Reactivity of imidazolidin-4-one derivatives of primaquine: implications for prodrug design

Article abstract of DOI:10.1016/j.tet.2006.08.026

In contrast to peptide-based imidazolidin-4-ones, those synthesized from N-(α-aminoacyl) derivatives of the antimalarial drug, primaquine and ketones are unexpectedly stable in pH 7.4 at 37 °C. The kinetics of hydrolysis of primaquine-based imidazolidin-4

Full text of DOI:10.1016/j.tet.2006.08.026

Tetrahedron 62 (2006) 9883–9891
Reactivity of imidazolidin-4-one derivatives of primaquine:
implications for prodrug design
a,b
a
a
c
b
Paula Chambel, Rita Capela, Francisca Lopes, Jim Iley, Jos ꢀe Morais,
b
d
d
a,
Lu ´ı s Gouveia, Jos ꢀe R. B. Gomes, Paula Gomes and Rui Moreira
*
a
Centro de Estudos de Ci e^ ncias Farmac e^ uticas, Faculdade de Farm aꢀ cia da Universidade de Lisboa, Av. For c¸ as Armadas,
P-1649-019 Lisboa, Portugal
b
UCTM, Faculdade de Farm aꢀ cia da Universidade de Lisboa, Av. For c¸ as Armadas, P-1649-019 Lisboa, Portugal
c
Department of Chemistry, The Open University, Milton Keynes, MK7 6AA, United Kingdom
d
Centro de Investiga c¸ a~ o em Quı´mica da Universidade do Porto, Departamento de Quı ´m ica da Faculdade de Ci e^ ncias do Porto,
Rua do Campo Alegre 687, P-4169-007 Porto, Portugal
Received 13 June 2006; revised 3 August 2006; accepted 7 August 2006
Available online 24 August 2006
Abstract—In contrast to peptide-based imidazolidin-4-ones, those synthesized from N-(a-aminoacyl) derivatives of the antimalarial drug,
ꢀ
primaquine and ketones are unexpectedly stable in pH 7.4 at 37 C. The kinetics of hydrolysis of primaquine-based imidazolidin-4-ones
ꢀ
were investigated in the pH range 0.3–13.5 at 60 C. The hydrolysis to the parent a-aminoacylprimaquine is characterized by sigmoidal-
shaped pH–rate profiles, reflecting the spontaneous decomposition of both unionized and protonated (at N-1) forms of the imidazolidin-4-
a a
one. The kinetically determined pK values are ca. 3.6–4.0, i.e., 4 pK units lower than those of amino acid amides, thus implying that
hydrolysis of imidazolidin-4-ones at pH 7.4 involves the unionized form. Reactivity of this form decreases with the steric crowding of the
amino acid a-substituent. In contrast, the rate constant for the spontaneous decomposition of the unionized form increases sharply for
imidazolidin-4-ones derived from cyclic ketones, an observation that can be explained by the I-strain (internal strain) effect. These results
are consistent with a mechanism of hydrolysis involving an S 1-type unimolecular cleavage of the imidazolidin-4-one C2–N3 bond with
N
departure of an amide-leaving group. The mechanism for the decomposition of the protonated imidazolidin-4-one is likely to involve an
amide-carbonyl oxygen protonated species, followed by the C2–N3 bond scission, as supported by computational studies. The results herein
presented suggest that imidazolidin-4-ones derived from simple N-alkyl a-aminoamides are too stable and therefore, may be useful as slow
drug release prodrugs.
Ó 2006 Elsevier Ltd. All rights reserved.
1
. Introduction
Peptide imidazolidin-4-one derivatives, 1, are readily hydro-
ꢀ
lyzed to the parent peptide in pH 7.4 buffer at 37 C, usually
5
–8
Compounds containing an a-aminoamide moiety, such as
peptides with a free N-terminal amino group, react with al-
dehydes and ketones to yield imidazolidin-4-ones, 1, as de-
with half lives ranging from 1 to 30 h (Scheme 1). When
compared with the parent peptides several imidazolidin-4-
one prodrugs of Met-enkephalin and Leu-enkephalin, e.g.,
2, display improved stability in human plasma, rabbit liver
1
–4
picted in Scheme 1. In aqueous solutions, compounds 1
revert back to the parent a-aminoamide (peptide) and alde-
hyde or ketone at a rate that is dependent on pH, on the struc-
7
homogenate, and toward aminopeptidase. This prodrug
strategy has been extended to improve the bioavailability
1
4
ture of the a-aminoamide substituents (R and R ), and on
the structure of the carbonyl component (R and R ). In
contrast to peptides, the hydrolysis of imidazolidin-4-ones
of ampicillin, a b-lactam antibiotic that also contains a
a-aminoamide backbone; the corresponding imidazolidin-
4-one, hetacillin (3), is rapidly hydrolyzed to ampicillin in
2
3
4,5
2,4
1
is not subjected to enzyme catalysis and thus imidazoli-
din-4-ones have been suggested as potentially useful pro-
drugs to protect the N-terminal amino acid residue of
peptides against aminopeptidase-catalyzed hydrolysis.
R1
O
6
–8
R1
O
R2
4
+
O
HN
R2
N
R3
R4
+
H O
2
H N
N
R
R3
2
H
Keywords: Imidazolidin-4-ones; Prodrugs; Primaquine; Kinetics; Substitu-
ent effects; Mechanism of hydrolysis.
1
*
Corresponding author. Tel.: +351 217946477; fax: +351 217946470;
e-mail: rmoreira@ff.ul.pt
Scheme 1.
0
040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.08.026

9884
P. Chambel et al. / Tetrahedron 62 (2006) 9883–9891
4
9
aqueous solutions and in vivo. More recently, the imidazo-
lidin-4-one approach has been proposed to develop oil-in-
jectable depots for the a-aminoanilide anesthetic agent
differences in reactivity, as previous authors had suggested
that substituents at the N-3 nitrogen atom in dipeptide and
tripeptide imidazolidin-4-ones exert a small effect on hydro-
1
0
7
prilocaine.
lysis rates. Obviously, understanding the reason for such
large differences in reactivity is fundamental to being able
to extend the imidazolidin-4-one prodrug approach to a-
aminoamides other than peptides. In this context, we now
present a kinetic study of hydrolysis of imidazolidin-4-
ones 4 undertaken to elucidate the structural factors that
affect their reactivity and to assess the usefulness of this pro-
drug type to other a-aminoamide drug moieties.
H
H
N
N
O
O
H
N
N
Ph
N
S
N
H
N
H
CO H
2
O
O
N
O
Ph
O
CO H
HO
2
2
3
We have recently synthesized the imidazolidin-4-ones 4 as
potential pro-prodrugs of the antimalarial primaquine, 5
1
1,12
2. Results and discussion
(
PQ).
Compounds 4 were designed with the aim of re-
ducing the metabolic inactivation pathway of primaquine
that involves oxidative deamination at the primary amino
2
.1. Kinetics and products of hydrolysis
1
3–16
group,
by primaquine, particularly its ability to induce oxidation
as well as to reduce the blood toxicity induced
As revealed by HPLC analysis, all compounds 4 hydrolyze
ꢀ
1
7
quantitatively at 60 C to the corresponding amino acid de-
of oxyhemoglobin to methemoglobin. The proposed
activation pathway of imidazolin-4-ones 4 implies the spon-
taneous hydrolysis to the corresponding amino acid deriva-
tives 6, which in turn can be enzymatically hydrolyzed to
the parent drug by parasitic aminopeptidases. In order to cir-
cumvent hydrolysis by the GI tract peptidases, the imidazo-
lidin-4-one pro-prodrug should hydrolyze at a rate that
allows quantitative formation of 6 after oral absorption.
Quite surprisingly, our initial studies showed that prima-
quine derived imidazolidin-4-ones 4 are reasonably stable
compounds, hydrolyzing to the corresponding amino acid
rivative 6 with first-order kinetics up to 4 half lives, over the
pH range 0.5–12. For the imidazolidin-4-one 4b it was also
possible to observe the hydrolysis of the corresponding ami-
no acid derivative 6 to primaquine at pH>10. An example of
product analysis is presented in Figure 1A for the hydrolysis
of compound 4b in pH 13.7, where the solid lines represent
the best computer fit to the experimental data of the consec-
utive first-order reactions model (i.e., 4/6/5) represented
1
8
by Eqs. 1–3.
ꢂk1t
ꢀ
½
½
4ꢁ ¼ ½4ꢁ e
ð1Þ
ð2Þ
derivatives, 6, in pH 7.4 buffer at 37 C with half lives rang-
ing from 9 to 30 days,
0
1
1,12
i.e., 50–100 times slower than
most of the imidazolidin-4-one counterparts derived from
di- and higher peptides. We were surprised by these large
½
4ꢁ k ꢀ
ꢂ eꢂk2tꢁ
0
1
ꢂk1t
e
6ꢁ ¼
k ꢂ k
2
1
MeO
½
4ꢁ ꢂ ꢀ1 ꢂ eꢂk1tꢁ
Here [4] is the concentration of the imidazolidin-4-one at
0
time zero, and k and k are the pseudo-first-order rate con-
1 2
ꢀ1 ꢂ eꢂk2tꢁꢃ
0
½
5ꢁ ¼ k
k
2
ꢂ k
1
ð3Þ
N
O
2
ꢂ k
1
HN
R1
N
R2
Me
N
R3
H
R1
R2
R3
stants for the hydrolysis of the starting material 4 and amino
acid derivative 6, respectively (data not shown). For 6b (pri-
maquine-Phe), values of k so-obtained can be plotted versus
4
a
b
H
Me
Me
Me
Me
Me
Me
2
4
CH Ph
2
ꢂ
ꢂ2
ꢂ1 ꢂ1
[
OH ] to obtain a value of k
ꢂ
¼ 3:6 ꢃ 10
M h ;
OH
4
c
CHMe2
H
this compares favorably with the value of kOHꢂ ¼ 2:2ꢃ
4d
(CH2)4
ꢂ2
of 6b in the same pH range (pH 11–13.7).
ꢂ1 ꢂ1
1
0
M h determined independently for the hydrolysis
4
e
CH Ph
(CH )
2
2 4
4
f
CHMe2
H
(CH )
2 4
4
g
h
(CH2)5
(CH2)5
At pH values between 2 and 11 no formation of primaquine
was observed over the time-scale for the complete hydroly-
sis of 4b. However, formation of primaquine was observed in
the hydrolysis of 4b at pH<2. The species profile for the hy-
drolysis at low pH values also fits the model for two consec-
utive first-order reactions (Fig. 1B). From the derived plot of
4
Me
4
i
CH Ph
(CH )
2
2 5
4
j
CHMe2
H
(CH )
2 5
4
k
(CH2)6
+
ꢂ1
ꢂ1 ꢂ1
k versus [H ], a value for k
þ
of 1.5ꢃ10
M h was
determined for the acid-catalyzed hydrolysis of 6b to prima-
2
H
MeO
MeO
ꢂ1
quine, which compares favorably with the value of 2.0ꢃ10
N
N
O
ꢂ1 ꢂ1
M
h
determined independently for the hydrolysis of 6b
HN
HN
NH2
NH2
N
H
over the same pH range (pH 0.3–2.0). The higher efficiency
of the acid-catalyzed hydrolysis of 6b when compared with
the alkaline hydrolysis can be ascribed to the stronger
Me
Me
R1
5
6

P. Chambel et al. / Tetrahedron 62 (2006) 9883–9891
9885
A
0
100
8
6
4
2
0
0
0
0
0
-
-
-
1
2
3
8.0
6.0
4
2
.0
.0
0.0
0
5
pH
10
0
50
100
150
t/h
0
2
4
6
8
10
12
14
pH
B
100
Figure 2. The pH–rate profiles for the hydrolysis of the imidazolidin-4-ones
ꢀ
4
b (-), 4c (B), 4d (C), and 4e (,) in aqueous solutions at 60 C; the
1
insert is a plot of k versus pH for 4e.
8
6
4
2
0
0
0
0
0
All pH–rate profiles have a sigmoid shape, with two pH-in-
dependent regions. Such sigmoid pH–rate profiles have been
reported for other imidazolidin-4-ones as well as for
their acyclic counterparts, N-Mannich bases, and can be
accounted for by assuming that both the protonated, SH ,
and the unionized, S, forms of the substrate undergo sponta-
neous hydrolysis (Scheme 2). The best computer fit (solid
line) to the experimental data for 4 in Figure 2 was achieved
using Eq. 4:
4
,5
2
1
+
ꢂ
ꢃ
0
30
60
90
þ
K
a
H
t/h
k
1
¼ kneut
ꢂ
ꢃ þ kprot
ꢂ
ꢃ
ð4Þ
þ
þ
K
a
þ H
K
a
þ H
ꢀ
Figure 1. Time profile for the hydrolysis at 60 C of 4b (-) into PhePQ
C) and PQ (B) in (A) 0.1 M NaOH and (B) 0.5 M HCl solutions.
(
where kneut and k are the apparent first-order rate con-
stants for the decomposition of neutral and protonated forms
prot
of 4, K is the acid dissociation constant of the protonated 4,
a
and [H ]/(K +[H ]) and K /(K +[H ]) represent, respec-
a a a
electron-withdrawing ability of the protonated amino group
+
+
+
1
9
(
(
pKa ca. 8.0) over the corresponding neutral form
s*NH ¼3.76; s*NH ¼0.62) that increases the reactivity
+
3
20
tively, the fractions of the protonated and neutral forms of
present in solution. The kneut and kprot values derived either
from the pH–rate profiles or determined at pH 11 (for kneut
2
4
of the amide-carbonyl carbon atom toward nucleophiles.
)
and pH 0.3 (for kprot) are presented in Table 2. Also included
in Table 2 are the pK values for compounds 4, derived from
the pH–rate profiles. The good agreement between the
The rates of hydrolyses of imidazolidin-4-ones 4 at a fixed
pH value were found to be independent of buffer concentra-
tion over a 10-fold buffer concentration range, indicating the
absence of general acid or base catalysis (Table 1). The in-
fluence of pH on the rate of hydrolysis of compounds 4 is
shown in Figure 2, where the logarithm of the observed
a
R1
+
R1
HN
O
O
K
a
pseudo-first-order rate constant, k , is plotted against pH.
1
H
+
H N
N
N
PQ
R2 R3
+
2
PQ
R2 R3
+
Table 1. First-order rate constants for the hydrolysis of 4b in acetate and
ꢀ
4, SH
4, S
ꢂ3
phosphate buffers at 60 C, with ionic strength maintained at 0.5 mol dm
by addition of NaClO
4
k
k
prot
neut
ꢂ3
2
ꢂ1
Buffer
[Buffer]/mol dm
pH
10 kobs/h
CH
3
CO
2
H
0.005
3.99
3.99
3.98
2.01
1.53
1.66
R1
H N
O
R2
0
0
.01
.05
+
O
N
H
PQ
R3
2
ꢂ
H
2
PO
4
0.002
6.01
6.05
5.95
1.75
1.65
1.72
6
0
0
.005
.01
Scheme 2.

9886
P. Chambel et al. / Tetrahedron 62 (2006) 9883–9891
ꢀ
Table 2. First-order rate constants for the hydrolysis of the neutral, kneut, and
ꢂ1 (at 37 C), and assuming that the equation log k¼
ꢂpK +C holds for compounds 4, then logðk =k Þ ¼
ꢀ
protonated, kprot, forms of the imidazolidin-4-ones 4, determined at 60 C,
ꢂ3
a
1 2
with ionic strength maintained at 0.5 mol dm with addition of NaClO
also included are the pK
4
,
2
a
1
a
pK ꢂ pK and it would be expected that compounds 4 hy-
a
values determined from the pH–rate profile
3
drolyze ca. 10 times slower than their counterparts derived
from dipeptides. The smaller differences reported previously
2
ꢂ1
2
ꢂ1
Compound
10 kneut/h
10 kprot/h
pK
a
ꢀ
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
g
h
i
1.80
1.90
1.32
35.2
5.02
4.61
nd
nd
at 37 C might be attributed to the fact that the amino acid
3.05
1.09
14.7
5.61
nd
nd
nd
8.34
nd
3.95
3.70
3.61
3.98
nd
nd
nd
nd
nd
chain affects both the amide anion leaving group ability as
well as the ability of the imidazolidin-4-one N-1 amino
nitrogen atom to expel the amide. Further support for the
unimolecular mechanism of hydrolysis of neutral imidazoli-
din-4-ones comes from the temperature dependence of k
for 4g (Table 2), which yielded an entropy of activation, DS ,
a
b
c
18.0; 0.160; 1.08; 2.55
neut
#
3.62
2.46
1.69
265
ꢂ1
ꢂ1
#
value of 25 J K mol . Positive DS values are consistent
with unimolecular mechanisms.
j
k
2
3
1.27
nd
Not determined, nd.
Despite the limited data for the pH-independent hydrolysis
of the protonated form of imidazolidin-4-ones 4, it is inter-
a
ꢀ
Reaction temperature: 37 C.
Reaction temperature: 45 C.
Reaction temperature: 50 C.
b
c
ꢀ
ꢂ2
ꢀ
esting to observe that k
h
values range from 1.3ꢃ10 to
prot
ꢂ1 ꢂ1
ꢀ
din-4-ones derived from peptides present k
1
.5ꢃ10
at 60 C (Table 2). In contrast, imidazoli-
values rang-
prot
ꢂ3
ꢂ2 ꢂ1
ꢀ
4–8,10
calculated and experimentally determined first-order rate
constants suggests that the degradation pathway presented
in Scheme 2 and Eq. 4 adequately describes the degradation
kinetics of compounds 4. The kinetically determined pKa
values are ca. 4 units lower than those of amino acid amides
ing from 8.4ꢃ10 to 6.6ꢃ10
h
at 37 C.
Thus,
the protonated form of primaquine derivatives 4 is also sig-
nificantly less reactive than the corresponding ionized form
of imidazolidin-4-ones derived from peptides. These results
are not consistent with a mechanism of decomposition in-
volving the rate-determining C2–N1 bond cleavage of the
imidazolidin-4-one with departure of protonated amine leav-
ing group, i.e., 7. If this were the case, then dipeptide imida-
zolidin-4-ones would be predicted to be less reactive than 4
at low pH as a result of electron-withdrawing effect of the C-
(pK ca. 7.5–8.0). A similar observation has been reported
a
for imidazolidin-4-ones derived from peptides and other
6
compounds containing an a-aminoamide moiety (e.g., prilo-
caine).
1
0
2
.2. Reactivity and mechanism of hydrolysis
terminal carboxyl group (7, R¼CO H) on N-1 nitrogen atom
2
nucleophilicity. An alternative mechanism might involve the
less favorable protonation of the carbonyl oxygen atom (the
A possible explanation for the unexpected chemical stability
of compounds 4 at physiological pH, when compared with
their peptide counterparts, may be found in their mechanism
of hydrolysis. According to Bundgaard and Rasmussen, imi-
dazolidin-4-ones hydrolyze at physiological pH, where the
2
4
pK for the O-protonated amides is ca. ꢂ1) , followed by
a
the C2–N3 bond scission, e.g., 8, as suggested for the hydro-
lysis of acyclic N-Mannich bases in the acidic region of
pH.
2
5
kneut pathway is dominant, via an S 1-type mechanism
N
that involves C2–N3 bond cleavage and departure of an
amide anion leaving group (Scheme 3). Thus, the observed
+
6
O
OH
differences in chemical stability may be ascribed to differ-
ences in the amide anion nucleofugacity. Indeed, amides re-
sulting from the rate-limiting ring opening of 4 are much
poorer leaving groups than those from dipeptide imidazoli-
din-4-ones, as suggested by the estimated difference of 3.3
H N
N
2
CH R
HN
N
+
2
CH R
2
7
8
2
2
pK units between the two amide types. Thus, how can
a
2.3. Structure and energetics
an apparently small difference in pK s dramatically affect
a
the reactivity? Let us assume that the pH-independent hydro-
lysis of imidazolidin-4-ones has the same susceptibility to
the leaving group effect as the acyclic N-Mannich base coun-
terparts, which hydrolyze via the same unimolecular mech-
Theoretical calculations involving two models have been
used to simulate the imidazolidin-4-one ring of 4, both re-
ducing the parent primaquine (5) building block to a single
methyl group, c.f. R in Scheme 3; the first and smaller
4
2
1
anism. Since the Br €o nsted b value for this reaction is ca.
model has substituents R ¼R ¼R ¼H and R ¼CH , the
lg
1
2
3
4
3
O
O
O
R4
R4
R1
O
R2
R3
R4
-
H O
N
2
N
R1
R1
N
H
+
O
R1
R2
+
fast
N
R4
R2
R2
H N
N
NH
2
NH
H
R3
H
R3
HO R3
10
9
11
Scheme 3.

P. Chambel et al. / Tetrahedron 62 (2006) 9883–9891
9887
A
B
C
1.463
1.445
1.467
1.380
1.463
1.373
1.433
1.481
1.299
1.205
.539
1
.536
.220
1
.308
1
1.464
1.531
1.458
1
1
.503
1.517
1.473
1.460
Figure 3. Optimized structures for the neutral (A), N-protonated (B), and O-protonated (C) species of the M1 model.
second resembles the imidazolidin-4-one ring of compound
4
species. The energetic barriers for the opening of the imida-
zolidin-4-one ring of the neutral M1 species are depicted in
Figure 4. These barriers are consistent with a unimolecular
pathway for the hydrolysis of neutral 4, since the energetic
barrier for the alternative pathway involving participation
of a water molecule is significantly higher than that com-
puted without water. For the neutral M2 species, AM1 and
b, i.e., with R ¼CH Ph and R ¼R ¼R ¼CH . These two
1
2
2
3
4
3
models are termed as M1 and M2 from now on. Selected
geometrical data for the neutral, N-protonated, and O-pro-
tonated M1 compounds are shown in Figure 3, while for
M2 compounds these parameters are given as Supplemen-
tary data.
ꢂ1
B3LYP energetic barriers are 172 and 186 kJ mol , respec-
The calculations performed on these models support the
hypothesis of alternative protonation sites, since the B3LYP/
tively. The transition state structures have only one imagi-
nary AM1 or B3LYP calculated frequency with values 196
or 81 cm , respectively. Again, as found for the neutral
M1 species, the AM1 energetic barrier is in good agreement
with that computed at the B3LYP/6-31G* level of theory.
ꢂ1
6
-31G* computed enthalpic difference between the most
stable N-protonated species and the O-protonated species
ꢂ1
is only 7.0 and 31.0 kJ mol for M1 and M2 models, re-
spectively. These values are equal to the differences between
the gas-phase acidities of the N-protonated and O-protonated
compounds.
The computed energetic barriers are much lower for the N-
protonated and O-protonated species as reported in Table 3.
For the M1 model, the B3LYP/6-31G* computed barrier for
ꢂ1
Much more interesting information is retrieved from AM1
and DFT calculations on the reaction of ring opening for
the neutral, N-protonated, and O-protonated M1 and M2
the ring opening in the N-protonated species is 74 kJ mol
while for the O-protonated species, a smaller barrier is
,
ꢂ1
found, c.f., 64 kJ mol . The order of the energetic barriers
350
300
250
200
150
100
3
04
220
219
2
09
+
H O
2
50
4
0
0
16
Figure 4. Energy profile for the imidazolidinone ring opening reaction, with and without a water molecule, computed at the AM1 and B3LYP/6-31G* levels of
theory. The zero of energy corresponds to the reactant with or without a water molecule. Full line: AM1 in the absence of water and numbers in italic; dashed
line: B3LYP in the absence of water with numbers underlined; dotted line: B3LYP in the presence of one water molecule with numbers in normal text.

9888
P. Chambel et al. / Tetrahedron 62 (2006) 9883–9891
ꢂ1
Table 3. Computed free energy differences (kJ mol ) of TS and final
product with respect to the initial (free energy¼0) neutral, N-protonated,
and O-protonated M1 and M2 imidazolidinone models, imaginary frequen-
0
0.4
0.8
1.2
1.6
ꢂ1
cies (cm ) of the TS are also given
-
-
-
-
Compound
AM1
B3LYP
TS Product Frequency TS Product Frequency
4g
M1 neutral+H
M1 neutral
M1 N-protonated 58 ꢂ7
M1 O-protonated 80
M2 neutral 172 151
2
O
— —
220 209
—
304
4
ꢂ629
ꢂ164
ꢂ186
ꢂ117
ꢂ81
ꢂ85
—
ꢂ247
ꢂ469
ꢂ465
ꢂ196
ꢂ387
ꢂ419
219 16
74 64
64 33
186 4
79 60
— —
2
M2 N-protonated 32 ꢂ30
M2 O-protonated 42 ꢂ77
4h
4i
4j
ꢂ1
is reversed using the AM1 approach, being 58 kJ mol for
the N-protonated species and 80 kJ mol for the O-proton-
ꢂ1
-
2
ated species. Nevertheless, the small difference between the
energetic barriers of these species gives further support to
the possibility of a reaction mechanism involving an O-pro-
0
0.2
0.4
0.6
0.8
2
5
1
tonated species, as suggested previously. Finally, for
the M2 model AM1 calculations reveal the energetic
barriers to be 32 and 42 kJ mol for the N-protonated and
Figure 5. Plot of log kneut versus Charton’s steric parameter, n, for the R
substituent in imidazolidin-4-ones 4g–j; the correlation equation for the
line is: log kneut ¼ ꢂ1:30n ꢂ 0:75 ðr ¼ 0:993; n ¼ 4Þ.
2
ꢂ1
O-protonated compounds, respectively, suggesting that the
reaction is faster in the case of the model resembling com-
pound 4b.
2
3
1
4
(1; R ¼R ¼Me; R ¼H, Me, CH Ph; R ¼phenylalanine;
2
ꢀ
6
data determined at 37 C ), we found a reasonable correla-
tion between log kneut and Charton’s steric parameter, n,
for the amino acid substituent:
At first glance, the differences between the computed energy
barriers for either the neutral or the protonated forms of sub-
strate indicate that protonated imidazolidinones would be
markedly less stable than their neutral forms. However,
ring opening of the neutral form leads to charge separation
in the gas-phase, i.e., to the formation of a highly disfavored
zwitterionic species in the gaseous state. Solvation effects in
aqueous media probably allow this species to be much more
stabilized, thus energetically closer to the cationic species
formed from the protonated substrate, which explains our
experimental observation of kneut and kprot of similar magni-
tude for imidazolidin-4-ones 4b,c,e.
ꢀ
ꢁ
2
log kneut ¼ ꢂ0:74n ꢂ 2:27 r ¼ 0:958; n ¼ 3
ð5Þ
The rate data presented in Table 2 show that the nature of the
substituents at C-2 of the imidazolidin-4-one moiety, and
thus the ketone starting material, affects the reactivity of
the neutral form of the imidazolidin-4-ones 4. Interestingly,
the order of reactivity for the glycine derivatives, according
to the ketone starting material, is: cycloheptanone>
cyclopentanone>cyclohexanone>acetone, the cyclohepta-
none compound 4k being ca. 150 times more reactive
than its acetone counterpart, 4a. A similar trend has
been reported for imidazolidin-4-ones derived from enkeph-
alins, where the order of reactivity is cyclopentanone>
2
.4. Structural effects on chemical reactivity
Inspection of the kinetic data presented in Table 2 reveals
that the pH-independent pathway rate constant predominant
at physiological pH, kneut, decreases with the increasing size
7
cyclohexanone>acetone.
1
of the amino acid substituent R . This effect is more evident
The higher reactivity of the seven- and five-membered ring
derivatives, 4d,k, when compared with the six-membered
ring derivative 4g, finds parallel in the solvolysis of cyclo-
alkyl halides and sulfonates and can be explained by
for the cyclopentanone, 4d–f, and cyclohexanone, 4g–j,
derived series than for the corresponding acetone derived se-
ries, 4a–c. Indeed a good correlation was obtained between
log kneut for the cyclohexanone, 4g–j, series and Charton’s
2
6–28
Brown’s I-strain (internal strain) effect.
Accordingly,
1
steric parameter, n, for the amino acid substituent R
seven- and five-membered rings are strained, and reactions
involving a change in coordination number of one of the car-
bon atoms from four to three, as in S 1-type reactions, will
lead to a decrease in internal strain. In contrast, six-mem-
bered halides and sulfonates are perfectly staggered and
strain-free, and changes in coordination number from four
to three will lead to an increase in internal strain and to a
decrease in reactivity. Thus, the smaller kneut value for the
cyclohexanone derivative 4g, when compared with the five-
and seven-membered ring derivatives 4d,k, is also consistent
(
Fig. 5). A good correlation, with a slope of ꢂ1.2, was
also determined for the three cyclopentanone derivatives
d–f. The negative sign of the steric term in the correlations
N
4
indicates that steric crowding of the amino acid substituent
retards the hydrolysis reaction of the neutral substrate. Al-
though this may seem unusual for an S 1-type reaction,
N
for which release of steric strain on going from reactant to
the transition state usually leads to rate enhancement, it
might be ascribed to unfavorable steric interactions between
2
3
the amino acid substituent and the R and R substituents in
the iminium ion 9 (Scheme 3). Interestingly, when we ana-
lyzed the published data on dipeptide imidazolidin-4-ones
with an S 1-type unimolecular mechanism. The reactivity of
N
the acetone imidazolidin-4-one 4a can be ascribed to the
lower stability of the corresponding ion 9 when compared

P. Chambel et al. / Tetrahedron 62 (2006) 9883–9891
9889
to those derived from cyclic ketones. It has been reported
that the isopropyl cation is ca. 30–40 kcal mol less stable
than methylcyclopentyl or methylcyclohexyl cation.
spectrometry (MS) was performed by the matrix-assisted la-
ser desorption ionization-time-of-flight (MALDI-TOF)
technique on an Applied Biosystems Voyager STR-DE spec-
trometer, using either anthracene or 2,5-dihydroxybenzoic
acid (DHB) as adjuvant matrices.
ꢂ1
2
9
Most of the kneut and kprot values for imidazolidin-4-ones 4
are of the same order of magnitude, with rate differences
less than two-fold for 4b,c,e (Table 2). In contrast, the
protonated form of the cycloheptanone derivative 4k is ca.
4.2. Synthesis of imidazolidin-4-ones 4
2
00 times less reactive than the corresponding neutral
The aminoacyl derivatives of primaquine 6 (2 mmol) were
mixed with an excess (4 mmol) of the appropriate ketone
(acetone, cyclopentanone, cyclohexanone, or cyclohepta-
none) and triethylamine (TEA, 2 mmol) in dry methanol
(10 ml) and the mixture was refluxed for 3 days in the pres-
form. Interestingly, the order of reactivity for the phenylala-
nine derivatives, according to the ketone starting material, is:
cyclohexanone>cyclopentanone>acetone, the cyclohexa-
none compound 4i being ca. 3 times more reactive than its
acetone counterpart, 4b. Although a similar order of reactiv-
ity has been reported for imidazolidin-4-ones derived from
˚
ence of 4 A molecular sieves (1 g). The reaction was moni-
tored by TLC and ketone was re-added (2 mmol) once per
day. The molecular sieves were removed by decantation
and the solution was evaporated to dryness. The oily residue
was submitted to column chromatography on silica gel,
eluted with DCM/THF (varying solvent proportions) or,
for compound 4a, DCM/ethanol 15:1 (v/v). Fractions
containing the chromatographically homogeneous product
were pooled and evaporated to dryness, yielding 4a–k as
yellow-orange oils (44 to 81%) that were analyzed
by high-resolution MS and NMR. Spectroscopic and analyt-
ical data for compounds 4a–k have been presented in
Ref. 11.
7
enkephalins, we cannot find an obvious explanation for
these observations.
3
. Implications in prodrug design
Imidazolidin-4-ones have been considered as useful chemi-
cally activated prodrugs for di- and larger peptides because
of their reasonably rapid hydrolysis to the parent peptides
in physiological conditions, i.e., pH 7.4 at 37 C. Since the
mechanism of hydrolysis of imidazolidin-4-ones is likely
ꢀ
to involve the S 1-type unimolecular cleavage of the C2–
N
N3 bond with departure of an amide-leaving group, the
rate of hydrolysis of this class of prodrugs will be highly de-
pendent on the pK of the amide, which, in turn, is largely
4.3. Kinetics of hydrolysis
The kinetics of hydrolysis of imidazolidin-4-ones 4 were
studied by HPLC using a Waters assembly equipped with a
a
Ò
dependent on the nature of the amide nitrogen atom sub-
stituent (Scheme 3, 11, R ). The results herein presented
indicate that simple N-alkyl a-aminoamide drugs (11,
4
model 600 controlled pump and a model 991 photodiode-
array detector set at 265 nm. The separation was performed
4
Ò
R ¼alkyl), in contrast to peptides, will lead to stable imida-
on a Purospher , 250ꢃ4.0-mm i.d. 5 mm analytical column.
zolidin-4-ones, which may be useful as slow drug release
prodrugs. Increasing the size of the substituents at the a-car-
bon of the a-aminoamide parent drug will also decrease the
rate of hydrolysis of the corresponding imidazolidin-4-one
prodrug. In contrast, substitution at C-2 of the imidazoli-
din-4-one moiety will increase the rate of hydrolysis, partic-
ularly when imidazolidin-4-one prodrugs derived from
cyclopentanone and cycloheptanone. In summary, the scope
of imidazolidin-4-ones as prodrugs is now expanded and can
encompass a-aminoamide drugs other than peptides.
The mobile phase consisted of a mixture of acetonitrile and
sodium acetate buffer (pH 4.75, 0.05 M) containing 10
ꢂ3
M
triethylamine. Two gradients were developed: one for the
imidazolidin-4-one derivatives of phenylalanine and the
other for the derivatives of valine. A linear gradient method
using 50–90% (v/v) acetonitrile over 20 min was used for
compounds 4b,e,i, while a linear gradient using 60–90%
(v/v) acetonitrile over 20 min was used for compounds
4c,f,j. For the remaining compounds, an isocratic method
was developed using a mixture of acetonitrile and pH 4.75
ꢂ3
acetate buffer (50:50 to 60:40%) containing 10 M triethyl-
amine.
4. Experimental
The kinetics of hydrolysis of imidazolin-4-ones 4 were stud-
ied at 60ꢄ0.1 C, in aqueous buffers with ionic strength kept
ꢀ
at 0.5 M by the addition of NaClO . The buffers used were
4
.1. General details
4
a
N -Protected amino acids were purchased from Bachem
(Switzerland). Solvents were of p.a. quality and bought
from Merck (Germany). Both thin layer chromatography
acetate (pH 4.0–5.0), phosphate (pH 6.0–7.5), and borate
(pH 8.0–11.5). For acetate and phosphate buffers the effect
of buffer concentration was studied in the range 0.01–
0.20 M. Sodium hydroxide and hydrochloric acid pseudo-
buffers were used at pH higher than 12.0 and lower
than 2.5, respectively. Typically, reactions were initiated
(
(
TLC) aluminum foil plates covered with silica 60 F254
0.25 mm) and silica gel 60 (70–230 mesh ASTM) for pre-
parative column chromatography were also purchased
from Merck. When required, solvents were previously dried
˚
with pre-activated molecular sieves (4 A) (Merck). Other
chemicals were obtained from Sigma–Aldrich.
ꢂ2
by injecting a 10 ml aliquot of a 10 M stock solution of
substrate in acetonitrile to 10 ml of the appropriate thermo-
stated buffer solution. At regular intervals, samples of
the reaction mixture were analyzed by HPLC. All reactions
followed first-order kinetics over four half lives.
Rate constants derived using this method were reproducible
to ꢄ6%.
NMR spectra were recorded on a Br €u ker AMX-300
spectrometer in deuterated chloroform (CDCl ) containing
tetramethylsilane (TMS) as an internal reference. Mass
3

9
890
P. Chambel et al. / Tetrahedron 62 (2006) 9883–9891
5. Klixbull, U.; Bundgaard, H. Int. J. Pharm. 1984, 20, 273–284.
4
.4. Computational details
6
. Rasmussen, G. J.; Bundgaard, H. Int. J. Pharm. 1991, 71,
45–53.
3
0
The semiempirical Austin Model 1, AM1, and the density-
functional theory, DFT, based B3LYP methods have
been used as included in the Gaussian 98 computer code.
3
1–33
7. Rasmussen, G. J.; Bundgaard, H. Int. J. Pharm. 1991, 76,
113–122.
3
4
These two methods have been parameterized in order to re-
produce experimentally measured molecular properties. The
simplest AM1 method is based on the neglect of differential
diatomic overlap formalism in which various Fock matrix el-
ements are set to zero or use parameters optimized to repro-
duce various properties such as molecular geometries and
standard gas-phase enthalpies of formation. This approach
is an evolution of the older Modified Neglect of Differential
Overlap, MNDO, method where some deficiencies, such as
poor reproduction of hydrogen bonds and too high reaction
activation energies, have been corrected. In the hybrid
B3LYP method, three parameters of the exchange functional
were optimized empirically in order to reproduce experi-
mental thermochemical data. The optimum mixing was
found forw20% Fock exchange in the exchange functional.
In the B3LYP calculations reported here, the atomic elec-
tronic density has been described by the standard 6-31G(d)
basis set.
8. Bak, A.; Fich, M.; Larsen, B. D.; Frokjaer, S.; Friis, G. J. Eur. J.
Pharm. Sci. 1999, 7, 317–323.
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Iley, J.; Chambel, P.; Morais, J.; Moreira, R. Tetrahedron 2004,
60, 5551–5562.
12. Ara ꢀu jo, M. J.; Bom, J.; Capela, R.; Casimiro, C.; Chambel, P.;
Gomes, P.; Iley, J.; Lopes, F.; Morais, J.; Moreira, R.; de
Oliveira, E.; do Ros ꢀa rio, V.; Vale, N. J. Med. Chem. 2005,
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tants) and open-ring (products) structures. Further, reactants
and products did not present any imaginary frequency while
only a single imaginary frequency was calculated for transi-
tion states. All figures with molecular structures have been
16. Constantino, L.; Paix ~a o, P.; Moreira, R.; Portela, M. J.;
Ros ꢀa rio, V. E.; Iley, J. Exp. Toxicol. Pathol. 1999, 51, 299–303.
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3
5
36
obtained with the XCrysDen and Molden programs.
Acknowledgements
2
a
0. Perrin, D. D.; Dempsey, B.; Serjeant, E. P. pK Prediction for
The authors thank Funda c¸ ~a o para a Ci ^e ncia e Tecnologia
Organic Acids and Bases; Chapman and Hall: London, 1981;
pp 109.
(
Portugal) for financial support through research project
POCTI/FCB/39218/2001 and pluriannual funding to re-
search units CECF and CIQUP. J.R.B.G. and P.C. thank
F.C.T. and the European Social Fund (ESF) under the
3rd Community Support Framework (CSF) for the award,
respectively, of a post-doctoral fellowship (SFRH/BPD/
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1
2
values for the dissociation of amides R CONHR can
22. The pK
a
P
be calculated using the equation pK
The difference between the amide derived from primaquine
2
and those from peptides is in the R group. For simplicity,
a
¼22ꢂ3.1 s* (Ref. 20).
1
2
1582/2002) and a Ph.D. research grant (SFRH/BD/11582/
002).
2
we considered the R group for primaquine to be butyl
(
s* can be estimated to be 0.84); thus, the difference in pK is
s*ꢂ0.23) and for the peptides to be MeNHCOCH
2
(for which
a
Supplementary data
3.1(0.84ꢂ(ꢂ0.23))z3.3.
2
3. Lopes, F.; Moreira, R.; Iley, J. J. Chem. Soc., Perkin Trans. 2
1999, 431–440.
Table S1 with the optimized Cartesian coordinates and Table
S2 with the energies of all compounds. Supplementary data
associated with this article can be found in the online
version, at doi:10.1016/j.tet.2006.08.026.
24. Homer, R. B.; Johnson, C. D. Acid–Base and Complexing
Properties of Amides. In The Chemistry of Amides; Zabicky,
J., Ed.; Interscience: London, 1970; p 187.
2
5. Loudon, G. M.; Almond, M. R.; Jacob, J. N. J. Am. Chem. Soc.
981, 103, 4508–4515.
1
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