Chemoselective synthesis of N-substituted α-amino-α′- chloro ketones via chloromethylation of glycine-derived Weinreb amides

Article abstract of DOI:10.1002/adsc.201201043

Functionalized α-arylamino-α′-chloro ketones are obtained in high yield via a straightforward homologation reaction of Weinreb amides derived from N-arylglycines using in situ generated chloromethyllithium. The use of the Weinreb amides is essential and allows the chemoselective homologation of N-aryl-N-substituted glycine analogues, a transformation which is not possible using similar glycine esters. The procedure is promising for the large-scale preparation of α-amino-α′-chloropropanones, which are valuable precursors for a variety of bioactive compounds. Copyright

Full text of DOI:10.1002/adsc.201201043

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988
Mini-Reviews in Medicinal Chemistry, 2013, 13, 988-996
ꢀ-Amino-ꢀ´-Halomethylketones:
Synthetic
Methodologies
and
Pharmaceutical Applications as Serine and Cysteine Protease Inhibitors
Vittorio Pacea,b*, Laura Castoldia,c and Massimo Pregnolatoc
aDepartment of Drug and Natural Product Synthesis, University of Vienna, Althanstrasse, 14-A-1090 Vienna, Austria,
bDepartment of Organic and Pharmaceutical Chemistry, Complutense University of Madrid, Pza. Ramón y Cajal s/n,
E-28040 Madrid, Spain, c Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100, Pavia, Italy
Abstract: ꢀ-Amino-ꢀ´-halomethylketones are interesting scaffolds bearing (at least) two sequential electrophilic carbons
that by interacting with the nucleophilic moieties of several enzymes, represent the ideal candidates for in vivo and in vitro
inhibition studies. In this work a summary of their use as optimal inhibitors of physiologically relevant serine and cysteine
proteases is given with a particular emphasis on recently established SAR studies. A brief survey of the most relevant
synthetic processes for their obtainment and the importance they possess in synthetic medicinal chemistry is reported.
Keywords: Aminoketones, Drug Synthesis, Haloketones, Inhibitors, Proteases, Structure-activity Relationship.
1. INTRODUCTION
aminohalohydrin (5) [9], which is the direct precursor of the
enantiopure epoxide (6). This oxirane, may be opened by a
nitrogenated nucleophile, thus providing the 1,3-
diaminopropan-2-ol fragment (7), that represents the
common feature of such class of inhibitors (Scheme 3)
[10-14].
ꢀ-Amino-ꢀ’-halomethylketones constitute
a versatile
class of organic structures [1] that may be viewed as acetone
derivatives bearing two distinct functions at the vicinal
positions of the carbonyl, namely the halogen atom and the
eventually substituted amino group (Scheme 1). Such a
feature governs all their chemical reactivity, as well as their
involvement as useful biologically active compounds:
effectively, from a reactivity perspective they present (at
least) two consequent electrophilic carbon atoms and thus, in
principle each of them may react with a given nucleophile [2,
3]. Both the halomethylenic fragment and the carbonyl one
are subject to nucleophilic reactions carried out with a
plethora of nucleophiles incorporated in enzymes and
peptides thus, allowing covalent bond formation responsible
for their use as enzyme-inhibitors [4].
R'
N
O
Nu
X
R
R, R' = H, alkyl, aryl, acyl
X = Cl, Br, I, F
Scheme. (1). General structure of ꢀ-amino-ꢀ’-halomethylketones.
H
Ph
O
N
NHBut
OH
O
H
N
H
H
N
O
N
Regardless the nature of the incorporated halogen atom,
the chemical properties displayed by them depend on the
nucleophilicity of the nitrogen atom which, in turn
determines the stability of the compound [5]. As a
consequence, they often present an electron-withdrawing
group (acyl type) on this nitrogen in order to modulate its
reactivity towards electrophiles [6]; in fact, the possibility to
suffer self-condensation is not remote and leads to the
destruction of the molecule.
HO
N
N
H
H
Me
O
O
NH2 OH
S
N
H
H
O
Saquinavir (1)
Nelfinavir (2)
Ph
N
O
O
O
O
S
NH OH
O
NH2
Amprenavir (3)
Effectively, much of the scientific attention they received
in the last decades is due to their usefulness as privileged
prochiral building blocks for the preparation of HIV protease
inhibitors [e.g. saquinavir (1), nelfinavir (2), amprenavir (3)]
(Scheme 2). [1, 7, 8] In fact, the enantioselective reduction
of the carbonyl of haloketone 4 leads to the homochiral
Scheme. (2). Examples of HIV protease inhibitors derived from ꢀ-
amino-ꢀ’-halomethylketones.
The particular route: ketone reduction - ring closure
-epoxide opening deserves some comment: although in
principle, the nucleophilic displacement may be carried out
on the aminohalohydrin (5), the reaction on the epoxide is
preferred for two main reasons: a) the reactivity of the latter
is evidently enhanced compared to the former as a
consequence of the ring-strain b) epoxides are easily
*Address Correspondence to this author at the Department of Drug and
Natural Product Synthesis, University of Vienna, Althanstrasse, 14-A-1090
Vienna, Austria; Tel: +34-91-3941820; Fax: +34-91-3941822;
E-mail: vpace@farm.ucm.es
1875-5607/13 $58.00+.00
© 2013 Bentham Science Publishers

ꢀ-Amino-ꢀ´-Halomethylketones: Synthetic Methodologies and Pharmaceutical
Mini-Reviews in Medicinal Chemistry, 2013, Vol. 13, No. 7 989
HIV protease inhibitors
Ar
R'
R'
N
OH
Ar
R'
N
O
O
O
RO
N
RO
X
RO
X
R'''
RO
N
N
O
O
O
R'
OH R''
Ar
Ar
(7)
(6)
(5)
(4)
Scheme. (3). Retrosynthesis of HIV protease inhibitors starting from ꢀ-amino-ꢀ’-halomethylketones.
chemoselectively obtained by base-promoted cyclization of
the aminohalohydrin [15].
amino-ꢀ’-halomethylketones is the acidolysis of easily
obtained ꢀ-amino-ꢀ’-diazoketones (9) [16-19] (Scheme 4);
although this procedure requires the use of the toxic and
dangerous reagent diazomethane, it is still currently used
because of its economical advantages and its high-yielding
performance [20, 21]. Effectively, to date it is possible to
minimize the amount of diazomethane allowing a safe scale-
up of the synthesis [22].
This review deals with the employment of the title
compounds in medicinal chemistry relevant processes: a
brief summary on the synthetic procedures, as well as their
importance as precursors of outstanding pharmaceuticals will
be presented. Subsequently, an overview of their capability
in inhibiting key-enzymes such as serine and cysteine
proteases will be discussed. Extensive surveys on the
synthesis and pharmaceutical employment of such
compounds as HIV protease inhibitors have been recently
published [1, 8] and thus, these aspects will just be briefly
mentioned in this work.
Nugent and coworkers proposed [23] the replacement of
diazomethane with the more environmental friendly
dimethylsulfoxonium methylylide, which in the presence of
a base converts the aminoacetic acid derivative 10, into the
ꢁ-ketodimethylsulfoxonium ylide 11, which is hydrolyzed to
give the desired ꢀ-chloroketone 11a (Scheme 5). Noticeably,
this technique can be applied to optically active starting
materials without loss of enantiopurity.
2. SYNTHESIS OF ꢀ-AMINO-ꢀ’-HALOMETHYL-
KETONES
As observed above, the crucial parameter in the
preparation of ꢀ-amino-ꢀ’-halomethylketones is the
electronic nature of the substituents on the nitrogen atoms: in
particular, the presence of alkylamino or acylamino
functions does not complicate their synthesis and different
methodologies have been developed so far. It is possible to
classify them on the bases of the chemical transformations
involved. So far the most direct approach involves the one-
carbon homologation of an appropriate precursor like a 2-
aminoacetic acid derivative (8) (acyl chloride or carboxylic
ester): in this case, the oldest and classical access to ꢀ-
However, the predominant approach to ꢀ-amino-ꢀ’-
halomethylketones is the use of lithium carbenoids
(chloromethyllithium or bromomethyllithium) developed by
Barluenga [24-27] such a protocol consists of the in situ
formation of the Li carbenoid at low temperature (-78ºC) by
the action of an alkyllithium (MeLi, n-BuLi) on
dihalomethane (CH2XY, X = Cl, Br; Y = Br, I) and
subsequent in situ trapping with the electrophilic 2-
aminoacetic acid derivative (12) which, upon aqueous acidic
treatment affords directly the haloketone (Scheme 6).
Although widely employed, this strategy presents serious
CH2N2 (excess)
Et2O
R'
HY
Y
N2
(PG)RN
(PG)RN
(PG)RN
O
O
O
8
9
9a
X = Cl, OCOR
PG = Moc, Boc, Cbz
Scheme. (4). Synthesis of ꢀ-amino-ꢀ’-halomethylketones via diazocarbonyl chemistry.
NHPG
NHPG
NHPG
OR'
LiCl, MsOH
Me3(O=)S
Cl
R
Cl
R
S
R
O
tBuOK, THF, 0˚C
O
O
70˚C
70-81%
O
85-95%
10
11
11a
R = CH3, iPr, Bn
R' = CH3, Np
PG = Boc, Cbz
Scheme. (5). Synthesis of ꢀ-amino-ꢀ’-halomethylketones via sulfur ylide chemistry.

990 Mini-Reviews in Medicinal Chemistry, 2013, Vol. 13, No. 7
Pace et al.
disadvantages as a consequence of the inherent high
response and apoptosis. On the other hand, uncontrolled
proteolytic processes often constitute the biochemical
rationale for different pathologies including viral infections,
cancer, inflammation and Alzheimer’s disease [36]. For
these reasons, the possibility to interfere on those
unregulated routes by means of protease inhibitors is of
outstanding importance in medicinal chemistry and herein an
overview of the use of the title compounds as irreversible or
covalent inhibitors is discussed.
reactivity of organolithiums that require
a
careful
optimization of the reaction conditions prior to the scale-up
[28, 29].
ClCH2I, MeLi
R'
R2N
Cl
R2N
12
O
O
LiBr, THF, -78˚C
NH4Cl (aq.)
12a
The active sites of serine, cysteine, and threonine
proteases show a high degree of similarity including an
active site nucleophile and a general base (Scheme 9), which
enhances the nucleophilicity of the heteroatom (17 to 18)
(oxygen for Ser, sulfur for Cys), thus allowing the attack on
the amidic carbonyl of 19 [36, 38, 39]. From a mechanistic
point of view, the transition state represented as a tetrahedral
adduct (the so-called oxyanion hole, 20), is stabilized by
different hydrogen bonds.
R' = OR, OCOR
R = alkyl, acyl
Scheme. (6). Synthesis of ꢀ-amino-ꢀ’-halomethylketones via
lithium carbenoids chemistry.
On the contrary, the presence of arylamino substituents
has been object of recent synthetic studies: it is conceivable
that the mesomeric effect played by the aniline-type
fragment further increases the chemical complexity of the ꢀ-
amino-ꢀ’-halomethylketones [30] and thus, the synthetic
approaches. To date it is possible to prepare such
arylaminoketones by the oxidative hydrolysis of easily
synthesized protected N-(2-haloallyl)anilines (13) [31-33]
with hypochlorite [34] (Scheme 7) or by the oxidation of the
corresponding N-protected aminohalohydrins with the Dess-
Martin periodinane (DMP) (15) (Scheme 8) [6, 35]. As it can
be observed, both strategies require the protection of the
amino function as acyl or alkyl derivative with the aim to
render make chemically inert the aromatic system.
The high electrophilicity of ꢀ-haloketones per se renders
them exceptional alkylating agents and thus, it is not
surprising why they are considered optimal enzymes
inhibitors candidates as described by Shaw et al. [40, 41]. In
fact, these Authors in their pioneering studies demonstrated
their potential as serine proteases inhibitors of trypsin-like
and chymotrypsin-like enzymes. Effectively, they developed
a series of peptidyl chloromethylketones including TPCK [1-
(tosylamino)-2-phenylethyl chloromethyl ketone] (21) which
showed a specific inhibitory activity towards the serine
protease chymotrypsin. Later, Poulos and coworkers
clarified the mechanism of such serine-inhibition [42], in
particular they showed the formation of the tetrahedral
adduct (23) formed by the attack of the serine hydroxyl
group to the carbonyl of the haloketone (22). Subsequently,
this adduct alkylates a residue of histidine of the protease
active site, giving the inactivated enzyme (26). It is unclear if
the alkylation occurs directly on the tetrahedral intermediate
(23 to 25) as proposed by Poulos, or it proceeds through a
two-steps sequence represented by the formation of a
reactive epoxide (24), followed by its opening mediated by
histidine, as conceived by Powers and coworkers (Scheme 10)
[43].
3. USE OF ꢀ-AMINO-ꢀ’-HALOMETHYLKETONES
AS
SERINE
AND
CYSTEINE
PROTEASES
INHIBITORS
Proteases are an ubiquitous class of enzymes deputed to
peptidic bonds hydrolysis [36]; a generally accepted
classification allows to distinguish four principal groups:
aspartic, serine, cysteine and metallo proteases [36, 37]. As a
consequence of the generality of this reaction in mammals, it
is clear their importance in regulating physiological
pathways such as digestion, wound healing, cell
differentiation and growth, blood coagulation, immune
CF3
Cl
O
CF3
O
Ca(ClO)2 (1.50 equiv.)
Acetone, H2O, AcOH
O
O
NaHCO3 (5% aq.)
H
N
N
N
Cl
Cl
Z
Z
Z
i-PrOH, 0.5 h
0˚C, 1 h, quant.
quant.
13
14
14a
Z = EWG
Scheme. (7). Synthesis of ꢀ-amino-ꢀ’-halomethylketones via oxidative hydrolysis of vinyl halides.
OR'
OH
OR'
O
O
TMSI, CH2Cl2
-20˚C-0˚C
O
O
DMP
H
N
Cl
N
Cl
N
Cl
Ar
Ar
Ar
CH2Cl2, 0.5 h
90-100%
3-24 h
71-83%
15
16
16a
Scheme. (8). Synthesis of ꢀ-amino-ꢀ’-halomethylketones via oxidation of N-protected halohydrins.

ꢀ-Amino-ꢀ´-Halomethylketones: Synthetic Methodologies and Pharmaceutical
Mini-Reviews in Medicinal Chemistry, 2013, Vol. 13, No. 7 991
O
H
Asp
O
(ArCH2CH2CO-) [47]. This simple transformation allows to
obtain an exceptional high activity against chymase (IC50
O.36 μM) compared to cathepsin G which was not inhibited
even at very high concentrations.
Ser
O
Ser
O
H
O
O
H
H
Asp
N
N
N
N
17
18
O
Cysteine proteases belong to a class of peptide-bond-
cleaving hydrolases, expressed in practically almost
organisms. The importance in humans is remarkable because
of their involvement in the pathogenesis of several diseases.
Accordingly, it is possible include them in the following sub-
classes calpains, cathepsins and caspases [38].
R'
R
N
H
19
O
O
Ser
O
R
R'
H
R'NH2
+
N
R
O
H
Further studies revealed that more nucleophilic mercapto
groups of cysteine proteases can be effectively inhibited by
such haloketones [48]. Analogously to the serine inhibition
described above, there are two different pathways leading to
the alkylation of cysteine proteases. As depicted in (Scheme 11),
the cysteine-inhibitor complex 26 can be formed by the
direct displacement of the chlorine (via A) or by the
tetrahedral thiohemiacetal (27) formed by the addition of the
thiolate to the carbonyl moiety (via B). Subsequently, this
tetrahedral intermediate evolves through an intramolecular
nucleophilic displacement operated by the alcoxy group to
afford the ꢀ-mercapto substituted epoxide (not shown),
which spontaneously rearranges to thiiranium (28). Finally,
this activated three-membered sulfoxonium ring, isomerizes
to the ꢀ-mercaptocarbonyl compound 26. The second
mechanism has not been fully clarified because of a lack of
evidence for the thiiranium ring formation [36, 49, 50].
20
Scheme. (9). Mechanistic pathway of the serine-mediated
proteolysis.
O
H
O
S
O
N
Cl
1-(tosylamino)-2-phenylethyl chloromethyl ketone, TPCK
(21)
H
N
O
O
H
N
O
H
N
O
Ser-OH
Cl
Ser
Cl
R
R
R
O
R'
R'
(24)
R'
(22)
Ser
(23)
Enz-His
Enz-His
Enz
N
Enz
N
H
N
O
H
N
O
H
N
O
O
H
O
Cys-SH
N
N
R
N
Cl
S
R
Cys-Enz
R
R
R'
R'
Ser
via A
R' (22)
R'
(26)
(25)
(26)
via B
Scheme. (10). Mechanism of the inhibition of ꢀ-
chloromethylketones against serine proteases.
Cys
S
H
H
O
S
Fundamental physiological enzymes, i.e. kallikrein,
plasmin, thrombin, and factor Xa, belong to these classes,
and important mechanistic understandings have been
achieved by their inhibition with chloromethylketones. In
fact, trypsin-type proteases are efficiently blocked by
tripeptides structures bearing an Arg residue at P1, [Phe-Pro-
Arg-CH2Cl (k2/Ki 107 M-1s-1 ca.)] [44]. On the contrary, the
inhibition of plasmin continues to be a challenge due to its
broad specificity and to date the best inhibitor tested is Ile-
Phe-Lys-CH2Cl (k2/Ki 105 M-1s-1ca.) [44].
N
N
Cl
Cys
R
R
O
R'
R'
(27)
(28)
Scheme. (11). Mechanism of the inhibition of ꢀ-
chloromethylketones against cysteine proteases.
In general, cysteine-protease inhibitors belong to the
peptidyl chloromethylketone class and possess a high degree
of homology with the optimal peptidic substrates. In fact,
inhibitors of cathepsin B should present a Phe fragment at
P2, as in the case of Cbz-Phe-Ala-CH2Cl (k2/Ki 104 M-1s-1 ca.)
[51]. It is interesting to note that the substitution of the
chlorine with the less reactive fluorine does not lower the
activity and thus, fluoromethyl ketones have been thoroughly
employed in SAR studies [52]. It should be pointed out that
the presence of a protonated residue at P1, as in the case Bz-
Phe-Arg-CH2F maximizes the inhibitory rate (k2/Ki of 105 M-1s-1).
Furthermore, variations at the N-terminal moiety are
admitted: by leaving untouched the dipeptidyl residue Phe-
Ala-CH2F, it is possible to install large substituents as
BnOCO CH2CH2CO on the N-terminal of Phe.
In addition, the common structural feature of
chymotrypsin inhibitors is the presence of a Phe residue at
P1. However, the deactivation of these proteases is
kinetically slower than the corresponding trypsin-like
enzymes: in fact, chymotrypsin is inhibited by the tripeptidyl
chloromethyl ketone Boc-Gly-Leu-Phe-CH2Cl (k2/Ki 60 M-1s-1
ca.) [45] or by Suc-Pro-Leu-Phe-CH2Cl (k2/Ki 4 M-1s-1 ca.)
[46]. It is remarkable how the former inhibitor rapidly
deactivates another serine protease such as the chymase
(k2/Ki 170 M-1s-1 ca.) [41]. Furthermore, Kiso and coworkers
designed a potent and selective chymase inhibitor with no
activity towards cathepsin G: in this case, the classical Cbz
group (PhCH2CO-) on Phe has been replaced by a homolog

992 Mini-Reviews in Medicinal Chemistry, 2013, Vol. 13, No. 7
Pace et al.
CO2H
The tripeptidyl chloromethylketones like the Leu-Leu-
Phe-CH2Cl series, have been evaluated as potent inhibitors
of calpains I and II [53]. Interestingly, the physiological role
of this class deals with the degradation of the myofibrils
proteins, responsible of damaging muscular and cardiac
tissue [54]. Furthermore, recently has been recognized a
significant activity in both cancer and neuronal cells [55].
Effectively, calpain I is the predominant form expressed in
damaged nervous tissue and for this reason its inhibition has
been object of several studies. In particular, Chatterije and
coworkers designed a series of potent and selective inhibitors
(dipeptide fluoromethylketones) (29a-d) (Scheme 12) in
which a significant influence in the potency is played by the
N-terminal capping group: more concretely, a substantial
improvement (up to 4 times) is observed when the benzyl
F
O
R'
NH
O
HN
O
R, R' = Me, R'' = i-Pr IDN1965 (30)
R, R = H, R'' = Me IDN1529 (31)
R''
N
R
Scheme. (13). Selective caspases inhibitors IDN1965 and IDN1529.
Effectively, several SAR studies have been performed
with the aim to improve the selectivity of these caspase
inhibitors. In this regard, Cai and coworkers showed that the
N-protecting groups of Val-Asp-CH2F fragments influence
the potency towards caspase 3 and, in general a broad range
of functionalities are well tolerated, although larger
substituents afford the best results (Cbz vs. Ac) (Scheme 14)
[61]. Val is the best aminoacidic residue for the P2 position
among the natural and non-natural aminoacids explored for
the purpose [62]. Interestingly, the free carboxylic acid in the
Asp residue is important not only for the activity as caspase
inhibitor, but also for the selectivity over non-caspase
proteases. It is worth observing how the hydrophobicity of
the molecule enhances the activity in cell apoptosis assays,
because of the increased permeability through the cell
membrane. Among the series of compounds screened by
these authors, compound MX1122 (2,4-dichloro-Cbz-Val-
CH2F), (Table 2, entry 1) showed a potent and broad caspase
inhibition spectrum, jointly with a good activity in the cell
apoptosis protection due to the afore mentioned high
apolarity [62]. Its high activity against several caspases
renders it particularly useful in the treatment of those
diseases such as brain ischemia and myocardial infarction
where it is desirable to have a fast and effcient inhibition of
all caspases. Further SAR studies aimed to evaluate the
effect of different N-protecting groups of the Val-Asp-
fluoromethylketone core (32), revealed that the
cyclopentymethoxycarbonyl- (entry 2), benzylglutaryl-
(entry 3), glutaryl- (entry 4), benzyloxy- (entry 5),
functionalities confer an interesting inhibitory action towards
apoptosis induced by TNF-ꢀ in HeLa cells. The substitution
pattern on the aromatic ring of aryloxymethyl compounds
plays a crucial role in maximizing the inhibitory effect: in
fact, by considering two regioisomers as the 2,5-
dichlorobenzyl and the 3,4-dichlorobenzyl, an improved
inhibition activity was observed for the former towards
caspase 3 (IC50 15 nM vs. 21 nM, entries 6-7). Interestingly,
these relatively apolar nitrogen protecting groups optimize
the hydrophobicity thus, providing the physic-chemical properties
required for a successful permeation into the cells [62].
urea
motif
is
replaced
by
the
constrained
tetrahydroisoquinolyl fragment (Table 1, entry 4).
Furthermore, such an inhibitor shows a considerable
specificity in inhibiting calpain I compared to other calpains
B and L [56].
R2
O
H
N
N
F
R1
H
O
Scheme. (12). Fluoromethylketones inhibitors of recombinant
human calpain I.
Table 1.
Recombinant Human Calpain I Inhibitory Activity
of Fluoromethylketones.
Entry
R1
R2
kobs/I M-1 s-1 (a)
-CO2CH2C6H5
1
2
3
-CH2CH3
-CH2C6H5
-CH2C6H5
24.250
(29a)
-CO2CH2C6H5
136.300
(29b)
-CONHCH2C6H5
67.200
(29c)
4
-CH2C6H5
276.000
(29d)
(a) Pseudo-first order rate constant for inactivation.
Effectively, the isosteric replacement of the amidic
function of 32 with the urethane moiety in 33, allows to
preserve the selective caspase inhibition activity, being
compound MX1153 the most effective inhibitor of this series
of dipeptidylaspartyl fluoromethyl ketones (Scheme 15).
Remarkably, also a significant apoptosis cell protection has
been noticed for some of these structures (Table 3) [63].
Interestingly, the use of such dipeptides offers the additional
advantage of a good stability and thus, they are particularly
suitable for in vivo studies.
Analogously, other cysteine-proteases such as the
caspases [57, 58] are the target of several fluoromethyl
ketones [59]. Of outstanding relevance are IDN1965 (30)
which has a moderate selectivity against caspases 6, 8 and 9
and IDN1529 (31) which is a broad-spectrum caspase
inhibitor [60]. Such structures present an analogous
fluoromethyl ketone with a Asp residue at P1 and a indoyl-
type substituent at the N-terminal (Scheme 13).

ꢀ-Amino-ꢀ´-Halomethylketones: Synthetic Methodologies and Pharmaceutical
Mini-Reviews in Medicinal Chemistry, 2013, Vol. 13, No. 7 993
CO2H
CO2H
O
H
O
H
R
N
R1
N
O
N
H
F
N
H
F
O
O
O
O
R2
32a-g
33a-d
Scheme. (14). General structure of fluoromethylketones with
Scheme. (15). General structure of ꢀ-carbamoyl-alkylcarbonyl-
selective caspase inhibition.
aspartyl-fluoromethyl ketones.
Table 2.
Caspase-3 Inhibiting activity of Dipeptide Fluoromethylketones-type.
Entry
R
IC50 (nM)
1
2,4-di-Cl-PhCH2O (32a) MX1122
21
¡
30
33
50
(32b)
Ph
O
3
4
O
(32c)
HO
O
(32d)
5
6
7
PhCH2O (32e)
30
15
21
2,5-chloroPhCH2O (32f)
3,4-chloroPhCH2O (32g)
Table 3.
Caspase-3 Inhibiting activity and Cell Apoptosis Protection activity of ꢀ-carbamoyl-alkylcarbonyl-aspartyl-fluoromethyl
ketones.
Entry
R1
R2
IC50(nM)
50% Cell Protection (nM)
1
2
3
4
Ph
Ph
Me (33a)
2-Pr (33b) MX1153
Me (33c)
66
17
20
12
1400
200
ND
PhCH2
2,5-DiCl-Ph
2-Pr (33d)
ND
Based on these experimental results, Anfantitis and
coworkers constructed a reliable protocol for the activity
prediction of such fluoromethylketones [64]. However, the
use of chloroketones as enzyme inhibitors is often associated
to the lack of selectivity towards a specific aminoacidic
residue (serine vs. cysteine) or to their tendency to alkylate
ubiquitous entities (e.g. glutathione) and thus, for such a
reason they are inappropriate for in vivo experiments [36].
versatile inhibitors due to their rapid degradation in an
aqueous medium. These studies on the effect of the nature of
the halogen atom demonstrated that a good alternative to ꢀ-
haloketone is represented by ꢀ-acyloxymethyl ketones [65,
66].
Moreover, the potential block of other cysteine protease,
such as the interleukin-1b-converting enzyme (ICE),
represents an useful pharmacological strategy in the
treatment of inflammation-type diseases. In fact, such an
enzyme can be efficiently alkylated by halomethylketones in
both reversible or irreversible manner. These kinds of
halomethylketone-type ICE-inhibitors, such as Boc-
Asp(benzyl)-chloromethyl ketone (34), were first reported
by Estrov and coworkers at Immunex [67]. Structural
modifications at the N-substituent (e.g. Ac-Tyr-Val-Ala-
Effectively, the absence of specificity observed with
chloroketones can be controlled by introducing a poor
leaving group: in fact, as showed above, the replacement of
chlorine with fluorine gives a specific and powerful
irreversible cysteine-inhibitor without any noticeable
inactivation of serine proteases. On the contrary, the
corresponding bromo and iodo-ketones do not represent

994 Mini-Reviews in Medicinal Chemistry, 2013, Vol. 13, No. 7
Pace et al.
H3C
CH3
CO2Bz
CH2Cl
O
H
H
N
O
N
N
H
N
CO2Bz
H
O
H
O
CH3
O
O
N
CH2Cl
N
H
O
CH3
O
OH
Boc-Ala-Asp(O-benzyl)-chloromethyl ketone
Ac-Tyr-Val-Ala-Asp-chloromethyl ketone
34
35
H3C
O
CH3
CO2CH3
CH2F
O
O
H
N
O
N
N
H
H
O
CH3
Cbz-Val-Ala-Asp(O-methyl)-fluoromethyl ketone
36
Scheme. (16). Series of halomethylketones cysteine protease inhibitors with interleukin-1b-converting enzyme activity.
[2]
[3]
[4]
Concellón, J. M.; Rodríguez-Solla, H. Synthesis and synthetic
applications of amino ketones derived from natural alpha-amino
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De Kimpe, N.; Verhé, R., The Chemistry of ꢀ-Haloketones, ꢀ-
Haloaldehydes and ꢀ-Haloimines. In The Chemistry of ꢀ-
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Hamada, T.; Torii, T.; Onishi, T.; Izawa, K.; Ikariya, T.
Asymmetric Transfer Hydrogenation of α-Aminoalkyl αâꢁ˜-
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E.; Appelt, K.; Burgess, J. A.; Campanale, K. M.; Chirgadze, N.
Y.; Clawson, D. K.; Dressman, B. A.; Hatch, S. D.; Khalil, D. A.;
Kosa, M. B.; Lubbehusen, P. P.; Muesing, M. A.; Patick, A. K.;
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Asp-chloromethyl ketone, 35) or at the P1 moiety (e.g. Cbz-
Val-Ala-Asp-fluoromethyl ketone, 36) allowed to obtain
highly efficient and specific inhibitors for clinical
employment (Scheme 16) [68].
Finally, the activity of these alkylating chloromethyl
ketones against cysteine protease enzymes like gingipains,
responsible for chronic periodontitis from Porphyromonas
gingivalis, leads Bialas and coworkers to develop a series of
inhibitors by applying a combined structure-based design
approach / 3D model using the X-ray structure of the enzyme
[69].
[5]
[6]
4. CONCLUSIONS
Although ꢀ-amino-ꢀ’-halomethylketones alkylate the
residues of serine and threonine mainly in an irreversible
manner and thus, their employment as drug candidates is
somewhat limited, undoubtedly they are exceptional agents
for in vitro and in vivo evaluations. In particular, these agents
contribute to understanding the bases of molecular
recognition between the enzyme and its optimal inhibitor.
Additionally, the recent synthetic studies allow to prepare
them in an efficient and safe way thus, facilitating their use
for in vitro studies.
[7]
[8]
[9]
CONFLICT OF INTEREST
[10]
[11]
The authors confirm that this article content has no
conflicts of interest.
ACKNOWLEDGEMENTS
The Authors warmly thank the University of Vienna and
Pavia for financial support. One of the Authors (V. P.) is
indebted to the OEAD for a postdoctoral Ernst Mach grant.
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Received: May 31, 2012
Revised: August 08, 2012
Accepted: August 08, 2012