Dehydro-β-amino acid containing peptides as promising sequences for drug development

Article abstract of DOI:10.1002/ejoc.200900870

In the course of a program, devoted to the synthesis of small peptidic molecules mimicking the RGD (Arg-Gly-Asp) motif, the allylic animation of enantiomerically pure carbonates with 4-substituted benzylamines afforded dehydro- β-amino esters through an S

Full text of DOI:10.1002/ejoc.200900870

FULL PAPER
DOI: 10.1002/ejoc.200900870
Dehydro-β-amino Acid Containing Peptides as Promising Sequences for Drug
Development
Giuliana Cardillo,*[a] Arianna Gennari,[a] Luca Gentilucci,[a] Elisa Mosconi,[a]
Alessandra Tolomelli,*[a] and Stefano Troisi[a]
Keywords: Amino acids / Enzymes / Chiral resolution / Peptidomimetics / Chemoselectivity / Regioselectivity
In the course of a program devoted to the synthesis of small
peptidic molecules mimicking the RGD (Arg-Gly-Asp) motif,
the allylic amination of enantiomerically pure carbonates
with 4-substituted benzylamines afforded dehydro-β-amino
esters through an SN2Ј mechanism. The reaction performed
with 4-aminobenzylamine occurred with complete chemose-
lectivity, as the aliphatic amine was much more reactive than
the aromatic one. This allowed useful precursors of biolo-
gically active compounds to be obtained, avoiding extra pro-
tection–deprotection steps. The same reaction performed on
glycine-derived amides gave similar results, allowing a novel
type of RGD mimetic to be prepared, whose ability to inhibit
cell adhesion was found to be very promising.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
electrostatic interactions with regions in the protein having
opposite charges: Arg interacts with two Asp situated in the
α unit of the protein and Asp with a metal cation.[7]
Several research groups have been recently interested in
the development of small constrained nonpeptidic mole-
cules, mimicking the RGD motif, whose enhanced bioavail-
ability could result in more promising drugs. We envisaged
the dehydro-β-amino acid as a rigid core that may be easily
linked to appendages corresponding to arginine and as-
partic acid side chains.
Recently, we reported a practical regio- and stereoselec-
tive synthesis of dehydro-β-amino esters through amination
of racemic and enantiomerically pure allylic carbonates.[8]
The uncatalyzed reaction, carried out with benzylamine as
nucleophile, proceeds by an SN2Ј mechanism. In contrast,
the palladium-catalyzed reaction shows a strong solvent-de-
pendent regiocontrol, affording exclusively one of the two
possible regioisomers with complete transfer of chirality
from the starting substrate to the product (Figure 1).
Introduction
Over the last few years, extensive studies have been un-
dertaken regarding the synthesis of unusual amino acids.[1]
Design and synthesis of new enantiopure nonproteinogenic
amino acids represent a central issue for chemists working
in the wide areas of pharmaceutical and medicinal chemis-
try.[2] Among them, unsaturated β-amino acids have at-
tracted high interest as valuable intermediates in the synthe-
sis of dehydropeptides, allowing the preparation of confor-
mationally constrained sequences, with improved biological
activity and selectivity.[3] The design of receptor-selective
peptide and peptidomimetic ligands with highly specific
biological properties has become one of the most important
areas in bioorganic and medicinal chemistry.[4] In fact, at
present there is a rapid growth in the number of biologically
active peptides under investigation. In the course of our
drug development program, we became interested in the
synthesis of small peptidic molecules mimicking the RGD
(Arg-Gly-Asp) motif, present in a wide number of extra-
cellular matrix (ECM) proteins.[5] These ligands bind to
αvβ3 and α5β1 integrins, a large family of heterodimeric
transmembrane glycoproteins, involved in the pathogenesis
of several diseases, such as atheroschlerosis, osteoporosis,
cancer and a variety of inflammatory disorders.[6] The re-
cognition sequence binds to the receptor mainly through
[a] Department of Chemistry “G. Ciamician” – University of Bolo-
gna
Via Selmi 2, 40126 Bologna Italy
Fax: +39-051-2099456
E-mail: giuliana.cardillo@unibo.it
alessandra.tolomelli@unibo.it
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900870.
Figure 1. Synthesis of dehydro-β-amino esters through amination
of racemic and enantiomerically pure allylic carbonates.
Eur. J. Org. Chem. 2009, 5991–5997
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G. Cardillo, A. Gennari, L. Gentilucci, E. Mosconi, A. Tolomelli, S. Troisi
FULL PAPER
Table 1. Amination reaction on compounds 1a–d.
Results and Discussion
We present here the development of the previously re-
ported results obtained by investigating the behaviour of
bifunctionalized nucleophiles and appropriate acceptors
under SN2Ј conditions with the aim to synthesize valuable
intermediates for potential RGD mimetic compounds. The
starting materials were prepared by treatment of aldehydes
with tert-butyl acetoacetate (1.5 equiv.) in the presence of
proline (0.5 equiv.), as catalyst for the Knoevenagel reac-
tion,[9] in DMSO whilst stirring at room temperature for
16 h. Under these conditions, 80:20 mixtures of Z/E ketones
were obtained in about 80% yield. The two stereoisomers
were easily separated by flash chromatography. In an alter-
native way, the reaction was carried out under microwave-
assisted conditions, affording similar results.[10]
[a] Reaction carried out in refluxing CH3CN. [b] Configuration Z/
E of the double bond 3:1. Yield of product purified by flash
chromatography on silica gel.
The reduction of ketones to alcohols, their resolution
with lipase from Pseudomonas Cepacia and their transfor-
mation into the corresponding methyl carbonates were car-
ried out following the procedure already reported by us
(Scheme 1).[10]
Scheme 2. Reaction of carbonates 1a–d with 4-substituted benzyl-
amines.
The regiochemistry of the products was easily assigned
1
on the basis of their H NMR spectra and accounts for
nucleophilic displacement occurring by an SN2Ј mecha-
nism. The good reactivity of the benzylamine function
prompted us to use 4-aminomethylaniline as a nucleophile
in refluxing CH3CN. Under these conditions, compounds
3a–d were isolated in 62–65% yield by flash chromatog-
raphy on silica gel (Table 1, Entries 6–9).
Although a long reaction time was required to convert
the starting material into the products, regioisomers 3a–d
were exclusively obtained, confirming the lower reactivity
of the aromatic amine relative to that of the aliphatic one.
Following the same protocol on chiral nonracemic (R)- or
(S)-1a–c, compounds (R)- or (S)-3a–c were obtained with
complete regio- and stereoselectivity (Scheme 3, Table 2).
Scheme 1. Synthesis of enantiomerically pure allylic carbonates 1a–
d.
In our initial experiments to displace the carbonate from
racemic starting material, we used methyl 4-aminobenzoate
in CH3CN at reflux. The substitution reaction did not oc-
cur due to the low reactivity of the selected aromatic amine
(Table 1, Entry 1). On changing the nucleophile with methyl
4-aminomethyl-benzoate in refluxing CH3CN (Scheme 2),
the corresponding dehydro-β-amino esters 2a–d were ob-
tained in good yield and complete regioselectivity (Table 1,
Entries 2–5). The reaction was followed by TLC and
stopped with the disappearance of the starting material; the
products were isolated and purified by flash chromatog-
raphy on silica gel.
Scheme 3. Reaction on enantiomerically pure carbonates 1a–c.
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Dehydro-β-amino Acid Containing Peptides in Drug Development
Table 2. Reaction of enantiomerically pure carbonates 1a–c with 4- tert-butyl ester allowed the orthogonal deprotection of one
aminomethylaniline.[a]
of the two moieties by selecting either basic or acidic condi-
tions.
Compound 4b was coupled with glycine tert-butyl ester
Entry
Substrate
Product
Yield[b] [%]
ee[c] [%]
1
2
3
4
5
6
(S)-1a
(R)-1a
(S)-1b
(R)-1b
(S)-1c
(R)-1c
(R)-3a
(S)-3a
(R)-3b
(S)-3b
(R)-3c
(S)-3c
65
63
60
65
63
60
Ͼ99
Ͼ99
Ͼ99
Ͼ99
Ͼ99
Ͼ99
to afford amide 5b. The SN2Ј displacement of the carbonate
function with 4-aminomethylaniline in refluxing CH3CN
failed and the unreacted starting material was recovered al-
most quantitatively (Scheme 4). To enhance the reactivity
of the amide by reducing the backdonation of nitrogen, an
electron-withdrawing tert-butyl carbamate group was intro-
duced onto the amide nitrogen, giving 6b in quantitative
yield. This substrate was much more reactive and could be
converted into the corresponding amino derivative 7b by
nucleophilic substitution with 4-aminomethylaniline under
the above-reported conditions. Finally, simultaneous aci-
dolysis of the tert-butyl ester and of the Boc protection with
H3PO4 in dichloromethane,[11] followed by treatment with
Dowex 50WX2–200 ion exchange resin, eluting with
NH4OH 0.5 , and removal of the aqueous solvent under
vacuum, afforded 8b in almost quantitative yield. The abil-
ity of this new ligand to inhibit integrin-mediated cell ad-
hesion gave promising results, having an IC50 value in the
micromolar range. On this basis, a library of derivatives will
be prepared and structure–activity relationship studies will
be performed and reported in due course.
[a] Reaction carried out in refluxing CH3CN. [b] Configuration Z/
E of the double bond 3:1. Yield of product purified by flash
chromatography on silica gel. [c] Determined by HPLC analysis on
a chiral column.
The enantiomeric excess values of the isolated products
(Table 2) were determined by HPLC analysis on a chiral
column and confirmed complete transfer of chirality of the
starting carbonates to the dehydroamino esters. The stereo-
chemistry of the products was unequivocally attributed on
the basis of previously reported research.[8]
Finally, we tried to perform the substitution reaction di-
rectly on amide 5b to obtain a peptidic sequence mimicking
the RGD integrin ligand. The amide was prepared in two
steps as reported in Scheme 4. By treatment of 1b with tri-
fluoroacetic acid, the corresponding acid 4b was obtained
in 95% yield. The presence of the methyl carbonate and
Conclusions
In the course of a program devoted to the synthesis of
small peptidic molecules mimicking the RGD (Arg-Gly-
Asp) motif, the allylic amination of enantiomerically pure
carbonates with 4-substituted benzylamines afforded dehy-
dro-β-amino esters by an SN2Ј mechanism. The reaction
performed with 4-aminobenzylamine occurred with com-
plete chemoselectivity, as the aliphatic amine was much
more reactive than the aromatic one. This allowed useful
precursors of biologically active compounds to be obtained,
avoiding extra protection–deprotection steps. The same re-
action performed on glycine-derived amides gave similar re-
sults, allowing a novel type of RGD mimetic to be prepare,
whose ability to inhibit cell adhesion was very promising.
Experimental Section
General: All chemicals were purchased from commercial suppliers
and used without further purification. Anhydrous solvents were
purchased in sureseal bottles over molecular sieves and used with-
out further drying. Flash chromatography was performed on silica
gel (230–400 mesh). DOWEX® 50WX2–200(H) ion-exchange resin
was used for purification of free amino acids or free amines. NMR
spectra were recorded with Varian Gemini 200, Mercury Plus 400
or Unity Inova 600 MHz spectrometers. Chemical shifts are re-
ported as δ values (ppm) relative to the solvent peak of CDCl3 set
at δ = 7.27 ppm (1H NMR) or at δ = 77.0 ppm (13C NMR) or of
CD3OD set at δ = 3.31 ppm (1H NMR) or at δ = 49.0 ppm (13C
NMR). Coupling constant are given in Hz. The enantiomeric ex-
cess values of the products were determined by HPLC analyses
performed with an HP1100 instrument with a UV/Vis detector and
Scheme 4.
Eur. J. Org. Chem. 2009, 5991–5997
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G. Cardillo, A. Gennari, L. Gentilucci, E. Mosconi, A. Tolomelli, S. Troisi
FULL PAPER
equipped with a Chiralcel ADcolumn (25ϫ0.46 cm), eluted with n-
hexane/2-propanol. Optical rotations were measured with a Perkin–
Elmer 343 polarimeter by using a 1-dm cuvette and are referenced
to the Na-D line value. Melting points were determined with a
Stuart Scientific SMP3 apparatus and are uncorrected. Com-
pounds 1a–d were prepared following a literature procedure.[8]
3 H, CH3CHN), 1.46 [s, 9 H, OC(CH3)3], 1.64 (d, J = 7.4 Hz, 3
H, CH3CHC), 2.45 (br. s, 1 H, NH), 3.54–3.79 (m, 3 H, CH2Ph,
NCHCH3), 3.86 (s, 3 H, CH3OCO), 6.77 (q, J = 7.4 Hz, 1 H,
CCHCH3), 7.36 (d, J = 8.4 Hz, 2 H, Ph), 7.94 (d, J = 8.6 Hz, 2 H,
1
Ph) ppm. H NMR (200 MHz, CDCl3) minor isomer: δ = 1.23 (d,
J = 6.6 Hz, 3 H, CH3CHN), 1.48 [s, 9 H, OC(CH3)3], 1.88 (d, J =
7.4 Hz, 3 H, CH3CHC), 3.31 (q, J = 6.6 Hz, 1 H, NCHCH3), 5.85
(q, J = 7.4 Hz, 1 H, CCHCH3) ppm. 13C NMR (75 MHz, CDCl3)
major isomer: δ = 13.7 (CH3), 20.6 (CH2), 28.3 (3 CH3), 50.1
(CH3), 51.1 (CH), 52.0 (CH3), 80.6 (C), 128.0 (2 CH), 128.7 (C),
129.7 (2 CH), 136.0 (C), 137.7 (CH), 146.2 (C), 166.5 (C), 167.1
(C) ppm. LC–ESI-MS (r.t.): tR = 11.04 min, m/z = 333 [], 356 [M
+ Na]. C19H27NO4 (333.19): calcd. C 68.44, H 8.16, N 4.20; found
C 68.33, H 8.17, N 4.20.
1a: Yellow oil. 1H NMR (200 MHz, CDCl3): δ = 1.45 (d, J =
6.6 Hz, 3 H, CH3CHO), 1.51 [s, 9 H, OC(CH3)3], 1.99 (d, J =
7.2 Hz, 3 H, CH3CHC), 3.76 (s, 3 H, CH3OCO), 5.48 (q, J =
6.6 Hz, 1 H, CH3CHO), 6.23 (q, J = 7.2 Hz, 1 H, CH3CHC) ppm.
13C NMR (75 MHz, CDCl3): δ = 15.3 (CH3), 20.0 (CH3), 28.3 (3
CH3), 54.6 (CH3), 74.1 (CH), 81.3 (C), 134.4 (C), 135.8 (CH), 155.0
(C), 165.5 (C) ppm. (S)-1a [α]D = –24.6 (c = 1, CHCl3); (R)-1a [α]
= +25.0 (c = 1, CHCl3). LC–ESI-MS (r.t.): tR = 9.77 min, m/z =
D
244 [M], 267 [M + Na]. C12H20O5 (244.13): calcd. C 59.00, H 8.25;
found C 59.09, H 8.28.
2b: Yield: 70%, 51 mg, isolated as a 3:1 mixture of Z/E isomers.
1H NMR (200 MHz, CDCl3) major isomer: δ = 0.74 (d, J = 6.6 Hz,
3 H, CH3CHCH3), 1.12 (d, J = 6.6 Hz, 3 H, CH3CHCH3), 1.50 [s,
9 H, OC(CH3)3], 1.60 (d, J = 7.0 Hz, 3 H, CH3CHC), 1.79–2.08
(m, 1 H, CH3CHCH3), 3.02 (d, J = 9.8 Hz, 1 H, CHCHN), 3.56
(d, J = 13.8 Hz, 1 H, NHCH2Ph), 3.86 (d, J = 13.8 Hz, 1 H,
NHCH2Ph), 3.91 (s, 3 H, CH3OCO), 6.92 (q, J = 7.0 Hz, 1 H,
CCHCH3), 7.43 (d, J = 8.0 Hz, 2 H, Ph), 7.98 (d, J = 8.0 Hz, 2 H,
1
1b: Pale yellow oil. H NMR (200 MHz, CDCl3): δ = 1.01 (d, J =
6.6 Hz, 6 H, CH3CHCH3), 1.42 (d, J = 6.6 Hz, 3 H, CH3CHO),
1.50 [s, 9 H, OC(CH3)3], 3.02–3.17 (m, 1 H, CH3CHCH3), 3.77 (s,
3 H, CH3OCO), 5.46 (q, J = 6.6 Hz, 1 H, CH3CHO), 5.84 (d, J =
9.6 Hz, 1 H, CHCHC) ppm. 13C NMR (75 MHz, CDCl3): δ = 19.8
(CH3), 22.5 (2 CH3), 26.2 (CH), 28.1 (3 CH3), 54.5 (CH3), 74.0
(CH), 81.2 (C), 131.3 (C), 146.7 (CH), 155.0 (C), 165.6 (C) ppm.
(S)-1b [α]D = –30.0 (c = 1, CHCl3); (R)-1b [α]D = +28.9 (c = 1,
CHCl3). LC–ESI-MS (r.t.): tR = 11.82 min, m/z = 272 [M], 295 [M
+ Na]. C14H24O5 (272.16): calcd. C 61.74, H 8.88; found C 61.76,
H 8.86.
1
Ph) ppm. H NMR (200 MHz, CDCl3) minor isomer: δ = 0.83 (d,
J = 6.6 Hz, 3 H, CH3CHCH3), 1.00 (d, J = 6.6 Hz, 3 H,
CH3CHCH3), 1.93 (d, J = 7.0 Hz, 3 H, CH3CHC), 2.68 (d, J =
8.4 Hz, 1 H, CHCHN), 5.72 (q, J = 7.0 Hz, 1 H, CCHCH3) ppm.
13C NMR (75 MHz, CDCl3) major isomer: δ = 13.7 (CH3), 20.0 (2
CH3), 27.9 (3 CH3), 31.7 (CH), 50.1 (CH2), 51.6 (CH3), 61.3 (CH),
80.0 (C), 127.7 (2 CH), 128.3 (C), 129.2 (2 CH), 133.9 (C), 138.5
1c: Pale yellow oil. 1H NMR (200 MHz, CDCl3): δ = 0.86–1.30 (m,
6 H, cyclohexyl), 1.39 (d, J = 6.2 Hz, 3 H, CH3CHO), 1.47 [s, 9 H,
OC(CH3)3], 1.66–1.79 (m, 4 H, cyclohexyl), 2.78 (m, CH cyclo-
hexyl), 3.74 (s, 3 H, CH3OCO), 5.43 (q, J = 6.2 Hz, 1 H,
CH3CHO), 5.83 (d, J = 9.4 Hz, 1 H, CHCHC) ppm. 13C NMR
(75 MHz, CDCl3): δ = 19.9 (CH3), 25.5 (2 CH2), 25.8 (CH2), 28.1
(3 CH3), 32.4 (2 CH2), 37.7 (CH), 54.5 (CH3), 74.0 (CH), 81.0 (C),
131.5 (C), 145.4 (CH), 154.8 (C), 165.5 (C) ppm. (S)-1c [α]D = –25.2
(c = 1, CHCl3); (R)-1c [α]D = +26.7 (c = 1, CHCl3). LC–ESI-MS
(r.t.): tR = 10.81 min, m/z = 312 [M], 335[M + Na]. C17H28O5
(312.19): calcd. C 65.36, H 9.03; found C 65.51, H 9.05.
(CH), 146.5 (C), 166.5 (C), 166.7 (C) ppm. LC–ESI-MS (r.t.): tR
=
13.87 min, m/z = 361 [M], 362 [M + 1]. C21H31NO4 (361.23): calcd.
C 69.78, H 8.64, N 3.87; found C 69.75, H 8.62, N 3.88.
2c: Yield: 65%, 52 mg, isolated as a 3:1 mixture of Z/E isomers.
1H NMR (200 MHz, CDCl3) major isomer: δ = 1.13–1.36 (m, 4 H,
cyclohexyl), 1.50 [s, 9 H, OC(CH3)3], 1.47–1.81 (m, 6 H, cyclohexyl),
1.59 (d, J = 7.2 Hz, 3 H, CH3CHC), 2.40 (m, 1 H, CH cyclohexyl),
3.13 (d, J = 9.4 Hz, 1 H, CHCHN), 3.57 (d, J = 13.8 Hz, 1 H,
NHCH2Ph), 3.81 (d, J = 13.8 Hz, 1 H, NHCH2Ph), 3.91 (s, 3 H,
CH3OCO), 6.92 (m, J = 7.2 Hz, 1 H, CCHCH3), 7.43 (d, J =
8.2 Hz, 2 H, Ph), 7.98 (d, J = 8.2 Hz, 2 H, Ph) ppm. 1H NMR
(200 MHz, CDCl3) minor isomer: δ = 1.92 (d, J = 7.0 Hz, 3 H,
CH3CHC), 2.72 (d, J = 8.8 Hz, 1 H, CHCHN), 5.68 (q, J = 7.0 Hz,
1 H, CCHCH3) ppm. 13C NMR (75 MHz, CDCl3) major isomer:
δ = 13.9 (CH3), 26.2 (CH2), 26.3 (CH2), 26.6 (CH2), 28.2 (3 CH3),
30.6 (CH2), 31.6 (CH2), 41.4 (CH), 50.6 (CH2), 51.8 (CH3), 60.0
(CH), 80.3 (C), 127.9 (2 CH), 128.4 (2 CH), 129.4 (CH), 133.6 (C),
138.8 (C), 146.7 (C), 166.8 (C), 167.1 (C) ppm. LC–ESI-MS (r.t.):
tR = 5.31 min, m/z = 401 [M], 424 [M + Na]. C24H35NO4 (401.26):
calcd. C 71.79, H 8.79, N 3.49; found C 71.83, H 8.78, N 3.48.
1d: Yellow oil. 1H NMR (200 MHz, CDCl3): δ = 1.49 [s, 9 H,
OC(CH3)3], 1.53 (d, J = 6.6 Hz, 3 H, CH3CHO), 3.78 (s, 3 H,
CH3OCO), 5.51 (q, J = 6.6 Hz, 1 H, CH3CHO), 6.72 (s, 1 H,
CCHC), 7.14–7.31 (m, 3 H, thiophenyl) ppm. 13C NMR (75 MHz,
CDCl3): δ = 20.1 (CH3), 28.0 (3CH3), 54.8 (CH3), 75.3 (CH), 82.1
(C), 125.3 (CH), 126.5 (CH), 126.7 (CH), 128.2 (C), 133.2 (CH),
136.1 (C), 155.0 (C), 166.6 (C) ppm. LC–ESI-MS (r.t.): tR
=
10.59 min, m/z = 312 [M], 335[M + Na]. C15H20O5S (312.1): calcd.
C 57.67, H 6.45, S 10.26; found C 57.85, H 6.44, S 10.29.
General Procedure for the Preparation of Dehydro-β-amino Esters
2a–d: To a stirred solution of carbonate 1a–d (0.2 mmol) in dry
CH3CN (2 mL), under a nitrogen atmosphere, was added methyl 4-
aminomethylbenzoate hydrochloride (1.5 equiv., 0.3 mmol, 61 mg)
and triethylamine (1.5 equiv., 0.3 mmol, 42 µL). The solution was
stirred at reflux for 4 d and then the solvent was removed under
reduced pressure. The residue was diluted with ethyl acetate
(10 mL) and washed twice with water (5 mL). The two phases were
separated, the organic layer was dried with Na2SO4 and solvent
was removed under reduced pressure. Compounds 2a–d were iso-
lated by flash chromatography on silica gel (cyclohexane/ethyl ace-
tate, 95:5).
2d: Yield: 35%, 28 mg, isolated as a 3:1 mixture of Z/E isomers.
1H NMR (200 MHz, CDCl3) major isomer: δ = 1.34 [s, 9 H,
OC(CH3)3], 1.75 (d, J = 7.2 Hz, 3 H, CH3CHC), 2.56 (br. s, 1 H,
NH), 3.77 (d, J = 13.8 Hz, 1 H, NHCH2Ph), 3.90 (s, 3 H,
CH3OCO), 3.94 (d, J = 13.8 Hz, 1 H, NHCH2Ph), 4.75 (s, 1 H,
CHNH), 6.95–7.05 (m, 2 H, CCHCH3, CH thiophenyl), 7.19–7.27
(m, 2 H, thiophenyl), 7.48 (d, J = 8.4 Hz, 2 H, Ph), 8.00 (d, J =
8.4 Hz, 2 H, Ph) ppm. 1H NMR (200 MHz, CDCl3) minor isomer:
δ = 1.99 (d, J = 7.0 Hz, 3 H, CH3CHC), 6.04 (q, J = 7.0 Hz, 1 H,
CCHCH3) ppm. 13C NMR (75 MHz, CDCl3) major isomer: δ =
13.9 (CH3), 28.1 (3 CH3), 50.6 (CH3), 52.1 (CH2), 54.4 (CH), 80.8
(C), 120.3 (CH), 125.2 (CH), 126.8 (CH), 128.0 (2 CH), 128.9 (C),
129.7 (2 CH), 134.4 (C), 139.0 (CH), 144.2 (C), 146.1 (C), 166.4
2a: Yield: 45%, 30 mg, isolated as a 3:1 mixture of Z/E isomers.
1H NMR (200 MHz, CDCl3) major isomer: δ = 1.29 (d, J = 7.0 Hz,
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Dehydro-β-amino Acid Containing Peptides in Drug Development
(C), 167.1 (C) ppm. LC–ESI-MS (r.t.): tR = 12.67 min, m/z = 401
[M], 424 [M + Na]. C22H27NO4S (401.17): calcd. C 65.81, H 6.78,
N 3.49, S 7.99; found C 65.77, H 6.80, N 3.50, S 7.98.
(CH2), 26.5 (CH2), 28.0 (3 CH3), 30.3 (CH2), 31.4 (CH2), 41.3
(CH), 50.2 (CH2), 59.6 (CH), 80.2 (C), 114.8 (2 CH), 129.0 (2 CH),
130.3 (C), 133.5 (C), 138.9 (CH), 144.9 (C), 166.9 (C) ppm. (S)-3c
[α]D = –9.0 (c = 1, CHCl3); (R)-3c [α]D = +13.4 (c = 1, CHCl3).
HPLC (n-hexane/2-propanol, 99:1 to 90:10 over 30 min;
1.0 mLmin–1; AD column; r.t.): tR = 12.44 [(S)-3c], 15.14 min [(R)-
3c]. LC–ESI-MS (r.t.): tR = 5.28 min, m/z = 358 [], 381 [M + Na].
C22H34N2O2 (358.26): calcd. C 73.70, H 9.56, N 7.81; found C
73.83, H 9.54, N 7.83.
General Procedure for the Preparation of Dehydro-β-amino Esters
3a–d: To a stirred solution of carbonate 1a–d (0.2 mmol) in dry
CH3CN (2 mL), under a nitrogen atmosphere, was added 4-amino-
methylaniline (1.5 equiv., 0.3 mmol, 34 µL). The solution was
heated at reflux for 3 d and then the solvent was removed under
reduced pressure. Compounds 3a–d were isolated by flash
chromatography on silica gel (cyclohexane/ethyl acetate, 85:15).
3d: Yield: 63%, 45 mg, isolated as a 3:1 mixture of Z/E isomers.
1H NMR (200 MHz, CDCl3) major isomer: δ = 1.35 [s, 9 H,
OC(CH3)3], 1.82 (d, J = 7.4 Hz, 3 H, CH3CHC), 3.61 (d, J =
13.0 Hz, 1 H, NHCH2Ph), 3.78 (d, J = 13.0 Hz, 1 H, NHCH2Ph),
4.81 (s, 1 H, CHNH), 6.66 (d, J = 8.4 Hz, 2 H, Ph), 6.96–7.06 (m,
2 H, CCHCH3, CH thiophenyl), 7.13–7.26 (m, 4 H, 2 CH thiophenyl
3a: Yield: 62–65%, 35–37 mg, isolated as a 3:1 mixture of Z/E iso-
1
mers. H NMR (200 MHz, CDCl3) major isomer: δ = 1.47 (d, J =
6.8 Hz, 3 H, CH3CHN), 1.49 [s, 9 H, OC(CH3)3], 1.73 (d, J =
7.4 Hz, 3 H, CH3CHC), 3.52 (d, J = 12.6 Hz, 1 H, NHCH2Ph),
3.68 (d, J = 12.6 Hz, 1 H, NHCH2Ph), 3.81 (q, J = 6.8 Hz, 1 H,
CH3CHN), 6.59 (d, J = 8.4 Hz, 2 H, Ph), 6.93 (q, J = 7.4 Hz, 1 H,
CCHCH3), 7.16 (d, J = 8.4 Hz, 2 H, Ph) ppm. 1H NMR (200 MHz,
CDCl3) minor isomer: δ = 1.37 (d, J = 6.8 Hz, 3 H, CH3CHN),
1.95 (d, J = 7.0 Hz, 3 H, CH3CHC), 4.02 (q, J = 6.8 Hz, 1 H,
CH3CHN), 6.23 (q, J = 7.0 Hz, 1 H, CCHCH3) ppm. 13C NMR
(75 MHz, CDCl3) major isomer: δ = 13.7 (CH3), 20.6 (CH3), 28.3
(3 CH3), 49.9 (CH), 50.9 (CH2), 80.5 (C), 115.2 (2 CH), 129.4 (2
CH), 130.5 (C), 136.3 (C), 137.7 (CH), 145.3 (C), 166.7 (C) ppm.
(S)-3a [α]D = –14.0 (c = 1, CHCl3); (R)-3a [α]D = +14.0 (c = 1,
CHCl3). HPLC (n-hexane/2-propanol, 99:1 to 96:4 over 30 min;
1.0 mLmin–1; AD column; r.t): tR = 23.52 [(R)-3a], 26.12 min [(S)-
3a]. LC–ESI-MS (r.t.): tR = 8.39 min, m/z = 290 [M], 313 [M +
Na]. C17H26N2O2 (290.2): calcd. C 70.31, H 9.02, N 9.65; found C
70.08, H 9.00, N 9.65.
1
and 2 CH, Ph) ppm. H NMR (200 MHz, CDCl3) minor isomer:
δ = 2.00 (d, J = 7.0 Hz, 3 H, CH3CHC), 4.51 (s, 1 H, CHNH),
6.09 (q, J = 7.0 Hz, 1 H, CCHCH3) ppm. 13C NMR (50 MHz,
CDCl3) major isomer: δ = 14.1 (CH3), 28.1 (3 CH3), 50.6 (CH2),
54.3 (CH), 80.7 (C), 115.2 (2 CH), 120.3 (CH), 125.0 (CH), 127.0
(CH), 129.4 (2 CH), 130.4 (C), 134.8 (C), 138.9 (CH), 144.6 (C),
145.4 (C), 166.6 (C) ppm. LC–ESI-MS (r.t.): tR = 10.22 min, m/z
= 358 [M], 381 [M + Na]. C20H26N2O2S (358.17): calcd. C 67.01,
H 7.31, N 7.81, S 8.94; found C 67.19, H 7.32, N 7.84, S 8.96.
Procedure for the Preparation of Acid 4b: To a stirred solution of
carbonate 1b (0.2 mmol) in CH2Cl2 (2 mL) at 0 °C, was added tri-
fluoroacetic acid (15 equiv., 3 mmol, 223 µL). After 2 h, the mix-
ture was washed twice with acidic water (5 mL), the organic layer
was dried with Na2SO4, and the solvent was removed under re-
duced pressure. Compound 4b was isolated in 95% yield (41 mg).
1H NMR (200 MHz, CDCl3): δ = 0.97 (d, J = 5.4 Hz, 6 H,
CH3CHCH3), 1.40 (d, J = 6.6 Hz, 3 H, CH3CHO), 3.25–3.42 (m,
1 H, CH3CHCH3), 3.70 (s, 3 H, CH3OCO), 5.46 (q, J = 6.6 Hz, 1
H, CH3CHO), 6.11 (d, J = 10.0 Hz, 1 H, CHCHC), 10.41 (br. s, 1
H, OCOH) ppm. 13C NMR (50 MHz, CDCl3): δ = 20.1 (CH3),
21.9 (2 CH3), 31.8 (CH), 54.4 (CH3), 73.3 (CH), 128.9 (C), 142.5
(CH), 154.7 (C), 171.4 (C) ppm. LC–ESI-MS (r.t.): tR = 6.65 min,
m/z = 216 [M], 239 [M + Na]. C10H16O5 (216.1): calcd. C 55.55, H
7.46; found C 55.59, H 7.49.
3b: Yield: 60–65%, 38–41 mg, isolated as a 3:1 mixture of Z/E iso-
1
mers. H NMR (200 MHz, CDCl3) major isomer: δ = 0.74 (d, J =
6.8 Hz, 3 H, CH3CHCH3), 1.10 (d, J = 6.8 Hz, 3 H, CH3CHCH3),
1.49 [s, 9 H, OC(CH3)3], 1.67 (d, J = 7.2 Hz, 3 H, CH3CHC), 1.80–
2.05 (m, 1 H, CH3CHCH3), 3.06 (d, J = 9.6 Hz, 1 H, CHCHN),
3.38 (d, J = 12.8 Hz, 1 H, NHCH2Ph), 3.70 (d, J = 12.8 Hz, 1 H,
NHCH2Ph), 6.63 (d, J = 8.2 Hz, 2 H, Ph), 6.92 (q, J = 7.2 Hz, 1 H,
CCHCH3), 8.20 (d, J = 8.2 Hz, 2 H, Ph) ppm. 1H NMR (200 MHz,
CDCl3) minor isomer: δ = 0.83 (d, J = 6.6 Hz, 3 H, CH3CHCH3),
0.97 (d, J = 6.6 Hz, 3 H, CH3CHCH3), 1.93 (d, J = 7.0 Hz, 3 H,
CH3CHC), 2.77 (d, J = 8.6 Hz, 1 H, CHCHN), 5.77 (q, J = 7.0 Hz,
1 H, CCHCH3) ppm. 13C NMR (50 MHz, CDCl3) major isomer:
δ = 14.0 (CH3), 20.1 (2 CH3), 28.3 (3 CH3), 32.1 (CH), 50.7 (CH2),
61.3 (CH), 80.2 (C), 114.9 (2 CH), 129.1 (2 CH), 131.1 (C), 134.4
(C), 138.7 (CH), 144.9 (C), 167.0 (C) ppm. (S)-3b [α]D = –11.7 (c
= 1, CHCl3); (R)-3b [α]D = +12.0 (c = 1, CHCl3). HPLC (n-hexane/
2-propanol, 99:1 to 90:10 over 30 min; 1.0 mLmin–1; AD column;
r.t.): tR = 10.39 [(R)-3b], 10.95 min [(S)-3b]. LC–ESI-MS (r.t.): tR
= 5.87 min, m/z = 318 [M], 341 [M + Na]. C19H30N2O2 (318.23):
calcd. C 71.66, H 9.50, N 8.80; found C 71.52, H 9.51, N 8.83.
Procedure for the Preparation of Amide 5b: To a stirred solution of
acid 4b (0.2 mmol) in dry CH2Cl2 (2 mL), under a nitrogen atmo-
sphere, was added EDCI (1.2 equiv., 0.24 mmol, 46 mg) and trieth-
ylamine (2.4 equiv., 0.48 mmol, 67 µL). After 30 min, HOBT
(1.2 equiv., 0.24 mmol, 33 mg) and glycine tert-butyl ester hydro-
chloride (1.2 equiv., 0.24 mmol, 41 mg) were added. The solution
was stirred overnight, and then the mixture was diluted with
CH2Cl2 and washed twice with acidic water (5 mL) and twice with
basic water (5 mL). The two phases were separated, the organic
layer was dried with Na2SO4 and the solvent was removed under
reduced pressure. Compound 5b was isolated in 65% yield (43 mg)
after flash chromatography on silica gel (cyclohexane/ethyl acetate,
80:20). 1H NMR (200 MHz, CDCl3): δ = 1.01 (d, J = 6.6 Hz, 6 H,
3c: Yield: 60–63%, 43–46 mg, isolated as a 3:1 mixture of Z/E iso-
mers. 1H NMR (200 MHz, CDCl3) major isomer: δ = 1.07–1.27
(m, 4 H, cyclohexyl), 1.41–1.81 (m, 6 H, cyclohexyl), 1.50 [s, 9 H,
OC(CH3)3], 1.65 (d, J = 7.2 Hz, 3 H, CH3CHC), 2.36 (m, 1 H, CH CH3CHCH3), 1.45 (d, J = 7.0 Hz, 3 H, CH3CHO), 1.49 [s, 9 H,
cyclohexyl), 3.17 (d, J = 9.6 Hz, 1 H, CHCHN), 3.39 (d, J =
13.2 Hz, 1 H, NHCH2Ph), 3.70 (d, J = 13.2 Hz, 1 H, NHCH2Ph),
OC(CH3)3], 2.70–2.88 (m, 1 H, CH3CHCH3), 3.79 (s, 3 H,
CH3OCO), 4.00 (d, J = 4.8 Hz, 2 H, NHCH2), 5.27 (q, J = 6.6 Hz,
6.64 (d, J = 8.4 Hz, 2 H, Ph), 6.92 (q, J = 7.2 Hz, 1 H, CCHCH3), 1 H, CH3CHO), 5.67 (d, J = 10.2 Hz, 1 H, CHCHC), 6.57 (br. t,
1
7.12 (d, J = 8.4 Hz, 2 H, Ph) ppm. H NMR (200 MHz, CDCl3) 1 H, NH) ppm. 13C NMR (50 MHz, CDCl3): δ = 19.9 (CH3), 22.7
minor isomer: δ = 1.93 (d, J = 7.4 Hz, 3 H, CH3CHC), 2.10 (m, 1 (2 CH3), 26.8 (CH), 28.0 (3 CH3), 41.9 (CH2), 54.8 (CH3), 76.0
H, CH cyclohexyl), 2.80 (d, J = 8.8 Hz, 1 H, CHCHN), 3.41 (d, J (CH), 82.2 (C), 131.8 (C), 137.3 (CH), 155.1 (C), 167.3 (C), 168.8
= 12.8 Hz, 1 H, NHCH2Ph), 3.72 (d, J = 12.8 Hz, 1 H,
NHCH2Ph), 5.73 (q, J = 7.4 Hz, 1 H, CCHCH3) ppm. 13C NMR
(75 MHz, CDCl3) major isomer: δ = 13.9 (CH3), 26.0 (CH2), 26.2
(C) ppm. LC–ESI-MS (r.t.): tR = 10.82 min, m/z = 329 [M], 352
[M + Na]. C16H27NO6 (329.18): calcd. C 58.34, H 8.26, N 4.25;
found C 58.31, H 8.24, N 4.25.
Eur. J. Org. Chem. 2009, 5991–5997
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5995

G. Cardillo, A. Gennari, L. Gentilucci, E. Mosconi, A. Tolomelli, S. Troisi
FULL PAPER
Procedure for the Preparation of the N-Boc-protected Amide 6b: To
a stirred solution of amide 5b (0.2 mmol) in dry THF (1 mL) was
added DMAP (0.2 equiv., 0.04 mmol, 5 mg), triethylamine
(1.2 equiv., 0.24 mmol, 34 µL) and (Boc)2O (1.3 equiv., 0.26 mmol,
60 µL). The solution was stirred overnight, and then the solvent
was removed under reduced pressure. The residue was diluted with
ethyl acetate (10 mL) and washed twice with water (5 mL). The two
phases were separated, the organic layer was dried with Na2SO4
and the solvent was removed under reduced pressure. Compound
6b was isolated in 90% yield (77 mg) by flash chromatography on
silica gel (cyclohexane/ethyl acetate, 95:5). 1H NMR (200 MHz,
CDCl3): δ = 0.99 (d, J = 6.6 Hz, 6 H, CH3CHCH3), 1.42 (d, J =
6.6 Hz, 3 H, CH3CHO), 1.48 [s, 18 H, OC(CH3)3], 2.41–2.49 (m, 1
H, CH3CHCH3), 3.75 (s, 3 H, CH3OCO), 4.28 (d, J = 16.8 Hz, 1
H, NCH2), 4.45 (d, J = 16.8 Hz, 1 H, NCH2), 5.42–5.53 (m, 2 H,
CH3CHO, CHCHC) ppm. 13C NMR (50 MHz, CDCl3): δ = 19.2
(CH3), 22.6 (2 CH3), 27.2 (CH), 27.8 (3 CH3), 28.0 (3 CH3), 45.9
(CH2), 54.5 (CH3), 74.7 (CH), 81.8 (C), 83.8 (C), 126.7 (C), 137.6
(CH), 151.4 (C), 155.1 (C), 167.6 (C), 169.8 (C) ppm. LC–ESI-MS
(r.t.): tR = 12.26 min, m/z = 429 [M], 452 [M + Na]. C21H35NO8
(429.24): calcd. C 58.72, H 8.21, N 3.26; found C 58.61, H 8.19, N
3.27.
Acknowledgments
This study was carried out with a fundamental contribution from
the Fondazione del Monte di Bologna e Ravenna. We also thank
MAE (Italian Minister for Foreign Affair, General Direction for
the Cultural Promotion and Cooperation) for financial support to
a bilateral project between Italy and Mexico. MIUR (PRIN 2006
prot.n. 2006030449_003) and the University of Bologna (Strategic
project ID 450) are also acknowledged for financial support. Mr.
Andrea Garelli is gratefully acknowledged for LC–ESI-MS analy-
sis.
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Procedure for the Preparation of Amino Derivative 7b: To a stirred
solution of amide 6b (0.2 mmol) in dry CH3CN (2 mL), under a
nitrogen atmosphere, was added 4-aminomethylaniline (1.5 equiv.,
0.3 mmol, 34 µL). The solution was heated at reflux for 16 h, and
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7b was isolated in 70% yield (66 mg) by flash chromatography on
silica gel (cyclohexane/ethyl acetate, 95:5). 1H NMR (200 MHz,
CDCl3): δ = 0.83 (d, J = 6.6 Hz, 6 H, CH3CHCH3), 1.42 (d, J =
7.4 Hz, 3 H, CH3CHC), 1.47 [s, 18 H, OC(CH3)3], 2.60–2.81 (m, 1
H, CH3CHCH3), 3.59 (br. s, 2 H, NH2), 3.83–3.98 (m, 3 H, NCH2,
CHNH), 4.34–4.61 (m, 2 H, CH2Ph), 6.61 (d, J = 8.0 Hz, 2 H, Ph),
6.83 (q, J = 7.4 Hz, 1 H, CCHCH3), 7.01 (d, J = 8.0 Hz, 2 H, Ph)
ppm. 13C NMR (50 MHz, CDCl3): δ = 13.5 (CH3), 20.2 (2 CH3),
28.1 (3 CH3), 28.5 (3 CH3), 31.8 (CH), 42.2 (CH2), 47.3 (CH), 50.1
(CH2), 79.8 (C), 82.1 (C), 115.0 (2 CH), 127.9 (2 CH), 130.7 (C),
131.3 (C), 139.6 (CH), 144.9 (C), 156.0 (C), 169.1 (C), 169.9 (C)
ppm. LC–ESI-MS (r.t.): tR = 9.15 min, m/z = 475 [M], 498 [M +
Na]. C26H41N3O5 (475.3): calcd. C 65.66, H 8.69, N 8.83; found C
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Procedure for the Preparation of Amino Acid 8b: To a stirred solu-
tion of compound 7b (0.2 mmol) in CH2Cl2 (0.5 mL) was added
H3PO4 85% (5 equiv., 1 mmol, 69 µL). After 3 h the solvent was
removed under reduced pressure, and the crude compound was
treated with Dowex 50WX2–200 ion-exchange resin, eluting with
0.5 NH4OH. Compound 8b was isolated after removal of the
aqueous solvent in 95% yield (61 mg). 1H NMR (200 MHz,
CD3OD) major isomer: δ = 0.93 (d, J = 6.6 Hz, 6 H, CH3CHCH3),
1.82 (d, J = 7.2 Hz, 3 H, CH3CHC), 2.18–2.28 (m, 1 H,
CH3CHCH3), 3.83–4.38 (m, 5 H, CHNH, NHCH2, CH2Ph), 6.74
(d, J = 8.4 Hz, 2 H, Ph), 6.90 (q, J = 7.2 Hz, 1 H, CCHCH3), 7.21
(d, J = 8.4 Hz, 2 H, Ph) ppm. 1H NMR (200 MHz, CD3OD) minor
isomer: δ = 2.06 (d, J = 7.2 Hz, 3 H, CH3CHC), 6.06 (q, J = 7.2 Hz,
1 H, CCHCH3) ppm. 13C NMR (50 MHz, CD3OD) major isomer:
δ = 14.3 (CH3), 19.9 (2 CH3), 31.9 (CH), 46.9 (CH2), 50.9 (CH2),
62.4 (CH), 116.3 (2 CH), 121.1 (C), 128.8 (CH), 132.0 (2 CH),
140.7 (C), 148.5 (C), 161.6 (C), 178.2 (C) ppm. LC–ESI-MS (r.t.):
tR = 1.06 min, m/z = 319 [M], 342 [M + Na]. C17H25N3O3 (319.19):
calcd. C 63.93, H 7.89, N 13.16; found C 63.99, H 7.90, N 13.11.
Supporting Information (see footnote on the first page of this arti-
1
cle): H NMR spectra of compounds 1a–d, 2a–d and 3a–d.
5996
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 5991–5997

Dehydro-β-amino Acid Containing Peptides in Drug Development
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Published Online: October 13, 2009
Eur. J. Org. Chem. 2009, 5991–5997
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5997