Series of Novel and Highly Potent Cyclic Peptide PCSK9 Inhibitors Derived from an mRNA Display Screen and Optimized via Structure-Based Design

Article abstract of DOI:10.1021/acs.jmedchem.0c01084

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein-protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.

Full text of DOI:10.1021/acs.jmedchem.0c01084

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Article
Series of Novel and Highly Potent Cyclic Peptide PCSK9 Inhibitors
Derived from an mRNA Display Screen and Optimized via Structure-
Based Design
Candice Alleyne, Rupesh P. Amin, Bhavana Bhatt, Elisabetta Bianchi, J. Craig Blain, Nicolas Boyer,
Danila Branca, Mark W. Embrey, Sookhee N. Ha, Kelli Jette, Douglas G. Johns, Angela D. Kerekes,
Kenneth A. Koeplinger, Derek LaPlaca, Nianyu Li, Beth Murphy, Peter Orth, Alonso Ricardo,
Scott Salowe, Kathleen Seyb, Aurash Shahripour, Joseph R. Stringer, Yili Sun, Rodger Tracy,
Chengwei Wu, Yusheng Xiong, Hyewon Youm, Hratch J. Zokian, and Thomas J. Tucker*
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ABSTRACT: Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C)
and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a
series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein−protein interaction between PCSK9
and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the
potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor
peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-
day PCSK9 therapeutics for the treatment of cardiovascular disease.
for LDL-C levels.1 Direct blockade of the LDL-receptor−
INTRODUCTION
■
PCSK9 interaction therefore serves to increase the steady-
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is
state levels of LDLR, making more LDLR available to assist in
a key regulator of plasma LDL-cholesterol (LDL-C) and an
the clearance of circulating LDL-C.1 There are currently two
important target for treating coronary artery disease.1 In
commercial therapeutic antibodies (alirocumab2 and evolocu-
genetic studies, it was demonstrated that humans hetero-
mab3) that have been approved for the treatment of familial
zygous for specific gain-of-function truncation or missense
hypercholesterolemia and for lowering LDL-C levels in
PCSK9 mutations (∼2−3% of the population) have a 15−
patients who are not at goal via statin therapy. Phase III
40% reduction in plasma LDL-C and a 50−90% reduction in
outcome studies with both have shown beneficial effects on
the risk of CHD.1 It has also been demonstrated via similar
cardiovascular events but demonstrated effects on lowering
genetic data that humans who express specific loss-of-function
mutations in PCSK9 have severe hypercholesterolemia and a
high risk of coronary artery disease.1
Received: August 3, 2020
The mechanism for the effect of PCSK9 on LDL-
cholesterol (LDL-C) is well defined. PCSK9 in circulation
decreases LDL-C levels by binding to the LDL-receptor
(LDLR) on the cell surface, and the resulting endocytosis and
lysosomal degradation serves as a key regulatory mechanism
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© XXXX American Chemical Society
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A

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Figure 1. mRNA display approach using Ra Pharma Platform.
Figure 2. Two lead series derived from mRNA display screening.
overall mortality await further data.2,3 These antibodies are
administered subcutaneously either biweekly or monthly, and
their initial uptake in the marketplace has not been as large as
anticipated largely because of pricing pressures.4
and level control compared to parenteral administration.
Another benefit would be the ease of therapy withdrawal by
simply discontinuing the dosing regimen.
Based on these factors, we chose to develop novel inhibitors
of PCSK9 with the goal of eventually developing oral
therapeutics for the treatment of hypercholesterolemia and
coronary artery disease. Strategically, our intent was to
develop PCSK9 inhibitors that generate equivalent LDL-C
lowering to the established mAb standards evolocumab2 and
alirocumab.3 The target of such inhibitors would be to
orthosterically block the protein−protein interaction (PPI)
between PCSK9 and LDLR by binding to the large, flat LDLR
binding surface on the PCSK9 protein, thus preventing the
direct interaction between the proteins.1 The proteins and
their interaction are well characterized in the literature,7 and
this provided a well-validated target for therapeutic
intervention.1 There are a large number of PCSK9 inhibitors
reported in the literature, with several recent descriptions of
novel small-molecule8−11 and peptide12−14 inhibitors, as well
It is estimated that of the approximately 70 million patients
treated with statins, as many as 50% do not reach their target
LDL-C levels.5,6 Of those that do achieve treatment goals,
another several million suffer major cardiovascular events
annually.5,6 The need exists for additional therapeutic options
for those that do not achieve their LDL-C goals, patients with
familial hypercholesterolemia, patients that do achieve goal
but have cardiovascular events, patients with other comorbid-
ities such as type 2-diabetes mellitus, and also in statin-
intolerant patient populations. An oral, once daily PCSK9
inhibitor therapy could potentially offer a more convenient
alternative for administration and better patient adherence.
The orally delivered therapy of this type would easily align
with other oral therapies that are standard of care in the at-
risk population and would provide the benefit of easy dosing
B
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Scheme 1. General Approach to the Synthesis of Cyclic Peptides
as siRNA-based therapeutics such as inclisiran.15 The PCSK9
inhibitor area has recently been the subject of several
comprehensive reviews.16−18 However, none of the known
inhibitors have the overall profile of potency and favorable
druglike properties necessary to be viable as oral therapeutic
agents.
The initial mRNA display libraries of linear peptides of
various lengths were prepared containing two reactive thiol
moieties, allowing for direct cyclization of the libraries using
the well-known 1,3-di(bromomethyl)benzene (DBX) as the
key cross-linking element (Figure 1).20 The DBX group is a
commonly used and highly efficient cross-linking element for
cyclization of peptide libraries derived from mRNA display.20
From an initial series of mRNA screens targeting cyclic
peptides of various sizes, we obtained two novel hit series
(Figure 2). Compound 1 (Figure 2) was identified as a potent
13 amino acid hit with good potency against PCSK9 using the
LDLR-FRET assay. A second, less potent but structurally
unique 12 amino acid series was also found, with compound 2
(Figure 2) emerging as the key lead molecule from this series.
The initial comparisons of the two series showed less overall
homology, and analysis strongly suggested differing binding
modes for each of the molecules. An early critical goal for the
team was to obtain crystal structures of each of these
representative structures bound to the LDLR binding site on
PCSK9. We quickly obtained a crystal structure of the
chemical series exemplified by 1 but were not able to initially
obtain a structure for 2 or any early analogues, likely because
of the large differences in potency between the two series, and
We chose to initially pursue macrocyclic peptides as our
modality of choice because of their potentially superior
stability characteristics19 as well as their proven ability to bind
to the large, flat surfaces that are characteristic of PPIs such as
the interaction between PCSK9 and LDLR. In collaboration
with our partner Ra Pharmaceuticals,20 we used the mRNA
display technology to find potent, cyclic peptide inhibitors of
PCSK9 that we could then engineer using internal medicinal
chemistry expertise to be deliverable via oral dosing. The use
of mRNA display as a proven and powerful platform for early
hit finding is well documented in the literature.20,21 Herein,
we detail our initial screening hit peptides and the
optimization of these macrocyclic peptides to provide highly
potent, novel, and specific inhibitors of PCSK9 that serve as
key starting points for the development of oral PCSK9
inhibitor therapeutics.
C
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Figure 3. Early progression of SAR from original mRNA display lead 2. Key structural changes are highlighted in red for clarity.
D
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a b c d e
, , , ,
Table 1. Mouse IV PK Parameters for Various Compounds
d
compound
number
clearance
(mL/min/kg)
protein binding
AUC0−inf (nM h)
AUC0−x (nM h)
Vdss (L/kg)
T1/2 (h)
(% bound, mouse plasma)
b
4
241.93 66.81
181.43 45.51
491.78 44.53
2505.11 164.56
171.52 3.03
240.9 42.20
184.73 45.34
490.65 43.34
2494.04 157.65
170.56 3.35
51.73 15.23
65.40 14.10
24.25 1.55
4.62 0.29
17.25 0.25
16.00 3.30
0.70 0.48
1.2 0.59
0.29 0.13
1.04 0.11
0.33 0.33
0.78 0.03
0.25 0.03
1.60 0.29
0.66 0.09
95.02
94.85
99.23
99.90
98.33
98.50
b
30
40
44
53
b
b
0.10
0.37
0
0
c
78
840.94 174.30
839.90 174.25
0.23 0.11
a
Compounds were tested in C57BL/6 mice at 1 mpk using a 30% propylene glycol/70% PBS vehicle at a concentration of 0.5 mg/mL or at 1 mpk
b
using a 20% DMSO/60% propylene glycol/20% water vehicle at a concentration of 1 mg/mL. For compounds 4, 30, 40, and 44, the values are the
average of three mice from a single experiment with the standard deviations noted. For compounds 53 and 78, the values are the average of two
mice from a single experiment, and the indicated errors are the difference between the mean and the individual values. All protein binding values
are derived from a single experiment. Raw concentration vs time profiles for all compounds are provided in the Supporting Information, along with
c
d
e
Tmax and Cmax data.
a
Table 2. Stability of Key Compounds against Gut Proteases and Whole Blood
chymotrypsin, 1 h
(% remaining)
elastase, 1 h
(% remaining)
pepsin, 1 h
(% remaining)
trypsin, 1 h
(% remaining)
mouse whole blood
Cyno whole blood
compound #
T1/2 (min)
T1/2 (min)
4
5
12
15.5
10.9
7.4
42.7
20.1
76.4
91
60.7
54.9
52.7
46.1
99.1
96.5
100
51
95
92.5
94
99.1
93.7
96.6
100
95
99.5
98.1
87.5
98.6
98.9
98.2
94.4
1.2
1.0
0.9
63.2
89.6
ND
ND
30
39
40
41
44
50
52
53
60
62
75
78
79
311.3
333.5
186.1
>360
>360
>360
62
>360
>360
>360
ND
>360
ND
>360
>360
ND
ND
186
ND
ND
0.8
79.5
91.0
94.3
1.9
91
56
1.2
100
24.2
39.5
91.8
2.5
100
68.2
96.3
96.6
100
98.8
95.9
98.4
96.0
99.2
100
97.6
ND
>360
113
>360
>360
95.7
>360
a
Compounds were incubated in duplicate in a single experiment for 1 h at 37 °C; ND indicates that the value was not determined for the specific
molecule.
had to rely primarily on empirical data and molecular
modeling based on that data. Details of the efforts around 1
will be described in another paper currently in preparation.22
The current manuscript will focus on the optimization of the
lead molecule 2, initially using standard empirical medicinal
chemistry strategies, which were later coupled with advanced
structure-based drug design approaches.
lasso “tail” region, we were quickly able to remove the amino
acids in the tail to provide 3 (Figure 3), which demonstrated a
20-fold improvement in potency in our LDLR-FRET assay.
Further truncation of the N-terminus to completely remove it
provided 4, which lost only about 2-fold potency versus 3
(Figure 3). Compound 4 became a key early analogue for our
program, and we decided to characterize overall properties in
more depth. We wanted to address the stability of our cyclic
peptide platform early on and chose to evaluate our peptides
against a series of important gastrointestinal proteases
(trypsin, chymotrypsin, pepsin, and elastase) for ultimate
oral dosing and in whole blood for intravenous (IV) dosing.
Early IV PK studies were primarily performed in C57BL/6
mice to keep the amount of compound needed from synthesis
at a minimum. Table 1 summarizes the IV PK for a number of
molecules discussed throughout this paper, and Table 2 details
the observed protease and whole blood stability. Compound 4
was shown to have a short half-life and moderately high
clearance in the mouse, and the whole blood stability
indicated a moderate half-life in mouse whole blood of
about 1 h. The protease panel clearly showed poor stability in
the presence of chymotrypsin, elastase, and trypsin. For 4, and
in general across all the peptides tested, full stability to pepsin
was observed. Metabolite identification (Met ID) studies after
incubation with each of the key proteases also showed major
RESULTS AND DISCUSSION
■
Screening and Early Structure−Activity Relationship.
Test peptides were initially evaluated for PCSK9 inhibition
activity using an LDLR-FRET assay (described in the
Experimental Section), and the results are initially reported
as IC50 values. In general, the peptides were synthesized using
standard solid-phase peptide synthesis (SPPS) approaches to
prepare the crude linear peptides after cleavage and
deprotection (Scheme 1). The crude peptide could then be
easily cyclized by addition of 1,3-di(bromomethyl)benzene
(DBX), followed by purification to give the desired cyclic
peptide products (Scheme 1). Specific synthetic details are
provided in the Experimental Section. Starting from
compound 2, we envisioned an approach by which we
would attempt to reduce the size and molecular weight of the
target peptides while also simultaneously addressing the
potency and peptide stability. Starting with the N-terminal
E
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Figure 4. Primary metabolic sites on a representative molecule based on met ID studies with key proteases and mouse whole blood.
metabolic vulnerabilities at four amide bonds in the N-
terminal region of the peptide (Figure 4), and we were able to
correlate the most vulnerable individual amide bonds with the
corresponding responsible protease. Met ID studies with the
peptide after incubation in mouse whole blood showed a
major metabolic site at the amide bond between the proline at
position 8 and the cysteine at position 9 (Figure 4).
Alanine, D-alanine, and N-methyl amino acid scans were
also performed using 4 as the scaffold. The results of these
scans are summarized in Table 3. The alanine scan (Table 3,
5−12) showed some tolerance for side chain modifications at
positions 1 (Lys; 5), 3 (5F-Trp at position 3; 7), 5(Asp; 9),
and 6 (His; 10). Interestingly, 5-fluoro-tryptophan (5F-Trp)
at position 3 appeared to be much more amenable to change
than the one at position 4. The D-amino acid scan (Table 3,
13−20) showed less tolerance for D-amino acids, although
position 2 (Gly) was not surveyed, and this would later
become of importance. Similarly, the N-methyl amino acid
scan (Table 3, 21−28) showed less tolerance for backbone N-
methylations, with the exceptions being positions 1 (Lys; 21)
and 7 (Tyr; 27) where N-methylation appeared to be
reasonably well tolerated.
Removal of the C-terminal amide of 4 provided 29 (Figure
3), which demonstrated a small 2-fold decrease in inhibitory
potency but removed two H-bond donors. As indicated above,
a metabolic liability was identified for the amide bond
between proline and cysteine that we theorized was likely due
to a prolyl-endopeptidase or similar enzyme, and we wanted
to address this issue immediately. For this reason, the proline
was replaced with an α-methyl proline to give 30. Peptide 30
turned out to be a key molecule for the evolution of our entire
program.
Tables 4 and 5 summarize the key SAR around positions 3
and 4 (5F-Trp moieties) and 8 (proline), respectively. Of
great interest were the unusual consecutive 5F-Trp moieties at
positions 3 and 4. From the data in Table 4, it became quite
clear that the 5F-Trp moiety at position 4 was playing a
crucial role in the binding of the peptide. Replacement of 5F-
Trp at position 3 with a simple tryptophan (Table 4, 31)
resulted in a small loss of potency. However, replacement of
5F-Trp at position 4 with a tryptophan (Table 4, 32) resulted
in a more pronounced 23-fold loss of potency. In addition,
5F-Trp at position 3 could be replaced with an alpha-
naphthylalanine (Table 4, 1-Nal; 33) with potency main-
tained. Replacement of 5F-Trp at position 3 with either a
larger 4-phenylphenylalanine (Table 4; 34) or a homopheny-
lalanine (Table 4; 35) demonstrated that while there was
some tolerability at this position, larger or longer side chains
were not well tolerated. Table 5 summarizes a few changes at
the Pro moiety at position 8.
As stated earlier, the addition of an alpha-Me proline to the
peptide provided an 8-fold potency enhancement versus 4 and
was a key SAR element at this position. Opening the proline
ring by replacement with a sarcosine provided 36, which
demonstrated a large potency loss. Replacement of the proline
with a four-membered azetidine amino acid 37 provided only
a slight loss of inhibitory potency versus 30, while enlarge-
ment of the ring to a six-membered ring in 38 showed a
substantial loss of inhibitory potency versus both 4 and 30.
Crystal Structure of Compound 30. The most
important aspect of the synthesis of compound 30 was that
we were able to use this compound to generate the first
cocrystal structure in this series with PCSK9. We were unable
to generate suitable crystals with earlier inhibitors in this
series, most likely because of the limited potency observed
with these molecules. The crystal structure of 30 was
incredibly informative in multiple dimensions and is shown
in Figure 5. The peptide assumes an overall “doughnut-like”,
circular binding conformation, with the side chain of 5F-Trp
at position 4 in the center of the doughnut, sticking into a
small pocket on the relatively flat surface of the binding site.
The fluorine sits nicely between the two H-bonded β-sheet
regions of the protein, with the shallow pocket defined by the
backbone NH and carbonyls of Gly 370, Val 380, Ser 381, and
Ile 369 and the side chains of Ile 369, Ser 381, and Phe 379
(Figure 6). The structure suggests possible favorable direct
We observed an 8-fold improvement in inhibitory potency
versus parent peptide 4 and much improved whole blood
stability (Table 2), suggesting alleviation of the metabolic
issue at this center. Despite having much improved stability in
mouse whole blood, compound 30 (Table 1) was shown in
the mouse to have a moderate clearance and extremely short
half-life. The molecule demonstrated poor stability against our
protease panel (Table 2).
In an attempt to explore some general structure−activity
relationship (SAR) around the peptides, we initiated a broader
survey of the SAR at several of the key positions along the
peptide, using the peptide backbones from peptides 3 and 4.
F
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a
Table 3. Alanine, D-Amino Acid, and N-Me Amino Acid Scans on 4
a
For all determinations, data is the average of n = 2, with the variability of the two determinations reported for molecules <1 μM. Data for the
parent molecule 4 is highlighted in green. Purity of all peptides >95%; all peptides exhibited correct MS; MPA = 3-mercaptopropionic acid, all
peptides cyclized from MPA to CYS using 1,3-di(bromomethyl)benzene (DBX). For peptides 14 and 15, D-Trp was substituted for the unavailable
D-5F-Trp; for peptides 23 and 24, racemic N-Me Trp was used in the scans, and two diastereomers were recovered and tested for each; all were
inactive.
interactions between the aryl fluorine atom and the NH of
Gly 370, the carbonyl oxygens of Val 380 and Ile 369, as well
as various interactions with the side chain carbons of the
aforementioned amino acid side chains. Such noncovalent
interactions between fluorine atoms and various amino acids
are well documented in the literature.23 The specific nature of
the fit of the fluorine into this small surface pocket along with
the previously described analogue work at this position
confirms the critical nature of the interaction of the aryl
fluoride with the small pocket on the PCSK9 surface. The
selection of the 5F-Trp amino acid as one of the amino acids
for the original mRNA display campaign was clearly critical to
the entire screening and hit finding process with these
peptides.
The lysine side chain at the N-terminal position 1 is fully
solvent-exposed and does not appear to be making any
specific interactions with the protein (Figure 5). The first 5F-
Trp in the sequence at position 3 lies outside of the
macrocyclic ring, opposite the inner 5F-Trp at position 4,
partially solvent-exposed and contacting a lipophilic region
consisting of the disulfide bond between Cys 375 and 378 and
the side chains of Val 380 and Asp 374. The Asp−His region
provides a turn-like geometry, with the Asp and His side
chains of the inhibitor forming an ion pair that helps to lock
this conformation. A key aspect of this entire N-terminal
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a b
,
Table 4. SAR for Peptide Amino Acid Positions 3 and 4
LDLR-FRET Ki
compound # N-terminus
X
Y
(nM)
3
4
AcNHCys
MPA
AcNHCys
AcNHCys
MPA
5F-Trp
5F-Trp
Trp
5F-Trp
1-Nal
5F-Trp
5F-Trp
5F-Trp
Trp
5F-Trp
5F-Trp
5F-Trp
47.2 1.6
111 1.3
103 1.6
1070 220
63 1.7
31
32
33
34
35
b
b
MPA
MPA
4Ph-Phe
homePhe
29,345 (n = 2)
2211 (n = 2)
b
a
FRET Ki values are the average of at least three determinations with
the standard deviation indicated unless otherwise noted; MPA = 3-
mercaptopropionic acid; alpha Nal = alpha-naphthylalanine; and 4-
b
PhPhe = 4-phenyl-phenylalanine. For n = 2, the error is reported for
Figure 6. Close-up of the shallow binding pocket occupied by 5F-
Trp in position 4 of 30. The pocket is defined by two hydrogen-
bonded beta sheets, with the aryl ring and 5-fluroro substituent of the
ligand fitting downward into the space between.
molecules <1 μM; error is defined as the difference between the mean
and the individual determinations.
a
Table 5. SAR for Peptide Amino Acid Position 8 (Proline)
compound #
X
LDLR-FRET Ki (nM)
4
Pro
115 1.4
30
36
37
38
alpha-Me Pro
14.0 1.7 (n = >200)
10,740 20.3
67.5 33.5
Sar
Aze
Pip
969 110
a
LDLR-FRET Ki values are the average of three determinations with
the standard deviation noted unless otherwise indicated, except for
30, which was used as an assay standard; Sar = sarcosine; Aze = 2(S)-
azetidinecarboxylic acid; and Pip = 2(S)-piperidinecarboxylic acid.
Figure 7. Schematic representation of the five key hydrogen bonding
interactions between inhibitor 30 (salmon) and the PCSK9 active
site β-sheet (white). The key interactions are described in detail in
the text.
1. The NH of the glycine moiety in position 2 of the
inhibitor makes an interaction with the side chain OH
of Ser 381.
2. The carbonyl of the same glycine on the inhibitor
makes an interaction with the backbone NH of Ser381.
3. The NH of 5F-Trp at position 3 of the inhibitor makes
an interaction with the backbone carbonyl of Phe379.
4. The carbonyl of 5F-Trp at position 4 of the inhibitor
makes an interaction with the backbone NH of Phe379.
5. The backbone carbonyl of the aspartic acid residue at
position 5 of the inhibitor makes an interaction with the
side chain OH of Thr377.
Figure 5. X-ray crystal structure of 30 (salmon) bound to the LDLR-
binding site of PCSK9 at 1.55 Angstrom resolution (PDB: 6XIB).
The molecule binds in an overall circular conformation, with 5F-Trp
at position 2 outside of the doughnut and 5F-Trp at position 3 inside
the macrocycle, with the fluorine atom in a small pocket on the
surface. The side chains of position 1 (Lys), 5 (Asp), and 6 (His) are
all solvent-exposed and do not directly interact with the protein. The
side chain of position 7 (Tyr) lies in a small groove on the surface,
and the aryl ring of the cross-linker lies directly on top of Ile 369.
These key H-bonding interactions are critical for the good
potency observed with these peptides. The tyrosine moiety of
the inhibitor lies in one of the few true pockets in the binding
surface (Figure 5). This shallow pocket is defined by the side
chains of Phe379, Glu195, Asp238, and Ala239 and has a
small, solvent-exposed opening near where the para-position
of the tyrosine of the inhibitor resides. Interestingly, the
proline ring sits quite close to the 5F-Trp side chain of the
inhibitor that lies inside the macrocycle, suggesting a
conformationally reinforcing intramolecular interaction be-
tween the two groups, likely reinforced further by the addition
region is the presence of five key hydrogen bonding
interactions with a β-sheet on the protein that lies directly
under the N-terminal region of the inhibitor. Figure 7
schematically details each of these H-bonding interactions,
which can be characterized as follows:
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Figure 8. Continued SAR evolution of key lead molecules.
of the potency enhancing alpha-methyl group to the proline.
These intramolecular interactions appear to be crucial for
locking the molecule into a desirable conformation for
binding. The C-terminal amide is largely solvent-exposed
and does not interact with the protein, suggesting that it is
likely not critical for binding, as suggested by data from
compound 29. Finally, to our surprise, we observed that the
DBX cross-linking group was contributing to potency and
directly interacting with the PCSK9 surface. The aryl ring lies
directly above the side chain of Ile 369, almost sitting directly
on top of the side chain in a manner analogous to a tabletop.
Expanded SAR around Compound 30. As we moved
forward with analogues of improved potency, we began to
observe a bottoming of the LDLR-FRET values and
differentiation between compounds below the 10 nM range
became difficult. We therefore established a second, more
accurate Alexa-FRET assay that was able to better distinguish
between compounds with more potent inhibitory potency
(Experimental Section). The values for both assays are
reported for a number of compounds to allow for comparison,
but we eventually moved completely to the Alexa-FRET assay
for more optimized molecules.
In addition to the key binding insights described above, the
crystal structure of 30 also provided several critical design
insights that would prove to be the key to driving subsequent
efforts (Figure 8). We removed the C-terminus of 30 to
provide 39, which demonstrated minimal loss of potency and
a similar behavior in our stability assays (Table 2). The β-
sheet binding region also contains the four protease-labile
amide bonds previously identified in these molecules (Figure
4). Although the alanine scan with 4 (Table 3) clearly
indicated that an alanine was not tolerated at position 2 (Gly)
because of a likely unfavorable interaction with 5F-Trp that
lies inside the macrocycle, the crystal structure of 30 strongly
suggested that a D-alanine would be well tolerated at this
position. The region that would be occupied by a substituent
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of a D-amino acid points away from the protein and into an
open, solvent-exposed region in parallel with the side chain of
Lys at position 1. We theorized that using a D-amino acid at
this position might also help to block some of the metabolism
observed in this region of the molecule. We therefore
prepared the analogue 40 containing a D-alanine at position
2 (Figure 8). This molecule was shown to be essentially
equipotent with 30 and 39. 40 was shown to have improved
stability versus both elastase and trypsin, the enzymes
responsible for proteolysis at the two amide bonds flanking
the D-alanine at position 2 (Table 2). Therefore, our strategy
to insert a D-amino acid at this position based on the
crystallographic data from 30 appeared to be successful,
allowing for the stabilization of two of the four vulnerable
amide bonds. Interestingly, the mouse whole blood stability of
40 decreased slightly but was still quite good. However, 40
still showed a short half-life after IV dosing to mice (Table 1).
Nonetheless, the noted improvement in protease stability
became a key feature for future compound design. Figure 9
D-alanine moiety at position 2. The methyl group of the D-
amino acid can be clearly seen pointing away from the protein
and pointing toward solvent. It is also easy to see why an L-
amino acid side chain is not tolerated at this position as
indicated in the alanine scan, as it would likely bump the
indole ring system of the key 5F-Trp moiety at position 4,
causing a displacement of this critical interaction.
Based on the crystal structure of 30, it was observed that
there was some space available on the aryl ring of the DBX
cross-linker to put a substituent in the position meta to the
two sulfurs. Adding a fluorine to this position provided 41
(Figure 8), which added a small amount of potency versus 40
but did not add any additional stability to the peptides (Table
2), with the peptide remaining vulnerable to chymotrypsin
proteolysis at the remaining two previously identified amide
bonds. In addition, we also capped the OH of the tyrosine aryl
ring at position 7 with a methyl group to give 42, which
retained similar potency to 40 but eliminated an H-bond
donating group, an SAR item that could be of interest for later
designs targeted at optimizing permeability/oral bioavailabil-
ity.
To more broadly understand the scope and limitations of
the D-amino acid side chain at position 2, we also prepared
several direct analogues of 40 with various D-amino acids
replacing the D-alanine. The results of this small SAR scan are
detailed in Table 6. Lipophilic side chains such as those on
43, 44, 47, and 48 were all well tolerated, as were polar side
chains such as 45 and 46. In addition, 46 demonstrates that
while the stereochemistry of the two centers is different, the
substitution on positions 1 and 2 is likely interchangeable
because both of these side chains point away from the protein
and into solvent. The SAR in general suggested that position 2
was extremely tolerant and could likely be exploited via a wide
variety of substituents and might later be useful as a handle for
modifying solubility and physical properties or for adding
various labels. In addition, 44 was screened for IV PK in the
mouse; however, it displayed a similar short half-life to earlier
analogues (Table 1). Compound 44 also showed a protease
stability profile similar to 40 (Table 2), although the bulkier
side chain of 44 did appear to be able to impact the
vulnerability to chymotrypsin as well.
Figure 9. X-ray crystal structure of 40 (lt. yellow) bound to the
LDLR binding site on the surface of PCSK9 at 1.38 Angstrom
resolution (PDB: 6XIC). The molecule binds in an identical manner
as 30, with the methyl group of the D-Ala at position two
(highlighted in the black box) pointing away from the protein
toward the solvent. The D-amino acid at this position also blocks
proteolytic degradation at the two flanking amide bonds caused by
trypsin and elastase activity.
Discovery of Pro−Thr Backbones to Replace Asp−
His. At this point, we wanted to take advantage of the finding
that the D-amino acid could add stability and wanted to
couple this with other changes in the backbone to address the
other metabolically labile amide bonds. In examining the
shows the crystal structure of 40; it is superimposable with the
previously shown structure of 30, with the addition of the key
a
Table 6. SAR for D-Amino Acids at Position 2
a
All values are the average of at least three determinations unless otherwise indicated; standard deviation is indicated for all n > 2; for n = 2, the
reported error is the difference between the mean and the individual; values. D-Nvl = D-norvaline; D-Val = D-valine; D-Thr = D-threonine; D-Lys = D-
lysine; D-Cpg = D-cyclopentyl gly; and D-Tbg = D-tert-butyl glycine. ND = not determined.
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Figure 10. Pro-Ala and Pro-Thr as replacements for Asp-His at positions 5 and 6 of the macrocyclic peptide.
crystal structures of compounds 30 and 40, it was apparent
that the Asp−His amino acids at positions 5 and 6 could be
replaced with amino acids that could similarly mimic the turn-
like geometry of this region. One simple replacement that
immediately came to mind was Pro−Ala. The Pro−Ala
dipeptide could serve as a simplified mimetic of Asp−His,
while also using the five-membered ring of the proline to mask
an additional NH, which we hoped would in turn stabilize one
of the remaining two metabolically labile amide bonds. Figure
10 details the progression of several analogues based on Asp−
His replacements. The simple Pro−Ala analogue 49 showed
the viability of this modification, although some inhibitory
potency was lost. The Pro−Thr analogue 50 regained some of
the lost potency. Combining this Pro−Thr backbone change
with the D-alanine at position 2 and the meta fluoro-DBX
linker (previously shown in 41) provided analogue 51. We
were able to obtain a crystal structure of compound 51
(Figure 11), which showed that Pro−Thr was a super-
imposable replacement for Asp−His with similar levels of
potency and the ability to mask an additional amide bond and
hopefully stabilize this region further against chymotrypsin.
The proline ring perfectly mimics the turn-like geometry of
Asp−His, and the side chain of Thr does not interact directly
with the protein and is solvent-exposed. The fluorine atom on
the aryl ring is shown to occupy open space in the center of
the macrocycle and may participate in and enhance the
intramolecular interactions between the α-Me Pro and the
indole ring of 5F-Trp at position 4. This intramolecular
interaction may in turn help to further stabilize the overall
conformation of the molecule.
Methylation of threonine OH to remove another H-bond
donor provided 52, which was equipotent with the
unmethylated analogue. This change was performed to
eliminate one H-bond donor from the molecule, and the
addition or subtraction of a methyl group here did not appear
to affect the potency or stability of the molecule greatly and as
such were used interchangeably. Guided by the crystal
structure, we were able to replace the proline in position 5
with the commercially available 4(S)-cis fluoroproline in an
attempt to further increase the chymotrypsin stability.
Combining this with the D-alanine at position 2 provided
53, which showed a 4-fold increase in inhibitory potency.
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Table 7. Rat Mast Cell Degranulation Activity for Select
a
Molecules
position 1 amino acid side
chain
rat mast cell degranulation
compound #
(EC50, μM)
4
30
53
Lys
Lys
Lys
positive@6.67
positive@6.67
positive@3.12
a
Histamine release activity was determined in rat peritoneal mast
cells, data for all compounds is the average of at least two
determinations. Rat peritoneal lavage contains about 5% mast cells,
which is sufficient to initiate a robust response (confirmed by the
positive control 48/80); assay end point is histamine release in
supernatant. Cells were incubated with test articles for 30 min in 1−
3% DMSO. For all tested molecules and DMSO controls, there was
no loss in cell viability up to 60 μM.
exhibited moderate to good inhibitory potency, again
confirming the solvent-exposed nature of this substituent
and the good tolerability to various changes at this position. A
strong correlation was found between the basicity of the
position 1 side chain and the observed mast cell degranulation
activity. More basic, free amine side chain-containing amino
acids such as ornithine (55) and epsilon N,N-dimethyl Lys
(61) demonstrated mast cell degranulation activity, while the
acidic side chain amino acids (54, 58, 59) and neutral side
chain amino acids (56, 57) were all negative in the assay.
Interestingly, the permanently positively charged epsilon
trimethyl Lys analogue 60 was also clean in our mast cell
degranulation assay. Despite the potential stability issues and
metabolic vulnerabilities based on the structure, 60 appeared
to have superior solubility characteristics and we decided to
evaluate the IV PK properties of the molecule. Peptide 60 was
dosed IV to mice and demonstrated improved pharmacoki-
netics (PKs) with a relatively moderate clearance and 1.2 h
half-life (Table 9).
Based on this result, 60 was also dosed IV in Wistar Han
rats, again demonstrating much improved IV PK with a low
clearance, 2.5 h half-life, and improved in vivo tolerability. This
molecule demonstrated the best overall IV PK in rodents of
any of the early molecules tested in this series. Acylating the
lysine epsilon amino group with a peg as in 62 (synthesized as
shown in Scheme 2) also eliminated mast cell degranulation
activity, as did the addition of moderately basic side chain
amines such as those in the PEGylated analogues 63
(synthesized as detailed in Scheme 2) and 66 (synthesized
as detailed in Scheme 3). The SAR at this position clearly
confirmed that free basic amino acid side chains such as Lys
and Orn were major contributors to the observed mast cell
degranulation activity and removing the basic amine or
substantially moderating the basicity remediated the undesired
activity. In general, the observed mast cell degranulation/
associated histamine release appears to be a compound/series
issue, directly related to the overall properties of the rest of
the molecule that contains the offending basic group.24,25 In
general, most amine-containing drug-like molecules do not
exhibit this activity; however, we have observed this
phenomenon internally with a number of diverse molecules
across various programs, suggesting that this behavior should
be suspected anytime a molecule containing a basic amine
side chain in a somewhat overall lipophilic framework
demonstrates an unusual or idiosyncratic toxicity in vivo.
Having obtained a reasonable understanding of this issue and
Figure 11. Crystal structure of 51 (gray) bound to the LDLR-
binding site on PCSK9 at 1.48 Angstrom resolution (PDB: 6XID).
The macrocyclic peptide binds similarly to the previously shown
crystal structures, with the Pro-Thr providing an excellent
replacement for the original Asp-His at positions 5 and 6. The Thr
side chain hydroxyl is solvent-exposed and does not make any direct
contact with the protein. The fluorine atom on the aryl ring is shown
to occupy open space in the center of the macrocycle and may
participate in and enhance the intramolecular interactions between
the alpha-Me Pro and the inner 5F-Trp aromatic.
Compound 53 demonstrated very good stability against the
entire panel of proteases including chymotrypsin and good
stability in mouse plasma (Table 2). However, after IV dosing
to mice, the compound was shown to have a moderate
clearance and a relatively short half-life (Table 1). The
introduction of a proline to this region appeared to help
stability against some proteases; however, it did not solve the
short in vivo half-life seen with these analogues despite their
apparent improved in vitro stability. We therefore chose to
pause work on Pro−Thr analogues.
Discovery and Remediation of Mast Cell Degranula-
tion Activity. At this point, based on the observations from
previous internal drug discovery programs, we became
concerned about the potential for mast cell degranulation
related to these peptides. We noted that all the molecules we
were synthesizing contained a highly basic lysine side chain in
an overall somewhat lipophilic framework. We had in previous
peptide and peptidomimetic drug discovery programs
correlated this generic structural phenotype in some cases
with histamine release in vivo, likely because of the extensive
mast cell degranulation activity.24,25 In an effort to confirm
this hypothesis, we tested several molecules in our rat mast
cell degranulation assay (Table 7). For the three molecules
initially screened, the results were striking. Moderately potent
mast cell degranulation activity was observed for 4, 30, and
53, all of which contained a lysine side chain in position 1 of
the peptides.
However, we needed to gather additional data to support or
disprove the mast cell degranulation hypothesis. We prepared
a series of analogues at the position 2 lysine to try to directly
confirm our initial hypothesis and also looked back at a few
earlier analogues that were made at this position as well. Table
8 details the potency and rat mast cell degranulation activity
for these Lys analogues (54−66). All these analogues
L
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a
Table 8. Lysine Analogues at Position 2: Inhibitory Potency and Rat Mast Cell Degranulation Activity for 70−63, 66
a
LDLR-FRET and Alexa-FRET data is the average of at least three determinations unless noted otherwise in table with standard error indicated; for
n = 2, the difference between the mean and the individual data points is noted as the error. The histamine release activity was determined in rat
peritoneal mast cells, data for all compounds is derived from at least two agreeing determinations. Rat peritoneal lavage contains about 5% mast
cells, which is sufficient to initiate a robust response; assay end point is histamine release in supernatant. Cells are incubated with test articles for 30
b
min in 1−3% DMSO. For all tested molecules and DMSO controls, there was no loss in cell viability up to 60 μM. 48/80 (refs 24 and 33) is used
as a positive control for the assay.
how to remediate it, we returned to our efforts to optimize the
overall properties of our molecules.
Novel Cyclizations. Upon further studying the crystal
structures of 30 and 51, we were struck by several potential
opportunities for novel cyclizations. From the crystal structure
of 51, it was apparent that there was more room in the space
occupied by the fluorine atom on the DBX linker. In fact, the
fluorine atom points directly at the NH of the indole of the
inner 5F-Trp, suggesting that the two groups could possibly
be linked together (Figure 12). We also chose to remove the
M
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a b
,
Table 9. Mouse and Rat IV Pharmacokinetics for 60
c
species
AUC0−inf (nM h)
AUC0−x (nM h)
clearance (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
protein binding (% bound)
a
b
C57BL/6 mice
Wistar Han rat
932.72 102.63
3648.33 422.20
895.96 70.06
3646.9 421.60
6.20 0.68
3.16 0.40
0.45 0.07
0.17 0.00
1.16 0.02
2.46 1.34
99.3
99.9
a
Compound was tested in C57BL/6 mice at 1 mpk using a 30% propylene glycol/70% PBS vehicle at a concentration of 0.5 mg/mL or at 1 mpk
using a 20% DMSO/60% propylene glycol/20% water vehicle at a concentration of 1 mg/mL. All determinations are the average of two mice from
b
a single experiment. The indicated errors are the difference between the mean and the individual values. Compound was tested in Wistar Han rats
at 1 mpk using a 20% DMSO/60% PEG/20% water vehicle at a concentration of 1 mg/mL. All determinations are the average of two rats from a
c
single experiment. The indicated errors are the difference between the mean and the individual values. Protein binding values are a single
determination.
Scheme 2. Synthesis of Compounds 62−63
C-terminal amide from this target molecule. We considered a
number of possible cross-linking strategies, but a copper-
catalyzed alkyne−azide cyclization (Click)26 reaction between
an N-propargyl-substituted 5F-Trp and an azido-substituted
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Scheme 3. Synthesis of Compound 66
associated functional groups to the target molecule. It was
necessary to synthesize the requisite N-propargyl 5F-Trp
amino acid and the azido-modified DBX cross-linker. Scheme
4 details the synthesis of the Fmoc-protected N-propargyl 5F-
Trp amino acid 70, starting from the commercially available
5F-Trp amino acid. The four-step synthesis was performed in
excellent overall yield to give the desired product, which was
used directly in solid-phase synthesis to assemble the linear
peptide. The aryl azide intermediate 73 was prepared in two
steps from a commercial starting material as shown in Scheme
5. The final peptide was assembled as shown in Scheme 6.
The linear peptide was assembled using standard SPPS, and
the crude linear peptide cleaved and deprotected. The crude
linear peptide intermediate was cyclized under standard
conditions using the DBX-azide described above (Scheme 5,
73) to give 74, which was then subjected to the second
cyclization26 conditions to provide the novel bicyclic peptide
75. Compound 75 showed similar potency (Alexa-FRET IC50
= 1.36 nM) to earlier analogues in the Asp−His series (such
as 30) and some improvement versus analogues in the Pro−
Thr series (such as 51). The molecule showed excellent
stability in our protease panel (Table 2) and was dosed IV to
both rats and cynomolgus monkeys (Table 10) to evaluate in
Figure 12. Proposed novel cross-link (green arrow) from bis-meta
position of the DBX linker to the indole nitrogen of the 5F Trp in
position 4.
linker aryl ring presented an opportunity to bridge the
distance with a novel spacer of the appropriate length.27 This
approach (Chart 1)27 also provided a very synthetically
accessible route and the appropriate orthogonality of the
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Chart 1. Retrosynthetic Analysis of the Proposed Bicyclic Target Peptide
Scheme 4. Synthesis of N-Propargyl Fmoc-Protected 5F-Trp 70
Scheme 5. Synthesis of Arylazide Intermediate 73
vivo PKs. Interestingly, the compound showed extremely poor
IV PK performance in rats; however, it was somewhat better
in monkeys, suggesting that the issues seen in vivo with
molecules containing a Pro at position 5 might be a rodent-
specific issue. Despite its poor PK performance in rats,
compound 75 represents a key, novel structure in the series,
demonstrating good potency, improved stability, and reason-
able IV PK performance in cynomolgus monkeys. Unfortu-
nately, we were unable to obtain suitable crystals to allow for
determination of a crystal structure of 75. However, based on
the structure shown in Figure 12 as well as the data for 75,
one can deduce that the triazolomethyl linker does not
drastically change the overall conformation of the molecule
P
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Scheme 6. Synthesis of Bicyclic Peptide 75
a b c
, ,
Table 10. Rat and Cyno Monkey IV PKs for 75
c
species
AUC0−inf (nM h)
AUC0−x (nM h)
clearance (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
protein binding (% bound)
a
cynomolgus monkey
Wistar Han rat
306.10 48.85
23.3 4.1
303.71 49.57
19.4 0.6
20.15 3.15
107 18
1.06 0.13
0.48 0.07
2.84 1.46
0.06 0.02
97.5
99.7
b
a
Compound was tested in cynomolgus monkeys at 0.5 mpk using a 30% propylene glycol/70% PBS vehicle at a concentration of 0.5 mg/mL. All
determinations are the average of two monkeys from a single experiment. The indicated errors are the difference between the mean and the
b
individual values. Compound was tested in Wistar Han rats at 1 mpk using a 20% DMSO/60% PEG/20% water vehicle at a concentration of 1
mg/mL. All determinations are the average of two rats from a single experiment. The indicated errors are the difference between the mean and the
c
individual values. Protein binding values are a single determination.
and likely serves as a simple direct link from the NH of the
Trp to the aryl ring of the DBX group.
There was also a second possible cyclization opportunity
that became apparent to us from the crystal structures of 30
and 51. We observed that the indole ring of 5F-Trp at
position 3 and the side chain of Asp at position 5 were
pointing outward into the solvent and were relatively close to
each other. This suggested the possibility of closing a ring
between the side chain of the Asp and the aryl ring of the side
chain of 5F-Trp, as illustrated in Figure 13. Such a ring could
Q
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the conformation of the molecule in its preferred binding
conformation, thereby providing potential entropic gains. Of
course, this would remove the existing intramolecular
interaction between the Asp and His side chains but would
constrain the overall conformation in a different manner.
We decided to strip off the side chain of Lys at position 1 to
a methyl group for ease of synthesis and to temporarily avoid
the mast cell degranulation issue. After scanning the literature
and available starting materials, we discovered that the meta-
(aminomethyl)phenylalanine was commercially available and
decided to use this as a replacement for 5F-Trp in position 3.
We could then use amino acids containing carboxylic acid side
chains at the Asp position 5 and attempt macrolactamization
of these two amino acids to provide an amide cross-linked
analogue. Scheme 7 details the synthesis of these lactam cross-
linked analogues, and Figure 14 details the SAR progression of
the bicyclic series of inhibitors. The linear peptides were
prepared on resin, followed by cleavage and deprotection to
provide 76. 76 was then cyclized using meta-dibromomethyl
benzene (DBX), and the crude product purified via reversed
phase prep HPLC to give the purified macrocycle 77. The
peptide was then treated with the coupling agent AOP28 in
the presence of DIEA to form the cyclic lactam, and the crude
material again purified via reverse-phase prep HPLC to give
Figure 13. Proposed cyclization between the side chain of position 3
and position 5. The side chains point in the same direction and are in
close proximity to each other, and the new bicyclic linker would lie in
a mostly solvent-exposed region.
potentially serve to block metabolism at most or all of the
remaining vulnerable amide bonds while also helping to lock
Scheme 7. Representative Synthesis of Lactam Analogues
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Figure 14. SAR and evolution of bicyclic amide PCSK9 inhibitors.
the desired bicyclic lactam analogue 78. This general synthetic
approach was used to prepare the various amide analogues in
this series. Compound 78 showed excellent potency in the
LDLR-FRET assay, thus confirming our hypothesis about
cyclization in this region. We were able to obtain a crystal
structure of 78, which is detailed in Figures 15 and 16. Other
than a slight twist of the DBX cross-linker group (there is
some enlargement in the density here, suggesting some
mobility for the aryl ring of the DBX cross-linking group), the
crystal structure of compound 78 is very similar to the
previous crystal structures in this series. Figure 15 shows that
the molecule retains all the interactions of the previous
inhibitors, with the addition of the lactam bicycle. Figure 16,
which shows an overlay of 78 versus 30, confirms the
superimposability of the two structures. The crystal structure
also suggests that all the vulnerable amide bonds are now in
some way shielded, suggesting the potential for better stability.
However, when we looked at the stability of compound 78 in
our panel of proteases and in whole blood, we noted that a
potential vulnerability to chymotrypsin remained (Table 2).
Despite marginal solubility, compound 78 was evaluated for
IV PK in the mouse (Table 2), and the compound was shown
to have a short half-life in vivo. In Figure 15 (highlighted in
the black box), one of the amide nitrogen atoms previously
identified as being susceptible to proteolysis by chymotrypsin
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Methylation of the amide nitrogen between positions 4 and 5
(synthesized via a procedure identical to the one detailed
earlier for 78 but using N-Me Glu at position 5 in place of
Glu) provided 79, which demonstrated good potency,
although somewhat reduced from that seen with the parent
(Figure 14). Note that in going from peptide 78 to 79, we
also introduced an OMe Thr moiety in place of Thr. As stated
earlier for compounds 50 versus 52, we had previously
established that Thr and OMe Thr provided identical
inhibitory potency and did not affect stability (Table 2),
and these amino acids were used interchangeably in several
cases. While the comparison between 78 and 79 is not
perfectly direct, the change from N-H to N-Me at this key
backbone position clearly provided a stability advantage
(Table 2) against chymotrypsin similar to that observed
when going from a Asp−His backbone like 30 to a Pro−Thr
backbone such as 50. Compound 79 showed full stability
against our panel of proteases as well as in the presence of
mouse and cyno monkey whole blood, suggesting that the
structure-guided changes we had made in the backbone were
able to effectively block potential metabolism by key
proteases. This was an important finding for our program
and shows that structural information is key in drug discovery
programs and can be used to not only enhance potency and
drive SAR but also to guide efforts to stabilize metabolically
vulnerable functionalities on key structures. Given the
extremely poor solubility of compound 79, we chose not to
dose it in vivo.
Figure 15. Crystal structure of 78 bound to the LDLR-binding site
of PCSK9 at 1.43 Angstrom resolution (PDB: 6XIE). The new
bicyclic ring between positions 3 and 5 is clearly visible. The
phenylalanine ring at position 3 occupies the same space as the fused
ring system of 5F-Trp it replaces. Other than a slight twist of the
DBX cross-linker aromatic ring, the rest of the molecule fits in a
virtually identical manner to previous crystal structures in this series.
The nitrogen of the amide bond between the inner 5F-Trp at
position 4 and the glutamic acid at position 5 (indicated by the black
box) remains exposed and vulnerable to chymotrypsin (see Table 2).
The crystal structure suggests that this nitrogen should be amenable
to methylation because it points upward and away from the protein
and does not participate in any interactions with the protein.
Additional studies were done around compounds 78−79 to
look at various ring sizes for the lactam bicycle (Figure 14).
The peptides were synthesized in an identical manner to 78,
substituting the appropriate amino acids in the SPPS.
Contracting the ring by one carbon (Asp at position 5) to
give 80 provided a substantial potency loss. Expanding the
ring by one carbon versus 78 (homoGlu at position 5) gave
81, which lost 20-fold in inhibitory potency. Further enlarging
the ring by one additional carbon versus 78 (bishomoGlu at
position 5) gave 82, which almost fully restored inhibitory
potency. Finally, moving the substitution pattern of the
aminomethyl group on the phenylalanine ring at position 2
from meta to para and cyclizing with the lengthened
bishomoGlu used in analogue 82 provided 83, which lost
about 100-fold in potency but still retained somewhat
interesting activity. We also continued to look for replace-
ments for the 5F-Trp moiety in position 4 that bound inside
the macrocycle with the fluorine embedded in a small pocket
on the surface of the binding site. We were able to purchase
the racemic 5F-4-quinoline amino acid, and synthesis of the
peptide using this amino acid gave 84 (Figure 14), which was
isolated as an approximately 1:2 mixture of isomers at the
quinoline amino acid center after chromatography and was
tested as a mixture. The mixture of diastereomers 84 showed a
slightly improved potency to the analogous 5F-Trp analogue
and represented the only amino acid side chain that we found
that was able to duplicate the activity seen with 5F-Trp at this
position. We were able to obtain a crystal structure of 84,
shown in Figure 17, overlaid with the crystal structure of 30.
Only the correct diastereomer is able to bind and form
suitable crystals and as such is the isomer represented in the
crystal structure. The overlay shows that the fluoroquinoline
ring of 84 appears to be a fully superimposable replacement
for the 5F-Trp moiety of most of the inhibitors. This data
once again reaffirmed the narrow SAR observed at this
Figure 16. Superposition of the crystal structures of 30 (salmon) and
78 (green). The two molecules bind in a similar fashion, with all of
the key hydrogen bonding and lipophilic interactions maintained.
There is a slight twist in the DBX cross-linker (extra density here
indicates likely some flexibility here). The new ring formed between
the side chains of position 3 and position 5 is clearly visible, with the
aryl ring sitting in a similar position as the indole of 5F-Trp and the
newly formed cyclic amide solvent-exposed.
remains unsubstituted in the structure and despite the
presence of the bicycle this remains a vulnerability.
Because this amide bond does not participate in any
hydrogen bonding interactions with the protein and points
upward and away from the protein, based on the crystal
structure, we rationalized that it should be amenable to N-
methylation. Our earlier N-methyl scan had suggested that N-
methylation at this position would not be well tolerated;
however, given the somewhat different nature of this bicyclic
structure, we decided to attempt N-methylation here. N-
T
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Method AWaters Acquity BEH C18 column (2.1 × 100 mm,
1.7 μm) eluting with 0.1% trifluoroacetic acid (TFA) in water
(solvent A) and 0.1% TFA in acetonitrile (solvent B) at a flow rate of
0.4 mL/min at 45 °C using a 0−4.0 min:25−45% B, 4.0−4.5 min
80% B gradient, detection via UV @ wavelengths of 215 nm. The
mass spectra (MS) were recorded on a Waters ZQ mass
spectrometer using the electrospray-positive ionization mode [ES+
to give (MH)+ molecular ions]. The cone voltage was 20 V.
Method BWaters Acquity BEH C18 column (2.1 × 100 mm,
1.7 μm) eluting with 0.1% TFA in water (solvent A) and 0.1% TFA
in acetonitrile (solvent B) at a flow rate of 0.4 mL/min at 45 °C
using a 0−4.0 min:20−40% B, 4.0−4.5 min 80% B. The mass spectra
(MS) were recorded on a Waters ZQ mass spectrometer using the
electrospray-positive ionization mode [ES+ to give (MH)+ molecular
ions]. The cone voltage was 20 V.
Method CWaters BEH 130 C18 Acquity 2.1 × 100 mm, 1.7 u
column, A/B mobile phasesA = 0.1% TFA in water; B = 0.1%
TFA in acetonitrile; flow rate = 0.4 mL/min at 45 °C using a 5−95%
B gradient over 10 min; and detection via UV @ wavelengths of 215
nm/254 nm (for some examples, 280 nm also), MS: API-ES. Cone
voltage = 20 V.
Method DWaters Acquity BEH C18 50 mm × 1 mm × 1.7 μm,
eluting with 0.05% TFA in water (solvent A) and 0.05% TFA in
acetonitrile (solvent B) at a flow rate of 0.3 mL/min, a column
temperature of 50 °C; 5−95% B (0.05% TFA) in 2 min. Detection at
215 nm. MS: API-ES. Cone voltage = 20 V.
Method EWaters BEH 130 C18 Acquity 2.1 × 100 mm, 1.7 u
column, A/B mobile phasesA = 0.1% TFA in water; B = 0.1%
TFA in acetonitrile; flow rate = 0.4 mL/min at 45 °C using a 5−95%
B gradient over 15 min; detection via UV @ wavelengths of 215 nm/
254 nm; the mass spectra were recorded on a Waters ZQ mass
spectrometer using the electrospray-positive ionization mode [ES+ to
give (MH)+ molecular ions]. The cone voltage was 20 V.
Method FPhenomenex EV C18 2.1 × 100 mm, 1.7 u; A/B
mobile phasesA = 0.1% TFA in water; B = 0.1% TFA in
acetonitrile; flow rate = 0.4 mL/min at 45 °C using a 20−65% B
gradient over 6 min. The mass spectra were recorded on a Waters
ZQ mass spectrometer using the electrospray-positive ionization
mode [ES+ to give (MH)+ molecular ions]. The cone voltage was 20
V.
Preparative HPLC was performed as indicated below. LC/MS data
for all final compounds is summarized in Table 11. The purities of all
final compounds were 95% or greater unless otherwise noted. 1H
NMR spectra were recorded in various solvents at ambient
temperature using a Varian 600 MHz NMR.
LDLR-FRET Assay. A time-resolved fluorescence resonance
energy (TR-FRET) assay was used to measure inhibition of
PCSK9-LDLR PPI. Briefly, 20 nM avitag-biotinylated human
PCSK9 was incubated with 20 nM His-tagged human LDLR EGFa
domain in the presence of 5 nM LANCE Ulight Streptavidin
(PerkinElmer) and 5 nM europium-Anti-6xHis (Perkin Elmer) for 2
h covered at room temperature in buffer containing 50 mM HEPES,
150 mM NaCl, 5 mM CaCl2, 0.01% BSA, and 0.01% surfactant P20.
Compounds were tested in dose−response and concentrations giving
half-maximal inhibition calculated using a four-parameter fit equation.
Dose−response curves consisted of 10-point, half-log dilutions over a
concentration range from 0.0015532 to 48.8 μM. The goodness of fit
for IC50 is a four-parameter logistic fit based on the Levenberg−
Marquardt algorithm. Ki was then calculated from the IC50 and KD of
PCSK9 to LDLR (480 nM, determined by SPR). The number of
replicates varies for the compounds, and full details are provided in
the table footnotes. For determinations of n < 3, the error is
characterized as the difference between the individual values and the
mean. For determinations of n = 3 or greater, the error is
characterized as the standard deviation. All n values are the result
of independent determinations.
Figure 17. Crystal structure of 84 at 1.77 Angstrom resolution
(green; PDB: 6XIF) overlaid with the crystal structure of 30
(salmon) bound to the LDLR binding site of PCSK9. The overall fit
of the molecule is identical to all of the previous inhibitor structures
described in this publication. The fluoroquinoline at position 4 is
shown to occupy the same pocket in an identical manner as 5F-Trp
in previous crystal structures.
position and the critical nature of the aryl fluorine substitution
here. Based on the combination of the marginal PK
demonstrated with 78 as well as the poor solubility in general
of these analogues, compounds 80−84 were not evaluated in
vivo.
CONCLUSIONS
■
In summary, by leveraging a structure-based design approach,
we have taken an initial lead from an mRNA display screen
and optimized the molecule, increasing the potency by almost
1000-fold, while finding two unique structural approaches to
stabilize the molecules to key gut proteases and also reducing
the molecular weight by 30%. We have also investigated an
observed off-target issue of mast cell degranulation and have
developed an SAR to mitigate this issue, which is a potential
issue across all peptide platforms and should be suspected
when the structural phenotype is present in the molecule or
idiosyncratic toxicity is observed. Having optimized potency
and stability to key gut enzymes and engineered out an
important off-target issue, we are continuing our efforts in this
series using these bicyclic platforms as a foundation, with the
goal of finding novel, orally bioavailable cyclic peptide PCSK9
inhibitors. Although these novel bicyclic peptide inhibitors are
not fully optimized, they provide a novel, potent, and
metabolically stable platform for continued lead optimization
in this space.
EXPERIMENTAL SECTION
■
General Methods. Amino acid building blocks and reagents were
obtained from various commercial sources such as Sigma-Aldrich,
Fisher, Novabiochem, Bachem, Chem-Impex, Iris, Genscript,
Frontier, or Nagase & Co. Ltd., WuXi Apptec. The noncanonical
amino acid Fmoc-5F-Trp was supplied by WuXi Apptec. SPPS resins
were obtained from EMD and Anaspec. The purities of intermediates
and final compounds were determined by LC/MS, conditions as
follows unless otherwise noted using these methods:
Alexa-FRET Assay. The PCSK9 TR-FRET Alexa-FRET Standard
assay measures the interaction between PCSK9 (biotinylated/C-
Avitagged) and an AlexaFluor647 (AF)-tagged cyclic peptide, reagent
A (KD = 83 nM). A solution containing 1 nM biotinylated PCSK9 +
U
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a b
,
Table 11. Tabulated LC/MS Data for All Final Molecules
MS
MS
LC
MS
MS
LC
compound #
MW
[M + H]+ [M + 2H]+ method tR (min)
compound #
MW
1402.64 1403.6
[M + H]+ [M + 2H]+ method tR (min)
2
3
4
5
6
7
8
9
1890.14
945.3
A
C
C
A
A
A
A
A
A
A
A
A
A
B
B
A
A
A
A
A
A
B
B
A
A
A
A
A
A
C
C
C
C
C
C
C
A
C
C
C
2.64
3.54
3.7
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C
D
C
C
C
C
C
C
C
C
D
D
D
D
C
D
D
C
C
C
E
4.91
0.82
3.06
5.29
5.3
4.98
4.53
5.23
5.4
1475.67 1476.6
1418.62 1419.4
1359.48 1360.7
1431.2
1283.9
1283.45 1284
1461.6
1416.6
1462
1417.6
1444.72 1445.7
1444.72 1445.7
1445.59 1446.6
732
681.1
716.1
642.6
642.8
687.1
676.1
663.1
696.2
709.2
700.1
700.3
709.6
709.6
709.1
709.4
709.1
716.4
716.2
707.3
707.3
707.1
707.4
716.1
716.3
716.3
716.7
3.38
2.61
2.79
2.47
2.51
3.18
2.31
2.34
2.5
2.54
3.67
4.07
2.56
2.78
3.04
2.42
2.29
2.5
3.67
3.67
2.51
2.56
2.77
2.76
2.7
2.83
4.15
3.84
3.5
1429.6
1282.5
1414.6
1468.7
1456.7
1415.7
1469.2
1457.7
1371.6
1372.9
1350.51 1351.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23a
23b
24a
24b
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
1323.5
1324.9
1332.57 1333.5
1376.62
4.9
1390.54 1391.5
1416.57 1417.8
1398.57 1399
1398.57 1398.8
1416.57 1417.3
1416.57 1417
1416.57 1417.7
1416.57 1416.9
1416.57 1417
688.31
0.85
0.98
0.95
0.98
5.38
0.66
0.94
5.77
5.26
5.38
4.52
1407.6
1408.8
1390.65 1391.9
1408.64 410
1402.58
1416.57 1417.8
1401.59 1402.8
701.9
708
701.4
1415.6
1416.6
1430.63 1431.1
1430.6
1430.6
1431.0
1431.1
1443.6
1457.7
1444.1
1457.64
(M+)
1412.63 1413.2
1412.63 1413.4
1412.63 1413.1
1412.63 1413.1
1430.62 1432
61
62
63
66
75
78
79
80
81
82
83
1441.7
1647.7
1676.7
1442.61
E
E
C
E
C
C
C
C
C
C
C
F
4.75
4.8
4.63
4.7
6.33
5.31
6.2
5.29
5.3
825.4
839.6
1517.61 1518.3
1383.62 1383.8
1290.56 1291.2
1318.2
1277.1
1304.58 1305.1
1318.61 1319.2
1317.6
1430.6
1431.1
1430.62 1431.1
1431.64 1432.7
1372.53 1372.2
1429.55 1429.2
1454.57 1454.2
1454.57 1454.2
1408.55 1408.2
1434.57 1434.2
1374.61
1317.6
1276.6
5.32
5.28
3.90,
659.5
3.52
4.04
4.42
3.64
3.4
4.04
4.04
4.25
b
84
1328.6
1329.57
4.11
a
LC methods are described in the Experimental Section; all molecules
demonstrated >95% purity by LC; representative LC traces for key
molecules are provided in the Supporting Information. Compound
84 was recovered as a mixture of diastereomers, combined purity >
99%; MS for each peak was identical.
1390.5
1390.9
969.1
b
1401.52 1401.2
1429.55 1429.2
1388.62 1389.6
2.5 nM Lance Streptavidin Europium (Strep-Eu) was made in 50
mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.01% BSA, and
0.01% surfactant P20. A separate solution containing 40 nM of the
AlexaFluor-tagged cyclic peptide was made in the same buffer system.
An Echo robot was used to transfer 0.750 μL of test compound to an
assay plate, followed by the addition of 15 μL of PCSK9+Stept-Eu
and 15 μL of AF peptide. The final assay volume was 30.750 μL
containing 0.5 nM PCSK9, 1.25 nM Strep-Eu, and 20 nM AF cyclic
peptide. The reaction was incubated at room temperature for at least
2 h prior to fluorescence measurements using an Envision Multilabel
Reader. IC50 values were determined by fitting data to a sigmoidal
dose−response curve using nonlinear regression. Compounds were
tested in dose−response and concentrations giving half-maximal
inhibition were calculated using a four-parameter fit equation. The
dose−response curves consisted of 10-point, half-log dilutions over a
concentration range from 0.0015532 to 48.8 μM. The goodness of fit
for IC50 is a four-parameter logistic fit based on the Levenberg−
Marquardt algorithm. Ki was then calculated from the IC50 and KD of
the AF cyclic peptide. Counts (B-counts) of the europium-labeled
PCSK9 were followed to observe if compounds were adversely
affecting PCSK9. A fall-off of the B-counts likely indicated a false
positive of inhibition. The number of replicates varies for the
compounds, and full details are provided in the table footnotes. For
determinations of n < 3, the error is characterized as the difference
between the individual values and the mean. For determinations of n
= 3 or greater, the error is characterized as the standard deviation. All
n values are the result of independent determinations.
Reagent A was prepared as shown below:
Synthesis of reagent A: compound 30 (15 mg) was dissolved in
0.2 mL of dry DMSO. Then, 15 mg of AlexaFluor 647NHS Ester
(A37566, Life Technology) dissolved in 1.5 mL of dry DMSO was
added. Dry DIEA (20 μL) was added. The reaction was left under
stirring at room temperature for 12 h under the nitrogen atmosphere
in the dark, quenched with TFA (pH to 3−4), and purified by RP-
HPLC (Dr Maish, Reprosil Gold C18, 250 × 20 mm, 120 c5, 10 μm;
20 to 35% of 0.1% TFA in ACN/0.1% TFA in H2O, over 20 min,
then 35 to 40% over 5 min at 20 mL/min flow rate). The collected
fractions were lyophilized to afford 16.1 mg of purified reagent A.
LCMS analysis was calcd for C105H122F2N17O26S63−, calc. 2268.58;
found 1135.8 [M + 2H]+.
V
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General Procedure for the Preparation of Peptides 2−61.
Methods A and B below were used interchangeably with similar
results obtained from each. In general, the yields of final peptides 2-
61 varied from 5 to 50% after SPPS, cleavage, cyclization, and
purification, and in all cases, the desired products were recovered
after purification in >95% purity. In the cases of compounds 41 and
51, the commercially available 1,3-di(bromomethyl)-5-fluorobenzene
was substituted for 1,3-di(bromomethyl)benzene (DBX) in step 3
(cyclization) using identical conditions.
Method A. Step 1: Peptide Synthesis. The peptides were
synthesized using commercial Fmoc-protected amino acids on a
solid-phase Rink Amide MBHA (NovaBiochem, ∼0.5 mequiv/g,
100−200 mesh) or AM (NovaBiochem, ∼0.73 mequiv/g, 100−200
mesh) or 2-aminoethanethiol-2-chlorotrityl resin (Anaspec, ∼0.9
mequiv/g, 200−400 mesh) with a CEM Liberty Blue automated
microwave peptide synthesizer. The peptides were typically
synthesized on a 0.1 mmol scale. Typical reaction conditions were
as follows: deprotection conditions: 20% piperidine (v/v) in
dimethylformamide (DMF) (2 × 2 min at 75 °C); residue coupling
conditions: 5 equiv (relative to resin) of activated amino acid (2.5
mL of a 0.2 M amino acid stock solution in DMF) was delivered to
the resin, followed by 5 equiv of DIC activator (1 mL of a 0.5 M
solution in DMF) and 5 equiv of Oxyma Pure (0.5 mL of a 1 M
solution in DMF) and allowed to react for 5 min at 75 °C. The
resultant product was used directly in the next step.
Step 2: Cleavage from Solid Support. The resin from step 1 was
transferred to either a 50 mL polypropylene centrifuge tube or a 10
mL fritted syringe. The peptides were cleaved from their solid
support using TFA/triisopropylsilane/DL-dithiothreitol/water (92.5/
2.5/2.5 (w/v)/2.5) mixture. For 0.1 mmol scale of resin, ∼10 mL
cleavage solution was used. The suspended resin was rocked in
cleavage solution for 3 h at room temperature. The filtrate was
collected in 50 mL polypropylene centrifuge tubes and precipitated
with chilled diethyl ether (∼50 mL per ∼5 mL cleavage filtrate). The
precipitated crude peptide was collected by centrifugation. The white
pellet was then subsequently suspended in chilled diethyl ether and
collected by centrifugation two additional times. The resulting solid
was air-dried to afford the crude peptide.
Step 3: Cyclization. For a 0.1 mmol scale, the linear peptide was
synthesized, cleaved from the solid support, and isolated according to
step 2. The solid crude peptide was dissolved with stirring in 30 mL
of a degassed water/acetonitrile (1:1) solution in a 50 mL
polypropylene centrifuge tube. Additional acetonitrile was added as
needed to ensure complete dissolution of the peptide. To this stirred
solution, aqueous ammonium bicarbonate (200 mM, degassed) was
added dropwise to bring the pH to ∼8. Alkylating reagent 1,3-
di(bromomethyl)benzene (0.1 mmol/1 equiv) in acetonitrile (1 mL)
was added dropwise to the reaction over ∼2 min. After complete
addition, the reaction was tested to ensure that the pH was
maintained at ∼8. If the pH was below ∼8, additional aqueous
ammonium bicarbonate (200 mM, degassed) was added dropwise to
the reaction until the pH was ∼8. The centrifuge tube was capped
and the resulting reaction mixture was stirred at room temperature
for 1 h. The reaction was then acidified to pH ∼1 with TFA and
lyophilized to afford the crude cyclic peptide as a powder. The
residue was redissolved in 19:1 DMSO−water (v/v) and purified
using gradient elution on reverse phase (50 × 250 mm Sunfire Prep
C18; 25−75% CH3CN/water w/0.1% TFA modifier over 30 min).
The fractions were then concentrated in vacuo to provide the cyclized
peptide.
Method B: Alternative HATU Method on CEM Liberty. Step 1:
Peptide Synthesis. The peptide was synthesized using Fmoc/t-Bu
chemistry on cysteamine 4-methoxytrityl resin (NovaBioChem, 0.82
mmol/g) with a CEM Liberty automated microwave peptide
synthesizer. The peptide sequence was typically synthesized on a
0.25 mmol scale. Typical reaction conditions were as follows: Fmoc
deprotections were performed using 20% (v/v) piperidine in DMF
(10 mL, 90 s at 90 °C). Residue coupling conditions: single
couplings of 4 equiv (relative to resin) of Fmoc-protected amino
acids (as a 0.2 M DMF solution) along with 3.6 equiv of HATU (as
a 0.45 M DMF solution) and 8 equiv of 2 M DIEA in NMP.
Coupling cycle: 300 s at 75 °C or for trityl side chain-protected
residues: 120 s at 20 °C, followed by 240 s at 50 °C.
Step 2: Cleavage from Solid Support. After isolation of the resin
via filtration, the peptide was cleaved from solid support using 15 mL
of TFA solution (v/v) (90.5% TFA:2.5% DODT:2.5% phenol:2%
triisopropylsilane:2.5% water) for approximately 4 h at room
temperature. The resin was filtered and washed with 5 mL of TFA
solution. Combined filtrate was concentrated and precipitated in
approximately 60 mL of cold ethyl ether (−78 °C). Crude peptide
pellet collected by centrifugation was washed in cold ethyl ether and
collected by centrifugation twice more. The resulting solid was air-
dried to afford the crude peptide.
Step 3: Cyclization. Crude linear peptide (0.25 mmol) was
redissolved in a solution of degassed acetonitrile (150 mL) and
degassed 20 mM aqueous solution of ammonium carbonate (63 mL,
1.26 mmol), a degassed solution of 1,3-di(bromomethyl)benzene (78
mg, 0.29 mmol) in ACN (5 mL total with rinse) was added, and the
reaction under an atmosphere of nitrogen for 18 h was stirred. The
reaction was filtered and then concentrated in vacuo. The residue was
redissolved in 19:1 DMSO−water and purified using gradient elution
on reverse phase (50 × 250 mm Sunfire Prep C18; 25−75%
CH3CN/water w/0.1% TFA modifier over 30 min). The fractions
were then concentrated in vacuo to provide the cyclized peptide.
W
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Synthesis of Compound 62 (Scheme 2). Compound 4 (4.4
mg, 3.08 μmol) was dissolved in 1.0 mL of DMF. 2,5-
Dioxopyrrolidin-1-yl 2,5,8,11-tetraoxatetradecan-14-oate (2.2 mg,
6.60 μmol) was dissolved in 100 μL of DMF and then was added
directly to the peptide solution. The reaction was stirred at room
temperature for 2 h, at which time LCMS showed the reaction was
completed. The reaction mixture was diluted with DMF and water to
3 mL, then purified on RP-HPLC using a Vydac protein & peptide
C18, 250 × 10 mm, wide pore; gradient: 20−60% over 50 min.;
mobile phases: A = 0.1% TFA in water; B = 0.1% TFA in
acetonitrile. Compound 62 (1.90 mg) was obtained with purity
>95% (HPLC), calcd C79H99F2N15O18S2, 1647.67; observed mass
[M + 2H]+.
Synthesis of Compound 63 (Scheme 2). Compound 4 (12
mg, 8.39 μmol) was dissolved in 0.5 mL of DMF. 2,2-Dimethyl-4-
oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid (6.13 mg, 0.017
mmol), (E)-ethyl-2-cyano-2-(hydroxyimino)acetate (Oxyma Pure;
0.017 mL, 0.017 mmol), and N,N′-methanediylidenebis (propan-2-
amine) (DIC; 0.034 mL, 0.017 mmol) were mixed together, allowed
to stand at room temperature for 30 min. Then, this mixture was
added to the peptide solution. LCMS showed the reaction was
ongoing. Preactivated Boc-PEG-COOH (0.017 mmol) was added
twice to the solution. After 48 h, the reaction was completed by
LCMS analysis. The reaction was concentrated, and 2 mL of 1:1
TFA in DCM was added directly to the residue. After 30 min, the
TFA solution was concentrated and suspended in cold diethyl ether.
The mixture was centrifuged, the precipitate was dissolved in
acetonitrile/water, and the solution was freeze-dried to a fluffy white
material. Purification was done with RP_HPLC using a Vydac
protein & peptide C18, 250 × 10 mm, wide pore, with a 20−60% A
gradient over 50 min; mobile phases: A = 0.1% TFA in water; B =
0.1% TFA in acetonitrile.
Approximately 40 mL of chilled diethyl ether was added to the
mixture to precipitate the peptide. The crude peptide was collected
by centrifugation on a Thermo ST40R at 2900 rpm for 15 min. The
pellet was then washed with 40 mL of chilled ether again. The
washed peptide pellet was dissolved in 0.1% TFA acetonitrile/water
solution and then was lyophilized to dryness. The correct linear
peptide 65 was confirmed. 65 was used directly as a crude in the next
step reaction.
LCMS anal calcd for C65H83F2N15O15S2, 1415.56; found m/z+2,
709.08.
Step 3Synthesis of Compound 66. Crude 65 (0.1 mmol) was
redissolved in a solution of degassed acetonitrile (60 mL) and 20
mM NH4HCO3 in water (90 mL). To this was slowly added a
degassed solution of 1,3-di(bromomethyl)benzene (52.8 mg, 0.2
mmol) in acetonitrile (60 mL total with rinse). The reaction was
stirred at room temperature until completed (18 h). The reaction
was lyophilized to dryness and then purified using gradient elution
on the reverse phase (30 × 150 mm OBD, Waters Xselect CSH130
C18, 5 μm; 31−36% ACN/water w/0.16% TFA modifier over 25
min). The fractions were lyophilized to provide 66 in 96% purity
(4.6 mg), along with 1.5 mg of a slightly earlier eluting second
product identified as a racemate at an undetermined stereocenter
(verified by Alexa-FRET assay = 385 nM).
LCMS anal calcd for C73H89F2N15O15S2, 1517.61; found, 1517.61.
Synthesis of Compound 75 (Schemes 4−6). Step 1
Synthesis of Intermediate Compound 67 (Scheme 4). In a flame-
dried flask under an atmosphere of nitrogen, (S)-2-amino-3-(5-
fluoro-1H-indol-3-yl)propanoic acid (10.8 g, 48.6 mmol) was
dissolved in anhydrous MeOH (200 mL) and cooled in an ice
bath. Trimethylchlorosilane (TMS-Cl) was added (35.6 mL, 279
mmol) dropwise, and the resulting solution was stirred at room
temperature overnight. An additional 4 mL of TMS-Cl was added,
and the reaction was allowed to stir for an additional 18 h to drive
the formation of the desired methyl ester.
Compound 63 (5.72 mg) was obtained with purity > 95%
(HPLC), calc. C80H102F2N16O18S2 1676.70; observed [M + 2H]+ =
839.64.
The reaction was cooled in an ice bath, and triethylamine (38 mL,
273 mmol) was added to raise the reaction pH to ∼9. A solution of
BOC-anhydride (12.98 mL, 55.9 mmol) dissolved in methanol (20
mL) was added, and the reaction was stirred at room temperature.
After 1 h, an additional 6 mL of triethylamine was added to make the
reaction basic, followed by stirring at room temperature for another
hour. The reaction was concentrated in vacuo and was partitioned
residue between water and ether. The aqueous layer was extracted
3× with ether, and the combined extracts were washed with brine
and dried over anhydrous Na2SO4. The solution was filtered through
a plug of silica gel and concentrated in vacuo. The residue was
concentrated in vacuo to dryness. The resultant crystalline solid was
triturated with hexanes 3× to provide 67 (15.5 g, 95%).
Synthesis of Compound 66 (Scheme 3). Step 1Synthesis of
Compound 64. The peptidyl resin intermediate was synthesized
using Fmoc/t-Bu chemistry on cysteamine 4-methoxytrityl resin
(Novabiochem, 0.82 mmol/g) with a CEM Liberty automated
microwave peptide synthesizer. The peptide sequence was synthe-
sized on a 1.0 mmol scale using single couplings of 3.3 equiv of
Fmoc-protected amino acids as a 0.2 M DMF solution along with 3.0
equiv of HATU as a 0.45 M DMF solution and 6.6 equiv of 2 M
DIEA in NMP. Fmoc deprotections were performed using 20% (v/v)
piperidine in DMF.
Compound 64 was sampled for cleavage in 2 mL of 88% TFA, 5%
DODT (3,6-dioxa-1,8-octanedithiol), 2% phenol, 2.5% triisopropyl-
silane, and 2.5% water to check the peptide quality. The correct
linear peptide 64 was confirmed. 64 was used as the starting resin in
the next step. LCMS anal calcd for C53H62F2N12O10S, 1096.44;
found, 1097.0 [M+H]+.
Step 2Synthesis of Compound 65. Synthesis of linear peptide
was continued on 64 (0.1 mmol) on a Biotage Alstra peptide
synthesizer (BiotageCorp.) with two cycles of elongation using 4
equiv of double coupling with DIC/Oxyma Pure as activators, and
the amino acids (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)-
amino)-3-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-
methylbutyl)amino)propanoic acid and 3-(tritylthio)propanoic acid,
respectively. Fmoc deprotections were performed using 20% (v/v)
piperidine in DMF.
The peptidyl resin was treated with 5% hydrazine in DMF for 10
min (repeated twice) to remove the side chain protection group
ivDde. The peptidyl resin was manually coupled with 4 equiv of 2,2-
dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (2.000 mL, 0.4
mmol) using DIC/Oxyma Pure for 1 h.
LCMS anal calcd for C17H21FN2O4, 336.4; found, 673.5 (2M +
1)+.
1H NMR (600 MHz, chloroform-d): δ 8.08 (s, 1H), 7.27−7.24
(m, 1H) (mixed with CHCl3 peak), 7.17 (d, J = 9.4 Hz, 1H), 7.04 (s,
1H), 6.96−6.90 (m, 1H), 5.07 (d, J = 7.5 Hz, 1H), 4.65−4.61 (m,
1H), 3.69 (s, 3H), 3.30−3.18 (m, 2H), 1.43 (s, 9H).
Step 2Synthesis of Intermediate Compound 68 (Scheme 4).
In a flame-dried flask under an atmosphere of nitrogen, 67 (1330 mg,
3.95 mmol) was dissolved in anhydrous DMF (30 mL) and the
resulting solution was cooled in an ice bath. Sodium hydride (60%
disp.) was added (125 mg, 4.94 mmol) portionwise. The mixture was
stirred for 20 min at 0 °C until it became a pale-colored solution. 3-
Bromoprop-1-yne (80% in xylene) (0.529 mL, 4.74 mmol) was
added dropwise, and the resulting solution was stirred at 0 °C for an
hour. Aqueous lithium hydroxide (1 M, 6 mL, 6.00 mmol) was
added dropwise to the solution and the mixture was stirred at room
temperature for 90 min. The solution was quenched with 6 mL of 1
N HCl to adjust the pH ∼3. The mixture was extracted 3× with
EtOAc, and the combined organic extracts were dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo to provide
68, which was used as a crude in the next step (1.425 mg, 100%).
LCMS anal. calcd for C19H21FN2O4, 360.4; found, 721.5 (2M +
1)+.
The fully assembled peptide on-resin was cleaved using 10 mL of
88% TFA, 5% DODT (3,6-dioxa-1,8-octanedithiol), 2% phenol, 2.5%
triisopropylsilane, and 2.5% water for 2 h at room temperature.
Another 5 mL of TFA solution was used to wash the peptidyl resin.
After filtering, combined TFA solutions were concentrated in vacuo.
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Step 3Synthesis of Intermediate Compound 69 (Scheme 4).
Crude 68 (1425 mg, 3.95 mmol) was dissolved in 4 M HCl in
dioxane (20 mL, 80 mmol) at room temperature. After 45 min, the
reaction was concentrated in vacuo. The residue was concentrated
twice from ether to form a yellow solid, which was dried in vacuo to
provide 69, which was used as a crude in the next step (1173 mg,
100%).
1,2,3-triazol-4-yl)methyl]amine and 49.5 mg (0.25 mmol) of sodium
ascorbate, followed by 18.6 mg (0.05 mmol) of tetrakis(acetonitrile)-
copper(I) hexafluorophosphate. The reaction was stirred at room
temperature, but there was no reaction observed after 90 min.
Additional portions of the three reagents were added again, and the
reaction was heated at 50 °C. The reaction was complete after 90
min. The precipitate was filtered off and washed with t-BuOH and
water. The resulting filtrate was concentrated in vacuo and
resuspended in a mixture of acetonitrile and methanol. The crude
material was purified by RP prep LC using a Vydac protein &
peptide C18, 250 × 10 mm, wide pore, with a 20−60% A gradient
over 50 min; mobile phases: A = 0.1% TFA in water; B = 0.1% TFA
in acetonitrile. The product fractions were combined and lyophilized
to give the desired product (17.5 mg) 75 as a white amorphous
powder. LC/MS purity = >95.5%; LC/MS calcd 1537.81; found,
1538.0 [M+H]+.
General Synthesis of Bicyclic Lactam Analogues (Example
for Compound 79). Crude linear peptide (0.25 mmol) was
redissolved in a solution of degassed acetonitrile (150 mL) and
degassed 20 mM aqueous solution of ammonium carbonate (63 mL,
1.26 mmol). To this solution was added a degassed solution of 1,3-
di(bromomethyl)benzene (78 mg, 0.29 mmol) in ACN (5 mL total
with rinse). The solution was stirred under an atmosphere of
nitrogen for 18 h. The reaction was filtered and then concentrated in
vacuo to give the crude cyclized peptide. The residue was redissolved
in 19:1 DMSO−water and purified using gradient elution on the
reverse phase (50 × 250 mm Sunfire Prep C18; 25−75% CH3CN/
water w/0.1% TFA modifier over 30 min). The fractions were then
concentrated in vacuo to provide the mono-cyclized peptide. To a
solution of AOP (19.9 mg, 45 μmol) and DIEA (30 μL, 172 μmol)
in DCM (100 mL) was added a solution of the above mono-cyclized
peptide (50 mg, 34 μmol) in DMF (6 mL) over 60 min via a syringe
pump. The syringe was rinsed with DMF (1.5 mL). The reaction was
stirred overnight at room temperature. The reaction was then
concentrated in vacuo to remove the DCM and the residue was
purified using gradient elution on the reverse phase (30 × 150 mm
Sunfire Prep OBD C18; 30−75% CH3CN/water w/0.1% TFA
modifier over 20 min). The fractions were lyophilized to provide 21
mg of compound 79 as a white powder. Dimeric lactam product was
also recovered as a byproduct.
LCMS anal. calcd for C14H13FN2O2, 260.3; found, 261.2 (M +
1)+.
Step 4Synthesis of Intermediate Compound 70 (Scheme 4).
Crude 69 (1173 mg, 3.95 mmol) was dissolved in acetone (30 mL)
along with a solution of sodium carbonate (838 mg, 7.91 mmol) in
water (60 mL). A solution of Fmoc-OSu (1334 mg, 3.95 mmol)
dissolved in 30 mL of acetone was added, and the solution was
stirred at room temperature for 1 h. The reaction was acidified by
addition of 1 N aq HCl (∼11 mL) dropwise to lower pH to ∼3. The
solution was extracted 3× EtOAc, and the combined organic extracts
were washed with brine and dried over anhydrous Na2SO4. The
solution was filtered, and the filtrate concentrated in vacuo. The
residue was dissolved in DCM and purified by flash chromatography
(120 g SiO2, 10−80% EtOAc/hexanes). The appropriate fractions
were concentrated in vacuo to provide 70 (1600 mg, 84%). 70 was
used in the assembly of the crude linear peptide 74.
LCMS anal calcd for C29H23FN2O4, 482.5; found, 483.3 (M + 1)+.
1H NMR (500 MHz, DMSO-d6): δ 12.71 (s, 1H), 7.88 (d, J = 7.5
Hz, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.66 (dd, J = 13.2, 7.5 Hz, 2H),
7.47 (dd, J = 8.9, 4.4 Hz, 1H), 7.44−7.36 (m, 3H), 7.34−7.23 (m,
3H), 7.08−7.00 (m, 1H), 5.08−5.00 (m, 2H), 4.27−4.15 (m, 4H),
3.38 (s, 1H), 3.15 (dd, J = 14.6, 4.6 Hz, 1H), 3.00 (dd, J = 14.6, 9.7
Hz, 1H).
Step 5Synthesis of Intermediate Compound 73 (Scheme 5).
Compound 71 (5-amino-1,3-phenylene) dimethanol (3 g, 19.59
mmol) was dissolved in acetonitrile (200 mL) under an atmosphere
of nitrogen. The solution was cooled in an ice bath and tert-butyl
nitrite (3.85 mL, 32.4 mmol) was added dropwise to form an orange
solution. After 15 min of stirring at 0 °C, trimethylsilyl azide (2.99
mL, 22.52 mmol) was added dropwise over 5 min. The reaction was
warmed to room temperature and stirred for 2 h and then
concentrated in vacuo. The residue was stirred in 2:1 toluene−
ether, filtered with 2:1 toluene−ether and 3:1 toluene−hexane rinses,
and dried to provide 72 (2.62 g, 74.7%)
LCMS: 95.6% purity. RT: 6.23 min (@215 nM on 5−95% ACN−
water (0.1% TFA) over 10 min on Sunfire C18) anal. calcd for
C67H84FN11O12S2, 1317.6; found, 1318.2 [M + H]+.
1H NMR (600 MHz, CD3OD): δ 7.12 (s, 1H); 6.96 (s, 2H); 4.59
(s, 4H).
Stability and Metabolite ID Studies. The metabolism of
various compounds was studied upon incubation with fully activated
porcine gastric pepsin, bovine pancreatic trypsin, porcine pancreatic
type IV elastase, and bovine pancreatic α-chymotrypsin. All reported
data is derived from a single determination.
Step 6Synthesis of Intermediate Compound 73 (Scheme 5).
Compound 72 (2.62 g, 14.62 mmol) was partially dissolved in DCM
(400 mL) under an atmosphere of nitrogen and cooled in an ice
bath. Triphenylphosphine dibromide (14.0 g, 33.2 mmol) was added
portionwise over 5 min. The resulting mixture was stirred at room
temperature for 90 min and the reaction eventually became a
solution. TLC (60% EtOAc/hexanes) analysis at this point indicated
that the reaction was not complete. An additional 600 mg of
triphenylphosphine dibromide was added and the reaction was
stirred for an additional 20 min. The reaction was filtered through a
120 g silica gel column, rinsing with 350 mL DCM. The filtrate was
concentrated in vacuo and dried to provide 73 as a crystalline tan
solid, which was stored in a freezer (3.78 g, 85%).
Pepsin. A 100 μg/mL solution of porcine pepsin was prepared by
dissolving lyophilized enzyme in 10 mM aqueous hydrochloric acid
(pH 2.0). Incubation mixtures were prepared in duplicate in a 96-
well propropylene plate by adding 5.0 μL aliquots of 100 μM
substrate in DMSO or DMSO only (no-substrate control) to 45 μL
of the 100 μg/mL solution of porcine pepsin in 10 mM aqueous
hydrochloric acid (pH 2.0), which had been prewarmed at 37 °C.
The reactions were terminated after an incubation time at 37 °C of
60 min by the addition of 2 volumes (100 μL) of acetonitrile
containing 4.5 μM of an internal standard peptide. True time zero
controls were prepared by adding the stopping solution prior to the
addition of the substrate. The plates were then vortex-mixed and
centrifuged at 2250g for 10 min. A 50 μL volume of the supernatant
from each well was added to 300 μL of 50/50 acetonitrile/20%
aqueous formic acid. The resulting supernatants were subjected to
LC−MS/MS analysis.
1H NMR (600 MHz, CDCl3): δ 7.18 (s, 1 H), 6.98 (s, 2 H), 4.43
(s, 4 H).
Step 7Synthesis of Compound 75 (Scheme 6). The linear
peptide intermediate 74 was prepared as described above in the
General Procedure using Method B, steps 1 and 2 incorporating the
above synthesized amino acid 70 at the appropriate point in the
peptide sequence. After SPPS and deprotection/cleavage as described
in Method B, steps 1 and 2, the crude linear peptide 74 (prepared on
0.25 mmol scale) was cyclized as described in Method B, step 3,
using 103 mg (0.34 mmol) of 73 (prepared above). The crude
product after lyophilization was used as isolated in the next reaction.
The crude product from the above was dissolved in a solution of
100 mL of t-BuOH/50 mL water and the soln. bubbled with N2. To
the solution was added 26.5 mg (0.05 mmol) of tris[(1-benzyl-1H-
Trypsin. A 100 μg/mL solution of bovine pancreatic trypsin was
prepared by dissolving lyophilized bovine pancreatic trypsin enzyme
(in 100 mM sodium phosphate buffer, pH 7.40). Incubation mixtures
were prepared in duplicate in a 96-well polypropylene plate by
adding 5.0 μL aliquots of 100 μM substrate in DMSO or DMSO
only (no-substrate control) to 45 μL of the 100 μg/mL solution of
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bovine pancreatic trypsin in 100 mM sodium phosphate buffer (pH
7.40), which had been prewarmed at 37 °C. The reactions were
terminated after an incubation time of 1 and 6 h at 37 °C by the
addition of 2 volumes (100 μL) of acetonitrile (with formic acid)
containing an internal standard peptide. True time zero controls were
prepared by adding the stopping solution prior to the addition of the
substrate. The plates were then vortex-mixed and centrifuged at
2250g for 10 min. A 50 μL volume of the supernatant from each well
was added to 300 μL of 50/50 acetonitrile/20% aqueous formic acid.
The resulting supernatants were subjected to LC−MS/MS analysis.
α-Chymotrypsin. A 100 μg/mL solution of bovine pancreatic α-
chymotrypsin was prepared by dissolving lyophilized enzyme in 100
mM sodium phosphate buffer, pH 7.40. Incubation mixtures were
prepared in duplicate in a 96-well polypropylene plate by adding 5.0
μL aliquots of 100 μM substrate in DMSO or DMSO only (no-
substrate control) to 45 μL of the 100 μg/mL solution of bovine
pancreatic α-chymotrypsin in 100 mM sodium phosphate buffer (pH
7.40), which had been prewarmed at 37 °C. The reactions were
terminated after an incubation time at 37 °C of 1 and 6 h by the
addition of 2 volumes(100 μL) of acetonitrile (with formic acid)
containing 4.5 μM of an internal standard peptide. True time zero
controls were prepared by adding the stopping solution prior to the
addition of the substrate. The plates were then vortex-mixed and
centrifuged at 2250g for 10 min. A 50 μL volume of the supernatant
from each well was added to 300 μL of 50/50 acetonitrile/20%
aqueous formic acid. The resulting supernatants were subjected to
LC−MS/MS analysis.
Type IV Pancreatic Elastase. A 100 μg/mL solution of porcine
type IV pancreatic elastase was prepared by dissolving lyophilized
enzyme in 100 mM sodium phosphate buffer, pH 7.40. Incubation
mixtures were prepared in duplicate in a 96-well polypropylene plate
by adding 5.0 μL aliquots of 100 μM substrate in DMSO or DMSO
only (no-substrate control) to 45 μL of the 100 μg/mL solution of
porcine type IV pancreatic elastase in 100 mM sodium phosphate
buffer (pH 7.40), which had been prewarmed at 37 °C. Reactions
were terminated after an incubation time at 37 °C of 1 and 6 h by
the addition of 2 volumes (100 μL) of acetonitrile (with formic acid)
containing 4.5 μM of an internal standard peptide. True time zero
controls were prepared by adding the stopping solution prior to the
addition of the substrate. The plates were then vortex-mixed and
centrifuged at 2250g for 10 min. A 50 μL volume of the supernatant
from each well was added to 300 μL of 50/50 acetonitrile/20%
aqueous formic acid. The resulting supernatants were subjected to
LC−MS/MS analysis.
Metabolic Stability with Mouse and Cynomolgus Monkey
Whole Blood. The metabolism of peptide candidates was studied
upon incubation with C57BL/6 mouse and cynomolgus monkey
whole blood. Incubation mixtures were prepared in duplicate in a 96-
well propropylene plate by adding 5.0 μL aliquots of either 100 μM
substrate peptide in DMSO or DMSO only (no-substrate control) to
45 μL of whole blood, which had been prewarmed at 37 °C.
Incubations were terminated after an incubation time at 37 °C of 1,
2, 4, and 6 h by the addition of 2 volumes (100 μL) of acetonitrile
containing 0.1% formic acid and 4.5 μM of an internal standard
peptide. True time zero controls were prepared by adding the
stopping solution prior to the addition of substrate. Plates were then
vortex-mixed and centrifuged at 2250g for 10 min. A 30 μL volume
of the supernatant from each well was added to 180 μL of 50/50
acetonitrile/20% aqueous formic acid. The resulting supernatants
were subjected to LC−MS/MS analysis.
Mouse Protein Binding. The binding of test molecules to
plasma proteins was determined by equilibrium dialysis using the HT
Dialysis apparatus (HT Dialysis LLC, Gales Ferry, CT). The peptide
was added to mouse plasma at a final concentration of 2.5 μM.
Phosphate-buffered saline (100 mM, pH 7.4) was added to one side
of the dialysis plate and an equal volume of plasma containing
peptide was added to the other side of the plate in replicates of six
for each drug concentration tested. The dialysis plates were
incubated at 37 °C under 5% CO2 for 4 h. Following the dialysis,
50 μL of plasma and buffer were removed from each dialysis well, a
matrix match was added to bring the pre-extracted samples to 50%
plasma and 50% buffer, and the analyte was extracted by the addition
of 250 μL acetonitrile containing 200 nM of the internal standard
mixture (labetalol, imipramine, and diclofenac). The samples were
vortex-mixed and centrifuged for 5 min at 3200g, and the resulting
supernatant fractions were analyzed by LC−MS/MS. The below
calculation was used to determine percent bound
% bound = [Cp]/[Cb] × 100
where Cb is the postincubation concentration of peptide in the buffer
side and Cp is the postincubation concentration in the plasma side.
LC−MS/MS analysis was performed on a LC−MS/MS system
equipped with an LEAP autosampler (Leap Technologies, Morris-
ville, NC) and a Thermo Scientific Dionex Ultimate 3000 RS Pump
(Thermo Fisher Scientific, Waltham, MA) interfaced to a Sciex 4500
mass spectrometer (Sciex, Framingham, MA) utilizing an electro-
spray source operating in the positive ion mode using multiple
reaction monitoring. Chromatographic separation was achieved on an
Acquity HSS T3 UPLC column 2.1 mm × 50 mm, 1.8 μm (Waters
Cat# 186003538, Milford, MA), using a mobile phase consisting of
0.1% formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B) at a flow rate of 0.75 mL/min. The
chromatography was run using a gradient elution as follows: the
column was equilibrated at 5% solvent B; after sample injection, the
gradient was held at 5% solvent B for 0.5 min. Solvent B was then
increased to 95% over a 1.5 min period. Solvent B was then
maintained at 95% for 0.42 min before it was returned to the initial
conditions for 0.83 min. The total run time was 3.25 min.
Crystallography. PCSK9 lacking the C-terminal domain
(PCSK9-ΔCRD) was produced and purified as described.12 The
final S60 gel filtration was performed in 20 mM Tris pH 8.0, 300
mM NaCl, 100 mM CaCl2, and 5% glycerol. Fractions containing
PCSK9-ΔCRD were combined, concentrated to 12 mg/mL, and
frozen in 50 mL aliquots till further use. Peptides were dissolved in
DMSO at 100 mM and added with a 2-fold molar excess to PCSK9-
ΔCRD. Complexes were incubated for 1 h on ice and screened
against commercially available crystallization screens. Suitable crystals
appeared with 20% PEG3350, 200 mM CaCl2, and 100 mM MES at
pH 6 as the precipitant. Crystals were harvested and cryoprotected
with an additional 10% glycerol. X-ray diffraction data were collected
at IMCA beamline ID17 and processed using the Globalphasing29
and CCP430 packages. Structures were solved by molecular
replacement using 4NMX12 and refined using BUSTER29 and
Coot.31 Peptides were modeled using standard amino acid residue
definitions or restrains generated using Flynn.32 Coordinates and
experimental data are available from the Protein Data Bank with the
accession codes: compound 30, 6XIB; compound 40, 6XIC;
compound 51, 6XID; compound 78, 6XIE; and compound 84,
6XIF. Authors will release the atomic coordinates and experimental
data upon article publication.
Rat Mast Cell Degranulation Assay. Isolation of rat peritoneal
lavage was conducted as described in the literature. In brief, Wistar
Han rats (males or females) were anesthetized with xylene. About 10
mL of Tyrode buffer (12 mM NaHCO3, 127 mM NaCl, 5 mM KCl,
0.5 mM NaH2PO4, 1 mM MgCl2, 5 mM glucose, and 10 mM
HEPES, pH 7.4) was injected into the peritoneal cavity, followed by
gentle abdominal massage for 30 s. Peritoneal lavage was then
collected and centrifuged at 350g for 10 min. After discarding the
supernatant, the cells are resuspended in Tyrode buffer to achieve a
final concentration of 1 × 106 cells/mL. For peptide treatment, 100
μL of freshly isolated rat peritoneal cells was added to a tissue-culture
treated 96-well plate. Cells were incubated with either positive
control (48/80; used to confirm assay responsiveness)33 or a titrating
concentration of various peptides for 30 min at 37 °C. After
incubation, the plate was centrifuged at 350g for 2 min at room
temperature. The supernatants were collected for histamine measure-
ment and cell pellets are resuspended in Tyrode buffer for cell
viability analysis. Histamine and cell viability were measured using
the commercially available kits, following vendor’s instructions. The
amount of histamine in the supernatant was presented as ng/mL, and
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cell viability was presented as percent viable cells compared to
vehicle-treated cells. Data were reported in for two determinations,
both of which were in close agreement in all cases.
PK Studies. All PK studies in preclinical species were conducted
according to the highest ethical standards in accordance with and
using procedures approved by the Institutional Animal Care and Use
Committee of Merck and Co., Inc., West Point, PA, USA.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01084.
■
sı
Representative LC traces for key molecules in the paper,
sample IC50 curves for LDLR-FRET and Alexa-FRET
assay control peptide, and raw concentration versus
time profiles for molecules tested in Mouse IV PK 3D
PDB files for each of the new crystal structures have
also been uploaded; PDB codes for the crystal
structures shown in the paper are as follows: compound
30, 6XIB; compound 40, 6XIC; compound 51, 6XID;
compound 78, 6XIE; and compound 84, 6XIF; and
authors will release the atomic coordinates and
experimental data upon article publication (PDF)
6xib (PDB)
Male C57BL/6 mice and male Wistar Hannover rats (n = 2−3 in
each arm) were used for IV PK studies. Mice and rats were allowed
to fast overnight prior to dosing and fed 2 h postdose. Dual jugular
vein and carotid artery surgically cannulated mice or rats were used
for dose administration and blood sampling, respectively. Blood
samples were collected using an automated blood sampling system
(Instech, Plymouth Meeting, PA) after IV administration. All blood
samples were collected in EDTA-coated tubes at predose, 0.08, 0.25,
0.5, 1, 2, 4, 7, 12, 18, and 24 h following IV dose administration. Rat
blood samples were centrifuged, and the supernatant plasma samples
were stored at −70 °C until bioanalysis. In mouse studies, whole
blood was diluted with citrate anticoagulant and whole blood PK
rather than plasma PK was measured.
Male cynomolgus monkeys (n = 2 per group) weighing 5−10 kg
were dosed IV. Monkeys were allowed to fast prior to dosing and fed
2−4 h postdose. IV dose was administered as a bolus via the cephalic
or saphenous vein. Blood samples were collected serially from the
saphenous, femoral, or cephalic veins in EDTA-coated tubes at
predose, 0.08, 0.25, 0.5, 1, 2, 4, 6, 24, 48, and 72 h. All blood samples
were centrifuged and the supernatant plasma samples were stored at
70 °C until bioanalysis.
Concentrations in mouse whole blood, rat plasma, and monkey
plasma were determined by LC−MS/MS assays following a protein
precipitation step. Aliquots (50 μL) of plasma were precipitated by
addition of 200 μL of methanol/acetonitrile (80:20) containing a
related macrocyclic peptide as an internal standard, followed by
centrifugation at 4000 rpm for 5 min. A 200 μL aliquot of the
supernatant was transferred into a 96-well plate injecting 2 μL of
each sample for analysis. Tandem LC−MS analysis was performed
on a Waters Acquity UPLC system interfaced to an AB Sciex
QTRAP-5500 or API6500 mass spectrometer utilizing the turbo ion
spray interface (AB Sciex, Framingham, MA). Separation was
achieved on a Waters Acquity HSS T3 C18 UPLC column (50 ×
2.1 mm, 1.8 μm) using a mobile phase consisting of water containing
0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic
acid (solvent B) at a flow rate of 0.75 mL/min. Quantification was
accomplished by monitoring the MS/MS transition of each peptide.
The concentration was determined across a linear concentration
range of compound matching standards (standard curve) in the
appropriate matrix.
PK parameters were calculated using noncompartmental methods
in Watson v.7.4. The area under the plasma concentration−time
curve (AUC0−t) was calculated from the first time point up to the last
time point using the linear trapezoidal method for ascending
concentrations and the log trapezoidal method for descending
concentrations. The concentration at 0 h after IV administration was
back-extrapolated using the first three time points (0.03, 0.13, and
0.25 h). The remaining area under the plasma concentration−time
curve (AUC t−∞) was estimated by dividing the observed
concentration at the last time point by the elimination rate constant.
This value was added to AUC0−t to estimate the AUC0−∞. The IV
6xic (PDB)
6xid (PDB)
6xie (PDB)
6xif (PDB)
AUTHOR INFORMATION
Corresponding Author
■
Thomas J. Tucker − Departments of Medicinal Chemistry,
Merck & Company, Inc., West Point, Pennsylvania 19486,
United States; orcid.org/0000-0001-5616-0802;
Email: tom_tucker@merck.com
Authors
Candice Alleyne − Discovery Pharmaceutical Sciences, Merck
& Company, Inc., Kenilworth, New Jersey 07033, United
States
Rupesh P. Amin − Safety Assessment, Merck & Comapny,
Inc., West Point, Pennsylvania 19486, United States
Bhavana Bhatt − Safety Assessment, Merck & Comapny, Inc.,
West Point, Pennsylvania 19486, United States
Elisabetta Bianchi − IRBM S.p.A., Pomezia, Rome 00071,
Italy
J. Craig Blain − UCB Ra Pharma, Cambridge, Massachusetts
02140, United States
Nicolas Boyer − UCB Ra Pharma, Cambridge, Massachusetts
02140, United States
Danila Branca − IRBM S.p.A., Pomezia, Rome 00071, Italy
Mark W. Embrey − Departments of Medicinal Chemistry,
Merck & Company, Inc., West Point, Pennsylvania 19486,
United States
Sookhee N. Ha − Modeling and Informatics, Merck &
Company, Inc., Kenilworth, New Jersey 07033, United States
Kelli Jette − UCB Ra Pharma, Cambridge, Massachusetts
02140, United States
Douglas G. Johns − Discovery Biology, Merck & Company,
Inc., Kenilworth, New Jersey 07033, United States
Angela D. Kerekes − Departments of Medicinal Chemistry,
Merck & Company, Inc., Kenilworth, New Jersey 07033,
United States
Kenneth A. Koeplinger − Pharmacokinetics
Pharmacodynamics and Drug Metabolism, Merck &
Company, Inc., West Point, Pennsylvania 19486, United
States
Derek LaPlaca − UCB Ra Pharma, Cambridge,
Massachusetts 02140, United States; orcid.org/0000-
0002-8513-2445
plasma clearance was calculated by dividing the dose by AUC0−∞
.
The terminal half-life of elimination was determined by unweighted
linear regression analysis of the log-transformed data. The volume of
distribution at steady state (Vdss) was obtained from the product of
plasma clearance and mean residence time (determined by dividing
the extrapolated area under the first moment curve by extrapolated
the area under the curve). The maximum plasma concentration
(Cmax) and the time at which maximum concentration occurred
(Tmax) were obtained by graphical inspection of the plasma
concentration−time data. For all n = 3, the error is characterized
as standard deviation. For n = 2, the error is characterized as the
difference between the mean and the individual determinations.
AA
https://dx.doi.org/10.1021/acs.jmedchem.0c01084
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Nianyu Li − Safety Assessment, Merck & Comapny, Inc., West
Point, Pennsylvania 19486, United States
subtilisin-like/kexin type 9; PK, pharmacokinetics; PPI,
protein−protein interaction; RP-HPLC, reverse phase high
performance liquid chromatography; SAR, structure−activity
relationship; SPPS, solid phase peptide synthesis; t-BuOH,
tert-butanol; TFA, trifluoroacetic acid; TMS-Cl, trimethyl-
chlorosilane
Beth Murphy − Discovery Biology, Merck & Company, Inc.,
Kenilworth, New Jersey 07033, United States
Peter Orth − Structural Sciences, Merck & Company, Inc.,
Kenilworth, New Jersey 07033, United States
Alonso Ricardo − UCB Ra Pharma, Cambridge,
Massachusetts 02140, United States
Scott Salowe − Discovery Biology, Merck & Company, Inc.,
Kenilworth, New Jersey 07033, United States
Kathleen Seyb − UCB Ra Pharma, Cambridge,
Massachusetts 02140, United States
Aurash Shahripour − Departments of Medicinal Chemistry,
Merck & Company, Inc., Kenilworth, New Jersey 07033,
United States
Joseph R. Stringer − UCB Ra Pharma, Cambridge,
Massachusetts 02140, United States
Yili Sun − UCB Ra Pharma, Cambridge, Massachusetts
02140, United States
Rodger Tracy − Pharmacokinetics Pharmacodynamics and
Drug Metabolism, Merck & Company, Inc., West Point,
Pennsylvania 19486, United States
Chengwei Wu − Departments of Medicinal Chemistry, Merck
& Company, Inc., West Point, Pennsylvania 19486, United
States
Yusheng Xiong − Departments of Medicinal Chemistry,
Merck & Company, Inc., Kenilworth, New Jersey 07033,
United States
Hyewon Youm − Departments of Medicinal Chemistry,
Merck & Company, Inc., Kenilworth, New Jersey 07033,
United States
Hratch J. Zokian − Discovery Biology, Merck & Company,
Inc., Kenilworth, New Jersey 07033, United States
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge Abbas Walji and H. Blair Wood for
helpful discussions and editing related to the manuscript and
scientific content. The authors are also grateful to Jennifer
Johnston for her excellent graphics work in support of the
manuscript.
ABBREVIATIONS
■
ACN, acetonitrile; AOP, tris(dimethylamino)(3H-1,2,3-
triazolo[4,5-b]pyridin-3-yloxy)phosphorus hexafluorophos-
phate; Boc, t-butoxycarbonyl; DBX, di-(1,3-bromomethyl)-
benzene; DCM, dichloromethane; DIC, N,N-diisopropylcar-
bodiimide; DIEA, diisopropylethyl amine; DMF, dimethylfor-
mamide; DMSO, dimethyl sulfoxide; DODT, 3,6-dioxa-1,8-
octanedithiol; EtOAc, ethyl acetate; 5F-Trp, 5-fluoro-trypto-
phan; Fmoc-OSu, Fmoc N-hydroxysuccinimide ester; HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate; IV, intravenous;
ivDde, [l-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-ethyl]-
protecting group; LC−MS, liquid chromatography−mass
spectrometry; LDL-C, LDL-cholesterol; LDLR, LDL receptor;
mAb, monoclonal antibody; Met ID, metabolite identification;
NMP, N-methyl pyrrolidinone; PCSK9, proprotein convertase
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