A facile one-pot synthesis of Nα-urethane protected 3-amino alkyl isoxazole-5-carboxylic acids and their utility for the preparation of isoxazole-linked peptidomimetics

Article abstract of DOI:10.1007/s10989-011-9256-x

Cu(I) catalyzed [3+2] cycloaddition of in situ generated N ^α-Fmoc/Boc/Z-protected amino alkyl nitrile oxide with propiolic acid has led to a new class of 3,5-disubstituted isoxazole-bearing amino acid derivatives. The click chemistry protocol described herein is efficient in furnishing the isoxazole embedded amino acids. These unnatural synthons were subjected to chain extension on N- as well as C-termini to obtain 3,5-disubstituted isoxazole bearing di and tripeptidomimetics. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s10989-011-9256-x

Int J Pept Res Ther (2011) 17:185–191
DOI 10.1007/s10989-011-9256-x
A Facile One-pot Synthesis of Na-Urethane Protected 3-Amino
Alkyl Isoxazole-5-Carboxylic Acids and their Utility
for the Preparation of Isoxazole-Linked Peptidomimetics
•
G. Chennakrishnareddy B. Vasantha
•
•
N. Narendra Vommina V. Sureshbabu
Accepted: 27 May 2011 / Published online: 7 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Cu(I) catalyzed [3?2] cycloaddition of in situ
generated Na-Fmoc/Boc/Z-protected amino alkyl nitrile
oxide with propiolic acid has led to a new class of
3,5-disubstituted isoxazole-bearing amino acid derivatives.
The click chemistry protocol described herein is efficient in
furnishing the isoxazole embedded amino acids. These
unnatural synthons were subjected to chain extension on
N- as well as C-termini to obtain 3,5-disubstituted isox-
azole bearing di and tripeptidomimetics.
1988) and isoxazoline (Kim et al. 1992; Annunziata et al.
1987) units has also been undertaken. Isoxazole and its
derivatives are an important class of N-heterocycles that
prevail in many medicinally useful compounds including
several natural products (Quilico 1962; Grunanger and
Vita-Finzi 2008).
Several classes of compounds bearing isoxazoles are
known to be selective modulators of the multidrug resis-
tance protein (Norman et al. 2005). Some of the com-
pounds bearing 3,5-disubstituted isoxazoles have been
found to activate neuronal nicotinic acetylcholine receptors
(Ebert et al. 1994), act as antipicornavirus agents (Bailey
et al. 1992) and possess ionophoric properties (Kantorow-
ski and Kurth 1997). Further, isoxazole derivatives having
adjacent amide groups display a2-adrenoceptor antagonis-
tic and serotonine reuptake inhibiting activities (Andre
et al. 2006). Kim’s group reported the preparation of
ketomethylene peptide isosteres through isoxazole ring
modification and cleavage (Kim et al. 1993; Chung et al.
1996).
Keywords Na-protected amino aldehyde Á Nitrile oxide Á
Click chemistry Á [2?3] cycloaddition Á Isoxazole Á One-pot
synthesis Á Peptidomimetics
Introduction
In recent years, peptidomimetics (Goodman et al. 2003)
have opened up new prospects in drug discovery. Inserting
urea (Patil et al. 2003; Sureshbabu et al. 2006), thiourea
(Vig et al. 1998; Shusheng et al. 2008), carbamate (Cho
et al. 1993), sulfonamide (Gennari et al. 1994) moieties in
place of peptide bond has led to pharmaceutically potent
compounds. Similarly, replacement of an amide bond with
heterocycles such as tetrazole (Yu and Johnson 1987),
triazole (Tam et al. 2007), oxazole (You et al. 2004), thi-
azole (You and Kelly 2005), imidazoline (Jones and Ward
Among synthetic reports for the preparation of
3,5-disubstituted isoxazoles, the predominantly used ones
include reaction of hydroxylamine with a,b-unsaturated
carbonyl compounds (Cuadrado et al. 2002), oximation of
chalcone epoxides (Elkasaby and Salem 1980), base pro-
moted cyclization of b,c-acetylinic oximes (Short and
Ziegler 1993), and ring opening and intramolecular
SNV reaction of cyclopropyl oximes (Wang et al. 2008).
Isoxazoles can also be obtained by the metal peroxides
mediated dehydrogenation of corresponding isoxazolines
(Easton et al. 1994). Recently, carboxamide functionalized
isoxazoles have been reported via condensation of a-acy-
lketene dithioacetals with hydroxylamine hydrochloride in
the presence of KOH under reflux conditions (Savant et al.
2010). Another commonly used method for their synthesis
G. Chennakrishnareddy Á B. Vasantha Á N. Narendra Á
V. V. Sureshbabu (&)
Department of Studies in Chemistry, Central College Campus,
Peptide Research Laboratory, Bangalore University, #109,
Dr. B. R. Ambedkar Veedhi, Bangalore 560 001, India
e-mail: sureshbabuvommina@rediffmail.com;
hariccb@hotmail.com; hariccb@gmail.com
123

186
Int J Pept Res Ther (2011) 17:185–191
is the [3?2] cycloaddition of alkynes and nitrile oxides
(Huisgen 1976). Solid-phase synthesis of isoxazoles has
also been reported employing either the nitrile oxide
(Cheng and Mjalli 1998) or the acetylenic component
bound to the resin support (Luca et al. 2001). Although,
1,3-dipolar cycloaddition reaction between dipolarophiles
and nitrile oxides gives the desired isoxazoles, most of
these protocols involve multi-step transformation, harsh
reaction conditions, moderate yields, low purity and regi-
oselectivity. Interestingly, the use of Cu(I) catalyst has
enabled the synthesis of 3,5-substituted isoxazole deriva-
tives regioselectivity (Himo et al. 2005).
CuSO4Á5H2O (0.5 mmol), NaAsc (0.05 mmol) and propi-
olic acid (1.5 mmol). The stirring was further continued at
room temperature for about 5 h (TLC analysis) and the
reaction mixture was diluted with 5% Na2CO3 and washed
with ether. The aqueous layer was acidified with 10% citric
acid solution and extracted into ethyl acetate. The organic
layer was washed with water, brine, and dried over anhy-
drous Na2SO4. The solvent was removed under reduced
pressure. Recrystallization from THF and n-hexane affords
3,5-disubstituted isoxazole bearing amino acid as solid 4.
General Experimental Procedure for the Preparation
of Na-Fmoc/Boc/Z-Protected 3,5-Disubstituted Isoxazole
Bearing di and Tripeptidomimetic 5a–d and 6a,
b Respectively
In view of the wide utility of isoxazole-tethered com-
pounds in biology, we envisaged that isoxazole bearing
amino acids would represent a useful class of synthons that
may contribute to generate interesting peptidomimetics.
Herein, we report an efficient one-pot protocol for the
synthesis of Na-Fmoc/Boc/Z-protected 3,5-disubstituted
isoxazole based unnatural amino acids and demonstrate
their utility as monomeric units for the preparation of
corresponding class of di as well as tripeptidomimetics.
To a solution of 4 (1.0 mmol) in dry CH2Cl2 (10 mL) was
added EDC (1.2 mmol), HOBt (1.2 mmol) followed by
amino methyl ester (1.2 mmol). The reaction mixture was
stirred for 3–4 h. After completion (TLC) the organic layer
was with H2O, brine and dried over anhyd. Na2SO4.
Evaporation of the solvent under vacuum followed by
recrystallisation using THF and n-hexane affords dipepti-
domimetics 5a–d as a white solid.
Materials and Methods
N-Fmoc protected dipeptidomimetic 5 (1 mmol) was
stirred in a mixture of DEA:CH2Cl2 (1:1, 2 mL) until
completion of deprotection (TLC). Repeated evaporation
of the solvent followed trituration with ether afforded the
deprotected intermediate. In the subsequent step, the free
amino derivative was coupled with an Fmoc-amino acid
(1.00 mmol) by employing EDC/HOBt protocol to afford
corresponding tripeptidomimetic 6.
Chemicals and Instruments
All solvents were freshly distilled before use. Amino acids
1
were used as received from Sigma-Aldrich. H and 13C
NMR were recorded on a Bruker AMX 300 MHz instru-
ment with tetramethylsilane (TMS) as an internal standard.
Mass spectra were recorded on high resolution mass
spectra (HRMS) Q-Tof micro mass spectrometer. All the
reactions were monitored using thin layer chromatography
(TLC) with precoated silica gel plates purchased from
Merck. Column Chromatography was performed with
Merck silica gel (100–200) at normal atmospheric pressure.
Rotations were recorded using JASCO P2000 (PC) digital
polarimeter.
1
4a: White solid, mp 138–139°C, H NMR (300 MHz,
DMSO-d6): d = 1.22 (3H, d, J = 4.8 Hz), 4.25 (1H, t,
J = 4.6 Hz,), 4.41 (2H, d, J = 5.8 Hz,), 4.96–4.99 (1H,
m), 6.13 (1H, s), 6.96 (1H, br), 7.25–7.71 (8H, m), 10.98
(1H, s); 13C NMR (75 MHz, DMSO-d6): d = 19.1 (CH3,
Ala), 47.5 (CH, Fmoc), 48.3 (CHa, Ala), 67.1 (OCH2,
Fmoc), 100.2 (C=C), 126. 0 (CH, Fmoc, 2C), 126.9
(CH, Fmoc, 2C), 127.5 (CH, Fmoc, 2C), 128.1 (CH, Fmoc,
2C), 141.3 (Cq, Fmoc, 2C), 144.5 (Cq, Fmoc, 2C), 150.1
(C=N), 155.3 (O–C=C), 156.1 (CO, Fmoc), 175.1 (COOH).
HRMS: m/z calcd for C21H18N2NaO5: 401.1113; found:
401.1108 [M ? Na].
Syntheses
General Experimental Procedure for the One-Pot
Preparation of Na-Fmoc/Boc/Z-Protected
3,5-Disubstituted Isoxazole Acids 4a–m
1
4b: White solid, mp 138–139°C, H NMR (300 MHz,
DMSO-d6): d = 1.24 (3H, d, J = 4.6 Hz), 4.27 (1H, t,
J = 4.6 Hz,), 4.42 (2H, d, J = 5.8 Hz,), 4.95–4.98 (1H, m),
6.14 (1H, s), 6.97 (1H, br), 7.25–7.75 (8H, m), 10.95 (1H, s);
13C NMR (75 MHz, DMSO-d6): d = 19.4 (CH3, Ala), 47.6
(CH, Fmoc), 48.5 (CHa, Ala), 67.4 (OCH2, Fmoc), 100.1
(C=C), 126. 1 (CH, Fmoc, 2C), 126.5 (CH, Fmoc, 2C),
127.7 (CH, Fmoc, 2C), 128.5 (CH, Fmoc, 2C), 141.7
(Cq, Fmoc, 2C), 144.6 (Cq, Fmoc, 2C), 150.5 (C=N), 155.8
To a solution of N-protected a-amino aldehyde (1.0 mmol)
in tert-BuOH/water (1:1) was added hydroxylamine
(1.1 mmol) (obtained by neutralizing the corresponding
hydrochloride salt using Na2CO3 (1.5 mmol)) and stirred at
room temperature for about 1 h. After the complete for-
mation of Na-protected amino aldoxime (monitored by
TLC), were added chloramine-T (1.5 mmol), followed by
123

Int J Pept Res Ther (2011) 17:185–191
187
(O–C=C), 156.6 (CO, Fmoc), 175.2 (COOH). HRMS: m/
z calcd for C21H18N2NaO5: 401.1113; found: 401.1108
[M ? Na].
29.8 (CH2b, Pro), 46.3 (CH2d, Pro), 47.2 (CH, Fmoc), 52.5
(CHa, Pro), 66.9 (OCH2, Fmoc), 100.3 (C=C), 125.1 (CH,
Fmoc, 2C), 126.2 (CH, Fmoc, 2C), 127.2 (CH, Fmoc, 2C),
128.7 (CH, Fmoc, 2C), 140.3 (Cq, Fmoc, 2C), 143.1 (Cq,
Fmoc, 2C), 150.1 (C=N), 156.7 (O–C=C), 158.4 (CO,
Fmoc), 174.9 (COOH). ESI-MS: m/z calcd for C23H20N2O5:
404.1; found: 403.1 [M - H].
1
4c: White solid, mp 135–137°C, H NMR (300 MHz,
DMSO-d6): d = 2.71 (2H, d, J = 5.2 Hz), 4.19 (1H, t,
J = 5.6 Hz), 4.42 (2H, d, J = 6.2 Hz), 5.35–5.37 (1H, m),
6.12 (1H, s), 6.99 (1H, br), 7.21–7.74 (13H, m), 11.2 (1H,
br); 13C NMR (75 MHz, DMSO-d6): d = 37.1, 45.2, 54.9,
66.5, 100.5, 125.3, 126.1, 126.9, 127.1,127.8, 128.1, 129.1,
138.9, 141.4, 144.2, 150.3, 154.5, 156.8, 173.1. ESI-MS:
m/z calcd for C27H22N2O5: 454.2; found: 453.2 [M–H].
1
4i: White solid, mp 132–134°C, H NMR (300 MHz,
DMSO-d6): d = 2.21–2.28 (4H, m), 4.21 (1H, t, J =
4.2 Hz), 4.44 (2H, d, J = 6.2 Hz), 4.93 (1H, m), 5.32 (2H,
s), 6.15 (1H, s) 6.53 (1H, br), 7.21–7.73 (13H, m), 11.10
(1H, br); 13C NMR (75 MHz, DMSO-d6): d = 25.8, 30.2,
47.1, 51.8, 67.5, 68.4, 100.6, 124.5, 125.2, 126.8, 127.5,
128.1, 128.9, 129.3, 141.1, 141.4, 144.5, 150.2, 155.3,
157.8, 174.8. HRMS: m/z calcd for C30H26N2O7: 526.2;
found: 527.2 [M ? H].
1
4d: White solid, mp 148–149°C, H NMR (300 MHz,
DMSO-d6): d = 1.91 (3H, s), 2.25 (2H, m), 2.38 (2H, t,
J = 4.8 Hz), 4.31 (1H, t, J = 4.8 Hz), 4.52 (2H, d,
J = 6.8 Hz), 4.85 (1H, m), 5.95 (1H, br), 6.23 (1H, s),
7.31–7.68 (8H, m), 10.92 (1H, br); 13C NMR (75 MHz,
DMSO-d6): d = 16.8, 28.3, 35.5, 47.1, 53.8, 67.3, 100.1,
124.8, 125.6, 126.8, 127.5, 141.5, 143.5, 152.5, 155.8,
157.9, 175.3. ESI-MS: m/z calcd for C23H22N2O5: 438.1;
found: 437.1 [M-H].
1
4j: White solid, mp 118–120°C, H NMR (300 MHz,
DMSO-d6): d = 1.34 (3H, d, J = 5.4 Hz), 4.98 (1H, m),
5.25 (2H, s), 5.98 (1H, br), 6.23 (1H, s), 7.12–7.31 (5H, m),
10.96 (1H, s); 13C NMR (75 MHz, DMSO-d6): d = 18.3
(CH3, Ala), 48.5 (CHa, Ala), 66.1 (OCH2, Z), 100.4 (C=C),
126.6 (CH, Z, 2C), 127.1 (CH, Z, 2C), 128.1 (CH, Z, 1C),
141.3 (Cq, Z, 1C), 150.2 (C=N), 156.9 (O–C=C), 158.5
(CO, Z), 175.5 (COOH). ESI-MS: m/z calcd for
C14H14N2NaO5: 313.1; found: 313.1 [M ? Na].
1
4e: White solid, mp 154–156°C, H NMR (300 MHz,
DMSO-d6): d = 2.90 (2H, d, J = 6.4 Hz), 4.21 (1H, t,
J = 4.2 Hz), 4.45 (2H, d, J = 5.6 Hz), 4.91 (1H, m), 5.21
(2H, s), 5.91 (1H, br), 6.21 (1H, s), 7.21–7.74 (13H, m),
11.21 (1H, s); 13C NMR (75 MHz, DMSO-d6): d = 37.5,
45.2, 48.5, 66.7, 68.1, 100.2, 125.1, 125.9, 126.4, 127.1,
127.9, 128.7, 129.3, 141.1, 144.2, 150.3, 155.4, 157.9,
175.5. HRMS: m/z calcd for C29H24N2NaO7: 535.1481;
found 535.1475 [M ? Na].
1
4k: White solid, mp 121–123°C, H NMR (300 MHz,
DMSO-d6): d = 1.22–1.35 (6H, m), 2.81–2.88 (2H, m),
4.12 (1H, m), 5.25 (2H, s), 5.99 (1H, br), 6.14 (1H, s), 6.93
(1H, br), 7.12–7.31 (5H, m), 11.31 (1H, s); 13C NMR
(75 MHz, DMSO-d6): d = 23.1, 28.9, 37.5, 41.8, 51.2,
65.1, 65.8, 100.3, 126.8, 127.5, 128.6, 141.3, 150.5, 154.6,
157.3, 175.1. HRMS: m/z calcd for C25H27N3NaO7:
504.1747; found: 504.1745 [M ? Na].
1
4f: White solid, mp 150–152°C, H NMR (300 MHz,
DMSO-d6): d = 1.25–1.38 (6H, m), 2.65–2.79 (2H, m),
3.98 (1H, m), 4.18 (1H, t, J = 4.4 Hz), 4.38 (2H, d,
J = 6.2 Hz), 5.22 (2H, s), 5.95 (2H, br), 6.22 (1H, s),
7.22–7.71 (13H, m), 10.99 (1H, br); 13C NMR (75 MHz,
DMSO-d6): d = 22.7, 29.2, 37.1, 42.7, 46.7, 50.3, 65.1,
66.9, 100.2, 125.1, 125.8, 126.6, 127.3, 128.1, 128.8,
129.5, 141.1, 141.4, 144.2, 150.3, 155.6, 155.9, 157.5,
173.9. HRMS: m/z calcd for C32H31N3NaO7: 592.2060;
found: 592.2055 [M ? Na].
4l: Gum, 1H NMR (300 MHz, DMSO-d6): d = 1.31
(3H, d, J = 5.2 Hz), 1.42 (9H, s), 4.98 (1H, m), 5.99 (1H,
br), 6.13 (1H, s), 11.01 (1H, s); 13C NMR (75 MHz,
DMSO-d6): d = 19.8 (CH3, Ala), 27.5 (CH3, Boc, 3C),
48.3 (CHa, Ala), 75.6 (Cq, Boc, 1C), 100.1 (C=C), 151.2
(C=N), 155.9 (O–C=C), 157.9 (CO, Boc), 175.6 (COOH).
HRMS: m/z calcd for C11H16N2O5: 256.1059; found:
257.1135 [M ? H].
1
4g: White solid, mp 122–123°C, H NMR (300 MHz,
DMSO-d6): d = 0.89 (6H, d, J = 6.2 Hz), 1.41 (2H, m),
1.81 (1H, m), 4.25 (1H, t, J = 5.6 Hz), 4.51 (2H, d,
J = 6.4 Hz), 4.75 (1H, m), 6.01 (1H, s), 6.93 (1H, br),
7.21–7.68 (8H, m), 10.85 (1H, s); 13C NMR (75 MHz,
DMSO-d6): d = 22.3, 24.5, 45.8, 47.5, 49.8, 67.3, 100.1,
125.2, 125.9, 126.8, 127.2, 141.6, 144.5, 150.3, 156.7,
158.2, 175.7. ESI-MS: m/z calcd for C24H24N2NaO5:
443.2; found: 443.2 [M ? Na].
1
4m: Gum, H NMR (300 MHz, DMSO-d6): d = 1.45
(9H, s), 2.56 (2H, d, J = 6.4 Hz), 4.91 (1H, m), 6.11 (1H,
s), 6.99 (1H, br), 7.12–7.36 (5H, m), 11.21 (1H, br); 13C
NMR (75 MHz, DMSO-d6): d = 28.1, 39.8, 52.2, 78.9,
100.2, 125.1, 126.8, 127.9, 139.5, 151.2, 156.5, 158.1,
174.9. ESI-MS: m/z calcd for C17H20N2NaO5: 355.1;
found: 355.1 [M ? Na].
1
1
4h: White solid, mp 143–145°C, H NMR (300 MHz,
5a: White solid, mp 136–138°C, H NMR (300 MHz,
DMSO-d6): d = 1.61–1.72 (4H, m), 3.21 (2H, m), 3.81
(1H, m), 4.12 (1H, t, J = 4.4 Hz), 4.42 (2H, d,
J = 5.8 Hz), 6.12 (1H, s), 7.31–7.76 (8H, m), 11.12 (1H,
s); 13C NMR (75 MHz, DMSO-d6): d = 22.3 (CH2c, Pro),
DMSO-d6): d = 0.92 (6H, d, J = 6.4 Hz), 1.21 (3H, d,
J = 6.2 Hz), 1.53 (1H, m), 1.82 (2H, m), 3.65 (3H, s), 4.25
(1H, t, J = 4.8 Hz), 4.53 (1H, m), 4.65 (2H, d,
J = 6.6 Hz), 5.12 (1H, m), 6.12 (1H, s), 6.95 (2H, br),
123

188
Int J Pept Res Ther (2011) 17:185–191
7.12–7.68 (8H, m); 13C NMR (75 MHz, DMSO-d6):
d = 19.1, 22.1, 24.5, 39.8, 47.5, 48.5, 51.2, 52.5, 67.1,
100.2, 125.1, 125.9, 126.5, 127.9, 141.3, 144.2, 150.1,
156.1, 158.1, 159.4, 171.2. HRMS: m/z calcd for
C28H31N3NaO6: 528.2111; found: 528.2115 [M ? Na].
(1H, m), 4.51 (2H, d, J = 5.2 Hz), 4.81–4.92 (2H, m), 6.02
(1H, s), 6.11–6.34 (2H, br), 6.98 (1H, br), 7.12–7.72 (13H,
m); 13C NMR (75 MHz, DMSO-d6): d = 10.6, 14.1, 19.2,
26.1, 38.1, 42.3, 45.6, 47.5, 49.8, 52.1, 55.1, 67.5, 100.5,
125.4, 126.2, 127.1, 127.9, 128.3, 128.9, 129.5, 139.5,
141.3, 144.4, 149.8, 155.1, 158.2, 160.3, 165.3, 171.2.
HRMS: m/z calcd for C37H40N4NaO7: 675.2795; found:
675.2791 [M ? Na].
1
5b: White solid, mp 168–171°C, H NMR (300 MHz,
DMSO-d6): d = 0.89 (6H, d, J = 5.8 Hz), 1.28–1.42 (6H,
m), 1.82 (1H, m), 3.65 (3H, s), 4.25 (1H, t, J = 4.2 Hz),
4.65 (2H, d, J = 6.2 Hz), 4.81–4.92 (2H, m), 5.25 (2H, s),
5.95–6.21 (br, 2H), 6.12 (1H, s), 6.91 (1H, br), 7.12–7.71
(13H, m); 13C NMR (75 MHz, DMSO-d6): d = 18.1, 22.1,
28.9, 31.3, 36.5, 41.8, 47.5, 49.2, 52.1, 55.5, 65.1, 67.5,
100.2, 125.1, 125.9, 126.5, 127.2, 128.1, 128.9, 129.5,
141.1, 141.5, 144.3, 150.1, 156.2, 156.7, 158.1, 160.2,
171.4. HRMS: m/z calcd for C38H42N4NaO8: 705.2900;
found: 705.2905 [M ? Na].
Results and Discussion
Though the ultimate step in an isoxazole synthesis is a
cycloaddition reaction between a nitrile oxide and an
alkyne, the generation of the required intermediates is a
multi-step process. It involves the preparation of aldoxime
which in turn should be prepared from the corresponding
aldehyde. This follows a series of reactions viz halogena-
tion of aldoxime to hydroximinoyl halide (Chiang 1971),
dehydrohalogenation into nitrile oxide (Liu et al. 1980),
and finally a cycloaddition leading to the corresponding
substituted isoxazole. The present study was initiated by
taking Na-Fmoc-Phe-OH as an example to optimize the
reaction conditions. Initially, the acid was converted to
alcohol by reducing the corresponding mixed anhydride
with moist sodium borohydride. Oxidation of the resulting
Na-Fmoc-Phe-w[CH2OH] employing Dess-Martin Period-
inane (DMP) (Myers et al. 2000) in CH2Cl2 afforded the
aldehyde Fmoc-Phe-H 1c in near quantitative yield.
Although many protocols are available for the conversion
of N-protected b-amino alcohols to corresponding alde-
hydes, we found that the only Dess-Martin periodinane
induced oxidation afforded the aldehyde derivatives with
maximal enantiomeric enrichment (up to 97–99% ee). To
determine the enantiomeric purity of the N-Fmoc-aminals,
they were converted to respective alcohols via the NaBH4
reduction and then analyzed through chiral HPLC
(chiralpak IC 250 9 4.6 mm. 5lL). An ee in the range of
97–99% was recorded in case of Fmoc-derivatives (Myers
et al. 2000). However, Boc and Z-protected aminals had ee
up to 94–96%.
1
5c: White solid, mp 157–159°C, H NMR (300 MHz,
DMSO-d6): d = 1.21 (3H, d, J = 5.8 Hz), 3.12 (2H, d,
J = 5.2 Hz), 3.66 (3H, s), 4.22 (1H, t, J = 4.8 Hz), 4.58
(1H, m), 4.67 (2H, d, J = 6.4 Hz), 5.02 (1H, m), 6.10 (1H,
s), 6.95–7.11 (2H, br), 7.12–7.68 (13H, m); 13C NMR
(75 MHz, DMSO-d6): d = 19.8, 35.8, 47.5, 48.8, 52.5,
53.6, 67.5, 100.1, 125.1, 125.9, 126.5, 127.1, 127.8,
128.1,128.8, 139.8, 141.2, 144.3, 150.5, 156.3, 158.4,
160.1, 171.2. HRMS: m/z calcd for C28H29N3NaO6:
562.1954; found: 562.1948 [M ? Na].
1
5d: White solid, mp 157–159°C, H NMR (300 MHz,
DMSO-d6): d = 1.23 (3H, d, J = 5.8 Hz), 3.16 (2H, d,
J = 5.2 Hz), 3.65 (3H, s), 4.21 (1H, t, J = 4.6 Hz), 4.56
(1H, m), 4.65 (2H, d, J = 6.2 Hz), 5.05 (1H, m), 6.12 (1H,
s), 6.95–7.08 (2H, br), 7.11–7.64 (13H, m); 13C NMR
(75 MHz, DMSO-d6): d = 19.7, 35.6, 47.3, 48.5, 52.2,
53.8, 67.4, 100.0, 125.3, 125.8, 126.4, 127.2, 127.7,
128.2,128.8, 139.5, 141.3, 144.5, 150.4, 156.5, 158.2,
160.3, 171.5. HRMS: m/z calcd for C28H29N3NaO6:
562.1954; found: 562.1948 [M ? Na].
1
6a: White solid, mp 144–145°C, H NMR (300 MHz,
DMSO-d6): d = 0.94 (6H, d, J = 5.8 Hz), 1.26–1.48 (6H,
m), 1.58–1.68 (4H, m), 1.81 (1H, m). 2.78 (2H, m), 3.25
(2H, m), 3.61 (3H, s), 3.91–4.20 (2H, m), 4.25 (1H, t,
J = 4.6 Hz), 4.51 (2H, d, J = 6.2 Hz), 4.81 (1H, m), 5.25
(2H, s), 5.62 (1H, br), 5.85–6.01 (2H, br), 6.21 (1H, s),
7.25–7.71 (13H, m); 13C NMR (75 MHz, DMSO-d6):
d = 17.6, 21.2, 22.5, 28.5, 29.8, 31.3, 35.3, 42.8, 47.5,
51.8, 52.5, 55.6, 58.1, 65.6, 67.5, 100.2, 124.5, 125.6,
126.2, 127.1, 127.8, 128.5, 129.3, 141.3, 141.5, 144.8,
150.3, 156.3, 157.1, 158.1, 159.5, 168.1, 171.5. HRMS:
m/z calcd for C43H49N5NaO9: 802.3428; found: 802. 3432
[M ? Na].
Na-Fmoc-phenylalaninal 1c thus obtained, was treated
with hydroxylamine hydrochloride (NH2OHÁHCl) in pres-
ence of Na2CO3 at room temperature for about an hour to
obtain the corresponding aldoxime 2b in 95% yield
(Scheme 1).
We then sought a mild protocol for the generation of
nitrile oxide from the corresponding aldoxime. Some of the
widely employed literature procedures include the combi-
nation of a halogenating agent and a strong base. However,
the harsh conditions requiring high temperatures and other
disadvantages did not encourage us to employ these pro-
tocols in peptide chemistry. In a report, Fokin et al.,
1
6b: White solid, mp 146–148°C, H NMR (300 MHz,
DMSO-d6): d = 0.89–1.01 (6H, m), 1.21 (2H, m), 1.32
(3H, d, J = 6.2 Hz), 2.62 (1H, m), 2.81 (2H, d,
J = 5.8 Hz), 3.65 (3H, s), 4.25 (1H, t, J = 4.6 Hz), 4.41
123

Int J Pept Res Ther (2011) 17:185–191
189
Scheme 1 Synthesis of N-
Fmoc/Boc/Z-protected 3,5-
disubstituted isoxazole based
d- amino acids
R1
R1
R1
TsN(Cl)Na.3H2O
-BuOH:H2O (1:1)
NH2OH
EtOH
H
H
PgHN
PgHN
N O
t
PgHN
NOH
O
2
3
1
R1
O
COOH
CuSo4.5H2O/NaAsc
PgHN
OH
N
O
4
demonstrated the utility of chloramine-T trihydrate as
halogenating agent as well as a base for the mild conver-
sion of oxime into nitrile oxide (Hansen et al. 2005).
Applying a similar protocol, N-Fmoc- Phe-aldoxime 2c
was treated with chloramine-T, initially to yield the cor-
responding hydroximinoyl chloride, which later underwent
dehydrohalogenation to form the nitrile oxide 3c. The
formation of nitrile oxide was confirmed by the IR analysis,
which showed a peak at around 2295 cm-1, characteristic
of -CN stretching frequency. This intermediate, without
isolation, was subjected to Cu(I) catalyzed cycloaddition
with propiolic acid in the presence of CuSO4/sodium
ascorbate (NaAsc) catalyst system. The reaction was
complete in 5 h at room temperature and the resulting
isoxazole 4 was isolated after a simple work up and
recrystallization in about 67% yield.
isolation of pure compound from unreacted aldoxime and
other impurities, thus avoiding time consuming column
chromatography. The protocol was successfully employed for
the preparation of several examples of Fmoc-protected 3,5-
substituted isoxazoles including those derived from bifunc-
tional amino acids 4a–i. When Boc and Z- groups were
employed for amino protection, the resulting 3,5-disubstituted
isoxazoles were isolated in affordable yields as gums
4j–m. All the compounds 4a–m were characterized
through NMR and mass spectrometry (Scheme 2; Table 1).
The chiral HPLC (chiralpak IC 250 9 4.6 mm. 5lL)
analyses of the two enantiomeric isoxazoles derived from
Ala 4a and D-Ala 4b had major peaks at Rt 12.351 and
7.100 min respectively, while the equimolar mixture of the
two enantiomers eluted at retention times 12.610 and
7.285 min (30–70% MeOH:H2O). This infers that the
isoxazole derivatives are optically homogeneous.
After establishing a pathway for accessing the target
molecule, we then turned our attention to simplify the
overall protocol. Since the conversion of aminal into
aldoxime was quantitative, we envisaged that its isolation
prior to the generation of nitrile oxide is not a mandatory.
Thus a one-pot synthesis of isoxazoles starting from
Na-protected amino aldehyde seemed achievable.
Next, we turned our attention to demonstrate the utility
of isoxazole-linked unnatural amino acids 4 as monomeric
building blocks for constructing corresponding di and tri-
peptidomimetics through N- as well as C-terminal exten-
sions. Initially, Na-Fmoc-protected 3-amino ethyl 5-carboxylic
acid 4a was coupled with leucine methyl ester in CH2Cl2 using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and
1-hydroxy benzotriazole (HOBt) system. The resulting di-
peptidomimetic 5a was isolated in 75% yield after workup.
Three more dipeptidomimetics 5b, 5c, 5d were also pre-
pared through similar protocol (Scheme 3).
Towards this goal, in a typical reaction, to a solution of
Fmoc-Phe-H 1c in tert-BuOH/water system, an equimolar
quantity of hydroxylamine was added. The resulting
aldoxime was treated with chloramine-T followed by the
addition of propiolic acid along with catalytic amount of
CuSO4 and NaAsc. The reaction mixture was stirred for
5 h at room temperature. The nitrile oxide 3 generated in
situ underwent cycloaddition with the propargyl group to
yield the corresponding isoxazolyl amino acid. The for-
mation of nitrile oxide intermediate 3 was traceable via
IR analysis. A strong signal at around 2295 cm-1 corre-
sponding to CN stretching frequency disappeared rapidly
upon addition of propiolic acid due to the cycloaddition
reaction. After acidification of the reaction mixture with
10% dil. HCl, the required isoxazole 2c precipitated out as
solid and was collected by filtration. Use of propiolic acid as
dipolarophile in the reaction led the isoxazole derivative
with free carboxy moiety. The acid base workup eased the
In the subsequent steps, N-terminal extension of dipep-
tidomimetics 5 leading to corresponding tripeptidomimetic
1. NH2OH.HCl
2. TsN(Cl)Na.3H2O
3. propiolic acid
4. CuSO4.5H2O
NaAsc
R1
O
R1
H
PgHN
OH
PgHN
t
-BuOH:H2O (1:1)
N
O
O
1a-m
4a-m
Pg = Fmoc, Boc, Z
R1 = alkyl, aryl
Scheme 2 One-pot synthesis of urethane protected 3,5-disubstituted
isoxazole based unnatural amino acids
123

190
Int J Pept Res Ther (2011) 17:185–191
Table 1 List of urethane protected 3,5-disubstituted isoxazole d-amino acid 4
Entry
Pg
R1
mp (8C)
Yield (%)
[a]2D9 (c 0.1, MeOH)
Mass found (calcd)
4a
4b
4c
4d
4e
4f
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Z
CH3
138–139
138–139
135–137
148–149
154–156
150–152
122–123
143–145
132–134
118–120
121–123
Gum
82
82
78
69
73
75
74
71
72
74
71
68
69
13.5
401.1108 (401.1113)a
401.1 (401.2)b
453.2 (454.2)c
CH3(D)
15.6
CH2C6H5
(CH2)2SCH3
CH2COOBzl
(CH2)4NHZ
CH2CH(CH3)2
–(CH2)3–
(CH2)2COOBzl
CH3
-11.1
-3.85
-5.7
437.1 (438.1)c
535.1475 (535.1481)a
592.2055 (592.2060)a
443.2 (443.2)b
403.1 (404.1)c
527.2 (526.2)d
-20.5
-19.65
-4.6
4g
4h
4i
8.9
4j
-6.7
313.1 (313.1)b
4k
4l
Z
(CH2)4NHZ
CH3
-15.6
-20.7
-17.8
504.1745 (504.1747)a
257.1135 (256.1059)e
355.1 (355.1)b
Boc
4m
Boc
CH2C6H5
Gum
a
b
c
d
e
HRMS [M ? Na], ESI-MS [M ? Na], ESI-MS [M - H], ESI-MS [M ? H], HRMS [M ? H]
R1
O
R1
O
R2
EDC, HOBt
OMe
FmocHN
OH
FmocHN
N
H2N-CH(R2)-COOMe
H
N
O
N
O
O
5
4
5a: R1 = CH3; R2 = CH2CH(CH3)2 (75%); mp = 136-138 °C; [α]D
(C
0.1, MeOH) -16.1
29
29
5b: R1 = (CH2)4NHCbz; R2 = CH(CH3)2 (71%); mp = 168-171 °C; [α]D
(C 0.1, MeOH) 1.7
29
5c: R1 = CH3; R2 = CH2C6H5 (79%); mp = 157-159 °C; [α]D
(C
0.1, MeOH) 8.5
29
5d: R1 = CH3; R2 = CH2C6H5 (72%); mp = 157-159 °C; [α]D
(C 0.1, MeOH) 5.6
O
R1 R2
O
50% (v/v) DEA/DCM
FmocHN
OMe
N
N
EDC, HOBt
FmocNH-CH(R3)-COOH
H
H
R3
O
N
O
6
29
6a: R1 = (CH2)4NHCbz; R2 = CH(CH3)2 ; R3 = -(CH2)3- (72%); mp = 144-145 °C; [α]D
(C 0.1, MeOH) -15.9
29
6b: R1 = CH(CH3)C2H5; R2 = CH3; R3 = CH2C6H5 (72%); mp = 146-148 °C; [α]D
(C 0.1, MeOH) 6.9
Scheme 3 Synthesis of di and tripeptidomimetics
was demonstrated. Starting with 5b, the amine was
deprotected using diethylamine (DEA)/CH2Cl2 (1:1) and
the resulting free amino dipeptidomimetic was coupled to
Fmoc-Pro-OH (EDC/HOBt method) to obtain the tripep-
tidomimetic 6a in 72% yield (Scheme 3). In a similar way,
one more tripeptidomimetic 6b was prepared and isolated.
To evaluate the possibility of racemization in the described
protocol, NMR and HPLC studies on a pair of epimeric
dipeptide mimetics FmocNH-L-Ala-w[C3H3NO]-CO-NH-
Phe-COOMe 5c and FmocNH-D-Ala-w[C3H3NO]-CO-NH-
Phe-COOMe 5d prepared by coupling of FmocNH-L-Ala-w
[(C3H3NO)-COOH] 4a and FmocNH-D-Ala-w[(C3H3NO)-
COOH] 4b respectively with H2N-Phe-COOMe was also
of these dipeptides 5c and 5d had sole peaks at Rt 15.953
and 10.354 min respectively (20–80% MeOH:H2O).
This clearly suggests the presence of a single epimer in
each sample, thus confirm the epimeric purity of the
compounds.
Conclusion
In summary, a regioselective, one-pot protocol for the
preparation of N-urethane protected 3,5-disubstituted
isoxazoles employing Cu(I) catalyst is described. Further,
the protocol has been extended for the synthesis of isox-
azole-linked di and tri peptidomimetics. All the compounds
were isolated as stable solids and characterized through 1H,
13C NMR and mass spectrometry.
1
considered. In the H NMR spectra, distinct doublets for
alanyl methyl group (d 1.20 and 1.22 for 5c and d 1.22 and
1.24 for 5d) were observed. Further, the chiral HPLC profiles
123

Int J Pept Res Ther (2011) 17:185–191
191
Acknowledgments We thank the Department of Science and
Technology (DST), Government of India (No. SR/S1/OC-26/2008)
for financial assistance. We also thank Professor Fred Naider, CUNY,
New York, for useful discussions.
Jones RCF, Ward GJ (1988) Amide bond isosteres: imidazolines in
pseudopeptide chemistry. Tetrahedron Lett 29:3853–3856
Kantorowski EJ, Kurth MJ (1997) Solid-phase synthesis of isoxazoles
and isoxazolines: en route to a new class of ionophores. J Org
Chem 62:6797–6803
Kim BH, Chung YJ, Keum G, Kim J, Kim K (1992) A new peptide bond
surrogate: 2- isoxazoline in pseudopeptide chemistry. Tetrahedron
Lett 33:6811–6814
References
Kim BH, Chung YJ, Ryu EJ (1993) Synthesis of a-hydroxy ketom-
ethylene dipeptide isosteres. Tetrahedron Lett 34:8465–8468
Liu K-C, Shelton BR, Howe RK (1980) A particularly convenient
preparation of benzohydroximinoyl chlorides (nitrile oxide
precursors). J Org Chem 45:3916–3918
Luca LD, Giacomelli G, Riu A (2001) Solid-phase synthesis of
isoxazole-based amino acids: a new scaffold for molecular diver-
sity. J Org Chem 66:6823–6825
Myers AG, Zhong B, Movassaghi M, Kung DW, Lanman BA, Kwon
S (2000) Synthesis of highly epimerizable N-protected a-amino
aldehydes of high enantiomeric excess. Tetrahedron Lett 41:
1359–1362
Norman BH, Lander PA, Gruber JM, Kroin JS, Cohen JD, Jungheim
LN, Starling JJ, Law KL, Self TD, Tabas LD, Williams DC, Paul
DC, Dantzig AH (2005) Cyclohexyl-linked tricyclic isoxazoles
are potent and selective modulators of the multidrug resistance
protein (MRP1). Bioorg Med Chem Lett 15:5526–5530
Patil BS, Vasanthakumar GR, Sureshbabu VV (2003) Isocyanates of
Na-[(9- Fluorenylmethyl)oxy]carbonyl amino acids: synthesis,
isolation, characterization, and application to the efficient
synthesis of urea peptidomimetics. J Org Chem 68:7274–7280
Quilico A (ed) (1962) Five and six membered compounds with
oxygen and nitrogen. Wiley Interscience, New York, pp 1–46
Savant MM, Pansuriya AM, Bhuva CV, Kapuriya N, Patel AS,
Audichya VB, Pipaliya PV, Naliapara YT (2010) Water mediated
construction of trisubstituted pyrazoles/isoxazoles library using
ketene dithioacetals. J Comb Chem 12:176–180
Andre I, Alcazar J, Alonso JM, Lucas AID, Iturrino L, Biesmansb I,
Megens AA (2006) Synthesis of 7-amino-3a,4-dihydro-3H-[1]benzo-
pyrano[4,3-c]isoxazole derivatives displaying combined a2-adreno-
ceptor antagonistic and 5-HT reuptake inhibiting activities. Bioorg
Med Chem 14:4361–4372
Annunziata R, Cinquini M, Cozzi F, Gennari C, Raimondi L (1987)
Stereoselectivity of intramolecular nitrile oxide cycloadditions to
Z and E chiral alkenes. J Org Chem 52:4674–4681
Bailey TR, Diana GD, Kowalczyk PJ, Akullian V, Eissenstatrt MA,
Cutcliffe D, Mallamo JP, Carabateas PM, Pevear DC (1992)
Antirhinoviral activity of heterocyclic analogs of Win 54954.
J Med Chem 35:4628–4633 and references cited therein
Cheng J-F, Mjalli AMM (1998) Solid-phase synthesis of D2-isox-
azolines. Tetrahedron Lett 39:939–942
Chiang YH (1971) Chlorination of oximes. Reaction and mechanism
of the chlorination of oximes in commercial chloroform and
methylene chloride. J Org Chem 36:2146–2155
Cho CY, Moran EJ, Cherry SR, Stephans JC, Fodor SPA, Adams CL,
Sundaran A, Jacobs JW, Schultz PG (1993) An unnatural
biopolymer. Science 261:1303–1305
Chung YJ, Ryu EJ, Keum G, Kim BH (1996) Synthesis of
2-isoxazoline and a- hydroxy ketomethylene dipeptide isosteres.
Bioorg Med Chem 4:209–225
Cuadrado P, Gonzalez-Nogal AM, Valero R (2002) Regiospecific
synthesis of 5-silyl azoles. Tetrahedron 58:4975–4980
Easton CJ, Hughes M, Tiekink ERT (1994) Reversal of regiochem-
istry in the synthesis of isoxazoles by nitrile oxide cycloaddi-
tions. Tetrahedron Lett 35:3589–3592
Short KM, Ziegler CB (1993) The synthesis of isoxazoles from b,
c-acetylinic oximes. Tetrahedron Lett 34:75–78
Ebert B, Lenz S, Brehm L, Bregnedal P, Hansen JJ, Frederiksen K,
Bogeso KP, Krogsgaard-Larsen P (1994) Resolution, absolute
stereochemistry, and pharmacology of the S-(?)- and R-(-)-
isomers of the apparent partial AMPA receptor agonist (R, S)-2-
amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid [(R,
S)-APPA]. J Med Chem 37:878–884
Elkasaby MA, Salem MAI (1980) Synthesis of new isoxazoles from
isoxazolines, chalkones, chalkone dibromides, epoxides and
acetylated ketoximes and their conversion into novel heterocy-
cles. Indian J Chem Section B 19B:571–575
Shusheng Z, Tianrong Z, Kun C, Youfeng X, Bo Y (2008) Simple and
efficient synthesis of novel glycosyl thiourea derivatives as
potential antitumor agents. Eur J Med Chem 43:2778–2783
Sureshbabu VV, Patil BS, Venkataramanarao R (2006) Preparation,
isolation, and characterization of Na-Fmoc-peptide isocyanates:
solution synthesis of oligo-a-peptidyl ureas. J Org Chem 71:
7697–7705 and references cited therein
Tam A, Arnold U, Soellner MB, Raines RT (2007) Protein prosthesis:
1,5-disubstituted[1,2,3]triazoles as cis-peptide bond surrogates.
J Am Chem Soc 129:12670–12671
Gennari C, Salom B, Potenza D, Williams A (1994) Synthesis of
sulfonamido- pseudopeptides: new chiral unnatural oligomers.
Angew Chem Int Ed Engl 33:2067–2069
Goodman M, Felix A, Moroder L, Toniolo C (eds) (2003) Synthesis
of peptides and peptidomimetics (Houben-Weyl), vol E22c.
George Thieme Verlag, Stuttgart and references cited therein
Grunanger P, Vita-Finzi P (eds) (2008) Chemistry of heterocyclic
compounds: isoxazoles, Part 1, vol 49. Wiley Interscience, New
York
Hansen TV, Wu P, Fokin VV (2005) One-pot copper(I)-catalyzed
synthesis of 3,5-disubstituted isoxazoles. J Org Chem 70:
7761–7764
Himo F, Lovell T, Hilgraf R, Rostovtsev VV, Noodleman L,
Sharpless KB, Fokin VV (2005) Copper(I)-catalyzed synthesis
of azoles. DFT study predicts unprecedented reactivity and
intermediates. J Am Chem Soc 127:210–216
Vig R, Mao C, Venkatachalam TK, Tuel-Ahlgren L, Sudbeck EA,
Uckun FM (1998) Rational design and synthesis of phenethyl-5-
bromopyridyl thiourea derivatives as potent non-nucleoside
inhibitors of HIV reverse transcriptase. Bioorg Med Chem 6:
1789–1797
Wang K, Xiang D, Liu J, Pan W, Dong D (2008) Efficient and
divergent synthesis of fully substituted 1H-pyrazoles and
isoxazoles from cyclopropyl oximes. Org Lett 10:1691–1694
You S-L, Kelly JW (2005) Total synthesis of didmolamides A and B.
Tetrahedron Lett 46:2567–2570
You S-L, Deechongkit S, Kelly JW (2004) Solid-phase synthesis and
stereochemical assignments of tenuecyclamides A–D employing
heterocyclic amino acids derived from commercially available
Fmoc a-amino acids. Org Lett 6:2627–2630
Yu K-L, Johnson RL (1987) Synthesis and chemical properties of
tetrazole peptide analogs. J Org Chem 52:2051–2059
Huisgen R (1976) 1,3-Dipolar cycloadditions. 76. Concerted nature of
1,3-dipolar cycloadditions and the question of diradical inter-
mediates. J Org Chem 41:403–419
123