Piperazinyl glutamate pyridines as potent orally bioavailable P2Y 12 antagonists for inhibition of platelet aggregation

Article abstract of DOI:10.1021/jm901518t

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl glutamate pyridine as a P2Y₁₂ antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized through modifications at the 4-position of the pyridine ring and the terminal nitrogen of the piperazine ring, leading to compound (4S)-4-[({4-[4-(methoxymethyl) piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy) carbonyl]piperazin-1-yl}pentanoic acid 47s with good human PRP potency, selectivity, in vivo efficacy, and oral bioavailability. Compound 47s was selected for further preclinical evaluations.

Full text of DOI:10.1021/jm901518t

2010 J. Med. Chem. 2010, 53, 2010–2037
DOI: 10.1021/jm901518t
Piperazinyl Glutamate Pyridines as Potent Orally Bioavailable P2Y12 Antagonists for Inhibition of
Platelet Aggregation
John J. Parlow,*,† Mary W. Burney,‡ Brenda L. Case,† Thomas J. Girard,‡ Kerri A. Hall,‡ Peter K. Harris,‡
Ronald R. Hiebsch,‡ Rita M. Huff,‡ Rhonda M. Lachance,‡ Deborah A. Mischke,† Stephen R. Rapp,‡
Rhonda S. Woerndle,† and Michael D. Ennis†
†Department of Medicinal Chemistry and ‡Department of Biology, Pfizer Global Research & Development, 700 Chesterfield Parkway West,
Chesterfield, Missouri 63017
Received October 12, 2009
Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl glutamate
pyridine as a P2Y12 antagonist. Exploitation of this lead provided compounds with excellent inhibition of
platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and
physiochemical properties were optimized through modifications at the 4-position of the pyridine ring and
the terminal nitrogen of the piperazine ring, leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-
yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 47s
with good human PRP potency, selectivity, in vivo efficacy, and oral bioavailability. Compound 47s was
selected for further preclinical evaluations.
Introduction
days to achieve its full effect.4 Once it is activated, the drug
becomes irreversibly bound to platelets. As a result, clopido-
grel has a slow onset and slow offset of pharmacological
action. This makes it less effective in acute settings and
difficult to manage if a patient bleeds, experiences a trauma,
or requires emergency surgery. In addition, there are subsets
of individuals either who do not metabolize the prodrug
adequately or who are resistant to the effects of clopidogrel.5
It is anticipated that a direct acting, orally bioavailable P2Y12
inhibitor will not be associated with such difficulties and will
therefore exhibit a significant improvement in efficacy while
maintaining a better safety profile. Accordingly, a need still
exists for new drug therapies for the treatment of subjects
suffering from or susceptible to a platelet aggregation
mediated condition. In particular, a need still exists for new
P2Y12 antagonists having one or more improved properties,
such as safety profile, efficacy, or physical properties, relative
to currently available P2Y12 antagonists. Several groups have
described their research efforts aimed toward discovering
ADP receptor antagonists, including some recent patents
issued by Actelion.6,7 Moreover, several new P2Y12 antago-
nists, including cangrelor (AR-C69931MX) and ticagrelor
(AZD-6140, 84),8 are currently in late stage clinical trials.6
Herein, we report our efforts to discover and develop potent,
selective P2Y12 antagonists to address the unmet medical need
for safe and effective oral antiplatelet agents.9
Cardiovascular and cerebrovascular diseases are still the
most common causes of mortality in the Western world.
Thrombotic complications of atherosclerosis such as acute
coronary syndrome (ACSa), and ischemic stroke are one of
the leading causes of this morbidity and mortality. Current
therapy includes antiplatelet agents such as aspirin, dipyrida-
mole, glycoproteinIIb/IIIa antagonists, and thienopyridines.1
Clopidogrel, a thienopyridine, is an oral prescription antipla-
telet drug approved for the reduction of atherosclerotic events
(stroke, myocardial infarction, and death) in patients with
ACS that acts by blocking adenosine diphosphate (ADP)-
stimulated platelet aggregation. ADP is an important platelet
agonist that induces a primary aggregation response and
contributes to secondary aggregation following release from
platelet dense granules upon activation by other agonists.
ADP-induced platelet aggregation is mediated by a dual
receptor system involving activation of P2Y1 and P2Y12
receptors, both members of the G-protein-coupled receptor
(GPCR) family.2 Experimental studies have demonstrated
that selective blockade of either receptor is sufficient to inhibit
platelet activation. However, P2Y1 has ubiquitous expression
whereasP2Y12 isprimarilyaplatelet specific receptor and thus
represents a more attractive therapeutic target for selective
modulation of ADP-induced platelet activation.
P2Y12 has been identified as the molecular target of clopi-
dogrel.3 Clopidogrel is a prodrug, the active metabolite of
which irreversibly and selectively inhibits the P2Y12 receptor;
thus, antiplatelet efficacy requires a loading dose and several
Results and Discussion
Quinoline derivatives 1 have been reported as antagonists
of the platelet P2Y12 receptor.10 The general structure is
shown in Figure 1, in which the lead compound from this
class is a prodrug where the R4 side chain possesses an ethyl
ester. Metabolism by systemic esterases of the ester prodrug to
its corresponding acid provides the active metabolite, which
*To whom correspondence should be addressed. Phone: 636-247-
3494. Fax: 636-247-5400. E-mail: john.j.parlow@pfizer.com.
a Abbreviations: PASP, polymer-assisted solution-phase; PRP, plate-
let rich plasma; ACS, acute coronary syndrome; ADP, adenosine dipho-
sphate; GPCR, G-protein-coupled receptor; b.i.d., twice daily.
r
pubs.acs.org/jmc
Published on Web 02/08/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2011
contains two carboxylic acid groups (R2 and R4 side chains).
It would be desirable to eliminate the need for a prodrug and
improve upon the physical chemical properties (diacid) as well
as potency. Interestingly, only quinolines and naphthalenes
were reported as the right-hand aryl moieties. We approached
analogue synthesis by considering the molecule as being
composed of three parts: the piperazine, the amino acid, and
the quinoline. Parallel syntheses allowing for variations of all
three parts of the molecule were then developed.
determined on each 96-well plate and the data for the test
compounds were normalized to that standard from that same
plate. Emphasis, in regard to the SAR, was placed on the PRP
potency as this was an indication of the functional activity
taking into account the effect of protein binding.
Many different heterocyclic ring systems were evaluated,
and an apparent trend was observed. Compounds with a
heteroatom, preferablya nitrogen, ortho tothe amideposition
and a phenyl ring ortho to the heteroatom exhibited P2Y12
binding activity. A second iteration with the heteroaryl acids
possessing this typeof substitutionpattern ledto the discovery
of compound 10a possessing a 4,6-diphenylpyridine ring
system with a P2Y12 binding Ki of 15 nM and a PRP IC50 of
76 μM (Table 1). In an effort to follow-up on the lead
compound 10a, a focused library was prepared that explored
elementary structural changes around the pyridine ring sys-
tem. A representative set of compounds from this library is
shown in Table 1. The unsusbstituted pyridine ring, com-
pound 10b, was devoid of any type of activity. Replacing the
6-phenyl ring with a hydrogen, compound10c, also resulted in
a loss of activity. However, when replacing the 4-phenyl ring
with a hydrogen, compound 10d, PRP activity was main-
tained. Removing the nitrogen from the pyridine ring system,
regardless of the phenyl substitution, resulted in loss of
binding activity with no PRP activity (10e,f). An additional
nitrogen at the 3-position with a 6-phenyl substitution, com-
pound 10i, maintained PRP activity, whereas the additional
nitrogen at the 5-position, compound 10h, resulted in no PRP
activity.
The SAR trends across the different amine templates were
consistent. The glycine analogues (9a,b, R2 = H) had reduced
binding activity and no PRP activity, indicating the S-gluta-
mic acid piece (R2 = (CH2)2CO2H) was required for PRP
activity. Replacing the ethyl carbamate group on the piper-
azine nitrogen of the lead (10a) with the m-tolyl (11a) resulted
in decreased binding activity and no PRP potency. While the
m-tolyl substitution provided compounds with good binding
activity (data not shown), no PRP activity was observed with
any of these analogues presumably because of high protein
binding.12 For example, when compound 11a was run in the
binding assay with 0.4% human serum albumin, no binding
activity was observed (Ki > 4.74 μM).
Initial efforts were focused at replacing the quinoline ring
with other heteroaryl and aryl ring systems. It was envisioned
that a PASP synthesis approach for the arylcarboxamides
could be accomplished through a simple amide bond-forming
reaction (Scheme 1). Thus, reacting arylcarboxylic acids with
the amine would afford analogues with alternative aryl ring
systems. Three amine templates 2-4 were selected for initial
library synthesis in which an ethyl carbamate and a m-tolyl
ring system were substituted on the terminal piperazine
nitrogen (R1) with either a glycine or S-glutamic acid as the
amino acid (R2). The aryl acid inputs 5 were initially biased
toward fused ring systems mimicking the quinoline and
naphthalene. The amines 2-4 were coupled to the aryl acids
5 using polymer-bound carbodiimide 6 as the coupling agent
with hydroxybenzotriazole to afford the crude amide pro-
ducts. Upon completion of the reaction, a mixed resin bed
containing polymer-bound amine 8 and polymer-bound iso-
cyanate 7 was added to the reaction mixture to sequester
hydroxybenzotriazole and any unreacted acid 5 or amine
starting materials 2-4 followed by filtration and evaporation
to afford purified products. Deprotection of the tert-butyl
ester using TFA in DCM provided the desired products 9-11.
Several hundred compounds were prepared by this synth-
esis and screened in a human P2Y12 receptor binding assay at
5-10 μM, and Ki’s were determined for those compounds
with g50% inhibition.11 These compounds were also tested as
antiplatelet agents by measuring their inhibitory action on the
in vitro aggregation of human platelet rich plasma (PRP)
stimulated by 20 μM ADP using a turbidity method.11 The
compounds were initially assayed at 100 or 50 μM, and
IC50 values were determined for compounds with g50%
inhibition of platelet aggregation. Historical analysis of hu-
man PRP potency data for P2Y12 antagonists revealed sig-
nificant donor-to-donor variability. To reduce this effect, the
potency data for a reference standard 84 (AZD-6140)8 were
Upon discovery of the pyridine ring system, modifications
to other parts of the molecule were explored. Efforts were
focused on derivatization of the piperazine nitrogen (R1).
With no difference in PRP activity of the 4,6-diphenyl (10a)
vs 6-phenylpyridine (10d) ring system, the 6-phenylpyridine
ring was used as the starting template for piperazine derivatiza-
tion. Scheme 2 shows the synthesis of substituted piperazine
analogues. The commercially available 6-phenylpicolinic acid
13 was coupled with 1-allyl 5-tert-butyl L-glutamate 12 using
Figure 1. Quinoline core structures.
Scheme 1. Polymer-Assisted Solution-Phase (PASP) Library Synthesisa
a Reagents and conditions: (a) 1.5 equiv of 6, NMM, HOBt, DCM/DMF; (b) excess 7 and 8, additional DCM; (c) 10% TFA/DCM.

2012 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
Table 1. Binding and PRP Activity Data for Selected Library Compounds 9-11
Cpd
R4
R6
X1
X3
X5
R1
R2
Ki a (nM)
IC50 b (μM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
Ph
H
Ph
H
N
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
EtOCO
EtOCO
EtOCO
EtOCO
EtOCO
EtOCO
EtOCO
EtOCO
EtOCO
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
15
>5400
>5700
209
76
>100
>100
71
N
Ph
H
H
N
Ph
Ph
Ph
Ph
Ph
Ph
N
Ph
H
CH
CH
CH
N
373
>100
>100
>50
>100
74
3640
484
Ph
H
N
CH
356
1190
H
N
9a
9b
Ph
H
Ph
Ph
N
N
CH
CH
CH
CH
EtOCO
EtOCO
H
H
733
1610
>50
>100
11a
11b
11c
11d
11e
Ph
Ph
H
Ph
H
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
m-tolyl
m-tolyl
m-tolyl
m-tolyl
m-tolyl
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
150
>5700
>5700
1430
>100
>100
>100
>100
>100
N
Ph
Ph
Ph
N
Ph
H
CH
CH
1310
a Membranes from CHO cells expressing recombinant human P2Y12 receptors incubated with 33P ADP and compound. Ki values are corrected from
IC50 using the Cheng and Prusoff equation and are the geometric mean of n = 2 or greater. b IC50 values are from human PRP incubated with 20 μM
ADP.
Scheme 2. Synthesis of Substituted Piperazine Analogues 20a
a Reagents and conditions: (a) 1.5-2.2 equiv of 6, NMM, HOBt, DCM/DMF; (b) Pd(PPh3)4, morpholine, MeCN; (c) H2, Pd/C, MeOH; (d) 4 equiv of
19, TEA, DCM; (e) excess 8, additional DCM; (f) 20% TFA/DCM.
polymer-bound carbodiimide6 with HOBt and NMM as base
to afford the allyl ester intermediate 14. Deprotection of the
allyl ester using tetrakis(triphenylphosphine)palladium as the
catalyst and morpholine provided the carboxylic acid inter-
mediate 15. A second coupling with the acid 15 and the Cbz
protected piperazine 16 followed by deprotection of the Cbz
using hydrogen with palladium on carbon afforded the piper-
azine intermediate 18. The piperazine 18 was reacted with an
excess of electrophile 19 in the presence of triethylamine. The
electrophiles used (alkyl halides, chloroformates, acid halides,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2013
isocyanates, thioisocyanates, and sulfonyl halides) were sel-
ected to represent a diverse set, affording products 20 contain-
ing alkyl, carbamate, amide, urea, thiourea, or sulfonamide
functionality. Upon completion of the reaction, polymer-
bound amine 8 was added to the reaction mixture to sequester
any unreacted electrophile 19 followed by filtration and
evaporation to afford purified products. Deprotection of the
tert-butyl ester using TFA in DCM provided the desired
substituted piperazine products 20.
aliphatic straight chains of four to six carbon length preferred
(26c-e). When unsaturation, branching, or a heteroatom
is introduced into the carbamate side chain, PRP activity is
lost.
Additional chemistry efforts explored replacing the 4-phenyl
substituent on the pyridine ring of lead 10a with other groups
such as substituted amines and ethers. Synthesis of the pyridine
intermediates is depicted in Scheme 4. The commercially
available 4-hydroxy-6-phenylpicolinic acid 27 was converted
to the methyl ester 28 followed by treatment with phosphorus
oxychloride to afford methyl 4-chloro-6-phenylpicolinate 29.
Hydrolysis of the methyl ester 29 with potassium hydroxide
provided the key 4-chloro-6-phenylpicolinic acid intermediate
30. Synthesis of the 4-aminopyridines and the intermediate for
the 4-oxygen substituted pyridine analogues is depicted in
Scheme 5. The BOC protected piperazine 32 was reacted with
the chloroformate 33 followed by BOC deprotection with
hydrochloric acid or TFA to afford the piperazine carbamate
35. Coupling of the piperazine 35 with N-benzyloxycarbonyl-
L-glutamic acid γ-tert-butyl ester 36 using carbodiimide
and N-methylmorpholine as base afforded the intermediate
37. Removal of the Cbz group via hydrogenation using
palladium on carbon provided the amine intermediate 38. A
second coupling using the amine 38 with the 4-chloro-6-phe-
nylpicolinic acid 30 afforded the 4-chloropyridine intermediate
43. The 4-chloropyridine intermediate 43 allows for substitution
at the 4-position with nitrogen nucleophiles. The 4-chloro was
Many substituted piperazine compounds were prepared,
and in general the unsubstituted compound (18), aliphatics,
amides, and sulfonamides were inactive while several of the
aliphatic ureas exhibited modest binding activity with little to
no PRP activity (Supporting Information). The thioureas
exhibited good binding activity and moderate PRP activity,
while the aliphatic carbamates were optimal with respect to
both binding and PRP activity. To further explore the carba-
mate SAR in a parallel format, a PASP library synthesis of
piperazine carbamate analogues was employed as shown in
Scheme 3.13 The synthesis allowed for the carbamate to be
prepared from the corresponding alcohol, as many of the
corresponding chloroformates were not commercially avail-
able. Polymer-supported N-hydroxysuccinimide 21 was trea-
ted with bistrichloromethyl carbonate 22 to afford polymer-
supported chloroformate 23. Treatment of 23 with the alcohol
24 provided, after removal of excess reagents by filtration, the
polymer-bound activated N-hydroxysuccinimide carbonate
25. The piperazine 18 was added to a slight excess of poly-
mer-bound carbonate 25 followed by filtration and evapora-
tion to afford the purified products. Deprotection of the tert-
butyl ester using TFA in DCM provided the desired carba-
mate products 26.
Table 2. Binding and PRP Activity Data for a Representative Set of
Piperazine Carbamate Analogues 26
Cpd
R
Ki a (nM)
IC50 b (μM)
26a
10d
26b
26c
26d
26e
26f
26g
26h
26i
Me
Et
2440
209
27
>100
71
62
A representative set of carbamate analogues 26 is shown in
Table 2. The SAR is very clear with the carbamates as
Pr
Bu
29
11
28
15
Pent
Hex
Hept
Oct
Scheme 3. PASP Synthesis of Piperazine Carbamate Analo-
gues 26a
43
19
56
7
14
>100
>100
>100
70
CH2CF3
i-Pr
94
280
18
26j
(CH2)3CF3
(CH2)2CCH
cPent
26k
26l
79
47
>100
>100
>100
>100
82
26m
26n
26o
26p
26q
26r
26s
26t
cHex
CH2cBu
44
28
CH2cPent
(CH2)2cPr
(CH2)2CH(CH3)2
(CH2)2C(CH3)3
(CH2)2OMe
CH2Ph
46
15
>100
88
33
42
>100
>100
>100
492
196
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP.
a Reagents and conditions: (a) 2.2 equiv of 22, pyridine, DCM; (b)
excess 24, pyridine, DCM; (c) 0.75 equiv of 18, DCM; (d) 20% TFA/
DCM.
Scheme 4. Preparation of 6-Phenylpyridine Intermediatesa
a Reagents and conditions: (a) H2SO4, MeOH, reflux; (b) POCl3, reflux; (c) KOH, H2O/dioxane; (d) NaN3, Aliquat 336, EtOH/H2O, 100 °C.

2014 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
Scheme 5. Synthesis of 4-Hydroxypyridines 39-42 and 4-Aminopyridine Analogues 45-48a
a Reagents and conditions: (a) 1.1 equiv of 33, DIEA, DCM; (b) 4 M HCl/dioxane or TFA, DCM; (c) EDC, HOBt, NMM, DCM; (d) H2, Pd/C,
MeOH; (e) excess 44, TEA, DMSO, 50-100 °C; (f) 10% TFA/DCM.
Scheme 6. Synthesis of 4-Ether Pyridines 50-53 and 4-Carbamate Pyridine Analogues 56 and 57a
a Reagents and conditions: (a) 2 equiv of 49, Cs2CO3, KI, DMF, room temp to 100 °C; (b) 2 equiv of 24, DEAD, PPh3, THF; (c) 10% TFA/DCM;
(d) 2.7 equiv of 54, TEA, DCM; (e) 1.1 equiv of 55, TEA, DCM, 1 h, 3.0 equiv of 44, TEA, DCM.
displaced by heating intermediate 43 with an excess of amine 44
and triethylamine in DMSO, followed by deprotection of the
tert-butyl ester group to afford the desired 4-aminopyridine
analogues 45-48. Displacement of the chlorine at the 4-position
afforded clean products with either the free carboxylic acid or
the tert-butyl ester group present. Initial concerns for racemiza-
tion under these conditions were eliminated by several studies,
one of which included heating enantiomerically pure (S)-45i at

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2015
Table 3. Binding and PRP Activity Data for a Representative Set of
4-Ether Pyridine Analogues 50-53
Table 4. Binding and PRP Activity Data for a Representative Set of
4-Carbamate Pyridine Analogues 56 and 57
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP. c (IC50 value of compound)/(IC50 value of
84 control from same plate) = normalized IC50 ratio.
Attempts to displace the 4-chloro with oxygen nucleophiles
provided limited success. As a result, the 4-hydroxypyridines
were prepared in a similar fashion by coupling the 4-hydroxy-
6-phenylpicolinic acid 27 with the amine 38 using carbodii-
mide and N-methylmorpholine as base to afford the 4-hydro-
xypyridine intermediates 39-42 (Scheme 5). Direct alkylation
or employing Mitsunobu conditions with the 4-hydroxypyr-
idine intermediates 39-42 provided 4-ether pyridine analo-
gues 50-53 as shown in Scheme 6. Direct alkylation of the
4-hydroxypyridines 39-42 using 2 equiv of the electrophile 49
with cesium carbonate as the base and a catalytic amount of
potassium iodide in DMF, followed by deprotection of the
tert-butyl ester group, afforded the desired 4-ether pyridine
analogues 50-53. Alternatively, employing Mitsunobu con-
ditions with 2 equiv of the alcohol 24 using DEAD and
triphenylphosphine in THF, followed by deprotection of the
tert-butyl ester group, afforded the desired 4-ether pyridine
analogues 50-53. Using both procedures allowed for a great-
er diversity of monomer inputs 24 and 49, ultimately provid-
ing a wide array of 4-ether pyridine analogues.
The 4-hydroxypyridine intermediates 40 and 41 were also
used to prepare 4-carbamate pyridine analogues 56-57 as
shown in Scheme 6. The 4-hydroxypyridines 40 and 41 were
reacted directly with a carbamoyl chloride 54 using triethyla-
mine as base in DCM to provide the carbamate. Deprotection
of the tert-butyl ester using TFA in DCM afforded the desired
4-carbamate pyridine products 56 and 57. Because of the
limited commercial availability of carbamoyl chlorides 54,
a second procedure was employed using the 4-hydroxypyr-
idines 40 and 41 and p-nitrophenylchloroformate 55 to
provide the activated carbonate followed by addition of the
amine 44 and triethylamine as base in DCM to afford the
carbamate. Deprotection of the tert-butyl ester using TFA in
DCM afforded the desired 4-carbamate pyridine products 56
and 57.
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP. c (IC50 value of compound)/(IC50 value of
84 control from same plate) = normalized IC50 ratio.
Table 3 shows the data for a representative set of 4-oxygen
analogues. The unsubstituted (hydroxy) analogues (50a-53a)
had modest binding and PRP potency, while substitution of
100 °C for 64 h with less than 6% of the R enantiomer detected
by chiral LC and circular dichroism.14

2016 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
the oxygen with alkyl chains of varying length (50b-53e)
tended to slightly decrease the potency. Comparable PRP
activity was observed with the benzyl ether analogue 50w,
and relatively flat SAR was observed across various substi-
tuted benzyl ether analogues with PRP activity in the range of
9-39 μM (data not shown). Extending a hydroxy or alkyl
ether two to four carbon atoms away from the 4-oxygen atom
increased the PRP activity (50f-51l). A similar trend was
observed by extending a primary or secondary amine two to
five carbon atoms away from the 4-oxygen atom (50m-53v).
In particular, the 4-oxypiperidine (50n-53n), 4-oxymethylpi-
peridine (50q-53q), and 4-oxyethylpiperidine (51t) analogues
were the most potent compounds of the series. Replacing the
4-oxypiperidine (51n) with 3-oxypyrrolidine (51p) or with the
regioisomer 3-oxypiperidine (51o) resulted in a slight decrease
in PRP activity. Similarly, replacing the 4-oxymethylpiperi-
dine (51q) with the 2- and 3-regioisomers (51r,s) resulted in a
slight loss in PRP activity. The same trend was observed with
the 4-oxyethylpiperidine regioisomers (51u,v). The nitrogen of
the 4-piperidine and 4-oxymethylpiperidine was essential
because when replaced with an oxygen a significant decrease
in PRP activity was observed (51k, 51l). Overall, the butyl and
pentyl carbamates of the piperazine tail (R1) were superior
relative to the ethyl and hexyl carbamates with respect to both
binding and PRP potency.
Table 4 shows the data for a representative set of 4-car-
bamate pyridine analogues 56 and 57. These compounds
when compared to the unsubstituted oxygen (51a, 52a,
Table 4) were considerably more potent in both the binding
and PRP assays. The carbamates had relatively flat SAR with
respect to the binding activity with most of them at the single
digit nanomolar level, but having an oxygen within the amine
side chain of the carbamate tended to increased the PRP
potency, with compounds 56b-57c having exceptional PRP
potency. Again, the butyl and pentyl carbamates of the
piperazine tail (R1) were optimal for both binding and PRP
activitywith no significant difference between the two in terms
of potency.
The 4-ether pyridine analogues, 51q and 52n, were two of
the most potent compounds of the series. Further efforts
focused on the synthesis of additional analogues with various
substitutions on the 4-nitrogen of the 4-oxypiperidine and
4-oxymethylpiperidine pyridine analogues. The synthesis is
depicted in Scheme 7. The 4-oxypiperidine and 4-oxymethyl
piperidine intermediates 60-63 were prepared under Mitsu-
nobu conditions, reacting the 4-hydroxy intermediates 40 and
Scheme 7. Synthesis of Substituted 4-Oxypiperidine 69 and 70 and 4-Oxymethylpiperidine Pyridine Analogues 71 and 72a
a Reagents and conditions: (a) 2 equiv of 58 or 59, DEAD, PPh3, THF; (b) H2, Pd/C, EtOH; (c) 2.0 equiv of 68, TEA or NMM, DCM or DMF;
(d) 10% TFA/DCM.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2017
41 with N-Cbz-4-hydroxy-1-piperidine 58 and 1-N-Cbz-4-
hydroxymethylpiperidine 59 to afford the Cbz protected
piperidines 60-63. Deprotection of the Cbz group by hydro-
genation using palladium on carbon afforded the unsubsti-
tuted nitrogen intermediates 64-67. The amines 64-67 were
reacted with an excess of electrophile 68 in the presence of a
base to afford the 4-nitrogen substituted analogues. The
electrophiles 68 used (alkyl halides, acid halides, sulfonyl
halides, and chloroformates) were selected to represent a
diverse set, affording products containing alkyl, amide, sulfo-
namide, or carbamate functionality. Deprotection of the tert-
butyl ester using TFA in DCM provided the desired substi-
tuted piperazine products 69-72.
A representative set of 4-substituted oxypiperidine analo-
gues 69, 70 is depicted in Table 5. The 4-substituted oxypiper-
idines had exceptional PRP potency with relatively flat SAR
across various functionalities at the 4-piperidine position with
the carbamates 69g, 70g and sulfonamides 69h, 70h slightly
less active in the PRP assay compared to the other analogues.
There was no significant difference in PRP activity between
the butyl carbamates and the corresponding pentyl carba-
mates (R1) for the 4-substitued piperidines.
The same holds true for the 4-substituted oxymethylpiper-
idine compounds 71, 72, with the amides and alkylated
analogues exhibiting exceptional binding and PRP activity,
while the carbamates 71g, 72g and sulfonamides 71h, 72h were
slightly less potent in the PRP assay (Table 6). The 4-sub-
stituted oxymethylpiperidine analogues 71, 72 were more
potent than the corresponding 4-substituted oxypiperidines
69-70, and there was no significant difference in PRP activity
between the butyl carbamates and the corresponding pentyl
carbamates.
Several libraries of the 4-aminopyridine analogues 45-48
were synthesized (Scheme 5), and the data for a representative
set are shown in Table 7. As has been the trend, the butyl and
pentyl carbamates were superior to the ethyl and hexyl
carbamates in terms of both binding and PRP potency. The
4-aminopyridine analogues substituted with two alkyl groups
(45b, 45d) were inactive in the PRP assay, while the secondary
amines with a single alkyl group (45a,c, 47c) had modest
potency. The 4-amine analogues substituted with alkyl groups
were less active than compounds with a heteroatom present in
the side chain. Compounds 45e,47e and 45g-48g provide
examples of secondary amine analogues with an oxygen
heteroatom, as a hydroxy or ether, as part of the amine group
with good binding and PRP activity. The corresponding
tertiary amine analogues 45f, 46h, and 47h were considerably
less active in the PRP assay. Interestingly, the piperidine
analogues (alkyl tertiary amines 45i and 47i) exhibited modest
PRP potency. Replacing the piperidine ring with morpholino
(45j and 46j) provided a slight increase in the PRP potency.
Substitution at the 4-position of the piperidine ring with
hydroxy (45k and 47k) or alkoxy (47l) increased both the
binding and PRP activity, while extending the hydroxy or
methoxy from the 4-position of the piperidine with a methy-
lene (47r,s) maintained binding and PRP activity. The hydroxy
analogues could be capped with a methyl and maintain PRP
activity, but capping withany alkylgrouplarger than a methyl
tended to decrease the PRP activity (47t). Direct substitution
from the 4-position of the piperidine with an amine increased
PRP activity with no significant difference in PRP activity
from the primary (45m-48m), secondary (46n and 47n), or
tertiary amines (46o, 47o, 45o-48p). Extending the amine
from the 4-position of the piperidine with a methylene
Table 5. Binding and PRP Activity Data for a Representative Set of
4-Substituted Oxypiperidine Pyridine Analogues 69 and 70
relative
Ki a (nM) IC50 b(μM) potency to 84c
Cpd
R1
R
51n Bu
52n Pent
69a Bu
H
12
4
1.1
0.50
2.0
1.8
0.88
0.48
1.2
1.7
1.2
1.8
2.1
4.7
0.52
1.5
3.7
8.2
3.3
5.3
0.99
0.64
1.9
H
Me
2.5
5.0
10
70a Pent Me
69b Bu i-Pr
70b Pent i-Pr
69c Bu (CH2)2OMe
70c Pent (CH2)2OMe
69d Bu COMe
70d Pent COMe
69e Bu COCF3
70e Pent COCF3
1.8
0.75
0.68
1.1
7.0
9.4
9.9
2.8
3.6
12
1.7
1.1
1.8
3.4
19
4.4
69f
Bu
Pent COCH2OMe
CO2Et
70g Pent CO2Et
69h Bu SO2Et
70h Pent SO2Et
COCH2OMe
2.4
11
0.78
1.5
70f
69g Bu
20
5.9
8.1
6.5
7.6
5.1
3.1
5.0
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP. c (IC50 value of compound)/(IC50 value of
84 control from same plate) = normalized IC50 ratio.
Table 6. Binding and PRP Activity Data for a Representative Set of
4-Substituted Oxymethylpiperidine Pyridine Analogues 71 and 72
relative
Ki a(nM) IC50 b(μM) potency to 84c
Cpd
R1
R
51q Bu
52q Pent
71a Bu
H
4.2
4.5
2.5
5.3
4.5
5.5
5.3
5.3
3.9
1.3
10
0.21
0.82
0.24
0.33
0.41
0.38
0.62
0.55
2.1
0.37
0.82
0.38
0.62
0.33
0.50
0.53
0.73
1.4
H
Me
72a Pent Me
71b Bu i-Pr
72b Pent i-Pr
71c Bu (CH2)2OMe
72c Pent (CH2)2OMe
71d Bu COMe
72d Pent COMe
71e Bu COCF3
72e Pent COCF3
1.3
3.0
1.8
2.8
4.5
2.6
12
3.5
1.5
4.8
1.4
71f
Bu
Pent COCH2OMe
CO2Et
72g Pent CO2Et
71h Bu SO2Et
72h Pent SO2Et
COCH2OMe
72f
1.3
4.0
1.3
2.2
71g Bu
7.6
1.3
5.3
7.4
7.4
3.0
5.1
2.8
2.8
1.9
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP. c (IC50 value of compound)/(IC50 value of
84 control from same plate) = normalized IC50 ratio.
(45u-47x) provided some of the most potent compounds of
the series in terms of both binding and PRP activity.
Other 4-substituted amines were also prepared as shown in
Scheme 8. Attempts to prepare the unsubstituted 4-aminopyr-
idine intermediate by simple displacement of the 4-chloropyr-
idine intermediate 43 with ammonia or ammonia equivalents
could not be efficiently accomplished. As a result, the 4-azido-
6-phenylpicolinic acid 31 was prepared from reacting sodium
azide with 4-chloro-6-phenylpicolinic acid 30 (Scheme 4).
Coupling of the 4-azido intermediate 31 with pentyl (S)-4-
(2-amino-4-tert-butoxycarbonylbutyryl)piperazine-1-carboxylate

2018 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
Table 7. Binding and PRP Activity Data for a Representative Set of
4-Aminopyridine Analogues 45-48
acid chlorides would not react and required using propylpho-
sphonic anhydride as a coupling reagent with the carboxylic
acid 74 to afford the desired amides. Deprotection of the tert-
butyl ester using TFA in DCM provided the desired 4-amide
pyridine products 75. The 4-carbamate and 4-urea analogues
were prepared from the 4-chloroformate intermediate. The
4-aminopyridine 73 was treated with bistrichloromethyl car-
bonate 22 to afford the chloroformate. Treatment of chlor-
oformate with the alcohol 24 provided the carbamates,
followed by deprotection of the tert-butyl ester to afford the
desired 4-carbamate pyridine products 77. Similarly, treat-
ment of chloroformate with the amine 44 provided the ureas,
followed by deprotection of the tert-butyl ester to afford the
desired 4-urea pyridine products 76.
The 4-amide pyridine analogues 75 provided compounds
with good binding activity. However, little to no PRP activity
was observed with these compounds. Similarly, the 4-carba-
mate pyridine analogues 77 exhibited good binding activity
but poor PRP activity, and the 4-urea pyridine analogues
76 showed good binding activity with poor to moderate
PRP activity. In general, these compounds (75-77) were
less active than the 4-aminopyridines (45-48) (Supporting
Information).
Thus far, substitutions at the 4-position of the pyridine
ring included nitrogen and oxygen. Other substitutions were
explored such as carbon-carbon to include carboxamides
and aliphatics. Scheme 9 shows the synthesis of 4-carboxa-
mide and 4-aliphatic pyridine analogues. The 4-hydroxypyr-
idine intermediate 41 was reacted with triflic anhydride to
afford the triflate intermediate 78. A palladium catalyzed
carbonylation reaction with the triflate 78 afforded the
4-carboxylic acid intermediate 79.15 The acid 79 was reacted
with various amines 44 using polymer-bound carbodiimide 6
with HOBt and NMM as base to afford the amide. Deprotec-
tion of the tert-butyl ester using TFA gave the desired 4-car-
boxamide pyridine products 80. The triflate intermediate was
also used for 4-aliphatic pyridine analogues. The triflate 78
was reacted with various substituted alkynes 82 using Sono-
gashira conditions followed by reduction of the triple bond
using hydrogen with palladium on carbon to provide the
4-aliphatic pyridines. Deprotection of the tert-butyl ester
using TFA provided the desired 4-aliphatic pyridine products
83. Alternatively, the triflate 78 could be reacted with various
substituted alkenes 81 using Heck reaction conditions fol-
lowed by reduction of the double bond using hydrogen with
palladium on carbon to provide the 4-aliphatic pyridines.
Deprotection of the tert-butyl ester using TFA provided the
desired 4-aliphatic pyridine products 83. Employing both
reaction procedures allowed for a greater diversity of mono-
mer inputs 81 and 82, providing a wide array of 4-aliphatic
pyridine products 83.
Table 8 shows a representative set of 4-carboxamide pyr-
idine products 80. In general, the secondary amides were less
potent in the PRP assay than the tertiary amides. The amides
with dimethyl substitution (80e) or methyl, ethyl substitution
(80f) were optimal for alkyl tertiary amides. When the alkyl
group was extended beyond a methyl or ethyl group, a
decrease in the PRP potency was observed (80g). The tertiary
amides, having a methyl as one substituent and extending a
methoxy or amine two to four carbon atoms away from the
amide nitrogen, provided compounds with excellent PRP
activity (80k-m). Cyclic tertiary amides also exhibited excel-
lent potency in terms of both binding and PRP activity. The
unsubstituted piperidine amide 80n exhibited good PRP
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP. c (IC50 value of compound)/(IC50 value of
84 control from same plate) = normalized IC50 ratio.
38c using EDC, HOBt, and N-methylmorpholine as base
afforded the azide intermediate. Reduction of the azide using
palladiumoncarbonaffordedthe 4-aminopyridine intermedi-
ate 73. The 4-aminopyridine 73 was reacted with acid chlor-
ides to afford the amides albeit in low yields. Because of the
poor nucleophilic nature of the 4-aminopyridine, many of the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2019
Scheme 8. Synthesis of 4-Amide, 4-Urea, and 4-Carbamate Pyridine Analogues 75-77a
a Reagents and conditions: (a) EDC, HOBt, NMM, DCM; (b) H2, Pd/C, MeOH; (c) 1.0 equiv of 22, pyridine, DCM, 0 °C; (d) 0.75 equiv of 44, DCM;
(e) 0.75 equiv of 24, DCM; (f) 10% TFA/DCM; (g) 1.4 equiv of 74, 1-methylimidazole, PPA, EtOAc.
Scheme 9. Synthesis of 4-Aliphatic 83 and 4-Carboxamide 80 Pyridine Analoguesa
a Reagents and conditions: (a) 3.0 equiv of Tf2O, pyridine, 0-20 °C; (b) CO, Pd(Ph3P)4, TEA, DMSO, 60 °C; (c) 1.5 equiv of 44, excess 6, NMM,
HOBt, DCM; (d) 10% TFA/DCM; (e) 2.8-4.0 equiv of 81, Cl2Pd (Ph3P)2, TEA, LiBr, DMF, 100 °C; (f) 2.8 equiv of 82, Pd(Ph3P)4, diethylamine, CuI,
THF; (g) H2, Pd/C, MeOH.

2020 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
Table 8. Binding and PRP Activity Data for a Representative Set of
4-Carboxamide Pyridine Analogues 80
Table 9. Binding and PRP Activity Data for a Representative Set of
4-Aliphatic Pyridine Analogues 83
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP. c (IC50 value of compound)/(IC50 value of
84 control from same plate) = normalized IC50 ratio.
a Membranes from CHO cells expressing recombinant human P2Y12
receptors incubated with 33P ADP and compound. Ki values are
corrected from IC50 using the Cheng and Prusoff equation and are the
geometric mean of n = 2 or greater. b IC50 values are from human PRP
incubated with 20 μM ADP. c (IC50 value of compound)/(IC50 value of
84 control from same plate) = normalized IC50 ratio.
three of the compounds had high clearance values. Another
concern with oral absorption was the molecular weight. The
core templates 26d and 26e have molecular weights over 500.
Substituting at the pyridine 4-position only adds to the
molecular weight, potentially resulting in the loss of good
bioavailability. Despite high molecular weight and, in numer-
ous cases, poor Caco-2permeability values, many compounds
had good bioavailability as exemplified by 51i (molecular
weight of 570, Papp = 1.1 ꢀ 10-6).
potency, while substitution at the 4-position of the piperidine
with methoxy (80p) or an amine (80q) provided very potent
compounds. Similarly, the 4-substituted piperazine amides
80r and 80s exhibited exceptional binding and PRP potency.
Table 9 contains analogues with aliphatic chains substi-
tuted at the 4-position of the pyridine. Alkyl chains of varying
length and/or branching exhibited binding activity but no
PRP activity (83a).12 The aliphatic chains extending a hydro-
xy, methoxy, or amine three to four carbon atoms away from
the pyridine ring (83b-j) and the propanamides 83k-l pro-
vided compounds with good to moderate PRP activity.
Many libraries containing several hundred compounds
were prepared with varying substituents at the 4-position of
the pyridine ring and varying carbamate chain lengths on the
piperazine terminal nitrogen. The SAR clearly showed that
butyl and pentyl carbamates of the piperazine terminal nitro-
gen were optimal and modulation of potency and other
properties could be obtained by variations at the 4-position
of the pyridine, providing compounds with submicromolar
PRP levels of activity.
Many compounds were profiled from the 4-oxygen pyri-
dine series which included the 4-ether and 4-carbamate pyr-
idine analogues. Included in this series was the general
template, the hydroxy intermediate 52a, which had high
clearance but good bioavailability. This was the general trend
for the ether series in which all of the compounds evaluated
had high clearance, oftentimes above the liver blood flow, yet
several had moderate to good bioavailability (51i, 52i). Some
of the most active compounds of the series, including the
4-oxypiperidine (52n, 53n) and 4-oxymethyl piperidine ether
analogues (52q), had high clearance. Capping the 4-nitrogen
of the 4-oxypiperidine (70c,d) and 4-oxymethyl piperidine
(72a-c) with various functionalities still resulted in com-
pounds with high clearance. All of the 4-carbamate pyridine
analogues (57b,d,f) profiled also exhibited high clearance
values.
Having identified many inhibitors with exceptional po-
tency, we sought to evaluate the chemical and in vivo phar-
macokinetic properties of selected analogues. In general, this
class of compounds had good solubility and was chemically
stable. These inhibitors also showed excellent metabolic sta-
bility in both the rat and human microsomal assays. Many
compounds were evaluated for their in vivo pharmacokinetic
characteristics in the rat, and a representative set of pharma-
cokinetic profiles is shown in Table 10. Compounds 10d, 26d,
and 26e each possessing a hydrogen at the 4-position of the
pyridine, with an ethyl, pentyl, and hexyl carbamate on the
piperazine terminal nitrogen, respectively, were selected to
profile the general template and to test if the carboxylic acid
would be detrimental to oral absorption. Gratifyingly, all of
the compounds were bioavailable with high bioavailabilities
for the pentyl and hexyl carbamate analogues. However, all
Several compounds from the carbon-carbon series at the
4-position were evaluated that included both the carboxa-
mides and aliphatics. The carboxamides (80o-q) had im-
proved, but still unacceptable, clearance values, while the
aliphatics (83g-i) had relatively high clearance.
Many compounds were profiled from the 4-aminopyridine
series. However, most of the initial compounds profiled had
high clearance values. The secondary alkyl (45c) and aliphatic
(47e) amines typically had high clearance, while the tertiary
amines, piperidine (45i) and morpholine (45j), were still high
but shifted in the right direction. The 4-piperidine analogues
typically provided the more potent compounds and provided
a handle to modulate the pharmacokinetic properties by
varying the substituent at the 4-position of the piperidine
ring. It was found that compounds with a basic amine directly
linked (47m,o,p) or extended by a methylene (47u,w) to the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2021
Table 10. Rat Pharmacokinetic Profiles of Selected P2Y12 Antagonistsa,b
Further evaluation of compound 47s showed good solubi-
lity and chemical stability with the crystalline form of the
compound. Compound 47s was found to have acceptable
margins vs hERG activity and a satisfactory CYP inhibition
profile. Further evaluation on the potency of compound 47s
was determined by measuring the IC50 in 13 human donors
using 20 μM ADP-stimulated PRP aggregation with the 4-well
Chronolog PRP aggregometry assay and found the average
IC50 to be 1.8 μM. Compound 47s was orally efficacious in the
rat ferric chloride model in which it dose-dependently pre-
vented thrombus formation in this model of injury induced
thrombosis.
In vitro receptor binding, signaling, and functional studies
have shown that 47s is a high affinity, selective, and compe-
titive antagonist at P2Y12 receptors. Compound 47s is more
than 340-fold selective for P2Y12 over the other purinergic
receptors tested, including the closest homologue, P2Y13
(48% homology to P2Y12), and a second platelet purinergic
GPCR, P2Y1 (19% homology to P2Y12). The Kb (8.5 nM) for
functional antagonism in the P2Y12 signaling assay is in good
agreement with the Ki (15 nM) for the receptor. The Kb in
human PRP platelet aggregation assays (300 nM) is higher
because of the presence of plasma proteins. Schild analysis
indicates that the interaction of 47s with the receptor in the
aggregation assays is competitive with ADP.
In summary, we have utilized PASP parallel library synth-
esis to identify a P2Y12 lead. This lead was quickly followed-
up with the generation of structure-activity relationships
leading to highly potent P2Y12 antagonists. With sufficient
levels of potency attained, pharmacokinetic and physiochem-
ical properties were modulated through various substituents
at the 4-pyridine position. Fine-tuning the PK properties led
to 47s, an orally bioavailable, direct acting, reversible P2Y12
antagonist. This compound is a selective antagonist of the
human P2Y12 receptor and demonstrates oral antiplatelet and
antithrombotic efficacy in preclinical species. No safety issues
were observed with 47s, as it was inactive in both the Ames
and micronucleus assays and has been evaluated in both rat
and dog safety studies (single dose and 7-day repeat dose)
without significant toxicological findings. The potency, selec-
tivity, safety, and overall pharmacokinetic profile for 47s
support advancement for clinical evaluation. Reports from
our backup program where efforts are focused on refining the
SAR for more potent analogues with improved pharmacoki-
netic properties will be forthcoming.
a Male Sprague-Dawley rats (n = 2-4 rats). b Dose: iv infusion at
2 mg/kg; po at 5 mg/kg. Vehicle: 50% PEG400/40% PBS/10% ethanol.
c Foral (n = 3): 92%, 95%, and 181%. d Foral (n = 1): 183%.
Experimental Section
4-position of the piperidine usually had high clearance values.
One strategy to reduce the basicity was to replace the basic
amine on the piperidine with an alkoxy group. Replacing the
amine group with a methoxy resulted in compounds with
acceptable clearance values, although a slight loss in potency
was observed. This exercise led to the discovery of compound
47s, with a low clearance of 3 (mL/min)/kg in rat (1 (mL/min)/
kg, dog) with good bioavailability (>90%, dog). Clearance of
47s in rats appears to be mediated through extensive biliary
excretion, with roughly 70% of an iv dose (2 mg/kg) recovered
in the bile within 4 h. Oral bioavailability in a definitive rat
study (2 mpk) averaged 25% (22%, 25%, and 27%), leading
to a predicted human bioavailability of 30% with the pro-
jected human dose of 250 mg b.i.d. Oral bioavailability in a
definitive dog study (0.2 mpk) averaged 67% (33%, 80%, and
87%), leading to a predicted human bioavailability of 70%
with the projected human dose of 85 mg b.i.d.
General Conditions. Solvents and chemicals were reagent
grade or better and were obtained from commercial sources.
All polymeric reagents and sequestering resins were obtained
from commercial sources. 1H and 13C NMR spectra were
recorded using a 300, 400, or 500 MHz NMR spectrometer.
Chemical shifts are reported in ppm, using the solvent as internal
standard. High-resolution mass spectral (HRMS) data were
recorded on a Mariner TOF from a Perseptive Biosystems
instrument with electrospray ionization of a loop inject with
10 mM ammonium acetate in methanol at 50 mL/min and a
lockmass of m/z 387 from AlaLeuAlaLeu. All tested compounds
were at least 95% pure as determined by HPLC analysis
equipped with a mass spectrometry detector using a C18 3.5 μm,
30 mm ꢀ 2.1 mm column, eluting with a gradient system of 5/95
to 95/5 acetonitrile/H2O with a buffer consisting of 0.1% TFA
over 4.5 min at 1 mL/min and detected by UV at 254 and 210 nm
using a diode array detector. Preparative column chromatogra-
phy was performed on a preparative liquid chromatography

2022 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
instrument using silica gel columns and/or on a HPLC system
using an 8 μm 60 A, C18 column (41 mm i.d. ꢀ 100 mm length).
Reported yields are not optimized, with emphasis on purity of
products rather than quantity.
(4S)-4-[(2,6-Diphenylisonicotinoyl)amino]-5-[4-(ethoxycarbo-
nyl)piperazin-1-yl]-5-oxopentanoic Acid (10g). General proce-
dures A and B afforded 54.0 mg (50%) of product 10g. HRMS
calcd for C30H32N4O6 (Mþ þ H) 545.2395, found 545.2432.
(S)-Ethyl 4-{4-Carboxy-2-[(2-phenylpyrimidine-4-carbonyl)-
amino]butyryl}piperazine-1-carboxylate (10h). General proce-
dures A and B afforded 15.8 mg (69%) of product 10h. Mp
60-68 °C; [R]2D5 þ23.9° (c 0.18); 1H NMR (DMSO-d6) δ 12.1 (br
s, 1H), 9.20 (d, 1H, J = 7.6 Hz), 9.15 (d, 1H, J = 5.0 Hz), 8.57
(m, 2H), 7.93 (d, 1H, J = 4.9 Hz), 7.60 (m, 3H), 5.05 (m, 1H),
4.06 (q, 2H, J = 7.1 Hz), 3.60 (m, 3H), 3.44 (m, 4H), 2.35 (m,
2H), 2.08 (m, 1H), 1.93 (m, 1H), 1.19 (t, 3H, J = 7.1 Hz); MS
(ESIþ) m/z 470 (M þ H). Anal. Calcd for C23H27N5O6-
General Procedure A. Coupling of Carboxylic Acid 5 with
Amine Templates 2-4 To Afford Products 9-11. To a mixture
of carboxylic acid 5 (0.26 mmol), 1-hydroxybenzotriazole
(0.02 mmol), and N-methylmorpholine (44.0 uL, 0.40 mmol)
in dichloromethane/dimethylformamide (4 mL, 7:1) in a 30 mL
vial were added amine templates 2-4 (0.38 M in dimethyl-
formamide) (526 uL, 0.20 mmol) and polymer-bound carbodii-
mide reagent 6 (1.17 mmol/g) (0.26 g, 0.30 mmol). The suspen-
sion was agitated 16 h. Upon completion of the reaction,
dichloromethane (6 mL) was added followed by polyamine
resin 8 (0.90 g, 2.5 mmol) (2.87 mmol/g) and isocyanate resin
7 (200 mg, 0.30 mmol) (1.47 mmol/g) and the suspension was
agitated for 1-20 h. The reaction mixtures were filtered and
rinsed with dichloromethane (1 ꢀ 4 mL). The combined filtrate
and washings were dried in vacuo to afford the tert-butyl ester
products.
General Procedure B. Deprotection of the tert-Butyl Ester
Group. A solution of 10% trifluoroacetic acid/dichloromethane
solution (2.0 mL) was added to the tert-butyl ester (∼0.20 mmol)
and agitated on an orbital shaker or stirred at room temperature
for 1-24 h. Evaporation of the solvents afforded the crude
products. The products were purified by HPLC to afford the
pure products 9-11.
S 0.35H2O: C, 58.06; H, 5.87; N, 14.72. Found: C, 58.17; H,
3
5.82; N, 14.56.
(S)-Ethyl 4-{4-Carboxy-2-[(4-phenylpyrimidine-2-carbonyl)-
amino]butyryl}piperazine-1-carboxylate (10i). General proce-
dures A and B afforded 55 mg (56%) of product 10i. 1H
NMR (CDCl3) δ 9.12 (s, 1H), 8.93 (d, 1H, J = 5.0 Hz), 8.19
(dd, 2H, J = 5.0, 1.5 Hz), 7.84 (d, 1H, J = 5.0 Hz), 7.84-7.53
(m, 3H), 5.33-5.30 (m, 1H), 4.16 (q, 1H, J = 7.0 Hz), 3.72-3.44
(m, 8H), 2.62-2.50 (m, 2H), 2.27-2.23 (m, 1H), 1.97-1.94 (m,
1H), 1.30 (t, 3H, J = 7.0 Hz); MS (ESIþ) m/z 470 (M þ H).
(4S)-4-{[(4,6-Diphenylpyridin-2-yl)carbonyl]amino}-5-[4-(3-met-
hylphenyl)piperazin-1-yl]-5-oxopentanoic Acid (11a). General
procedures A and B afforded 126.0 mg (62%) of product 11a.
HRMS calcd for C34H34N4O4 (Mþ þ H) 563.2653, found
563.2655.
Ethyl 4-{N-[(4,6-Diphenylpyridin-2-yl)carbonyl]glycyl}piper-
azine-1-carboxylate (9a). General procedure A afforded 61.0 mg
(65%) of product 9a. HRMS calcd for C27H28N4O4 (Mþ þ H)
473.2183, found 473.2151.
(4S)-5-[4-(3-Methylphenyl)piperazin-1-yl]-5-oxo-4-{[(4-phen-
ylpyridin-2-yl)carbonyl]amino}pentanoic Acid (11b). General
procedures A and B afforded 131.0 mg (91%) of product 11b.
HRMS calcd for C28H30N4O4 (Mþ þ H) 487.2340, found
487.2377.
Ethyl 4-{N-[(6-Phenylpyridin-2-yl)carbonyl]glycyl}piperazine-1-
carboxylate (9b). General procedure A afforded 240 mg (92%) of
1
(4S)-5-[4-(3-Methylphenyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
pyridin-2-yl)carbonyl]amino}pentanoic Acid (11c). General proce-
dures A and B afforded 28.0 mg (19%) of product 11c. HRMS
calcd for C28H3N4O4 (Mþ þ H) 487.2340, found 487.2379.
(4S)-5-[4-(3-Methylphenyl)piperazin-1-yl]-5-oxo-4-[(1,10:30,100-
terphenyl-50-ylcarbonyl)amino]pentanoic Acid (11d). General pro-
cedures A and B afforded 52.0 mg (38%) of product 11d. HRMS
calcd for C35H35N3O4 (Mþ þ H) 562.2700, found 562.2715.
(4S)-4-[(1,10-Biphenyl-3-ylcarbonyl)amino]-5-[4-(3-methylphenyl)-
piperazin-1-yl]-5-oxopentanoic Acid (11e). General procedure A
afforded 21.0 mg (33%) of product 11e. HRMS calcd for
C29H31N3O4 (Mþ þ H) 486.2387, found 486.2418.
product 9b. H NMR (CD3OD) δ 1.24 (t, 3H, J = 8.0 Hz),
3.48-3.62 (m, 8H), 4.12 (q, 2H, J = 12.0 Hz), 4.35 (s, 2H),
7.42-7.50 (m, 3H), 7.99-8.05 (m, 3H), 8.16 (d, 2H, J = 8.0 Hz);
MS (ESIþ) m/z 397 (M
þ
H). Anal. Calcd for
C21H24N4O4 0.2H2O: C, 63.05; H, 6.15; N, 14.00. Found: C,
3
62.88; H, 5.94; N, 13.91.
(4S)-4-{[(4,6-Diphenylpyridin-2-yl)carbonyl]amino}-5-[4-(eth-
oxycarbonyl)piperazin-1-yl]-5-oxopentanoic Acid (10a). General
procedures A and B afforded 107.0 mg (66%) of product 10a.
HRMS calcd for C30H32N4O6 (Mþ þ H) 545.2395, found
545.2375.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-[(pyridin-
2-ylcarbonyl)amino]pentanoic Acid (10b). General procedures A
and B afforded 19 mg (22%) of product 10b. HRMS calcd for
C18H24N4O6 (Mþ þ H) 393.1769, found 393.1765.
1-Allyl 5-tert-Butyl-N-[(4-phenylpyridin-2-yl)carbonyl]-L-glu-
tamate (14). To a mixture of 6-phenylpyridine-2-carboxylic acid
13 (11.3 g, 56.7 mmol), hydroxybenzotriazole (763 mg, 5.7 mmol),
and N-methylmorpholine (24.9 mL, 226 mmol) in dichloro-
methane/dimethylformamide (140 mL, 5:2) were added 1-allyl
5-tert-butyl L-glutamate hydrochloride 12 (10.7 g, 38.2 mmol)
and polymer-bound carbodiimide reagent 6 (1.27 mmol/g) (66.9 g,
85.0 mmol). The suspension was agitated 16 h, and the resin was
filtered and rinsed with dichloromethane (4 ꢀ 10 mL). The filtrate
and washings were dried in vacuo to afford 16.3 g (68%) of product
(42S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(4-phenyl-
pyridin-2-yl)carbonyl]amino}pentanoic Acid (10c). General proce-
dures A and B afforded 82.0 mg (63%) of product 10c. 1H NMR
(DMSO-d6) δ 8.85 (d, 1H, J = 8.1 Hz), 8.71 (dd, 1H,
J = 5.2 Hz, J’ = 0.5 Hz), 8.28 (d, 1H, J = 1.2 Hz), 7.95 (dd,
1H, J = 5.2 Hz, J’ = 1.9 Hz), 7.85 (dd, 2H, J = 8.2 Hz, J’ = 1.5
Hz), 7.51 (m, 3H), 5.03 (m, 1H), 4.05 (q, 2H, J = 7.0 Hz), 3.64 (br,
2H), 3.42 (m, 6H), 2.30 (m, 2H), 2.05 (m, 1H), 1.85 (m, 1H), 1.17 (t,
3H, J = 7.0 Hz); HRMS calcd for C24H28N4O6 (Mþ þ H)
469.2082, found 469.2051.
1
14. H NMR (CDCl3) δ 1.33 (s, 9H), 2.08-2.46 (m, 4H), 4.63
(d, 2H), 4.80 (m, 1H), 5.19-5.32 (m, 2H), 5.83-5.96 (m, 1H),
7.40-7.50 (m, 3H), 7.85-7.87 (m, 2H), 7.94 (s, 1H), 8.01-8.04 (m,
2H), 8.77 (d, NH).
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
pyridin-2-yl)carbonyl]amino}pentanoic Acid (10d). General proce-
dures A and B afforded 37.9 mg (43%) of product 10d. HRMS
calcd for C24H28N4O6 (Mþ þ H) 469.2082, found 469.2082.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-[(1,10:30,100-
terphenyl-50-ylcarbonyl)amino]pentanoic Acid (10e). General pro-
cedures A and B afforded 36.0 mg (29%) of product 10e. HRMS
calcd for C31H33N3O6 (Mþ þ H) 544.2442, found 544.2420.
(4S)-4-[(1,10-Biphenyl-3-ylcarbonyl)amino]-5-[4-(ethoxycarbonyl)-
piperazin-1-yl]-5-oxopentanoic Acid (10f). General procedures A and
B afforded 45.0 mg (41%) of product 10f. HRMS calcd for
C25H29N3O6 (Mþ þ H) 468.2129, found 468.2141.
5-tert-Butyl N-[(4-phenylpyridin-2-yl)carbonyl]-L-glutamic Acid
(15). Tetrakis(triphenylphosphine)palladium(0) (300 mg,
0.25 mmol) was added to a solution of 1-allyl 5-tert-butyl-
N-[(4-phenylpyridin-2-yl)carbonyl]-L-glutamate 14 (16.3 g,
38.4 mmol) and morpholine (3.36 mL, 38.4 mmol) in acetonitrile
(200 mL) under nitrogen. The mixture was stirred at room
temperature for 1 h. Upon completion, the product mixture
was filtered through Celite, and the filtrate was concentrated in
vacuo. The residue was purified by reverse-phase HPLC
(10-60% acetonitrile/water with 0.1% TFA modifier) to afford
11.5 g (78%) of product 15. 1H NMR (CDCl3) δ 1.36 (s, 9H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2023
2.11-2.51 (m, 4H), 4.85 (m, 1H), 7.39-7.47 (m, 3H), 7.85 (m,
2H), 8.02-8.01 (m, 3H), 8.91 (d, NH).
Carbamate Formation with tert-Butyl (4S)-5-Oxo-4-{[(6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-piperazin-1-ylpentanoate 18
and Polymer-Supported NHS-Carbonate 25. tert-Butyl (4S)-5-
oxo-4-{[(6-phenylpyridin-2-yl)carbonyl]amino}-5-piperazin-1-
ylpentanoate 18 (0.25 M in dichloromethane) (1 mL, 0.25
mmol) was added to the preswelled polymer-supported NHS-
carbonate 25 in dichloromethane (2 mL). The reaction mixtures
were agitated for 16 h before the vessels were drained and rinsed
with dichloromethane (4 ꢀ 3 mL), and the combined filtrate
and washings were dried in vacuo to afford the tert-butyl ester
products.
Deprotection of the tert-Butyl Ester Group. A solution of 20%
trifluoroacetic acid/dichloromethane solution (8.0 mL) was
added to the residue (∼0.25 mmol) and agitated on an orbital
shaker or stirred at room temperature for 1-24 h. Evaporation
of the solvents afforded the products 26. The products were
purified by HPLC to afford the pure products 26.
Benzyl 4-((2S)-5-tert-Butoxy-5-oxo-2-{[(6-phenylpyridin-2-yl)-
carbonyl]amino}pentanoyl)piperazine-1-carboxylate (17). To a
mixture of 5-tert-butyl N-[(4-phenylpyridin-2-yl)carbonyl]-L-
glutamic acid 15 (11.5 mL, 29.9 mmol), hydroxybenzotriazole
(1.2 g, 3.0 mmol), and N-methylmorpholine (13.1 mL, 119.6
mmol) in dichloromethane/dimethylformamide (140 mL, 13:1)
were added benzyl 1-piperazinecarboxylate 16 (5.8 mL, 29.9
mmol) and polymer-bound carbodiimide reagent 6 (1.27 mmol/
g) (35.3 g, 44.8 mmol). The suspension was agitated for 16 h, and
the resin was filtered and rinsed with dichloromethane (4 ꢀ 10
mL). The filtrate and washings were dried in vacuo to afford
15.8 g (90%) of product 17. 1H NMR (CDCl3) δ 1.42 (s, 9H),
1.90-2.18 (m, 2H), 2.29-2.41 (m, 2H), 3.62 (m, 8H), 5.14 (s,
2H), 5.22 (m, 1H), 7.34 (m, 4H), 7.47 (m, 3H), 7.88 (m, 3H), 8.07
(m, 3H), 8.96 (d, NH).
tert-Butyl (4S)-5-Oxo-4-{[(6-phenylpyridin-2-yl)carbonyl]amino}-
5-piperazin-1-ylpentanoate (18). Benzyl 4-((2S)-5-tert-butoxy-5-
oxo-2-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoyl)pip-
erazine-1-carboxylate 17 (15.8 g, 26.9 mmol) was dissolved in
methanol (200 mL), and the reaction vessel was flushed with
(4S)-5-[4-(Methoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
pyridin-2-yl)carbonyl]amino}pentanoic Acid (26a). General proce-
dure D afforded 28.1 mg (41%) of product 26a. HRMS calcd for
C23H26N4O6 (Mþ þ H) 455.1925, found 455.1912.
(4S)-5-Oxo-4-{[(6-phenylpyridin-2-yl)carbonyl]amino}-5-[4-
(propoxycarbonyl)piperazin-1-yl]pentanoic Acid (26b). General
procedure D afforded 38.9 mg (44%) of product 26b. HRMS
calcd for C25H30N4O6 (Mþ þ H) 483.2238, found 483.2254.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
pyridin-2-yl)carbonyl]amino}pentanoic Acid (26c). General pro-
cedure D afforded 17.3 mg (23%) of product 26c. HRMS calcd
for C26H32N4O6 (Mþ þ H) 497.2395, found 497.2403.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-{[(6-
phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26d). Gener-
al procedure D afforded 17.4 mg (23%) of product 26d. HRMS
calcd for C27H34N4O6 (Mþ þ H) 511.2551, found 511.2543.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-{[(6-
phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26e). Gener-
al procedure D afforded 61.6 mg (64%) of product 26e. HRMS
calcd for C28H36N4O6 (Mþ þ H) 525.2708, found 525.2705.
(4S)-5-{4-[(Heptyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-{[(6-
phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26f). Gener-
al procedure D afforded 45.2 mg (46%) of product 26f. HRMS
calcd for C29H38N4O6 (Mþ þ H) 539.2864, found 539.2901.
(4S)-5-{4-[(Octyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-{[(6-
phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26g). Gener-
al procedure D afforded 66 mg (66%) of product 26g. HRMS
calcd for C30H40N4O6 (Mþ þ H) 553.3021, found 553.3045.
(4S)-5-Oxo-4-{[(6-phenylpyridin-2-yl)carbonyl]amino}-5-{4-
[(2,2,2-trifluoroethoxy)carbonyl]piperazin-1-yl}pentanoic Acid (26h).
General procedure D afforded 110.0 mg (83%) of product 26h.
HRMS calcd for C24H25F3N4O6 (Mþ þ H) 523.1799, found
523.1791.
N
2(g). Then 10% Pd/C (1.6 g, 10% by weight) was added to the
solution, and the reaction vessel was flushed with N2(g) then
2(g). The mixture was stirred for 16 h under a balloon of H2(g)
H
.
The mixture was filtered through a pad of Celite. The filtrate and
cake rinses were dried in vacuo to afford 11.0 g (81%) of product
18. 1H NMR (CDCl3) δ 1.40 (s, 9H), 1.83-2.42 (m, 12H), 5.19
(m, 1H), 7.41-7.50 (m, 3H), 7.82-8.09 (m, 3H), 8.94 (d, NH).
General Procedure C. Reaction of Amine 18 with Electrophiles
19 To Afford Products 20. A solution of tert-butyl (4S)-5-oxo-
4-{[(6-phenylpyridin-2-yl)carbonyl]amino}-5-piperazin-1-ylpen-
tanoate 18 (0.15 M in dichloromethane) (1 mL, 0.15 mmol)
was added to each reaction well containing the electrophile 19
(0.6 mmol) (which included chloroformates, acid chlorides,
isocyanates, thioisocyanates, sulfonyl isocyanates, and sulfonyl
chlorides), triethylamine (125 μL, 0.9 mmol) (wells with chlor-
oformates and acid chlorides only) and dichloromethane (6 mL).
The vials were agitated for 1 to several hours. Upon completion
of the reaction, the product mixture was dried under a stream of
nitrogen gas and, if necessary, diluted with dichloromethane
(8 mL) and scavenged with PS-diethylenetriamine 8 (0.6 mmol).
The resin was filtered and rinsed with dichloromethane (2 ꢀ 6 mL).
The filtrate and washings were dried in vacuo to afford the tert-
butyl ester products.
Deprotection of the tert-Butyl Ester Group. A solution of 20%
trifluoroacetic acid/dichloromethane solution (8.0 mL) was
added to the tert-butyl ester (∼0.15 mmol) and agitated on an
orbital shaker or stirred at room temperature for 1-24 h.
Evaporation of the solvents afforded the crude products. The
products were purified by HPLC to afford the pure products 20.
General Procedure D. Preparation of Piperazine Carbamate
Analogues 26. Preparation of Polymer-Supported NHS-Chloro-
formate (23). Polymer supported N-hydroxysuccinimide resin
21 (1.09 mmol/g) (300 mg, 0.33 mmol) was placed in a Quest 210
(Argonaut) disposable, fritted reaction vessel and swelled with
(4S)-5-[4-(Isopropoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
pyridin-2-yl)carbonyl]amino}pentanoic Acid (26i). General procedure
D afforded 11.5 mg (16%) of product 26i. HRMS calcd for
C25H30N4O6 (Mþ þ H) 483.2238, found 483.2213.
(4S)-5-Oxo-4-{[(6-phenylpyridin-2-yl)carbonyl]amino}-5-{4-
[(4,4,4-trifluorobutoxy)carbonyl]piperazin-1-yl}pentanoic Acid (26j).
General procedure D afforded 89.4 mg (64%) of product 26j.
HRMS calcd for C26H29F3N4O6 (Mþ þ H) 551.2112, found
551.2075.
dichloromethane (2 mL). Triphosgene 22 (0.72
M in
dichloromethane) (1 mL, 0.72 mmol) and pyridine (32 uL,
0.40 mmol) were delivered to the reaction vessels followed by
agitation for 1 h. Upon completion of the reaction, the vessels
were drained and rinsed with dichloromethane (4 ꢀ 3 mL) to
remove excess reagents to afford polymer-supported NHS-
chloroformate 23.
Preparation of Polymer-Supported NHS-Carbonates (25).
The alcohol 24 (0.6 M in dichloromethane) (4 mL, 2.4 mmol)
and pyridine (32 μL, 0.40 mmol) were added to the polymer-
supported NHS-chloroformate 23. The reaction mixtures were
agitated for 2 h before the vessels were drained and rinsed with
dichloromethane (4 ꢀ 3 mL) to afford polymer-supported NHS-
carbonate 25.
(4S)-5-{4-[(But-3-yn-1-yloxy)carbonyl]piperazin-1-yl}-5-oxo-
4-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26k).
General procedure D afforded 47.3 mg (52%) of product 26k.
HRMS calcd for C26H28N4O6 (Mþ þ H) 493.2082, found
493.2094.
(4S)-5-{4-[(Cyclopentyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-
{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26l). Gen-
eral procedure D afforded 38.0 mg (30%) of product 26l. HRMS
calcd for C27H32N4O6 (Mþ þ H) 509.2395, found 509.2405.
(4S)-5-{4-[(Cyclohexyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-{[(6-
phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26m). General

2024 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
procedure D afforded 54.3 mg (41%) of product 26m. HRMS calcd
for C28H34N4O6 (Mþ þ H) 523.2551, found 523.2530.
(4S)-5-{4-[(Cyclobutylmethoxy)carbonyl]piperazin-1-yl}-5-oxo-
4-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26n).
General procedure D afforded 38.5 mg (30%) of product 26n.
HRMS calcd for C27H32N4O6 (Mþ þ H) 509.2395, found
509.2432.
(4S)-5-{4-[(Cyclopentylmethoxy)carbonyl]piperazin-1-yl}-5-
oxo-4-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26o).
General procedure D afforded 55.2 mg (42%) of product 26o.
HRMS calcd for C28H34N4O6 (Mþ þ H) 523.2551, found
523.2541.
(4S)-5-{4-[(2-Cyclopropylethoxy)carbonyl]piperazin-1-yl}-5-
oxo-4-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26p).
General procedure D afforded 20.9 mg (16%) of product 26p.
HRMS calcd for C27H32N4O6 (Mþ þ H) 509.2395, found
509.2400.
(4S)-5-{4-[(3-Methylbutoxy)carbonyl]piperazin-1-yl}-5-oxo-
4-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26q).
General procedure D afforded 90.2 mg (16%) of product 26q.
HRMS calcd for C27H34N4O6 (Mþ þ H) 511.2551, found
511.2559.
(4S)-5-{4-[(3,3-Dimethylbutoxy)carbonyl]piperazin-1-yl}-5-oxo-
4-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26r).
General procedure D afforded 80.7 mg (61%) of product 26r.
HRMS calcd for C28H36N4O6 (Mþ þ H) 525.2708, found
525.2696.
(4S)-5-{4-[(2-Methoxyethoxy)carbonyl]piperazin-1-yl}-5-oxo-
4-{[(6-phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26s).
General procedure D afforded 23.6 mg (32%) of product 26s.
HRMS calcd for C25H30N4O7 (Mþ þ H) 499.2187, found
499.2182.
the mixture was stirred for 30 min, the slurry was cooled in an
ice-water bath and then acidified with 3 N HCl to pH 5. The
crude product was isolated by vacuum filtration and washed
with water (1 ꢀ 100 mL) and diethyl ether (3 ꢀ 50 mL). Residual
solvents were removed in vacuo to afford 7.83 g (95%) of
product 30. 1H NMR (DMSO-d6) δ 8.31 (d, 1H, J = 1.7 Hz),
8.20 (m, 2H), 7.97 (d, 1H, J = 1.7 Hz), 7.52 (m, 3H); MS (ESIþ)
m/z 234 (M þ H).
4-Azido-6-phenylpyridine-2-carboxylate (31). To a solution of
4-chloro-6-phenylpyridine-2-carboxylate 30 (200 mg, 0.83 mmol)
and Aliquat 336 (33.5 mg, 0.083 mmol) in ethanol/water (2 mL/
1 mL) was slowly added sodium azide (65.0 mg, 1.0 mmol), and
the mixture was heated to 100 °C for 1 h. After cooling, the
mixture was poured onto ice/water to provide a solid. The
precipitatewas filtered, washed withwater, anddried ina vacuum
oven overnight to provide 163 mg (79%) of product 31. 1H NMR
(methanol-d4) δ 4.53 (br s, 1H), 7.35-7.43 (m, 4H), 7.61 (m, 1H),
8.04 (d, 2H, J = 4.0 Hz).
General Procedure E. Coupling of tert-Butyl Piperazine-1-
carboxylate 32 with Chloroformates 33. Piperazine-1,4-dicar-
boxylic Acid tert-Butyl Ester Butyl Ester (34b). To a mixture
of tert-butyl piperazine-1-carboxylate 32 (5.0 g, 26.8 mmol) and
N,N-diisopropylethylamine (5.14 mL, 29.5 mmol) in anhydrous
methylene chloride (50 mL) at 0 °C was added n-butyl chlor-
oformate 33b (4.45 g, 29.5 mmol) dropwise over 15 min. The
mixture warmed to room temperature and stirred for 3 h. The
mixture was poured into 10% aqueous citric acid (200 mL), and
the organic layer was isolated, dried over sodium sulfate,
filtered, and concentrated to give 8.05 g (99%) of product 35b.
1H NMR (CDCl3) δ 4.08 (t, 2H, J = 6.7 Hz), 3.42 (m, 8H), 1.63
(m, 2H), 1.47 (s, 9H), 1.33 (m, 2H), 0.91 (t, 2H, J = 6.5 Hz).
Piperazine-1,4-dicarboxylic Acid tert-Butyl Ester Pentyl Ester
(34c). General procedure E afforded 3.40 g (100%) of product
33. 1H NMR (CDCl3) δ 4.09 (m, 2H), 3.43 (m, 8H), 1.63 (m, 2H),
1.47 (s, 9H), 1.26-1.36 (m, 4H), 0.91 (m, 3H).
(4S)-5-{4-[(Benzyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-{[(6-
phenylpyridin-2-yl)carbonyl]amino}pentanoic Acid (26t). Gen-
eral procedure D afforded 20 mg (45%) of product 26t. 1H
NMR (CD3OD) 1.94-1.99 (m, 1H), 2.17-2.20 (m, 1H),
2.41-2.45 (m, 2H), 3.40-3.74 (m, 6H), 3.76 (br s 2H), 5.11 (s,
2H), 5.21 (m, 1H), 7.25-7.33 (m, 5H), 7.44-7.50 (m, 3H),
7.99-8.03 (m, 3H), 8.12 (d, 2 H, J = 8.0 Hz); HRMS calcd for
C29H30N4O6 (Mþ þ H) 531.2244, found 531.2258.
Methyl 4-Hydroxy-6-phenylpyridine-2-carboxylate (28). A
solution of 4-hydroxy-6-phenylpyridine-2-carboxylic acid 27
(15.1 g, 70.2 mmol), concentrated sulfuric acid (14 mL), and
methanol (400 mL) was heated to reflux for 24 h with the
condensate passing through a 100 mL addition funnel filled
Piperazine-1,4-dicarboxylic Acid tert-Butyl Ester Hexyl Ester
(34d). General procedure E afforded crude product 34d. 1H
NMR (CDCl3) δ 4.06 (m, 2H), 3.40 (m, 8H), 1.62 (m, 2H), 1.44
(s, 9H), 1.26-1.36 (m, 6H), 0.87 (m, 3H).
General Procedure F. Boc Deprotection. Butyl Piperazine-1-
carboxylate (35b). Method A. To a solution of piperazine-1,4-
dicarboxylic acid tert-butyl ester butyl ester 34b (7.67 g,
26.8 mmol) in methylene chloride (50 mL) was added trifluor-
oacetic acid (25.0 mL, 336 mmol). The mixture was stirred for 3
h at room temperature. The mixture was concentrated to an oily
residue in vacuo and dissolved in methylene chloride (20 mL),
and sodium carbonate (25% by weight in water) was added with
vigorous stirring until the aqueous phase reached pH 10. The
organic phase was separated, dried over sodium sulfate, filtered,
˚
with 3 A molecular sieves. Upon completion of the reaction, the
solution was cooled to ambient temperature and neutralized
with solid sodium bicarbonate (80 g) and then filtered. The
filtrate was dried in vacuo and purified on silica gel to afford 15.3
g (95%) of product 28. Mp 49-52 °C; 1H NMR (DMSO-d6) δ
11.19 (s, 1H), 8.05 (m, 2H), 7.42-7.54 (m, 5H), 3.90 (s, 3H); MS
(ESIþ) m/z 230 (M þ H).
1
and dried in vacuo to afford 4.94 g (99%) of product 34b. H
NMR (CDCl3) δ 4.08 (t, 2H, J = 6.7 Hz), 3.43 (t, 4H, J = 5.0
Hz), 2.82 (t, 4H, J = 5.0 Hz), 1.63 (m, 2H), 1.37 (m, 2H), 0.93 (t,
3H, J = 6.4 Hz); MS (ESIþ) m/z 187 (M þ H).
Methyl 4-Chloro-6-phenylpyridine-2-carboxylate Hydrochlor-
ide (29). A mixture of methyl 4-hydroxy-6-phenylpyridine-2-
carboxylate 28 (7.56 g, 33.0 mmol) and phosphorus oxychloride
(70 mL) was heated to reflux under nitrogen for 20 h. The
reaction mixture was cooled to ambient temperature and dried
in vacuo. In an ice-water bath, methanol (70 mL) was slowly
added to the reaction residue. Once this addition was complete,
the resulting solution was concentrated to ∼30 mL and diluted
with water (100 mL). The resulting precipitate was collected by
vacuum filtration, washed with water (50 mL), and dried in
Method B: Pentyl Piperazine-1-carboxylate Hydrochloride
(35c). To a solution of piperazine-1,4-dicarboxylic acid tert-
butyl ester pentyl ester 34c (1.77 g, 5.90 mmol) in methylene
chloride (15 mL) was added hydrochloric acid (4 M in 1,4-
dioxane) (15.0 mL, 60.0 mmol). The mixture was stirred at room
temperature for 2 h and was dried in vacuo to afford 1.20 g
(87%) of product 35c. 1H NMR (CDCl3) δ 10.00 (br s, 1H), 4.12
(m, 2H), 3.84 (s, 4H), 3.21 (s, 4H), 1.64 (m, 2H), 1.33 (m, 4H),
0.91 (m, 3H); MS (ESIþ) m/z 201 (M þ H).
1
vacuo to give 7.68 g (82%) of product 29. Mp 86-88 °C; H
Hexyl Piperazine-1-carboxylate Hydrochloride (35d). General
procedure F, method B, afforded 41.7 g (99%) of product 35d.
1H NMR (CDCl3) δ 4.09 (m, 2H), 3.83 (s, 4H), 3.19 (s, 4H), 1.71
(m, 2H), 1.61 (m, 2H), 1.29 (m, 4H), 0.88 (m, 3H).
NMR (CDCl3) δ 8.03 (m, 3H), 7.88 (d, 1H, J = 1.8 Hz), 7.48
(m, 3H), 4.03 (s, 3H); MS (ESIþ) m/z 248 (M þ H).
4-Chloro-6-phenylpyridine-2-carboxylate (30). To a solution
of methyl 4-chloro-6-phenylpyridine-2-carboxylate hydrochlor-
ide 29 (10.0 g, 35.2 mmol) in dioxane (50 mL) was slowly added
potassium hydroxide (0.26 M in water) (40 mL, 105.6 mmol).
Additional water (50 mL) was added to facilitate stirring. After
General Procedure G. Coupling of Piperazines 35 with
N-Benzyloxycarbonyl-L-glutamic Acid γ-tert-Butyl Ester 36.
Ethyl 4-((2S)-2-{[(Benzyloxy)carbonyl]amino}-5-tert-butoxy-
5-oxopentanoyl)piperazine-1-carboxylate (37a). To a solution

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2025
of N-benzyloxycarbonyl-L-glutamic acid γ-tert-butyl ester 36
(53.0 g, 0.16 mol) in dichloromethane (250 mL) chilled to 5 °C
with an ice/water bath were added hydroxybenzotriazole
(24.4 g, 0.18 mol) and 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide hydrochloride (36.0 g, 0.19 mol). N-Methyl-
morpholine (21.0 mL, 0.19 mol) was introduced into the flask
in three portions, and stirring continued for 20 min. Ethyl 1-
piperazinecarboxylate 35a (27.6 mL, 0.19 mol) was added
dropwise to the reaction mixture, keeping the temperature
between 5 and 11 °C. The mixture came to room temperature,
was stirred for 16 h, and was dried in vacuo. The residue was
diluted with ethyl acetate (500 mL) and then washed with
water (2 ꢀ 250 mL), citric acid (10% by weight in water) (2 ꢀ
150 mL), sodium bicarbonate (saturated in water) (2 ꢀ 500
mL), and brine (2 ꢀ 150 mL). The organic phase was dried
over sodium sulfate and dried in vacuo. The crude residue
was purified on silica gel (50-100% ethyl acetate/heptane) to
afford 62.3 g (83% yield) of product 37a. 1H NMR (DMSO-
d6) δ 7.54 (d, 1H, J = 8.2 Hz), 7.33 (m, 5H), 5.02 (s, 2H), 4.47
(m, 1H), 4.03 (q, 2H, J = 7.1 Hz), 3.26-3.51 (m, 8H), 2.25 (t,
2H, J = 7.0 Hz), 1.80 (m, 1H), 1.67 (m, 1H), 1.39 (s, 9H), 1.19
(t, 3H, J = 7.1 Hz); MS (APCIþ) m/z 478 (M þ H).
(t, 2H, J = 6.7 Hz), 3.76 (d, 1H, J = 6.0 Hz), 3.40-3.72 (m, 8H),
2.54 (m, 1H), 2.35 (m, 1H), 1.90 (br s, 3H), 1.64 (m, 2H), 1.55 (m,
1H), 1.44 (s, 9H), 1.34 (m, 4H), 0.91 (t, 3H, J = 6.4 Hz); MS
(ESIþ) m/z 386 (M þ H).
Hexyl 4-[(2S)-2-Amino-5-tert-butoxy-5-oxopentanoyl]pipera-
zine-1-carboxylate (38d). General procedure H afforded 85.0 g
(85%) of a clear oil of product 38d. 1H NMR (DMSO-d6) δ ppm
0.83 (t, 3H, J = 6.7 Hz), 1.24 (m, 6H), 1.36 (s, 9H), 1.53 (m, 3H),
1.68 (m, 1H), 2.30 (m, 2H), 3.47 (m, 10H), 3.71 (m, 1H), 3.97 (t,
2 H, J = 6.6 Hz).
General Procedure I. Coupling of Amines 38 with Pyridine
Acids. Ethyl 4-((2S)-5-tert-Butoxy-2-{[(4-hydroxy-6-phenylpyr-
idin-2-yl)carbonyl]amino}-5-oxopentanoyl)piperazine-1-carbox-
ylate (39). To a mixture of 4-hydroxy-6-phenylpyridine-2-
carboxylic acid hydrochloride salt 27 (3.0 g, 7.95 mmol), 1-
hydroxybenzotriazole (0.215 g, 1.59 mmol), and N-methylmor-
pholine (2.6 mL, 23.8 mmol) in anhydrous dichloromethane/
dimethylformamide (100 mL, 20:1) were added ethyl 4-[(2S)-2-
amino-5-tert-butoxy-5-oxopentanoyl]piperazine-1-carboxylate
38a (3.0 g, 7.95 mmol) and polymer-bound carbodiimide reagent
6 (1.3 mmol/g, 9.9 g, 12.7 mmol). The suspension was agitated
for 16 h. Upon completion of the reaction, the react-
ion mixture was filtered and rinsed with dichloromethane (4 ꢀ
10 mL) and concentrated in vacuo. Compound was purified on
silica gel (30-50% ethyl acetate/hexanes with 5.0% methanol)
Butyl (S)-4-(2-Benzyloxycarbonylamino-4-tert-butoxycarbo-
nylbutyryl)piperazine-1-carboxylate (37b). General procedure
G afforded 4.27 g (86%) of product 37b. 1H NMR (CDCl3) δ
7.32 (m, 5H), 5.79 (d, 1H, J = 8.3 Hz), 5.10 (m, 2H), 4.73 (m,
1H), 4.10 (t, 2H, J = 6.7 Hz), 3.40-3.72 (m, 8H), 2.30 (m, 2H),
1.97 (m, 1H), 1.70 (m, 1H), 1.64 (m, 2H), 1.45 (s, 9H), 1.38 (m,
2H), 0.95 (t, 3H, J = 6.4 Hz); MS (ESIþ) m/z 506 (M þ H).
Pentyl (S)-4-(2-Benzyloxycarbonylamino-4-tert-butoxycarbo-
nylbutyryl)piperazine-1-carboxylate (37c). General procedure G
afforded 4.24 g (92%) of product 37c. 1H NMR (CDCl3) δ 7.32
(m, 5H), 5.69 (d, 1H, J = 8.3 Hz), 5.10 (m, 2H), 4.73 (m, 1H),
4.10 (t, 2H, J = 6.7 Hz), 3.40-3.70 (m, 8H), 2.30 (m, 2H), 1.97
(m, 1H), 1.70 (m, 1H), 1.64 (m, 2H), 1.45 (s, 9H), 1.34 (m, 4H),
0.92 (t, 3H, J = 6.4 Hz); MS (ESIþ) m/z 520 (M þ H).
1
to afford 3.72 g (87%) of product 39. H NMR (DMSO-d6) δ
ppm 1.19 (t, 3H, J = 7.12 Hz), 1.34 (s, 9H), 1.77-1.96 (m, 1H,
J = 7.79 Hz), 1.93-2.12 (m, 1H), 2.19-2.39 (m, 2H), 3.20-3.54
(m, 5H), 3.52-3.77 (m, 3H), 4.06 (q, 2H, J = 6.98 Hz),
4.80-5.15 (m, 1H), 7.34-7.63 (m, 5H), 8.01-8.23 (m, 2H),
8.95 (d, 1H, J = 8.06 Hz), 11.13 (s, 1H); HRMS calcd for
C28H35N4O7 (Mþ þ H) 541.2657, found 541.2628.
Butyl 4-((2S)-5-tert-Butoxy-2-{[(4-hydroxy-6-phenylpyridin-
2-yl)carbonyl]amino}-5-oxopentanoyl)piperazine-1-carboxylate
(40). General procedure I afforded 11.5 g (74%) of product 40.
1H NMR (DMSO-d6) δ ppm 0.72-1.07 (m, 3H), 1.19-1.45 (m,
11H), 1.47-1.66 (m, 2H), 1.69-1.94 (m, 1H), 1.97-2.16 (m,
1H), 2.15-2.43 (m, 2H), 3.19-3.79 (m, 8H), 4.01 (t, 2H, J =
6.44 Hz), 4.90-5.14 (m, 1H), 7.34-7.64 (m, 5H), 8.06-8.24 (m,
2H), 8.95 (d, 1H, J = 8.32 Hz), 11.11 (s, 1H); HRMS calcd for
C30H41N4O7 (Mþ þ H) 569.2970, found 569.2957.
Hexyl 4-[(2S)-2-{[(Benzyloxy)carbonyl]amino}-5-tert-butoxy-
5-oxopentanoyl]piperazine-1-carboxylate (37d). General proce-
dure G afforded 95.5 g (99%) of product 37d. 1H NMR
(DMSO-d6) δ ppm 0.84 (t, 3H, J = 6.7 Hz), 1.25 (m, 6 H),
1.36 (s, 9 H), 1.54 (m, 2 H), 1.65 (m, 1 H), 1.80 (m, 1 H), 2.23 (m,
2H), 3.48 (m, 8H), 4.00 (t, 2H, J = 6.6 Hz), 4.44 (m, 1H), 4.99 (s,
2H), 7.31 (m, 5H), 7.50 (d, 1H, J = 8.2 Hz).
Pentyl 4-((2S)-5-tert-Butoxy-2-{[(4-hydroxy-6-phenylpyridin-
2-yl)carbonyl]amino}-5-oxopentanoyl)piperazine-1-carboxylate
(41). General procedure I afforded 0.45 g (59%) of product 41.
1H NMR (DMSO-d6) δ ppm 0.84 (t, 3H), 1.15-1.41 (m, 13H),
1.44-1.65 (m, 2H), 1.72 1.89 (m, 1H), 1.90-2.11 (m, 1H),
2.16-2.36 (m, 2H), 3.05-3.70 (m, 8H), 3.97 (t, 2H, J = 6.58
Hz), 4.84-5.16 (m, 1H), 7.23-7.62 (m, 5H), 7.99-8.21 (m, 2H),
8.91 (d, 1H, J = 8.06 Hz), 11.10 (s, 1H); HRMS calcd for
C31H43N4O7 (Mþ þ H) 583.3126, found 583.3094.
General Procedure H. Cbz Deprotection. Ethyl 4-[(2S)-2-
Amino-5-tert-butoxy-5-oxopentanoyl]piperazine-1-carboxylate
(38a). To a 2.5 L Parr shaker bottle were added ethyl 4-((2S)-
2-{[(benzyloxy)carbonyl]amino}-5-tert-butoxy-5-oxopentanoyl-
piperazine-1-carboxylate 37a (62.3 g, 130 mmol), ethanol (700 mL),
and 10% Pd/C (3.6 g, 5% by weight). The mixture was stirred for
16 h under 40 psi of H2(g). The reaction mixture was filtered through
Celite and the filtrate dried in vacuo, dissolved in dichloromethane
(500 mL), and treated with hydrochloric acid (1 N in diethyl ether)
(144 mL, 144 mmol) under N2(g) at 0 °C. The reaction mixture was
concentrated in vacuo and the residue triturated with heptane. A
solid was collected by vacuum filtration, washed with heptane, and
dried in vacuo to afford 43.1 g (92% yield) of product 38a. 1H NMR
(DMSO-d6) δ 4.05 (q, 2H, J = 7.1 Hz), 3.61 (m, 1H), 3.32-3.51
(m, 8H), 2.30 (m, 2H), 1.68 (m, 1H), 1.44 (m, 1H), 1.41 (s, 9H), 1.19
(t, 3H, J = 7.1 Hz); MS (APCIþ) m/z 344 (M þ H); mp =
137.5-141.2 °C (dec).
Hexyl 4-((2S)-5-tert-Butoxy-2-{[(4-hydroxy-6-phenylpyridin-
2-yl)carbonyl]amino}-5-oxopentanoyl)piperazine-1-carboxylate
(42). General procedure I afforded 1.35 g (71%) of product 42.
1H NMR (DMSO-d6) δ ppm 0.86 (t, 3H), 1.03-1.43 (m, 15H),
1.46-1.66 (m, 2H), 1.84 (d, 1H, J = 6.98 Hz), 1.91-2.14 (m,
1H), 2.20-2.40 (m, 2H), 3.20-3.73 (m, 8H), 4.00 (t, 2H, J =
6.71 Hz), 4.85-5.14 (m, 1H), 7.38-7.60 (m, 5H), 8.05-8.18 (m,
2H), 8.95 (d, 1H, J = 8.32 Hz), 11.13 (s, 1H); HRMS calcd for
C32H45N4O7 (Mþ þ H) 597.3283, found 597.3276.
Ethyl 4-[(2S)-5-tert-Butoxy-2-{[(4-chloro-6-phenylpyridin-2-
yl)carbonyl]amino}-5-oxopentanoyl]piperazine-1-carboxylate (43a).
General procedure I using 4-chloro-6-phenylpyridine-2-car-
boxylic acid 30 (0.68 g, 2.90 mmol), 1-hydroxybenzotriazole
(3.68 g, 4.70 mmol), polymer-bound carbodiimide reagent 6
(1.3 mmol/g, 3.68 g, 4.70 mmol), ethyl 4-[(2S)-2-amino-5-tert-
butoxy-5-oxopentanoyl]piperazine-1-carboxylate 38a (84 mg,
0.62 mmol), and N-methylmorpholine (1.0 mL, 9.1 mmol) to
afford 0.98 g (61%) of product 43a. 1H NMR (CDCl3) δ ppm
1.26 (t, 3H, J = 7.12 Hz), 1.43 (s, 9H), 1.89 (m, 1H), 2.14 (m,
1H), 2.34 (m, 2H), 3.58 (m, 8H), 4.15 (q, 2H, J = 7.25 Hz), 5.20
Butyl (S)-4-(2-Amino-4-tert-butoxycarbonylbutyryl)piperazine-
1-carboxylate (38b). General procedure H afforded 3.11 g (99%)
1
of a light-yellow oil of product 38b. H NMR (CDCl3) δ 4.11
(t, 2H, J = 6.7 Hz), 3.76 (d, 1H, J = 6.0 Hz), 3.40-3.72 (m, 8H),
2.54 (m, 1H), 2.35 (m, 1H), 1.99 (br s, 3H), 1.90 (m, 1H), 1.64 (m,
2H), 1.44 (s, 9H), 1.40 (m, 2H), 0.95 (t, 3H, J = 6.4 Hz); MS
(ESIþ) m/z 372 (M þ H).
Pentyl (S)-4-(2-Amino-4-tert-butoxycarbonylbutyryl)piperaz-
ine-1-carboxylate (38c). General procedure H afforded 3.24 g
(94%) of a yellow oil of product 38c. 1H NMR (CDCl3) δ 4.10

2026 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
(m, 1H), 7.49 (m, 3H), 7.85 (d, 1H, J = 1.61 Hz), 8.03 (m, 2H),
8.09 (d, 1H, J = 1.61 Hz), 8.86 (d, 1H, J = 8.59 Hz).
Butyl 4-[(2S)-5-tert-Butoxy-2-{[(4-chloro-6-phenylpyridin-2-
yl)carbonyl]amino}-5-oxopentanoyl]piperazine-1-carboxylate (43b).
General procedure I afforded 5.47 g (92%) of product 43b. 1H
NMR (CDCl3) δ ppm 0.96 (t, 3H, J = 7.25 Hz), 1.35-1.44 (m,
2H), 1.46 (s, 9H), 1.58-1.71 (m, 2H), 1.86-1.98 (m, 1H),
2.11-2.24 (m, 1H), 2.28-2.50 (m, 2H), 3.41-3.79 (m, 8H),
4.12 (t, 2H, J = 6.58 Hz), 5.18-5.28 (m, 1H), 7.45-7.57 (m,
3H), 7.88 (d, 1H, J = 1.88 Hz), 8.03-8.09 (m, 2H), 8.11 (d, 1H,
J = 1.88 Hz), 8.88 (d, 1H, J = 8.59 Hz).
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-[({4-[(2-hydroxy-
ethyl)(methyl)amino]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-
pentanoic Acid (45f). General procedure J afforded 41.7 mg (39%)
of product 45f. HRMS calcd for C27H35N5O7 (Mþ þ H) 542.2609,
found 542.2582.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-[({4-[(2-methoxy-
ethyl)amino]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxopenta-
noic Acid (45g). General procedure J afforded 81.9 mg (76%) of
product 45g. HRMS calcd for C27H35N5O7 (Mþ þ H) 542.2609,
found 542.2574.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
4-piperidin-1-ylpyridin-2-yl)carbonyl]amino}pentanoic Acid (45i).
General procedure J afforded 103 mg (94%) of product 45i.
HRMS calcd for C29H37N5O6 (Mþ þ H) 552.2817, found
552.2800.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-{[(4-morpholin-
4-yl-6-phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid
(45j). General procedure J afforded 35.2 mg (75%) of product
45j. HRMS calcd for C28H35N5O7 (Mþ þ H) 554.2609, found
554.2628.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-({[4-(4-hydroxy-
piperidin-1-yl)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopen-
tanoic Acid (45k). General procedure J afforded 83.2 mg (74%)
of product 45k. HRMS calcd for C29H37N5O7 (Mþ þ H)
568.2766, found 568.2758.
(4S)-4-({[4-(4-Aminopiperidin-1-yl)-6-phenylpyridin-2-yl]car-
bonyl}amino)-5-[4-(ethoxycarbonyl)piperazin-1-yl]-5-oxopenta-
noic Acid (45m). General procedure J afforded 20.6 mg (56%)
of product 45m. HRMS calcd for C29H38N6O6 (Mþ þ H)
567.2931, found 567.2927.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(4-pyrrolidin-1-ylpiperidin-1-yl)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (45p). General procedure J afforded 33.2 mg (82%) of product
45p. HRMS calcd for C33H44N6O6 (Mþ þ H) 621.3400, found
621.3428.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-[({4-[4-(hydroxy-
methyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-
oxopentanoic Acid (45r). General procedure J afforded 60.2 mg
(52%) of product 45r. HRMS calcd for C30H39N5O7 (Mþ þ H)
582.2922, found 582.2940.
Pentyl 4-((2S)-5-tert-Butoxy-2-{[(4-chloro-6-phenylpyridin-2-
yl)carbonyl]amino}-5-oxopentanoyl)piperazine-1-carboxylate (43c).
General procedure I afforded 12.2 g (96%) of product 43c. 1H
NMR (CDCl3) δ ppm 0.89 (t, 3H, J = 6.8 Hz), 1.21-1.40 (m,
4H), 1.42 (s, 9H), 1.59-1.66 (m, 2H), 1.85-1.91 (m, 1H),
2.11-2.43 (m, 3H), 3.42-3.69 (m, 8H), 4.08 (t, 2H, J = 6.58
Hz), 5.17-5.22 (m, 1H), 7.43-7.52 (m, 3H), 7.84-8.08 (m, 4H),
8.90 (d, 1H, J = 8.40 Hz); HRMS calcd for C31H41ClN4O6
(Mþ þ H) 601.2787, found 601.2750.
Hexyl 4-[(2S)-5-tert-Butoxy-2-{[(4-chloro-6-phenylpyridin-2-
yl)carbonyl]amino}-5-oxopentanoyl]piperazine-1-carboxylate (43d).
General procedure I afforded 8.02 g (65%) of product 43d. 1H
NMR (CDCl3) δ ppm 0.84 0.96 (m, 3H), 1.26-1.43 (m, 6H),
1.46 (s, 9H), 1.56-1.72 (m, 2H), 1.84-2.00 (m, 1H), 2.11-2.25
(m, 1H), 2.27-2.51 (m, 2H), 3.39-3.81 (m, 8H), 4.11 (t, 2H, J =
6.71 Hz), 5.15-5.29 (m, 1H), 7.46-7.57 (m, 3H), 7.88 (d, 1H,
J = 1.88 Hz), 8.04-8.08 (m, 2H), 8.12 (d, 1H, J = 1.61 Hz), 8.88
(d, 1H, J = 8.59 Hz).
General Procedure J. Displacement of 4-Chloropyridines
43a-d with Amine 44. A solution of the 4-chloropyridine inter-
mediate 43 (0.09 M in DMSO) (2 mL, 0.18 mmol) was added to
each secondary amine (0.80 mmol) or primary amine (2.0 mmol)
in a vial, followed by the addition of triethylamine (with
secondary amine, 0.1 mL, 0.72 mmol) (with primary amine,
0.3 mL, 2.2 mmol). The mixtures were agitated at 50-100 °C for
16-168 h until judged complete or no longer progressing by LC/
MS analysis. The reaction solutions were concentrated in vacuo.
If necessary, the residue was purified by HPLC.
Deprotection of the tert-Butyl Ester Group. A solution of 10%
trifluoroacetic acid/dichloromethane solution (1.5 mL) was
added to the tert-butyl ester agitated on an orbital shaker or
stirred at room temperature for 1-24 h. Evaporation of the
solvents afforded the crude products. The products were pur-
ified by HPLC to afford the pure products 45-48.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-[({4-[4-(methoxy-
methyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-
oxopentanoic Acid (45s). General procedure J afforded 91.7 mg
(78%) of product 45s. HRMS calcd for C31H41N5O7 (Mþ þ H)
596.3079, found 596.3062.
(4S)-4-[({4-[4-(Aminomethyl)piperidin-1-yl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-[4-(ethoxycarbonyl)piperazin-1-yl]-5-ox-
opentanoic Acid (45u). General procedure J afforded 93.8 mg
(82%) of product 45u. HRMS calcd for C30H40N6O6 (Mþ þ H)
581.3082, found 581.3080.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-({[4-(methylamino)-
6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic Acid (45a).
General procedure J afforded 33.0 mg (85%) of product 45a.
HRMS calcd for C25H31N5O6 (Mþ þ H) 498.2347, found
498.2316.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[(2-methoxy-
ethyl)amino]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxopenta-
noic Acid (46g). General procedure J afforded 46.1 mg (67%) of
product 46g. HRMS calcd for C29H39N5O7 (Mþ þ H) 570.2928,
found 570.2953.
(4S)-4-({[4-(Dimethylamino)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-[4-(ethoxycarbonyl)piperazin-1-yl]-5-oxopentanoic Acid
(45b). General procedure J afforded 36.1 mg (82%) of product
45b. HRMS calcd for C26H33N5O6 (Mþ þ H) 512.2504, found
512.2512.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[(2-methoxy-
ethyl)(methyl)amino]-6-phenylpyridin-2-yl}carbonyl)amino]-5-
oxopentanoic Acid (46h). General procedure J afforded 62.5
mg (90%) of product 46h. HRMS calcd for C30H41N5O7
(Mþ þ H) 584.3084, found 584.3068.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phen-
yl-4-(propylamino)pyridin-2-yl]carbonyl}amino)pentanoic Acid (45c).
General procedure J afforded 101.7 mg (97%) of product 45c.
HRMS calcd for C27H35N5O6 (Mþ þ H) 526.2660, found
526.2631.
(4S)-4-({[4-(Dipropylamino)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-[4-(ethoxycarbonyl)piperazin-1-yl]-5-oxopentanoic Acid
(45d). General procedure J afforded 21.4 mg (19%) of product
45d. HRMS calcd for C30H41N5O6 (Mþ þ H) 568.3130, found
568.3120.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-morpholin-
4-yl-6-phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid
(46j). General procedure J afforded 54.6 mg (78%) of product
46j. HRMS calcd for C30H39N5O7 (Mþ þ H) 582.2928, found
582.2923.
(4S)-4-({[4-(4-Aminopiperidin-1-yl)-6-phenylpyridin-2-yl]car-
bonyl}amino)-5-[4-(butoxycarbonyl)piperazin-1-yl]-5-oxopenta-
noic Acid (46m). General procedure J afforded 18 mg (%) of
product 46m. HRMS calcd for C31H42N6O6 (Mþ þ H)
595.3244, found 595.3206.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-[({4-[(2-hydro-
xyethyl)amino]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxopen-
tanoic Acid (45e). General procedure J afforded 62.7 mg (75%)
of product 45e. HRMS calcd for C26H33N5O7 (Mþ þ H)
528.2453, found 528.2477.

Article
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[4-(methyl-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2027
2.28 (m, 2H), 3.05 (t, 2H), 3.33-3.99 (m, 10H), 3.97 (t, 2H), 4.18
(d, 2H), 4.99 (m, 1H), 7.43-7.51 (m, 5H), 7.91 (d, 2H), 8.12 (d,
2H), 8.98 (d, 2H); HRMS calcd for C32H44N6O6 (Mþ þ H)
609.3395, found 609.3361.
amino)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-
oxopentanoic Acid (46n). General procedure J afforded 107.6
mg (100%) of product 46n. HRMS calcd for C32H44N6O6
(Mþ þ H) 609.3400, found 609.3439.
(4S)-4-[({4-[4-(Methylamino)piperidin-1-yl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]pipera-
zin-1-yl}pentanoic Acid (47n). General procedure J afforded
64.2 mg (58%) of product 47n. HRMS calcd for C33H46N6O6
(Mþ þ H) 623.3552, found 623.3531.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[4-(dimethyl-
amino)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-
oxopentanoic Acid (46o). General procedure J afforded 33.8
mg (46%) of product 46o. HRMS calcd for C33H46N6O6
(Mþ þ H) 623.3557, found 623.3586.
(4S)-4-[({4-[4-(Dimethylamino)piperidin-1-yl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (47o). General procedure J afforded 40.2 mg
(27%) of product 47o. 1H NMR (DMSO-d6) δ 0.86 (t, 3H), 1.28
(m, 4H), 1.56 (m, 2H), 2.05 (m, 4H), 2.29 (m, 2H), 2.68 (m, 1H),
2.74 (s, 3H), 2.76 (s, 3H), 2.98 (t, 2H), 3.38-3.64 (m, 10H), 3.99
(t, 2H), 4.30 (m, 2H), 5.00 (m, 1H), 7.47-7.53 (m, 5H), 8.16 (d,
2H), 8.97 (d, 1H); HRMS calcd for C34H48N6O6 (Mþ þ H)
637.3708, found 637.3721.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(4-pyrrolidin-1-ylpiperidin-1-yl)pyridin-2-yl]carbonyl}amino)-
pentanoic Acid (46p). General procedure A afforded 31.6 mg
(63%) of product 46p. HRMS calcd for C35H48N6O6 (Mþ þ H)
649.3713, found 649.3708.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{4-[(methyl-
amino)methyl]piperidin-1-yl}-6-phenylpyridin-2-yl)carbonyl]amino}-
5-oxopentanoic Acid (46v). General procedure J afforded 92.7
mg (100%) of product 46v. HRMS calcd for C33H46N6O6
(Mþ þ H) 623.3552, found 623.3560.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-({[6-
phenyl-4-(4-pyrrolidin-1-ylpiperidin-1-yl)pyridin-2-yl]carbonyl}-
amino)pentanoic Acid (47p). General procedure J afforded
66.4 mg (57%) of product 47p. HRMS calcd for C36H50N6O6
(Mþ þ H) 663.3865, found 663.3860.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{4-[(dimethyl-
amino)methyl]piperidin-1-yl}-6-phenylpyridin-2-yl)carbonyl]amino}-
5-oxopentanoic Acid (46w). General procedure J afforded 46.5 mg
(77%) of product 46w. HRMS calcd for C34H48N6O6 (Mþ þ H)
637.3708, found 637.3684.
(4S)-4-({[4-(4-Morpholin-4-ylpiperidin-1-yl)-6-phenylpyridin-
2-yl]carbonyl}amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]pipera-
zin-1-yl}pentanoic Acid (47q). General procedure J afforded 31.2
mg (26.2%) of product 47q. 1H NMR (DMSO-d6) δ 0.84 (t, 3H),
1.26 (m, 4H), 1.54 (m, 4H), 1.48-1.65 (m, 4H), 1.81 (m, 1H),
1.97-2.31 (m, 6H), 2.92-3.15 (m, 6H), 3.29-3.68 (m, 12H),
3.97 (t, 2H), 4.30 (m, 2H), 4.99 (m, 1H), 7.44-7.51 (m, 5H), 8.14
(d, 2H), 8.96 (d, 1H); HRMS calcd for C36H50N6O7 (Mþ þ H)
679.3814, found 679.3828.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-[({6-phenyl-
4-[4-(pyrrolidin-1-ylmethyl)piperidin-1-yl]pyridin-2-yl}carbonyl)
amino]pentanoic Acid (46x). General procedure J afforded 86.1
mg (100%) of product 46x. HRMS calcd for C36H50N6O6
(Mþ þ H) 663.3870, found 663.3882.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-({[6-
phenyl-4-(propylamino)pyridin-2-yl]carbonyl}amino)pentanoic Acid
(47c). General procedure J afforded 59.1 mg (26%) of product
47c. HRMS calcd for C30H41N5O6 (Mþ þ H) 568.3130, found
568.3130.
(4S)-4-[({4-[4-(Hydroxymethyl)piperidin-1-yl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (47r). General procedure J afforded 17 mg
(45%) of product 47r. HRMS calcd for C33H45N5O7 (Mþ þ H)
624.3397, found 624.3342.
(4S)-4-[({4-[(2-Hydroxyethyl)amino]-6-phenylpyridin-2-yl}-
carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-
pentanoic Acid (47e). General procedure J afforded 420 mg
(4S)-4-[({4-[4-(Methoxymethyl)piperidin-1-yl]-6-phenylpyri-
din-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]pipe-
razin-1-yl}pentanoic Acid (47s). General procedure J afforded
60 mg (40%) of product 47s. 1H NMR (DMSO-d6) δ 0.84 (t, 3H),
1.14-1.28 (m, 6H), 1.54 (m, 2H), 1.71-1.85 (m, 4H), 2.01 (m, 1H),
2.93 (t, 2H), 3.17 (d, 2H), 3.20 (s, 3H), 3.37-3.63 (m, 8H), 3.97 (t,
2H), 4.10 (m, 2H), 5.00 (m, 1H), 7.37-7.49 (m, 5H), 8.14 (d, 2H),
8.83 (d, 1H); 13C NMR (DMSO-d6) δ 14.27, 22.21, 27.99, 28.30,
28.58, 29.20, 31.83, 35.96, 41.79, 44.96, 46.34, 48.99, 58.57, 65.40,
77.03, 105.23, 106.64, 127.26, 128.94, 129.42, 139.14, 150.44,
155.05, 156.29, 156.61, 164.37, 170.42, 175.65; HRMS calcd for
C34H47N5O7 (Mþ þ H) 638.3548, found 638.3529; mp = 173 °C.
Optical rotation: [R]2D5 þ32.1° (c 0.01 M, MeOH).
(74%) of product 47e. HRMS calcd for C29H39N5O7 (Mþ
H) 570.2922, found 570.2940.
þ
(4S)-4-[({4-[(2-Methoxyethyl)amino]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-
pentanoic Acid (47g). General procedure J afforded 144.0 mg
(56%) of product 47g. HRMS calcd for C30H41N5O7 (Mþ þ H)
584.3079, found 584.3070.
(4S)-4-[({4-[(2-Methoxyethyl)(methyl)amino]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (47h). General procedure J afforded 55 mg
(77%) of product 47h. HRMS calcd for C31H43N5O7 (Mþ þ H)
598.3240, found 598.3252.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-{[(6-
phenyl-4-piperidin-1-ylpyridin-2-yl)carbonyl]amino}pentanoic Acid
(47i). General procedure J afforded 54.2 mg (23%) of product
47i. HRMS calcd for C32H43N5O6 (Mþ þ H) 594.3286, found
594.3280.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-[({6-
phenyl-4-[4-(propoxymethyl)piperidin-1-yl]pyridin-2-yl}carbonyl)-
amino]pentanoic Acid (47t). General procedure J afforded 30 mg
(39%) of product 47t. HRMS calcd for C36H52N5O7 (Mþ þ H)
666.3861, found 666.3843.
(4S)-4-({[4-(4-Hydroxypiperidin-1-yl)-6-phenylpyridin-2-yl]-
carbonyl}amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-
pentanoic Acid (47k). General procedure J afforded 93.6 mg
(65%) of product 47k. HRMS calcd for C32H43N5O7 (Mþ þ H)
610.3213, found 610.3235.
(4S)-4-[({4-[4-(Aminomethyl)piperidin-1-yl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (47u). General procedure J afforded 109.2
mg (74%) of product 47u. HRMS calcd for C33H46N6O6 (Mþ þ
H) 632.3552, found 623.3549.
(4S)-4-({[4-(4-Methoxypiperidin-1-yl)-6-phenylpyridin-2-yl]-
carbonyl}amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-
pentanoic Acid (47l). General procedure J afforded 68.6 mg
(4S)-4-{[(4-{4-[(Methylamino)methyl]piperidin-1-yl}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (47v). General procedure
afforded 40.2 mg (67%) of product 47v. HRMS calcd for
J
(47%) of product 47l. HRMS calcd for C33H45N5O7 (Mþ
þ
C34H48N6O6 (Mþ þ H) 637.3708, found 637.3695.
H) 624.3392, found 624.3388.
(4S)-4-({[4-(4-Aminopiperidin-1-yl)-6-phenylpyridin-2-yl]car-
bonyl}amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-
pentanoic Acid (47m). General procedure J afforded 97.7 mg
(68%) of product 47m. 1H NMR (DMSO-d6) δ 0.84 (t, 3H), 1.26
(m, 4H), 1.46-1.57 (m, 4H), 1.80 (m, 1H), 1.94-2.04 (m, 3H),
(4S)-4-{[(4-{4-[(Dimethylamino)methyl]piperidin-1-yl}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
zpiperazin-1-yl}pentanoic Acid (47w). General procedure J
afforded 27.6 mg (45%) of product 47w. HRMS calcd for
C35H50N6O6 (Mþ þ H) 651.3865, found 651.3875.

2028 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-[({6-
phenyl-4-[4-(pyrrolidin-1-ylmethyl)piperidin-1-yl]pyridin-2-yl}-
carbonyl)amino]pentanoic Acid (47x). General procedure J
afforded 84.7 mg (100%) of product 47x. HRMS calcd for
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-{[(4-ethoxy-6-
phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid (50c).
General procedure K afforded 89 mg (81%) of product 50c.
HRMS calcd for C26H32N4O7 (Mþ þ H) 513.2344, found
513.2350.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
4-propoxypyridin-2-yl)carbonyl]amino}pentanoic Acid (50d).
General procedure K afforded 90 mg (95%) of product 50d.
HRMS calcd for C27H34N4O7 (Mþ þ H) 527.2500, found
527.2460.
C
37H52N6O6 (Mþ þ H) 677.4026, found 677.4036.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-4-[({4-[(2-meth-
oxyethyl)amino]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxope-
ntanoic Acid (48g). General procedure J afforded 219.7 mg
(83%) of product 48g. HRMS calcd for C31H43N5O7 (Mþ
H) 598.3235, found 598.3260.
þ
(4S)-4-({[4-(4-Aminopiperidin-1-yl)-6-phenylpyridin-2-yl]car-
bonyl}amino)-5-{4-[(hexyloxy)carbonyl]piperazin-1-yl}-5-oxo-
pentanoic Acid (48m). General procedure J afforded 18.9 mg
(47%) of product 48m. HRMS calcd for C33H46N6O6 (Mþ þ
H) 623.3557, found 623.3551.
(4S)-4-{[(4-Butoxy-6-phenylpyridin-2-yl)carbonyl]amino}-5-
[4-(ethoxycarbonyl)piperazin-1-yl]-5-oxopentanoic Acid (50e).
General procedure K afforded 52 mg (93%) of product 50e.
HRMS calcd for C28H36N4O7 (Mþ þ H) 541.2657, found
541.2635.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-({[6-
phenyl-4-(4-pyrrolidin-1-ylpiperidin-1-yl)pyridin-2-yl]carbonyl}amino)-
pentanoic Acid (48p). General procedure J afforded 31.4 mg (71%) of
product 48p. HRMS calcd for C37H52N6O6 (Mþ þ H) 677.4026,
found 677.3999.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-({[4-(2-hydroxy-
ethoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (50f). General procedure K afforded 93 mg (82%) of
product 50f. HRMS calcd for C26H32N4O8 (Mþ þ H)
529.2293, found 529.2271.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-4-[({4-[4-(met-
hoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-
5-oxopentanoic Acid (48s). General procedure J afforded 110 mg
(48%) of product 48s. HRMS calcd for C35H49N5O7 (Mþ þ H)
652.3705, found 652.3706.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-({[4-(3-hydroxy-
propoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (50g). General procedure K afforded 65 mg (56%) of
product 50g. HRMS calcd for C27H34N4O8 (Mþ þ H) 543.2449,
found 543.2431.
(4S)-4-[({4-[4-(Aminomethyl)piperidin-1-yl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-{4-[(hexyloxy)carbonyl]piperazin-1-yl}-
5-oxopentanoic Acid (48u). General procedure J afforded 150.3
mg (66%) of product 48u. HRMS calcd for C34H48N6O6 (Mþ þ
H) 637.3708, found 637.3723.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-({[4-(4-hydroxy-
butoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (50h). General procedure K afforded 49 mg (96%) of
product 50h. HRMS calcd for C28H36N4O8 (Mþ þ H) 557.2606,
found 557.2585.
General Procedure K. Alkylation of the 4-Hydroxypyridine
Intermediates 39-42. To a 2 dram vial with 4-hydroxypyridine
intermediates 39-42 (0.20 mmol) dissolved in dimethylforma-
mide (1 mL) were added cesium carbonate (0.13 g, 0.4 mmol), a
catalytic amount of potassium iodide, and electrophile 49 (0.40
mmol). The mixtures were stirred at 50-100 °C for 1-16 h until
judged complete. The mixtures were filtered, dried in vacuo, and
purified by HPLC if necessary.
Deprotection of the tert-Butyl Ester Group. A solution of 10%
trifluoroacetic acid/dichloromethane solution (2.0 mL) was
added to the tert-butyl ester (∼0.20 mmol) and agitated on an
orbital shaker or stirred at room temperature for 1-24 h.
Evaporation of the solvents afforded the crude products. The
products were purified by HPLC to afford pure products 50-53.
General Procedure L. Mitsunobu Reaction with 4-Hydroxy-
pyridines 39-42. To a 2 dram vial with 4-hydroxypyridine
intermediates 39-42 (0.5 mmol) dissolved in anhydrous tetra-
hydrofuran (2 mL) were added the alcohol 24 (1.0 mmol) and
triphenylphosphine (0.26 g, 1.0 mmol). The mixtures were
chilled to 5 °C, and diethylazodicarboxylate (158 μL, 1.0 mmol)
was added dropwise. The mixtures were stirred at ambient
temperature for 1-24 h, solvents were evaporated, and the
residue was purified by HPLC if necessary.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-({[4-(2-methoxy-
ethoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (50i). General procedure K afforded 86 mg (74%) of
product 50i. HRMS calcd for C27H34N4O8 (Mþ þ H) 543.2449,
found 543.2476.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-({[4-(3-methoxy-
propoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (50j). General procedure K afforded 80 mg (96%) of
product 50j. HRMS calcd for C28H36N4O8 (Mþ þ H) 557.2606,
found 557.2592.
(4S)-4-({[4-(2-Aminoethoxy)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-[4-(ethoxycarbonyl)piperazin-1-yl]-5-oxopentanoic
Acid (50m). General procedure K afforded 95 mg (91%) of
product 50m. HRMS calcd for C26H33N5O7 (Mþ þ H) 528.2453,
found 528.2497.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(piperidin-4-yloxy)pyridin-2-yl]carbonyl}amino)pentanoic Acid
(50n). General procedure K afforded 29 mg (91%) of product
50n. HRMS calcd for C29H37N5O7 (Mþ þ H) 568.2766, found
568.2727.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(piperidin-4-ylmethoxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (50q). General procedure K afforded 99 mg (88%) of
product 50q. HRMS calcd for C30H39N5O7 (Mþ þ H) 582.2922,
found 582.2894.
(4S)-4-({[4-(Benzyloxy)-6-phenylpyridin-2-yl]carbonyl}amino)-
5-[4-(ethoxycarbonyl)piperazin-1-yl]-5-oxopentanoic Acid (50w).
General procedure K afforded 99 mg (99%) of product 50w.
HRMS calcd for C31H34N4O7 (Mþ þ H) 575.2500, found
575.2496.
Deprotection of the tert-Butyl Ester Group. A solution of 10%
trifluoroacetic acid/dichloromethane solution (2.0 mL) was
added to the tert-butyl ester (∼0.20 mmol) and agitated on an
orbital shaker or stirred at room temperature for 1-24 h.
Evaporation of the solvents afforded the crude products. The
crude products were purified by HPLC to afford pure products
50-53.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-{[(4-hydroxy-6-
phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid (50a).
General procedure B afforded 57 mg (55%) of product 50a.
HRMS calcd for C24H28N4O7 (Mþ þ H) 485.2031, found
485.1991.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-hydroxy-6-
phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid (51a).
General procedure B afforded 41 mg (80%) of product 51a.
HRMS calcd for C26H33N4O7 (Mþ þ H) 513.2344, found
513.2318.
(4S)-5-[4-(Ethoxycarbonyl)piperazin-1-yl]-4-{[(4-methoxy-6-
phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid (50b).
General procedure K afforded 78 mg (73%) of product 50b.
HRMS calcd for C25H30N4O7 (Mþ þ H) 499.2187, found
499.2198.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-butoxy-6-
phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid (51e).
General procedure K afforded 86 mg (71%) of product 51e.
HRMS calcd for C30H40N4O7 (Mþ þ H) 569.2970, found
569.2990.

Article
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-(2-hydroxy-
ethoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic Acid
(51f). General procedure K afforded 53 mg (100%) of product
51f. HRMS calcd for C28H36N4O8 (Mþ þ H) 557.2606, found
557.2600.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2029
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(2-piperidin-3-ylethoxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (51u). General procedure L afforded 107 mg (100%) of
product 51u. HRMS calcd for C33H45N5O7 (Mþ þ H) 624.3392,
found 624.3389.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-(3-hydroxy-
propoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (51g). General procedure K afforded 38.1 mg (31%) of
product 51g. HRMS calcd for C29H38N4O8 (Mþ þ H) 571.2762,
found 571.2798.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(2-piperidin-2-ylethoxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (51v). General procedure L afforded 112 mg (100%) of
product 51v. HRMS calcd for C33H45N5O7 (Mþ þ H) 624.3392,
found 624.3401.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-(4-hydroxy-
butoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (51h). General procedure K afforded 31.6 mg (25%) of
product 51h. HRMS calcd for C30H40N4O8 (Mþ þ H) 585.2919,
found 585.2952.
(4S)-4-{[(4-Hydroxy-6-phenylpyridin-2-yl)carbonyl]amino}-5-
oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic Acid
(52a). General procedure B afforded 52 mg (92%) of product
52a. HRMS calcd for C27H34N4O7 (Mþ þ H) 527.2500, found
527.25500.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-(2-methoxy-
ethoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (51i). General procedure K afforded 17 mg (70%) of
product 51i. HRMS calcd for C29H38N4O8 (Mþ þ H) 571.2762,
found 571.2728.
(4S)-4-{[(4-Ethoxy-6-phenylpyridin-2-yl)carbonyl]amino}-5-
oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic Acid
(52c). General procedure K afforded 38 mg (32%) of product
52c. HRMS calcd for C29H38N4O7 (Mþ þ H) 555.2813, found
555.2801.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-(3-methoxy-
propoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (51j). General procedure K afforded 95.4 mg (55%) of
product 51j. HRMS calcd for C30H40N4O8 (Mþ þ H) 585.2919,
found 585.2922.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-{[(6-
phenyl-4-propoxypyridin-2-yl)carbonyl]amino}pentanoic Acid
(52d). General procedure K afforded 102.6 mg (85%) of
product 52d. HRMS calcd for C30H40N4O7 (Mþ þ H) 569.2970,
found 569.2970.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(tetrahydro-2H-pyran-4-yloxy)pyridin-2-yl]carbonyl}amino)-
pentanoic Acid (51k). General procedure L afforded 85 mg
(100%) of product 51k. HRMS calcd for C31H40N4O8 (Mþ þ
H) 597.2919, found 597.2925.
(4S)-4-{[(4-Butoxy-6-phenylpyridin-2-yl)carbonyl]amino}-5-
oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic Acid
(52e). General procedure K afforded 39.0 mg (95%) of
product 52e. HRMS calcd for C31H42N4O7 (Mþ þ H) 583.3126,
found 583.3118.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phen-
yl-4-(tetrahydro-2H-pyran-4-ylmethoxy)pyridin-2-yl]carbonyl}-
amino)pentanoic Acid (51l). General procedure L afforded 84 mg
(100%) of product 51l. HRMS calcd for C32H42N4O8 (Mþ þ H)
611.3081, found 611.3052.
(4S)-4-({[4-(2-Hydroxyethoxy)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (52f). General procedure K afforded 64 mg (70%) of
product 52f. HRMS calcd for C29H38N4O8 (Mþ þ H) 571.2762,
found 571.2790.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(piperidin-4-yloxy)pyridin-2-yl]carbonyl}amino)pentanoic Acid
(51n). General procedure K afforded 34 mg (58%) of product
51n. HRMS calcd for C31H41N5O7 (Mþ þ H) 596.3079, found
596.3246.
(4S)-4-({[4-(2-Methoxyethoxy)-6-phenylpyridin-2-yl]carbo-
nyl}amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pen-
tanoic Acid (52i). General procedure K afforded 63 mg (68%) of
product 52i. HRMS calcd for C30H40N4O8 (Mþ þ H) 585.2919,
found 585.2880.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(piperidin-3-yloxy)pyridin-2-yl]carbonyl}amino)pentanoic Acid
(51o). General procedure L afforded 63 mg (100%) of product
51o. HRMS calcd for C31H41N5O7 (Mþ þ H) 596.3082, found
596.3075.
(4S)-4-({[4-(2-Aminoethoxy)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (52m). General procedure K afforded 90.1 mg (74%) of
product 52m. HRMS calcd for C29H39N5O7 (Mþ þ H) 570.2922,
found 570.2887.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(pyrrolidin-3-yloxy)pyridin-2-yl]carbonyl}amino)pentanoic Acid
(51p). General procedure L afforded 33 mg (35%) of product 51p.
HRMS calcd for C30H39N5O7 (Mþ þ H) 582.2922, found
582.2921.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-({[6-
phenyl-4-(piperidin-4-yloxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (52n). General procedure K afforded 26 mg (62%) of product
52n. HRMS calcd for C32H43N5O7 (Mþ þ H) 610.3235, found
610.3180.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(piperidin-4-ylmethoxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (51q). General procedure K afforded 109.2 mg (85%) of
product 51q. HRMS calcd for C32H43N5O7 (Mþ þ H) 610.3235,
found 610.3191.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-({[6-
phenyl-4-(piperidin-4-ylmethoxy)pyridin-2-yl]carbonyl}amino)-
pentanoic Acid (52q). General procedure K afforded 133.5 mg
(100%) of product 52q. HRMS calcd for C33H45N5O7 (Mþ þ
H) 624.3392, found 624.3355.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(piperidin-3-ylmethoxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (51r). General procedure L afforded 118 mg (100%) of
product 51r. HRMS calcd for C32H43N5O7 (Mþ þ H) 610.3240,
found 610.3259.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-4-{[(4-hydroxy-
6-phenylpyridin-2-yl)carbonyl]amino}-5-oxopentanoic Acid (53a).
General procedure B afforded 45 mg (79%) of product 53a.
HRMS calcd for C28H36N4O7 (Mþ þ H) 541.2657, found
541.2660.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(piperidin-2-ylmethoxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (51s). General procedure L afforded 14.7 mg (81%) of
product 51s. HRMS calcd for C32H43N5O7 (Mþ þ H) 610.3240,
found 610.3226.
(4S)-4-{[(4-Butoxy-6-phenylpyridin-2-yl)carbonyl]amino}-5-
{4-[(hexyloxy)carbonyl]piperazin-1-yl}-5-oxopentanoic Acid (53e).
General procedure K afforded 35 mg (43%) of product 53e.
HRMS calcd for C32H44N4O7 (Mþ þ H) 597.3283, found
597.3283.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-({[6-phenyl-
4-(2-piperidin-4-ylethoxy)pyridin-2-yl]carbonyl}amino)pentanoic
Acid (51t). General procedure L afforded 119 mg (100%) of
product 51t. HRMS calcd for C33H45N5O7 (Mþ þ H) 624.3397,
found 624.3410.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-4-({[4-(2-hydro-
xyethoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopenta-
noic Acid (53f). General procedure K afforded 46 mg (99%) of
product 53f. HRMS calcd for C30H40N4O8 (Mþ þ H) 585.2919,
found 585.2900.

2030 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-4-({[4-(2-meth-
oxyethoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxopentanoic
Acid (53i). General procedure K afforded 13 mg (100%) of
product 53i. HRMS calcd for C31H42N4O8 (Mþ þ H) 599.3075,
found 599.3040.
(4S)-4-({[4-(2-Aminoethoxy)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-{4-[(hexyloxy)carbonyl]piperazin-1-yl}-5-oxopentanoic
Acid (53m). General procedure K afforded 92.9 mg (75%) of
product 53m. HRMS calcd for C30H41N5O7 (Mþ þ H) 584.3079,
found 584.3101.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-({[6-
phenyl-4-(piperidin-4-yloxy)pyridin-2-yl]carbonyl}amino)penta-
noic Acid (53n). General procedure K afforded 22 mg (36%) of
product 53n. HRMS calcd for C33H45N5O7 (Mþ þ H) 624.3392,
found 624.3356.
(4S)-5-{4-[(Hexyloxy)carbonyl]piperazin-1-yl}-5-oxo-4-({[6-
phenyl-4-(piperidin-4-ylmethoxy)pyridin-2-yl]carbonyl}amino)-
pentanoic Acid (53q). General procedure K afforded 73.9 mg
(55%) of product 53q. HRMS calcd for C34H47N5O7 (Mþ þ H)
638.3548, found 638.3539.
General Procedure M. Method A: Preparation of 4-Pyridine-
carbamates 56 and 57. To a 2 dram vial with carbamoyl chlorides
54 (0.40 mmol) were added the 4-hydroxypyridine intermediate
40 or 41 (0.5 M in dichloromethane) (3 mL, 0.15 mmol) and
triethylamine (112 uL, 0.80 mmol). The mixtures were stirred at
room temperature for 18 h. If judged incomplete, the mixture
continued to be stirred at 35 °C until complete. The solvents
were evaporated, and if necessary the residue was purified by
HPLC.
Deprotection of the tert-Butyl Ester Group. A solution of 10%
trifluoroacetic acid/dichloromethane solution (2.0 mL) was
added to the tert-butyl ester (∼0.15 mmol) and agitated on an
orbital shaker or stirred at room temperature for 1-24 h.
Evaporation of the solvents afforded the crude products. The
products were purified by HPLC to afford the pure products
56-57.
General Procedure M. Method B: Preparation of 4-Carba-
mates 56 and 57. To a mixture of 4-hydroxypyridine intermedi-
ate 40 or 41 (2.79 g, 4.8 mmol) and triethylamine (2.04 mL, 27.8
mmol) in dichloromethane (48 mL) was added 4-nitrophenyl
chloroformate 55 (1.06 g, 5.3 mmol). The mixture was stirred at
room temperature for 1 h. An aliquot of the 4-nitrophenylcar-
bonate intermediate (0.1 M reaction mixture) (2 mL, ∼0.2
mmol) was added to a 2 dram vial with amine 44 (0.60 mmol).
The mixture was stirred at ambient temperature for 1 h, solvents
were evaporated, and the residue was purified by HPLC if
necessary.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[(morpholin-
4-ylcarbonyl)oxy]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-
pentanoic Acid (56f). General procedure M, method B, afforded
19.4 mg (19%) of product 56f. HRMS calcd for C31H39N5O9
(Mþ þ H) 626.2821, found 626.2820.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-({[(3R)-3-
methoxypyrrolidin-1-yl]carbonyl}oxy)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxopentanoic Acid (56g). General procedure M, method B,
afforded 27.8 mg (26%) of product 56g. HRMS calcd for
C32H41N5O9 (Mþ þ H) 640.2977, found 640.2971.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[(4-ethoxy-
piperidin-1-yl)carbonyl]oxy}-6-phenylpyridin-2-yl)carbonyl]amino}-
5-oxopentanoic Acid (56h). General procedure M, method B,
afforded 37.6 mg (34%) of product 56h. HRMS calcd for
C34H45N5O9 (Mþ þ H) 668.3290, found 668.3249.
(4S)-4-{[(4-{[(Dimethylamino)carbonyl]oxy}-6-phenylpyridin-
2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-
yl}pentanoic Acid (57a). General procedure M, method A,
afforded 23.6 mg (17%) of product 57a. HRMS calcd for
C30H39N5O8 (Mþ þ H) 598.2871, found 598.2846.
(4S)-4-({[4-({[Methyl(tetrahydro-2H-pyran-4-ylmethyl)amino]-
carbonyl}oxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxo-
5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic Acid (57b).
General procedure M, method B, afforded 36.3 mg (30%) of
product 57b. HRMS calcd for C35H47N5O9 (Mþ þ H) 682.3447,
found 382.3448.
(4S)-4-({[4-({[Methyl(tetrahydrofuran-3-yl)amino]carbonyl}
oxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxo-5-{4-[(pentyloxy)
carbonyl]piperazin-1-yl}pentanoic Acid (57c). General procedure
M, method B, afforded 48.8 mg (42%) of product 57c. HRMS
calcd for C33H43N5O9 (Mþ þ H) 654.3134, found 654.3130.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-[({6-
phenyl-4-[(pyrrolidin-1-ylcarbonyl)oxy]pyridin-2-yl}carbonyl)amino]
pentanoic Acid (57d). General procedure M, method A, afforded
60.9 mg (41%) of product 57d. HRMS calcd for C32H41N5O8
(Mþ þ H) 624.3028, found 624.3063.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-[({6-
phenyl-4-[(piperidin-1-ylcarbonyl)oxy]pyridin-2-yl}carbonyl)amino]-
pentanoic Acid (57e). General procedure M, method A, afforded
46.2 mg (31%) of product 57e. HRMS calcd for C33H43N5O8
(Mþ þ H) 638.3184, found 631.3191.
(4S)-4-[({4-[(Morpholin-4-ylcarbonyl)oxy]-6-phenylpyridin-2-
yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (57f). General procedure M, method A,
afforded 83.9 mg (56%) of product 57f. HRMS calcd for
C
32H41N5O9 (Mþ þ H) 640.2977, found 640.2988.
(4S)-4-({[4-({[(3S)-3-Methoxypyrrolidin-1-yl]carbonyl}oxy)-
Deprotection of the tert-Butyl Ester Group. A solution of 10%
trifluoroacetic acid/dichloromethane solution (2.0 mL) was added
to the tert-butyl ester (∼0.22 mmol) and agitated on an orbital
shaker or stirred at room temperature for 1-24 h. Evaporation of
the solvents afforded the crude products. The products were
purified by HPLC to afford the pure products 56 and 57.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[(dimethyl-
amino)carbonyl]oxy}-6-phenylpyridin-2-yl)carbonyl]amino}-5-
oxopentanoic Acid (56a). General procedure M, method B,
afforded 12.6 mg (13%) of product 56a. HRMS calcd for
6-phenylpyridin-2-yl]carbonyl}amino)-5-oxo-5-{4-[(pentyloxy)-
carbonyl]piperazin-1-yl}pentanoic Acid (57g). General proce-
dure M, method A, afforded 37.1 mg (32%) of product 57g.
HRMS calcd for C33H43N5O9 (Mþ þ H) 654.3134, found
654.3114.
(4S)-4-{[(4-{[(4-Ethoxypiperidin-1-yl)carbonyl]oxy}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (57h). General procedure M,
method B, afforded 27.3 mg (23%) of product 57h. HRMS
calcd for C35H47N5O9 (Mþ þ H) 682.3447, found 682.3453.
Butyl 4-[(2S)-2-({[4-({1-[(Benzyloxy)carbonyl]piperidin-4-yl}oxy)-
6-phenylpyridin-2-yl]carbonyl}amino)-5-tert-butoxy-5-oxopent-
anoyl]piperazine-1-carboxylate (60). General procedure L af-
forded 1.5 g (77%) of product 60. MS (ESIþ) m/z 786 (M þ H).
Pentyl 4-[(2S)-2-({[4-({1-[(Benzyloxy)carbonyl]piperidin-4-yl}-
oxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-tert-butoxy-5-ox-
opentanoyl]piperazine-1-carboxylate (61). General procedure L
afforded 287 mg (72%) of product 61. HRMS calcd for C44-
H57N5O9 (Mþ þ H) 800.4227, found 800.4224.
C
29H37N5O8 (Mþ þ H) 584.2715, found 584.2720.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-({[methyl-
(tetrahydro-2H-pyran-4-ylmethyl)amino]carbonyl}oxy)-6-phenyl-
pyridin-2-yl]carbonyl}amino)-5-oxopentanoic Acid (56b). General
procedure M, method B, afforded 41.1 mg (38%) of product
56b. HRMS calcd for C34H45N5O9 (Mþ þ H) 668.3290, found
668.3319.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-({[4-({[methyl-
(tetrahydrofuran-3-yl)amino]carbonyl}oxy)-6-phenylpyridin-2-
yl]carbonyl}amino)-5-oxopentanoic Acid (56c). General proce-
dure M, method B, afforded 42.8 mg (41%) of product 56c.
HRMS calcd for C32H41N5O9 (Mþ þ H) 640.2977, found
640.2940.
Butyl 4-[(2S)-2-({[4-({1-[(Benzyloxy)carbonyl]piperidin-4-yl}-
methoxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-tert-butoxy-5-
oxopentanoyl]piperazine-1-carboxylate (62). General procedure
L afforded 1.0 g (52%) of product 62. MS (ESIþ) m/z800 (M þ H).

Article
Pentyl 4-[(2S)-2-({[4-({1-[(Benzyloxy)carbonyl]piperidin-4-yl}-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2031
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
4-{[1-(trifluoroacetyl)piperidin-4-yl]oxy}pyridin-2-yl)carbonyl]-
amino}pentanoic Acid (69e). General procedure N afforded 101
mg (100%) of product 69e. 1H NMR (DMSO-d6) δ 0.89 (t, 3H,
J = 7.32 Hz), 1.26-1.40 (m, 2H), 1.48-1.61 (m, 3H), 1.69-
1.95 (m, 4H), 2.07 (d, 4H, J = 9.52 Hz), 2.23-2.39 (m, 2H),
3.36-3.92 (m, 1 H), 4.01 (t, H, J = 6.41 Hz), 4.92-5.22 (m, 1H),
7.37-7.66 (m, 4H), 7.72 (d, 1H, J = 1.83 Hz), 8.20 (d, 2H, J =
7.32 Hz), 8.96 (d, 1H, J = 8.05 Hz); HRMS calcd for
C33H40F3N5O8 (Mþ þ H) 692.2907, found 692.2881.
oxy)-6-phenylpyridin-2-yl]carbonyl}amino)-5-tert-butoxy-5-ox-
opentanoyl]piperazine-1-carboxylate (63). General procedure L
afforded 1.33 g (77%) of product 63. HRMS calcd for C45H59-
N5O9 (Mþ þ H) 814.4386, found 814.4393.
Butyl 4-[(2S)-5-tert-Butoxy-5-oxo-2-({[6-phenyl-4-(piperidin-
4-yloxy)pyridin-2-yl]carbonyl}amino)pentanoyl]piperazine-1-
carboxylate (64). General procedure H afforded 1.2 g (98%)
of product 64. MS (ESIþ) m/z 652 (M þ H).
Pentyl 4-[(2S)-5-tert-Butoxy-5-oxo-2-({[6-phenyl-4-(piperidin-
4-yloxy)pyridin-2-yl]carbonyl}amino)pentanoyl]piperazine-1-car-
boxylate (65). General procedure H afforded 196 mg (82%) of
product 65. HRMS calcd for C36H51N5O7 (Mþ þ H) 666.3861,
found 666.3881.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(methoxy-
acetyl)piperidin-4-yl]oxy}-6-phenylpyridin-2-yl)carbonyl]amino}-
5-oxopentanoic Acid (69f). General procedure N afforded 86 mg
(100%) of product 69f. 1H NMR (DMSO-d6) δ 0.88 (t, 3H, J =
7.32 Hz), 1.26-1.42 (m, 3H), 1.48-1.77 (m, 7H), 1.79-2.15 (m,
7H), 2.23-2.40 (m, 3H), 3.36-3.73 (m, 7H), 3.78-3.93 (m, 1H),
4.01 (t, 2H, J = 6.59Hz), 4.10(s, 3H), 5.04 (s, 1H), 7.39-7.63(m,
4H), 7.71 (s, 1H), 8.20 (d, 2H, J = 7.69 Hz), 8.96 (d, 1H, J = 8.05
Hz); HRMS calcd for C34H45N5O9 (Mþ þ H) 668.3290, found
668.3272.
Butyl 4-[(2S)-5-tert-Butoxy-5-oxo-2-({[6-phenyl-4-(piperidin-
4-ylmethoxy)pyridin-2-yl]carbonyl}amino)pentanoyl]piperazine-
1-carboxylate (66). General procedure H afforded 830 mg
(100%) of product 66. MS (ESIþ) m/z 666 (M þ H).
Pentyl 4-[(2S)-5-tert-Butoxy-5-oxo-2-({[6-phenyl-4-(piperidin-
4-ylmethoxy)pyridin-2-yl]carbonyl}amino)pentanoyl]piperazine-
1-carboxylate (67). General procedure H afforded 507 mg
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(ethoxy-
carbonyl)piperidin-4-yl]oxy}-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-oxopentanoic Acid (69g). General procedure N
afforded 121 mg (100%) of product 69g. 1H NMR
(DMSO-d6) δ 0.89 (t, 3H, J = 7.32 Hz), 1.19 (t, 3H, J =
7.14 Hz), 1.26-1.44 (m, 2H), 1.49-1.72 (m, 6H), 1.76-2.16
(m, 6H), 2.22-2.41 (m, 3H), 3.37-3.82 (m, 8H), 3.95-4.12
(m, 4H), 4.90-5.14 (m, 1H), 7.37-7.60 (m, 4H), 7.70 (d, 1H,
J = 2.20 Hz), 8.20 (d, 2H, J = 7.32 Hz), 8.96 (d, 1H, J = 8.05
Hz); HRMS calcd for C34H45N5O9 (Mþ þ H) 668.3295,
found 668.3322.
(55%) of product 67. HRMS calcd for C37H53N5O7 (Mþ
H) 680.4018, found 680.4305.
þ
General Procedure N. Reaction of Amines 64-67 with Elec-
trophiles 68 To Afford Products 69-72. A solution of the amine
64-67 (0.15 M in dichloromethane or dimethylformamide) (1
mL, 0.15 mmol) was added to each reaction well containing the
electrophile 68 (0.30 mmol) (which included alkyl halides,
chloroformates, acid chlorides, and sulfonyl chlorides). Triethy-
lamine or N-methylmorpholine (0.75 mmol) and dichloro-
methane or dimethyl formamide (2 mL) were added to each
reaction well. Mixtures in dichloromethane were agitated for 1
to several hours. Mixtures in dimethylformamide were heated to
75 °C and agitated for 1 to several hours. The product mixtures
were evaporated to afford the tert-butyl ester product.
Deprotection of the tert-Butyl Ester Group. A solution of 10%
trifluoroacetic acid/dichloromethane solution (1.5 mL) was
added to the tert-butyl ester (∼0.15 mmol) and agitated on
an orbital shaker or stirred at room temperature for 1-24 h.
Evaporation of the solvents afforded the crude products.
The products were purified by HPLC to afford pure products
69-72.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(ethyl-
sulfonyl)piperidin-4-yl]oxy}-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-oxopentanoic Acid (69h). General procedure N
1
afforded 82 mg (100%) of product 69h. H NMR (DMSO-
d6) δ 0.89 (t, 3H, J = 7.32 Hz), 1.15-1.42 (m, 6H), 1.50-1.61
(m, 3H), 1.66-2.17 (m, 8H), 2.2 -2.41 (m, 3H), 3.09 (q, 2H,
J = 7.32 Hz), 3.36-3.78 (m, 8H), 4.01 (t, 2H, J = 6.41 Hz),
4.89-5.13 (m, 1H), 7.35-7.61 (m, 4H), 7.71 (d, 1H, J = 1.83
Hz), 8.20 (d, 2H, J = 7.32 Hz), 8.96 (d, 1H, J = 8.05 Hz);
HRMS calcd for C33H45N5O9S (Mþ þ H) 688.3016, found
688.3040.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[(1-methyl-
piperidin-4-yl)oxy]-6-phenylpyridin-2-yl}carbonyl)amino]-5-ox-
opentanoic Acid (69a). General procedure N afforded 90 mg
(92%) of product 69a. 1H NMR (DMSO-d6) δ 0.89 (t, 3H, J =
7.50 Hz), 1.25-1.40 (m, 3H), 1.48-1.61 (m, 3H), 1.77-2.39 (m,
13H), 2.79 (s, 3H), 3.40-3.73 (m, 6H), 5.04 (s, 1H), 7.38-7.65
(m, 4H), 7.73 (d, 1H, J = 16.84 Hz), 8.20 (d, 2H, J = 7.32 Hz),
8.97 (d, 1H, J = 8.05 Hz); HRMS calcd for C32H43N5O7 (Mþ þ
H) 610.3240, found 610.3195.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[(1-isopro-
pylpiperidin-4-yl)oxy]-6-phenylpyridin-2-yl}carbonyl)amino]-5-
oxopentanoic Acid (69b). General procedure N afforded 28 mg
(72%) of product 69b. HRMS calcd for C34H47N5O7 (Mþ þ H)
638.3548, found 638.3507.
(4S)-4-[({4-[(1-Methylpiperidin-4-yl)oxy]-6-phenylpyridin-2-
yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (70a). General procedure N afforded 36 mg
(88%) of product 70a. HRMS calcd for C33H45N5O7 (Mþ þ H)
624.3392, found 624.3416.
(4S)-4-[({4-[(1-Isopropylpiperidin-4-yl)oxy]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (70b). General procedure N afforded 43 mg
(83%) of product 70b. HRMS calcd for C35H49N5O7 (Mþ þ H)
652.3705, found 652.3724.
(4S)-4-{[(4-{[1-(2-Methoxyethyl)piperidin-4-yl]oxy}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (70c). General procedure N af-
forded 19 mg (79%) of product 70c. HRMS calcd for C35H50-
N5O8 (Mþ þ H) 668.3654, found 668.3632.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(2-meth-
oxyethyl)piperidin-4-yl]oxy}-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-oxopentanoic Acid (69c). General procedure N
(4S)-4-[({4-[(1-Acetylpiperidin-4-yl)oxy]-6-phenylpyridin-2-
yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (70d). General procedure N afforded 20 mg
(18%) of product 70d. HRMS calcd for C34H45N5O8 (Mþ þ H)
652.3341, found 652.3336.
afforded 41 mg (83%) of product 69c. HRMS calcd for C34
H
-
47N5O8 (Mþ þ H) 654.3497, found 654.3498.
(4S)-4-[({4-[(1-Acetylpiperidin-4-yl)oxy]-6-phenylpyridin-2-
yl}carbonyl)amino]-5-[4-(butoxycarbonyl)piperazin-1-yl]-5-ox-
opentanoic Acid (69d). General procedure N afforded 113 mg
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-{[(6-
phenyl-4-{[1-(trifluoroacetyl)piperidin-4-yl]oxy}pyridin-2-yl)car-
bonyl]amino}pentanoic Acid (70e). General procedure N af-
1
(100%) of product 69d. H NMR (DMSO-d6) δ 0.88 (t, 3H,
1
J = 7.32 Hz), 1.26-1.41 (m, 2H) 1.47-1.76 (m, 6H),
1.76-2.13 (m, 9H), 2.24-2.39 (m, 2H), 3.36-3.76 (m, 8H),
3.84 (s, 1H), 5.03 (s, 1H), 7.41-7.64 (m, 4H), 7.70 (s, 1H), 8.20
(d, 2H, J = 8.05 Hz), 8.96 (d, 1H, J = 8.05 Hz); HRMS calcd
for C33H43N5O8 (Mþ þ H) 638.3190, found 638.3174.
forded 85 mg (100%) of product 70e. H NMR (DMSO-d6) δ
0.87 (t, 3H, J = 6.77 Hz), 1.19-1.37 (m, 5H), 1.50-1.64 (m, 2H),
1.67-1.94 (m, 4H), 1.99-2.18 (m, 4H), 2.22-2.40 (m, 2H),
3.36-3.72 (m, 8H), 3.86 (s, 2H), 4.00 (t, 2H, J = 6.41 Hz),
4.94-5.21 (m, 1H), 7.41-7.66 (m, 4H), 7.72 (d, 1H, J = 1.83

2032 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
Hz), 8.20 (d, 2H, J = 6.95 Hz), 8.97 (d, 1H, J = 8.05 Hz); HRMS
calcd for C34H42F3N5O8 (Mþ þ H) 706.3063, found 706.3056.
(4S)-4-{[(4-{[1-(Methoxyacetyl)piperidin-4-yl]oxy}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (70f). General procedure N afforded
80 mg (91%) of product 70f. HRMS calcd for C35H47N5O9 (Mþ þ
H) 682.3447, found 682.3485.
3H), 2.63 (s, 1H), 3.01 (s, 1H), 3.35-3.71 (m, 4H), 3.79 (s, 1H),
3.85-4.23 (m, 7H), 4.34 (s, 1H), 5.03 (d, 1H, J = 3.29 Hz),
7.35-7.61 (m, 4H), 7.68 (s, 1H), 8.20 (d, 2H, J = 7.69 Hz), 8.96
(d, 1H, J = 8.05 Hz); HRMS calcd for C35H47N5O9 (Mþ þ H)
682.3452, found 682.3430.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(ethoxy-
carbonyl)piperidin-4-yl]methoxy}-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-oxopentanoic Acid (71g). General procedure N af-
forded 97 mg (96%) of product 71g. 1H NMR (DMSO-d6) δ
0.89 (t, 3H, J = 7.32 Hz), 1.08-1.45 (m, 6H), 1.44-1.63 (m, 2H),
1.68-2.43 (m, 10H), 2.71-2.90 (m, 1H), 2.91-3.06 (m, 1 H),
3.36-3.77 (m, 5H), 3.89 (s, 1H), 3.95-4.08 (m, 4H), 4.07-4.22
(m, 3H), 4.33 (d, 1H, J = 13.18 Hz), 4.96-5.12 (m, 1H),
7.40-7.60 (m, 4H), 7.68 (s, 1H), 8.20 (d, 2H, J = 7.32 Hz),
8.96 (d, 1H, J = 8.05 Hz); HRMS calcd for C35H47N5O9 (Mþ þ
H) 682.3452, found 682.3433.
(4S)-4-{[(4-{[1-(Ethoxycarbonyl)piperidin-4-yl]oxy}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (70g). General procedure N afforded
120 mg (90%) of product 70g. HRMS calcd for C35H47N5O9 (Mþ
þ H) 682.3447, found 682.3430.
(4S)-4-{[(4-{[1-(Ethylsulfonyl)piperidin-4-yl]oxy}-6-phenylpyridin-
2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-
yl}pentanoic Acid (70h). General procedure N afforded 113 mg
(100%) of product 70h. 1H NMR (DMSO-d6) δ 0.86 (t, 3H, J =
6.41 Hz), 1.13-1.36 (m, 9H), 1.49-1.64 (m, 3H), 1.68-1.94 (m,
5H), 2.06 (d, 5H, J = 7.69 Hz), 2.21-2.37 (m, 4H), 3.08 (q, 1H,
J = 7.32 Hz), 3.36-3.73 (m, 5H), 4.00 (t, 2H, J = 6.59 Hz),
4.90-5.11 (m, 1H), 7.36-7.61 (m, 4H), 7.71 (s, 1H), 8.20 (d, 2H,
J = 7.69 Hz), 8.96 (d, 1H, J = 8.05 Hz); HRMS calcd for
C34H47N5O9S (Mþ þ H) 702.3167, found 702.3152.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(ethyl-
sulfonyl)piperidin-4-yl]methoxy}-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-oxopentanoic Acid (71h). General procedure N afforded
52 mg (100%) of product 71h. 1H NMR (DMSO-d6) δ 0.89 (t, 3H,
J = 7.32 Hz), 1.21 (t, 4H, J = 7.50 Hz), 1.26-1.46 (m, 6H),
1.49-1.64 (m, 2H), 1.78-2.17 (m, 6H), 2.23-2.40 (m, 2H), 2.85 (t,
2H, J = 11.16 Hz), 3.03 (q, 2H, J = 7.32 Hz), 3.37-3.78 (m, 6H),
4.01 (t, 2H, J = 6.41 Hz), 4.15 (d, 2H, J = 6.22 Hz), 4.94-5.19 (m,
1H), 7.42-7.57 (m, 4H), 7.68 (d, 1H, J=1.83Hz), 8.21(d, 2H, J=
6.95 Hz), 8.97 (d, 1H, J= 8.05 Hz); HRMS calcd for C34H47N5O9S
(Mþ þ H) 702.3173, found 702.3176.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[(1-methyl-
piperidin-4-yl)methoxy]-6-phenylpyridin-2-yl}carbonyl)amino]-
5-oxopentanoic Acid (71a). General procedure N afforded 61 mg
(87%) of product 71a. 1H NMR (DMSO-d6) δ 0.88 (t, 3H, J =
7.32 Hz), 1.24-1.43 (m, 2H), 1.45-1.71 (m, 6H), 1.74-2.18 (m,
7H), 2.21-2.42 (m, 3H), 2.73 (s, 3H), 2.97 (s, 1H), 3.37-3.76 (m,
5H), 4.01 (t, 2H, J = 6.59 Hz), 4.09-4.40 (m, 3H), 4.98-5.16 (m,
1H), 7.39-7.61 (m, 4H), 7.68 (s, 1H), 8.20 (d, 2H, J = 7.69 Hz),
8.97 (d, 1H, J = 8.05 Hz); HRMS calcd for C33H45N5O7 (Mþ þ
H) 624.3392, found 624.3365.
(4S)-4-[({4-[(1-Methylpiperidin-4-yl)methoxy]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-
yl}pentanoic Acid (72a). General procedure N afforded 26 mg
(19%) of product 72a. HRMS calcd for C34H47N5O7 (Mþ þ H)
638.3548, found 638.3530.
(4S)-4-[({4-[(1-Isopropylpiperidin-4-yl)methoxy]-6-phenylpyr-
idin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (72b). General procedure N
afforded 52 mg (93%) of product 72b. HRMS calcd for
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-[({4-[(1-isopropyl-
piperidin-4-yl)methoxy]-6-phenylpyridin-2-yl}carbonyl)amino]-
5-oxopentanoic Acid (71b). General procedure N afforded 18
mg (93%) of product 71b. HRMS calcd for C35H49N5O7 (Mþ
þ H) 652.3705, found 652.3701.
C
36H52N5O7 (Mþ þ H) 666.3861, found 666.3864.
(4S)-4-{[(4-{[1-(2-Methoxyethyl)piperidin-4-yl]methoxy}-6-
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(2-meth-
oxyethyl)piperidin-4-yl]methoxy}-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-oxopentanoic Acid (71c). General procedure N afforded
25 mg (93%) of product 71c. HRMS calcd for C35H49N5O8 (Mþ
þ H) 668.3654, found 668.3653.
phenylpyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)-
carbonyl]piperazin-1-yl}pentanoic Acid (72c). General proce-
dure N afforded 53 mg (91%) of product 72c. HRMS calcd
for C36H52N5O8 (Mþ þ H) 682.3810, found 682.3833.
(4S)-4-[({4-[(1-Acetylpiperidin-4-yl)methoxy]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (72d). General procedure N afforded 53 mg
(84%) of product 72d. 1H NMR (DMSO-d6) δ 0.87 (t, 3H, J =
6.77 Hz), 1.04-1.39 (m, 8H), 1.49-1.64 (m, 3H), 1.73-1.94 (m,
4H), 1.95-2.15 (m, 5H), 2.24-2.40 (m, 2H), 2.55-2.67 (m, 1H),
3.07 (s, 1H), 3.65 (s, 4H), 3.85 (d, 1H, J = 13.91 Hz), 4.00 (t, 2H,
J = 6.59 Hz), 4.13 (d, 2H, J = 6.59 Hz), 4.40 (d, 1H, J = 12.45
Hz), 4.95-5.12 (m, 1H), 7.37-7.62 (m, 4H), 7.68 (d, 1H, J =
2.20 Hz), 8.21 (d, 2H, J = 6.96 Hz), 8.97 (d, 1H, J = 8.05 Hz);
HRMS calcd for C35H47N5O8 (Mþ þ H) 666.3503, found
666.3560.
(4S)-4-[({4-[(1-Acetylpiperidin-4-yl)methoxy]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-[4-(butoxycarbonyl)piperazin-1-yl]-5-
oxopentanoic Acid (71d). General procedure N afforded 49
mg (89%) of product 71d. 1H NMR (DMSO-d6) δ 0.89 (t, 3H,
J = 7.32 Hz), 1.06-1.44 (m, 6H), 1.46-1.69 (m, 2H), 1.68-
2.17 (m, 9H), 2.24-2.42 (m, 2H), 2.58 (s, 1H), 3.07 (s, 1H),
3.36-3.73 (m, 5H), 3.83 (s, 1H), 4.01 (t, 2H, J = 6.59 Hz),
4.12 (d, 2H, J = 6.59 Hz), 4.40 (d, 1H, J = 12.08 Hz), 4.95-
5.13 (m, 1H), 7.39-7.60 (m, 4H), 7.68 (d, 1H, J = 2.20
Hz), 8.21 (d, 2, H, J = 6.96 Hz), 8.96 (d, 1H, J = 8.05 Hz);
HRMS calcd for C34H45N5O8 (Mþ þ H) 652.3347, found
652.3367.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-{[(6-
phenyl-4-{[1-(trifluoroacetyl)piperidin-4-yl]methoxy}pyridin-2-
yl)carbonyl]amino}pentanoic Acid (72e). General procedure N
afforded 39 mg (74%) of product 72e. 1H NMR (DMSO-d6) δ
0.87 (t, 3H, J = 6.77 Hz), 1.20-1.45 (m, 8H), 1.49-1.66 (m,
3H), 1.78-2.40 (m, 9H), 2.97 (s, 1H), 3.65 (s, 4H), 3.91 (d, 1H,
J = 13.18 Hz), 4.00 (t, 2H, J = 6.59 Hz), 4.15 (d, 2H, J = 6.22
Hz), 4.33 (d, 1H, J = 12.81 Hz), 4.94-5.14 (m, 1H), 7.41-7.62
(m, 4H), 7.68 (d, 1H, J = 2.20 Hz), 8.21 (d, 2H, J = 6.96 Hz),
8.97 (d, 1H, J = 8.42 Hz); HRMS calcd for C35H44F3N5O8
(Mþ þ H) 720.3220, found 720.3201.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-5-oxo-4-{[(6-phenyl-
4-{[1-(trifluoroacetyl)piperidin-4-yl]methoxy}pyridin-2-yl)carbonyl]-
amino}pentanoic Acid (71e). General procedure N afforded 86 mg
(100%) of product 71e. 1H NMR (DMSO-d6) δ 0.88 (t, 3H, J =
7.50 Hz), 1.12-1.44 (m, 6H), 1.42-1.65 (m, 2H), 1.68-2.41 (m,
10H), 2.84-3.08 (m, 1H), 3.37-3.76 (m, 4H), 3.79-4.09 (m, 3H),
4.15 (d, 2H, J = 6.22 Hz), 4.94-5.11 (m, 1H), 7.42-7.59 (m, 4H),
7.68 (s, 1H), 8.20 (d, 2H, J = 7.69 Hz), 8.96 (d, 1H, J = 8.05 Hz);
HRMS calcd for C34H42F3N5O8 (Mþ þ H) 706.3058, found
706.3070.
(4S)-5-[4-(Butoxycarbonyl)piperazin-1-yl]-4-{[(4-{[1-(methoxy-
acetyl)piperidin-4-yl]methoxy}-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-oxopentanoic Acid (71f). General procedure N af-
forded 78 mg (100%) of product 71f. 1H NMR (DMSO-d6) δ
0.88 (t, 3H, J = 7.50 Hz), 1.00-1.40 (m, 7H), 1.43-1.65 (m,
2H), 1.66-1.92 (m, 5H), 1.95-2.17 (m, 2H), 2.18-2.39 (m,
(4S)-4-{[(4-{[1-(Methoxyacetyl)piperidin-4-yl]methoxy}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (72f). General procedure N afforded
88 mg (100%) of product 72f. HRMS calcd for C36H50N5O9 (Mþ þ
H) 696.3603, found 696.3631.

Article
(4S)-4-{[(4-{[1-(Ethoxycarbonyl)piperidin-4-yl]methoxy}-6-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2033
General Procedure P. Preparation of 4-Urea and 4-Carbamate
Pyridine Products 76 and 77. A solution of pentyl 4-[(2S)-2-{[(4-
amino-6-phenylpyridin-2-yl)carbonyl]amino}-5-tert-butoxy-5-
oxopentanoyl]piperazine-1-carboxylate 73 (58 mg, 0.10 mmol)
and pyridine (91 uL, 1.12 mmol) in dichloromethane (2.0 mL)
was chilled to 0 °C. Bis(trichloromethyl)carbonate 22 (0.1 M in
DCM) (1 mL, 0.1 mmol) was added, the mixture was stirred for
1 min, and the reaction mixture was added to a chilled solution
of amine 44 or alcohol 24 (0.75 mmol) in dichloromethane
(1.0 mL). The mixtures were stirred at ambient temperature
for 0.5-16 h, the solvents were evaporated, and if necessary, the
residue was purified by HPLC.
Deprotection of the tert-Butyl Ester Group. A solution of 10%
trifluoroacetic acid/dichloromethane solution (1.5 mL) was
added to the tert-butyl ester (∼0.10 mmol) and agitated on an
orbital shaker or stirred at room temperature for 1-24 h.
Evaporation of the solvents afforded the crude products. The
products were purified by HPLC to afford pure products 76
or 77.
phenylpyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)-
carbonyl]piperazin-1-yl}pentanoic Acid (72g). General proce-
dure N afforded 104 mg (100%) of product 72g. H NMR
1
(DMSO-d6) δ 0.87 (t, 3H, J = 6.59 Hz), 1.09-1.38 (m, 11H),
1.50-1.64 (m, 2H), 1.70-2.14 (m, 7H), 2.24-2.39 (m, 2H),
2.83 (s, 2H), 3.40-3.71 (m, 4H), 3.94-4.22 (m, 8H),
4.92-5.15 (m, 1H), 7.36-7.59 (m, 4H), 7.67 (d, 1H, J =
2.20 Hz), 8.20 (d, 2H, J = 7.32 Hz), 8.96 (d, 1H, J = 8.05 Hz);
HRMS calcd for C36H49N5O9 (Mþ þ H) 696.3608, found
696.3634.
(4S)-4-{[(4-{[1-(Ethylsulfonyl)piperidin-4-yl]methoxy}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (72h). General procedure N afforded
86 mg (100%) of product 72h. 1H NMR (DMSO-d6) δ 0.87 (t, 3H,
J = 6.59 Hz), 1.08-1.44 (m, 11H), 1.46-1.65 (m, 2H), 1.70-2.13
(m, 7H), 2.20-2.40 (m, 2H), 2.85 (t, 2H, J= 11.35 Hz), 3.03 (q, 2H,
J = 7.32 Hz), 3.36-3.77 (m, 7H), 4.00 (t, 2H, J = 6.41 Hz), 4.15 (d,
2H, J = 6.59 Hz), 4.88-5.18 (m, 1H), 7.38-7.59 (m, 4H), 7.68 (d,
1H, J = 2.20 Hz), 8.21 (d, 2H, J = 7.32 Hz), 8.97 (d, 1H, J = 8.05
Hz); HRMS calcd for C35H49N5O9S (Mþ þ H) 716.3329, found
716.3333.
Pentyl 4-[(2S)-2-{[(4-Amino-6-phenylpyridin-2-yl)carbonyl]-
amino}-5-tert-butoxy-5-oxopentanoyl]piperazine-1-carboxylate
(73). To a mixture of 4-azido-6-phenylpyridine-2-carboxylic
acid 31 (4.79 g, 19.9 mmol), hydroxybenzotriazole (2.90 g,
21.5 mmol), and N-methylmorpholine (6.36 mL, 57.8 mmol)
dissolved in DMF (160 mL) were added pentyl (S)-4-(2-amino-
4-tert-butoxycarbonylbutyryl)piperazine-1-carboxylate 38c (7.67
g, 19.9 mmol) and 1-ethyl-3-[3-(dimethylamino)propyl]carbo-
diimide hydrochloride (5.8 g, 30.2 mmol). The mixture was stirred
at room temperature for 23 h and then diluted with ethyl acetate
(140 mL) and water (140 mL). The aqueous phase was extracted
with ethyl acetate (1 ꢀ 100 mL) and the combined organics were
washedwithaqueoussaturatedNaHCO3 (1ꢀ 100 mL), brine (1 ꢀ
75 mL), dried over magnesium sulfate, filtered, and concentrated
to provide the crude product. The residue was dissolved in
methanol (250 mL), and the reaction vessel was flushed with
N2(g). Then 10% Pd/C (50% water-wet, 2.07 g, 0.97 mmol) was
added to the solution, and the reaction vessel was flushed with
N2(g) and then H2(g). The mixture was stirred for 19 h under a
balloon of H2(g). The mixture was filtered through a pad of Celite,
and the filtrate and cake rinses were dried in vacuo. The crude
residue was purified on silica gel (50-80% ethyl acetate:hexanes)
to afford 7.26 g (55% yield) of product 73. 1H NMR (CDCl3) δ
0.86-0.97 (m, 3H), 1.28-1.41 (m, 4H), 1.45 (s, 9H), 1.57-1.71
(m, 2H), 1.86-2.01 (m, 1H), 2.09-2.24 (m, 1H), 2.27-2.51 (m,
2H), 3.37-3.81 (m, 8H), 4.10 (t, 2H, J = 6.71 Hz), 4.44 (s, 2H),
5.15-5.26 (m, 1H), 7.06 (d, 1H, J = 2.15 Hz), 7.37-7.53 (m, 4H),
7.97-8.03 (m, 2H), 8.92 (d, 1H, J = 8.86 Hz); HRMS calcd for
C31H44N5O6 (Mþ þ H) 582.3286, found 582.3273.
Pentyl 4-[(2S)-5-tert-Butoxy-5-oxo-2-{[(6-phenyl-4-{[(trifluoro-
methyl)sulfonyl]oxy}pyridin-2-yl)carbonyl]amino}pentanoyl]piper-
azine-1-carboxylate (78). A solution of pentyl 4-((2S)-5-tert-
butoxy-2-{[(4-hydroxy-6-phenylpyridin-2-yl)carbonyl]amino}-5-
oxopentanoyl)piperazine-1-carboxylate 43c (13.0 g, 22.3 mmol)
was dissolved in pyridine (90 mL), and the solution was chilled in
an ice bath. Triflic anhydride (11.55 mL, 68.6 mmol) was added
slowly, keeping the temperature below 20 °C. The solvent was
reduced in vacuo and the residue dissolved in ethyl acetate. The
solids were removed by vacuum filtration, and the filtrate was
purified on silica gel (30-70% ethyl acetate:hexanes) to afford
12.4 g (78%) of product 78. 1H NMR (CDCl3) δ 0.85-0.95 (m,
3H), 1.28-1.39 (m, 4H), 1.43 (s, 9H), 1.57-1.70 (m, 2H),
1.84-1.99 (m, 1H), 2.09-2.23 (m, 1H), 2.27-2.48 (m, 2H),
3.38-3.79 (m, 8H) 4.09 (t, 2H, J = 6.71 Hz), 5.14-5.28 (m,
1H), 7.48-7.57 (m, 3H), 7.75 (d, 1H, J = 2.42 Hz), 8.00 (d, 1H,
J = 2.15 Hz), 8.05 (dd, 2H, J = 7.79, 1.61 Hz), 8.87 (d, 1H, J =
8.32 Hz); 19F NMR (376 MHz, CDCl3) δ ppm -73.08 (s).
2-((4-Carboxy-1-oxo-1-(4-(pentyloxycarbonyl)piperazin-1-yl)-
butan-2-yl)carbamoyl)-6-phenylisonicotinic Acid (79). A solution
of pentyl 4-[(2S)-5-tert-butoxy-5-oxo-2-{[(6-phenyl-4-{[(tri-
fluoromethyl)sulfonyl]oxy}pyridin-2-yl)carbonyl]amino}-
pentanoyl]piperazine-1-carboxylate 78 (∼9.4 mmol) in DMSO
(100 mL) was added to Pd(PPh3)4 (2.68 g, 2.3 mmol) and
triethylamine (9.16 mL, 66 mmol) under an inert atmosphere.
The reaction solution was evacuated and flushed with N2(g) and
then with CO(g). Themixture wasstirred under a CO(g) balloon at
60 °C for 27 h and then was diluted with dichloromethane (200
mL) and washed with HCl (1 N in water) (1 ꢀ 200 mL). The
aqueous phase was extracted with dichloromethane (150 mL)
and the combined organics were washed with brine (200 mL),
dried over MgSO4, filtered, concentrated, and purified by re-
verse-phase HPLC (30-95% acetonitrile/water gradient with
0.1% TFA modifier) to afford 4.58 g (53% yield) of 75% pure
product 79. 1H NMR (CDCl3) δ 0.87-0.98 (m, 3H), 1.29-1.43
(m, 4H), 1.45 (s, 9H), 1.60-1.72 (m, 2H), 1.93-2.08 (m, 1H),
2.15-2.28 (m, 1H), 2.33-2.56 (m, 2H), 3.42-3.90 (m, 8H), 4.13
(t, 2H, J = 6.71 Hz), 5.25-5.37 (m, 1H), 7.41-7.54 (m, 3H),
8.03-8.11 (m, 2H), 8.36 (d, 1H, J = 1.34 Hz), 8.62 (d, 1H, J =
1.34 Hz), 8.96 (d, 1H, J = 8.32 Hz); HRMS calcd for
C32H43N4O8 (Mþ þ H) 611.3075, found 611.3089.
General Procedure O. Preparation of 4-Amide Pyridine Pro-
ducts 75. To a solution of pentyl 4-[(2S)-2-{[(4-amino-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-tert-butoxy-5-oxopentanoyl]-
piperazine-1-carboxylate 73 (116 mg, 0.20 mmol) in ethyl
acetate (1.5 mL) were added the carboxylic acid 74 (0.28
mmol), 1-methylimidazole (128 μL, 1.56 mmol), and propyl-
phosphonic anhydride (50% in EtOAc) (298 μL, 0.5 mmol).
The mixtures were stirred at 55 °C for 1-16 h, cooled to room
temperature, and diluted with water (1 mL) and dichloro-
methane (2 mL). The phases were partitioned, the organic
solvents were evaporated, and if necessary, the residue was
purified by HPLC.
General Procedure Q. Preparation of 4-Carboxamide Pyridine
Compounds 80. A solution of 2-((4-carboxy-1-oxo-1-(4-(pentyl-
oxycarbonyl)piperazin-1-yl)butan-2-yl)carbamoyl)-6-phenyli-
sonicotinic acid 79 (3.6 g, 6 mmol), 1-hydroxybenzotriazole (0.2
g, 1.48 mmol), and N-methylmorpholine (1.8 mL, 18 mmol) in
dichloromethane (96 mL) was aliquoted (1 mL per well) between
96 1-dram vials containing PS-carbodiimide 6 (∼0.10 mmol per
well). The mixtures were agitated for 10 min, and then amines 44
(0.09 mmol) were added to the vials. The mixtures were agitated
at room temperature for 12-72 h. The reaction mixtures were
Deprotection of the tert-Butyl Ester Group. A solution of
10% trifluoroacetic acid/dichloromethane solution (1.5 mL)
was added to the tert-butyl ester (∼0.20 mmol) and agitated
on an orbital shaker or stirred at room temperature for
1-24 h. Evaporation of the solvents afforded the crude
products. The products were purified by HPLC to afford
pure products 75.

2034 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
filtered and filtrates evaporated to afford the tert-butyl ester
intermediates.
Q afforded 22.6 mg (36%) of product 80m. HRMS calcd for
C35H48N6O7 (Mþ þ H) 665.3663, found 665.3654.
Deprotection of the tert-Butyl Ester Group. A solution of 15%
trifluoroacetic acid/dichloromethane (0.65 mL) was added to
each tert-butyl ester (∼0.063 mmol) and agitated on an orbital
shaker or stirred at room temperature for 16 h. Purification by
HPLC provided the desired pure products 80.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-({[6-
phenyl-4-(piperidin-1-ylcarbonyl)pyridin-2-yl]carbonyl}amino)-
pentanoic Acid (80n). General procedure Q afforded 13.7 mg
(21%) of product 80n. HRMS calcd for C33H43N5O7 (Mþ þ H)
622.3240, found 622.3239.
(4S)-4-({[4-(Morpholin-4-ylcarbonyl)-6-phenylpyridin-2-yl]car-
bonyl}amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pent-
anoic Acid (80o). General procedure Q afforded 22.1 mg (34%) of
product 80o. HRMS calcd for C32H41N5O8 (Mþ þ H) 624.3033,
found 624.3050.
(4S)-4-({[4-(Aminocarbonyl)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (80a). General procedure Q afforded 15.5 mg (14%) of
product 80a. HRMS calcd for C28H35N5O7 (Mþ þ H) 554.2615,
found 554.2606.
(4S)-4-[({4-[(4-Methoxypiperidin-1-yl)carbonyl]-6-phenylpyr-
idin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]pipe-
razin-1-yl}pentanoic Acid (80p). General procedure Q afforded
25.1 mg (44%) of product 80p. HRMS calcd for C34H45N5O8
(Mþ þ H) 652.3347, found 652.3327.
(4S)-4-[({4-[(Methylamino)carbonyl]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}penta-
noic Acid (80b). General procedure Q afforded 42 mg (37%) of
product 80b. HRMS calcd for C29H37N5O7 (Mþ þ H) 568.2771,
found 568.2775.
(4S)-4-[({4-[(Ethylamino)carbonyl]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}penta-
noic Acid (80c). General procedure Q afforded 18.3 mg (16%) of
product 80c. HRMS calcd for C30H39N5O7 (Mþ þ H) 582.2928,
found 582.2953.
(4S)-4-{[(4-{[4-(Dimethylamino)piperidin-1-yl]carbonyl}-6-phenyl-
pyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]-
piperazin-1-yl}pentanoic Acid (80q). General procedure Q afforded
10 mg (15%) of product 80q. HRMS calcd for C35H48N6O7 (Mþ þ
H) 665.3663, found 665.3665.
(4S)-4-[({4-[(4-Methylpiperazin-1-yl)carbonyl]-6-phenylpyri-
din-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]pipe-
razin-1-yl}pentanoic Acid (80r). General procedure Q afforded
44.7 mg (36%) of product 80r. HRMS calcd for C33H44N6O7
(Mþ þ H) 637.3350, found 637.3352.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-[({6-
phenyl-4-[(propylamino)carbonyl]pyridin-2-yl}carbonyl)amino]-
pentanoic Acid (80d). General procedure Q afforded 22.7 mg
(45%) of product 80d. HRMS calcd for C31H41N5O7 (Mþ þ H)
596.3084, found 596.3115.
(4S)-4-[({4-[(Dimethylamino)carbonyl]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}penta-
noic Acid (80e). General procedure Q afforded 50.1 mg (43%) of
product 80e. HRMS calcd for C30H39N5O7 (Mþ þ H) 582.2928,
found 582.2913.
(4S)-4-[({4-[(4-Acetylpiperazin-1-yl)carbonyl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (80s). General procedure Q afforded 13.4 mg
(17%) of product 80s. HRMS calcd for C34H44N6O8 (Mþ þ H)
665.3293, found 665.3292.
General Procedure R. Method A (Heck Coupling): Preparation
of 4-Aliphatic Pyridine Compounds 83. To a solution of pentyl
4-[(2S)-5-tert-butoxy-5-oxo-2-{[(6-phenyl-4-{[(trifluoromethyl)-
sulfonyl]oxy}pyridin-2-yl)carbonyl]amino}pentanoyl]piperazine-
1-carboxylate 78 (250 mg, 0.35 mmol), trans-dichlorobis-
(triphenylphosphine)Pd(II) (12.6 mg, 0.018 mmol), and LiBr
(97.2 mg, 1.12 mmol) in DMF (3.5 mL) was added alkene 81
(1.0-1.4 mmol). The mixtures were stirred at 100 °C for 1 h, then
cooled to room temperature. Triethylamine (54 uL, 0.49 mmol)
was added, and the mixtures were stirred at 100 °C for 1.5-18 h.
The crude reaction solution was diluted with R,R,R-trifluoroto-
luene (4 mL) and washed with water (4 mL). The organic phase
was isolated, and the aqueous phase was extracted with R,R,R-
trifluorotoluene (4 mL). The combined organics were dried in
vacuo to afford the tert-butyl ester products.
(4S)-4-{[(4-{[Ethyl(methyl)amino]carbonyl}-6-phenylpyridin-2-
yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (80f). General procedure Q afforded 25.7
mg (42%) of product 80f. HRMS calcd for C31H41N5O7 (Mþ þ
H) 596.3084, found 596.3046.
(4S)-4-{[(4-{[Butyl(methyl)amino]carbonyl}-6-phenylpyridin-
2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]pipera-
zin-1-yl}pentanoic Acid (80g). General procedure Q afforded
19.5 mg (52%) of product 80g. HRMS calcd for C33H45N5O7
(Mþ þ H) 624.3397, found 624.3412.
(4S)-4-{[(4-{[(2-Methoxyethyl)amino]carbonyl}-6-phenylpyri-
din-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]pipe-
razin-1-yl}pentanoic Acid (80h). General procedure Q afforded
25.4 mg (39%) of product 80h. HRMS calcd for C31H41N5O8
(Mþ þ H) 612.3033, found 612.3063.
Deprotection of the tert-Butyl Ester Group. A solution of 15%
trifluoroacetic acid/dichloromethane (3.5 mL) was added to the
tert-butyl ester (∼0.35 mmol) agitated on an orbital shaker or
stirred at room temperature for 12-60 h. Evaporation of the
solvents and purification by reverse-phase HPLC afforded the
pure alkene intermediates.
(4S)-4-{[(4-{[(3-Methoxypropyl)amino]carbonyl}-6-phenylpyr-
idin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)carbonyl]pipe-
razin-1-yl}pentanoic Acid (80i). General procedure Q afforded
19.3 mg (31%) of product 80i. HRMS calcd for C32H43N5O8
(Mþ þ H) 626.3190, found 626.3193.
(4S)-5-Oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}-4-[({6-
phenyl-4-[(tetrahydro-2H-pyran-4-ylamino)carbonyl]pyridin-2-
yl}carbonyl)amino]pentanoic Acid (80j). General procedure Q
afforded 15.5 mg (42%) of product 80j. HRMS calcd for
Reduction of the Alkene. The alkenes (∼0.35 mmol) were
dissolved in EtOAc (2.5 mL), and the reaction vessel was flushed
with N2(g). A slurry of 10% Pd/C (0.016 mmol) in EtOAc (0.5
mL) was added to the solution, and the reaction vessels were
flushed with N2(g) and then H2(g). The mixtures were stirred for
2-18 h under a balloon of H2(g) and then filtered through a pad
of Celite. The filtrate and cake rinses were dried in vacuo, and
the residues were purified by HPLC to afford pure products 83.
General Procedure R. Method B (Sonogashira): Preparation of
4-Aliphatic Pyridine Compounds 83. A solution of pentyl 4-[(2S)-
5-tert-butoxy-5-oxo-2-{[(6-phenyl-4-{[(trifluoromethyl)sulfonyl]-
oxy}pyridin-2-yl)carbonyl]amino}pentanoyl]piperazine-1-carboxyl-
ate 78 (386 mg, 0.54 mmol), CuI (23 mg, 0.12 mmol), Pd(PPh3)4
(39 mg, 0.034 mmol), and diethylamine (165 μL, 1.6 mmol) in THF
(5.4 mL) was added to each alkyne 82 (1.5 mmol). The mixtures
were agitated at room temperature for 2-48 h and then diluted
with R,R,R-trifluorotoluene (4 mL) and aqueous saturated NH4Cl
C
33H43N5O8 (Mþ þ H) 638.3190, found 638.3191.
(4S)-4-{[(4-{[(3-Methoxypropyl)(methyl)amino]carbonyl}-6-
phenylpyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)-
carbonyl]piperazin-1-yl}pentanoic Acid (80k). General pro-
cedure Q afforded 23.3 mg (42%) of product 80k. HRMS
calcd for C33H45N5O8 (Mþ þ H) 640.3347, found 640.3323.
(4S)-4-({[4-({[3-(Dimethylamino)propyl](methyl)amino}carbo-
nyl)-6-phenylpyridin-2-yl]carbonyl}amino)-5-oxo-5-{4-[(pentyl-
oxy)carbonyl]piperazin-1-yl}pentanoic Acid (80l). General pro-
cedure Q afforded 31 mg (55%) of product 80l. HRMS calcd
for C34H48N6O7 (Mþ þ H) 653.3663, found 653.3646.
(4S)-4-{[(4-{[Methyl(1-methylpiperidin-4-yl)amino]carbonyl}-
6-phenylpyridin-2-yl)carbonyl]amino}-5-oxo-5-{4-[(pentyloxy)-
carbonyl]piperazin-1-yl}pentanoic Acid (80m). General procedure

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2035
(4 mL). The organic phase was isolated and evaporated to afford the
tert-butyl ester intermediates.
(4S)-4-({[4-(3-Morpholin-4-yl-3-oxopropyl)-6-phenylpyridin-
2-yl]carbonyl}amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (83l). General procedure R, method A,
afforded 49 mg (18%) of product 83l. HRMS calcd for
C34H45N5O8 (Mþ þ H) 652.3347, found 652.3336.
Deprotection of the tert-Butyl Ester Group. A solution of 15%
trifluoroacetic acid/dichloromethane (2.2 mL) was added to the tert-
butyl ester (∼0.27 mmol) agitated on an orbital shaker or stirred at
room temperature for 24 h. Evaporation of the solvents and purifica-
tion by reverse-phase HPLC afforded the pure alkyne intermediates.
Reduction of the Alkyne. The alkynes (∼0.25 mmol) were
dissolved in EtOAc (2 mL), and the reaction vessel was flushed
with N2(g). A slurry of 10% Pd/C (0.025 mmol) in EtOAc (0.4
mL) was added to the reaction solution, and the reaction vessels
were flushed with N2(g) and then H2(g). The mixtures were stirred
for 1.5 h under a balloon of H2(g) and then filtered through a pad
of Celite. The filtrate and cake rinses were dried in vacuo, and
the residues were purified by HPLC to afford products 83.
(4S)-4-({[4-(Cyclohexylmethyl)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (83a). General procedure R, method A, afforded 48.4 mg
(19%) of product 83a. HRMS calcd for C34H46N4O6 (Mþ þ H)
607.3495, found 607.3529.
Biology. P2Y12 Radioligand Binding Assay. CHO (Chinese
hamster ovary) cells transfected with a synthetic human P2Y12
gene were cultured in R-MEM containing 10% dialyzed FBS,
0.05% Geneticin, 1ꢀ GlutaMAX, and 1ꢀ penicillin-strepto-
mycin. Washed membranes prepared from near confluent cells
were stored at -80 °C. Binding reactions were conducted in
polypropylene assay plates in a volume of 150 μL of assay buffer
(50 mM Tris, 100 mM NaCl, 1 mM EDTA) including 0.3 μg/well
membrane protein, 0.2 nM 33P-MeSADP, and serial dilutions of
test compounds, vehicle, or 300 nM 2-MeSADP for the defini-
tion of nonspecific binding. Dry compounds were prepared as 10
mM DMSO stocks and were diluted in seven-point, 3-fold
dilution series in assay buffer with 0.02% BSA. Each concentra-
tion was run in triplicate beginning at 10 mM, final concentra-
tion in the assay.
Binding reactions were incubated at room temperature for 1 h
and stopped by dilution and transfer/aspiration of the mixture
onto GF/B UniFilter 96-well plates (Perkin-Elmer) and washed
3ꢀ with ice-cold 50 mM Tris, pH 7.4, aspirating between each
wash. The filter plates were counted on a Top Count (Perkin-
Elmer), and data were analyzed using GraphPad Prism using a
single site binding equation.
Blood Donors and Draws. The donors consisted of males and
females of 18-65 years, and those with sickle cell anemia were
excluded. Blood donors were asked to abstain from aspirin and
nonsteroidal anti-inflammatory drugs (NSAIDs) usage 7-10
days prior to donation, but fasting was not required. A total of
100 mL each per donor (ꢀ4 donors) was collected into five
individual 20 mL syringes (Terumo brand, VWR, St. Louis,
MO) filled with 2 mL of 0.105 M buffered sodium citrate
anticoagulant (Sigma, St. Louis, MO) using a 19 gauge butterfly
needle.
(4S)-4-({[4-(3-Hydroxypropyl)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (83b). General procedure R, method B, afforded 8.3 mg
(5%) of product 83b. HRMS calcd for C30H40N4O7 (Mþ þ H)
569.2970, found 569.2971.
(4S)-4-({[4-(4-Hydroxybutyl)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (83c). General procedure R, method B, afforded 28 mg
(17%) of product 83c. HRMS calcd for C31H42N4O7 (Mþ þ H)
583.3126, found 583.3102.
(4S)-4-({[4-(5-Hydroxypentyl)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (83d). General procedure R, method B, afforded 49.3 mg
(28%) of product 83d. HRMS calcd for C32H44N4O7 (Mþ þ H)
619.3102, found 619.3073.
(4S)-4-({[4-(3-Methoxypropyl)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (83e). General procedure R, method B, afforded 81 mg
(45%) of product 83e. HRMS calcd for C31H42N4O7 (Mþ þ H)
583.2126, found 583.3124.
(4S)-4-({[4-(4-Methoxybutyl)-6-phenylpyridin-2-yl]carbonyl}-
amino)-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic
Acid (83f). General procedure R, method B, afforded 20.4 mg
(11%) of product 83f. HRMS calcd for C32H44N4O7 (Mþ þ H)
597.3283, found 597.3270.
(4S)-4-[({4-[3-(Isobutylamino)propyl]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pent-
anoic Acid (83g). General procedure R, method B, afforded 22 mg
(13%) of product 83g. HRMS calcd for C34H49N5O6 (Mþ þ H)
624.3756, found 624.3728.
(4S)-4-[({4-[3-(Dimethylamino)propyl]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pent-
anoic Acid (83h). General procedure R, method B, afforded 58 mg
(34%) of product 83h. HRMS calcd for C32H45N5O6 (Mþ þ H)
596.3443, found 596.3481.
(4S)-4-[({4-[3-(Diethylamino)propyl]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pent-
anoic Acid (83i). General procedure R, method B, afforded 40 mg
(23%) of product 83i. HRMS calcd for C34H49N5O6 (Mþ þ H)
624.3756, found 624.3732.
Chemicals and Agonists. Dilutions were prepared in analytical
grade (ACS) reagents such as PBS (Gibco, Carlsbad, CA),
DMSO, and human albumin serum (Sigma). Stocks of a 1
mM adenosine diphosphate (ADP) (Chrono-Log, Havertown,
PA) were prepared in PBS for a final agonist concentration of 20
μM in assay per 96 reaction well.
Human Platelet-Rich Plasma Preparation. Platelet-rich plas-
ma (PRP) was prepared from whole blood by diluting prepared
PRP with prepared platelet poor plasma (PPP) to achieve a final
concentration of 330 000 platelets per microliter for use in assay.
All centrifugation steps were performed at room temperature in
a Beckman Allegra X-12 benchtop centrifuge using rotor
SX4750A. Whole blood collected from syringes was transferred
into 50 mL VWR conical tubes. Any whole blood with evidence
of clotting was excluded from preparation. Whole blood sam-
ples containing an acutely dense appearance of lipemia were also
excluded from PRP preparations, but minor lipemic conditions
were allowed. PRP was obtained by a two-step centrifugation
process: Centrifugation at 910g (average) for 10 s was immedi-
ately followed by centrifugation at 220g (average) for 15 min.
The upper layer was collected using a 10 mL pipet, into a
polypropylene bottle (Corning, Acton, MA), leaving a 5 mL
layer of liquid behind to avoid disturbing the buffy coat and red
blood cells layers in the conical tubes. For platelet poor plasma,
the lower phase and remaining PRP in the conicals was cen-
trifuged at 2380g for 15 min. The upper layer was collected
again, taking care not to disturb the RBC layer, into a clean
polypropylene bottle. PRP platelets counts were determined
using a Z1 Coulter particle counter (Beckman Coulter, Full-
erton, CA), and PRP was adjusted to 330 000 cells per microliter
by dilution with PPP.
(4S)-4-[({4-[4-(Dimethylamino)butyl]-6-phenylpyridin-2-yl}car-
bonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pent-
anoic Acid (83j). General procedure R, method A, afforded 62.4
mg (24%) of product 83j. HRMS calcd for C33H47N5O6 (Mþ þ H)
610.3605, found 610.3608.
(4S)-4-[({4-[3-(Dimethylamino)-3-oxopropyl]-6-phenylpyridin-
2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-
1-yl}pentanoic Acid (83k). General procedure R, method A,
afforded 88.4 mg (35%) of product 83k. HRMS calcd for
C32H43N5O7 (Mþ þ H) 610.3240, found 610.3248.
ADP-Induced Human PRP Aggregation Assay. Platelet ag-
gregation was measured with a microplate spectrophotometer

2036 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Parlow et al.
(SpectraMax Plus 384, SoftMax Pro software, Molecular De-
vices, Sunnyvale, CA). First, a dilution plate at 5% DMSO/
4.2% HSA in PBS was prepared by 2-fold serial dilution of
DMSO stocks of test compounds to yield 7 concentrations per
96-well plate (500-7.813 μM). The last plate row contains buffer
alone for effect control purposes. Next, assay plates are pre-
pared from the dilution plate by pipetting 18 μL (1/10 of 180 μL
total assay reaction volume) in duplicate columns on an empty
assay plate. Final assay dose response concentrations for test
compound and reference control AZD-6140 were 10, 5, 2.5,
1.25, 0.625, 0.313, 0.156 μM at 0.5% DMSO. The reference
compound AZD-6140 was included on every plate for normal-
ization purposes to minimize donor-to-donor variability. For
preincubation of PRP and vehicle or vehicle plus compound,
144 μL of PRP was added to the assay plate containing diluted
analogues, then mixed 30 s on titer plate shaker (setting 3), then
warmed for 5 min at 37 °C on heater block. Reverse pipetting
(Rainin LTS multichannel pipet) prevented the formation of air
bubbles on the surface of plasma that otherwise would obscure
reader function, giving rise to false values. Aggregation was then
initiated by addition of 18 μL of 200 μM ADP (20 μM final
concentration in assay) to assay plate. The last row on each plate
containing PRP plus vehicle was divided by half, so the first six
wells represented 100% effect aggregation to which 18 μL of
ADP was added and the last six wells represented 0% effect
aggregation to which 18 μL of PBS was added. Immediately,
aggregation response was measured by kinetic readings of the
optical density at 626 nm at 37 °C for 15 min using a SpectraMax
Plus spectrophotometer. The kinetic program was set for auto-
mix function (oscillatory bidirectional motion) for 45 s between
the 1 min reading intervals during the 15 min duration. Assay
plate preincubations and incubations were staggered in order to
have one plate at the end point read while another plate was
ready for kinetic read. Concentration-response data from the
15 min end point read were analyzed using BioAssay Solver to
afford IC50 values. Final volume and concentration of assay
were as follows: total volume per well 180 μL, 0.5% final
DMSO, 20 μM ADP, 4.752 ꢀ 107 platelets/well, and 50 or 10
μM test compounds.
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Article
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(14) Piperazinyl glutamate pyridines with the R-glutamic acid com-
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showed a significant decrease in binding activity (>30-fold less
active) and did not exhibit any PRP activity at 50 uM. The R
enantiomer of 47s has a Ki of 510 nM and is inactive in the PRP
assay, IC50 > 50 μM.
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(11) Compounds 9-11; 26a-g; 45a,c,f,g,k,m,p,s,u; 47k,s,u; 48s,u; 50b,e,
f,h,i,m,n,q,w; 51e,f,i,n; and 52e,f,i,n and their corresponding P2Y12
binding Ki values and human PRP IC50 values have been previously
published.9a
(12) In most cases, the difference in Ki value from the binding assay to
the IC50 value of the functional PRP assay was attributed to protein
binding. These compounds showed significantly reduced binding
affinity when 0.4% human serum albumin was added to the
binding assay.
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