aza-PGE1 Series EP4 Receptor Binding and Activity - ACS Publications

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Difluoromethylene at the #-Lactam #-Position Improves 11-Deoxy-8aza-PGE Series EP Receptor Binding and Activity: 11-Deoxy-10,10difluoro-8-aza-PGE Analog (KMN-159) as a Potent EP Agonist 1

4

1

4

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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Journal of Medicinal Chemistry

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

ABSTRACT: 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 against 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.

H H

O

F F

N

O N

O OH

HO

Potency

O

Selectivity

OH

HO

KMN-80

KMN-159

Solubility

Ki: 2.35 nM (EP4) Ki: >10,000 nM (EP2)

Ki: 0.38 nM (EP4) Ki: 4,971 nM (EP2)

Lipophilicity Permeability

Prostaglandin & -Lactam Ring Scaffolds & Analogs

INTRODUCTION Prostaglandin E2 (PGE2) is a potent endogenous molecule that 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 sometimes divergent physiological and clinical effects of PGE2.1 In particular, the EP4 receptor is widely expressed in the gastrointestinal track, uterus, hematopoietic tissues, bone, and skin. Immune modulation, regulation of inflammatory responses and vascular tone, preservation of renal and bone homeostasis as well as mucosal integrity have been all proposed functions for the EP4 receptor.2 The 2-pyrrolidinone (-lactam, or E ring) scaffold is a known synthetic PGE structural mimic deployed in EP receptor-based SAR programs for decades (Figure 1)3-5. The relative ease with which a diverse array of - and -chains can be introduced on the core ring makes this an attractive template from a synthetic enablement perspective.

O

HO

O

-chain

E

E

-chain

O 9

11

X

8 12

13

O

2-Pyrrolidinone (-Lactam) Ring System O

OH 10

7

O

5 6 14

HO 15

-chain

-chain

Prostaglandin E Cyclopentanone Ring System 10

N

3

O

4 16

17

N8

12 13

7 5 6 14

3

OH

18

HO

HO 15

16

17

X=OH PGE1 (1a) X=H 11-Deoxy-PGE1 (1b)

PGE2 (2)

1

2

OH

18 19

20

O

4

1

2

19

HO

9

11

20

11-Deoxy-8-aza-PGE1 (3)

Figure 1. Prostaglandin and -lactam E ring-scaffolds and numbering. Various potent prostanoid- and -lactam-based EP4 receptor agonists, such as Ono Pharmaceutical’s Rivenprost (ONO4819) free acid,6 Merck-Frosst’s L-902,688,7 and Kaken Pharmaceutical’s KAG-3088 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).912 O

Ono Pharmaceuticals

O

Merck-Frosst

N N NH N

Kaken Pharmaceuticals

N S

HO

O HO

OR

F

HO

F

H N N N N

F OH OH

OMe

R= Me Rivenprost (ONO-4819)6 (4a) R = H Rivenprost free acid6 (4b)

F O

L-902,6887 (5a)

KAG-3088 (5b)

Figure 2. Published PG and -lactam EP4 receptor-selective agonists. Although the -lactam and PGE scaffolds share common ring size, relative carbonyl and side-chain positioning, and ring C12 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 (e.g. 11-deoxy-PGE1, (1b))13, 14 and its corresponding -lactam derivative (e.g. 11-deoxy-8-aza-PGE1, (3)).3, 15, 16 Differences 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 sp2 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 sp3 hybridized, tetrahedral, and possesses no valence electrons in conjugation with the carbonyl  system. These distinguishing structural and electronic features between a PGE ring and its 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, activity, physicochemical, and pharmacokinetic properties of small molecule drug candidates.17, 18 Although fluoro substituents have relatively low steric demand, they are highly electronegative and can influence electronics, molecular conformation, and other properties of organic molecules.19, 20 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(sp3)-F bond is remarkably stable in contrast to the corresponding bonds substituting the other halogens. The highly-electronegative fluoro substituent stabilizes the carbon bonding sp3 orbital to which it is bound to expose an accessible (due to fluorine’s low steric demand) low-lying antibonding orbital (*C-F) that can participate in stabilizing 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 shape, conformation, and flexibility.21 In our quest to identify novel selective EP4 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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structure, and Drug Metabolism and Pharmacokinetics (DMPK). RESULTS AND DISCUSSION 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 extensive SAR program; each pair including both a nonfluorinated -lactam species and its corresponding 3,3difluoro-2-pyrrolidinone (“difluorolactam”) analog. Figure 3 illustrates two such nonfluoro-difluoro pairs we will highlight in this report. 11-Deoxy-8-aza-PGE1 (3)3 is the closest -lactam structural analog to prostaglandin 11-deoxy-PGE1. We prepared both this compound and its difluorolactam analog 6 (KMN-165). The other analog pair we scrutinized incorporates the acetylene moiety of the prostacyclin analog cicaprost22 into the -chain, providing nonfluoro/difluoro pair 7 (KMN-80)/8 (KMN-159). KMN-159 is a current lead compound that has emerged from this series. O

X X

X X

N

O N

O

O

OH

HO

(3) KMN-165 (6)

X=H X=F

OH

HO

X = H KMN-80 (7) X = F KMN-159 (8)

Figure 3. -Lactam analog nonfluoro-difluoro pairs. Scheme 1 shows that our final assembly strategy involved carbon-carbon bond formation by way of Horner-EmmonsWadsworth (HEW) chemistry utilizing aldehyde 9 to install the -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 originating from commercially available 12a.23 Scheme 1. Retrosynthetic Analysis of Difluorolactam F F

O

R

F

N

O

F

R

N

R HO

8b

O

F F

OH

12a

NH

O

OH

H

9

12b

O

NH

O

F F

N

O N

O

11

O

10

Both lactam 7 and difluorolactam 8 syntheses followed a seven-step parallel route from corresponding (R)-5(hydroxymethyl)pyrrolidine-2-one (12a24, 25/12b26) starting points (Scheme 2). Tert-Butyldimethylsilyl (TBDMS) 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


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starting material during the alkylation over that of the nonfluorinated 13a. Scheme 2. Synthesis of KMN-80 and KMN-159 OMe O

X X

a

NH

O

X X

b

NH

OH

X X

X=H 13a X=F 13b

O

OTBDMS

X=H 14a X=F 14b

54% 67%

OMe O

X X

O

O

X X

O

+

N

OH

X

H

94% 91%

f

O

X=H 18a X=F 18b

e 17

57% 55%

X=H 16a X=F 16b OMe

O

N

Table 1. Binding affinities to human EP receptor subtypes Human EP Receptor Binding Ki (nM) Cmpd hEP2 hEP3 hEP4 1.03+0.13 2.17+0.21 0.110+0.020 PGE2

O

OMe O

MeO MeO P O

O

X=H 15a X=F 15b

X

66% 81%

OMe

d

N

c

N

OTBDMS

X=H 12a 91% X=F 12b 99%

O

X X

O

O

X

N

HO

21% 20%

X

X=H 19a X=F 19b

O

O

N

HO

74% 80%

(7)b,c 12,173+1,265 (3)a,b,c 4,971+879 (3)a,c 1,459+123 (3)a,b,c 329+26 (3)a,b

(6) 2.349+0.658 (4)a,b,c,d 0.378+0.105 8 (5) 1.606+0.061 3 (3)a,b,c,d 0.200+0.019 6 (3) 0.396 + 0.121 4b nd nd (4) Ki 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. P10,000 >10,000 (3) (3) >10,000 7,358+2,998 (3) (2)a,c,d >10,000 >10,000 (3) (2) >10,000 2,266+78 (3) (2)a,b,d 10,000 610+145 (3) (2)b,c,d

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. P10,000 nM)

Receptor Docking. Recently published crystal structures of EP3 and EP4 have provided a wealth of data for the binding



mode of PGE2.29-32 The EP4 receptor was crystallized in the inactive conformation bound to an antagonist, ONO-AE3-208 (PDB: 5YWY, ligand ID: 7UR).29 The EP3 receptor was crystallized separately with two different agonists bound, one of which was PGE2.30, 31 To compare our ligands with a known binding mode of PGE2, docking was performed with the active EP3 structure (PDB: 6AK3, ligand ID: P2E)30 using an InducedFit-Docking (IFD) protocol within Maestro 11 (Schrödinger, LLC, New York, NY, 2018.).33-38 Relative to EP4, EP3 possesses 35% sequence identity and 48.2% sequence similarity. Docking to EP3 suggested that our agonists bind in like manner to PGE2 within EP3 (Figure 4). The model showed clear overlap between PGE2 and the 7/8 pair, supporting a similar binding mode between the molecules, despite the rigidity of the acetylene ω-chain. The 3/6 pair, which bears the identical saturated -chain terminus as PGE2, likewise docked in a nearly identical position to PGE2 (Figure S1). A. W169ECL2

T168ECL2 T206ECL2

R3167.37 R3337.40

S732.58 T1072.58

S3367.43 Q3397.46

B. W169ECL2 W207ECL2

T692.54 Q1032.54

N3217.45 Q3397.46

Figure 5. Molecular Mechanics with generalized Born and surface

D652.50 D992.50 T168ECL2 T206ECL2

area solvation calculations of docked poses to human EP3. Significance determined via unpaired t-test with two-tailed p-value within GraphPad Prism v. 6.07.

Y802.65 Y1142.65

R3167.37 R3337.40 S3367.43

R316(7.37), T69(2.54), D65(2.50) and N321(7.45). Residues D65(2.50) and N321(7.45) make up part of a putative Na2+ binding pocket, which has been speculated to be involved with receptor activation.30 Molecular-Mechanics with generalized Born and Surface Area Solvation (MM-GBSA) scoring, a simplified computation methodology for determining binding free energies, was applied to the binding poses within EP3.41, 42 The results predict increased potency arising from 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 scoring function versus EP3 Ki shows that the rank-order is well maintained and results in an R2 of 0.69 (Figure S2).43

Y802.65 Y1142.65

W207ECL2

N3217.45

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S732.58 T1072.58 T692.54 Q1032.54 D652.50 D992.50

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 PGE2. (B) Docked pose of 8 (black stick & ball 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

To surmise residues of importance within EP4, a binding site alignment was performed between EP3 and EP4, using a homology model of EP4 generated within the RaptorX Structure prediction suite (raptorx.uchicago.edu/).40 Essential residues for 7 and 8 binding were predicted to include Y80(2.65),

Crystallography/Quantum-Mechanics. To further understand the mechanism of the difluoromethylene-driven potency shift, we obtained small molecule crystal structures of 7 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, 2018).44 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 (Figure S3). We calculated entropy at standard conditions to be 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 a decreased C7-N8-C9-O dihedral angle. Structural observations of a trigonal planar nitrogen within 8 indicate a fully sp2-hybridized center and signify increased delocalization 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-


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gem-difluoro-γ-lactams exist within the Cambridge Crystallographic Data Center (CCDC), displaying both C11 exo and C11 endo E-ring pucker between the enteries.45-47 Increased sp2 character of the γ-lactam nitrogen atom appears to be conserved (Figure S4 and Figure S5).45, 47 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 conformation (Figure S6).48 Consequently, we postulate that the effects of difluoromethylene incorporation appear to be mediated through inductive effects of the fluorine atoms within this system. A.

B.

C.

8.8

D. 

0.21Å

N8 C9

C10

0.2



0.48Å

N8 C9

C10

E.

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 xray 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. Partial charges were calculated for atoms within 7 and 8, with expected significant differences, where partial positive hydrogen atoms (0.2463 e ± 0.0014, n=2) in 7 were exchanged for negative fluorine (0.3548 e ± 0.0038, n=2) in 8. The presence of the fluorine substituents significantly inverted the partial charge centered on C10 from a partial negative (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 carbon (C9) was reduced from 0.6582 e ± 0.0050 (n=2) in 7 to 0.5796 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

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). 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 with the EP3 crystal structures displaying no H-bonding with the carbonyl of PGE2 or Misoprostol.30, 31 Electronic and 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 (p=0.0002, n=2) over a 100 ns MD simulation.49 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 8 in human plasma coincides with the on- and off-target potency shift observed with gem-difluoro substitution. Table 3. Drug-like profile of compounds 7 and 8 7 (KMN-80) Physicochemical properties * Aqueous Solubility (PBS, pH 7.4) 192.3+7.2 M * Log D (n-octanol/PBS, pH 7.4) -0.07 * Chemical Stability (% recovery) n.d. aqueous solution (PBS, pH 7.4, 4oC) n.d. autoclave (PBS, pH 7.4, 4oC) n.d. -irradiation (29.7 Kga Co-60) * Protein binding (10 M in plasma) 44.2+0.1% In vitro Absorption * Caco-2 Permeability A-to-B Papp (10-6 cm/s) 6.38+0.59 Percent Recovery 103% -6 B-to-A Papp (10 cm/s) 10.10 + 1.11 Percent Recovery 97% Efflux Ratio 1.6 * MDR1-MDCK Permeability A-to-B Papp (10-6 cm/s) n.d. n.d. Percent Recovery B-to-A Papp (10-6 cm/s) n.d. n.d Percent Recovery Efflux Ratio n.d. In vitro Metabolism 115.5 + 10.5 min * Half-life (1M in human plasma) > 120 min * Half-life (1M in human blood) * Intrinsic Clearance (0.1M in human liver microsomes) > 60 min Half-life (min) < 115.5 CLint (L/min/mg) * Intrinsic Clearance (0.1M in human liver hepatocytes) > 120 min Half-life (min) < 8.2 CLint (L/min/Mcell) * Cytochrome P450 Inhibition All negative (10M in human liver microsomes) Genetic Toxicity * Bacterial cytotoxicity All negative * Ames fluctuation test All negative * Micronucleus CHO cells -/+ S9 Negative Cardiac Toxicity


8 (KMN-159) 197.0+0.4 M 0.11 100% 100% 95% 58.4+4.1%a

9.93+0.23 94% 14.50 + 2.05 93% 1.5 0.59 + 0.36 84% 11.8 + 0.14 93% 20.0 > 120 min > 120 min > 60 min < 115.5 > 120 min < 8.2 All negative All negative All negative Negative


* hERG IC50 (patch-clamp) a

29.0 + 11.9 M

25.9 + 8.5 M

p < 0.05 by Unpaired t-test; n.d. not determined

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). CONCLUSION In summary, a novel pair of human EP4 selective full agonists are presented featuring a 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 EP4 with >5,000 times selectivity over EP2. A leftward shift of Ki of 4.6 times was observed for gem-difluoro analog 8 (KMN-159) relative to corresponding nonfluoro analog 7 (KMN-80). We also observed this effect between the 3/6 pair and without exception within our broader SAR series, where the difluoromethylene moiety was associated with 3-10fold 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. EXPERIMENTAL SECTION 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 obtained on a Cary 50 Bio UV-visible spectrophotometer. 1H, 13C, and 19F 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 1200 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 1100 preparative HPLC system and followed by an Agilent 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 acidmethanol-water gradient or ammonium acetate-acetonitrilewater gradient. All final compounds gave satisfactory purity (≥

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95%) by HPLC and by 1H NMR spectroscopy. Thin layer chromatography (TLC) analyses were performed on Uniplate™ 250 silica gel plates (Analtech, Inc. Catalog No. 02521) and 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-6yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoic acid (KMN-80, 7) (R)-5-(((tert-Butyldimethylsilyl)oxy)methyl)pyrrolidin-2one (13a). To a stirring solution consisting of (R)-5(hydroxymethyl)pyrrolidin-2-one (12a24, 25) (0.759 g, 5.02 mmol) in dry DMF (10 mL) was added imidazole (0.581 g, 8.53 mmol). The mixture was cooled to 0 °C and tertbutyldimethylchlorosilane (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 heptaneisopropanol (95:5 v/v) to afford the title compound as a white solid (1.21 g, 91%); 1H NMR (400 MHz, CDCl3) δ 5.84 (br s, 1H), 3.6-3.8 (m, 1H), 3.57 (dd, 1H, J=7.7, 3.9 Hz), 3.38 (dd, 1H, J=7.7, 10.0 Hz), 2.9-2.9 (m, 1H), 2.8-2.9 (m, 1H), 2.2-2.4 (m, 2H), 2.12.2 (m, 1H), 1.68 (dddd, 1H, J=5.5, 7.5, 9.4, 12.9 Hz), 0.8-0.8 (m, 9H), 0.00 (s, 6H); MS (ESI+) m/z 230.1 (M+H). Methyl (R)-7-(2-(((tert-butyldimethylsilyl)oxy)methyl)-5oxopyrrolidin-1-yl)heptanoate (14a). To a 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)5-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-2-one (13a) (10.0 g, 43.6 mmol) in DMF (120 mL). The ice bath was 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, 43.5 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%); 1H NMR (400 MHz, CDCl3) δ 3.62 (s, 3H), 3.5-3.7 (m, 4H), 2.92 (ddd, 1H, J=5.1, 8.8, 13.7 Hz), 2.3-2.5 (m, 1H), 2.25 (t, 2H), 2.2-2.3 (m, 1H), 2.0-2.1 (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-1yl)heptanoate (15a). To a solution consisting of methyl (R)-7(2-(((tert-butyldimethylsilyl)oxy)methyl)-5-oxopyrrolidin-1yl)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


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filtration and the filtrate was applied to a silica column. Elution 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%); TLC Rf 0.24 (solvent system: ethyl acetate-methanol 97:3 v/v); 1H NMR (400 MHz, CDCl ) δ 3.7-3.8 (m, 1H), 3.6-3.7 (m, 1H), 3 3.65 (s, 3H), 3.5-3.6 (m, 2H), 2.9-3.0 (m, 1H), 2.71 (br s, 1H), 2.42.6 (m, 1H), 2.3-2.4 (m, 1H), 2.29 (t, 2H), 2.0-2.2 (m, 1H), 1.92.0 (m, 1H), 1.5-1.7 (m, 3H), 1.4-1.5 (m, 1H), 1.2-1.4 (m, 4H); MS (APCI+) m/z 258 (M+H). Methyl (R)-7-(2-formyl-5-oxopyrrolidin-1-yl)heptanoate (16a). To a solution consisting of methyl (R)-7-(2(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 intermediate 18a. TLC Rf 0.24 (solvent system: 2-propanolheptane 15:85 v/v). Methyl 7-((R)-2-((S,E)-4-methyl-3-oxonon-1-en-6-yn-1yl)-5-oxopyrrolidin-1-yl)heptanoate (18a). A solution consisting of (S)-(+)-dimethyl (3-methyl-2-oxooct-5-yn-1yl)phosphonate (17) (3.5 g, 14 mmol), lithium chloride (2.1 g, 50 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-5oxopyrrolidin-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 acetateheptane 7:3 to 9:1 v/v) afforded the title compound as clear oil (3.14 g, 57%); TLC Rf 0.33 (solvent system: ethyl acetateheptane 4:1 v/v); 1H NMR (400 MHz, CD3OD) 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, 3H), 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, 4H), 1.3-1.4 (m, 4H), 1.15 (d, 3H, J=6.6 Hz), 1.06 (t, 3H, J=7.4Hz); MS (ESI+) m/z 376.2 (M+H). Methyl 7-((R)-2-((3S,4S,E)-3-hydroxy-4-methylnon-1-en6-yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoate (19a). To a mixture consisting of methyl 7-((R)-2-((S,E)-4-methyl-3oxonon-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 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,

0.426 mmol) was added and the reaction mixture stirred for 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 Rf 0.36 (solvent system: 2-propanol-heptane 30:70 v/v); 1H 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-en6-yn-1-yl)-5-oxopyrrolidin-1-yl)heptanoate. TLC Rf 0.45 (solvent system: 2-propanol-heptane 30:70 v/v); 1H NMR (400 MHz, CD3OD) δ 5.77 (dd, 1H, J=5.7, 15.4 Hz), 5.55 (dd, 1H, J=8.8, 15.4 Hz), 4.19 (q, 1H, J=7.6 Hz), 4.11 (t, 1H, J=4.9 Hz), 3.64 (s, 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, J=7.4 Hz), 0.98 (d, 3H, J=6.6 Hz); MS (ESI+) m/z 378.3 (M+H). 7-((R)-2-((3S,4S,E)-3-Hydroxy-4-methylnon-1-en-6-yn-1yl)-5-oxopyrrolidin-1-yl)heptanoic acid (KMN-80, 7). To a solution consisting of methyl 7-((R)-2-((3S,4S,E)-3-hydroxy-4methylnon-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 brinewater (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%); melting point 142.5-143.5 °C; TLC Rf 0.17 (solvent system: ethyl acetate-heptane-acetic acid 80:20:1 v/v); 1H NMR (400 MHz, CD3OD) δ 5.75 (dd, 1H, J=6.96, 15.38 Hz), 5.54 (dd, 1H, J=8.79, 15.38 Hz), 4.15-4.22 (m, 1H), 4.02 (t, 1H, J=6.77 Hz), 3.42-3.51 (m, 1H), 2.94 (ddd, 1H, J=5.49, 8.70, 13.64 Hz), 2.342.41 (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 Hz), 0.96 (d, 3H, J=6.96 Hz); 1H NMR (400 MHz, CDCl3) δ 5.71 (dd, 1H, J=6.54, 15.33 Hz), 5.57 (dd, 1H, J=8.42, 15.37 Hz), 4.285.29 (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), 2.122.29 (m, 4H), 1.70-1.85 (m, 2H), 1.58-1.69 (m, 2H), 1.23-1.58 (m, 6H), 1.09-1.17 (m, 3H), 0.98 (d, 3H, J=6.77 Hz); MS (ESI+) m/z 364.2 (M+H), (ESI-) 362.2 (M-H); HRMS (ESI-): m/z calculated for C21H33NO4 – H+ [M-H]: 362.2337, found 362.2342; specific rotation: []21.9D = +0.068/(0.01)(0.5) = +13.6°; HPLC 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, 99.4%. Synthesis of 7-((R)-3,3-difluoro-5-((3S,4S,E)-3-hydroxy-4methylnon-1-en-6-yn-1-yl)-2-oxopyrrolidin-1-yl)heptanoic acid (KMN-159, 8) (R)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-3,3difluoropyrrolidin-2-one (13b). To a solution consisting of (R)-3,3-difluoro-5-(hydroxymethyl)pyrrolidin-2-one (12b26) (0.880 g, 5.82 mmol) in DMF (10 mL) and THF (10 mL) was 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, 99%); TLC Rf 0.60 (solvent system: dichloromethane-methanol 95:5 v/v); 1H NMR (400 MHz, CDCl3) δ 6.91 (br s, 1H), 3.74 (m, 1H), 3.63 (dd, 1H, J=4.2, 10.4 Hz), 3.45 (dd, 1H, J=7.0, 10.3 Hz), 2.52-2.67 (m, 1H), 2.19 (dtd, 1H, J=4.9, 15.1, 16.8 Hz), 0.82 (s, 9H), 0.00 (2s, 6H); 1H NMR (400 MHz, CD3OD) δ 3.75-3.83 (m, 1H), 3.67-3.73 (m, 1H), 3.57-3.64 (m, 1H), 2.53-2.70 (m, 1H), 2.33-2.49 (m, 1H), 0.86-0.94 (s, 9H), 0.08 (2s, 6H); 19F NMR (376 MHz, CDCl3) δ -103.77 (dt, J=271, 14 Hz) -104.88 (dt, J=271, 18 Hz); 13C NMR (100 MHz, CDCl3) 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)3,3-difluoro-2-oxopyrrolidin-1-yl)heptanoate (14b). To a 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-difluoropyrrolidin2-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 7bromoheptanoate (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-propanolheptane 5:95 v/v afforded the title compound (13.6 g, 67%); TLC Rf 0.41 (solvent system: 2-propanol-heptane 1:9 v/v); 1H NMR (400 MHz, CDCl3) 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), 0.07 (s, 6H); MS (ESI+) m/z 408.2 (M+H). Methyl (R)-7-(3,3-difluoro-5-(hydroxymethyl)-2oxopyrrolidin-1-yl)heptanoate (15b). To a solution consisting of methyl (R)-7-(5-(((tertbutyldimethylsilyl)oxy)methyl)-3,3-difluoro-2-oxopyrrolidin1-yl)heptanoate (14b) (13.6 g, 33.4 mmol) in methanol (100 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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and washed with brine (150 mL). 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 dichloromethane-methanol 97:3 v/v afforded the title compound as a white solid (7.9 g, 81 %); TLC Rf 0.30 (solvent system: 2-propanol-heptane 1:4 v/v); 1H NMR (400 MHz, CDCl3) 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 Hz), 1.44-1.70 (m, 4H), 1.21-1.40 (m, 4H); 19F NMR (376 MHz, CDCl3) δ -102.63 (dt, J=271, 15 Hz), -103.46 (dt, J=271, 19 Hz); 19F NMR (376 MHz, CD OD) δ -103.73 (dt, J=271, 15 Hz), 3 104.60 (dt, J=271, 15 Hz); 13C NMR (100 MHz, CD3OD) 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+H2O). Methyl (R)-7-(3,3-difluoro-5-formyl-2-oxopyrrolidin-1yl)heptanoate (16b). To a solution consisting of (R)-methyl 7(3,3-difluoro-5-(hydroxymethyl)-2-oxopyrrolidin-1yl)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 dichloromethanemethanol 200:1 v/v afforded the title intermediate as a paleyellow oil (0.077 g, 91%), which was used immediately to prepare compound 18b; TLC Rf 0.60 (solvent system: dichloromethane-methanol 93:7 v/v). Methyl 7-((R)-3,3-difluoro-5-((S,E)-4-methyl-3-oxonon-1en-6-yn-1-yl)-2-oxopyrrolidin-1-yl)heptanoate (18b). A mixture consisting of (S)-(+)-dimethyl (3-methyl-2-oxooct-5yn-1-yl)phosphonate (17) (4.5 g, 18 mmol), methyl (R)-7-(3,3difluoro-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) afforded the title compound (4.5 g, 55%); 1H NMR (400 MHz, CD3OD) δ 6.6-6.7 (dd, 1H), 6.5-6.6 (d, 1H), 4.47 (tt, 1H, J=3.9, 8.0 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 Hz); 19F NMR (376 MHz, CD3OD) δ -104.33 (dt, J=271, 15 Hz), 107.07 (ddd, J=274, 19, 15 Hz); MS (ESI+) m/z 412.2 (M+H) 429.3 (M+OH), (ESI-) m/z 410.2 (M-H). Methyl 7-((R)-3,3-difluoro-5-((3S,4S,E)-3-hydroxy-4methylnon-1-en-6-yn-1-yl)-2-oxopyrrolidin-1yl)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


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sodium borohydride (46 mg, 1.21 mmol) was added as stirring 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, ethanolheptane 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 compound (49.2 mg, 20%); TLC Rf 0.36 (solvent system: 2propanol-heptane 15:85 v/v); 1H NMR (400 MHz, CD3OD) δ 5.90 (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), 3.0-3.1 (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, J=7.4 Hz), 0.96 (d, 3H, J=7.0 Hz); 19F NMR (376 MHz, CD3OD) δ -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). Methyl 7-((R)-3,3-difluoro-5-((3R,4S,E)-3-hydroxy-4methylnon-1-en-6-yn-1-yl)-2-oxopyrrolidin-1yl)heptanoate. TLC Rf 0.43 (solvent system: 2-propanolheptane 15:85 v/v); 1H NMR (400 MHz, CD3OD) δ 5.93 (dd, 1H, 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, 13.0 Hz), 2.7-2.9 (m, 1H), 2.31 (br t, 2H, J=7.4 Hz), 1.9 – 2.4 (m, 2H), 2.14 (br d, 2H, J=7.4 Hz), 2.02 (br dd, 1H, J=7.4, 16.4 Hz), 1.7-1.7 (m, 1H), 1.5-1.7 (m, 4H), 1.2-1.4 (m, 5H), 1.09 (t, 3H, J=7.4 Hz), 0.98 (d, 3H, J=6.6 Hz), 7-((R)-3,3-Difluoro-5-((3S,4S,E)-3-hydroxy-4-methylnon1-en-6-yn-1-yl)-2-oxopyrrolidin-1-yl)heptanoic acid (KMN-159, 8). To a mixture consisting of methyl 7-((R)-3,3difluoro-5-((3S,4S,E)-3-hydroxy-4-methylnon-1-en-6-yn-1yl)-2-oxopyrrolidin-1-yl)heptanoate (19b) (6.5 g, 16 mmol) in methanol (325 mL) at room temperature was added 1 N NaOH (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 Rf 0.50 (solvent system: dichloromethane-methanol-acetic acid 94:5:1 v/v); 1H NMR (400 MHz, CDCl3) 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), 2.6-2.8 (m, 1H), 2.33 (t, 2H, J=7.2 Hz), 2.1-2.3 (m, 5H), 1.79 (td, 1H, J=6.5, 12.7 Hz), 1.4-1.7 (m, 4H), 1.2-1.4 (m, 4H), 1.11 (t, 3H, J=7.4 Hz), 0.97 (d, 3H, J=7.0 Hz); 19F NMR (376 MHz, CDCl3) δ 103.57 (dt, J=271, 15 Hz), -105.58 (dt, J=271, 15 Hz); 13C NMR (100 MHz, CDCl3) δ 178.7, 163.5 (t, J=30 Hz), 137.3, 129.0, 117.5 (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 (ESI+) m/z 398.2 (M+H); HRMS (ESI-): m/z calculated for C21H31F2NO4 – H+ [M-H]: 398.21484, found 398.21573; specific rotation: []21.9D = +0.17/(0.810)(0.5) = +4.19°; HPLC Gemini 5 C18 250x4.6 mm, methanol-water-acetic acid 70:30:0.1 v/v, 1mL/min, 210nm, 99.3%.

Synthesis of 7-((R)-3,3-Difluoro-5-((S,E)-3-hydroxyoct-1-en-1yl)-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 (R,E)-7-(3,3-difluoro-2-oxo-5-(3-oxooct-1-en-1yl)pyrrolidin-1-yl)heptanoate. clear oil; 1H NMR (400 MHz, CD3OD) δ 6.61 (dd, 1H, J=8.8, 15.8 Hz), 6.39 (d, 1H, J=16.0 Hz), 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), 2.32.5 (m, 1H), 2.30 (t, 2H, J=7.4 Hz), 1.5-1.6 (m, 6H), 1.2-1.4 (m, 8H), 0.90 (t, 3H, J=6.8 Hz); 19F NMR (376 MHz, CD3OD) 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-1yl)-2-oxopyrrolidin-1-yl)heptanoate. HPLC (Luna 5 silica 250x4.6 mm, ethanol-heptane 10:90) of the crude product indicated a diastereomeric ratio of 68:32 R/S at C15-OH. TLC Rf 0.38 (solvent system: 2-propanol-heptane 15:85 v/v); 1H NMR (400 MHz, CD3OD) δ 5.89 (dd, 1H, J=6.1, 15.4 Hz), 5.47 (dd, 1H, 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 (m, 3H); 19F NMR (376 MHz, CD3OD) δ -104.93 (dt, J=271, 13 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-1yl)-2-oxopyrrolidin-1-yl)heptanoate. TLC Rf 0.40 (solvent system: 2-propanol-heptane 15:85 v/v); 1H NMR (400 MHz, CD3OD) δ 5.92 (dd, 1H, J=5.5, 15.2 Hz), 5.4-5.5 (m, 1H), 4.2-4.3 (m, 1H), 4.09 (q, 1H, J=5.7 Hz), 3.64 (s, 3H), 3.5-3.6 (m, 1H), 3.13.1 (m, 1H), 2.7-2.9 (m, 1H), 2.2-2.3 (m, 3H), 1.4-1.7 (m, 7H), 1.32 (br s, 10H), 0.91 (br t, 3H, J=6.4 Hz); 19F NMR (376 MHz, CD3OD) δ -104.95 (dt, J=267, 15 Hz), -107.03 (dt, J=271, 15 Hz); 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)-2oxopyrrolidin-1-yl)heptanoic acid (KMN-165, 6). white solid; 1H NMR (400 MHz, CDCl3) 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); 19F NMR (376 MHz, CDCl ) δ -103.61 (dt, J=267, 15 Hz), -105.61 3 (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 C19H31F2NO4 + H+ [M+H]: 376.2294, found 376.2297; HPLC 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%. ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. 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 EP3 Ki, MacroModel conformation search results and MD simulation of KMN-80 (7) and KMN-159 (8), comparison of -lactam dihedrals with CCDC reference molecules, Ering crystallographic bond lengths, biological assay procedures, x-ray crystallographic procedures, physiochemical procedures, DMPK procedures, safety assessment procedures, docked poses (.PDB), NBO partial



charge predictions and MM-GBSA ΔG values (.xlsx) and Molecular formula strings (CSV). Accession Codes 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. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Phone: (734)975-3986. #E-mail: [email protected]. Phone: (734)929-1647. ORCID 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. 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. ABBREVIATIONS PGE2, Prostaglandin E2; EP1-4, E-type prostanoid receptor subtypes 1-4; PGE, Prostaglandin type E class molecules; DMPK, Drug Metabolism and Pharmacokinetics; HEW, HornerEmmons-Wadsworth; TBDMS, Tert-Butyldimethylsilyl; DMF, N,N-Dimethylformamide; THF, Tetrahydrofuran; MeOH, Methanol; FP, Prostaglandin F2α receptor; DP1, 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. REFERENCES

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177x109mm (300 x 300 DPI)


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Figure 1. Prostaglandin and γ-lactam E ring-scaffolds and numbering. 152x102mm (300 x 300 DPI)



Figure 2. Published PG and γ-lactam EP4 receptor-selective agonists. 175x65mm (300 x 300 DPI)


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Figure 3. γ-Lactam analog nonfluoro-difluoro pairs. 111x51mm (300 x 300 DPI)



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 PGE2. (B) Docked pose of 8 (black stick & ball 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 69x119mm (300 x 300 DPI)


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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. 115x147mm (300 x 300 DPI)


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Scheme 1. Retrosynthetic Analysis of Difluorolactam 121x79mm (300 x 300 DPI)



Scheme 2. Synthesis of KMN-80 and KMN-159 189x194mm (300 x 300 DPI)


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aza-PGE1 Series EP4 Receptor Binding and Activity - ACS Publications

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