Synthesis of Variously Functionalized Azabicycloalkane Scaffolds by Domino Metathesis Reactions

Article abstract of DOI:10.1021/acs.joc.7b02047

7,5-Fused azabicycloalkane scaffolds, carrying a quaternary stereocenter at C3 position of the lactam ring, can act as effective reverse-turn mimics and have proven to be useful intermediates for the preparation of Arg-Gly-Asp (RGD)-based cyclopentapeptides (cRGD) with nanomolar activity as α_vβ₃/α_vβ₅ integrin antagonists. Here, we report the synthesis of new azabicycloalkane scaffolds endowed at the C6 position with a para-substituted phenethyl side chain, which could be exploited to obtain cRGD-based bioconjugates that may find promising applications in anticancer therapy. By performing a domino cross enyne metathesis/ring-closing metathesis (CEYM/RCM) in the presence of styrene derivatives, followed by catalytic hydrogenation of the diene system, we easily converted a dipeptide precursor into the desired C6-functionalized azabicycloalkane scaffolds. The presence of a suitably protected p-amino group on the styrene moiety could be exploited, after deprotection, either to directly conjugate a bioactive compound or to introduce a suitable spacer between the cRGD unit and the bioactive compound.

Full text of DOI:10.1021/acs.joc.7b02047

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Article
Synthesis of Variously Functionalized Azabicycloalkane
Scaffolds by Domino Metathesis Reactions
Massimo Serra, Elena Giulia Peviani, Eric Bernardi, and Lino Colombo
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02047 • Publication Date (Web): 27 Sep 2017
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Synthesis of Variously Functionalized Azabicycloalkane Scaffolds by
Domino Metathesis Reactions
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Massimo Serra*, Elena Giulia Peviani, Eric Bernardi, and Lino Colombo*
Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section,
University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
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E-mail: massimo.serra@unipv.it
lino.colombo@unipv.it
Abstract: 7,5-fused azabicycloalkane scaffolds, carrying a quaternary stereocenter at C3 position of
the lactam ring, can act as effective reverse-turn mimics and have proved to be useful intermediates
for the preparation of Arg-Gly-Asp (RGD)-based cyclopentapeptides (cRGD) with nanomolar
activity as αvβ3/αvβ5 integrin antagonists. Here we report the synthesis of new azabicycloalkane
scaffolds endowed at C6 position with a p-substituted phenethyl side chains, which could be
exploited to obtain cRGD-based bioconjugates that may find promising applications in anticancer
therapy. By performing a domino Cross Enyne Metathesis/Ring Closing Metathesis (CEYM/RCM)
in the presence of styrene derivatives, followed by catalytic hydrogenation of the diene system, we
easily converted a dipeptide precursor into the desired C6-functionalized azabicycloalkane scaffolds.
The presence of a suitably protected p-amino group on the styrene moiety could be exploited, after
deprotection, either to directly conjugate a bioactive compound or to introduce a suitable spacer
between the cRGD unit and the bioactive compound.
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Introduction
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One of the main issues concerning anticancer therapy is the selectivity of the active species with
respect to the target. Most chemotherapeutics, albeit being potent cytotoxic, cytostatic or
antiangiogenic agents, often exhibit low selectivity for their respective targets causing severe side
effects, which highly reduce the efficacy and sustainability of the therapy. In light of this,
significant efforts are being pursued in order to develop targeted delivery systems1.
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By exploiting the presence of a homing device, it is possible to deliver cytotoxic drugs selectively
to the target, allowing a reduction of the active ingredient doses and side effects. In this field,
integrin antagonists may play a pivotal role because of their overexpression on many tumour cell
types and on neoangiogenic vessels with respect to their healthy counterparts. Through binding
with extracellular matrix (ECM) components, integrins regulate a wide range of processes like cell
adhesion, proliferation and differentiation2. In particular integrins αvβ3 and αvβ5 were found to be
interesting targets because of their key role in metastatic spreading and neoangiogenesis3, which are
the most harmful processes for diffusion and progression in colon cancer, melanoma, breast cancer,
and brain cancers, such as glioblastoma (GBM)4.
The common recognition motif for the interaction of these receptors with their endogenous ligands
is the tripeptide sequence Arg-Gly-Asp (RGD)5. Cilengitide, a cyclic RGD-based pentapeptide, was
the first αvβ3/αvβ5 integrin antagonist to be tested in clinical trials for the treatment of GBM6. Even
though its use as an anticancer therapeutic has been recently discontinued due to the failure in phase
III clinical trials7, the important biological roles played by αvβ3 and αvβ5 integrin subtypes and the
low toxicity of RGD-based antagonists stimulated the development of new high affinity ligands8
suitable for conjugation with bioactive compounds. These RGD-based bioconjugates may find
promising application for targeted drug delivery, theranostic, and general cancer-cell labeling9.
Our continuous efforts in this field have been focused on the synthesis of azabicycloalkane
scaffolds, which can act as effective reverse-turn mimetics in such a way that a properly embedded
RGD sequence could be forced to adopt conformations suitable for receptor binding10.
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In particular 7,5-fused bicyclic systems of the type 1 (Figure 1), carrying a quaternary stereocenter
at C3 position of the lactam ring, proved to be useful intermediates for the synthesis of RGD-based
cyclopentapeptides such as 1a-RGD, a nanomolar inhibitor of αvβ3 and αvβ5 integrin receptors11.
Moreover, 1a-RGD is one of the few RGD-like antagonists capable of inhibiting cell migration and
attachment, and inducing anoikis in glioblastoma cell lines12. The relevant biological activity shown
by 1a-RGD prompted us to develop a synthetic protocol that would allow further functionalization
of the bicyclolactam scaffold, in order to obtain bioconjugates that may find application for targeted
drug delivery systems, as theranostic agents or for cancer cell labeling.
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Figure 1.
In a recent work we reported a synthetic strategy that allowed the attainment of 7,5-fused
azabicycloalkane scaffolds of type 2 endowed with an exocyclic double bond. The C5 vinyl moiety
turned out to be highly reactive towards Heck-coupling reactions, thus demonstrating its usefulness
and versatility for the introduction of suitable linkers to which bioactive compounds could be
conjugated13.
As a part of our research project, we were interested in extending our studies towards the synthesis
of C6-functionalizable azabicycloalkane scaffolds (Scheme 1) in order to broaden the scope of our
library of structures. Indeed, the preparation of a set of further functionalizable dipeptide mimics
could be useful to gain detailed information about the conformation specifically required by the
peptide to effectively bind and activate the receptor14. Literature precedents by Lubell et al. reported
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the synthesis of a series of C6-functionalized azabicyclo[5.3.0]alkanone amino acids exploiting a
diastereoselective SN1 displacement of an iodide precursor15. This synthetic approach, which
allowed also the introduction of a hydroxyl and an azido group at C6, has as its main limitation the
possibility of introducing only O- or N-linked side-chains. To the best of our knowledge, no
examples of installation of C-linked side-chains at 6-position onto azabicyclo[5.3.0]alkanone amino
acids have been previously reported. These considerations, together with the need of a more rapid
synthetic protocol to prepare “easy-to-functionalize” azabicycloalkane scaffolds, led us to consider
the synthetic approach depicted in Scheme 1, exploiting as a key reaction a ring closing enyne
metathesis (RCEYM). The intramolecular bond reorganization involved in the reaction would allow
to convert in one step the dipeptide 5 into the corresponding cyclic diene 6, endowed with an
exocyclic vinyl moiety at C6 position of the seven-membered lactam ring. As highlighted by the
retrosynthetic analysis, the dipeptide 5 could be obtained by a condensation reaction between the α-
allyl quaternary α-amino acid 4 and the 5-ethynylproline derivative 3. After a regioselective
functionalization of the exocyclic double bond with an appropriate linker, followed by reduction of
the diene system, it would be possible to use the functionalized azabicycloalkane scaffolds as
templates for the attainment of cRGD-based bioconjugates.
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Scheme 1. RCEYM approach for the preparation of C6-functionalized azabicycloalkane scaffolds
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Results and discussion
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The synthesis of the first building block, namely the (2S,5R)-5-ethynylpyrrolidine-2-methyl ester 3,
was performed following the synthetic route depicted in Scheme 2. The readily available methyl
(S)-pyroglutamate was N-protected as methyl carbamate16 and converted into the corresponding
methoxylated derivative 8 by reduction with Super-Hydride (LiBEt3H) followed by treatment with
trimethyl orthoformate in the presence of pyridinium p-toluenesulfonate (PPTS). This optimized
procedure allowed us to obtain the desired methoxylated carbamate 8 in yields higher than those
previously reported in literature17.
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The Lewis acid-catalyzed bistrimethylsilylacetylene addition to the N-acyliminium intermediate
derived from 8 afforded a cis/trans mixture of alkynylated derivatives 918. The chromatography
purification of the diastereomeric mixture allowed the isolation of the cis stereoisomer 9a which,
after removal of the trimethylsilyl group with tetrabutylammonium fluoride and subsequent
deprotection of the methyl carbamate with iodotrimethylsilane, gave the desired 5-ethynylproline
methyl ester 3 in good overall yield.
Scheme 2. Synthesis of the 5-ethynylproline methyl ester
Reagents and conditions: (i) NaHMDS, ClCO2CH3, THF, -78 °C then 0 °C; (ii) a) LiEt3BH, THF,
-78 °C; b) CH(OCH3)3, PPTS, MeOH; (iii) bistrimethylsilylacetylene, SnCl4, AlCl3, CH2Cl2, -45 °C;
(iv) TBAF, THF, -20°C then 0 °C; (v) TMSI, CH2Cl2, 65 °C
On the basis of our long term interest on the study of the reactivity of 5-alkyl-oxazol-5-(4H)-ones19
to establish quaternary stereocenters in α,α-disubstituted α-amino acids, we designed the synthesis
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of the N-protected α-allyl-α-benzyl amino acid 4 (Scheme 1) starting from the corresponding 2-
phenyl-4-benzyl-oxazol-5-(4H)-one through an electrophilic allylation of the C4 position of the
oxazolone. In addition, this procedure should have allowed an enantioselective version by only
marginal modification of the synthetic protocol. It follows that a reaction promoted by a modifiable
catalyst would meet this need. Several endeavors in this direction have been reported in the
literature, e.g. Trost et al. performed the direct installation of an allyl moiety at the C-4 position of
the oxazolone ring by exploiting an asymmetric intermolecular allylic alkylation reaction in the
presence of chiral palladium catalysts20. The highest levels of ee’s (≥90%) were obtained with
substituted allyl derivatives, while the use of allyl acetate gave the desired 4-alkyl-4-allyl
oxazolones with ee’s not higher than 40%. A different strategy made use of a diazaphosphorane
(BEMP) as a base to deprotonate various oxazolones and subsequent allylation of the resulting
enolates with allyl bromide to give racemic quaternary allyl oxazolones21. The same products were
enantioselectively obtained using Cinchona-derived dimeric ammonium salts (80% ee)22, or chiral
tetraaminophosphonium salts (91% ee)23 as phase-transfer organocatalysts.
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Among the various methods able to introduce an allyl group on the oxazolone C-4 stereocenter, we
opted for a protocol based on a Tsuji decarboxylative allylation24. Four main considerations
directed our efforts towards this choice: a) the catalytic nature of the process; b) the possibility to
exploit readily accessible and cheap starting materials; c) the opportunity of rapidly obtaining the
racemic quaternary amino acid in order to test the feasibility and the “robustness” of our synthetic
strategy; d) the perspective to devise, as a future goal, an enantioselective variant of this
transformation by the use of chiral Pd catalysts. It is worth mentioning that even though a catalytic
enantioselective Tsuji allylation of simple enolizable carbonyl derivatives was reported by Stoltz et
al.25, this reaction has remained essentially unexplored on oxazol-5-(4H)-ones. To the best of our
knowledge only 4-allyl-4-methyl-oxazol-5-one was prepared in a very low 2% ee employing (S)-t-
Bu-phosphinooxazoline as chiral ligand26.
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Racemic phenylalanine-derived azlactone 11 was easily converted to the allyl enol carbonate 12 by
formation of the corresponding enolate and subsequent treatment with allyl chloroformate (Scheme
3). For the key Tsuji allylation step, we opted for a catalytic system previously utilized by us in the
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amino acids19a. Adding a Pd/diphosphine 1:4 complex, prepared by mixing in a separate flask
Pd2(dba)3 with 1,2-bis(diphenylphosphino)ethane (dppe), to a solution of 12, we obtained the
desired 4-allyl-oxazolone 13 in quantitative yields. The subsequent opening of the oxazolone ring
with trifluoroacetic acid afforded the free quaternary amino acid 14, which was converted into the
corresponding N-protected derivatives 15-17. Notably, this protocol was successfully applied to the
gram-scale synthesis of the zwitterionic amino acid 14 without any decrease in the overall yield.
Scheme 3. Synthesis of the N-protected quaternary amino acids 15-17
Reagents and conditions: (i) allyl chloroformate, Et3N, THF, 0 °C; (ii) Pd2(dba)3*CHCl3, 1,2-
bis(diphenylphosphino)ethane (dppe), THF; (iii) TFA, 100 °C; (iv) compound 15: (Cbz)2O,
CH3CN; compound 16: (Boc)2O, THF; compound 17: Fmoc-Cl, THF/H2O.
The subsequent condensation reaction turned out to be troublesome. To the best of our knowledge
no examples of condensation between 5-alkynyl proline esters and quaternary N-protected α-amino
acids have been previously reported in literature, whereas N-Boc-α-cyclopentyl glicine and a N-
Boc-valine were shown to couple in good yield under standard conditions27. Extending the search to
5-substituted proline derivatives, apart from our works on 5-allyl proline10a, 13, no examples of
condensation with quaternary amino acids were known, while tertiary amino acids carrying an allyl
or a benzyl moiety at Cα were shown to couple both with 5-vinyl and 5-allyl proline esters28.
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Following our previous experience on similar substrates13, the first experiments were carried out by
performing a condensation reaction between (2S,5R)-5-ethynylpyrrolidine-2-methylester 3 and N-
Cbz-α-allyl-α-benzyl amino acid 15 in the presence of PyBrop as condensing agent (Scheme 4).
Despite the screening of various solvents [CH2Cl2, tetrahydrofuran (THF), N,N-dimethylformamide
(DMF)], the application of conventional and microwave-assisted heating, and attempts to force the
reaction conditions by increasing the temperature (from room temperature to 100 °C), and
prolonging the reaction time (up to 48 hours), it has never been possible to isolate the desired
dipeptide 18. We recovered only unreacted starting material along with variable amounts (8–40%)
of by-products I and II. The N-carboxyanhydride I was likely formed by nucleophilic attack of the
carbamate carbonyl oxygen onto the activated carboxylic group of 15 followed by water addition on
the intermediate imminium ion and final elimination of benzyl alcohol, while the symmetric
anhydride II results from self-condensation of the quaternary amino acid. These by-products turned
out to be stable towards flash chromatography on silica gel, and fully unreactive in our attempts to
separately condense them with 5-ethynyl proline 3.
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To overcome these drawbacks, we performed an extensive screen of condensing agents, such as
N,N-dicyclohexylcarbodiimide (DCC) alone or in the presence of N-hydroxysuccinimide (NHS),
isobutyl chloroformate (IBCF), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole
(HOAt), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), and (1-cyano-2-ethoxy-2-
oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU).
Unfortunately, the outcome of the reaction remained unchanged, since only DCC afforded traces (<
4%) of a product whose 1H-NMR, albeit complicated by the presence of conformers, was in
agreement with the structure of dipeptide 18.
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Scheme 4. Condensation reaction
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In order to understand whether the difficulties encountered in the condensation reaction were
ascribable to the quaternary amino acid 15 or to the 5-alkynylproline 3, the latter derivative was
reacted with enantiopure (S)-N-Cbz-α-vinyl phenylalanine 2129. Applying our standard reaction
condition, i.e. the use of PyBrop in CH2Cl2 at rt, the reaction proceeded smoothly, allowing the
isolation of the dipeptide 22 in unoptimized 50% yield. This result indicates that probably the
presence of the allyl moiety could make the amino acid 15 less prone to condensation by shielding
the carbonyl face from attack of the N-nucleophile. By reasoning that different protecting groups on
the amine function could also affect the conformational properties of the quaternary amino acid, we
synthesized the N-(tert-butoxycarbonyl)- and N-(fluorenyl-9-methoxycarbonyl)-protected
derivatives 16 and 17.
Although the N-Boc derivative 16 was shown to be unreactive towards the condensation with 5-
alkynyl proline 3, giving only the N-carboxyanhydride side product I, the first attempt to react the
N-Fmoc-protected amino acid 17 allowed the isolation of the desired dipeptide 20 with an
encouraging 42% yield. By carrying out a study of the reaction parameters similar to that
previously reported, we eventually found experimental conditions that allowed to improve the yield
of the condensation up to 70% by the use of BOP-Cl as condensing agent and a reaction
temperature of 50 °C.
The following key reaction, i.e. the ring closing enyne metathesis of dipeptide 20 turned out to be a
further obstacle to overcome (Scheme 5). An extensive experimentation was carried out by the
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screening of the Grubbs catalysts highlighted in Scheme 5, varying reaction parameters such as
catalyst loading (5–30%), temperature (from rt to 120 °C), heating mode (conventional or MW-
assisted), solvent, and concentration (0.01–0.1M). Moreover, in order to preserve the catalyst
activity, especially when heating the reactions in high-boiling aprotic solvents (dichloroethane,
toluene), a slow addition of the catalyst to the reaction mixture by the use of a syringe pump was
tested. Unfortunately, we have never been able to isolate the desired cyclic diene 23 in yields higher
than 10%, achieved only by the use of the phosphine-free Hoveyda-Grubbs catalyst HG2 in
refluxing toluene.
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Scheme 5. RCEYM experimentation
Ph
CO2Me
CO2Me
CO2Me
"Ru"
catalyst
N
N
N
O
O
O
NHFmoc
NHFmoc
NHFmoc
23
Ph
24
Ph
Ph
20
(10% with HG2)
traces (with G1 and G2)
MesN
Cl
NMes
MesN NMes
Cl
PCy3
Ru
PCy3
G1
MesN
Cl
NMes
Cl
Cl
N
Ru
O
Ru
Ph
Ru
Cl
Ph
Ph
Br
Cl
Cl
N
PCy3
G2
Br
G3-BrPy
HG2
Mes =
Grubbs Catalysts
The use of third-generation catalyst G3-BrPy, reported to be a fast metathesis initiator30, resulted in
the complete recovery of unreacted starting material, whilst G1 and G2 afforded only traces of aryl-
functionalized diene 24. The isolation of this side-product led us to reason about a plausible
mechanism leading to its formation. Though the diene 24 could formally be obtained by a cross
metathesis reaction of the desired scaffold 23 with the benzylidene residue of G1 and G2 Grubbs
catalysts, the low reactivity showed by the dipeptide 20 in the RCEYM step suggested us an
alternative synthetic pathway (Scheme 6). We speculated that the side-product 24 could also be
generated by an initial conversion of the dipeptide 20 into the more reactive triene M2 through an
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“exo” cross enyne metathesis (CEYM) reaction31 between the proline triple bond and the
benzylidene moiety of the catalyst. The hypothesized pathway was in agreement with the results
reported by Lee and co-workers in the tandem CM (cross metathesis)/RCM reaction of alkynyl
silyloxy-tethered enynes32. They observed that, employing sterically hindered mono-, di-, and
trisubstituted alkenes, the initial CM reaction was shown to occur on the alkyne moiety. This
literature precedent could be an indication that the allyl group in dipeptide 20 is sterically hindered,
plausibly by some conformational effect. On the other hand, one can not exclude that the
conformation of the peptide bond in 20 could turn from the usually more stable s-trans to s-cis after
the addition of the Ru-carbene on the double bond, thus inhibiting or slowing down the following
ene-yne metathesis and favoring the alternative yne-ene mechanism. The subsequent RCM of the
intermediate M2, probably more prone to cyclize than the starting dipeptide 20, could allow the
attainment of 24. In order to get some insight into this hypothesis, we reacted 20 in the presence of
10% Hoveyda-Grubbs catalyst HG2 and a stoichiometric amount of styrene in order to maximize
the formation of the benzylidene Ru catalyst as the active species of the catalytic cycle. It was
indeed fruitful that this control experiment was performed, as the TLC monitoring of the reaction,
after few minutes, showed the formation of the aryl-functionalized derivative 24, which was
isolated in 42% yield. The reaction produced also a small amount of diene 23 (<10%), unreacted
starting material (15%), and 1,2-diphenylethene (12%), which arises from homocoupling of styrene,
while the expected triene 25 was not isolated, thus supporting our speculation that once formed, it
immediately reacts to give the RCM product 24.
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Scheme 6. Hypothesized mechanism for the formation of compound 24
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In the light of these findings we next tried to extend the scope of the reaction to p-functionalized
styrene derivatives. If successful, this could pave the way to a direct preparation of seven-
membered lactam rings endowed with variously substituted aromatic linkers that could be
conjugated with differently functionalized compounds. As a proof of concept we repeated the same
procedure with 4-vinylaniline derivatives, which can be regarded as models of “versatile linkers”.
Indeed, an opportunely protected p-amino group on the newly introduced aromatic moiety could be
exploited, after deprotection, both to directly conjugate a bioactive compound or to introduce a
suitable spacer between the RGD-based unit and the bioactive compound. Alternatively, the
aromatic amine could be easily converted into an azide and submitted to a Huisgen azide-alkyne
1,3-dipolar cycloaddition33. We initially decided to explore the feasibility of the domino
CEYM/RCM protocol in the presence of N-(4-vinylphenyl)acetamide as a test compound. Applying
our optimized reaction conditions, i.e. slow addition of the HG2 catalyst (0.05 eq.) to a solution of
dipeptide 20 in the presence of stoichiometric amount of the styrene derivative 26, we isolated the
desired functionalized azabicycloalkane scaffold 27 in 54% yield (Scheme 7). This result is
particularly remarkable, since the preparation of our previously reported 5-functionalized scaffold
S213, differing substantially only for the position of the diene system, required 7 synthetic linear
steps, and was obtained in 32% overall yield starting from the corresponding dipeptide precursor
S134.
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Scheme 7. Domino CEYM/RCM protocol for the preparation of functionalized azabicycloalkane
4
5
scaffolds
6
7
H3COCHN
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CO2Me
N
O
(54%)
O
H3COCHN
H
N
26
TBDMSO
O
27
NHFmoc
CO2Me
Ph
N
H
HG2-catalyzed
CEYM/RCM
CO2Me
H
N
N
H
O
TBDMSO
H2
Pd/C(en)
NHFmoc
O
Ph
NHFmoc
(77%)
20
MeOH
(56%)
CO2Me
Ph
N
30
O
3
O
TBDMSO
N
H
2.0%
H6
NHFmoc
31
29a (3R)
29b (3S)
28
H7
Ph
CO2Me
6.5%
10
7
6
N
H
Ref 13
3.0%
O
Hb
Hb
7 steps
yield: 32%
CO2Me
CO2Me
1.7%
N
N
NHCbz
O
CbzN
2'
2'
O2N
O
CH2Ph
NHCbz
O
Ph
S1
S2
NOESY correlations observed
for 31 (curved arrows)
Eventually, in order to explore the feasibility of introducing a spacer between the RGD-based
azabicycloalkane scaffolds and the bioactive compounds, the experimentation was extended to the
use of the O-protected 2-hydroxy-N-(4-vinylphenyl)acetamide derivative 28. This choice was
mainly dictated by the prospect of modulating the length of the spacer and, at the same time,
changing the nature of the functional group acting as a handle for further synthetic manipulations.
Reaction of 4-vinylaniline with O-TBS-α-hydroxy acetyl chloride gave the N-acetyl derivative 28,
which reacted smoothly with dipeptide 20 using the same experimental conditions as before giving
the desired scaffolds 2935 in almost unchanged yields. In order to study the reactivity of the diene
system towards the reduction reaction required for the attainment of the corresponding saturated
bicyclolactam scaffolds, compound 29a was submitted to catalytic hydrogenation. Given the high
synthetic value of 29a, to avoid the known undesiderable deprotection of the O-TBDMS protecting
group in the presence of Pd/C36, we performed the reaction using Pd/C poisoned with
ethylenediamine (Pd/C(en)). This Pd/C–ethylenediamine complex, which was successfully
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employed in the chemoselective reduction of various reducible functionalities leaving intact the
TBDMS group37, allowed the isolation of compound 30, as a single stereoisomer, with 77% yield.
The configurational assignment of the newly created C6 stereocenter was carried out on the N-Cbz
derivative 31, which was synthesized, exclusively for analytical purpose, in order to get an easier
analysis of NOESY spectrum. The magnetization transfer between H7 and Hb and H7 and H2’
allowed to verify the absolute R-configuration at the ring fusion carbon (Scheme 7). In a similar
way, the NOE between H10 and H2’ confirmed the S absolute configuration at C10. Diagnostic for
the absolute configuration at C6 is the presence of an NOE between H6 and H7 (see supporting
information for details). This data was in agreement with the results obtained by Lubell et al. on
similar azabicycloalkane scaffolds15. Indeed, they observed that a magnetization transfer between
vicinal protons only occurred when the vicinal protons were oriented towards the same side of the
seven-membered lactam ring.
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Computational studies
Computational studies designed to investigate the ability of the C6-functionalized azabicycloalkane
scaffolds to adopt reverse-turn conformations were performed on the N-acetyl-N’-methylamide
dipeptide analogues 32a and 32b, differing for the absolute configuration at C6 (Supporting
Information, Figure S4). Both compounds were characterized by the presence of capping groups on
the N- and C-termini suitable for the definition of the various turns and H-bond parameters. The
preferred conformations of each stereoisomeric scaffold were compared to the structures of the
parent 7,5-fused azabicyclolactam derivative 33.
The quantitative characterization of the turn propensity of the functionalized azabicycloalkane
scaffolds showed that these systems are more effective as reverse-turn mimetics than as β- or γ-turn
mimics, in agreement with the results obtained for the unsubstituted bicyclic lactam 33. In fact, the
percentages of conformers with a torsion angle β (absolute value) of less than 60° are within the
range 90-97%, whereas the percentages of conformers forming intramolecular hydrogen bonds
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(stabilizing either a γ-turn or a β-turn) are significantly lower (0-25% for γ-turn, 0-2% for β-turn,
see Table S2). The analysis of the Cαi-Cαi+3 distance (dα) confirms that the reverse-turn mimetic
scaffolds induce rather ‘open’ turns. Accordingly, the lowest energy conformer of 33, 32a and 32b
features an open turn, which induces the peptide backbone to reverse direction (Figure S5).
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Conclusions
We have reported a new synthetic approach to obtain 7,5-fused 2-oxo-1-azabicycloalkane scaffolds
characterized by the presence of a p-substituted phenethyl side-chain at C6 position of the seven-
membered lactam ring. The synthetic sequence is based on a domino CEYM/RCM reaction that
allowed the one-pot conversion of the dipeptide 20 into the corresponding C6-functionalized
bicyclic systems 27 and 29 in 54-56% overall yield. As a proof of concept, compound 29a,
endowed with a further functionalizable side-chain, was submitted to catalytic hydrogenation and
converted to the saturated azabicyclolalkane derivative 30. Computational studies proved that the
functionalization of the seven-membered lactam ring at C6 position does not alter the
conformational properties of the scaffolds. Thus the newly synthesized azabicycloalkane scaffolds
can be viewed as useful templates for the synthesis of cRGD-based bioconjugates that may find
promising applications for targeted drug delivery, theranostic or cancer cell labelling.
Experimental Section
General remarks
All chemicals were of reagent grade and were used without further purification. Solvents were
purified according to the guidelines in Purification of Laboratory Chemicals38. All solvents were
freshly distilled from the appropriate drying agent. THF, and toluene were distilled from
sodium/benzophenone ketyl; TEA and DCM from CaH2. Reactions requiring anhydrous conditions
were performed under N2. Yields were calculated for compounds purified by flash chromatography
and judged homogeneous by thin-layer chromatography, NMR, and mass spectrometry. Thin layer
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chromatography was performed on Kieselgel 60 F254 (Merck) glass Plate eluting with solvents
indicated, visualized by a 254 nm UV lamp, and stained with aqueous ceric molybdate solution or
iodine and a solution of 4,4’-methylenebis-N,N-dimethylaniline, ninhydrin, and KI in an aqueous
ethanolic solution of AcOH. Flash chromatography was performed on Merck Kieselgel 60 (230–
400 mesh). Optical rotations [α]D were measured in a cell of 5 cm path length and 1 mL capacity
with a Jasco DIP-1000 polarimeter. Infrared spectra were recorded on a Perkin–Elmer ATR-FTIR
1600 series spectrometer using neat samples. Mass spectra were acquired with a Thermo Finnigan
Q Exactive (API- HESI) FT Orbitrap mass spectrometer. Glassware for all reactions was oven-dried
at 110 °C and cooled in a desiccator, or flame-dried and cooled under inert atmosphere prior to use.
Liquid reagents and solvents were introduced by oven-dried syringes through septa-sealed flasks
under an inert atmosphere.
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NMR spectroscopic methods
Nuclear magnetic resonance spectra were acquired using a Bruker Avance 400 MHz spectrometer
equipped with Bruker’s TopSpin 1.3 software package and a Bruker Avance III HD 500 MHz
spectrometer equipped with Bruker’s TopSpin 3.5 (patch level 7) and provided with a high-
sensitivity 5mm TCI cryoprobe. The abbreviatons s, d, t, q, br s, and m stand for the resonance
multiplicities singlet, doublet, triplet, quartet, broad singlet, and multiplet, respectively. In the peak
listing of 13C spectra abbreviations s and t refer to zero and two protons attached to the carbons, as
determined by DEPT-135 experiments. Phase-sensitive 2D-NOESY experiments were performed at
298K by using noesygpph pulse program from the Bruker library (mixing time of 0.5s or 0.8s).
Sample temperatures were controlled with the variable-temperature unit of the instrument.
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Synthesis of (2S)-dimethyl 5-oxopyrrolidine-1,2-dicarboxylate (7)
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8
(S)-methyl 5-oxopyrrolidine-2-carboxylate (4.00 g, 28.0 mmol) was solubilized in dry THF (70.0
mL, 0.40 M) under nitrogen atmosphere and the reaction mixture was cooled at -78 °C. Methyl
chloroformate (2.38 mL, 30.8 mmol) and a 1 M solution of LiHMDS in THF (30.8 mL, 30.8 mmol)
were added in three portions at 40 minutes intervals. Formation of the product was monitored by
TLC analysis, Rf = 0.58 (AcOEt/MeOH 90:10). After the last addition, the mixture was stirred for
15 minutes at -78 °C and then was warmed to 0 °C. Afterward 56 mL of a 10% HCl aqueous
solution were added at 0 °C and the mixture was allowed to warm to room temperature. The organic
solvent was evaporated under reduced pressure, 84 mL of 10% HCl solution were added and the
aqueous phase was extracted three times with AcOEt. The organic phases were collected, dried with
anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The crude
mixture was purified by flash chromatography (AcOEt/hexane, 70:30) to obtain pure compound 7
as a yellow oil (4.67 g, 83 %). Rf = 0.27; [α]D22 –37.0 (c = 0.5, CDCl3); IR (neat, cm-1) 2957, 1796,
1716, 1200, 1179, 1047, 1032; 1H NMR (CDCl3, 400 MHz) δ 2.11 (dddd, J = 2.6, 3.2, 9.4, 13.3 Hz,
1H), 2.39 (dddd, J = 9.3, 9.4, 10.6, 13.3 Hz, 1H), 2.54 (ddd, J = 3.2, 9.3, 17.6 Hz, 1H), 2.68 (ddd, J
= 9.4, 10.6, 17.6 Hz, 1H), 3.81 (s, 3H), 3.89 (s, 3H), 4.72 (dd, J = 2.6, 9.4 Hz, 1H); 13C{1H} NMR
(CDCl3, 100 MHz) δ 22.2 (t), 31.5 (t), 53.2, 54.3, 59.0, 152.3 (s), 171.9 (s), 173.1 (s); HRMS (ESI)
calculated for C8H12NO5 [M+H] + 202.0710, found 202.0709 (ꢀ = 0.7 ppm).
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Synthesis of dimethyl (2S)-5-methoxypyrrolidine-1,2-dicarboxylate (8)
Compound 7 (6.43 g, 32.0 mmol) was solubilized in dry THF (80.0 mL, 0.40 M) under nitrogen
atmosphere and the reaction mixture was cooled at -78 °C. A 1 M solution of Li(Et)3BH in THF
(35.2 mL, 35.2 mmol) was added dropwise. Formation of the product was monitored by TLC
analysis, Rf = 0.25 (Toluene/AcOEt 60:40). After 10 minutes the reaction was warmed to 0 °C and
45 mL of a NaHCO3 saturated aqueous solution and 7.80 mL of a 30% wt H2O2 aqueous solution
were added. Afterwards the mixture was allowed to warm to room temperature and was stirred for
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30 minutes. The organic solvent was evaporated under reduced pressure, the residue was recovered
with a NaCl saturated solution and was extracted three times with AcOEt. The organic phases were
collected, dried with anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced
pressure to obtain the product as a colorless oil without further purification.
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The crude mixture (5.54 g, 27.3 mmol) was dissolved in dry MeOH (90.9 mL, 0.30 M) under
nitrogen atmosphere and added with trimethyl orthoformate (22.4 mL, 205 mmol) and Pyridinium
p-toluenesulfonate (PPTS) (6.87 g, 27.3 mmol). Formation of the product was monitored by TLC
analysis, Rf = 0.35 (hexane/AcOEt 60:40). After reaction completion the solvent was evaporated
under reduced pressure, the residue was recovered with a NaHCO3 saturated aqueous solution and
was extracted three times with Et2O. The organic phases were collected, dried with anhydrous
Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The crude product 8 was
reacted in the next step without further purification. (5.62 g, 81 % over two steps). Rf = 0.35.
Synthesis of (2S)-dimethyl 5-((trimethylsilyl)ethynyl)pyrrolidine-1,2-dicarboxylate (9)
Compound 8 (4.18 g, 19.2 mmol) and bis(trimethylsilyl)acetylene (6.56g, 38.5 mmol) were
solubilized in dry DCM (96.2 mL, 0.20 M) under nitrogen atmosphere and the reaction mixture was
cooled at -45 °C. A 1M solution of SnCl4 in DCM (25.0 mL, 25.0 mmol) and AlCl3 (3.60 g, 26.9
mmol) were added. The reaction mixture was allowed to warm gradually to room temperature.
Formation of the products was monitored by TLC analysis, Rf = 0.35, Rf = 0.26 (hexane/AcOEt
70:30). After 12 hours the mixture was slowly added to a NaHCO3 saturated aqueous solution (128
mL). Afterwards 64 mL of a 1 M HCl solution were added and the aqueous phase was extracted
three times with AcOEt. The organic phases were collected, dried with anhydrous Na2SO4, filtered,
and the solvent was evaporated under reduced pressure. The crude mixture was purified by flash
chromatography (hexane/AcOEt, 70:30) to obtain diastereoisomers 9.
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Diastereoisomer 9a (2S,5R): Colorless oil (2.24 g, 41%). Rf = 0.26 (AcOEt/hexane 70:30); [α]D
+22.4 (c = 1.0, CDCl3); IR (neat, cm-1) 2956, 2900, 2174, 1741, 1704, 1195, 1171, 1038; 1H NMR
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(dmso-d6, 90 °C, 400 MHz) δ 0.15 (s, 9H), 1.93 (m, 1H), 2.03 (m, 1H), 2.16 (m, 1H), 2.27 (m, 1H),
3.63 (s, 3H), 3.67 (s, 3H), 4.30 (app t, J = 7.2 Hz, 1H), 4.57 (dd, J = 3.4, 7.3 Hz, 1H); 13C{1H}
NMR (dmso-d6, 90 °C, 100 MHz) δ 0.8 (3C), 29.6 (t), 33.4 (t), 50.5, 52.5, 53.0, 60.2, 87.4 (s),
107.1 (s), 154.9 (s), 172.7 (s); HRMS (ESI) calculated for C13H21NNaO4Si [M+Na] + 306.1132,
found 306.1130 (ꢀ = 0.8 ppm). Diastereoisomer 9b (2S,5S): Colorless oil (1.85 g, 34%). Rf = 0.35
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(AcOEt/hexane 70:30); [α]D –41.5 (c = 1.0, CDCl3); IR (neat, cm-1) 2956, 2900, 2171, 1748,
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1706, 1192, 1173, 1069; H NMR (dmso-d6, 90 °C, 400 MHz) δ 0.15 (s, 9H), 1.92 (m, 1H), 2.01
(m, 1H), 2.17 (m, 1H), 2.38 (m, 1H), 3.63 (s, 3H), 3.66 (s, 3H), 4.35 (dd, J = 1.5, 8.9 Hz, 1H), 4.62
(dd, J = 1.4, 7.9 Hz, 1H); 13C{1H} NMR (dmso-d6, 90 °C, 100 MHz) δ; 0.7, 29.4 (t), 32.4 (t), 50.2,
52.7, 53.0, 59.6, 87.2 (s), 107.1 (s), 154.8 (s), 172.9 (s); HRMS (ESI) calculated for
C13H21NNaO4Si [M+Na] + 306.1132, found 306.1129 (ꢀ = 0.9 ppm).
Synthesis of (2S,5R)-dimethyl 5-ethynylpyrrolidine-1,2-dicarboxylate 10a
Compound 9a (1.50 g, 5.29 mmol) was solubilized in dry THF (88.2 mL, 0.06 M) under nitrogen
atmosphere and the reaction mixture was cooled at -20 °C. A 1 M solution of tetrabutylammonium
fluoride in THF (5.82 mL, 5.82 mmol) was added and the reaction mixture was warmed to 0 °C in
20 minutes. Formation of the product was monitored by TLC analysis, Rf = 0.41 (hexane/AcOEt
50:50). After 30 minutes 132 mL of an NH4Cl saturated aqueous solution were added and the
mixture was extracted three times with Et2O. The organic phases were collected, dried with
anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The crude
mixture was purified by flash chromatography (hexane/AcOEt, 60:40) to obtain pure compound
10a as a colorless oil (1.01 g, 90 %). Rf = 0.35 (AcOEt/hexane 60:40); [α]D22 +9.3 (c = 1.0, CDCl3);
1
IR (neat, cm-1) 3240, 2955, 2919, 2850, 2117, 1736, 1689, 1189, 1120, 1038; H NMR (dmso-d6,
90 °C, 400 MHz) δ 1.92–2.08 (m, 2H), 2.15–2.34 (m, 2H), 2.98 (s, 1H, partially masked by H2O
signal), 3.63 (s, 3H), 3.68 (s, 3H), 4.3 (app t, J = 6.8 Hz, 1H), 4.57 (m, 1H); C{1H} NMR (dmso-
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d6, 90 °C, 100 MHz) δ 29.7 (t), 33.1 (t), 49.7, 52.5, 53.1, 60.3, 73.5, 84.5 (s), 154.9 (s), 172.8 (s);
HRMS (ESI) calculated for C10H13NNaO4 [M+Na] + 234.0737, found 234.0736 (ꢀ = 0.4 ppm).
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Synthesis of (2S,5R)-methyl 5-ethynylpyrrolidine-2-carboxylate (3)
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Compound 10a (1.00 g, 4.73 mmol) was solubilized in dry DCM (4.73 mL, 1.00 M) under nitrogen
atmosphere. A solution of iodotrimethylsilane (TMSI) in DCM (14.2 mL, 5.68 mmol) was added
and the mixture was heated to 65 °C. Formation of the product was monitored by TLC analysis, Rf
= 0.30 (hexane/AcOEt 50:50). After 7 hours the solvent was evaporated under reduced pressure and
crude mixture was purified by flash chromatography (hexane/AcOEt, 50:50) to obtain pure
compound 3 as a yellow oil (543 mg, 75 %). Rf = 0.30; [α]D22 +16.0 (c = 1.0, CDCl3); IR (neat, cm-
1
1) 3274, 2952, 2114, 1733, 1621, 1211, 1171, 1076; H NMR (CDCl3, 400 MHz) δ 1.89 (m, 1H),
2.06–2.25 (m, 3H), 2.35 (d, J = 2.1 Hz, 1H), 3.10 (s, 1H, exchanges with D2O), 3.77 (s, 3H), 3.86
(app t, J = 6.8 Hz, 1H), 3.94 (m, 1H); C{1H} NMR (CDCl3, 100 MHz) δ 29.8 (t), 33.5 (t), 49.5,
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52.8, 59.9, 72.1, 84.9 (s), 175.0 (s); HRMS (ESI) calculated for C8H12NO2 [M+H] + 154.0863,
found 154.0862 (ꢀ = 0.3 ppm).
Synthesis of 4-benzyl-2-phenyloxazol-5(4H)-one (11)
A solution of N-benzoylphenylalanine (10.0 g, 37.2 mmol) in dry DCM (200 mL) and Et2O (80
mL) was added dropwise over 20 minutes to a stirred solution of DCC (7.67 g, 37.2 mmol) in dry
DCM (124 mL, 0.30 M), at 0 °C, under nitrogen atmosphere. Reaction completion was monitored
by TLC analysis Rf = 0.55 (hexane/AcOEt 85:15). While keeping the reaction mixture at 0 °C, a
vacuum filtration with Gooch apparatus was performed. The residue was washed with Et2O and the
solvent was evaporated under reduced pressure. The crude mixture was purified by crystallization
with light petroleum ether to obtain 11 as white solid (7.46 g, 80%). The product was matched with
reported data39.
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Synthesis of allyl (4-benzyl-2-phenyloxazol-5-yl) carbonate (12)
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To a solution of compound 11 (4.00 g, 15.9 mmol) in dry THF (159 mL, 0.10 M) under nitrogen
atmosphere at 0 °C, NEt3 (2.44 mL, 17.5 mmol) and allyl chloroformate (1.86 mL, 17.5 mmol)
were added. Afterwards, the reaction mixture was warmed to room temperature and the formation
of the product was monitored by TLC analysis, Rf = 0.25 (hexane/AcOEt 95:05). After 24 hours 80
mL of H2O were added, the organic solvent was evaporated under reduced pressure and the aqueous
layer was extracted three times with Et2O. The organic phases were collected, dried with anhydrous
Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The crude mixture was
purified by flash chromatography (hexane/AcOEt, 95:05) to obtain pure compound 12 as an
amorphous white solid (5.13 g, 96 %). Rf = 0.25; IR (neat, cm-1) 3031, 2934, 1781, 1664, 1217,
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1119; H NMR (CDCl3, 400 MHz) δ 3.91 (s, 2H), 4.72 (app d, J = 5.9 Hz, 2H), 5.39 (dd, J = 0.9,
10.4 Hz, 1H), 5.45 (dd, J = 0.9, 17.2 Hz, 1H), 5.96 (ddt, J = 5.9, 10.4, 17.2 Hz, 1H), 7.19–7.38 (m,
5H), 7.35–7.51 (m, 3H), 7.92–8.03 (m, 2H); 13C{1H} NMR (CDCl3, 100 MHz) δ 31.9 (t), 70.9 (t),
120.9 (t), 123.8 (s), 126.4, 126.9, 127.5 (s), 128.9, 129.1, 129.3, 130.6, 130.8, 137.9 (s), 146.7 (s),
151.6 (s), 155.6 (s); HRMS (ESI) calculated for C20H18NO4 [M+H] + 336.1230, found 336.1229 (ꢀ
= 0.3 ppm).
Synthesis of 4-allyl-4-benzyl-2-phenyloxazol-5(4H)-one (13)
1,2-bis(diphenylphosphino)ethane (DPPE) (286 mg, 0.725 mmol) and Tris(dibenzylideneacetone)
dipalladium(0)-chloroform adduct (Pd2(dba)3CHCl3) (250 mg, 0.241 mmol) were solubilized in the
least amount of dry THF under nitrogen atmosphere and were maintained under stirring for 30
minutes. Afterwards the mixture was added dropwise to a solution of compound 12 (1.62 g, 4.83
mmol) in dry THF (48.3 mL, 0.10 M) under nitrogen atmosphere. Formation of the product was
monitored by TLC analysis, Rf = 0.59 (hexane/AcOEt 85:15). After 30 minutes phosphate buffer
was added until the formation of a black precipitate was observed. The organic solvent was
evaporated under reduced pressure and the aqueous layer was extracted three times with DCM. The
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organic phases were collected, dried with anhydrous Na2SO4, filtered, and the solvent was
evaporated under reduced pressure. The crude mixture was purified by flash chromatography
(hexane/AcOEt, 90:10) to obtain pure compound 13 as a pale yellow oil (1.41 g, quantitative). The
product was matched with reported data23.
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Synthesis of 2-amino-2-benzylpent-4-enoic acid (14)
Compound 13 (1.40 g, 4.80 mmol) was dissolved in TFA/water 90:10 (48.0 mL, 0.10 M) and
heated to 100 °C for 12h. After reaction completion, the solvent was evaporated under reduced
pressure, the residue was recovered with water and washed three times with DCM. The aqueous
phase was evaporated under reduced pressure and the crude mixture was purified by ion-exchange
chromatography with AMBERLITE IR-120(PLUS)® resin as stationary phase. The crude mixture
was dissolved in H2O and loaded onto the column. The stationary phase was washed with H2O until
the eluted solution turned from acidic to neutral, then elution with NH3 10% aqueous solution and
evaporation of the solvent under reduced pressure afforded 14 as a white amorphous solid (985 mg,
quantitative). IR (neat, cm-1) 3242, 3032, 1607, 1584, 1082; H NMR (CD3OD, 400 MHz) δ 2.46
1
(dd, J = 8.4, 14.4 Hz, 1H), 2.80 (dd, J = 6.3, 14.4 Hz, 1H), 2.99 (d, J = 14.1 Hz, 1H), 3.31 (d, J =
14.1 Hz, 1H), 5.22–5.30 (m, 2H), 5.90 (dddd, J = 6.3, 8.4, 10.3, 16.7 Hz, 1H), 7.28–7.36 (m, 5H);
13C{1H} NMR (CD3OD, 100 MHz) δ 41.1 (t), 42.0 (t), 64.8 (s), 120.2 (t), 127.5, 128.7, 130.5,
+
131.5, 134.9 (s), 173.6 (s); HRMS (ESI) calculated for C12H16NO2 [M+H] 206.1176, found
206.1177 (ꢀ = 0.4 ppm).
Synthesis of 2-benzyl-2-(((benzyloxy)carbonyl)amino)pent-4-enoic acid (15)
A solution of 14 (60 mg, 0.29 mmol) in dry CH3CN (2.9 mL, 0.10 M) under nitrogen atmosphere,
was added with tetramethyl ammonium hydroxide pentahydrate (105 mg, 0.580 mmol). The
reaction mixture was stirred for 45 minutes, then Cbz2O (166 mg, 0.580 mmol ) was added.
Formation of the N-protected derivative was monitored by TLC analysis, Rf = 0.35 (AcOEt/MeOH
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95:05). After 24 hours, the solvent was evaporated under reduced pressure, the residue was
recovered with a saturated aqueous solution of Na2CO3 and washed three times with Et2O. The pH
of the aqueous phase was adjusted to 1 with a HCl 1N solution and extracted three times with
AcOEt. The organic layer was dried with anhydrous Na2SO4, filtered and evaporated under reduced
pressure to obtain compound 15 as a colorless oil without further purification (94 mg, 96%). The
product was matched with reported data40.
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Synthesis of 2-benzyl-2-((tert-butoxycarbonyl)amino)pent-4-enoic acid (16)
To a solution of 14 (40 mg, 0.19 mmol) in dry THF (1.90 mL, 0.10 M) under nitrogen atmosphere,
Di-tert-butyl dicarbonate (56 mg, 0.25 mmol) and catalytic amount of DMAP were added.
Formation of the N-protected derivative was monitored by TLC analysis, Rf = 0.45 (hexane/AcOEt
60:40 + 2% AcOH). After 24 hours, the solvent was evaporated under reduced pressure and the
crude mixture was purified by flash chromatography (hexane/AcOEt 60:40 + 2% AcOH) to obtain
pure compound 16 as a white amorphous solid (57 mg, 98%). The product was matched with
reported data41.
Synthesis of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-benzylpent-4-enoic acid (17)
Compound 14 (1.50 g, 7.31 mmol) was suspended in 1:1 THF/H2O (43.0 mL, 0.17 M) at 0 °C and
was added with N-(9-Fluorenylmethoxycarbonyloxy)succinimide (2.74 g, 8.04 mmol) and Na2CO3
(1.55 g, 14.6 mmol). After 30 minutes the reaction mixture was warmed to room temperature.
Formation of the N-protected derivative was monitored by TLC analysis, Rf = 0.35 (AcOEt/MeOH
95:05). After 24 hours the pH was adjusted to 2 with a HCl 1N solution and extracted three times
with AcOEt. The organic layer was dried with anhydrous Na2SO4, filtered and evaporated under
reduced pressure. The crude mixture was purified by flash chromatography (AcOEt/MeOH, 95:05)
to obtain pure compound 17 as a white amorphous solid (3.12 g, quantitative). Rf = 0.35. IR (neat,
1
cm-1) 3404, 3067, 3031, 1696, 1686, 1593, 1223, 1076; H NMR (dmso-d6, 90 °C, 400 MHz,
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mixture of rotamers) δ 2.94–3.19 (m, 4H, partially masked by H2O signal), 4.25 (t, J = 6.2 Hz, 1H),
4.39–4.51 (m, 2H), 4.95–5.08 (m, 2H), 5.66 (m, 1H), 6.20–6.37 (m, 1H), 6.96–7.08 (m, 2H), 7.15–
7.23 (m, 3H), 7.33 (td, J = 1.0, 7.4 Hz, 2H), 7.42 (td, J = 1.3, 7.4 Hz, 2H), 7.67 (t, J = 7.2 Hz, 2H),
7.87 (d, J = 7.5 Hz, 2H), the chemical shift of the acid proton could not be unequivocally assigned;
13C{1H} NMR (dmso-d6, 90 °C, 100 MHz, only major rotamer peaks were reported) δ 41.6 (t), 42.7
(t), 47.9, 64.1 (s), 66.2 (t), 109.8 (s), 120.8, 125.8, 127.1, 127.8, 128.4, 128.6, 130.7, 141.7 (s),
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–
144.8 (s), 155.0 (s), 174.1 (s); HRMS (ESI) calculated for C27H24NO4 [M] 426.1711, found
426.1717 (ꢀ = 1.4 ppm).
Synthesis of (2S,5R)-methyl 1-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-benzylhexa-
1,5-dien-2-yl)-5-ethynylpyrrolidine-2-carboxylate (20)
To a solution of 17 (1.00 g, 2.34 mmol) and 3 (358 mg, 2.34 mmol) in dry THF (47.0 mL, 0.05 M),
bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl) (595 mg, 2.34 mmol) and NaHCO3 (197
mg, 2.34 mmol) were added. The reaction mixture was heated to 50 °C and monitored with TLC
analysis, Rf = 0.41 (hexane/AcOEt 60:40). Progressive additions of BOP-Cl (up to 1 eq.) and
NaHCO3 (up to 2 eq.) were performed. After 48 hours the solvent was evaporated under reduced
pressure and the crude mixture was purified by flash chromatography (hexane/AcOEt 65:35) to
obtain pure compound 20 as a white amorphous solid (922 mg, 70%). Rf = 0.36. IR (neat, cm-1)
1
3375, 3286, 3030, 2948, 2254, 2116, 1784, 1754, 1719, 1632, 1167, 1077; H NMR (CDCl3, 400
MHz, mixture of diastereoisomers and rotamers) δ 1.59–1.81 (m, 1.3H), 1.90–2.48 (m, 4.7H), 2.52–
2.74 (m, 0.6H) 2.84–3.46 (m, 2.4H), 3.54–4.00 (m, 4H), 4.18–4.30 (m, 1H), 4.32–4.57 (m, 2.6H),
4.65–4.76 (m, 0.4H), 5.04–5.32 (m, 2.3H), 5.64–5.93 (m, 1.4H), 6.03–6.25 (m, 0.3H), 7.02–7.15
(m, 1H), 7.16–7.27 (m, 4H), 7.29–7.37 (m, 2H), 7.38–7.47 (m, 2H), 7.54–7.66 (m, 2H), 7.75–7.84
(m, 2H); 13C{1H} NMR (CDCl3, 100 MHz, mixture of diastereoisomers and rotamers) δ 26.8 (br, t),
34.6 (br, t), 40.0 (t), 40.3 (t), 40.5 (t), 47.6, 50.1, 50.6, 52.7, 61.9 (br), 65.4 (br, s), 66.8 (t), 74.1
(br), 82.5 (s), 119.8 (t), 119.9 (t), 120.4, 125.4, 125.6, 127.2, 127.4, 127.5 (2C), 128.1, 128.2, 128.4,
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128.8, 130.1 (br), 130.9, 132.8, 133.0, 136.1 (br, s), 136.9 (s), 141.7 (s), 144.1 (s), 144.3 (2C, s),
155.1 (s), 170.3 (s), 170.6 (s), 172.3 (s); HRMS (ESI) calculated for C35H34N2NaO5 [M+Na] +
585.2360, found 585.2349 (ꢀ = 1.8 ppm).
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Synthesis of methyl (2S,5R)-1-((S)-2-benzyl-2-(((benzyloxy)carbonyl)amino)but-3-enoyl)-5-
ethynylpyrrolidine-2-carboxylate (22)
To a solution of 21 (115 mg, 0.354 mmol) and 3 (54 mg, 0.35 mmol) in dry DCM (1.20 mL, 0.30
M), Bromotripyrrolidinophosphonium hexafluorophosphate (PyBrop; 182 mg, 0.396 mmol), N,N-
diisopropylethylamine (DIEA; 0.15 mL, 0.89 mmol) and DMAP (17 mg, 0.14 mmol) were added.
After 26 hours the solvent was evaporated under reduced pressure and the crude mixture was
purified by flash chromatography (hexane/AcOEt 70:30) to obtain pure compound 22 as a white
amorphous solid (83 mg, 50%). Rf = 0.31 (hexane/AcOEt 70:30); [α]D22 +18.70 (c = 0.25, CDCl3);
1
IR (neat, cm-1) 3292, 2952, 2925, 1722, 1638, 1255, 1197, 1166; H NMR (dmso-d6, 400 MHz) δ
1.69 (m, 1H), 1.84–2.00 (m, 2H), 2.29 (m, 1H), 3.07 (d, J = 13.0 Hz, 1H), 3.22 (d, J = 1.8 Hz, 1H),
3.49 (d, J = 13.1 Hz, 1H), 3.66 (s, 3H), 4.30 (m, 1H), 4.85 (d, J = 12.3 Hz, 1H), 4.86 (m, 1H), 5.03
(d, J = 17.8 Hz, 1H), 5.14 (d, J = 10.9 Hz, 1H), 5.26 (d, J = 12.3 Hz, 1H), 5.84 (dd, J = 10.9, 17.7
Hz, 1H), 6.89–7.05 (m, 2H), 7.11–7.25 (m, 3H), 7.29–7.49 (m, 5H), 7.71 (s, 1H); 13C{1H} NMR
(dmso-d6, 75 MHz) δ 27.6 (t), 33.9 (t), 42.5 (t), 50.4, 52.4, 61.4, 64.5 (s), 66.2 (t), 74.5, 83.7 (s),
115.1 (t), 127.1, 128.5, 128.7, 129.2, 131.7, 137.1 (s), 138.0 (s), 139.0, 155.0 (s), 170.5 (s), 172.5
(s); HRMS (ESI) calculated for C27H28KN2O5 [M+K] + 499.1630, found 499.1625 (ꢀ = 1.1 ppm).
General Procedure for domino cross-enyne metathesis/ring-closing metathesis
Compound 20 (1 eq.) and the styrene derivative (2 eq.) were solubilized in Toluene (31.0 mL, 0.017
M) and the solution was heated to 80 °C. Hoveyda Grubbs II generation catalyst (33.4 mg, 10%
mol) was dissolved in Toluene (22.3 mL) and slowly added to the reaction mixture over 7 hours
through the use of a syringe pump. After 1 hour from the end of the addition the solvent was
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evaporated under reduced pressure. Formation of the product was monitored by TLC analysis
(hexane/AcOEt 60:40).
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Synthesis of methyl (3S,9aR)-6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-benzyl-5-oxo-
9-vinyl-2,3,5,6,7,9a-hexahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate (23)
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The title compound was obtained from compound 20 (100 mg, 0.177 mmol) as a side product of the
domino cross-enyne metathesis/ring-closing metathesis reaction. The crude mixture was purified by
flash chromatography (hexane/AcOEt 70:30) to obtain 23 as a yellow amorphous solid (10 mg,
10%). Rf = 0.34 (hexane/AcOEt 70:30); [α]D -11.4 (c = 1.0, CDCl3); IR (neat, cm-1) 3371, 2984,
22
1
2941, 1720, 1694, 1613, 1241, 1158, 1054; H NMR (CDCl3, 400 MHz, mixture of conformers) δ
1.88–2.05 (m, 3H), 2.32 (m, 1H), 2.42 (dd, J = 7.2, 18.3 Hz, 1H), 2.63 (d, J = 18.4 Hz, 1H), 3.03 (d,
J = 14.1 Hz, 1H), 3.62 (d, J = 14.1 Hz, 1H), 3.74 (s, 3H), 4.23 (t, J = 5.7 Hz, 1H), 4.33 (m,1 H),
4.57–4.70 (m, 2H), 4.75–4.86 (m, 2H), 4.99 (d, J = 10.9 Hz, 1H), 5.12 (d, J = 17.2 Hz, 1H), 5.56 (d,
J = 6.4 Hz, 1H), 6.11 (dd, J = 11.0, 17.1 Hz, 1H), 6.96–7.07 (m, 2H), 7.22–7.27 (m, 3H), 7.35 (t, J
= 7.3 Hz, 2H), 7.44 (t, J = 7.3 Hz, 2H), 7.63 (app t, J = 6.4 Hz, 2H), 7.81 (d, J = 7.4 Hz, 2H);
13C{1H} NMR (CDCl3, 100 MHz, mixture of conformers) δ 26.6 (t), 33.3 (t, 2C), 40.1 (t), 47.9,
52.8, 59.5, 59.7 (s), 62.0, 66.3 (t), 115.1 (t), 120.4, 121.6, 125.2, 125.3, 127.0, 127.4, 127.5, 128.2
(2C), 128.6, 131.7, 136.9 (s), 137.7, 140.8 (s), 141.8 (s), 141.9 (s), 144.0 (s), 144.1 (s), 155.1 (s),
+
171.6 (s), 173.3 (s); HRMS (ESI) calculated for C35H34N2NaO5 [M+Na] 585.2360, found
585.2354 (ꢀ = 1.1 ppm).
Synthesis of (3S,9aR)-methyl 6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-benzyl-5-oxo-
9-((E)-styryl)-2,3,5,6,7,9a-hexahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate (24)
The title compound was prepared from compound 20 (100 mg, 0.177 mmol) and styrene (37 mg,
0.36 mmol) according to the general procedure. The crude mixture was purified by flash
chromatography to obtain diastereoisomers 24 (48 mg, 42%).
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Diastereoisomer 24a: Flash chromatography (hexane/AcOEt 65:35). Rf = 0.27. H NMR (CDCl3,
400 MHz) δ 1.91–2.10 (m, 3H), 2.60 (dd, J = 8.7, 13.7 Hz, 1H), 2.68 (dd, J = 11.1, 13.7 Hz, 1H),
2.92 (m, 1H), 3.09 (d, J = 15.1 Hz, 1H), 3.78 (d, J = 15.1 Hz, 1H), 3.79 (s, 3H), 3.91 (dd, J = 4.1,
12.2 Hz, 1H), 4.02 (app t, J = 9.4 Hz, 1H), 4.17–4.25 (m, 2H), 4.79 (dd, J = 5.3, 11.1 Hz, 1H), 4.84
(dd, J = 4.9, 11.1 Hz, 1H), 6.19 (d, J = 16.7 Hz, 1H), 6.49 (d, J = 16.7 Hz, 1H), 7.08–7.25 (m, 10H),
7.29–7.63 (m, 6H), 7.67–7.82 (m, 2H). Diastereoisomer 24b: Flash chromatography
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(hexane/AcOEt 65:35). Rf = 0.26. H NMR (CDCl3, 400 MHz) δ 1.83–2.16 (m, 4H), 2.30 (dd, J =
6.8, 13.8 Hz, 1H), 2.67 (dd, J = 8.5, 13.8 Hz, 1H), 2.92 (m, 1H), 3.17 (d, J = 15.0 Hz, 1H), 3.65 (d,
J = 15.0 Hz, 1H), 3.78 (s, 3H), 3.93 (dd, J = 4.4, 11.6 Hz, 1H), 4.16 (app t, J = 5.1 Hz, 1H), 4.24
(app d, J = 8.2 Hz, 1H), 4.66 (dd, J = 5.3, 11.0 Hz, 1H), 4.81 (dd, J = 6.9, 8.5 Hz, 1H), 4.87 (dd, J =
5.0, 11.0 Hz, 1H), 6.15 (d, J = 17.0 Hz, 1H), 6.32 (d, J = 17.0 Hz, 1H), 7.12–7.25 (m, 10H), 7.27–
7.36 (m, 3H), 7.40 (t, J = 7.4 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.68 (d, J
= 7.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H). 13C{1H} NMR (CDCl3, 100 MHz, mixture of
diastereoisomers 24a and 24b) δ 26.7 (t), 30.1 (t), 33.5 (t), 40.3 (t), 47.9 (t), 52.7, 59.7, 59.8 (s),
62.1, 66.3 (t), 120.4, 120.5, 121.7, 125.2, 125.4, 126.8, 127.1, 127.5 (2C), 128.2 (2C), 128.6, 129.1,
129.3, 130.1, 131.8, 136.9 (s), 137.4 (s), 140.4 8s), 141.7 (s) 141.9 (s), 144.1 (s), 155.1 (s), 171.7
(s), 173.3 (s); IR (neat, cm-1) 2963, 2922, 1725, 1628, 1259, 1078, 1017; HRMS (ESI) calculated
for C41H38N2NaO5 [M+Na] + 661.2673, found 661.2666 (ꢀ = 1.0 ppm).
Synthesis of N-(4-vinylphenyl)acetamide (26)
A solution of vinyl aniline (1.00 g, 8.34 mmol) in dry DCM (83.4 mL, 0.10 M) under nitrogen
atmosphere at 0 °C, was added with acetic anhydride (7.86 mg, 83.4 mmol) and DMAP (1.02 g,
8.34 mmol). The pH was monitored with litmus paper and TEA was used to basify the solution
when necessary. After 10 minutes the reaction mixture is warmed to room temperature and the
formation of the product was monitored by TLC analysis, Rf = 0.37 (hexane/AcOEt 60:40). After 15
minutes the mixture was washed three times with phosphate buffer. The organic layer was dried
with anhydrous Na2SO4, filtered and evaporated under reduced pressure. The crude mixture was
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purified by flash chromatography (hexane/AcOEt 60:40) to obtain compound 26 as a white
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amorphous solid (1.06 g, 79%). The product was matched with reported data42.
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Synthesis of (3S,9aR)-methyl 6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-9-((E)-4-
acetamidostyryl)-6-benzyl-5-oxo-2,3,5,6,7,9a-hexahydro-1H-pyrrolo[1,2-a]azepine-3-
carboxylate (27)
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The title compound was prepared from compound 20 (100 mg, 0.177 mmol) and styrene derivative
26 (57 mg, 0.36 mmol) according to the general procedure. The crude mixture was purified by flash
chromatography to obtain diastereoisomers 27. Diastereoisomer 27a: White amorphous solid (30
mg, 24%). Rf = 0.24 (AcOEt/hexane 60:40); [α]D22 +85.22 (c = 1.00, CDCl3) 1H NMR (CDCl3, 400
MHz, mixture of rotamers) δ 2.08–2.22 (m, 6H), 2.62 (m, 1H), 2.85 (d, J = 17.4 Hz, 1H), 3.41 (d, J
= 13.9 Hz, 1H), 3.54 (m, 1H), 3.69 (d, J = 13.4 Hz, 1H), 3.75 (s, 3H), 4.17–4.29 (m, 2H), 4.51 (m,
1H), 4.75 (d, J = 6.2 Hz, 1H), 4.88 (m,1H), 6.10 (m, 1H), 6.50–6.67 (m, 3H), 6.99–7.11 8m, 2H),
7.16–7.25 (m, 3H), 7.27–7.48 (m, 7H), 7.52 (app t, J = 7.6 Hz, 3H), 7.61 (d, J = 7.1 Hz, 1H), 7.79
(d, J = 7.4 Hz, 2H); 13C{1H} NMR (CDCl3, 100 MHz, mixture of rotamers) δ 25.1, 26.7 (t), 30.1
(t), 33.9 (t), 36.1 (t), 47.6, 52.8, 62.3, 62.7, 62.9 (s), 66.8 (t), 124.8, 125.6, 125.8, 127.3, 127.4 (2C),
127.5, 128.0 82C), 128.6, 128.8, 129.3, 129.6, 133.3 (s), 136.8 (s), 137.2 (s, 2C), 137.9 (s), 141.6
(s), 141.7 (s), 144.3 (s), 144.5 (s), 154.7 (s), 168.7 (s), 172.3 (s), 172.7 (s); IR (neat, cm-1) 3349,
2963, 1741, 1716, 1670, 1260, 1075, 1019; HRMS (ESI) calculated for C43H41N3NaO6 [M+Na] +
718.2888, found 718.2876 (ꢀ = 1.6 ppm). Diastereoisomer 27b: White amorphous solid (38 mg,
30%). Rf = 0.16 (AcOEt/hexane 60:40); [α]D +21.98 (c = 1.00, CDCl3) 1H NMR (CDCl3, 400
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MHz, mixture of rotamers) δ 1.94–2.09 (m, 3H), 2.20 (s, 3H), 2.37 (m, 1H), 2.46 (dd, J = 6.1, 18.2
Hz, 1H), 2.67 (d, J = 18.2 Hz, 1H), 3.05 (d, J = 14.1 Hz, 1H), 3.61 (d, J = 14.1 Hz, 1H), 3.74 (s,
3H), 4.23 (t, J = 5.6 Hz, 1H), 4.39 (br s, 1H), 4.64 (d, J = 5.5 Hz, 2H), 4.78–4.90 (m, 2H), 5.66 (d, J
= 6.1 Hz, 1H), 6.38 (d, J = 16.1 Hz, 1H), 6.43 (d, J = 16.1 Hz, 1H), 6.97–7.05 (m, 2H), 7.22–7.35
(m, 7H), 7.37–7.45 (m, 2H), 7.46–7.56 (m, 3H), 7.62 (app t, J = 6.2 Hz, 2H), 7.77 (app t, J = 6.3
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Hz, 2H); C{1H} NMR (CDCl3, 100 MHz, mixture of rotamers) δ 25.1, 26.7 (t), 30.1 (t), 33.5 (t),
40.3 (t), 47.9, 52.6, 59.7, 59.8 (s), 62.1, 66.3 (t), 120.3, 120.4 (2C), 121.4, 125.2, 125.3, 127.0,
127.3, 127.4, 127.5, 128.2 (2C), 128.4, 128.6, 129.4, 131.7, 133.4 (s), 136.8 (s), 137.9 (s), 140.4 (s),
141.8 (s), 141.9 (s), 144.0 (s), 155.1 (s), 168.8 (s), 171.7 (s), 173.3 (s); IR (neat, cm-1) 3301, 2962,
2923, 1720, 1672, 1629, 1260, 1076, 1021; HRMS (ESI) calculated for C43H41N3NaO6 [M+Na] +
718.2888, found 718.2880 (ꢀ = 1.1 ppm).
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Synthesis of 2-((tert-butyldimethylsilyl)oxy)-N-(4-vinylphenyl)acetamide (28)
To a solution of 2-(tert-Butyldimethylsilyloxy)acetic acid (1.00 g, 5.26 mmol) in dry DCM (6.60
mL, 0.80 M) under nitrogen atmosphere, N-hydroxysuccinimide (667 mg, 5.78 mmol) and DCC
(1.20 g, 5.78 mmol) were added. The formation of the product was monitored by TLC analysis, Rf =
0.35 (hexane/AcOEt 70:30). After 1 hour, while keeping the reaction mixture at 0 °C, a vacuum
filtration with Gooch apparatus was performed. The solvent was evaporated under reduced pressure
and the crude was used in the following reaction without further purification. The crude mixture
was solubilized in dry DCM (52.6 mL, 0.10 M) under nitrogen atmosphere. Vinyl aniline (0.616
mL, 5.26 mmol) was added and the reaction mixture was heated to 40 °C. The formation of the
product was monitored by TLC analysis, Rf = 0.74 (hexane/AcOEt 70:30). After 16 hours the
solvent was evaporated and the crude mixture was purified by flash chromatography to obtain
compound 28. Flash chromatography (hexane/AcOEt 95:05). Pale yellow oil (858 mg, 56%). Rf =
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0.35; H NMR (CDCl3, 400 MHz) δ 0.18 (s, 6H), 1.00 (s, 9H), 4.21 (s, 2H), 5.22 (d, J = 10.9 Hz,
1H), 5.71 (d, J = 17.6 Hz, 1H), 6.70 (dd, J = 10.9, 17.6 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.55 (d, J
= 8.5 Hz, 2H), 8.50 (s, 1H); C{1H} NMR (CDCl3, 100 MHz) δ 5.1, 18.6 (s), 26.2, 63.7 (t), 113.5
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(t), 119.8, 127.3, 134.3 (s), 136.5, 137.1 (s), 169.4 (s); IR (neat, cm-1) 3385, 2952, 2928, 1694,
1628, 1608, 1253, 1102; HRMS (ESI) calculated for C16H25NNaO2Si [M+Na] + 314.1547, found
314.1545 (ꢀ = 0.4 ppm).
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