SAR studies of 4-acyl-1,6-dialkylpiperazin-2-one arenavirus cell entry inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.08.024

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R¹ and R⁴ scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R⁶ substituents and iterative R¹, R⁴ and R⁶ substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.

Full text of DOI:10.1016/j.bmcl.2019.08.024

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SAR studies of 4-acyl-1,6-dialkylpiperazin-2-one arenavirus cell entry
inhibitors
Michael B. Plewea, Landon R. Whitbyb, Shibani Naika, Eric R. Browna, Nadezda V. Sokolovaa,
Vidyasagar Reddy Gantlaa, Joanne Yorkd, Jack H. Nunbergd, Lihong Zhange, Birte Kalverame,
⁎
Alexander N. Freiberge,f, Dale L. Bogerb,c, Greg Henkela, Ken McCormacka,
a Arisan Therapeutics, 11189 Sorrento Valley Rd, Suite 104, San Diego, CA 92054, United States
b Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
c The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States
d Montana Biotechnology Center, The University of Montana, Missoula, MT 59812, United States
e Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555, United States
f Center for Biodefense and Emerging Infectious Diseases, The University of Texas Medical Branch, Galveston, TX 77555, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Arenavirus
Lassa
Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers.
Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-
dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I stu-
dies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In
subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated
to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory
activity, in vitro human liver microsome metabolic stability and aqueous solubility.
Junin
Machupo
Piperazinone
Entry inhibitor
The Arenaviridae family of viruses is comprised of a large number of
species, some of which are associated with acute hemorrhagic fevers
(HF) in humans.1–3 HF arenaviruses, including Lassa (LASV) and the
New World species Machupo (MACV) or Junin (JUNV), represent a
serious public health risk, especially in endemic regions of Africa and
South America.4–6 Due to the high mortality rates and limited ther-
apeutic options these HF viruses are classified as Category A pathogens
by the Centers for Disease Control (CDC).7 LASV, in particular, may be
responsible for up to 300,000 human disease cases per year.8–10 Mor-
tality rates for hospitalized LASV HF patients are 15–20% and survivors
often suffer permanent sequelae including bilateral hearing da-
mage.11–14 The nonspecific antiviral Ribavirin currently offers the sole
therapeutic option, however, its use is restricted to high-risk patients
due to its variable efficacy and potentially serious adverse effects.15–17
The development of potent and specific agents to treat Lassa and other
arenavirus hemorrhagic fevers is therefore, urgently needed.
that is highly-related to Category BSL4 arenaviruses including JUNV
and MACV, was used to identify a novel chemical series (including ST-
294) of arenavirus inhibitors.18 Cell-based assays specifically targeting
the arenavirus glycoprotein (GP) and cell entry, including pseudotyped
virus and cell–cell membrane fusion assays, have also been used to
identify or characterize the benzimidazole and 4-acyl-1,6-dialkylpi-
perazin-2-one chemical series, which share a common GP binding site
and mechanism of action.19–23
The initial report of the 4-acyl-1,6-dialkylpiperazin-2-one inhibitors
identified a set of substituents at the R1, R4 and R6 positions of the 4-
acyl-piperazin-2-one core (Fig. 1) that exhibited low micromolar to
submicromolar potency against LASV glycoprotein expressed in vesi-
cular stomatitis virus (VSV) pseudotyped viruses (pLASV).20,24,25 Addi-
tional investigation of the structure–activity relationships (SAR) of this
chemical series identified the carbonyl groups as significant activity
determinants and that the enantiomer possessing (S) configuration at C6
(R6 group attachment point) was approximately 15-fold more potent
than the (R)-enantiomer.26 The (S)-enantiomers of our initial 4-acyl-
1,6-dialkylpiperazin-2-one compounds 1 (16G8) and 2 (17D1) re-
present eutomers with pLASV EC50 values: rac-16G8 600 nM vs (S)-
16G8 300 nM and rac-17D1 500 nM vs (S)-17D1 250 nM.26,27 We
To mitigate the significant safety and logistical challenges asso-
ciated with utilizing Biosafety Level 4 (BSL4) arenaviruses in drug
discovery, a number of surrogate BSL2 assays have been developed. For
example, a screening campaign using the New World arenavirus
Tacaribe virus (TCRV), which is a BSL2 Clade B New World arenavirus
⁎ Corresponding author.
E-mail address: kenm@arisanthera.com (K. McCormack).
https://doi.org/10.1016/j.bmcl.2019.08.024
Received 20 April 2019; Received in revised form 10 August 2019; Accepted 11 August 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:MichaelB.Plewe,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.08.024

M.B. Plewe, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
1
(16G8)
2
(
17D1)
R6
R6
R4
R4
R1
R1
R1
R4
Phase I
Scheme 2. Reagents and conditions: (i) RCHO, MeOH, rt, 1 h, then NaBH4, 0 °C
to rt, 2 h; (ii) 2-(2-nitrophenylsulfonamido)acetyl chloride, NaHCO3, CH2Cl2, rt,
3 h; (iii) PPh3, DIAD, THF, under N2, rt, 2 h; (iv) PhSH, K2CO3, DMF, under N2,
rt, 3 h; (v) R4COOH, HOAt, 2,6-lutidine, EDCI, DMF, rt, 14 h.
LASV GP cell
fusion assay
R1 x R4
R6 x R1
position (Scheme 2) started with commercially available aminoalcohol
G. The R1 group was added using a stepwise reductive amination pro-
cedure with appropriate aldehyde entailing pre-formation of imine
followed by NaBH4 reduction (MeOH) to give intermediate H. Sub-
sequent N-acylation by treatment of H with the acid chloride of no-
sylglycine (NaHCO3, CH2Cl2) provided I. The 6-membered ring was
closed using an intramolecular Mitsunobu alkylation of the sulfona-
mide28 (PPh3, DIAD, THF) to give J. Nosyl deprotection using thio-
phenol and K2CO3 (DMF) and subsequent acylation of the free amine
with the appropriate carboxylic acid R4COOH (EDCI, HOAt, 2,6-luti-
dine, DMF) furnished compounds of type K. All compounds were pur-
ified by PTLC or flash chromatography and the purity and identity of all
compounds was assessed by LC/MS.
R3 R5
Phase II
pLASV, pMACV, pJUNV
HLM stability, solubility,
pTCRV vs Naꢀve TCRV
R1
R4
R6 x R4
R6
R1 x R4
Fig. 1. Initial 4-acyl-1,6-dialkylpiperazin-2-one starting points and optimiza-
tion flow scheme.
therefore utilized the (S)-enantiomers of R6 benzyl, phenethyl and other
substituents as starting points for further SAR exploration of this che-
mical series.
At R1 benzyl was favored over phenethyl or phenylpropyl sub-
stituents (Table 1, compare 6, 14 and 16), indicating a defined pre-
ference for the linker length at R1. A single aryl ring demonstrated
greater activity than naphthalene and biaryl groups (compare 6 with
10, 12, 13 and 15) and the identity of aryl substituents significantly
impacted activity while their positional effects were relatively weaker.
The incorporation of single methoxy or hydroxy groups revealed a
preference for meta-substitution but were well tolerated at any ring
position (4 vs. 8 and 9, 5 vs. 7). In contrast, halogen, phenoxy, cyano,
nitro, or basic nitrogen substitutions in the ring significantly reduced
activity at any position (see Supplementary data, Fig. S1). Overall, the
results indicated significant flexibility for incorporation of small lipo-
philic substituents or polar alcohols on the R1 benzyl ring.
In Phase I exploration R1 and R4 scanning libraries were synthesized
and screened for inhibition of LASV cell entry as determined in a
membrane cell–cell fusion assay at 50 nM (Fig. 1). In Phase II studies,
iterative SAR modifications at substituent positions R1, R4 and R6 were
evaluated in assays with pseudotyped pLASV, pMACV or pJUNV virions
expressing LASV, MACV or JUNV glycoproteins, respectively, in order
to identify broad-spectrum arenavirus cell entry inhibitors. Selected
compounds were subsequently characterized in human liver microsome
(HLM) metabolism and solubility assays (Fig. 1).
For R1 scanning library compounds we devised a synthetic route
enabling final step diversification at the R1 position starting with an
intermolecular Mitsunobu alkylation28 of t-butyl nosylglycine with Boc
protected aminoalcohol A to provide B (Scheme 1). The nosyl group
was cleaved (thiophenol, K2CO3, DMF) to yield the secondary amine C,
which was then coupled with the appropriate carboxylic acid R4COOH
(EDCI, HOAt, 2,6-lutidine, DMF) to give the tertiary amide D. Hydro-
lysis of the t-butyl ester (LiOH, t-BuOH/H2O/THF) followed by Boc-
deprotection with TFA (CH2Cl2) provided the amino acid E as the TFA
salt. Final step diversification at R1 was completed through a one-pot
reductive amination29/EDCI-mediated ring closure starting with inter-
mediate E.
As opposed to R1 substituents, the R4 acyl substituents demonstrated
SAR with strong preferences in both the identity and position of aryl
functionality (Table 1, Supplementary data, Fig. S2). The two most
active classes of aryl groups observed in the original piperazinone study
(the 2-heteroaryl (17–19, 22) and the 4-substituted phenyl (20, 21, 23)
groups) proved optimal in the R4 scanning library.20 Of note, in-
corporation of an alkoxy or dimethylamino group at the 4-position of
the phenyl ring resulted in a notable increase in potency for compounds
20, 21, and 23 while the absence of a 4-position substituent (28) and/
or the placement of a dimethylamino (29) or alkoxy (30) group at the 3-
position resulted in dramatic losses in activity. The addition of a 5-
methoxy group to the 2-indole substituent (compounds 17 vs. 26) also
provided a notable increase in potency. In contrast, the addition of a
single carbon linker (27) to the phenyl ring significantly decreased
activity.
A synthetic route enabling final step diversification at the R4
Following the individual R1 and R4 replacements, a small combi-
natorial library was synthesized containing three of the most active R1
substituents (compounds 3–5, Table 1) and R4 acyl groups (compounds
17, 18, and 20, Table 1). These compounds showed low-nanomolar
inhibition of LASV GP fusion with no loss of activity when the more
polar optimal substituents were combined in the same structure (com-
pounds 31–33, Table 2) suggesting that optimization for lipophilic ef-
ficiency (LipE) may lead to potent compounds with improved drug-like
properties. These initial scanning libraries allowed the identification of
promising R1 and R4 substituents as starting points to initiate optimi-
zation of broad-spectrum arenavirus inhibitory activity and drug-like
Scheme 1. Reagents and conditions: (i) PPh3, DIAD, THF, under N2, rt, 2 h; (ii)
PhSH, K2CO3, DMF, under N2, rt, 3 h; (iii) R4COOH, HOAt, 2,6-lutidine, EDCI,
DMF, rt, 16 h; (iv) LiOH, t-BuOH/H2O/THF, rt, 1 h, then 1 N aq. HCl; (v) TFA,
CH2Cl2, rt; (vi) RCHO, NaBH(OAc)3, Et3N, ClCH2CH2Cl, rt, 18 h, then EDCI,
NaHCO3, CH2Cl2, rt, 8 h.
2

M.B. Plewe, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 1
fold greater potency over benzyl (Table 3, compare 35, 40, 41 and 42).
However, R6 benzyl compounds exhibited greater solubility and meta-
bolic stability in HLM assays. Thus, while 42 was 2–4 fold less potent in
pLASV, pMACV and pJUNV assays than 41 it exhibited a substantially
increased HLM half-life of 59 vs 10 min and solubility of 36 vs 4 μM.
For Phase II SAR expansion we synthesized R6 (S) benzyl analogs
with a limited number of R4 substituents (5-methoxy, 5-methyl, and 5-
cyclopropyl indoles) to further explore R1 SAR (80 analogs) as well as a
limited number of R1 substituents (benzyl, m-methoxybenzyl, m-iso-
propoxybenzyl, or m-difluoromethoxybenzyl) to explore additional R4
SAR (68 analogs), respectively. Novel R6 SAR was also explored uti-
lizing a limited number of R1 and R4 substituents (64 analogs). In total
212 additional analogs were synthesized in Phase II modifications at R1,
R4 and R6.
SAR data of selected R1 (left panel) and R4 (right panel) substituent Phase I
scanning libraries indicating their % inhibition at 50 nM in the LASV GPC
cell–cell fusion assay.
Compd. R1
% inh. Compd. R4
50 nM
% inh.
50 nM
3
86
17
66
All Phase II analogs were first screened against pLASV and pMACV.
Those exhibiting EC50 values < 25 nM in both assays were subsequently
tested against pJUNV. Of the 132 of compounds displaying < 25 nM
potency in the pLASV assay, 118 were also < 25 nM against pMACV
and of those 108 were also < 25 nM against pJUNV. Comparative EC50
values and the range of activity spectrums for these Phase II analogs are
illustrated in Fig. 2. While a number of interesting pseudotyped-specific
SAR trends were observed, our focus was on the identification of
compounds exhibiting broad spectrum activity with EC50 values < 10
nM in all three assays (64 compounds).
4
82
18
61
5
6
7
8
9
81
71
64
61
60
19
20
21
22
23
59
58
56
54
50
To confirm that the activities observed in pseudotyped virus assays
translate to the inhibition of replicative arenaviruses the activities of
two compounds (35 and 42, Table 3) were initially tested and con-
firmed against BSL4 replicative LASV with EC50 values of 33 and
113 nM, respectively. To extend this observation to a broader range of
compounds we subsequently tested compounds exhibiting EC50 va-
lues < 25 nM against pLASV, pMACV and pJUNV in assays with both
pseudotyped (p-TCRV) and replicative Tacaribe (TCRV) virus, a BLS2
virus that is non-pathogenic to humans but highly related to JUNV.18 As
shown in Fig. 3 compounds exhibiting < 25 nM EC50 values against the
BSL4 pseudotyped viruses also exhibited comparable activities against
pTCRV. These data confirm comparable inhibition between the glyco-
protein-mediated cell entry of both the BSL4 and BSL2 viruses. Fur-
thermore, the data confirm the translation between pseudotyped and
replicative virus inhibition (R2 = 0.61, Fig. 3). Thus, the data further
validate the selection of cell entry inhibitors based on their activities in
pseudotyped virus assays.
10
58
24
45
11
12
13
53
51
36
25
26
27
40
26
18
At R1, SAR trends were observed in both antiviral activity and HLM
stability (Table 4). Benzyl groups with less polar meta- or ortho- sub-
stituents are consistently potent and broad spectrum (compounds 40,
43, 44, 45, 48, and 49) with a particular preference for lipophilic
ethers. Some polar R1 meta-benzyl substituents groups including alco-
hols (compounds 52, 56, and 59), retain broad spectrum activity de-
spite their increased polarity while others such as sulfonamides (54),
and moderately polar groups like 1H-pyrazolyl (53) and alkyl esters
(55) retain pMACV and pJUNV activity but lose activity against pLASV.
Both carboxylic acids and amides (compounds 60 and 61) are not tol-
erated for any viral species tested (see data for more analogs in
Supplementary data, Table S1).
14
15
34
33
28
29
15
14
16
31
30
0
Unfortunately, alkyl ethers at R1 pose a significant metabolic lia-
bility as demonstrated by the relative HLM stability of compounds 42
and 43 vs. 40, 44 and 45. Stabilized ethers with oxidation-resistant
alkyl groups (compounds 46 and 47) have more favorable microsome
stability but only the m-difluoromethoxy (46) group maintains broad-
spectrum activity. The broad-spectrum nature of m-difluoromethoxy
group is interesting in light of the poor pJUNV and pMACV activity of
more lipophilic m-trifluoromethoxy (47) analog and the general pre-
ference for alcohols as R1 substituents, as the difluoromethoxy group
has been reported to act as a hydrogen-bond donor and potential iso-
stere for alcohols.30
properties.
Phase II SAR exploration studies, including characterization of
broad-spectrum arenavirus (pseudotyped pLASV, pMACV and pJUNV)
activity, human liver microsome (HLM) metabolic stability and solu-
bility, were initiated with the synthesis of analogs containing benzyl or
phenethyl at R6, benzyl or m-methoxy benzyl at R1 and 5-methox-
yindole at R4. R6 phenethyl was found to exhibit approximately 2–4
3

M.B. Plewe, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 2
Analogs incorporating combinations of the most active substituents from the R1
and R4 scanning libraries and their respective EC50 values in the LASV GPC
cell–cell fusion assay.
R4
R1
Compd. EC50
(nM)
Compd. EC50 (nM) Compd. EC50
(nM)
Fig. 2. A 3-dimensional plot of EC50 values obtained in the pLASV, pMACV and
pJUNV assays for the analogs exhibiting < 25 nM (dark circles) or < 10 nM
(yellow circles) EC50 values in each of the pseudtoyped virus assays.
31
5
34
4
37
5
32
8
35
7
38
7
demonstrated sufficiently increased metabolic stability over 5-meth-
oxyindole analog (42), when paired with a benzyl R1. Of note, com-
pound 66 having 5-cyclopropylindole at R4 and m-difluoromethoxy
benzyl at R1 showed some improvement in metabolic stability but at the
same time was slightly less potent than the 5-methoxyindole analog 46
(see Table 4).
33
7
36
6
39
5
The 7-fold difference in potency against pLASV between 66 and 69
strongly suggested that it should be possible to pair 5-cyclopropylindole
and a suitable R1 group to obtain a broadly potent compound that
maintained the cyclopropylindole’s metabolic stability gains. While
other heteroaryl groups showed acceptable-to-good broad-spectrum
potency, such as quinolines 67 and 73 and benzothiophene 71, none of
them were found to endow stability or solubility advantages over the
indole. Further, the wide range of 4-substituted phenyls synthesized
showed significantly reduced broad-spectrum activity with few excep-
tions, e.g., the 4-piperidinylphenyl group (75), which while potent,
exhibited an HLM half-life of only 5 min. Polar substituents such as
sulfones (72, 74), nitriles (70), acetyl (65, 76) and a variety of other
groups were not tolerated on either phenyl or heteroaryl rings at any
position and lead to dramatically reduced potency (see Supplementary
data, Table S2). 5-methyl and 5-cyclopropyl indoles were thus selected
as the preferred R4 groups to generate R1 and R6 combinatorial com-
pounds optimized for stability and potency.
Both 6-indolyl (50) and N-alkyl 6-indolyl groups (51, 57, 58) also
demonstrate good broad-spectrum activity, but the highest activity is
derived from the unalkylated analog 50. This is likely due to the hy-
drogen bonding of indole NeH, similar to the phenols and –OCF2H
groups. The unalkylated indoles also possess above average metabolic
stability with 50 having an HLM half-life (T1/2) of 36 min, while in-
troduction of alkyl groups causes significant metabolic liability.
Novel R4 analogs were synthesized using the metabolically more
stable R1 groups benzyl and m-difluoromethoxy benzyl to further
evaluate potential metabolic liabilities stemming from the R4 sub-
stituent (Table 5). The majority of Phase II R4 analogs were designed to
explore substituents on the most potent aryl and heteroaryl R4 groups
(indoles, phenyls, and quinolines) identified in the Phase I exploration,
with the intention of further improving metabolic stability while re-
taining broad-spectrum potency. Although some sterically bulky 5-al-
koxy indoles showed dramatic improvements in metabolic stability
(compounds 62 and 63) broad-spectrum potency was lost. 5-methy-
lindole and 5-cyclopropylindole substituents (compounds 68 and 69)
In parallel with expansion of R1 and R4 SAR we explored additional
Table 3
Comparison of activity of R6 linker length and R1 substituents. Activity is given
as EC50 (nM) in the indicated pseudotyped virus assays. Cytotoxicity is given as
CC50 in Vero cells (7 days). HLM data is the observed half-life (T1/2 (min)) in a
human liver microsome assay. Aqueous solubility (µM) was measured at pH 7.4.
Compd.
n
X
p-LASV p-MACV p-JUNV CC50 (nM) HLM Solubility
35
40
41
42
2
1
2
1
OMe 1.74
OMe 6.9
2.39
3.6
1.77
2.52
5.8
4,500
3,300
7.7
9.8
4.9
Fig. 3. Logarithmic plot of pTCRV vs replicative TCRV (immunostaining assay)
EC50 values (R2 = 0.61) for analogs selected for EC50 values < 25 nM against
each of the pseudotyped viruses (pLASV pMACV and pJUNV) and HLM half-life
data.
7.4
H
H
3.3
2.13
11.52
> 30,000 10
> 10,000 59
4.4
8.64
4.87
36.3
4

M.B. Plewe, et al.
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Table 4
Phase II SAR of R1 substituents. Activities provided are EC50 values (nM) in the indicated pseudotyped virus assays. Cytotoxicity is given as CC50 in Vero cells
(7 days). HLM data is the observed half-life (minutes) in a human liver microsome assay. Aqueous solubility (µM) was measured at pH 7.4.
Compd.
R1
p-LASV
6.9
p-MACV
3.6
p-JUNV
2.52
CC50 (nM)
3,300
HLM
9.8
Solubility
7.4
40
42
43
44
45
46
47
48
8.64
2.92
1.33
2.37
0.94
5.8
11.52
2.08
1.54
2.88
1.85
> 25
2.85
4.87
3.04
1.91
3.78
0.75
12.23
1.56
> 10,000
> 10,000
41,000
59
37
12
8
36.3
5.8
6.9
49,000
5.7
> 30,000
> 30,000
> 30,000
59
72
25
7.2
5.6
5.45
11.3
49
50
5.56
6.26
1.73
11.16
2.68
> 100,000
5,000
27
36
24.2
0.733
10.21
51
52
53
4.44
2.81
22.1
1.32
3.17
1.38
3.14
6.91
1.65
> 30,000
79,500
13
9.2
11
48.8
6.8
> 30,000
N/A
54
55
> 25
23.6
5.71
5.4
3.27
1.8
N/A
N/A
N/A
12
> 30,000
6.9
56
57
2.42
3.7
2.50
2.88
5.38
N/A
10,200
18
17.2
3.9
> 30,000
5.3
58
2.7
2.03
2.6
> 100,000
13
7.5
59
60
4.09
1.01
1.85
N/A
39,000
N/A
29
30.2
N/A
> 25
> 25
N/A
61
> 25
> 25
N/A
N/A
N/A
N/A
R6 substituents whereby R4 (5-methoxy indole) and R1 (benzyl, m-
methoxybenzyl and m-difluoromethoxybenzyl) substituents were re-
stricted and novel R6 substituent analogs synthesized. The R6 position is
the most tolerant of different substitutions and a wide variety of either
lipophilic or polar charged groups were synthesized using either
Scheme 1, starting with the appropriate commercially available chiral
aminoalcohol, or Scheme 3 with the intention of increasing solubility
and metabolic stability concurrently. Thus, methyl (O-t-Bu)serinate (L)
was first acylated with R1COOH and EDCI (Scheme 3). Subsequent re-
duction of M using lithium aluminum hydride afforded aminoalcohol N.
Acylation of the amine with nosylglycine acyl chloride and magnesium
oxide gave intermediate O, which was cyclized under Mitsunobu
5

M.B. Plewe, et al.
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Table 5
Phase II SAR of R4 substituents. Activities provided are EC50 values (nM) in the indicated pseudotyped virus assays. Cytotoxicity is given as CC50 in Vero cells
(7 days). HLM data is the observed half-life (T1/2 (min)) in a human liver microsome assay. Aqueous solubility (µM) was measured at pH 7.4.
Compd.
R4
X
p-LASV
8.64
p-MACV
11.52
p-JUNV
4.87
CC50 (nM)
> 10,000
HLM
59
Solubility
36.3
42
H
62
63
H
H
9.38
> 25
> 25
> 25
4.235
> 30,000
N/A
137
246
8.6
> 25
14.6
64
65
OCF2H
H
2.34
25.4
19.52
> 25
3.8
> 100,000
> 10,000
43
5.3
> 25
120
15.5
66
OCF2H
2.12
6.81
4.05
10,400
77
6.2
67
68
69
OCF2H
2.29
1.95
14.6
2.99
0.52
2.68
> 25
29,700
13
10
H
H
21.52
> 25
11,200
119
125
7.7
8.3
> 100,000
70
H
> 25
> 25
> 25
16,000
N/A
10.8
71
72
H
H
5.96
12.53
> 25
4.46
9,040
N/A
42
5.7
> 25
> 25
N/A
67.7
73
OCF2H
2.31
3.59
4.78
> 100,000
16
9.5
74
75
76
OMe
OCF2H
H
> 25
4.68
> 25
3.98
> 25
8.82
N/A
N/A
5
> 100
8.8
46,700
> 100,000
> 25
> 25
> 25
31
> 100
conditions to afford piperazinone P. Treatment of the t-butyl ether with
4 N HCl in 1,4-dioxane gave free alcohol Q, which was first triflated
using triflic anhydride and finally reacted with a desired amine NHR'R''
to afford intermediate R. Finally, nosyl deprotection followed by the
amide coupling with the appropriate carboxylic acid R4COOH furnished
compounds of type S.
Additionally, the basicity of the amine plays an important role in de-
termining activity, as difluorinated piperidine analogs, which are more
lipophilic yet less basic, exhibit decreased activity compared to the
unfluorinated parent piperidine (compound 78 vs. 86 and 93). If the
site of fluorination is sufficiently removed from the basic nitrogen, as in
the 4-methylpiperidine (85) and its 4-trifluoromethyl analog (89), the
inductive withdrawal effect on basicity is minimized and the increased
lipophilicity makes 89 more potent.
For non-polar R6 substituents (Table 6) potency tracks well with
lipophilicity and the physical length of the group is unimportant
(compounds 77, 81, 82, 83, and 84). For R6 analogs bearing a basic
amine, a linker length of one carbon between the piperazinone and
amine (e.g., 79 vs 94) is preferred for activity. Overall lipophilicity of
the R6 amine is important for activity, as tertiary amines (90) with
fewer carbons than piperidine, and thus lower lipophilicity, resulted in
complete loss of potency. Larger amine groups themselves bearing polar
substituents such as sulfones and sulfonamides (88, 91), nitriles (92),
and even methoxy (87) groups all show significant losses in activity.
The HLM half-life of all compounds in this R6 exploration series
was < 50 min, except 79, which exhibited a half-life of 52 min. Pairing
the cyclopropylpiperidine of 79 with an R4 group that is less metabo-
lically labile than 5-methoxy indole could provide further improvement
in HLM stability. A number of additional R6 analogs did not exhibit
broad-spectrum potency and/or improved HLM stability (see
Supplementary data, Table S3).
Initial exploration at positions R3 and R5, as well as attempts to
6

M.B. Plewe, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 3. Reagents and conditions: (i) R1COOH,
EDCI, Et3N, DCM, rt, 16 h; (ii) LAH, THF, reflux, 16 h;
(iii) 2-(2-nitrophenylsulfonamido)acetyl chloride,
MgO, 4:1 THF/H2O, rt, 4 h; (iv) PPh3, DIAD, THF,
under N2, rt, 16 h; (v) 4 N HCl/1,4-dioxane, 70 °C,
4 h; (vi) Tf2O, Et3N, DCM, under N2, 0 °C, 1 h, then rt,
3 h; (vii) NHR’R’’, Et3N, THF, rt, 16 h; (viii) PhSH,
K2CO3, DMF, under N2, rt, 16 h; (ix) R4COOH, 2,6-
lutidine, EDCI, DMF, rt, 4 h.
Table 6
Phase II SAR of R6 substituents. R6 groups bearing amines were synthesized according to Scheme 3. Activities provided are EC50 values (nM) in the indicated
pseudotyped virus assays. Cytotoxicity is given as CC50 in Vero cells (7 days). HLM data is the observed half-life (T1/2 (min)) in a human liver microsome assay.
Aqueous solubility (µM) was measured at pH 7.4.
Compd.
R6
X
p-LASV
5.53
p-MACV
20.75
p-JUNV
5.66
CC50 (nM)
> 10,000
HLM
39
Solubility
12.2
77
H
78
79
H
H
4.76
2.99
4.75
4.53
5.35
1.25
76,900
17,100
17
52
> 100
28.2
80
81
H
12.56
2.7
11.24
3.38
12.6
4.65
4,900
N/A
N/A
11.8
11.7
OMe
> 10,000
82
OMe
1.34
2.66
1.14
> 10,000
N/A
3.8
83
84
OMe
H
5.56
5.53
5.08
8.05
4.3
> 10,000
> 30,000
N/A
N/A
11.8
8.8
4.285
85
86
H
H
11.2
> 25
20.5
3.15
23.6
> 30,000
95,300
16
23
93.1
72.4
11.34
87
88
H
H
> 25
> 25
> 25
> 25
N/A
N/A
N/A
N/A
N/A
N/A
> 100
> 100
89
H
3.47
8.925
3.92
17,300
32.5
16.5
90
91
H
H
> 25
21.1
> 25
> 25
N/A
16.2
N/A
N/A
N/A
N/A
> 100
16.9
92
93
OCF2H
OCF2H
20.69
20.25
23.24
25.52
> 25
32.7
N/A
N/A
N/A
27.9
53.1
> 100,000
94
H
> 25
> 25
> 25
N/A
N/A
> 100
7

M.B. Plewe, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
conformationally lock the position of the R4 or R1 substituents (see
Supplementary data, Table S4 and Fig. S3) were unable to identify
suitably active chemical subseries. Thus, final analog series were syn-
thesized to identify additional R1 (Table 7, Supplementary data, Table
S5) and R6 (Table 8) modifications in combination with the most stable,
broadly potent complementary R4, R6 and R1 groups. Combining pre-
ferred R1 substituents with the 5-methylindole and 5-cyclopropylindole
moieties gave a series of compounds with attractive broad-spectrum
potency and improved stability.
from > 10- (98) to nearly 1000-fold (46). Interestingly, the attenuating
mutation (F427I) in the GP of Junin (Candid-1) virus also exhibits re-
sistance to benzimidazole cell entry inhibitors.31,32 Consistent with
earlier reports that the 4-acyl-1,6-dialkylpiperazin-2-one and benzimi-
dazole chemical series share a common GP binding site and mechanism
of action our results indicate that these distinct chemical series also
share at least some resistance variants associated with attenua-
tion.22,23,31
In summary, the SAR exploration and optimization of 4-acyl-1,6-
dialkylpiperazin-2-one chemical series was initiated with the (S)-en-
antiomers of compounds 1 and 2 exhibiting pLASV EC50 values in the
250–300 nM range.26 In the current study we generated analogs with up
to ~100-fold greater potency including low to sub-nanomolar broad
spectrum activity against Old (pLASV) and New World (pMACV, pJUNV
and pTCRV) pseudotyped viruses that translate to activity against re-
plicative Lassa and Tacaribe viruses. Attempts to substantially increase
solubility while maintaining broad-spectrum potency and other drug
like properties met with limited success. However, novel R1, R4 and R6
substituent combinations that improved the HLM metabolic half-lives
from < 10 min in our initial broad-spectrum compounds to > 45 min
were identified for a subset of analogs. Those compounds exhibiting
broad-spectrum arenavirus activity with EC50 values ≤10 nM against
each of the pseudotyped viruses and HLM half-lives of > 45 min
(Table 9) may be prioritized for further evaluation and advancement.
The most active polar compounds in this set (Table 7, compounds
99, 100, 103, and 104) contain R1 groups with benzyl alcohol sub-
stituents, as previously suggested by the Phase I and II R1 explorations.
While the benzyl alcohols proved to be metabolically labile m-alkyl
substituted groups (96, 101) provided moderate HLM stability. Com-
pound 102, containing the 6-indolyl group, demonstrated broad-spec-
trum potency and an excellent HLM half-life of 187 min. Unfortunately,
additional combinations of selected metabolically stable R1 (benzyl)
and R4 groups (5-methylindole and 5-cyclopropylindole) with R6
amines intended to increase aqueous solubility did not prove ad-
vantageous for either solubility or broad-spectrum antiviral activity
(Table 8, compounds 105–108).
In a final functional assay the prioritized compounds (Table 9) were
also tested against pseudotyped virus expressing the GP of an atte-
nuated JUNV strain (Candid-1). Each of these analogs exhibited de-
creased potencies against pJUNV (Candid-1) vs pJUNV, ranging
Table 7
Secondary Phase II synthesis and SAR of R1 analogs combined with selected R4 and benzyl R6 groups. Activities provided are EC50 values (nM) in the indicated
pseudotyped virus assays. Cytotoxicity is given as CC50 in Vero cells (7 days). HLM data is the observed half-life (T1/2 (min)) in a human liver microsome assay.
Aqueous solubility (µM) was measured at pH 7.4.
Compd.
R1
X
p-LASV
7.72
p-MACV
1.08
p-JUNV
2.16
CC50 (nM)
4,300
HLM
50
Solubility
9.4
95
96
97
0.94
2.16
1.38
1.62
1.24
0.98
9,770
46
35
3.2
3.6
> 100,000
98
99
6.40
7.05
3.97
4.54
3.51
3.75
8,170
102
20
11.7
28.5
23,950
100
101
3.57
3.15
3.05
1.14
1.53
0.71
5,560
22
54
11.75
3.25
> 100,000
102
103
104
3.91
1.00
0.89
3.35
2.79
2.04
3.50
1.03
0.65
9,210
27,830
4,960
187
19
12
11.34
7.39
6.8
8

M.B. Plewe, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 8
Secondary Phase II synthesis and SAR of R6 analogs combined with best R4 and benzyl R1 groups. Activities provided are EC50 values (nM) in the indicated
pseudotyped virus assays. Cytotoxicity is given as CC50 in Vero cells (7 days). HLM data is the observed half-life (T1/2 (min)) in a human liver microsome assay.
Aqueous solubility (µM) was measured at pH 7.4.
Compd.
R6
X
p-LASV
11.87
p-MACV
17.18
p-JUNV
> 25
CC50 (nM)
7,800
HLM
N/A
Solubility
12.30
105
106
107
108
10.50
15.10
4.13
15.58
5.09
2.33
5.55
3.64
3,400
5,500
24,450
156
N/A
23
7.70
12.86
12.35
11.32
Table 9
Collection of prioritized compounds expressing potent, broad spectrum activity (< 10 nM EC50 activity against pLASV, pMACV and pJUNV) and HLM metabolic half-
life (T1/2) > 45 min with indicated aqueous solubility (µM) at pH 7.4 and pJUNV (Candid-1) EC50 activity.
Compd.
p-LASV
2.12
p-MACV
6.81
p-JUNV
4.05
p-JUNV (Candid-1)
N/A
HLM
77
Solubility
6.2
66
46
79
0.94
2.99
1.85
4.53
0.75
1.25
710.3
56.2
59
52
7.2
28.2
95
7.72
0.94
6.40
3.15
3.91
1.08
1.38
3.97
1.14
3.35
2.16
1.24
3.51
0.71
3.5
272.9
356
50
9.4
96
46
3.2
98
48.4
102
54
11.7
3.25
12
101
102
173.2
181.5
187
9

M.B. Plewe, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Acknowledgements
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Appendix A. Supplementary data
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