Movement to the Clinic of Soluble Epoxide Hydrolase Inhibitor EC5026 as an Analgesic for Neuropathic Pain and for Use as a Nonaddictive Opioid Alternative

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Drug Annotation

Movement to the Clinic of Soluble Epoxide Hydrolase Inhibitor

EC5026 as an Analgesic for Neuropathic Pain and for Use as a

Nonaddictive Opioid Alternative

Bruce D. Hammock, Cindy B. McReynolds, Karen Wagner, Alan Buckpitt, Irene Cortes-Puch,

Glenn Croston, Kin Sing Stephen Lee, Jun Yang, William K. Schmidt, and Sung Hee Hwang*

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sı Supporting Information

ABSTRACT: This report describes the development of an orally active

analgesic that resolves inﬂammation and neuropathic pain without the addictive

potential of opioids. EC5026 acts on the cytochrome P450 branch of the

arachidonate cascade to stabilize epoxides of polyunsaturated fatty acids

(

EpFA), which are natural mediators that reduce pain, resolve inﬂammation,

and maintain normal blood pressure. EC5026 is a slow-tight binding transition-

state mimic that inhibits the soluble epoxide hydrolase (sEH) at picomolar

concentrations. The sEH rapidly degrades EpFA; thus, inhibiting sEH increases

EpFA in vivo and confers beneﬁcial eﬀects. This mechanism addresses disease

states by shifting endoplasmic reticulum stress from promoting cellular senescence and inﬂammation toward cell survival and

homeostasis. We describe the synthesis and optimization of EC5026 and its development through human Phase 1a trials with no

drug-related adverse events. Additionally, we outline fundamental work leading to discovery of the analgesic and inﬂammation-

resolving CYP450 branch of the arachidonate cascade.

INTRODUCTION

establishing EpFA as key chemical mediators. In contrast to the

cyclooxygenase (COX) and lipoxygenase (LOX) branches that

are predominately but not exclusively pro-inﬂammatory, it is

generally accepted that a third branch (cytochrome P450s or

CYP450s) of the arachidonic acid cascade is largely anti-

■

There is a pressing need for new pharmaceuticals to treat pain

in general and neuropathic pain speciﬁcally in view of the

opioid epidemic discussed below. Here we provide an overview

of the discovery and development of a new class of nonopioid

analgesics that appear devoid of addictive potential, resolve

inﬂammation, and reduce endoplasmic reticulum (ER) stress.

EC5026, a small molecule drug candidate disclosed in this

study, is an exceptionally potent transition-state mimic

inhibitor of the soluble epoxide hydrolase (sEH) enzyme

3

inﬂammatory and pro-resolving. The discovery that ureas,

amides, carbamates, and various heterocycles could act, as not

only epoxide mimics but also transition-state mimics, started

the ﬁeld down a path to several exceptionally potent sEHI

4

,5

including EC5026 described here. The physical, pharmaco-

kinetic (PK), and biological properties of sEHI yielded a

unique set of advantages and challenges in their development

to the clinic as described below. In addition, EC5026 has been

successfully moved to the clinic through a unique and largely

publicly funded clinical development path.

1

which is widely distributed in mammalian tissues. Interest-

ingly, the sEH was found while studying fundamental insect

developmental biology where the hydration of epoxides of

juvenile hormones is a key reaction in metamorphosis. This

biology was exploited for the rational development of a class of

green pesticides termed insect growth regulators (IGRs), and

the mammalian sEH was discovered while studying the

metabolism of one of these IGRs in mammals. Following the

ﬁrst report of the sEH in 1972, the full metabolism of the

terpene epoxide developed as a vector control and agricultural

chemical was described in 1974, and the properties of the sEH

In the following paragraphs, we cover the early chemistry

and structure−activity relationship studies of sEHI, and the

path to drug candidates for human, equine, and companion

animals. We also present the synthetic pathway to EC5026 and

its full characterization (see Supporting Information). In

2

in mammals were investigated. Over the past several decades,

Received: October 31, 2020

Published: February 7, 2021

a primary role of this enzyme was found to be the rapid

conversion of anti-inﬂammatory, antihypertensive, and analge-

sic chemical mediators, the fatty acid epoxides also named

3

epoxy-fatty acids (EpFA), into their respective 1,2-diols.

Therefore, several classes of sEH inhibitors (sEHI) have been

developed over the past several decades and were useful in

©

2021 American Chemical Society

https://dx.doi.org/10.1021/acs.jmedchem.0c01886

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Journal of Medicinal Chemistry

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Drug Annotation

Figure 1. Chemical structures of early stage sEHI. A commonly used four-letter abbreviation and a University of California (UC) number are

provided. Our lead compound in this series was DCU. The presence of an adamantyl group made the sEHI more potent and easier to detect by

LC-MS in blood. They also tended to mimic EpFA, which were the presumptive substrates.

Figure 2. Chemical structures of late stage sEH inhibitors. A commonly used four-letter abbreviation and University of California numbers are

provided.

parallel to the medicinal chemistry, advances were made in

understanding the action of sEHI with the target enzyme and

on mammalian physiology. Although there are a variety of

possible indications and clinical paths for sEHI, neuropathic

pain has been selected for multiple reasons including lack of

eﬀective therapies for this pressing need, the reluctance of large

pharmaceutical companies to enter the ﬁeld, and the possibility

that a small company could take a new product through trials

demonstrating eﬃcacy. We present an update on the Phase 1a

clinical trial and our plans for the immediate future. We

conclude with a brief discussion of moving the compounds to

the clinic largely with the support of the NIH.

a hydrated form of dicyclohexyl carbodiimide (DCC). DCU is

a byproduct from amide coupling reactions with DCC. This

urea was our initial lead compound, and throughout the

chemical history of this series, the goal has been to maximize

potency and other pharmacological properties while dealing

with these high melting, poorly soluble lipophilic molecules.

The redeeming features of these transition-state mimics were

their exceptionally high potency on the target enzyme and their

high selectivity. A variety of structures have been used as

mimics of epoxides at putative receptors including ureas,

amides, and carbamates, so these functional groups were

examined as central pharmacophores of the inhibitors. Since

epoxides of long chain polyunsaturated fatty acids were the

hypothetical natural substrates of sEH, many early compounds

were designed to mimic these substrates while incorporating a

pharmacophore that mimics the epoxide group of the substrate

or the putative transition-state(s) of the epoxide ring opening.

This resulted in a series of compounds such as 12-(3-

adamantan-1-yl-ureido)dodecanoic acid (AUDA, Figure 1),

which were exceptionally potent enzyme inhibitors but also

suﬀered from being good mimics of the biological active EpFA

CHEMISTRY

■

Early sEH Chemistry. Early substrate mimics and

irreversible inhibitors of the sEH were useful biochemical

probes but too metabolically unstable to be useful as

4

pharmaceuticals. A series of fundamental studies in enzymol-

ogy discovered that the sEH belongs to the α/β-fold hydrolase

class of enzymes and that the key catalytic residue is a

6

nucleophilic aspartic acid. This class of enzymes has been

8

inhibited by carbodiimides, which initially appeared to be the

at their putative receptors. In addition, they were rapidly

7

9

case with sEH as well. However, Morisseau et al. found the

metabolized to inactive degradation products, and, as

actual compound that acts as a potent, time-dependent

inhibitor against sEH is dicyclohexyl urea (DCU, Figure 1),

expected based on their physical properties such as high

lipophilicity and high melting points as well as diﬃculty in

1

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Drug Annotation

Table 1. Physical Properties, Potency, and Target Occupancy of AR9281, TPPU, EC5023, EC5024, and EC5026

a

b

c

Solubility was measured with sodium phosphate buﬀer (0.1 M, pH 7.4). LogP was measured by HPLC. K and t (which is a reciprocal of k )

i

R

off

were determined against the aﬃnity puriﬁed recombinant human sEH (hsEH) by a FRET-displacement assay.

formulation to obtain high bioavailability. Despite these

diﬃculties, a number of biologists used these compounds to

demonstrate multiple roles of EpFA in reducing hypertension

and vascular inﬂammation. In early stages of the develop-

ment of sEHI, the adamantyl group used in AUDA and AEPU

group such as the adamantyl group by more stable aryl groups

such as the p-triﬂuoromethoxyphenyl group as in TPPU and t-

16,17

TUCB (UC1770, EC1728, Figure 2).

from the conformationally restricted ureas was AR9281

Figure 1) that was proven to be safe even at high doses in

One compound

10

(

(Figure 1) yielded exceptionally high detection sensitivity on

a Phase 2a clinical trial but failed to show adequate eﬃcacy for

further development. Although it is surprisingly water-soluble,

it contained an adamantyl group that resulted in complex

metabolites and a short half-life due to rapid hydroxylation of

an LC-MS as well as high potency on the target sEH. This

detection sensitivity aﬀorded by the adamantyl group allowed

rapid PK studies of these sEHI in rodents where PK-

absorption, distribution, metabolism, and excretion (ADME)

could be rapidly determined using just a few microliters of

18

the adamantyl group. One of its human metabolites was only

found in primates requiring expensive primate safety studies.

This, coupled with mediocre potency on the human

recombinant sEH and a short drug-target residence time on

the human enzyme, probably contributed to its clinical failure.

Because once or twice a day dosing is the norm for

hypertension and diabetes drugs, exceptionally high levels of

1

blood. The ease of determining oral bioavailability and PK

parameters as well as availability of rapid quantitative enzyme

assays resulted in rapid ﬁlters to select progressively more

12

potent orally bioavailable sEHI. Kim et al. found that the

presence of a polar group approximately 7 Å from the carbonyl

of the central pharmacophore dramatically increased solubility

without loss of potency as in AEPU shown in Figure 1.

These leads were exploited aggressively in both industrial

18

AR9281 were required to maintain adequate blood levels.

Interestingly, it has never been clear why AR9281 was selected

for development when the related TPPU which demonstrated

much greater potency on the recombinant human enzyme and

1

3

and academic laboratories. Among these, an excellent

14

compound was GSK2256294 (Figure 2). A major success

was the discovery of ureas that are constructed with simple

cyclicalkyl groups on a nitrogen atom of the urea combined

with conformationally restricted substituents on the other

nitrogen atom of the urea. This approach resulted in

compounds such as APAU (AR9281) and t-AUCB (Figure

17,19

a 100-fold higher PK-AUC in mice was already reported.

In pain assays in rodents, TPPU was of similar potency to

AR9281 on the recombinant murine enzyme but was more

17

potent than morphine. As mentioned above, AR9281 is quite

potent on the recombinant mouse sEH, but it is quite a weak

inhibitor with a rapid oﬀ rate on the recombinant human

enzyme as discussed below. TPPU has emerged as the

standard in the ﬁeld used in many biological studies to inhibit

1

) possessing drug-like properties, high potency, and improved

1

5,16

PK proﬁles.

These series of compounds were further

optimized by replacing the metabolically unstable cyclicalkyl

1

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Drug Annotation

the sEH due to its good PK and high potency on both primate

and rodent sEH enzymes.

Polypharmacy with sEHI. sEHI have been found to

synergize with several other pharmaceuticals. In several cases,

the mechanism of this synergism is quite well understood often

based on monitoring the proﬁles of blood regulatory lipids and

key regulatory pathways. The best studied to date are

structures combining pharmacophores that inhibit COX-2.

This synergism was predicted when lipid proﬁles in murine

sepsis models showed that EpFA were increased and diols

decreased in the blood as expected with sEHI, but the sEHI

quickly and dramatically resolved the high levels of PGE and

2

other inﬂammatory mediators. The eﬀect on products the

COX branch of the arachidonate cascade was far more

dramatic than on the CYP450 branch in plasma analysis. Even

in the early study, this was shown to be via transcriptional

down regulation of COX-2 induced in the sepsis model.

There is now evidence that this is due to reduction of ER stress

Figure 3. Chemical properties impact ease of formulation. Generally,

the more potent the compound the more facile the formulation.

There are relatively routine procedures to formulate high melting or

poorly water-soluble materials, but high melting poorly soluble

crystals are a challenge. This problem was addressed by synthesis to

increase potency on the human target enzyme, increase water

solubility, and reduce the stability of the crystal structure.

20

and inﬂammatory pathways leading to prostaglandin synthases

2

1

and COX-2. This synergism of COX and sEHI when

combined is fortunate since in addition to synergism many of

the cardiovascular and gastrointestinal side eﬀects of COX

22

5

inhibitors are blocked by sEHI. sEHI have been found to

synergize with PPAR agonists, FLAP and FAAH inhibitors,

phosphodiesterase inhibitors, and others which can be

state resulting in a drop in the melting point. In addition, a

19

previous SAR study suggested that introduction of an extra

methyl group at the α-carbon of the amide group of the

inhibitor could signiﬁcantly improve the potency of the

inhibitor. Therefore, a signiﬁcant improvement of potency

was expected with EC5019 when we introduced an extra

methyl group on the α-carbon of the amide group of EC5023.

As expected, EC5019 improved the potency but showed poor

water solubility due probably to the increased melting points.

Therefore, we further modiﬁed EC5019 by performing another

23

exploited by drug combinations or integrated molecules.

A

summary of this class of dual inhibitors/modulators is listed in

Table S4.

SAR Leading to IND Candidate EC5026. On the basis of

investigations of compounds such as TPPU, it became clear

that the drug-target residence time with sEHI was a better

indicator of inhibitor’s in vivo eﬃcacy than its potency in

19,24

5

enzyme assays (IC or K ).

The drug-target residence time

SAR study and found that introduction of another extra

50

i

and potency were correlated but not tightly correlated,

methyl group at the β-carbon of EC5019 could lower melting

points, which in turn improve water solubility (EC5019 vs

EC5026 in Table 1) and also further improve potency. To

further test whether we could improve the potency and water

solubility of EC5026 by reducing its size, we replaced the 4-

triﬂuoromethoxy group on the phenyl of EC5026 by 4-

triﬂuoromethyl group (EC5034). However, this attempt failed,

resulting in lower potency and poorer water solubility

compared to EC5026. Therefore, EC5026 was selected as a

drug candidate for further development. The increase in

potency coupled with a low melting point reduces the

likelihood of oﬀ-target toxicity and makes the compounds a

remarkably simple formulate for oral administration (Table

especially for very tight-binding sEHI (IC or K ≤ 5

5

0

i

1

9,24

nM).

sEHI with a urea pharmacophore like TPPU work

well in vivo, but a careful formulation is critical for reproducible

results, particularly for experiments requiring high doses due to

the high lipophilicity, high melting point, and poor water-

solubility of some sEHI. Formulation of either high melting or

lipophilic compounds can normally be addressed particularly

with highly potent compounds. However, high melting

lipophilic compounds such as some of the potent sEHI

present a challenge for the ﬁeld (Figure 1). Although replacing

the central urea pharmacophore by an amide group lowered

the melting point and increased the water solubility in general,

such replacement also reduced the potency of the inhibitors by

5

1).

1

an order of magnitude (EC5026 vs EC5049 in Table 1).

The selection of an IND candidate is based on multiple

factors ranging from the potency to the cost of synthesis.

Unique drivers in this series included long drug-target

Within EicOsis, the goal was to ﬁnd sEHI that were more

potent with a lower melting point, and/or more water-soluble.

A new series of compounds was designed based on the scaﬀold

residence time (reciprocal of k of the inhibitor from the

off

5

19

shown in TPPU.

enzyme), the moderate in vivo half-life needed for a ﬁrst-in-

Within the series of inhibitors synthesized by EicOsis, the

incorporation of a ﬂuorine atom in the meta-position of the p-

triﬂuoromethoxyphenyl group of sEHI (Table 1) not only

increased the potency against human sEH (dramatically in

some cases) but also reduced the melting point signiﬁcantly

while maintaining the drug-target residence time (e.g., TPPU

vs EC5023 in Table 1). This came with the cost of reduced

water solubility and increased LogP. It was assumed that an

addition of a meta-ﬂuoro substituent on the phenyl group of

the inhibitors broke the overall symmetry of the chemical

structure of the inhibitors and thus destabilized the crystalline

class compound, and sometimes a compromise between

reduced melting point and potency in favor of ease of

formulation. With many highly potent compounds in hand,

plasma protein binding was considered as an asset to facilitate

a rapid drug distribution of inhibitors. Among the most potent

inhibitors possessing the meta-ﬂuoro substituent on the phenyl

ring, the PK proﬁles in mice were found to vary dramatically.

In some cases, the compounds such as EC5023 and EC5024

and even TPPU were considered too stable following oral

administration in preclinical species for development for use in

humans (Table 2). Thus, the slow, tight-binding EC5026 was

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Drug Annotation

Table 2. PK Proﬁles of EC5023, EC5024, and EC5026 in Rats

a

Sprague−Dawley rats (male, n = 6) received the compounds by oral gavage with a single dose at 0.1 mg/kg (inhibitors were dissolved in PEG

3

00). PK parameters of inhibitors were calculated by WinNonlin based on the best ﬁtted one compartment model. Note that the driver was a lower

AUC and shorter half-life for a ﬁrst-in-class drug.

selected as a leading candidate for the clinic because it had a

signiﬁcantly shorter in vivo half-life than other analogues, yet a

suﬃcient half-life to avoid break-through pain should a patient

miss a daily dose.

Formulation for Human Phase 1a. The EC5026 drug

product was initially developed as an immediate-release oral

dosage form in a gelatin capsule using liquid formulation by

considering the following attributes. First, EC5026 is an

uncharged molecule with a low pH-independent solubility of

that PEG 400 in combination with even very low

concentrations of PEG 3350 produces a semisolid matrix

26

which is hard and not easily deformed. The semisolid matrix

forms after a heated solution of the two PEGs cools to room

temperature. The combination of PEG compounds in a ratio of

80% PEG 400 and 20% PEG 3350 was chosen for the

semisolid matrix formulation because the ratio of the two PEG

compounds provides the physical rigidity to prevent leaking,

analysis showed no crystal formation, EC5026 remained in

solution in the dosage form, and the process was suitable for

transfer to a compounding pharmacy where the clinical drug

product was to be made for Phase 1a clinical trials. Therefore,

the EC5026 drug product for use in initial clinical studies was

an extemporaneously compounded immediate-release capsule

for oral administration. Brieﬂy, EC5026 capsules were

prepared by ﬁlling a solution heated at 70 °C consisting of

EC5026 drug substance dissolved in 80% PEG 400/20% PEG

<

0.1 mg/mL in aqueous systems. Therefore, the equilibrium

solubility of EC5026 in various media was screened to aid the

development of preclinical formulations and a dissolution

method (Table S1). Second, the permeability (P_a_p_p) of

EC5026 using a colorectal adenocarcinoma cell line (Caco-

−

6

2

) is 26.4 × 10 cm/s indicated that the compound is well

absorbed (Table S2). Therefore, per the Biopharmaceutics

Classiﬁcation System (BCS), EC5026 is classiﬁed as a BCS

Class 2 compound (high permeability and poor solubility over

the physiologic pH range), and its absorption rate is limited by

3350 (w/w) into size 0, white opaque/white opaque, hard-

gelatin capsules. The capsules were hand ﬁlled by volume with

the heated solution and allowed to cool prior to capping. On

the basis of an in-use stability study at both accelerated (40

25

either solubility or dissolution or both.

Poor solubility of BCS Class 2 compounds limits the

formulation choices and drug product dosage forms that can

deliver the drug to the systemic circulation. Prior experience

with structurally similar compounds to EC5026 indicated the

drugs need to be in solution when dosed otherwise they will

not be absorbed, which is directly related to poor solubility.

This directed a formulation eﬀort seeking a fully solubilized

drug substance in a dosage form for both preclinical toxicology

studies and use in the clinic. If dissolution is too fast, the

compounds can also be recrystallized in the stomach.

Therefore, liquid-ﬁlled hard capsules were the initial target

for development. Since EC5026 has good solubility in PEG

°

4

C/75% RH) and long-term (25 °C/60% RH) conditions for

weeks, these EC5026 drug products were found to be stable,

but capsules were stored at 15−30 °C at the clinical site until

dosing following preparation at the clinical pharmacy in

accordance with pharmacy compounding procedures. Compo-

sitions of the EC5026 capsules, as well as the function and

quality standard for each component of the dosage forms, are

given in Table 3 (the detailed preparation of EC5026 is

described in the Supporting Information). This procedure

could be considered a low technology hot melt where, even if

the compound were not soluble in the PEG matrix, its glass-

like consistency precludes crystal formation.

4

00 (Table S1), eﬀorts focused on capsules ﬁlled with the drug

substance EC5026 dissolved in PEG 400. Several diﬀerent

capsules were evaluated, but all of the ones acceptable to the

contract laboratory were aﬀected by long-term exposure to

PEG 400. The most prevalent incompatibility was leaking, with

capsule deformation as a distant second problem. Overall, the

output from these studies showed that none of the capsules

tested were compatible with PEG 400. To address leaking of

PEG 400 from capsules, we investigated novel PEG-based

formulation using combinations of various PEG compounds

based our prior experience and that of others which suggested

BIOCHEMCAL MECHANISM OF ACTION

■

Degradation of Fatty Acid Epoxides (EpFA). By weight,

well over 50% of the world’s drugs act on the arachidonic acid

cascade; only the COX and LOX pathways of the cascade have

been exploited, and these drugs act by blocking the production

or action of predominantly inﬂammatory chemical mediators.

Of these, the COX branch has been the most studied and

predominantly exploited by NSAIDs and COXIBs. In contrast,

the sEHI work on the more recently discovered and largely

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Table 3. Compositions of 0.5 mg and 8 mg EC5026 Product

located at C-terminus, while the phosphatase domain is located

at N-terminus.

quantity per capsule

In addition, the urea of the inhibitors mimics the transition

19

0

.5 mg

8 mg

quality

state of the epoxide hydration in sEH (Figure 5 A). The

component

EC5026

strength

function

API

standard

0.50 mg

8.0 mg

in house

PEG 400

435.6 mg

429.6 mg solubilizer

Ph Eur,

JP

PEG 3350

108.9 mg

1 each

107.4 mg matrix former

USP

in house

Licaps capsules

1 each

dosage form

encapsulation

white opaque, size

a

0

total capsule ﬁll

weight

545.0 mg

a

Manufactured by Capsugel (Greenwood, SC). Abbreviations: API =

active pharmaceutical ingredient, JP = Japanese pharmacopeia; PEG =

polyethylene glycol; Ph Eur = European Pharmacopeia; USP =

United States Pharmacopeia

Figure 5. (A) The crystal structure (PDB ID: 4OD0) of human sEH

bound with TPPU (cyan, stick) showed that Tyr383 and Tyr466

formed tight hydrogen bonding with the carbonyl oxygen of the urea

and Asp336 formed a tight hydrogen bonding with the urea

hydrogens. (B) The crystal structure revived the long hydrophobic

binding channel at the C-terminus domain of human sEH.

analgesic and inﬂammation-resolving CYP450 branch of the

cascade. Of these, the epoxides of arachidonic acid,

eicosapentaenoic acid, and docosahexaenoic acid, abbreviated

EETs, EEQs, and EDPs, respectively, are generally analgesic

and anti-inﬂammatory mediators known collectively as EpFA.

One could alter the pathway by increasing precursor

polyunsaturated fatty acids, by stimulating the biosynthesis of

EpFA or the release of EpFA from phospholipids, by using

mimic natural bioactive EpFA, or by stabilizing the biologically

oxygen of the urea forms two tight hydrogen bonds with

tyrosine 383 and tyrosine 466. In addition, both urea

hydrogens strongly interact with aspartate 336 (Figure 5A).

The resulting polarization of the urea may lead to an

aspartate−urea salt bridge stabilized by the two hydrogen

bonds with the urea. The binding pocket of sEH resembles a

long hydrophobic channel (Figure 5B); therefore, the binding

pocket generally cannot tolerate the polar or charged

substituent, such as a pyridyl group, on 1,3-disubstituted

3

active EpFA as reported here with sEHI. The sEH is a very

active enzyme with a high k , a low K , and in some tissues of

cat

m

higher abundance than its putative EpFA substrates, and thus a

powerful inhibitor with high target occupancy is needed. The

sEHI discussed here ﬁt these criteria resulting in inhibition of

the target sEH and the resulting increase in EpFA (Figure 4).

Thus, the sEHI are increasing tissue levels of the natural

EpFA resulting in a variety of generally positive eﬀects. Among

these positive eﬀects are a reduction in both inﬂammatory and

5

urea. Interestingly, the recent crystal structure of human sEH

with TPPU also showed an extra binding pocket next to the α

position of the piperidinyl amide of the TPPU as predicted

12

earlier by Kim. This observation was further supported from

5

,19

SAR studies (EC5019 vs EC5023 in Table 1).

Also, Lee et

2

7

30

neuropathic pain.

al. revealed that the binding pocket of sEH is promiscuous,

and the left side of the pocket can tolerate chemical structures

of various sizes. The tunnel appears to breathe or even open

to accept bulky groups. Therefore, EicOsis synthesized a new

series of sEHI with a methyl substituent at the alpha-carbon of

Structure and Inhibitor Binding to Enzyme. Several X-

31

ray crystal structures of human sEH complexed with urea-

19,28,29

based sEHI have been determined.

The human sEH

structures revealed that the epoxide hydrolase domain is

Figure 4. Roles of EETs and sEH in the arachidonic acid cascade. Similar products arise from other polyunsaturated fatty acids. EETs are epoxides

of arachidonic acid, and DHETs are the corresponding diols.

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3

4

the piperidinyl amide of EC5023, resulting in a very potent

EC5026 (Table 1).

that they were exceptionally potent. We have since observed

a lack of eﬃcacy in preclinical models with high reactive

oxygen species. Later studies resulted in the discovery that

stabilization of EpFA by sEHI moves the ER stress response

back toward cell survival and homeostasis and away from acute

Catalytic Enzyme Assays. Multiple assays have been

developed to determine the potency of the sEHI, which

includes early radiolabel assays, high-throughput colorimetric

and ﬂuorescent substrate assays, an LC-MS/MS assay with

35

inﬂammation and cell death. Thus, a unifying mechanism

was found for the combined action of EpFA and sEHI. ER

stress has been found to be an underlying contributor to every

disease state where EpFA are eﬀective to date, and therefore,

the presence of ER stress in a disease suggests that sEHI

should be eﬀective.

32

natural substrates, and FRET displacement assays. These and

4

,33

other assays have been discussed in reviews. In EicOsis, the

ﬂuorescent substrate assay was used as a primary screening

assay because of the robustness and throughput of the assay.

However, the ﬂuorescent substrate screening assay has limits in

distinguishing extremely potent compounds (IC ≤ 5 nM)

Selecting a Clinical Path. The sEHI stabilize whatever

EpFA are released and biosynthesized at the time they are

administered. Since various EpFA have slightly diﬀerent

biological action and potencies, the eﬀects of sEHI are

anticipated to vary somewhat with the individual treated, their

diet, and their physiological state. However, reversing the shift

of the ER stress pathway away from initiation of inﬂammation,

pain, and other disease states and toward resolution seems a

common eﬀect of stabilizing and increasing EpFA. As indicated

by Table S6 and in several review articles, this fundamental

mechanism underlies many disease states ranging from hepatic

steatosis to senescence and aging, and more possible clinical

targets are being found as the role of ER stress in disease

becomes better understood. Thus, each of the companies

involved in the development of sEHI or mimics of EpFA has

the “problems of the rich” in selecting a good commercial

target and a viable clinical path from among many possibilities.

50

3

2

eﬀectively. Therefore, the potencies of the identiﬁed sEHI

were further validated with a FRET displacement assay,

radiometric assay, and LC-MS/MS based assay with natural

substrates. Recent studies revealed that drug-target residence

time is a better predictor of the in vivo activity of sEHI. This is

particularly important when natural substrates have a very high

apparent aﬃnity for the enzyme. Therefore, the drug-target

residence time of the sEHI which can be determined by the

FRET displacement assay was also included in our inhibitor

19,32

selection criteria.

The availability of recombinant sEH of

human and other preclinical species and the robust high-

throughput assays allowed rapid SAR study of sEHI.

Combining these data with a rapid determination of PK/

ADME identiﬁed a promising candidate for eﬃcacy testing in

1

vivo.

PHYSIOLOGICAL MECHANISM OF ACTION

̂

For example, Arete Therapeutics initially selected hypertension

for a short acting drug candidate, although the standard at the

time in the ﬁeld of hypertension was once or twice a day

dosing. Numerous published studies demonstrated the eﬃcacy

of sEHI in treating hypertension driven by the renin−

■

History of EpFA Action. sEHI allowed for the elucidation

of the biological activity of the transient EpFA, and it was

discovered that EpFA are anti-inﬂammatory, pro-resolving,

antihypertensive, and analgesic. Although the receptor for

EpFA has not been identiﬁed, preclinical models of disease

have identiﬁed multiple biological actions that contribute to

the eﬃcacious properties of EpFA. These actions include

preventing or reversing endothelial cell dysfunction, altering

the immune response, and reducing ER stress. These activities

result in the regulation of cellular stress by stabilizing

mitochondrial function, reducing reactive oxygen species, and

shifting the ER response toward maintenance of homeostasis

and away from severe activation of inﬂammatory pathways and

cell death.

10,36

angiotensin system.

However, for many patients, hyper-

tension can be eﬀectively controlled by several combination

therapies delivered by drugs that appear quite safe and often

inexpensive. Thus, this type of hypertension is a very hard

target to address commercially with new chemistries. Since

sEHI control many of the comorbidities of diabetes, Arete

̂

Therapeutics thought diabetes may be a good target and tried

in their Phase 2a clinical trial to pick up eﬃcacy on both

diabetes and hypertension but failed to get clinically and

economically relevant eﬃcacy in either indication. In

retrospect, we now know that ER stress is stimulated by high

glucose. Diabetes is an attractive target as a growing and

massive market, and certainly sEHI can address many of the

comorbidities of diabetes, but diabetes itself is a diﬃcult

clinical path and comes with expensive and long-term clinical

trials.

ER Stress and Mitochondrial Dysfunction as a

Unifying Mechanism. This mechanism explains why there

are so many possible indications for sEHI. We predict when

ER stress and/or mitochondrial dysfunction is involved, sEHI

administration will be beneﬁcial. The original biological

10

activity found for sEHI was the reduction of blood pressure.

Considering that a search for endothelium derived hyper-

polarizing factors had been in progress for years, EETs

appeared to ﬁt the criteria for an endothelium-derived

hyperpolarizing factor (EDHF). However, with the ability to

block the rapid hydrolysis of EpFA with sEHI, new roles for

both were quickly found including attenuating inﬂammation,

and beneﬁcial eﬀects in treating COPD, sepsis, stroke, cardiac

contractile function, cardiac hypertrophy, and others, which is

Problems caused by cardiac ischemia have long been

considered potential targets. EicOsis initially considered

COPD and asthma as indications as well, but GSK was

already moving down this path. Atrial ﬁbrillation also was

considered since it is common, and one can envision relatively

37

short and well-deﬁned clinical trials. Atrial ﬁbrillation also is a

precursor to atrial ﬁbrosis and cardiac hypertrophy, which are

both major markets but involve expensive and long clinical

1

37

summarized in a recent review paper. This of course raised the

trials. However, we were swayed toward pain by the

concern among critics if there was any disease where the sEHI

were ineﬀective. Since neuropathic pain remains largely an

unmet medical need, and we needed to demonstrate there was

a disease where the sEHI had no eﬃcacy, we evaluated them

on neuropathic pain rodent models and were surprised to ﬁnd

observation that sEHI not only were powerful analgesics in

inﬂammatory pain models where they synergized with and

reduced the gastrointestinal toxicity of NSAIDs and COXIBs,

but they also were analgesic in neuropathic pain models.

Neuropathic pain remains an unmet medical need, and it is

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Figure 6. (A) In a chronic constriction injury model of neuropathy in male SD rats, EC5026 blocked pain measured with a von Frey assay

mechanical withdrawal thresholds). The eﬃcacy of EC5026 was superior to pregabalin at 10−20 fold lower doses over the time course

(

represented as area under the response curve (AUC) over 6 h post dose. The AUC was calculated with the trapezoidal method for each animal and

the average of the group reported ± SEM (n = 6−8/group). Kruskal−Wallis one way analysis of variance on ranks, H = 21.011 with 3 degrees of

freedom with Dunn’s Method post hoc. EC5026 vs PEG 300 (p ≤ 0.001), EC5026 vs pregabalin (30 mg/kg) (p = 0.039). (B) The same eﬀective

dose of 3 mg/kg EC5026 was able to block withdrawal pain in morphine dependent rats (male SD rats, n = 4/group). Rats were made morphine

tolerant with twice daily 10 mg/kg subcutaneous injections of morphine for 10 days. Morphine was stopped and after 18 h of withdrawal the rats

were assessed and administered EC5026 or vehicle by oral gavage and tested at 30 min and 1 h post treatment. Painful withdrawal baseline scores

(

von Frey assay) were normalized to zero (zero is the painful state). EC5026 relieved opioid withdrawal pain compared to vehicle controls. For

both graphs scores are calculated as the percent improvement over the painful baseline: the score per time point/painful baseline score *100

calculated for each animal and averaged ± SEM.

poorly handled by existing medications. There is a long history

of pharma failures with the neuropathic pain indication.

However, we were encouraged by the evidence that we could

successfully treat severe equine laminitis, a neuropathic pain

condition, with sEHI. This gave us conﬁdence in moving from

models into patients and from rodents into humans. Some

neuropathic pain conditions also lend themselves to relatively

short-term trials. With both in-house expertise and strong

collaborators in the neuropathic pain area, we selected this

indication. Because of the increasing appreciation that the

opioid crisis in the United States is devastating both on a

personal basis and economically, EicOsis was lucky in ﬁnding

nondilutive resources to follow the neuropathic pain

indication.

mouse model, where TPPU has shown brain-to-plasma ratio of

17.2−21.7% in mice.

38

The available treatments for neuropathic pain have, at most,

modest eﬃcacy, and dose-limiting and/or serious side eﬀects,

including somnolence, decreased cognition, dizziness, and, in

some cases, respiratory depression. The commonly prescribed

drugs, such as gabapentin and pregabalin, can produce

39

signiﬁcant CNS side eﬀects. In addition, 50% or more of

patients with neuropathic pain and painful diabetic peripheral

neuropathy, in particular, are prescribed opioids to manage

their symptoms, with the associated risk of opioid misuse and

40

addiction. Patients requiring more than one drug class are at

a higher risk for these central eﬀects and for respiratory

depression, especially with opioids. CNS adverse eﬀects are

common to most currently available neuropathic pain drugs

and because of their impact on the day-to-day functioning of

patients often result in discontinuation of treatment. Even

NSAIDs, often prescribed for inﬂammatory pain including

osteoarthritis, present a signiﬁcant risk of GI-associated adverse

events. Novel drugs able to demonstrate eﬃcacy in

inﬂammatory and neuropathic pain without these side eﬀects

would provide an important advance for patients.

CLINICAL PATH TO TREATING INFLAMMATORY

AND NEUROPATHIC PAIN

■

Importance of Pain Drugs. We chose diabetic peripheral

neuropathy (DPN) for our initial neuropathic pain trials due to

the prevalence and growing incidence in the U.S. population,

but also because DPN is a serious condition that causes

substantial disability and often leads patients to use opioid

analgesics when they fail to get adequate pain relief from

currently approved neuropathic pain drugs. We are following

this with several other pain indications selected based on the

eﬃcacy of sEHI in rodent and nonrodent species and where

the indications seem to lead to opioid dependence. Our

biological data support the hypothesis that the sEHI can

control both inﬂammatory and neuropathic pain by acting

sEHI in general and EC5026, in particular, are devoid of the

side eﬀects produced by other drugs used for this therapeutic

41

indication. sEHI have a unique mechanism, blocking ER

stress and inﬂammation in the diseased state to shift responses

toward resolution and repair, providing eﬃcacy without the

side eﬀects seen with other mechanisms. On the basis of data

from preclinical studies, EC5026 has the potential to provide

eﬃcacy comparable to available treatments while avoiding

serious side eﬀects observed with other pain drugs, including

CNS-related adverse events (Figure 6). Previous sEHI,

including AR9281 and GSK2256294, that were tested in

2

1

peripherally. Therefore, our conclusion is that central

nervous system penetration is not necessary for analgesic

action, which also explains why sEHI work topically. However,

it does not exclude action at the dorsal root ganglia or the

CNS. Recently, it was reported that TPPU and EC5026

prevent neuroinﬂammation in the CNS of an LPS-induced

14

clinical trials showed minor adverse events. Unlike opioid

analgesics, sEH inhibition has shown no evidence thus far of

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4

tolerance, dependence, or additional liability with chronic

exposure in animals, avoiding the risk of addiction. sEHI do

not disturb cognition, or produce sedation preclinically, and

they do not produce the GI side eﬀects of NSAIDs, and

synergize with NSAIDs to block their side eﬀects, further

of EC5026 after a single oral dose of [ C]EC5026 at 3 mg/kg

in male and 1.5 mg/kg in female Sprague−Dawley rats. 96.5%

of the dose was recovered in the urine and feces through 168 h

postdose. On the basis of these data, it is believed that the

elimination of EC5026 is mainly attributed to hepatic

metabolism, likely through CYP450 metabolism.

14

supporting their unique potential as pain drugs.

Other important aspects of drug safety include the selectivity

of drugs for the target and the avoidance of drug−drug

interactions. Several of the available therapies for neuropathic

pain have been shown to cause drug−drug interactions, and

patients with neuropathic pain often require more than one

Safety Studies. EC5026 has been assessed in a series of

nonclinical toxicology studies consistent with ICH M3(R2):

Guidance on Nonclinical Safety Studies for the Conduct of Human

Clinical Trials and Marketing Authorization for Pharmaceuticals

(January 2010) including in vitro metabolism, genotoxicity

studies, CNS eﬀects in a modiﬁed Irwin assay, and single and

multiple repeat dose studies in the most sensitive species in

male and female rats and dogs. Twenty-eight day repeat-dose

GLP toxicity studies were also conducted to estimate safety in

humans.

Three dose levels of EC5026 were tested via oral gavage

were tested in GLP toxicity studies conducted in rat and dog

for 28 consecutive days in independent groups of male and

female Beagle dogs and Sprague−Dawley rats (6 dogs and 15

rats/sex). A control group was administered the vehicle (PEG

400) at an equivalent volume. Animals were sacriﬁced on Days

29 and 57 (4 dogs and 10 rats/sex each at “Main” and 2 dogs

and 5 rats/sex each at “Recovery” necropsies). The following

potential toxicities were evaluated: plasma drug levels and

toxicokinetics, mortality/morbidity, clinical observations in-

cluding electrocardiograms (dog only) and ophthalmologic

exams, functional observational battery (male rats only), body

weights, micronucleus evaluation for genetic toxicity (rats

only), food consumption, clinical pathology (hematology,

clinical chemistry, and coagulation), urinalysis, gross necropsy

observations, organ weights, and microscopic evaluation of

tissues.

4

2

drug class. EC5026 has proven selective for sEH when

screened against a large receptor panel in vitro, and additional

in vitro data described below suggest that the potential of

drug−drug interactions for EC5026 is low, further supporting

the safety of EC5026 and its unique proﬁle as a therapeutic for

5

neuropathic pain.

IND-Enabling Studies. Species selection for dose-range

ﬁnding toxicology studies is generally determined based on

species comparisons of rates of metabolism and on whether

unique metabolites are generated in humans. These studies

involve the use of microsomal enzymes and/or hepatocytes

from rodents, canine, and primates. Rat and dog were selected

as the toxicological species due to similarities in PK and

metabolite formation. Studies to identify clinically relevant

toxicological species based on metabolism proﬁles and to

predict human equivalent doses based on clearance rates are

described below.

In vitro metabolism studies were conducted in mouse, rat,

dog, monkey, and human hepatocyte incubations at SEKISUI

XenoTech and demonstrated the formation of seven putative

metabolites (hydroxylated EC5026 and their corresponding

dehydrated products) that were separated by HPLC and

detected by mass spectrometry. All seven metabolites were

detected in the mouse, rat, monkey, and human hepatocyte

incubations, while only six metabolites were observed in dog

hepatocytes. On the basis of the in vitro metabolite proﬁles, it

is likely that EC5026 is mainly metabolized by CYP450

enzymes.

Considering the main route of metabolism is likely through

CYP450s, inhibition and induction of CYP450s were measured

after incubation of 7 μM EC5026. Of seven major CYP450

isoforms assessed for inhibition, EC5026 had little to no

inhibitory eﬀects except for CYP2C9 and CYP2C19. At 7 μM,

EC5026-mediated inhibition was about 25% and 34% for

CYP2C9 and CYP2C19, respectively. An in vitro study was

conducted to assess the inductive eﬀects of EC5026 on

CYP1A2, CYP2B6, and CYP3A4 by using three diﬀerent

batches of cryopreserved human hepatocytes. CYP1A2,

CYP2B6, and CYP3A4 are known to be induced via three

mechanistically distinct mechanisms. Treatment of cultured

human hepatocytes with EC5026 up to 30 μM had little or no

eﬀect (i.e., < 2-fold change and/or <20% of the positive

control) on CYP1A2, CYP2B6, and CYP3A4 mRNA levels.

These results strongly suggest that EC5026 is not a potent

CYP inhibitor or inducer. Clinically eﬀective concentrations

measured at the highest dose tested (24 mg) in human clinical

trials achieved 301 ng/mL, or 9.4× lower than the

concentrations used in this study. Therefore, potential CYP-

mediated drug interactions caused by EC5026 are not

expected.

With the available animal data via the 28 Day GLP toxicity

studies and eﬃcacy studies in preclinical species, a therapeutic

index (TI) was estimated based blood level exposures to

EC5026. For this estimation there was no toxic dose in 50% of

the population (TD50) observed so the dose for the No

Observed Adverse Eﬀect Level (NOAEL) was used. To

correlate to this level the minimum dose with observed eﬃcacy

was used rather than the eﬃcacious dose in 50% of subjects

(ED ). The Area Under the Curve after the last dose

5

0

(AUC ) in ng*h/mL exposure for the NOAEL was divided

last

by the AUC_l_a_s_tof the minimum eﬃcacious dose previously

determined in a rat chronic constriction injury model of

neuropathic pain.

^T^I⁼^A^U^Clast^N^O^A^E^L^/^A^U^ClastEfficacy_m_i_n_i_m_u_m

This resulted in a 37−111× therapeutic index based on rat

and dog exposures, respectively.

In addition, Panigrahy et al. reported that EETs promote

tumor growth. In particular, EETs can stimulate multiorgan

metastasis and escape from tumor dormancy in several tumor

43

mouse models. EETs promoted metastasis by triggering

secretion of VEGF by the endothelium, which was critical for

EETs’ cancer-stimulating activity. The stimulation of meta-

stasis by EETs is due to their action at the secondary

(metastasis) site and not due to their eﬀect on the cells of the

primary tumor. The authors failed to show EETs-promoted

growth of the primary tumor and at the site of metastasis using

sEH-null mice while exogenously administrated EETs promote

them where systemic EET level is twice than the sEH-null

Excretion: Distribution studies were contracted with QPS

Newark, DE) to characterize the rate and extent of excretion

(

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Figure 7. (A) C_m_a_xand (B) area under the curve (AUC) of EC5026 after single oral dose administration in humans. Following single oral

administration of EC5026 in healthy male and female subjects, plasma concentrations peaked rapidly with T_m_a_xobserved at 1.25 h across all dose

groups and declined slowly through the end of sampling (14 days, data not shown). Plasma concentrations following 0.5 mg EC5026 were below

the limit of quantiﬁcation for all subjects at all time. Linear exposure was observed in the highest concentration measured at a speciﬁc time.

mice. However, such high EET level in the sEH-null mice

cannot be achieved by sEHI.

subjects randomly assigned to receive EC5026 and two

subjects the placebo. Dose escalation was done in a stepwise

fashion following acceptable safety and tolerability of the

preceding dose(s), as determined by the safety review

committee. EC5026 was monitored in urine and plasma over

14 days. The study was completed with no drug-related safety

concerns or adverse eﬀects, and all doses were very well

tolerated. EC5026 was well absorbed with linear dose-

proportionality (Figure 7). Safety was demonstrated at ∼12×

the predicted eﬃcacious dose, and concentrations were 600×

higher than the predicted eﬃcacious concentrations deter-

mined from preclinical pain studies. The mean terminal half-

life of EC5026 in plasma was between 42 and 59 h at doses of

8−24 mg, consistent with predictions based on IND-enabling

toxicity studies, and suitable for once daily dosing. These

results are reassuring for the advancement of EC5026 into

multiple ascending dose (MAD) Phase 1b studies. EicOsis is

planning to start a Phase 1b MAD clinical trial in healthy

volunteers in the second half of 2021, followed by two nested

Phase 1b MAD studies in patients with two chronic pain

conditions that are anticipated to start in 2021. These MAD

Phase 1b studies will evaluate the safety and tolerability of

three escalating dose regimens of EC5026, administered as a

single oral dose daily, for 7 consecutive days. Each dose

regimen will be tested ﬁrst in the healthy volunteer population

before being tested in the two chronic pain populations.

Rationale for the Anticipated Therapeutic Index.

With the assumption that the in vivo IC₉₀of EC5026 in

humans is similar to the in vitro IC₉₀(0.62 nM), the eﬀective

plasma AUC required to maintain target inhibition over 24 h is

estimated to be ≥892 nM·min (362 ng·min/mL). For a daily

dosing regimen, the eﬀective total AUC of EC5026 can then

be calculated to be 6 ng h/mL. After a single oral dose,

administration of 2 mg EC5026 exceeded these concen-

trations; therefore, we predict that the eﬃcacious dose level

will be <2 mg administered once daily and the therapeutic

index >12x based on the highest concentration tested to date

in human, 24 mg.

More importantly, GSK was also aware of this issue and

concerned about the role of EETs and sEH in VEGF signaling

and expression. Therefore, GSK measured VEGF concen-

trations to ensure human safety in their Phase 1 clinical trial

and found no signiﬁcant changes in VEGF concentrations from

baseline to day 15 in the repeat dose subjects. Overall, serum

VEGF concentrations were not increased and actually trended

lower in subjects who received the higher dose. Meanwhile,

near maximal sEH inhibition (98−99%) was observed for the

14b

doses following two-week repeated dosing. Considering the

reasons listed above, the results from using mouse models of

cancer are not likely to translate to human cancers, and no

such concern was raised by the FDA when EC5026 moved on

in the IND application process to Phase 1a clinical trial.

Despite this, long-term studies are still necessary to fully assess

the potential eﬀects.

Fast Track. In April 2020, the FDA granted Fast Track

designation to EC5026 for the treatment of neuropathic pain.

The FDA’s Fast Track process is intended to facilitate the

development and expedite the review of new therapies to

address an unmet medical need in the treatment of a serious

condition. Neuropathic pain is estimated to aﬀect 7−10% of

the general population, substantially aﬀecting day-to-day

functioning and quality of life of patients. Currently available

treatments for neuropathic pain remain ineﬀective and are

associated with frequent adverse eﬀects and poor tolerability.

The FDA’s Fast Track designation recognizes the potential of

EC5026 to address the pressing need for eﬀective, non-

addictive alternative drugs for pain management, decrease the

use of prescription opioids, and reduce the opioid abuse

epidemic. The Fast Track program enables drug companies to

have early and frequent communication with the FDA

throughout the drug development and review process, and

confers important beneﬁts, including the potential eligibility

for Priority Review of a New Drug Application.

Phase 1a Clinical Trial. A Phase 1a single ascending dose

study to investigate the safety, tolerability, and PK of EC5026

in healthy volunteers was recently completed at PPD in Austin,

TX (ClinicalTrials.gov Identiﬁer: NCT04228302). Oral

EC5026 was administered in ﬁve escalating single dose

regimens (0.5−24 mg) in 40 healthy male and female subjects,

eight subjects in each cohort, equal male and female with six

Metabolism. The abundant metabolites found in human

blood after a single oral dose of EC5026 in Phase 1a trial were

hydroxylated products on carbons that are susceptible to

oxidation by CYPs as predicted from a previous metabolism

44

study of the related sEHI compound TPPU and previous

microsomal and hepatocyte metabolism studies prepared from

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Figure 8. Chemical structures of metabolites of EC5026 identiﬁed from human blood in Phase 1a clinical trial. Amide or urea cleavage was not

observed.

Figure 9. Hulahalla (4-year old female thoroughbred), shown above, before (left) and after (right) a ﬁve-day treatment of once daily t-TUCB at 0.1

mg/kg. Hulahalla presented with acute laminitis and was treated with standard of care for 3 days with little eﬀect. Her condition became extremely

painful, and given the lack of response to the medications and worsening of the condition, humane euthanasia was being considered as the last

resort to relieve pain and suﬀering. At this point, daily t-TUCB was administered in addition to her standard of care treatment. Over the course of

the ﬁve-day treatment, her pain level decreased, and no signs of adverse eﬀects were observed both from clinical exams and evaluation of blood

work. Hulahalla continued to do well, and laminitis has not reoccurred.

rat, dog and monkey with EC5026 (Figure 8). Of note, there

were no detectable cleavage products of the urea moiety or

deacylation products of the hindered amide. Neither

glucuronides formed from conjugation of the oxidative

metabolites nor the N,N’, or diglucuronides formed from N-

glucuronidation of the ureas as seen with the urea

triclocarban were detected in human blood. These

glucuronide metabolites would be predicted to be rapidly

excreted into the urine where they are found.

Candidates” speciﬁcally for new human health drugs; however,

EicOsis is also pursuing a second sEHI in a diﬀerent patent

class, t-TUCB or EC1728 (Figure 2), for eﬃcacy in horses and

companion animals with neuropathic and arthritic pain.

Animal health represents a major market, but more relevant

to human health, demonstration of eﬃcacy in these studies

validates the ability to translate preclinical eﬃcacy studies to a

heterogeneous human patient population with diverse disease

ideologies. Translating success in rodent pain models to

humans has proven diﬃcult in the past. This may be due to

vast genetic diﬀerences in species, or possibly the use of

models where pain is generated mechanically, genetically, or

clinically. At EicOsis, we have had the advantage of close

collaboration with a veterinary school where we can evaluate

sEHI on veterinary patients with real disease states rather than

models, patients presenting with complex comorbidities, and

patients of four signiﬁcantly diﬀerent mammalian orders. The

sEHI successfully relieving neuropathic and inﬂammatory pain

certainly raises conﬁdence that translation of this work to pain

patients in another mammalian orders will be successful.

Possibly the most visual demonstration of the use of sEHI in

4

5

44

Future Plans. As a small company, EicOsis must focus on a

path to the clinic targeting neuropathic and inﬂammatory pain.

However, stabilization of EpFA regulates a homeostatic

mechanism of ER stress and has the potential for treating

multiple disease states as mentioned above. Hopefully, given

the extensive library of highly potent compounds held by

EicOsis, other promising indications can be followed. In

addition to the conditions discussed previously, additional

indications could include reduction of postsurgical pain,

chemotherapy, and CAR-T induced tumor growth and

46

metastasis due to cell debris, modulation of cytokine storms

47

associated with sepsis and COVID-19, and inﬂammation of

48

49

the central nervous system.

animals resulted from research by Guedes in horses with

Equine laminitis and Treating Companion Animals.

The purpose of the ACS symposium this report is associated

with was to highlight “First-Time Disclosures of Clinical

laminitis. Laminitis is a painful inﬂammatory condition that

shifts to a neuropathic pain resulting from initial inﬂammation

of the hoof laminae. It is often of idiopathic origin and presents

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as both chronic and acute duration, either of which often ends

in euthanasia of the horse due to pain and inability to stand.

After discovering that sEH is upregulated in laminitic joints,

expressing the relevant enzymes to antibodies and enzyme

assays for monitoring them supported this biological work.

Demonstration of the in vivo eﬃcacy of sEHI by a variety of

international scientists demonstrated their potential roles as

pharmaceuticals. As indicated by the patent literature,

numerous companies followed these leads with both classical

SAR driven and very innovative high-throughput approaches,

leading to potent sEHI. The extensive information on the

biology and chemistry of the EpFA came largely from

government support through competitive funding and a spirit

of openness and collaboration among academic and govern-

ment scientists.

The development of the sEHI class was based on knowledge

of enzymology and a methodical stepwise synthesis and

screening approach over many years enabling the discovery of

the initial lead sEHI. Parallel tracks were taken in the academic

and industrial paths toward structural optimization. Key to the

success of the academic path in medicinal chemistry was

simple synthesis, rapid feedback from quantitative enzyme

assays, rapid analysis of parent molecules in the blood

following oral administration, technology to monitor blood

eicosanoids as indicators of eﬃcacy and target engagement,

and, of course, a rigorous ﬁlter to only move the most

promising compounds forward into animal pain models. The

resulting university use patents were supplemented by later

composition of matter patents allowing EicOsis to spin out of

the University of California. With several SBIR grants from

NIEHS, then the NINDS Blueprint Development Grant, and

now a NIDA Heal Grant and NCI SBIR, EicOsis was able to

develop the independent composition of matter IP described

here, obtain an IND and FDA Fast Track Status, and complete

human Phase 1a human trials. This example of sEHI

development is among the ﬁrst successful examples to date

of the NIH-academic collaborative approach to drug develop-

ment. The NIH-academic-industry collaboration illustrated

here will add to the range of current approaches in drug

development and to the diversity of new therapeutics that are

so desperately needed.

5

0

Guedes implemented emergency measures to treat a

laminitic horse scheduled for euthanasia with t-TUCB after

all available treatments failed. After 5 days of treatment, this

horse made a complete recovery with no recurrent laminitis

4

9

(

Figure 9). Since this initial case, seven other laminitic horses

showed improvement in disease after having been treated with

50

t-TUCB when all other treatments failed.

In another example, dogs with naturally occurring osteo-

arthritis were treated with sEHI for 5 days and had signiﬁcant

reduction in pain, as determined by a questionnaire quantifying

pain behaviors and recorded by a blinded technician,

51

compared to placebo-treated animals. t-TUCB was selected

for use in animals over EC5026 due to potency on companion

animal enzymes.

Fundamental Concepts Advanced by This sEHI

Project. The mammalian sEH was discovered investigating

the rodent metabolism chemistry of an agricultural chemical.

Although it was discovered in an applied project, the enzyme

research was driven by fundamental curiosity primarily in the

academic sector with inhibitors of sEH and mimics of EpFA

initially prepared as research tools. However, with translating

the sEHI to the clinic, the scientists involved have made a

number of fundamental advances. Since the sEH is so eﬃcient

at hydrolyzing most EpFA, the ﬁrst application of this work

was in allowing researchers to evaluate the role of EpFA in a

number of physiological systems where previously the

metabolism was so fast the biological results were not

reproducible. As the sEHI were increasingly used in vivo,

they illustrated new and often unexpected involvement of

EpFA in biological systems. These discoveries continue a

broad front. For example, in nutrition they illustrate that many

of the attributes of dietary omega-3 fatty acids may be through

their epoxide metabolites. The most recent example of

unexpected activities comes from the observation that sEHI

prevent and ameliorate several CNS diseases in rodent models.

They were also valuable in dissection of the crosstalk between

predominantly inﬂammatory pathways such as those driven by

the COX and the LOX enzymes. Using dual inhibitors and

inhibitor combinations, this has been extended to the

interaction of sEHI with the PPARs. The sEHI are an

excellent example of mimicking the transition state(s) along

reaction coordinates to design inhibitors and the value of

kinetic treatments to describe this interaction. The slow-tight-

binding nature of sEHI and often their slow kinetic oﬀ rates

illustrated the value of target occupancy in describing potent

inhibitors. This slow oﬀ rate of sEHI from the target enzyme

has been used to illustrate the role of target mediated drug

EXPERIMENTAL SECTION

■

General Synthetic Procedures. This synthesis was performed by

Adesis, Inc. All reagents and solvents were obtained from commercial

suppliers and were used without further puriﬁcation. All reactions,

unless otherwise described, were performed under an inert

atmosphere of dry nitrogen. Melting points were determined on an

1

13

OptiMelt melting point apparatus and are uncorrected. H NMR,

C

1

9

NMR, and F NMR spectra were recorded at 600 or 400, 150, and

1

2

376 MHz, respectively. H− H D NMR was recorded at 400 MHz.

ATR FT-IR, UV−vis, and elemental analyses were determined at

Robertson Microlit laboratories, Ledgewood, NJ. Ultraviolet

absorbance spectrum was obtained on a PerkinElmer Lambda 35

UV−vis spectrometer. Mass spectra were measured by an Agilent

Inﬁnity 1290 LC with Agilent 6150 Quadrupole MS using

electrospray (+) ionization. All ﬁnal products synthesized to >93%

purity as determined by HPLC or LC/MS. HPLC analysis was

performed using Agilent 1100 LC. HPLC conditions; wavelength 210

nm, bandwidth 4, column: SorbTech C18AQ, 2.1 × 50 mm, 3 μm;

gradient method from 95:5 to 5:95 water/MeCN (0.1% formic acid in

both) in 14 min with a 4 min hold at 95% acetonitrile, 0.7 mL/min

52

disposition (TMDD) in explaining both eﬃcacy and PK.

Recent work illustrates a key role of lipid chemical mediators in

regulating endoplasmic reticulum stress, resolving inﬂamma-

tion and controlling pain in general. Hopefully, there will be

more fundamental insights as EC5026 and other sEHI move

toward the clinic.

CONCLUSION

■

(retention time 6.81 min). LC-MS analysis was performed using

Research by numerous academic scientists around the world

discovered a plethora of biological eﬀects mediated by the

CYP450 branch of the arachidonate cascade due, in part, to the

availability of high-quality EpFA and sEH inhibitors to stabilize

them. A variety of technologies ranging from cloning and

Agilent 1260 with Bruker timeTOF instrument. HPLC conditions;

column: Phenomenex Kinetex C18, 21. X150 m, 1.7 μm; gradient

method from 98:2 to2:98 water/MeCN (0.1% formic acid in both) in

10 min. EC5026 was synthesized to >99% ee as determined by chiral

HPLC Analysis (Agilent 1100 LC with a Chiralpak OD column, 25

1

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J. Med. Chem. 2021, 64, 1856−1872

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

Scheme 1. Scale-up Non-GMP Synthesis of EC5026

a

Reagents and conditions: (a) triphosgene, tert-butyl 4-amino-piperidine-1-carboxylate, Et N, DCM, −78 °C to rt, overnight; (b) 4 N HCl in

3

MeOH, rt, overnight; (c) 7, Jones reagent, acetone, 0 °C to rt, 2.4 h; (d) EDCI, Et N, DCM, rt, overnight.

3

cm x 4.6 mm, 10 μm). Optical rotation was measured using

PerkinElmer polarimeter 343. Experimental details for the synthesis

and characterization of standards of EC5026 metabolites 1 and 2 are

available in the Supporting Information .

Scale-Up Synthesis of EC5026. The scale-up non-GMP

synthesis of EC5026 is shown in Scheme 1

solution of CrO (263 g, 2.63 mol) in 500 mL of water and 250 mL of

3

conc. H SO ) while maintaining the temperature below 5 °C. During

2

4

the addition, chromium salts began to precipitate out, either forming a

gum on the bottom of the ﬂask of a golf size ball. The addition was

considered complete when an orange/brown color persisted (620 mL

of Jones reagent)). After removal of the cooling bath, the reaction

mixture was stirred for additional 2.5 h at ambient temperature. The

reaction was quenched by adding 50 mL of IPA (50 mL), and the

reaction mixture was stirred overnight. The solution was decanted and

concentrated under reduced pressure. All solids were dissolved in

water (1.3 L) and transferred to a separatory funnel. After tert-butyl

methyl ether (750 mL) was added, the layers were separated. The

remained organic layer was further washed with water (250 mL). The

combined aqueous layer was extracted with tert-butyl methyl ether

tert-Butyl 4-(3-(3-ﬂuoro-4-(triﬂuoromethoxy)phenyl)ureido)-

piperidine-1-carboxylate (4). A solution of 3-ﬂuoro-4-

(

triﬂuoromethoxy)aniline (3) (250 g, 1.28 mol) and Et N (260

3

mL, 1.86 mol) in DCM (500 mL) was added dropwise over 4 h to a

thick slurry of triphosgene (171 g, 0.576 mol) in DCM (500 mL) in a

dry ice/acetone bath while maintaining the temperature below −64

°

C. The cold bath was removed, and the mixture was warmed to rt

and stirred for 30 min. The reaction was cooled to −78 °C, and a

slurry of tert-butyl 4-amino-piperidine-1-carboxylate (386.4 g, 1.92

(750 mL), and the organic layer was washed with water (200 mL). All

mol) and Et N (260 mL, 1.86 mol) in DCM (1.2 L) was added in

3

combined organic layers were dried over anhydrous Na SO and

2

4

small portions over 3 h while maintaining the temperature below −65

concentrated under reduced pressure. The material from two

reactions of equal size was combined and distilled under reduced

pressure (Teﬂon pump, 20 Torr, 80−85 °C) to give (S)-2-

°C. The reaction was warmed to rt and stirred overnight, at which

point LCMS indicated a mixture of compound 3 (major), as well as

both symmetrical ureas. The reaction mixture was washed with 10%

aq HCl (4 × 1 L) The combined organic layer was dried over Na SO

methylbutanoic acid (7, 234 g, 72% yield) as a colorless liquid,

2

4

20

[

∝] = +19.2 (c = 1.671 g/100 mL chloroform).

D

and concentrated under reduced pressure to give a crude compound 4

as a viscous yellow oil (800 g) which was used in the next step

without further puriﬁcation.

(

S)-1-(3-Fluoro-4-(triﬂuoromethoxy)phenyl)-3-(1-(2-

methylbutanoyl)piperidin-4-yl)urea (EC5026). A mixture of (S)-2-

methylbutanoic acid 7 (175 g, 1.71 mol, 1.3 equiv). EDCI (327 g,

1

-(3-Fluoro-4-(triﬂuoromethoxy)phenyl)-3-(piperidin-4-yl)urea

1

.71 mol, 1.3 equiv), Et N (260 mL, 1.86 mol, 1.4 equiv), and

3

(

5). to a solution crude compound 4 (800 g) in MeOH (1.5 L) was

compound 5 (425 g, 1.32 mol, 1 equiv) in DCM (6 L) was stirred

overnight at room temperature, at which point LC-MS indicated

added a solution of 4 N HCl in MeOH (1.8 L), which was prepared

with 600 mL of conc. HCl and 1.2 L of MeOH. The reaction mixture

was stirred at room temperature overnight at which point LC-MS

indicated the reaction was complete. The MeOH was removed under

reduced pressure and the residue was slurried in a mixture of water

∼

75% conversion to the desired product. 10% aqueous HCl solution

2 L) was added to the reaction ﬂask, and stirring was continued for 5

(

min. The mixture was transferred to a 22 L separatory funnel

containing 4 L of 10% aq HCl solution. After stirring for 10 min, the

layers were separated. The organic layer was washed with 10% HCl (4

(500 mL) and DCM (250 mL) and ﬁltered. The ﬁltrate was

transferred to a separatory funnel and the layers were separated. The

aqueous layer was transferred to a 5 L 4-neck ﬂask and basiﬁed to pH

L) followed by saturated aq. Na CO solution (2 × 6 L). The organic

2

3

layer from two reactions of the same scale were combined, dried over

anhydrous Na SO , and concentrated to dryness under reduced

1

2 by adding 300 mL of 50% aq NaOH solution dropwise. After

stirring for 10 min, the precipitates were ﬁltered and washed with

water (500 mL) followed by heptanes (500 mL). The product was

initially dried in a convection oven overnight at 40 °C. The material

from two reactions of similar size was combined and triturated with

2

4

pressure. The residue was slurried in EtOAc (2 L) and

reconcentrated. EtOAc (2 L) was added to the residue, the vacuum

was stopped, and the bath temperature was raised to 60 °C. A clear

yellow solution was obtained. The heat was turned oﬀ, and the

solution was cooled with rotation to ambient temperature. After no

visible solids were conﬁrmed, EtOAc was removed in vacuo until

solids began to form. The vacuum was terminated, and the mixture

was stirred overnight at ambient temperature and pressure. The solids

were collected by ﬁltration and dried in a convection oven at 40 °C

overnight to give EC5026 (526.6 g, 49% yield, > 99% purity) as a

white solid. The ﬁltrate was concentrated under reduced pressure to

give a yellow waxy solid. After triturating with heptanes (400 mL), the

resulting solid was recrystallized twice with EtOAc (250 and 150 mL)

1

5% DCM in heptanes (2 L). The solid was dried overnight in a

convection oven at 40 °C to give compound 5 (778 g, 96% yield over

2

1

steps) as a white powder. H NMR (400 MHz, CDCl ) δ 7.43 (dd,

3

J = 12.1, 2.6 Hz, 1H), 7.17 (t, J = 8.6 Hz, 1H), 6.97 (ddd, J = 8.8, 2.5,

1

3

1

.5 Hz, 1H), 6.69 (br s, 1H), 4.77 (br s, 1H), 3.81−3.70 (m, 1H),

.07 (td, J = 12.6, 3.3 Hz, 2H), 2.73−2.65 (m, 2H), 1.98 (br dd, J =

2.3, 2.8 Hz, 2H), 1.38−1.28 (m, 2H).

(

S)-2-Methylbutanoic acid (7). To a solution of (S)-2-methyl-

butan-1-ol 6 (140 g, Sigma 65980-100 ML, ≥ 95%ee, 1.59 mol, 1

equiv) in 1.4 L of acetone was added dropwise Jones reagent (a

1

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J. Med. Chem. 2021, 64, 1856−1872

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

to give additional EC5026 (63.6 g, > 99% purity) for an overall yield

of 55%.

Pain Attenuation in a Chronic Constriction Injury Model of

Neuropathy. Male SD rats underwent a chronic constriction injury

surgery where 4 ligatures were loosely tied around the sciatic nerve.

The rats were allowed to heal for 14 days and tested for allodynia in a

von Frey assay. At 21 days post injury rats were treated via oral gavage

with PEG 300 (vehicle), EC5026 or pregabalin as indicated and

followed over a 6-h time course. The AUCs were calculated with the

trapezoidal method for each animal and the average of the group

reported ± SEM (n = 6−8/group). Kruskal−Wallis One Way

Analysis of Variance on Ranks, H = 21.011 with three degrees of

freedom with Dunn’s Method post hoc. EC5026 vs Peg300 (p ≤

Final Puriﬁcation. A 12 L 4-neck ﬂask was rinsed with absolute

EtOH before use. Absolute EtOH (1 L) was added to the ﬂask, the

heat was turned on, and EC5026 (1034 g) was added in portions with

stirring. Once all the solid had been added, additional EtOH was

added in portions until a clear solution was obtained (1.45 L). The

heat was turned oﬀ and DIUF water was added dropwise until a

cloudy solution was obtained (1.38 L). The mixture was then

reheated to obtain a clear solution and left to cool gradually with

stirring. Once the solution became cloudy, the mixture was seeded

and stirring was continued for 2 h. The resulting solids were ﬁltered

and washed with water (500 mL). The solids were dried in a

convection oven at 40 °C overnight to give EC5026 (854 g, 82.6%

0.001), EC5026 vs pregabalin 30 (p = 0.039).

Blocking Withdrawal Pain in Morphine-Dependent Rats. Male

SD rats were made morphine tolerant with twice daily treatment of 10

mg/kg s.c. morphine. After 10 days the morphine was withheld, and

opioid withdrawal pain was measured with a von Frey assay 18 h after

the last morphine dose. The opioid withdrawal baselines decreased

recovery, 99.7% purity, 97.5% ee) as a white solid. mp 148.8−150.0

1

°

(

2

6

1

C, H NMR (600 MHz, CDCl ) δ 8.04 (d, J = 38.3 Hz, 1H), 7.53

3

ddd, J = 12.4, 6.0, 2.5 Hz, 1H), 7.15 (t, J = 8.6 Hz, 1H), 7.04−7.00

m, 1H), 5.42 (d, J = 8.1 Hz, 1H), 4.66−4.46 (m, 1H), 4.05−3.85 (m,

̈

49% from pain-free naive baseline scores. Rats were then dosed with

vehicle or EC5026 (n = 4/group) via oral gavage and assessed for

mechanical withdrawal thresholds at 30 min and 1 h post dose.

Mann−Whitney Rank Sum Test, U Statistic = 10.0, T = 90.0 n(small)

H), 3.21 (td, J = 12.1, 2.7 Hz, 1H), 2.90−2.73 (m, 1H), 2.68 (p, J =

.8 Hz, 1H), 2.33−2.17 (m, 1H), 2.04−1.88 (m, 1H), 1.71−1.58 (m,

H), 1.47−1.37 (m, 1H), 1.24−1.15 (m, 2H), 1.11 (dd, J = 27.9, 6.7

=

8, n(big) = 8, P(exact) = 0.021.

1

3

Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). C NMR (151 MHz, DMSO-d ) δ

6

1

2

73.54, 154.01, 153.6 (d, J = 246.1 Hz), 141.24 (d, J = 10.4 Hz),

28.39 (d, J = 13.3 Hz), 120.12 (q, J = 256.8 Hz), 113.57, 113.55,

05.65 (d, J = 23.6 Hz), 46.36, 43.48, 35.60, 32.77, 31.86, 31.66,

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01886.

■

*

1

9

6.59, 17.17, 11.54. F NMR (CDCl3) δ −59.25 (d, J = 3.8 Hz),

+

−

127.19 (t, J = 7.5 Hz). MS (ESI) m/z = 406.2 (M + H ). Anal.

(

C H F N O ·0.95H O): cald. C, 51.17; H, 5.94; N, 9.95. found. C,

18

23

4

3

2

0

Data for the full characterization of EC5026; EC5026

drug product preparation; P-gp inhibition and in vitro

absorption of EC5026; CYP induction and inhibition by

EC5026; Tables S1−S7 (PDF )

51.11; H, 5.80; N, 9.80. [∝] = +10.4 (c = 0.506 g/100 mL EtOH).

D

EC5026 Metabolite Standard Synthesis. 1-(3-Fluoro-4-

(

triﬂuoromethoxy)phenyl)-3-(1-(3-hydroxy-2-methylbutanoyl)-

piperidin-4-yl)urea (1, Most Abundant EC5026 Metabolite). To a

solution of the 1-(3-ﬂuoro-4-(triﬂuoromethoxy)phenyl)-3-(piperidin-

Molecular formula strings (CSV)

4-yl)urea 5 (100 mg, 0.31 mmol) and 3-hydroxy-2-methylbutanoic

acid (40 mg, 0.34 mmol) in DMF (3 mL) were added PyBOP (208

mg, 0.40 mmol) followed by Et N (66 μL, 0.47 mmol) at 0 °C. The

3

AUTHOR INFORMATION

Corresponding Author

■

reaction mixture was allowed to slowly warm to room temperature

and stirred overnight. The solvent was removed in vacuo, and the

residue was puriﬁed by column chromatography (3:7 hexanes-EtOAc

containing 5% MeOH) to give the title compound, 111 mg (85%,

Sung Hee Hwang − EicOsis Human Health Inc., Subsidiary of

EicOsis LLC, Davis, California 95616, United States;

orcid.org/0000-0002-9891-928X ; Phone: (530) 338-

0070; Email: shhwang@eicosis.com

1

purity = 93.75%) as a white solid. mp 53.9−63.5 °C. H NMR (600

MHz, DMSO-d ): δ 8.77 (d, J = 30.9 Hz, 1H), 7.67 (d, J = 13.3 Hz,

6

1

3

1

H), 7.39 (t, J = 8.9 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 6.43−6.35 (m,

H), 4.65−4.53 (m, 1H), 4.27−4.16 (m, 1H), 3.96−3.86 (m, 1H),

.75−3.62 (m, 2H), 3.22−3.11 (m, 1H), 2.84−2.65 (m, 2H), 1.92−

Authors

Bruce D. Hammock − EicOsis Human Health Inc., Subsidiary

of EicOsis LLC, Davis, California 95616, United States;

orcid.org/0000-0003-1408-8317

Cindy B. McReynolds − EicOsis Human Health Inc.,

Subsidiary of EicOsis LLC, Davis, California 95616, United

States; orcid.org/0000-0003-3309-1773

Karen Wagner − EicOsis Human Health Inc., Subsidiary of

EicOsis LLC, Davis, California 95616, United States;

orcid.org/0000-0002-5238-7847

Alan Buckpitt − EicOsis Human Health Inc., Subsidiary of

EicOsis LLC, Davis, California 95616, United States;

orcid.org/0000-0001-6643-5208

Irene Cortes-Puch − EicOsis Human Health Inc., Subsidiary

of EicOsis LLC, Davis, California 95616, United States;

orcid.org/0000-0002-3639-5046

Glenn Croston − EicOsis Human Health Inc., Subsidiary of

EicOsis LLC, Davis, California 95616, United States;

orcid.org/0000-0002-6352-0922

.73 (m, 2H), 1.52−1.12 (m, 2H), 1.09−0.88 (m, 6H). MS (ESI) m/

−

z: 420.1557 (M − H) .

1

-(3-Fluoro-4-(triﬂuoromethoxy)phenyl)-3-(1-(2-hydroxy-2-

methylbutanoyl)piperidin-4-yl)urea (2, Minor EC5026 Metabolite).

To a solution of the 1-(3-ﬂuoro-4-(triﬂuoromethoxy)phenyl)-3-

(piperidin-4-yl)urea 5 (100 mg, 0.31 mmol) and 2-hydroxy-2-

methylbutanoic acid (40 mg, 0.34 mmol) in DMF (3 mL) was

added PyBOP (208 mg, 0.40 mmol) followed by Et N (66 μL, 0.47

3

mmol) at 0 °C. The reaction mixture was allowed to slowly warm to

room temperature and stirred overnight. The solvent was removed in

vacuo ,and the residue was puriﬁed by column chromatography (3:7

Hexanes-EtOAc containing 5% MeOH) to give the title compound,

1

07 mg (82%, purity = 98.92) as a white solid. mp 188.2−189.9 °C.

1

H NMR (600 MHz, DMSO-d ): δ H NMR (599 MHz, DMSO-d )

6

δ 8.75 (s, 1H), 7.67 (dd, J = 13.3, 2.5 Hz, 1H), 7.39 (t, J = 9.0 Hz,

1

4

H), 7.11 (d, J = 8.9 Hz, 1H), 6.38 (d, J = 7.6 Hz, 1H), 5.22 (s, 1H),

.75−4.13 (m, 2H), 3.75−3.66 (m, 1H), 3.25−3.18 (m, 1H), 2.82 (br

s, 1H), 1.91−1.77 (m, 3H), 1.74−1.53 (m, 2H), 1.35−1.22 (m, 4H),

−

0.80 (t, J = 7.3 Hz, 3H). MS (ESI) m/z: 420.1566 (M − H) .

Kin Sing Stephen Lee − Synthia LLC, Gualala, California

Chronic Constriction Injury Model of Neuropathy. All animal

95445, United States; orcid.org/0000-0003-4541-3063

experiments were performed based on protocols approved by the

Animal Use and Care Committee of Antibody, Inc. Sprague−Dawley

rats (male, 250−300 g) were purchased from Charles River

Laboratories.

Jun Yang − EicOsis Human Health Inc., Subsidiary of EicOsis

LLC, Davis, California 95616, United States; orcid.org/

0000-0001-8126-1728

1

869

https://dx.doi.org/10.1021/acs.jmedchem.0c01886

J. Med. Chem. 2021, 64, 1856−1872

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

William K. Schmidt − EicOsis Human Health Inc., Subsidiary

of EicOsis LLC, Davis, California 95616, United States;

orcid.org/0000-0001-5841-4810

(4) Morisseau, C.; Hammock, B. D. Epoxide hydrolases:

mechanisms, inhibitor designs, and biological roles. Annu. Rev.

Pharmacol. Toxicol. 2005, 45, 311−333.

(5) Lee, K. S. S.; Ng, J. C.; Yang, J.; Hwang, S. H.; Morisseau, C.;

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01886

Wagner, K.; Hammock, B. D. Preparation and evaluation of soluble

epoxide hydrolase inhibitors with improved physical properties and

potencies for treating diabetic neuropathic pain. Bioorg. Med. Chem.

2020, 28 (22), 115735.

Notes

(6) Pinot, F.; Grant, D. F.; Beetham, J. K.; Parker, A. G.; Borhan, B.;

This work was presented as Paper MEDI 217 in the “First-

Time Disclosure of Clinical Candidates” at the American

Chemical Society Meeting in San Diego, CA, 2019. The

authors declare no competing ﬁnancial interest other than

obvious association with EicOsis Human Health and the

University of California in some cases.

The authors declare the following competing ﬁnancial

interest(s): BDH, CBM, KW, AB, IC-P, GC, JY, WKS and

SHH are employees of EicOsis LLC and KSSL is an employee

of Synthia LLC.

Landt, S.; Jones, A. D.; Hammock, B. D. Molecular and biochemical

evidence for the involvement of the Asp333-His523 pair in the

catalytic mechanism of soluble epoxide hydrolase. J. Biol. Chem. 1995,

2

(

70 (14), 7968−7974.

7) Morisseau, C.; Goodrow, M. H.; Dowdy, D.; Zheng, J.; Greene,

J. F.; Sanborn, J. R.; Hammock, B. D. Potent urea and carbamate

inhibitors of soluble epoxide hydrolases. Proc. Natl. Acad. Sci. U. S. A.

1999, 96 (16), 8849−8854.

(8) Olearczyk, J. J.; Field, M. B.; Kim, I.-H.; Morisseau, C.;

Hammock, B. D.; Imig, J. D. Substituted adamantyl-urea inhibitors of

the soluble epoxide hydrolase dilate mesenteric resistance vessels. J.

Pharmacol. Exp. Ther. 2006, 318 (3), 1307−1314.

ACKNOWLEDGMENTS

■

(9) Liu, J. Y.; Tsai, H. J.; Morisseau, C.; Lango, J.; Hwang, S. H.;

This work was supported by the National Institute of

Environmental Health Sciences (NIEHS) SBIR Program

R43/R44 ES025598, National Institute of Neurological

Disorders and Stroke (NINDS) Blueprint Neurotherapeutics

Network UG3/UH3NS094258, National Cancer Institute

Watanabe, T.; Kim, I. H.; Hammock, B. D. In vitro and in vivo

metabolism of N-adamantyl substituted urea-based soluble epoxide

hydrolase inhibitors. Biochem. Pharmacol. 2015, 98 (4), 718−731.

(10) Imig, J. D.; Zhao, X.; Capdevila, J. H.; Morisseau, C.;

Hammock, B. D. Soluble epoxide hydrolase inhibition lowers arterial

blood pressure in angiotensin II hypertension. Hypertension 2002, 39

(

NCI) R43CA233371, and National Institute on Drug Abuse

NIDA) Award Number UG3DA048767.

(

2), 690−694.

(

11) Watanabe, T.; Schulz, D.; Morisseau, C.; Hammock, B. D.

High-throughput pharmacokinetic method: cassette dosing in mice

associated with minuscule serial bleedings and LC/MS/MS analysis.

Anal. Chim. Acta 2006, 559, 37−44.

ABBREVIATIONS USED

■

AEPU, 1-adamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]-

pentyl]}urea; t-AUCB, trans-4-[4-(3-adamantan-1-yl-ureido)-

cyclohexyloxy]-benzoic acid; AUDA, 12-(3-adamantan-1-yl-

ureido)dodecanoic acid; BCS, biopharmaceutics classiﬁcation

system; sEH, soluble epoxide hydrolase; DCC, dicyclohexyl

carbodiimide; DCU, dicyclohexyl urea; DPN, diabetic

peripheral neuropathy; ECD, endothelial cell dysfunction;

EDHF, endothelium-derived hyperpolarizing factor; EETs, cis-

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Movement to the Clinic of Soluble Epoxide Hydrolase Inhibitor EC5026 as an Analgesic for Neuropathic Pain and for Use as a Nonaddictive Opioid Alternative

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