Synthesis of 5'-amino-5'-phosphonate analogues of pyrimidine nucleoside monophosphates.

Article abstract of DOI:10.1021/jo030050x

The 5'-amino-5'-phosphono derivatives of cytidine, cytosine arabinoside (ara-C), and uridine have been prepared via the corresponding nucleoside aldehydes. Phosphite addition to imines derived from the nucleoside aldehydes and p-methoxybenzylamine was eff

Full text of DOI:10.1021/jo030050x

Syn th esis of 5′-Am in o-5′-p h osp h on a te An a logu es of P yr im id in e
Nu cleosid e Mon op h osp h a tes
Xuemei Chen and David F. Wiemer*
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242-1294
david-wiemer@uiowa.edu
Received February 11, 2003
The 5′-amino-5′-phosphono derivatives of cytidine, cytosine arabinoside (ara-C), and uridine have
been prepared via the corresponding nucleoside aldehydes. Phosphite addition to imines derived
from the nucleoside aldehydes and p-methoxybenzylamine was efficient, and use of this amine
allowed cleavage of the products to the parent amino phosphonic acids. The phosphite additions
proved to be diastereoselective, with the cytidine and uridine derivatives favoring the 5′S
stereochemistry and the ara-C derivative favoring the 5′R isomer. The stereochemistry of one
cytidine derivative was established by single-crystal diffraction analysis, and detailed comparisons
of the 13C NMR data allowed assignments of the other amino phosphonates.
Cytosine arabinoside (1, ara-C, Figure 1) is an effective
agent for treatment of myelogenous and other leukemias.
Unfortunately remissions obtained through ara-C treat-
ments tend to be short-lived, and ultimately patients
relapse with highly resistant disease refractory to all
subsequent forms of therapy.1 We have prepared ara-C
hydroxy phosphonates (e.g., 2 and 3) designed to mimic
ara-C monophosphate (ara-CMP, 4) and perhaps circum-
vent resistance on the basis of current understanding of
its metabolism.2 Formal replacement of the hydroxyl
group in a 5′-hydroxy-5′-phosphonate with an amino
functionality would create a new series of nucleoside
derivatives, 5′-amino-5′-phosphonates (5), which also
could serve as analogues of ara-CMP. Because the amino
group is inductively electron-withdrawing, the nucleoside
R-amino phosphonates might be expected to have similar
advantages to the R-hydroxy phosphonates in structure,
acidity, and metabolic stability. In addition, the basicity
F IGURE 1.
of the amino group may lead to a zwitterionic structure
at physiological pH and partially balance the negative
result, synthesis of phosphonate analogues of R-amino
charge of the phosphonic acid.
acids has been investigated extensively. The more gen-
Although there are no reports on synthesis and bio-
eral methods for preparation of chiral, nonracemic R-ami-
activity of nucleoside R-amino phosphonates, R-amino
no phosphonic acids include enantioselective addition of
phosphonic acids are commonly viewed as analogues of
phosphite to achiral cyclic imines,5 addition of phosphite
to nonracemic chelating imines3 (Figure 2, eq 1) and
nitrones,6 alkylation of phosphonamides with a stereo-
genic center either on the phosphonate ester7a or on an
imine functionality,7b preparation from chiral, nonrace-
mic R-hydroxy phosphonates via R-azidophosphonates8
the natural amino acids,3 and an interesting spectrum
of biological activities has been observed in peptidomim-
ics that incorporate an R-amino phosphonic acid.4 As a
* To whom correspondence should be addressed. Tel: 319-335-1365.
Fax: 319-335-1270.
(1) Grant, S. Adv. Cancer Res. 1998, 72, 197-233.
(2) Chen, X.; Wiemer, A. J .; Hohl, R. J .; Wiemer, D. F. J . Org. Chem.
2002, 67, 9331-9339.
(3) (a) Yager, K. M.; Taylor, C. M.; Smith, A. B., III. J . Am. Chem.
Soc. 1994, 116, 9377-9378. (b) Smith, A. B., III; Yager, K. M.; Taylor,
C. M. J . Am. Chem. Soc. 1995, 117, 10879-10888.
(4) (a) Atherton, F. R.; Hassall, C. H.; Lambert, R. W. J . Med. Chem.
1986, 29, 29-40. (b) Mcleod, D. A.; Brinkworth, R. I.; Ashley, J . A.;
J anda, K. D.; Wirsching, P. Bioorg. Med. Chem. Lett. 1991, 1, 653-
658. (c) Hirschmann, R.; Smith, A. B., III; Taylor, C. M.; Benkovic, P.
A.; Taylor, S. D.; Yager, K. M.; Sprengeler, P. A.; Benkovic, S. J . Science
1994, 265, 234-237.
(5) Groger, H.; Saida, Y.; Sasai, H.; Yamaguchi, K.; Martens, J .;
Shibasaki, M. J . Am. Chem. Soc. 1998, 120, 3089-3103.
(6) De Risi, C.; Dondoni, A.; Perrone, D.; Pollini, G. P. Tetrahedron
Lett. 2001, 42, 3033-3036.
(7) (a) Hanessian, S.; Bennani, Y. L. Tetrahedron Lett. 1990, 31,
6465-6468. (b) Song, Y.; Niederer, D.; Lane-Bell, P. M.; Lam, L. K.
P.; Crawley, S.; Palcic, M. M.; Pickard, M. A.; Pruess, D. L.; Vederas,
J . C. J . Org. Chem. 1994, 59, 5784-5793.
(8) Hammerschmidt, F.; Wuggenig, F. Tetrahedron: Asymmetry.
1999, 10, 1709-1721.
10.1021/jo030050x CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/08/2003
6108
J . Org. Chem. 2003, 68, 6108-6114

Analogues of Pyrimidine Nucleoside Monophosphates
SCHEME 2a
F IGURE 2.
SCHEME 1a
a
(i) Tf2O, pyr, CH2Cl2, -20 °C to rt; (ii) NaN3, DMF, 70 °C.
(eq 2), and phosphoramidate-amino phosphonate rear-
rangements9 (eq 3).
The requirement for strong base to induce the phos-
phoramidate rearrangement was not attractive in the
presence of the extensive functionality of the nucleoside
system, and so this approach was not explored. Because
the 5′-hydroxy phosphonates of cytidine and ara-C can
be prepared in a stereoselective fashion,2 the method
described in eq 2 initially was most attractive. Prepara-
tion of 5′-azido phosphonate 7 was attempted via syn-
thesis of the corresponding triflate from the 5′-hydroxy
phosphonate 6 followed by nucleophilic displacement of
triflate with NaN3 (Scheme 1), but unfortunately the
desired compound 7 was not obtained. When this at-
tempted transformation of a 5′-hydroxy phosphonate to
a 5′-azido phosphonate was not immediately successful,
efforts to synthesize the 5′-amino phosphonate nucleo-
sides were focused on addition of phosphite to nucleoside
imines. Addition of the lithium salt of diethyl phosphite
to nucleoside aldehydes readily affords R-hydroxy phos-
phonates, which also encouraged exploration of the
reactivity of nucleoside imines with phosphite anion.
As reported by Smith,3 a nonracemic imine prepared
by condensation of a nonracemic amine with an aldehyde
can give high stereocontrol to lithium-mediated phos-
phonylation of the corresponding imine. However, given
the several stereogenic centers inherently contained in
nucleoside aldehydes, in this case chirality in the amine
may not be required for stereochemical control. Among
achiral amines, 4-methoxybenzylamine (PMBNH2) ap-
peared to offer real advantage because well-studied PMB
deprotection methods allow choices for removal of the
PMB group in the later stage of the synthesis.
a
(i) DMSO, EDC, pyr, TFA, benzene; (ii) PMBNH2, benzene,
reflux with Dean-Stark apparatus; (iii) nBuLi, HP(O)(OEt)2, THF,
0 °C to rt.
2), initially was not as straightforward as precedents
suggested.10 In the presence of different dehydrating
agents, such as freshly dried Na2SO4, MgSO4, or 4 Å
molecular sieves, the reaction of aldehyde 9 with
PMBNH2 at room temperature did not afford the desired
nucleoside imine 10. However, when the aldehyde 9 was
treated with PMBNH2 in benzene and the reaction
mixture was heated at reflux with a Dean-Stark ap-
paratus, condensation was apparent. After removal of the
1
solvent, the H NMR spectrum of the resulting residue
showed a resonance at 7.9 ppm as a broad single peak
corresponding to the imine hydrogen. Without further
purification, this nucleoside imine (10) was treated with
the lithium salt of diethyl phosphite prepared as de-
scribed3 by reaction of diethyl phosphite and n-butyl-
lithium. In addition to the desired PMB-protected amino
phosphonate 11 and the corresponding deacylated prod-
uct 13, compound 12 (31P resonance at 51.5 ppm) also
was obtained in a substantial amount and could not be
separated readily from phosphonate 11. Instead of pur-
suing purification and characterization at this stage, it
was assumed that separation might be achieved more
readily after removal of the PMB group and that a
structure determination could be completed at that time.
Oxidative cleavage of a PMB group through reaction
with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)11 or
ceric ammonium nitrate (CAN)12 is attractive for depro-
Condensation of PMBNH2 with the nucleoside alde-
hyde 9, prepared from the protected cytidine 8 (Scheme
(10) (a) Boehm, J . C.; Kingsbury, W. D. J . Org. Chem. 1986, 51,
2307-2314. (b) Masson, G.; Compain, P.; Martin, O. Org. Lett. 2000,
2, 2971-2974. (c) Feldman, K. S.; Mingo, P. A.; Hawkins, P. C. D.
Heterocycles 1999, 51, 1283-1293.
(9) Hammerschmidt, F.; Hanbauer, M. J . Org. Chem. 2000, 65,
6121-6131.
J . Org. Chem, Vol. 68, No. 16, 2003 6109

Chen and Wiemer
SCHEME 3a
nance at 7.6 ppm was observed during the reaction, and
a sharp singlet at 8.0 ppm was observed after the reaction
was quenched, which corresponds to recovered phosphite.
For the parallel reaction with n-BuLi, four broad reso-
nances were detected at 49.2, 38.9, 35.6, and 6.3 ppm
during the reaction, and three sharp resonances were
found after NH4Cl was added, at 49.4, 35.2, and 8.0 ppm
in a ratio of 1.5:4.6:1.0. This result indicated that
exchange between the n-butyl and ethoxy groups can
occur when n-butyllithium and diethyl phosphite are
allowed to react and suggests that this exchange occurs
prior to addition of phosphorus to the nucleoside imine.
When diethyl phosphite is treated with LHMDS under
the same conditions, there is no evidence of exchange
with the ethoxy groups.15
On the basis of the above observations, the lithium salt
of diethyl phosphite, prepared in situ by treatment of
diethyl phosphite with LHMDS, was used in phospho-
nylation of the nucleoside imines below. The imines 10
and 22 (Scheme 5), prepared from protected cytidine 82
and uridine 21,16 respectively, via oxidation of the
primary alcohol and condensation of the resulting alde-
hyde with PMBNH2, were treated with the lithium salt
of diethyl phosphite to afford two sets of epimeric amino
phosphonates in very good yield. In the cytidine series,
the amino phosphonates 11 and 24 were obtained in a
ratio of 6:1. In the uridine series, the major amino
phosphonate 23 was preferred by ratio of 2:1 to the minor
isomer 25, and a third isomer (26) was observed with a
31P NMR resonance at 26.4 ppm and a significant
downfield shifted resonance for C-4′ compared to phos-
phonates 23 and 25. Extensive NMR experiments have
been done to clarify the stereochemistry of the third
isomer. The C-4′ resonance is easily identified in the 13C
NMR spectrum on the basis of C-P coupling constants.
Therefore assignment of the H-4′ resonance could be
made through an HMQC experiment. Once this reso-
nance was identified, NOE experiments revealed a
significant correlation between H-4′ and H-6 in isomer
26, whereas no NOE effect was observed in isomer 23.
On the basis of these data, the uridine derivatives 23 and
25 were assigned as 4′â isomers and phosphonate 26 was
assigned as a 4′R isomer.
a
(i) DDQ, CH2Cl2/H2O (20:1).
tection in the presence of the other aromatic moieties that
may be labile to Pd-catalyzed hydrogenolysis. These two
oxidants have been widely used for deprotection of PMB
groups from alcohol,11 tertiary amine,13 and amide func-
tionalities.12 Reports of oxidative cleavage from secondary
amines are more rare14 and may require hydrolysis of
an imine intermediate14b or addition of a reagent to trap
the benzaldehyde byproduct.14a
As expected, attempted deprotection of secondary
amine 13 through reaction with DDQ provided imine 14
instead of complete removal of the 4-methoxybenzyl
group (Scheme 3). The mixture of phosphonate 11 and
compound 12 also was treated with DDQ to give imines
15 and 16, which could not be separated readily by flash
chromatography (Scheme 4). Hydrolysis of these two
conjugated imines led to a mixture of amino phosphonate
17 and compound 18 and the corresponding deacylated
compounds 19 and 20. The H, 13C (with 1H decoupled,
1
and with both 1H and 31P decoupled), and 31P NMR
spectra and the HR mass spectrum of compound 20
allowed its assignment as an R-amino phosphine oxide
derivative of cytidine. The structure assignment for
compound 20 also allowed identification of compounds
12, 16, and 18, as the PMB-protected amino phosphine
oxide, the conjugated imine phosphine oxide, and the
deprotected amino phosphine oxide, respectively (Schemes
2 and 4).
To clarify the reaction pathway to formation of the
phosphine oxide, parallel experiments have been con-
ducted by treatment of diethyl phosphite (2 equiv) with
n-BuLi or with LHMDS in THF from 0 °C to room
temperature. The 31P NMR spectra of the two reaction
mixtures were recorded both during the reaction and
after the reaction was quenched by addition of NH4Cl.
For the experiment with LHMDS, a broad single reso-
A similar phosphonylation procedure was employed to
synthesize amino phosphonate derivatives 29 and 30
from a protected ara-C (27), and the 5′ isomers were
obtained in a ratio of ∼ 5:2 (Scheme 6). The two possible
4′R-isomers also were observed in the reaction mixture
with 31P NMR resonance at 25.2 and 24.7 ppm. However,
only trace amounts of these products were generated and
their isolation was not pursued.
To remove the PMB protecting groups from these
amino phosphonates, a CAN oxidation was employed on
the basis of the premise that this acidic reagent might
also cleave the imine bond in the intermediate benzyl
imines. Therefore, the 5′-amino phosphonate 23 was
treated with CAN in a mixture of acetonitrile and water
(Scheme 7). After several hours, a very polar spot was
(11) (a) Horita, K.; Yoshioka, T.; Tanaka, T.; Oikawa, Y.; Yonemitsu,
O. Tetrahedron 1986, 42, 3021-3028. (b) J ohansson R.; Samuelsson,
B. J . Chem. Soc., Perkin Trans. 1 1984, 2371-2374.
(12) (a) Yoshimura, J .; Yamaura, M.; Suzuki, T.; Hashimoto, H.
Chem. Lett. 1983, 1001-1002. (b) Yamaura, M.; Suzuki, T.; Hashimoto,
H.; Yoshimura, J .; Okamoto, T.; Shin, C. Bull. Chem. Soc. J pn. 1985,
58, 1413-1420.
(13) (a) Singh, S. B. Tetrahedron Lett. 1995, 36, 2009-2012. (b) Bull,
S. D.; Davies, S. G.; Fenton, G.; Mulvaney, A. W.; Prasad, R. S.; Smith,
A. D. J . Chem. Soc., Perkin Trans. 1 2000, 3765-3774.
(14) (a) Pierce, M. E.; Parsons, R. L, J r.; Radesca, L. A.; Lo, Y. S.;
Silverman, S.; Moore, J . R.; Islam, Q.; Choudhury, A.; Fortunak, J .
M. D.; Nguyen, D. Luo, C.; Morgan, S. J .; Davis, W. P.; Confalone, P.
N.; Chen, C. Y.; Tillyer, R. D.; Frey, L.; Tan, L.; Xu, F.; Zhao, D.;
Thompson, A. S.; Corley, E. G.; Grabowski, E. J . J .; Reamer, R.; Reider,
P. J . J . Org. Chem. 1998, 63, 3, 8536-8543. (b) Gunji, H.; Vasella, A.
Helv. Chim. Acta 2000, 83, 3229-3245. (c) Adams, H.; Anderson, J .
C.; Peace, S.; Pennell, A. M. K. J . Org. Chem. 1998, 63, 9932-9934.
(15) (a) McEldoon, W. L.; Wiemer, D. F. Tetrahedron 1995, 51,
7131-7148. (b) Lefebvre, I. M.; Evans, S. A., J r. J . Org. Chem. 1997,
62, 7532-7533. (c) Davis, F. A.; Lee, S.; Yan, H.; Titus, D. D. Org.
Lett. 2001, 3, 1757-1760.
(16) Zhu, X. F.; Williams, H. J .; Scott, A. I. J . Chem. Soc., Perkin
Trans. 1 2000, 2305-2306.
6110 J . Org. Chem., Vol. 68, No. 16, 2003

Analogues of Pyrimidine Nucleoside Monophosphates
SCHEME 4a
a
(i) DDQ, CH2Cl2/H2O (20:1); (ii) 1 N HCl aq, MeOH.
SCHEME 5a
SCHEME 6a
a
(i) DMSO, EDC, pyr, TFA, benzene; (ii) PMBNH2, benzene,
reflux with Dean-Stark apparatus; (iii) LHMDS, HP(O)(OEt)2,
THF, 0 °C to rt.
could be realized by treatment of TBAF, the excess TBAF
could not be completely removed by flash chromatogra-
phy. Bubenik17 reported that the TBS groups and ethyl
groups in phosphonate ethyl esters could be removed by
initial treatment with TMSBr and subsequent acidic
workup at high temperature. Therefore phosphonate 31
was treated with TMSBr and the reaction was quenched
by addition of MeOH to afford the phosphonic acid 32
directly in an excellent yield. Presumably, HBr generated
in situ from the excess TMSBr was sufficient to remove
the TBS groups.
These results encouraged efforts to simplify the depro-
tection procedure because the imine moiety is an acid-
sensitive functionality. Accordingly after DDQ oxidation
of the PMB-protected amino phosphonate 11, complete
deprotection transformed imine phosphonate 15 to the
phosphonic acid 33 in excellent yield. In this reaction,
the benzylic imine, the phosphonate esters, and the N4-
a
(i) DMSO, EDC, pyr, TFA, benzene; (ii) PMBNH2, benzene,
reflux with Dean-Stark apparatus; (iii) LHMDS, HP(O)(OEt)2,
THF, 0 °C to rt.
observed by TLC corresponding to the free amino phos-
phonate 31. To our surprise, after the reaction mixture
was neutralized and extracted with EtOAc and the
organic phase was dried over NaSO4 and concentrated,
the amino phosphonate 31 had condensed with benzal-
dehyde to form the corresponding imine. This adduct was
observed by TLC as a less polar spot than phosphonate
31. Acidic hydrolysis of the imine product and subsequent
workup with isopropylamine as a benzaldehyde scaven-
ger afforded the free amino phosphonate 31 in good yield.
Although removal of TBS groups from compound 31
(17) Bubenik, M.; Rej, R.; Nguyen-Ba, N.; Attardo, G.; Ouellet, F.;
Chan, L. Bioorg. Med. Chem. Lett. 2002, 12, 3063-3066.
J . Org. Chem, Vol. 68, No. 16, 2003 6111

Chen and Wiemer
SCHEME 7a
TABLE 1. Syn th esis of 5′-Am in o P h osp h on ic Acid s of
Cytid in e, Ur id in e, a n d Ar a -C
a
(i) CAN, CH3CN/H2O; (ii) 1 N HCl, MeOH; (iii) TMSBr,
CH2Cl2, then MeOH; (iv) DDQ, CH2Cl2/H2O (20:1).
acetyl group were cleaved cleanly in one flask. To our
delight, the imine phosphonate 15 could be obtained as
a single crystal and therefore the stereochemistry at the
5′ position was established as the S configuration through
an X-ray diffraction analysis.18
Following parallel procedures, the minor amino phos-
phonate derivative of cytidine 24 and the uridine deriva-
tive 25 (Table 1) and the major and minor ara-C
derivatives 29 and 30 were oxidized with DDQ to the
corresponding imine adducts 34-37 in good yields.
Complete deprotection of these imines provided, respec-
tively, the phosphonic acids 38-41 as their hydrogen
bromide salts in excellent yield.
TABLE 2. 13C Ch em ica l Sh ifts (p p m ) for Ur id in e,
Cytid in e, Ar a -C, a n d Th eir 5′-Am in o P h osp h on ic Acid
Der iva tivesa ,b
The 13C chemical shifts of uridine, cytidine, and ara-C
and their amino phosphonic acids are listed in Table 2.
For uridine and cytidine, the phosphonic acids 39 and
38 derived from the minor phosphite addition adducts
of uridine and cytidine showed resonances for C-4′ with
upfield shifts of 4.8 and 3.9 ppm, respectively, relative
to their parent nucleosides, while the phosphonic acids
32 and 33 derived from the major phosphite products
have no significant change in the resonances for C-4′ but
appear with C-1′ shifted 5.3 and 5.1 ppm downfield
respectively relative to their parent nucleosides. For ara-
C, the stereochemical preference appeared in an opposite
sense. The resonance for C-4′ in phosphonic acid 40,
generated from the major phosphite product, shifted
upfield 5.7 ppm. The minor phosphonic acid 41 displayed
a resonance for C-4′ with an upfield shift of 3.3 ppm,
much less than the major phosphonic acid 40, and the
C-1′ resonance shifted downfield by 3.8 ppm. With the
X-ray analyses for imine 15 and the above NMR data,
the amino phosphonic acids 32 and 41 were assigned the
C-1′
C-2′
C-3′
C-4′
C-5′
uridine
32
39
cytidine
33
38
ara-C
41
40
90.7
96.0
92.6
91.6
96.7
93.8
87.2
91.0
89.6
74.9
74.8
77.8
75.3
75.2
78.4
76.8
78.2
77.8
70.8
74.1
75.9
70.6
73.9
75.9
76.8
79.9
84.2
85.4
84.1
80.6
85.0
84.4
81.1
84.5
81.2
78.8
62.1
53.7
53.1
62.1
53.8
53.0
62.1
52.4
52.6
a
Data for cytidine and ara-C measured in D2O relative to
internal dioxane at 67.86 ppm.19 Data for compounds 32, 33,
b
and 38-41 measured in D2O relative to DSS at 0 ppm.
same 5′S configuration as phosphonic acid 33, and the
amino phosphonic acids 39 and 40 were assigned as the
5′R isomers, the same configuration as acid 38.
In summary, the 5′-amino-5′-phosphonate derivatives
of uridine, cytidine, and ara-C can be prepared through
addition of the lithium salt of diethyl phosphite to
nucleoside imines protected with a PMB group. The
configuration of the new stereogenic center at C-5′ of the
major 5′-amino-5′-phosphonate cytidine derivative was
(18) The single-crystal X-ray diffraction data for compound 15 is
available from Cambridge Crystallographic Data Centre with reference
number CCDC 197774.
(19) Hruska, F. E.; Blonski, W. J . P. Can. J . Chem. 1982, 60, 3026-
3032.
6112 J . Org. Chem., Vol. 68, No. 16, 2003

Analogues of Pyrimidine Nucleoside Monophosphates
3.95 (dd, J ) 20.8, 1.5 Hz, 1H), 3.88 (s, 3H), 3.79 (dd, J ) 7.6,
4.0 Hz, 1H), 1.30 (t, J ) 7.0 Hz, 3H), 1.29 (t, J ) 7.2 Hz, 3H),
0.90 (s, 9H), 0.84 (s, 9H), 0.25 (s, 3H), 0.09 (s, 3H), -0.04 (s,
3H), -0.18 (s, 3H); 13C NMR δ 166.5 (d, J CP ) 12.0 Hz), 165.9,
162.6, 155.9, 143.3, 130.2 (2C), 128.6 (d, J CP ) 2.8 Hz), 114.5
determined as S through an X-ray diffraction analysis
of a single crystal of the corresponding 5′-phenylmeth-
ylene amino phosphonate. The stereochemistry for the
uridine and ara-C 5′-amino-5′-phosphonic acids could be
assigned by comparison of their 13C NMR data with that
of the cytidine 5′-amino phosphonic acid. The stereose-
lectivity observed in phosphonylation of cytidine and
uridine 5′-imines favored the 5′S isomers, whereas for
ara-C the 5′R isomer was greatly favored. After separa-
tion of the diastereomeric adducts, the PMB group can
be removed through reaction with DDQ and an acidic
hydrolysis, conditions that also cleave all other protecting
groups to afford the parent phosphonic acids.
(2C), 93.2, 91.4, 81.3 (d, J CP ) 6.8 Hz), 75.6, 71.2 (d, J CP
)
12.3 Hz), 67.0 (d, J CP ) 151.3 Hz), 63.1 (d, J CP ) 7.2 Hz, 2C),
55.7, 26.1 (3C), 26.0 (3C), 18.3, 18.1, 16.7 (d, J CP ) 6.1 Hz,
2C), -3.7, -3.8, -4.8, -5.1; 31P NMR δ 21.1; HRMS (ESI) m/z
calcd for C33H58N4O8PSi2 (M + H)+ 725.3531, found 725.3562.
5′S -[(p -Me t h oxyp h e n yl)m e t h yle n e ]a m in o-5′-d ie t h -
ylp h osp h on yl-2′,3′-d i-O-ter t-bu tyld im eth ylsilyl-N4-a cetyl
Cytid in e (15). DDQ (20 mg, 0.09 mmol) was added to a
solution of the protected amino phosphonate 11 (55 mg, 0.07
mmol) in CH2Cl2 and water (20:1). The reaction mixture was
stirred for 7 h and then was diluted with EtOAc. The organic
phase was washed with NaHCO3 and brine, dried (Na2SO4),
and filtered. The filtrate was concentrated and purified by
flash chromatography (EtOAc) to give imine phosphonate 15
(43 mg, 77%) as a white solid: 1H NMR δ 9.33 (d, J ) 7.6 Hz,
1H), 8.44 (d, J ) 4.6 Hz, 1H), 7.78 (d, J ) 8.6 Hz, 2H), 7.60 (d,
J ) 7.6 Hz, 1H), 7.07 (d, J ) 8.9 Hz, 2H), 5.96 (d, J ) 2.7 Hz,
1H), 4.70 (ddd, J ) 6.2, 4.5, 1.6 Hz, 1H), 4.27 (m, 1H), 4.23-
4.11 (m, 4H), 4.05 (dd, J ) 20.1, 1.8 Hz, 1H), 3.90 (dd, J )
5.7, 3.7 Hz, 1H), 3.88 (s, 3H), 2.21 (s, 3H), 1.32 (t, J ) 7.2 Hz,
3H), 1.30 (t, J ) 7.2 Hz, 3H), 0.90 (s, 9H), 0.89 (s, 9H), 0.13 (s,
3H), 0.03 (s, 6H), -0.05 (s, 3H); 13C NMR δ 173.3, 169.0 (d,
J CP ) 12.8 Hz), 164.6, 164.5, 158.2, 147.5, 131.5 (d, J CP ) 1.4
Hz, 2C), 129.6 (d, J CP ) 3.0), 115.7 (2C), 98.0, 92.4, 83.9 (d,
Exp er im en ta l Section
5′S-(p -Met h oxyb en zyl)a m in o-5′-d iet h ylp h osp h on yl-
2′,3′-d i-O-ter t-bu tyld im eth ylsilyl Cytid in e (13). The alde-
hyde 9 was prepared from alcohol 8 (319 mg, 0.62 mmol)
through a modified Moffatt oxidation.2 Without further puri-
fication, the aldehyde 9 was dissolved in benzene and treated
with 4-methoxybenzylamine (84 mg, 0.61 mmol). The reaction
mixture was heated at reflux with a Dean-Stark apparatus
for 4 h. After the reaction mixture was allowed to cool to room
temperature, the solvent was removed under vacuum to give
the crude imine 10, which was used in the next step without
further purification.
To a solution of diethyl phosphite (0.25 mL, 2.73 mmol) in
THF at 0 °C was added dropwise n-BuLi (2.26 M in hexane,
0.74 mL). The mixture was stirred for 30 min, allowed to warm
to room temperature, and added via a cannula to a solution of
imine 10 in THF (8 mL) at 0 °C. The reaction mixture was
allowed to warm to room temperature and was monitored by
TLC. After the mixture was stirred for 5 h, water (5 mL) was
added and THF was removed in vacuo. The aqueous layer was
saturated with NaCl and extracted with EtOAc. The combined
EtOAc extract was dried (NaSO4) and filtered. The filtrate was
concentrated and purified by flash chromatography (MeOH
gradient in EtOAc) to give a mixture of amino phosphonate
11 and amino phosphine oxide 12 (ratio based on 31P NMR is
1:0.7, 203 mg, 42% in total from alcohol 31) and amino
phosphonate 13 (40 mg, 9% from alcohol 31). For phosphonate
11: 31P NMR δ 27.1. For phosphine oxide 12: 31P NMR δ 51.5.
For phosphonate 13: 1H NMR δ 7.84 (d, J ) 7.6 Hz, 1H), 7.28
(d, J ) 8.7 Hz, 2H), 6.91 (d, J ) 8.7 Hz, 2H), 5.90 (d, J ) 7.6
Hz, 1H), 5.88 (d, J ) 7.4 Hz, 1H), 4.45 (dd, J ) 7.0, 5.2 Hz,
1H), 4.23-4.10 (m, 5H), 4.05 (d, J ) 12.9 Hz, 1H), 3.81 (dd, J
) 12.9, 3.5 Hz, 1H), 3.80 (s, 3H), 3.73 (dd, J ) 5.1, 2.3 Hz,
1H), 3.00 (dd, J ) 17.0, 2.1 Hz, 1H), 1.37 (t, J ) 7.2 Hz, 3H),
1.32 (t, J ) 7.1 Hz, 3H), 0.88 (s, 9H), 0.86 (s, 9H), 0.10 (s, 3H),
0.00 (s, 3H), -0.05 (s, 3H), -0.13 (s, 3H); 13C NMR δ 167.5,
160.8, 158.3, 144.2, 133.0, 131.7 (2C), 115.2 (2C), 96.7, 90.9,
86.9, 75.7 (d, J CP ) 3.6 Hz), 75.0 (d, J CP ) 15.3 Hz), 64.1 (d,
J CP ) 7.0 Hz), 63.8 (d, J CP ) 8.3 Hz), 55.9, 55.0 (d, J CP ) 136.9),
52.4, 26.6 (3C), 26.5 (3C), 19.0, 19.0, 17.1 (d, J CP ) 4.2 Hz),
17.0 (d, J CP ) 3.7 Hz), -3.9, -4.0, -4.2, -4.2; 31P NMR δ 27.6;
HRMS (ESI) m/z calcd for C33H60N4O8PSi2 (M + H)+ 727.3687,
found 727.3688.
5′S -[(p -Me t h oxyp h e n yl)m e t h yle n e ]a m in o-5′-d ie t h -
ylp h osp h on yl-2′,3′-d i-O-ter t-bu tyld im eth ylsilyl Cytid in e
(14). DDQ (82 mg, 0.4 mmol) was added to a solution of the
amino phosphonate 13 (218 mg, 0.3 mmol) in CH2Cl2 and
water (19 mL, 20:1). The reaction mixture was stirred for 6 h,
and EtOAc (10 mL) was added. The organic phase was washed
with saturated NaHCO3 and brine, dried (Na2SO4), and
filtered. Flash chromatography of the concentrated filtrate
(10% MeOH in EtOAc) gave imine phosphonate 14 as a light
yellow solid (124 mg, 57%): 1H NMR (CDCl3) δ 8.86 (d, J )
7.5 Hz, 1H), 8.36 (d, J ) 4.6 Hz, 1H), 7.66 (d, J ) 8.4 Hz, 2H),
6.96 (d, J ) 8.8 Hz, 2H), 5.84 (d, J ) 1.3 Hz, 1H), 5.69 (d, J )
7.3 Hz, 1H), 4.69 (ddd, J ) 7.7, 4.0, 1.7 Hz, 1H), 4.17 (m, 5H),
J CP ) 6.5 Hz), 77.2, 73.2 (d, J CP ) 13.6 Hz), 68.4 (d, J CP
)
154.0 Hz), 64.8 (d, J CP ) 7.2 Hz), 64.6 (d, J CP ) 6.5 Hz), 56.2,
26.6 (3C), 26.5 (3C), 24.8, 19.1, 19.0, 17.0 (d, J CP ) 2.1 Hz),
16.9 (d, J CP ) 3.0 Hz), -3.7, -3.8, -4.5, -4.7; 31P NMR δ 21.8;
HRMS (ESI) m/z calcd for C35H60N4O9PSi2 (M + H)+ 767.3637,
found 767.3620.
5′S-Am in o-5′-d ieth ylp h osp h on yl-2′,3′-d i-O-ter t-bu tyld i-
m eth ylsilyl Cytidin e (19) an d 5′S-Am in o-5′-di-n -bu tylph os-
p h in yl-2′,3′-d i-O-ter t-bu tyld im eth ylsilyl Cytid in e (20). To
a solution of phosphonate 11 and phosphine oxide 12 (1:0.7,
202 mg, 0.26 mmol) in CH2Cl2 and H2O (20:1, 10 mL) was
added DDQ (71 mg, 0.30 mmol). The reaction mixture was
stirred at room temperature and monitored by TLC. After 6
h, the reaction mixture was filtered through filter paper and
rinsed with CH2Cl2. The combined CH2Cl2 solution was washed
with NaHCO3 and brine, dried (Na2SO4), and filtered. The
filtrate was purified by flash chromatography (EtOAc) to give
a mixture of imine phosphonate 15 and phosphine oxide 16
(89 mg, ratio based on 31P NMR was 1:0.5).
The mixture of imine phosphonate 15 and phosphine oxide
16 (1:0.5, 85 mg) was dissolved in MeOH and treated with 1
N HCl (1 mL). The mixture was shaken for 5 min, and then
TLC showed the hydrolysis was complete. After concentration
under vacuum, the aqueous residue was extracted with EtOAc,
and the organic phase was washed with saturated NaHCO3
and dried (Na2SO4). After concentration, the residue was
purified by flash chromatography (MeOH gradient in EtOAc)
to afford a mixture of amino phosphonate 17 and phosphine
oxide 18 (ratio based on 31P NMR was 1:0.7, 43 mg), amino
phosphonate 19 (15 mg), and phosphine oxide 20 (9 mg). For
amino phosphonate 17: 31P NMR δ27.1. For phosphine oxide
18: 31P NMR δ56.9. For amino phosphonate 19: 1H NMR δ
7.86 (d, J ) 7.5 Hz, 1H), 5.90 (d, J ) 7.6 Hz, 1H), 5.71 (d, J )
5.7 Hz, 1H), 4.63 (dd, J ) 5.7, 4.8 Hz, 1H), 4.36 (dd, J ) 4.7,
3.7 Hz, 1H), 4.26 (ddd, J ) 5.1, 3.7, 3.4 Hz, 1H), 4.21-4.09
(m, 4H), 3.31 (dd, J ) 16.8, 3.4 Hz, 1H), 1.34 (t, J ) 7.1 Hz,
3H), 1.30 (t, J ) 7.1 Hz, 3H), 0.95 (s, 9H), 0.89 (s, 9H), 0.15 (s,
3H), 0.14 (s, 3H), 0.08 (s, 3H), 0.03 (s, 3H); 13C NMR δ 167.8,
158.4, 145.2, 96.3, 94.0, 85.3, 75.0, 74.2 (d, J CP ) 11.7 Hz),
64.4 (d, J CP ) 7.1 Hz), 64.0 (d, J CP ) 7.1 Hz), 50.5 (d, J CP
)
168.0 Hz), 26.6 (6C), 19.1 (2C), 16.9 (J CP ) 5.2 Hz, 2C), -3.9,
-4.2 (2C), -4.3; 31P NMR δ 26.9; HRMS (ESI) m/z calcd for
C
25H52N4O7PSi2 (M + H)+ 607.3112, found 607.3117. Phos-
J . Org. Chem, Vol. 68, No. 16, 2003 6113

Chen and Wiemer
phine oxide 20: 1H NMR δ 7.84 (d, J ) 7.4 Hz, 1H), 5.90 (d,
J ) 7.3 Hz, 1H), 5.54 (d, J ) 5.4, 1H), 4.77 (dd, J ) 5.2, 5.2,
1H), 4.40 (m, 2H), 3.28 (dd, J ) 13.4, 1.4 Hz, 1H), 1.87 (m,
4H), 1.50 (m, 8H), 0.97 (s, 9H), 0.97 (t, J ) 7.2 Hz, 3H), 0.91
(s, 9H), 0.91 (t, J ) 7.2 Hz, 3H), 0.18 (s, 3H), 0.16 (s, 3H), 0.11
(s, 3H), 0.04 (s, 3H); 13C NMR δ 167.9, 158.3, 146.2, 96.2, 96.1,
84.9, 74.2, 74.2 (d, J CP ) 10.9), 51.7 (d, J CP ) 71.1), 26.6 (6C),
5′S-Am in o-5′-d ieth ylp h osp h on yl-2′,3′-d i-O-ter t-bu tyld i-
m eth ylsilyl Ur id in e (31). CAN (1.9 g, 3.5 mmol) was added
to a solution of amino phosphonate 23 (732 mg, 1.0 mmol) in
acetonitrile (7 mL) and distilled water (7 mL). After the
reaction mixture was stirred for 9 h, water (5 mL) was added
and the aqueous layer was extracted with EtOAc. The com-
bined organic extract was washed with Na2SO3, dried (Na2-
SO4), and filtered. The filtrate was concentrated, and the
residue was dissolved in MeOH. To this solution was added 1
N HCl (3 mL), and the reaction mixture was shaken for 4 min.
After dilution with EtOAc, isopropylamine (1 mL) was added
to trap the resulting 4-methoxybenzylaldehyde and neutralize
the reaction mixture. The aqueous layer was extracted with
EtOAc, and the combined organic layers were dried (Na2SO4)
and filtered. The filtrate was concentrated, and the residue
was purified by flash chromatography (MeOH gradient in
EtOAc) to give amino phosphonate 31 (369 mg, 61%) as a light
brown solid: 1H NMR δ 7.91 (d, J ) 7.8 Hz, 1H), 5.78 (d, J )
5.9 Hz, 1H), 5.73 (d, J ) 7.8 Hz, 1H), 4.56 (dd, J ) 5.9, 4.7 Hz,
1H), 4.35 (dd, J ) 4.7, 3.0 Hz, 1H), 4.26 (ddd, J ) 4.6, 3.1, 3.0,
1H), 4.20-4.11 (m, 4H), 3.30 (dd, J ) 17.4, 3.1 Hz, 1H), 1.34
(t, J ) 7.2 Hz, 3H), 1.31 (t, J ) 7.1 Hz, 3H), 0.95 (s, 9H), 0.89
26.4 (d, J CP ) 62.8 Hz), 25.9 (d, J CP ) 61.0), 25.4 (d, J CP
)
14.2), 25.4 (d, J CP ) 14.2 Hz), 24.7 (d, J CP ) 4.2 Hz), 24.6 (d,
J CP ) 2.6 Hz), 19.1, 19.0, 14.1, 14.0, -3.9, -4.1 (2C), -4.3; 31
P
NMR δ 57.1; HRMS (ESI) m/z calcd for C29H60N4O5PSi2 (M +
H)+ 631.3840, found 631.3838.
5′S-(p-Meth oxyben zyl)am in o-5′-dieth ylph osph on yl-2′,3′-
d i-O-ter t-bu tyld im eth ylsilyl-N4-a cetyl Cytid in e (11) a n d
5′R-(p-Meth oxyben zyl)a m in o-5′-d ieth ylp h osp h on yl-2′,3′-
d i-O-ter t-bu tyld im eth ylsilyl-N4-a cetyl Cytid in e (24). The
aldehyde was prepared in situ from alcohol 8 (463 mg, 0.9
mmol) through a modified Moffatt oxidation. The crude alde-
hyde, without further purification, was dissolved in benzene
and treated with 4-methoxybenzylamine (123 mg, 0.9 mmol).
The reaction mixture was heated at reflux with Dean-Stark
apparatus for 4 h. After the reaction mixture was allowed to
cool to room temperature, the solvent was removed under
vacuum to give the crude imine 10.
(s, 9H), 0.16 (s, 3H), 0.14 (s, 3H), 0.09 (s, 3H), 0.04 (s, 3H); 13
C
NMR δ 166.1, 152.4, 144.4, 103.1, 92.1, 85.9, 75.1 (d, J CP
)
Lithium diethyl phosphite was prepared from diethyl phos-
phite (0.41 mL, 4.5 mmol) and LHMDS (1.0 M in THF, 3.6
mL) in THF from -50 °C to room temperature. To a solution
of compound 10 in THF at -78 °C was added the lithium
diethyl phosphite dropwise through a cannula. The reaction
mixture was allowed to warm to room temperature and stirred
for 24 h. The reaction was stopped by addition of saturated
NH4Cl. The aqueous layer was extracted with EtOAc, and the
combined organic layer was dried (Na2SO4) and filtered. The
filtrate was concentrated under vacuum, and the residue was
purified by flash chromatography (EtOAc gradient in hexane)
to give amino phosphonates 11 (330 mg, 48% from compound
8) and 24 (55 mg, 8% from compound 8) as white solids. For
amino phosphonate 11: 1H NMR δ 8.43 (d, J ) 7.7 Hz, 1H),
7.40 (d, J ) 7.5 Hz, 1H), 7.29 (d, J ) 8.4 Hz, 2H), 6.93 (d, J )
8.4 Hz, 2H), 5.91 (d, J ) 5.3 Hz, 1H), 4.45 (dd, J ) 5.0, 5.0 Hz,
1H), 4.30 (m, 1H), 4.22-4.07 (m, 5H), 3.82 (m, 2H), 3.81 (s,
3H), 3.06 (dd, J ) 17.5, 1.7 Hz, 1H), 2.18 (s, 3H), 1.38 (t, J )
6.9 Hz, 3H), 1.32 (t, J ) 7.0 Hz, 3H), 0.89 (s, 9H), 0.88 (s, 9H),
0.11 (s, 3H), 0.03 (s, 3H), -0.01 (s, 3H), -0.08 (s, 3H); 13C NMR
δ 173.1, 164.5, 160.8, 158.2, 147.7, 133.1, 131.5 (2C), 115.3
2.4 Hz), 74.3 (d, J CP ) 12.4 Hz), 64.4 (d, J CP ) 6.1 Hz), 64.0 (d,
J CP ) 7.2 Hz), 50.7 (d, J CP ) 147.3 Hz), 26.6 (3C), 26.5 (3C),
19.1, 19.0, 16.9 (d, J CP ) 5.9 Hz, 2C), -4.0, -4.1, -4.1, -4.4;
31P NMR δ 26.7; HRMS (ESI) m/z calcd for C25H51N3O8PSi2
(M + H)+ 608.2952, found 608.2953.
5′-[1′-(5′(S)-Am in o-â-D-r ib o-p en t a -1′,4′-fu r a n osyl)u r a -
cil] P h osp h on ic Acid (32). To a solution of amino phospho-
nate 31 (163 mg, 0.27 mmol) in CH2Cl2 (5 mL) was added
TMSBr at 0 °C. The reaction mixture was allowed to warm to
room temperature and stirred for 24 h. The volatile compo-
nents were removed in vacuo, and the residue was coevapo-
rated with MeOH and then with water. The resulting residue
was dissolved in minimum amount of MeOH and precipitated
with EtOAc. After centrifugation, the precipitate was sepa-
rated from the supernant. The solid was dissolved in water
and lyophilized to give amino phosphonic acid 32 as its
hydrogen bromide salt (100 mg, 92%) as a white solid: 1H
NMR (D2O, DSS standard) δ 7.66 (d, J ) 7.8 Hz, 1H), 5.87 (d,
J ) 7.8 Hz, 1H), 5.69 (d, J ) 5.1 Hz, 1H), 4.70 (dd, J ) 5.1,
5.1 Hz, 1H), 4.50 (dd, J ) 5.1, 4.6 Hz, 1H), 4.31 (ddd, J ) 9.3,
4.7, 4.5 Hz, 1H), 3.61 (dd, J ) 13.3, 9.3 Hz, 1H); 13C NMR δ
169.2, 154.5, 147.3, 105.2, 96.0, 84.1 (d, J CP ) 3.1 Hz), 74.8,
74.1 (d, J CP ) 1.5 Hz), 53.7 (d, J CP ) 136.7 Hz); 31P NMR δ
9.2; HRMS (ESI) m/z calcd for C9H14N3O8PNa (M + Na)+
346.0416, found 346.0406.
(2C), 98.5, 92.0, 86.4, 76.3 (d, J CP ) 3.7 Hz), 74.2 (d, J CP
)
13.6 Hz), 64.1 (d, J CP ) 7.6 Hz), 63.9 (d, J CP ) 7.7 Hz), 55.9,
54.9 (d, J CP ) 138.8 Hz), 52.9, 26.6 (3C), 26.5 (3C), 24.7, 19.0,
19.0, 17.1 (d, J CP ) 5.1 Hz), 17.0 (d, J CP ) 5.3 Hz), -3.9, -4.1,
-4.1, -4.3; 31P NMR δ 27.5; HRMS (ESI) m/z calcd for
C
35H62N4O9PSi2 (M + H)+ 769.3793, found 769.3805. Anal.
Calcd for C35H61N4O9PSi2: C, 54.66; H, 8.00; N, 7.29. Found:
C, 54.66; H, 8.01; N, 7.14. For amino phosphonate 24: 1H NMR
δ 8.09 (d, J ) 7.6 Hz, 1H), 7.46 (d, J ) 7.5 Hz, 1H), 7.26 (d, J
) 8.6 Hz, 2H), 6.87 (d, J ) 8.6 Hz, 2H), 5.95 (d, J ) 6.3 Hz,
1H), 4.73 (m, 2H), 4.36 (dd, J ) 3.0, 2.8 Hz, 1H), 4.20-4.06
(m, 4H), 3.98 (dd, J ) 12.4, 2.2 Hz, 1H), 3.85 (dd, J ) 12.4,
2.2 Hz, 1H), 3.77 (s, 3H), 3.71 (dd, J ) 10.1, 10.1 Hz, 1H),
2.18 (s, 3H), 1.34 (t, J ) 7.1 Hz, 3H), 1.29 (t, J ) 7.1 Hz, 3H),
0.94 (s, 9H), 0.89 (s, 9H), 0.20 (s, 3H), 0.15 (s, 3H), 0.02 (s,
3H), -0.05 (s, 3H); 13C NMR δ 173.2, 164.6, 160.4, 158.3, 148.2,
134.1, 130.4 (2C), 114.9 (2C), 98.6, 93.5, 81.3, 77.8, 75.5 (d,
J CP ) 10.2 Hz), 63.8 (d, J CP ) 7.4 Hz), 63.6 (d, J CP ) 7.2 Hz),
55.9 (d, J CP ) 143.2 Hz), 55.8, 51.5, 26.9 (3C), 26.8 (3C), 24.7,
19.6, 19.2, 17.1 (d, J CP ) 5.7 Hz), 17.0 (d, J CP ) 5.6 Hz), -3.2,
-3.9, -4.1, -4.4; 31P NMR δ 28.1; HRMS (ESI) m/z calcd for
Ack n ow led gm en t. We would like to thank Dr. Dale
Swenson for his assistance with the diffraction analyses,
the Center for Biocatalysis and Bioprocessing for a
predoctoral fellowship, and the Leukemia and Lym-
phoma Society (LLS99-6059) and The Roy J . Carver
Charitable Trust for financial support.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal protocols; additional examples of reactions described in the
Experimental Section; 1H and 13C NMR spectra for compounds
13-15, 19, 20, 24-26, and 29-41; and an ORTEP drawing
for compound 15. This material is available free of charge via
the Internet at http://pubs.acs.org.
C
35H62N4O9PSi2 (M + H)+ 769.3793, found 769.3775.
J O030050X
6114 J . Org. Chem., Vol. 68, No. 16, 2003