalog no. 14 276, lot no. D8MN034U;

Dundee, Scotland) or Invitrogen (Akt2, catalog no. PV3184, lot no.

28770P; Akt3, catalog no. PV3185, lot no. 28771K; Madison, WI). The

PKA enzyme employed in these studies consisted of the recombinant

untagged human isolated catalytic subunit of PKA (GenBank accession

number X07767) expressed in Escherichia coli obtained from Invitrogen

(catalog no. 14-440, lot no. 26698U). Inhibitor, enzyme (9 nM Akt1 or

100 pM PKA), and substrate (100 nM Crosstide, catalog no. R7110,

Molecular Devices) were incubated with 5 μM ATP in assay buﬀer (10

mM Tris−HCl (pH 7.2), 10 mM MgCl₂, 0.1% BSA (w/v), ﬁnal DMSO

2% (v/v)) for 60 min at ambient temperature in a 5 μL reaction volume.

Reactions were initiated by addition of enzyme + peptide substrate to

ATP solutions. IMAP binding reagent (15 μL) was added to terminate

the reaction, and the stopped reactions were incubated for a minimum of

30 min at room temperature (rt).

Cellular Assays. Phosphorylation of PRAS40 at Thr246 was

measured in situ in LNCaP cells (American Type Culture Collection,

catalog no. CRL-1740). The cells were plated in 96-well plates (Grenier,

catalog no. 655946) at a density of 20 000 cells/well and incubated for

16−24 h at 37 °C and 5% CO₂. The cells were treated with 0−25 μM

inhibitor for 1.5 h at 37 °C. Medium above the cells was removed, and

each well was supplemented with ﬁxation solution (3.7% (v/v)

formaldehyde in phosphate-buﬀered saline (PBS)) for 20 min at rt.

The cells were permeabilized with a 10 min exposure to 100% methanol

(−20 °C) and subsequently rehydrated in PBS and blocked in blocking

buﬀer (catalog no. 927-40000, LI-COR Inc., Lincoln, NE) for 60 min at

rt. A primary antibody solution consisting of an antibody speciﬁc for

Thr246-phosphorylated PRAS40 (rabbit polyclonal antibody, 1:500

dilution, catalog no. AS1011, Calbiochem, San Diego, CA) and a signal-

normalizing antibody against glyceraldehyde 3-phosphate dehydrogen-

ase (GAPDH; mouse monoclonal, 1 μg/mL ﬁnal concentration, catalog

no. RDI-TRK5-6C5, Fitzgerald Industries Inc., Concord, MA) in

blocking buﬀer was applied to each of the wells and incubated overnight

at 4 °C. The wells were then washed with PBS containing 0.05% (v/v)

Tween-20, treated with a secondary antibody solution containing

ﬂuorophore-conjugated antibodies speciﬁc for rabbit (Alexa680

ﬂuorophore-conjugated goat antirabbit immunoglobulin G (IgG),

catalog no. AS21109, Invitrogen) and mouse IgG (IRDye800

ﬂuorophore-conjugated goat antimouse IgG, catalog no. 610-132-121,

Rockland Inc., Gilbertsville, PA), and incubated for 1 h at rt. The wells

were washed in PBS with 0.05% (v/v) Tween-20 and then imaged and

quantiﬁed on an LI-COR Aerius imager (LI-COR Inc.). The phospho-

PRAS40 signal was normalized to the GAPDH signal to control for well-

to-well variation in cell number.

Inhibition of cellular viability was measured in LNCaP cells

(American Type Culture Collection, catalog no. CRL-1740) plated in

black, clear-bottomed 96-well plates (Grenier, catalog no. 655946) at a

density of 5000 cells/well and subsequently treated with 0−10 μM 28

for 72 h at 37 °C and 5% CO₂. The extent of cell proliferation was

determined by measuring the reduction of resazurin to resoruﬁn as

described in the manufacturer’s protocol (CellTiterBlue Cell Viability

Determination Kit, catalog no. G8082, Promega, Madison, WI) using an

excitation wavelength of 560 nm and an emission wavelength of 590 nm.

Dose−response curves were generated using the four-parameter logistic

model, and 50% inhibitory concentration (IC₅₀) values were determined

from these curve ﬁts.

Inhibition of cellular proliferation was measured in PC3-NCI, MCF7-

neo/HER2-neo/Her2, and BT474M1 cells plated in black, clear-

bottomed 384-well plates (catalog no. 353962, Becton Dickinson,

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with a Zorbax SB C18 column (3.0 × 100 mm, 3.5 μm) at 40 °C with a

ﬂow rate of 0.7 mL/min. Mobile phase A was 0.05% TFA in water, and

mobile phase B was 0.05% TFA in acetonitrile. The gradient was 2% B to

98% B in 25.5 min. Mass spectral analysis was conducted on a Thermo

Separation Products (TSP) HPLC or Waters Micromass ZQ instru-

ment.

were washed with brine, dried over solid MgSO₄, and concentrated in

vacuo to give the desired (R)-ethyl 2-amino-5-methylcyclopent-1-

enecarboxylate (136.2 g, 91% yield) as a brown oil. This material was

used without further puriﬁcation: ¹H NMR (CDCl₃, 400 MHz) δ 5.5 (br

s, 2H), 4.22−4.10 (m, 2H), 2.98−2.94 (m, 1H), 2.64−2.55 (m, 1H),

2.37−2.29 (m, 1H), 2.09−1.99 (m, 1H), 1.45−1.38 (m, 1H), 1.28 (t, J =

6.4 Hz, 3H), 1.96 (d, J = 6.4 Hz, 3H); LC/MS APCI (+) m/z 170.1 (M +

H)⁺.

(2R)-Ethyl 2-Methyl-5-oxocyclopentanecarboxylate (5a). To a 5 L

round-bottom ﬂask were added (R)-pulegone (600.0 g, 3.94 mol),

anhydrous NaHCO₃(165.0 g, 1.97 mol), and ether (2.0 L). The mixture

was cooled to 0 °C using an ice bath, and bromine (206.0 mL, 4.02 mol)

was added dropwise over 1 h. The reaction was allowed to stir for an

additional 30 min after bromine addition was complete, and the mixture

was ﬁltered to give ﬁltrate A. To a separate 12 L round-bottom reactor

equipped with a mechanical stirrere and a thermocouple were charged

21% NaOEt (3.2 L, 8.7 mol) and EtOH (2.0 L). This reaction mixture

was cooled to 0 °C, and ﬁltrate A was added dropwise at a rate which

maintained an internal temperature below 40 °C. Caution: Addition is

exothermic, and adequate cooling is required! After complete addition of

ﬁltrate A, the reaction was allowed to warm to rt. The reaction was

quenched by the addition of 1 N HCl (1.0 L) and water (1.5 L), followed

by the addition of methyl tert-butyl ether (MTBE; 1.0 L). The organic

layer was separated and the aqueous phase extracted with MTBE (3 ×

1.5 L). The combined organic layers were concentrated to give a brown

oil. To a second 12 L round-bottom reactor equipped with a mechanical

stirrer were charged semicarbazide hydrochloride (300.0 g, 2.6 mol),

NaOAc (300.0 g, 3.6 mol), and water (3.0 L). The crude oil from above

was added slowly as a solution in ethanol (1.5 L). The mixture was then

reﬂuxed for 3 h and stirred at rt overnight. The mixture was treated with

water (1.0 L) and MTBE (1.0 L). The organic layer was separated, and

the aqueous phase was extracted with MTBE (3 × 1.5 L). The combined

organic layer was washed with brine and concentrated to give a brown

oil. The oil was distilled under vacuum to give (2R)-ethyl 2-methyl-5-

propan-2-ylidenecyclopentanecarboxylate (497 g, 64% yield) collected

at 73−76 °C at 0.5 mm as a clear oil: ¹H NMR (CDCl₃, 400 MHz) δ

4.17−4.07 (m, 2H), 3.39 (d, J = 8.0 Hz, 0.5 H), 2.93 (d, J = 6.0 Hz, 0.5

H), 2.48−2.15 (m, 3 H), 2.03−1.98 (m, 1H), 1.79−1.72 (m, 1H), 1.65−

1.59 (m, 6H), 1.25 (t, J = 8.0 Hz, 3H), 1.03 (dd, J = 12.0, 6.8 Hz, 3H).

A solution of (2R)-ethyl 2-methyl-5-propan-2-ylidenecyclopentane-

carboxylate (220.0 g, 1.1 mmol) in EtOAc (1.0 L) was cooled to −78 °C

using a dry ice/2-propanol bath. Ozone was bubbled into the reaction

mixture until it turned purple in color. At this point ozone generation

was stopped, and the reaction mixture was removed from the dry ice

bath. Nitrogen was bubbled through the reaction solution until it turned

yellow. The reaction was concentrated and the resulting residue

dissolved in glacial acetic acid (200 mL). The solution was cooled to 0

°C, and zinc dust (113.0 g, 1.7 mol) was added in small portions over a

30 min period. The reaction was allowed to stir for 1.5 h, at which point

the reaction mixture was ﬁltered through Celite. The resulting solution

was concentrated in vacuo to remove acetic acid, and the residue was

diluted with MTBE (500 mL). The mixture was neutralized to pH 7.0 by

careful addition of aqueous 6 N NaOH. The organic layer was separated,

and the aqueous layer was extracted with MTBE (2 × 250 mL). The

combined organics were washed with brine, dried with solid MgSO₄, and

concentrated by rotary evaporation to give a dark brown liquid. This

liquid was passed through a plug of silica gel eluting with a small amount

of MTBE. The combined ﬁltrate was again concentrated by rotary

evaporation to give the desired (2R)-ethyl 2-methyl-5-oxocyclopenta-

necarboxylate (180.1 g, 94% yield) as a light brown liquid which was

used without any further puriﬁcation: ¹H NMR (CDCl₃, 400 MHz) δ

4.21 (q, J = 6.4 Hz, 2H), 2.75 (d, J = 11.2 Hz, 1H), 2.64−2.56 (m, 1H),

2.46−2.30 (m, 2H), 2.24−2.16 (m, 1H), 1.53−1.42 (m, 1H), 1.29 (t, J =

6.4 Hz, 3H), 1.19 (d, J = 6.4 Hz, 3H).

To a 2 L three-necked magnetically stirred round-bottom reactor

equipped with a condenser and thermocouple were added (R)-ethyl 2-

amino-5-methylcyclopent-1-enecarboxylate (308.0 g, 1.8 mol), ammo-

nium formate (172.0 g, 2.7 mol), and formamide (504 mL, 12.7 mol).

The mixture was heated to an internal temperature of 150 °C for 24 h.

Note: Sublimed ammonium formate could build up in the condenser.

Addition of 25 mL of a lower boiling solvent such as o-xylene helps keep the

condenser clear. The reaction mixture was cooled and transferred to 2 L

single-neck ﬂask, and the excess formamide was removed by distillation

under vacuum. Once removal of the formamide was complete, the ﬂask

was cooled, and the resulting oil was dissolved in DCM (2.0 L) and

washed with brine (3 × 200 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated by rotary evaporation. The resulting

brown oil was dissolved in a small amount of DCM and slowly added to

a stirring solution of ether (ca. 5× volume of ether vs DCM), resulting in

a brown slurry. The slurry was ﬁltered, and the resulting wet cake was

rinsed with ether. The brown ﬁltrate was concentrated by rotary

evaporation and dried under high vacuum to give crude (R)-5-methyl-

6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (180.1 g, 66% yield). This

1

material was used without further puriﬁcation: H NMR (CDCl₃, 400

MHz) δ 12.8 (br s, 1H), 8.07 (s, 1H), 3.35−3.29 (m, 1H), 2.98−2.91

(m, 1H), 2.85−2.78 (m, 1H), 2.36−2.26 (m, 1H), 1.71−1.64 (m, 1H),

1.31 (d, J = 6.4 Hz, 3H); LC/MS APCI (+) m/z 151.1 (M + H)⁺.

General Procedure for the Incorporation of Boc-Protected

Piperazine. (R)-tert-Butyl 4-(5-Methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazine-1-carboxylate (7a). To a solution of (R)-5-

methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (150.1 g, 998

mmol) in 1,2-dichloroethane (DCE; 500 mL) was added POCl₃(233

mL, 2.5 mol) dropwise. The reaction mixture was heated to reﬂux for 6 h

and concentrated by rotary evaporation. The crude oil was diluted with a

small amount of DCM to give a suspension, which was added to a

stirring solution of 6 M aqueous NaHCO₃(more NaHCO₃was added as

needed to keep the solution basic). The organic layer was separated, and

the aqueous layer was extracted with DCM (2 × 250 mL). The

combined organics were dried over MgSO₄, ﬁltered, and concentrated in

vacuo. The crude brown oil was puriﬁed by passing through a silica plug

eluting with hexanes−EtOAc (4:1) to give the desired (R)-4-chloro-5-

methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine (81.2 g, 48% yield) as

a brown liquid: ¹H NMR (CDCl₃, 400 MHz) δ 8.77 (s, 1H), 3.46−3.41

(m, 1H), 3.20−3.11 (m, 1H), 2.98 (ddd, J = 10.2, 6.4, 6.4, 1H), 2.42−

2.32 (m, 1H), 1.86−1.78 (m, 1H), 1.34 (d, J = 6.4 Hz, 3H); LC/MS

APCI (+) m/z 169.3 (M + H)⁺.

A solution of (R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidine (73.3 g, 434.7 mmol), tert-butyl piperazine-1-carboxylate

(85.0 g, 456.4 mmol), and N,N-diisopropylethylamine (DIEA; 227.0

mL, 1.30 mol) in 1-BuOH (720 mL) was heated to reﬂuxing under a

nitrogen atmosphere for 16 h. The reaction mixture was concentrated by

rotary evaporation, and the crude residue was puriﬁed by silica gel

chromatography eluting with hexanes−EtOAc (2:1) to EtOAc to give a

brown solid. The solid was recrystallized from heptane to give the

desired pure (R)-tert-butyl 4-(5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazine-1-carboxylate (112.6 g, 81% yield) as an oﬀ-

white solid: ¹H NMR (CDCl₃, 400 MHz) δ 8.47 (s, 1H), 3.72−3.68 (m,

2H), 3.57−3.44 (m, 7H), 2.96−2.84 (m, 2H), 2.32−2.26 (m, 1H),

1.72−1.67 (m, 1H), 1.48 (s, 9H), 1.79 (d, J = 7.2 Hz, 3H); LC/MS APCI

(+) m/z 319.1 (M + H)⁺.

General Procedure for Formation of the Cyclopenta[d]pyrimidine

Core: (R)-5-Methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol

(6a). To a solution of 5a (150.1 g, 881 mmol) in MeOH (2.0 L) was

added NH₄OAc (268.2 g, 3.5 mol). The reaction mixture was stirred

overnight and concentrated under reduced pressure. The resulting

residue was dissolved in dichloromethane (DCM; 1.0 L) and partitioned

with water (2.0 L). The organic layer was separated, and the aqueous

layer was extracted with DCM (3 × 750 mL). The combined organics

tert-Butyl 4-((5R)-7-Acetoxy-5-methyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate (9). To a 3 L

three-necked round-bottom reactor equipped with a mechanical stirrer,

nitrogen inlet, and thermocouple at 0 °C containing a mixture of 7a

(50.1 g, 157.0 mmol), solid NaHCO₃(46.2 g, 550.4 mmol), and CHCl₃

(700 mL) was added m-chloroperoxybenzoic acid (m-CPBA; 45.0 g,

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197.2 mmol, 75% by weight) in small portions. After the addition was

complete, the reaction mixture was stirred at 0 °C for 10 min and then

warmed to rt for 4.5 h. The reaction mixture was cooled to 0 °C, and a

solution of Na₂S₂O₃(49.7 g, 314 mmol) in water (70 mL) was added

slowly. After complete addition, the reaction mixture was stirred at 0 °C

for 10 min. The pH of the mixture was adjusted by the dropwise addition

of Na₂CO₃(66.6 g, 628.0 mmol) as a solution in water (100 mL). The

reaction mixture was stirred for 20 min and then warmed to rt for 10

min. The mixture was diluted with water (200 mL) and partitioned with

CHCl₃(200 mL). The organic layer was separated, and the aqueous

layer was extracted with CHCl₃(2 × 400 mL). The combined organics

were washed with saturated Na₂CO₃(500 mL), dried over Na₂SO₄,

ﬁltered through Celite, and concentrated in vacuo to give the crude (R)-

4-(4-(tert-butoxycarbonyl)piperazin-1-yl)-5-methyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidine 1-oxide (52.5 g, 100% yield), which was used

without puriﬁcation in the next step: LC/MS APCI (+) m/z 335.1 (M +

H)⁺.

1.73−1.65 (m, 1H), 1.48 (s, 9H), 1.17 (d, J = 7.2 Hz, 3H); LC/MS APCI

(+) m/z 233.1 [M − C₅H₉O₂+ H].

General Procedure for Asymmetric Hydrogenation. tert-Butyl 4-

((5R,7R)-7-Hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazine-1-carboxylate (12a). A round-bottom ﬂask

was charged with 10 (17.1 g, 51.4 mmol), DCM (400 mL), formic acid

(2.4 mL, 63.3 mmol), and TEA (7.6 mL, 54.5 mmol) and ﬂushed with

nitrogen for 15 min. To this solution was added RuCl(p-cymene)-

[(R,R)-TsDPEN] (0.163 g, 0.257 mmol) in one portion, and the

reaction mixture was stirred under a nitrogen atmosphere overnight.

The reaction mixture was concentrated by rotary evaporation and

puriﬁed directly by silica gel chromatography eluting with DCM−

EtOAc (1:4) to DCM−MeOH (9:1) to give crude tert-butyl 4-

((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazine-1-carboxylate (15.2 g, 88.2) as a tan foam.

The material was dissolved in DCM (300 mL), cooled to 0 °C under

nitrogen, and treated with TEA (18.9 mL, 136.2 mmol) and 4-

nitrobenzoyl chloride (12.6 g, 68.1 mmol), respectively. The reaction

was stirred, warming to rt for 3 h. The reaction was quenched with

saturated aqueous NaHCO₃and stirred for 5 min before separation of

the organic layer. The aqueous layer was extracted with DCM (2 × 200

mL), and the combined organic extracts were washed with brine, dried

over Na₂SO₄, and concentrated in vacuo. The crude material was

puriﬁed by silica gel chromatography eluting with hexanes−EtOAc

(5:1) to EtOAc to provide the desired tert-butyl 4-((5R,7R)-5-methyl-7-

((4-nitrobenzoyl)oxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-

To a round-bottom reactor containing the (R)-4-(4-(tert-

butoxycarbonyl)piperazin-1-yl)-5-methyl-6,7-dihydro-5H-cyclopenta-

[d]pyrimidine 1-oxide (42.0 g, 126.0 mmol) was slowly added acetic

anhydride (178 mL, 1.8 mol) (note: mild exotherm). After complete

addition, the reaction mixture was heated to 100 °C and stirred under a

nitrogen atmosphere for 2 h. The reaction mixture was cooled to rt and

concentrated by rotary evaporation to remove excess acetic anhydride.

The resulting residue was dissolved in DCM (1.0 L), and the solution

was poured into ice and aqueous saturated Na₂CO₃(500 mL). The

organic layer was separated, and the aqueous layer was extracted with

DCM (2 × 200 mL). The combined organic extracts were washed with

brine, dried over Na₂SO₄, and concentrated in vacuo. The crude material

was passed through a plug of silica gel eluting with hexanes−EtOAc

(1:1) to give the desired tert-butyl 4-((5R)-7-acetoxy-5-methyl-6,7-

dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate

(42.0 g, 89% yield) as a brown foam after rotary evaporation: ¹H NMR

(CDCl₃, 400 MHz) δ 8.60 (S, 1H), 6.04 (t, J = 7.2 Hz, 1H), 3.80−3.76

(m, 2H), 3.74−3.46 (m, 7H), 2.31−2.23 (m, 2H), 2.14 (s, 3H), 1.49 (s,

9H), 1.21 (d, J = 7.2 Hz, 3H); LC/MS APCI (+) m/z 333.1 (M + H)⁺.

(R)-tert-Butyl 4-(5-Methyl-7-oxo-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazine-1-carboxylate (10). To a solution of the

tert-butyl 4-((5R)-7-acetoxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazine-1-carboxylate (16.5 g, 43.8 mmol) in THF

(200 mL) was added 3 M LiOH (40 mL, 120 mmol). The reaction

mixture was stirred at rt for 16 h and then neutralized with the addition

of 2 N HCl (60 mL). The mixture was concentrated by rotary

evaporation, and the residue was puriﬁed by silica gel chromatography

eluting with DCM−MeOH (10:1) to give the desired tert-butyl 4-((5R)-

7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-

1

piperazine-1-carboxylate (19.2 g, 87% yield) as a tan foam: H NMR

(CDCl₃, 400 MHz) δ 8.60 (s, 1H), 8.28−8.22 (ABq, 4H), 6.35 (t, J = 7.2

Hz, 1H), 3.83−3.78 (m, 2H), 3.78−3.49 (m, 7H), 2.40−2.36 (m, 2H),

1.49 (s, 9H), 1.27 (d, J = 7.2 Hz, 3H); LC/MS APCI (+) m/z 484.1 (M

+ H)⁺.

tert-Butyl 4-((5R,7R)-5-methyl-7-((4-nitrobenzoyl)oxy)-6,7-dihy-

dro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate (19.2 g,

39.7 mmol) was dissolved in THF (150 mL) and water (75 mL). The

reaction was cooled to 0 °C and treated with solid LiOH−H₂O (4.2 g,

99.3 mmol). The mixture was stirred for 1 h and concentrated by rotary

evaporation. The residue was partitioned between EtOAc (300 mL) and

saturated aqueous NaHCO₃(300 mL). The organic layer was separated,

and the aqueous layer was extracted with EtOAc (2 × 150 mL). The

combined organic layer was washed with brine, dried over Na₂SO₄,

ﬁltered through Celite, and concentrated in vacuo to provide the desired

tert-butyl 4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta-

[d]pyrimidin-4-yl)piperazine-1-carboxylate (11.5 g, 87% yield) as a

tan powder: ¹H NMR (CDCl₃, 400 MHz) δ 8.57 (s, 1H), 5.12 (t, J = 7.2

Hz, 1H), 4.04 (br s, 1H), 3.81−3.75 (m, 2H), 3.67−3.46 (m, 7H), 2.21−

2.16 (m, 2H), 1.48 (s, 9H), 1.20 (d, J = 7.2 Hz, 3H); LC/MS APCI (+)

m/z 335.2 (M + H)⁺.

tert-Butyl 4-((5R,7S)-7-Hydroxy-5-methyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate (13a). Com-

pound 13a was prepared from 10 by the same procedure as for

compound 12a, using RuCl(p-cymene)[(S,S)-TsDPEN]: ¹H NMR

(CDCl₃, 400 MHz) δ 8.56 (s, 1H), 5.04−5.00 (dd, 1H), 3.72−3.67 (m,

2H), 3.63−3.43 (m, 6H), 3.33−3.23 (m, 1H), 2.72 (dt, J_d= 13.5 Hz, J_t=

8.0 Hz, 1H), 1.66−1.58 (dt, 1H), 1.48 (s, 9H), 1.29 (d, J = 6.8 Hz, 3H);

LC/MS (APCI) m/z 335.2 (M + H)⁺.

1

piperazine-1-carboxylate (14.5 g, 99% yield) as a clear oil: H NMR

(CDCl₃, 400 MHz) δ 8.47 (s, 1H), 3.72−3.68 (m, 2H), 3.57−3.43 (m,

7H), 2.96−2.81 (m, 2H), 2.32−2.26 (m, 1H), 1.73−1.65 (m, 1H), 1.48

(s, 9H), 1.17 (d, J = 7.2 Hz, 3H); LC/MS APCI (+) m/z 335.2 (M +

H)⁺.

To a 1 L three-necked round-bottom reactor containing a solution of

oxalyl chloride (21.2 mL, 243.4 mmol) in DCM (150 mL) at −78 °C

was added a solution of dimethyl sulfoxide (DMSO; 34.5 mL, 486.7

mmol) in DCM (50 mL) dropwise. The reaction mixture was stirred for

30 min before a solution of the (R)-tert-butyl 4-(7-hydroxy-5-methyl-

6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate

(58.1 g, 173.8 mmol) in DCM (80 mL) was added slowly. After

complete addition, the reaction mixture was stirred for 1 h at −78 °C

before triethylamine (TEA; 114 mL, 817.0 mmol) was added slowly.

The reaction mixture was then allowed to warm to rt and stirred for 30

min before being quenched with water (200 mL). The organic layer was

separated, and the aqueous layer was extracted with DCM (3 × 200

mL). The combined extracts were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The residue was puriﬁed by silica gel

chromatography eluting with DCM−EtOAc (2:1 to 1:3) to give the

desired (R)-tert-butyl 4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta-

[d]pyrimidin-4-yl)piperazine-1-carboxylate (41.0 g, 71% yield) as a

brown foam: ¹H NMR (CDCl₃, 400 MHz) δ 8.47 (s, 1H), 3.72−3.68

(m, 2H), 3.57−3.43 (m, 7H), 2.96−2.81 (m, 2H), 2.32−2.26 (m, 1H),

General Procedure for Preparation of Fluorinated Cores: tert-Butyl

4-((5R,7S)-7-Fluoro-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazine-1-carboxylate (14a). To a solution of 12a

(1.2 g, 3.6 mmol) in DCM (55 mL) at −20 °C was added DAST (1.4

mL, 10.7 mmol). After being stirred for 1 h at −20 °C, the reaction was

quenched with ice and warmed to rt. The mixture was diluted with

saturated aqueous NH₄Cl, and the layers were separated. The aqueous

phase was extracted with DCM (2 × 25 mL), and the combined organic

layer was dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The

crude residue was puriﬁed by silica gel chromatography eluting with

hexanes−EtOAc (2:1) to give the desired tert-butyl 4-((5R,7S)-7-ﬂuoro-

5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-

1

carboxylate (0.73 g, 61% yield) as a dark oil: H NMR (CDCl₃, 400

MHz) δ 8.62 (s, 1H), 5.80 (ddd, J = 560, 7.2, 3.2 Hz, 1H), 3.78−3.72 (m,

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2H), 3.62−3.46 (m, 7H), 2.59−2.46 (m, 1H), 2.08−1.98 (m, 1H), 1.48

(s, 9H), 1.20 (d, J = 7.2 Hz, 3H).

concentrated by rotary evaporation to remove THF before extraction of

the remaining aqueous layer with ether (3 × 150 mL). The aqueous layer

was partitioned with EtOAc (150 mL) and acidiﬁed to pH 1 with 1 N

HCl. The organic layer was separated, and the aqueous layer was

extracted with EtOAc (2 × 150 mL). The combined organic was dried

over MgSO₄and concentrated in vacuo to give the desired (S)-3-((tert-

butoxycarbonyl)isopropylamino)-2-(4-chlorophenyl)propanoic acid

General Procedure for Removal of the Boc Group on Piperazine.

(5R,7R)-5-Methyl-4-(piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-7-ol Dihydrochloride (12b). To a solution of 12a (11.5 g,

34.4 mmol) in dioxane (100 mL) and DCM (10 mL) was added 4 N

HCl in dioxane (117 mL, 471 mmol) by addition funnel at 0 °C. The

reaction mixture was warmed to rt and stirred under a nitrogen

atmosphere overnight before concentration to dryness by rotary

evaporation. The resulting solid was dissolved in a small amount of

MeOH (60 mL) and added slowly to a stirring solution of ether (500

mL), resulting in a slurry. The slurry was stirred for 5 min before

ﬁltration under a nitrogen atmosphere (hygroscopic). The wet cake was

dried under high vacuum to give the desired (5R,7R)-5-methyl-4-

(piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-ol dihydro-

chloride (9.3 g, 89% yield) as a white solid: ¹H NMR (D₂O, 400 MHz) δ

8.51 (s, 1H), 5.32 (t, J = 7.2 Hz, 1H), 4.27−4.23 (m, 2H), 4.09−4.06 (m,

2H), 3.58 (ddd, J = 7.2 Hz, 7.2 Hz, 7.2 Hz, 1H), 3.35−3.32 (m, 4H),

2.27−2.22 (m, 1H), 2.11−2.06 (m, 1H), 1.07 (d, J = 7.2 Hz, 3H); LC/

MS APCI (+) m/z 235.0 (M + H)⁺.

1

(17.4 g, 77% yield), which was used without further puriﬁcation: H

NMR (CDCl₃, 400 MHz) δ 7.34 (ABq, 4H), 4.16−4.09 (m, 1H), 4.07

(br s, 1H), 3.75 (dd, J = 14.0, 7.2 Hz), 1H), 3.30 (dd, J = 14.0, 7.2 Hz,

1H), 1.45 (s, 9H), 1.04 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz, 3H); LC/

MS APCI (+) m/z 242.1 [M − C₅H₉O₂+ H].

tert-Butyl (S)-(3-((R)-4-Benzyl-2-oxooxazolidin-3-yl)-2-(4-chloro-

phenyl)-3-oxopropyl)(2,4-dimethoxybenzyl)carbamate (19f). To a

−78 °C solution of (R)-4-benzyl-3-(2-(4-chlorophenyl)acetyl)-

oxazolidin-2-one (6.00 g, 18.2 mmol) in DCM (180 mL) was added 1

M TiCl₄in toluene (22.7 mL, 22.7 mmol), followed by diisopropylethyl-

amine (3.30 mL, 19.1 mmol), giving rise to a dark purple mixture. The

reaction mixture was stirred for 20 min before a solution of tert-butyl

(2,4-dimethoxybenzyl)(methoxymethyl)carbamate (6.80 g, 21.8 mmol)

in DCM (30 mL) was added dropwise. After complete addition, the

reaction mixture was stirred for 10 min at −78 °C and then warmed to

−10 °C over 3 h. The reaction mixture was quenched with the addition

of saturated NH₄Cl solution (50 mL). The resulting mixture was

separated, and the aqueous layer was extracted with DCM (3 × 100

mL). The combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The crude residue was puriﬁed by silica gel

chromatography eluting with hexanes−EtOAc (6:1 to 4:1) to provide

the pure tert-butyl (S)-(3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-

chlorophenyl)-3-oxopropyl)(2,4-dimethoxybenzyl)carbamate (7.30 g,

66% yield) as a white foam: HPLC (method A) purity 95%; LC/MS

APCI (+) m/z 509 (M − Boc + H).

tert-Butyl (S)-(3-((R)-4-Benzyl-2-oxooxazolidin-3-yl)-2-(4-chloro-

phenyl)-3-oxopropyl)carbamate (19h). To a solution of 19f (5.30 g,

8.70 mmol) in DCM (80 mL) and H₂O (5 mL) was added 2,3-dichloro-

5,6-dicyano-1,4-benzoquinone (DDQ; 2.57 g, 11.3 mmol). The reaction

mixture was stirred vigorously at rt for 19 h. The mixture was quenched

with saturated NaHCO₃(10 mL), and the organic layer was washed with

saturated NaHCO₃(2 × 10 mL). The combined aqueous layer was

extracted with DCM (2 × 50 mL), and the combined organic layers were

dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude was

puriﬁed by silica gel chromatography eluting with hexanes−EtOAc (9:1

to 5:1) to give the desired tert-butyl (S)-(3-((R)-4-benzyl-2-

oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)carbamate (4.0 g,

100% yield) as a yellow oil: ¹H NMR (CDCl₃, 400 MHz) δ 10.3 (s, 1H),

7.81 (d, J = 8.6 Hz, 1H), 7.38−7.19 (m, 6H), 6.58−6.53 (m, 1H), 6.47−

6.43 (m, 1H), 5.19−5.11 (m, 1H), 4.84−4.77 (m, 1H), 4.67−4.58 (m,

1H), 4.11−4.03 (m, 1H), 3.76−3.65 (m, 1H), 3.58−3.47 (m, 1H),

3.36−3.26 (m, 1H), 2.90−2.78 (m, 1H), 1.43 (s, 9H); LC/MS APCI

(+) m/z 359 (M − Boc + H).

(S)-3-((tert-Butoxycarbonyl)amino)-2-(4-chlorophenyl)propanoic

Acid (20h). To a solution of LiOH−H₂O (0.73 g, 17 mmol) in THF−

H₂O (2:1, 83 mL) was added 35 wt % aqueous H₂O₂(1.8 mL, 22

mmol). The reaction mixture was stirred for 30 min at rt before being

cooled to 0 °C. A solution of 19h (4.0 g, 8.7 mmol) in THF (40 mL) was

added dropwise over a 25 min period, and the reaction mixture was

warmed to rt for 12 h. The reaction mixture was cooled to 0 °C again and

treated with 1 M Na₂SO₃(35 mL). The mixture was stirred for 15 min

before concentration in vacuo. The mixture was diluted with water (40

mL) and washed twice with ether (2 × 25 mL). The aqueous layer was

acidiﬁed with solid KHSO₄and extracted with DCM (2 × 25 mL). The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo to give the desired (S)-3-(tert-butoxycarbonyl)-

2-(4-chlorophenyl)propanoic acid (2.8 g, 100% yield) as a crude pale

yellow foam. This material was used without further puriﬁcation: HPLC

purity 80%; LC/MS APCI (+) m/z 200 (M − Boc + H).

(R)-4-Benzyl-3-(2-(4-chlorophenyl)acetyl)oxazolidin-2-one (17).

To a stirred solution of (R)-4-benzyloxazolidin-2-one (16) (48.7 g,

275.0 mmol) in THF (700 mL) at −78 °C was added 2.5 M n-BuLi in

toluene (107 mL, 267.5 mmol) dropwise by syringe. After complete

addition, the reaction was warmed to −20 °C for 1 h. The reaction

mixture was cooled again to −78 °C, and 2-(4-chlorophenyl)acetyl

chloride (47.2 g, 250 mmol) in THF (50 mL) was added slowly. After

complete addition, the mixture was warmed to rt overnight. The

reaction was quenched with 1 N HCl, and the partitioned organic layer

was separated. The aqueous layer was extracted with EtOAc (2 × 250

mL), and the combined organic layer was dried over MgSO₄, ﬁltered,

and concentrated in vacuo. The crude material was puriﬁed by column

chromatography eluting with hexanes−EtOAc (4:1) to give the desired

(R)-4-benzyl-3-(2-(4-chlorophenyl)acetyl)oxazolidin-2-one (64.7 g,

1

79% yield) as a white solid: H NMR (CDCl₃, 400 MHz) δ 7.33−

7.26 (m, 7H), 7.13 (d, J = 6.4 Hz, 2H), 4.69−4.66 (m,1H), 4.33−4.16

(m, 4H), 3.60 (dd, J = 10.2, 3.2 Hz), 1H), 2.76 (dd, J = 10.2, 9.6 Hz, 1H).

General Procedure for the Asymmetric Mannich Reaction. tert-

Butyl (S)-(3-((R)-4-Benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-

3-oxopropyl)isopropylcarbamate (19a). To a −78 °C solution of (R)-

4-benzyl-3-(2-(4-chlorophenyl)acetyl)oxazolidin-2-one (10.0 g, 30.3

mmol) in DCM (300 mL) was added 1 M TiCl₄in toluene (31.8 mL,

31.8 mmol), resulting in an orange solution. Subsequent addition of

DIEA (5.8 mL, 33.4 mmol) by syringe gave a dark purple reaction

mixture. The reaction was stirred for 15 min before a solution of tert-

butyl isopropyl(methoxymethyl)carbamate (8.0 g, 39.4 mmol) in DCM

(20 mL) was added slowly by syringe. The reaction mixture was stirred

for 15 min at −78 °C and then warmed to rt. The reaction was allowed to

stir for 1.5 h before being quenched with saturated aqueous NH₄Cl (50

mL). The organic layer was separated, and the aqueous layer was

extracted with DCM (2 × 50 mL). The combined organics were washed

with brine, dried over Na₂SO₄, and concentrated in vacuo. The crude

material was puriﬁed by column chromatography eluting with hexanes−

EtOAc (5:1) to give the desired tert-butyl (S)-(3-((R)-4-benzyl-2-

oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)-

1

isopropylcarbamate (10.9 g, 72% yield) as a white foam: H NMR

(CDCl₃, 400 MHz) δ 7.35−7.22 (m, 9H), 5.54 (br s, 1H), 4.62−4.58

(m, 1H), 4.13−3.95 (m, 4H), 3.41−3.36 (m, 2H), 2.76 (br s, 1H), 1.48

(s, 9H), 1.08 (d, J = 6.4 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H); LC/MS APCI

(+) m/z 359.0 [M − C₅H₉O₂+ H].

(S)-3-((tert-Butoxycarbonyl)isopropylamino)-2-(4-chlorophenyl)-

propanoic Acid (20a). To a 0 °C solution of LiOH−H₂O (3.1 g, 131.7

mmol) in THF (750 mL) and H₂O (250 mL) was added 35 wt %

aqueous hydrogen peroxide (19.2 mL, 197.6 mmol). This solution was

stirred for 10 min before a solution of tert-butyl (S)-(3-((R)-4-benzyl-2-

oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)-

isopropylcarbamate (33.0 g, 65.8 mmol) in THF (50 mL) was added.

The reaction was warmed to rt overnight. The reaction was quenched by

the addition of 10 wt % aqueous Na₂SO₃(10 mL) and saturated aqueous

NaHCO₃(10 mL). After being stirred for 10 min, the reaction was

To a solution of (S)-3-(tert-butoxycarbonyl)-2-(4-chlorophenyl)-

propanoic acid (2.8 g, 9.4 mmol) in dioxane−DCM (2:1, 90 mL) was

added slowly 4 N HCl in dioxane (70.8 mL, 283.1 mmol). The reaction

mixture was stirred at rt for 17 h before concentration to dryness. The

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residue was dissolved in DCM (15 mL) and MeOH (5 mL). The

resulting solution was added dropwise to a solution of vigorously stirring

ether (300 mL), resulting in a slurry. The slurry was ﬁltered under a

nitrogen atmosphere, rinsed with ether (200 mL), and dried in vacuo to

give (S)-3-amino-2-(4-chlorophenyl)propanoic acid hydrochloride (1.7

g, 79% yield) as a white solid: ¹H NMR (CD₃OD, 400 MHz) δ 7.42 (d, J

= 8.6 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 3.97 (dd, 1H), 3.57−3.54 (m,

1H), 3.24−3.17 (m, 1H); HPLC (method A) purity 97%; LC/MS APCI

(+) m/z 200 (M + H).

(S)-2-(4-Chlorophenyl)-3-(isopropylamino)-1-(4-((R)-5-methyl-

6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-

1-one Dihydrochloride Salt (3). Compound 3 was prepared from 8a

and 20a by the same procedure as described for 28: ¹H NMR (D₂O, 400

MHz) δ 8.30 (s, 1H), 7.36 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H),

4.31 (dd, J = 8.2, 4.7 Hz, 1H), 4.16−4.04 (m, 1H), 3.92−3.82 (m, 1H),

3.80−3.70 (m, 1H), 3.70−3.58 (m, 1H), 3.58−3.40 (m, 6H), 3.35 (d, J =

6.5 Hz, 1H), 3.21 (dd, J = 12.9, 4.7 Hz, 1H), 2.97 (d, J = 9.0 Hz, 1H),

2.82 (ddd, J = 18.3, 9.8, 2.7 Hz, 1H), 2.30−2.16 (m, 1H), 1.78−1.68 (m,

1H), 1.20 (d, J = 4.4 Hz, 3H), 1.19 (d, J = 4.0 Hz, 3H), 0.94 (d, J = 6.6

Hz, 3H); LC/MS APCI (+) m/z 442 (M + H)⁺; HPLC (method A)

>99% purity, t_R= 1.86 min.

(S)-2-(4-Chlorophenyl)-1-(4-(5,5-dimethyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)-

propan-1-one Bis(triﬂuoroacetate) Salt (25). Compound 25 was

prepared from 8b and 20a by the same procedure described for 28: ¹H

NMR (DMSO-d₆, 400 MHz) δ 8.45 (s, 1H), 7.36 (dd, J = 20.9, 8.5 Hz,

4H), 4.17 (dd, J = 8.2, 5.6 Hz, 1H), 3.74−3.55 (m, 3H), 3.51−3.38 (m,

1H), 3.27−3.04 (m, 4H), 2.95−2.57 (m, 6H), 1.81 (t, J = 7.2 Hz, 2H),

1.32 (d, J = 4.9 Hz, 6H), 0.93 (dd, J = 7.4, 6.4 Hz, 6H); LC/MS m/z 456

(M + H)⁺; HPLC (method E) 97.9% purity, t_R= 3.38 min.

To a thin slurry of (S)-3-amino-2-(4-chlorophenyl)propanoic acid

hydrochloride (1.7 g, 7.4 mmol) and tetramethylammonium hydroxide

pentahydrate (3.4 g, 18.5 mmol) in 10:1 MeCN−H₂O (80 mL) was

added Boc₂O (3.2 g, 14.8 mmol). The reaction mixture was stirred at rt

for 8 h and concentrated by rotary evaporation. The mixture was diluted

with 0.5 M NaOH (10 mL) and washed with ether (2 × 25 mL). The

aqueous layer was acidiﬁed with solid KHSO₄and extracted with DCM

(2 × 50 mL). The combined organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. The resulting residue was

concentrated from DCM−hexanes mixtures twice to give the desired

(S)-3-((tert-butoxycarbonyl)amino)-2-(4-chlorophenyl)propanoic acid

1

(2.07 g, 93% yield) as a white foam: H NMR (CDCl₃, 400 MHz,

(S)-2-(4-Chlorophenyl)-3-(isopropylamino)-1-(4-((S)-5-vinyl-6,7-

dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-

one (26). Compound 26 was prepared from 8c and 20a by the same

procedure described for 28: ¹H NMR (DMSO-d₆, 400 MHz) δ 8.35 (s,

1H), 7.36 (dd, J = 8.0, 8.5 Hz, 4H), 5.85 (ddd, J = 17.2, 10.2, 7.0 Hz, 1H),

4.99 (d, J = 10.2 Hz, 1H), 4.81 (d, J = 17.2 Hz, 1H), 4.18 (dd, J = 8.4, 5.6

Hz, 1H), 4.04 (m, 1H), 3.45−3.70 (m, 7H), 3.13 (dd, J = 11.6, 8.6 Hz,

2H), 3.06 (d, J = 10.0 Hz, 1H), 2.80−2.61 (m, 4H), 2.21 (m, 1H), 1.74

(m, 1H), 0.95 (t, J = 6.6 Hz, 6H); LC/MS APCI (+) m/z 454.2 (M + 1);

HPLC (method D) 98% purity, t_R= 3.88 min.

(S)-2-(4-Chlorophenyl)-1-(4-((R)-5-(hydroxymethyl)-6,7-dihydro-

5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)-

propan-1-one (27). Compound 27 was prepared from 8d and 20a by

the same procedure described for 28: ¹H NMR (CD₃OD, 500 MHz) δ

8.39 (s, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 4.18 (m,

1H), 4.08 (m, 1H), 3.90 (m, 1H), 3.75 (m, 1H), 3.69−3.48 (m, 7H),

3.42 (m, 3H), 3.27 (m, 1H), 3.07−2.82 (m, 2H), 2.25 (m, 1H), 2.04 (m,

1H), 1.25 (t, J = 6.6 Hz, 6H); LC/MS APCI (+) m/z 458.2 (M + 1);

HPLC (method E) 100% purity, t_R= 6.47 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7S)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

(isopropylamino)propan-1-one Dihydrochloride Salt (29). Com-

pound 29 was prepared from 13b and 20a by the same procedure

described for 28: ¹H NMR (D₂O, 400 MHz) δ 8.39 (s, 1H), 7.36 (d, J =

8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 5.08 (dd, J = 8.4, 4.8 Hz, 1H), 4.30

(dd, J = 8.0, 4.8 Hz, 1H), 4.15 (m, 1H), 3.84 (m, 1H), 3.69 (m, 1H),

3.56−3.43 (m, 5H), 3.41−3.32 (m, 2H), 3.22 (dd, J = 12.8, 4.8 Hz, 1H),

2.67 (ddd, J = 14.4, 8.0, 4.0 Hz, 1H), 1.51 (ddd, J = 14.0, 10.0, 4.0 Hz,

1H), 1.38 (d, J = 7.2 Hz, 1H), 1.19 (dd, J = 6.4, 4.4 Hz, 6H), 1.04 (d, J =

7.2 Hz, 3H); LC/MS APCI (+) m/z 458 (M + H)⁺; HPLC (method A)

>95% purity, t_R= 1.82 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-ﬂuoro-5-methyl-6,7-dihy-

dro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

(isopropylamino)propan-1-one Bis(triﬂuoroacetate) Salt (30). Com-

pound 30 was prepared from 15b and 20a by the same procedure

described for 28: ¹H NMR (D₂O, 400 MHz) δ 8.52 (s, 1H), 7.50 (d, J =

8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 5.71 (td, J = 56.0, 8.0 Hz, 1H), 4.42

(m, 2H), 3.93−3.83 (m, 1H), 3.78−3.68 (m, 1H), 3.60−3.30 (m, 7H),

3.20−3.00 (m, 2H), 2.65−2.50 (m, 1H), 1.77 (dd, J = 28, 8 Hz, 1H),

1.23 (m, 6H), 1.14 (d, J = 8.0 Hz, 3H); LC/MS APCI (+) m/z 460 (M +

H)⁺; HPLC (method C) 99% purity, t_R= 3.84 min.

rotamers present) δ 7.32 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H),

3.96−3.85 (m, 0.2H), 3.82−3.73 (m, 0.8 H), 3.67−3.54 (m, 0.4H),

3.54−3.38 (m, 1.6H), 1.48 (s, 5.8H), 1.42 (s, 3.2H); HPLC (method B)

purity 97%, >99% ee by chiral HPLC; LC/MS APCI (+) m/z 200 (M −

Boc + H).

General Procedure for Amide Coupling: tert-Butyl (S)-(2-(4-

Chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)-

isopropylcarbamate (24a). To a solution of 12b (2.0 g, 6.5 mmol), 20a

(2.2 g, 6.5 mmol), and DIEA (3.6 mL, 20.8 mmol) in DCM (55 mL) at 0

°C was added N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-

uronium hexaﬂuorophosphate (HBTU; 2.4 g, 6.5 mmol). The reaction

mixture was warmed to rt over a 4 h period. The reaction was quenched

by the addition of 2 M Na₂CO₃(5 mL), and the organic layer was

separated. The aqueous layer was extracted with DCM (2 × 25 mL), and

the combined organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The crude material was puriﬁed by silica gel

chromatography eluting with DCM−EtOAc (1:1 to 1:9) and then

DCM−MeOH (30:1). The crude product was dissolved in DCM (10

mL) and hexanes (100 mL). The resulting slurry was cooled to 0 °C and

ﬁltered to give the desired tert-butyl (S)-(2-(4-chlorophenyl)-3-(4-

((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl)isopropylcarbamate (3.2 g,

87% yield) as a white powder: ¹H NMR (CDCl₃, 400 MHz) δ 8.49 (s,

1H), 7.34 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 5.09 (t, J = 7.2 Hz,

1H), 3.78−3.31 (m, 15H), 2.18−2.14 (m, 2H), 1.47 (s, 9H), 1.13 (d, J =

7.2 Hz, 3H), 0.97−0.95 (m, 3H), 0.68−0.67 (m, 2H); LC/MS APCI (+)

m/z 458.2 [M − C₅H₉O₂+ H].

General Procedure for Boc Deprotection. (S)-2-(4-Chlorophenyl)-

1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one Di-

hydrochloride Salt (28). To a solution of 24a (2.5 g, 4.5 mmol) in

dioxane (25 mL) was added 4 M HCl in dioxane (22.4 mL, 89.6 mmol).

The resulting solution was stirred overnight at rt before concentration

by rotary evaporation to a gel. This gel was dissolved in a small amount

of MeOH (10 mL) followed by addition to ether (300 mL) to give a

white slurry. The resulting slurry was ﬁltered under a nitrogen

atmosphere and subsequently dried under a nitrogen ﬂow to give the

desired (S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-

6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

(isopropylamino)propan-1-one dihydrochloride salt (2.1 g, 90% yield)

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7S)-7-ﬂuoro-5-methyl-6,7-dihy-

dro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

(isopropylamino)propan-1-one Bis(triﬂuoroacetate) Salt (31). Com-

pound 31 was prepared from 14b and 20a by the same procedure

described for 28: ¹H NMR (D₂O, 400 MHz) δ 8.54 (s, 1H), 7.50 (d, J =

8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 5.86 (dt, J = 56.0, 4.0 Hz, 1H), 4.47

(m, 2H), 3.90−3.78 (m, 3H), 3.83 (m, 1H), 3.70 (m, 1H), 3.62−3.50

(m, 5H), 3.18−3.00 (m, 2H), 2.45−2.30 (m, 1H), 2.12−1.98 (m, 1H),

1

as a light yellow solid: H NMR (CD₃OD, 400 MHz) δ 8.58 (s, 1H),

7.45 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 5.31 (appt, J = 8.0 Hz,

1H), 4.58 (dd, J = 9.6, 4.0 Hz, 1H), 4.19 (m, 1H), 4.05−3.89 (m, 3H),

3.82−3.65 (m, 5H), 3.48−3.41 (m, 2H), 3.17 (dd, J = 12.4, 3.6 Hz, 1H),

2.30 (dd, J = 12.8, 7.6 Hz, 1H), 2.19 (ddd, J = 16.4, 12.8, 8.0 Hz, 1H),

1.37 (d, J = 6.4 Hz, 6H), 1.19 (d, J = 6.8 Hz, 3H); LC/MS APCI (+) m/z

458 (M + H)⁺; HPLC (method B) >99% purity, t_R= 1.83 min.

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1.23 (m, 6H), 1.06 (d, J = 8.0 Hz, 3H); LC/MS APCI (+) m/z 460 (M +

H)⁺; HPLC (method C) >99% purity, t_R= 3.92 min.

1H), 7.36 (d, J = 5.6 Hz, 1H), 5.07 (m, 1H), 4.50 (m, 1H), 3.91 (m, 2H),

3.80−3.60 (m, 4H), 3.40−3.22 (m, 5H), 3.04 (m, 2H), 2.02 (m, 2H),

1.22 (m, 6H), 1.04 (d, J = 8.0 Hz, 6H); LC/MS APCI (+) m/z 540 (M +

H)⁺; HPLC (method C) 98.3% purity, t_R= 3.90 min.

(S)-2-(4-Chlorophenyl)-1-(4-((R)-7,7-diﬂuoro-5-methyl-6,7-dihy-

dro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

(isopropylamino)propan-1-one Dihydrochloride Salt (32). Com-

pound 32 was prepared from 11b and 20a by the same procedure

described for 28: ¹H NMR (D₂O, 400 MHz) δ 8.28 (s, 1H), 7.32 (d, J =

8.6 Hz, 2H), 7.18 (d, J = 8.6 Hz, 2H), 4.28 (dd, J = 8.4, 4.9 Hz, 1H),

3.94−3.80 (m, 2H), 3.58−3.38 (m, 7H), 3.33 (d, J = 6.6 Hz, 1H), 3.19

(dd, J = 12.8, 4.8 Hz, 1H), 3.12−3.02 (m, 1H), 2.76−2.56 (m, 1H),

2.20−2.04 (m, 1H), 1.19 (d, J = 4.0 Hz, 3H), 1.17 (d, J = 4.0 Hz, 3H),

0.98 (d, J = 7.0 Hz, 3H); LC/MS APCI (+) m/z 478 (M + H)⁺; HPLC

(method B) >99% purity, t_R= 2.27 min.

(S)-2-(4-Chlorophenyl)-1-(4-((R)-5-(ﬂuoromethyl)-6,7-dihydro-

5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)-

propan-1-one (33). Compound 33 was prepared from 8e and 20a by

the same procedure described for 28: ¹H NMR (CD₃OD, 400 MHz) δ

8.59 (s, 1H), 7.41 (m, 4H), 4.20 (m, 1H), 4.07 (m, 1H), 3.98−3.84 (m,

3H), 3.62−3.80 (m, 5H), 3.60−3.40 (m, 4H), 3.27−2.92 (m, 4H), 2.40

(m, 1H), 2.15 (m, 1H), 1.37 (m, 6H); LC/MS APCI (+) m/z 460.2 (M

+ 1); HPLC (method C) 88% purity, t_R= 3.48 min.

(S)-2-(4-Chlorophenyl)-3-(cyclohexylamino)-1-(4-((5R,7R)-7-hy-

droxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-

piperazin-1-yl)propan-1-one Dihydrochloride Salt (41). Compound

41 was prepared from 12b and 20e by the same procedure described for

28: ¹H NMR (D₂O, 400 MHz) δ 8.37 (s, 1H), 7.28 (ABq, 4H), 5.29 (t, J

= 7.2 Hz, 1H), 4.32−4.27 (m, 1H), 4.18−4.10 (m, 1H), 3.88−3.78 (m,

2H), 3.71−3.62 (m, 1H), 3.56−3.44 (m, 5H), 3.28−3.20 (m, 2H),

3.04−2.98 (m, 1H), 2.21−2.16 (m, 1H), 2.05−1.99 (m, 1H), 1.94−1.88

(m, 2H), 1.70−1.68 (m, 2H), 1.53−1.50 (m, 1H), 1.24−1.56 (m, 4H),

1.06−1.03 (m, 1H), 0.96 (d, J = 7.2 Hz, 3H); LC/MS APCI (+) m/z

498.3 (M + H)⁺; HPLC (method B) >99% purity, t_R= 2.01 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

(isopropylmethylamino)propan-1-one Dihydrochloride Salt (42). A

solution of 28 (0.040 g, 0.075 mmol), DIEA (0.039 mL, 0.23 mmol),

and 37% formaldehyde (0.056 mL, 0.75 mmol) in 1:1 DCE−THF (0.8

mL) was stirred for 10 min. NaBH(OAc)₃(0.024 g, 0.11 mmol) was

added. The reaction mixture was stirred at rt for 6 h. Saturated NaHCO₃

was added, and the mixture was extracted with DCM. The combined

extracts were dried (Na₂SO₄), ﬁltered, and concentrated. The crude was

puriﬁed by ﬂash column chromatography with silica gel (6:1 DCM−

MeOH) to give the free base, which was converted to (S)-2-(4-

chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-

c y c l o p e n t a [ d ] p y r i m i d i n - 4 - y l ) p i p e r a z i n - 1 - y l ) - 3 -

(isopropylmethylamino)propan-1-one dihydrochloride (0.031 g, 76%

yield) by treatment with 2 M HCl in diethyl ether: ¹H NMR (D₂O, 400

MHz, note that rotamers were observed) δ 8.37 (s, 1H), 7.38 (d, J = 8.6

Hz, 0.4H), 7.35 (d, J = 8.6 Hz, 1.6H), 7.27 (d, J = 8.6 Hz, 0.4H), 7.21 (d,

J = 8.6 Hz, 1.6H), 5.26 (t, J = 8.0 Hz, 1H), 4.47 (dd, J = 11.0, 3.5 Hz, 1H),

4.18−4.06 (m, 1H), 3.92−3.42 (m, 9H), 3.34−3.20 (m, 1H), 3.00 (dd, J

= 12.9, 3.5 Hz, 1H), 2.74 (s, 2.3H), 2.68 (s, 0.7H), 2.19 (dd, J = 13.1, 7.6

Hz, 1H), 2.08−1.98 (m, 1H), 1.26 (d, J = 6.7 Hz, 2.3H), 1.20 (d, J = 6.7

Hz, 0.7H), 1.18 (d, J = 6.7 Hz, 2.3H), 1.15 (d, J = 6.7 Hz, 0.7H), 0.97 (d,

J = 7.0 Hz, 3H); LC/MS APCI (+) m/z 472 (M + H)⁺; HPLC (method

B) 99% purity, t_R= 1.89 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-((S)-pyrroli-

din-2-yl)ethanone Dihydrochloride Salt (43). Compound 43 was

prepared from 12b and 23i by the same procedure described for 28: ¹H

NMR (CD₃OD, 400 MHz) δ 8.57 (s, 1H), 7.45 (d, J = 8.6, 2H), 7.41 (d,

J = 8.6, 2H), 5.31 (t, J = 8.0, 1H), 4.51−4.45 (m, 1H), 4.23−3.28 (mm,

11H), 2.33−2.26 (m, 1H), 2.23−2.16 (m, 1H), 2.16−2.06 (m, 1H),

1.98−1.84 (m, 1H), 1.84−1.72 (m, 1H), 1.40−1.34 (m, 2H), 1.18 (d, J =

7.0, 3H); LC/MS APCI (+) m/z 456.1, 458.1 (M + H)⁺; HPLC

(method A) >97% purity, t_R= 2.56 min.

General Procedure for Reductive Amination. (S)-2-(4-Chlorophen-

yl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazin-1-yl)-3-((tetrahydro-2H-pyran-4-yl)amino)-

propan-1-one Dihydrochloride Salt (44). A solution of 34 (0.075 g,

0.15 mmol) in 2 M Na₂CO₃(20 mL) was saturated with NaCl and

extracted three times with DCM. The combined extracts were dried

(Na₂SO₄), ﬁltered, and concentrated. The resulting residue was

dissolved in DCE (2.5 mL). Tetrahydropyran-4-one (0.021 mL, 0.23

mmol) was added, and the solution was stirred at rt for 5 min.

NaBH(OAc)₃(0.065 g, 0.31 mmol) was then added, and the reaction

mixture was stirred at rt for 13 h. Saturated NaHCO₃was added, and the

mixture was extracted with DCM. The combined extracts were dried

(Na₂SO₄), ﬁltered, and concentrated. The crude material was puriﬁed by

ﬂash column chromatography with silica gel (6:1 DCM−MeOH) to

give the free base, which was converted to (S)-2-(4-chlorophenyl)-1-(4-

((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazin-1-yl)-3-((tetrahydro-2H-pyran-4-yl)amino)-

propan-1-one dihydrochloride (0.041 g, 47% yield) by treatment with 2

M HCl in diethyl ether: ¹H NMR (D₂O, 400 MHz) δ 8.36 (s, 1H), 7.35

(d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 5.24 (t, J = 7.8 Hz, 1H), 4.31

(dd, J = 8.4, 4.9 Hz, 1H), 4.14−4.04 (m, 1H), 3.93 (d, J = 11.7 Hz, 2H),

3.90−3.82 (m, 1H), 3.82−3.72 (m, 1H), 3.70−3.60 (m, 1H), 3.60−3.40

(S)-3-Amino-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-meth-

yl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-

propan-1-one Dihydrochloride Salt (34). Compound 34 was prepared

1

from 12b and 20f by the same procedure described for 28: H NMR

(D₂O, 400 MHz) δ 8.39 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4

Hz, 2H), 5.27 (appt, J = 7.6 Hz, 1H), 4.30 (dd, J = 6.4 Hz, 1H), 4.11 (m,

1H), 3.88−3.80 (m, 2H), 3.70 (dd, J = 6.4, 4.4 Hz, 1H), 3.69−3.65 (m,

1H), 3.63−3.60 (m, 2H), 3.56−3.53 (m, 2H), 3.26 (dd, J = 13.2, 5.6 Hz,

1H), 2.20 (dd, J = 13.2, 8.0 Hz, 1H), 2.03 (ddd, J = 16.0, 12.8, 8.0 Hz,

1H), 1.21 (d, J = 6.4 Hz, 1H), 0.97 (d, J = 6.4 Hz, 3H); LC/MS APCI

(+) m/z 416 (M + H)⁺; HPLC (method B) 98.5% purity, t_R= 1.72 min.

(S)-2-(4-Chlorophenyl)-3-((cyclopropylmethyl)amino)-1-(4-

((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazin-1-yl)propan-1-one Dihydrochloride Salt

(35). Compound 35 was prepared from 12b and 20b by the same

procedure described for 28: ¹H NMR (D₂O, 400 MHz) δ 0.38 (2H, d, J

= 5.3 Hz), 0.71 (2H, d, J = 7.2 Hz), 1.18 (3H, t, J = 6.9 Hz), 2.19 (1H, dd,

J = 8.0, 14.6 Hz), 2.36 (1H, dd, J = 7.7, 13.0 Hz), 3.01 (2H, d, J = 7.2 Hz),

3.30 (1H, m), 3.49 (1H, dd, J = 5.1, 12.9 Hz), 3.57−3.75 (m, 8H), 3.92

(1H, m), 4.07−4.19 (3H, m), 4.52 (1H, dd, J = 5.5, 7.8 Hz), 5.43 (1H, t, J

= 7.9 Hz), 7.39 (2H, d, J = 8.4 Hz), 7.53 (2H, d, J = 8.4 Hz), 8.55 (1H, s);

LC/MS APCI (+) m/z 470 [M + H]⁺; HPLC (method B), 97% purity,

t_R= 1.86 min

(S)-2-(4-Chlorophenyl)-3-((2-ﬂuoroethyl)amino)-1-(4-((5R,7R)-7-

hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)-

piperazin-1-yl)propan-1-one (36). Compound 36 was prepared from

12b and 20c by the same procedure described for 28: ¹H NMR

(CD₃OD, 400 MHz) δ 8.58 (s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.39 (d, J =

8.4 Hz, 2H), 5.31 (t, J = 8.0 Hz, 1H), 4.72 (t, J = 4.4 Hz, 1H), 4.61 (dd, J

= 8.4, 4.4 Hz, 1H), 4.01−3.66 (m, 8H), 3.51−3.41 (m, 4H), 2.32−2.27

(m, 1H), 2.22−2.17 (m, 1H), 1.39−1.35 (m, 3H), 1.18 (d, J = 6.8 Hz,

3H); LC/MS APCI (+) m/z 462.2 (M + H)⁺; HPLC (method B) 99%

purity, t_R= 2.24 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-((2-

methoxyethyl)amino)propan-1-one Dihydrochloride Salt (38). Com-

pound 38 was prepared from 12b and 20d by the same procedure

described for 28: ¹H NMR (CD₃OD, 400 MHz) δ 8.58 (s, 1H), 7.46 (d,

J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 5.32 (appt, J = 8.0 Hz, 1H), 4.56

(dd, J = 9.6, 4.0 Hz, 1H), 4.25−4.18 (m, 1H), 4.01−3.91 (m, 2H), 3.84−

3.82 (m, 1H), 3.75−3.65 (m, 8H), 3.48 (q, J = 6.8 Hz, 2H), 3.41 (s, 3H),

3.31−3.26 (m, 2H), 2.30 (dd, J = 13.2, 7.6 Hz, 1H), 2.19 (ddd, J = 13.2,

8.4, 3.6 Hz, 1H), 1.18 (d, J = 6.8 Hz, 3H); LC/MS APCI (+) m/z 474.1

(M + H)⁺; HPLC (method B) >99% purity, t_R= 1.81 min.

(S)-2-(3-Fluoro-4-(triﬂuoromethyl)phenyl)-3-((1-hydroxy-2-meth-

ylprop-2-yl)amino)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-

5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one Bis-

(triﬂuoroacetate) Salt (39). Compound 39 was prepared from 12b

and 20e by the same procedure described for 28: ¹H NMR (D₂O, 400

MHz) δ 8.58 (s, 1H), 7.86 (dd, J = 8.0, 5.6 Hz, 1H), 7.49 (d, J = 8.0 Hz,

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(m, 5H), 3.40−3.30 (m, 3H), 3.30−3.20 (m, 2H), 2.18 (dd, J = 13.3, 7.4

Hz, 1H), 2.08−1.99 (m, 1H), 1.99−1.87 (m, 2H), 1.59 (dd, J = 12.1, 4.7

Hz, 2H), 0.96 (d, J = 7.0 Hz, 3H); LC/MS APCI (+) m/z 500 (M + H)⁺;

HPLC (method B) 99% purity, t_R= 1.86 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(((1R,4S)-4-

methoxycyclohexyl)amino)propan-1-one (45). Compound 45 was

prepared from 12b and 20g by the same procedure described for 28: ¹H

NMR (CDCl₃, 400 MHz) δ 11.11 (br s, 1H), 8.45 (s, 1H), 7.20 (ABq,

4H), 6.62 (br s, 1H), 5.44−5.42 (m, 1H), 4.78−4.67 (m, 1H), 4.14−

3.48 (m, 9H), 3.34 (s, 3H), 3.21−3.06 (m, 4H), 2.27−2.18 (m, 6H),

1.60−1.56 (m, 2H), 1.34−1.28 (m, 2H), 1.16 (t, J = 6.0 Hz, 3H); LC/

MS APCI (+) m/z 528 (M + H)⁺; HPLC (method B) 98% purity, t_R=

1.95 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(((tetrahy-

dro-2H-pyran-4-yl)methyl)amino)propan-1-one (46). Compound 46

was prepared from 34 and tetrahydro-2H-pyran-4-carbaldehyde by the

same procedure described for 44: ¹H NMR (D₂O, 400 MHz) δ 8.34 (s,

1H), 7.28 (ABq, 4H), 5.21 (t, J = 7.2 Hz, 1H), 4.30 (dd, J = 5.2, 5.2 Hz,

1H), 4.11−4.05 (m, 1H), 3.87−3.83 (m, 3H), 3.76−3.73 (m, 1H),

3.66−3.62 (m, 1H), 3.52−3.42 (m, 5H), 3.37−3.24 (m, 4H), 2.93−2.82

(m, 2H), 2.19−2.13 (m, 1H), 2.05−1.92 (m, 2H), 1.58−1.55 (m, 2H),

1.27−1.16 (m, 2H), 0.95 (d, J = 7.2 Hz, 3H); LC/MS APCI (+) m/z

514.3 (M + H)⁺; HPLC (method B) >99% purity, t_R= 1.88 min.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-((S)-piperi-

din-2-yl)ethanone Dihydrochloride Salt (47). Compound 47 was

prepared from 12b and 23j by the same procedure described for 28: ¹H

NMR (D₂O, 400 MHz) δ 8.36 (s, 1H), 7.35 (d, J = 8.6 Hz, 2H), 7.20 (d,

J = 8.6 Hz, 2H), 5.26 (t, J = 7.8 Hz, 1H), 4.16−4.06 (m, 2H), 3.92−3.84

(m, 1H), 3.84−3.75 (m, 1H), 3.70−3.61 (m, 1H), 3.61−3.45 (m, 4H),

3.36−3.18 (m, 2H), 2.87 (t, J = 13.1 Hz, 1H), 2.19 (dd, J = 13.3, 7.8 Hz,

1H), 2.08−1.97 (m, 1H), 1.73 (app. t, J = 16.8 Hz, 2H), 1.54 (app. t, J =

16.4 Hz, 2H), 1.48−1.25 (m, 3H), 0.96 (d, J = 6.6 Hz, 3H); LC/MS

APCI (+) m/z 470 (M + H)⁺; HPLC (method B) 99% purity, t_R= 1.87

min.

2H), 3.65−3.60 (m, 1H), 3.27−3.18 (m, 3H), 2.86−2.80 (m, 1H); LC/

MS APCI (+) m/z 282 (M + H)⁺.

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-((2,2,2-

triﬂuoroethyl)amino)propan-1-one Dihydrochloride Salt (37). To a

solution of 12a (0.163 g, 0.531 mmol), 58a (0.170 g, 0.531 mmol), and

DIEA (0.323 mL, 1.86 mmol) in 1:1 DCM−DMF (5 mL) was added

HBTU (0.201 g, 0.531 mmol). The reaction mixture was stirred at rt for

1 h, after which 2 M Na₂CO₃was added. The mixture was extracted with

DCM, and the combined extracts were dried (Na₂SO₄), ﬁltered, and

concentrated. The crude was puriﬁed by silica gel column chromatog-

raphy (9:1 DCM−MeOH) to give the 1:1 mixture of diastereomers,

which was separated by chiral HPLC to give (S)-2-(4-chlorophenyl)-1-

(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazin-1-yl)-3-((2,2,2-triﬂuoroethyl)amino)propan-

1-one dihydrochloride (0.062 g, 20% yield): ¹H NMR (D₂O, 400 MHz)

δ 8.36 (s, 1H), 7.35 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 5.25 (t, J

= 8.0 Hz, 1H), 4.34 (dd, J = 8.2, 5.2 Hz, 1H), 4.16−4.04 (m, 1H), 3.90−

3.40 (m, 10H), 3.32 (dd, J = 12.8, 5.4 Hz, 2H), 2.18 (dd, J = 12.8, 8.0 Hz,

1H), 2.08−1.96 (m, 1H), 0.96 (d, J = 7.0 Hz, 3H); LC/MS APCI (+) m/

z 478.2 (M + H)⁺; HPLC (method B) 98% purity, t_R= 2.21 min.

General Procedure for Preparation of Compounds with Cyclic

Tertiary Amines and tert-Butylamines. (S)-2-(4-Chlorophenyl)-3-(4-

ﬂuoropiperidin-1-yl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-

5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one (40). A

solution of 4-ﬂuoropiperidine hydrochloride (1.065 g, 7.629 mmol) in

THF (10 mL) was treated with TEA (1.134 mL, 8.137 mmol) followed

by the addition of methyl 2-(4-chlorophenyl)acrylate (57a; 1.000 g,

5.086 mmol). The reaction mixture was stirred at rt for 48 h. The

reaction was diluted with EtOAc, washed with NaHCO₃and brine, dried

over MgSO₄, ﬁltered, and concentrated to give methyl 2-(4-

chlorophenyl)-3-(4-ﬂuoropiperidin-1-yl)propanoate (1.521 g, 100%

yield): ¹H NMR (CDCl₃, 400 MHz) δ 7.31−7.23 (m, 4H), 4.73−4.66

(m, 0.5H), 4.60−4.54 (m, 0.5H), 3.83−3.77 (dd, 1H), 3.68 (s, 3H),

3.15−3.07 (dd, 1H), 2.73−2.64 (m, 1H), 2.62−2.43 (m, 3H), 2.40−

2.32 (m, 1H), 1.92−1.75 (m, 4H); LC/MS APCI (+) m/z 300.1 (M +

H)⁺.

To a solution of methyl 2-(4-chlorophenyl)-3-(4-ﬂuoropiperidin-1-

yl)propanoate (0.380 g, 1.27 mmol) in THF (10 mL) was added

KO(TMS) (0.199 g, 1.39 mmol). The reaction mixture was stirred at rt

overnight. Additional KO(TMS) (20 mg) was added. The reaction was

concentrated, and the crude product (0.232 g, 0.716 mmol) was added

to a solution of 12b (0.200 g, 0.651 mmol) in DCM (4 mL). DIEA

(0.363 mL, 2.08 mmol) and HBTU (0.272 g, 0.716 mmol) were added

to the reaction mixture. The reaction was stirred at rt overnight and then

partitioned between DCM and saturated NaHCO₃. The aqueous layer

was extracted with DCM. The combined extracts were washed with

brine, dried over MgSO₄, ﬁltered, and concentrated. The residue was

puriﬁed by silica gel column chromatography (12:1 DCM−MeOH) to

give the 1:1 mixture of diastereomers, which was separated by chiral SFC

to give (S)-2-(4-chlorophenyl)-3-(4-ﬂuoropiperidin-1-yl)-1-(4-

((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

(S)-2-(4-Chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-((R)-mor-

pholin-3-yl)ethanone Hydrochloride Salt (48). Compound 48 was

prepared from 12b and 23k by the same procedure described for 28: ¹H

NMR (CD₃OD, 400 MHz) δ 8.57 (s, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.40

(d, J = 8.6 Hz, 2H), 5.28 (t, J = 7.8, 1H), 4.51 (d, J = 9.0 Hz, 1H), 4.17−

4.10 (m, 1H), 4.02−3.19 (m, 15H), 2.32−2.24 (m, 1H), 2.22−2.13 (m,

1H), 1.17 (d, J = 7.0 Hz, 3H); LC/MS APCI (+) m/z 472.1, 474.1 (M +

H)⁺; HPLC (method A) >95% purity, t_R= 2.40 min.

Potassium 2-(4-Chlorophenyl)-3-((2,2,2-triﬂuoroethyl)amino)-

propanoate (58a). A solution of methyl 3-amino-2-(4-chlorophenyl)-

propanoate hydrochloride (215 mg, 0.860 mmol) in 1:1 THF−DMF

(3.0 mL) was treated with DIEA (389 μL, 2,23 mmol) at rt.

Triﬂuoroethyl triﬂate (299 mg, 1.29 mmol) was added to the mixture,

and the reaction mixture was stirred for 20 h. The mixture was

partitioned between ethyl acetate and diluted NaHCO₃solution. The

aqueous portion was extracted twice, and the combined organics were

washed with water (3×). The organic portion was washed with brine,

separated, dried over MgSO₄, ﬁltered, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (4:1 hexanes−

EtOAc) to aﬀord methyl 2-(4-chlorophenyl)-3-((2,2,2-triﬂuoroethyl)-

1

pyrimidin-4-yl)piperazin-1-yl)propan-1-one (0.032 g, 10% yield): H

NMR (DMSO-d₆, 400 MHz) δ 8.44 (s, 1H), 7.39 (d, J = 9.2 Hz, 2H),

7.36 (d, J = 9.2 Hz, 2H), 5.41 (d, J = 5.6 Hz, 1H), 4.84 (dd, J = 12.0, 6.4

Hz, 1H), 4.62 (dd, J = 49.2, 3.2 Hz, 1H), 4.31 (dd, J = 12.0, 6.4 Hz, 1H),

3.74−3.72 (m, 1H), 3.64−3.60 (m, 2H), 3.54−3.28 (m, 5H), 3.02 (dd, J

= 12.4, 8.0 Hz, 1H), 2.59−2.54 (m, 2H), 2.44 (dd, J = 12.8, 6.0 Hz, 1H),

2.36−2.34 (m, 2H), 1.95 (ddd, J = 27.6, 13.2, 6.4 Hz, 1H), 1.92 (ddd, J =

20.4, 13.2, 3.2 Hz, 1H), 1.79−1.76 (m, 1H), 1.73−1.70 (m, 1H), 1.13 (d,

J = 6.4, 1H), 1.05 (d, J = 6.8 Hz, 3H); LC/MS APCI (+) m/z 502 (M +

H)⁺; HPLC (method B) >99% purity, t_R= 1.89 min.

(S)-2-(4-Chlorophenyl)-3-((1-hydroxy-2-methylpropan-2-yl)-

amino)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one Bis-

(triﬂuoroacetate) Salt (39). Compound 39 was prepared from 57a

and 2-amino-2-methylpropan-1-ol by the same procedure described for

40: ¹H NMR (CD₃OD, 400 MHz) δ 8.40 (s, 1H), 7.39 (d, J = 8.0 Hz,

2H), 7.32 (d, J = 8.0 Hz, 2H), 5.36 (d, J = 6.2 Hz, 1H), 4.84−4.79 (m,

1H), 4.39−4.36 (m, 1H), 4.30 (d, J = 4.5 Hz, 2H), 4.09−4.05 (m, 1H),

3.70−3.55 (m, 4H), 3.50−3.43 (m, 3H), 3.20−3.00 (m, 4H), 2.60−2.57

1

amino)propanoate (235 mg, 93% yield) as a colorless oil: H NMR

(CDCl₃, 400 MHz) δ 7.33−7.29 (m, 2H), 7.24−7.20 (m, 2H), 3.76−

3.71 (dd, 1H), 3.69 (s, 3H), 3.38−3.30 (m, 1H), 3.24−3.12 (m, 2H),

3.05−2.97 (m, 1H); LC/MS APCI (+) m/z 296 (M + H)⁺.

A solution of methyl 2-(4-chlorophenyl)-3-((2,2,2-triﬂuoroethyl)-

amino)propanoate (235 mg, 0.795 mmol) in THF (3.0 mL) was treated

with KO(TMS) (153 mg, 1.19 mmol) at rt. The reaction mixture was

stirred for 18 h before the mixture was diluted with diethyl ether. The

resulting precipitate was isolated by ﬁltration and dried in vacuo to give

potassium 2-(4-chlorophenyl)-3-((2,2,2-triﬂuoroethyl)amino)-

propanoate (299 mg, 100% yield) as a white solid: ¹H NMR

(CD₃OD, 400 MHz) δ 7.33 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz,

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(m, 1H), 2.06−1.94 (m, 2H), 1.09−1.04 (m, 3H), 0.89 (d, J = 8.0 Hz,

6H); LC/MS APCI (+) m/z 488 (M + H)⁺; HPLC (method C) >99%

purity, t_R= 3.42 min.

ASSOCIATED CONTENT

* Supporting Information

Enzyme inhibition results (expressed as a percentage of the

control) for compounds 2, 3, and 28 tested against a panel of 226,

225, and 230 kinases, respectively, performed at Upstate,

Charlottesville, VA.¹⁵This material is available free of charge

via the Internet at http://pubs.acs.org.

■

S

(S)-3-Amino-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-(4-(triﬂuoromethyl)-

phenyl)propan-1-one Bis(triﬂuoroacetate) Salt (49). Compound 49

was prepared from 2-(4-triﬂuoromethyl)acetyl chloride by the same

procedure described for 34: ¹H NMR (DMSO-d₆, 400 MHz) δ 8.80 (s,

1H), 7.92 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 5.08 (m, 1H),

4.44−4.40 (m, 1H), 3.89−3.84 (m, 2H), 3.76−3.30 (m, 8H), 3.12 (m,

2H), 2.08 (m, 2H), 1.07 (d, J = 8.0 Hz, 3H); LC/MS APCI (+) m/z 450

(M + H)⁺; HPLC (method C) >99% purity, t_R= 3.30 min.

(S)-2-(4-Chloro-3-ﬂuorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-meth-

yl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

(isopropylamino)propan-1-one Dihydrochloride Salt (50). Com-

pound 50 was prepared from 12b and (S)-2-(4-chloro-3-ﬂuorophen-

yl)-3-(isopropylamino)propanoic acid by the same procedure described

for 28: ¹H NMR (D₂O, 400 MHz) δ 8.35 (s, 1H), 7.44 (t, J = 7.2 Hz,

1H), 7.14 (d, J = 9.2 Hz, 1H), 7.04 (d, J = 7.2 Hz, 1H), 5.28−5.16 (m,

1H), 4.38−4.28 (m, 1H), 4.10−3.98 (m, 1H), 3.88−3.72 (m, 2H),

3.72−3.16 (m, 9H), 2.22−2.12 (m, 1H), 2.10−1.96 (m, 1H), 1.32−1.08

(m, 6H), 0.96 (d, J = 5.6 Hz, 3H); LC/MS APCI (+) m/z 476 (M + H)⁺;

HPLC (method A) 98% purity, t_R= 2.38 min.

AUTHOR INFORMATION

Corresponding Author

*Phone: (303) 386-1262. E-mail: jblake@arraybiopharma.com.

Notes

■

The authors declare no competing ﬁnancial interest.

ABBREVIATIONS USED

■

AGC, cAMP-dependent protein kinase/protein kinase G/

protein kinase C extended family; DAST, diethylaminosulfur

triﬂuoride; HER2, human epidermal growth ractor receptor 2;

LNCaP, lymph node carcinoma of the prostate; MCF7,

Michigan Cancer Foundation-7; MCT, methylcellulose with

0.2% Tween-80; mTOR, mammalian target of rapamycin;

p70S6K, phosphoprotein 70 ribosomal protein S6 kinase;

PBSF, n-perﬂuorobutanesulfonyl ﬂuoride; PC3, prostate cancer

cell line; PRAS40, proline-rich Akt substrate; PRKG, cyclic

GMP-dependent protein kinase; PTEN, phosphatase and tensin

homologue; ROCK1, ρ-associated coiled coil containing protein

kinase 1; RPS6, ribosomal protein S6; shRNA, short hairpin

RNA; TsDPEN, tosyl-1,2-diphenylethylene

(S)-3-(tert-Butyl)-2-(3-ﬂuoro-4-(triﬂuoromethyl)phenyl)-1-(4-

((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]-

pyrimidin-4-yl)piperazin-1-yl)propan-1-one Bis(triﬂuoroacetate)

Salt (51). Compound 51 was prepared from 57b and tert-butylamine

1

by the same procedure described for 40: H NMR (DMSO-d₆, 400

MHz) δ 8.80 (s, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H),

5.08 (m, 1H), 4.44−4.40 (m, 1H), 3.89−3.85 (m, 2H), 3.76−3.30 (m,

8H), 3.12 (m, 2H), 2.08 (m, 2H), 1.07 (d, J = 8.0 Hz, 3H); LC/MS

APCI (+) m/z 524 (M + H)⁺; HPLC (method C) 95.3% purity, t_R=

3.95 min.

(S)-2-(3-Fluoro-4-(triﬂuoromethyl)phenyl)-3-((1-hydroxy-2-meth-

ylpropan-2-yl)amino)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihy-

dro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one

Bis(triﬂuoroacetate Salt (52). Compound 52 was prepared from 57b

and 2-amino-2-methylpropan-1-ol by the same procedure described for

40: ¹H NMR (D₂O, 400 MHz) δ 8.58 (s, 1H), 7.86 (dd, J = 8.0, 5.6 Hz,

1H), 7.49 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 5.6 Hz, 1H), 5.07 (m, 1H),

4.50 (m, 1H), 3.91 (m, 2H), 3.80−3.60 (m, 4H), 3.40−3.22 (m, 5H),

3.04 (m, 2H), 2.02 (m, 2H), 1.22 (m, 6H), 1.04 (d, J = 8.0 Hz, 6H); LC/

MS APCI (+) m/z 540 (M + H)⁺; HPLC (method C) 98.3% purity, t_R=

3.90 min.

REFERENCES

■

(1) (a) Cheng, J. Q.; Lindsley, C. W.; Cheng, G. Z.; Yang, H.; Nicosia,

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(S)-3-((Cyclopropylmethyl)amino)-2-(3-ﬂuoro-4-(triﬂuoromethyl)-

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̀

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1

described for 28: H NMR (DMSO-d₆, 400 MHz) δ 8.32 (s, 1H),

7.78 (dd, J = 8.0, 6.2 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 6.2

Hz, 1H), 5.10−5.08 (m, 1H), 4.62−4.60 (m, 1H), 3.90−3.20 (m, 8H),

2.86 (m, 2H), 2.06−2.02 (m, 2H), 1.18−1.15 (m, 1H), 1.09 (d, J = 8.0

Hz, 3H), 0.59−0.54 (m, 2H), 0.33−0.29 (m, 2H); LC/MS APCI (+) m/

z 522 (M + H)⁺; HPLC (method C) >99% purity, t_R= 4.08 min.

(S)-2-(4-Chloro-3-ﬂuorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-meth-

yl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-

((tetrahydro-2H-pyran-4-yl)amino)propan-1-one Dihydrochloride

Salt (54). Compound 54 was prepared from (S)-3-amino-2-(3-ﬂuoro-

4-(triﬂuoromethyl)phenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-di-

hydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one

and tetrahydro-2H-pyran-4-carbaldehyde by the same procedure

described for 44: ¹H NMR (D₂O, 400 MHz) δ 8.37 (s, 1H), 7.44 (t, J

= 7.6 Hz, 1H), 7.15 (d, J = 9.6 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 5.24 (t, J

= 7.8 Hz, 1H), 4.38−4.30 (m, 1H), 4.12−4.04 (m, 1H), 4.00−3.88 (m,

2H), 3.88−3.75 (m, 2H), 3.75−3.64 (m, 1H), 3.64−3.42 (m, 5H),

3.42−3.18 (m, 5H), 2.22−2.18 (m, 1H), 2.08−1.86 (m, 3H), 1.68−1.52

(m, 2H), 0.97 (d, J = 6.4 Hz, 3H); LC/MS APCI (+) m/z 518 (M + H)⁺;

HPLC (method A) 98% purity, t_R= 2.37 min.

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Article Doi

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors

DOI: 10.1021/jm301024w

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Article abstract of DOI:10.1021/jm301024w

Full text of DOI:10.1021/jm301024w

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Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors

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