Synthesis of conformationally locked versions of puromycin analogues

Article abstract of DOI:10.1021/jo8016132

(Chemical Equation Presented) Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger-Vilarrasa coupling of the corresponding North (2 and 3) and South (6 and 7) 3′-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-L-tyrosine. North azides 2 and 3 were reported earlier. The 3′-azido intermediates 6 and 7 that are necessary for the synthesis of the South puromycin analogues are described herein for the first time.

Full text of DOI:10.1021/jo8016132

SCHEME 1
Synthesis of Conformationally Locked Versions
of Puromycin Analogues
Hisao Saneyoshi,§,† Benoˆıt Y. Michel,§,‡ Yongseok Choi,†
Peter Strazewski,*,‡ and Victor E. Marquez*,†
Laboratory of Medicinal Chemistry, Center for Cancer
Research, NCI-Frederick, Maryland 21702, and Laboratoire
de Synthe`se de Biomole´cules, Institut de Chimie et Biochimie
Mole´culaires et Supramole´culaires (UMR 5246), UniVersite´
Claude Bernard Lyon 1, 69622 Villeurbanne, France
marquezV@mail.nih.goV; strazewski@uniV-lyon1.fr
ReceiVed July 23, 2008
independent of the codon, it enters the A site because of its
molecular resemblance to the 3′ end of the normal ester-linked
3′-aminoacyl tRNA. Its primary amino group is capable of
taking over the nascent peptide chain; however, the resulting
more stable amide bond is resistant to further nucleophilic attack.
This causes the ribosome to stop peptide chain elongation and
to release a truncated C-terminal puromycyl peptide, which may
be lethal to the organism.
Conformationally locked North and South versions of puro-
mycin analogues built on a bicyclo[3.1.0]hexane pseudosugar
template were synthesized. The final assembly of the products
was accomplished by the Staudinger-Vilarrasa coupling of
the corresponding North (2 and 3) and South (6 and 7) 3′-
azidopurine carbanucleosides with the Fmoc-protected 1-hy-
droxybenzotriazole ester of 4-methoxy-L-tyrosine. North
azides 2 and 3 were reported earlier. The 3′-azido intermedi-
ates 6 and 7 that are necessary for the synthesis of the South
puromycin analogues are described herein for the first time.
Because of the important role of sugar puckering in nucleo-
sides, we have proposed the use of conformationally locked
carbocyclic nucleosides built on a rigid bicyclo[3.1.0]hexane
scaffold to restrict the embedded cyclopentane pseudosugar ring
to either a North-type (2E) or a South-type (3E) conformation
as defined in the pseudorotational cycle.4 These types of
modified nucleoside analogues have been used to study the role
of sugar conformation in the processes of recognition and
binding of nucleosides, nucleotides, and oligonucleotides to their
target enzymes.5
In the present work, we have expanded the scope of our
investigations in this area by synthesizing the puromycin
analogues in both North-type (4) and South-type flavors (8),
plus analogues 5 and 9 containing the 6-aminopurine base
(Scheme 1). The latter compounds are also valid probes because
Puromycin (1) is a 3′-amino-3′-deoxynucleoside antibiotic
produced by Streptomyces alboniger1 that specifically inhibits
peptidyl transfer on both prokaryotic and eukaryotic ribosomes
by interfering with RNA function and leading to premature chain
termination during translation.2,3 Like the natural substrate, but
† Center for Cancer Research.
‡ Universite´ Claude Bernard Lyon 1.
§ These two authors contributed equally to this work.
(1) Vara, J.; Pe´rez-Gonza´lez, J.; Jime´nez, A. Biochemistry 1985, 24, 8074–
8081.
(2) Kaji, A.; Kiel, M. C.; Hirokawa, G.; Muto, A. R.; Inokuchi, Y.; Kaji, H.
Cold Spring Harbor Symp. Quant. Biol. 2001, 66, 515–529.
(3) Azzam, M. E.; Algranati, I. D. Proc. Natl. Acad. Sci. U.S.A. 1973, 70,
3866–3869.
(4) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205–8212.
(5) Marquez, V. E. In Modified Nucleosides in Biochemistry, Biotechnology
and Medicine; Herdewijn, P., Ed.; Wiley-VCH: Weinheim, 2008; Chapter 12,
pp 307-341.
10.1021/jo8016132 CCC: $40.75
Published on Web 11/08/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 9435–9438 9435

SCHEME 2
SCHEME 3
SCHEME 4
the methyl substituents on the dimethylamino group are not
essential for puromycin-like activity.6
These novel puromycin ensembles (Scheme 1) should help
determine the preferred conformation of the 3′-terminal ribosyl
residue of peptide-accepting aminoacyl-tRNA at the critical A
site. Although the proposed target compounds lack the 2′-OH,
the bicyclo[3.1.0]hexane ring system is able to precisely confine
the conformation of the pseudosugar regardless of the presence
of the 2′-OH. In 1990 Koizumi et al. synthesized 2′-deoxy-
puromycin and suggested that the weaker antimicrobial proper-
ties of the compound were due to a conformational dominance
of the South conformation in contrast to puromycin′s North
conformation that was revealed by proton NMR.7 Hence, the
target compounds (4 and 5, 8 and 9) are proposed to reliably
test whether the puckering of the sugar ring changes the
orientation of the aminoacyl moiety of puromycin and how it
influences the recognition by peptidyl transferase at the active
site.
series using Mitsunobu reaction conditions.8 Formation of the
mesylate esters and a second inversion of configuration resulting
from the nucleophilic attack with sodium azide produced
compounds 18 and 19, which after the removal of the silyl ether
provided the key target 3′-azides 6 and 7 in satisfactory yields.
In order to make the corresponding 6-aminopurine puromycin
analogues, the amino group required protection, which was
accomplished by reacting with N,N-di(n-butyl)formamide di-
methyl acetal prepared as described by Froehler and Matteucci10
and reacted with North and South 6-aminopurine analogues, 38
and 7 to give the 6-N-[(di-n-butylamino)methylene]-protected
compounds 20 and 21 (Scheme 3).
Compounds 2 and 3 in the North hemisphere, which are
necessary for the synthesis of 4 and 5, have already been made
and reported.8 For the South puromycin analogues a similar
strategy required the synthesis of the critical 3′-azido intermedi-
ates 6 and 7 as shown in Scheme 2.
Starting from known cyclopentenol 10, the synthesis of 11
was performed in 12 steps (4.2% yield) as previously described.9
Conversion of the 6-chloro derivative to either the 6-dimethy-
lamino- or 6-aminopurine carbobicyclic nucleosides 12 and 13
was easily accomplished after treatment with dimethylamine
or ammonium hydroxide, respectively. After protection of the
primary alcohol as silyl ethers 14 and 15, the strategy to invert
the configuration of the 3′-OH (nucleoside numbering) was
performed by a tandem oxidation-reduction methodology using
Dess-Martin oxidation followed by reduction with L-selectride
to give 16 and 17. This process was more efficient than the
inversion of configuration approach reported earlier for the North
Having the necessary 3′-azide precursors at hand, we then
employed the very efficient, one-step Vilarrasa-modified11
Staudinger coupling utilizing the activated amino acid, 4-meth-
oxy-L-tyrosine, protected as the N-(9-fluorenylmethyl)-carbamate
(N-Fmoc-OMe-L-Tyr) as shown in Scheme 4. Previously, our
group had successfully employed and optimized this approach
for the synthesis of a number of noncarbocyclic puromycin
analogues.12-15 Thus, a mixture of N-Fmoc-OMe-L-Tyr and
anhydrous 1-hydroxybenzotriazole (HOBT) was treated with
1,3-diisopropylcarbodiimide (DIC) and then chemoselectively
added to the phosphine imines that were generated from the
reaction between the azides and trimethylphosphine. The final
coupling yields after deprotection were ca. 78%.
In summary, we have achieved the syntheses of conforma-
tionally locked North and South versions of puromycin ana-
(10) Froehler, B. C.; Matteucci, M. D. Nucleic Acids Res. 1983, 11, 8031–
8036.
(11) Ariza, X.; Urp´ı, F.; Viladomat, C.; Vilarrasa, J. Tetrahedron Lett. 1998,
(6) Lichtenthaler, F. W.; Cuny, E.; Morino, T.; Rychlik, I. Chem. Ber. 1979,
39, 9101–9102.
112, 2588–2601.
(12) Chapuis, H.; Strazewski, P. Tetrahedron 2006, 62, 12108–12115.
(13) Charafeddine, A.; Dayoub, W.; Chapuis, H.; Strazewski, P. Chem. Eur.
(7) Koizumi, F.; Oritani, T.; Yamashita, K. Agric. Biol. Chem. 1990, 54,
3093–3097.
(8) Choi, Y. S.; George, C.; Strazewski, P.; Marquez, V. E. Org. Lett. 2002,
4, 589–592.
J. 2007, 13, 5566–5584.
(14) Charafeddine, A.; Chapuis, H.; Strazewski, P. Org. Lett. 2007, 9, 2787–
2790.
(9) Ezzitouni, A., Jr.; Marquez, V. E. J. Chem. Soc., Perkin Trans 1 1997,
1073–1078.
(15) Michel, B. Y.; Krishnakumar, K. S.; Strazewski, P. Synlett 2008, 2461–
2464.
9436 J. Org. Chem. Vol. 73, No. 23, 2008

mixture was stirred for 1 min at room temperature. The amino acid
solution was warmed to room temperature during 1 min and then
added to the iminophosphorane solution. The reaction mixture was
stirred at root temperature overnight. The solution was concentrated
under reduced pressure, coevaporated from CHCl3 (10 mL),
dissolved in EtOAc (30 mL), and extracted with saturated NaHCO3
(15 mL). The organic layer was extracted twice with EtOAc, washed
with H2O (2 × 10 mL), dried (MgSO4), filtered, and evaporated in
vacuo. The residue was purified by silica gel column chromatog-
raphy (EtOAc/cyclohexane/MeOH, 3/1/0 f 4/1/0.5 f 4/1/0.7, v/v)
to give the coupling product 23 (115 mg, 82%) as a solid, mp
99-102 °C.
logues on a bicyclo[3.1.0]hexane pseudosugar template. These
compounds were designed to investigate the ideal conformation
of the 3′-terminal ribofuranose ring of 3′-aminoacyl tRNAs
during takeover of the nascent peptide in the ribosome. The
biological evaluation of these compounds will be reported
elsewhere.
Experimental Section
(2S)-N-{(1R,2S,4S,5S)-4-[6-(Dimethylamino)purin-9-yl)-1-
(hydroxymethyl)bicyclo[3.1.0]hex-2-yl}-2-[(fluoren-9-ylmethoxy)-
carbonylamino]-3-(4-methoxyphenyl)propanamide (22). A mixture
of N-Fmoc-O-Me-L-Tyr (22 mg, 0.063 mmol) and HOBT (8.6 mg,
0.053 mmol) was coevaporated three times from anhydrous THF
(3 mL). The residue was dissolved in THF (1.0 mL), and the
solution was cooled down to 0 °C under N2 for 10 min. Then, DIC
(34 µL, 0.212 mmol) was added, and the reaction mixture was
stirred for 10 min at the same temperature. Nearby, (Me)3P (1 M
in THF, 80 µL, 0.080 mmol) was added to a solution of the azide
28 (12 mg, 0.038 mmol) in THF (1.0 mL), which was stirred for 1
min at room temperature. The amino acid solution was warmed to
room temperature during 1 min and then added to the iminophos-
phorane solution. The reaction mixture was stirred at room
temperature overnight. The solution was concentrated under reduced
pressure, coevaporated from CHCl3 (5 mL), then dissolved in
EtOAc (15 mL), and extracted with saturated NaHCO3 (8 mL).
The organic layer was extracted twice with EtOAc, washed with
H2O (2 × 5 mL), dried (MgSO4), filtered, and evaporated under
reduced pressure. The oily residue was purified by silica gel column
chromatography (EtOAc/cyclohexane/MeOH, 2/1/0 f 3/1/0 f
4/1/0 f 5/1/0 f 4/1/0.7, v/v) to give the coupling product 22 (23
mg, 89%) as a colorless resin.
(2S)-N-[(1R,2S,4S,5S)-4-(6-Aminopurin-9-yl)-1-(hydroxymethyl)-
bicyclo[3.1.0]hex-2-yl]-2-amino-3-(4-methoxyphenyl)propana-
mide (5). Compound 23 (57 mg, 0.071 mmol) was dissolved in
33% CH3NH2/EtOH (7 mL). The reaction mixture was stirred at
room temperature overnight in a closed vessel. The solution was
concentrated under reduced pressure and coevaporated from CHCl3
(7 mL). The oily residue was purified by silica gel column
chromatography (EtOAc/toluene/MeOH, 5/1/0.5 f 4/1/0.5; EtOAc/
MeOH/H2O, 8/1/0.5 f 6/1/0.5 f 4 /1 /0.5, v/v) to give after
evaporation the target compound (5) as a fluffy solid (30 mg, 96%),
which after lyophilization from H2O/TFA (pH 3.0) gave the more
1
water-soluble salt 5·TFA: mp 140-142 °C; H NMR (D2O, 500
MHz) δ 0.69 (1H, irregular t, J ) 7.0, 8.0, H-6′a), 0.81 (1H, dd, J
) 6.0, 4.0 Hz, H-6′b), 1.66-1.74 (2H, m, H-5′, H3′a), 2.11 (1H,
dd, J ) 10.0, 8.0 Hz, H-3′b), 2.82 (1H, dd, 1 H, J ) 13.5, 6.9 Hz,
p-MeOPhCHH), 3.02-3.08 (1H, m, p-MeOPhCHH), 3.07 (1H, d,
J ) 12.3 Hz, CHHOH), 3.49 (1H, d, J ) 12.3 Hz, CHHOH),
3.75-3.82 (1H, m, HR-tyrosyl), 3.82 (3H, s, OCH3), 4.61 (1H,
irregular t, J ) 9.5, 85 Hz, H-2′), 4.86 (1H, d, J ) 8.5 Hz, H-4′),
6.95 (2H, d, J ) 8.5 Hz, Ph(OMe)), 7.18 (2H, d, J ) 8.5 Hz,
Ph(OMe)), 8.10 (1H s,, H-2), 8.34 (1H, s, H-8); 13C NMR (C5D5N/
few drops D2O, 75 MHz) δ 10.6, 25.8, 35.8, 36.3, 41.3 49.7, 55.8,
(2S)-N-{(1R,2S,4S,5S)-4-[6-(Dimethylamino)purin-9-yl]-1-
(hydroxymethyl)bicyclo[3.1.0]hex-2-yl}-2-amino-3-(4-methoxyphe-
nyl)propanamide (4). Compound 22 (20 mg, 0.071 mmol) was
dissolved in 33% CH3NH2/EtOH (3 mL). The reaction mixture was
stirred at room temperature overnight in a closed vessel. The
solution was concentrated in vacuo and coevaporated from CHCl3
(5 mL). The oily residue was purified by silica gel column
chromatography (EtOAc/toluene/MeOH, 5 /1 /0.5 f 4 /1 /0.5, v/v;
EtOAc/MeOH/H2O, 8/1/0.5 f 6/1/0.5 f 4/1/0.5, v/v) to yield after
evaporation the target compound 4 (13.5 mg, 99%) and, after
lyophilisation from H2O/TFA (pH 3.0), the more water-soluble salt
55.9, 57.4, 63.0, 114.7, 119.7, 130.6, 131.4, 139.9, 149.2, 153.0,
+
156.5, 158.9, 176.9; HRMS (ESI+) calcd for (C22H28N7O3
)
438.2254 (MH+), found 438.2252.
(2S)-N-{(1S,3S,4S,5S)-1-[6-(Dimethylamino)purin-9-yl]-4-
(hydroxymethyl)bicyclo[3.1.0]hex-3-yl}-2-(fluoren-9-ylmethoxy)-
carbonylamino]-3-(4-methoxyphenyl)propanamide (24). A mixture
of N-Fmoc-O-Me-L-Tyr (106 mg, 0.263 mmol) and HOBT (42 mg,
0.263 mmol) was coevaporated three times from anhydrous THF
(2 mL). The residue was dissolved in THF (2.0 mL), and the
solution was cooled to 0 °C under N2 for 10 min. Then, DIC (36
µL, 0.225 mmol) was added, and the reaction mixture was stirred
for 10 min at the same temperature. Nearby, (Me)3P (1 M in THF,
375 µL, 0.375 mmol) was added to a solution of the azide 6 (59
mg, 0.188 mmol) in THF (2.0 mL) and stirred for 1 min at room
temperature. The amino acid was warmed to room temperature
during 1 min and then added to the iminophosphorane solution.
The reaction mixture was stirred at room temperature overnight.
The solution was concentrated under reduced pressure, coevaporated
from CHCl3 (10 mL), then dissolved in EtOAc (30 mL), and
extracted with saturated NaHCO3 (15 mL). The organic layer was
extracted twice with EtOAc, washed with H2O (2 × 10 mL), dried
(MgSO4), filtered, and evaporated in vacuo. The oily residue was
purified by silica gel column chromatography (EtOAc/cyclohexane/
MeOH), 2/1/0 f 3/1/0 f 4/1/0 f 5/1/0 f 4/1/0.5, v/v) to give
the coupling product 24 (105 mg, 81%) as a colorless resin.
(2S)-N-{(1S,3S,4S,5S)-1-[6-(Dimethylamino)purin-9-yl]-4-
(hydroxymethyl)bicyclo[3.1.0]hex-3-yl}-3-(4-methoxyphenyl)pro-
panamide (8). The protected coupling product 24 (60 mg, 0.087
mmol) was dissolved in 33% CH3NH2/EtOH (5 mL). The reaction
mixture was stirred at room temperature overnight in a closed vessel.
The solution was concentrated under reduced pressure and co-
evaporated from CHCl3 (7 mL), and the oily residue was purified
by silica gel column chromatography [(EtOAc/toluene/MeOH), 5/1/
0.5 f 4/1/0.5, v/v; (EtOAc/MeOH/H2O), 8/1/0.5 f 6/1/0.5 f 4/1/
0.5, v/v] to give after evaporation the target compound 8 (39 mg,
1
4·TFA as a hygroscopic solid: H NMR (CD3OD, 300 MHz) δ
0.73 (1H, dd, J ) 6.0, 3.9 Hz, H-6′a), 0.90 (1H, dd, J ) 6.0, 3.9
Hz, H-6′b), 1.68-1.79 (2H, m, H-3′a, H-5′), 2.07 (1H, dd, J )
14.7, 8.1 Hz, H-3′b), 2.87 (1H, dd, J ) 13.6, 6.8 Hz, p-
MeOPhCHH), 2.95 (1H, dd, J ) 13.6, 7.5 Hz, p-MeOPhCHH),
3.16 (1H, d, J ) 11.9 Hz, CHHOH), 3.49 (6H, br. s, NMe2), 3.68
(1H, irregular t, J ) 7.5, 6.8 Hz,, HR-tyrosyl), 3.77 (3H, s, OCH3),
3.86 (1H, d, J ) 11.9 Hz, CHHOH), 4.77-4.87 (1H, m, H-2′),
5.01 (1H, d, J ) 6.6 Hz, H-4′), 6.88 (2H, d, J ) 8.6, Ph(OMe),
7.16 (2H, d, J ) 8.6 Hz, Ph(OMe)), 8.19 (1H, s, H-2), 8.55 (1H,
s, H-8); 13C NMR (CD3OD, 75 MHz) 10.7, 26.5, 36.0, 37.0, 39.1,
40.6, 50.2, 55.8, 56.4, 57.1, 63.5, 115.2, 121.2, 129.5, 131.5, 139.2
150.7, 152.8, 156.2, 160.3, 174.9; HRMS (ESI+) calcd for
(C24H32N7O3+) 466.2567 (MH+), found 466.2566.
(2S)-N-((1R,2S,4S,5S)-4-{6-[1-Aza-2-(dibutylamino)-vinyl]pu-
rin-9-yl}-1-(hydroxymethyl)bicyclo-[3.1.0]hex-2-yl)-2-[(fluoren-
9-ylmethoxy)carbonylamino]-3-(4-methoxyphenyl)propana-
mide (23). A mixture of N-Fmoc-O-Me-L-Tyr (99 mg, 0.247 mmol)
and HOBT (40 mg, 0.247 mmol) was coevaporated three times
with anhydrous THF (3 mL). The residue was dissolved in THF
(2.5 mL), and the solution was cooled to 0 °C under N2 for 10
min. Then, DIC (34 µL, 0.212 mmol) was added, and the reaction
mixture was stirred for 10 min at the same temperature. Nearby,
(Me)3P (1 M in THF, 282 µL, 0.282 mmol) was added to a solution
of the azide 20 (75 mg, 0.176 mmol) in THF (2.5 mL), and the
J. Org. Chem. Vol. 73, No. 23, 2008 9437

96%) and, after lyophilization from H2O/TFA (pH 3.0), the more
propanamide (9). Compound 25 (59 mg, 0.074 mmol) was
dissolved in 33% CH3NH2/EtOH (5 mL). The reaction mixture was
stirred at room temperature overnight in a closed vessel. The
solution was then concentrated under reduced pressure and co-
evaporated from CHCl3 (8 mL). The oily residue was purified by
silica gel column chromatography (EtOAc/toluene/MeOH, 5 /1 /0.5
f 4 /1 /0.5, v/v; EtOAc/ MeOH/H2O, 8 /1 /0.5 f 6 /1 /0.5 f 4
/1/0.5, v/v) to give after evaporation the target compound 9 (31
mg, 96%) and, after lyophilization from H2O/TFA (pH 3.0), the
more water-soluble salt 9·TFA as an off-white and hygroscopic
amorphous solid: 1H NMR (CD3OD, 300 MHz) δ 0.80 (1H,
irregular t, J ) 5.1, 6.3 Hz, H-6′a), 1.14-1.27 (1H, m, H-6′b);
1.69 (1H, dd, J ) 4.8, 9.6 Hz, H-5′), 1.85-1.89 (1H, m, H-4′),
2.16 (1H, d, J ) 13.8 Hz, H-2′a, 2.46-2.54 (1H, m, H-2′b);
2.84-3.00 (2H, m, p-MeOPhCH2), 3.68 (3H, s, OCH3), 3.75 (1H,
d, J ) 11.3, 4.1 Hz, CHHOH), 3.80-3.90 (1H, m, HR-tyrosyl),
3.86 (1H, dd, J ) 11.3, 3.0 Hz, CHHOH); 4.13 (1H, d, J ) 8.7
Hz, H-3′), 6.82 (2H, d, J ) 8.4 Hz, Ph(OMe)), 7.11 (2H, d, J )
8.4 Hz, Ph(OMe)), 8.04 (1H, s, H-8), 8.08 (1H, s, H-2); 13C NMR
(CD3OD, 75 MHz) δ 18.1, 28.3, 39.1, 40.5, 44.8, 51.6, 54.9, 55.8,
56.2, 66.3, 115.4, 120.2, 128.3, 131.7, 143.2, 150.9, 153.5, 157.5,
160.6, 171.3; HRMS (ESI+) calcd for (C22H27N7NaO3+) 460.2073
(MNa+), found 460.2072.
water-soluble salt 8·TFA as a off-white amorphous and hygroscopic
1
solid: H NMR (CD3OD, 500 MHz) δ 0.68 (1H, dd, J ) 5.4, 6.3
Hz, H-6′a), 1.23-1.32 (1H, m, H-6′b), 1.79-1.90 (2H, m, H-5′,
H-4′), 2.24 (1 H, d, J ) 13.5 Hz, H-2′a), 2.56 (1H, dd, J ) 13.5,
9.0 Hz, H-2′b), 2.99 (1H, dd, J ) 9.6, 12.4 Hz, p-MeOPhCHH),
3.18 (1H, dd, J ) 5.7, 12.4 Hz, p-MeOPhCHH), 3.55 (6H, br s,
NMe2), 3.80 (3H, s, OCH3), 3.83 (1H, dd, J ) 4.8, 11.4 Hz,
CHHOH), 3.91 (1H, dd, J ) 3.9, 11.4 Hz, CHHOH), 3.96 (1H,
dd, J ) 5.7, 9.6 Hz, HR-tyrosyl), 4.14 (1H, d, J ) 9.0 Hz, H-3′),
6.88 (2H, d, J ) 8.6 Hz, Ph(OMe)), 7.13 (2H, d, J ) 8.6 Hz,
Ph(OMe)), 7.98 (1H, s, H-8), 8.19 (1H, s, H-2); 13C NMR (CD3OD,
125 MHz) δ 18.0, 28.4, 39.1, 39.8, 40.6, 44.7, 51.6, 54.9, 55.8,
56.5, 66.4, 115.3, 121.2, 128.8, 131.7, 141.2, 151.7, 152.8, 156.3,
160.6, 172.3; HRMS (ESI+) calcd for (C24H31N7NaO3+) 488.2386
(MNa+), found 488.2389.
(2S)-N-((1S,3S,4S,5S)-1-{6-[1-Aza-2-(dibutylamino)vinyl]pu-
rin-9-yl}-4-(hydroxymethyl)bicyclo-[3.1.0]hex-3-yl)-2-[(fluoren-
9-ylmethoxy)carbonylamino]-3-(4-methoxyphenyl)propana-
mide (25). N-Fmoc-O-Me-L-Tyr (106 mg, 0.263 mmol) and HOBT
(42 mg, 0.263 mmol) was coevaporated three times from anhydrous
THF (3 mL). The mixture was dissolved in THF (2.0 mL), and the
solution was cooled to 0 °C under N2 for 10 min. Then, DIC (36
µL, 0.226 mmol) was added, and the reaction mixture was stirred
for 10 min at the same temperature. Nearby, (Me)3P (1 M in THF,
376 µL, 0.376 mmol) was added to a solution of the azide 21 (79
mg, 0.186 mmol) in THF (2.0 mL) and stirred for 1 min at room
temperature. The aminoacid solution was warmed to room tem-
perature during 1 min and then added to the iminophosphorane
solution. The reaction mixture was stirred at room temperature
overnight. The solution was concentrated in vacuo, coevaporated
from CHCl3 (10 mL), then dissolved in EtOAc (30 mL), and
extracted with saturated NaHCO3 (15 mL). The organic layer was
extracted twice with EtOAc, washed with H2O (2 × 10 mL), dried
(MgSO4,), filtered, and evaporated under reduced pressure. The oily
residue was purified by silica gel column chromatography (EtOAc/
cyclohexane/MeOH, 2/1/0f3/1/0f 4/1/0.5f4/1/0.7, v/v) to give
the coupling product 25 (120 mg, 81%) as a white solid, mp 91-93
°C.
Acknowledgment. This research was supported in part by
the Intramural Research Program of the NIH, Center for Cancer
Research, NCI-Frederick. We gratefully acknowledge James A.
Kelley, Christopher Lai, D. Bouchu and C. Duchamp for mass
spectrometry results; B. Fenet and C. Gilbert for NMR analyses;
and Dina M. Sigano for her help during the preparation of this
manuscript. B.Y.M. is thankful for the Ministe`re de la Recherche
for his graduate fellowship.
Supporting Information Available: General experimental
details, procedures for the synthesis of compounds in Schemes
1
2 and 3, and record of H NMR, 13C NMR, and HRMS for
compounds 22-25, plus full NMR spectral characterization of
all compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(2S)-N-[(1S,3S,4S,5S)-1-(6-Aminopurin-9-yl)-4-(hydroxy-
methyl)bicyclo[3.1.0]hexan-3-yl]-2-amino-3-(4-methoxy-phenyl)-
JO8016132
9438 J. Org. Chem. Vol. 73, No. 23, 2008