Difluoromethylene at the γ-Lactam α-Position Improves 11-Deoxy-8-aza-PGE1 Series EP4 Receptor Binding and Activity: 11-Deoxy-10,10-difluoro-8-aza-PGE1 Analog (KMN-159) as a Potent EP4 Agonist

Article abstract of DOI:10.1021/acs.jmedchem.9b00336

A series of small-molecule full agonists of the prostaglandin E₂ type 4 (EP₄) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

Full text of DOI:10.1021/acs.jmedchem.9b00336

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Article
Difluoromethylene at the #-Lactam #-Position Improves 11-Deoxy-8-
1
4
aza-PGE Series EP Receptor Binding and Activity: 11-Deoxy-10,10-
1
4
difluoro-8-aza-PGE Analog (KMN-159) as a Potent EP Agonist
Stephen Douglas Barrett, Melissa C Holt, James B Kramer, Bradlee Germain,
Chi S Ho, Fred L. Ciske, Andrei Kornilov, Joseph M Colombo, Adam Uzieblo,
James P O'Malley, Thomas A Owen, Adam J Stein, and Maria Morano
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00336 • Publication Date (Web): 09 Apr 2019
Downloaded from http://pubs.acs.org on April 9, 2019
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of their duties.

Page 1 of 20
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Difluoromethylene at the -Lactam -Position Improves 11-Deoxy-8-
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aza-PGE Series EP Receptor Binding and Activity: 11-Deoxy-10,10-
1
4
difluoro-8-aza-PGE Analog (KMN-159) as a Potent EP Agonist
1
4
†
,⊥,*
†,⊥,#
†
†
†
†
†
Stephen D. Barrett,
Melissa C. Holt,
James B. Kramer, Bradlee Germain, Chi S. Ho, Fred L. Ciske,
†
†
†
‡
‡,§
Andrei Kornilov, Joseph M. Colombo, Adam Uzieblo, James P. O’Malley, Thomas A. Owen, Adam J. Stein,
†
and Maria I. Morano
†
Cayman Chemical Company, Inc., 1180 East Ellsworth Road, Ann Arbor, Michigan, 48108, United States
‡
Myometrics, LLC, 216 Howard Street, New London, Connecticut, 06320, United States
§
Ramapo College of New Jersey, 505 Ramapo Valley Road, Mahwah, New Jersey, 07430, United States
O
O
H
F
ABSTRACT: A series of small molecule full agonists of the
prostaglandin E type 4 (EP ) receptor have been generated and
H
F
N
N
2
4
Potency
evaluated for binding affinity and cellular potency. KMN-80 and its
gem-difluoro analog KMN-159 possess high selectivity against
other prostanoid receptors. Difluoro substitution is positioned
alpha to the lactam ring carbonyl and results in KMN-159's five-
fold increase in potency versus KMN-80. The two analogs exhibit
electronic and conformational variations including altered
nitrogen hybridization and lactam ring puckering that may drive
the observed difluoro-associated increased potency within this
four-compound series.
O
O
Selectivity
Solubility
OH
OH
HO
HO
KMN-80
KMN-159
K : 2.35 nM (EP )
i
4
K : 0.38 nM (EP )
i
4
Ki: >10,000 nM (EP2)
Ki: 4,971 nM (EP2)
Lipophilicity
Permeability
Prostaglandin & -Lactam Ring Scaffolds & Analogs
O
O
INTRODUCTION
2 2
Prostaglandin E (PGE ) is a potent endogenous molecule that
E
-chain
-chain
E
N
-chain
mediates a large variety of biological functions in the human
body by interaction with four different G-protein coupled
receptors known as E-type prostanoid receptors: EP1-4. Each EP
receptor subtype shows specific patterns of tissue distribution
and signal transduction pathways, explaining the complex and
HO

-chain
2-Pyrrolidinone
(-Lactam) Ring System
Prostaglandin E
Cyclopentanone Ring System
O
9
O
OH
O
O
9
N8
12
1
0
10
1
7
7
sometimes divergent physiological and clinical effects of PGE
2
.
1
1
8
5
11
5
12
In particular, the EP receptor is widely expressed in the
4
X
6
3
HO
6
3
1
3
14
O
13
14
O
1
OH
4
4
gastrointestinal track, uterus, hematopoietic tissues, bone, and
skin. Immune modulation, regulation of inflammatory
responses and vascular tone, preservation of renal and bone
1
1
6
2
16
2
HO 15
OH
HO
HO 15
18
19
18
19
1
7
17
20
20
homeostasis as well as mucosal integrity have been all
X=OH PGE1 (1a)
X=H 11-Deoxy-PGE1
(1b)
PGE2 (2)
11-Deoxy-8-aza-PGE1 (3)
2
proposed functions for the EP
4
receptor.
The 2-pyrrolidinone (-lactam, or E ring) scaffold is a known
Figure 1. Prostaglandin and -lactam E ring-scaffolds and numbering.
synthetic PGE structural mimic deployed in EP receptor-based
3-5
SAR programs for decades (Figure 1) . The relative ease with
Various potent prostanoid- and -lactam-based EP
4
receptor
which a diverse array of - and -chains can be introduced on
the core ring makes this an attractive template from a synthetic
enablement perspective.
agonists, such as Ono Pharmaceutical’s Rivenprost (ONO-
6
7
4
819) free acid, Merck-Frosst’s L-902,688, and Kaken
8
Pharmaceutical’s KAG-308 were discovered through rational
design and have been assessed as potential therapies for bone
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-
healing, ischemia, glaucoma, and ulcerative colitis (Figure 2).
structure, and Drug Metabolism and Pharmacokinetics
DMPK).
12
(
N
N
O
O
NH
Kaken Pharmaceuticals
N
Ono Pharmaceuticals
Merck-Frosst
H
RESULTS AND DISCUSSION
N
S
Chemistry. To explore the yet untested effects of
difluoromethylene incorporation into the E ring, we prepared
and screened multiple pairs of analogs as part of our more
HO
O
N
F
F
N
N
F
F
O
N
HO
OR
HO
OH
extensive SAR program; each pair including both
a
OH
KAG-3088 (5b)
OMe
nonfluorinated -lactam species and its corresponding 3,3-
difluoro-2-pyrrolidinone (“difluorolactam”) analog. Figure 3
illustrates two such nonfluoro-difluoro pairs we will highlight
R= Me Rivenprost (ONO-4819)6 (4a) L-902,6887 (5a)
R = H Rivenprost free acid6 (4b)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Figure 2. Published PG and -lactam EP receptor-selective agonists.
3
in this report. 11-Deoxy-8-aza-PGE
structural analog to prostaglandin 11-deoxy-PGE
prepared both this compound and its difluorolactam analog 6
KMN-165). The other analog pair we scrutinized incorporates
1
(3) is the closest -lactam
1
.
We
Although the -lactam and PGE scaffolds share common ring
size, relative carbonyl and side-chain positioning, and ring C-
12 stereochemistry, the -lactam nitrogen atom substitution of
the corresponding PGE ring C-8 atom imposes structural and
electronic variations responsible for any biological and
physicochemical property differences between a PGE analog
(
2
2
the acetylene moiety of the prostacyclin analog cicaprost into
the -chain, providing nonfluoro/difluoro pair 7 (KMN-80)/8
(
KMN-159). KMN-159 is a current lead compound that has
1
3, 14
emerged from this series.
(
e.g. 11-deoxy-PGE
1
, (1b))
and its corresponding -lactam
3
, 15, 16
derivative (e.g. 11-deoxy-8-aza-PGE
1
, (3)).
Differences
O
X
O
X
between carbon and nitrogen atom valence, electronegativity,
hybridization, and geometry all play expected roles in ring
conformation, electronic, and side chain spatial relationship
variations between the two systems. The lactam nitrogen atom
possesses mainly sp character and is trigonal planar. Its lone
pair valence electrons occupy the p-orbital oriented to overlap
with the carbonyl  system that allows delocalization of charge
and electron density. The PGE ring C-8 atom is sp hybridized,
tetrahedral, and possesses no valence electrons in conjugation
with the carbonyl  system. These distinguishing structural
X
N
X
N
O
O
2
OH
OH
HO
HO
3
X = H (3)
X = F KMN-165 (6)
X = H KMN-80 (7)
X = F KMN-159 (8)
Figure 3. -Lactam analog nonfluoro-difluoro pairs.
and electronic features between
a PGE ring and its
Scheme 1 shows that our final assembly strategy involved
carbon-carbon bond formation by way of Horner-Emmons-
Wadsworth (HEW) chemistry utilizing aldehyde 9 to install the
corresponding lactam would be expected to affect free energies
of the ligand both unbound and bound to the receptor due to
both enthalpic and entropic variations and thus impact EP
receptor binding affinity and associated biological activity.
Fluorine substitution of aliphatic and aromatic hydrocarbon
hydrogen atoms can be a useful strategy for improving binding,
-chain of final product 8b. Alkylation of the lactam nitrogen
of O-protected 12b installs the -chain. A published procedure
describes the [3.3.0] bicyclic bis fluorination protocol of 11
23
originating from commercially available 12a.
activity, physicochemical, and pharmacokinetic properties of
17, 18
small molecule drug candidates.
Although fluoro
Scheme 1. Retrosynthetic Analysis of Difluorolactam
substituents have relatively low steric demand, they are highly
electronegative and can influence electronics, molecular
O
F
R
O
O
F
F
F
R
1
9, 20
N
conformation, and other properties of organic molecules.
F
F
N
NH
OH
Polarization of the C-F bond is reverse that of the C-H bond,
with fluorine possessing a partial negative charge as opposed
to the partial positive charge localized at the hydrogen atom.
The C(sp )-F bond is remarkably stable in contrast to the
corresponding bonds substituting the other halogens. The
highly-electronegative fluoro substituent stabilizes the carbon
R
O
HO
H
8b
9
12b
3
O
O
O
N
F
3
NH
OH
N
F
bonding sp orbital to which it is bound to expose an accessible
(due to fluorine’s low steric demand) low-lying antibonding
O
O
orbital (*C-F
)
that can participate in stabilizing
12a
11
10
hyperconjugative interactions, thereby lending favor to
stereoelectronic orientations that affect molecular shape,
conformation, bond lengths, and electron density distribution.
Difluoro substitution of one or more methylene groups in large
carbocyclic rings, for example, modifies the macrocycle’s
Both lactam 7 and difluorolactam 8 syntheses followed a
seven-step parallel route from corresponding (R)-5-
2
4, 25
26
(hydroxymethyl)pyrrolidine-2-one (12a
/12b ) starting
points (Scheme 2). Tert-Butyldimethylsilyl (TBDMS)
21
shape, conformation, and flexibility.
In our quest to identify novel selective EP
protection of hydroxymethyl function provided corresponding
protected intermediates 13a/13b which, upon alkylation of
the lactam ring nitrogen to incorporate a methyl heptanoate -
chain, gave 14a/14b. We note that the presence of the fluoro
substituents in 13b slightly accelerated consumption of the
4
receptor agonists
with high potency, desired functional activity, and drug-like
properties, we prepared parallel (nonfluoro/difluoro) -lactam
series and studied the impact of difluoromethylene
incorporation on receptor binding and activation, crystal
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2
starting material during the alkylation over that of the
this difluorolactam pan-EP receptor binding superiority over
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nonfluorinated 13a.
their corresponding nonfluorolactams without exception for
all such analog pairs we generated in our broader SAR program
(data unpublished). The general potency shift in this already
Scheme 2. Synthesis of KMN-80 and KMN-159
OMe
highly EP
benefits related to an expected lower dosing requirement.
Difluorolactams 6 and 8 are essentially equipotent EP binders
with PGE and Rivenprost free acid (4b).
4
-selective series offers immediate potential drug
O
O
O
O
X
X
X
a
b
c
X
NH
OH
X
NH
OTBDMS
X
N
4
OTBDMS
2
X=H 12a 91%
X=F 12b 99%
X=H 13a 54%
X=F 13b 67%
X=H 14a
X=F 14b
66%
81%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OMe
OMe
Table 1. Binding affinities to human EP receptor subtypes
MeO
O
X
O
O
O
MeO
Human EP Receptor Binding K (nM)
X
P
i
d
e
X
N
X
N
+
O
Cmpd
hEP
2
hEP
3
hEP
4
O
OH
O
17
1.03+0.13
2.17+0.21
0.110+0.020
(6)
PGE
2
H
X=H 15a
X=F 15b
94%
91%
X=H 16a
X=F 16b
57%
55%
(7)b,c
(3)b,c
7
12,173+1,265
880+14
(3)
2.349+0.658
(4)
OMe
O
a,b,c
a,b,c
a,b,c,d
OMe
OH
O
(
3)
O
X
O
O
O
4,971+879
258+8
(3)
0.378+0.105
(5)
X
X
8
f
g
X
N
a,c
a
X
N
X
N
(3)
3
1,459+123
1,175+63
1.606+0.061
O
(3)a,b,c
(3)a,b,c
(3)a,b,c,d
HO
HO
6
329+26
(3)
350+25
(3)
0.200+0.019
(3)
X=H 18a
X=F 18b
21%
20%
X=H 19a
X=F 19b
74%
80%
X=H 7 (KMN-80)
X=F 8 (KMN-159)
a,b
a
4b
0.396 + 0.121
aR e a g e na tn sac o n ai t i(oan T) sB :D TS C li ,m i aa z oD lTeF ,/ TH F( ;b m) e t h y7 l-
nd
nd
(
4)
b bo m o - h e p t a Nn ao Ia N,t ae H, ,D TF ; ( c )3 N H C l ,T e O H ;( a) D e s s - Ta bt
Pe bi o ai n Ca Hn 2eC ,l2; ( e L) i C lE t,3 N , TH F ;( f C) e C 3 l• 7 H 2O , N a B H4, Te O H ,- 7 8
i
K values were calculated using GraphPad Prism 7.02 software and are
presented as mean + SEM of (n) replicates. Statistical analysis of the
differences was performed using ANOVA followed by Tukey Test.
°
C ;( g N) a O H( a q, T) e O H .
(
a)
(b)
(c)
(d)
P<0.01 as compared to PGE
2
,
8, 6, and 4b. nd: not determined.
Subsequent deprotection of intermediates 14a/14b afforded
corresponding hydroxymethyl intermediates 15a/15b with
good yields. Dess-Martin oxidation of 15a/15b produced the
corresponding HEW partner aldehydes 16a/16b with
excellent yields. HEW coupling of 16a/16b with known 2-
ketophosphonate ester, (+)-dimethyl (S)-(3-methyl-2-oxooct-
The EP and EP receptors activate adenylyl cyclase resulting
4
2
in the increased formation of intracellular cyclic 3,5-adenosine
monophosphate (cAMP). Endogenous PGE2 or exogenous
compound agonists activate the transcription factor cAMP-
responsive element-binding protein (CREB) upon binding to
these receptors. Table 2 depicts compound cAMP and CREB
2
7
5-yn-1-yl)phosphonate (17), provided enone intermediates
8a/18b, which underwent subsequent Luche reduction to
1
activation EC s in HEK-293 cells overexpressed with the
50
provide 19a/19b, each as about a 3:7 OH mixture revealed
by HPLC. The diastereomeric pairs were separated using silica
column chromatography to provide the respective purified C15
human receptors. All four of our prepared lactam analogs 7, 8,
3 and 6 are full, selective EP
binding affinities, difluorolactam analogs 6 and 8 are
significantly more potent EP and EP activators over their
corresponding nonfluoro analogs across all assays.
4
agonists. Mirroring their relative

OH diastereomers as penultimate ester intermediates
4
2
19a/19b in low yields. Saponification afforded target free acid
compounds 7 and 8 in high yields from their respective methyl
esters. Small molecule x-ray crystallography of 8 confirmed the
absolute stereochemical assignment of C15 (S-configuration)
as corresponding to the C15 OH stereochemistry of 19b.
Compound 6 was prepared from 16b in similar fashion to 7
and 8 except dimethyl (2-oxoheptyl)phosphonate was used
instead of phosphonate 17.
Table 2. Biological activation of EP
4
and EP
2
receptors
hEP
4
EC50 (nM)
CREB
hEP
2
EC50 (nM)
Cmpd
cAMP
cAMP
CREB
activation
0.020+0.001
(14)
activation
PGE
7
2
0.583+0.078
331+61
(6)
>10,000
(3)
>10,000
(3)
9.3+0.8
b
b,c,d
(
30)
(7)
19.920+4.280
0.476+0.072
>10,000
(3)
a,b,c,d
a,b,c,d
Pharmacology. Our lactam/difluorolactam pairs were
screened for prostanoid receptor binding and were compared
(5)
2.631+0.319
(3)
8
0.088+0.019
7,358+2,998
a,c,d
a,c,d
a,c,d
(
3)
(3)
(2)
with PGE
subtypes with low nanomolar potencies and has relatively
weak affinity for FP, DP , and IP receptors. Lactam analogs
showed low- and sub-nanomolar potencies for EP and
relatively weaker affinities for EP and EP (Table 1); however,
they showed no measurable binding affinity for EP nor the
non-EP prostanoid receptors (Table S1). Interestingly, a
notable improvement in EP selectivity over EP was shown by
2 2
and 4b (Table 1). PGE binds to all four EP receptor
3
1.765 + 0.314
0.065 + 0.015
>10,000
(3)
>10,000
(2)
a,c,d
a,c,d
(
3)
(3)
1
6
0.348+0.040
0.021+0.002
>10,000
(3)
2,266+78
(2)
b
b
a,b,d
(3)
(4)
4
4b
0.142+0.013
0.010+0.003
10,000
(3)
610+145
(2)
2
3
b
b
b,c,d
(
3)
(3)
1
EC50 values were calculated using GraphPad Prism 7.02 software and
are presented as mean + SEM of (n) replicates. Statistical analysis of the
differences was performed using ANOVA followed by Tukey Test.
4
2
the acetylene-bearing pair 7/8 relative to the 3/6 pair. This
result is consistent with published SAR that show lactam -
chain rigidity leads to high EP
Difluoromethylene incorporation into the E ring significantly
improved binding potencies across all EP receptor subtypes for
which we obtained measurable binding (EP2-4). We observed
(a)
(b)
(c)
(d)
P<0.01 as compared to PGE
2
,
8, 6, and 4b. No EC50 was
calculated for compounds with stimulation lower than 50% at 10 M
(results indicated as >10,000 nM)
2
8
4
selectivity over EP
2
.
Receptor Docking. Recently published crystal structures of
EP and EP have provided a wealth of data for the binding
3 4
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6
(2.50)
mode of PGE
2
.29-32 The EP
4
receptor was crystallized in the
R316(7.37), T69(2.54), D65(2.50) and N321(7.45). Residues D65
and N321
(7.45)
2+
inactive conformation bound to an antagonist, ONO-AE3-208
make up part of a putative Na binding pocket,
which has been speculated to be involved with receptor
29
(
PDB: 5YWY, ligand ID: 7UR). The EP
3
receptor was
3
0
crystallized separately with two different agonists bound, one
activation.
3
0, 31
of which was PGE
binding mode of PGE
EP
Fit-Docking (IFD) protocol within Maestro 11 (Schrödinger,
2
.
To compare our ligands with a known
Molecular-Mechanics with generalized Born and Surface Area
Solvation (MM-GBSA) scoring, a simplified computation
methodology for determining binding free energies, was
The results predict
difluoromethylene
incorporation into the E ring, as seen with the calculated
energies of the 3/6 and 7/8 pairs (Figure 5). The predictions
are consistent with biological data (Table 1), which show
increased potency across EP2-4 receptor subtypes with the
presence of the fluoro substitutions. Linear regression of the
2
, docking was performed with the active
structure (PDB: 6AK3, ligand ID: P2E)30 using an Induced-
3
4
1, 42
applied to the binding poses within EP
3
.
3
3-38
LLC, New York, NY, 2018.).
5% sequence identity and 48.2% sequence similarity. Docking
to EP suggested that our agonists bind in like manner to PGE
within EP (Figure 4). The model showed clear overlap
between PGE and the 7/8 pair, supporting a similar binding
4 3
Relative to EP , EP possesses
increased potency arising from
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
2
3
2
mode between the molecules, despite the rigidity of the
acetylene ω-chain. The 3/6 pair, which bears the identical
saturated -chain terminus as PGE , likewise docked in a
2
nearly identical position to PGE
scoring function versus EP
maintained and results in an R of 0.69 (Figure S2).
3 i
K shows that the rank-order is well
2
43
2
(Figure S1).
A.
ECL2
T168
ECL2
T206
2.65
W169ECL2
Y80
Y1142
.65
W207ECL2
R3167.37
R3337.40
2.58
.58
S73
T1072
S3367.43
N3217.45
2.54
T69
Q1032.54
2.50
Figure 5. Molecular Mechanics with generalized Born and surface
area solvation calculations of docked poses to human EP . Significance
determined via unpaired t-test with two-tailed p-value within
Q3397.46
D65
D99
2
.50
3
GraphPad Prism v. 6.07.
ECL2
ECL2
T168
B.
T206
Y802.65
ECL2
Crystallography/Quantum-Mechanics.
To
further
W169
2.65
Y114
ECL2
understand the mechanism of the difluoromethylene-driven
potency shift, we obtained small molecule crystal structures of
and 8 (Figure 6). Quantum-mechanical calculations were
performed for each compound in duplicate using the crystal
structure-determined coordinates for both molecules of the
asymmetric unit using Jaguar (Schrödinger, LLC, New York, NY,
018).
We observed several conformational and electronic
differences between 7 and 8. The difluorolactam 8 E ring
displayed both C11 endo- and exo-puckers for each molecule
within the asymmetric unit (Figure 6). Lactam 7 displayed only
an E ring C11 endo-pucker of the lactam ring for each molecule
within the asymmetric unit. Differences in conformational
preference within the solution state were likewise identified by
MD simulation and force-field based conformation search
W207
7
R3167
.37
S732.58
R3337
.40
2.58
T107
44
2
S3367.43
2.54
.54
T69
Q1032
N3217.45
Q3397.46
D652.50
D992.50
Figure 4. Crystal structure of PGE
bound to EP receptor, residues shown in line representation in orange
PDB: 6AK3, ligand ID: P2E). (A) Docked pose of 7 (gray stick & ball
representation) in EP receptor overlaid overtop of PGE . (B) Docked
pose of 8 (black stick & ball representation) overlaid on top of PGE
2
(green stick & ball representation)
3
3
0
(
3
2
(
Figure S3). We calculated entropy at standard conditions to be
2
significantly higher (p=0.0035) in 8 (126.8 kcal/mol ± 0.4090,
n=2) than in 7 (120.1 kcal/mol ± 0.3975, n=2). These values
suggest a higher number of conformational and vibrational
states for the difluorolactam. γ-Lactam nitrogen atom planarity
furthermore appears to be increased within 8, as observed by
decreased C7-N8-C9-O dihedral angle. Structural
observations of a trigonal planar nitrogen within 8 indicate a
(
green stick & ball representation). Binding site alignment with human
4
EP receptor homology model performed with aligning residues shown
in line representation in blue. Residues numbered with one letter
amino acid code and sequence number with Ballesteros-Weinstein
39
number in superscript.
a
To surmise residues of importance within EP
alignment was performed between EP and EP
homology model of EP generated within the RaptorX Structure
prediction suite (raptorx.uchicago.edu/).40 Essential residues
4
, a binding site
3
4
, using a
2
fully sp -hybridized center and signify increased delocalization
4
of the nitrogen lone pair relative to 7. This is consistent with
Jaguar pKa prediction of decreased γ-lactam nitrogen basicity
of 8 (pKa=11.7) versus 7 (pKa=3.8). Several examples of α-
(
2.65)
for 7 and 8 binding were predicted to include Y80
,
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Journal of Medicinal Chemistry
1
2
-
gem-difluoro-γ-lactams exist within the Cambridge
Crystallographic Data Center (CCDC), displaying both C11 exo
oxygen also displays this decrease in charge in 8 (0.5888 e ±
0.00043, n=2) versus 7 (0.6252 e ± 0.003735, n=2).
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-
45-47
and C11 endo E-ring pucker between the enteries.
In general, decreased electron density of an H-bond accepting
carbonyl oxygen would reduce intermolecular bond potential
and potency. While the reduction in carbonyl oxygen-hydrogen
bonding potential of our difluoromethylene compounds does
not explain the observed increase in potency, it is consistent
3
2
Increased sp character of the γ-lactam nitrogen atom appears
4
5, 47
to be conserved (Figure S4 and Figure S5).
Interestingly, no significant differences
between
corresponding E ring atom bond lengths of the fluorinated and
nonfluorinated molecules exist, indicating hyperconjugation
may not play a significant role in 8 ring size, shape, and
with the EP crystal structures displaying no H-bonding with
3
0, 31
the carbonyl of PGE2 or Misoprostol.
Electronic and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
8
conformation (Figure S6). Consequently, we postulate that
the effects of difluoromethylene incorporation appear to be
mediated through inductive effects of the fluorine atoms within
this system.
conformational differences between 7 and 8 and other
nonfluoro-difluoro analog pairs may explain the observed
difluoromethylene-driven potency increases instead through
modifications in ligand desolvation or receptor dewetting. This
is supported by a calculated 1.8±.026 kcal/mol difference in
average potential energy of solvation between 7 and 8
A.
B.
4
9
(
p=0.0002, n=2) over a 100 ns MD simulation.
Physicochemical Properties/DMPK/Safety
Profile.
Compounds 7 and 8 possess key drug-like properties (Table 3).
The aqueous solubility for 7 and 8 are equivalent and suitable
for good bioavailability. The slight increase in lipophilicity of 8
over 7 is expected given the addition of the fluoro substituents.
Lipophilic ligand efficiency (LLE) is higher for 8 relative to 7
indicating that the potency shift is a consequence of more than
hydrophobic effect. The modest increase in protein binding of
C.
D.
0
8
in human plasma coincides with the on- and off-target

N8
C9
0.48Å
potency shift observed with gem-difluoro substitution.
0.21Å

N8
C9
8.8
.2
C10
C10
Table 3. Drug-like profile of compounds 7 and 8
7
(KMN-80) 8 (KMN-159)
Physicochemical properties
E.
*
Aqueous Solubility (PBS, pH 7.4)
192.3+7.2 M
197.0+0.4 M
* Log D (n-octanol/PBS, pH 7.4)
-0.07
0.11
*
Chemical Stability (% recovery)
o
aqueous solution (PBS, pH 7.4, 4 C)
n.d.
n.d.
n.d.
100%
100%
95%
o
autoclave (PBS, pH 7.4, 4 C)
-irradiation (29.7 Kga Co-60)
Protein binding (10 M in plasma)
a
*
44.2+0.1%
58.4+4.1%
In vitro Absorption
*
Caco-2 Permeability
A-to-B Papp (10 cm/s)
-6
6.38+0.59
103%
10.10 + 1.11
97%
9.93+0.23
94%
14.50 + 2.05
93%
Percent Recovery
-6
B-to-A Papp (10 cm/s)
Percent Recovery
Efflux Ratio
Figure 6. (A) Ellipsoid plots of 7 KMN-80 and (B) 8 KMN-159 as
determined by x-ray crystallography displaying two molecules within
asymmetric unit in tail-tail and head-head orientations. (C) Edge-on
views of 7 and (D) 8 revealing puckered conformations observed by x-
ray data collection. 8 displays both exo & endo C11 pucker of lactam
ring. 7 shows only endo C11 pucker. (E) C7-N8-C9-C10 dihedral angles
of 7 and 8, significance determined via unpaired t-test with one-tailed
p-value within GraphPad Prism v. 6.07.
1.6
1.5
* MDR1-MDCK Permeability
-
6
A-to-B Papp (10 cm/s)
Percent Recovery
n.d.
n.d.
n.d.
n.d
0.59 + 0.36
84%
11.8 + 0.14
93%
-6
B-to-A Papp (10 cm/s)
Percent Recovery
Efflux Ratio
n.d.
20.0
In vitro Metabolism
* Half-life (1M in human plasma)
115.5 + 10.5 min
> 120 min
> 120 min
> 120 min
Partial charges were calculated for atoms within 7 and 8, with
*
Half-life (1M in human blood)
expected significant differences, where partial positive
* Intrinsic Clearance
(0.1M in human liver microsomes)
Half-life (min)

hydrogen atoms (0.2463 e ± 0.0014, n=2) in 7 were exchanged

> 60 min
< 115.5
> 60 min
< 115.5
for negative fluorine (0.3548 e ± 0.0038, n=2) in 8. The
CLint (L/min/mg)
presence of the fluorine substituents significantly inverted the
partial charge centered on C10 from a partial negative
*
Intrinsic Clearance
(
0.1M in human liver hepatocytes)
Half-life (min)
CLint (L/min/Mcell)


(0.5175 e ± 0.0044, n=2) in 7 to a partial positive (0.7072 e
0.0010, n=2) in 8. The partial positive value for carbonyl
> 120 min
< 8.2
> 120 min
< 8.2
±

* Cytochrome P450 Inhibition
10M in human liver microsomes)
Genetic Toxicity
Bacterial cytotoxicity
* Ames fluctuation test
Micronucleus CHO cells -/+ S9
Cardiac Toxicity
carbon (C9) was reduced from 0.6582 e ± 0.0050 (n=2) in 7 to
.5796 e ± 0.0006 (n=2) in 8. Most importantly, the nitrogen
showed lower partial charge in 8 (0.4314 e ± 0.0014, n=2)
than in 7 (0.4478 e ± 0.0043, n=2). In addition, the carbonyl
All negative
All negative
(

0

*
All negative
All negative
Negative
All negative
All negative
Negative

*
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Journal of Medicinal Chemistry
Page 6 of 20
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
*
hERG IC50 (patch-clamp)
29.0 + 11.9 M
p < 0.05 by Unpaired t-test; n.d. not determined
25.9 + 8.5 M
95%) by HPLC and by
chromatography (TLC) analyses were performed on Uniplate™
50 silica gel plates (Analtech, Inc. Catalog No. 02521) and
1
H NMR spectroscopy. Thin layer
a
2
Both 7 and 8 show high absorption potential according to
their high apparent permeability in the Caco-2 assay. Low
blood-brain-barrier penetration potential of 8 was determined
by MDR1-MDCK permeability assay. Compounds 7 and 8
possess oxidative metabolic stability as seen in microsomes
and hepatocytes across species. No cytochrome P450
inhibition was detected for CYP1A, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, or CYP3A. Both compounds tested negative
in gene and cardiac toxicity screens (Table S2).
were typically developed for visualization using 50 volume %
concentrated sulfuric acid in water spray or Hanessian’s Stain.
Synthesis of 7-((R)-2-((3S,4S,E)-3-hydroxy-4-methylnon-1-en-6-
yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoic acid (KMN-80, 7)
R)-5-(((tert-Butyldimethylsilyl)oxy)methyl)pyrrolidin-2-
one (13a). To a stirring solution consisting of (R)-5-
hydroxymethyl)pyrrolidin-2-one (12a
mmol) in dry DMF (10 mL) was added imidazole (0.581 g, 8.53
mmol). The mixture was cooled to °C and tert-
butyldimethylchlorosilane (0.969 g, 6.43 mmol) was added.
The reaction mixture was stirred at room temperature
overnight and was diluted with ethyl acetate (150 mL). The
organic phase was washed with brine (2 x 75 mL), dried over
anhydrous sodium sulfate, and concentrated under reduced
pressure to provide a transparent oil (1.68 g). The oil was
purified by silica gel chromatography eluting with heptane-
isopropanol (95:5 v/v) to afford the title compound as a white
solid (1.21 g, 91%); H NMR (400 MHz, CDCl
.6-3.8 (m, 1H), 3.57 (dd, 1H, J=7.7, 3.9 Hz), 3.38 (dd, 1H, J=7.7,
0.0 Hz), 2.9-2.9 (m, 1H), 2.8-2.9 (m, 1H), 2.2-2.4 (m, 2H), 2.1-
.2 (m, 1H), 1.68 (dddd, 1H, J=5.5, 7.5, 9.4, 12.9 Hz), 0.8-0.8 (m,
H), 0.00 (s, 6H); MS (ESI ) m/z 230.1 (M+H).
Methyl (R)-7-(2-(((tert-butyldimethylsilyl)oxy)methyl)-5-
oxopyrrolidin-1-yl)heptanoate (14a). To stirring
suspension comprising sodium iodide (7.2 g, 48 mmol) and
sodium hydride (1.7 g, 44 mmol, 60% dispersion in mineral oil)
in DMF (60 mL) at 0 °C was added a solution consisting of (R)-
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24, 25
(
) (0.759 g, 5.02
0
CONCLUSION
In summary, a novel pair of human EP
are presented featuring unique acetylene ω-chain.
Compounds 7 and 8 possess excellent drug-like properties.
Both molecules present low-nM to sub-nM binding and
activation of EP
leftward shift of K
analog 8 (KMN-159) relative to corresponding nonfluoro
analog 7 (KMN-80). We also observed this effect between the
4
selective full agonists
a
4
with >5,000 times selectivity over EP
2
. A
i
of 4.6 times was observed for gem-difluoro
1
3
) δ 5.84 (br s, 1H),
3/6 pair and without exception within our broader SAR series,
3
1
2
9
where the difluoromethylene moiety was associated with 3-10-
fold potency increases regardless of α- or ω-chain substituents.
Difluoromethylene incorporation produced electronic and
conformational differences, including changes in charge
distribution, γ-lactam nitrogen hybridization, and E-ring
puckering. These findings suggest -lactam gem-difluoro
substitution is a successful strategy for enhancing potency
which may find utility for related chemical motifs and other
biological targets.
+
a
5
(
-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-2-one
13a) (10.0 g, 43.6 mmol) in DMF (120 mL). The ice bath was
EXPERIMENTAL SECTION
removed, and the mixture was stirred at room temperature for
one hour and was subsequently warmed to 40 °C for one hour,
then 50 °C for 30 minutes. To the warm mixture was slowly
added methyl 7-bromoheptanoate (11.7 g, 52.3 mmol) and the
mixture was stirred overnight at 50 °C. The mixture was cooled
with an ice-water bath and treated with 0.21 N HCl (207 mL,
Refer to the Supporting Information for details pertaining to
modeling, x-ray structure determination, and in vitro biological
and pharmacological evaluation of all pertinent compounds.
Materials and Methods
Chemistry
Liquid chromatography – mass spectra (LC/MS) were obtained
using an Agilent LC/MSD G1946D or an Agilent 1100 Series
LC/MSD Trap G1311A or G2435A. Quantifications were
4
3.5 mmol) by slow addition. The mixture was twice extracted
with ethyl acetate (500 mL and 100 mL). The combined organic
phase was twice washed with water, then brine, and was dried
over magnesium sulfate, filtered, and concentrated under
reduced pressure to provide a brown oil (16 g). The crude oil
was purified by silica gel chromatography. Elution with a
gradient (3:2 v/v heptane-ethyl acetate to 100% ethyl acetate,
then 9:1 to 4:1 v/v ethyl acetate-methanol) afforded the title
compound as a light brown oil (8.8 g, 54%); H NMR (400 MHz,
3
) δ 3.62 (s, 3H), 3.5-3.7 (m, 4H), 2.92 (ddd, 1H, J=5.1, 8.8,
3.7 Hz), 2.3-2.5 (m, 1H), 2.25 (t, 2H), 2.2-2.3 (m, 1H), 2.0-2.1
1
obtained on a Cary 50 Bio UV-visible spectrophotometer. H,
1
3
19
C, and F Nuclear magnetic resonance (NMR) spectra were
obtained using a Varian INOVA nuclear magnetic resonance
spectrometer at 400, 100, and 376 MHz, respectively. High
performance liquid chromatography (HPLC) analytical
separations were performed on an Agilent 1100 or Agilent
1
CDCl
1
1
200 HPLC analytical system and followed by an Agilent
Technologies G1315B Diode Array Detector set at or near the
UVmax @ 210 nm. HPLC preparatory separations were
performed on a Gilson preparative HPLC system or an Agilent
(
(
(
m, 1H), 1.7-1.8 (m, 1H), 1.4-1.6 (m, 4H), 1.2-1.3 (m, 4H), 0.83
s, 9H), 0.00 (s, 6H).
R)-Methyl
7-(2-(hydroxymethyl)-5-oxopyrrolidin-1-
1
100 preparative HPLC system and followed by an Agilent
yl)heptanoate (15a). To a solution consisting of methyl (R)-7-
(2-(((tert-butyldimethylsilyl)oxy)methyl)-5-oxopyrrolidin-1-
yl)heptanoate (14a) (8.8 g, 24 mmol) in methanol (80 mL) was
added 1 N HCl (2 mL) and the mixture was stirred for 2.5 hours.
Additional 1 N HCl (1 mL) was added and the mixture was
stirred overnight. Triethylamine (0.56 mL) was added and the
mixture was concentrated under reduced pressure to produce
an oil which was subsequently chased with toluene. The
resulting residue was dissolved in a minimal volume of 4:1 v/v
ethyl acetate-heptane. Remaining solids were removed by
Technologies G1315B Diode Array Detector set at or near the
UVmax @ 210 nm. Analytical chiral HPLC separations were
performed on an Agilent 1100 analytical system and followed
by an Agilent Technologies G1315B Diode Array Detector set at
or near the UVmax @ 210 nm. The separations were
accomplished with a Gemini 3 or 5 C18 50x2.5 mm or
250x4.6 mm solid phase column eluting with acetic acid-
methanol-water gradient or ammonium acetate-acetonitrile-
water gradient. All final compounds gave satisfactory purity (≥
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Journal of Medicinal Chemistry
1
2
filtration and the filtrate was applied to a silica column. Elution
0.426 mmol) was added and the reaction mixture stirred for
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
stepwise with 7:3 v/v ethyl acetate-heptane, 100% ethyl
acetate, and continued with a gradient (98:2 to 9:1 v/v ethyl
acetate-methanol) afforded the title compound (4.05 g, 66%);
one additional hour while maintaining at -78 °C. Acetone was
added and the cold mixture continued stirring for five minutes
before removing the cold bath. The room temperature reaction
mixture was treated with saturated aqueous ammonium
chloride. The aqueous phase was made acidic with 1 N HCl and
was extracted with ethyl acetate (100 mL). The organic layer
was separated and washed sequentially with water and brine,
was dried over anhydrous magnesium sulfate, filtered, and
concentrated under reduced pressure to provide a crude
residue. HPLC (Luna 5 silica 250x4.6 mm, ethanol-heptane
10:90) of the crude product indicated a diastereomeric ratio of
approximately 73:27 R/S at C15-OH. The residue was purified
by silica gel chromatography. Elution with 2-propanol-heptane
10:90 to 17:83 v/v afforded the title compound (16.8 mg, 21%).
TLC R
f
0.24 (solvent system: ethyl acetate-methanol 97:3 v/v);
1
H NMR (400 MHz, CDCl ) δ 3.7-3.8 (m, 1H), 3.6-3.7 (m, 1H),
3
3
2
2
.65 (s, 3H), 3.5-3.6 (m, 2H), 2.9-3.0 (m, 1H), 2.71 (br s, 1H), 2.4-
.6 (m, 1H), 2.3-2.4 (m, 1H), 2.29 (t, 2H), 2.0-2.2 (m, 1H), 1.9-
.0 (m, 1H), 1.5-1.7 (m, 3H), 1.4-1.5 (m, 1H), 1.2-1.4 (m, 4H); MS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
(
APCI ) m/z 258 (M+H).
Methyl (R)-7-(2-formyl-5-oxopyrrolidin-1-yl)heptanoate
(16a). To solution consisting of methyl (R)-7-(2-
a
(hydroxymethyl)-5-oxopyrrolidin-1-yl)heptanoate (15a) (4.0
g, 16 mmol) in dichloromethane (30 mL) at room temperature
was added Dess-Martin periodinate (7.2 g, 17.1 mmol) in small
portions. The reaction mixture was additionally stirred for 30
minutes and was subsequently concentrated under reduced
pressure to provide a crude residue. The residue was
suspended in diethyl ether and filtered through a pad of Celite.
The solids were washed copiously with diethyl ether and the
filtrate was concentrated under reduced pressure. The
resulting residue was passed through a short plug of silica gel
flushing stepwise with 100% diethyl ether, 4:1, 9:1, and 95:5
v/v diethyl ether-methanol while collecting fractions. Select
fractions were combined and concentrated to afford the title
compound as a white semisolid (3.8 g, 94%). The product was
carried on rapidly to the next step used to prepare
TLC R 0.36 (solvent system: 2-propanol-heptane 30:70 v/v);
f
1
H NMR (400 MHz, CD OD) δ 5.75 (dd, 1H, J=7.0, 15.2 Hz), 5.54
3
(dd, 1H, J=8.6, 15.6 Hz), 4.18 (q, 1H, J=7.4 Hz), 4.01 (t, 1H, J=6.8
Hz), 3.64 (s, 3H), 3.4-3.5 (m, 1H), 2.9-3.0 (m, 1H), 2.2-2.4 (m,
6H), 2.1-2.2 (m, 3H), 1.7-1.8 (m, 2H), 1.4-1.6 (m, 4H), 1.2-1.4
+
(m, 5H), 1.09 (t, 3H, J=7.4 Hz), 0.95 (d, 3H, J=6.6 Hz); MS (ESI )
m/z 378.3 (M+H).
Methyl 7-((R)-2-((3R,4S,E)-3-hydroxy-4-methylnon-1-en-
6-yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoate. TLC R 0.45
f
1
(solvent system: 2-propanol-heptane 30:70 v/v); H NMR (400
MHz, CD OD) δ 5.77 (dd, 1H, J=5.7, 15.4 Hz), 5.55 (dd, 1H, J=8.8,
3
15.4 Hz), 4.19 (q, 1H, J=7.6 Hz), 4.11 (t, 1H, J=4.9 Hz), 3.64 (s,
intermediate 18a. TLC R
f
0.24 (solvent system: 2-propanol-
3H), 3.4-3.5 (m, 1H), 2.95 (ddd, 1H, J=5.3, 8.4, 13.7 Hz), 2.2-2.4
(m, 6H), 2.14 (q, 2H, J=7.6 Hz), 2.00 (br dd, 1H, J=7.4, 16.4 Hz),
1.7-1.8 (m, 2H), 1.4-1.6 (m, 4H), 1.2-1.4 (m, 5H), 1.09 (t, 3H,
heptane 15:85 v/v).
Methyl
7-((R)-2-((S,E)-4-methyl-3-oxonon-1-en-6-yn-1-
solution
+
yl)-5-oxopyrrolidin-1-yl)heptanoate (18a).
A
J=7.4 Hz), 0.98 (d, 3H, J=6.6 Hz); MS (ESI ) m/z 378.3 (M+H).
consisting of (S)-(+)-dimethyl (3-methyl-2-oxooct-5-yn-1-
yl)phosphonate (17) (3.5 g, 14 mmol), lithium chloride (2.1 g,
7-((R)-2-((3S,4S,E)-3-Hydroxy-4-methylnon-1-en-6-yn-1-
yl)-5-oxopyrrolidin-1-yl)heptanoic acid (KMN-80, 7). To a
solution consisting of methyl 7-((R)-2-((3S,4S,E)-3-hydroxy-4-
methylnon-1-en-6-yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoate
(19a) (0.22 g, 0.58 mmol) in methanol (6 mL) at room
temperature was added 2 N NaOH (1.2 mL) and the mixture
was stirred overnight. The reaction mixture was poured into a
solution consisting of 1 N HCl and saturated ammonium
chloride. The organic material was twice extracted with ethyl
acetate and the organic phase was twice washed with brine-
water (1:1 v/v) and once with brine. The organic phase was
dried over anhydrous magnesium sulfate, filtered, and
concentrated under reduced pressure to provide a residue. The
residue was crystallized from ethyl acetate-heptane 1:1 v/v to
afford the title compound as a colorless solid (0.157 g, 74%);
5
0 mmol) and triethylamine (3.1 mL, 23 mmol) in THF (250
mL) was stirred at 0 °C for 15 minutes. To the reaction mixture
was added a solution consisting of methyl (R)-7-(2-formyl-5-
oxopyrrolidin-1-yl)heptanoate (16a) (3.8 g, 15 mmol) in THF
(100 mL). The reaction mixture was allowed to warm to room
temperature overnight, after which time it was quenched with
a solution consisting of saturated ammonium chloride (200
mL) and 6 N HCl (2 mL) and extracted with diethyl ether. The
organic phase was washed sequentially with water and brine,
and was dried over anhydrous magnesium sulfate, filtered, and
concentrated under reduced pressure to provide a crude
yellow oil. The crude product was purified by silica gel
chromatography. Elution with a gradient (ethyl acetate-
heptane 7:3 to 9:1 v/v) afforded the title compound as clear oil
melting point 142.5-143.5 °C; TLC R 0.17 (solvent system:
f
1
(
3.14 g, 57%); TLC R
f
0.33 (solvent system: ethyl acetate-
heptane 4:1 v/v); H NMR (400 MHz, CD OD) δ 6.73 (dd, 1H,
J=8.4, 15.8 Hz), 6.41 (d, 1H, J=15.6 Hz), 4.3-4.4 (m, 1H), 3.64 (s,
H), 3.4-3.5 (m, 1H), 2.95-3.05 (m, 1H), 2.93 (dt, 1H, J=6.7, 13.9
Hz), 2.2-2.5 (m, 7H), 2.0-2.1 (m, 2H), 1.8-1.9 (m, 1H), 1.4-1.7 (m,
H), 1.3-1.4 (m, 4H), 1.15 (d, 3H, J=6.6 Hz), 1.06 (t, 3H, J=7.4Hz);
ethyl acetate-heptane-acetic acid 80:20:1 v/v); H NMR (400
1
3
MHz, CD OD) δ 5.75 (dd, 1H, J=6.96, 15.38 Hz), 5.54 (dd, 1H,
3
J=8.79, 15.38 Hz), 4.15-4.22 (m, 1H), 4.02 (t, 1H, J=6.77 Hz),
3
3.42-3.51 (m, 1H), 2.94 (ddd, 1H, J=5.49, 8.70, 13.64 Hz), 2.34-
2.41 (m, 2H), 2.19-2.32 (m, 4H), 2.08-2.18 (m, 3H), 1.67-1.82
(m, 2H), 1.51-1.63 (m, 3H), 1.24-1.51 (m, 6H), 1.09 (t, 3H, J=7.51
4
+
1
MS (ESI ) m/z 376.2 (M+H).
Hz), 0.96 (d, 3H, J=6.96 Hz); H NMR (400 MHz, CDCl ) δ 5.71
3
Methyl 7-((R)-2-((3S,4S,E)-3-hydroxy-4-methylnon-1-en-
(dd, 1H, J=6.54, 15.33 Hz), 5.57 (dd, 1H, J=8.42, 15.37 Hz), 4.28-
5.29 (br, 2H), 4.04-4.15 (m, 2H), 3.54 (td, 1H, J=7.81, 13.93 Hz),
2.89 (ddd, 1H, J=5.40, 8.46, 13.68 Hz), 2.31-2.50 (m, 4H), 2.12-
2.29 (m, 4H), 1.70-1.85 (m, 2H), 1.58-1.69 (m, 2H), 1.23-1.58
6
-yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (19a). To a
mixture consisting of methyl 7-((R)-2-((S,E)-4-methyl-3-
oxonon-1-en-6-yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoate
+
(
18a) (80 mg, 0.213 mmol) in methanol (3 mL) at -40 °C was
(m, 6H), 1.09-1.17 (m, 3H), 0.98 (d, 3H, J=6.77 Hz); MS (ESI )
-
-
added cerium (III) chloride heptahydrate (79 mg, 0.213 mmol).
The reaction mixture was stirred until all the cerium (III)
chloride heptahydrate was dissolved (20 minutes) then cooled
to -78 °C and stirred for 15 min. Sodium borohydride (16 mg,
m/z 364.2 (M+H), (ESI ) 362.2 (M-H); HRMS (ESI ): m/z
+
calculated for C H NO – H [M-H]: 362.2337, found
362.2342; specific rotation: []
2
1
33
4
21.9
D
= +0.068/(0.01)(0.5) =
+13.6°; HPLC Gemini 3 C18 50x2.5 mm, methanol-water-
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Journal of Medicinal Chemistry
Page 8 of 20
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2
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1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
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5
5
5
6
acetic acid 90:10:0.1 to 10:90:0.1 v/v, 0.4 mL/min, 210 nm,
9.4%.
and washed with brine (150 mL). The organic phase was dried
9
over anhydrous sodium sulfate, filtered, and concentrated
under reduced pressure to provide a residue. The residue was
purified by silica gel chromatography. Elution with
dichloromethane-methanol 97:3 v/v afforded the title
Synthesis
methylnon-1-en-6-yn-1-yl)-2-oxopyrrolidin-1-yl)heptanoic acid
KMN-159, 8)
R)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-3,3-
difluoropyrrolidin-2-one (13b). To a solution consisting of
of
7-((R)-3,3-difluoro-5-((3S,4S,E)-3-hydroxy-4-
(
(
compound as a white solid (7.9 g, 81 %); TLC R
f
0.30 (solvent
system: 2-propanol-heptane 1:4 v/v); H NMR (400 MHz,
CDCl ) δ 3.72-3.85 (m, 2H), 3.59-3.72 (m, 5H), 3.13 (ddd, 1H,
J=5.22, 8.71, 13.79 Hz), 2.36-2.71 (m, 3H), 2.29 (t, 2H, J=7.41
1
3
2
6
(
(
R)-3,3-difluoro-5-(hydroxymethyl)pyrrolidin-2-one (12b )
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
0.880 g, 5.82 mmol) in DMF (10 mL) and THF (10 mL) was
Hz), 1.44-1.70 (m, 4H), 1.21-1.40 (m, 4H); F NMR (376 MHz,
CDCl ) δ -102.63 (dt, J=271, 15 Hz), -103.46 (dt, J=271, 19 Hz);
F NMR (376 MHz, CD OD) δ -103.73 (dt, J=271, 15 Hz), -
added tert-butyldimethylchlorosilane (1.40 g, 9.23 mmol)
followed by imidazole (0.800 g, 6.55 mmol). The reaction
mixture was stirred at room temperature for 16 hours and was
subsequently diluted with water (10 mL) and extracted thrice
with ethyl acetate (55 mL, 2x25 mL). The combined organics
were washed with water-brine 1:1 v/v (3x10 mL) and brine (5
mL) and were then dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure to provide a
crude residue. The residue was purified by silica gel
chromatography. Elution with dichloromethane-methanol
50:1 v/v afforded the title intermediate as a clear oil (1.5 g,
3
19
3
1
3
3
104.60 (dt, J=271, 15 Hz); C NMR (100 MHz, CD OD) δ 174.5,
164.8 (t, J=31 Hz), 118.4 (t, J=250 Hz), 59.8, 54.0 (t, J=4 Hz),
50.5, 40.9, 33.2, 31.2 (t, J=23 Hz), 28.2, 26.0, 25.9, 24.4; MS
+
(ESI ) m/z 294.1 (M+H), 311.2 (M+H
2
O).
Methyl
(R)-7-(3,3-difluoro-5-formyl-2-oxopyrrolidin-1-
yl)heptanoate (16b). To a solution consisting of (R)-methyl 7-
(3,3-difluoro-5-(hydroxymethyl)-2-oxopyrrolidin-1-
yl)heptanoate (15b) (0.085 g, 0.29 mmol) in dichloromethane
(10 mL) was added Dess-Martin periodinate (0.15 g, 0.35
mmol), and the reaction mixture was stirred for 4 hours. The
reaction mixture was filtered, and the filtrate concentrated
under reduced pressure to provide a residue that was purified
by silica gel chromatography. Elution with dichloromethane-
methanol 200:1 v/v afforded the title intermediate as a pale-
yellow oil (0.077 g, 91%), which was used immediately to
f
99%); TLC R 0.60 (solvent system: dichloromethane-methanol
1
9
1
2
9
1
2
3
5:5 v/v); H NMR (400 MHz, CDCl ) δ 6.91 (br s, 1H), 3.74 (m,
H), 3.63 (dd, 1H, J=4.2, 10.4 Hz), 3.45 (dd, 1H, J=7.0, 10.3 Hz),
.52-2.67 (m, 1H), 2.19 (dtd, 1H, J=4.9, 15.1, 16.8 Hz), 0.82 (s,
H), 0.00 (2s, 6H); H NMR (400 MHz, CD
1
3
OD) δ 3.75-3.83 (m,
H), 3.67-3.73 (m, 1H), 3.57-3.64 (m, 1H), 2.53-2.70 (m, 1H),
.33-2.49 (m, 1H), 0.86-0.94 (s, 9H), 0.08 (2s, 6H); 19F NMR
prepare compound 18b; TLC R
dichloromethane-methanol 93:7 v/v).
Methyl 7-((R)-3,3-difluoro-5-((S,E)-4-methyl-3-oxonon-1-
en-6-yn-1-yl)-2-oxopyrrolidin-1-yl)heptanoate (18b). A
f
0.60 (solvent system:
3
(376 MHz, CDCl ) δ -103.77 (dt, J=271, 14 Hz) -104.88 (dt,
1
3
3
J=271, 18 Hz); C NMR (100 MHz, CDCl ) δ 65.45, 50.34, 32.92
+
(
t, J=23 Hz), 25.72, 18.16, -5.54; MS (ESI ) m/z 266.1 (M+H).
Methyl (R)-7-(5-(((tert-butyldimethylsilyl)oxy)methyl)-
,3-difluoro-2-oxopyrrolidin-1-yl)heptanoate (14b). To a
mixture consisting of (S)-(+)-dimethyl (3-methyl-2-oxooct-5-
yn-1-yl)phosphonate (17) (4.5 g, 18 mmol), methyl (R)-7-(3,3-
difluoro-5-formyl-2-oxopyrrolidin-1-yl)heptanoate (16b) (5.9
g, 20 mmol) and lithium chloride (2.8 g, 67 mmol) in THF (200
mL) was stirred at room temperature until the lithium chloride
was completely dissolved. The reaction mixture was cooled to
0 °C and triethylamine (4.25 mL, 30.5 mmol) in THF (17 mL)
was added dropwise. The reaction mixture was allowed to
reach room temperature overnight, after which time saturated
aqueous ammonium chloride was added. The mixture was
extracted three times with ethyl acetate and the combined
organic phase was dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure to provide a
residue. The residue was purified by silica gel chromatography.
Elution with a gradient (heptane-ethyl acetate 4:1 to 7:3 v/v)
3
suspension consisting of sodium hydride (60% dispersion in
oil, 1.99 g, 49.5 mmol) and sodium iodide (8.16 g, 54.5 mmol)
in dimethylformamide (100 mL) at 0 °C was added (R)-5-
(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluoropyrrolidin-
2-one (13b) (13.1 g, 49.5 mmol). The reaction mixture was
allowed to warm to room temperature with stirring over one
hour and was then heated at 50 °C for 30 minutes. Methyl 7-
bromoheptanoate (13.2 g, 59.4 mmol) was added to the
reaction mixture, which was stirred and maintained at 50 °C
overnight. The mixture was diluted with ethyl acetate (200
mL), washed with 1 N HCl (200 mL) and 5% aqueous sodium
thiosulfate (200 mL) and brine. The organic phase was dried
over anhydrous sodium sulfate, filtered, and concentrated
under reduced pressure to provide a residue. The residue was
purified by silica gel chromatography. Elution with 2-propanol-
1
afforded the title compound (4.5 g, 55%); H NMR (400 MHz,
3
CD OD) δ 6.6-6.7 (dd, 1H), 6.5-6.6 (d, 1H), 4.47 (tt, 1H, J=3.9, 8.0
heptane 5:95 v/v afforded the title compound (13.6 g, 67%);
Hz), 3.63 (s, 3H), 3.5-3.6 (m, 1H), 3.07 (dddd, 1H, J=2.0, 5.9, 8.1,
13.8 Hz), 3.0-3.0 (m, 1H), 2.85 (dddd, 1H, J=7.8, 13.7, 14.8, 17.2
Hz), 2.4-2.5 (m, 2H), 2.2-2.3 (m, 3H), 2.1-2.1 (m, 1H), 1.5-1.7 (m,
4H), 1.3-1.4 (m, 5H), 1.16 (d, 3H, J=7.0 Hz), 1.06 (t, 3H, J=7.6
1
TLC R
f
0.41 (solvent system: 2-propanol-heptane 1:9 v/v); H
NMR (400 MHz, CDCl
3
) δ 3.7-3.8 (m, 2H), 3.67 (s, 3H), 3.6-3.7
m, 1H), 3.1-3.2 (m, 1H), 2.5-2.6 (m, 1H), 2.3-2.4 (m, 1H), 2.31
t, 2H, J=7.4 Hz), 1.5-1.7 (m, 5H), 1.3-1.4 (m, 4H), 0.89 (s, 9H),
(
(
19
3
Hz); F NMR (376 MHz, CD OD) δ -104.33 (dt, J=271, 15 Hz), -
+
+
0.07 (s, 6H); MS (ESI ) m/z 408.2 (M+H).
107.07 (ddd, J=274, 19, 15 Hz); MS (ESI ) m/z 412.2 (M+H)
-
Methyl
(R)-7-(3,3-difluoro-5-(hydroxymethyl)-2-
429.3 (M+OH), (ESI ) m/z 410.2 (M-H).
oxopyrrolidin-1-yl)heptanoate (15b). To
a
solution
(R)-7-(5-(((tert-
butyldimethylsilyl)oxy)methyl)-3,3-difluoro-2-oxopyrrolidin-
-yl)heptanoate (14b) (13.6 g, 33.4 mmol) in methanol (100
Methyl
7-((R)-3,3-difluoro-5-((3S,4S,E)-3-hydroxy-4-
consisting
of
methyl
methylnon-1-en-6-yn-1-yl)-2-oxopyrrolidin-1-
yl)heptanoate (19b). To a mixture consisting of methyl 7-
((R)-3,3-difluoro-5-((S,E)-4-methyl-3-oxonon-1-en-6-yn-1-yl)-
2-oxopyrrolidin-1-yl)heptanoate (18b) (250 mg, 0.607 mmol)
in methanol (50 mL) at -40 °C was added cerium (III) chloride
heptahydrate (226 mg, 0.607 mmol). The reaction mixture was
cooled to -78 °C with stirring for 15 min, after which time
1
mL) at room temperature was added 1 N HCl (20 mL). The
reaction mixture was allowed to stir for 8 hours, after which
time it was concentrated under reduced pressure to about half
volume. The mixture was diluted with ethyl acetate (300 mL)
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Journal of Medicinal Chemistry
1
2
sodium borohydride (46 mg, 1.21 mmol) was added as stirring
Synthesis of 7-((R)-3,3-Difluoro-5-((S,E)-3-hydroxyoct-1-en-1-
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
continued for an additional two hours maintaining at -78 °C.
Acetone (3 mL) was added and the stirring mixture maintained
at -78 °C for 20 minutes. The mixture was allowed to warm to
room temperature and was treated with a saturated solution of
ammonium chloride (30 mL). The aqueous phase was made
acidic with 1N HCl and was extracted with ethyl acetate (100
mL). The organic phase was washed sequentially with water
then brine, dried over magnesium sulfate, filtered, and
concentrated under reduced pressure to provide a crude
residue (240 mg). HPLC (Luna 5 silica 250x4.6 mm, ethanol-
heptane 10:90) of the crude product indicated a diastereomeric
ratio of 73:27 R/S at C15-OH. The crude residue was purified
by silica gel chromatography. Elution with a stepwise gradient
of 2-propanol-heptane (1:99 to 6:94) afforded the title
yl)-2-oxopyrrolidin-1-yl)heptanoic acid (KMN-165, 6)
Compound 6 was synthesized from intermediate 16b using the
three-step procedure described for the synthesis of 8, except
dimethyl (2-oxoheptyl)phosphonate was used instead of
phosphonate intermediate 17.
Methyl
yl)pyrrolidin-1-yl)heptanoate. clear oil; H NMR (400 MHz,
CD OD) δ 6.61 (dd, 1H, J=8.8, 15.8 Hz), 6.39 (d, 1H, J=16.0 Hz),
(R,E)-7-(3,3-difluoro-2-oxo-5-(3-oxooct-1-en-1-
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4.4-4.5 (m, 1H), 3.64 (s, 3H), 3.5-3.6 (m, 1H), 3.06 (td, 1H, J=6.5,
13.5 Hz), 2.85 (dq, 1H, J=8.0, 15.2 Hz), 2.63 (t, 2H, J=7.2 Hz), 2.3-
2.5 (m, 1H), 2.30 (t, 2H, J=7.4 Hz), 1.5-1.6 (m, 6H), 1.2-1.4 (m,
1
9
3
8H), 0.90 (t, 3H, J=6.8 Hz); F NMR (376 MHz, CD OD) δ -
107.00 (dt, J=271, 15 Hz), -104.38 (dt, J=271, 15 Hz); MS (ESI+)
m/z 388.3 (M+1) 405.3 (M+OH).
Methyl 7-((R)-3,3-difluoro-5-((S,E)-3-hydroxyoct-1-en-1-
yl)-2-oxopyrrolidin-1-yl)heptanoate. HPLC (Luna 5 silica
250x4.6 mm, ethanol-heptane 10:90) of the crude product
compound (49.2 mg, 20%); TLC R
f
0.36 (solvent system: 2-
propanol-heptane 15:85 v/v); H NMR (400 MHz, CD OD) δ
.90 (dd, 1H, J=7.0, 15.2 Hz), 5.51 (dd, 1H, J=9.4, 15.2 Hz), 4.29
br s, 1H), 4.03 (t, 1H, J=6.6 Hz), 3.64 (s, 3H), 3.5-3.6 (m, 1H),
.0-3.1 (m, 1H), 2.7-2.9 (m, 1H), 2.31 (br t, 2H), 2.2-2.4 (m, 2H),
2.1-2.2 (m, 3H), 1.5-1.8 (m, 5H), 1.2-1.4 (m, 4H), 1.09 (t, 3H,
1
3
5
(
3
indicated a diastereomeric ratio of 68:32 R/S at C15-OH. TLC R
f
1
0.38 (solvent system: 2-propanol-heptane 15:85 v/v); H NMR
(400 MHz, CD OD) δ 5.89 (dd, 1H, J=6.1, 15.4 Hz), 5.47 (dd, 1H,
3
1
9
J=7.4 Hz), 0.96 (d, 3H, J=7.0 Hz); F NMR (376 MHz, CD
3
OD) δ
J=9.4, 14.8 Hz), 4.27 (br s, 1H), 4.09 (q, 1H, J=6.4 Hz), 3.64 (s,
3H), 3.5-3.6 (m, 1H), 3.06 (td, 1H, J=6.4, 13.4 Hz), 2.7-2.9 (m,
1H), 2.2-2.3 (m, 3H), 1.4-1.7 (m, 7H), 1.32 (br s, 10H), 0.9-0.9
+
-
104.90 (dt, J=271, 16 Hz), -107.09 (dt, J=271, 17 Hz); MS (ESI )
-
m/z 414.1 (M+H), (ESI ) m/z 412.1(M-H).
19
Methyl
7-((R)-3,3-difluoro-5-((3R,4S,E)-3-hydroxy-4-
3
(m, 3H); F NMR (376 MHz, CD OD) δ -104.93 (dt, J=271, 13
methylnon-1-en-6-yn-1-yl)-2-oxopyrrolidin-1-
Hz), -107.04 (dt, J=271, 16 Hz); MS (ESI+) m/z 390.3 (M+1)
407.3 (M+OH).
Methyl 7-((R)-3,3-difluoro-5-((R,E)-3-hydroxyoct-1-en-1-
yl)heptanoate. TLC R
heptane 15:85 v/v); H NMR (400 MHz, CD OD) δ 5.93 (dd, 1H,
f
0.43 (solvent system: 2-propanol-
1
3
J=5.5, 15.2 Hz), 5.51 (dd, 1H, J=9.8, 16.0 Hz), 4.30 (br s, 1H), 4.15
(t, 1H, J=4.9 Hz), 3.64 (s, 3H), 3.5-3.6 (m, 1H), 3.09 (td, 1H, J=6.6,
yl)-2-oxopyrrolidin-1-yl)heptanoate. TLC R
f
0.40 (solvent
system: 2-propanol-heptane 15:85 v/v); H NMR (400 MHz,
CD OD) δ 5.92 (dd, 1H, J=5.5, 15.2 Hz), 5.4-5.5 (m, 1H), 4.2-4.3
1
1
2
1
3.0 Hz), 2.7-2.9 (m, 1H), 2.31 (br t, 2H, J=7.4 Hz), 1.9 – 2.4 (m,
H), 2.14 (br d, 2H, J=7.4 Hz), 2.02 (br dd, 1H, J=7.4, 16.4 Hz),
.7-1.7 (m, 1H), 1.5-1.7 (m, 4H), 1.2-1.4 (m, 5H), 1.09 (t, 3H,
3
(m, 1H), 4.09 (q, 1H, J=5.7 Hz), 3.64 (s, 3H), 3.5-3.6 (m, 1H), 3.1-
3.1 (m, 1H), 2.7-2.9 (m, 1H), 2.2-2.3 (m, 3H), 1.4-1.7 (m, 7H),
1
9
J=7.4 Hz), 0.98 (d, 3H, J=6.6 Hz),
1.32 (br s, 10H), 0.91 (br t, 3H, J=6.4 Hz); F NMR (376 MHz,
CD OD) δ -104.95 (dt, J=267, 15 Hz), -107.03 (dt, J=271, 15 Hz);
7
1
-((R)-3,3-Difluoro-5-((3S,4S,E)-3-hydroxy-4-methylnon-
-en-6-yn-1-yl)-2-oxopyrrolidin-1-yl)heptanoic acid
3
MS (ESI+) m/z 390.2 (M+1) 407.3 (M+OH).
7-((R)-3,3-Difluoro-5-((S,E)-3-hydroxyoct-1-en-1-yl)-2-
(KMN-159, 8). To a mixture consisting of methyl 7-((R)-3,3-
difluoro-5-((3S,4S,E)-3-hydroxy-4-methylnon-1-en-6-yn-1-
yl)-2-oxopyrrolidin-1-yl)heptanoate (19b) (6.5 g, 16 mmol) in
methanol (325 mL) at room temperature was added 1 N NaOH
oxopyrrolidin-1-yl)heptanoic acid (KMN-165, 6). white
1
solid; H NMR (400 MHz, CDCl
3
) δ 5.85 (dd, 1H, J=5.8, 15.4 Hz),
5.49 (dd, 1H, J=9.1, 15.1 Hz), 4.20 (q, 1H, J=6.2 Hz), 4.0-4.2 (m,
1H), 3.5-3.6 (m, 1H), 2.9-3.1 (m, 1H), 2.6-2.8 (m, 1H), 2.35 (t,
2H, J=7.2 Hz), 2.2-2.3 (m, 1H), 1.2-1.7 (m, 17H), 0.8-1.0 (m, 3H);
(
120 mL). The reaction mixture was stirred at room
temperature for one hour and was cooled and maintained at 0
C for 18 hours. The reaction was slowly and carefully
neutralized with 3 N HCl (120 mL) then diluted with water
700 mL). The resulting precipitate was filtered and dried to
afford the title compound as a white solid (5.0 g, 80%); melting
point 144-146 °C; TLC 0.50 (solvent system:
dichloromethane-methanol-acetic acid 94:5:1 v/v); H NMR
400 MHz, CDCl ) δ 5.83 (dd, 1H, J=6.6, 15.2 Hz), 5.53 (dd, 1H,
J=9.0, 15.2 Hz), 4.1-4.2 (m, 2H), 3.5-3.6 (m, 1H), 2.9-3.0 (m, 1H),
1
9
°
3
F NMR (376 MHz, CDCl ) δ -103.61 (dt, J=267, 15 Hz), -105.61
+
+
(dt, J=271, 15 Hz);MS (ESI ) m/z 398.2 (M+Na ), 358.2 (M-OH),
(ESI ) m/z 374.1 (M-H); HRMS (ESI ): m/z calculated for
C H F NO + H [M+H]: 376.2294, found 376.2297; HPLC
19 31 2 4
-
+
(
+
R
f
Gemini 3 C18 50x2.5 mm, methanol-water-acetic acid
90:10:0.1 to 10:90:0.1 v/v, 0.4 mL/min, 210 nm, 100.0%.
1
(
3
ASSOCIATED CONTENT
Supporting Information.
This material is available free of charge via the Internet at
http://pubs.acs.org.
2
1
.6-2.8 (m, 1H), 2.33 (t, 2H, J=7.2 Hz), 2.1-2.3 (m, 5H), 1.79 (td,
H, J=6.5, 12.7 Hz), 1.4-1.7 (m, 4H), 1.2-1.4 (m, 4H), 1.11 (t, 3H,
19
J=7.4 Hz), 0.97 (d, 3H, J=7.0 Hz); F NMR (376 MHz, CDCl
3
) δ -
103.57 (dt, J=271, 15 Hz), -105.58 (dt, J=271, 15 Hz); C NMR
(100 MHz, CDCl ) δ 178.7, 163.5 (t, J=30 Hz), 137.3, 129.0,
17.5 (t, J=247 Hz), 84.0, 76.9, 74.9, 55.0, 41.1, 38.1, 36.3 (t,
J=21 Hz), 33.7, 28.4, 26.5, 26.2, 24.3, 22.1, 15.7, 14.2, 12.4; MS
1
3
Binding affinities to human prostanoid receptor subtypes,
additional drug-like profile measurements, docked pose of
KMN-165 (6), linear regression of MM-GBSA scores versus
EP K , MacroModel conformation search results and MD
3
1
3
i
+
-
(
ESI ) m/z 398.2 (M+H); HRMS (ESI ): m/z calculated for
simulation of KMN-80 (7) and KMN-159 (8), comparison
of -lactam dihedrals with CCDC reference molecules, E-
ring crystallographic bond lengths, biological assay
+
21 31 2 4
C H F NO – H [M-H]: 398.21484, found 398.21573; specific
2
1.9
rotation: []
D
= +0.17/(0.810)(0.5) = +4.19°; HPLC Gemini
5
1
 C18 250x4.6 mm, methanol-water-acetic acid 70:30:0.1 v/v,
mL/min, 210nm, 99.3%.
procedures,
x-ray
crystallographic
procedures,
physiochemical procedures, DMPK procedures, safety
assessment procedures, docked poses (.PDB), NBO partial
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charge predictions and MM-GBSA ΔG values (.xlsx) and
Molecular formula strings (CSV).
(1) Markovic, T.; Jakopin, Z.; Dolenc, M. S.; Mlinaric-Rascan, I. Structural
features of subtype-selective EP receptor modulators. Drug Discovery
Today 2017, 22, 57-71.
(
2) Konya, V.; Marsche, G.; Schuligoi, R.; Heinemann, A. E-type
Accession Codes
prostanoid receptor 4 (EP4) in disease and therapy. Pharmacology &
Therapeutics 2013, 138, 485-502.
(3) Spisani, S.; Vicenzi, E.; Traniello, S.; Pollini, G. P.; Barco, A. Synthetic
prostaglandin E1 analog: in vitro studies on human neutrophils.
Immunopharmacology 1982, 4, 323-330.
Coordinates and structure factors have been deposited within
the Cambridge Crystallographic Data Center: deposition codes
CCDC-1877890 and CCDC-1877891. The authors will release
the atomic coordinates upon article publication.
(
4) Zoretic, P. A.; Branchaud, B.; Sinha, N. D. Synthesis of 11-deoxy-8-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
azaprostaglandin E1. Journal of Organic Chemistry 1977, 42, 3201-
3203.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: sbarrett@caymanchem.com. Phone: (734)975-3986.
E-mail: mcholt@caymanchem.com. Phone: (734)929-1647.
(
5) Bruin, J. W.; De Koning, H.; Huisman, H. O. Synthesis of 8-
azaprostaglandin E1 and E2. Tetrahedron Letters 1975, 1975, 4599-
602.
#
4
ORCID
(6) Maruyama, T.; Asada, M.; Shiraishi, T.; Sakata, K.; Seki, A.; Yoshida,
H.; Shinagawa, Y.; Maruyama, T.; Ohuchida, S.; Nakai, H.; Kondo, K.;
Toda, M. Design and synthesis of a highly selective EP4-receptor
agonist. Part 2: 5-thia and 9β-haloPG derivatives with improved
stability. Bioorganic & Medicinal Chemistry Letters 2001, 11, 2033-
2035.
Stephen D. Barrett: 0000-0001-7003-0051
Melissa C. Holt: 0000-0002-7883-924X
Author Contributions
⊥
SDB and MCH contributed equally. The manuscript was
written by SDB, MCH, JBK, and MIM along with contributions of
all authors. SDB, JBK, BG, FLC, AK, JMC, and AU contributed to
the organic synthesis. Synthetic enablement was enhanced by
SDB, JBK, BG, AK, JMC, and AU. Difluoromethylene conception
was made by FLC and AK. Conception of the acetylene was
made by AU and SDB. MCH conducted QM, MM, docking, and
interpreted crystallographic results with guidance from AJS.
JPO and TAO provided biological context. CSH, and MIM
conducted biological studies. MIM oversaw DMPK studies. SDB,
AJS and MIM oversaw this work. All authors have given
approval to the final version of the manuscript.
(
7) Young, R. N.; Billot, X.; Han, Y.; Slipetz, D. A.; Chauret, N.; Belley, M.;
Metters, K.; Mathieu, M. C.; Greig, G. M.; Denis, D.; Girard, M. Discovery
and synthesis of a potent, selective and orally bioavailable EP4 receptor
agonist. Heterocycles 2004, 64, 437-446.
(8) Watanabe, Y.; Murata, T.; Amakawa, M.; Miyake, Y.; Handa, T.;
Konishi, K.; Matsumura, Y.; Tanaka, T.; Takeuchi, K. KAG-308, a newly-
identified EP4-selective agonist shows efficacy for treating ulcerative
colitis and can bring about lower risk of colorectal carcinogenesis by
oral administration. European Journal of Pharmacology 2015, 754,
1
79-189.
(
9) Kambe, T.; Maruyama, T.; Nakai, Y.; Yoshida, H.; Oida, H.; Maruyama,
T.; Abe, N.; Nishiura, A.; Nakai, H.; Toda, M. Discovery of novel
prostaglandin analogs as potent and selective EP2/EP4 dual agonists.
Bioorganic & Medicinal Chemistry 2012, 20, 2235-2251.
(10) Akram, A.; Gibson, C. L.; Grubb, B. D. Neuroprotection mediated by
the EP4 receptor avoids the detrimental side effects of COX-2 inhibitors
following ischaemic injury. Neuropharmacology 2013, 65, 165-172.
(11) DeMars, K. M.; McCrea, A. O.; Siwarski, D. M.; Sanz, B. D.; Yang, C.;
Candelario-Jalil, E. Protective effects of L-902,688, a prostanoid EP4
receptor agonist, against acute blood-brain barrier damage in
experimental ischemic stroke. Frontiers in Neuroscience 2018, 12, 89.
Notes
Authors SDB, MCH, JBK, BG, CSH, FLC, AK, JMC, AU, AJS, MIM
are employees of Cayman Chemical Company, Inc.
ACKNOWLEDGMENT
We thank Richard Staples for x-ray crystallographic data
studies. We thank Paige Heiple and Kirk Olson for significant
contributions. We thank Kirk Maxey for continued guidance
and support. All funds were R&D expenditures of Cayman
Chemical Company, Inc.
(
12) Luu, K. T.; Zhang, E. Y.; Prasanna, G.; Xiang, C.; Anderson, S.;
Fortner, J.; Vicini, P. Pharmacokinetic-pharmacodynamic and response
sensitization modeling of the intraocular pressure-lowering effect of
the EP4 agonist 5-{3-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)
phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl}thiophene-2-carboxylate
(PF-04475270). Journal of Pharmacology and Experimental
Therapeutics 2009, 331, 627-635.
ABBREVIATIONS
PGE , Prostaglandin E ; EP1-4, E-type prostanoid receptor
2 2
subtypes 1-4; PGE, Prostaglandin type E class molecules;
DMPK, Drug Metabolism and Pharmacokinetics; HEW, Horner-
Emmons-Wadsworth; TBDMS, Tert-Butyldimethylsilyl; DMF,
N,N-Dimethylformamide; THF, Tetrahydrofuran; MeOH,
(
13) Bushueva, G. I.; Batrameeva, L. A.; Manukina, T. A.; Dikovskaia, K.
I. Biological properties of 11-deoxyprostaglandin E1. Problemy
Endokrinologii 1982, 28, 63-68.
(14) Leduc, M.; Breton, B.; Galés, C.; Le Gouill, C.; Bouvier, M.; Chemtob,
S.; Heveker, N. Functional selectivity of natural and synthetic
prostaglandin EP4 receptor ligands. Journal of Pharmacology and
Experimental Therapeutics 2009, 331, 297-307.
1
Methanol; FP, Prostaglandin F2α receptor; DP , Prostaglandin
D2 receptor subtype 1; IP, Prostaglandin I2 receptor; SAR,
Structure activity relationship; cAMP, cyclic 3,5-adenosine
monophosphate; CREB, cAMP-responsive element-binding
protein; HEK-293, Human embryonic kidney 293; ANOVA,
Analysis of variance; SEM, Standard error of the mean; PDB,
Protein data bank; IFD, Induced fit docking; MM-GBSA,
Molecular-Mechanics with generalized Born and Surface Area
Solvation; QM, Quantum Mechanical; MD, Molecular dynamics;
CCDC, Cambridge crystallographic data center; Caco-2, human
epithelial colorectal adenocarcinoma cells; Papp, apparent
permeability; MDR1-MDCK, MDR1 gene (encoding P-gp
protein) transfected Madin Darby canine kidney cells; CYP,
cytochrome p450; hERG, human Ether-à-go-go-Related Kv11.1
ion channel; BBB, Blood brain barrier.
(15) Saijo, S.; Wada, M.; Himizu, J.; Ishida, A. Heterocyclic
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V.
Synthesis
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(12R,15S)-(-)-11-deoxy-8-
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16) Vicenzi, E.; Spisani, S.; Traniello, S. Effects of natural and synthetic
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Bollettino della Societa Italiana di Biologia Sperimentale 1980, 56,
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17) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N.
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(
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chemistry. Journal of Enzyme Inhibition and Medicinal Chemistry 2007,
2, 527-540.
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32) Hollenstein, K. Structures shed light on prostanoid signaling.
Nature Chemical Biology 2019, 15, 3-5.
33) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.;
(49) Foloppe, N.; Chen, I. J. Towards understanding the unbound state
of drug compounds: Implications for the intramolecular reorganization
energy upon binding. Bioorganic & Medicinal Chemistry 2016, 24,
2159-2189.
(
Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw,
D. E.; Francis, P.; Shenkin, P. S. Glide:ꢀ a new approach for rapid, accurate
docking and scoring. 1. Method and assessment of docking accuracy.
Journal of Medicinal Chemistry 2004, 47, 1739-1749.
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Figure 1. Prostaglandin and γ-lactam E ring-scaffolds and numbering.
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Figure 2. Published PG and γ-lactam EP4 receptor-selective agonists.
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Figure 3. γ-Lactam analog nonfluoro-difluoro pairs.
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Figure 4. Crystal structure of PGE2 (green stick & ball representation) bound to EP3 receptor, residues
shown in line representation in orange (PDB: 6AK3, ligand ID: P2E).30 (A) Docked pose of 7 (gray stick &
ball representation) in EP3 receptor overlaid overtop of PGE . (B) Docked pose of 8 (black stick & ball
2
representation) overlaid on top of PGE2 (green stick & ball representation). Binding site alignment with
human EP4 receptor homology model performed with aligning residues shown in line representation in blue.
Residues numbered with one letter amino acid code and sequence number with Ballesteros-Weinstein
number in superscript.39
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Figure 6. (A) Ellipsoid plots of 7 KMN-80 and (B) 8 KMN-159 as determined by x-ray crystallography
displaying two molecules within asymmetric unit in tail-tail and head-head orientations. (C) Edge-on views
of 7 and (D) 8 revealing puckered conformations observed by x-ray data collection. 8 displays both exo &
endo C11 pucker of lactam ring. 7 shows only endo C11 pucker. (E) C7-N8-C9-C10 dihedral angles of 7 and
8
, significance determined via unpaired t-test with one-tailed p-value within GraphPad Prism v. 6.07.
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Scheme 1. Retrosynthetic Analysis of Difluorolactam
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Scheme 2. Synthesis of KMN-80 and KMN-159
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