Tetrahydro-4 H-(pyrrolo[3,4- d]isoxazol-3-yl)methanamine: A Bicyclic Diamino Scaffold Stabilizing Parallel Turn Conformations

Article abstract of DOI:10.1021/acs.joc.8b01299

A tetrahydro-4H-(pyrrolo[3,4-d]isoxazol-3-yl)methanamine scaffold was designed as a diamino derivative to stabilize parallel turn conformations. Its synthesis took advantage of a [1,3]-dipolar cycloaddition reaction between the nitrile oxide derived from

Full text of DOI:10.1021/acs.joc.8b01299

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Article
Tetrahydro-4H-(pyrrolo[3,4-d]isoxazol-3-yl)methanamine: a
bicyclic diamino scaffold stabilizing parallel turn conformations
Raffaella Bucci, Sabrina Giofré, Francesca Clerici, Alessandro Contini, Andrea
Pinto, Emanuela Erba, Raffaella Soave, Sara Pellegrino, and Maria Luisa Gelmi
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01299 • Publication Date (Web): 07 Sep 2018
Downloaded from http://pubs.acs.org on September 7, 2018
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Tetrahydro-4H-(pyrrolo[3,4-d]isoxazol-3-yl)methanamine: a bicyclic diamino scaffold
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stabilizing parallel turn conformations
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Raffaella Bucci,† Sabrina Giofré,† Francesca Clerici,† Alessandro Contini,† Andrea Pinto,#
Emanuela Erba,† Raffaella Soave, ‡ Sara Pellegrino,† Maria Luisa Gelmi†*
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†DISFARM, Sezione di Chimica Generale e Organica “A. Marchesini” Università degli Studi
Milano Via Venezian 21, 20133 Milano, Italy
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DeFENS, Sezione di Scienze Chimiche e Biomolecolari, Università degli Studi Milano, Via
Celoria 2, 20133 Milano, Italy
‡CNRꢀ Istituto di Scienze e Tecnologie Molecolari, Via Golgi 19 20133, Milano, Italy
Eꢀmail: marialuisa.gelmi@unimi.it
Abstract. Tetrahydroꢀ4Hꢀ(pyrrolo[3,4ꢀd]isoxazolꢀ3ꢀyl)methanamine scaffold was designed as
diamino derivative to stabilize parallel turn conformations. Its synthesis took advantage of a [1,3]ꢀ
dipolar cycloaddition reaction between the nitrile oxide derived from the inexpensive enantiopure
Lꢀphenylalanine, and Nꢀbenzylꢀ3ꢀpyrroline. Two diastereoisomers were formed whose distribution
depends on the selected base. 3aR,6aSꢀIsomer is favoured in organic bases which formation is
driven by πꢀinteractions. On the other hand, the above interactions were significantly prevented
using an inorganic base due to the chaotropic effect of the cation, decreasing the amount of the
above isomer. Finally, we demonstrated that this isomer is able of stabilizing parallel turn
conformations when inserted in short peptide sequences.
Introduction
The use of nonꢀcoded amino acids or scaffolds for the synthesis of peptidomimetics mimicking the
natural structures opens the door to a huge number of different artificial shapes such as helixes,
different kind of turns, and stabilized βꢀstrand.1 Focusing on βꢀstrand natural peptides, they can
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assemble into either antiparallel or parallel βꢀsheets,2 very important motifs for the protein folding
and fibril formations. Several proteins are characterized by parallel βꢀsheet motif ranging from
C
amyloid3a,b,d,f to prion protein (PrP )3c, leucineꢀrich repeat (LRR)ꢀcontaining proteins,3h shikimate
kinase (SK) involved in tubercolosys3g and many others.
Strategies designed to stabilize this motif mostly consist in the use of an artificial scaffold that can
be used to link two βꢀstrands by Nꢀ or Cꢀterminus.4 Other strategies are reported such as the
formation of cyclic peptides,4a and the stabilization via disulfide crossꢀlink between cysteins.5
These strategies are particularly profitable not only to stabilize intermolecular interactions for bioꢀ
molecular recognition, but also to improve metabolic stability necessary in the biological field.
Our interest toward the preparation of nonꢀcoded amino acids6 and their use for peptidomimetic
synthesis and their applications7 is well documented. In this respect, click reactions were used both
to stabilize βꢀhairpin structures,8a and to prepare a new scaffold useful in the preparation of βꢀ
hairpin mimics targeting the Alzheimer disease.8b
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Here we focused on the design and synthesis of a new ꢁ2ꢀisoxazoline scaffold, fused with a
pyrrolidine ring, of general structure 3 to be used to stabilize parallel turn conformations (Scheme
1).
Scheme 1. Retrosynthetic pathway to obtain isoxazolineꢀcontaining scaffold and peptidomimetics.
This scaffold shows appealing features such as the presence of two amino groups, making it a good
candidate for the above application. Furthermore, due to its calix conformational constraint, it could
better orient the peptide arms, thus improving intraꢀstrand Hꢀbonds. The presence of
oxygen/nitrogen atoms in the isoxazoline ring, as well as the aromatic moieties make this turn
region appealing for possible intermolecular Hꢀbonds and πꢀinteractions, respectively, when it is
used to build peptidomimetics targeting PPI interactions.
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Isoxazoline derivatives are often prepared using, as key step, a [1,3]ꢀdipolar cycloaddition reaction
between nitrile oxides and dipolarophiles.9 The main drawback in using isolated nitrile oxides
regards their instability.10 In fact, they easily rearrange to isocyanate, generally by heating, or
dimerize giving furoxanes at room or lower temperatures. The formation of these side products
affects the yield of the cycloaddition reaction, resulting in a difficult isolation and purification of the
reaction products. For these reasons, nitrile oxides are usually generated in situ in the presence of a
dipolarophile. They can be obtained in different ways according to Mukaiyama’s,11 Huisgen’s
protocols,12 and MachettiꢀDe Sarlo reactions.13 Huisgen’s protocol consists in a basic
dehydrohalogenation of a hydroximoyl halide, easily obtained from an aldoxime, giving the nitrile
oxide. We envisaged in this procedure the best protocol to obtain an isoxazoline ring functionalized
with a nitrogen containing chain. As shown in the retrosynthetic Scheme 1, chlorooxime 1 was
selected as a chiral starting material to generate the corresponding nitrile oxide because it can be
obtained from an inexpensive commercially available chiral amino acid.14 As dipolarophile, 3ꢀ
pyrroline 2 was selected giving access to a second nitrogen atom in the final scaffold. Thanks to its
symmetry, the formation of regioisomeric mixtures is avoided. A couple of diastereoisomeric
isoxazolines could be obtained due to the presence of a stereocenter in the nitrile oxide.
As reported in the literature,9c the cycloaddition of chiral nitrile oxides to achiral alkenes generally
results in poor stereoselection. Considering the unfavourable chirality of our reagents (chiral nitrile
oxide and achiral alkene) and also the low reactivity of the dienophile,15 our challenge has been the
preparation of scaffold 3 in satisfying yields and with a control of the diastereoselection. To reach
this target, we envisaged in the use of two aryl substituted reagents, able to stabilize the transition
state through π interactions, the key step to drive the diastereoselection. As a result, the nitrile oxide
derived from Lꢀphenylalanine and the Nꢀbenzylꢀ3ꢀpyrroline were used. Our hypothesis has also
been supported by computational studies.
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Both diastereoisomers of 3 were used for the preparation of short peptides (Scheme 1) whose
conformation was studied by NMR spectroscopy. We found that the 3aR,6aSꢀisomer is able to
stabilize parallel turn conformations when combined with natural amino acids.
Results and discussion
Synthesis of scaffold 3. Chlorooxime 1, the precursor of nitrile oxide, was synthesized starting
from the enantiopure cheap commercially available (L)ꢀPhe. (Scheme S1, Supporting Information).
Its synthesis, via oxime intermediate 7, was revisited due to a partial racemization of the precursor
aldehyde 6. All the screened procedures and the corresponding HPLC are reported in the
Supporting Information (Figure S1, Supporting Information).
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The cycloaddition reaction was performed by using Nꢀbenzylꢀ3ꢀpyrroline (2) as dipolarophile. Two
diastereoisomeric compounds could be expected, i.e. ꢀ2ꢀisoxazoline derivatives 3a and 3b (Scheme
2). To optimize the yield and to control the diastereoselection, several reaction conditions were
tested and the overall results are reported in the Table 1.
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Scheme 2. Cycloaddition reaction between 1 and 2.
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Table 1. Reaction conditions tested for the cycloaddition reaction between 1 and 2 affording compounds 3.
3a/3b, %b
Entry
Solvent
(25 °C)
CHCl3
MeCN
MeCN
AcOEt
MeCN
Base
Time (h)
3a:3b d.r.a
1c
2d
3e
4d
5e
TEA
TEA
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5
ꢀ
6%
38%
24%
32%
51%
50%
50%
53%
29%
Traces
31%
14%
1:3
1:2.5
1:2
1:2
1:1
1:3
1:2.5
1:2
ꢀ
DABCO
NaHCO3
NaHCO3
Li2CO3
TEA
6f
7e
8e
9e
10e
11h
12
MeCN
20
16
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16
4
MeCN/DMFg
MeCN/DMFg
MeCN/NMPg
MeCN/DMFg
MeCN/DMFg
DABCO
TEA
NaHCO3
NaHCO3
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16
1:2
1:1
MeCN/DMFg TEA (2 eq.)
LiCl (2 eq.)
aCalculated by 1H NMR analysis; bIsolated producs; c1:2:TEA in 1:1:1.2 ratio at reflux; d1:2:base in 2.2:1:2.2 ratio at 25
°C, slowly addition of 1 (1 h); e1:2:base in 2:1:2 ratio at 25 °C and slowly addition of 1 (2 h); f 1:2:base in 2:1:1 ratio at
25 °C and slowly addition of 1 (2 h); gMeCN/DMF (5:1); hThe reaction was performed as described in the note e) but at
0°C.
First, we performed the cycloaddition reaction using the standard conditions reported in the
literature when nitrile oxide derived from amino acids were used.14b The reaction was performed
using a mixture of 1 and 2 (1:1) in CHCl3 at reflux after which TEA (1.2 equiv.) was added as base
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to generate the nitrile oxide (Table 1, entry 1). A mixture of 3a and 3b was obtained in very low
yield. The temperature (from 0 to 25 °C) and the amount of TEA (2 equiv.) were changed, as well
the base (1/2/DABCO, 1:1:2), but the reaction failed.
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MeCN was then tested instead of CHCl3 for its higher polarity. Operating at 25 °C and increasing
both the amount of 1 and TEA (entry 2), a mixture of 3a and 3b (1:3 ratio) was obtained in 38%
yield. Worse results were achieved when changing the addition order of the reagents, being the
addition of the chloroxime the last one. Lower yield or trace amounts of compounds 3 were
detected when changing the base (DABCO, entry 3; DIPEA or DBU, data not reported) and
operating as reported above.
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Then we switched to inorganic bases. NaHCO3 was used both in AcOEt (entry 4) and MeCN (entry
5). A dramatic decrease of the reaction time and an increase of yields (51%) but with a loss of
diastereoselectivity (1:2) was found with MeCN. Similar results were achieved with Li2CO3 with a
further decrease of the diastereoselection (entry 6). A phaseꢀtransfer catalysis protocol in
heterogeneous conditions was also attempted using K2CO3 and tetrabutylammonium bromide as the
catalyst in MeCN (1/2/Bu4NBr/K2CO3, 1:0.5:0.1:2) affording only trace amounts of compounds 3.
Since 3b was the diastereoisomer of interest for the preparation of parallel sheets (see below),
further efforts were done to improve its yield. Considering the beneficial result in terms of yield
when the more polar MeCN was used, we tested a mixture of MeCN/DMF (5:1 ratio) and TEA as
the base. The reaction was successful and compounds 3a/3b were obtained in the same ratio but
with improved yield (50%, entry 7). Similar results were found with DABCO (entry 8) with a small
decrease in diastereoselection. On the other hand, when NMP was tested as a coꢀsolvent, a decrease
of yield was found (entry 9).
We also evaluated the use of NaHCO3 in MeCN/DMF at both 25 °C (entry 10) and at 0 °C (entry
11). The reaction failed in the first case and gave lower yields in the second one, maintaining the
same diastereoisomeric ratio (compare entries 5 and 11).
Overall, our results indicate that more polar solvents (MeCN/CHCl3; compare entries 1, 2 and 7) are
beneficial to the reaction yields when a weak organic base (pKb 5ꢀ3) is used. The use of inorganic
bases in MeCN heterogeneous conditions give good results too. On the other hand. in this case the
higher is the polarity of the solvent the lower are the yields (compare entries 10, 11 with 5). This is
probably due to the improved solubility of the base. As a result, the formation of nitrile oxide
strongly increases that partially gave byꢀproducts because of the low reactivity of the dienophile.15b
By taking these data together, it has to be concluded that the rate of the nitrile oxide generation
from 1 is crucial to avoid sideꢀreactions, in agreement to the reported results.15 It has to be pointed
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out that a 50% yield represents a rather good result, considering the sluggish reactivity of our
dipolarophile.
Interestingly, we observed that the diastereoisomeric ratio is affected by the choice of the organic or
inorganic base, moving from 1:3 ratio in favour of adduct 3b, to 1:1 ratio, respectively.
To clarify the above results, computational studies were performed. First, diastereoiosmers 3a and
3b were evaluated by a conformational search at the molecular mechanics level. All the
conformations found in the range of 2 kcal/mol (20 and 25 for 3a and 3b, respectively) were then
selected for DFT optimization and energy evaluation at the CPCMꢀHCTC/6ꢀ311+G(d,p)//HCTH/6ꢀ
31+G(d) level,16 accordingly to a protocol previously adopted for similar studies, using MeCN as
the solvent. The geometries of the most stable conformation for each compound is presented in
supplementary Figure S9 (Supporting Information), while the energies obtained for all 3a and 3b
conformations are reported in Tables S5 and S6 (Supporting Information), respectively.
Since the observed diastereoselectivity is likely to be determined at the transition state level, the
most stable conformations of 3a and 3b were used to generate the corresponding transition
structures TS-3a and TS-3b, that were successively optimized using the same scheme as above.
Single point energies were then calculated at the HCTH/6ꢀ311+g(d,p) level, using the CPCM
solvent model for MeCN. The optimized transition structures of TSꢀ3a and TSꢀ3b are shown in
Figure 1.
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Figure 1. Optimized structures of TSꢀ3a and TSꢀ3b. Selected distances are reported in Angstrom.
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Although the enthalpy difference between the two transition states (TS) is quite low (0.2 kcal/mol),
TSꢀ3b is favoured. Based on the Arrhenius equation (see Table S7 and Equation ES1 in Supporting
Information), the predicted ratio for 3a and 3b is 28:72. This ratio is very close to that
experimentally determined (1:3, when an organic base is used), confirming that the reaction is under
kinetic control and that the chosen level of theory is correct. The analysis of the geometry of the
two TSs shows that TSꢀ3b can be stabilized by a Tꢀshaped CH/π interaction between the two
aromatic rings. In TSꢀ3a, the phenylꢀphenyl interaction is replaced by a CH/π interaction between
the Boc moiety and the pyrroline benzyl group that, being mediated by hydrogens linked to a sp3
carbon is probably weaker, thus the slightly lower stabilization observed.
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Therefore, based on our computational results, we can hypothesize that when an organic base is
used, πꢀinteractions between the aryl moieties of pyrroline and nitrile oxide derivatives drive the
diastereoselectivity. As a result, even if we are not in a favourable stereochemical environment (see
introduction9c), the formation of 3b increases, supporting our strategy in selecting two arylꢀ
functionalized reagents to drive the diastereoselection.
When using the inorganic base, the decrease in diastereoselection could be due to the presence of a
metal cation able to disrupt the πꢀinteractions, thus acting as “chaotropic agent”.17 Indeed, it has
been reported that metal cations can destroy week interaction with a trend Li+ > Na+. In our case the
distribution between 3a and 3b is confirmed (compare entries 5 and 6, Table 1). To further verify
this hypothesis, we performed the cycloaddition reaction using TEA, in the presence of LiCl as the
cationic donor (Table 1, entry 16). It was found that a decrease in the yields occurred together with
a decrease in the diastereoselection (1:1 ratio instead of 1:3, found in absence of LiCl). This last
result confirms that the stereochemical results might be driven by πꢀinteractions, as suggested by
the computational study.
Even if diastereoisomers 3a and 3b were collected as a rather inseparable mixture, their purification
on silica gel column chromatography allowed the isolation and characterization of a pure amount of
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diastereoisomer 3b. H NOESY analysis (CD3CN) of the major diastereoisomer 3b shows some
significant spatial proximities (Figure 2A), allowing us to assign the 3aR,6aSꢀconfiguration to the
new generated stereocenters with respect to the S configuration of the side chain.
As a further confirmation, suitable needleꢀshape crystals of 9b, the debenzylated compound of 3b
obtained as described in Scheme 3, were achieved (CDCl3/Et2O, 1:10). Their singleꢀXꢀrayꢀcrystal
analysis confirmed the (3aR,6aS) absolute configuration of the isoxazoline derivative 9b and,
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indirectly, of 3b (Figure 2B). Crystal structure data of 9b was deposited at the Cambridge
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Crystallographic Data Centre and allocated the deposition number CCDC 1841009.
2.36
5
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A)
5.02
H
3.06
H
1
H
O
3.25
6
2
2.94
6a S
N
H
S
5
N
H
3a
H 3.63
R
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3
3.55
H
9
Ph
4.53H
H
H
3.06
H
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3.79
2.27
NBoc
H
5.54
3b
B)
9b
Figure 2. A) NOE signals of compound 3b. B) ORTEP plot of 9b at 293 K with atom numbering
scheme; ellipsoids of nonꢀH atoms are at 15% probability level.
Peptidomimetic synthesis. Both diasteroisomers 3a and 3b were used for the preparation of
peptidomimetic models (Scheme 3). Their orthogonal deprotection at nitrogen of the pyrrolidine
ring was first studied. Since the isoxazoline nucleus is not stable to the catalytic reduction with
H2/Pd, a “oneꢀpot” debenzylation/acylation reaction was selected according to a known procedure.18
Starting from of a mixture of 3a and 3b and by using 2,2,2ꢀtrichloroethyl chloroformate (TrocCl;
MeCN, 2 h, 98%), diastereoisomers 8a and 8b were efficiently obtained and easily separated by
flash chromatography.
The nitrogen of pyrrolidine ring of compounds 8 was orthogonally deprotected with Zn powder in
an aqueous solution of AcOH (90%) at 25 °C giving Bocꢀderivatives 9a (80%) and 9b (70%).
Two small peptides models, i.e. 11a and 11b, were prepared to evaluate the ability of our scaffolds
to stabilize a parallel turn conformation when inserted in a natural sequence. The dipeptide NꢀAcꢀ
LeuꢀValꢀOH (10) was made to react with amines 9a and 9b. Compounds 11a (65%) and 11b (59%)
were obtained in very good yield using the solution phase protocol and the classic coupling agents
[EDC (1.1 equiv.), HOBT, (1.1 equiv.), DIEA (1.1 equiv.) in CH2Cl2].
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Scheme 3. Synthesis of peptides 11-15. Reaction conditions: i) TrocCl (1 eq.), MeCN, 25 °C, 2 h;
ii) Zn, AcOH, 25 °C; iii) EDC (1.1 equiv.)/HOBT (1.1 equiv.)/DIEA (1.1 equiv.), CH2Cl2.
NMR spectroscopy characterization of peptides. Peptides 11 were fully characterized by NMR
(1H, 13C, HMBC, HSQC, NOESY; MeCN, 300 MHz; for details see Tables S1ꢀS4 in Supporting
Information).
1H NMR analysis of compound 11b revealed the presence of two main isomers in 60:40 ratio.
NOESY experiments showed that similar NOEs are present for the same proton in the two isomers
proving an equilibrium between them. Wellꢀdistributed chemical shifts of amide protons were
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detected. Furthermore, JHN/CH are major than 8 Hz both for Val and Leu as well as for the residue
linked to the isoxazoline ring that could be considered the equivalent of Phe. These data suggest
that the peptide chain could assume a wellꢀordered conformation.
The main observed NOEs for both isomers of compound 11b were reported in Figure 3. A medium
NOE was detected between H4y and CHBn in both isomers (Figure S8A, Supporting Information)
as well as a spatial proximity between CHLeu/NHVal (s; Figure 4). A week NHLeu/NHVal NOE
(Figure S7B) was found for 11b due to the conformational freedom of Leu. Of relevance,
intrastrand proximities were detected between the phenyl ring and MeVal (Figure 4) and between
Boc and the alkyl chain of Leu.19 These NOEs support the idea that a parallel turn conformation
could be induced by our scaffold.
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Figure 3. NOEs (red lines; 300 MHz, 500 ms) and Hꢀbond (dotted black lines) of peptide 11b and
11’b.
Figure 4. CH/NH NOEs regions (CD3CN, 20 mM, 300 MHz, 500 ms): black*, main isomer 11b;
red°, minor isomer 11b’; blu, overlapped signals.
The δꢀ/δΤ analyses showed that NHBoc is involved in a Hꢀbond (ꢀ4.5 ppb) for isomer 11b and
NHLeu (ꢀ4 ppb) for isomer 11b’ (Figure 5). Since these isomers are in equilibrium, these values are
consistent with a medium δꢀ/δΤ value. Coalescence of NHBoc signal occurs at 313K and a
completed coalescence for all NHs takes place at 323K. We hypothesize that NHBoc forms an Hꢀ
bond with C=OLeu in rotamer 11b and NHLeu with C=OBoc in isomer 11b’. The formation of a Hꢀ
bond between NHBoc and C=OVal was excluded because is not consistent with the detected NOEs
(H4y/CHBn proximity).
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11b
11b'
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NHVal-2
NHBoc
NHLeu-3
Figure 5. ꢀδ/ꢀT NH values (273ꢀ323 K; 300 MHz) for conformers 11b and 11b’.
A mixture of two conformers (about 1:1 ratio), with a minor amount of a third one, was detected in
1
the H NMR spectrum of diastereoisomer 11a. Several signals are overlapped preventing the fine
characterization of the main isomers. With respect to 11b, larger ꢀδ/ꢀT values were observed
(Figure
S4, Supporting Information). A complete coalescence for all NHs takes place at 313K.
Several NOEs are present between CHVal with both protons Hꢀ4 and Hꢀ6 as well as between CHLeu
with Hꢀ4x and Hꢀ6y (Figure S5, Supporting Information). Differently to 11b, intrastrand NOEs
were not detected. Taken together these data suggest that a random conformation for this peptide is
more likely.
Conclusion
In conclusion, the synthesis of two diastereoisomeric tetrahydroꢀ4Hꢀ(pyrrolo[3,4ꢀd]isoxazolꢀ3ꢀ
yl)methanamine derivatives was performed using a [1,3]ꢀdipolar cycloaddition reaction between the
nitrile oxide derived from the cheap enantiopure Lꢀphenylalanine and Nꢀbenzylpyrroline.
Computational studies confirmed that πꢀinteractions between the phenyl substituents of the reagents
favour the formation of the 3aR,6aSꢀisomer 3b. NMR studies of model peptides containing the
molecular scaffolds 3 proved that 3b, the main isomer obtained in the cycloaddition reaction,
stabilizes a parallel turn conformation. Thus, 3b could be used as a profitable scaffold for the
stabilization of parallel βꢀsheet secondary structures.
Experimental
General Information. Melting points were determined in a Stuart Scientific melting point apparatus
in open capillary tubes and are certified. Chemicals were obtained from Sigma Aldrich and used
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without further purification. HPLC analyses were carried out on Jasco PUꢀ980 pump equipped with
a UV–vis detector Jasco UVꢀ975 (wavelength: 220 nm) and on a Kromasil 5ꢀAmyCoat column (4.6
mm i.d. × 250 mm, 5 ꢂm, AkzoNobel). ESI mass spectra were recorded on an LCQESI MS were
recorded on a LCQ Advantage spectrometer from Thermo Finningan and a LCQ Fleet spectrometer
from Thermo Scientific. The NMR spectroscopic experiments were carried out on a Varian
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OXFORD 300 MHz (300 and 75 MHz for H and 13C, respectively). Optical rotations were
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measured on a Perkin–Elmer 343 polarimeter at 20°C (concentration in g/100 mL). Chemical shifts
δ are given in ppm relative to the CHCl3 internal standard, and the coupling constants J are reported
in Hertz (Hz).
tꢀButyl (S)ꢀ(1ꢀhydroxyꢀ3ꢀphenylpropanꢀ2ꢀyl)carbamate (5). Operating in a threeꢀneck roundꢀbottom
flask, equipped with magnetic stirrer, thermometer, nitrogen inlet and dropping funnel, NaBH4 (400
mg, 10.5 mmol) was suspended in THF (20 mL). The solution was cooled to 0°C and a solution of
I2 (1.5 g, 6 mmol) in THF (5 mL) was added very slowly under vigorous stirring. Lꢀphenylalanine
(4) (1 g, 6 mmol) was added in one portion and the reaction mixture was stirred at reflux for 16 h.
After cooling at 25 °C, MeOH (13 mL) was added until discoloration and the stirring was continued
for further 30 min. The solution was cooled to 0°C and TEA (1.67 mL, 12 mmol) was added
dropwise. After 10 min., a solution of diꢀtertꢀbutyl dicarbonate (1.4 g, 6.4 mmol) in THF (4 mL)
was dropped. The reaction mixture was stirred for additional 20 h. The solvent was removed under
reduced pressure and the crude reaction mixture was dissolved in ethyl acetate (15 mL). The
organic layer was washed with saturated solution of NH4Cl (10 mL) and dried over Na2SO4. The
solvent was removed under reduced pressure and the crude product was crystallized from
hexane/AcOEt (12:1), affording compound 5 (1.5 g, 5.85 mmol, 97%) as a white solid. TLC:
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hexane/AcOEt, 7:3 (Rf: 0.18). Mp 92°C; [α] D: ꢀ28.5° (c 0.6 in CHCl3); H NMR (300 MHz,
CDCl3) δ 7.32ꢀ7.20 (m, 5H), 4.80 (br s, 1H), 3.86 (br s, 1H), 3.66ꢀ3.63 (m, 1H), 3.55ꢀ3.52 (m, 1H),
2.83 (d, J 7 Hz, 2H), 2.08 (br s, 1H), 1.41 (s, 9H) ppm; MS (ESI) m/z: Calcd for C14H21NO3 251.15;
Found 274.40 [M+Na]+. The analytical and spectroscopical data are in agreement with the literature
data.20
tꢀButyl (S)ꢀ(1ꢀoxoꢀ3ꢀphenylpropanꢀ2ꢀyl)carbamate (6). Method A: Swern Oxidation. Operating in a
threeꢀneck roundꢀbottom flask, equipped with magnetic stirrer, thermometer and nitrogen inlet, a
solution of dry (COCl)2 (0.25 mL, 3 mmol) in dry CH2Cl2 (2.5 mL) was added and cooled to ꢀ78°C.
A solution of dry DMSO (0.24 mL, 3.4 mmol) in CH2Cl2 (1.0 mL) was dropped over 5 min, and
then the reaction was stirred for 15 min. A solution of 5 (500 mg, 2.0 mmol) in dry CH2Cl2 (2.5
mL) was dropped over 10 min. The reaction mixture was stirred for 30 min. and then a solution of
dry TEA (1.12 mL, 8.0 mmol) was added dropwise over 10 min. The reaction was slowly heated to
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25 °C and was stirred for 1 hour. The reaction mixture was diluted with CH2Cl2 (16 mL) and
washed with 10% citric acid (15 mL), H2O (15 mL), a saturated solution of NaHCO3 (15 mL) and
dried over Na2SO4. The solvent was removed under reduced pressure. The crude compound (493
mg, 1.98 mmol, 99%) was immediately transformed into oxime 7 without further purifications (for
HPLC analysis see Figures S1X and S1Y). Method B: DessꢀMartin oxidation. Operating in a roundꢀ
bottom flask, equipped with magnetic stirrer, DessꢀMartin periodinane (551 mg, 1.3 mmol) was
suspended in a mixture of CH2Cl2 (5 mL) and dioxane (3mL). A solution of alcohol 5 (300 mg, 1.2
mmol) in CH2Cl2 (1 mL) was added dropwise over a period of 20 min. The reaction was stirred at
25°C for 20 min. When the reaction was complete, Et2O (2 mL) and sodium thiosulfate (0.5 g)
dissolved in a saturated NaHCO3 solution (2 mL) were added. The reaction was stirred for further 5
min and then Et2O (2 mL) was added. The layers were separated and the organic one was washed
with a saturated solution NaHCO3 (2 x 4 mL), H2O (2 x 4 mL) and dried over Na2SO4. The solvent
was removed under reduced pressure, affording 6 (237 mg, 0.95 mmol, 93%) as a paleꢀyellow oil.
1H NMR (300 MHz, CDCl3) δ 9.64 (s, 1H), 7.37ꢀ7.28 (m, 3H), 7.20ꢀ7.17 (m, 2H), 5.04 (s, 1H),
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4.44 (d, J 6.4 Hz, 1H), 3.13 (d, J 6.4 Hz, 2 H), 1.45 (s, 9 H). H NMR data are in agreement with
the reported data.21 The aldehyde was immediately transformed into oxime 7 without further
purifications.
tꢀButyl (S)ꢀ(1ꢀ(hydroxyimino)ꢀ3ꢀphenylpropanꢀ2ꢀyl)carbamate (7). Operating in a bottom flask,
equipped with magnetic stirrer and thermometer, aldehyde 6 (500 mg, 2 mmol) was dissolved in
THF (7.5 mL) at 0°C. NaHCO3 (102.5 mg, 1.22 mmol) and H2NOHꢃHCl (148 mg, 2.2 mmol) were
added to the solution. The reaction mixture was heated to 25 °C and stirred overnight (TLC:
hexane/AcOEt, 7:3). After solvent evaporation, the residue was extracted with ethyl acetate (4 x 4.5
mL). The combined organic layers were washed with brine (4.5 mL), dried over Na2SO4 and
concentrated in vacuo. Purification of the crude product by flash chromatography (hexane/AcOEt
8:2) afforded 7 (385 mg, 83% as a white solid). After crystallization pure isomer Eꢀ7 was isolated.
Mp 120.3 °C (hexane/AcOEt, 10:1); [α]20D + 28.3° (c 1 in CHCl3);22 1H NMR (300 MHz, CD3CN):
δ 8.90 (s, 1H), 7.48ꢀ7.12 (m, 5H), 6.67 (d, J 6.1 Hz, 1H), 5.44 (brs, 1H), 4.90 (m, 1H), 2.99 (dd, J
4.9, 13.7 Hz, 1H), 2.81 (dd, J 9.3, 13.7, Hz 1H), 1.39 (s, 9H) ppm; MS (ESI) m/z: Calcd for
C14H20N2O3 264.15; Found 286.9 [M+Na]+. Zꢀ7 (small amount isolated by chromatography): mp
1
118.5 °C (hexane/AcOEt, 15:1); H NMR (300 MHz, CD3CN) δ 8.59 (s, 1H), 7.49ꢀ7.26 (m, 6H),
5.26 (br s, 1H), 4.40 (ddd, J 14.4, 8.4, 6.0 Hz, 1H), 2.99 (dd, J 13.7, 6.3, Hz, 1H), 2.86 (dd, J 13.7,
8.24 Hz, 1H), 1.39 (s, 9H) ppm.
NꢀtꢀButoxycarbonyl [1ꢀchloroꢀ1ꢀ(hydroxyimino)ꢀ3ꢀphenylpropanꢀ2ꢀyl)]carbamate (1). Operating in
a bottom flask, equipped with magnetic stirrer and thermometer, oxime 7 (550 mg, 2.1 mmol) was
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suspended in CCl4 (20 mL) and Nꢀchlorosuccinimide (278 mg, 2.1 mmol) was added portion wise
over a period of 60 min at 0°C. The reaction mixture was refluxed overnight. The solution was
cooled to 0°C and filtered to remove the succinimide (TLC: hexane/AcOEt, 7:3; Rf 0.45). The
solvent was removed under reduced pressure, affording product 7 (601.2 mg, 97%) as yellow oil.
1H NMR (300 MHz, CD3CN) δ 9.34 (s, 1H), 7.38ꢀ7.23 (m, 5H), 5.75 (br s, 1H), 4.63 (dd, J 15.1,
7.7 Hz, 1H), 3.07 (dd, J 13.8, 7.7 Hz, 1H), 2.94 (dd, J 13.7, 8.5 Hz, 1H), 1.39 (s, 9H) ppm;24 MS
(ESI) m/z: Calcd for C14H19ClN2O3 298.11; Found 299.5 [M+H]+.
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tꢀButyl
1ꢀ(5ꢀbenzylꢀ3a,5,6,6aꢀtetrahydroꢀ4Hꢀpyrrolo[3,4ꢀd]isoxazolꢀ3ꢀyl)ꢀ2Sꢀ
phenylethyl)carbamate (3). Method A. Chloroꢀoxime 1 (200 mg, 0.67 mmol) was dissolved in
MeCN (5 mL) and DMF (1 mL). Distilled Nꢀbenzylꢀ3ꢀpyrroline (2) (106.7 mg, 0.13 mL, 0.67
mmol) was added in one portion at 25 °C, followed by the addition of TEA (67.9 mg, 0.67 mmol)
or DABCO (75.1 mg, 0.67 mmol). The reaction mixture was stirred for 1 h. Afterwards, chloroꢀ
oxime 2 (200 mg, 0.67 mmol) and TEA (67.9 mg, 0.67 mmol) or and DABCO (75.1 mg, 0.67
mmol) were added portionwise over 60 min. The reaction mixture was stirred for additional 16 h at
25 °C. The solvent was removed under reduced pressure. Purification of the crude product by flash
chromatography (hexane/AcOEt 9:1) afforded the diastereoisomeric mixture 3a/3b (TEA: 1:3, 132
mg, 49%; DABCO: 1:2.5, 151.6 mg, 53%) as a white solid. The same reaction performed in the
presence of LiCl (58.8 mg, 1.34 mmol) gave a 1:1 mixture of 3a/3b (39.15 mg, 0.09 mmol, 14%).
Method B. Chloroꢀoxime 1 (100 mg, 0.33 mmol) was dissolved in MeCN (3 mL). Distilled Nꢀ
benzylꢀ3ꢀpyrroline (2) (66.9 mg, 79.9 µL, 0.42 mmol) was added in one portion at 25 °C. NaHCO3
(26.9 mg, 0.33 mmol) or Li2CO3 (12.2 mg, 0.165 mmol) was added and the reaction mixture was
stirred for 1 h. Chloroꢀoxime 1 (152.04 mg, 0.51 mmol) and NaHCO3 (42.85 mg, 0.51 mmol) or
(12.2 mg, 0.165 mmol) were subsequently added portionwise over 60 min. The reaction mixture
was stirred for additional 4 h at 25 °C. The solvent was removed under reduced pressure.
Purification of the crude product by flash chromatography (hexane/AcOEt 9:1) afforded the
diastereoisomeric mixture 3a/3b (NaHCO3 1:2, 90 mg, 51%; Li2CO3 1:1, 66.55 mg, 48%) as a
white solid.
3aS,6aR 3a: mixture with 3b. 1H NMR (300 MHz, CD3CN) main signal: δ 4.70ꢀ4.60 (m, 1H) ppm;
13C NMR (75 MHz, CD3CN) δ 159.2, 155.3, 138.8, 137.7, 129.4, 128.5, 128.3, 128.2, 126.9, 126.5,
84.3, 78.8, 61.7, 58.4, 56.2, 53.5, 49.7, 38.7, 27.5 ppm.
3aR,6aSꢀ3b. TLC: hexane/AcOEt, 1:1; Rf 0.57. M.p. 99.3 °C; IR (NaCl) νmax 3374, 1712 cmꢀ1; H
1
NMR (300 MHz, CD3CN) δ 7.42ꢀ7.13 (m, 10H), 5.53 (d, J 8.8 Hz, 1H), 5.01 (dd, J 9.4, 4.6 Hz,
1H), 4.53 (dd, J 14.1, 9.9 Hz, 1H), 3.83ꢀ3.69 (m, 1H), 3.55 (dd, J 22.4, 13.3 Hz, 2H); 3.25 (dd, J
14.2, 5.2 Hz, 1H), 3.06 (d, J 9.6 Hz, 2H), 2.94 (dd, J 13.9, 9.7 Hz 1H), 2.36 (dd, J 11.0, 4.8 Hz,
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1H), 2.27 (dd, J 10.0, 7.1 Hz, 1H), 1.40 (s, 9H) ppm; 13C NMR (75 MHz, CD3CN) δ 159.4, 138.9,
132.4, 129.3, 128.5, 128.4, 128.3, 128.2, 127.0, 126.6, 126.3, 83.9, 61.9, 58.3, 56.0, 53.0, 48.8, 37.7
ppm; Anal. Calcd for C25H31N3O3: C, 71.23; H, 7.41; N, 9.97. Found: C, 71.06; H, 7.65; N, 9.80;
MS (ESI) m/z: Calcd for C25H31N3O3 421.24; Found 422.24 [M+H]+; 444.26 [M+Na]+.
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2,2,2ꢀTrichloroethyl
3ꢀ(2Sꢀ1ꢀtertꢀbutoxycarbonylaminoꢀ2ꢀphenylethyl)ꢀ6,6aꢀdihydroꢀ3aHꢀ
pyrrolo[3,4ꢀd]isoxazoleꢀ5(4H)ꢀcarboxylate (8). A mixture of 3a/3b (1:3 ratio; 50 mg, 0.12 mmol)
was dissolved in MeCN (2 mL) under stirring. 2,2,2ꢀTrichloroethylchloroformate (29.7 mg, 19.3
µL, 0.14 mmol) was added dropwise to the solution at 25 °C. The reaction mixture was stirred for 2
h. The solvent was removed under reduced pressure. Purification of the crude mixture by flash
chromatography (hexane/AcOEt, 3:1) afforded the products 8a (19.7 mg, 0.04 mmol, 32.5%) and
8b (39.5 mg, 0.08 mmol, 65%) as white solids.
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3aS,6aRꢀ8a: TLC: hexane/AcOEt, 7:3; Rf 0.23. Mp 125.5 °C; [α]20D: ꢀ 68.5 (c 1, MeOH); IR (NaCl)
1
νmax 3355, 3061, 1717, 1690 cmꢀ1; H NMR (300 MHz, CDCl3) δ 7.36ꢀ7.23 (m, 5H), 5.13 (brs,
2H), 4.76ꢀ4.66 (m, 3H), 3.98 (brs, 1H), 3.85 (brs, 1H), 3.64 (brs, 1H), 3.47ꢀ3.42 (m, 2H), 3.22ꢀ3.05
(m, 2H), 1.43 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3) δ 159.7, 155.0, 152.5, 136.4, 129.2, 128.7,
127.1, 110.0, 85.0, 80.2, 75.0, 53.4, 52.9, 49.8, 49.2, 48.4, 40.1 ppm; Anal. Calcd for
C21H26Cl3N3O5: C, 49.77; H, 5.17; N, 8.29. Found: C, 49.61; H, 5.30; N, 8.10; MS (ESI) m/z: Calcd
for C21H26Cl3N3O5 505.9; Found 528.62 [M+Na]+.
3aR,6aSꢀ8b: TLC: hexane/AcOEt 7:3; Rf 0.19. M.p. 104.1 °C; [α]20D: + 31 (c 1, MeOH); IR (NaCl)
ν
max 3376, 3065, 1724, 1684 cmꢀ1; 1H NMR (300 MHz, CDCl3) δ 7.34ꢀ7.23 (m, 5H), 5.22ꢀ5.17 (m,
1H), 4.76 ꢀ 4.66 (m, 4H), 3.96ꢀ3.77 (m, 3 H), 3.67ꢀ3.54 (m, 2H), 3.44ꢀ3.32 (m, 1H), 3.04ꢀ3.11 (m,
1H), 1.38 (s, 1H) ppm; 13C NMR (75 MHz, CDCl3) 159.5, 155.2, 152.4, 136.8, 129.4, 128.6, 126.9,
95.4, 84.4, 80.3, 75.0, 53.7,52.5, 48.6, 48.3, 38.5, 28.2 ppm; Anal. Calcd for C21H26Cl3N3O5: C,
49.77; H, 5.17; N, 8.29. Found: C, 49.68; H, 5.25; N, 8.15; MS (ESI) m/z: Calcd for C21H26Cl3N3O5
505.9; Found 528.48 [M+Na]+.
tꢀButyl (2S)ꢀphenylꢀ1ꢀ(4,5,6,6aꢀtetrahydroꢀ3aHꢀpyrrolo[3,4ꢀd]isoxazolꢀ3ꢀyl)ethyl)carbamate (9).
Compound 8 (40 mg, 0.08 mmol) was dissolved in an aqueous solution of AcOH (90%, 0.4 mL).
Zn (80 mg, 2.6 mmol) was added at 0 °C and the reaction was left under stirring at 25 °C for 2 h
(TLC: CH2Cl2/MeOH 40:1; Rf 0.12). Zinc was filtered and AcOH was removed under vacuum. The
crude mixture was dissolved in AcOEt (4 mL) and the white solid was filtered. The mother liquor
was extracted with NaHCO3 (2 x 4 mL) then dried over Na2SO4 and concentrated in vacuum. The
crude was purified by flash chromatography (CH2Cl2/MeOH; 40:1) affording pure compound 9 as
white foam (9a: 21 mg, 80%; 9b: 18.6 mg, 0.056 mmol, 70% yield).
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3aS,6aRꢀ9a: [α]20D: ꢀ4 (c 1, MeOH); IR (NaCl) νmax 3358, 1682 cmꢀ1; H NMR (300 MHz, CDCl3):
δ 7.35ꢀ7.22 (m, 5H), 5.27ꢀ5.24 (m, 1H), 5.11ꢀ5.09 (m, 1H), 4.70ꢀ4.63 (m, 1H), 3.43 – 3.30 (m, 3H),
3.31ꢀ3.09 (m, 3H), 3.07ꢀ2.82 (m, 2H), 1.42 (s, 9H) ppm; 13C NMR (75 MHz, CDCl3): 159.4, 155.2,
136.4, 129.3, 128.7, 127.0, 86.7, 80.1, 56.2, 55.9, 51.3, 49.7, 40.2, 28.3 ppm; Anal. Calcd for
C18H25N3O3: C, 65.23; H, 7.60; N, 12.68. Found: C, 65.00; H, 7.83; N, 12.47; MS (ESI) m/z: Calcd
for C18H25N3O3 331.19; Found 332.2 [M+H]+.
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3aR,6aSꢀ9b: [α]20D: +12 (c 1, MeOH); IR (NaCl) νmax 3355, 1687 cmꢀ1; H NMR (300 MHz,
CDCl3) δ 7.20ꢀ7.40 (m, 5H), 5.10ꢀ5.20 (m, 1H), 4.87ꢀ4.45 (m, 2H), 3.63 (m, 3H), 3.40ꢀ3.21 (m,
2H), 3.00ꢀ3.15 (m, 1H), 2.6ꢀ 2.8 (m, 2H), 2.0ꢀ1.8 (m, 2H), 1.2 (s, 9H) ppm; 13C NMR (75 MHz,
CDCl3) δ 158.9, 155.2, 137.1, 129.3, 127.0, 126.6, 87.1, 80.3, 56.7, 55.9, 51.5, 49.1, 39.3, 28.2
ppm; Anal. Calcd C18H25N3O3: C, 65.23; H, 7.60; N, 12.68. Found: C, 65.04; H, 7.79; N, 12.52; MS
(ESI) m/z: Calcd for C18H25N3O3: 331.19; Found 332.18 [M+H]+.
General Procedure for Peptide Synthesis. Dipeptide AcꢀNꢀLeuꢀValꢀOH (10) (46.4 mg, 0.17 mmol)
was dissolved in CH2Cl2 (3 mL) and CH2Cl2 (1 mL)/DMF (0.2 mL), respectively. The solution was
cooled to 0 °C under stirring. HOBt (25.6 mg, 0.19 mmol) and EDC (36.8 mg, 0.19 mmol) were
added. After 1 h, compound 9 (56.3 mg, 0.17 mmol) in CH2Cl2 (1 mL) was dropped, followed by
the addition of DIEA (22 mg, 29 µL, 0.17 mmol). The reaction mixture was stirred for 24 h and
then it was washed with a aqueous solution of KHSO4 (5%, 5 mL), a saturated solution of NaHCO3
(5 mL) and brine (5 mL). After drying over Na2SO4, the solvent was removed under reduced
pressure. Purification of the crude mixture by flash chromatography (CH2Cl2/MeOH; 40:1) afforded
compound 11 (11a: 64.4 mg, 65%; 11b: 59.2 mg, 0.10 mmol, 59%) as a white solid.
11a: TLC: CH2Cl2/MeOH, 40:1; Rf 0.20. [α]20D: ꢀ85 (c 0.3, MeOH); IR (NaCl) νmax 3437, 3305,
1709, 1635 cmꢀ1; Anal. Calcd C31H47N5O6 63.57; H, 8.09; N, 11.96; Found: 63.31; H, 8.40; N,
11.78; MS (ESI): m/z calcd for C31H47N5O6: 585.36; found: m/z 586.58 [M+H]+. For NMR data see
Tables S1 and S2.
11b: TLC: CH2Cl2/MeOH, 40:1; Rf 0.20. Rf 0.20. [α]20D: +25 (c 0.3, MeOH). IR (NaCl) νmax 3298,
1699,1637 cmꢀ1; Anal. Calcd C31H47N5O6 63.57; H, 8.09; N, 11.96; Found: 63.37; H, 8.38; N,
11.80; MS (ESI): m/z calcd for C31H47N5O6: 585.36; found: m/z 586.36 [M+H]+. For NMR data see
Tables S3 and S4.
Computational Methods. After designing the molecular structures using MOE software,25
compounds 3a and 3b were submitted to a conformational search via Low Mode MD simulation,
implemented in MOE, setting a Rejection Limit of 100, an Iteration Limit of 100000 and a MM
Iteration Limit of 1000.
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MMFF94x was used as the force field, while the Born solvent model with the dielectric constant of
acetonitrile (ε 37.5) was used to include solvation effects. For compound 3a, 203 different
conformations were obtained in an energy range of 7 kcal/mol; 240 conformations were instead
obtained for the compound 3b, in the same energy range. For each diastereoisomer all the
conformations that were within 2 kcal/mol from the more stable one (20 conformations for 3a, 25
for 3b) were subjected to a geometry optimization at the DFT level of theory, using the HCTH
functional and the 6ꢀ31+G(d) basis set. Indeed, this protocol was successfully used in previous
studies on [1,3]ꢀdipolar cycloadditions where πꢀinteractions resulted relevant in determining the
regiochemical outcome of the reaction.14 A vibrational analysis was then performed on the
optimized geometries and to calculate the zero point and thermochemical corrections to the
electronic energies (1 atm, 298.15 K) and no imaginary frequencies were observed. Single point
energies were then computed at the HCTH/6ꢀ311+g(d,p) level, using CPCM solvent model for
MeCN.26 The most stable conformation of 3a and 3b was used to generate a starting guess for the
corresponding transition states (TS-3a and TS-3b, respectively), that were treated with the same
protocol described above. The vibrational analysis, performed on the optimized geometries at the
same level of theory, confirmed the presence of a single imaginary frequency (ꢀ362.52 and ꢀ357.85
cmꢀ1 for TS-3a and TS-3b, respectively) corresponding to the stretching of the forming CꢀC and Cꢀ
O bonds. All quantum mechanical calculations were performed using the Gaussian09 package.27
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ASSOCIATED CONTENTS
Supporting Information. Synthesis for chloroxime 1 and HPLC data for oxime 7. Spectroscopical
1
13
data for 3b. NMR characterization of peptides 11a, 11b. Computational Section. H and C NMR
spectra for all new compounds. Xꢀray data for 9b (ORTEP plot, atomic coordinates, selected bond
distances and angles…). The Supporting Information is available free of charge on the ACS
Publications website at DOI:.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: marialuisa.gelmi@unimi.it.
Fax: (+39)02ꢀ50314481
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to MIUR for funding (2015 PRIN project Prot. 20157WW5EH)
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