Scholarly article on 4-benzylpiperazine-1-carboxylic acid butyl ester 1019928-96-1 from Journal of Medicinal Chemistry p. 9133

Article

pubs.acs.org/jmc

4‑((R)‑2-{[6-((S)‑3-Methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-

carbonyl]amino}-3-phosphonopropionyl)piperazine-1-carboxylic

Acid Butyl Ester (ACT-246475) and Its Prodrug (ACT-281959), a Novel

P2Y₁₂Receptor Antagonist with a Wider Therapeutic Window in the

Rat Than Clopidogrel

Eva Caroﬀ,* Francis Hubler, Emmanuel Meyer, Dorte Renneberg, Carmela Gnerre, Alexander Treiber,

Markus Rey, Patrick Hess, Beat Steiner, Kurt Hilpert, and Markus A. Riederer

Drug Discovery and Preclinical Research and Development, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil,

Switzerland

S

* Supporting Information

ABSTRACT: Recent post hoc analyses of several clinical trials

with P2Y₁₂antagonists showed the need for new molecules

being fully eﬃcacious as antiplatelet agents and having a

reduced propensity to cause major bleeding. We have

previously reported the discovery of the 2-phenylpyrimidine-

4-carboxamide analogs as P2Y₁₂antagonists with nanomolar

potency in the disease-relevant platelet aggregation assay in

human plasma. Herein we present the optimization steps that

led to the discovery of clinical candidate ACT-246475 (30d). The key step was the replacement of the carboxylic acid

functionality by a phosphonic acid group which delivered the most potent molecules of the program. In addition, low in vivo

clearance in rat and dog was achieved for the ﬁrst time. Since the bioavailability of 30d was low in rat and dog, we developed the

bis((isopropoxycarbonyl)oxy)methyl ester prodrug (ACT-281959, 45). Compound 30d showed eﬃcacy in the rat ferric chloride

thrombosis model when administered intravenously as parent or orally as its prodrug 45. Moreover, 30d displays a wider

therapeutic window as compared to clopidogrel in the rat surgical blood loss model.

for the initial activation at low levels of ADP, it appears that

P2Y₁₂is required to amplify and sustain the responses leading

to stable thrombus formation. The P2Y₁₂receptor is a validated

target in antiplatelet therapy for patients with acute coronary

syndrome (ACS) or undergoing percutaneous coronary

intervention (PCI).⁶

INTRODUCTION

■

Following a lesion of the vascular wall, multiple signals will

trigger activation of circulating platelets that subsequently

adhere and aggregate at the site of injury, forming a seal to

prevent blood loss. Similarly, rupture of an atherosclerotic

plaque may lead to uncontrolled platelet thrombus formation.

Vessel occlusion might occur followed by ischemia of the

downstream located tissue with consecutive myocardial

infarction (MI).¹Inhibiting the activation and aggregation of

platelets is the recommended therapeutic option to prevent

atherothrombotic events in patients with atherosclerotic disease

in the coronary, peripheral, or cerebrovascular circulation.^2,3

The ADP receptor P2Y₁₂is a key player in the process of

activating platelets and aggregation.^4,5Platelet activation is

initially triggered by the interaction of glycoproteins on the

surface of platelets with components of the subendothelial

matrix or ruptured atherosclerotic plaque and is ampliﬁed by

the release of soluble mediators, which also recruit new

platelets. One of the most important soluble mediators is ADP,

which is an agonist at the two G protein-coupled receptors

(GPCRs), P2Y₁, and P2Y₁₂. Both P2Y₁and P2Y₁₂participate in

the activation of integrin glycoprotein IIb/IIIa, which binds

ﬁbrinogen, contributing to the creation of the cross-linked

aggregates that form a thrombus. Whereas P2Y₁is important

In patients with ACS, the current guidelines recommend a

dual antiplatelet therapy with acetylsalicylic acid and a P2Y₁₂

receptor antagonist, such as clopidogrel, prasugrel, or

ticagrelor.⁷⁻⁹Clopidogrel is an irreversible P2Y₁₂receptor

antagonist with proven antithrombotic eﬃcacy.^10,11Never-

theless, the antiplatelet eﬀect is delayed by the need for

conversion to the active metabolite, and additionally about 14−

30% of the patients are resistant to clopidogrel treatment due to

genetic polymorphisms of the CYP-450 enzymes responsible

for its metabolism.^12,13Prasugrel is also an irreversible P2Y₁₂

receptor antagonist from the thienopyridine class, with a

diﬀerent mechanism for the formation of the active

metabolite.¹⁴It achieves stronger inhibition of platelet

aggregation, but the superior eﬃcacy versus clopidogrel in

moderate- to high-risk patients with ACS undergoing PCI^15,16

Received: June 17, 2015

A

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Figure 1. (a) Previous work: 2-phenylpyrimidine-4-carboxamide analogs 1 and 2 where the central core was set as L-glutamic acid. (b) This

disclosure: general structure of (S)-6-(3-methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carboxamide analogs. R¹is ethyl or n-butyl. R²and

stereochemistry at the α carbon of the amino acid core are detailed in the tables.

a

Scheme 1. Preparation of Building Block 8

a

Reagents and conditions: (a) see ref 28, 4 steps, 51%; (b) BOC₂O, Et₃N, DCM, 100% (crude); (c) NaH, MeI, THF, 93% (crude); (d) HCl in EA,

95%; (e) DIPEA, THF, 60 °C, 4 days, 93%.

is associated with an increased bleeding risk.¹⁷Ticagrelor, the

ﬁrst reversible P2Y₁₂receptor antagonist tested in clinical trials,

demonstrated superiority over clopidogrel in the prevention of

ischemic events with no statistically signiﬁcant increase of

bleeding.¹⁸Nevertheless, it is still discussed which P2Y₁₂

antagonist is the best choice and which subgroups of patients

should be treated with clopidogrel, prasugrel, or ticagrelor.¹⁹

Consequently, new P2Y₁₂inhibitors combining high inhibition

of platelet aggregation with low risk of bleeding are still actively

searched.²⁰Recent publication of X-ray structural informa-

tion^21,22as well as molecular modeling studies docking known

P2Y₁₂antagonists (including a close analog of the molecules

disclosed herein)²³will likely support this dynamic research

ﬁeld.

responsive to high local concentrations of ADP.^25,26In

addition, some of the excess bleeding associated with

thienopyridines may be due to oﬀ-target eﬀects, as shown by

comparing clopidogrel and prasugrel treatment with genetic

ablation of the P2Y₁₂receptor in mice.²⁷

We have previously reported the discovery of the 2-

phenylpyrimidine-4-carboxamide series and the SAR of the

substituent on position 6 of the pyrimidine ring.²⁸We

identiﬁed two sets of highly potent P2Y₁₂antagonists, of

which compounds 1 and 2 are the most active representatives

(Figure 1a). The best molecules showed IC₅₀values in the low

nanomolar range in the binding assay and in the light

transmission aggregometry (LTA) assay. No inhibition of the

major P450 enzymes or of the hERG channel was observed.

Moderate to high clearance in the rat represented the main

caveat of the two chemical classes. We set the substituent in

position 6 of the pyrimidine ring to (S)-3-methoxypyrrolidin-1-

yl as in analog 2 and the SAR of the R²group was explored

(Figure 1b). In the present paper we report the properties of

At Actelion, we aimed at discovering a reversible and

selective P2Y₁₂antagonist, which may provide an improved

therapeutic window²⁴compared to the benchmark drug

clopidogrel. Indeed, reversible antagonism may cause less

bleeding than irreversible blockade, since the receptor remains

B

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Scheme 2. Preparation of P2Y₁₂Antagonist Analogs 13−22, 28, 29a−f, 30a−k, 31a,b, 32a,b, 33−37, and 38a,b (for R¹and R²,

a

See Tables 1, 2, and 4)

a

Reagents and conditions: (a) HOBt/EDC-Cl or HATU or PyBOP, DIPEA, DCM or DCM/THF, rt. (b) PG = BOC: TFA/DCM, rt, or HCl in EA

or EA/dioxane. PG = Cbz: Pd/C (wet, 5%), H₂, EtOH or EtOH/AcOH, rt. PG = Fmoc: Et₂NH, DCM, rt. (c) 8, similar conditions as described in

(a); (d) TFA, DCM, rt, 22−100%; (e) TMSBr, MeCN, 0 °C to rt overnight, then DCM, water, rt, 1 h, 17−98%; (f) 4 M HCl in dioxane, rt, 30 min,

99−100%; (g) ZnBr₂, NaN₃, H₂O, 100 °C, 20 h, 22% (32a,b), or NH₂OH−HCl, NaHCO₃, MeOH, 70 °C, 24 h, then CDI, DBU, dioxane, 105 °C,

2 h, 2% (33); (h) Me₄NCl, DMF, 150 °C, overnight, 23%; (i) trichloroacetyl isocyanate, EtOH, 0 °C, 20 min, then NaBH₄, EtOH, 0 °C to rt

overnight, 5%; (j) SOCl₂, rt, 1 h, then Et₃N, CF₃SO₂NH₂, DCM, rt, overnight, 6% (35), or MeSO₂NH₂, DCC, DMAP, DCM, 0 °C, 1 h, then rt, 20

h, 36% (36); (k) CF₃SO₂Cl, Et₃N, THF, 0 °C to rt, overnight, 11%; (l) 3,4-diethoxy-3-cyclobutene-1,2-dione, Et₃N, EtOH, rt, overnight, 52−65%,

then 4 M HCl in dioxane, THF, 50 °C, 2 days, 52−56%.

(S)-6-(3-methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbox-

amide analogs and the path that led to the discovery of a new

and potent P2Y₁₂antagonist with a unique eﬃcacy/safety ratio.

second amide coupling step assembled the amine 12 and the

acid building block 8 to give the ﬁnal scaﬀold in good overall

yield. Analogs 13−22 were directly obtained without any

additional transformation. Compound 28 was obtained by

reacting the hydroxyl group of analog 22 with trichloroacetyl

isocyanate, followed by NaBH₄mediated trichloroacetamide

cleavage.²⁹

RESULTS AND DISCUSSION

■

Chemistry. Retrosynthetically, the target molecules were

divided into three parts, the piperazine carbamate, the central

amino acid core, and the pyrimidine moiety. Scheme 1 shows

the synthesis of the pyrimidinecarboxylic acid building block 8.

Intermediate 5 was obtained in four consecutive steps starting

from commercially available ethyl 4-methoxy-3-oxobutanoate 3

and benzamidine 4. Chloropyrimidine 5 was reacted with (S)-

3-methoxypyrrolidine 7 at 60 °C in THF in the presence of

DIPEA to give the acid 8.

When R²contained a tert-butyl protected acidic function

such as in intermediates 23a−f, TFA treatment in DCM

aﬀorded analogs 29a−f in high yields. Hydrolysis of the diethyl

phosphonate group of intermediates 24a−k was achieved using

TMSBr in anhydrous MeCN and provided analogs 30a−k.³⁰

Analogs 31a,b were quantitatively obtained upon treatment of

BOC-protected amino derivatives 25a,b with HCl in dioxane.

The cyano group of intermediates 26a,b was transformed into a

tetrazole ring upon treatment with NaN₃in the presence of

ZnBr₂to provide analogs 32a,b.³¹Furthermore, the cyano

group was reacted with NH₂OH and the resulting hydroxy-

amidine was subsequently cyclized to the oxadiazolone analog

33 upon treatment with CDI.³²Analog 34 was obtained by

cleavage of the isobutyl sulfonate using Me₄NCl.³³Carboxylic

acid analogs 29a,b were further transformed into sulfonylamide

analogs 35 and 36 using the corresponding primary

The synthesis of the P2Y₁₂antagonists was designed and

carried out as shown in Scheme 2. The chirality at the amino

acid Cα was introduced with intermediates 10 and was

conserved throughout the synthesis. The piperazine carbamate

9 was coupled to the amino acid 10 using as activating agent

EDC-Cl/HOBt or HATU or PyBOP. The deprotection step

performed on the resulting intermediate 11 was a HCl- or

TFA-mediated BOC cleavage or a palladium catalyzed

hydrogenolysis of Cbz or a Fmoc removal with Et₂NH. The

C

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a

Scheme 3. Preparation of Intermediates 10a−g for the Synthesis of Phosphonic Acid-Containing Analogs 30a−k

a

Reagents and conditions: (a) isobutyl chloroformate, NMM, THF, −15 °C, 14 h, then NaBH₄, MeOH, −10 °C, 100% (crude) (41f−g); (b) CBr₄,

PS−PPh₃, DCM, 0 °C, 2−3 h, 25−52% (40d−e), or imidazole, PPh₃, I₂, THF, 0 °C to rt, 11 h, 65−100% (40f−g); (c) P(OEt)₃, 120−130 °C,

overnight, 57−100% (crude); (d) LiOH, MeOH, EtOH, H₂O, 0 °C, 1 h, 100% (10a), or rt, 3 h to overnight, 48−100% (10e−g), or LiOH, THF,

H₂O, rt, 3 h to overnight, 72−100% (10b−d).

sulfonamide reagent in combination with DCC/DMAP or

SOCl₂. Compound 37 was prepared starting from analog 31b

and reacted with CF₃SO₂Cl. Analogs 38a,b were obtained by

reacting 3,4-diethoxy-3-cyclobutene-1,2-dione with analogs

31a,b.^34,35

bulk at the amino acid Cβ such as tert-butyl (17) led to

micromolar activities. Polar functional groups such as carboxyl

(2, 29a−f), amine (31a,b), hydroxyl (22, 43), ether (19),

sulfone (20), primary amide, and carbamate (21, 28) were

introduced, varying the length of the linker to the amino acid

Cα. Among those, the most potent analogs with binding IC₅₀

values below 100 nM were hydroxymethyl (22), aminomethyl

(31a), and primary carbamate (28) derivatives and carboxylic

acids (2, 29a,b, 29e,f). Interestingly, analog 29c, where the

stereochemistry at the amino acid core is reversed to (R), was

11-fold less active than the corresponding (S) analog 29a. The

trend favoring the (S) orientation was even more striking when

the linker was longer, as shown with analogs (S)-29b and (R)-

29d. In the LTA assay in human plasma, only carboxyl-

containing analogs showed IC₅₀values below 100 nM. Finally,

elongating R¹from ethyl to n-butyl led to analogs with better

aﬃnity, increased lipophilicity, and slightly higher potency in

human plasma (see 16 vs 15, 2 vs 29b, 29e vs 29a).

These results supported the prevalence of the carboxyl group

to reach good potency in human plasma. Furthermore, the

carboxyl group was most likely responsible for the medium

clearance observed in the rat via excretion as unchanged drug in

the bile (data not shown). Therefore, we synthesized a number

of carboxylic acid bioisosteres^39,40with the goal to achieve

superior PK behavior while keeping the high potency.

Biological data are reported in Table 2 (PK data are discussed

in Table 4).

Indeed, apart from the sulfonic acid analog 34, all carboxylic

acid bioisosteres (30f, 32a−38b, 44) were potent in the

binding assay ranging from 3.7 to 43 nM. In the LTA assay,

analog 35 came out as an outlier with an IC₅₀value of 2100

nM. High lipophilicity might account for this low aﬃnity, since

the two analogs 35 and 37 having the highest clogP (>3.2) also

show the highest plasma shift and modest potencies in the LTA

assay. All other molecules showed medium to good potencies in

the LTA assay similar to carboxyl-containing derivatives.

Notably, analogs 30f and 44 showed two of the lowest plasma

shifts ever encountered in the course of our medicinal

chemistry program, 1.4 and 1.7, respectively. In the next step,

we focused on analog 30f and expanded the phosphonic acid-

containing compound class by varying the linker length and the

stereochemistry at the amino acid core. The biological data are

listed in Table 3.

The key intermediates 10 leading to intermediates 23a−f,

25a,b, 26a,b, and 27 and to ﬁnal analogs 13−22 were

commercially available. For the preparation of diethyl

phosphonate derivatives 24a−k leading to analogs 30a−k,

intermediates 10a−g were synthesized as shown in Scheme 3.

When the phosphonic acid group was directly attached to the

amino acid backbone, racemic intermediate 10a was easily

prepared from commercially available racemic dimethyl

phosphonate 39a by saponiﬁcation with aqueous LiOH. For

the enantiomers 10b,c, application of the Arbuzov protocol on

the enantiomerically pure iodoserine derivatives 40b,c gave the

corresponding phosphonate analogs 39b,c.³⁶Whenever the

appropriate protected amino acid derivative bearing a

halogenated side chain was not commercially available, it was

prepared starting from the hydroxyl analog. Enantiomers 41d,e

were reacted with CBr₄in the presence of PPh₃, leading to

enantiomers 40d and 40e, respectively. Likewise, hydroxyl

precursors 41f−g were reacted with iodine in the presence of

PPh₃and imidazole leading to enantiomers 40f and 40g.³⁷In

case the phosphonic acid group was attached to the amino acid

backbone through a n-propyl linker (10f−g), the side chain

carboxylic groups of protected (^L)- and (D)-glutamic acid

derivatives 42f−g were reduced to the corresponding alcohols

by activation with isobutyl chloroformate followed by NaBH₄

treatment aﬀording enantiomers 41f−g.³⁸

Analogs 43 and 44 were prepared following a diﬀerent route

(not shown) and the synthetic protocols and schemes can be

found in the Experimental Section and Supporting Information.

Structure−Activity Relationships (SARs). The aﬃnity of

the synthesized analogs to the P2Y₁₂receptor was measured in

the binding assay. The most potent compounds (generally IC₅₀

below 100 nM) were further proﬁled ex vivo; i.e., inhibition of

ADP-induced platelet aggregation was measured in human

platelet-rich plasma (PRP) using light transmission aggrego-

metry (LTA). An extensive screening of the amino acid side

chain R²was undertaken, and a panel of representative

examples are shown in Table 1. Generally, most R²residues

were well tolerated, leading to moderate to good IC₅₀values in

the binding assay. R²being hydrogen (13) and alkyl groups of

moderate size (14−16 and 18) provided IC₅₀values in the

hundred nanomolar range. Lipophilic groups with more steric

Analog 30a (epimeric mixture) with the phosphonic acid

directly attached to the amino acid backbone displayed

moderate activity in the binding assay. The inﬂuence of the

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Table 1. Screening of the Amino Acid Side Chain R²: Binding Aﬃnity and LTA IC₅₀Values

a

b

Absolute stereochemistry at the amino acid Cα. CHO cells expressing recombinant P2Y₁₂receptors incubated with [³³P]-2MeSADP and

c

d

compound. Data are presented as the mean SEM of n = 2−4. Incubation for 2 min in human PRP with 3 μM ADP. n = 1. See ref 28.

stereocenter was analogous to what was observed with

carboxyl-containing analogs 29a−d favoring the forward

orientation of R², irrespective of the linker length. Indeed,

analogs extending R²in the backward direction (as depicted in

Table 3), 30e, 30g, 30i, and 30k, were at least 10 times less

active in the binding assay than the corresponding analogs 30d,

30f, 30h, and 30j. Unexpectedly, 30c was only twice less active

as 30b. In general, elongation of R¹from ethyl to butyl led to

increased binding aﬃnity. However, notable exceptions are 30e

and 30i wherein R¹is butyl which are equally or less active than

30c and 30g wherein R¹is ethyl. Overall, reﬁnements of the

SAR led to ﬁve phosphonic acid-containing analogs 30b−d,

30h, and 30j that were extremely potent in human plasma.

Pharmacokinetic Analysis. On the basis of in vitro and ex

vivo potencies, a selection of analogs from Tables 1, 2, and 3

were progressed for evaluation of PK parameters in rat (see

Table 4; data for 1 and 2 are indicated for comparison).

Systemic clearance was close to liver blood ﬂow for

hydroxymethyl 22, benzoic acid-containing analog 29f,

compounds 32a,b, 36, 38b, and 44 containing a carboxylic

acid bioisostere other than a phosphonic acid and analog 30j

extending a phosphonic acid group on a n-propyl linker.

However, compounds 30c, 30d, and 30h extending a

phosphonic acid group on shorter linkers showed low

clearances of 3.7, 2.1, and 5.4 mL/(min·kg), respectively.

With the exception of hydroxyl-containing analog 22, volumes

of distribution at steady state were in the range of total body

water. Consequently half-lives were short, with a maximum of

1.8 h for 30d. Oral bioavailability was 16% and 28% for 22 and

29f, respectively, but below 3% for all molecules containing a

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Table 2. Replacement of the Carboxylic Acid by Acid Bioisosteres: Binding Aﬃnity and LTA IC₅₀Values, Plasma Shift, and

clogP

a

b

Absolute stereochemistry at the amino acid Cα is (S). CHO cells expressing recombinant P2Y₁₂receptors incubated with [³³P]-2MeSADP and

c

d

compound. Data are presented as the mean SEM of n = 2−4. Incubation for 2 min in human PRP with 3 μM ADP. n = 1. Plasma shift = (LTA

IC₅₀)/(binding IC₅₀).

carboxylic acid bioisostere including a phosphonic acid.

Whether the poor oral exposure of analogs 32b, 36, 38b, and

44 is the result of high clearance combined with limited oral

absorption cannot be concluded from these basic pharmaco-

kinetic experiments. However, considering the predicted pK_aof

the carboxylic acid bioisosteric moiety of the four analogs (32b,

4.86; 36, 4.45; 38b, 1.23; 44, 8.06),⁴¹one can hypothesize that

the oral exposure of 38b below limit of quantiﬁcation is likely

due to the ionized state of the molecule at the pH of the upper

intestine (6.8) leading to poor permeability, and to a clearance

close to liver blood ﬂow. Likewise, since the phosphonic acid

group bears at least one negative charge at intestinal pH,⁴²

analogs 30c−d and 30h may not readily undergo passive

diﬀusion across cellular membranes and intestinal mucosa,

resulting in low oral bioavailability, despite a low in vivo

clearance. As a conclusion, the new P2Y₁₂antagonists described

in Table 4 show poorer bioavailabilities and shorter half-lives

compared to compound 1. Interestingly, the phosphonic acid-

containing analogs 30c, 30d, and 30h exhibit markedly lower

clearances than compound 1. Among these three phosphonates,

analog 30d was deemed the best combination of potency,

clearance, and exposure in the rat. Therefore, it was further

progressed together with compound 1.

eﬃcacy of the two P2Y₁₂antagonists. The deposition of a 20%

aqueous ferric chloride solution onto the right carotid artery of

the rat induced a rapid decrease in carotid blood-ﬂow velocity.

Within 11 1 min, the ﬂow was not measurable anymore due

to histologically conﬁrmed thrombotic occlusion of the artery

(data not shown). In vehicle treated rats, the vessel injury

resulted in 100% thrombotic occlusion (Figure 2, vehicle). Oral

treatment with clopidogrel as well as intravenous bolus

injection of compound 1 dose-dependently prevented the

ferric chloride-induced thrombus formation (Figure 2A,B). At

the highest doses of 30 mg/kg of clopidogrel and 1 mg/kg of

compound 1, thrombus formation was fully blocked. The

observed increase of 26% in blood ﬂow at 30 mg/kg of

clopidogrel was attributed to oﬀ-target eﬀects at the vessel

wall.²⁷Intravenous bolus injection of compound 30d dose-

dependently prevented the ferric chloride-induced thrombotic

occlusions (Figure 2C). At the highest dose tested (0.1 mg/kg),

full antithrombotic eﬃcacy was achieved and no increase in

carotid blood ﬂow versus baseline ﬂow was measured. Thus, the

P2Y₁₂antagonists 1 and 30d were shown to be potent

antithrombotic agents. Compound 30d showed maximal

eﬃcacy in this rat model at about 10-fold lower dose than

compound 1.

In Vivo Eﬃcacy/Safety Assessment. Compounds 1 and

30d were tested for in vivo eﬃcacy and safety and were

compared with clopidogrel. The ferric chloride model was

performed in Wistar rats to determine the antithrombotic

To determine the eﬃcacy/safety ratio, the two compounds

and clopidogrel were proﬁled in a rat surgical blood loss model

at doses inducing the maximal antithrombotic eﬀect (Figure 3).

The surgical blood loss was deﬁned as the amount of blood

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Table 3. Replacement of the Carboxylic Acid by a Phosphonic Acid: Binding Aﬃnity and LTA IC₅₀Values

a

b

Absolute stereochemistry at the amino acid Cα. CHO cells expressing recombinant P2Y₁₂receptors incubated with [³³P]-2MeSADP and

c

compound. Data are presented as the mean SEM, with n = 2−4. Incubation for 2 min in human PRP with 3 μM ADP. n = 1.

oozing out of the rat spleen after placing a standardized surgical

wound. In the absence of any antithrombotic treatment

(vehicle group), the surgical blood loss was quantiﬁed at 0.16

and 0.18 g of blood, after oral gavage and intravenous bolus

injection, respectively. At maximal antithrombotic dose of

clopidogrel (30 mg/kg), the surgical blood loss was increased

from 0.16 to 2.8 g of blood. At maximal antithrombotic dose of

compound 1 (1 mg/kg), the surgical blood loss was increased

from 0.18 to 1.5 g of blood. In striking contrast, at maximal

antithrombotic dose of compound 30d (0.1 mg/kg), the

observed surgical blood loss was only 0.4 g. On the basis of

these in vivo data, compound 30d, at maximal antithrombotic

eﬃcacy, displayed a wider therapeutic window in the rat than

clopidogrel and compound 1. The clear diﬀerence in blood loss

between compound 30d and clopidogrel at maximal eﬃcacious

doses warranted further characterization of 30d toward clinical

development.

Preclinical Characterization of Compound 30d. In

radioligand displacement assays, [³³P]-2MeSADP was able to

displace prebound compound 30d and to reach maximal

binding capacity, hence conﬁrming 30d as a reversible P2Y₁₂

antagonist. During SAR optimization, inhibition of platelet

aggregation was assessed in human PRP, with incubation time

of 2 min and 3 μM ADP for platelet activation. In this setting

compound 30d displayed an IC₅₀value of 8.0 nM. As a

preparation for clinical proﬁling, assay conditions were adapted

to the protocols used in hospital setting. Using a PAP-8

aggregometer with a 20 min incubation time and 20 μM ADP,

the IC₅₀value was conﬁrmed to be 14 nM (n = 8).⁴³No

antagonistic activity on genetically related P2Y receptors was

measured. Up to 10 μM concentration of 30d did not interfere

with agonist binding to P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, and

P2Y₁₃. No antagonistic activity was detected on P2X₁, P2X₂,

P2X₃, P2X₄, and P2X₇receptors. Furthermore, compound 30d

was tested for potential oﬀ-target activity against a broad panel

of receptors, transporters, and enzymes. Radioligand displace-

ment assays were performed against 68 targets, including

GPCRs, ion channels, and transporters, and activity assays were

performed with 30 enzymes. To expand the proﬁling and

include potential agonism, PathHunter technology was used on

a panel of 161 recombinant GPCRs. All assays were performed

in duplicate using a ﬁnal concentration of 10 μM. Compound

30d did not interact with the target in any of these assays. At 50

μM concentration of 30d, less than 10% reduction of the hERG

K⁺current was observed.⁴⁴Compound 30d did not inhibit

cytochrome P450 enzymes in assays assessing competitive

inhibition using human liver microsomes up to 50 μM and did

not exert time-dependent inhibition of CYP2C9, CYP2D6, or

CYP3A4.⁴⁵Plasma protein binding in animal species used for

preclinical safety testing, i.e., rat, dog, as well as in man, was

above 99%. In liver microsomes of human, rat, dog, rabbit, and

cynomolgus monkey, the turnover was very low and the

cleavage of the −CO−N− bond of the carbamate moiety was

not observed. Similar to rat (Table 4), compound 30d showed

a low plasma clearance and volume of distribution in the dog,

resulting in a half-life of 4.8 h. Oral bioavailability in the dog

was around 1%. Compound 30d successfully passed preclinical

toxicology and safety studies.

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c

Table 4. Rat Pharmacokinetic Proﬁles of Selected P2Y₁₂Antagonists

a

Low clearance: CL < 30% of liver blood ﬂow (LBF). Moderate clearance: 30% < CL < 70% LBF. High clearance: >70% LBF. LBF in rat is 55−90

mL/(min·kg). For correct classiﬁcation, plasma clearance needs to be converted to blood clearance considering the blood/plasma partitioning ratio,

b

c

which in the absence of data is assumed to be 1. See ref 28. Male Wistar rats. Dose: iv infusion at 1 mg/kg (n = 1 or 2); po at 10 mg/kg (n = 2 or

3). BLQ: below limit of quantiﬁcation (∼1 ng/mL). SDs and ranges are provided as Supporting Information.

Figure 2. Eﬀects of (A) clopidogrel, (B) compound 1, and (C) compound 30d on thrombotic occlusion after FeCl₃-induced arterial injury in

anesthetized Wistar rats (n = 3−4 per group (A), n = 5−11 per group (B), and n = 3−11 per group (C)). Doses are in mg/kg. Thrombotic occlusion

is deﬁned as % reduction in blood ﬂow velocity from baseline, 30 min after arterial injury. Data are presented as the mean SEM. (A) Vehicle and

clopidogrel were administered orally as single dose 2 h before arterial injury. Vehicle was saline 9 g/L, pH adjusted to 2.5 with 3 M HCl. (B, C)

Vehicle and compounds 1 and 30d were administered as intravenous bolus injection 5 min before arterial injury. Vehicle was PEG 400 (7.5%), PG

(7.5%), Cremophor (5%), phosphate buﬀer, pH = 7.4.

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Figure 3. Eﬀects of (A) clopidogrel and (B) compounds 1 and 30d on surgical blood loss after spleen puncture in anesthetized Wistar rats (n = 8−

14 per group (A) or n = 5−20 per group (B)). Surgical blood loss was measured 30 min after spleen puncture. Data are presented as the mean

SEM. (A) Vehicle and clopidogrel were administered orally as single dose 2 h before spleen puncture. Vehicle was saline 9 g/L, pH adjusted to 2.5

with 3 M HCl. Open bar = vehicle. Hatched bar = clopidogrel at 30 mg/kg. (B) Vehicle and compounds 1 and 30d were administered as intravenous

bolus injection 5 min before spleen puncture. Vehicle was PEG 400 (7.5%), PG (7.5%), Cremophor (5%), phosphate buﬀer, pH = 7.4. Open bar =

vehicle. Gray bar = compound 1 at 1 mg/kg. Black bar = compound 30d at 0.1 mg/kg.

In Vivo Eﬃcacy of Compound 45 as Prodrug of

Compound 30d. On the basis of in vitro metabolic studies

and PK data in rat and dog, we expected a low oral absorption

of compound 30d in human. Therefore, we decided to mask

the phosphonic acid group as a double ester prodrug.^46,47

Brieﬂy, we synthesized a variety of phosphonate ester prodrugs

and evaluated the chemical stability, metabolic stability in

plasma and liver microsomes, and PK in rat and dog. Finally,

the bis((isopropoxycarbonyl)oxy)methyl ester prodrug 45

(Figure 4) was selected for preclinical development. An

overview of the synthetic procedures and optimization program

toward this prodrug will be published separately.

by 65 16% (Figure 5A). A similar reduction in blood ﬂow

velocity 30 min after injury of 47 18% (i.e., inhibition of

thrombotic occlusion by 53 18%) was measured after oral

administration of a 1 mg/kg dose of analog 45 (Figure 5B).

During the 30 min time period of the experiments, the plasma

concentration of 30d was between 72 and 141 ng/mL for the

0.01 mg/kg intravenous dose of 30d (Figure 6A) and between

101 and 190 ng/mL for the 1 mg/kg oral dose of 45 (Figure

6B). Plasma concentrations of the parent drug 30d were

comparable in both experiments, which supports the observed

similar antithrombotic eﬃcacy. Therefore, one can reasonably

conclude that (1) the prodrug 45 liberates the parent drug 30d

in circulation, (2) the observed antithrombotic eﬀect after oral

administration of 45 is entirely due to the parent drug 30d, and

(3) a 1 mg/kg oral dose of 45 leads to a comparable

antithrombotic eﬃcacy as a 0.01 mg/kg iv dose of 30d in the

ferric chloride rat thrombosis model. Compound 45 showed no

toxicity or safety signals in preclinical studies.

CONCLUSION

■

The ﬁrst optimization sequence of the 2-phenylpyrimidine-4-

carboxamide scaﬀold led to analogs 1 and 2 showing high

potencies in both the binding and LTA assays albeit with

moderate PK proﬁles in rat and dog (data not shown). In a

second step, keeping the newly discovered (S)-3-methoxypyr-

rolidinyl substituent in position 6 of the pyrimidine ring, we

focused on the amino acid core. Introduction of various

functional groups on the amino acid side chain conﬁrmed our

initial observation that an acidic group was needed to reach

high potency in human plasma. We also suspected the acidic

group to be responsible for the medium clearance observed in

vivo in rat and dog. Therefore, we embarked on a third

optimization sequence with the goal to identify a bioisosteric

replacement for the carboxylic acid functionality. The

phosphonic acid group was found beneﬁcial, since it delivered

the most potent molecules in the LTA assay as well as low in

vivo clearance in the rat. Consequently, the phosphonic acid-

containing analog 30d was selected for preclinical development.

In vitro, it was shown to be a reversible and highly selective

P2Y₁₂antagonist, potent in human plasma at a low nanomolar

concentration. 30d dose-dependently blocked thrombus

formation in the rat ferric chloride model and displayed a

wide therapeutic window. Overall, 30d exhibited a very safe

Figure 4. Compound 45, bis((isopropoxycarbonyl)oxy)methyl ester

prodrug of 30d.

To conﬁrm the oral antithrombotic eﬃcacy of compound 45,

the ferric chloride model in Wistar rats was repeated comparing

oral administration of the prodrug 45 with intravenous

infusion⁴⁸of the parent drug 30d. The prodrug 45 was

below analytical detection limits in rat blood after oral gavage.

Plasma concentration of the active 30d was measured in the

two experiments at the time of vessel injury and 30 min later. In

vehicle treated rats, the vessel injury resulted in complete

thrombotic occlusion of the artery, i.e., 100% decrease of blood

ﬂow (Figure 5, vehicle). Intravenous infusion of 0.01 mg/kg/50

min of analog 30d led to a blood ﬂow velocity decrease 30 min

after injury of 35

16% compared to baseline ﬂow in the

absence of vessel injury; i.e., thrombotic occlusion was inhibited

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Figure 5. Eﬀects of (A) compound 30d and (B) compound 45 on thrombotic occlusion after FeCl₃-induced arterial injury in anesthetized Wistar

rats (n = 10−35 per group (A) or n = 6 per group (B)). Thrombotic occlusion is deﬁned as % reduction in blood ﬂow velocity from baseline, 30 min

after arterial injury. Data are presented as the mean SEM. (A) Vehicle and compound 30d were administered as continuous intravenous infusion

over 50 min at the rate of 1 (mL/kg)/20 min, starting 20 min before arterial injury. Vehicle was PEG 400 (7.5%), PG (7.5%), Cremophor (5%),

phosphate buﬀer, pH = 7.4. (B) Vehicle and compound 45 were administered orally as single dose 1 h before arterial injury. Vehicle was PEG 400

(7.5%), PG (7.5%), Cremophor (5%), water, 5 mL/kg.

Figure 6. Plasma concentrations of the active drug 30d (A) at 20 and 50 min after starting iv infusion of 30d in the experiment described in Figure

5A and (B) at 60 and 90 min after oral gavage of 45 in the experiment described in Figure 5B. Plasma was obtained from arterial blood sampling

(EDTA). Experimental details are given in Figure 5.

expression the human P2Y₁₂gene were grown in Ham’s F12 medium

with phenol red and ^L-glutamine (Gibco no. 21765-029) containing

250 μg/mL Geneticin (Gibco), 100 μg/mL gygromycin B

(Invitrogen), and 10% FCS Sera Plus (PAN Biotech; Aidenbach,

DE). Cells were cultivated in 96-well plates at a density of 75 000

cells/well in medium supplemented with apyrase 0.1 U/mL and grown

overnight at 37 °C and 5% CO₂. The next day, the cells were washed

three times with binding buﬀer (Ham’s F12 medium (Gibco) with

phenol red and ^L-glutamine (Gibco no. 21765-029), supplemented

with 0.01% protease-free BSA). The assay was performed as follows:

First, 100 μL of binding buﬀer was added to each well. Second, 50 μL

of binding buﬀer containing 5% DMSO or compound was added.

Third, 100 μL of 0.25 nM radioligand [³³P]-2MeSADP (PerkinElmer

CUS55563) was added to each well and incubated for 90 min at room

proﬁle in in vitro and in vivo preclinical experiments. However,

on the basis of in vitro metabolic studies and PK data in rat and

dog, we expected a low oral absorption of compound 30d in

human. Therefore, we developed the bis((isopropoxycarbonyl)-

oxy)methyl ester prodrug 45 which showed antithrombotic

eﬃcacy after oral administration in the rat ferric chloride model.

Both compounds 45 and the parent drug 30d entered clinical

studies in healthy volunteers.⁴³

EXPERIMENTAL SECTION

■

P2Y₁₂Radioligand Displacement Assay (Binding Assay).

Recombinant Chinese hamster ovary (CHO) cells with recombinant

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temperature, with gentle agitation. The assay was stopped by three

washes with binding buﬀer, followed by addition of 200 μL of

scintillation liquid (Microscint 40, Packard) and quantiﬁcation in a

scintillation counter (Packard, Top Count NXT).

inserted into the right femoral vein for bolus injection or continuous

infusion at a rate of 1 (mL/kg)/20 min of compounds 1 and 30d or

vehicle. A second catheter was inserted in the right femoral artery to

measure mean arterial pressure (MAP) and heart rate (HR). The right

carotid artery was gently dissected free of connective tissue, and a

silastic tubing ﬂow transducer (D-20-0.8mm, Triton Technologies

Inc.) was placed on the artery for blood ﬂow velocity measurement.

Ferric chloride (FeCl₃, Fluka no. 44943) was dissolved in distilled

water at a concentration of 200 mg/mL (20%). At 5 min after bolus

injection or 20 min after infusion start of vehicle or compound, two

ﬁlter papers (Tempo handkerchief, 4 mm × 4 mm) were soaked in this

solution and placed on the carotid artery anterior to the ﬂow

transducer for 5 min to induce thrombus formation. Mean arterial

pressure (MAP), heart rate (HR), and carotid blood ﬂow velocity were

continuously recorded on a PowerLab data acquisition system (IOX

data acquisition, Emka Technologies, France) during 30 min after

removal of the ferric chloride ﬁlter papers by using a IOX software

(Emka Technologies, France). Data were exported to a Dell

Dimension 733R computer for analysis (Datanalyst, version 2.10.17,

Emka Technologies, France). At the end of the experiment, rats were

euthanized by an intravenous infusion of pentobarbital (100 mg/kg,

Esconarkon, Streuli Pharma AG, Switzerland). Clopidogrel and its

vehicle were administered by oral gavage at 5 mL/kg 2 h before FeCl₃-

soaked ﬁlter papers were applied to the carotid artery. The time for

clopidogrel gavage was determined in a pilot experiment where the

FeCl₃arterial induced injury was performed 2 and 4 h after oral

administration of 10 mg/kg clopidogrel. The same antithrombotic

eﬃcacy was observed in both cases; therefore, the time for clopidogrel

administration was set to 2 h before arterial injury.⁵³Compound 45

and its vehicle were administered by oral gavage at 5 mL/kg 1 h before

FeCl₃-soaked ﬁlter papers were applied to the carotid artery. An

arterial blood sample (EDTA) was withdrawn immediately before

FeCl₃-soaked ﬁlter papers were applied to the carotid artery and 30

min after. Blood was centrifuged, and supernatant plasma was removed

and stored at −20 °C until plasma concentration measurements.

Rat Blood Loss Model. The blood loss model was developed in

house. After an acclimatization period of at least 7 days, the rats were

anesthetized by an intraperitoneal injection of thiobutabarbital sodium

salt hydrate at a dose of 150 mg/kg (Inactin, Sigma-Aldrich GmbH).

The animals were then placed on a thermostatically controlled heating

table to maintain body temperature at 36−38 °C. After tracheotomy, a

catheter was inserted into the right jugular vein for injection of

compound. A laparotomy was performed and vehicle or compounds 1

and 30d were intravenously injected as a bolus of 1 mL/kg. Five

minutes later, the spleen was punctured with a 4 mm biopsy punch

(Stiefel) to induce a surgical wound, from which blood was collected

for 30 min. The amount of blood loss was measured and reported in

gram. At the end of the experiment, rats were euthanized by an

intravenous infusion of pentobarbital (100 mg/kg, Esconarkon, Streuli

Pharma AG, Switzerland). Clopidogrel and its vehicle were

administered by oral gavage at 5 mL/kg 2 h before the spleen was

punctured.

In Vitro Light Transmission Aggregometry (LTA) Assay.⁴⁹

Preparation of Platelet-Rich Plasma (PRP). Blood was collected from

healthy, aspirin-free volunteers, after obtaining informed consent, by

venipuncture of the large arm vein. The blood was immediately

anticoagulated with 100 μM direct thrombin inhibitor napsagatran⁵⁰

(RO 46-6240, kindly provided by Hoﬀmann La Roche Ltd.). With this

anticoagulant, the physiological calcium concentration is maintained.

10 mL aliquots of blood were centrifuged at 200g (1000 rpm) for 10

min. The top layer, representing the platelet-rich plasma (PRP), was

carefully transferred into a fresh tube. The remaining blood was again

centrifuged at 1800g (3000 rpm) for 10 min. The top layer

constituting the platelet-poor plasma (PPP) was carefully transferred

into a new tube. Finally, platelet numbers in PRP were determined

using a CASY cell counter system (Roche Diagnostics (Schweiz) AG)

and, if necessary, platelet count was adjusted to 314 × 106 platelets/

mL using PPP from the same donor.

In Vitro Platelet Aggregation Assay Using LTA. All experiments were

performed with a four-channel aggregometer (Chrono-Log Corpo-

ration, Lumi-Aggregometer 490-4D) with the AggroLink software

package. First, 400 μL of PRP at 314 × 10⁶Plts/mL were placed into a

siliconized glass cuvette (ref 70180 from Probe and Go). With

napsagatran-anticoagulated PRP, 85 μL of Tyrode’s solution (NaCl

133 mM, KCl 2.7 mM, NaHCO₃11.9 mM, NaH₂PO₄−H₂O 0.36 mM,

Hepes 10 mM, glucose 5 mM, BSA 0.1%, CaCl₂2 mM, MgCl₂1 mM,

pH 7.4) was added, mixed, and equilibrated at 37 °C for at least 15

min without stirring. Second, 5 μL of DMSO or test compound in

DMSO was added and incubated for 2 min at 37 °C with brief stirring

for 2 min only. The samples were stirred with siliconized magnetic stir

bars (ref 70188 from Probe and Go) at 1100 rpm at 37 °C. In parallel,

the aggregometer system was calibrated with PRP (0% aggregation

control = baseline) and PPP (100% aggregation control). Third,

aggregation was started by addition of 10 μL of ADP (during SAR

optimization at 3 μM ﬁnal concentration) to the cuvette. The addition

of these various volumes resulted in ﬁnal concentration of 250 × 10⁶

Plts/mL. Aggregation was monitored for up to 8 min. The results are

expressed as the maximal change in light transmission at peak response

(=maximal aggregation response) or as the change in light

transmission at 6 min after ADP addition (=ﬁnal aggregation

response).⁵¹IC₅₀values were calculated using XLﬁt software. All

compounds were tested in parallel with one reference compound. The

geometric mean IC₅₀value for this reference compound was 167 nM

with a 95% conﬁdence interval of 143−196 nM over 19 independent

IC₅₀determinations.

Pharmacology. Normotensive male Wistar rats delivered from

Harlan Netherlands were group-housed in climate-controlled con-

ditions with a 12 h light/dark cycle in accordance with the guidelines

of the Basel Cantonal Veterinary Oﬃce, with appropriate environ-

mental enrichment (shelter, polycarbonate rat tunnel, chew blocks,

aspen bricks). All animals were maintained under identical conditions

and had free access to drinking water and normal pelleted food (no.

3336, Provimi Kliba SA, Switzerland). Plavix, 75 mg, from Sanoﬁ/BMS

(clopidogrel) was purchased in a pharmacy and stored at room

temperature (max, 25 °C) protected from light. Clopidogrel was

dissolved in saline (9 g/L, containing 3 μL/mL of 3 M HCl) at a

concentration of 6 mg/mL pH = 2.5. Compounds 1 and 30d were

dissolved in PEG 400 (7.5% v/v), PG (7.5% v/v), Cremophor (5% v/

v) (Cremophor EL, Fluka no. 27963), in phosphate buﬀer pH = 7.4.

Compound 45 was dissolved in PEG 400 (7.5%), PG (7.5%),

Cremophor (5%), water.

Chemistry. All reagents and solvents were used as purchased from

commercial sources. Moisture sensitive reactions were carried out

under an argon atmosphere. Progress of the reactions was followed

either by thin-layer chromatography (TLC) analysis (Merck, 0.2 mm

silica gel 60 F₂₅₄on glass plates) or by liquid chromatography−mass

spectrometry (LC−MS). LC−MS: Thermo Finnigan MSQ Plus, with

Agilent G4220A binary pump and DAD Agilent G4212A; column,

Zorbax SB-AQ, 5 μm, 120 Å, 4.6 mm × 50 mm (Agilent); gradient, 5−

95% acetonitrile in water containing 0.04% of TFA, within 1 min, then

95% acetonitrile in water containing 0.04% of TFA for 0.5 min; ﬂow,

4.5 mL/min; 40 °C; t_Ris given in min. Purity of all target compounds

was checked by an additional LC−MS analysis on a Waters Acquity

UPLC system equipped with an ACQ-PDA detector, an ACQ-ESLD

detector, and an ACQ-SQD detector; column, ACQUITY UPLC CSH

C18 1.7 μm, 2.1 mm × 50 mm or ACQUITY UPLC HSS T3 C18 1.8

μm, 2.1 mm × 50 mm; gradient, 2−98% acetonitrile containing

0.045% formic acid in water containing 0.05% formic acid over 2 min;

ﬂow, 1.0 mL/min; 60 °C. Chiral HPLC (chiral stationary phase):

Rat FeCl₃Thrombosis Model. The FeCl₃model was performed

based on previous reports.⁵²Speciﬁcally, after an acclimatization

period of at least 7 days, the rats were anesthetized by an

intraperitoneal injection of thiobutabarbital sodium salt hydrate at a

dose of 150 mg/kg (Inactin, Sigma-Aldrich GmbH). The animals were

then placed on a thermostatically controlled heating table to maintain

body temperature at 36−38 °C. After tracheotomy, a catheter was

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hardware from UltiMate instrument series (Dionex), HPG-3200SD

binary pump, WPS-3000 autosampler, TCC-3200 thermostated

column compartment, DAD-3000 detector, SRD-3400 degasser.

Column, solvent, and retention time (t_R) are as indicated, at 25 °C,

ﬂow 1 mL/min. High resolution LC−MS (LC−HRMS): analytical

pump, Waters Acquity Binary; MS, SYNAPT G2 MS; source

temperature, 150 °C; desolvation temperature, 400 °C; desolvatation

gas ﬂow, 700 L/h; cone gas ﬂow, 10 L/h; extraction cone, 4; RF lens,

0.1 V; sampling cone, 30; capillary, 3 kV; high-resolution mode; gain,

1.0; MS function, 0.2 s per scan, 120−1000 amu in full scan, centroid

mode; lock spray, leucine enkephalin 2 ng/mL (556.2771 Da) scan

time 0.2 s with interval of 10 s and average of ﬁve scans; DAD, Acquity

UPLC PDA detector; column, Acquity UPLC CSH C18 1.7 μm, 2.1

mm × 50 mm from Waters; gradient, 2−98% acetonitrile containing

0.05% formic acid in water containing 0.05% formic acid over 2 min;

ﬂow, 0.6 mL/min; 60 °C; detection, UV 214 nm and MS, t_Ris given in

min. NMR spectroscopy: Bruker Avance II 400 MHz Ultrashield or

Bruker Ascend 500. Chemical shifts are reported in parts per million

(ppm) relative to tetramethylsilane (TMS), and multiplicities are given

as s (singlet), d (doublet), t (triplet), q (quartet), quint (quint), or m

(multiplet), br = broad, and coupling constants are given in Hz.

Compound puriﬁcation: compounds were puriﬁed by either ﬂash

column chromatography (CC) on silica gel 60 (Fluka Sigma−Aldrich,

Switzerland) or preparative LC−MS (column, solvent and retention

time (t_R) as indicated). Purity of all target compounds was assessed

using the two independent LC−MS methods described above: (1) a

Zorbax SB-AQ, 5 μm, 120 Å, 4.6 mm × 50 mm (Agilent) column,

eluting with a gradient of 5−95% acetonitrile in water containing

0.04% of TFA, within 1 min, ﬂow of 4.5 mL/min at 40 °C; (2) an

ACQUITY UPLC CSH C18 1.7 μm, 2.1 mm × 50 mm column or an

ACQUITY UPLC HSS T3 C18 1.8 μm, 2.1 mm × 50 mm column,

eluting with a gradient of 2−98% acetonitrile containing 0.045%

formic acid in water containing 0.05% formic acid over 2 min, ﬂow of

1.0 mL/min at 60 °C. In addition, target compounds were analyzed by

LC−HRMS as described above. Purity and identity of the target

compounds were further corroborated by NMR spectroscopy, and

chiral integrity was proven by HPLC using chiral stationary phases.

According to these LC−MS analyses, target compounds showed a

purity of greater than or equal to 95% (UV at 230 and at 214 nm).

Clopidogrel.⁵⁴Plavix 75 mg (6.3 g, 84 tablets) was grounded in a

mortar, and the powder was suspended in Et₂O (160 mL) and sat. aq

Na₂CO₃(160 mL). The resulting mixture was vigorously stirred for 30

min, ﬁltered oﬀ, and the phases were separated. The organic layer was

washed with sat. aq Na₂CO₃, dried (Na₂SO₄), and evaporated in

vacuo. The obtained colorless paste was dissolved in Et₂O (137 mL),

and HCl (2 M, 16 mL) was added. The resulting suspension was

stirred at rt for 5 min and ﬁltered oﬀ. The white powder was washed

with Et₂O and dried in high vacuum to aﬀord 5.1 g of clopidogrel as

HCl salt. LC−MS: t_R= 0.72 min, [M + H]⁺= 321.94. ¹H NMR (400

MHz, CDCl₃): δ 8.37 (d, J = 7.8 Hz, 1 H), 7.55−7.44 (m, 3 H), 7.30

(m, 1 H), 6.78 (br, 1 H), 5.56 (s, 1 H), 4.44 (br, 1 H), 3.54 (s, 3 H),

phases were dried over Na₂SO₄and evaporated oﬀ to give (S)-3-

methoxypyrrolidine-1-carboxylic acid tert-butyl ester (23.0 g, 93%) as a

yellow oil. H NMR (400 MHz, CDCl₃): δ 3.93 (s, 1 H), 3.50−3.35

(m, 4 H), 3.34 (s, 3 H), 2.04−1.88 (m, 2 H), 1.47 (s, 9 H).

1

(c) (S)-3-Methoxypyrrolidine-1-carboxylic acid tert-butyl ester (23.0

g) was dissolved in EA (100 mL), and 3 M HCl in EA (115 mL) was

added. The reaction mixture was stirred at rt for 20 h, and the solvent

was evaporated oﬀ. The residue was taken up in Et₂O and the

compound precipitated out. The suspension was ﬁltered oﬀ and the

powder washed with Et₂O. High vacuum drying aﬀorded title

1

compound 7 (15.2 g, 95%) as hydrochloride salt, brown solid. H

NMR (400 MHz, DMSO-d₆): δ 9.71 (br, 1 H), 9.37 (br, 1 H), 4.07 (s,

1 H), 3.23 (s, 3 H), 3.18−3.04 (m, 4 H), 3.34 (s, 3 H), 2.05−1.99 (m,

1 H), 1.93−1.84 (m, 1 H).

6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-car-

boxylic Acid (8). (a) Benzamidine hydrochloride (4) (6.00 g), ethyl

4-methoxy-3-oxobutanoate (3) (7.30 g), and NaOMe (30% in MeOH,

18.0 g) were suspended in EtOH (30.0 mL). The reaction mixture was

reﬂuxed overnight, cooled down, and water (50.0 mL) was added. The

resulting suspension was ﬁltered oﬀ and the solid was washed with

Et₂O and dried to give 6-methoxymethyl-2-phenylpyrimidin-4-ol (3.81

g). The remaining aq liquors were extracted three times, and the

combined organic layers were dried over MgSO₄, ﬁltered, and

evaporated oﬀ. The resulting crude product was puriﬁed by CC on

silica gel (using a gradient from heptane/EA 1:0 to heptane/EA 0:1)

to give additional 2.20 g. An amount of 6.01 g (65%) of 6-

methoxymethyl-2-phenylpyrimidin-4-ol was obtained as a brown solid.

1

LC−MS: t_R= 0.68 min, [M + H]⁺= 217.11. H NMR (400 MHz,

CDCl₃): δ 13.1 (br, 1 H), 8.24 (m, 2 H), 7.58−7.54 (m, 3 H), 6.60 (s,

1 H), 4.46 (s, 2 H), 3.54 (s, 3 H).

(b) POCl₃(40.0 mL) was slowly added to a stirred powder of 6-

methoxymethyl-2-phenylpyrimidin-4-ol (8.64 g). The resulting

mixture was reﬂuxed for 2 h, cooled down, and carefully added onto

crushed ice. After 30 min of stirring, the obtained suspension was

extracted twice with EA. The organic layers were washed twice with aq

NaHCO₃, dried over Na₂SO₄, and evaporated oﬀ to aﬀord 4-chloro-6-

methoxymethyl-2-phenylpyrimidine (8.81 g, 94%) as a brown oil. LC−

MS: t_R= 1.00 min, [M + H]⁺= 235.08. ¹H NMR (400 MHz, DMSO-

d₆): δ 8.30 (m, 2 H), 7.56−7.49 (m, 3 H), 7.45 (s, 1 H), 4.57 (s, 2 H),

3.42 (s, 3 H).

(c) A solution of BBr₃(2.51 mL) in DCM (20.0 mL) was syringed

into a solution of the above material (4.85 g) in DCM (100 mL) under

argon at 0 °C. After 30 min of stirring at 0 °C, the reaction was

complete. It was quenched by the addition of Et₂O (100 mL), water

(100 mL), and 1 M NaOH (100 mL). After 1 h of stirring at rt, the

mixture was extracted three times with DCM and the organic layers

were washed with water, dried over Na₂SO₄, and evaporated oﬀ. The

resulting oil crushed out and the solid was washed with heptane to give

(6-chloro-2-phenylpyrimidin-4-yl)methanol (4.58 g, 100%) as a brown

oil. LC−MS: t_R= 0.88 min, [M + H]⁺= 221.29. ¹H NMR (400 MHz,

CDCl₃): δ 8.38 (m, 2 H), 7.43 (m, 3 H), 7.20 (s, 1 H), 4.74 (d, J = 5.1

Hz, 2 H), 3.08 (t, J = 5.3 Hz, 1 H).

(d) The above alcohol (4.58 g) was dissolved in dioxane (170 mL),

and a solution of NaOH (0.829 g) in water (400 mL) was added,

followed by KMnO₄(9.82 g). The mixture was stirred at rt for 3 h.

Then 2 M HCl (100 mL) was added and the resulting mixture was

stirred for 1 h and ﬁltered oﬀ. The solution was extracted twice with

EA. The organic phases were dried over Na₂SO₄and evaporated oﬀ to

aﬀord 5 (4.04 g, 83%) as a white solid. LC−MS: t_R= 0.87 min, [M +

H]⁺= 235.28. ¹H NMR (400 MHz, DMSO-d₆): δ 8.37 (m, 2 H), 7.95

(s, 1 H), 7.55 (m, 3 H).

(e) A solution of intermediates 5 (3.20 g) and 7 (2.25 g) and

DIPEA (5.14 mL) in THF (40.0 mL) was stirred at 60 °C for 20 h.

Intermediate 7 (0.375 g) and DIPEA (0.467 mL) were added, and the

reaction mixture was further stirred at 60 °C for 72 h. Water and DCM

were added, and the phases were separated. The aq phase was

extracted three times with DCM and the combined organic layers were

dried over Na₂SO₄and evaporated oﬀ to give title compound 8 (3.78

g, 93%) as a beige solid. LC−MS: t_R= 0.74 min, [M + H]⁺= 300.42.

¹H NMR (400 MHz, CDCl₃): δ 8.39 (m, 2 H), 7.56−7.48 (m, 3 H),

24.5

3.85 (s, 3 H), 3.80 (m, 1 H), 3.19 (m, 1 H).** [α]_D+57 (c 1,

MeOH).

(S)-3-Methoxypyrrolidine Hydrochloride (7). (a) Di-tert-butyl-

dicarbonate (27.5 g) was added portionwise to a solution of (S)-3-

hydroxypyrrolidine (6) (10.0 g) and Et₃N (32.0 mL) in DCM (240

mL). The reaction mixture was stirred overnight at rt. Water was

added, and the phases were separated. The organic layer was washed

with water and saturated aq NaCl, dried over Na₂SO₄, and evaporated

oﬀ to give (S)-3-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester

(23.0 g, 100%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 4.45 (s,

1 H), 3.49−3.32 (m, 4 H), 2.17 (s, 1 H), 2.07−1.81 (m, 2 H), 1.47 (s,

9 H).

(b) To an ice-cold solution of (S)-3-hydroxypyrrolidine-1-

carboxylic acid tert-butyl ester (23.0 g) in THF (230 mL), NaH

(7.37 g, 60% dispersion in mineral oil) was added portionwise. The

reaction mixture was stirred for 30 min at rt, and MeI (11.5 mL) was

added dropwise. Stirring was continued for additional 20 h at rt. Water

was added to the reaction mixture. The layers were separated, and the

aq phase was extracted with DCM three times. The combined organic

L

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Article

7.09 (d, 1 H), 4.22−4.01 (m, 2 H), 3.86−3.66 (m, 1 H), 3.62 (br, 2

H), 3.41 (s, 3 H), 2.37−2.06 (m, 2 H).

DMSO-d₆): δ 168.2, 163.1, 162.5, 161.4, 155.1, 137.6, 131.2, 128.9,

128.4, 99.8, 79.6, 78.8, 72.4, 65.2, 59.1, 56.4, 52.0, 51.7, 49.4, 45.3,

45.1, 44.8, 44.0, 43.6, 42.1, 31.1, 30.6, 30.1, 19.1, 14.1. LC−HRMS: t_R

= 1.42 min, [M + H]/z = 569.3087, found 569.3087.

Piperazine-1-carboxylic Acid Butyl Ester (9b). (a) To a

solution of 1-benzylpiperazine (1.97 mL) and Et₃N (1.90 mL) in

DCM (100 mL), butyl chloroformate (1.47 mL) was added. The

mixture was stirred at rt for 2 h. Water was added, and the organic

layer was separated, dried (Na₂SO₄), and evaporated oﬀ to give 4-

benzylpiperazine-1-carboxylic acid butyl ester (3.13 g, 100%) as a

4-((S)-3-Carbamoyloxy-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-

2-phenylpyrimidine-4-carbonyl]amino}propionyl)piperazine-

1-carboxylic Acid Butyl Ester (28). To a solution of 22 (0.160 g) in

THF (2.00 mL) at 0 °C, trichloroacetyl isocyanate (0.089 g) was

added. After 20 min of stirring at rt, EtOH (0.200 mL) was added and

the reaction mixture was concentrated to dryness. The residue was

dissolved in EtOH (10.0 mL), cooled to 0 °C, and treated with NaBH₄

(0.123 g). The reaction mixture was stirred at rt overnight. Aqueous

Na₂CO₃was added, and the mixture was stirred for 3 h and extracted

with DCM three times. The combined organic phases were dried over

MgSO₄and concentrated to dryness. The crude product was puriﬁed

by CC on silica gel (using a gradient from heptane/EA 1:0 to heptane/

EA 0:1) and by preparative TLC (DCM/MeOH, 97:3) to give title

compound 28 (0.006 g, 5%) as a white solid. LC−MS: t_R= 1.18 min,

1

colorless oil. H NMR (400 MHz, CDCl₃): δ 7.35−7.25 (m, 5 H),

4.10 (t, J = 6.5 Hz, 2 H), 3.53 (s, 2 H), 3.49 (br, 4 H), 2.41 (br, 4 H),

1.63 (m, 2 H), 1.40 (m, 2 H), 0.95 (t, J = 7.2 Hz, 3 H).

(b) The above material (3.13 g) was hydrogenated in EtOH (100

mL) with Pd/C (wet, 5%, 0.480 g) for 24 h. The mixture was ﬁltered

through Celite and evaporated oﬀ to give title compound 9b (2.04 g,

96%) as a pale yellow liquid. ¹H NMR (400 MHz, CDCl₃): δ 5.54 (s,

1 H), 4.06 (t, J = 6.6 Hz, 2 H), 3.45 (m, 4 H), 2.83 (m, 4 H), 1.78 (s, 1

H), 1.66−1.59 (quint, J = 6.7 Hz, 2 H), 1.45−1.35 (m, 2 H), 0.95 (t, J

= 7.4 Hz, 3 H).

1

[M + H]⁺= 598.5. H NMR (400 MHz, CDCl₃): δ 9.07 (m, 1 H),

4-((S)-3-Methoxy-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phe-

nylpyrimidine-4-carbonyl]amino}propionyl)piperazine-1-car-

boxylic Acid Butyl Ester (19). (a) A solution of N-Boc-^L-serine

(2.00 g), HOBt (1.97 g), and EDC-Cl (2.24 g) in DCM (20.0 mL)

was stirred for 15 min at rt. 9b (1.81 g) was added, and the reaction

mixture was stirred overnight at rt. Precipitation occurred upon

addition of 1 M NaHSO₄. After 10 min of stirring, the suspension was

ﬁltered oﬀ. The layers were separated, and the aq phase was extracted

three times with DCM. The combined organic layers were dried over

MgSO₄and concentrated to dryness. The resulting crude product was

puriﬁed by CC on silica gel (using a gradient from heptane/EA 1:1 to

heptane/EA 0:1) to give 4-((S)-2-tert-butoxycarbonylamino-3-

hydroxypropionyl)piperazine-1-carboxylic acid butyl ester (0.500 mg,

14%) as a colorless oil. LC−MS: t_R= 0.82 min, [M + H]⁺= 374.34. ¹H

NMR (400 MHz, CDCl₃): δ 5.64 (d, J = 8.0 Hz, 1 H), 4.64 (m, 1 H),

4.13 (t, J = 6.7 Hz, 2 H), 3.87 (m, 1 H), 3.77−3.65 (m, 3 H), 3.63−

3.40 (m, 6 H), 3.26 (br, 1 H), 1.63 (m, 2 H), 1.46 (s, 9 H), 1.39

(quint, J = 7.5 Hz, 2 H), 0.96 (t, J = 7.4 Hz, 3 H).

(b) To a solution of the above compound (0.900 g) in THF (10.0

mL), NaH (0.174 g, 60% dispersion in mineral oil) was added at rt.

After stirring at rt for 15 min, MeI (0.150 mL) was added and the

stirring was continued for 4 h. Water was added, the organic layer was

separated, dried (Na₂SO₄), and evaporated oﬀ to give 4-((S)-2-tert-

butoxycarbonylamino-3-methoxypropionyl)piperazine-1-carboxylic

acid butyl ester (0.500 g, 54%) as a yellow oil. LC−MS: t_R= 0.91 min,

[M + H]⁺= 388.35.

8.49 (m, 2 H), 7.48 (m, 3 H), 7.05 (s, 1 H), 5.43 (m, 1 H), 4.80 (br, 2

H), 4.48 (m, 1 H), 4.28 (m, 1 H), 4.15 (m, 1 H), 4.12 (t, J = 6.3 Hz, 2

H), 4.02 (m, 1 H), 3.84−3.46 (m, 11 H), 3.40 (s, 3 H), 2.31−2.04 (m,

2 H), 1.64 (m, 2 H), 1.40 (m, 2 H), 0.96 (t, J = 7.3 Hz, 3 H). ¹³C

NMR (125 MHz, DMSO-d₆): δ 167.5, 163.4, 162.5, 161.3, 156.9,

155.1, 137.7, 131.2, 128.9, 128.5, 99.9, 99.8, 79.6, 78.8, 65.2, 63.2, 56.4,

52.0, 51.7, 49.7, 45.2, 44.8, 43.8, 43.5, 42.2, 31.1, 30.6, 30.1, 19.1, 14.1.

LC−HRMS: t_R= 1.29 min, [M + H]/z = 598.2989, found 598.2991.

4-((R)-3-Carboxy-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phe-

nylpyrimidine-4-carbonyl]amino}propionyl)piperazine-1-car-

boxylic Acid Ethyl Ester (29c). (a) A suspension of Cbz-^D-

Asp(O^tBu)-OH (3.00 g), HOBt (0.500 g), EDC-Cl (0.356 g), and

DIPEA (0.291 mL) in THF/DCM (6.00 mL, 5:1) was stirred for 15

min at rt. 1-(Ethoxycarbonyl)piperazine (0.244 g) was added, and the

reaction mixture was stirred for 24 h at rt. The reaction mixture was

partitioned between water and EA. The organic layer was washed with

2 M Na₂CO₃, with 1 M NaHSO₄, dried over MgSO₄, and

concentrated to dryness to give 4-((R)-2-benzyloxycarbonylamino-3-

tert-butoxycarbonylpropionyl)piperazine-1-carboxylic acid ethyl ester

1

(0.800 g, 100%) as a pale yellow oil. H NMR (400 MHz, CDCl₃): δ

7.35 (m, 5 H), 5.70 (m, 1 H), 5.11 (s, 2 H), 5.00 (m, 1 H), 4.18 (q, J =

7.1 Hz, 2 H), 3.70−3.38 (m, 8 H), 2.76 (dd, J = 7.2 and 15.8 Hz, 1 H),

2.55 (dd, J = 5.6 and 15.8 Hz, 1 H), 1.43 (s, 9 H), 1.29 (t, J = 7.1 Hz, 3

H).

(b) The above material (0.800 g) was hydrogenated in EtOH (20.0

mL) with Pd/C (wet, 5%, 0.080 g) for 24 h. The mixture was ﬁltered

through Celite and evaporated oﬀ to give 4-((R)-2-amino-3-tert-

butoxycarbonylpropionyl)piperazine-1-carboxylic acid ethyl ester

(c) A mixture of the above material (0.500 g) in DCM/TFA (6.00

mL, 5:1) was stirred at rt for 4 h and concentrated to dryness. The

residue was taken up in DCM and washed with aq Na₂CO₃. The layers

were separated, and the aq phase was extracted three times with DCM.

The combined organic layers were dried over MgSO₄and

concentrated to dryness to give 4-((S)-2-amino-3-methoxypropionyl)-

piperazine-1-carboxylic acid butyl ester (0.120 g, 32%) as a yellow oil.

1

(0.600 g, 100%) as a yellow oil. H NMR (400 MHz, CDCl₃): δ

4.18 (q, J = 7.0 Hz, 2 H), 3.68−3.46 (m, 8 H), 2.63 (dd, J = 4.9 and

16.2 Hz, 1 H), 2.49 (dd, J = 8.0 and 16.3 Hz, 1 H), 1.46 (s, 9 H), 1.29

(t, J = 7.1 Hz, 3 H).

1

LC−MS: t_R= 0.62 min, [M + H]⁺= 288.23. H NMR (400 MHz,

(c) A solution of 8 (0.363 g) and PyBOP (0.695 g) in DCM (10.0

mL) was stirred at rt for 10 min. DIPEA (0.173 mL) and the above

material (0.400 g) were added. After 24 h of stirring at rt, the reaction

mixture was diluted with DCM and 2 M Na₂CO₃. The organic layer

was washed with 1 M NaHSO₄, brine, dried over Na₂SO₄, and

concentrated to dryness. The resulting crude product was puriﬁed by

CC on silica gel (using a gradient from heptane/EA 1:0 to heptane/

EA 0:1) to give 4-((R)-3-tert-butoxycarbonyl-2-{[6-((S)-3-methox-

ypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}propionyl)-

piperazine-1-carboxylic acid ethyl ester (23c) (0.250 g, 34%) as a

CDCl₃): δ 4.13 (t, J = 6.7 Hz, 2 H), 3.95 (m, 1 H), 3.76 (br, 1 H),

3.64−3.40 (m, 8 H), 3.36 (s, 3 H), 2.86 (br, 1 H), 1.90 (br, NH₂), 1.64

(m, 2 H), 1.46 (s, 9 H), 1.41 (m, 2 H), 0.97 (t, J = 7.4 Hz, 3 H).

(d) A solution of 8 (0.120 g), HOBt (0.081 g), and EDC-Cl (0.092

g) was stirred for 15 min at rt. The above material (0.115 g) was

added, and the reaction mixture was stirred overnight at rt. Addition of

1 M NaHSO₄was followed by 20 min of stirring and ﬁltration of the

resulting suspension. The layers were separated, and the aq phase was

extracted three times with DCM. The combined organic layers were

dried over MgSO₄and concentrated to dryness. The resulting crude

product was puriﬁed by CC on silica gel (using a gradient from

heptane/EA 1:1 to heptane/EA 0:1) to give title compound 19 (0.074

g, 32%) as a pale yellow foam. LC−MS: t_R= 1.3 min, [M + H]⁺=

569.5. ¹H NMR (400 MHz, CDCl₃): δ 8.97 (d, J = 8.3 Hz, 1 H), 8.48

(s, 2 H), 7.47 (s, 3 H), 7.05 (br, 1 H), 5.33 (m, 1 H), 4.15 (m, 1 H),

4.12 (t, J = 6.7 Hz, 2 H), 4.01 (br, 1 H), 3.88−3.54 (m, 11 H), 3.47

(m, 2 H), 3.39 (s, 6 H), 2.32−2.05 (m, 2 H), 1.64 (quint, J = 6.8 Hz, 2

H), 1.40 (m, 2 H), 0.96 (t, J = 7.4 Hz, 3 H). ¹³C NMR (125 MHz,

1

colorless oil. LC−MS: t_R= 1.09 min, [M + H]⁺= 611.12. H NMR

(400 MHz, CDCl₃): δ 8.91 (d, J = 9.4 Hz, 1 H), 8.47 (m, 2 H), 7.48

(m, 3 H), 7.05 (br, 1 H), 5.47 (m, 1 H), 4.16 (m, 2 H), 4.08−3.97 (m,

1 H), 3.84−3.44 (m, 12 H), 3.41 (s, 3 H), 2.99 (dd, J = 7.8 and 16.0

Hz, 1 H), 2.71 (dd, J = 5.0 and 16.0 Hz, 1 H), 2.32−2.08 (m, 2 H),

1.46 (s, 9 H), 1.28 (t, J = 7.1 Hz, 3 H).

(d) A solution of the above compound (0.250 g) in DCM/TFA

(2.00 mL/1.20 mL) was stirred at rt for 4 h and concentrated to

dryness. The crude product was puriﬁed by preparative LC−MS

M

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Article

(Gemini C18 110A Ax, 10 μm, 21.2 mm × 50 mm, 38.8−95% MeCN

in water containing 0.2% formic acid, 45 mL/min) to aﬀord the title

compound 29c (0.090 g, 40%) as a white powder. LC−MS: t_R= 1.07

43.6, 42.3, 31.0, 30.6, 30.1, 19.1, 14.1. LC−HRMS: t_R= 0.93 min, [M

+ H]/z = 554.3091, found 554.3094.

4-((S)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimi-

dine-4-carbonyl]amino}-4-triﬂuoromethanesulfonylamino-

butyryl)piperazine-1-carboxylic Acid Butyl Ester (37). To a

suspension of 31b (0.053 g) in DCM (1.00 mL) at 0 °C, Et₃N (0.368

mL) was added, followed by triﬂuorosulfonyl chloride (0.112 mL).

The reaction mixture was stirred at rt and concentrated to dryness.

The crude product was puriﬁed by CC on silica gel (using a gradient

from DCM/MeOH 1:0 to DCM/MeOH 9:1) to give title compound

37 (0.007 g, 11%) as a white solid. LC−MS: t_R= 1.38 min, [M + H]⁺

1

min, [M + H]⁺= 555.4. H NMR (400 MHz, CDCl₃): δ 9.11 (m, 1

H), 8.44 (m, 2 H), 7.46 (m, 3 H), 7.02 (br, 1 H), 5.52 (m, 1 H), 4.15

(q, J = 7.0 Hz, 3 H), 4.01 (m, 1 H), 3.84−3.44 (m, 11 H), 3.40 (s, 3

H), 3.09 (dd, J = 7.2 and 16.5 Hz, 1 H), 2.86 (dd, J = 4.6 and 16.5 Hz,

1 H), 2.30−2.07 (m, 2 H), 1.26 (t, J = 7.1 Hz, 3 H). ¹³C NMR (125

MHz, DMSO-d₆): δ 173.6, 169.8, 163.3, 162.5, 161.3, 155.2, 155.0,

137.7, 131.2, 128.8, 128.7, 100.0, 79.6, 78.8, 61.4, 56.4, 51.9, 51.7, 46.6,

45.2, 45.1, 44.8, 43.6, 42.2, 36.8, 30.6, 30.1, 15.0. LC−HRMS: t_R= 1.19

min, [M + H]/z = 555.2567, found 555.2569.

1

= 700.5. H NMR (400 MHz, CD₃OD): δ 8.50 (m, 2 H), 7.48 (m, 3

H), 7.01 (s, 1 H), 5.22 (m, 1 H), 4.58 (br, 1 H), 4.19 (m, 1 H), 4.11

(t, J = 6.5 Hz, 2 H), 3.99 (br, 1 H), 3.80−3.45 (m, 11 H), 3.40 (s, 3

H), 3.38 (m, 2 H), 2.23 (m, 2 H), 2.16−2.00 (m, 2 H), 1.64 (m, 2 H),

1.41 (m, 2 H), 0.96 (t, J = 7.4 Hz, 3 H). ¹³C NMR (125 MHz, DMSO-

d₆): δ 169.1, 163.7, 162.5, 161.4, 155.1, 137.7, 131.2, 128.8, 128.5,

123.9, 121.4, 118.8, 99.8, 79.6, 78.8, 65.2, 56.4, 52.0, 51.7, 47.3, 45.1,

44.8, 43.9, 43.6, 42.1, 40.9, 33.0, 31.0, 30.7, 30.1, 19.1, 14.1. LC−

HRMS: t_R= 1.49 min, [M + H]/z = 700.2740, found 700.2742.

4-((S)-3-(2-Hydroxy-3,4-dioxocyclobut-1-enylamino)-2-{[6-

((S)-3-methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-

carbonyl]amino}propionyl)piperazine-1-carboxylic Acid Butyl

Ester (38a). (a) To a solution of 3,4-diethoxy-3-cyclobutene-1,2-

dione (0.022 g) in EtOH (0.500 mL), a solution of 31a (0.075 g) and

Et₃N (0.188 mL) in EtOH (0.500 mL) was added dropwise. The

reaction mixture was stirred overnight at rt and concentrated to

dryness. The crude product was puriﬁed by CC on silica gel (using a

gradient from heptane/EA 1:0 to heptane/EA 0:1) to give 4-((S)-3-(2-

ethoxy-3,4-dioxocyclobut-1-enylamino)-2-{[6-((S)-3-methoxypyrroli-

din-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}propionyl)piperazine-

1-carboxylic acid butyl ester (0.045 g, 52%) as a white foam. LC−MS:

t_R= 1.01 min, [M + H]⁺= 678.25.

(b) 4 M HCl in dioxane (0.500 mL) was added to a solution of the

above compound (0.045 g) in THF (1.00 mL). The reaction mixture

was stirred at 50 °C for 48 h and concentrated to dryness. The crude

product was puriﬁed by CC on silica gel (using a gradient from

heptane/EA 1:0 to heptane/EA 0:1) to give title compound 38a

(0.024 g, 56%) as a gray solid. LC−MS: t_R= 1.38 min, [M + H]⁺=

650.5. ¹H NMR (400 MHz, CD₃OD): δ 8.44 (m, 2 H), 7.46 (m, 3 H),

6.91 (s, 1 H), 5.36 (m, 1 H), 4.17−3.99 (m, 2 H), 4.10 (t, J = 6.4 Hz, 2

H), 3.89 (m, 2 H), 3.78 (m, 2 H), 3.68−3.45 (m, 9 H), 3.37 (s, 3 H),

2.25−2.02 (m, 2 H), 1.64 (m, 2 H), 1.41 (m, 2 H), 0.96 (t, J = 7.4 Hz,

3 H). ¹³C NMR (125 MHz, DMSO-d₆): δ 200.1, 189.1, 168.4, 163.7,

162.5, 161.3, 155.4, 155.1, 137.7, 131.1, 128.9, 128.6, 99.7, 79.6, 78.8,

65.2, 56.4, 51.8, 45.2, 44.1, 42.0, 31.1, 19.2, 14.1. LC−HRMS: t_R= 1.15

min, [M + H]/z = 650.2938, found 650.2939.

4-((S)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimi-

dine-4-carbonyl]amino}-4-oxo-4-triﬂuoromethane-

sulfonylaminobutyryl)piperazine-1-carboxylic Acid Ethyl Ester

(35). A solution of 29a (0.153 g) in SOCl₂(1.00 mL) was stirred at rt

for 1 h and concentrated to dryness. The residue was taken up in

DCM (4.00 mL), and Et₃N (0.115 mL) was added, followed by

CF₃SO₂NH₂(0.041 g). The reaction mixture was stirred overnight at

rt and poured onto aq NaHCO₃. The layers were separated, and the aq

phase was acidiﬁed to pH 1 with 2 M HCl and extracted with DCM.

The combined organic layers were dried over MgSO₄and

concentrated to dryness. The crude product was puriﬁed by

preparative LC−MS (Zorbax PrepHT SB.Aq, 5 μm, 21.2 mm × 50

mm, 31.5−100% MeCN in water containing 0.2% formic acid, 100

mL) and by preparative TLC (DCM/MeOH, 97:3) to aﬀord title

compound 35 (0.012 g, 6%) as a white foam. LC−MS: t_R= 1.61 min,

1

[M + H]⁺= 686.4. H NMR (400 MHz, CDCl₃): δ 9.17 (br, 1 H),

8.44 (m, 2 H), 7.46 (m, 3 H), 7.01 (br, 1 H), 5.52 (s, 1 H), 4.13 (m, 2

H), 3.95 (m, 1 H), 3.82−3.42 (m, 11 H), 3.39 (s, 3 H), 3.04 (m, 1 H),

2.83 (m, 1 H), 2.30−2.00 (m, 2 H), 1.25 (m, 3 H). ¹³C NMR (125

MHz, DMSO-d₆): δ 174.9, 169.8, 163.1, 162.5, 161.4, 155.3, 155.1,

137.7, 131.1, 128.8, 128.6, 122.1, 119.5, 99.8, 79.6, 78.8, 61.4, 56.4,

46.8, 45.4, 45.1, 44.8, 43.6, 42.1, 30.6, 30.1, 29.5, 29.2, 22.6, 15.0, 14.5.

LC−HRMS: t_R= 1.20 min, [M + H]/z = 686.2220, found 686.2231.

4-((S)-5-Methanesulfonylamino-2-{[6-((S)-3-methoxypyrroli-

din-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-5-

oxopentanoyl)piperazine-1-carboxylic Acid Ethyl Ester (36).

To a solution of 29b (0.325 g) in DCM (10.0 mL), DMAP (0.076 g)

and MeSO₂NH₂(0.059 g) were added, followed by DCC (0.128 g) at

0 °C. The reaction mixture was stirred at 0 °C for 1 h and at rt for 20 h

and was ﬁltered oﬀ. The ﬁltrate was concentrated to dryness and the

crude product was puriﬁed by CC on silica gel (eluting with DCM/

MeOH 10:1) and preparative TLC (DCM/acetone, 1:5) to give title

compound 36 (0.111 g, 36%) as a white solid. LC−MS: t_R= 1.1 min,

1

[M + H]⁺= 646.4. H NMR (500 MHz, DMSO-d₆): δ 11.6 (s, 1 H),

9.8 (d, J = 7.8 Hz, 1 H), 8.49 (m, 2 H), 7.54 (m, 3 H), 6.97 (s, 1 H),

5.01 (m, 1 H), 4.14 (m, 1 H), 4.07 (q, J = 7.0 Hz, 2 H), 3.88 (m, 1 H),

3.74−3.34 (m, 11 H), 3.30 (s, 3 H), 3.21 (s, 3 H), 2.18−2.06 (m, 3

H), 1.93 (m, 1 H), 1.20 (t, J = 7.1 Hz, 3 H). ¹³C NMR (125 MHz,

DMSO-d₆): δ 172.6, 169.6, 163.5, 162.5, 161.4, 155.2, 155.1, 137.7,

131.2, 128.9, 128.4, 99.8, 99.7, 79.6, 78.8, 61.5, 56.4, 52.0, 51.7, 48.7,

45.1, 44.8, 43.9, 43.5, 42.0, 41.4, 40.9, 31.6, 30.6, 30.1, 26.3, 15.0. LC−

HRMS: t_R= 1.33 min, [M + H]/z = 646.2659, found 646.2659.

4-((S)-3-Amino-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phe-

nylpyrimidine-4-carbonyl]amino}propionyl)piperazine-1-car-

boxylic Acid Butyl Ester (31a). A mixture of 4-((S)-3-tert-

butoxycarbonylamino-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phenyl-

pyrimidine-4-carbonyl]amino}propionyl)piperazine-1-carboxylic acid

butyl ester (25a) (0.305 g) in 4 M HCl in dioxane (1.50 mL) was

stirred at rt for 30 min and concentrated to dryness to give title

compound 31a (0.250 g, 99%) as hydrochloride salt, as a beige solid.

4-[(S)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimi-

dine-4-carbonyl]amino}-4-(1H-tetrazol-5-yl)butyryl]-

piperazine-1-carboxylic Acid Ethyl Ester (32b). (a) A solution of

Cbz-Gln-OH (25.0 g), HOBt (14.5 g), and EDC-Cl (20.5 g) in THF/

DCM (750 mL, 4:1) was stirred for 5 min at rt. 1-(Ethoxycarbonyl)-

piperazine (14.1 g) was added, and the reaction mixture was stirred at

rt for 24 h. Water and EA were added, and the layers were separated.

The organic phase was washed with 2 M Na₂CO₃and 1 M NaHSO₄,

was dried (Na₂SO₄) and evaporated oﬀ to give 4-((S)-2-

benzyloxycarbonylamino-4-carbamoylbutyryl)piperazine-1-carboxylic

acid ethyl ester (37.5 g, 100%) as a yellow oil. LC−MS: t_R= 0.74 min,

[M + H]⁺= 421.49.

(b) Benzenesulfonyl chloride (13.8 mL) was added to a solution of

the above compound (37.4 g) in pyridine (29.6 mL), and the resulting

reaction mixture was stirred at 50 °C for 1 h. After cooling down, 2 N

HCl was added to pH 7 and the mixture was extracted three times

with EA. The combined organic layers were washed with 1 N HCl, aq

NaHCO₃, and water, dried (Na₂SO₄), and concentrated to dryness to

give 4-((S)-2-benzyloxycarbonylamino-4-cyanobutyryl)piperazine-1-

carboxylic acid ethyl ester (30.0 g, 84%) as a yellow oil. LC−MS: t_R

= 0.85 min, [M + H]⁺= 403.48.

1

LC−MS: t_R= 0.86 min, [M + H]⁺= 554.5. H NMR (400 MHz,

CDCl₃+ drop of D₂O): δ 8.48 (m, 2 H), 7.51 (m, 3 H), 6.96 (s, 1 H),

5.20 (m, 1 H), 4.13 (m, 1 H), 3.94 (t, J = 5.8 Hz, 2 H), 3.86 (m, 1 H),

3.69−3.35 (m, 10 H), 3.30−3.15 (m, 3 H), 3.26 (s, 3 H), 2.12 (m, 2

H), 1.47 (m, 2 H), 1.25 (m, 2 H), 0.81 (m, 3 H). ¹³C NMR (125

MHz, DMSO-d₆): δ 167.3, 164.4, 162.5, 161.3, 155.1, 155.0, 137.6,

131.2, 128.7, 100.1, 79.6, 78.8, 65.2, 56.4, 52.0, 51.7, 48.0, 45.2, 44.8,

(c) The above material (10.0 g) was hydrogenated in EtOH (40.0

mL) with Pd/C (wet, 5%, 1.22 g) for 24 h. The mixture was ﬁltered

N

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through Celite and evaporated oﬀ to give 4-((S)-2-amino-4-

cyanobutyryl)piperazine-1-carboxylic acid ethyl ester (6.99 g, 100%)

as a brown oil. LC−MS: t_R= 0.51 min, [M + H]⁺= 269.39.

29.5, 28.5, 28.2, 26.0, 15.0. LC−HRMS: t_R= 1.21 min, [M + H]/z =

595.2628, found 595.2635.

4-((S)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimi-

dine-4-carbonyl]amino}-4-sulfobutyryl)piperazine-1-carbox-

ylic Acid Ethyl Ester (34). A mixture of 27 (0.404 g) and Me₄NCl

(0.402 g) in DMF (6.00 mL) was heated at 150 °C overnight and

partitioned in DCM/water. The aq phase was extracted with DCM,

and the combined organic layers were dried over Na₂SO₄and

concentrated to dryness. The crude product was puriﬁed by

preparative LC−MS (XBridge Prep C18, 10 μm, OBD 30 mm × 75

mm, 5−95% MeCN in water containing 0.5% concentrated ammonia,

75 mL/min) to aﬀord title compound 34 (0.082 g, 23%) as a beige

(d) A solution of 8 (0.200 g), HOBt (0.108 g), and EDC-Cl (0.154

g) in THF/DCM (5.00 mL, 4:1) was stirred for 5 min at rt. The above

product (0.179 g) was added, and the reaction mixture was stirred at rt

for 24 h. Water and EA were added, and the layers were separated. The

organic phase was washed with 2 M Na₂CO₃and 1 M NaHSO₄, was

dried (Na₂SO₄) and evaporated oﬀ to give 4-((S)-4-cyano-2-{[6-((S)-

3-methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-

butyryl)piperazine-1-carboxylic acid ethyl ester (26b) (0.290 g, 79%)

as a brown foam. LC−MS: t_R= 0.97 min, [M + H]⁺= 550.56.

(e) A mixture of 26b (0.250 g), ZnBr₂(0.102 g), and NaN₃(0.032

g) in water (2.00 mL) was stirred at 100 °C in a sealed tube overnight.

After cooling down, 2 N HCl was added to pH 1, followed by DCM

(100 mL), EA (50.0 mL), and water (30.0 mL). The layers were

separated, and the aq phase was extracted with DCM. The combined

organic layers were dried (Na₂SO₄) and concentrated to dryness. The

crude product was puriﬁed by CC on silica gel (using a gradient from

DCM/MeOH 1:0 to DCM/MeOH 7:3) to give title compound 32b

(0.060 g, 22%) as a beige solid. LC−MS: t_R= 1.04 min, [M + H]⁺=

1

powder. LC−MS: t_R= 1.43 min, [M + H]⁺= 605.30. H NMR (500

MHz, DMSO-d₆): δ 9.01 (m, 1 H), 8.49 (m, 2 H), 7.54 (m, 3 H), 7.20

(s, 1 H), 7.09 (s, 1 H), 6.99 (s, 1 H), 6.98 (s, 1 H), 5.14 (m, 1 H),

4.17−4.10 (m, 1 H), 4.07 (q, J = 7.0 Hz, 2 H), 3.91−3.86 (m, 1 H),

3.74−3.35 (m, 11 H), 3.30 (s, 3 H), 2.53 (m, 1 H), 2.40 (m, 1 H),

2.18−1.95 (m, 4 H), 1.20 (t, J = 7.1 Hz, 3 H). ¹³C NMR (125 MHz,

DMSO-d₆): δ 169.9, 163.2, 162.4, 161.4, 155.3, 155.1, 137.7, 131.2,

128.9, 128.4, 99.8, 99.7, 79.6, 78.8, 61.4, 56.4, 51.9, 51.7, 48.9, 47.7,

45.1, 44.8, 44.0, 43.6, 42.0, 30.6, 30.1, 29.0, 15.0. LC−HRMS: t_R= 1.19

min, [M + H]/z = 605.2393, found 605.2394.

1

593.4. H NMR (500 MHz, DMSO-d₆): δ 9.15 (d, J = 7.9 Hz, 1 H),

(R)-2-tert-Butoxycarbonylamino-3-(diethoxyphosphoryl)-

propionic Acid Methyl Ester (39b). A mixture of Boc-3-iodo-^L-Ala-

OMe (40b) (4.00 g) in P(OEt)₃(40.4 g) was stirred at 150 °C

overnight and was concentrated to dryness to give title compound 39b

(3.95 g, 96%) as crude, as a yellow oil. LC−MS: t_R= 0.85 min, [M +

H]⁺= 340.09. ¹H NMR (400 MHz, CDCl₃): δ 7.37 (m, 5 H), 5.41 (m,

1 H), 5.14 (s, 2 H), 4.53 (m, 1 H), 3.78 (s, 3 H), 3.45 (t, J = 6.9 Hz, 2

H), 2.46 (m, 1 H), 2.27 (m, 1 H).

8.49 (m, 2 H), 7.53 (m, 3 H), 6.96 (s, 1 H), 5.11 (m, 1 H), 4.13 (m, 1

H), 4.06 (q, J = 7.0 Hz, 2 H), 3.89 (m, 1 H), 3.73−3.33 (m, 11 H),

3.30 (s, 3 H), 3.02−2.89 (m, 2 H), 2.36 (m, 1 H), 2.20−2.04 (m, 3 H),

1.20 (t, J = 7.1 Hz, 3 H). ¹³C NMR (125 MHz, DMSO-d₆): δ 169.4,

163.6, 162.5, 161.4, 156.1, 155.2, 155.1, 137.7, 131.2, 128.9, 128.4,

99.8, 79.6, 78.8, 61.5, 56.4, 52.0, 51.7, 48.9, 45.1, 44.8, 44.0, 43.5, 42.0,

30.6, 30.1, 30.0, 19.8, 15.1. LC−HRMS: t_R= 1.19 min, [M + H]/z =

593.2948, found 593.2959.

(R)-2-tert-Butoxycarbonylamino-3-(diethoxyphosphoryl)-

propionic Acid (10b). A solution of LiOH (monohydrate, 0.418 g)

in water (11.8 mL) was added to a solution of 39b (3.95 g) in THF

(35.0 mL). After stirring at rt for 2 h, LiOH (monohydrate, 0.139 g)

was added and the stirring was continued for 2 h. Water and Et₂O

were added, and the layers were separated. The aq phase was acidiﬁed

with 2 M HCl to pH 1 and extracted with DCM. The combined

organic layers were dried over MgSO₄and concentrated to dryness to

give title compound 10b (2.03 g, 53%) as a yellow solid. LC−MS: t_R=

4-[(S)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimi-

dine-4-carbonyl]amino}-3-(5-oxo-4,5-dihydro[1,2,4]oxadiazol-

3-yl)propionyl]piperazine-1-carboxylic Acid Ethyl Ester (33).

(a) Hydroxylamine hydrochloride (0.233 g) and NaHCO₃(0.279 g)

were added to a solution of 4-((S)-3-cyano-2-{[6-((S)-3-methox-

ypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}propionyl)-

piperazine-1-carboxylic acid ethyl ester (26a) (1.60 g) in MeOH (5.50

mL), and the reaction mixture was stirred at reﬂux for 20 h.

Hydroxylamine hydrochloride (0.062 g) and NaHCO₃(0.076 g) were

added, the reﬂux was continued for 3 h, and the reaction mixture was

concentrated to dryness. The residue was taken up in EA/water, and

the resulting suspension was ﬁltered oﬀ. The ﬁltrate was decanted, and

the organic layer was washed with water, dried (MgSO₄), and

evaporated oﬀ. The crude product was puriﬁed by preparative LC−MS

(Zorbax PrepHT SB.Aq, 5 μm, 21.2 mm × 50 mm, 21−100% MeCN

in water containing 0.2% formic acid, 100 mL/min) to aﬀord 4-((S,Z)-

4-amino-4-(hydroxyimino)-2-(6-((S)-3-methoxypyrrolidin-1-yl)-2-

phenylpyrimidine-4-carboxamido)butanoyl)piperazine-1-carboxylic

acid ethyl ester (0.046 g, 3%) as a white solid. LC−MS: t_R= 0.47 min,

1

0.77 min, [M + H]⁺= 326.13. H NMR (400 MHz, CDCl₃): δ 8.75

(br, 1 H), 5.75 (d, J = 7.2 Hz, 1 H), 4.51 (dd, J = 6.4 and 27.8 Hz, 1

H), 4.12 (m, 4 H), 2.47 (m, 2 H), 1.32 (t, J = 7.1 Hz, 6 H).

4-((R)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimi-

dine-4-carbonyl]amino}-3-phosphonopropionyl)piperazine-1-

carboxylic Acid Butyl Ester (30d). A solution of 4-((R)-3-

(diethoxyphosphoryl)-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phenyl-

pyrimidine-4-carbonyl]amino}propionyl)piperazine-1-carboxylic acid

butyl ester (24d) (2.66 g) in MeCN (11.8 mL) was cooled down to

0 °C, and TMSBr (10.2 mL) was added dropwise. The reaction

mixture was stirred at rt overnight. Water (20.0 mL) was added, and

the reaction mixture was stirred at rt for 2 h and extracted with DCM

ﬁve times. The combined organic phases were dried over Na₂SO₄and

concentrated to dryness. The crude product was puriﬁed by CC on

reverse phase (using a gradient from water/MeCN/TFA 95:5:1 to

water/MeCN/TFA 10:90:1) to give title compound 30d (1.54 g,

63%) as a white powder. LC−MS: t_R= 1.37 min, [M + H]⁺= 619.4.

¹H NMR (400 MHz, CD₃OD): δ 8.38 (d, J = 7.1 Hz, 2 H), 7.55 (m, 3

H), 7.16 (s, 1 H), 5.39 (m, 1 H), 4.27−4.19 (m, 1 H), 4.13 (t, J = 6.5

Hz, 2 H), 3.89−3.67 (m, 7 H), 3.63−3.55 (m, 3 H), 3.53−3.47 (m, 2

H), 3.41 (s, 3 H), 2.45−2.17 (m, 4 H), 1.66 (m, 2 H), 1.43 (m, 2 H),

0.98 (t, J = 7.3 Hz, 3 H). ¹³C NMR (125 MHz, DMSO-d₆): δ 170.9,

163.3, 162.4, 161.4, 155.1, 137.7, 130.9, 128.7, 128.4, 99.5, 79.6, 78.8,

65.1, 56.4, 51.8, 51.6, 47.1, 45.4, 44.9, 44.7, 43.7, 42.1, 31.0, 30.6, 30.1,

19.1, 14.1. LC−HRMS: t_R= 1.14 min, [M + H]/z = 619.2645, found

647.2651. HPLC with chiral stationary phase (ChiralCel OZ-H 4.6

mm × 250 mm, 5 μm; 60% heptane/TFA 100:0.5, 40% EtOH/

MeOH/TFA 50:50:0.5): t_R= 14.1 min, 94% de.

1

[M + H]⁺= 569.32. H NMR (400 MHz, CDCl₃): δ 9.07 (m, 1 H),

8.47 (m, 2 H), 7.48 (m, 3 H), 7.03 (s, 1 H), 5.32 (m, 1 H), 5.17 (br, 1

H), 4.17 (q, J = 7.0 Hz, 2 H), 4.08−3.95 (m, 1 H), 3.84−3.70 (m, 2

H), 3.67−3.47 (m, 8 H), 3.41 (s, 3 H), 3.02−2.85 (m, 2 H), 2.82−2.75

(m, 1 H), 2.70−2.63 (m, 1 H), 2.30−2.05 (m, 2 H), 1.28 (t, J = 7.1

Hz, 3 H).

(b) 1,1′-Carbonyldiimidazole (0.016 g) and DBU (0.013 mL) were

added to a solution of the above material (0.045 g) in dioxane (0.100

mL). The reaction mixture was reﬂuxed for 2 h and poured onto

DCM/water. pH was adjusted to 2 with 1 N HCl, and the layers were

separated. The organic phase was dried over Na₂SO₄and concentrated

to dryness. The crude product was puriﬁed by preparative TLC

(DCM/acetone/acetic acid, 10:4:0.1) to give title compound 33

(0.026 g, 55%) as a yellow oil. LC−MS: t_R= 1.1 min, [M + H]⁺=

1

595.4. H NMR (400 MHz, DMSO-d₆): δ 12.3 (br, 1 H), 9.34 (m, 1

H), 8.52 (m, 2 H), 7.52 (m, 3 H), 6.94 (s, 1 H), 5.29 (m, 1 H), 4.13

(m, 1 H), 4.05 (m, 2 H), 3.88 (m, 1 H), 3.72 (m, 1 H), 3.65−3.40 (m,

10 H), 3.30 (s, 3 H), 3.10 (m, 1 H), 2.90 (m, 1 H), 2.19−2.05 (m, 2

H), 1.17 (t, J = 7.2 Hz, 3 H). ¹³C NMR (125 MHz, DMSO-d₆): δ

167.9, 163.6, 162.5, 161.4, 155.0, 137.7, 131.2, 128.8, 128.6, 99.9, 79.6,

78.8, 62.5, 61.5, 56.4, 52.0, 51.7, 47.1, 45.2, 44.8, 43.5, 42.3, 30.6, 30.1,

(S)-2-Benzyloxycarbonylamino-4-hydroxybutyric Acid

Methyl Ester (41d). (a) To an ice-cold solution of H-Hse-OH

(5.00 g) in dioxane (168 mL) and 2 M NaOH (42.0 mL), Cbz-Cl

O

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Journal of Medicinal Chemistry

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(8.12 g) was added portionwise. The reaction mixture was stirred at rt

overnight, and dioxane was removed in vacuo. The resulting aq

solution was extracted with Et₂O, acidiﬁed with 2 M HCl to pH 1, and

extracted with DCM. The combined organic layers were dried over

Na₂SO₄and concentrated to dryness to give (S)-2-benzyloxy-

carbonylamino-4-hydroxybutyric acid (8.90 g, 84%) as a beige solid.

separated. The aq phase was acidiﬁed with 2 M HCl to pH 3 and

extracted with Et₂O. The combined organic layers were dried over

MgSO₄and concentrated to dryness to give title compound 10g

(0.277 g, 48%) as a colorless oil. LC−MS: t_R= 0.80 min, [M + H]⁺=

388.09. ¹H NMR (400 MHz, CDCl₃): δ 7.36 (m, 5 H), 5.64−5.51 (m,

1 H), 5.12 (br, 2 H), 4.43 (m, 2 H), 4.12 (m, 4 H), 2.03 (m, 1 H),

1.90−1.66 (m, 3 H), 1.35 (t, J = 7.1 Hz, 6 H).

1

LC−MS: t_R= 0.71 min, [M + H]⁺= 254.41. H NMR (400 MHz,

4-((2S,3R)-3,4-Dihydroxy-2-{[6-((S)-3-methoxypyrrolidin-1-

yl)-2-phenylpyrimidine-4-carbonyl]amino}butyryl)piperazine-

1-carboxylic Acid Ethyl Ester (43). (a) Imidazole (1.83 g) was

added to a solution of (R)-3,4-dihydroxybutyric acid methyl ester⁵⁵

(2.40 g) in DMF (40.0 mL) at rt. After 15 min of stirring,

chlorotriisopropylsilane (3.79 mL) was added and the stirring was

pursued overnight at rt. Additional chlorotriisopropylsilane (0.758

mL) was added, and the mixture was stirred at 35 °C for 2 days and

concentrated to dryness. The crude product was puriﬁed by CC on

silica gel (eluting with heptane/EA 95:5) to give (R)-3-hydroxy-4-

triisopropylsilanyloxybutyric acid methyl ester (2.61 g, 50%) as a

CD₃OD): δ 7.34 (m, 5 H), 5.09 (s, 2 H), 4.34 (m, 1 H), 3.66 (m, 2

H), 2.09 (m, 1 H), 1.88 (m, 1 H).

(b) Dicyclohexylamine (6.37 g) was added portionwise to a solution

of the above material (8.90 g) in EtOH (77.0 mL). The mixture was

concentrated leading to a white suspension which was ﬁltered oﬀ. The

white solid was suspended in Et₂O, ﬁltered oﬀ, and dried under

vacuum to give (S)-2-benzyloxycarbonylamino-4-hydroxybutyric acid

1

as dicyclohexylamine salt (12.2 g, 80%), as a white powder. H NMR

(400 MHz, CD₃OD): δ 7.35 (m, 5 H), 5.10 (s, 2 H), 4.12 (m, 1 H),

3.65 (m, 2 H), 2.07 (m, 5 H), 1.88 (m, 5 H), 1.74 (m, 2 H), 1.45−1.18

(m, 12 H).

1

colorless oil. H NMR (400 MHz, CDCl₃): δ 4.13 (m, 1 H), 3.80−

(c) MeI (2.10 mL) was added dropwise at rt to a suspension of the

above material (12.2 g) in DMF (196 mL). The reaction mixture was

stirred overnight at rt. MeI (1.75 mL) was added, and the stirring was

continued at rt for 6 h. MeI (3.50 mL) was added, and the stirring was

continued at rt overnight. The solvent was removed in vacuo, and the

residue was taken up in water and EA. The layers were separated, the

organic phase was washed with saturated NaCl, dried over Na₂SO₄,

and concentrated to dryness. The crude product was puriﬁed by CC

on silica gel (eluting with Et₂O) to give title compound 41d (4.82 g,

64%) as a colorless resin. LC−MS: t_R= 0.79 min, [M + H]⁺= 268.30.

¹H NMR (400 MHz, CDCl₃): δ 7.38 (m, 5 H), 5.66 (m, 1 H), 5.15

3.67 (m, 2 H), 3.73 (s, 3 H), 2.89 (m, 1 H), 2.63−2.51 (m, 2 H), 1.14

(m, 3 H), 1.08 (d, 18 H).

(b) Reference 56. ZnBr₂(0.733 g) was charged into a heat gun

dried ﬂask. The solid was heated to 120 °C under vacuum for 2 h and

ﬁlled with argon. The ﬂask was cooled down to −50 °C, and THF

(3.00 mL) was added, followed by dropwise addition of MeLi (1.6 M

in Et₂O, 2.00 mL). The reaction mixture was allowed to warm up to 0

°C. A ﬂask was charged with THF (6.00 mL) and diisopropylamine

(0.963 mL) under argon. The solution was cooled to −78 °C, and

BuLi (2.5 M in hexane, 2.80 mL) was added dropwise. The resulting

solution was allowed to warm up to 0 °C over 15 min and was cooled

down to −78 °C. To a third ﬂask containing (R)-3-hydroxy-4-

triisopropylsilanyloxybutyric acid methyl ester (0.900 g) in THF (3.00

mL) under argon at 0 °C was added the MeZnBr solution. After

stirring at 0 °C for 1 h, the mixture was cooled down to −78 °C and

the −78 °C LDA solution was added. The dark blue reaction mixture

was stirred at −78 °C for 1 h, and a solution of dibenzylazocarboxylate

(1.85 g) in THF (3.00 mL) was added dropwise. The reaction mixture

was stirred for 40 min at −78 °C, was quenched with saturated aq

NH₄Cl and allowed to warm up to rt and extracted with Et₂O three

times. The combined organic layers were dried over MgSO₄, ﬁltered

oﬀ, and concentrated to dryness. The crude product was puriﬁed by

CC on silica gel (eluting with heptane/EA 1:1) to give dibenzyl 1-

((2S,3R)-3-hydroxy-1-methoxy-1-oxo-4-((triisopropylsilyl)oxy)butan-

2-yl)hydrazine-1,2-dicarboxylate (0.480 g, 26%) as a yellow oil. LC−

MS: t_R= 1.19 min, [M + H]⁺= 589.37.

(c) 2,6-Lutidine (0.178 mL) was added to a solution of the above

material (0.450 g) in DCM (1.00 mL) at 0 °C. After 15 min of stirring

at 0 °C, tert-butyldimethylsilyltriﬂuoromethanesulfonate (1.15 mL)

was added dropwise and the reaction mixture was stirred at 0 °C for 2

h. Aqueous saturated NH₄Cl was added, the aqueous phase was

extracted with EA three times, the combined organic layers were dried

over MgSO₄and concentrated to dryness. The crude product was

puriﬁed by CC on silica gel (using a gradient from heptane/EA 1:0 to

heptane/EA 9:1) to give dibenzyl 1-((2S,3R)-3-((tert-

butyldimethylsilyl)oxy)-1-methoxy-1-oxo-4-((triisopropylsilyl)oxy)-

butan-2-yl)hydrazine-1,2-dicarboxylate (0.490 g, 91%) as a colorless

oil. LC−MS: t_R= 1.33 min, [M + H]⁺= 703.64.

(m, 2 H), 4.58 (m, 1 H), 4.45 (br, 0.5 H), 4.29 (br, 0.5 H), 3.79 (s, 3

H), 3.73 (m, 1 H), 2.19 (m, 1 H), 1.74 (m, 1 H).

(S)-2-Benzyloxycarbonylamino-4-bromobutyric Acid Methyl

Ester (40d). To an ice-cold solution of 41d (2.41 g) and CBr₄(6.75

g) in DCM (120 mL), PPh₃on resin (12.4 g, 1.60 mmol/g) was

added. The ice bath was removed, the reaction mixture was stirred at rt

for 2.5 h, ﬁltered oﬀ, and concentrated to dryness. The crude product

was puriﬁed by CC on silica gel (eluting with heptane/EA 3:1) to give

title compound 40d (1.54 g, 52%) as a colorless resin. LC−MS: t_R=

1

0.98 min, [M + H]⁺= 330.33. H NMR (400 MHz, CDCl₃): δ 7.37

(m, 5 H), 5.41 (m, 1 H), 5.14 (s, 2 H), 4.53 (m, 1 H), 3.78 (s, 3 H),

3.45 (t, J = 6.9 Hz, 2 H), 2.46 (m, 1 H), 2.27 (m, 1 H).

(R)-2-Benzyloxycarbonylamino-5-hydroxypentanoic Acid

Methyl Ester (41g). To a −15 °C solution of Cbz-^D-Glu-OMe

(42g, 0.700 g) in THF (35.0 mL), NMM (0.264 g) was added

followed by dropwise addition of isobutyl chloroformate (0.341 mL).

The reaction mixture was stirred at −15 °C for 14 h. NaBH₄(0.269 g)

was added followed by dropwise addition of MeOH (23.0 mL). The

reaction mixture was stirred at 0 °C for 50 min and quenched by

adding 1 M KHSO₄. The mixture was extracted with EA three times,

the combined organic layers were washed with water, dried over

Na₂SO₄and concentrated to dryness to give title compound 41g

(0.647 g, 100%) as a colorless gel. LC−MS: t_R= 0.82 min, [M + H]⁺=

282.11. ¹H NMR (400 MHz, CDCl₃): δ 7.37 (m, 5 H), 5.46 (m, 1 H),

5.13 (m, 2 H), 4.44 (m, 1 H), 3.80 (m, 2 H), 3.76 (s, 3 H), 1.96 (m, 1

H), 1.78 (m, 1 H), 1.63 (m, 2 H).

(R)-2-Benzyloxycarbonylamino-5-iodopentanoic Acid Meth-

yl Ester (40g). PPh₃(0.905 g) and imidazole (0.251 g) were added to

a solution of 41g (0.647 g) in THF (25.0 mL). At 0 °C iodine (0.876

g) was added portionwise. The reaction mixture was stirred at rt for 3

h. Aqueous Na₂S₂O₃was added to quench the excess iodine, followed

by Et₂O and water. The layers were separated, and the organic phase

was dried over MgSO₄and concentrated in vacuo. Et₂O (20.0 mL)

was added to the residue. Precipitation occurred upon standing. The

suspension was decanted, the solution was separated and concentrated

to dryness to give title compound 40g (0.586 g, 65%) as a brown oil.

LC−MS: t_R= 1.04 min, [M + H]⁺= 391.84.

(d) To a solution of the above material (0.220 g) in EtOH (5.00

mL), Pd/C (wet, 5%, 0.033 g) was added. The ﬂask was evacuated and

backﬁlled with argon, evacuated, and backﬁlled with hydrogen. The

reaction mixture was stirred for 1 h, and Raney nickel (suspension in

water, 5.00 mL) was added. The reaction mixture was stirred at rt

under hydrogen overnight and ﬁltered oﬀ over Celite. The Celite was

washed with DCM/MeOH/Et₃N 9:1:0.1 and the ﬁltrate was

concentrated to dryness to give (2S,3R)-2-amino-3-(tert-

butyldimethylsilanyloxy)-4-triisopropylsilanyloxybutyric acid methyl

ester (0.280 g, 100%) as crude, as a colorless oil. LC−MS: t_R= 1.05

min, [M + H]⁺= 420.56.

(R)-2-Benzyloxycarbonylamino-5-(diethoxyphosphoryl)-

pentanoic Acid (10g). A mixture of 39g (0.602 g) and LiOH (0.126

g) in water/EtOH/MeOH (5.00 mL, 1:2:2) was stirred at 0 °C for 1 h

and at rt for 2 h. Water and Et₂O were added. The layers were

(e) PyBOP (0.287 g) was added to a solution of 8 (0.150 g) in

DCM (2.00 mL). The reaction mixture was stirred at rt for 15 min,

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and the above material (0.210 g) and DIPEA (0.094 mL) were added.

The resulting mixture was stirred at rt overnight and partitioned in

saturated aq NH₄Cl and EA. The aq phase was extracted with EA, and

the combined organic layers were dried over MgSO₄and concentrated

to dryness. The crude product was puriﬁed by CC on silica gel (using a

gradient from heptane/EA 1:0 to heptane/EA 4:6) to give (2S,3R)-3-

(tert-butyldimethylsilanyloxy)-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-

phenylpyrimidine-4-carbonyl]amino}-4-triisopropylsilanyloxybutyric

acid methyl ester (0.170 g, 48%) as a colorless oil. LC−MS: t_R= 1.37

min, [M + H]⁺= 701.30. ¹H NMR (400 MHz, CDCl₃): δ 9.02 (d, J =

8.7 Hz, 1 H), 8.51 (m, 2 H), 7.46 (m, 3 H), 7.11 (br, 1 H), 5.18 (dd, J

= 2.0 and 8.7 Hz, 1 H), 4.22 (m, 1 H), 4.16−3.98 (m, 2 H), 3.93 (m, 1

H), 3.80 (s, 3 H), 3.79 (m, 2 H), 3.58 (m, 2 H), 3.38 (s, 3 H), 2.29−

2.03 (m, 2 H), 1.15 (m, 3 H), 1.13 (s, 12 H), 1.12 (s, 6 H), 0.90 (s, 9

H), 0.09 (s, 3 H), 0.07 (s, 3 H).

on silica gel (eluting with heptane/EA 5:1) to give 3-benzyloxyisox-

azole-5-carboxylic acid methyl ester (1.60 g, 100%) as a beige oil. LC−

MS: t_R= 0.97 min, [M + H]⁺= 234.24.

(b) NaBH₄(0.353 g) was added to a solution of the above material

(1.60 g) in MeOH (49.0 mL) at 0 °C, and the reaction mixture was

stirred at 0 °C for 4 h. MeOH (100 mL) and 1 M HCl (200 mL) were

added, and the resulting mixture was extracted three times with DCM.

The combined organic layers were dried over MgSO₄and

concentrated to dryness. The crude product was puriﬁed by CC on

silica gel (eluting with heptane/EA 3:1) to give (3-benzyloxyisoxazol-

5-yl)methanol (1.23 g, 87%) as a yellow oil. LC−MS: t_R= 0.81 min,

[M + H]⁺= 206.22.

(c) Reference 58. A solution of the above material (1.23 g) and

pyridine (0.989 mL) in DCM (22.5 mL) was added dropwise to a 0

°C suspension of triphenylphosphine dibromide (4.10 g) in DCM

(30.0 mL). The reaction mixture was stirred at 0 °C for 1 h and

washed twice with 10% aq sodium bisulﬁte. The aq layers were

extracted with Et₂O. The combined organic layers were dried over

MgSO₄and concentrated to dryness. The resulting solid was triturated

with Et₂O, the suspension was ﬁltered oﬀ, and the solution was

concentrated to dryness. The crude product was puriﬁed by CC on

silica gel (eluting with heptane/EA 2:1) to give 3-benzyloxy-5-

(f) 2 M LiOH (0.350 mL) was added to a solution of the above

material (0.170 g) in THF (5.00 mL) and water (0.700 mL). The

reaction mixture was stirred overnight at rt and then quenched with aq

NH₄Cl followed by addition of 2 M HCl until pH 3. The mixture was

extracted with EA three times, and the combined organic layers were

dried over MgSO₄and concentrated to dryness to give (2S,3R)-3-(tert-

butyldimethylsilanyloxy)-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phe-

nylpyrimidine-4-carbonyl]amino}-4-triisopropylsilanyloxybutyric acid

(0.160 g, 96%) as a white foam. LC−MS: t_R= 1.30 min, [M + H]⁺

bromomethylisoxazole (1.54 g, 96%) as a yellow oil. LC−MS: t_R

=

0.99 min, [M + H]⁺= 268.03.

1

(d) Reference 59. BuLi (1.6 M in hexane, 2.53 mL) was added to a

solution of (R)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (1.06

mL) in THF (53.0 mL) cooled at −78 °C, and the reaction mixture

was stirred at −78 °C for 30 min. A solution of 3-benzyloxy-5-

bromomethylisoxazole (1.54 g) in THF (42.0 mL) was added, and the

reaction mixture was stirred at −78 °C for 4 h. A solution of (R)-2,5-

dihydro-3,6-dimethoxy-2-isopropylpyrazine (1.06 mL) and BuLi (1.6

M in hexane, 2.53 mL) in THF (53.0 mL) prepared as before was

added three more times at 1 h intervals to the reaction mixture stirred

at −78 °C. The reaction mixture was allowed to warm to 0 °C, aq

NH₄Cl was added, and the mixture was concentrated in vacuo. The

residue was extracted with Et₂O three times. The combined organic

layers were dried over MgSO₄and concentrated to dryness. The crude

product was puriﬁed by CC on silica gel (eluting with heptane/EA

9:1) to give (2S,5R)-2-(3-benzyloxyisoxazol-5-ylmethyl)-5-isopropyl-

3,6-dimethoxy-2,5-dihydropyrazine (2.14 g, 100%) as a yellow oil.

= 687.63. H NMR (400 MHz, CDCl₃): δ 9.24 (m, 1 H), 8.51 (m, 2

H), 7.47 (m, 3 H), 7.07 (br, 1 H), 5.04 (m, 1 H), 4.32 (m, 1 H), 4.19−

4.00 (m, 2 H), 3.97 (dd, J = 4.5 and 10.3 Hz, 1 H), 3.87−3.55 (m, 4

H), 3.40 (s, 3 H), 2.30−2.08 (m, 2 H), 1.23 (m, 3 H), 1.15 (s, 12 H),

1.14 (s, 6 H), 0.89 (s, 9 H), 0.05 (s, 3 H), 0.04 (s, 3 H).

(g) A solution of the above material (0.160 g), EDC-Cl (0.536 g),

and HOBt (0.472 g) in DCM (2.00 mL) was stirred at rt for 15 min,

and 9a (0.368 g) was added. The reaction mixture was stirred at rt

overnight, and aq NH₄Cl was added. The resulting mixture was

extracted three times with EA. The combined organic layers were dried

over MgSO₄and concentrated to dryness. The crude product was

puriﬁed by CC on silica gel (using a gradient from heptane/EA 1:0 to

heptane/EA 1:1) to give 4-((2S,3R)-3-(tert-butyldimethylsilanyloxy)-

2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]-

amino}-4-triisopropylsilanyloxybutyryl)piperazine-1-carboxylic acid

ethyl ester (0.130 g, 67%) as a white foam. LC−MS: t_R= 1.35 min,

1

LC−MS: t_R= 1.06 min, [M + H]⁺= 372.24. H NMR (400 MHz,

1

[M + H]⁺= 827.80. H NMR (400 MHz, CDCl₃): δ 8.77 (d, J = 9.5

CDCl₃): δ 7.45−7.32 (m, 6 H), 5.66 (s, 1 H), 5.23 (s, 2 H), 4.30 (m, 1

H), 4.03−3.98 (m, 2 H), 3.88 (t, J = 3.5 Hz, 1 H), 3.72 (s, 5 H), 3.69

(s, 2 H), 3.66 (s, 3 H), 3.22 (dd, J = 4.3 and 15 Hz, 1 H), 3.10 (dd, J =

6.3 and 15 Hz, 1 H), 2.24 (m, 2 H), 3.88 (t, J = 6.4 Hz, 6 H).

(e) A mixture of the above compound (2.14 g) and 1 M HCl (11.5

mL) in MeCN (57.5 mL) was stirred at rt for 2 h and evaporated in

vacuo. Concentrated ammonia was added to the residue until pH 9,

and the mixture was extracted with DCM. The organic phase was dried

(MgSO₄) and concentrated to dryness to give (S)-2-amino-3-(3-

benzyloxyisoxazol-5-yl)propionic acid methyl ester (1.85 g, 100%) as a

beige solid. LC−MS: t_R= 0.69 min, [M + H]⁺= 277.28.

Hz, 1 H), 8.47 (m, 2 H), 7.48 (m, 3 H), 7.04 (br, 1 H), 5.40 (t, J = 8.8

Hz, 1 H), 4.19−4.00 (m, 4 H), 4.17 (q, J = 7.0 Hz, 2 H), 3.85−3.71

(m, 2 H), 3.83 (d, J = 3.8 Hz, 2 H), 3.63−3.52 (m, 3 H), 3.41 (s, 3 H),

3.37−3.23 (m, 3 H), 2.30−2.09 (m, 2 H), 1.29 (m, 6 H), 1.02 (br, 18

H), 0.90 (s, 9 H), 0.13 (s, 3 H), 0.08 (s, 3 H).

(h) TBAF (1 M in THF, 0.363 mL) was added to a solution of the

above material (0.120 g) in THF (3.00 mL). The reaction mixture was

stirred at rt for 4 h and concentrated to dryness. The crude product

was puriﬁed by CC on silica gel (using a gradient from DCM/MeOH

1:0 to DCM/MeOH 9:1) to give title compound 43 (0.061 g, 76%) as

1

a beige foam. LC−MS: t_R= 1.39 min, [M + H]⁺= 557.5. H NMR

(f) A solution of 8 (0.861 g), HOBt (0.497 g), and EDC-Cl (0.619

g) in DCM (16.0 mL) was stirred for 5 min at rt. The above product

(0.794 g) was added, and the reaction mixture was stirred at rt

overnight. Aqueous NaHSO₄was added, and the layers were

separated. The organic phase was washed with aq NH₄Cl, dried

(Na₂SO₄), and evaporated oﬀ. The crude product was puriﬁed by CC

on silica gel (eluting with heptane/EA 1:1) to give (S)-3-(3-

benzyloxyisoxazol-5-yl)-2-{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phe-

nylpyrimidine-4-carbonyl]amino}propionic acid methyl ester (0.447 g,

28%) as a beige powder. LC−MS: t_R= 1.10 min, [M + H]⁺= 558.20.

¹H NMR (400 MHz, CDCl₃): δ 8.89 (s, 1 H), 8.46 (s, 2 H), 7.49−

7.34 (m, 8 H), 7.05 (s, 1 H), 5.84 (s, 1 H), 5.25 (s, 2 H), 5.09 (m, 1

H), 4.20−3.94 (m, 2 H), 3.84 (br, 3 H), 3.78−3.53 (m, 3 H), 3.49−

3.36 (m, 2 H), 3.41 (br, 3H), 2.33−2.07 (m, 2 H).

(400 MHz, DMSO-d₆): δ 9.27 (d, J = 7.7 Hz, 1 H), 8.47 (m, 2 H),

7.53 (dd, J = 1.8 and 5.2 Hz, 3 H), 6.96 (s, 1 H), 5.29 (d, J = 5.6 Hz, 1

H), 5.01 (t, J = 7.6 Hz, 1 H), 4.96 (t, J = 4.9 Hz, 1 H), 4.13 (d, J = 23.6

Hz, 1 H), 4.07 (q, J = 7.1 Hz, 2 H), 3.88 (m, 1 H), 3.80 (m, 1 H),

3.72−3.41 (m, 11 H), 3.34 (m, 2 H), 3.30 (s, 3 H), 2.16−2.01 (m, 2

H), 1.21 (t, J = 7.1 Hz, 3 H). ¹³C NMR (125 MHz, DMSO-d₆): δ

169.4, 163.3, 162.4, 161.4, 155.1, 137.6, 131.2, 128.9, 128.4, 99.7, 79.6,

78.8, 72.3, 63.9, 61.4, 56.4, 52.1, 52.0, 51.7, 45.7, 45.1, 44.8, 43.9, 43.6,

42.1, 30.6, 30.1, 15.1. LC−HRMS: t_R= 1.14 min, [M + H]/z =

557.2723, found 557.2728.

4-((S)-3-(3-Hydroxyisoxazol-5-yl)-2-{[6-((S)-3-methoxypyrro-

lidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}propionyl)-

piperazine-1-carboxylic Acid Ethyl Ester (44). (a) Reference 57.

A suspension of methyl 3-hydroxyisoxazole-5-carboxylate (1.00 g) and

K₂CO₃(1.97 g) in acetone (20.0 mL) was heated at 70 °C for 1 h.

Benzyl bromide (1.27 mL) was added, and the reaction mixture was

stirred at 70 °C for 3 h and at rt for 2 h. The mixture was ﬁltered oﬀ

and concentrated to dryness. The crude product was puriﬁed by CC

(g) A solution of the above compound (0.447 g) and LiOH·H₂O

(0.168 g) in THF/H₂O (10.0 mL, 3:1) was stirred at rt for 1 h. The

solvent was removed in vacuo, and 1 M HCl and EA were added to the

residue. The layers were separated, and the aq phase was further

Q

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Journal of Medicinal Chemistry

Article

extracted with EA. The combined organic layers were dried (Na₂SO₄)

and evaporated oﬀ to give (S)-3-(3-benzyloxyisoxazol-5-yl)-2-{[6-((S)-

3-methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-

propionic acid (0.430 g, 98%) as a beige foam which was used without

further puriﬁcation. LC−MS: t_R= 1.03 min, [M + H]⁺= 544.21.

(h) A solution of the above material (0.430 g), HOBt (0.139 g),

EDC-Cl (0.164 g), and DIPEA (0.282 mL) in DCM (9.00 mL) was

stirred for 15 min at rt. 9a (0.129 g) was added, and the reaction

mixture was stirred at rt overnight. Aqueous NaHSO₄was added, and

the layers were separated. The aq phase was extracted with EA, and the

combined organic layers were dried (Na₂SO₄) and evaporated oﬀ. The

crude product was puriﬁed by CC on silica gel (eluting with DCM/

MeOH 9:1) to give 4-((S)-3-(3-benzyloxyisoxazol-5-yl)-2-{[6-((S)-3-

methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-

propionyl)piperazine-1-carboxylic acid ethyl ester (0.542 g, 99%) as a

2 H), 3.89 (m, 1 H), 3.80−3.37 (m, 11 H), 3.30 (s, 3 H), 2.66−2.54

(m, 1 H), 2.21−2.04 (m, 2 H), 1.54 (m, 2 H), 2.33 (m, 2 H), 1.23 (m,

12 H), 1.04 (d, J = 6.1 Hz, 3 H). ¹³C NMR (125 MHz, DMSO-d₆): δ

168.4, 163.4, 162.6, 161.4, 155.1, 153.1, 137.7, 131.2, 128.8, 128.6,

99.9, 84.6, 79.6, 78.8, 73.3, 65.2, 62.5, 56.4, 51.9, 51.7, 47.9, 45.3, 45.1,

44.8, 44.6, 43.7, 43.3, 42.3, 35.6, 31.0, 30.6, 30.1, 29.5, 28.3, 26.0, 22.3,

21.8, 19.1, 14.1. LC−HRMS: t_R= 1.29 min, [M + H]/z = 851.3514,

found 851.3585.

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b00933.

1

beige powder. LC−MS: t_R= 1.09 min, [M + H]⁺= 684.28. H NMR

Molecular formula strings (CSV)

(400 MHz, CDCl₃): δ 8.90 (m, 1 H), 8.45 (br, 2 H), 7.50−7.34 (m, 8

H), 7.04 (s, 1 H), 5.84 (s, 1 H), 5.48 (m, 1 H), 5.25 (s, 2 H), 4.15 (m,

2 H), 4.03 (m, 1 H), 3.86−3.43 (m, 12 H), 3.41 (s, 3 H), 3.34 (m, 1

H), 3.20 (m, 1 H), 2.32−2.08 (m, 2 H), 1.28 (m, 3 H).

Characterization of all target compounds not described

in the main text, synthetic schemes for analogs 43 and

44, and SDs and ranges for the data in Table 4 (PDF)

(i) Pd on barium sulfate (5%, 0.098 g) was added to a solution of

the above material (0.393 g) in MeOH (13.0 mL). The ﬂask was

evacuated and backﬁlled with argon, evacuated, and backﬁlled with

hydrogen. The reaction mixture was stirred for 3.5 h, and additional Pd

on barium sulfate (5%, 0.098 g) was added. The reaction mixture was

stirred at rt under hydrogen overnight and ﬁltered oﬀ over Celite. The

Celite was washed with MeOH, and the ﬁltrate was concentrated to

dryness. The crude was puriﬁed by preparative LC−MS (Gemini C18

110A Ax, 10 μm, 21.2 mm × 50 mm, 27.7−95% MeCN in water

containing 0.2% formic acid, 45 mL/min) to give title compound 44

(0.070 g, 21%) as a white powder. LC−MS: t_R= 1.10 min, [M + H]⁺=

594.4. ¹H NMR (500 MHz, CDCl₃): δ 8.91 (d, J = 8.6 Hz, 1 H), 8.45

(m, 2 H), 7.48 (m, 3 H), 7.25 (m, 0.5 H), 7.19 (m, 0.5 H), 7.05 (s, 1

H), 5.83 (s, 1 H), 5.49 (m, 1 H), 4.19−4.10 (m, 3 H), 4.02 (m, 1 H),

3.85−3.70 (m, 2 H), 3.68−3.43 (m, 9 H), 3.40 (s, 3 H), 3.35−3.27

(m, 1 H), 3.22−3.14 (m, 1 H), 2.31−2.10 (m, 2 H), 1.27 (m, 3 H).

¹³C NMR (125 MHz, DMSO-d₆): δ 170.7, 169.8, 168.4, 163.4, 162.5,

161.3, 155.0, 137.7, 131.2, 128.8, 128.5, 99.8, 95.0, 79.6, 78.8, 61.5,

56.4, 52.0, 51.7, 48.0, 45.2, 44.8, 43.6, 42.2, 30.6, 29.9, 15.0. LC−

HRMS: t_R= 1.21 min, [M + H]/z = 594.2676, found 594.2671.

4-((R)-3-(Bis-isopropoxycarbonyloxymethoxyphosphoryl)-2-

{[6-((S)-3-methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-

carbonyl]amino}propionyl)piperazine-1-carboxylic Acid Butyl

Ester (45). (a) Reference 60. Chloromethyl chloroformate (2.07 mL)

and isopropyl alcohol (1.75 mL) were dissolved in Et₂O (34.0 mL).

The solution was cooled down to 0 °C, and pyridine was added

dropwise. The ice bath was removed, and the reaction mixture was

stirred at rt overnight. The suspension was ﬁltered oﬀ, and the ﬁltrate

was washed with 10% aq citric acid, water, sat. aq NaHCO₃, and brine.

The organic phase was dried (Na₂SO₄) and concentrated in vacuo to

give carbonic acid chloromethyl ester isopropyl ester (3.03 g, 87%) as

a colorless liquid. ¹H NMR (400 MHz, CDCl₃): δ 5.74 (s, 2 H), 4.98

(m, 1 H), 1.35 (d, J = 6.3 Hz, 6 H).

(b) Reference 61. Et₃N (0.101 mL) was added to a solution of 30d

(0.150 g) in anhydrous DMPU (0.44 mL). The mixture was stirred at

rt for 10 min, and carbonic acid chloromethyl ester isopropyl ester

(0.416 mL) followed by NaI (0.0441 g) was added. The reaction

mixture was stirred at 45 °C overnight. TEA (0.101 mL) and carbonic

acid chloromethyl ester isopropyl ester (0.416 mL) were added, and

the mixture was stirred for 3.5 h at 45 °C and partitioned between

toluene and water/brine. The layers were separated, the aq phase was

extracted with toluene, and the combined organic layers were dried

(Na₂SO₄), ﬁltered oﬀ, and concentrated to dryness. The resulting

crude product was puriﬁed by CC on silica gel (using a gradient from

heptane/EA 1:0 to heptane/EA 0:1) to give a yellow oil contaminated

with residual DMPU. The oil was lyophilized twice to give title

compound 45 (0.094 g, 45%) as a white powder. LC−MS: t_R= 1.38

min, [M + H]⁺= 851.1. ¹H NMR (500 MHz, DMSO-d₆): δ 9.27 (m, 1

H), 8.54 (m, 2 H), 7.52 (m, 3 H), 6.96 (s, 1 H), 5.63−5.52 (m, 4 H),

5.24 (m, 1 H), 4.79 (m, 2 H), 4.19−4.09 (m, 1 H), 4.00 (t, J = 6.5 Hz,

AUTHOR INFORMATION

Corresponding Author

*Phone: +41 61 565 62 31. Fax: +41 61 565 65 00. E-mail: eva.

caro ﬀ @actelion.com.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

The authors thank Raimund Brauner, Simon Buetikofer, Rem

Castro, Dominik Fosshag, Yannick Gallin, Marie Idoux, Petar

́

Maric, Valerie Siefken, and Gina Wintenberger for the synthetic

chemistry work, Martine Baumann, Brigitte Butscha, Isabelle

Guillaumat, and Benoit Lack for biological testing, Markus

Kramberg for technical assistance for in vivo experiments,

Fabienne Drouet, Rolf Wuest, Isabelle Weber, Aude Weigel,

and Nathalie Jaouen for generating DMPK data, Eric Ertel for

providing hERG data, Bruno Capeleto for early formulation,

and Henri Ramuz for stimulating discussions.

■

́

y

ABBREVIATIONS USED

■

CDI, 1,1′-carbonyldiimidazole; DIPEA, diisopropylethylamine;

DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

EA, ethyl acetate; EDC-Cl, N-(3-dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride; HATU, 1-[bis-

(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-

pyridinium 3-oxide hexaﬂuorophosphate; HOBt, 1-hydroxy-

benzotriazole hydrate; NMM, N-methylmorpholine; PS,

polymer-supported; PyBOP, (benzotriazol-1-yloxy)-

tripyrrolidinophosphonium hexaﬂuorophosphate; [³³P]-2Me-

SADP, ³³P-labeled 2-methylthioadenosine 5′-diphosphate

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Article Doi

4-((R)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-3-phosphonopropionyl)piperazine-1-carboxylic Acid Butyl Ester (ACT-246475) and Its Prodrug (ACT-281959), a Novel P2Y12 Receptor Antagonist with a Wider Therapeutic Window in the Rat Than Clopidogrel

DOI: 10.1021/acs.jmedchem.5b00933

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