Towards optimization of arylamides as novel, potent, and brain-penetrant antiprion lead compounds

Article abstract of DOI:10.1021/ml300454k

The prion diseases caused by PrP^Sc, an alternatively folded form of the cellular prion protein (PrP^C), are rapidly progressive, fatal, and untreatable neurodegenerative disorders. We employed HTS ELISA assays to identify compounds that lower the level of PrP^Sc in prion-infected mouse neuroblastoma (ScN2a-cl3) cells and identified a series of arylamides. Structure-activity relationship (SAR) studies indicated that small amides with one aromatic or heteroaromatic ring on each side of the amide bond are of modest potency. Of note, benzamide (7), with an EC₅₀ of 2200 nM, was one of only a few arylamide hits with a piperazine group on its aniline moiety. The basic piperazine nitrogen can be protonated at physiologic pH, improving solubility, and therefore, we wanted to exploit this feature in our search for a drug candidate. An SAR campaign resulted in several key analogues, including a set with biaryl groups introduced on the carbonyl side for improved potency. Several of these biaryl analogues have submicromolar potency, with the most potent analogue 17 having an EC₅₀ = 22 nM. More importantly, 17 and several biarylamides (20, 24, 26, and 27) were able to traverse the blood-brain barrier (BBB) and displayed excellent drug levels in the brains of mice following oral dosing. These biarylamides may represent good starting points for further lead optimization for the identification of potential drug candidates for the treatment of prion diseases.

Full text of DOI:10.1021/ml300454k

Letter
pubs.acs.org/acsmedchemlett
Towards Optimization of Arylamides As Novel, Potent, and Brain-
Penetrant Antiprion Lead Compounds
Zhe Li,†,‡,∥ Satish Rao,†,‡ Joel R. Gever,†,‡ Kartika Widjaja,† Stanley B. Prusiner,*,†,‡
and B. Michael Silber†,‡,§,⊥
‡
§
†Institute for Neurodegenerative Diseases, Department of Neurology, Department of Bioengineering and Therapeutic Sciences,
University of California, San Francisco, California 94143, United States
S
* Supporting Information
ABSTRACT: The prion diseases caused by PrPSc, an alternatively folded form of
the cellular prion protein (PrPC), are rapidly progressive, fatal, and untreatable
neurodegenerative disorders. We employed HTS ELISA assays to identify
compounds that lower the level of PrPSc in prion-infected mouse neuroblastoma
(ScN2a-cl3) cells and identified a series of arylamides. Structure−activity
relationship (SAR) studies indicated that small amides with one aromatic or
heteroaromatic ring on each side of the amide bond are of modest potency. Of note,
benzamide (7), with an EC50 of 2200 nM, was one of only a few arylamide hits with
a piperazine group on its aniline moiety. The basic piperazine nitrogen can be protonated at physiologic pH, improving solubility,
and therefore, we wanted to exploit this feature in our search for a drug candidate. An SAR campaign resulted in several key
analogues, including a set with biaryl groups introduced on the carbonyl side for improved potency. Several of these biaryl
analogues have submicromolar potency, with the most potent analogue 17 having an EC50 = 22 nM. More importantly, 17 and
several biarylamides (20, 24, 26, and 27) were able to traverse the blood−brain barrier (BBB) and displayed excellent drug levels
in the brains of mice following oral dosing. These biarylamides may represent good starting points for further lead optimization
for the identification of potential drug candidates for the treatment of prion diseases.
KEYWORDS: Neurodegenerative diseases, prion disease, Creutzfeldt−Jakob disease, amide, arylamide, SAR
lzheimer’s, Parkinson’s, and Creutzfeldt−Jakob (CJD)
prion diseases, we focused on finding compounds that lower
Adiseases as well as the frontotemporal dementias are the levels of PrPSc in RML prion-infected neuroblastoma
(ScN2a-cl3) cells.17 HTS of ∼53 000 compounds resulted in
the identification of 14 antiprion chemotypes, among which
arylamides were the largest class of hits (Silber et al., in
preparation). Herein, we report structure−activity relationship
(SAR) analysis and preliminary lead optimization efforts aimed
at improving the potency and physicochemical properties of the
original arylamide hits. This effort led to several potent
antiprion leads 17, 20, 24, 26, and 27, which also exhibited
excellent in vitro metabolic stability and excellent in vivo brain
exposure when dosed orally in mice.
To accompany all EC50 measurements, compound toxicity
toward ScN2a-cl3 cells was also evaluated using the fluorescent
probe calcein-AM.13 Several brain-penetrant leads from the 2-
aminothiazole (2-AMT) series, one of the 14 scaffolds, were
identified after preliminary SAR studies.18 Despite this success,
we discovered that the 2-AMTs suffered from generally poor
aqueous solubility, resulting in the need to use PEG400 in our
in vivo studies.19 To find superior leads, special attention was
devoted to improving the physicochemical properties,
especially solubility, of other chemical classes identified from
our screening campaign. Arylamides represent the largest single
considered prion disorders.1,2 An expanding body of evidence
argues that prions, or self-propagating proteins, cause many
different neurodegenerative illnesses. The human prion diseases
caused by the aberrant prion protein (PrPSc), which is formed
posttranslationally from the cellular prion protein (PrPC),
include kuru, CJD, fatal insomnia, and Gerstmann−Straussler−
̈
Scheinker disease.2,3 The conversion of the α-helical-rich PrPC
into an aberrant β-rich PrPSc conformer and accumulation of
PrPSc are primary pathogenic events in prion disease.4,5 PrPC-
to-PrPSc conversion can occur spontaneously, result from
inherited mutations in the gene encoding PrPC, or be triggered
by infection with exogenous PrPSc. Despite research efforts to
find treatments for CJD, no therapy exists to date.6,7 While the
exact mechanism of PrPC misfolding is unclear, it is known that
infectious prions can be transmitted and propagated in cell
cultures such as murine neuroblastoma (ScN2a) cell lines.8
Screening efforts using these cell cultures have been carried out
to discover antiprion compounds of different chemical
classes.9−15 However, most of these antiprion leads are not
drug-like and only demonstrate in vitro activity. To the best of
our knowledge, Compd B, a phenyl hydrazone analogue, is the
only small molecule reported to have efficacy in Rocky
Mountain Laboratory (RML) prion−infected mouse models.16
As part of our ongoing medicinal chemistry program toward
the screening and identification of potential therapeutics for
Received: December 17, 2012
Accepted: April 16, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml300454k | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
compound class from the ∼53 000 compounds screened. Many
of these simple aryl or heteroaryl amides (Supplementary
Figure 1) displayed only moderate potency (EC50 = 1000−10
000 nM). Additionally, the aryl/heteroaryl rings of these
amides form a highly conjugated system via the center amide
bond and likely adopt a flat coplanar conformation, a structural
feature often associated with poor solubility.20,21
One amide, 7, with moderate antiprion activity (EC50 = 2200
nM), has a basic N-benzylpiperazine substituent at the para
position of its aniline moiety (Figure 1). The piperazine group
than compound 7, having an EC50 = 520 nM. The N-
ethylpiperazine analogue (16) demonstrated further improve-
ment in antiprion potency (EC50 = 221 nM). These results
suggest that a larger N-alkyl group on the piperazine ring may
be favorable for potency. The next analogue in the series, N-
isopropylpiperazine analogue (17), was synthesized via stand-
ard amide coupling of commercially available 4-biphenylcarbox-
ylic acid (18a) and 4-(4-isopropylpiperazin-1-yl)aniline (19b)
(Scheme 1). As we had hoped, 17 showed further enhancement
of antiprion activity, displaying an EC50 = 22 nM, a 100-fold
increase over the original lead 7.
a
Scheme 1. Synthesis of Biarylamides
Figure 1. Selected arylamide hits and their antiprion potency.
has a pKa ≈ 8.5 and is protonated at physiologic pH, and thus
confers superior solubility for the piperazine-containing
molecule. We decided to explore the SAR on both sides of
the amide bond of 7 with the initial goal of improving its
potency (Figure 2). A closer survey of the initial actives showed
a
Reaction conditions: (a) HATU, DIEA, anhydrous DMF, rt, 12 h.
Given the promising potency of 17, we sought to explore the
SAR around the A- and C-rings (Figure 3; Table 1; 20−27)
Figure 3. SAR strategy for modifying A-ring and C-ring of biphenyl
lead 17.
Figure 2. SAR strategy for HTS hit compound 7.
that amides with one aryl/heteroaryl group on both sides of the
and initiated the synthesis of heterocyclic A-ring analogues in
order to examine the effects of pyridyl biphenyls on antiprion
potency. Three regioisomeric pyridyl congeners (20−22)
amide bond had only moderate antiprion potency (EC50
>
1000 nM; compounds 1−6 (Supplementary Figure 1) and 7−
10 (Figure 1)). In contrast, amides with at least one biaryl in a
fused or linear fashion displayed more potent antiprion activity
(EC50 < 1000 nM; e.g., compounds 11−14, Figure 1).
Hence, we decided to introduce a biaryl group to 7 on the
carbonyl side and preserve the piperazine group on the aniline
side of the amide for solubility considerations (Figure 2). In
addition to introducing the biaryl moiety, we also sought to
replace the bulky N-benzyl group on the piperazine with a
smaller alkyl group, such as methyl or ethyl. The reasons for
replacing the N-benzyl group are 2-fold. First, the N-benzyl
group is flexible and likely adopts a noncoplanar conformation,
which may be detrimental to antiprion potency (Silber et al., in
preparation). Second, the replacement of the bulky N-benzyl
with smaller alkyl groups should result in lead compounds with
lower molecular weight.
Considering the 10-fold improvement in potency demon-
strated by replacing the 4-ethoxy in 6 with a 4-phenyl to give
the biaryl 14, we decided to further explore this scaffold. In
addition, the inactivity of both the 3-ethoxyphenyl amide (8)
and the 3-bromophenyl amide (9) relative to the 4-
bromophenyl amide (10) also suggested that substitution at
the 4-position (i.e., 4-biphenyl analogues) might be favored
over the corresponding 3-biphenyl congeners. Consistent with
this SAR analysis, the N-methylated 15 was 4-fold more potent
Table 1. Antiprion Potency (ELISA) and Cell Viability
(Calcein AM) for Arylamide Analogues
ELISA
EC50 SEM calcein AM
R1
Y
Z
R2
(nM)
LD50 (nM)
n
15
16
17
20
21
22
23
24
25
26
27
phenyl
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
N
Me
Et
520 173
221 95
>5000
3
3
4
3
3
3
3
3
4
3
3
phenyl
>9000
phenyl
iPr
Et
22
4
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
2-pyridyl
3-pyridyl
4-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
365 70
243 29
743 25
165 31
348 129
677 195
411 123
334 59
Et
Et
iPr
iPr
iPr
iPr
iPr
CH
CF
CH
CF
CH
B
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ACS Medicinal Chemistry Letters
Letter
derived from 16 were synthesized according to Scheme 1 by
direct amide couplings of the corresponding pyridylbenzoic
acids (18b−d) and N-ethylpiperazineaniline (19a) (Supporting
Information). All three pyridyl analogues (20−22) suffered up
to a 3-fold potency loss compared to the parent biphenyl (16),
suggesting that an electron-deficient group is not preferred at
the terminal phenyl position. We also aimed to modify the C-
ring of 17 because the electron-rich aniline moiety may be
susceptible to oxidation mediated by CYP450 isozymes. Hence,
reducing the electron density of the phenyl ring may alleviate
the potential oxidation liability. Additionally, we also replaced
the terminal phenyl of the biphenyl with a 2-pyridyl group in
this C-ring pyridyl and fluoro-substituted C-ring series. To gain
access to the key N-isopropylpiperazinylpyridinylamines (19c
and 19d) and fluoro N-isopropylpiperazinylaniline intermedi-
ates (19e and 19f), appropriate halonitropyridines (28a and
28b) or difluoronitrobenzenes (28c and 28d) were reacted first
with N-isopropylpiperazine (29) to give the corresponding N-
isopropyl-4-(nitropyridinyl)piperazines (30a and 30b) or N-
(fluoro,nitrophenyl)-4-isopropylpiperazines (30c and 30d)
(Scheme 2). The nitro group (30a−d) was then selectively
to evaluate their PK profile and blood−brain barrier (BBB)
permeability, thus assessing their potential as CNS drugs. We
administered 8 compounds (16, 17, 20, 21, 23, 24, 26, and 27)
to mice, utilizing an abbreviated study protocol to obtain key
parameters such as Cmax and AUC in the brain, important
criteria for initial compound selection and prioritization for
efficacy studies. In general, for our single-dose PK studies, 10
mg/kg of each compound was administered by oral gavage.
Brain and plasma concentration were measured at 0.5, 2, 4, and
6 h after administration. The Cmax and AUC from time zero to
6 h (AUC0−6h) for both brain and plasma were obtained (Table
2). To further aid compound prioritization, in vitro microsomal
stability studies were also conducted using a standard protocol.
For biphenyl analogues, both the N-ethyl 16 (EC50 = 221
nM) and the N-isopropyl 17 (EC50 = 22 nM) displayed high
concentrations in brain, with 16 having 3-fold higher exposure
(Cmax and AUC) than 17. Because the N-isopropyl congener 17
is more stable (t1/2 > 60 min) in mouse hepatic microsomes
than the smaller N-ethyl analogue 16 (t1/2 = 19.0 min), the
lower brain exposure of 17 is likely caused by an absorption
and/or permeability difference between the two congeners.
Despite its lower brain exposure, 17 possesses higher Cmax
/
a
Scheme 2. Synthesis of 19c−f
EC50 and AUC/EC50 ratios than 16 due to its superior potency.
More importantly, both analogues displayed at least 10-fold
higher concentrations in brain than in plasma, typical of an
effective CNS drug. An intriguing difference was found between
the two close analogues 20 and 21; the 2-pyridyl congener 20
had much higher brain exposure (>4-fold Cmax and >7-fold
AUC) than the 3-pyridyl congener 21. Considering both
analogues had similar stability in mouse hepatic microsomal
preparations (t1/2 = 27.0 min for 20 and t1/2 = 29.2 min for 21)
(Table 2), this brain exposure difference may be due to
absorption differences. Surprisingly, while the terminal 2-
pyridyl biphenyl analogue (A-ring analogue) 23 exhibited much
lower brain exposure than either 16 or 20, it had superior
hepatic mouse microsomal stability (t1/2 > 60 min); the
reduced brain exposure may be due to poor BBB permeability
and/or poor dissolution of compound 23.
In summary, we have optimized a series of benzamides,
typified by 7 with moderate antiprion potency (EC50 = 2200
nM), to a series of potent biaryl amide leads, one (17) having
an EC50 of 22 nM. Additionally, several compounds (e.g., 17,
24, 26, and 27) demonstrated excellent metabolic stability in
hepatic microsomal preparations, superior in vivo brain
exposure, and highly favorable brain-to-plasma drug ratios.
These piperazine-bearing biarylamides represent an exciting
a
Reaction conditions: (a) CH3CN, reflux, 5 h (for 30a and 30b) or
K2CO3, HMPA, rt, 2 d (for 30c and 30d); (b) Pd/C, H2, MeOH, rt,
12 h.
reduced to the corresponding amines 19c−f by catalytic
hydrogenation over palladium carbon in methanol. Finally,
amide coupling of amines 19b−f with [1,1′-biphenyl]-4-
carboxylic acid (18a) or 4-(pyridin-2-yl)benzoic acid (18b)
furnished the C-ring-modified products 24−27 (see Supporting
Information). Unfortunately, these analogues showed much
reduced antiprion potency, with their EC50 values at least 15-
fold greater than the parent biphenyl analogue 17 (Table 1).
As a prelude to in vivo efficacy studies, we performed in vivo
mouse PK studies on the most active compounds in the series
Table 2. In vivo Pharmacokinetic Parameters (Single Oral Dose of 10 mg/kg) and in vitro Microsomal Stability for Arylamide
Analogues
microsomal stability
t1/2 in min (% remaining after 60 min
brain exposure
AUC(μM·h)
plasma exposure
incubation)
compd
Cmax (μM)
Cmax (μM)
AUC (μM·h)
mouse
human
16
17
20
21
23
24
26
27
7.95 2.40
2.52 0.26
6.23 0.43
1.44 0.13
0.09 0.05
6.00 0.75
17.3 1.18
6.75 0.95
25.4 6.23
7.48 0.77
24.2 4.39
3.32 0.05
0.28 0.11
22.3 1.20
39.8 9.84
28.7 7.43
0.37 0.20
0.27 0.01
0.28 0.03
0.21 0.02
0.32 0.05
0.34 0.18
0.65 0.07
0.46 0.04
1.09 0.40
0.97 0.03
1.05 0.21
0.25 0.03
1.20 0.16
1.26 0.46
1.46 0.00
2.28 0.09
19.0 (12)
>60 (51)
27.0 (23)
29.2 (24)
>60 (61)
>60 (57)
57.3 (48)
>60 (58)
>60 (66)
>60 (64)
>60 (50)
>60 (53)
>60 (77)
>60 (71)
>60 (76)
>60 (83)
C
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ACS Medicinal Chemistry Letters
Letter
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental and analytical data for compounds 17, 20−27,
and 30a−d and intermediates 19c−f; biological methods;
Supporting Figure 1. This material is available free of charge via
the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(S.B.P.) Phone: (415) 476-4482. Fax: (415) 476-8386. E-
mail: stanley@ind.ucsf.edu.
■
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Present Addresses
∥Global Blood Therapeutics, Inc., South San Francisco,
California 94080, United States.
⊥ELMEDTECH, LLC, San Francisco, California 94123, United
States.
Author Contributions
All authors have given approval to the final version of this
manuscript.
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Funding
This work was funded by grants from the NIH (AG021601,
AG031220, AG002132, and AG010770) and by gifts from the
Sherman Fairchild, Larry L. Hillblom, Rainwater Charitable,
and Lincy foundations as well as from the Fight for Mike
Homer Program, Robert Galvin, and Mary Jane Brinton.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Phillip Benner for preparing the animal dosing
solution and collecting samples for PK studies; Ms. Priya
Jaishankar for experimental assistance; Dr. Kurt Giles and the
staff of the Hunter’s Point animal facility for overseeing animal
studies; Dr. Adam Renslo for helpful discussions; Dr. John
Nuss for reviewing the manuscript; and Ms. Hang Nguyen for
editorial assistance.
ABBREVIATIONS
■
2-AMT, 2-aminothiazole; CJD, Creutzfeldt−Jakob disease;
DIEA, diisopropylethylamine; HATU, 2-(1H-7-azabenzotria-
zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
PrPSc, pathogenic isoform of the prion protein; RML, Rocky
Mountain Laboratory; ScN2a-cl3, murine neuroblastoma
clone3 cells
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