Two-pronged attack: Dual inhibition of Plasmodium falciparum M1 and M17 metalloaminopeptidases by a novel series of hydroxamic acid-based inhibitors

Article abstract of DOI:10.1021/jm501323a

Plasmodium parasites, the causative agents of malaria, have developed resistance to most of our current antimalarial therapies, including artemisinin combination therapies which are widely described as our last line of defense. Antimalarial agents with a novel mode of action are urgently required. Two Plasmodium falciparum aminopeptidases, PfA-M1 and PfA-M17, play crucial roles in the erythrocytic stage of infection and have been validated as potential antimalarial targets. Using compound-bound crystal structures of both enzymes, we have used a structure-guided approach to develop a novel series of inhibitors capable of potent inhibition of both PfA-M1 and PfA-M17 activity and parasite growth in culture. Herein we describe the design, synthesis, and evaluation of a series of hydroxamic acid-based inhibitors and demonstrate the compounds to be exciting new leads for the development of novel antimalarial therapeutics.

Full text of DOI:10.1021/jm501323a

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Article
A Two-pronged Attack: Dual Inhibition of Plasmodium
falciparum M1 and M17 Metalloaminopeptidases by
a Novel Series of Hydroxamic acid-based Inhibitors.
Shailesh N Mistry, Nyssa Drinkwater, Chiara Ruggeri, Komagal Kannan
Sivaraman, Sasdekumar Loganathan, Sabine Fletcher, Marcin Drag, Alessandro
Paiardini, Vicky M. Avery, Peter J. Scammells, and Sheena McGowan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501323a • Publication Date (Web): 09 Oct 2014
Downloaded from http://pubs.acs.org on October 10, 2014
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Published by American Chemical Society. Copyright © American Chemical Society.
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produced by employees of any Commonwealth realm Crown government in the course
of their duties.

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A Twoꢀpronged Attack: Dual Inhibition of
Plasmodium falciparum M1 and M17
Metalloaminopeptidases by a Novel Series of
Hydroxamic acidꢀbased Inhibitors.
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§†
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‡$
‡
Shailesh N. Mistry , Nyssa Drinkwater , Chiara Ruggeri , Komagal Kannan Sivaraman ,
#
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@
$
Sasdekumar Loganathan , Sabine Fletcher , Marcin Drag , Alessandro Paiardini , Vicky M.
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†
‡
Avery , Peter J. Scammells* and Sheena McGowan*
§
Contributed equally to this work.
†
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University,
Parkville Campus, VIC 3052, Australia.
‡
Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus,
Melbourne, VIC 3800, Australia.
$
Dipartmento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Universita di Roma, 00185
Roma, Italy.
#
Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan,
Queensland 4111, Australia
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Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology,
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KEYWORDS falciparum malaria, aminopeptidase, inhibitors, hydroxamic acid, zinc binding
group
ABSTRACT: Plasmodium parasites, the causative agents of malaria, have developed resistance
to most of our current antimalarial therapies, including artemisinin combination therapies which
are widely described as our last line of defense. Antimalarial agents with a novel mode of action
are urgently required. Two Plasmodium falciparum aminopeptidases, PfAꢀM1 and PfAꢀM17,
play crucial roles in the erythrocytic stage of infection, and have been validated as potential
antimalarial targets. Using compoundꢀbound crystal structures of both enzymes, we have used a
structureꢀguided approach to develop a novel series of inhibitors capable of potent inhibition of
both PfAꢀM1 and PfAꢀM17 activity, and parasite growth in culture. Herein we describe the
design, synthesis, and evaluation of a series of hydroxamic acidꢀbased inhibitors, and
demonstrate the compounds to be exciting new leads for the development of novel antimalarial
therapeutics.
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INTRODUCTION
Malaria, caused by parasites of the genus Plasmodium (of which Plasmodium falciparum, Pf, is
the most lethal), is the world’s most prevalent parasitic disease. The spread of drugꢀresistant
parasites has rendered most of the current antimalarial treatments ineffective; consequently,
prevention and treatment of malaria is becoming increasingly difficult. The most recent WHO
World Malaria Report indicated that in 2012 alone, there were approximately 627,000 deaths
from malaria. There is an urgent need for next generation antimalarial agents with novel modes
of action.
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During the symptomatic erythrocytic stage of Pf infection, parasites degrade
1
, 2
haemoglobin into free amino acids. These free amino acids are essential in a variety of critical
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processes; including parasite growth and development, protein synthesis, and maintenance of
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osmotic pressure within the infected red blood cell to prevent premature cell lysis. The final
stage of haemoglobin digestion is mediated by metalloaminopeptidases (MAPs), which act to
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, 5
degrade small peptide fragments into free amino acids.
Inhibitors of these enzymes, thus
represent attractive leads for the development of novel antimalarial therapeutic agents. Pf
employs two neutral MAPs, PfAꢀM1 and PfAꢀM17. Both enzymes bear zinc bindingꢀmotifs that
6
have essential roles in both substrate and drug binding.
Inhibition of PfAꢀM1 and PfAꢀM17 has been shown to control both Pf laboratory and P.
7, 8
chabaudi chabaudi murine models of malaria, and while potent inhibitors of either PfAꢀM1 or
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ꢀ12
PfAꢀM17 have been identified,
meant that compound elaboration generally favored selectivity for one enzyme over the other.
Previous work within our group identified ((4ꢀ(1Hꢀpyrazolꢀ1ꢀ
differences in the compound binding profiles of the enzymes
yl)phenyl)(amino)methyl)phosphonic acid (1) (Figure 1) as a lead for the development of a dual
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inhibitor of both PfAꢀM1 and PfAꢀM17. Xꢀray crystal structures of both enzymes in complex
with 1 showed that the compound sits within the S1 pocket and coordinates the catalytic zinc
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ion/s through the aminophosphonate moiety. Although aminophosphonates are an established
zincꢀbinding group (ZBG), the phosphonic acid group would exist as a dianion at physiological
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pH (pK 2.35 and ~6) with potentially detrimental effects on membrane permeability. To
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generate dual PfAꢀM1/PfAꢀM17 inhibitors and address potential permeability issues conferred
by the aminophosphonate moiety, we sought to employ an alternative ZBG. Hydroxamic acid
based compounds that are capable of reaching the site of action within infected red blood cells
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and inhibiting PfAꢀM1 have been described previously. Therefore, we replaced the phosphonic
acid moiety of 1 with a hydroxamic acid to generate compounds that anticipated that installation
of a hydroxamic acid group to replace the aminophosphonate phosphonic acid moiety of our
previous series might confer improved inhibitory activity through improved coordination of the
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zinc ion. In addition, these compounds would have a more desirable permeability
and plasma
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stability profile. The hydroxamic acid group has been successfully used in inhibitors for several
proteases, the most attractive being Tosedostat that shows promising efficacy in phase II clinical
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trials for patients with myelodysplastic syndrome and / or acute myeloid leukemia. Herein, we
report the design, synthesis and inhibitory activity of novel carboxylic and hydroxamic acid
derivatives of 1, leading to the discovery of compound 7 as a potent dual inhibitor of PfAꢀM1/ꢀ
M17. Utilizing compound 7 as our new lead, we investigated structureꢀactivity relationships
(
SAR) of this scaffold further, through a series of Nꢀacyl derivatives in replacement of the NꢀBoc
group. Finally, we demonstrate parasiticidal activity of our more potent dual inhibitors.
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RESULTS AND DISCUSSION
Chemistry. It was envisaged that the desired αꢀaminohydroxamic acid functionality could be
accessed from the corresponding NꢀBoc protected amino acid by first coupling with
hydroxylamine before NꢀBoc deprotection to unmask the final compound. Our initial strategy
employed 2ꢀaminoꢀ2ꢀ(4ꢀbromophenyl)acetic acid as a starting material. NꢀBocꢀprotection of the
amine and esterification of the carboxylic acid functionality were performed, prior to attempted
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1
9ꢀ21
Ullmannꢀtype coupling with pyrazole under a variety of conditions,
none of which were
successful (data not shown). Suspecting the presence of the bulky protected amine and
carboxylic acid groups to be responsible for the failed coupling (either through a steric or
potentially catalystꢀchelating mechanism), an alternative strategy was devised, whereby Ullmanꢀ
type Nꢀarylation of pyrazole could be carried out with a simple coupling partner, with subsequent
installation of the αꢀaminoꢀacid functionality (Scheme 1).
Literature precedent for the synthesis of 1ꢀ(4ꢀ(1Hꢀpyrazolꢀ1ꢀyl)phenyl)ethanꢀ1ꢀone (3) in
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2ꢀ33
good to excellent yield is widespread.
In our hands, Nꢀarylation of pyrazole with 4’ꢀ
iodoacetophenone (2) to give 3, was achieved in near quantitative yield using copper (I)ꢀ
2
1
catalysed, ligand free Ullmanꢀtype coupling. Subsequent oxidation of 3 to the corresponding αꢀ
3
4
keto acid 4 was achieved with ease using SeO /pyridine as the oxidation system. Installation of
2
the racemic amino acid system was carried out through initial reductive amination of the ketone
3
5
moiety of 4 with N,Nꢀdibenzylamine and Na(OAc) BH under standard conditions. The
3
intermediate N,Nꢀdibenzylꢀprotected amino acid then underwent complete debenzylation in the
presence of 10% Pd/C in acidified methanol under a hydrogen atmosphere. Finally, NꢀBoc
protection of the HCl salt of the intermediate amino acid was carried out using Boc O under
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mildly basic conditions. After purification, 2ꢀ(4ꢀ(1Hꢀpyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀ((tertꢀ
butoxycarbonyl)amino)acetic acid (5), was obtained in 56 % yield over three steps.
The hydroxamic acid ZBG has commonly been derived from the corresponding
carboxylic acid precursor. In many cases the carboxylic acid group has also been found to
possess the ability to bind zinc ions, albeit generally more weakly than the corresponding
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hydroxamic acid.
carboxylic acid and hydroxamic acid derivatives of 1. NꢀBoc deprotection of 5 in the presence of
M HCl/dioxane and DCM afforded the HCl salt of the αꢀaminocarboxylic acid derivative 6, in
With this in mind, we chose to evaluate the inhibitory activity of both the
4
nearꢀquantitative yield. In contrast the desired hydroxamic acid derivative was accessed via a
twoꢀstep procedure. Initial conversion of NꢀBocꢀprotected αꢀaminocarboxylic acid 5 to the
corresponding NꢀBocꢀprotected αꢀaminohydroxamic acid 7 was achieved in good yield by CDIꢀ
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mediated activation followed by condensation with NH OH.HCl, under reported conditions.
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Finally, the desired αꢀaminohydroxamic acid 8 was obtained in the HCl salt form, through NꢀBoc
deprotection of 7.
Evaluation of the inhibitory activity of 5–8 against PfAꢀM1 and PfAꢀM17 revealed Nꢀ
Boc protected αꢀaminohydroxamic acid 7 to be a potent, subꢀmicromolar inhibitor of both
enzymes (Table 1). We were therefore curious to investigate the effect that other moieties would
have at this position. To this end, we developed a series of Nꢀacyl derivatives, based on 7,
comprising of simple linear and branched alkyl groups as well as substituted aryl moieties.
Access to these compounds was achieved from the previously synthesized compound 4 in five to
six steps (Scheme 2).
As described above, αꢀketo acid 4 underwent reductive amination with benzylamine
rather than N,Nꢀdibenzylamine in order to improve the rate and reaction outcome (the
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corresponding reaction with N,Nꢀdibenzylamine proceeded slowly and complete conversion was
not achieved). The new conditions were successful, and the reaction was complete after
overnight stirring, generating the Nꢀbenzylamino acid 9 in good yield. This was subsequently
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esterified with MeOH in the presence of catalytic concentrated H SO4 (aq), to give the
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corresponding methyl ester 10. Attempts to obtain 10 through a reversal of the reaction sequence
(i.e. esterification followed by reductive amination) proved unsuccessful. Heating 4 under the
esterification conditions described above resulted in both methyl ester formation as well as
conversion of the ketone to the corresponding dimethyl ketal. Although selective deprotection of
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the ketal was achieved by standing in TFA, attempted reductive amination of the resulting αꢀ
keto ester produced several sideꢀproducts by LCMS analysis, including the ketone 3, the αꢀketo
alcohol (total reduction of the ester), αꢀhydroxy ester (ketone reduction) and the Nꢀbenzylamino
acid 9.
NꢀDebenzylation of 10 was achieved as described under acidic hydrogenation conditions
in excellent yield, affording αꢀamino ester 11 as the HCl salt. Compound 11 underwent acylation
with a selection of alkyl and substitutedꢀbenzoyl chlorides, benzoic acids and tertꢀbutyl
isocyanate. Where acid chlorides or tertꢀbutyl isocyanate were used as the electrophile, these
were simply stirred with 11 in the presence of a slight excess of TEA, to give the corresponding
amides or urea in excellent yield, with only aqueous workup required to isolate the desired
product cleanly. Coupling to substitutedꢀbenzoic acids was achieved in the presence of
HCTU/DIPEA in DMF at room temperature, once again with only simple aqueous workup
necessary to afford high purity amide product. The nitrobenzamides 12jꢀk were reduced to the
corresponding aminobenzamides over 10% Pd/C in MeOH under a hydrogen atmosphere in
excellent yield.
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Evaluation of the literature indicated successful aminolysis of simple esters on similar
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scaffolds with NH OH.HCl directly.
Such a conversion is attractive, as it removes the need
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to hydrolyse the ester prior to a coupling step. After conducting successful pilot reactions, we
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opted to employ the combination of NH OH.HCl and methanolic KOH. These conditions gave
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mixed results for the conversion of methyl esters 12aꢀi and 12lꢀm to the corresponding
hydroxamic acids. Following the literature procedure, we obtained desired compounds in modest
yield in the majority of cases, with the remainder of reactions affording the corresponding
carboxylic acid (through basic hydrolysis) as the major product. Even in cases where the desired
hydroxamic acid was successfully isolated, generation of the carboxylic acid product was also
noted. Where only the carboxylic product was isolated, this was converted to the corresponding
hydroxamic acid product using CDIꢀmediated activation as described earlier in the synthesis of
7
.
Further optimization of the aminolysis conditions allowed more consistent conversion to
the desired hydroxamic acid product, with a lower proportion of hydrolysis to the carboxylic acid
being observed by LCMS analysis. These conditions involved the use of anhydrous MeOH as the
reaction solvent and to make up the methanolic KOH solution. In addition, the KOH stock
solution was stored under a septum under an atmosphere of nitrogen to exclude exposure to
moisture. Finally, rather than sequential addition of NH OH.HCl and methanolic KOH, the two
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were premixed before addition to the appropriate ester reagent. This was done with a view to
freshly generate the free NH OH nucleophile prior to addition, thus reducing the concentration of
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circulating hydroxide ions and reducing the risk of ester hydrolysis.
Hydroxamic acid compound series probes the S1’ cavity of PfA-M1 and PfA-M17
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In synthesizing 8, the aminohydroxamic acid analogue of aminophosphonic acid 1, we identified
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, the NꢀBoc precursor of 8, as a potent inhibitor of both PfAꢀM1 and PfAꢀM17 (Table 1,
Supplementary Figure 1). The PfAꢀM1 inhibitory activity of 7 (K = 0.8 μM) is approximately
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1
00 fold higher than that of 1 (K = 104 μM), while the PfAꢀM17 activity is comparable (K of 1
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is 0.011 μM and 7 is 0.03 μM), making 7 the most potent dual PfAꢀM1/M17 inhibitor described
to date. To assess the selectivity of 7 for PfAꢀM1 and PfAꢀM17 over other MAPs, the inhibitory
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activity against PfM18AAP, another P. falciparum MAP was determined. The observed K for
i
PfM18AAP, 49.6 μM, showed that 7 is > 60ꢀfold and 1600ꢀfold selective for PfAꢀM1 and PfAꢀ
M17 respectively. To investigate the structural mechanism behind the improved PfAꢀM1 and
PfAꢀM17 potency, we determined the coꢀcrystal structure of compound 7 bound to PfAꢀM1
(PfAꢀM1:7) and PfAꢀM17 (PfAꢀM17:7). Similarly to 1, compound 7 coordinates strongly to the
catalytic zinc ion/s of both PfAꢀM1 and PfAꢀM17, and probes the hydrophobic S1 pocket with
the aromatic regions of the compound (namely, a 4ꢀ(1Hꢀpyrazolꢀ1ꢀyl)phenyl moiety). However,
the NꢀBoc group, which is not present on 1, was observed binding within the S1’ cavity. Since
the S1’ cavity has not previously been explored with this series of inhibitors, we chose to probe
the region with a focused library of Nꢀacyl derivatives, assessing dual PfAꢀM1/M17 inhibitory
activity, and using structural information obtained from a combination of Xꢀray crystallography
and docking analysis to guide inhibitor development.
The crystal structures of PfAꢀM1 and PfAꢀM17 bound to selected compounds in the
series were determined by either coꢀcrystallization or soaking experiments. Similarly to previous
ligandꢀbound PfAꢀM1 and PfAꢀM17 crystal structures, the only structural differences observed
on compound binding occurred within proximity of the active sites, while the overall protein
folds and domain arrangements were unchanged. In all structures, PfAꢀM1 crystallized as a
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monomer, with one copy in the asymmetric unit. In accordance with previous observations, PfAꢀ
M17 crystallized as a functional hexamer, with two copies in the asymmetric unit; each structure
of PfAꢀM17 therefore contains a total of twelve binding sites. Although minor differences in
quality of the ligand electron density is observed between the sites, the compound bindingꢀmode
is largely conserved. Therefore, for clarity, when referring to structural features, we refer to the
chain that showed the clearest electron density only (PfAꢀM17:7, chain B; PfAꢀM17:13l, chain
C; PfAꢀM17:13m, chain C). The electron density was such that while racemic mixes of
compounds were used for crystallization, only the Sꢀenantiomer of 7, and the Rꢀenantiomer of
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13d, 13l, and 13m could be modeled for both enzymes (Supplementary Figure 2).
Geometry of the ZBG dictates ligand binding position
The current series of compounds was anticipated to improve inhibitory potency by
replacing the phosphonic acid ZBG present on 1, a potent PfAꢀM1/PfAꢀM17 inhibitor (Figure
1
), with a hydroxamic acid moiety. As has been observed for all ligandꢀbound PfAꢀM1 crystal
structures to date, PfAꢀM1:7 revealed the zinc ion to be coordinated by residues of the catalytic
triad: His496, His500 and Glu519. In addition, both oxygen atoms of the hydroxamic acid
moiety also coordinate the zinc ion. The hydroxamic acid moiety takes part in extensive
hydrogenꢀbonding interactions with active site components: the OH with His500 and Glu497, the
NH with Glu497 and an ordered water molecule (W1), and the carbonyl with Glu519, Tyr580,
and W1 (Figure 2a). In total, the hydroxamic acid forms 12 metalloꢀ or hydrogenꢀbonds with
PfAꢀM1, in comparison to the 7 interactions made by the phosphonic acid of the parent
compound 1.
PfAꢀM17 has two catalytic zinc ions, which are coordinated by Asp459, Lys374, Asp379,
Asp399 and Glu461. The crystal structure of PfAꢀM17:7 shows the hydroxamic acid moiety
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coordinates both catalytic zinc ions, as well as forming hydrogen bonds to five active site
residues: Leu487, Lys374, Asp379, Asp459 and Lys386. Additionally, the moiety forms a waterꢀ
mediated hydrogen bond with Asp399, and interacts with the active site carbonate ion present in
all PfAꢀM17 structures (Figure 2b). Overall the hydroxamic acid of 7 can make fourteen
potential interactions with PfAꢀM17, whereas the aminophosphonic acid moiety present on 1
makes only eight. While the hydroxamic acid of 7 and phosphonic acid of 1 are bound in
comparable positions in both PfAꢀM1 and PfAꢀM17, the orientation of the 4ꢀ(1Hꢀpyrazolꢀ1ꢀ
yl)phenyl system, which is common to both compounds, varies considerably (Figure 2c and 2d).
In the active sites of both enzymes, the 4ꢀ(1Hꢀpyrazolꢀ1ꢀyl)phenyl system of 7 is substantially
translated and additionally rotated compared to 1. This places the biaryl system of 7 in a different
region of the pockets, stacking between Tyr575 and Val459 in PfAꢀM1:7, and Phe398, Met396,
and Gly489 in PfAꢀM17:7. While few other direct interactions are observed between the 4ꢀ(1Hꢀ
pyrazolꢀ1ꢀyl)phenyl system and enzyme in either the PfAꢀM1:7 or PfAꢀM17:7 structures, the
binding pose of 7 is more reminiscent of that observed for Bestatin than that of the parent
aminophosphonic acid 1. Bestatin is a PheꢀLeu analogue inhibitor based on the transition state of
the proteolysis reaction performed by PfAꢀM1 and PfAꢀM17. When the Bestatinꢀbound PfAꢀM1
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(
3EBH) and PfAꢀM17 (3KR4) structures are overlaid with the corresponding structures bound
to 7, the benzyl group (Phe sidechain) of Bestatin sits in the same position as the 4ꢀ(1Hꢀpyrazolꢀ
1ꢀyl)phenyl moiety, and the NꢀBoc group of 7 occupies the same pocket as the Leu sidechain of
Bestatin (Supplementary Figure 3a and b). It is the altered binding position of 7 compared to
that of 1 that allows the NꢀBoc group access to the S1’ pocket. In PfAꢀM1:1 and PfAꢀM17:1, the
nitrogen atom of the 2ꢀamino group points away from the S1’ pocket and forms hydrogen bonds
with Glu463 and Glu319 in PfAꢀM1:1, and Asp399 in PfAꢀM17:1, whereas in PfAꢀM1:7 and
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PfAꢀM17:7, the corresponding nitrogen atom (now part of a carbamate group), is positioned to
allow the NꢀBoc group to access the S1’ pockets (Fig 2c and d). The altered binding pose
conferred by the scaffold has different effects on PfAꢀM1 and PfAꢀM17 inhibitory activity. The
aminophosphonic acid 1 and its structural analogue from the hydroxamic acid series, 8, have
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similar PfAꢀM1 inhibitory potencies (K = 104 and 74.5 μM respectively, Table 1), suggesting
i
that the different binding pose does not come at any cost to binding. In contrast, direct exchange
of the phosphonic acid for the hydroxamic acid ZBG has a detrimental effect on PfAꢀM17
inhibitory activity; 1 has a K of 0.011 μM compared to 86 μM for 8.
i
The change in binding pose of 1 versus 7 could result from either the geometry of the
different ZBG and the nature of its interaction with the zinc ion(s), and/or, steric limitations in
the PfAꢀM1 and PfAꢀM17 S1 site for accommodation of the NꢀBoc group. To determine whether
one or both of these factors promotes the altered binding pose, we docked 8 into both enzymes
(
Supplementary Figure 4a and 5a). In PfAꢀM1:8dock, the ligand has a similar binding pose to 7
Figure 2e), with the amine directed towards the S1’ pocket and forming hydrogen bonds with
(
the main chain nitrogen and oxygen atoms of Ala461. Thus, in PfAꢀM1 it appears that the
distinct interaction of the hydroxamic acid ZBG with the zinc ion may be responsible for the
translated binding pose, relative to that of 1. The structure of PfAꢀM17:8dock positions the ligand
further from the S1’ pocket, towards the S1 pocket (Figure 2f). This suggests that the NꢀBoc
group present on 7, functions as an S1’ꢀanchor, and in its absence there may be some mobility of
compound binding that contributes to the reduced affinity of this inhibitor. Regardless, similarly
to PfAꢀM1:8dock, the binding pose of 8 in PfAꢀM17:8dock is similar to that observed for 7, again
suggesting that it is the geometry of the ZBG that dictates the binding pose of 7, not the NꢀBoc
group. The altered binding pose observed upon substitution of the phosphonic acid for a
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hydroxamic acid suggests that exchange of a ZBG late in the lead optimization process may not
be practical for the retention of other binding interactions in the active site. However, for this
compound series, the altered binding pose fortuitously offers the opportunity to exploit further
interaction with the S1’ pocket.
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Compound SAR for the S1’ pocket is different for PfA-M1 versus PfA-M17
The consequence of the altered binding pose brought about by substitution of the series’ ZBG is
that further functionalization of the free amine of 8 offers the potential to exploit binding
interactions with the S1’ pocket which were previously unavailable to this compound series. In
both PfAꢀM1:7 and PfAꢀM17:7, the hydrophobic NꢀBoc group sits in the S1’ pocket, in the
position occupied by the side chain of Leu in Bestatin, forming favorable interactions with
hydrophobic residues (Val493 in PfAꢀM1; Ala460 and Ile547 in PfAꢀM17) (Figure 2a and 2b).
To probe the steric tolerance of these pockets, compounds with a range of aliphatic Nꢀacyl
groups (13a-e) as well as substituted Nꢀbenzoyl groups (13fꢀi) were tested for inhibitory activity
at both PfAꢀM1 and PfAꢀM17; their capability to occupy the S1’ pocket examined using a
combination of crystal and docked structures.
The Nꢀacetyl derivative 13a was found to have opposing effects on the inhibition of PfAꢀ
M1 and PfAꢀM17. Whilst its PfAꢀM1 inhibitory potency is roughly comparable to 8 (8 K = 74.5
i
μM, 13a K = 113 μM), it exhibited a considerable improvement in inhibitory potency for PfAꢀ
i
M17 (8 K = 86 μM, 13a K = 0.019 μM) (Table 1). The docked structure of PfAꢀM17:13a
i
i
suggests that the additional carbonyl group is capable of picking up a hydrogen bonding
interaction with the backbone amine of Gly489 (Supplementary Figure 5). This interaction is
also observed in the structure of Bestatin bound to PfAꢀM17, suggesting that it is also common
to substrates of the enzyme, and therefore a good target in inhibitor design.
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As the size and branching of the Nꢀacyl group increases, from acetyl (13a), to propionyl
13b), to isovaleryl (13c), through to pivaloyl (13d), a corresponding improvement in PfAꢀM1
(
inhibitory potency is observed (K = 113 μM, 41.3 μM, 12.3 μM and 0.7 μM respectively, Table
i
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). This indicates that increasing the steric bulk of the Nꢀacyl substituent is favorable for binding
to the PfAꢀM1 S1’ pocket. Docking analysis of each of these compounds indicates that their
binding modes are highly conserved (Supplementary Figure 4), suggesting that the additional
space occupied by the larger substituents within the hydrophobic pocket contributes to improved
binding. Compound 13d and 7, the most potent PfAꢀM1 inhibitors of the series, both bear tertꢀ
butyl moieties; however 13d bears an amide linker, compared to the corresponding carbamate
linker present on 7. The tertꢀbutyl moiety of 13d is therefore relatively closer to the ZBG. In the
structure of PfAꢀM1:7, the tertꢀbutyl group sits deep within the S1’ pocket and forms
hydrophobic contacts with Val493 at the base. It was therefore not surprising that in the structure
of PfAꢀM1:13ddock the shorter substituent of 13d does not occupy the same space as the
equivalent group in 7, and is unable to form hydrophobic interactions with Val493. However,
given the lack of any additional interactions capable of compensating for this loss, it was curious
that both 7 and 13d demonstrated equivalent inhibitory potencies (K values of 0.8 μM and 0.7
i
μM respectively). We therefore determined the coꢀcrystal structure of PfAꢀM1 in complex with
13d (PfAꢀM1:13d). While a racemic mixture of the ligand was used for crystallization, only the
Rꢀenantiomer of 13d was observed to bind PfAꢀM1. This observation is in contrast to the PfAꢀ
M1:7 structure, which showed only the Sꢀenantiomer bound (Figure 3a). The switch in
enantiomeric preference allows the tertꢀbutyl group of 13d to occupy a similar space to that of 7,
and to maintain the hydrophobic interactions with Val493. This adjustment to maintain the
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interaction with Val493 emphasizes the importance of the interaction in the S1’ pocket of PfAꢀ
M1.
4
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Catalytically, PfAꢀM17 has a clear preference for bulky hydrophobic residues, therefore
it was anticipated that compounds bearing increasingly hydrophobic Nꢀacyl moieties would
exhibit improved inhibitory potency. However, in contrast to the SAR observed with PfAꢀM1, an
increase in hydrophobic steric bulk from 13a through to 13d showed very little effect on
inhibition of PfAꢀM17 (Table 1). When docked into the active site (Supplementary Figure 5),
it was observed that these compounds all form a hydrogen bond with Gly489 through the amide
carbonyl oxygen atom. This hydrogen bond orientates short alkyl substituents out of the main
hydrophobic S1’ cavity (Figure 3b), explaining why additional steric presence in this region
does not contribute to binding affinity.
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The tolerance of the PfAꢀM1 S1’ pocket to a variety of substituted aromatic moieties was
then examined with Nꢀbenzoyl (13f) and Nꢀ(fluoro)benzoylꢀsubstituted derivatives (13g-i).
Docking analysis showed that the phenyl group of 13f cannot be accommodated within
interaction distance to Val493, and is instead directed to solvent, accounting for the reduced PfAꢀ
M1 inhibitory potency. Although the Nꢀ(fluorobenzoyl)ꢀsubstituted compounds regained some of
this potency, none inhibited PfAꢀM1 as well as 7 or 13d. However, beyond the hydrophobic edge
of the S1’ pocket of PfAꢀM1, the region becomes increasingly solvated within the large Cꢀ
terminal channel. This is the putative substrate entry channel, where both hydrophilic residues
and ordered water molecules are observed. To take advantage of this dual nature of the pocket,
Nꢀ(amino)benzoyl substituted derivatives 13l and 13m were also examined. Both compounds
demonstrated moderate inhibitory activity (5.5 μM and 7.4 μM respectively, Table 1), and
comparison of the crystal structure of PfAꢀM1:13l and the docked structure of PfAꢀM1:13mdock
,
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showed that the aminoꢀsubstituent is placed in a similar position for both binding poses, where it
is able to form a direct hydrogen bond with Arg489, in addition to waterꢀmediated hydrogen
bonds with Glu526 (Figure 3c). Elaboration of this moiety to take advantage of additional
hydrogen bonding partners may lead to an improved PfAꢀM1 inhibitory potency of these
compounds.
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The same series of substituted Nꢀbenzoyl compounds (13g-i, and 13l-m), was tested for
PfAꢀM17 inhibitory activity, and was found to effectively recover the loss of potency exhibited
by 13f. To determine the structural rationale for this difference, both 13l and 13m were coꢀ
crystallized with the enzyme. The two compounds bound with nearꢀidentical binding poses, with
only the Rꢀenantiomers of each observed. Similarly to 13a-d at PfAꢀM1, these enantiomers
allows the Nꢀacylꢀsubstituent to probe deeper into the S1’ pocket. This region of the pocket
contains more hydrophilic residues than the neck of the pocket, making it more favorable for the
electronegative fluoroꢀ or polar aminoꢀ substituents to occupy (Figure 3d). However, no direct
interactions with the pocket were observed, and little variation was observed in compound
inhibitory potencies. There does appear to be additional space in the PfAꢀM17 S1’ pocket,
specifically a small auxiliary cavity, that is not occupied by 13l-m (Figure 3d); further extension
of this region of the scaffold to better fit the pocket may offer improvements in PfAꢀM17
inhibition. Conversely, such extension is likely to be detrimental to PfAꢀM1 inhibitor potency, as
the PfAꢀM1 S1’ pocket is less tolerant to aromatic moieties in this region.
Hydroxamic acid compound series inhibit the growth of P. falciparum 3D7
The described series of hydroxamic acidꢀbased compounds contain the most potent dual
PfAꢀM1/PfAꢀM17 inhibitors described to date. We therefore determined the ability of these
compounds to inhibit the growth of the asexual erythrocytic stages of Pfꢀ3D7 parasites, utilizing
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a wellꢀestablished Pf imaging assay (Supplementary Figure 6). Briefly, parasite number (and
thus growth inhibition) was determined through measurement of the incorporation of the DNAꢀ
intercalating dye 4',6ꢀdiamidinoꢀ2ꢀphenylindole into the parasite nucleus following exposure to
compounds. Fluorescent images acquired on the PerkinElmer’s Opera® were analyzed with a
spot detection algorithm using the Acapella® software. Previous potent PfAꢀM1/PfAꢀM17
inhibitors Bestatin and CHRꢀ6768 have been reported to demonstrate inhibitory potencies
between 0.4 – 5 μM for Pfꢀ3D7 parasite growth. Compounds from our present series inhibit
parasite growth with IC values between 0.2 – 0.7 μM (Table 1), with the variation in potency
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in general agreement with changes in PfAꢀM1/PfAꢀM17 inhibitory activity. Compound 13d, the
most effective PfAꢀM1 inhibitor (K = 0.7 μM) and one of the more potent PfꢀM17 inhibitors (K
i
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=
0.028 μM), is the most effective compound against 3D7 malaria parasites, with an IC of 227
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nM (Table 1). Although the in vitro inhibitory properties of compound 7 are comparable to 13d,
it has an IC for parasite growth of only 783 nM, suggesting that compounds substituted with
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the NꢀBoc group are either less capable of cellular penetration, or less stable than the
corresponding Nꢀacyl derivatives. Compounds 13l and 13m are also highly effective against the
malaria parasites (IC values of 233 and 228 nM respectively), despite slightly reduced affinity
5
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for PfꢀM1 compared to 13d (and comparable activity for PfꢀM17). This could be attributed to
indirect factors, such as improved cellular penetration, or protozoan cytotoxicity arising from
toxic metabolites produced by the parasite, which has the potential to be metabolically active
during the 3D7 assay. We additionally screened all of the compounds in the series for
cytotoxicity against human embryonic kidney cells (HEK293), and saw no observable
cytotoxicity at concentrations up to 40 μM (Supplementary Figure 6). Although we are yet to
perform stability and distribution studies, previous analyses of hydroxamic acid based inhibitors
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suggest that the scaffold is capable of reaching the site of action within cultured erythrocytes,
making this a promising scaffold for further development.
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8
Hydroxamateꢀcontaining inhibitors of human Aminopeptidase N have been identified,
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and Bestatin has undergone clinical trials as an immunomodulating agent.
Therefore, the
potential for the compounds described here to act upon the human isoforms of PfAꢀM1 and / or
PfAꢀM17 in vitro cannot be discounted. However, the lack of any observed toxicity of our
compounds in HEK293 cells is encouraging for the continued development of the series.
51, 52
Homologous M1 and M17 aminopeptidases also function as key enzymes in other parasites
5
3, 54
and bacterial pathogens.
This compound series may therefore have potential applications for
the development of other MAP inhibitors for the treatment of a range of diseases.
CONCLUSIONS
The Pf aminopeptidases M1 and M17 are validated drug targets for antimalarial agents with a
novel mode of action. While previous inhibitor series’ demonstrated that crossꢀinhibition was
possible, differences in the compound binding profiles of the enzymes meant that compound
elaboration tended towards improved inhibition of one enzyme target at the expense of the other.
We report here the first series of compounds capable of potent dual inhibition of both PfAꢀM1
and PfAꢀM17. The series additionally possesses activity against 3D7 malaria parasites and no
observable cytotoxicity. This hydroxamic acid based series of compounds therefore provides
extremely attractive lead molecules for further development into antimalarial compounds with a
novel mode of action.
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EXPERIMENTAL SECTION
Chemistry. Chemicals and solvents were purchased from standard suppliers and used without
further purification. Davisil® silica gel (40ꢀ63 µm), for flash column chromatography (FCC)
was supplied by Grace Davison Discovery Sciences (Victoria, Australia) and deuterated solvents
were purchased from Cambridge Isotope Laboratories, Inc. (USA, distributed by Novachem
PTY. Ltd, Victoria, Australia).
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Unless otherwise stated, reactions were carried out at ambient temperature. Reactions
were monitored by thin layer chromatography on commercially available preꢀcoated aluminiumꢀ
backed plates (Merck Kieselgel 60 F ). Visualisation was by examination under UV light (254
2
54
and 366 nm). General staining carried out with KMnO or phosphomolybdic acid. A solution of
4
Ninhydrin (in ethanol) was used to visualize primary and secondary amines. A solution of
Fe(III)Cl (5% in 0.5 M HCl ) was used to visualise hydroxamic acids. All organic extracts
3
(aq)
collected after aqueous workꢀup procedures were dried over anhydrous Na SO before gravity
2
4
filtering and evaporation to dryness. Organic solvents were evaporated in vacuo at ≤ 40°C
water bath temperature). Purification using preparative layer chromatography (PLC) was
carried out on Analtech preparative TLC plates (200 mm x 200 mm x 2 mm).
(
1
13
H NMR and C NMR spectra were recorded on a Bruker Avance Nanobay III 400 MHz
Ultrashield Plus spectrometer at 400.13 MHz and 100.6 MHz respectively. Chemical shifts (δ)
are recorded in parts per million (ppm) with reference to the chemical shift of the deuterated
solvent. Coupling constants (J) and carbonꢀfluorine coupling constants (J ) are recorded in Hz
CF
and the significant multiplicities described by singlet (s), doublet (d), triplet (t), quadruplet (q),
broad (br), multiplet (m), doublet of doublets (dd), doublet of triplets (dt). Spectra were assigned
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using appropriate COSY, distortionless enhanced polarisation transfer (DEPT), HSQC and
HMBC sequences.
LCMS were run to verify reaction outcome and purity using either system A or B.
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System A: an Agilent 6100 Series Single Quad coupled to an Agilent 1200 Series HPLC. The
following buffers were used; buffer A: 0.1% formic acid in H O; buffer B: 0.1% formic acid in
2
MeCN. The following gradient was used with a Phenomenex Luna 3µM C8(2) 15 x 4.6 mm
column, and a flow rate of 0.5 mL/min and total run time of 12 min; 0–4 min 95% buffer A and
5% buffer B, 4–7 min 0% buffer A and 100% buffer B, 7–12 min 95% buffer A and 5% buffer
B. Mass spectra were acquired in positive and negative ion mode with a scan range of 0–1000
m/z at 5V. UV detection was carried out at 254 nm. System B: an Agilent 6120 Series Single
Quad coupled to an Agilent 1260 Series HPLC. The following buffers were used; buffer A: 0.1%
formic acid in H O; buffer B: 0.1% formic acid in MeCN. The following gradient was used with
2
a Poroshell 120 ECꢀC18 50 x 3.0 mm 2.7 micron column, and a flow rate of 0.5 mL/min and
total run time of 5 min; 0–1 min 95% buffer A and 5% buffer B, from 1ꢀ2.5 min up to 0% buffer
A and 100% buffer B, held at this composition until 3.8 min, 3.8–4 min 95% buffer A and 5%
buffer B, held until 5 min at this composition. Mass spectra were acquired in positive and
negative ion mode with a scan range of 100–1000 m/z. UV detection was carried out at 214 and
2
54 nm. All retention times (t ) are quoted in minutes.
R
Preparative HPLC was performed using an Agilent 1260 infinity coupled with a binary
preparative pump and Agilent 1260 FCꢀPS fraction collector, using Agilent OpenLAB CDS
software (Rev C.01.04), and an Altima 5 µM C8 22 x 250 mm column. The following buffers
were used; buffer A: H O; buffer B: MeCN, with sample being run at a gradient of 5% buffer B
2
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to 100% buffer B over 20 min, at a flow rate of 20 mL/min All screening compounds were of >
95% purity unless specified in the individual monologue.
General Procedure A: Condensation of methyl 2-(4-(1H-pyrazol-1-yl)phenyl)-2-
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aminoacetate hydrochloride (11) with acid chlorides and isocyanates. Methyl 2ꢀ(4ꢀ(1Hꢀ
pyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀaminoacetate hydrochloride (11) (134 mg, 0.50 mmol) and TEA (0.154
mL, 1.10 mmol, 2.2 eq in the case of acid chlorides or 0.077 mL, 0.55 mmol, 1.1 eq in the case
of isocyanates) were dissolved in DCM (3 mL). To this was added the appropriate acid chloride
(0.55 mmol, 1.1 eq), and the mixture stirred at room temperature for 18 h. Reaction progress was
monitored by TLC (EtOAc/DCM 1:1). On completion, the reaction mixtures were diluted with
DCM (10 mL) and washed with water (10 mL). Concentration of the organic layer afforded the
product, which was carried through to the next step with no further purification necessary.
General Procedure B: Direct aminolysis of methyl esters to corresponding
hydroxamic acids by sequential addition of reagents. The appropriate methyl ester (0.40–
0
.55 mmol) and NH OH.HCl (139 mg, 2 mmol) were dispersed in MeOH (2 mL) at room
2
temperature. To this was added in a dropwise fashion, 5M KOH/MeOH (0.5 mL, 2.5 mmol)
further diluted in MeOH (to 10 mL). The mixtures were stirred at room temperature and
monitored by LCMS analysis. The mixtures were concentrated before acidification with either
1M HCl (aq) (10 mL) or AcOH/water (1:1, 10 mL, for more acid sensitive compounds). The
aqueous slurry was then extracted with EtOAc (3 x 10 mL), and the combined organic layers
washed with brine (10 mL), then dried of Na SO . Concentration of the organic layers gave the
2
4
crude product, which was further purified by FCC (eluent MeOH/DCM 0:100 to 10:90).
General Procedure C: Direct aminolysis of methyl esters to corresponding
hydroxamic acids with premixing of reagents using anhydrous MeOH. The appropriate
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methyl ester (0.40–0.55 mmol) was dissolved in anhydrous MeOH (0.5 mL) at room temperature
in a nitrogen flushed vessel. In a separate nitrogen flushed vial, NH OH.HCl (139 mg, 2 mmol)
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and 5M KOH/anhydrous MeOH (0.5 mL, 2.5 mmol) were mixed, then sonicated for 30 sec. The
resulting suspension was added to the methanolic ester solution with washings of anhydrous
MeOH (1 mL). The mixtures were stirred at room temperature overnight and monitored by
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®
LCMS analysis. The mixtures were directly dryꢀloaded on to Isolute HMꢀN (Biotage), before
purification by FCC (eluent MeOH/DCM 0:100 to 10:90).
General Procedure D: CDI-mediated coupling of carboxylic acids to hydroxylamine
to give the corresponding hydroxamic acid. The appropriate carboxylic acid (1 eq) was
dissolved in anhydrous THF (1ꢀ5 mL/mmol) at room temperature under an atmosphere of
nitrogen, and CDI (1.5 eq) was added. The mixture was stirred at room temperature for 1 h,
before adding in NH OH.HCl (2 eq). Stirring was continued at room temperature for 24 to 48 h,
2
before diluting the mixture with sat. NH Cl (aq) (20 mL), then extracting with EtOAc (3 x 20
4
mL). The combined organic layers were washed with sat. NaHCO3 (aq) (20 mL) and brine (20
mL), before concentration under reduced pressure. The resulting crude product was further
purified by FCC (eluent MeOH/DCM 0:100 to 10:90).
3
1
1
-(4-(1H-Pyrazol-1-yl)phenyl)ethan-1-one (3). 4’ꢀIodoacetophenone (2) (16.15 g,
6
5.63 mmol), pyrazole (4.92 g, 72.19 mmol, 1.1 eq) and Cs CO (42.77 g, 131.26 mmol, 2.0 eq)
2
3
were placed in a dry flask under an atmosphere of nitrogen. To this was added dry DMF (100
mL). The solution was degassed by sonication before adding Cu O (939 mg, 6.56 mmol, 0.1 eq)
2
and heating at 100°C for 17.5 h. The mixture was cooled, before diluting with water (400 mL),
then extracting with EtOAc (4 x 100 mL). The combined organic layers were washed with brine
(
100 mL), before drying over MgSO , then passing the organic solution through a plug of silica
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gel. The filtrate was concentrated under reduced pressure, and the resulting residue dissolved in
DCM and passed through a second plug of silica gel, initially washing with DCM, followed by
elution with EtOAc/DCM (1:1). On concentration of the filtrate 11.8 g of pale blue solid was
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1
obtained (97%). H NMR (CDCl ) δ 8.10 – 8.04 (m, 2H), 8.01 (d, J = 2.5 Hz, 1H), 7.85 – 7.79
3
1
3
(m, 2H), 7.77 (d, J = 1.5 Hz, 1H), 6.52 (dd, J = 2.4/1.9 Hz, 1H), 2.62 (s, 3H); C NMR (CDCl )
3
+
δ 197.0, 143.4, 142.2, 135.0, 130.1, 127.1, 118.5, 108.7, 26.7; m/z MS (TOF ES ) C H N O
1
1
11
2
+
[
MH] calcd 187.1; found 187.2; LCꢀMS t : 3.50.
R
2
-(4-(1H-Pyrazol-1-yl)phenyl)-2-oxoacetic
acid
(4).
1ꢀ(4ꢀ(1HꢀPyrazolꢀ1ꢀ
yl)phenyl)ethanꢀ1ꢀone (3) (11.8 g, 63.37 mmol) and SeO (9.6 g, 86.52 mmol, 1.37 eq) were
2
dispersed in anhydrous pyridine (300 mL) under an atmosphere of nitrogen gas, and heated
under reflux at 110°C for 20 h. LCMS analysis indicated almost complete conversion to the
desired product, so SeO (3.516 g, 31.69 mmol, 0.5 eq) was added before continuing heating for
2
a further 5 h. LCMS analysis indicated the reaction was complete so the reaction was cooled to
room temperature with stirring overnight. The mixture was filtered, and the filtrate concentrated
under reduced pressure to remove pyridine. The resulting residue was taken up in 2M HCl(aq)
(
200 mL) and extracted with EtOAc (300 mL, 2 x 200 mL, 100 mL). The combined organic
layers were then extracted with 2M NaOH(aq) (2 x 100 mL). The combined aqueous extracts were
reacidified with conc. HCl(aq) and reextracted with EtOAc (3 x 200 mL). Finally the combined
organic layers were washed with brine (100 mL). The combined organic layers were
concentrated until precipitation was observed. At this point, excess hexanes were added to the
flask and the resultant precipitate collected by filtration (vacuum). After drying, 12.58 g (92%)
1
of pale yellow solid were obtained. H NMR (DMSO) δ 14.75 (s, 1H), 8.77 – 8.65 (m, 1H), 8.17
13
– 8.02 (m, 4H), 7.87 (d, J = 1.5 Hz, 1H), 6.65 (dd, J = 2.6/1.7 Hz, 1H); C NMR (DMSO) δ
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+
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84.3, 163.0, 141.1, 139.6, 128.5, 126.2, 125.7, 115.3, 106.2; m/z MS (TOF ES ) C H N O
11 9 2 3
+
[
MH] calcd 217.1; found 217.1; LCꢀMS t : 2.67.
R
2
-(4-(1H-Pyrazol-1-yl)phenyl)-2-((tert-butoxycarbonyl)amino)acetic acid (5). 2ꢀ(4ꢀ
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(1HꢀPyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀoxoacetic acid (4) (500 mg, 2.31 mmol) was dispersed in dry 1,2ꢀ
dichloroethane (20 mL) under an atmosphere of nitrogen. To this was added N,Nꢀdibenzylamine
(
913 mg, 0.890 mL, 4.63 mmol, 2 eq). The mixture was stirred at room temperature for 5 min,
before addition of Na(OAc) BH (980 mg, 4.63 mmol, 2 eq). The mixture was then stirred at
3
room temperature overnight. Workup of a small sample of reaction mixture in water/EtOAc and
LCMS analysis of each layer showed starting material and N,Nꢀdibenzylamine partitioned into
the aqueous layer, whereas the EtOAc layer contained product and some N,Nꢀdibenzylamine.
Further Na(OAc) BH (490 mg, 2.31 mmol, 1 eq) was added and stirring continued at room
3
temperature overnight. The mixture was diluted with water (50 mL), and stirred for 5 min, before
extraction with EtOAc (3 x 50 mL). The combined organic layers were then washed with brine
(
50 mL), with the brine washings kept separate from the initial water layer. On washing with
brine, precipitation of a white solid occurred. This was collected by filtration (vacuum) of both
organic and brine layers, and washing the collected solid with water. LCMS analysis of this salt
indicated it was the acetate salt of N,Nꢀdibenzylamine. The organic layers were concentrated
under reduced pressure, and further purified by FCC (eluent MeOH/DCM 0:100 to 5:95), to give
8
9 mg of Nꢀ(4ꢀ(1Hꢀpyrazolꢀ1ꢀyl)benzyl)ꢀNꢀbenzylꢀ1ꢀphenylmethanamine (decarboxylated
product) and 702 mg of desired product contaminated with N,Nꢀdibenzylamine.
The crude desired product was dissolved in MeOH/water (9:1, 20 mL), before adding
1
0% Pd/C (70 mg). The mixture was hydrogenated (balloon) at room temperature overnight.
LCMS analysis indicated almost complete monoꢀdebenzylation, so water (15 mL) and conc.
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HCl(aq) (1 mL) were added, along with further 10% Pd/C (70 mg), before continued
hydrogenation at room temperature for 3.5 h. LCMS analysis after this time indicated complete
Nꢀbenzyl deprotection to the primary amine had occurred. The mixture was filtered through a
bed of Celite (vacuum), with washings of MeOH, before concentration under reduced pressure.
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The resulting crude residue was taken up in water (20 mL) and washed with Et O (20 mL),
2
before basifying with sat. NaHCO3 (aq). The aqueous layer was washed again with Et O (20 mL)
2
and the organic layer discarded. Boc O (555 mg, 2.54 mmol) was added to the aqueous layer,
2
and the mixture stirred at room temperature for 30 min, before addition of further Boc O (252
2
mg, 1.15 mmol) and THF (10 mL). The mixture was allowed to stir at room temperature
overnight. After this time the mixture was carefully acidified to pH 4 with 2M HCl , before
(aq)
extraction with DCM (3 x 50 mL). The combined organic layers were concentrated under
reduced pressure, before further purification be FCC (eluent MeOH/DCM 0:100 to 10:90) to
1
give 410 mg of foamy white solid (56% over 3 steps). H NMR (DMSO) δ 12.86 (s, 1H), 8.49
(d, J = 2.4 Hz, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 1.5 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H),
13
7
.51 (d, J = 8.6 Hz, 2H), 6.54 (dd, J = 2.3/1.9 Hz, 1H), 5.15 (d, J = 8.2 Hz, 1H), 1.39 (s, 9H); C
NMR (DMSO) δ 172.2, 155.2, 141.1, 139.2, 135.3, 128.9, 127.8, 118.3, 107.9, 78.5, 57.1, 28.2;
+
+
m/z MS (TOF ES ) C H N O [MH] calcd 318.1; found 318.2; LCꢀMS t : 3.18.
1
6
20
3
4
R
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-(4-(1H-Pyrazol-1-yl)phenyl)-2-aminoacetic acid hydrochloride (6). 2ꢀ(4ꢀ(1Hꢀ
Pyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)acetic acid (5) (38 mg, 0.12 mmol) was
dissolved in DCM (3 mL), before adding 4M HCl/1,4ꢀdioxane (1 mL). The mixture was stirred
at room temperature for 1 h, before concentrating under reduced pressure to give 29 mg of white
1
solid (97%). H NMR (DMSO) δ 13.96 (s, 1H), 8.91 (s, 3H), 8.55 (d, J = 2.4 Hz, 1H), 7.94 (d, J
= 8.7 Hz, 2H), 7.78 (d, J = 1.5 Hz, 1H), 7.63 (d, J = 8.7 Hz, 2H), 6.58 (dd, J = 2.4/1.8 Hz, 1H),
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13
5
.18 (s, 1H); C NMR (DMSO) δ 169.7, 141.4, 140.2, 130.9, 129.5, 128.0, 118.6, 108.3, 54.9;
+
+
m/z MS (TOF ES ) C H N O [MH] calcd 218.1; found 218.1; LCꢀMS t : 1.39.
11 12
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tert-Butyl (1-(4-(1H-pyrazol-1-yl)phenyl)-2-(hydroxyamino)-2-oxoethyl)carbamate
(7). Pyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)acetic acid (5) (100 mg, 0.32 mmol)
was dissolved in dry THF (1 mL) under an atmosphere of nitrogen at room temperature. To this
was added CDI (76 mg, 0.47 mmol, 1.5 eq), and the mixture stirred for 1 h at room temperature.
Hydroxylamine hydrochloride (44 mg, 0.63 mmol, 2 eq) was added and the mixture was then
stirred at room temperature overnight. LCMS analysis indicated approximately 75% conversion
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had taken place, so stirring was continued for a further 3 days. Sat. NH Cl(aq) (20 mL) was added,
4
before extracting with EtOAc (3 x 20 mL). The combined organic layers were then washed with
brine (20 mL). After concentration of the organic layers, the crude residue was further purified
by PLC (NH OH/MeOH/DCM 1:10:89), with the plate being run twice, to give 73 mg of white
4
1
solid (69%). H NMR (DMSO) δ 10.91 (s, 1H), 9.00 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 7.79 (d, J
=
8.7 Hz, 2H), 7.73 (d, J = 1.4 Hz, 1H), 7.58 – 7.45 (m, 3H), 6.54 (dd, J = 2.4/1.8 Hz, 1H), 5.08
1
3
(d, J = 8.6 Hz, 1H), 1.38 (s, 9H); C NMR (DMSO) δ 171.7, 166.9, 141.0, 139.1, 134.2, 128.3,
ꢀ
ꢀ
1
27.8, 118.2, 107.9, 80.5, 54.9, 28.2; m/z MS (TOF ES ) C H N O [MꢀH] calcd 331.1; found
16 19 4 4
3
31.2; LCꢀMS t : 3.02.
R
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-(4-(1H-Pyrazol-1-yl)phenyl)-2-amino-N-hydroxyacetamide hydrochloride (8). tertꢀ
Butyl (1ꢀ(4ꢀ(1Hꢀpyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀ(hydroxyamino)ꢀ2ꢀoxoethyl)carbamate (7) (62 mg, 0.19
mmol) was dissolved in MeOH/DCM (1:1, 4 mL). To this was added 4M HCl/1,4ꢀdioxane (2
mL) and the mixture stirred at room temperature for 2 h. The mixture was concentrated under
reduced pressure, to give 47 mg of hygroscopic yellow solid. This was further purified by
1
preparative HPLC to give 13 mg of colourless semiꢀsolid (25%). H NMR (MeOD) δ 8.29 (d, J =
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.5 Hz, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 1.5 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H), 6.56 (dd,
1
3
J = 2.4/1.9 Hz, 1H), 4.86 (s, 1H); C NMR (MeOD) δ 167.0, 142.7, 142.3, 133.5, 130.3, 129.1,
+
+
1
20.7, 109.3, 55.6; m/z MS (TOF ES ) C H N O [MH] calcd 233.1; found 233.1; LCꢀMS t :
11 13 4 2 R
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.49.
2
-(4-(1H-Pyrazol-1-yl)phenyl)-2-(benzylamino)acetic acid (9). 2ꢀ(4ꢀ(1HꢀPyrazolꢀ1ꢀ
yl)phenyl)ꢀ2ꢀoxoacetic acid (4) (6.00 g, 27.75 mmol) was dispersed in anhydrous 1,2ꢀ
dichloroethane (240 mL) under an atmosphere of nitrogen at room temperature. To this stirred
suspension, was added benzylamine (5.95 g, 6.06 mL, 55.51 mmol, 2 eq), and stirring continued
at room temperature for 5 min. Na(OAc) BH (11.77 g, 55.51 mmol, 2 eq) was added in portions,
3
and stirring was continued at room temperature overnight. The mixture was concentrated, and
the resulting residue partitioned between water (200 mL) and EtOAc (100 mL), and stirred for 30
min at room temperature. The organic solvent was then removed under reduced pressure, and the
resulting solid collected by filtration (vacuum) and washed with water. After drying, 6.45 g
1
(76%) of offꢀwhite solid was obtained, which was used without further purification. HꢀNMR
analysis proved difficult due to poor solubility in a variety of solvents, including DMSO,
obtained spectra were difficult to interpret due to peak broadness. LCꢀMS analysis gave a single
peak with the correct mass, so NMR analysis was carried out in the subsequent step to confirm
+
+
structure. m/z MS (TOF ES ) C H N O [MH] calcd 308.1; found 308.2; LCꢀMS t : 3.13.
18
18
3
2
R
Methyl 2-(4-(1H-pyrazol-1-yl)phenyl)-2-(benzylamino)acetate (10). 2ꢀ(4ꢀ(1HꢀPyrazolꢀ
ꢀyl)phenyl)ꢀ2ꢀ(benzylamino)acetic acid (9) (6.34 g, 20.61 mmol) was dispersed in MeOH (200
1
mL). To this was added concd H SO
(2 mL), and was boiled under reflux for 24 h, before
4 (aq)
2
adding in further conc. H SO4 (aq) (1 mL). Heating was continued for a further 24 h period.
2
LCMS analysis indicated the reaction was progressing slowly, so MeOH (100 mL) was added
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and heating continued for a further 72 h. LCMS analysis indicated the reaction was complete, so
the mixture was cooled, before concentrating under reduced pressure. The resulting residue was
basified with sat. NaHCO3 (aq) (250 mL), before extraction with DCM (3 x 100 mL). The
combined organic layers were concentrated under reduced pressure, and the resulting residue
0
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0
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purified by FCC (eluent EtOAc/PE 0:100 to 30:70, with a plateau at 15:85) to give 5.90 g (89%)
1
of pale yellow oil. H NMR (CDCl ) δ 7.96 – 7.90 (m, 1H), 7.90 – 7.70 (m, 3H), 7.69 – 7.53 (m,
3
2H), 7.49 – 7.28 (m, 5H), 6.54 – 6.40 (m, 1H), 4.70 – 4.45 (m, 1H), 4.44 – 4.02 (m, 1H), 3.95 –
1
3
3.75 (m, 2H), 3.75 – 3.64 (m, 2H); C NMR (CDCl ) δ 172.2, 141.7, 140.0, 139.9, 138.1, 130.1,
3
+
1
29.53, 129.4, 129.0, 128.5, 126.9, 119.7, 108.2, 63.6, 53.2, 52.7; m/z MS (TOF ES )
+
1
13
C H N O [MH] calcd 322.2; found 322.2; LCꢀMS t : 3.21. H/ C NMR spectra indicate
19 20
3
2
R
multiple conformational states.
Methyl 2-(4-(1H-pyrazol-1-yl)phenyl)-2-aminoacetate hydrochloride (11). Methyl 2ꢀ
4ꢀ(1Hꢀpyrazolꢀ1ꢀyl)phenyl)ꢀ2ꢀ(benzylamino)acetate (10) (6.047 g, 18.82 mmol) was dissolved
(
in MeOH (250 mL) and conc. (32%) HCl (aq) (4 mL), before adding 10% Pd/C (600 mg, 0.1 eq
by weight). The mixture was hydrogenated (balloon) at room temperature for 48 h. LCMS
analysis indicated complete conversion had taken place. The mixture was filtered through a bed
of Celite, with washings of MeOH. The filtrate was concentrated under reduced pressure, and the
resulting residue dispersed in sat. NaHCO3 (aq), before extracting with DCM (3 x 100 mL). The
combined organic layers were concentrated under reduced pressure to give 5.507 g of crude
product. This was taken up in Et O/DCM, before adding a solution of 1M HCl/Et O (25 mL).
2
2
This was stirred at room temperature for 30 min, before diluting with Et O and filtering the
2
1
resulting precipitate, to give 4.407 g (87%) white amorphous solid. H NMR (DMSO) δ 9.13 (d,
J = 3.3 Hz, 3H), 8.56 (d, J = 2.3 Hz, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 1.5 Hz, 1H), 7.63
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Journal of Medicinal Chemistry
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(
d, J = 8.7 Hz, 2H), 6.58 (dd, J = 2.4/1.8 Hz, 1H), 5.36 (q, J = 5.0 Hz, 1H), 3.73 (s, 3H); C
NMR (DMSO) δ 168.8, 141.5, 140.4, 130.1, 129.7, 128.0, 118.6, 108.3, 54.7, 53.3; m/z MS
+
+
(TOF ES ) C H N O [MH] calcd 232.1; found 232.2; LCꢀMS t : 3.02.
12 14 3 2 R
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Methyl 2-(4-(1H-pyrazol-1-yl)phenyl)-2-acetamidoacetate (12a). Acetyl chloride
(
0.039 mL, 0.55 mmol, 1.1 eq) was reacted as described in General Procedure A. After 24 h,
further acetyl chloride (0.4 eq) and triethylamine (0.5 eq) were added and stirring continued for
1
2
4 h prior to workup, to give 128 mg (93%) of offꢀwhite solid. H NMR (CDCl ) δ 7.91 (d, J =
3
2
.4 Hz, 1H), 7.73 (d, J = 1.5 Hz, 1H), 7.72 – 7.65 (m, 2H), 7.51 – 7.41 (m, 2H), 6.57 (d, J = 6.7
Hz, 1H), 6.48 (dd, J = 2.4, 1.9 Hz, 1H), 5.62 (d, J = 7.1 Hz, 1H), 3.75 (s, 3H), 2.31 (s, 1H), 2.06
13
(
s, 3H); C NMR (CDCl ) δ 171.4, 169.5, 141.4, 140.3, 135.0, 128.6, 127.0, 119.8, 108.0, 56.0,
3
+
+
5
3.1, 23.2; m/z MS (TOF ES ) C H N O [MH] calcd 274.1; found 274.1; LCꢀMS t : 3.30.
14 16 3 3 R
Methyl 2-(4-(1H-pyrazol-1-yl)phenyl)-2-propionamidoacetate (12b). Propionyl
chloride (0.048 mL, 0.55 mmol, 1.1 eq) was reacted as described in General Procedure A, to give
1
1
35 mg (93%) of pale pink solid. H NMR (CDCl ) δ 7.95 – 7.89 (m, 1H), 7.75 (d, J = 1.5 Hz,
3
1
H), 7.73 – 7.66 (m, 2H), 7.50 – 7.42 (m, 2H), 6.52 (d, J = 6.7 Hz, 1H), 6.48 (dd, J = 2.4, 1.9 Hz,
1H), 5.62 (d, J = 7.0 Hz, 1H), 3.75 (s, 3H), 2.30 (dd, J = 7.6/3.1 Hz, 2H), 2.23 (s, 1H), 1.17 (t, J
1
3
=
7.6 Hz, 3H); C NMR (CDCl ) δ 173.2, 171.5, 141.3, 140.2, 135.2, 128.6, 127.1, 119.8, 108.0,
3
+
+
5
5.9, 53.1, 29.5, 9.6; m/z MS (TOF ES ) C H N O [MH] calcd 288.1; found 288.2; LCꢀMS
15 18 3 3
tR: 3.40.
Methyl 2-(4-(1H-pyrazol-1-yl)phenyl)-2-isobutyramidoacetate (12c). Isobutyryl
chloride (0.058 mL, 0.55 mmol, 1.1 eq) was reacted as described in General Procedure A, to give
1
1
29 mg (85%) of offꢀwhite solid. H NMR (CDCl ) δ 7.95 – 7.90 (m, 1H), 7.74 (d, J = 1.7 Hz,
3
1H), 7.73 – 7.66 (m, 2H), 7.50 – 7.41 (m, 2H), 6.54 (d, J = 6.7 Hz, 1H), 6.48 (dd, J = 2.4/1.9 Hz,
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