Synthesis, antimalarial properties, and sar studies of alkoxyurea-based HDAC inhibitors

Article abstract of DOI:10.1002/cmdc.201300469

Histone deacetylase (HDAC) inhibitors are an emerging class of potential antimalarial drugs. We investigated the antiplasmodial properties of 16 alkoxyurea-based HDAC inhibitors containing various cap and zinc binding groups (ZBGs). Ten compounds displayed sub-micromolar activity against the 3D7 line of Plasmodium falciparum. Structure-activity relationship studies revealed that a hydroxamic acid ZBG is crucial for antiplasmodial activity, and that the introduction of bulky alkyl substituents to cap groups increases potency against asexual blood-stage parasites. We also demonstrate that selected compounds cause hyperacetylation of P.a falciparum histone H4, indicating inhibition of one or more PfHDACs. To assess the selectivity of alkoxyurea-based HDAC inhibitors for parasite over normal mammalian cells, the cytotoxicity of representative compounds was evaluated against neonatal foreskin fibroblast (NFF) cells. The most active compound, 6-((3-(4-(tert-butyl)phenyl)ureido)oxy)- N-hydroxyhexanamide (1 e, Pf3D7 IC₅₀: 0.16a μM) was 31-fold more toxic against the asexual blood stages than towards normal mammalian cells. Moreover, a subset of four structurally diverse HDAC inhibitors revealed moderate activity against late-stage (IV-V) gametocytes.

Full text of DOI:10.1002/cmdc.201300469

CHEMMEDCHEM
FULL PAPERS
DOI: 10.1002/cmdc.201300469
Synthesis, Antimalarial Properties, and SAR Studies of
Alkoxyurea-Based HDAC Inhibitors
Finn K. Hansen,[a] Tina S. Skinner-Adams,[b] Sandra Duffy,[b] Linda Marek,[a]
Subathdrage D. M. Sumanadasa,[b] Krystina Kuna,[a] Jana Held,[c] Vicky M. Avery,[b]
Katherine T. Andrews,*[b] and Thomas Kurz*[a]
Histone deacetylase (HDAC) inhibitors are an emerging class of
potential antimalarial drugs. We investigated the antiplasmo-
dial properties of 16 alkoxyurea-based HDAC inhibitors con-
taining various cap and zinc binding groups (ZBGs). Ten com-
pounds displayed sub-micromolar activity against the 3D7 line
of Plasmodium falciparum. Structure–activity relationship stud-
ies revealed that a hydroxamic acid ZBG is crucial for antiplas-
modial activity, and that the introduction of bulky alkyl sub-
stituents to cap groups increases potency against asexual
blood-stage parasites. We also demonstrate that selected com-
pounds cause hyperacetylation of P. falciparum histone H4, in-
dicating inhibition of one or more PfHDACs. To assess the se-
lectivity of alkoxyurea-based HDAC inhibitors for parasite over
normal mammalian cells, the cytotoxicity of representative
compounds was evaluated against neonatal foreskin fibroblast
(NFF) cells. The most active compound, 6-((3-(4-(tert-butyl)phe-
nyl)ureido)oxy)-N-hydroxyhexanamide (1e, Pf3D7 IC50: 0.16 mm)
was 31-fold more toxic against the asexual blood stages than
towards normal mammalian cells. Moreover, a subset of four
structurally diverse HDAC inhibitors revealed moderate activity
against late-stage (IV–V) gametocytes.
Introduction
Malaria, a parasitic infection caused by Plasmodium spp., has
been among mankind’s deadliest diseases. Over 3.3 billion
people living in tropical and subtropical regions were at risk in
2011.[1] Children from sub-Saharan Africa take the heaviest
burden, and, despite large investments to develop other inter-
ventions such as malaria vaccines,[2,3] it is unlikely that malaria
can be controlled effectively without the development of new
antimalarial drugs. A major limitation of antimalarial drug treat-
ment is the rapid spread of drug-resistant P. falciparum para-
sites.[4] Alarming signs of emerging resistance to artemisinin
derivatives in Southeast Asia are now threatening the widely
accepted artemisinin combination therapies (ACTs).[5,6] For in-
stance, a recent study conducted in Cambodia revealed that
the percentage of patients who were still parasitemic after
three days of treatment with dihydroartemisinin–piperaquine
increased from 26% in 2008 to 45% in 2010.[7] Accordingly, to
tackle drug resistance, there is an urgent need for new antima-
larial drug classes with novel modes of action.
Histone deacetylase (HDAC) inhibitors are an emerging class
of antiparasitic compounds.[8,9] HDACs play a key role in the
epigenetic modulation of gene expression by altering chroma-
tin structure,[10] and HDAC inhibitors have been widely studied
and developed as effective treatments for human cancers.[8]
HDAC gene homologues have been discovered in all Plasmodi-
um species that can infect humans, and five HDAC-encoding
genes have been identified in the P. falciparum genome.[11]
Three of these PfHDACs show homology to human class I
(PfHDAC1) or class II HDACs (PfHDAC2,3), whereas two genes
are class III homologues (PfSir2A and B).[8] The enzyme P. falci-
parum histone deacetylase 1 (PfHDAC1) is considered an
emerging target for malaria intervention strategies.[12] The po-
tential of HDAC inhibitors for treating parasitic diseases was
first reported in 1996 when the cyclic tetrapeptide apicidin
was discovered to have broad-spectrum antiprotozoal activi-
ty.[13] Apicidin was also parenterally and orally active in vivo
against P. berghei malaria in mice.[13] However, apicidin shows
high toxicity towards normal mammalian cells and poor para-
site selectivity (Selectivity Index (SI): 0.2–0.5).[8] Pan-inhibitors of
human HDACs, such as vorinostat (SAHA) and trichostatin A
(TSA), have potent antimalarial activity in vitro (Figure 1). These
compounds cause hyperacetylation of P. falciparum histones,
indicating inhibition of one or more PfHDAC enzymes. TSA suf-
fers from metabolic instability, but it has been considerably
useful in helping our understanding of how HDAC inhibition
affects malarial parasite growth, development, and transcrip-
tional control.[8,14] WR301801 and SB939 (Figure 1) are two of
the most promising antimalarial HDAC inhibitors studied to
date, particularly in terms of in vivo efficacy in mouse malaria
[a] Dr. F. K. Hansen, L. Marek, K. Kuna, Prof. Dr. T. Kurz+
Institute of Pharmaceutical and Medicinal Chemistry
Heinrich Heine University Dꢀsseldorf, 40225 Dꢀsseldorf (Germany)
E-mail: thomas.kurz@uni-duesseldorf.de
[b] Dr. T. S. Skinner-Adams, S. Duffy, S. D. M. Sumanadasa, Prof. Dr. V. M. Avery,
Assoc. Prof. Dr. K. T. Andrews+
Eskitis Institute for Drug Discovery
Griffith University, QLD 4111 (Australia)
E-mail: k.andrews@griffith.edu.au
[c] Dr. J. Held
Institut fꢀr Tropenmedizin
Eberhard-Karls-Universitꢁt Tꢀbingen, 72074 Tꢀbingen (Germany)
[+] K.T.A. and T.K. contributed equally to this work as senior authors.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 665 – 670 665

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
were prepared from the readily available starting ma-
terial 6 using an optimized synthetic protocol. Briefly,
deprotection of the phthaloyl group was accom-
plished by treatment of 6 with methylhydrazine to
furnish the aminoxy intermediate 7. The respective
aniline derivatives were activated with 1,1’-carbonyl-
diimidazole (CDI), and the resulting imidazolides
were subsequently treated with 7 to provide the pro-
tected alkoxyureas 8a–d. Finally, the O-benzyl pro-
tecting groups were cleaved by catalytic hydrogena-
tion to afford HDAC inhibitors 1a–d (Scheme 1).
Figure 1. Selected antimalarial HDAC inhibitors. Pf3D7 data from references [9] (TSA), [16]
(WR301801), and [17] (SAHA, SB939).
In vitro activity against asexual-stage and gameto-
cyte-stage parasites
The antimalarial activity of HDAC inhibitors 1a–l and
2–5 was first tested against the chloroquine-sensitive
models. WR301801, identified by screening a panel of 50 phe-
nylthiazolylhydroxamate-based HDAC inhibitors,[15] was able to
cure mice when administered by intraperitoneal injection.[16]
SB939 has also demonstrated in vivo efficacy in a murine
model of cerebral malaria when administered orally to P. ber-
ghei ANKA-infected mice, preventing the development of cere-
bral malaria-like symptoms.[17] These data underscore the po-
tential of HDAC inhibitors as novel leads for malaria drug re-
search.
Structurally, HDAC inhibitors are characterized by a widely
accepted pharmacophore model. Most HDAC inhibitors con-
Scheme 1. Synthesis of alkoxyurea-based HDAC inhibitors 1a–d. Reagents
and conditions: a) methylhydrazine, CH2Cl2, ꢁ108C, 2 h, 57%; b) 1. aniline de-
rivative, CDI, CH2Cl2, RT, 2 h, 2. 7, Et3N, RT, 18 h, 23–70%; c) Pd/C, H2 (1 bar),
THF, RT, 2 h, 41–83%.
tain a cap group, a connecting-unit linker region, as well as
a zinc binding group (ZBG).[18] The design of antimalarial HDAC
inhibitors is challenging because of the lack of recombinant
PfHDAC2 and 3 enzymes and PfHDAC crystal structures. It is
therefore particularly important to develop comprehensive
structure–activity relationships (SARs). The vast majority of an-
timalarial HDAC inhibitors contain hydroxamic acids as ZBGs,
and only limited data are available regarding the antiplasmo-
dial activity of HDAC inhibitors with alternative ZBGs.
3D7 line of P. falciparum using a [3H]hypoxanthine incorpora-
tion assay. The calculated physicochemical properties of our al-
koxyurea-based HDAC inhibitors indicates that all compounds
comply with Lipinski’s rules (logP<5, HBA<10, HBD<5, Mr <
500 Da).[20] All compounds had calculated logP values between
0.49 and 2.85 (Table 1) and were thus expected to be cell per-
meable. When we examined the activity of alkoxyurea-based
HDAC inhibitors against asexual blood-stage parasites, we
found that 10 of 16 compounds tested (1a,b and 1d–k) had
IC50 values ꢀ1 mm, three had less potent activity with IC50
values between 1.4 and 2.6 mm (1c, 1l, and 2), while three (3–
5) were inactive (IC50 >5 mm, Table 1). These data provided
a number of valuable SARs. In particular, all compounds with
a classical hydroxamic acid as ZGB were active, whereas all
compounds with alternative ZBGs were inactive. The data for
the N-methylhydroxamic acid 3, o-hydroxyanilide 4, and o-ami-
noanilide 5 suggest that an unsubstituted hydroxamic acid
group is crucial for potent activity against asexual blood-stage
parasites. The chain-shortened derivative 2 (n=4) displayed
only moderate activity against the 3D7 line (IC50: 1.86 mm),
demonstrating that an elongated linker is required to efficient-
ly access the active site zinc ion. All HDAC inhibitors of type
1 (n=5), except compounds 1c (R=PhO) and 1l (R=2-CH3),
were active against asexual blood-stage parasites at nanomolar
concentrations. Five compounds (1a, 1e–h) revealed potent
In a previous study we discovered the promising anticancer
activity of a new class of human HDAC inhibitors containing
a novel alkoxyurea connection-unit linker region.[19] In this
work, we explore the antimalarial properties of a panel of al-
koxyurea-based HDAC inhibitors containing various cap groups
and ZBGs. This allowed us to identify a number of notable
SARs to establish design principles for the development of
potent antimalarial HDAC inhibitors. All compounds were eval-
uated for in vitro activity against asexual blood-stage P. falcipa-
rum parasites. Representative compounds were then screened
for gametocytocidal properties. In addition, selected compounds
were examined for their cytotoxicity against a normal mammali-
an cell line and their ability to hyperacetylate histones in asex-
ual-stage P. falciparum parasites as a marker of HDAC inhibition.
Results and Discussion
Synthesis
HDAC inhibitors 1e–l and 2–5 were synthesized as described
previously.[19] The novel alkoxyurea-based compounds 1a–d
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 665 – 670 666

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
drug design to develop alkoxyurea-based HDAC inhibitors with
improved gametocytocidal properties.
Table 1. Calculated logP values of alkoxyurea-based HDAC inhibitors and
the in vitro activity against asexual blood stages of P. falciparum 3D7 and
gametocyte stage parasites.
Cytotoxicity and selectivity indices
To assess the selectivity of alkoxyurea-based HDAC inhibitors
for P. falciparum parasites, the cytotoxicity of five representa-
tive compounds was determined with a normal human cell
line (neonatal foreskin fibroblast (NFF) cells). As shown in
Table 2, asexual-stage SIs ranged from 9 to 33, with the highest
parasite-specific selectivity observed for 1a and 1e (SI: 33 and
31, respectively). Accordingly, the SIs of 1a and 1e are similar
to those previously reported for SB939 and SAHA (SB939 SINFF/
Pf3D7: 19, SAHA SINFF/Pf3D7: 41; Table 2). Compound 1e is also the
most potent against asexual-stage 3D7 parasites. In contrast to
the asexual-stage data, the gametocyte selectivity of the alkox-
yurea-based HDAC inhibitors was poor, with all compounds
having SIꢀ5.
IC50 [mm]
Compd
R
logP[a]
Pf3D7[b]
PfLSG[c]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
1j
1k
1l
2
4-PrO
1.56ꢂ0.55
0.36ꢂ0.05
0.69ꢂ0.17
1.41ꢂ0.19
0.90ꢂ0.12
0.16ꢂ0.03
0.34ꢂ0.06
0.44ꢂ0.08
0.48ꢂ1.12
0.73ꢂ0.06
0.74ꢂ0.09
0.78ꢂ0.02
2.59ꢂ0.62
1.86ꢂ0.16
5.49ꢂ0.38
4-C2H5O 1.02ꢂ0.55
ND
ND
4-PhO
4-Ph
2.57ꢂ0.59
2.61ꢂ0.56
2.23ꢂ0.54
1.88ꢂ0.54
1.00ꢂ0.54
0.49ꢂ0.55
1.46ꢂ0.54
1.00ꢂ0.54
0.78ꢂ0.02
1.00ꢂ0.54
0.32ꢂ0.54
1.71ꢂ0.34
4-tBu
4-iPr
3.45ꢂ0.76
ND
ND
ND
Table 2. Cytotoxicity and selectivity indices.
4-CH3
4-CH3O
3,5-CH3
3-CH3
4-F
SI[b]
3.33ꢂ0.34
ND
Compd
R
IC50 [mm][a]
Asexual stage
Gametocyte
ND
ND
ND
ND
ND
ND
ND
ND
1a
1d
1e
1 f
1i
SAHA
SB939
4-PrO
4-Ph
4-tBu
4-iPr
11.9ꢂ3.2
7.9ꢂ1.3
4.9ꢂ2.0
7.6ꢂ1.1
7.7ꢂ1.9
4.9ꢂ1.2[c]
1.5ꢂ0.6[c]
33
9
31
22
2
5
2
ND
2
ND
ND
2-CH3
H
3
4
5
1.24ꢂ0.61 >5
2.85ꢂ0.55 >5
2.26ꢂ0.55 >5
3,5-CH3
10
41[c]
19[c]
chloroquine
SAHA
methylene blue
pyrimethamine
artesunate
0.006ꢂ0.002
0.13ꢂ0.02
[a] NFF cell line, data are the mean ꢂSD for n=3 experiments. [b] SI=
(mammalian cell IC50)/(P. falciparum IC50 (Table 1)); larger values indicate
greater malaria parasite selectivity. [c] Data from reference [17]. ND: not
determined.
ND
ND
ND
0.23ꢂ0.015
>120
0.0023ꢂ0.0009
[a] logP values were calculated using ACD/ChemSketch freeware version
12.01. [b] P. falciparum 3D7 asexual-stage assays, n=3, each in triplicate
wells. [c] P. falciparum NF54 late-stage gametocytes (IV–V), n=2, each in
duplicate wells. ND: not determined.
Mode of action studies
The mode of action of five representative alkoxyurea-based
HDAC inhibitors in asexual-stage parasites was determined by
using an in situ histone hyperacetylation assay. Trophozoite-
stage P. falciparum 3D7 parasites were incubated with 3ꢁIC50
concentrations of 1a, 1d–f, or 1i, or control compounds chlor-
oquine (CQ) or SAHA. Following 3 h exposure, protein lysates
were prepared and analyzed by western blot using antisera
specific to anti-(tetra)acetyl histone H4. In comparison with ve-
hicle controls (Figure 2; C-0h and C-3h) the control HDAC in-
hibitor SAHA, but not the antimalarial drug CQ, caused hyper-
acetylation of histone H4. Likewise, all five alkoxyurea-based
HDAC inhibitors caused hyperacetylation of histone H4, con-
firming a mode of action against HDAC activity. Equivalent
loading was confirmed by probing the same membrane with
antisera that recognizes P. falciparum RAP2.
activity with IC50 values ꢀ0.5 mm (Table 1). Interestingly, a bulky
alkyl group at the 4-position emerged as being beneficial to
activity, as indicated by the potent activity of compounds 1e
and 1 f (Table 1). Notably, compound 1e, bearing a tert-butyl
group at the 4-position, was the most active compound in this
series (IC50: 0.16 mm). The activity of 1e against the 3D7 line of
P. falciparum is similar to the activity of the reference com-
pound SAHA (IC50: 0.13 mm, Table 1).
The focus in malaria drug discovery is moving from preven-
tion and treatment towards eradication. It is therefore of par-
ticular importance to identify new antimalarial agents with ac-
tivity against sexual (gametocyte) stages and the ability to
block the transmission to the mosquito vector. Thus, four
structurally diverse HDAC inhibitors of type 1 were screened
for activity against late-stage (IV–V) gametocytes using an
imaging viability assay. All compounds (1a, 1d, 1e, and 1i)
showed moderate activity against late-stage gametocytes (IC50:
1.71–5.49 mm, Table 1). Compound 1d, bearing a biphenyl cap
group revealed the highest activity (IC50: 1.71 mm), which indi-
cates that 1d might be a suitable starting point for further
Conclusions
In summary, 16 HDAC inhibitors containing an alkoxyurea con-
necting-unit linker region were evaluated in vitro against the
P. falciparum 3D7 line. From the data obtained, some SARs can
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 665 – 670 667

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
a solution of compound 6 (7.65 g, 20 mmol) in dry CH2Cl2 (50 mL).
The reaction mixture was stirred for 2 h, and the precipitate was
removed by filtration. The filtrate was evaporated under reduced
pressure and treated with Et2O (5 mL). The precipitate was re-
moved by filtration, and a saturated solution of HCl in Et2O was
added to the filtrate to obtain the hydrochloride of the product.
The solid was collected by filtration and subsequently dissolved in
H2O (15 mL). A saturated Na2CO3 solution was added until pH>8,
and the aqueous layer was extracted with EtOAc (3ꢁ30 mL). The
combined organic layers were dried over Na2SO4 and evaporated
under reduced pressure to provide 7 as a white solid (2.88 g, 57%):
mp: 1058C; 1H NMR (500.13 MHz, [D6]DMSO): d=10.94 (s, 1H),
7.42–7.27 (m, 5H), 5.86 (s, 2H), 4.77 (s, 2H), 3.48 (t, J=6.5 Hz, 2H),
1.94 (t, J=7.2 Hz, 2H), 1.50–1.35 (m, 4H), 1.30–1.20 ppm (m, 2H);
13C NMR (125.76 MHz, [D6]DMSO): d=169.20, 136.00, 128.65,
128.18, 128.09, 76.63, 74.61, 32.10, 27.59, 25.04, 24.75 ppm; Anal.
calcd for C13H20N2O3: C 61.88, H 7.99, N 11.10, found: C 61.82, H
7.81, N 10.83.
Figure 2. Hyperacetylation of P. falciparum histone H4 by alkoxyurea-based
HDAC inhibitors. Trophozoite-stage P. falciparum 3D7 parasites were treated
for 3 h with 3ꢁIC50 concentrations of chloroquine (CQ), SAHA, or the alkox-
yurea-based HDAC inhibitors 1a, 1d, 1e, 1 f, or 1i. Protein lysates were ana-
lyzed by SDS-PAGE and western blot using antisera that recognizes acetylat-
ed forms of histone H4 (anti-(tetra)acetyl H4 (lysine 5, 8, 12, 16)). Antisera
recognizing PfRAP2 was used to assess loading.
be drawn: 1) a hydroxamic acid ZBG is crucial for antiplasmo-
dial activity, 2) linker length appears to be critical for activity,
and 3) bulky alkyl substituents at the 4-position of the cap
group improve potency. When screened in vitro for their activi-
ty against sexual-stage P. falciparum parasites (stage IV–V ga-
metocytes), compounds 1a, 1d, 1e, and 1i showed moderate
gametocytocidal properties (IC50: 1.71–5.49 mm). Selected com-
pounds were shown to cause hyperacetylation of P. falciparum
histone H4, and the most potent asexual stage inhibitor 1e
(Pf3D7 IC50: 0.16 mm) was >30-fold more cytotoxic against the
P. falciparum 3D7 line compared to normal mammalian cells.
Thus, compound 1e will serve as a starting point for future
work to develop alkoxyurea-based HDAC inhibitors with en-
hanced parasite selectivity and improved antiplasmodial prop-
erties.
General procedure for the preparation of O-benzyl-protected
derivatives 8a–d: A solution of the respective aniline derivative
(2 mmol) in dry CH2Cl2 (4 mL) was added dropwise to a suspension
of CDI (0.357 g, 2.2 mmol) in dry CH2Cl2 (10 mL). The reaction mix-
ture was stirred for 2 h at room temperature. Afterwards, 6-(amino-
oxy)-N-(benzyloxy)hexanamide (7) (0.505 g, 2 mmol) was added,
and the reaction mixture was stirred overnight and subsequently
evaporated under reduced pressure. The residue was dissolved in
EtOAc (30 mL), and the solution was extracted with a saturated so-
lution of NaHCO3 (3ꢁ10 mL), H2O (10 mL), 1m HCl (10 mL), H2O
(10 mL), dried over Na2SO4, and evaporated under reduced pres-
sure. The residue was purified by column chromatography using
hexane/EtOAc (gradient 50:50!0:100) as eluent to afford com-
pounds 8a–d.
N-(Benzyloxy)-6-((3-(4-propoxyphenyl)ureido)oxy)hexanamide
8a: white solid (0.198 g, 23%): mp: 1018C; 1H NMR (500.13 MHz,
[D6]DMSO): d=10.96 (s, 1H), 9.31 (s, 1H), 8.53 (s, 1H), 7.49–7.28 (m,
7H), 6.89–6.77 (m, 2H), 4.78 (s, 2H), 3.86 (t, J=6.5 Hz, 2H), 3.73 (t,
J=6.7 Hz, 2H), 1.97 (t, J=7.3 Hz, 2H), 1.76–1.66 (m, 2H), 1.65–1.56
(m, 2H), 1.56–1.46 (m, 2H), 1.35–1.24 (m, 2H), 0.96 ppm (t, J=
7.4 Hz, 3H); 13C NMR (125.76 MHz, [D6]DMSO): d=169.17, 157.25,
154.18, 135.98, 131.72, 128.65, 128.17, 128.08, 121.32, 114.09, 76.64,
75.47, 68.92, 32.05, 27.13, 24.70, 24.64, 21.99, 10.32 ppm; Anal.
calcd for C23H31N3O5: C 64.32, H 7.27, N 9.78, found: C 64.34, H
7.20, N 9.66.
Experimental Section
Synthesis
All solvents and chemicals were used as purchased without further
purification. The progress of all reactions was monitored on Merck
pre-coated silica gel plates (with fluorescence indicator UV254)
using EtOAc/n-hexane as solvent system. Column chromatography
was performed with Macherey–Nagel silica gel 60M (0.04–
0.063 mm) with the solvent mixtures specified in the correspond-
ing experiment. Spots were visualized by irradiation with ultraviolet
light (254 nm). Melting points (mp) were taken in open capillaries
on a Mettler FP 5 melting-point apparatus and are uncorrected.
Proton (1H) and carbon (13C) NMR spectra were recorded on
N-(Benzyloxy)-6-((3-(4-ethoxyphenyl)ureido)oxy)hexanamide 8b:
white solid (0.382 g, 46%): mp: 958C; 1H NMR (500.13 MHz,
[D6]DMSO): d=10.95 (s, 1H), 9.31 (s, 1H), 8.52 (s, 1H), 7.53–7.30 (m,
7H), 6.82 (d, J=8.9 Hz, 2H), 4.78 (s, 2H), 3.96 (q, J=6.9 Hz, 2H),
3.74 (t, J=6.8 Hz, 2H), 1.97 (t, J=7.4 Hz, 2H), 1.68–1.57 (m, 2H),
1.56–1.46 (m, 2H), 1.37–1.22 ppm (m, 5H); 13C NMR (125.76 MHz,
[D6]DMSO): d=169.19, 157.26, 154.04, 136.00, 131.72, 128.65,
128.17, 128.09, 121.35, 114.06, 76.65, 75.48, 62.94, 32.06, 27.13,
24.71, 24.64, 14.61 ppm; Anal. calcd for C22H29N3O5: C 63.60, H 7.04,
N 10.11, found: C 63.88, H 7.02, N 9.82.
a Bruker Avance 500 (500.13 MHz for H, 125.76 MHz for 13C) using
[D6]DMSO as solvent. Chemical shifts are given in parts per million
(ppm), (d relative to residual solvent peak for H and 13C or to tet-
1
1
ramethylsilane). Elemental analysis was performed on a PerkinElmer
PE 2400 CHN elemental analyzer. HPLC purity determination was
carried out using a Phenomenex Luna C-18(2) 1.8 mm particle
(250 mmꢁ4.6 mm) column, supported by Phenomenex Security
Guard Cartridge Kit C18 (4.0 mmꢁ3.0 mm), isocratic (eluent: H2O/
CH3CN 15:85 (v/v) containing 0.05% TFA) over 20 min at a flow
rate of 0.8 mLminꢁ1. The purity of all final compounds was 98% or
higher. HRMS analysis was performed on a UHR-TOF maXis 4G,
Bruker Daltonics, Bremen (Germany). The synthesis of compounds
1e–l, 2–5, and 6 was described previously by us.[19]
N-(Benzyloxy)-6-((3-(4-phenoxyphenyl)ureido)oxy)hexanamide
8c: white solid (0.442 g, 53%): mp: 1098C; 1H NMR (500.13 MHz,
[D6]DMSO): d=10.96 (s, 1H), 9.44 (s, 1H), 8.73 (s, 1H), 7.64–7.53 (m,
2H), 7.44–7.30 (m, 7H), 7.15–7.03 (m, 1H), 7.00–6.88 (m, 4H), 4.78
(s, 2H), 3.75 (t, J=6.7 Hz, 2H), 1.97 (t, J=7.3 Hz, 2H), 1.67–1.57 (m,
2H), 1.57–1.47 (m, 2H), 1.38–1.25 ppm (m, 2H); 13C NMR
(125.76 MHz, [D6]DMSO): d=169.17, 157.47, 157.09, 151.01, 135.98,
134.88, 129.80, 128.65, 128.16, 128.08, 122.66, 121.24, 119.26,
Synthesis of 6-(aminooxy)-N-(benzyloxy)hexanamide (7): Methyl-
hydrazine (1.68 mL, 32 mmol) was added dropwise at ꢁ108C to
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 665 – 670 668

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
117.52, 76.63, 75.54, 32.05, 27.11, 24.69, 24.63 ppm; Anal. calcd for
C26H29N3O5: C 67.37, H 6.31, N 9.07, found: C 67.10, H 6.42, N 9.18.
2H), 1.43–1.26 ppm (m, 2H); 13C NMR (125.76 MHz, [D6]DMSO): d=
169.39, 157.41, 140.20, 138.97, 134.46, 129.22, 127.22, 127.03,
126.53, 120.18, 76.09, 32.58, 27.63, 25.31, 25.30 ppm; HRMS-ESI: m/
z [M+H]+ calcd for C19H24N3O4: 358.1761, found: 358.1763. HPLC
purity: 99.04%.
6-((3-([1,1’-Biphenyl]-4-yl)ureido)oxy)-N-(benzyloxy)hexanamide
1
8d: yellow solid (0.627 g, 70%): mp: 1488C; H NMR (500.13 MHz,
[D6]DMSO): d=10.98 (s, 1H), 9.50 (s, 1H), 8.80 (s, 1H), 7.71–7.66 (m,
2H), 7.64 (d, J=7.5 Hz, 2H), 7.61–7.56 (m, 2H), 7.50–7.28 (m, 8H),
4.79 (s, 2H), 3.78 (t, J=6.7 Hz, 2H), 1.98 (t, J=7.1 Hz, 2H), 1.70–
1.58 (m, 2H), 1.58–1.48 (m, 2H), 1.38–1.25 ppm (m, 2H); 13C NMR
(125.76 MHz, [D6]DMSO): d=169.66, 157.42, 140.19, 138.96, 136.45,
134.46, 129.22, 129.13, 128.65, 128.56, 127.22, 127.03, 126.53,
120.17, 77.12, 76.06, 32.54, 27.60, 25.19, 25.12 ppm; Anal. calcd for
C26H29N3O4: C 69.78, H 6.53, N 9.39, found: C 69.54, H 6.37, N 9.11.
Biological assays
Asexual in vitro antimalarial assays: Asexual-stage growth inhibition
assays were carried out using P. falciparum line 3D7 parasites and
the [3H]hypoxanthine incorporation method, essentially as previ-
ously described.[17,21] Test compounds diluted in parasite culture
media (RPMI 1640 supplemented with 10% heat-inactivated
human serum) were incubated with synchronous ring-stage para-
sites (0.25% parasitemia and 2.5% hematocrit) for 48 h at 378C
under standard parasite culture conditions, followed by the addi-
tion of [3H]hypoxanthine (0.5 mCi per well). After a further 24 h cul-
ture, [3H]hypoxanthine incorporation was measured by harvesting
onto 1450 MicroBeta filter mats (Wallac) and counting using
a 1450 MicroBeta liquid scintillation counter. Percentage inhibition
of growth relative to vehicle controls (0.5% DMSO) was deter-
mined for three independent experiments, carried out in triplicate
wells. Chloroquine was included in each assay as a positive control.
IC50 values were calculated using log–linear interpolation of inhibi-
tion curves[22] and are presented as mean ꢂSD for the three inde-
pendent assays.
General procedure for the preparation of HDAC inhibitors 1a–d:
A solution of the respective O-benzyl-protected hydroxamic acid
8a–d (0.5 mmol) in dry THF (50 mL) was hydrogenated (1 bar) at
room temperature in the presence of a catalytic amount of Pd/C
(10 wt%). Upon completion, the crude mixture was filtered
through Celite to remove the catalyst, and the filtrate was concen-
trated under reduced pressure. The residue was purified by
column chromatography using EtOAc or EtOAc/MeOH (9:1) as
eluent.
N-Hydroxy-6-((3-(4-propoxyphenyl)ureido)oxy)hexanamide 1a:
beige solid (0.070 g, 41%): mp: 1058C; 1H NMR (500.13 MHz,
[D6]DMSO): d=10.36 (s, 1H), 9.31 (s, 1H), 8.69 (s, 1H), 8.53 (s, 1H),
7.46–7.38 (m, 2H), 6.88–6.80 (m, 2H), 3.87 (t, J=6.6 Hz, 2H), 3.74 (t,
J=6.7 Hz, 2H), 1.96 (t, J=7.4 Hz, 2H), 1.75–1.65 (m, 2H), 1.65–1.56
(m, 2H), 1.56–1.46 (m, 2H), 1.39–1.24 (m, 2H), 0.96 ppm (t, J=
7.4 Hz, 3H); 13C NMR (125.76 MHz, [D6]DMSO): d=169.42, 157.73,
154.66, 132.17, 121.82, 114.57, 75.97, 69.40, 32.56, 27.62, 25.30,
25.27, 22.45, 10.79 ppm; HRMS-ESI: m/z [M+H]+ calcd for
C16H26N3O5: 340.1867, found: 340.1865; Anal. calcd for C16H25N3O5:
C 56.62, H 7.42, N 12.38, found: C 56.45, H 7.39, N 12.27.
Late-stage P. falciparum (IV–V) anti-gametocyte assays: This assay
was undertaken as described previously.[23] Briefly, highly synchro-
nous stage IV gametocytes, induced from transgenic NF45 P. falci-
parum parasites expressing the gametocyte-specific protein Pfs16,
fused to green fluorescent protein (NF-54-Pfs16-GFP), were isolated
by magnetic column on day 8 post-induction. Gametocytes were
added to 384-well imaging plates, containing test and control
compounds, at 33000 gametocytes per well, and the plates were
incubated for 72 h under reduced oxygen tension (5% CO2, 5% O2,
80% N2). Mitotracker Red CM-H2XRos (0.07 mgmLꢁ1, 5 mL) was
added to each well and incubated overnight as described above.
Gametocyte viability was evaluated on an OPERA (PerkinElmer)
High-Content Screening System. Images acquired for GFP and Mi-
totracker Red CM-H2XRos were overlaid, and the number of elon-
gated viable gametocytes per image was determined using
a script based on Acapella software, developed for use with the
OPERA imaging system. Relative percent inhibition relative to 0.4%
DMSO (vehicle control) and 5 mm puromycin was calculated, and
the mean IC50 values ꢂSD were determined by four-parameter
nonlinear regression analysis, sigmoidal dose–response (variable
slope) fit using Prism 4.0 for two separate experiments in quadru-
plicate point.
6-((3-(4-Ethoxyphenyl)ureido)oxy)-N-hydroxyhexanamide
1b:
white solid (0.073 g, 45%): mp: 938C; 1H NMR (500.13 MHz,
[D6]DMSO): d=10.35 (s, 1H), 9.31 (s, 1H), 8.67 (s, 1H), 8.53 (s, 1H),
7.42 (d, J=8.6 Hz, 2H), 6.83 (d, J=8.6 Hz, 2H), 3.97 (q, J=6.9 Hz,
2H), 3.74 (t, J=6.7 Hz, 2H), 1.96 (t, J=7.5 Hz, 2H), 1.68–1.57 (m,
2H), 1.56–1.47 (m, 2H), 1.39–1.25 ppm (m, 5H); 13C NMR
(125.76 MHz, [D6]DMSO): d=168.93, 157.25, 154.03, 131.73, 121.35,
114.06, 75.51, 62.94, 32.09, 27.16, 24.84, 24.82, 14.61 ppm; HRMS-
ESI: m/z [M+H]+ calcd for C15H24N3O5: 326.1710, found: 326.1708;
Anal. calcd for C15H23N3O5: C 55.37, H 7.13, N 12.91, found: C 55.12,
H 7.00, N 12.72.
N-Hydroxy-6-((3-(4-phenoxyphenyl)ureido)oxy)hexanamide 1c:
grey solid (0.187 g, 63%): mp: 1078C; 1H NMR (500.13 MHz,
[D6]DMSO): d=10.36 (s, 1H), 9.44 (s, 1H), 8.74 (s, 1H), 8.68 (s, 1H),
7.64–7.52 (m, 2H), 7.40–7.32 (m, 2H), 7.09 (t, J=7.4 Hz, 1H), 7.00–
6.88 (m, 4H), 3.75 (t, J=6.7 Hz, 2H), 1.96 (t, J=7.4 Hz, 2H), 1.69–
1.57 (m, 2H), 1.56–1.46 (m, 2H), 1.40–1.24 ppm (m, 2H); 13C NMR
(125.76 MHz, [D6]DMSO): d=168.90, 157.47, 157.08, 151.01, 134.88,
129.80, 122.67, 121.25, 119.26, 117.52, 75.57, 32.09, 27.14, 24.82,
24.80 ppm; HRMS-ESI: m/z [M+H]+ calcd for C19H24N3O5: 374.1710,
found: 374.1712; Anal. calcd for C19H23N3O5: C 61.11, H 6.21, N
11.25, found: C 61.01, H 6.39, N 11.15.
Principle of script used for image analysis: Maximal fluorescent pixel
intensities are identified for the MTR-acquired image, and the aver-
age intensity for designated objects calculated. Objects with an
average fluorescent MTR intensity, above an assay-specified classifi-
cation of minimal MTR signal, are identified as viable objects. GFP-
positive objects are identified and overlaid with the MTR-positive
objects. The GFP objects with an MTR-positive signal are then eval-
uated for the characteristic of being at least fourfold longer than
they are wide. Objects that are both MTR-positive and GFP-object-
elongated are identified as viable late-stage gametocytes.
6-((3-([1,1’-Biphenyl]-4-yl)ureido)oxy)-N-hydroxyhexanamide
1
(1d): beige solid (0.148 g, 83%): mp: 1418C; H NMR (500.13 MHz,
[D6]DMSO): d=10.37 (s, 1H), 9.50 (s, 1H), 8.80 (s, 1H), 8.69 (s, 1H),
7.68 (d, J=8.3 Hz, 2H), 7.64 (d, J=7.7 Hz, 2H), 7.59 (d, J=8.3 Hz,
2H), 7.44 (t, J=7.6 Hz, 2H), 7.32 (t, J=7.4 Hz, 1H), 3.78 (t, J=
6.7 Hz, 2H), 1.98 (t, J=7.4 Hz, 2H), 1.71–1.60 (m, 2H), 1.58–1.47 (m,
In vitro mammalian cell toxicity assays: Cytotoxicity assays were car-
ried out essentially as previously described.[24] Briefly, neonatal fore-
skin fibroblast (NFF) cells were cultured in RPMI media (Life Tech-
nologies) supplemented with 10% heat-inactivated fetal calf serum
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 665 – 670 669

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
M. C. Tahita, W. Kabore, S. Ouedraogo, Y. Sandrine, R. T. Guiguemde, J. B.
Ouedraogo, M. J. Hamel, S. Kariuki, C. Odero, M. Oneko, K. Otieno, N.
Awino, J. Omoto, J. Williamson, V. Muturi-Kioi, K. F. Laserson, L. Slutsker,
W. Otieno, L. Otieno, O. Nekoye, S. Gondi, A. Otieno, B. Ogutu, R.
Wasuna, V. Owira, D. Jones, A. A. Onyango, P. Njuguna, R. Chilengi, P.
Akoo, C. Kerubo, J. Gitaka, C. Maingi, T. Lang, A. Olotu, B. Tsofa, P. Bejon,
N. Peshu, K. Marsh, S. Owusu-Agyei, K. P. Asante, K. Osei-Kwakye, O.
Boahen, S. Ayamba, K. Kayan, R. Owusu-Ofori, D. Dosoo, I. Asante, G.
Adjei, D. Chandramohan, B. Greenwood, J. Lusingu, S. Gesase, A. Mala-
beja, O. Abdul, H. Kilavo, C. Mahende, E. Liheluka, M. Lemnge, T. The-
ander, C. Drakeley, D. Ansong, T. Agbenyega, S. Adjei, H. O. Boateng, T.
Rettig, J. Bawa, J. Sylverken, D. Sambian, A. Agyekum, L. Owusu, F. Mar-
tinson, I. Hoffman, T. Mvalo, P. Kamthunzi, R. Nkomo, A. Msika, A.
Jumbe, N. Chome, D. Nyakuipa, J. Chintedza, W. R. Ballou, M. Bruls, J.
Cohen, Y. Guerra, E. Jongert, D. Lapierre, A. Leach, M. Lievens, O. Ofori-
Anyinam, J. Vekemans, T. Carter, D. Leboulleux, C. Loucq, A. Radford, B.
Savarese, D. Schellenberg, M. Sillman, P. Vansadia, N. Engl. J. Med. 2011,
365, 1863–1875.
(CSL Biosciences), and 1% penicillin–streptomycin (Life Technolo-
gies). Cells were seeded into wells of 96-well tissue culture plates
(3000 cells per well), cultured for 24 h at 378C and 5% CO2, then
treated with a dilution series of each test or control compound.
After 72 h, the medium was removed, plates were washed in PBS,
and cells fixed with denatured alcohol. After washing with water,
sulforhodamine B (50 mL, 0.4% in 1% acetic acid; Sigma) was
added. Plates were incubated for 1 h prior to washing three times
with 1% acetic acid. Each well then received 100 mL of 10 mm Tris
base (unbuffered, pH>9) prior to reading at 564 nm in an ELISA
microplate reader. Percent growth inhibition relative to matched
DMSO controls (0.5%) was then determined. Assays were carried
out on three separate occasions, each with triplicate test wells.
Chloroquine was included as an internal control in each assay. IC50
values were determined by log–linear interpolation of inhibition
curves,[22] and are presented as mean ꢂSD of the three independ-
ent assays.
[4] S. Turschner, T. Efferth, Mini-Rev. Med. Chem. 2009, 9, 206–214.
[5] A. M. Dondorp, F. Nosten, P. Yi, D. Das, A. P. Phyo, J. Tarning, K. M. Lwin,
F. Ariey, W. Hanpithakpong, S. J. Lee, P. Ringwald, K. Silamut, M.
Imwong, K. Chotivanich, P. Lim, T. Herdman, S. S. An, S. Yeung, P. Sin-
ghasivanon, N. P. Day, N. Lindegardh, D. Socheat, N. J. White, N. Engl. J.
Med. 2009, 361, 455–467.
[6] A. M. Dondorp, S. Yeung, L. White, C. Nguon, N. P. Day, D. Socheat, L.
von Seidlein, Nat. Rev. Microbiol. 2010, 8, 272–280.
[7] WHO Global Plan for Artemisinin Resistance Containment (GPARC),
World Health Organization, Geneva, 2011.
[8] K. T. Andrews, T. N. Tran, D. P. Fairlie, Curr. Pharm. Des. 2012, 18, 3467–
3479.
[9] K. T. Andrews, T. N. Tran, N. C. Wheatley, D. P. Fairlie, Curr. Top. Med.
Chem. 2009, 9, 292–308.
[10] O. Witt, H. E. Deubzer, T. Milde, I. Oehme, Cancer Lett. 2009, 277, 8–21.
[11] P. Horrocks, E. Wong, K. Russell, R. D. Emes, Mol. Biochem. Parasitol.
2009, 164, 9–25.
[12] V. Patel, R. Mazitschek, B. Coleman, C. Nguyen, S. Urgaonkar, J. Cortese,
R. H. Barker, E. Greenberg, W. Tang, J. E. Bradner, S. L. Schreiber, M. T. Du-
raisingh, D. F. Wirth, J. Clardy, J. Med. Chem. 2009, 52, 2185–2187.
[13] S. J. Darkin-Rattray, A. M. Gurnett, R. W. Myers, P. M. Dulski, T. M. Crum-
ley, J. J. Allocco, C. Cannova, P. T. Meinke, S. L. Colletti, M. A. Bednarek,
S. B. Singh, M. A. Goetz, A. W. Dombrowski, J. D. Polishook, D. M.
Schmatz, Proc. Natl. Acad. Sci. USA 1996, 93, 13143–13147.
[14] K. T. Andrews, A. P. Gupta, T. N. Tran, D. P. Fairlie, G. N. Gobert, PLoS One
2012, 7, e31847.
Histone hyperacetylation assays: The effect of compounds on his-
tone acetylation was determined as previously described.[17] Briefly,
trophozoite-stage P. falciparum 3D7 parasites were incubated for
3 h with 3ꢁIC50 concentrations of the control HDAC inhibitor
SAHA, the antimalarial drug chloroquine, or compounds 1a, 1d–f,
or 1i. Vehicle control (0.05% DMSO) samples were taken as the
start (t=0 h) and end (t=3 h) of the treatment. Protein lysates
were prepared and resuspended in SDS-PAGE loading dye before
heat denaturing at 968C, then separating by 15% SDS-PAGE. Two-
color western blot was carried out by using Odyssey reagents (LI-
COR Biosciences) according to the manufacturers’ instructions. The
following primary antisera were used: anti-(tetra)acetyl H4 (Milli-
pore) which recognizes lysines 5, 8, 12, and 16 of histone H4, and
anti-IC3/94 monoclonal antisera which was used as a loading con-
trol and recognizes P. falciparum rhoptry-associated protein 2
(RAP2).[25] IRDye secondary antibodies (LI-COR Biosciences) were
used, and membranes imaged using an Odyssey infrared imaging
system (LI-COR biosciences).
Acknowledgements
The Deutsche Forschungsgemeinschaft (DFG) is acknowledged
for funds used to purchase the UHR-TOF maXis 4G (Bruker Dal-
tonics, Bremen) HRMS instrument used in this research. The Aus-
tralian Research Council is acknowledged for research support
(FT0991213 to K.T.A. and LP120200557 to V.M.A.). We thank the
Australian Red Cross Blood Service for provision of human blood
and sera.
[15] G. S. Dow, Y. Chen, K. T. Andrews, D. Caridha, L. Gerena, M. Gettayaca-
min, J. Johnson, Q. Li, V. Melendez, N. Obaldia, T. N. Tran, A. P. Kozikow-
ski, Antimicrob. Agents Chemother. 2008, 52, 3467–3477.
[16] S. Agbor-Enoh, C. Seudieu, E. Davidson, A. Dritschilo, M. Jung, Antimi-
crob. Agents Chemother. 2009, 53, 1727–1734.
[17] S. D. M. Sumanadasa, C. D. Goodman, A. J. Lucke, T. Skinner-Adams, I.
Sahama, A. Haque, T. A. Do, G. I. McFadden, D. P. Fairlie, K. T. Andrews,
Antimicrob. Agents Chemother. 2012, 56, 3849–3856.
[18] P. Bertrand, Eur. J. Med. Chem. 2010, 45, 2095–2116.
[19] L. Marek, A. Hamacher, F. K. Hansen, K. Kuna, H. Gohlke, M. U. Kassack, T.
Kurz, J. Med. Chem. 2013, 56, 427–436.
[20] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug Delivery
Rev. 2001, 46, 3–26.
Keywords: gametocytocides
inhibitors · malaria · Plasmodium falciparum
·
histone deacetylases
·
[21] R. E. Desjardins, C. J. Canfield, J. D. Haynes, J. D. Chulay, Antimicrob.
Agents Chemother. 1979, 16, 710–718.
[22] W. Huber, J. C. Koella, Acta Trop. 1993, 55, 257–261.
[1] World Health Organization, World Malaria Report 2011, WHO Press,
Geneva, 2012.
[2] S. Abdulla, N. Salim, F. Machera, R. Kamata, O. Juma, M. Shomari, S. Kub-
hoja, A. Mohammed, G. Mwangoka, T. Aebi, H. Mshinda, D. Schellen-
berg, T. Carter, T. Villafana, M.-C. Dubois, A. Leach, M. Lievens, J. Veke-
mans, J. Cohen, W. R. Ballou, M. Tanner, Malar. J. 2013, 12, 11.
[3] S. T. Agnandji, B. Lell, S. S. Soulanoudjingar, J. F. Fernandes, B. P. Abosso-
lo, C. Conzelmann, B. G. Methogo, Y. Doucka, A. Flamen, B. Mordmꢂller,
S. Issifou, P. G. Kremsner, J. Sacarlal, P. Aide, M. Lanaspa, J. J. Aponte, A.
Nhamuave, D. Quelhas, Q. Bassat, S. Mandjate, E. Macete, P. Alonso, S.
Abdulla, N. Salim, O. Juma, M. Shomari, K. Shubis, F. Machera, A. S.
Hamad, R. Minja, A. Mtoro, A. Sykes, S. Ahmed, A. M. Urassa, A. M. Ali,
G. Mwangoka, M. Tanner, H. Tinto, U. D’Alessandro, H. Sorgho, I. Valea,
[23] S. Duffy, V. M. Avery, Malar. J. 2013, 12, 408.
[24] R. A. Davis, A. R. Carroll, K. T. Andrews, G. M. Boyle, T. L. Tran, P. C. Healy,
J. A. Kalaitzis, R. G. Shivas, Org. Biomol. Chem. 2010, 8, 1785–1790.
[25] D. L. Baldi, K. T. Andrews, R. F. Waller, D. S. Roos, R. F. Howard, B. S.
Crabb, A. F. Cowman, EMBO J. 2000, 19, 2435–2443.
Received: November 15, 2013
Revised: January 6, 2014
Published online on February 4, 2014
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 665 – 670 670