1,3,5-triazepan-2,6-diones as structurally diverse and conformationally constrained dipeptide mimetics: Identification of malaria liver stage inhibitors from a small pilot library

Article abstract of DOI:10.1002/chem.200600560

The development of the 1,3,5-triazepan-2,6-dione system as a novel, conformationally restricted, and readily accessible class of dipeptidomimetics is reported. The synthesis of the densely functionalized 1,3,5-triazepan-2,6- dione skeleton was achieved in

Full text of DOI:10.1002/chem.200600560

DOI: 10.1002/chem.200600560
1,3,5-Triazepan-2,6-diones as Structurally Diverse and Conformationally
Constrained Dipeptide Mimetics: Identification of Malaria Liver Stage
Inhibitors from a Small Pilot Library
[a]
Gersande Lena,[a] Eliette Lallemand,[b] Anne Charlotte Gruner,[b] JoelBoegiln,
Solveig Roussel,[a] Arnaud-Pierre Schaffner,[a] AndrØ Aubry,[c] Jean-FranÅois Franetich,[d]
Dominique Mazier,[d] Ir›ne Landau,[e] Jean-PaulBriand, Claude Didierjean,[c]
[a]
Laurent RØnia,*[b] and Gilles Guichard*[a]
Dedicated to the memory of the late Professor Murray Goodman
Abstract: The development of the
1,3,5-triazepan-2,6-dione system as a
novel, conformationally restricted, and
readily accessible class of dipeptidomi-
metics is reported. The synthesis of the
densely functionalized 1,3,5-triazepan-
2,6-dione skeleton was achieved in only
four steps from a variety of simple
linear dipeptide precursors. To extend
the practical value of 1,3,5-triazepane-
2,6-diones, a general polymer-assisted
solution-phase synthesis approach ame-
nable to library production in a multi-
parallel format was developed. The
conformational preferences of the
1,3,5-triazepan-2,6-dione skeleton were
investigated in detail by NMR spectros-
copy and X-ray diffraction. The ring
exhibits a characteristic folded confor-
mation which was compared to that of
related dipeptide-derived scaffolds in-
cluding the more planar 2,5-diketopi-
perazine (DKP). Molecular and struc-
tural diversity was increased further
through post-cyclization appending op-
erations at urea nitrogens. Preliminary
biological screens of a small collection
of 1,3,5-triazepan-2,6-diones revealed
inhibitors of the underexplored malaria
liver stage and suggest strong potential
for this dipeptide-derived scaffold to
interfere with and to modulate biologi-
cal pathways.
Keywords: heterocycles · molecular
diversity · peptides · peptidomimet-
ics · solid-phase synthesis
Introduction
ciently distribute selected pharmacophores in the 3D space
is an important method to identify small-molecules capable
of modulating biological processes and for dissecting biolog-
ical pathways.[1–3] In this context, molecules incorporating
The design and synthesis by combinatorial chemistry techni-
ques of cyclic/polycyclic molecular frameworks that can effi-
[d]J.-F. Franetich, Dr. D. Mazier
[a]G. Lena, J. Boeglin, Dr. S. Roussel, Dr. A.-P. Schaffner,
Dr. J.-P. Briand, Dr. G. Guichard
INSERM U511, Groupe Hospitalier PitiØ-SalpÞtri›re
UniversitØ Pierre et Marie Curie, 91 Bld de l’Hôpital
75013 Paris (France)
Immunologie et Chimie ThØrapeutiques (ICT), UPR CNRS 9021
Institut de Biologie MolØculaire et Cellulaire, 15
rue Descartes, 67084 Strasbourg (France)
Fax : (+33)388-610-680
[e]Prof. I. Landau
Equipe Parasitologie ComparØe et Mod›les ExpØrimentaux USM0307
MusØum National d’Histoire Naturelle
64 rue Buffon, 75005, Paris (France)
E-mail: g.guichard@ibmc.u-strasbg.fr
[b]Dr. E. Lallemand, Dr. A. C. Gruner, Dr. L. RØnia
Immunohepatology Group, Institut Cochin
INSERM U567, CNRS UMR 8104
UniversitØ RenØ Descartes, Bat Gustave Roussy
27 rue du Faubourg Saint Jacques
75014 Paris (France)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author and contains a
1H NMR spectrum of crude TFA·H2N-Phe-gSar-COOSu (3a), an illus-
tration of polymer-supported quenching for removal of electrophiles
after alkylation of urea nitrogens, partial 500 MHz ROESY spectra of
1,3,5-triazepin-2,6-dione 4a, 4i, 4p, and 10a, stability of 1,3,5-triaze-
Fax : (+33)140-516-535
E-mail: renia@cochin.inserm.fr
1
pin-2,6-diones in [D6]DMSO and H2O, C18 RP-HPLC traces, FTIR, H
and 13C NMR spectra for all compounds 4–13, and toxicity of the dif-
[c]Dr. A. Aubry, Dr. C. Didierjean
ferent 1,3,5-triazepin-2,6-diones towards hepatocytes in vitro.
LCM3B, UMR-CNRS 7036, UniversitØ Henri PoincarØ
BP 239, 54506 Vandoeuvre (France)
8498
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Chem. Eur. J. 2006, 12, 8498 – 8512

FULL PAPER
small or medium rings derived from peptides are of particu-
lar interest owing to their facile access, the chemical and
stereochemical diversity of these peptide derivatives, and
enhanced diversity resulting from post-cyclization appending
operations. In addition, such molecules are potentially
useful to constrain intrinsically short linear peptide chains
and for mimicking local folded polypeptide structures, such
from the remarkable biological activities exhibited by mole-
cules with diazepine and triazepine skeletons. In particular,
seven-membered cyclic ureas have attracted much attention
with application in the development of HIV-protease[12] and
reverse transcriptase[13] inhibitors, Factor Xa inhibitors,[14] b-
lactamases inhibitors,[15] phospholipase C inhibitors,[16] and
chemokine receptor antagonists.[17] A polymer-assisted solu-
tion-phase synthesis appropriate for library construction has
been developed, and post-cyclization diversification at urea
nitrogens examined. Conformational and structural features
of the 1,3,5-triazepan-2,6-dione were investigated in detail
with comparison to related peptide-derived heterocyclic sys-
tems and the utility of the scaffold was demonstrated by
screening a small “prospecting” library against the malaria
liver stage (LS) which holds great promise for drug target-
ing.
Results and Discussion
Solution-phase synthesis: As shown in Scheme 1, 1,3,5-tria-
zepan-2,6-diones 4[18] are constructed in only four steps by
head-to-tail cyclization of simple activated dipeptide deriva-
Figure 1. Skeletal diversity from simple dipeptide substrates.
as b-turns. The piperazin-2,5-dione or 2,5-diketopiperazine
(DKP) A (Figure 1) is the archetype of a peptide-derived
heterocycle and has been the subject of considerable interest
and numerous studies.[4] This essentially planar head-to-tail
cyclodipeptide structure characterizes the core of many nat-
ural products (for example, demethoxyfumitremorgin C, try-
prostatins, brevianamides, (iso)roquefortine C, rosstratins A,
and vertihemiptellides)[4b,5] and represents a versatile scaf-
fold for new drug discovery.[6] Several routes suitable for li-
brary production have been developed, making the DKP
scaffold readily available in combinatorial and multiparallel
format for biological screens.[7] In recent years, elegant syn-
thetic pathways amenable to polymer-supported chemistry
have been explored to transform short peptide fragments
into novel heterocyclic scaffolds through backbone cycliza-
tion.[8–10] However, the possibility to rapidly generate confor-
mationally defined DKP-related dipeptidomimetics with en-
larged ring and nonplanar geometry through simple synthet-
ic manipulation of the dipeptide backbone has remained un-
derused.[11]
Scheme 1. Solution-phase synthesis of 1,3,5-triazepan-2,6-diones 4.
a) EtOCOCl, NMM, THF, ꢀ208C, then NaN3 in H2O; b) toluene, 658C,
then HOSu, py; c) TFA, 30 min; d) DIEPA, MeCN; e) PS-DIPEA,
CH2Cl2.
tives, and the approach benefits from the considerable diver-
sity of commercial a-amino acids.[19] The general cyclization
strategy parallels our previously reported approach to the
preparation of enantiopure macrocyclic oligoureas.[20]
It involves a key linear diamino precursor activated at
one end as a succinimidyl carbamate and protected at the
other end by a Boc group. Succinimidyl carbamates 2 were
prepared from Boc-dipeptides 1 as previously described for
N-protected a- and b-amino acids.[21] Selective removal of
the Boc group by treatment of crude 2 with trifluoroacetic
acid (TFA) afforded 3[23] which cyclized to 4 in the presence
of diisopropylethylamine (DIPEA). The cyclization pro-
ceeded rapidly with concomitant release of N-hydroxysucci-
nimide (HOSu) and formation of the TFA salt of DIPEA.
These byproducts were removed by either recrystallization
of crude 4, liquid/liquid extraction, or polymer supported se-
We report a general synthetic approach leading to the
densely functionalized (five points of diversity) seven-mem-
bered 1,3,5-triazepan-2,6-dione scaffold B that further ex-
pands the skeletal and structural diversity attainable with
simple dipeptide substrates. Our interest in developing and
evaluating this previously unreported scaffold stemmed
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G. Guichard, L. RØnia et al.
questration (PSS) with tris-(2-aminoethyl)amine polystyrene
(PS-triamine).[23] Alternatively, polymer-supported DIPEA
(PS-DIPEA) was found to be very effective in promoting
cyclization and simultaneously removing byproducts (see
Supporting Information).
A set of 18 monocyclic to tetracyclic 1,3,5-triazepin-2,6-
diones 4a–s were synthesized by this approach in moderate
to high overall yield starting from a variety of dipeptide se-
quences (Table 1, Figure 2).
Table 1. Solution-phase synthesis of 1,3,5-triazepan-2,6-diones 4 from di-
peptides 1.
Entry
1
Xaa
Xbb
Yield 3 [%][a] Yield 4 [%][b,c]
1
2
3
4
5
6
7
8
a
b
c
d
e
f
g
h
i
j
k
l
m
o
p
q
r
l-Phe
d-Phe
l-Val
l-Leu
l-Dap
l-Phg
l-2-Nal
l-Tic
l-Phe
l-2-Nal
l-Tic
l-Phe
l-Phe
l-Ala
d-Phe
d-Phe
l-Pro
l-Pro
Sar
Sar
Sar
Sar
93
78
95
86
72
58
50
59
90
82
77
34
73
54
80
60
66
58
71[d](82[e]
74[d]
)
67[f]
34[f]
A
43[f]
41[f](67[e]
77[f]
)
Sar
Sar
l-Pro
l-Pro
68[d]
66[d]
78[d]
57[d]
97[d]
99[f,g]
97[i]
9
10
11
12
13
14
15
16
17
18
l-Pro
l-N-MePhe
l-Hyp(Bn)
l-Tic
l-Pro
l-N-MeAla
l-Val
66[h]
48
Figure 2. C18-RP-HPLC traces of crude cyclization products before and
after PSS with PS-triamine: a) cyclo(l-Phe-gSar-CO) (4a) and b) cyclo(l-
Phe-gN-MePhe-CO) (4l).
11[h,j,k]
7[h,j,l]
s
l-Leu
[a]Overall yields from 1. [b]Yields from 3. [c]Cyclizations performed by
using DIPEA unless otherwise stated. [d]Purification by recrystallization.
[e]PS-DIPEA used for cyclization. [f]Purification by flash-column chro-
matography (CH2Cl2/MeOH/AcOH 97:3:1). [g]Hydrogenation of the
benzyl group in 4m afforded triazepandione 4n with a hydroxyl side
chain in 70% yield. [h]Purification by C 18 RP-HPLC. [i]Purification by
using PS-triamine. [j]Cyclization performed with N-methyl morpholine
at a concentration of 10ꢀ3 m. The formation of 4 was accompanied by the
formation of the corresponding cyclodimer 5. [k] 4r/5r 54:46. 5r isolated
in 35% yield. [l] 4s/5s 50:50. 5s isolated in 10% yield. Abbreviations:
Dap=Diaminopropanoic, Phg=Phenylglycine, 2-Nal=2-naphtylphenyl-
alanine, Tic=Tetrahydroisoquinoline-3-carboxylic acid, Hyp=cis-4-Hy-
droxyproline.
4:5 ratio was obtained when cyclization was conducted at
10ꢀ3 m in the presence of N-methyl morpholine (NMM) in-
stead of DIPEA as the base. Cyclooligomers of higher order
were not observed under these conditions. The 1,3,5-triaze-
pan-2,6-dione 4 and cyclodimer 5 were purified by prepara-
tive C18 RP-HPLC which may explain the somewhat low
yield of recovered product.
Polymer-assisted synthesis: To extend the practical value of
1,3,5-triazepane-2,6-diones as dipeptide mimetics, a poly-
mer-assisted solution-phase synthesis amenable to large
scale library production was developed. The selected strat-
egy which combines catch and release and cyclative cleav-
age[24] is outlined in Scheme 2. The key intermediate in this
concise procedure is the polymer-bound carbamate 6 acces-
sible in a one pot sequence from protected dipeptides. Sev-
eral resins including polymer-supported tetrafluorophenol
(PS-TFP), N-hydroxysuccinimide (PS-HOSu), and oxime
(PS-NOH) resins were initially considered for the synthesis
of the polymer-bound carbamate. PS-HOSu was finally
chosen based on our solution-phase synthesis experience.
PS-HOSu was prepared according to the method reported
by Adamczyk and coworkers.[25] Treatment of (mercaptome-
thyl)polystyrene (PS-SH) (2 mmolgꢀ1) with N-hydroxyma-
leimide in presence of pyridine yielded PS-HOSu with quan-
titative capping (1.6 mmolgꢀ1). After the reaction, the resin
Carbamates 2a–m and 2o bearing a tertiary amide N-ter-
minal to the gXbb residue (R3 ¼H) were expected to give
an equilibrium mixture between trans and cis isomers (a
~1:1 ratio was observed for 3a by H NMR spectroscopy in
CD3CN, see the Supporting Information) and as a result
1
readily cyclize to 4 (entries 1–14).
In the case of carbamates derived from sequences com-
posed of heterochiral residues (entries 15 and 16), cycliza-
tion to 4 was also found to proceed readily. Minor epimeri-
zation (approximately 5–10%) was observed at the gem-dia-
mino residue during cyclization of 3p. In contrast, cycliza-
tion of precursors 3 with a secondary amide N-terminal to
gXbb (R3 =H, entries 17 and 18) gave 4 together with the
corresponding 14-membered ring cyclodimer 5. The ratio of
monomer versus dimer was found to be strongly influenced
by the concentration and the nature of the base. A higher
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1,3,5-Triazepan-2,6-dione Dipeptide Mimetics
FULL PAPER
approach enabled for easy removal of soluble impurities
and excess reagents by filtration. Evidence for the formation
of the polymer-bound carbamates 6 came from FTIR spec-
troscopic observation of characteristic stretches at 3300,
1740 and 1165 cmꢀ1. Removal of the Boc group by treat-
ment with TFA, followed by neutralisation of the TFA salt 7
and slight heating afforded the desired product 4 in solution.
Optimized conditions for the synthesis of 4 were investi-
gated by systematically varying the solvent in both the catch
and release steps. Three solvents were evaluated for the
preparation of polymer-bound carbamates 6a and 6l derived
from Boc-Phe-Sar-OH (1a) and Boc-Phe-N-MePhe-OH
(1l), namely ethyl acetate, toluene, and THF. Experiments
were carried out in small flasks equipped with cooling sys-
tems, for both of the model compounds and the reactions
were monitored by FTIR spectroscopy. Each sample was
then divided into three portions and subjected to TFA treat-
ment and cyclative cleavage. Again three solvents (CH2Cl2,
THF, and MeCN) were investigated in the cyclorelease step.
In CH2Cl2, cyclization was generally completed in less than
30 minutes at 408C. The best average purity (90% purity
for 4a and 84% purity for 4l) was obtained when the catch
and cyclorelease steps were conducted in THF and CH2Cl2,
respectively (see Figure 3).
Scheme 2. Polymer-assisted synthesis of 1,3,5-triazepan-2,6-diones 4.
a) (i) DPPA (1.1 equiv), Et3N (1.1 equiv), RT, 30 min; (ii) 708C for 1.5 h;
(iii) PS-HOSu (0.3 equiv), 708C, 40 min; b) TFA/CH2Cl2 (1:1), 2”5 min,
RT, washings; c) DIPEA (1.1 equiv), 40–608C, 4”30 min.
was controlled by IR spectroscopy. A characteristic stretch
was present at 1720 cmꢀ1 corresponding to n(O=C N C=
O).
ꢀ ꢀ
Starting from the N-Boc-protected dipeptide 1, the acyl
azide was generated in situ by reaction with diphenylphos-
phoryl azide (DPPA) in the presence of Et3N.[26] It was then
rearranged under heating to the corresponding isocyanate
which was trapped with PS-HOSu to give the polymer-sup-
ported carbamate 6 in a one pot process (Scheme 2). This
This optimized polymer-assisted three-step procedure de-
veloped for 4a and 4l should greatly facilitate the access to
Figure 3. Solvent optimization in polymer-assisted synthesis of a) 4a and b) 4l.
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G. Guichard, L. RØnia et al.
Table 2. Diversification of parent scaffold 4 by di- and mono-N alkylation of urea.
column
chromatography
(Table 2, entries 1–4). Alterna-
tively, liquid/liquid extraction
followed by PSS of excess elec-
trophile with N-(2-mercapto-
ethyl)aminomethyl-PS afforded
pure compounds (Supporting
Information). Desymmetriza-
tion of urea in 1,3,5-triazepan-
2,6-diones was also investigat-
ed. Direct monoalkylation of
C2-symmetric cycloureas in the
presence of one equivalent of
electrophile is often inconsis-
tent, proceeding in low yield
due to both incomplete reaction
and competing dialkylation.[28]
Alternative methods include
the formation of O-alkylated
isourea derivatives,[29] the mon-
odeprotection of symmetric bis-
[b]
Entry
4
R5X
Product
Method[a]
T [8C]Yield [%]
Ratio[c]
10:8 or 11:9
1
2
3
4
5
6
7
8
a
i
a
i
a
c
i
a
c
f
BnBr
BnBr
BrCH2COOtBu
BrCH2COOtBu
BnBr
BnBr
BnBr
BrCH2COOtBu
BrCH2COOtBu
BrCH2COOtBu
BrCH2COOtBu
8a
8i
9a
9i
10a
10c
10i
11a
11c
11 f
11i
A
A
A
A
B
B
C
B
B
B
C
25
60
25
60
25
25
40
25
25
25
40
50
66
82
73
68
94
50
65
92
79
64
0:100
0:100
0:100
0:100
>99:1
>99:1
55:45
>99:1
>99:1
97:3
9
10
11
i
79:21
[a]Synthetic methods: method A: NaH (4 equiv), R 5X (4 equiv), THF, 48 h; method B: KF/Al2O3 40% wt
(10 equiv), R5X (1.5 equiv), CH2Cl2, 24–48 h; method C: NaH (2 equiv), R5X (1.5 equiv), CH2Cl2, 24 h.
[b]Yields calculated for pure compounds after purification by flash-column chromatography. [c]Ratio deter-
mined by C18 RP-HPLC of crude products. In all cases, there was no starting cycle 4 remaining.
the 1,3,5-triazepan-2,6-dione skeleton in multiparallel library
format.
Post-cyclization appending operations: The urea moiety in
the 1,3,5-triazepan-2,6-dione ring offers one or two addition-
al sites for post-cyclization appending operations. Further
diversification of parent scaffold 4 was achieved by either al-
kylation (Table 2, Scheme 3) or acylation (Scheme 4) of the
urea moiety.
Scheme 4. Monoacylation reactions of 1,3,5-triazepan-2,6-dione 4a.
benzylated derivatives,[30] or the discrimination of urea nitro-
gens by selective introduction of an alkoxycarbonyl protect-
ing group.[31] Interestingly, we found that direct alkylation of
cyclo(Xaa-gSar-CO) 4a, 4c, 4 f, and cyclo(Phe-gPro-CO) 4i,
in the presence of KF/Al2O3 40% wt (10 equiv)[32] or NaH
(2 equiv), respectively, reliably provided 10 and 11 monoal-
kylated at the gem-diamino urea nitrogen (Table 2, en-
tries 5–11).[33] Mono-N-alkylation reaction occurred exclu-
sively on the N3-atom. It is worth noting that excellent se-
lectivities of mono versus dialkylated products (>99:1) were
generally observed in KF/Al2O3-mediated alkylation
(Table 2, entries 5, 6, and 8–10). 1,3,5-Triazepane-2,6-diones
containing trisubstituted ureas with either N3H or N1H can
be further manipulated to generate dissymmetrically N-alky-
lated rings as exemplified for 14 and 15 (Scheme 3).
Scheme 3. Synthesis of dissymmetrically N-alkylated 1,3,5-triazepan-2,6-
diones.
Conditions for acylation of triazepandiones 4 were also in-
vestigated. Again differential reactivity of urea nitrogens
was observed. Treatment of 4a with four equivalents of ben-
zoyl chloride afforded 16 monoacylated at the N1 position
with minor amounts of the corresponding diacylated deriva-
tive and no sign of acylation at N3. The optimized proce-
dure for the acylation reaction entailed the use of collidine
as the base and THF as the solvent. Under these conditions,
Di-N-alkylation of seven-membered cycloureas (for exam-
ple, 1,3-diazepan-2-diones) which has been used extensively
in the preparation of C2-symmetric HIV protease inhibitors
generally proceeds in good yields.[27] Similarly, treatment of
scaffold 4 with NaH (5 equiv) and various electrophiles
(3 equiv) afforded dialkylated cycloureas (for example, 8
and 9) in good yields and purities after purification by flash-
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1,3,5-Triazepan-2,6-dione Dipeptide Mimetics
FULL PAPER
1,3,5-triazepan-2,6-dione 4a was cleanly converted to mono-
acylated derivatives 16–19 in yields ranging from 71 to 85%
(Scheme 4).
The use of other bases, such as pyridine, DMAP
(DMAP=4-dimethylaminopyridine), and 2,6-di-tert-butyl-4-
methylpyridine proved less satisfactory with much slower
conversion. Alternatively, inorganic bases (for example,
K2CO3) were also found to be effective in promoting clean
monobenzoylation of 4a. Mono-
by 4k(I) follows the general tendency discussed above with
side chains R2 and R4 in an equatorial position. However, in
the second molecule 4k(II), the substituent R2 adopts a
pseudoaxial orientation thus forcing the urea to deviate
from planarity (T1=ꢀ52.88, T2=ꢀ26.78) and leading to a
higher a angle value (1588). This deviation is associated with
a strong pyramidalization of N1 in 4k(II) which is apparent
when the sum of the angles formed by the substituents at-
acylation thus offers another
interesting possibility to desym-
metrize the urea in 1,3,5-triaze-
pan-2,6-diones and expands fur-
ther the diversity accessible
from post-cyclization opera-
tions.
Structuralfeatures and compar-
ison with related dipeptide-de-
rived skeletons: The conforma-
tional preferences of 1,3,5-tri-
azepan-2,6-dione were investi-
gated by detailed X-ray diffrac-
1
tion and H NMR spectroscopy
studies of representative mem-
bers of the library. In the solid
state, compounds
4
derived
from homochiral sequences
generally adopt a rigid nonpla-
nar conformation with side
chains R2 and R4 in the pseu-
doequatorial
position
(Figure 4).[34,35]
The “V-shape” of the mole-
cules can be described by the
measure of the angle (a) be-
tween the mean amide and
urea plans (Table 3).
The angle a is either close to
1208 (4j, 4k(I), 4l, 4o and 4 s)
or 1608 (4k(II)) showing that
1,3,5-triazepane-2,6-diones
4
can adopt two different two-
folded conformations in the
solid state,
A and B (vide
infra), respectively. Examina-
tion of torsion angles T1, T2,
and T5 (see Table 3) revealed
that in all triazepanediones 4
the amide moiety is essentially
planar and that the urea is
nearly planar when a is close to
1208. In the crystal structure of
tetracyclic cyclo(Tic-gPro-CO)
(4k), the two conformations A
and B of the ring are coexisting.
The conformation A adopted Figure 4. X-ray crystal structures of selected 1,3,5-triazepan-2,6-diones 4 and 9–11.
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G. Guichard, L. RØnia et al.
Table 3. Torsion angles and geometrical data for 1,3,5-triazepan-2,6-diones.
Entry
Compound
Form
Space group
Crystallization Solvent
Torsion angle [8][a]
a [8][b]
T1
T2
15.2
ꢀ14.3
ꢀ26.7
4.4
T3
T4
T5
T6
69.6
74.9
ꢀ14.3
67.8
61.9
69.2
ꢀ25.4
ꢀ20.8
62.8
T7
1
2
4j
4k
C2
P21
CH2Cl2/DIPE or dioxane
AE/Hex
3.8
3.3
35.4
66.6
80.9
61.5
50.8
65.8
88.5
83.5
52.7
ꢀ64.9
ꢀ59.6
ꢀ47.5
ꢀ72.1
ꢀ70.9
ꢀ62.9
ꢀ52.6
ꢀ41.6
ꢀ75.7
70.2
4.5
ꢀ67.6
ꢀ52.7
65.3
ꢀ44.1
ꢀ60.9
ꢀ48.4
74.6
117.4
120.0
158.5
115.9
119.5
122
170.9
162.9
117.9
125.5
117.6
124.4
I
II
ꢀ13.9
ꢀ52.8
ꢀ14.3
5.8
3.9
3
4
5
6
7
8
4l
4o
4s
9a
9i
10a
P212121
P212121
P21
P212121
P212121
P1
AE/Hex
CH2Cl2
CH2Cl2/DIPE
CH2Cl2/DIPE
CH2Cl2/DIPE
CH2Cl2/DIPE
ꢀ4.7
7.7
ꢀ12.6
11.1
0.7
0.8
1.4
ꢀ13.6
ꢀ33.3
ꢀ34.5
6.6
5.2
11.2
2.0
ꢀ50.1
ꢀ52.0
ꢀ2.3
18.6
76.3
I
II
I
5.9
3.4
8.2
1.4
ꢀ56.2
ꢀ68.9
ꢀ53.0
25.1
9
11c
P21
CH2Cl2/DIPE
6.3
42.2
ꢀ71.9
67.2
ꢀ68.9
25
II
21.2
ꢀ67.1
72.2
ꢀ52.5
[a]The dihedral angles T1–7 refer to the torsion angles of the sequence of atoms C7-N1-C2-N3, N1-C2-N3-C4, C2-N3-C4-N5, N3-C4-N5-C6, C4-N5-C6-
C7, N5-C6-C7-N1, C6-C7-N1-C2, respectively (see Figure 5 for labeling of atoms). According to the IUPAC-IUB rules for the conformation of polypep-
tide chains, T7 can also be defined as f1, T6 as y1, and T5 as w. [b] a is measured between the mean urea and amide planes. Abbreviations: DIPE=diso-
propylether.
Figure 5. Structural comparison of 2,5-diketopiperazine (DKP), 1,3,5-triazepan-2,6-diones, and 1,2,5-triazepan-3,6-dione skeletons. For each skeleton, in-
ternal angles are shown in orange and bond lengths (Š) in black. Backbone torsion angles are in blue and italicized. The a angle between amide, amide/
urea planes are indicated for the three molecules.
tached to N1 are compared: 358.78 for 4k(I) versus 341.58
in 4k(II). The folded conformation A induces an unusual
close proximity of the hydrogens H4 and H7 (refer to
Figure 5 for numbering of atoms). In 4l the distance be-
pseudoaxial orientation of the R2 side chain. The conforma-
tion of 4a and 4i in solution was investigated by H NMR
1
spectroscopy in [D6]DMSO. The observation of a strong
ROE correlation in ROESY experiments between H4 and
H7 suggests a short interproton distance characteristic of
the folded conformation A with R2 (phenylalanine) side
chain in pseudoequatorial orientation (Supporting Informa-
tion).
[36]
tween H4 and H7 is 1.92 Š which is less than the sum of
the Van der Waals radii (ꢀ=2.4 Š) for two hydrogen atoms.
When comparing the two structures 4k(I) and 4k(II) dis-
cussed above, the distance between H4 and H7 changes
from 2.14 to 3.71 Š as a result of the reorganization in the
8504
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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 8498 – 8512

1,3,5-Triazepan-2,6-dione Dipeptide Mimetics
FULL PAPER
Structural features of 1,3,5-triazepane-2,6-dione 4 and
DKP originating from a common dipeptide precursor (l-
Pro-l-Leu) were compared (Figure 5).
The structures of DKPs in the solid state have been exten-
sively studied and reviewed.[4c,37,38] Depending on the pep-
tide sequence and stereochemistry, the DKP ring has been
shown to adopt several conformations ranging from planar
some pyramidalization of the urea nitrogens. Both crystal
structures of N3-alkylated 1,3,5-triazepane-2,6-diones 10a
and 11c show two different conformations coexisting in the
crystal cell. One molecule in the asymmetric unit (10a(I)
and 11c(I)) adopts the standard folded conformation A
commonly found for compounds 4, with the R2 substituent
in pseudoequatorial position and an a angle close to 1208.
In the second conformation observed (10a(II) and 11c(II)),
the puckering of the ring is reversed thus keeping the a
angle close to 1208 but causing the R2 substituent to adopt a
quasi-axial orientation. This overall inversion of the ring ge-
ometry results in pulling apart the two hydrogens H4 and
or nearly planar (for example, in cycloACHTREU(NG Gly-Gly) and
cyclo(d-Ala-l-Ala) the a angle between amide planes is
~1808) to folded (twist boat) (for example, cyclo(l-Ala-l-
Ala), a=~1518). The substituents on the Ca atom have
been observed to be either in the axial or most of the time
in quasi-equatorial position depending on their nature and
the stereochemistry. With a values as low as 1178, the 1,3,5-
triazepan-2,6-dione ring is much more folded than the corre-
sponding 2,5-diketopiperazine ring. In the Pro-Leu series, a
values of 143 and 1228 are observed, respectively, for the
DKP[39] and the triazepanedione (Figure 5). The inter CaH
H7 which adopt quasi-equatorial positions. The dACHTREUNG(H4,H7)
increases from 1.98 in 11c(I) to 4.67 Š in 11c(II). The con-
formation of 10a was investigated by NMR spectroscopy in
[D6]DMSO. The presence of a single set of sharp peaks and
the strong ROE correlation observed between H4 and H7
may suggest that conformation A is mainly populated in sol-
ution.
proton distance (dACHTREUNG(H3,H6) of 2.81 Š in the DKP) is much
larger than that observed in the corresponding triazepan-
dione ring and reflects the more planar conformation. The
quasi-equatorial position of the side chains on Ca carbons is
common to both scaffolds.[40]
First biological screens: Identification of Malaria liver stage
inhibitors: Because they are structurally diverse and rapidly
accessible in a library format from simple dipeptides, 1,3,5-
triazepan-2,6-diones have a strong potential for use in bio-
logical screens. Here, we report a preliminary account of
our screening efforts to identify malaria liver stage inhibi-
tors.
In 1996 Lenman et al. reported the design and synthesis
of cis-peptidyl prolinamide mimetics based on a 4,5-fused
1,2,5-triazepane-3,6-dione skeleton that differ from the orig-
inal cis-proline peptide by only two hydrogens.[11b] These
molecules were obtained by cyclization of a-bromoacetyl
proline hydrazide intermediates. Comparison of 1,2,5-triaze-
pane-3,6-dione and 1,3,5-triazepane-2,6-dione-ring structures
(cyclo(Pro-gLeu-CO) versus cyclo(Sar-Pro-NH) respectively,
Figure 5) reveals a very close match with almost perfect su-
perimposition of backbone traces, the urea nitrogen N3 in
4s overlying the a-carbon C7 in cyclo(Sar-Pro-NH).
The 1,2,5-triazepan-3,6-diones thus exhibit the folded con-
formation A defined above with a a angle slightly lower
(1108) than that observed in 1,3,5-triazepan-2,6-diones. The
major difference in the ring geometry is observed between
the bond angles (C4-N3-C2) in 4s and (N1-C7-C6) in
cyclo(N-MeSar-Pro-NH) which differ by approximately 108
(Figure 5). This similarity suggests that the 1,3,5-triazepan-
2,6-dione scaffold alkylated on the N3 urea nitrogen could
be used to design mimetics of cis-peptidyl prolinamides.
The crystal structures solved for several di- and mono-N-
alkylated compounds (9a, 9i, 10a, and 11c) further highlight
the structural diversity among 1,3,5-triazepan-2,6-diones
(Figure 4). The overall ring geometry undergoes important
changes upon alkylation of urea nitrogen N3. Di-N-alkylat-
ed compounds 9a and 9i exhibit the characteristics of the
folded conformation B defined above and observed for com-
pound 4k(II): the R2 substituent (phenylalanine side chain
for both 9a and 9i) adopts a quasi-axial orientation; the
values of T1 and T2 torsion angles (approximately, ꢀ50 and
ꢀ358) show that the urea is no longer planar and that the
geometry of the ring is highly distorted; the sums of the
angles formed by the substituents attached to N1 (approxi-
mately 3508) and N3 (approximately 3568, Table 3) indicate
Invasion of host hepatocytes by sporozoites represents an
early step in the life cycle of the malaria parasite. Sporo-
zoites develop in the liver and give rise to merozoites which
subsequently invade erythrocytes after release into the
bloodstream. This blood phase is responsible for the pathol-
ogy of the malaria infection. Malaria liver stages (LS) hold
great promise for drug targeting because 1) they possess a
more complex and distinct metabolism than their blood
stage counterpart and 2) they precede the pathogenic blood
stage, thus offering prophylaxis possibilities against malaria.
However, very few drugs against LS parasites are available
and most of them like primaquine display severe side effects
or loose efficiency due to the development of parasite resist-
ance.[41] The discovery of new and specific inhibitors of the
LS would lead eventually to the identification of novel bio-
logical targets, thus revealing new therapeutic horizons.
Seventeen different 1,3,5-triazepan-2,6-diones were ran-
domly selected and were first tested at different doses for
toxicity on primary mouse hepatocyte culture. From these,
nine were shown to be toxic at all doses tested (6.25–
100 mgmLꢀ1) and two at high doses (over 50 mgmLꢀ1, see
Supporting Information).[42] The six remaining nontoxic mol-
ecules were thus tested to evaluate their effects on sporo-
zoite invasion and development in hepatocytes. Four of
these 4p, 9a, 5r, and 13a had no significant effect on liver
parasites (Figure 5a, 5c and data not shown). In contrast,
two molecules, 4b and 10a, were shown to inhibit signifi-
cantly and repeatedly LS development (Figures 6a and b),
4b being the most potent.
Chem. Eur. J. 2006, 12, 8498 – 8512
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8505

G. Guichard, L. RØnia et al.
Mass spectra have been recorded by using a MALDI-TOF apparatus
(BRUKER Protein-TOF) and an ESI-TOF apparatus (Bruker micro-
TOF).
Generalprocedure for the soul tion-phase synthesis of 1,3,5-triazepan-2,6-
diones 4 (exemplified for 4a): N-methyl morpholine (NMM, 2.3 mL,
21.4 mmol, 1.2 equiv) was added to Boc-l-Phe-Sar-OH (1a, 6.0 g,
17.8 mmol) dissolved in dry THF (60 mL) under argon. The mixture was
placed into
a
bath at ꢀ208C and ethyl chloroformate (2.05 mL,
21.4 mmol, 1.2 equiv) was added. The mixture was stirred at ꢀ208C for
25 min, then the bath was removed and a solution of NaN3 (2.9 g,
44.6 mmol, 2.5 equiv) in water was added. The mixture was stirred at
room temperature for 5 min. The reaction was quenched by addition of
brine and extracted by ethyl acetate (1”240 mL). The organic layer was
dried over Na2SO4 and concentrated under vacuum without any heating.
The resulting residue was dissolved in dry toluene (40 mL) under argon
and heated at 708C. The Curtius rearrangement was followed by gas evo-
lution (bubbling). When gas emission decreased, HOSu (2.05 g,
17.8 mmol, 1 equiv) and pyridine (1 mL, 12.3 mmol, 0.7 equiv) were
added. The reaction was stirred at 708C for 40 additional minutes and al-
lowed to cool to room temperature. Solvents were concentrated under
vacuum to give succinimidyl carbamate 2a as a yellow foam which was
used in the next step without purification. The crude succinimidyl carba-
mate 2a was dissolved in trifluoroacetic acid (8 mL). After the reaction
had been stirred at room temperature for 30 min, a mixture of cyclohex-
ane and diethyl ether (1:4 v/v) was slowly added to the reaction to pre-
cipitate the TFA salt. This heterogeneous system was stored at ꢀ208C
for 4 h, triturated, and sonicated prior to filtration. The precipitate was
washed several times with a cyclohexane/diethyl ether mixture (1:4 v/v)
and dried under high vacuum overnight. Compound 3a was obtained as a
pale yellow solid (7.6 g, 16.5 mmol, 93%). The TFA salt 3a (4 g,
8.66 mmol) was dissolved in CH3CN (200 mL) and the resulting solution
Figure 6. Effect of selected 1,3,5-triazepan-2,6-diones on malaria liver
stage development. Compounds were added at the time of sporozoite in-
vasion and during liver stage development. Schizont mber was estimated
by counting this mature parasite forms in 48 h cultures. Results are ex-
pressed as the mean ꢁSD of 3 triplicate cultures. Data are representative
of 2 to 6 experiments per compounds tested; *=P<0.05, one-way analy-
sis of variance, followed by the post-hoc Tukey test.
was added dropwise to
a solution of DIPEA (1.66 mL, 9.5 mmol,
1.1 equiv) in acetonitrile (400 mL). When the addition was completed,
the mixture was stirred for an additional 30 minutes at room temperature
and solvent was evaporated. The desired compound was recrystallized
from a mixture of CH2Cl2 and DIPE. After filtration and drying under
high vacuum, the filtrate was concentrated and the recrystallization pro-
cess was repeated several times. Finally, cyclo(l-Phe-gSar-CO) 4a was
obtained (2.72 g, 11.7 mmol, 71%).
Conclusion
We have presented the design and construction of a structur-
ally diverse small library of dipeptide-derived 1,3,5-triaze-
pan-2,6-diones, which led to the identification of two mole-
cules active against the malaria liver stages, which do not
show any toxicity on mouse hepatocytes. Because of its
simple and rapid elaboration, the 1,3,5-triazepan-2,6-dione
system nicely complements the existing collection of skele-
tons accessible from small linear dipeptide precursors.
Large-scale library construction by integrating a more com-
prehensive set of dipeptides (including side-chain and ste-
Preparation of PS-HOSu:[25] PS-SH (5 g, 10 mmol) was swollen in dry
DMF (150 mL) and N-hydroxymaleimide (2.26 g, 20 mmol) followed by
pyridine (4.85 mL, 60 mmol) were added. The mixture was stirred for
24 h at room temperature and then heated at 558C for 3 h under an inert
atmosphere. The resin was filtered and washed with DMF (”3), H2O
(”3), MeOH (”3), and CH2Cl2 (”4). Once the washings were complete,
the resin was dried under high vacuum for 16 h (mass=5.99 g, 98%).
Based on mass recovery the loading of PS-HOSu was determined to be
1.60 mmolgꢀ1
.
ACHTREUNGreochemical diversity) and appending processes is ongoing
and should help for the rapid identification of more potent
molecules for use in the underexplored stages of malaria.
Generalprocedure for the poylmer-assisted synthesis of 1,3,5-triazepan-
2,6-diones (exemplified for 4a): A round-bottomed flask (10 mL) equip-
ped with cooling system was charged with Boc-l-Phe-Sar-OH (1a,
245 mg, 0.73 mmol) and THF (1.5 mL) under Ar. Et3N (110 mL,
0.79 mmol) and DPPA (157 mL, 0.73 mmol) were added. The mixture was
stirred for 30 min at RT, followed by heating at 708C for 1.5 h. Addition
of PS-HOSu (150 mg, 0.24 mmol) was followed by gentle stirring at 708C
for 40 min. The content of the flask was transferred into a polypropylene
syringe equipped with a filter. Filtration and washes (dry DMF ”3, THF
”3, and CH2Cl2 ”3) afforded the substituted resin which was dried under
high vacuum for 16 h. The reaction was monitored by using IR spectros-
ExperimentalSection
Generalmethods : THF was distilled from Na/benzophenone. CH2Cl2 and
cyclohexane were distilled from CaH2. TLC was performed on silica gel
60 F254 (Merck) with detection by UV light and charring with 1% w/w
ninhydrin in ethanol followed by heating. Flash-column chromatography
was carried out on silica gel (0.063–0.200 nm). HPLC analysis was per-
formed on a Nucleosil C18 column (5 mm, 3.9 ’ 150 mm) by using a linear
gradient of A (0.1% TFA in H2O) and B (0.08% TFA in CH3CN) at a
flow rate of 1.2 mLminꢀ1 with UV detection at 214 nm. Optical rotations
were recorded with a Perkin–Elmer polarimeter. IR spectra were record-
ed on a Perkin–Elmer Spectrum One in four scans with a ATR (attenu-
ated total reflection) accessory. 1H and 13C NMR spectra were recorded
by using Bruker Advance DPX-300, DPX-400, and DPX-500 instruments.
AHCTREcUNG opy. Removal of the Boc group was performed with TFA/CH2Cl2 (1:1
v/v, 1.5 mL) for 5 min at RT. The resin was then washed with dry CH2Cl2
(”4). This step was repeated twice and the resin was swollen in dry
CH2Cl2 (2 mL). DIPEA (45 mL, 0.26 mmol) was added and the syringe
was vortexed at 408C for 1 h. The resin was filtered and rinsed with dry
CH2Cl2 (2”1mL). The filtrate was evaporated to give 4a in 77% yield
and 90% purity as determined by HPLC analysis.
Cyclo(l-Phe-gSar-CO) (4a): White solid; C18-RP-HPLC (A: 0.1% TFA
in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
8506
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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 8498 – 8512

1,3,5-Triazepan-2,6-dione Dipeptide Mimetics
FULL PAPER
10.38 min; [a]2D0 =ꢀ41.3 (c=1.1 in MeOH); 1H NMR (500 MHz, CDCl3,
298 K): d=2.81 (dd, J=8.9, 14.4 Hz, 1H; CH2Ph), 3.08 (s, 3H; NCH3),
3.35 (dd, J=5.2, 14.4 Hz, 1H; CH2Ph), 4.00 (dd, J=7.0, 15.6 Hz, 1H;
NCH2N), 4.62–4.65 (m, 1H; NCHCO), 4.87 (brs, 1H; NH), 5.20 (dd, J=
5.1, 15.5 Hz, 1H; NCH2N), 6.97 (brs 1H; NH), 7.24–7.27 (m, 3H; H-Ar),
7.31–7.34 ppm (m, 2H; H-Ar); 13C NMR (125 MHz, CDCl3, 298 K): d=
33.8 (CH3), 37.3 (CH2), 54.5 (CH), 56.0 (CH2), 127.2, 129.0, 129.3 (CH-
Ar), 136.2 (Cq-Ar), 157.8, 169.9 ppm (CO); IR (solid): n˜ =3182 (NH),
3049 (Csp2), 2928 (Csp3), 1651 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for
C12H16N3O2: 234.1242; found: 234.1281.
n˜ =3402, 3233 (NH), 3058 (Csp2), 2921 (Csp3), 1658 cmꢀ1 (CO); HRMS
(ESI): m/z: calcd for C16H17N3O2: 384.1394; found: 384.1382.
Cyclo(Tic-gSar-CO) (4h): White solid, C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
10.91 min; [a]2D0 =ꢀ78.7 (c=0.60 g in MeOH); 1H NMR (300 MHz,
CDCl3, 298 K): d=3.02–3.09 (m, 4H; NCH3, CH2CH), 3.34 (dd, J=11.1,
15.6 Hz, 1H; CH2CH), 4.37 (dd, J=6.9, 15 Hz, 1H; NCH2N), 4.47 (d, J=
15.6 Hz, 1H; CH2N), 4.53 (dd, J=4.2, 11.1 Hz, 1H; CH), 4.72 (d, J=
15.6 Hz, 1H; CH2N), 4.83 (dd, J=5.7, 15 Hz, 1H; NCH2N), 7.08 (brs;
NH), 7.14–7.24 ppm (m, 4H; H-Ar); 13C NMR (75 MHz, CDCl3, 298 K):
d=32.4 (CH2), 34.1 (NCH3) 46.5, 56.6 (CH2) 57.7 (CH), 126.0, 126.7,
127.3, 127.6 (CH-Ar), 134.3, 134.4 (Cq-Ar), 159.1, 170.0 ppm (CO); IR
(solid): n˜ =3337, 3297 (NH), 3078 (Csp2), 2956, 2922 (Csp3), 1651,
1641 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for C13H16N3O2: 246.1237;
found: 246.1229.
Cyclo(d-Phe-gSar-CO) (4b): White solid; [a]2D0 =36.5 (c=0.81 in
MeOH); other spectroscopic data corresponding to 4a.
Cyclo(Val-gSar-CO) (4c): White solid; C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
8.50 min; [a]2D0 =ꢀ53.5 (c=0.63 in MeOH); 1H NMR (300 MHz, CDCl3,
298 K): d=0.98 (d, J=6.6 Hz, 3H; CH3), 1.02 (d, J=6.6 Hz, 3H; CH3),
Cyclo(Phe-gPro-CO) (4i): White solid; C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
10.91 min; [a]2D0 =ꢀ18.2 (c=1.06 in MeOH); 1H NMR (500 MHz,
[D6]DMSO, 298 K): d=1.77–2.01 (m, 4H; 2CH2), 2.71 (dd, J=7.2,
14.1 Hz, 1H; CH2Ph), 3.05 (dd, J=6.5, 14.1 Hz, 1H; CH2Ph), 3.22–3.27
(m, 1H; CH2), 3.55–3.60 (m, 1H; CH2), 4.65–4.68 (m, 1H; NCHCO),
5.56–5.58 (m, 1H; NCHN), 6.14 (brs; NH), 6.98 (brs; NH), 7.19–7.21 (m,
1H; H-Ar), 7.26–7.29 (m, 2H; H-Ar), 7.34–7.36 ppm (m, 2H; H-Ar);
13C NMR (75 MHz, CDCl3, 298 K): d=22.6, 33.5, 36.5, 46.2 (CH2), 55.00,
64.2 (CH), 127.2, 128.9, 129.3 (CH-Ar), 136.3 (Cq-Ar), 157.5, 167.8 ppm
(CO); IR (solid): n˜ =3196 (NH), 3076 (Csp2), 2961, 2890 (Csp3),
1651 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for C14H18N3O2: 260.1399;
found: 260.1394.
2.03–2.14 (m, 1H; CHACHTREUNG(CH3)2), 3.01 (s, 3H; NCH3), 3.88 (dd, J=4.8,
8.1 Hz, 1H; NCHCO), 4.25 (dd, J=6.3, 15.6 Hz, 1H; CH2NH), 4.91 (dd,
J=5.1, 15.6 Hz, 1H; NCH2N), 5.59 (brs; NH), 7.46 ppm (brs; NH);
13C NMR (75 MHz, CDCl3, 298 K): d=18.4, 19.8 (CH3), 29.9 (CH), 33.7
(NCH3), 56.0 (CH2), 60.2 (CH), 158.9, 170.2 ppm (CO); IR (solid): n˜ =
3238 (NH), 2961, 2926, (Csp3), 1652 cmꢀ1 (CO); HRMS (ESI): m/z: calcd
for C8H16N3O2: 186.1234; found: 186.1237.
Cyclo(Leu-gSar-CO) (4d): Pale yellow solid; C18-RP-HPLC (A: 0.1%
TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLmin-1, 20 min):
tR =9.37 min; [a]2D0 =ꢀ29.2 (c=0.85 in MeOH); 1H NMR (300 MHz,
CDCl3, 298 K): d=0.94 (d, J=2.7 Hz, 3H; CH3), 0.95 (d, J=2.7 Hz, 3H;
CH3), 1.39–1.48 (m, 1H; CHCATHER(UGN CH3)2), 1.70–1.88 (m, 2H; CHCH2CH), 3.07
(s, 3H; NCH3), 4.06 (dd, J=6.9, 15.6 Hz, 1H; NCH2N), 4.35–4.40 (m,
1H; NCHCO), 4.96 (brs; NH), 5.25 (dd, J=5.1, 15.6 Hz, 1H; NCH2N),
6.85 ppm (brs; NH); 13C NMR (75 MHz, CDCl3, 298 K): d=22.02, 22.82
(CH3), 24.38 (CH), 33.75 (CH3), 39.97 (CH2), 51.00 (CH), 56.17 ppm
(CH2); IR (solid): n˜ =3579, 3219 (NH), 2927 (Csp3), 1676, 1646 cm-1
(CO); HRMS (ESI): calcd for C9H18N3O2: 200.1399; found: 200.1420.
Cyclo(2-Nal-gPro-CO) (4j): Pale yellow solid; C18-RP-HPLC (A: 0.1%
TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min):
tR =13.29 min; [a]2D0 =ꢀ4.0 (c=0.99 in MeOH); 1H NMR (300 MHz,
CDCl3, 298 K): d=1.87–1.95 (m, 2H; CH2), 2.01–2.25 (m, 2H; CH2), 2.95
(dd, J=8.7, 14.4 Hz, 1H; CH2-Nal), 3.44–3.57 (m, 2H; CH2), 3.77–3.85
(m, 1H; CH2-Nal), 4.63–4.69 (m, 1H; NCHCO), 4.96 (brs; NH), 5.45 (d,
J=4.8 Hz, 1H; NCHN), 6.53 (brs; NH), 7.38 (dd, J=1.5, 8.4 Hz, 1H; H-
Ar), 7.45–4.49 (m, 2H; H-Ar), 7.74 (s, 1H; H-Ar), 7.79–7.81 ppm (m,
3H; H-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=22.6, 33.3, 36.7, 42.2
(CH2), 54.9, 64.3 (CH), 125.9, 126.4, 127.0, 127.7, 128.3, 128.7 (CH-Ar),
132.5, 133.5, 133.7 (Cq-Ar), 157.6, 167.7 ppm (CO); IR (solid): n˜ =3269
(NH), 3057 (Csp2), 2951, 2926, 2886 (Csp3), 1650 cmꢀ1 (CO); HRMS
(ESI): calcd for C18H20N3O2: 310.1550; found: 310.1556.
CycloACHTREUNG(DapACHTREUNG(Fmoc)-gSar-CO) (4e): White solid, C18-RP-HPLC (A: 0.1%
TFA in H2O, B: 0.08% TFA in MeCN, 0-100% B, 1.2 mLmin-1, 20 min):
tR =13.74 min; 1H NMR (400 MHz, CD3CN, 298 K): d= 2.95 (s, 3H;
CH3), 3.26–3.33 (m, 1H; CHCH2NH), 3.44–3.51 (m, 1H; CHCH2NH),
3.94–3.99 (m, 1H; NCH2N), 4.19–4.22 (m, 1H; NCH2N), 4.34 (s, 2H; CH-
ACHTREUNG(Fmoc), CH2Fmoc), 4.50 (s, 1H; CH2Fmoc), 5.08–5.13 (m, 1H;
NCHCO), 5.87 (brs, 1H; NH), 6.05 (brs, 2H; NH), 7.26–7.42 (m, 4H;
CH-Ar), 7.57–7.64 (m, 2H; CH-Ar), 7.75–7.82 ppm (m, 2H; CH-Ar);
13C NMR (75 MHz, CDCl3, 298 K): d=33.24 (CH2), 42.51 (CH3), 48.08,
54.25 (CH2), 56.33, 67.29 (CH), 120.97, 126.15, 128.12, 128.69 (CH-Ar),
142.16, 145.10 (Cq-Ar), 158.33, 171.12 ppm (CO); HRMS (ESI): calcd for
C21H23N4O4: 395.1714; found: 395.1704.
Cyclo(Tic-gPro-CO) (4k): White solid; C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLmin-1, 20 min): tR =
11.23 min; [a]2D0 =17.5 (c=0.51 in MeOH); 1H NMR (300 MHz, CDCl3,
298 K): d=1.72–1.97 (m, 2H; CH
2.25–2.34 (m, 1H; CH2A(gPro)), 3.08 (dd, J=5.1, 15.7 Hz, 1H;
CHCH2Ph), 3.33 (dd, J=10.5, 15.6 Hz, 1H; CHCH2Ph), 3.52–3.60 (m,
1H; CH2A(gPro)), 3.73–3.81 (m, 1H; CH2A(gPro)), 4.36 (d, J=15.3 Hz, 1H;
2ACHRTU(NEG gPro)), 2.02–2.13 (m, 1H; CH2ACHTREUNG(gPro)),
CTHREUNG
Cyclo(Phg-gSar-CO) (4 f): White solid; C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
9.26 min; [a]2D0 =ꢀ86.9 (c=0.64 in MeOH); 1H NMR (300 MHz, CDCl3,
298 K): d=3.02 (s, 3H; CH3N), 3.87 (dd, J=6.9, 15.6 Hz, 1H; NCH2N),
4.54 (dd, J=5.7, 15.6 Hz, 1H; NCH2N), 5.26 (d, J=6.9 Hz, 1H;
NCHCO), 6.29 (d, J=6.6 Hz, 1H; CHNH), 6.99 (brs; NH), 7.32–
7.44 ppm (m, 5H; H-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=33.2
(NCH3), 54.1 (CH2), 61.2 (CH), 124.3, 127.4, 128.4 (CH-Ar), 137.2 (Cq-
Ar), 158.2, 168.9 ppm (CO); IR (solid): n˜ =3278 (NH), 3108 (Csp2), 2956,
2934 (Csp3), 1674, 1639 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for
C11H14N3O2: 220.1081; found: 220.1069.
C
CHTREUNG
NCH2Ph), 4.56 (dd, J=5.1, 10.5 Hz, 1H; NCHCO), 4.87 (d, J=15.2 Hz,
1H; NCH2Ph), 5.41–5.46 (m, 1H; NCHN), 5.61 (brs; NH), 7.18–
7.25 ppm (m, 4H; CH-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=22.40,
30.57, 34.08, 46.28 (CH2), 56.00, 65.35 (CH), 125.83, 126.69, 127.40, 127.55
(CH-Ar), 137.77, 135.15 (Cq-Ar), 158.17, 167.80 ppm (CO); IR (solid):
n˜ =3265, 3221 (NH), 3067 (Csp2), 2966, 2916 (Csp3), 1658, 1636 cmꢀ1
(CO); HRMS (ESI): calcd for C15H18N3O2: 272.1399; found: 272.1405.
Cyclo(Phe-gN-MePhe-CO) (4l): White solid; C18-RP-HPLC (A: 0.1%
TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min):
tR =14.06 min; [a]2D0 =ꢀ18.8 (c=0.73 in MeOH); 1H NMR (300 MHz,
CDCl3, 298 K): d=2.80 (dd, J=8.1, 14.7 Hz, 1H; CH2Ph), 2.96 (s, 3H;
NCH3), 2.98–3.09 (m, 2H; CH2Ph), 3.38 (dd, J=5.1, 14.4 Hz, 1H;
CH2Ph), 4.74–4.79 (m, 1H; NCHCO), 5.06 (brs; NH), 5.64–5.71 (m, 1H;
NCHN), 7.13 (brs; NH), 7.19–7.35 ppm (m, 10H; CH-Ar); 13C NMR
(75 MHz, CDCl3, 298 K): d=27.1 (CH3), 37.5, 37.9 (CH2), 54.6, 63.9
(CH), 127.3, 127.7, 128.7, 129.1, 129.2, 129.3 (CH-Ar), 134.7, 136.2 (Cq-
Ar), 156.8, 170.5 ppm (CO); IR (solid): n˜ =3402, 3301, 3224 (NH), 3064
(Csp2), 2965 (Csp3), 1652 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for
C19H22N3O2: 324.1707; found: 324.1701.
Cyclo(2-Nal-gSar-CO) (4g): White solid; C18-RP-HPLC (A: 0.1% TFA
in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
1
12.69 min; [a]2D0 =ꢀ17.9 (c=0.92 in MeOH); H NMR (300 MHz, CDCl3,
298 K): d=2.97 (dd, J=8.7, 14.4 Hz, 1H; CH2-Nal), 3.09 (s, 3H; NCH3),
3.51 (dd, J=5.1, 14.4 Hz, 1H; CH2-Nal), 4.00 (dd, J=6.9, 15.6 Hz, 1H;
NCH2N), 4.71–4.76 (m, 1H; NCHCO), 4.82 (brs; NH), 5.21 (dd, J=5.1,
15.6 Hz, 1H; NCH2N), 6.57 (brs; NH), 7.36 (dd, J=1.5, 8.4 Hz, 1H; H-
Ar), 7.45–7.49 (m, 2H; H-Ar), 7.72 (s, 1H; H-Ar), 7.78–7.83 ppm (m,
3H; H-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=33.9 (NCH3), 37.6
(CH2), 54.4(CH), 56.1 (CH2), 125.9, 126.4, 126.9, 127.6, 127.6, 128.2, 128.8
(CH-Ar), 132.5, 133.5, 133.6 (Cq-Ar), 157.7, 169.9 ppm (CO); IR (solid):
Chem. Eur. J. 2006, 12, 8498 – 8512
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8507

G. Guichard, L. RØnia et al.
Cyclo
A
Cyclo(Pro-gVal-CO-Pro-gVal-CO) (5r): White solid; C18-RP-HPLC (A:
0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1
0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1
,
20 min): tR =14.05 min; [a]2D0 =ꢀ4.5 (c=1.19 in MeOH); 1H NMR
(300 MHz, CDCl3, 298 K): d=1.97–2.03 (m, 1H; CHCH2CH), 2.48–2.56
(m, 1H; CHCH2CH), 2.79 (dd, J=9.6, 14.7 Hz, 1H; CHCH2Ph), 3.38
(dd, J=9.6, 14.7 Hz, 1H; CHCH2Ph), 3.71 (dd, J=4.5, 12.6 Hz, 1H;
CHCH2N), 3.87–3.91 (m, 1H; CHCH2N), 4.24 (m, 1H; OCH), 4.45–4.55
(m, 3H; PhCH2O, NCHCO), 4.66 (brs; NH), 5.59–5.60 (m, 1H; NCHN),
6.62 (brs; NH), 7.25–7.32 ppm (m, 10H; CH-Ar); 13C NMR (75 MHz,
CDCl3, 298 K): d=36.7, 39.6, 50.5 (CH2), 54.5, 63.5 (CH), 71.4 (CH2),
74.9 (CH), 127.2, 127.8, 128.0, 128.6, 129.0, 129.2 (CH-Ar), 136.1, 137.2
(Cq-Ar), 157.5, 168.4 ppm (CO); IR (solid): n˜ =3207 (NH), 3064 (Csp2),
20 min): tR =10.74 min; 1H NMR (500 MHz, CDCl3, 273 K; signals of ro-
tamers are in italic): d=0.93 (d, J=6.7 Hz, 3H; CH3), 1.00 (d, J=6.7 Hz,
3H; CH3), 1.87–2.28 (m, 5H; CHMe2, bCH2 Pro, gCH2 Pro), 3.35 (m,
1H; dCH Pro), 3.51, 3.64 (m, 1H; dCH Pro), 4.21, 4.29 ppm (dd, J=2.7,
8.6 Hz, J=5.5, 7.9 Hz, 1H; aCH Pro); HRMS (ESI): m/z: calcd for
C20H35N6O4: 423.2720; found: 423.2754.
Cyclo(Pro-gLeu-CO-Pro-gLeu-CO) (5s): White solid; C18-RP-HPLC (A:
0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1
,
20 min): tR =9.86 min; 1H NMR (300 MHz, CD3CN, 298 K): d=0.91 (m,
3H; CH3), 0.94 (m, 3H; CH3), 1.49–1.65 (m, 3H; CH2, CH2CHMe2),
1.77–2.02 (m, 3H; CH2, CH2CHMe2), 2.14–2.21 (m, 1H; CH2,
CH2CHMe2), 3.22–3.29 (m, 1H; NCH2), 3.55–3.63 (m, 1H; NCH2), 4.14–
4.18 (m, 1H; NCHCO), 5.28–5.36 (m, 1H; NCHN), 5.75 (d, J=9.0 Hz,
1H; NH), 7.28–7.31 ppm (brs, 1H; NH); IR (solid): n˜ =3313 (NH), 2957,
2930 (Csp3), 1651 (CO), 1091 cmꢀ1 (C O C); HRMS (ESI): m/z: calcd
ꢀ
ꢀ
for C21H24N3O3: 366.1817; found: 366.1839.
Cyclo(Ala-gTic-CO) (4o): White solid; C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
10.43 min; [a]2D0 =89.0 (c=0.30 in MeOH); 1H NMR (300 MHz, CDCl3,
298 K): d=1.38 (d, J=6.6 Hz, 3H; CH3), 2.87–2.94 (m, 1H; CH2CH),
3.19–3.26 (m, 1H; CH2CH), 4.53–4.56 (m, 1H; NCHCO), 4.59 (d, J=
15 Hz, 1H; CH2N), 4.71 (d, J=15 Hz, 1H; CH2N), 5.04 (brs; NH), 5.73
(m, 2H; NCHN, NH), 7.21–7.29 ppm (m, 4H; H-Ar); 13C NMR
(75 MHz, CDCl3, 298 K): d=16.7 (CH3), 33.9 (CH2N), 42.3 (CH2), 48.8,
59.4 (CH), 126.4, 127.8, 127.9, 128.0 (CH-Ar), 131.2, 134.6 (Cq-Ar),
156.9, 169.7 ppm (CO); IR (solid): n˜ =3456, 3394, 3237 (NH), 3053
(Csp2), 2926 (Csp3), 1641 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for
C13H16N3O2: 246.1242; found: 246.1295.
2870 (Csp3), 1651 (CO), 1511 cmꢀ1 (HN CO); HRMS (ESI): m/z: calcd
ꢀ
for C22H37N6O4: 451.3027; found: 451.3023.
1,3,7-Tribenzyl-5-methyl-[1,3,5]triazepane-2,6-dione (8a): BnBr (204 mL,
1.72 mmol) and NaH (95% wt, 42 mg, 1.72 mmol) were added to a solu-
tion of 4a (100 mg, 0.43 mmol) in THF (8 mL) under Ar. The reaction
was stirred at RT under Ar for 48 h. After this time, the reaction mixture
was dissolved in ethyl acetate and washed with H2O. The organic layer
was dried over Na2SO4 and evaporated. The crude residue was dissolved
in CH2Cl2 and purified by addition of N-(2-mercaptoethyl)aminomethyl-
polystyrene (645 mg, 1.29 mmol, used as supplied). The mixture was
gently stirred for 24 h. The resin was removed by filtration. The filtrate
was evaporated and concentrated under vacuum. Purification by silica-
gel chromatography (ethyl acetate/cyclohexane 1:1 v/v) yielded 8a
(89 mg, 50%) as a colorless oil. C18-RP-HPLC (A: 0.1% TFA in H2O, B :
0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =16.82 min;
[a]2D0 =2.4 (c=0.75 in MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=
2.68 (s, 3H; NCH3), 3.16–3.33 (m, 2H; CHCH2Ph), 3.51 (d, J=15.0 Hz,
1H; NCHN), 4.01 (d, J=13.9 Hz, 1H; NCH2Ph), 4.13–4.17 (m, 1H;
NCHCO), 4.29 (d, J=14.9 Hz, 1H; NCH2Ph), 4.53 (d, J=14.9 Hz, 1H;
NCH2Ph), 4.74 (d, J=15.0, 1H; NCHN), 4.90 (d, J=13.9 Hz, 1H;
NCH2Ph), 7.11–7.14 (m, 2H; H-Ar), 7.22–7.37 ppm (m, 13H; H-Ar);
13C NMR (75 MHz, CDCl3, 298 K): d=34.5 (CH3), 39.7, 51.4, 52.5, 62.4
(CH2), 66.7 (CH), 126.8, 127.3, 127.7, 127.9, 128.3, 129.5, 128.6, 129.8
(CH-Ar), 136.7, 137.3 (Cq-Ar), 163.2, 169.0 ppm (CO); IR (solid): n˜ =
3062, 3029 (Csp2), 2926 (Csp3), 1666, 1638 cmꢀ1 (CO); HRMS (ESI): m/z:
calcd for C26H28N3O2: 414.2176; found: 414.2176.
Cyclo(d-Phe-gPro-CO) (4p): White solid; C18-RP-HPLC (A: 0.1% TFA
in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
10.63 min; 1H NMR (500 MHz, [D6]DMSO, 298 K): d=1.63–1.67 (m,
1H; CH2), 1.79–1.83 (m, 1H; CH2), 1.88–1.90 (m, 1H; CH2), 1.98–2.02
(m, 1H; CH2), 2.99–3.08 (m, 2H; CH2Ph), 3.23–3.28 (m, 1H; CH2), 3.47–
3.51 (m, 1H; CH2), 3.95 (q, J=6.6 Hz, 1H; NCHCO), 4.90–4.92 (m, 1H;
NCHN), 6.29 (d, J=5.7 Hz, 1H; NH), 7.16–7.18 (m, 2H; NH, H-Ar),
7.20–7.23 (m, 1H; H-Ar), 7.26–7.29 ppm (m, 3H; H-Ar); 13C NMR
(75 MHz, CDCl3, 298 K): d=21.9, 32.7, 42.2, 46.7 (CH2), 61.3, 63.8 (CH),
127.4, 128.8, 129.6 (CH-Ar), 136.1 (Cq-Ar), 160.7, 167.4 ppm (CO); IR
(solid): n=3331, 3240 (NH), 2925, 2882 (Csp3), 1669, 1639 cmꢀ1 (CO);
HRMS (ESI): m/z: calcd for C14H18N3O2: 260.1379; found: 260.1394.
Cyclo(D-Phe-gN-MeAla-CO) (4q): White solid; C18-RP-HPLC (A:
0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1
,
1
20 min): tR =11.34 min; H NMR (300 MHz, CDCl3, 298 K): d=1.43–1.45
(d, J=6.4 Hz, 3H; CH3), 2.80 (dd, J=8.4, 14.5 Hz, 1H; CH2Ph), 2.96 (s,
3H; NCH3), 3.39 (dd, J=5.3, 14.5 Hz, 1H; CH2Ph) 4.74–4.79 (m, 2H;
NCHCO, NH), 5.62–5.66 (m, 1H; NCHN), 6.42–6.43 (brs; NH), 7.05–
7.35 ppm (m, 5H; H-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=17.6,
26.4 (CH3), 37.3 (CH2), 54.3, 58.8 (CH), 127.2, 129.0, 129.3 (CH-Ar),
145.5 (Cq-Ar), 157.0, 170.3 ppm (CO); IR (solid): n˜ =3301, 3188 (NH),
3069 (Csp2), 2927 (Csp3), 1652 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for
C13H18N3O2: 248.1394; found: 248.1400.
5,6,8-Tribenzyl-hexahydro-3a,6,8-triaza-azulene-4,7-dione (8i): Colorless
oil; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–
100% B, 1.2 mLminꢀ1, 20 min): tR =17.30 min; [a]2D0 =57.2 (c=0.57 in
MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=1.65–2.12 (m, 4H;
CH2), 3.07 (dd, J=8.6, 13.9 Hz, 1H; CHCH2Ph), 3.30 (dd, J=3.6,
13.9 Hz, 1H; CHCH2Ph), 3.41 (d, J=14.8 Hz, 1H; NCH2Ph), 3.46–3.53
(m, 1H; CH2), 3.69–3.77 (m, 1H; CH2), 4.12 (dd, J=3.6, 8.6 Hz, 1H;
NCHCO), 4.35 (d, J=16.6 Hz, 1H; NCH2Ph), 4.50 (d, J=16.6 Hz, 1H;
NCH2Ph), 4.65 (d, J=14.9 Hz, 1H; NCH2Ph), 5.57 (dd, J=6.4, 9.0 Hz,
1H; NCHN), 7.10–7.12 (m, 2H; H-Ar), 7.20–7.35 ppm (m, 13H; H-Ar);
13C NMR (75 MHz, CDCl3, 298 K): d=21.7, 30.8, 39.7, 46.1, 47.6, 53.3
(CH2), 66.2, 71.2 (CH), 1126.8, 126.9, 127.0, 127.4, 128.3, 128.4, 128.6,
128.6, 130.0 (CH-Ar), 137.5, 137.8, 139.0 (Cq-Ar), 165.0, 168.0 ppm
(CO); IR (solid): n˜ =3083, 3058, 3029 (Csp2), 2951, 2926, 2881 (Csp3),
1667, 1634 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for C28H30N3O2:
440.2333; found: 440.2342.
Cyclo(Pro-gVal-CO) (4r): White solid; C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
1
7.76 min; H NMR (500 MHz, [D6]DMSO, 298 K): d=0.94 (d, J=6.7 Hz,
3H; CH3), 0.95 (d, J=6.7 Hz, 3H; CH3), 1.67–1.71 (m, CH2, 3H; CH-
ACHTREUNG(CH3)2), 1.79–1.89 (m, 2H; CH2), 2.22–2.29 (m, 1H; CH2), 4.70 (t, J=
7.1 Hz, 1H; CHCH2), 4.82 (dd, J=7.2, 13.1 Hz, 1H; CHCH), 6.72 (d, J=
5.6 Hz; NH), 8.34 ppm (d, J=7.6 Hz; NH); HRMS (ESI): m/z: calcd for
C10H18N3O2: 212.1393; found: 212.1394.
Cyclo(Pro-gLeu-CO) (4s): White solid; C18-RP-HPLC (A: 0.1% TFA in
H2O, B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =
(7-Benzyl-3-tert-butoxycarbonylmethyl-5-methyl-2,6-dioxo-
AHCTRE[UNG 1,3,5]triazepan-1-yl)acetic acid tert-butylester (9a) : White solid; C18-
1
8.74 min; H NMR (300 MHz, CDCl3, 298 K): d=0.92 (d, J=5.3 Hz, 3H;
RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B,
CH3), 0.95 (d, J=5.3 Hz, 3H; CH3), 1.47–1.52 (t, J=6.9 Hz, 2H;
CHCH2CH), 1.60–1.91 (m, 3H; (CH3)2CH, CH2), 1.96–2.07 (m, 1H;
CH2), 2.44–2.56 (m, 1H; CH2), 3.49–3.56 (m, 2H; CH2), 4.57–4.61 (t, J=
6.9 Hz, 1H; NHCHCO), 5.09–5.13 (m, 1H; NHCHNH), 5.55 (d, J=
4.6 Hz; NH), 6.50 ppm (d, J=7.3 Hz; NH); IR (solid): n˜ =3260, 3207
(NH), 2957, 2925, 2871 (Csp3), 1689, 1661, 1620 (CO), 1453 cmꢀ1 (NH
lactam); HRMS (ESI): m/z: calcd for C11H20N3O2: 226.1555; found:
226.1554.
1.2 mLminꢀ1, 20 min): tR =16.55 min; [a]2D0 =ꢀ15.0 (c=1.05 in MeOH);
1H NMR (300 MHz, CDCl3, 298 K): d=1.39 (s, 9H; C
ACHTRE(UGN CH3)3), 1.47 (s,
9H; CACHTREU(GN CH3)), 2.91 (d, J=17.7 Hz, 1H; CHCH2Ph), 3.06 (s, 3H; NCH3),
3.19–3.24 (m, 2H; CHCH2Ph, NCH2tBu), 3.92 (q, J=17.4 Hz, 2H;
NCH2tBu), 4.02 (d, J=17.7 Hz, 1H; NCH2tBu), 4.08 (dd, J=4.9, 8.4 Hz,
1H; NCHCO), 4.24 (d, J=14.1 Hz, 1H; NCH2N), 5.21 (d, J=14.1 Hz,
1H; NCH2N), 7.22–7.33 ppm (m, 5H; H-Ar); 13C NMR (75 MHz, CDCl3,
8508
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 8498 – 8512

1,3,5-Triazepan-2,6-dione Dipeptide Mimetics
FULL PAPER
298 K): d=28.0, 28.0 (CH3, 6C), 35.2 (NCH3), 40.4, 51.2, 52.0, 65.2
(CH2), 69.7 (CH), 81.8, 82.2 (Cq), 127.0, 128.6, 129.7 (CH-Ar), 137.2 (Cq-
Ar), 162.0, 168.6, 168.8, 169.2 ppm (CO); IR (solid): n˜ =3032 (Csp2),
2976, 2921, 2850 (Csp3), 1739, 1682, 1637 cmꢀ1 (CO); HRMS (ESI): m/z:
calcd for C24H36N3O6: 462.2599; found: 462.2593.
ꢀ53.0 (c=0.84 in MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=1.45
(s, 9H; CACTHRE(UGN CH3)3), 2.81 (dd, J=9.2, 14.3 Hz, 1H; CH2Ph), 3.13 (s, 3H;
NCH3), 3.36 (dd, J=4.5, 14.3 Hz, 1H; CH2Ph), 3.86 (d, J=17.7 Hz, 1H;
NCH2CO), 4.17 (d, J=15.7 Hz, 1H; NCH2N), 4.28 (d, J=17.7 Hz, 1H;
NCH2CO), 4.58–4.63 (m, 2H; NCHCO, NH), 5.44 (d, J=15.7 Hz, 1H;
NCH2N), 7.23–7.36 ppm (m, 5H; H-Ar); 13C NMR (75 MHz, CDCl3,
298 K): d=28.1 (CH3, 3C), 34.7 (NCH3), 38.2, 51.5 (CH2), 56.0 (CH),
62.8 (CH2), 82.4 (Cq), 127.3, 129.0, 129.4 (CH-Ar), 136.1 (Cq-Ar), 157.3,
168.9, 169.8 ppm (CO); IR (solid): n˜ =3291, 3221 (NH), 3063 (Csp2),
2975, 2925, 2850 (Csp3), 1741, 1653 cmꢀ1 (CO); HRMS (ESI): m/z: calcd
for C18H26N3O4: 348.1918; found: 348.1917.
(5-Benzyl-8-tert-butoxycarbonylmethyl-4,7-dioxo-octahydro-3a,6,8-triaza-
azulen-6-yl)acetic acid tert-butylester (9i) : Pale yellow solid; C18-RP-
HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B,
1.2 mLminꢀ1, 20 min): tR =17.00 min; [a]2D0 =ꢀ26.8 (c=1.18 in MeOH);
1H NMR (300 MHz, CDCl3, 298 K): d=1.37 (s, 9H; C
ACHTRE(UGN CH3)3), 1.46 (s,
9H; CACHTREUNG(CH3)3), 1.80–1.99 (m, 3H; CH2), 2.25–2.32 (m, 1H; CH2), 2.78 (d,
J=17.7 Hz, 1H; NCH2CO), 3.18 (dd, J=9.6, 13.9 Hz, 1H; CH2Ph), 3.28
(dd, J=3.5, 13.9 Hz, 1H; CH2Ph), 3.50–3.60 (m, 1H; CH2), 3.76 (d, J=
17.9 Hz, 1H; NCH2CO; m, 1H; CH2), 3.97 (d, J=17.9 Hz, 1H;
NCH2CO; m, 1H; NCHCO), 4.07 (d, J=17.7 Hz; NCH2CO), 5.70 (dd,
J=6.0, 8.7 Hz, 1H; NCHN), 7.20–7.32 ppm (m, 5H; H-Ar); 13C NMR
(75 MHz, CDCl3, 298 K): d=21.4 (CH2), 28.0 (CH3, 6C), 30.7, 40.5, 46.4,
46.9, 52.7 (CH2), 69.7, 71.6 (CH), 81.7, 81.9 (Cq), 126.9, 128.5, 129.8 (CH-
Ar), 137.7 (Cq-Ar), 163.9, 167.7, 168.6, 169.2 ppm (CO); IR (solid): n˜ =
2977, 2956, 2922, 2853 (Csp3), 1739, 1679, 1638 (CO), 1221, 1146 cmꢀ1
(6-Isopropyl-1-methyl-4,7-dioxo
ACHTRE[UNG 1,3,5]triazepan-3-yl)acetic acid tert-butyl
ester (11c): White solid; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08%
TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =12.18 min; [a]2D0
=
ꢀ49.1 (c=0.43 in MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=1.02
(d, J=7.0 Hz, 3H; CH3), 1.04 (d, J=7.0 Hz, 3H; CH3), 1.43 (s, 9H;
CH3), 2.15–2.22 (m, 1H; CHCATHER(UGN CH3)2), 3.07 (s, 3H; NCH3), 3.91–3.97 (m,
2H; NCH2CO, NCHCO), 4.19 (d, J=17.7 Hz, 1H; NCH2CO), 4.47 (d,
J=15.6 Hz, 1H; NCH2N), 4.84 (d, J=3.8 Hz, 1H; NH), 5.09 ppm (d, J=
15.6 Hz, 1H; NCH2N); 13C NMR (75 MHz, CDCl3, 298 K): d=18.0, 19.7,
28.0 (CH3), 30.6 (NCH3), 34.7 (CH), 51.3 (CH2), 61.4 (CH), 62.8 (CH2),
83.1 (Cq), 157.9, 168.9, 170.0 ppm (CO); IR (solid): n˜ =3392, 3296, 3226
(NH), 2921, 2850 (Csp3), 1734, 1651 cmꢀ1 (CO); HRMS (ESI): m/z: calcd
for C14H26N3O4: 300.1918; found: 300.1907.
ꢀ
(C O); HRMS (ESI): m/z: calcd for C26H38N3O6: 488.2755; found:
488.2755.
3,7-Dibenzyl-5-methylACHTREUNG[1,3,5]triazepane-2,6-dione (10a): BnBr (80 mL,
0.67 mmol) and KF/Al2O3 40% wt (930 mg, 6.4 mmol) were added to a
solution of 4a (150 mg, 0.64 mmol) in CH2Cl2 (6 mL) under Ar. The mix-
ture was stirred for 24 h at RT under Ar. After this time, the reaction
mixture was diluted with CH2Cl2, washed with H2O, dried over Na2SO4,
and concentrated. The crude residue was purified by silica-gel chroma-
tography (ethyl acetate/cyclohexane/AcOH 50:50:1 v/v/v) to yield 10a
(141 mg, 68%) as a white powder. C18-RP-HPLC (A: 0.1% TFA in H2O,
(5-Benzyl-4,7-dioxo-octahydro-3a,6,8-triaza-azulen-8-yl)acetic acid tert-
butylester (11i) : Colorless oil; C18-RP-HPLC (A: 0.1% TFA in H2O, B :
0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =14.33 min;
1H NMR (300 MHz, CDCl3, 298 K): d=1.45 (s, 9H; C
ACHTRE(UNG CH3)3), 1.91–2.21
(m, 4H; CH2), 2.81 (dd, J=9.7, 14.3 Hz, 1H; CH2Ph), 3.44 (dd, J=4.2,
14.3 Hz, 1H; CH2Ph), 3.56–3.69 (m, 2H; CH2), 3.74 (d, J=18.1 Hz, 1H;
NCH2CO), 4.43 (d, J=18.1 Hz, 1H; NCH2CO; m, 1H; NCHCO), 4.57
(brs; NH), 5.76 (dd, J=3.9, 6.3 Hz, 1H; NCHN), 7.22–7.37 ppm (m, 5H;
H-Ar); IR (solid): n˜ =3272 (NH), 3058 (Csp2), 2927, 2874 (Csp3), 1735,
1705, 1674 (CO), 1612 cmꢀ1 (C=C); HRMS (ESI): m/z: calcd for
C20H27N3O4: 374.2074; found: 374.2086.
B: 0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1
, 20 min): tR =
14.07 min; [a]2D0 =ꢀ74.1 (c=0.63 in MeOH); H NMR (500 MHz, CDCl3,
298 K): d=2.65 (s, 3H; NCH3), 2.83 (dd, J=9.1, 14.3 Hz, 1H;
CHCH2Ph), 3.36 (dd, J=4.5, 14.3 Hz, 1H; CHCH2Ph), 4.13 (d, J=
15.6 Hz, 1H; NCH2N), 4.39 (d, J=15.1 Hz, 1H; NCH2Ph), 4.55–4.59 (m,
2H; NCHCO, NH), 4.79 (d, J=15.1 Hz, 1H; NCH2Ph), 5.15 (d, J=
15.6 Hz, 1H; NCH2N), 7.26–7.36 ppm (m, 10H; H-Ar); IR (solid): n˜ =
3296, 3199 (NH), 3063 (Csp2), 2956, 2923, 2853 (Csp3), 1653 cmꢀ1 (CO);
HRMS (ESI): m/z: calcd for C19H22N3O2: 324.1707; found: 324.1702.
1
(7-Benzyl-1-carboxymethyl-5-methyl-2,6-dioxoACHTREU[NG 1,3,5]triazepan-3-yl)acetic
acid (12): Pale yellow solid; C18-RP-HPLC (A: 0.1% TFA in H2O, B :
0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =11.74 min;
1H NMR (300 MHz, CDCl3, 298 K): d=2.91 (s, 3H; CH3), 3.10 (dd, J=
7.5, 13.6 Hz, 1H; CH2Ph), 3.22 (dd, J=6.9, 13.6 Hz, 1H; CH2Ph), 3.52 (d,
J=17.5 Hz, 1H; NCH2), 3.79 (d, J=17.5 Hz, 1H; NCH2), 3.92 (d, J=
17.6 Hz, 1H; NCH2), 4.05 (d, J=17.6 Hz, 1H; NCH2), 4.15 (t, J=7.2 Hz,
1H; NCHCO), 4.82 (q, J=17.9 Hz, 1H; NCH2N), 7.18–7.30 ppm (m,
5H; CH-Ar) ; 13C NMR (75 MHz, CDCl3, 298 K): d=34.58 (CH3), 39.6,
50.6, 50.8, 62.8 (CH2), 68.8 (CH), 127.0, 128.6, 130.0 (CH-Ar), 137.5 (Cq-
Ar), 160.3, 169.0, 171.5, 171.7 ppm (CO); IR (solid): n˜ =3448 (OH), 2927
(Csp3), 2622, 2531 (OH), 1713, 1918 cmꢀ1 (CO); HRMS (ESI): m/z: calcd
for C16H20N3O6: 350.1347; found: 350.1343.
3-Benzyl-7-isopropyl-5-methylACHTRE[UGN 1,3,5]triazepane-2,6-dione (10c): White
solid; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–
100% B, 1.2 mLminꢀ1, 20 min): tR =12.45 min; [a]2D0 =ꢀ54.4 (c=0.82 in
MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=1.03 (d, J=6.7 Hz, 3H;
CH3), 1.06 (d, J=6.7 Hz, 3H; CH3), 2.14–2.25 (m, 1H; CHACHTRE(UNG CH3)2), 2.62
(s, 3H; NCH3), 3.93 (dd, J=4.2, 6.8 Hz, 1H; NCHCO), 4.43 (d, J=
15.3 Hz, 1H; NCH2Ph), 4.50 (d, J=15.0 Hz, 1H; NCH2N), 4.71 (d, J=
15.0 Hz, 1H; NCH2N), 4.83–4.88 (m, 2H; NCH2Ph, NH), 7.26–7.37 ppm
(m, 5H; H-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=18.1, 19.8 (CH3),
31.0 (CH), 34.2 (NCH3), 51.5, 60.7 (CH2), 127.9, 128.3, 128.9 (CH-Ar),
137.6 (Cq-Ar) 158.6, 170.0 ppm (CO); IR (solid): n˜ =3393, 3199 (NH),
3063 (Csp2), 2959, 2920, 2850 (Csp3), 1647 cmꢀ1 (CO); HRMS (ESI): m/z:
calcd for C15H22N3O2: 276.1680; found: 276.1691.
(6-Benzyl-1-methyl-4,7-dioxo
solid; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–
100% B, 1.2 mLminꢀ1 20 min): tR =10.35 min; 1H NMR (300 MHz,
ACHTRE[UNG 1,3,5]triazepan-3-yl)acetic acid (13): White
,
[D6]DMSO, 298 K): d=2.78 (dd, J=6.8, 14.1 Hz, 1H; CH2Ph), 2.95, (s,
3H; NCH3), 3.06 (dd, J=6.6, 14.1 Hz, 1H; CH2Ph), 4.03 (q, J=17.6 Hz,
2H; NCH2), 4.35 (d, J=15.6 Hz, 1H; NCH2N), 4.77–4.80 (m, 1H;
NCHCO), 5.56 (d, J=15.6 Hz, 1H; NCH2N), 6.28 (s; NH), 7.16–
7.32 ppm (m, 5H; H-Ar); 13C NMR (75 MHz, [D6]DMSO, 298 K): d=
33.4 (CH3), 36.2, 50.3 (CH2), 54.1 (CH), 61.5 (CH2), 126.2, 129.0, 129.4
(CH-Ar), 138.1 (Cq-Ar), 156.3, 170.5, 171.6 ppm (CO); IR (solid): n˜ =
3277 (NH), 2924 (Csp3), 2455 (OH), 1702, 1672, 1608 cmꢀ1 (CO); HRMS
(ESI): m/z: calcd for C14H18N3O4: 292.1292; found: 292.1294.
5,8-Dibenzyl-hexahydro-3a,6,8-triaza-azulene-4,7-dione
solid; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–
100% B, 1.2 mLminꢀ1 20 min): tR =14.57 min; 1H NMR (300 MHz,
(10i): White
,
CDCl3, 298 K): d=1.76–1.86 (m, 2H; CH2), 1.96–2.06 (m, 1H; CH2),
2.18–2.28 (m, 1H; CH2), 2.84 (dd, J=9.2, 14.2 Hz, 1H; CHCH2Ph), 3.44–
3.68 (m, 3H; CH2, CHCH2Ph), 4.38–4.44 (m, 1H; NCHCO), 4.54 (d, J=
16.8 Hz, 1H; NCH2Ph), 4.80 (d, J=16.8 Hz, 1H; NCH2Ph), 5.12 (d, J=
2.4 Hz, 1H; NH), 5.69 (dd, J=4.4, 6.8 Hz, 1H; NCHN), 7.19–7.35 ppm
(m, 10H; CH-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=22.6, 30.6,
38.6, 45.7, 47.5 (CH2), 58.0, 69.2 (CH), 126.3, 127.1, 127.1, 128.8, 128.8,
129.6 (CH-Ar), 136.8, 138.8 (Cq-Ar), 160.4, 163.4 ppm (CO); IR (solid):
n˜ =3211 (NH), 3029 (Csp2), 2925 (Csp3), 1663, 1635 cmꢀ1 (CO); HRMS
(ESI): m/z: calcd for C21H24N3O2: 350.1863; found: 350.1866.
(9-Methyl-6,10-dioxo-5,8,9,10,10a,11-hexahydro-5a,7,9-triazacyclohep-
ta[b]naphthalen-7-yl)acetic acid tert-butylester (14) : White solid; C18-
RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0–100% B,
1.2 mLminꢀ1
,
20 min): tR =14.92 min; 1H NMR (300 MHz, CDCl3,
298 K): d=1.46 (s, 9H; CH3), 3.11–3.31 (m, 3H; NCH3, CHCH2Ph), 4.07
(s, 2H; NCH2Ph), 4.29–4.39 (m, 2H; NCH2CO, NCHCO), 4.59 (d, J=
15.1 Hz, 1H; NCH2N), 4.72 (d, J=16.4 Hz, 1H; NCH2CO), 4.98 (d, J=
(6-Benzyl-1-methyl-4,7-dioxo
ester (11a): White solid; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08%
TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =13.82 min; [a]2D0
ACHTRE[UNG 1,3,5]triazepan-3-yl)acetic acid tert-butyl
=
15.1 Hz, 1H; NCH2N), 7.12–7.20 ppm (m, 4H; H-Ar)
;
13C NMR
Chem. Eur. J. 2006, 12, 8498 – 8512
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8509

G. Guichard, L. RØnia et al.
(75 MHz, CDCl3, 298 K): d=28.1 (CH3), 33.9 (CH2), 35.3 (CH3), 49.2,
51.3 (CH2), 62.6 (CH), 63.5 (CH2), 82.3 (Cq), 126.3, 126.6, 126.8, 128.3
(CH-Ar), 133.2, 133.5 (Cq-Ar), 160.7, 168.8, 170.1 ppm (CO); IR (solid):
n˜ =2980 , 2926 (Csp3), 1747, 1673, 1629, 1617 cmꢀ1 (CO); HRMS (ESI):
m/z: calcd for C19H26N3O4: 360.1918; found: 360.1907.
7-Benzyl-1-(2,2-dimethyl-propionyl)-5-methylACHTERUN[G 1,3,5]triazepane-2,6-dione
(19): In this case, trimetylacetyl chloride (4 equiv, 100 mL 1.72 mmol) was
used and the reaction mixture was stirred for 30 h at 508C. White solid;
Yield: 109 mg, 80%; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08%
TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =12.67 min; [a]2D0
=
156.2 (c=0.73 in MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=1.16
(s, 9H; (CH3)3CCO), 2.91 (dd, J=14.4 Hz, 10.2 Hz, 1H; CHCH2Ph), 3.04
(s, 3H; NCH3), 3.43 (dd, J=14.1 Hz, 3,9 Hz, 1H; CHCH2Ph), 4.11 (dd,
J=13.8 Hz, 6.9 Hz, 1H; NCH2N), 5.07 (dd, J=14.1 Hz, 5.7 Hz, 1H;
NCH2N), 5.72 (dd, J=10.2 Hz, 3,8 Hz, 1H; CHCH2Ph), 6.67 (brt, J=
6.3 Hz, 1H; NH), 1.17–7.35 ppm (m, 5H; H-Ar); 13C NMR (75 MHz,
CDCl3, 298 K): d=27.8, 35.4 (CH3), 39.6 (CH2), 42.0 (Cq), 56.9 (CH2),
60.1 (CH), 126.6, 127.9, 129.8 (CH-Ar), 136.7 (Cq-Ar), 158.3, 169.2,
178.8 ppm (CO); IR (solid): n˜ =3157 (NH), 2927 (Csp2), 2866 (Csp3),
1735, 1675, 1615 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for C17H24N3O3:
318.1812; found: 318.1801.
(3,7-Dibenzyl-5-methyl-2,6-dioxoACHTREU[NG 1,3,5]triazepan-1-yl)acetic acid tert-
butylester (15) : White solid; C18-RP-HPLC (A: 0.1% TFA in H2O, B :
0.08% TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =16.63 min;
1H NMR (300 MHz, CDCl3, 298 K): d=1.40 (s, 9H; (CH3)3), 2.69 (s, 3H;
NCH3), 3.00 (d, J=17.6 Hz, 1H; NCH2CO), 3.16 (dd, J=8.6, 13.8 Hz,
1H; CH2Ph), 3.25 (dd, J=4.1, 13.8 Hz, 1H; CH2Ph), 4.03–4.10 (m, 3H;
NCH2CO, NCH2N, NCHCO), 4.32 (d, J=15.0 Hz, 1H; NCH2Ph), 4.55
(d, J=15.0 Hz, 1H; NCH2Ph), 5.03 (d, J=13.9 Hz, 1H; NCH2N), 7.22–
7.34 ppm (m, 10H; CH-Ar); 13C NMR (75 MHz, CDCl3, 298 K): d=28.0,
34.7 (CH3), 40.6, 51.7, 52.2, 62.7 (CH2), 69.9 (CH), 81.8 (Cq), 127.1,
127.9, 128.5, 128.6, 128.8, 129.9 (CH-Ar), 136.9, 137.1 (Cq-Ar), 162.4,
168.9, 169.2 ppm (CO); IR (solid): n˜ =3075 (Csp2), 2977, 2930 (Csp3),
1736, 1672, 1641 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for C25H32N3O4:
438.2387; found: 438.2366.
Mice and parasites: BALB/c female mice were purchased from Harlan
Laboratories (OrlØans, France). All experiments and procedures con-
formed to the French Ministry of Agriculture regulations for animal ex-
perimentation (1987). Sporozoites of the uncloned line of the 265BY
strain of P. yoelii yoelii were obtained by dissection of the salivary glands
of infected Anopheles stephensi mosquitoes, 16 to 21 days after an infect-
ed blood meal.
1-Benzoyl-7-benzyl-5-methylCAHTRE[UNG 1,3,5]triazepane-2,6-dione (16): Cyclo(l-
Phe-gSar-CO) (4a, 100 mg, 0.43 mmol) was dissolved in distilled THF
(10 mL) and then 2,4,6-collidine (113 mL, 0.86 mmol) was added, followed
by the dropwise addition of BzCl (100 mL, 0.86 mmol). The reaction was
stirred at RT under inert atmosphere for 7 h. After this time, the solvent
was evaporated and the crude residue was purified by flash-column chro-
matography (EtOAc/cyclohexane 3:2 v/v) to yield 16 (103 mg, 71%) as a
white solid. C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in
MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =12.01 min; [a]2D0 =121.8
(c=0.63 in MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=3.02 (s, 3H;
NCH3), 2.95–3.07 (m, 1H; CHCH2Ph), 3.45 (dd, J=14.1, 3.9 Hz, 1H;
CHCH2Ph), 4.10 (dd, J=14.1, 7.5 Hz, 1H; NCHN), 5.15 (dd, J=14.1,
5.4 Hz, 1H; NCHN) 5.69 (dd, J=9.6, 3.9 Hz, 1H; NCH2Ph), 6.68–6.75
(m, 1H; NH), 7.18–7.50 ppm (m, 10H; H-Ar); 13C NMR (75 MHz,
CDCl3, 298 K): d=35.5 (CH3), 40.0 (CH2), 57.0 (CH2), 60.7 (CH), , 126.9,
127.0, 128.1, 128.3, 130.0, 131.3 (CH-Ar), 135.0, 136.5 (Cq-Ar), 158.4,
168.8, 170.0 ppm (CO); IR (solid): n˜ =3261 (NH), 2927 (Csp2), 2851
(Csp3), 1728, 1674, 1634 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for
C19H20N3O3: 338.1499; found: 338.1497.
Hepatocyte culture: Mouse hepatocytes were prepared as described, with
minor modifications.[43] Cells were isolated by collagenase perfusion
(Boehringer Mannheim) of liver fragments and were further purified
over a 60% Percoll gradient (Pharmacia Biotech, Uppsala, Sweden).
Hepatocyte purity and viability were >95% as assessed by Trypan blue
dye exclusion. Cells were cultured in eight-chamber plastic Lab-Teck
slides (Nunc, Naperville, IL) or in flat bottom 96 wells (Costar) in Wil-
liamꢁs E medium (Life Technologies, Edinburgh, Scotland) supplemented
with 5% FCS (Life Technologies), 100 UImlꢀ1 of penicillin-streptomycin
(Life Technologies) and incubated at 378C in 3.5% CO2 for 24 h.
Evaluation of drug toxicity: Drugs were diluted first in DMSO then in in-
complete medium. Toxicity of the drugs to primary culture of hepato-
cytes (20”103 I flat bottom 96 wells) was evaluated in duplicate by
adding the drugs at decreasing concentrations. Medium was changed 3
and 24 h after with fresh medium containing the drug at the same con-
centration. After 48 h, 110 ml of a solution containing 10 ml of a MTT sol-
ution (5 mgmLꢀ1) and 100 ml of medium was added to dissolve formazan
crystals. Optical density was read immediately at 570 nm with a reference
wavelength at 630 nm.[44]
1-Benzoyl-7-benzyl-5-methyl
Yield: 123 mg, 79%; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08%
TFA in MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =13.52 min; [a]2D0
ACHTRE[UNG 1,3,5]triazepane-2,6-dione (17): White solid;
=
180.4 (c=0.68 in MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=3.06
(s, 3H; NCH3), 2.97–3.10 (m, 1H; CHCH2Ph), 3.51 (dd, J=14.3, 3.04 Hz,
1H; CHCH2Ph), 4.13 (dd, J=14.1, 6.9 Hz, 1H; NCH2N), 5.08 (dd, J=
14.1, 5.1 Hz, 1H; NCHCO), 6.66–6.75 (m, 1H; NH), 6.77 (d, J=15.3 Hz,
1H; CHCHPh), 7.15–7.49 (m, 10H; H-Ar), 7.62 ppm (d, J=15. Hz, 1H;
CHCHPh); 13C NMR (75 MHz, CDCl3, 298 K): d=35.8 (CH3), 40.4, 57.4
(CH2), 59.8 (CH), 117.7 (CH), 127.2, 128.5, 128.5, 128.6, 129.1, 129.1,
130.0, 130.0, 130.7 (CH-Ar), 134.7, 136.8 (Cq-Ar), 158.7, 165.7, 169.3 ppm
(CO); IR (solid): n˜ =3121 (NH), 2926 (Csp2), 2851 (Csp3), 1719, 1679,
1620 cmꢀ1 (CO); HRMS (ESI): m/z: calcd for C21H22N3O3: 364.1656;
found: 364.1632.
In vitro assay of sporozoite invasion of and development in hepatocytes:
After removal of medium from the culture chambers, 104 sporozoites
were added in 100 ml of fresh supplemented medium with the various
drugs tested at different concentrations. Medium was replaced 3 and 24 h
after with fresh medium containing the drug tested. Experimental deter-
minations were performed in triplicate. Control cultures were incubated
with medium containing 10% DMSO (the highest concentration used).
Cultures were stopped 48 h after sporozoite infection, fixed with cold
methanol and schizonts numbers were assessed by immunofluorescence
by using a monoclonal antibody recognizing P. yoelii liver stages.[45, 46]
7-Benzyl-1-(4-bromo-benzoyl)-5-methylCAHTRE[UNG 1,3,5]triazepane-2,6-dione (18):
In this case, 4-bromo-benzoyl chloride (3.5 equiv, 330 mg, 1.51 mmol) was
used and the reaction was stirred for 30 h at 408C. White solid; Yield:
152 mg, 85%; C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in
MeCN, 0–100% B, 1.2 mLminꢀ1, 20 min): tR =13.60 min; [a]2D0 =64.7 (c=
0.83 in MeOH); 1H NMR (300 MHz, CDCl3, 298 K): d=3.07 (s, 3H;
NCH3), 2.99–3.08 (m, 1H; CHCH2Ph), 3.48 (dd, J=14.4, 3.9 Hz, 1H;
CHCH2Ph), 4.17 (dd, J=14.1, 7.2 Hz, 1H; NCHN), 5.16 (dd, J=14.4,
5.4 Hz, 1H; NCHN) 5.70 (dd, J=9.6, 3.9 Hz, 1H; NCH2Ph), 6.63–6.74
(m, 1H; NH), 7.08–7.37 (m, 7H; H-Ar), 7.46 ppm (d, J=8.4 Hz, 2H; H-
ArBr); 13C NMR (75 MHz, CDCl3, 298 K): d=35.8 (CH3), 40.3, 57.3
Acknowledgements
This research was supported in part by Centre National de la recherche
Scientifique (CNRS), Institut National de la SantØ et de la Recherche
MØdicale, Minist›re de la Recherche (PAL+, grant no.RPC03033 KKA),
RØgion Alsace, and ImmuPharma France. CIFRE fellowships from Im-
muPharma France and the Association Nationale pour la Recherche
Technique (ANRT) for G.L. and J.B. are gratefully acknowledged. We
thank Roland Graff and Lionel Allouche for their assistance with
2D NMR measurements.
ꢀ
(CH2), 61.2 (CH), 126.3 (CH-Ar), 127.4 (C Br), 128.5, 128.9, 130.4,
132.0, (CH-Ar), 134.2, 136.6 (Cq-Ar), 158.5, 168.9, 169.5 ppm (CO); IR
(solid): n˜ =3266 (NH), 2925 (Csp2), 2852 (Csp3), 1726, 1674, 1633 cmꢀ1
(CO); HRMS (ESI): m/z: calcd for C19H19Br1N3O3: 416.0604; found:
416.0592; calcd for C19H19Br1N3Na1O3: 438.0424; found: 438.0415.
8510
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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 8498 – 8512

1,3,5-Triazepan-2,6-dione Dipeptide Mimetics
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2
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the chemical stability of the triazepandione skeleton was assessed in
this solvent. Randomly selected Compounds 4i and 8a did not show
any sign of degradation after more than two years in [D6]DMSO at
RT. In addition, there was no sign of hydrolysis of the 1,3,5-triaze-
pan-2,6-dione skeleton upon prolonged storage (up to 30 d) in water
as measured by C18-RP-HPLC of
a solution of 4a in water
(1 mgmLꢀ1, see Supporting Information).
[33]Quantitative removal of protecting groups in 9 and 11 by treatment
with TFA afforded an additional set of triazepandiones with free
carboxylic side chains (12 and 13, respectively).
[34]CCDC-283640 to -28346 contain the crystal structures of 4j, 4k, 4l,
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ꢀ
calculated positions and refined by using a riding model, with C H
Received: April 20, 2006
Published online: August 22, 2006
ꢀ
distances 0.93–0.98 Š and an N H distance of 0.86 Š. G. M. Shel-
8512
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Chem. Eur. J. 2006, 12, 8498 – 8512