Evaluation of spiropiperidine hydantoins as a novel class of antimalarial agents

Article abstract of DOI:10.1016/j.bmc.2015.02.050

Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease β-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC₅₀ values against Plasmodium falciparum 3D7 of 0.310 μM and 0.099 μM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study.

Full text of DOI:10.1016/j.bmc.2015.02.050

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of spiropiperidine hydantoins as a novel class
of antimalarial agents
a
a
a
a
Marvin J. Meyers a, , Elizabeth J. Anderson , Sarah A. McNitt , Thomas M. Krenning , Megh Singh ,
Jing Xu b, Wentian Zeng b, Limei Qin c, Wanwan Xu c, Siting Zhao c, Li Qin c, Christopher S. Eickhoff a,
Jonathan Oliva a, Mary A. Campbell a, Stacy D. Arnett a, Michael J. Prinsen a, David W. Griggs a,
Peter G. Ruminski a, Daniel E. Goldberg d, Ke Ding e, Xiaorong Liu b, Zhengchao Tu b, Micky D. Tortorella b,
⇑
Francis M. Sverdrup a, Xiaoping Chen c,
⇑
a Center for World Health and Medicine, Saint Louis University School of Medicine, 1402 South Grand Blvd, M132 Schwitalla Hall, Saint Louis, MO 63104, USA
b Drug Discovery Pipeline at the Guangzhou Institutes for Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
c Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health, Chinese Academy
of Sciences, #190, Kaiyuan Avenue, Guangzhou Science Park, Guangzhou 510530, China
d Departments of Medicine and Molecular Microbiology, Washington University in St. Louis, Saint Louis, MO, USA
e Key Laboratory of Regenerative Biology, Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel
chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses
a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-
like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bear-
ing similarity to known inhibitors of the human aspartic protease b-secretase (BACE), as new leads for
antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure–activity relationship
profile with positions identified as being tolerant of a variety of substitution patterns as well as a key
piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505)
Received 23 January 2015
Revised 23 February 2015
Accepted 25 February 2015
Available online xxxx
Keywords:
Aspartic protease inhibitors
Spiropiperidine hydantoins
Antiplasmodial
are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310
lM and
0.099 M, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2
l
Antimalarial
strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and
E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the
current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space
and provide a new class of potent antimalarial agents for further study.
Ó 2015 Published by Elsevier Ltd.
1. Introduction
species, has developed varying degrees of resistance to all cur-
rently used antimalarial drugs.2–5 Approaches to combat parasite
Malaria is caused by the parasite Plasmodium. In 2013, there
were approximately 198 million cases of malaria leading to
ꢀ584,000 deaths, being particularly deadly to young children
in sub-Saharan Africa.1 Plasmodium falciparum, the most lethal
resistance include combination of antimalarial drugs as standard
treatment regimens, as well as identification of new antimalarial
drugs with unique mechanisms of action that can be combined
with existing antimalarial drugs.
Plasmodium expresses a number of aspartic proteases necessary
for its survival, including essential aspartic proteases plasmepsin V
(PMV or PM-5) and signal peptide peptidase (PfSPP).6–11 While a
number of potent peptidomimetic inhibitors of Plasmodium aspar-
tic proteases have been identified,7,12–14 we have focused on repur-
posing classes of drug-like aspartic protease inhibitors developed
by the pharmaceutical industry for human aspartic proteases such
as b-secretase (BACE)15,16 or renin.17
Abbreviations: PM, plasmepsin; SPP, signal peptide peptidase; HIV, human
immunodeficiency virus; SAR, structure–activity relationships; BACE, beta-site APP
cleaving enzyme 1 or beta-secretase; TCAMS, Tres Cantos Antimalarial Set; RBC, red
blood cells; CatD, cathepsin D; CatE, cathepsin E; MLM, mouse liver microsomes;
RLM, rat liver microsomes; HLM, human liver microsomes; PK, pharmacokinetics.
⇑
Corresponding authors.
E-mail addresses: mmeyers8@slu.edu (M.J. Meyers), chen_xiaoping@gibh.ac.cn
(X. Chen).
http://dx.doi.org/10.1016/j.bmc.2015.02.050
0968-0896/Ó 2015 Published by Elsevier Ltd.
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M. J. Meyers et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 1. Strategy to identify drug-like aspartic protease inhibitors as novel antimalarials.
aspartic protease inhibitors (Fig. 1b). The reported x-ray crystal
structure of 2 (3FKT)16 demonstrates the mechanism by which
the protonated piperidine nitrogen forms a salt bridge with a water
molecule in the active site. Similarly, other related piperidine and
pyrrolidine BACE, renin and HIV protease inhibitor crystal struc-
tures demonstrate similar binding modes,17,23 leading us to
hypothesize that the spiropiperidine scaffold may be an appropri-
ate core for mining antimalarial phenotypic screening databases.
Substructure-based searching of the TCAMS revealed a single hit,
TCMDC-124587 (4a), with a reported XC50 of 0.840 lM. Given its
modest molecular weight, favorable CLogP, and submicromolar
antimalarial potency, an effort to validate this hit and evaluate
the potential of this class of spiropiperidines as antimalarials was
initiated.
Figure 2. Preliminary R8 structure–activity relationships. Reported potencies are
IC50 values in P. falciparum 3D7 infected erythrocytes.
2. Results and discussion
2.1. Validation of hit and initial SAR
We have hypothesized that maintaining core structural motifs
known to bind the aspartate residues in the active site may allow
identification and optimization of novel classes of antimalarial
compounds. Accordingly, we mined the Tres Cantos Anti-Malarial
dataset (TCAMS) representing thousands of compounds18 for
drug-like aspartic protease inhibitors. For example, we recently
reported our identification and initial optimization of
aminohydantoins as novel antimalarial compounds with selectiv-
ity for Plasmodium and in vivo antimalarial efficacy (e.g., CWHM-
117) originating from BACE inhibitor 1 and database hit TCMDC-
136879 (Fig. 1a).19
Searches of commercially available compound databases
revealed that TCMDC-124587 and closely-related analogs could
be purchased from ChemBridge. Most commercially-available
compounds were derivatized at the R8 position. Two iterations of
sets of six spiropiperidines each, including TCMDC-124587, were
purchased and evaluated for inhibition of parasite growth in P. fal-
ciparum 3D7-infected red blood cells. Key structure–activity
relationships are shown in Figure 2. Of foremost importance, 4a
was found to have similar 3D7 potency (IC50 = 0.940 lM) as
reported in the screening dataset. Substituent position was found
to be important. For example, moving the methoxy group from
the 40- to the 30- or 50-positions resulted in 6-fold loss or 2-fold
improvement in potency, respectively (4b,c). While deletion of
the methoxy group (4d) did not have a significant impact on
Spiropiperidine-containing compounds such as 2 and 3 have
been reported as non-peptidomimetic BACE inhibitors16,20–22 and
represent a novel scaffold for development of new antimalarial
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M. J. Meyers et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
potency, replacement with chlorine (4e) gave about a five-fold
improvement in potency. Most striking is the dependence of
potency on the presence of the phenol moiety. Capping the phenol
with a methyl group (4g) or deletion (4f,h) led to 8- to 60-fold
losses in potency.
analogs such as 4e, 10a–b, 10d–e, 10g–i, 11a–d and 12a–u.
Additionally, some R8 analogs were prepared from 8 standard
amide coupling or alkylation conditions.
Sterically hindered or otherwise incompatible R1 analogs were
prepared instead by the approach reported by Carrera and
Garvey26 shown in Scheme 2. In brief, the primary R1 amine is con-
The antimalarial activity of lead compound was determined to
not be due to general cytotoxicity (HepG2 72 h cytotoxicity
densed with ketone 13 to furnish
a-amino nitriles 14.
IC50 = 37
l
M), having a selectivity index of >100-fold. We were fur-
Condensation with potassium cyanate and ring closure gives
access to mono-substituted hydantoins 15. Subsequent alkylation,
deprotection and reductive amination provided the final com-
pounds 10c and 10f.
ther encouraged by identification of 4e in the Novartis-GNF
antimalarial screening hit collection (GNF-Pf-5345, reported
EC50 = 0.349 lM), although only a few related compounds were
present in this collection.24 These data, along with the
demonstration of a discrete SAR, encouraged us to investigate this
class further by resynthesizing lead compound 4e and broadening
the SAR.
2.3. Structure–activity relationship studies
Structure–activity relationships in the Pf3D7-infected red blood
cell assay for R1 and R3 derivatives are shown in Figures 3 and 4.
Resynthesized 4e was found to have a similar IC50 value as the
commercial lot and was used a reference compound for subse-
quent SAR studies. Keeping R8 constant as the 50-chloro-20-hydoxy-
benzyl group, R1 and R3 were varied independently. R1 proved
tolerant of modification provided that the substituents remained
suitably large and flexible. For example, replacement of the isopen-
tyl (4e) with hydrogen (10a) or ethyl (10b) led to a 20-fold or
greater reduction in potency. Similarly, substitution with a phenyl
2.2. Synthesis
The synthesis of 4e and related analogs is shown in Scheme 1.
The spirohydantoin core 6 was prepared as previously described.25
The R3 alkyl group was then incorporated by simple alkylation
with potassium carbonate. Subsequent alkylation with sodium
hydride and R1X, followed by deprotection of the BOC group
afforded intermediate 9. Finally, reductive amination provided R8
Scheme 1. Reagents and conditions: (a) KCN, (NH4)2CO3, aq MeOH; (b) R3X, K2CO3, DMF; (c) NaH, R1X, DMF; (d) ArI, CuI, 2,2,6,6-tetramethyl-3,5-heptanedione, Cs2CO3; (e)
HCl or TFA; (f) RCHO, Na(OAc)3BH, DMF; (g) RCO2H, EDC, HOBt, NEM, DCM; (h) R8Br, K2CO3, CH3CN.
Scheme 2. Reagents and conditions: (a) R1NH2-HCl, KCN, aq MeOH; (b) (i) KOCN, AcOH, H2O; (ii) aq HCl; (c) NaH, EtI, DMF; (d) KOH, EtOH; (e) RCHO, Na(OAc)3BH, DMF.
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M. J. Meyers et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
a dramatic loss in activity. Conversely, the ethyl group could be
replaced with larger groups such as isopropyl (11b), which
improved potency three-fold, or CH2-pyridyl (11c) and (CH2)3-pyr-
idyl (11d) with only modest two-three-fold losses of potency.
Given the relative flexibility in SAR in the R1 and R3 positions,
we elected to focus on expanding the more sensitive SAR in the
R8 position (Fig. 5). Moving the phenolic hydroxyl group to the
30- or 40-positions proved to be increasingly detrimental to 3D7
potency (12a,b) but not as dramatically as deletion of the hydroxyl
group (4f). In contrast, the chlorine atom could be replaced by a
variety of substituents. For example, while F is not an equivalent
replacement (12c), bromo, methyl, and phenyl replacements are
all well tolerated (12d,e,f).
Positioning of the chlorine atom was found to be important for
optimal 3D7 potency for this series. 60-Chloro analog 12g was
found to be essentially equipotent to the 50-chloro 4e. In contrast,
the 30-chloro and 40-chloro analogs (12h,i) were three- to seven-
fold less potent. 30,50-Dichloro and 50,60-dichloro analogs (12j,l)
were equipotent to the 50-chloro compound 4e. Interestingly, with
Figure 3. R1 structure–activity relationships. Reported potencies are IC50 values in
P. falciparum 3D7 infected erythrocytes.
an IC50 of 0.090 l
M, 40,50-dichloro analog 12k proved to be the
most potent compound we identified in this series.
To further refine our understanding of the importance of the
basic amine and benzylic phenol functionalities, we prepared
amide analog 12m. The lack of 3D7 potency of analog 12m, a direct
comparator to lead 4e, demonstrates the importance of basic
amine to the anti-plasmodium pharmacophore. This result is con-
sistent with the hypothesis that these compounds may be inhibit-
ing an aspartic protease, similar to their BACE-inhibiting
predecessors 2 and 3. However, the basic amine functionality alone
is not sufficient for potency as demonstrated by deletion analog
12n and phenol replacements and isosteres (12o–u) which were
Figure 4. R3 structure–activity relationships. Reported potencies are IC50 values in
P. falciparum 3D7 infected erythrocytes.
not efficacious up to 10 lM. The only phenol replacement to give
a hint of potency was indole 12u.
ring (10d) proved detrimental to potency while cyclopentyl (10c)
was well tolerated. This is presumably due to the rigidity of the
phenyl ring since extension with one (10e) or two (10f) methylene
linkers were roughly equipotent with the isopentyl group.
Benzyl analogs with electron donating (10g) or withdrawing
(10h,i) groups were tolerated with some modest effects on
potency.
2.4. Inhibition of aspartic proteases, cytotoxicity and
mechanism of action
We evaluated twelve of these compounds for inhibition against
a panel of aspartic proteases: human proteases BACE-1, cathepsin
D (CatD) and cathepsin E (CatE), as well as plasmodium proteases
SAR at the R3 position also showed tolerance for a variety of
lipophilic groups (Fig. 4). Deletion of the ethyl group (11a) led to
Figure 5. Preliminary R8 structure–activity relationships. Reported potencies are IC50 values in P. falciparum 3D7 infected erythrocytes.
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M. J. Meyers et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
plasmepsin II and IV (PM-II and PM-IV). Remarkably, all twelve
compounds had no inhibitory activity on these aspartic proteases
up to 10
M. Our previously described series,19 the aminohydan-
toins, represented by CWHM-117, are also not potent against
human aspartic proteases, but exhibit low nanomolar inhibition
of plasmepsins II and IV. This selectivity result is encouraging from
a toxicology and drug discovery standpoint but calls into question
whether these compounds are acting through an aspartic protease
mechanism.
(A)
(B)
(C)
100.00
10.00
1.00
l
0.10
In addition to compound 4e, compounds 12f, 12h, and 12k were
evaluated for cytotoxicity in HepG2 cells. All three compounds
exhibited IC50 values >50
lM, corresponding to a selectivity index
0.01
of greater than 500 for 12k.
0
1
2
3
4
5
The mechanism of action of these compounds is currently
unknown. Notably, spiropiperidine 4e is equipotent against both
chloroquine-sensitive (3D7) and multidrug-resistant (Dd2) strains
CLogP
200
180
160
140
120
100
80
of P. falciparum (3D7 IC50 = 0.31 lM; Dd2 IC50 = 0.22 lM). The Dd2
12n
12o
12r
strain is resistant to antimalarial drugs chloroquine, quinine, pyri-
methamine, and sulfadoxine.27 Thus, the mechanism of action of
these spiropiperidine hydantoins appears to be unique and is not
due to pathways affected by these existing agents.
HLM
RLM
MLM
12o
2.5. Pharmacokinetic profile
60
Finally, we evaluated the pharmacokinetic (PK) properties of
this novel series of antimalarials. A subset of analogs were assessed
for metabolic stability in mouse, rat and human liver microsome
assays (MLM, RLM and HLM, respectively). Figure 6 depicts
relationships between antimalarial activity, metabolic stability
and lipophilicity. All of the compounds evaluated herein fall within
generally acceptable lipophilicity ranges for druglike compounds
(Fig. 6a). Not surprisingly, there is a correlation between lipophilic-
ity and 3D7 potency with the more lipophilic compounds being
generally more potent.
Most of the compounds evaluated have poor metabolic stability
in liver microsomes with half-lives typically less than 10 min,
regardless of species (Fig 6b). A handful of analogs, however, are
metabolically stable (12n,o,r) but are not potent in the 3D7 assay.
These stable compounds all have CLogP values of approximately
1.0 and do not contain an N-benzyl group. This data suggests that
the metabolic stability for this series of compounds requires either
compounds with low CLogP (less than 2), non-benzylic amines or a
combination of both features.
40
20
0
0.01
0.1
1
10
100
200
180
160
140
120
100
80
HLM
RLM
MLM
60
40
20
0
0
1
2
3
4
5
Lead compound 4e was also evaluated in vivo in a rat PK experi-
ment to determine if in vitro liver microsome assays are predictive
of in vivo PK properties. Compound 4e has short half-lives in vitro
(MLM, RLM and HLM t1/2 = 4.0, 3.0 and 9.0 min, respectively) and
in vivo (rat PK t1/2 = 0.28 h, CL = 152 mL/min/kg, Vd = 3.7 L/kg and
F = 10%). However, the modest oral bioavailability is encouraging.
Figure 6. Relationship between 3D7 potency, metabolic stability and lipophilicity.
(A) Plot of Pf 3D7 potency as a function of CLogP. (B) Plot of Pf 3D7 potency versus
metabolic stability in mouse, rat and human liver microsomes (MLM, RLM and
HLM). (C) Plot of metabolic stability in liver microsomes as a function of CLogP.
compounds that face significant metabolic stability challenges that
will need to be overcome in lead optimization. Progress towards
understanding the mechanism of action and identification of more
metabolically stable compounds will be reported in due course.
3. Conclusions
We have identified spiropiperidine hydantoins as a novel series
of antimalarial compounds with oral bioavailability but short half-
lives. We have explored structure–activity relationships for the
three pendant groups and found that the R1 and R3 positions toler-
ate a variety of functionality, suggesting that modulation of these
positions should allow modulation of physiochemical properties
without detrimental effects on potency. However, the R8 benzylic
phenol was found to be very sensitive to modification. We were
able to demonstrate ꢀ10-fold improvement in antimalarial activity
through addition of chlorine atoms in the 40 and 50 positions, but
replacement of the phenol or basic amine functionality resulted
in dramatic losses in antimalarial potency. Unfortunately, this
dependency on a benzylic amine for potency results in a series of
4. Experimental
4.1. General
Commercially available reagents and solvents were used with-
out further purification unless stated otherwise. LC–MS analyses
were performed on an Agilent 1100 or 1200HPLC/MSD electro-
spray mass spectrometer in positive ion mode with scan range
was 100–1000d. Preparative normal phase chromatography was
performed on a Biotage SP1 with prepacked Biotage or Varian silica
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M. J. Meyers et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
gel cartridges. Preparative reverse phase HPLC was performed on a
Shimadzu LC-20AP or Biotage SP1 equipped with a C18 column and
a methanol/water or acetonitrile/water/0.05% TFA gradient. The
purity of tested compounds was P95% as determined by HPLC
analysis conducted on an Agilent 1100 or 1260 system using a
reverse phase C18 column with diode array detector unless stated
otherwise. NMR spectra were recorded on a Bruker 400 MHz spec-
trometer. The signal of the deuterated solvent was used as internal
reference. Chemical shifts (d) are given in ppm and are referenced
to residual not fully deuterated solvent signal. Coupling constants
(J) are given in Hz.
(dt, J = 14.1, 9.2 Hz, 2H), 1.86 (d, J = 14.4 Hz, 2H), 1.55 (spt,
J = 1.0 Hz, 1H), 1.45 (q, J = 1.0 Hz, 2H), 1.08 (t, J = 1.0 Hz, 3H), 0.91
(d, J = 1.0 Hz, 6H).
4.3.4. 8-(5-Chloro-2-hydroxybenzyl)-3-ethyl-1-isopentyl-1,3,8-
triazaspiro[4.5]decane-2,4-dione hydrochloride (CWHM-123;
4e)
A mixture of 299 hydrochloride (200 mg, 0.658 mmol) and 5-
chloro-2-hydroxybenzaldehyde (113 mg, 0.724 mmol) in DCE
(2 mL) was treated with sodium triacetoxyborohydride (279 mg,
1.32 mmol) followed by DMF (1 mL). The suspension was stirred
at room temp overnight. The reaction was quenched with water,
acidified with TFA, concentrated and purified by reverse phase
HPLC (10–70% acetonitrile/water/0.05% TFA). Excess acetonitrile
was removed under vacuum and the resultant aqueous solution
was neutralized with saturated sodium bicarbonate, extracted
with EtOAc, washed with brine, dried over sodium sulfate and con-
centrated to give a white foam (191 mg). The white foam was dis-
solved in diethyl ether and treated with 1 N HCl in diethyl ether
(0.6 mL). The white ppt was filtered, washed with ether and dried
to give the title compound a white HCl salt (194 mg, 0.436 mmol,
66% yield). HPLC purity >95%. ES-MS m/z 408 (M+H). 1H NMR
(400 MHz, DMSO-d6) d 10.61 (s, 1H), 7.63 (d, J = 2.45 Hz, 1H),
7.34 (dd, J = 2.73, 8.69 Hz, 1H), 7.00 (d, J = 8.85 Hz, 1H), 4.20–4.32
(m, 2H), 3.34–3.47 (m, 6H), 3.13–3.20 (m, 2H), 2.42–2.48 (m,
2H), 1.90–1.98 (m, 2H), 1.49–1.62 (m, 1H), 1.39–1.48 (m, 2H),
1.08 (t, J = 7.18 Hz, 3H), 0.90 (d, J = 6.59 Hz, 6H).
4.2. Purchased compounds
Compounds 4a–g and 11c–d were purchased from ChemBridge
(www.hit2lead.com). Compound 4e was resynthesized as
described in Section 4.3.
4.3. Synthesis of CWHM-123 (4e)
4.3.1. tert-Butyl 2,4-dioxo-1,3,8-triazaspiro[4.5]decane-8-
carboxylate (6)
A solution of 11.25 g KCN in 25 mL water was added drop wise
to a suspension of 16.24 g 1-Boc-piperidone (Oakwood; 80 mmol)
and 16.9 g ammonium carbonate in 45 mL water and 55 mL MeOH.
During the addition, everything dissolved. The reaction flask was
closed up with a balloon. After several hours a ppt began to form.
The mixture was stirred at room temp over the weekend. After
72 h, the thick reaction mixture was filtered, washed with small
portions of water and dried to give the desired product as a white
solid: 16.1 g, 59.8 mmol, 75% yield. HPLC purity >98%. ES-MS m/z
292 (M+Na).
Author contributions
All authors have given approval to the final version of the
manuscript. The authors declare no competing financial interest.
Funding sources
4.3.2. tert-Butyl 3-ethyl-2,4-dioxo-1,3,8-triazaspiro[4.5]decane-
8-carboxylate (7a)
Research reported in this publication at Saint Louis University,
was supported by Saint Louis University and the National
Institute Of Allergy And Infectious Diseases of the National
Institutes of Health under Award Number R01AI106498. Research
reported in this publication at the Guangzhou Institutes of
Biomedicine and Health, Chinese Academy of Sciences, was sup-
ported by Bureau of Science and Information Technology of
Guangzhou Municipality under Grant Number 2009Z1-E841 and
Natural Science Foundation of China under Grant Number
81361120380. D.E.G. is supported by the National Institute of
Allergy and Infectious Diseases of the National Institutes of
Health under Award Number AI047798. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of Saint Louis University, the Guangzhou
Institutes of Biomedicine and Health, Chinese Academy of
Sciences, the Natural Science Foundation of China, Washington
University or the National Institutes of Health.
A mixture of 6 (3.03 g, 11.3 mmol) and potassium carbonate in
DMF (30 mL) was treated with ethyl iodide (1.0 mL, 12.4 mmol)
dropwise at room temp. After 5 h, the reaction was diluted with
EtOAc, washed with brine, dried over sodium sulfate, filtered and
concentrated to give the crude product. The crude solid was recrys-
tallized from EtOAc/heptane to give the title compound as a white
solid (2.53 g, 8.51 mmol, 75% yield). HPLC purity >98%. ES-MS m/z
298 (M+H).
4.3.3. 3-Ethyl-1-isopentyl-1,3,8-triazaspiro[4.5]decane-2,4-
dione hydrochloride (12n)
7a (3.16 g, 10.6 mmol) was dissolved in 30 mL DMF (anhy-
drous) and was treated with NaH (60% disp. in mineral oil,
483 mg, 12.1 mmol) at room temp. After 15 min of stirring, 1-
iodo-3-methylbutane (1.7 mL, 12.9 mmol) was added dropwise.
After 5 days, the reaction was quenched with water and parti-
tioned between EtOAc and satd ammonium chloride. The organic
layer was washed with satd ammonium chloride twice, and was
dried with sodium sulfate, filtered and concentrated. The crude
product was purified by silica gel chromatography (0–30% EtOAc/
hexane) to give 3.1 g of tert-butyl 3-ethyl-1-isopentyl-2,4-dioxo-
1,3,8-triazaspiro[4.5]decane-8-carboxylate as a clear oil. The oil
was then dissolved in 10 mL of diethyl ether, and 35 mL of 1 M
HCl was added. The reaction was stirred at room temp for 48 h.
The precipitate was then filtered and washed with diethyl ether,
yielding the title compound as a white, powdery HCl salt. (2.13 g,
7.02 mmol, 66% yield). HPLC purity >90%. ES-MS m/z 268 (M+H).
1H NMR (400 MHz, DMSO-d6) d ppm 9.26 (br s, 1H), 3.39 (q,
J = 7.2 Hz, 2H), 3.32 (d, J = 6.9 Hz, 4H), 3.18 (t, J = 1.0 Hz, 2H), 2.36
Acknowledgments
The authors would like to thank Eva Istvan for supplying the
PM-II and PM-IV DNA constructs used to express PM-II and PM-
IV and Anna Oksman for supplying red blood cells for the 3D7
assay.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.02.050.
Please cite this article in press as: Meyers, M. J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.02.050

M. J. Meyers et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Shaw, P. J.; Hodder, A. N.; Smith, B. J.; Cowman, A. F.; Boddey, J. A. PLoS Biol.
2014, 12, e1001897.
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