Comparative Study of the Synthesis and Structural and Physicochemical Properties of Diketopiperazines vs Aza-diketopiperazines

Article abstract of DOI:10.1021/acs.joc.6b02895

Aza-diketopiperazines (aza-DKPs) represent an underprivileged motif obtained by scaffold hopping of 2,5-diketopiperazines (2,5-DKPs). Herein, we compare the synthesis and the structural and physicochemical properties of aza-DKP 4 vs 2,5-DKP 7. Thus, X-ray and ¹H NMR studies show that aza-DKP 4 is a rigid and nonflat scaffold like the 2,5-DKP 7. Moreover, the replacement of one C_α-stereogenic center by a nitrogen atom results in a significant improvement of both the water solubility and the microsomal stability.

Full text of DOI:10.1021/acs.joc.6b02895

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Note
A Comparative Study of the Synthesis, Structural and Physicochemical
Properties of Diketopiperazines vs Aza-Diketopiperazines
Pierre Regenass, Damien Bosc, Stéphanie Riché, Patrick Gizzi,
Marcel Hibert, Lydia Brelot, A. Ganesan, and Dominique Bonnet
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02895 • Publication Date (Web): 23 Feb 2017
Downloaded from http://pubs.acs.org on February 24, 2017
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A Comparative Study of the Synthesis, Structural and Physicochemical Properties of
Diketopiperazines vs Aza-Diketopiperazines
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Pierre Regenass‡, Damien Bosc‡, Stéphanie Riché‡, Patrick Gizzi†, Marcel Hibert‡, Lydia
Brelot§, A. Ganesan||, Dominique Bonnet‡*
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‡Laboratoire d’Innovation Thérapeutique, UMR7200 CNRS/Université de Strasbourg,
LabEX MEDALIS, Faculté de Pharmacie, 74 route du Rhin, 67412 Illkirch, France.
§Service de Cristallographie, Institut de Chimie de Strasbourg, BP 296/R8, 67008
Strasbourg, France.
||School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ,
UK.
†PCBIS, FMTS, UMS 3286 CNRS/Université de Strasbourg, ESBS Pôle API, Bld Sébastien
Brant, 67401 Illkirch, France
*Corresponding author: Dr Dominique BONNET; Phone: + 33 1 44 27 16 63; Email:
dominique.bonnet@unistra.fr
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ABSTRACT
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Azaꢀdiketopiperazines (AzaꢀDKP) represent an underprivileged motif obtained by scaffold
hopping of 2,5ꢀdiketopiperazines (2,5ꢀDKP). Herein, we compare the synthesis but also the
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structural and the physicochemical properties of azaꢀDKP
1H NMR studies show that azaꢀDKP
4 vs 2,5ꢀDKP 7. Thus, Xꢀray and
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4
is a rigid and nonꢀflat scaffold like the 2,5ꢀDKP 7.
Moreover, the replacement of one Cαꢀstereogenic center by a nitrogen atom results in a
significant improvement of both the water solubility and the microsomal stability.
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Highꢀthroughput screening is nowadays one of the most efficient methods to find new leads in
medicinal chemistry. Since the advent of combinatorial chemistry associated with solid
support synthesis, thousands of derivatives with the same scaffold have been synthesized
leading to scarce originality in chemical libraries.1 Many libraries used in screening are often
populated by aromatic compounds which are planar, with low stereochemical complexity.2
This class of compounds may be suitable to target proteins such as kinases3 but scaffolds with
high content of sp2 and sp hybridized carbons do not fit optimally with the spatial
configuration required for interaction with most biological receptors.4,5 Moreover, flat
aromatic structures are correlated with poorer specificity and physicochemical properties.
Lovering et al. have shown that compounds possessing a high fraction of sp3 hybridized
carbons (Fsp3) and chirality have a higher likelihood of progressing through the hitꢀtoꢀdrug
process.6 Therefore, there is a crucial need to enrich the chemical diversity of screening
libraries to facilitate the discovery of new drugs. In this context, scaffold hopping approaches
consisting in replacing the central core of a known active compound by a new scaffold able to
mimic the same biological interactions with the target could be used to improve both
pharmacodynamic and pharmacokinetic properties.7 In the course of the discovery of new
scaffolds relevant for medicinal chemistry, we can also list as selection criteria a low
molecular weight to allow substitutions while keeping the molecular weight below 500 g.molꢀ
1, and the rigidity of these platforms that can adopt a wellꢀdefined and threeꢀdimensional (3D)
orientation of their substituents. 2,5ꢀDiketopiperazines (2,5ꢀDKP) fulfill these criteria since
they have a low molecular weight and a constrained structure providing a nonꢀplanar system
that directs its four potential substituents in different vectors in 3Dꢀspace. 2,5ꢀDKP is a wellꢀ
known and privileged scaffold in medicinal chemistry as examples are able to interact with
many biological targets.8 While this scaffold has received great attention and is thus described
in numerous patents making its exploitation difficult, scaffold hopping by introduction of an
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additional nitrogen gives the much less studied heterocycle 1,2,4ꢀtriazineꢀ3,6ꢀdione or azaꢀ
diketopiperazine (azaꢀDKP, Figure 1). Indeed, very few efficient syntheses with broad scope
are available for the preparation of this rare heterocycle.9–14 AzaꢀDKPs have been obtained
from Nꢀ(mesyloxy)malonamide reacting with hydrazines,9 by using triphosgene and gaseous
HCl to form azaꢀDKPs from NꢀBoc hydrazides,10 from semicarbazone in acidic conditions11
and by a recent oneꢀpot sequence involving nitrogenꢀsubstituted isocyanates as key
intermediates.12 However, these approaches enable the synthesis of disubstituted azaꢀDKPs
only and/or very limited trisubstituted analogs. Thereby, to prepare more complex azaꢀDKPs
and facilitate the preparation of diversityꢀoriented libraries, our group has recently reported a
convenient solutionꢀ and solidꢀphase strategy from αꢀaminoesters and NꢀBoc hydrazides,13
and a stepꢀeconomical multicomponent process driven by Rh(I)ꢀcatalyzed hydroformylation
of alkenylsemicarbazides.14 Interestingly, the replacement of one carbon by a planar nitrogen
atom may have a deep influence on the chemical and pharmacochemical properties of DKP.
Herein, we evaluate the synthesis and the structural and physicochemical properties of azaꢀ
DKP as compared to 2,5ꢀDKP.
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Figure 1. Generic structure of 2,5ꢀDKP and azaꢀDKP
In order to study the impact of the substitution of the αꢀcarbon by a nitrogen atom on both the
physicochemical and structural parameters, we compared azaꢀDKP
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and its 2,5ꢀDKP analog
ꢀphenylalanine which was first protected using nosyl
1 was monoalkylated with propyl iodide under mild
7. The azaꢀDKP synthesis began with
L
chloride (Scheme 1). Then, the amine
conditions using Fukuyama’s sulfonamide method.15 Deprotection of
2 with thiophenol gave
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with an overall yield of 73% from L 3 with
ꢀphenylalanine.16 A subsequent, activation of
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triphosgene followed by a treatment with 1ꢀNꢀBocꢀ2ꢀmethylhydrazine provided the
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corresponding semicarbazide. In the presence of TFA/water (95/5, v/v), the crude mixture was
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then readily converted into azaꢀDKP
synthesize the 2,5ꢀDKP analog, alkyl amine
resulting linear dipeptide was then treated with a solution of TFA/H2O (95:5) to initiate the
deprotection/cyclization towards the corresponding 2,5ꢀDKP. However, the acidic treatment
only resulted in the quantitative deprotection to amine which did not cyclize. This difference
of reactivity between the linear dipeptide and the deprotected semicarbazide may be due to
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following a deprotection/cyclization process.14 To
was coupled with NꢀBocꢀ ꢀalanine. The
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3
L
5
6
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the αꢀeffect and lower basicity of the hydrazine moiety leading to a more nucleophilic βꢀ
nitrogen that favors the cyclization in acidic catalysis. Nevertheless, a basic treatment with
triethylamine in DMF at 60 °C resulted in the cyclization of
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affording the 2,5ꢀDKP
7
in a
is higher than that
, the
is more rapid and performed at room temperature that makes easier
good yield (76%).17 The overall yield for the synthesis of azaꢀDKP
7
obtained for 2,5ꢀDKP
synthesis of azaꢀDKP
7 (54% vs 32% from Lꢀphenylalanine). As compared to 2,5ꢀDKP 7
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the access to chemical libraries.
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Scheme 1. Synthesis of azaꢀDKP
4 (A) and 2,5ꢀDKP 7 (B)
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A
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thiophenol
1-iodiopropane
Ns
O2N
SO2Cl
N
CO2Me
+
HN
Ns
1, 79%
CO2Me
H3N
CO2Me
K2CO3, DMF,
rt, 3 h
K2CO3, DMF,
rt, 16 h
Cl-
CH2Cl2, rt, 3 h
2, 93%
a)
b)
triphosgene,
DIEA, THF,
rt, 15 min
TFA/H2O
(95:5),
O
O
rt, 1 h
N
N
BocHN
HN
CO2Me
NH
N
O
N
CO2Me
CH3NHNHBoc,
DIEA, THF,
70 °C, 16 h
O
3, 100%
4, 74% (2 steps)
B
a) triphosgene, THF,
2,4,6-trimethylpyridine,
rt, 1 h
O
R1
triethylamine
N
L-Boc-Ala-OH
N
CO2Me
NH
b)
3, rt, 16 h
DMF, 60 °C,
16 h
O
5, R1=NHBoc, 58%
TFA/H2O (95/5)
rt, 1 h
7, 76%
6, R1=NH2+CF3CO2 , 100%
-
Both azaꢀDKP
4
and 2,5ꢀDKP 7 were crystalline materials that were suitable for obtaining
singleꢀcrystal Xꢀray diffraction structures.18 Despite the substitution of a carbon by a nitrogen
atom in the central core, both compounds clearly display similarities in the spatial orientation
of the three substituents in space (Figure 2). However, some subtle structural differences were
observed. Indeed, the conformation of the azaꢀDKP
N2ꢀN1ꢀC1ꢀN3 and N2ꢀC2ꢀC3ꢀN3 dihedral angles of ꢀ4.2(3)° and ꢀ22.2(2)°, respectively
whereas the conformation of the 2,5ꢀDKP is a flattened boat shape with N1ꢀC2ꢀC1ꢀN2 and
4 is a flattened pseudoꢀboat shape with
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N1ꢀC3ꢀC4ꢀN2 dihedral angles of ꢀ10.1(3)° and ꢀ11.1(3)°, respectively. In both compounds,
the benzyl moiety is in an axial orientation on the same side as the methyl substituent while
the propyl group is orientated on the other side to avoid a steric clash.
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Moreover, the Xꢀray structure of azaꢀDKP 4 shows that the methyl moiety is notably deviated
from the N2ꢀN1ꢀC1 plane with an angle of 27.1(2)° and adopts a pseudoꢀaxial position. The
propyl moiety linked to N3 is diverted with an angle of 18.7(2)° from the C1ꢀN3ꢀC3 plane
and the hydrogen atom on N2 is diverted with an angle of 6.0(2)° from the N1ꢀN2ꢀC2 plane.
For the 2,5ꢀDKP 7, the deviation for the methyl moiety is more important (48.1(2)° from the
C1ꢀC2ꢀN1 plane), the other deviations are moderate (2.7(2)° for the propyl moiety from the
C1ꢀN2ꢀC4 plane, and 12.2(19)° for the hydrogen atom carried by N1 from the C2ꢀN1ꢀC3
plane) relative to the azaꢀDKP 4. Interestingly N1 of the azaꢀDKP ring adopts a partial sp3
hybridization as observed for azaꢀpeptides.19 Examination of the distance between N1 and the
plane formed by N2, C1, C2 and C3 indicates a Sꢀconfiguration with a 0.229(2) Å distortion
from planarity.
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Figure 2. Xꢀray structures of azaꢀDKP 4 and 2,5ꢀDKP 7
The spatial configuration of 2,5ꢀDKP 7 has previously been studied and shown to be a rigid
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structure.20 Malavašič et al. have published that according to H NMR, the chemical shift of
the methyl group protons at C2 in 2,5ꢀDKP 7 is unusually low, around 0.8 ppm in DMSO at
298 K, whereas the signal of these protons in 2,5ꢀDKP without the benzyl group appears
between 1.40 ppm and 1.54 ppm. These data are in accordance with the crystallographic
structure of 2,5ꢀDKP 7 which has displayed a spatial proximity of the methyl protons with the
aromatic ring allowing πꢀσ interaction.20 In order to investigate the spatial organization of the
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azaꢀDKP 4, a 1H NMR study was performed in CDCl3, a nonꢀpolar solvent, and in DMSO, a
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polar aprotic solvent (Figure 3).
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Figure 3. 1H NMR spectra of azaꢀDKP 4 in DMSO at 363 K (A), in DMSO at 298 K (B) and
in CDCl3 at 298 K (C). .
In the spectra, the two protons H12 (H12a and H12b) of the propyl chain appear as an AX2Y
spin system. Furthermore, their chemical shifts were significantly different as their signals
were at 3.75 ppm and 2.51 ppm in CDCl3 and at 3.55 ppm and 2.62 ppm in DMSO at 298 K.
The increase of the temperature from 298 K to 363 K had no effect on the chemical shift of
these protons. Thus, these two protons are in a different magnetic environment indicating that
the rotation of the propyl group is slow on the NMR timeꢀscale. In addition, the chemical shift
of the methyl group (H4) is shifted to low field (2.73 ppm in CDCl3 and 2.49 ppm in DMSO
at 298 K). As observed for 2,5ꢀDKP 7, this could be explained by shielding of these protons
by the proximal benzyl moiety. The chemical shift of these methyl protons was slightly
affected by the increase of temperature (2.49 ppm at 298 K to 2.64 ppm at 363 K).
Thus, the nature of the solvent and the temperature have a low impact on the chemical shift of
the protons and consequently on the conformation of the azaꢀDKP scaffold. According to our
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Xꢀray and NMR data, azaꢀDKP 4 displays a rigid structure that prevents the propyl chain
rotation and blocks the methyl group on the same side as the benzyl ring. Thus, the ability of
azaꢀDKP scaffold to efficiently orientate its substituents in different planes of 3Dꢀspace
should be an advantage for drug discovery by optimizing interaction with biological targets.21
On the other hand, in comparison to DKP, the azaꢀDKP scaffold may undergo adaptive
chirality as previously reported.22
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Regarding the drugꢀlike properties, azaꢀDKP 4 and 2,5ꢀDKP 7 have a low polar surface area
and molecular weight, less than five hydrogen bond acceptors and only one hydrogen bond
donor in accordance with Lipinski rules and other guidelines for oral bioavailability.23 Some
physicochemical properties of azaꢀDKP 4 and 2,5ꢀDKP 7 were then assessed (Table 1).
Thermodynamic solubility studies in a pH 7.4 PBS buffer showed that the additional nitrogen
atom on azaꢀDKP 4 had a significant effect on the compound solvation. Indeed, a 17ꢀfold
increase was observed on the aqueous solubility of azaꢀDKP 4 as compared to 2,5ꢀDKP 7
(16.5 mM vs 0.98 mM, respectively). Moreover, logD7.4 was lowered enabling the
introduction of nonꢀpolar substitutions on this scaffold while keeping an acceptable
distribution coefficient during the optimization process for drug discovery purposes.
Table 1. Drugꢀlike Properties and Physicochemical Parameters of azaꢀDKP 4 and 2,5ꢀDKP 7
AzaꢀDKP 4 2,5ꢀDKP 7
Parameters
Molecular weight (g/mol)
Polar surface area (Ų)a
Hꢀbond acceptor
Hꢀbond donor
261.3
260.3
52.65
49.41
5
4
1
4
1
4
Rotatable bond
Solubility (mM)b
LogD7.4
16.5 ± 0.6
0.98 ± 0.06
1.06 ± 0.03 1.38 ± 0.13
b
aPolar surface area calculated by ChemAxon software. Thermodynamic solubility measured
in a pH 7.4 phosphateꢀbuffered saline.
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The chemical and metabolic stabilities of AzaꢀDKP 4 and 2,5ꢀDKP 7 were also studied (Table
2). After 24 h of incubation in PBS buffer, these two compounds were fully stable. Similarly,
AzaꢀDKP 4 and 2,5ꢀDKP 7 remained perfectly intact in plasma after 6 h of exposure in spite
of the presence of amidases. In mouse liver microsomes, the azaꢀDKP 4 was found to be more
stable than the 2,5ꢀDKP 7 after 30 min of incubation (83% vs 51% remaining) indicating the
biological robustness of this new scaffold.
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Table 2. Chemical and Metabolic Stability of azaꢀDKP 4 and 2,5ꢀDKP 7
AzaꢀDKP 4 2,5ꢀDKP 7
Stabilitya
%
%
PBSb
> 98
> 98
83 ± 3
> 98
> 98
51 ± 1
Plasmac
Microsomesd
aPercent of remaining starting compounds. pH 7.4 phosphateꢀbuffered saline after 24 h.
b
cMouse plasma after 6 h at 37 °C. dMouse hepatocytes after 30 min at 37 °C.
In conclusion, the comparison between 2,5ꢀDKP and azaꢀcounterpart offers an interesting
insight regarding the synthesis, the conformation and pharmacokinetic properties of the latter.
Relying on an efficient and rapid deprotection/cyclization process, azaꢀDKP was obtained at
room temperature and with a higher yield than 2,5ꢀDKP. As shown by Xꢀray analysis and 1H
NMR study, azaꢀDKP displayed a structure close to 2,5ꢀDKP despite the replacement of one
tetrahedral carbon by a nitrogen atom. Interestingly, this azaꢀheterocycle exhibited a rigid
backbone directing its substituents in different directions of 3Dꢀspace. This nonꢀplanar
scaffold provides a new opportunities to improve drugꢀtarget interactions and success in
clinical studies.6 Furthermore, the presence of the nitrogen atom significantly improved
aqueous solubility and distribution coefficient compared to its 2,5ꢀDKP analog allowing the
substitution with more hydrophobic groups during the optimization process to increase the
biological activity. Metabolic stability was also significantly enhanced thanks to this scaffold
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as compared to the 2,5ꢀDKP analog. In summary, we demonstrated that azaꢀDKP is an
original heterocycle, readily accessible from simple building blocks, giving new opportunities
in medicinal chemistry by mimicking the structural architecture of 2,5ꢀDKP while it may
improve patentability and physicochemical and pharmacokinetic properties.
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EXPERIMENTAL SECTION
General methods. Reagents, dry solvents and deuterated solvents were obtained from
commercial sources and used without any further purification. Thinꢀlayer chromatography
was performed on silica gel 60 F254 plates.
Flash chromatography was performed on silica gel preꢀpacked columns RP18 (30 mm).
Analytical reverseꢀphase highꢀperformance liquid chromatography (RPꢀHPLC) separations
were performed on a C18 column (2.7 µm, 4.6 mm x 75 mm) using a linear gradient (5 % to
95 % of solvent B in solvent A in 7 min, flow rate 1.6 mL/min), detection at 220 nm, solvent
A : water/0.1 % trifluoroacetic acid (TFA), solvent B : acetonitrile/0.1 % TFA).
Semiꢀpreparative reverseꢀphase highꢀperformance liquid chromatography (RPꢀHPLC)
separations were performed on a RPꢀC18 column (7 µm, 19 mm x 300 mm).
1H and 13C NMR spectra were recorded on a 500 MHz/125 MHz and 400 MHz/100 MHz
spectrometer. Conditions are specified for each spectrum (temperature 25 °C unless
specified). Chemical shifts are reported in parts per million (ppm) relative to residual solvent
and coupling constants (J) are reported in hertz (Hz). Signals are described as s (singlet), d
(doublet), t (triplet), m (multiplet), dd (doublet of doublets), ddd (doublet of doublet of
doublets), qd (quartet of doublets) and br s (broad singlet).
HRMS were obtained on a QꢀTof LC/MC apparatus using electrospray ionization mode and
timeꢀof flight analyzer (ESIꢀTOF).
Melting points were determined in open capillary tubes. Specific rotations were measured
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using a 10 cm cell with a Na 589 nm filter.
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Methyl ((2-nitrophenyl)sulfonyl)-L-phenylalaninate 1. To a solution of methyl Lꢀ
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phenylalaninate hydrochloride (2.40 g, 11.1 mmol, 1 equiv) and NEt3 (4.64 mL, 33.4 mmol, 3
equiv) in CH2Cl2 (60 mL) was added 2ꢀnitrobenzenesulfonyl chloride (3.70 g, 16.7 mmol, 1.5
equiv). The mixture was stirred for 3 h at rt. The organic layer was washed with a solution of
NaHCO3, water and NaCl. The organic layer was dried on anhydrous Na2SO4 and evaporated.
The residue was purified on silica gel column eluting with 20ꢀ40% EtOAc in nꢀheptane (Rf
0.18, 40% EtOAc in nꢀheptane) to afford the desired product (3.20 g, 8.78 mmol, 79%) as a
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light yellow oil: [∝]ꢁꢀꢂ = −89.2 °(ꢃ 0.19, CDClꢄ); H NMR (400 MHz, CDCl3) δ 7.93ꢀ7.90
(m, 1H), 7.81ꢀ7.78 (m, 1H), 7.68ꢀ7.59 (m, 2H), 7.21ꢀ7.04 (m, 5H), 6.02 (d, J = 8.7 Hz, 1H),
4.44 (ddd, J = 8.7, 7.1, 5.6 Hz, 1H), 3.50 (s, 3H), 3.14 (dd, J = 13.9, 5.6 Hz, 1H), 3.04 (dd, J =
13.9, 7.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 170.9, 147.5, 135.0, 134.0, 133.7, 133.1,
130.4, 129.3, 128.7, 127.5, 125.6, 57.9, 52.6, 39.2; HRMS (ESIꢀTOF) calcd for
C16H16N2O6SNa [M+Na]
+ 387.06214, found 387.06233.
Methyl N-((2-nitrophenyl)sulfonyl)-N-propyl-L-phenylalaninate 2. To a mixture of methyl
((2ꢀnitrophenyl)sulfonyl)ꢀLꢀphenylalaninate (3.00 g, 8.23 mmol, 1 equiv) and K2CO3 (2.28 g,
16.5 mmol, 2 equiv) in DMF (40 mL), 1ꢀiodopropane (1.61 mL, 16.5 mmol, 2 equiv) was
added. The mixture was stirred for 16 h at rt and evaporated. The residue was purified on
silica gel column eluting with 20ꢀ40% EtOAc in nꢀheptane (Rf 0.38, 40% EtOAc in nꢀ
heptane) to afford the desired product (3.12 g, 7.68 mmol, 93%) as a light yellow oil:
1
[∝]ꢁꢀꢂ = −25.8 ° ꢃ 0.22, CDClꢄ ; H NMR (400 MHz, CDCl3) δ 7.82ꢀ7.78 (m, 1H), 6.66ꢀ6.51
(m, 3H), 7.28ꢀ7.16 (m, 5H), 4.86 (t, J = 7.6 Hz, 1H), 3.52 (s, 3H), 3.45ꢀ3.32 (m, 2H), 3.26ꢀ
3.16 (m, 1H), 3.01 (dd, J = 14.1, 7.2 Hz, 1H), 1.70ꢀ1.52 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H); 13C
(
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NMR (100 MHz, CDCl3) δ 170.8, 148.3, 136.6, 133.7, 133.3, 131.6, 130.9, 129.3, 128.7,
127.1, 124.1, 61.4, 52.4, 48.4, 36.9, 23.8, 11.5; HRMS (ESIꢀTOF) calcd for C19H22N2O6SNa
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[
M+Na]
+ 429.10908, found 429.11023.
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Methyl propyl-L
-phenylalaninate 3.20 To a solution of methyl Nꢀ((2ꢀnitrophenyl)sulfonyl)ꢀ
NꢀpropylꢀLꢀphenylalaninate (3.10 g, 7.63 mmol, 1 equiv) and thiophenol (1.17 mL, 11.4
mmol, 1.5 equiv) in DMF (41 mL), K2CO3 (2.11 g, 15.3 mmol, 2 equiv) was added. The
mixture was stirred at rt for 3 h and evaporated. Purification on silica gel column eluting with
20ꢀ40% EtOAc in nꢀheptane (Rf 0.32, 30% EtOAc in nꢀheptane) afforded the desired product
1
(1.69 g, 7.63 mmol, 100%) as a clear oil. H NMR (400 MHz, CDCl3) δ 7.34ꢀ7.15 (m, 5H),
3.66 (s, 3H), 3.54 (t, J = 7.0 Hz, 1H), 2.97 (dd, J = 7.0, 2.9 Hz, 2H), 2.60ꢀ2.53 (m, 1H), 2.49ꢀ
2.41 (m, 1H), 1.55ꢀ1.41 (m, 3H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
175.2, 137.4, 129.1, 128.4, 126.7, 63.2, 51.6, 50.2, 39.8, 23.2, 11.6; LRMS (ESIꢀTOF) m/z =
222.2 [M+H]+.
(S
)-5-Benzyl-2-methyl-4-propyl-1,2,4-triazinane-3,6-dione 4. To a solution of triphosgene
(270 mg, 0.91 mmol, 0.335 equiv) in anhydrous THF (16.3 mL), under argon, a solution of Nꢀ
propyl Lꢀphenylalanine methyl ester (601 mg, 2.72 mmol, 1 equiv) and DIEA (520 L, 2.99
µ
mmol, 1.1 equiv) in anhydrous THF (6.8 mL) was added dropwise (over 5 minutes). The
mixture was stirred at rt for 15 minutes. A solution of tertꢀbutyl 2ꢀmethylhydrazineꢀ1ꢀ
carboxylate (397 mg, 2.72 mmol, 1 equiv) and DIEA (520
µL, 2.99 mmol, 1.1 equiv) in
anhydrous THF (4.0 mL) was added dropwise. The mixture was heated at 40 °C overnight
and evaporated in vacuo. The crude was then treated with a solution of TFA/H2O (95:5, 2
mL) for 1 h at room temperature and concentrated under reduced pressure. Purification on
silica gel column eluting with a gradient of 20% EtOAc in nꢀheptane (Rf 0.22) afforded 525
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mg (2.01 mmol, 74%) of the title compound as a white solid: mp 140.7ꢀ142.7 °C; [∝]ꢀꢁꢂ
=
+9.8 ꢃ 0.2, MeOH ; 1H NMR (400 MHz, CDCl3) δ 7.43ꢀ7.16 (m, 5H), 4.07ꢀ4.01 (m, 1H),
3.82ꢀ3.70 (m, 1H), 3.11ꢀ2.98 (m, 2H), 2.74 (s, 3H), 2.57ꢀ2.47 (m, 2H), 1.61ꢀ1.43 (m, 2H),
0.89ꢀ0.82 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 165.3, 153.8, 135.4, 129.9, 128.9, 127.5,
61.8, 48.1, 36.2, 34.8, 21.5, 11.3; HRMS (ESIꢀTOF) calcd for C14H20N3O2 (M+H)+
262.15500, found 262.15485.
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Methyl
N
-((tert-butoxycarbonyl)-
L-alanyl)-
N
-propyl-
L
-phenylalaninate
5.
(tertꢀ
Butoxycarbonyl)ꢀLꢀalanine (339 mg, 1.79 mmol, 1 equiv) was dissolved in anhydrous THF (5
mL). 2,4,6ꢀCollidine (954 ꢁL, 7.17 mmol, 4 equiv) and BTC (213 mg, 0.72 mmol, 0.4 equiv)
were added to the mixture, which was allowed to stir at rt for 5 min. A solution of methyl
propylꢀLꢀphenylalaninate (462 mg, 1.79 mmol, 1 equiv) in anhydrous THF (5 mL) was added
and the mixture was stirred at rt for 16 h. The mixture was evaporated under reduced pressure.
The residue was purified on silica gel eluting with a gradient of 30ꢀ60% EtOAc in nꢀheptane
followed by a purification on flash chromatography with 5ꢀ100% MeCN in water (Rf 0.29,
50% EtOAc in nꢀheptane) to afford the desired product as a white solid (406 mg, 1.03 mmol,
1
58%): mp 99.8 ꢀ 101.8 °C; [∝]ꢁꢀꢂ = −181.4 °(ꢃ 0.17, MeOH); H NMR (500 MHz, DMSOꢀ
d6, 70 °C) δ 7.33ꢀ7.13 (m, 5H), 6.44 (br s, 1H), 4.36ꢀ4.21 (m, 2H), 3.59 (s, 3H), 3.25 (dd, J =
14.0, 5.0 Hz, 1H), 3.17ꢀ3.00 (m, 2H), 2.72ꢀ2.62 (m, 1H), 1.45ꢀ1.34 (m, 11H), 1.11 (d, J = 6.9
Hz, 3H), 0.74 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, DMSOꢀd6) δ 172.6, 170.8, 154.8,
138.1, 129.3, 128.1, 126.4, 77.8, 61.6, 51.5, 51.1, 46.1, 34.0, 28.1, 21.3, 17.9, 10.8; HRMS
(ESIꢀTOF) calcd for C21H32N2O5Na (M+Na)+ 415.22035, found 415.22041.
(S)-1-(((S)-1-Methoxy-1-oxo-3-phenylpropan-2-yl)(propyl)amino)-1-oxopropan-2-
aminium 2,2,2-trifluoroacetate 6. Methyl Nꢀ((tertꢀbutoxycarbonyl)ꢀLꢀalanyl)ꢀNꢀpropylꢀLꢀ
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phenylalaninate (373 mg, 0.95 mmol, 1 equiv) was solubilized in a mixture of trifluoroacetic
acid and water (2.1 mL, 95:5). The mixture was allowed to stir at rt at 0 °C for 1 h and was
then evaporated to a slurry (386 mg, 0.95 mmol, 100%). Analysis of the crude product by
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NMR showed only trifluoroacetate 6 without product from cyclisation. H NMR (400 MHz,
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CDCl3) δ 7.71 (br s, 3H), 7.26ꢀ6.96 (m, 5H), 4.31ꢀ4.20 (m, 1H), 3.86 (dd, J = 10.9, 4.8 Hz,
1H), 3.60 (s, 3H), 3.31ꢀ3.12 (m, 2H), 2.93ꢀ2.81 (m, 1H), 2.37ꢀ2.25 (m, 1H), 1.42ꢀ1.26 (m,
5H), 0.67 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 170.9, 169.6, 161.2 (q, J = 38
Hz), 136.9, 129.3, 129.0, 127.5, 115.5 (q, J = 288 Hz), 63.6, 52.9, 52.5, 48.0, 34.3, 21.5, 17.0,
10.6; HRMS (ESIꢀTOF) calcd for C16H25N2O3 (M+H)+ 293.186518, found 293.186546.
(3
S,6S
)-6-Benzyl-3-methyl-1-propylpiperazine-2,5-dione 7.20 (S)ꢀ1ꢀ(((S)ꢀ1ꢀMethoxyꢀ1ꢀ
oxoꢀ3ꢀphenylpropanꢀ2ꢀyl)(propyl)amino)ꢀ1ꢀoxopropanꢀ2ꢀaminium 2,2,2ꢀtrifluoroacetate
4
(227 mg, 0.68 mmol, 1 equiv) and Et3N (380 ꢁL, 2.74 mmol, 4 equiv) were dissolved in DMF
(5 mL). The mixture warmed to 60 °C and stirred overnight. The mixture was then evaporated
to a slurry. The residue was purified on flash chromatography eluting with 5ꢀ100% MeCN in
water (Rf 0.34, 5% MeOH/CH2Cl2) to afford 135 mg (0.52 mmol, 76%) of the desired
product as
a
white solid: mp 218.3–220.3 °C, lit 221
ꢀ
223 °C;
[∝]ꢁꢀꢂ = +56.0 (ꢃ 0.50, CHꢁClꢁ); 1H NMR (400 MHz, DMSOꢀd6) δ 8.12 (br s, 1H), 7.33ꢀ7.20
(m, 3H), 7.07ꢀ7.02 (m, 2H), 4.21 (t, J = 4.2 Hz, 1H), 3.84 (ddd, J = 13.4, 9.2, 6.3 Hz, 1H),
3.64 (qd, J = 6.9 Hz, 2.8 Hz, 1H), 3.17 (dd, J = 13.9, 4.8 Hz, 1H, AB system), 3.08 (dd, J =
13.9 Hz, 3.6 Hz, 1H, AB system), 2.90 (ddd, J = 13.4, 9.0, 5.5 Hz, 1H), 1.67ꢀ1.44 (m, 2H),
0.84 (t, J = 7.4 Hz, 3H), 0.26 (d, J = 7.1 Hz, 3H); 13C NMR (100 MHz, DMSOꢀd6) δ 166.1,
165.2, 135.6, 130.2, 128.3, 126.9, 59.8, 50.0, 44.8, 36.2, 20.0, 19.5, 11.1; HRMS (ESIꢀTOF)
calcd for C15H21N2O2 (M+H)+ 261.15975, found 261.15949.
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Solubility. Thermodynamic solubility was measured by dissolving compounds 4 or 7 up to
saturation in a pH 7.4 phosphateꢀbuffered saline (PBS) with the following composition: 137.5
mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4. Samples were shaken for
24 h at 20 °C. Saturation was confirmed by the presence of undissolved powder. After
ultracentrifugation, the concentration in the supernatant was measured by an HPLC procedure
using a calibration curve established for each compound by diluting a 10 mM DMSO stock
solution to adapted concentrations.
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Stability in mouse plasma. Stability was determined in mouse plasma at 37 °C after 6 h. For
each compound, the 10 mM DMSO stock solution was diluted in MeCN to a final
concentration of 100 ꢁM. This solution was again diluted in plasma to a final concentration of
1 ꢁM. The mixture was divided into two aliquots. The incubation of each aliquot was stopped
at t0 and 6 h by adding one volume of ice cold acetonitrile. Samples were stirred for 3 min,
sonicated for 3 min, and then centrifuged at 4 °C before HPLC injection. The percentage of
remaining test compound relative to t0 was measured by monitoring the peak area on the
chromatogram.
Microsomal stability in mouse hepatocytes. Microsomal stability was determined in mouse
hepatocytes at 37 °C after 30 min. For each compound, the 10 mM DMSO stock solution was
diluted in DMSO to a final concentration of 100 ꢁM. This solution was again diluted to a final
concentration of 1 ꢁM in pH 7.4 phosphateꢀbuffered saline (PBS) containing mouse liver
microsomes (0.5 mg/mL), 1 mM of NaDPH, and 3 mM of MgCl2. The mixture was divided
into two aliquots. The incubation of each aliquot was stopped at t0 and 30 min by adding one
volume of iceꢀcold acetonitrile. Samples were stirred for 3 min, sonicated for 3 min, and then
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centrifuged at 4 °C before HPLC injection. The percentage of remaining test compound
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relative to t0 was measured by monitoring the peak area on the chromatogram.
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ACKNOWLEDGMENTS
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This work was supported by the Centre National de la Recherche Scientifique, the Université
de Strasbourg (UDS) and the LabEX Medalis (ANRꢀ10ꢀLABXꢀ0034). We are grateful to
Barbara Lamure and Justine Viéville for NMR experiments and Patrick Wehrung for MS
analyses (Service Commun d’Analyse, Unistra).
SUPPORTING INFORMATION
1H NMR and 13C NMR spectra for all compounds, Xꢀray crystal structures and
crystallographic data for 4 and 7. This material is available free of charge via the Internet at
http://pubs.acs.org/.
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REFERENCES
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