Synthesis of nucleoside boranophosphoramidate prodrugs conjugated with amino acids

Article abstract of DOI:10.1021/jo0481248

(Chemical Equation Presented) Nucleoside boranophosphates and nucleoside amino acid phosphoramidates have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside prodrugs. A combination of these two types of compoun

Full text of DOI:10.1021/jo0481248

Synthesis of Nucleoside Boranophosphoramidate Prodrugs
Conjugated with Amino Acids
Ping Li and Barbara Ramsay Shaw*
Department of Chemistry, Box 90346, Duke University, Durham, North Carolina 27708-0346
brshaw@chem.duke.edu.
Received October 22, 2004
Nucleoside boranophosphates and nucleoside amino acid phosphoramidates have been shown to
be potent antiviral and anticancer agents with the potential to act as nucleoside prodrugs. A
combination of these two types of compounds results in a boranophosphoramidate linkage between
the nucleoside and amino acid. This new class of potential prodrugs is expected to possess advantages
conferred by both types of parent compounds. Two approaches, specifically the H-phosphonate and
oxathiaphospholane approaches, are described here to synthesize nucleoside boranophosphoramidate
prodrugs conjugated with amino acids. The H-phosphonate approach involves a key intermediate,
silylated nucleoside amino acid phosphoramidite 6, prepared from a series of reactions starting
from nucleoside H-phosphonate in the presence of condensing reagent DPCP. Due to the lengthy
procedure and the difficulties in removing DPCP from the final products, we switched to the
oxathiaphospholane approach in which the DBU-assisted oxathiaphospholane ring-opening process
constituted a key step for the generation of nucleoside amino acid boranophosphoramidates 24.
We demonstrate that this key step did not cause any measurable C-racemization of boranophos-
phorylated amino acids 22. Diastereomers of compounds 24a-f were separated by RP-HPLC. An
“adjacent”-type mechanism is proposed to explain the diastereomer ratio in the final products
obtained via the oxathiaphospholane approach. A tentative assignment of configuration for the
diastereomers was carried out based on the mechanism, molecular modeling, and 1H NMR.
Conclusively, the oxathiaphospholane methodology proved to be more facile and efficient than
H-phosphonate chemistry in the preparation of the nucleoside amino acid boranophosphoramidate
analogues that are promising as a new type of antiviral prodrugs.
Introduction
phorylated forms. Specifically, for d4T the first phospho-
rylation step is the rate-limiting step, whereas for AZT
phosphorylation to the nucleoside 5′-diphosphate is the
slowest step in the phosphorylation pathway. Several
of these types of nucleoside analogues are known to be
The development of nucleoside prodrugs capable of
undergoing intracellular activation to the corresponding
nucleotide has become an area of intense interest.
3
1
Various 2′,3′-dideoxy nucleosides, including 3′-azido-2′,3′-
poor substrates for nucleoside kinases and 5′-nucleo-
2a,b
dideoxythymidine (AZT, zidovudine) and 2′,3′-dideoxy-
′,3′-didehydrothymidine (d4T, stavudine),2c,d are known
to be potent inhibitors of HIV-1, the causative agent of
2
(
2) (a) Mitsuya, H.; Weinhold, K. J.; Furman, P. A.; Stclair, M. H.;
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1
,2
AIDS.
In general, the biological activity of these
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(
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1
0.1021/jo0481248 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/18/2005
J. Org. Chem. 2005, 70, 2171-2183
2171

Li and Shaw
4
tidases. In addition, the long-term administration of
tronic with sulfur found in the phosphorothioate, and
isosteric with the methyl group present in the meth-
ylphosphonate. These chemical similarities imply that
boranophosphates will share a number of chemical and
biochemical properties with the aforementioned ana-
logues yet will have other unique properties. Nucleoside
boranophosphates have recently drawn the attention of
medicinal chemists due to their therapeutic applications
in antiviral drug design.11 It has been found that an
R-boranophosphate group in 2′,3′-dideoxynucleoside tri-
phosphates (ddNTPs) increases their selectivity to viral
nucleoside-based drugs can result in decreased kinase
activity, thus reducing their efficacy. For example, HIV
5
5
a,b
resistance to the antiviral activity of AZT,
2′,3′-
dideoxycytidine (ddC),5 and acyclovir (ACV) has been
shown to arise from the decreased activity of the prereq-
uisite first phosphorylating enzyme. These findings have
prompted the development of prodrug or “pronucleotide”
strategies that bypass initial kinase dependency by the
intracellular delivery of the monophosphorylated nucleo-
c
5d,e
1
side analogue.
Nucleoside amino acid phosphoramidates show prom-
reverse transcriptases (RTs) relative to bacterial DNA
1a,b
11a,b
ise as a potential pronucleotide strategy.
Studies by
polymerases.
For example, steady-state and pre-
McGuigan et al. demonstrated that the phosphoramidate
steady-state kinetic analyses indicate that whereas the
efficiency of the single nucleotide incorporation for ddCTP
by moloney murine leukemia virus (MMLV) RT is nearly
two orders lower than for natural dCTP, the introduction
of an R-boranophosphate group increases the efficiency
of incorporation for ddCTPRB by 30-fold.1 It also has
been shown that the presence of the R-boranophosphate
diester prodrugs of 2′,3′-dideoxy-2′,3′-didehydroadenosine
6
a
6a
6b-d
(
d4A), 2′,3′-dideoxyadenosine (ddA), and d4T
ex-
hibited greatly enhanced activity against HIV compared
to their parent nucleoside analogues in vitro and, in
contrast to the parent nucleoside analogues, full activity
1a
6
was retained in kinase-deficient cell lines. Moreover,
nucleoside phosphoramidate monoesters are potent an-
tiviral and/or anticancer agents with enhanced activity
group in AZTDP,1 d4TDP,
1c
11d
and ddADP improves
11e
both the phosphorylation of these diphosphates by nu-
cleoside diphosphate kinase and incorporation of their
7
and reduced cytotoxicity. Specifically, prodrugs of AZT
11c
amino acid phosphoramidate derivatives described by
Wagner et al. exhibited potent antiviral and anticancer
activity with less cytotoxicity and improved plasma half-
corresponding ddNTPs by wild-type and mutant mul-
tidrug-resistant HIV-RT.1
1d,e
Moreover, after these
ddNTPRB analogues were incorporated into DNA, repair
of the blocked DNA chains by pyrophosphorolysis was
reduced significantly by mutant HIV-RT enzymes from
drug-resistant viruses.1 These results, combined with
the distinct properties of boranophosphates that are more
8
life compared to AZT. These nucleoside phosphoramidate
monoesters are thought to exert their biological functions
1b
through a P-N bond cleavage by phosphoramidases to
6c,7d,9
yield the corresponding nucleoside monophosphates.
Boranophosphates, in which one of the nonbridging
oxygen atoms in the phosphate group is replaced by a
lipophilic and nuclease-resistant than the normal phos-
phates,1
0e,15c
suggest that boranophosphates may be
12
borane (BH
3
) group, were first introduced by our group
potentially useful in a prodrug approach.
1
0
a decade ago. The borane group is isoelectronic with
oxygen occurring in normal phosphate, pseudoisoelec-
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2172 J. Org. Chem., Vol. 70, No. 6, 2005

Nucleoside Boranophosphoramidate Prodrugs
SCHEME 1. Synthesis of Nucleoside Boranophosphoramidate via an H-Phosphonate Approach
It could be foreseen that the combination of nucleoside
amino acid phosphoramidates and nucleoside borano-
phosphates would create a new type of antiviral and
anticancer prodrug with the advantages conferred by
both phosphoramidates and boranophosphates. In this
paper we present the synthesis of nucleoside amino acid
boranophosphoramidates through H-phosphonate meth-
odology as well as an oxathiaphospholane approach. The
availability of these boranophosphoramidate analogues
would permit the investigations of their chemical and
enzymatic stabilities, substrate properties, and delivery
into cells compared to their parent compounds of nucleo-
side amino acid phosphoramidates.
Results and Discussion
Synthesis of Nucleoside Amino Acid Borano-
phosphoramidates via an H-Phosphonate Approach.
H-Phosphonate chemistry has been widely used in the
synthesis of phosphorus-containing compounds, especial-
1
3
ly for the synthesis of nucleotides and oligonucleotides.
For example, it has been applied to synthesize nucleoside
boranophosphate14 and oligothymidine boranophosphate
analogues in good yields via silylation of an H-phospho-
nate precursor followed by boronation. Meanwhile, Staw-
inski et al. reported the synthesis of P3′ f N5′ H-phos-
phonamidate internucleotide linkage through an aryl
H-phosphonate.16 After an extensive literature search
and study we decided to carry out the synthesis of
nucleoside amino acid boranophosphoramidate via an
H-phosphonate approach as outlined in Scheme 1.
15
(
14) (a) Sergueev, D.; Hasan, A.; Ramaswamy, M.; Shaw, B. R.
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11579.
J. Org. Chem, Vol. 70, No. 6, 2005 2173

Li and Shaw
SCHEME 2. Proposed Mechanism for the
Formation of Products When Pv-Cl Is Used as
Condensing Reagent
FIGURE 1. Structures of condensing reagents 2 and their
abbreviations.
TABLE 1. Summary of the Generation of
H-Phosphonamidate 7 with Different Condensing
Reagents 2
entry
condensing reagent 2
equiv of 2
yield (%) of 7a
2
a
Pv-Cl
1.1
2.5
1.0
1.0
0
<10
25
2
b
NEP-Cl
OXP-Cl
DPCP
2
c
2d
55
a
Yield was calculated by peak integration in 31P NMR.
To investigate the viability of this synthetic approach,
we started with the commercially available H-phospho-
nate, i.e., 5′-O-dimethoxytrityl (DMT) thymidine 3′-H-
phosphonate 1. After reacting with 2,4,6-trichlorophenol
in the presence of condensing reagent 2, a highly reactive
intermediate, thymidine aryl H-phosphonate diester 3,
was formed. Different condensing reagents, including
pivaloyl chloride (Pv-Cl), 2a, 2-chloro-5,5,-dimethyl-2-
oxo-1,3,2-dioxaphosphinane (NEP-Cl), 2b, bis(2-oxo-3-
oxazolidinyl)phosphinic chloride (OXP-Cl), 2c, and di-
phenyl chlorophosphate (DPCP), 2d, were used in this
step; their structures are depicted in Figure 1. Although
all these reactions cleanly produced the desired aryl
H-phosphonate 3, the yield of the nucleoside amino acid
H-phosphonamidate 7 (Route A, Scheme 1) upon addition
of L-tryptophan methyl ester 5b differed significantly
depending on the type of condensing agent that was used
for the generation of 3. The yields are summarized in
Table 1.
To avoid the problem associated with the use of Pv-
Cl we decided to try chlorophosphates as condensing
agents for the formation of aryl H-phosphonate 3. To
minimize the side reaction of amino acid with chloro-
phosphates (or with the corresponding pyrophosphates
produced during the course of condensation), sterically
hindered chlorophosphates, including NEP-Cl 2b, OXP-
Cl 2c, and DPCP 2d, were chosen as the condensing
reagents. Whereas the aminolysis of 3 by 5b (Scheme 3)
in the presence of NEP-Cl produced less than 10% of
the desired H-phosphonamidate 7 after the reaction
workup, the yields of 7 increased to 25% and 55% when
OXP-Cl and DPCP, respectively, were used as condens-
ing reagents. According to the proposed mechanism
shown in Scheme 3 there exists a two-step reaction
including the activation of H-phosphonate 1 and the
subsequent reaction to produce aryl H-phosphonate di-
ester 3. With the increasing reactivity of 2b < 2c < 2d
toward starting material 1, the first step to activate
H-phosphonate 1 becomes faster than the second step to
generate 3, and thus a smaller amount of pyrophosphate
15 is generated due to consumption of condensing re-
agent. Therefore, the amount of the reactive intermediate
3 increases with the condensing reagents 2b < 2c < 2d,
which results in the increased yield for the formation of
When Pv-Cl 2a was used as the condensing reagent
3
1
and after the addition of water, both TLC and P NMR
(
7: δ 15 ppm) indicated that no desired H-phosphona-
midate 7 was formed. However, this reaction produced
N-pivaloylated L-tryptophan methyl ester 12 (Scheme 2),
which was isolated in 25% yield along with the 50%
recovery of starting material H-phosphonate 1. According
to the products isolated in this reaction and studies
reported by Stawinski et al.,16 a reaction mechanism
could be proposed as shown in Scheme 2. Nucleoside aryl
H-phosphonates 3 can be involved in equilibrium with
pivaloate anion 11,17 which may produce detectable
amounts of pivalic-phosphonic mixed anhydride 10.
Assuming that the mixed anhydride 10 could react with
amines with incomplete chemoselectivity, attack from the
16
H-phosphonamidate 7. Moreover, the partial decompo-
sition of H-phosphonamidate 7 during silica gel chroma-
tography led to a search for a condensing reagent that
would not only provide efficient formation of 7 but also
enable purification of the product via aqueous extraction.
Thus, although the yield was only satisfactory, DPCP was
selected as the condensing reagent for the production of
reactive aryl H-phosphonate diester 3.
R-NH
2
of the L-tryptophan methyl ester 5b on the
carbonyl center in 10 would result in the formation of
both starting material 1 and N-pivaloylated product 12.
After following route A as depicted in Scheme 1 the
anhydrous nucleoside amino acid H-phosphonamidate 7
was subjected to silylation in the presence of triethyl-
amine (TEA) followed by boronation with borane-di-
methyl sulfide. As deduced from 31P NMR spectra of
(17) Kers, A.; Kers, I.; Kraszewski, A.; Stawinski, J. Collect. Czech.
Chem. Commun. 1996, 61, 1, S246.
2174 J. Org. Chem., Vol. 70, No. 6, 2005

Nucleoside Boranophosphoramidate Prodrugs
SCHEME 3. Proposed Mechanism for the
Formation of Products Using Chlorophosphate as
the Condensing Reagent
SCHEME 4. Two Possible Tautomeric
Equilibriums for H-Phosphonamidate 7
two diastereomers (two singlets, δ ) 139.5, 139.7 ppm)
for intermediate 6 became a broad peak centered at δ
the mixtures, the reaction did not proceed as expected
to form borane-phosphoramidate complex 8. Other si-
lylation reagents, such as N,O-bis(trimethylsilyl)aceta-
mide (BSA) and bis(trimethylsilyl)trifluoroacetamide
1
18.0 ppm corresponding to the borane complex 8.
Treatment of 8 with H O resulted in the formation of
desired compound boranophosphoramidate 9, whose
NMR showed a characteristic broad peak at δ 93.5 ppm.
2
3
1
P
18
(
BSTFA), were also tried but without success. After
further addition of H O to the reaction mixtures, most
of the H-phosphonamidate 7 was recovered, as indicated
Due to the presence of the DMT group in 9 and its
hydrophobicity, condensing reagent DPCP could easily
be removed by repeatedly washing the reaction mixture
in dichloromethane with brine. However, when this
method was applied to synthesize 3′-O-acetylthymidine
boranophosphoramidate 24a, the condensing reagent
DPCP and the desired compound 24a could not be
separated by aqueous extraction between dichloromethane
and brine because a large amount of 24a was distributed
to the brine layer together with the hydrolysis products
from DPCP. Further purification with reverse-phase
HPLC did not resolve the problem.
2
31
by P NMR. It can be proposed that H-phosphonamidate
could undergo two different tautomeric equilibriums
7
as shown in Scheme 4. In such a case if tautomer 17 were
the major form, it would lack the lone-pair electrons that
are required for the boronation step. Hydrolysis of 18
2
with H O would then result in the formation of H-
phosphonamidate 7. This is in accordance with the
observance in the experiments.
To avoid the aforementioned problems associated with
route A, we decided to follow route B. Instead of removing
condensing reagent DPCP via aqueous extraction at an
early stage, silylated phosphoramidite diester 6 (Scheme
Synthesis of Nucleoside Amino Acid Borano-
phosphoramidates via an Oxathiaphospholane Ap-
1
) was directly boronated with borane-dimethyl sulfide.
The reaction was completed within 30 min as monitored
by P NMR, where the two peaks corresponding to the
(
18) Lin, J. L.; Shaw, B. R. Nucleosides Nucleotides Nucleic Acids
3
1
2001, 20, 587.
J. Org. Chem, Vol. 70, No. 6, 2005 2175

Li and Shaw
TABLE 2. Reactivity of Boronating Reagentsa
borane-amine complex
SCHEME 5. Synthesis of Nucleoside Amino Acid
Boranophosphoramidates via an
Oxathiaphospholane Approach
borane
BH3: BH3:
BH3:
BH3: BH3:
An Py
BH3:
Py-2-Cl
b
complex
Me2 S THF DIPEA
conversion of
100% none 20%
15% none
60%
c,d
2
1bf22b
a
N-(1,3,2-Oxathiaphospholanyl)-L-tryptophan methyl ester 21b
b
was used as a model compound. Ten molar equivalents of borane
c 31
complex were used.
P NMR samples were prepared at 30 min
d
after addition of the borane complex. Conversion was calculated
as the integration percentage of compound 22b relative to the total
3
1
integration of compounds 21b and 22b in the P NMR spectra.
pylethylamine (DIPEA) in dichloromethylene solution to
give the N-(1,3,2-oxathiaphospholanyl)amino acid methyl
ester 21. This phosphitylation reaction was completed in
3
0 min as evidenced by 31P NMR spectra, where the
singlet at δ 207 ppm for 2-chloro-1,3,2-oxathiaphos-
pholane 20 was transformed into two singlets around δ
1
29.5 ppm corresponding to the diastereomers of 21 (ratio
2
:3) in about 70% yield along with some unidentified
products. Attempts to purify compound 21 by silica gel
column chromatography failed as it appeared to decom-
pose quickly on the column.
Thus, without isolation of N-(1,3,2-oxathiaphosphola-
nyl)amino acid methyl ester 21, in situ boronation was
carried out by reacting with boronating reagents to
provide oxathiaphospholane-borane complex 22. Its
formation was confirmed by the presence of a character-
istic broad resonance peak centered at δ 136 ppm in its
proach. Due to the difficulties encountered by employing
H-phosphonate chemistry, we decided to explore an alter-
native synthetic method to obtain nucleoside amino acid
boranophosphoramidates. The oxathiaphospholane ap-
proach, first developed by Stec for the synthesis of ster-
eoregular oligo(nucleoside phosphorothioate)s,1 recently
has been adopted to synthesize phosphorothioylated pro-
ducts of biologically relevant alcohols and amino acids.20
The oxathiaphospholane approach involves tricoordinate
phosphorus compounds as the precursors and therefore,
as demonstrated earlier,21 is suitable for the introduction
of a borane group. By employing this approach, which is
superior to the procedure using the phosphoramidate
31
P NMR spectrum. According to molecular orbital (MO)
theory and valence shell electron pair repulsion (VSEPR)
theory, the boron atom in BH has an empty 2p orbital
9
3
2
4
and, therefore, is labile to attack by nucleophiles. The
tervalent phosphorus atom with lone-pair electrons could
act as a Lewis-base donor of electrons to an electron-
3
deficient BH . The reactivities of a series of borane-group
transferring reagents, including borane-dimethyl sulfide
and borane-tetrahydrofuran, to intermediate 21 were
examined as well as three types of borane-amine com-
plexes: aliphatic-borane, aromatic-borane, and hetero-
cyclic-borane. The results are summarized in Table 2,
where the most active boronating reagent is borane-
dimethyl sulfide. Experiments on varying the equivalency
of borane-dimethyl sulfide were also carried out to
determine the suitable amount of the boronating reagent
necessary for the reaction. It turned out that 5 mol equiv
of borane-dimethyl sulfide was sufficient for completion
of the reaction in 30 min. Increasing the amount to 10
mol equiv did not significantly reduce the completion time
as monitored by 31P NMR spectrum. However, with 2 mol
equiv the reaction proceeded slowly and went to comple-
tion in about 2 h. Thus, 5 mol equiv of boron-dimethyl
sulfide was selected as the choice of optimal amount and
reagent to complete the boronation step in 30 min.
An attempt to purify the oxathiaphospholane-borane
complex 22 by silica gel column chromatography was not
successful due to its instability in the presence of
moisture. Therefore, the crude product 22 was reacted
with 0.8 mol equiv of nucleoside dissolved in anhydrous
acetonitrile in the presence of 1,8-diazabicyclo[5.4.0]-
2
2
R
approach, nucleoside 5′-P -boranodiphosphates were
23
recently obtained in good yields. To further investigate
the application of an oxathiaphospholane approach in
borane-containing nucleotide chemistry, we synthesized
nucleoside boranophosphoramidates conjugated with
amino acids via an oxathiaphospholane ring-opening
condensation as depicted in Scheme 5.
Amino acid methyl ester 5 was reacted with 2-chloro-
,3,2-oxathiaphospholane 20 in the presence of diisopro-
1
(19) (a) Stec, W. J.; Grajkowski, A.; Kobylanska, A.; Karwowski, B.;
Koziolkiewicz, M.; Misiura, K.; Okruszek, A.; Wilk, A.; Guga, P.;
Boczkowska, M. J. Am. Chem. Soc. 1995, 117, 12019. (b) Guga, P.;
Okruszek, A.; Stec, W. J. Top. Curr. Chem. 2002, 220, 169.
(
20) (a) Baraniak, J.; Kaczmarek, R.; Stec, W. J. Tetrahedron Lett.
2
000, 41, 9139. (b) Baraniak, J.; Kaczmarek, R.; Korczynski, D.;
Wasilewska, E. J. Org. Chem. 2002, 67, 7267. (c) Baraniak, J.;
Kaczmarek, R.; Wasilewska, E.; Stec, W. J. Phosphorus Sulfur Silicon
Relat. Elem. 2002, 177, 1667. (d) Olesiak, M.; Krajewska, D.; Wasilews-
ka, E.; Korczynski, D.; Baraniak, J.; Okruszek, A.; Stec, W. J. Synlett
2
002, 967-971. (e) Zmudzka, K.; Nawrot, B.; Chojnacki, T.; Stec, W.
J. Org. Lett. 2004, 6, 1385.
(
21) (a) Okruszek, A.; Sierzchala, A.; Zmudzka, K.; Stec, W. J.
Nucleosides Nucleotides Nucleic Acids 2001, 20, 1843. (b) Baraniak,
J.; Kaczmarek, R.; Wasilewska, E. Tetrahedron Lett. 2004, 45, 671.
(22) Lin, J. L.; He, K. Z.; Shaw, B. R. Helv. Chim. Acta 2000, 83,
1
392.
(24) Housecroft, C. E. Boranes and Metalloboranes Structure, Bond-
ing and Reactivity; John Wiley & Sons: New York, 1990; pp 18-30.
(23) Li, P.; Shaw, B. R. J. Org. Chem. 2004, 69, 7051.
2176 J. Org. Chem., Vol. 70, No. 6, 2005

Nucleoside Boranophosphoramidate Prodrugs
TABLE 3. Conditions for the Oxathiaphospholane
Ring-Opening Condensationa
TABLE 4. HPLC Separation Conditions for Nucleoside
Amino Acid Boranophosphoramidates 24a
molar equiv reaction completion
retention time (min)
[area percentage]
amino acid nucleoside product of DBU
temp. (°C) time (h)
L-alanine
3-OAc-T
AZT
24a
24c
24e
24b
24d
24f
24 g
24h
9
3
40
45
40
60
60
60
40
40
50
2
5
1
3
8
2
2
2
5
compound
4a
4b
bufferb
isomer I [%] isomer II [%]
20.55 [59.7] 24.00 [40.3]
5
2
5-40% CH
3
CN in TEAB
d4T
1.5
3
5
1.5
3
3
5
over 60 min
L-tryptophan 3-OAc-T
2
2
22% CH
3
CN/78% TEAB
10.19 [55.5] 11.41 [44.5]
9.91 [63.3] 11.52 [36.7]
AZT
d4T
3-OAc-T
4c
4d
24e
4f
3
5-30% CH CN in TEAB
over 60 min
D-alanine
D,L-alanine 3-OAc-T
L-tryptophan 5-DMT-T
2
3
15-50% CH CN in TEAB 22.61 [56.9] 24.80 [43.1]
over 60 min
3-15% CH CN in TEAB
3
7.47 [61.6]
9.62 [38.4]
over 60 min
a
The reaction was monitored by TLC with developing system
MeOH/CH2Cl2 (8/92).
2
3
12-40% CH CN in TEAB 21.40 [57.6] 25.78 [42.4]
over 60 min
a
Waters Delta-Pak C18 column: 15 µm, 100 Å, 25 × 100 mm,
b
with Z-module. 100 mM TEAB (triethylammonium bicarbonate,
pH 8.0) and acetonitrile (CH3CN).
undec-7-ene (DBU). The reaction underwent ring-opening
condensation to form the boranophosphoramidate inter-
mediate 23 followed by fast DBU-assisted elimination of
ethylene episulfide to provide the desired nucleoside
amino acid boranophosphoramidate 24. It should be
pointed out that the amount of DBU and the reaction
temperature required for the ring-opening condensation
depended on the specific amino acid methyl ester 5 used
in the reaction and the corresponding nucleoside. Gener-
ally, the more hindered the amino acid methyl ester and
nucleoside, the greater the amount of DBU required and
the higher the necessary reaction temperature. The
reaction conditions are described in Table 3. The crude
product boranophosphoramidate 24 was isolated by silica
gel column chromatography. To ensure that the nitrogen
3
atom from the amino acid was free of BH possibly
introduced during the boronation step, the reaction
mixture should be treated with water/triethylamine
mixtures for 15 min before loading onto the column for
purification.
The purified products, nucleoside amino acid borano-
phosphoramidates 24a-h and 9, were obtained in 35-
4
5% overall yields from 19 to 24 (or 9) after silica gel
purification. Their structures were confirmed by 1D- and
1
31
2
D- H NMR, P NMR, FAB-MS, and HRMS. It has to
31
be pointed out that the P NMR of boranophosphorami-
date 24 (or 9) has a characteristic broad peak around δ
1
8
9
2 ppm. This result does not agree with the reported
3
1
P NMR around δ 105 ppm by Baraniak et al. in a recent
communication on the synthesis of boranophosphorami-
1
date in which neither H NMR nor HRMS data were
provided.
FIGURE 2. Compounds of nucleoside amino acid borano-
phosphoramidates synthesized via an oxathiaphospholane
approach.
2
1b
As seen in the other cases,10,12,22 modification of a
phosphoramidate at the phosphorus atom results in a
noticeable change of its phosphorus chemical shift: from
3
ramidates. Introduction of a borane group (BH ) to
8
,25
δ ≈ 7 ppm for a normal phosphoramidate
to δ ≈ 60
replace one of the oxygen atoms on the phosphoramidate
produces a pair of diastereomers for compound 24. These
diastereomers of 24a-f have been separated by semi-
preparative reverse-phase HPLC (RP-HPLC) and named
isomer I and isomer II in order of their retention time.
Their tentative configuration assignment will be de-
scribed in the following section. The HPLC separation
conditions are summarized in Table 4. The isomeric
purity of each of the individual diastereomers was
determined by RP-HPLC under the same conditions used
for separation. The HPLC profiles for 2(S)-[(2′,3′-dideoxy-
ppm for a phosphorothioamidate2
0a,b
and, as we have
found in our studies, to δ ≈ 92 ppm for a boranophos-
phoramidate. Notably, the residence peak for nucleoside
amino acid boranophosphoramidates in P NMR is about
the same as that for dinucleoside boranophosphates,14
which indicates a similar chemical environment for
phosphorus in both types of compounds.
18
31
Studies on the HPLC Separation of Diastereo-
mers of Nucleoside Amino Acid Boranophospho-
(
25) Shirokova, E. A.; Jasko, M. V.; Khandazhinskaya, A. L.; Ivanov,
2
′,3′-didehydrothymidin-5′-yl)boranophosphorylamino]-
A. V.; Yanvarev, D. V.; Skoblov, Y. S.; Mitkevich, V. A.; Bocharov, E.
V.; Pronyaeva, T. R.; Fedyuk, N. V.; Kukhanova, M. K.; Pokrovsky, A.
G. J. Med. Chem. 2004, 47, 3606.
proprionic acid methyl ester 24e before and after pre-
parative HPLC separation are shown in Figure 3. The
J. Org. Chem, Vol. 70, No. 6, 2005 2177

Li and Shaw
SCHEME 6. Proposed “Adjacent”-Type
Mechanism in the 1,3,2-Oxathiaphospholane Ring-
Opening Process during the Synthesis of Nucleo-
side Amino Acid Boranophosphoramidate 24
FIGURE 3. Gradient separation of diastereomers of 2(S)-
(2′,3′-dideoxy-2′,3′-didehydrothymidin-5′-yl)boranophosphor-
[
ylamino]proprionic acid methyl ester 24e by RP-HPLC (A), and
analysis of purity of HPLC-isolated isomer I (B) and isomer
II (C). The separation was carried out on the Waters Delta-
Pak C18 column with gradient 3-15% acetonitrile in 100 mM
TEAB (pH 8.0) over 60 min at a flow rate of 8.0 mL/min. The
absorbance was monitored at λ 260 nm. The retention times
for the two diastereomers were 7.47 and 9.62 min, respectively.
Peak 1: isomer I of compound 24e. Peak 2: isomer II of
compound 24e.
oxygen atom, resulting in a trigonal bipyramidal inter-
mediate, which before collapse must undergo pseudoro-
tation, placing the cleavable P-S bond in the apical
position, to form the ring-opened product. As shown in
Scheme 6, the formation of the oxathiaphospholane-
borane complex 22 produces a pair of diastereomers
designated as Rp-22 and Sp-22. Except for an oxygen
atom in the apical position, all the other atoms and
groups are in equatorial positions. Nucleoside ROH
approaches complex 22 from the side opposite the en-
docyclic P-O bond, resulting in the formation of inter-
mediate 25, which has the two most electronegative
elements (two oxygen atoms) in apical positions. Because
the principle of microreversibility requires that the
structure of each diastereomer of 24a-f was confirmed
1
31
by 1D- and 2D- H NMR, P NMR, FAB-MS, and HRMS.
Tentative Configuration Assignment for Diaster-
eomers of Nucleoside Amino Acid Boranophospho-
ramidates. Noticeably, for each pair of diastereomers
in Table 3 the ratio between isomer I and II was
approximately 3 to 2, which was understandable because
the oxathiaphospholane approach was first introduced
as a way of stereocontrolled synthesis of oligo(nucleoside
phosphorothioate)s.19 An explanation proposed to ratio-
nalize the ratio of isomer I to II is depicted in Scheme 6.
Stec et al. have shown that an “adjacent”-type mechanism
is associated with the 1,3,2-oxathiaphospholane ring-
opening process,26 which involves a nucleophilic attack
from the side opposite the most apicophilic endocyclic
2
7
leaving group is located in the apical position, a single
pseudorotation process (with the phosphorus atom as
-
“pivot”) leads to intermediate 26. Since BH
3
has a strong
preference for the equatorial position as calculated by
(
26) Uznanski, B.; Grajkowski, A.; Krzyzanowska, B.; Kazmierkows-
ka, A.; Stec, W. J.; Wieczorek, M. W.; Blaszczyk, J. J. Am. Chem. Soc.
992, 114, 10197.
(27) Westheimer, F. H. Rearrangements in Ground & Excited States;
Academic Press: New York, 1980; Vol. 2, p 229.
1
2178 J. Org. Chem., Vol. 70, No. 6, 2005

Nucleoside Boranophosphoramidate Prodrugs
FIGURE 4. 1H NMR for different diastereomers of compound 2(S)-[(3′-O-acetylthymidin-5′-yl)boranophosphorylamino]proprionic
acid methyl ester 24a. Spectra were recorded on 500 MHz spectrometer in D O.
2
Thatcher et al.,28 isomer 26b is favored over 26a. Collapse
of 26 via cleavage of the apical P-S bond and the follow-
up elimination of ethylene episulfide from 23 gives
boranophosphoramidate 24. It has to be pointed out that
the function of DBU is neglected in this proposed mech-
anism. This is mainly because the driving force for the
overall process derives from the ring-strain relief arising
from the opening of the oxathiaphospholane ring, fol-
lowed by fast elimination of episulfide.26 Thus, from the
mechanistic point of view, Rp-24 is favored over Sp-24.
Accordingly, isomer I of boranophosphoramidate, whose
configuration is in large excess in final product, would
have an Rp configuration for the phosphorus center,
whereas isomer II corresponds to Sp.
Studies on the Racemization of Amino Acid dur-
ing Oxathiaphospholane Ring-Opening Process. A
major problem in amino acid chemistry is associated with
racemization of the amino acid or its derivative. Since
the oxathiaphospholane ring-opening process in 22 is
catalyzed by a strong base such as DBU, it was necessary
to check if racemization of phosphorylated derivatives
31
occurred during this reaction. P NMR was considered
as a technique for this purpose.2 However, due to the
0b
quadrapole broadening caused by 10B and B nuclei, it
11
3
1
is difficult to assign the different diastereomers by
P
NMR spectra. Therefore, HPLC was adopted to identify
the formation of different diastereomers of 24 after ring-
opening condensation with nucleoside.
It is noteworthy that chemical shifts and splitting
patterns for the 2′-position protons on the ribose in
compound 2(S)-[(3′-O-acetylthymidin-5′-yl)boranophos-
phorylamino]proprionic acid methyl ester 24a were quite
different in each diastereomer. As shown in Figure 4,
isomer I had a chemical shift with multiplet at δ 2.32
ppm for the two 2′-position protons whereas isomer II
had distinct chemical shifts with a multiplet at δ 2.35
and 2.24 ppm for the two 2′-position protons. According
to the preliminary results from the spatial model of
compound 24a constructed with the CHEM3D ULTRA
Compounds 22, specifically, 2-borano-1,3,2-oxathia-
phospholane derivatives of L-alanine, D-alanine, and
racemic D,L-alanine, were prepared as the model com-
pounds. When the derivative 22 of L-alanine reacted with
3
2
′-O-acetylthymidine/DBU, a pair of P-diastereomers of
4a was formed and could be separated by RP-HPLC
(Figure 5A). When the derivative 22 of D-alanine reacted
with 3′-O-acetylthymidine/DBU, a pair of P-diastereo-
1
mers of 24g was also formed as seen from H NMR, but
they could not be separated by RP-HPLC in the same
HPLC conditions (Figure 5B). When the derivative 22 of
racemic D,L-alanine was used, two enantiomeric pairs
were formed and the HPLC profile equaled the combina-
tion of HPLC profiles for 24a and 24g (Figure 5C). Every
peak shown in the HPLC profiles in Figure 5 was
subjected to electronic ionization mass (EI-MS) analysis,
which indicated that they all had the same molecular
6
.0 program, the chemical environments for the two
protons on the 2′-position in Rp-24 are similar because
all the neighboring groups are far away from them.
However, in Sp-24 the chemical environments for the two
protons on the 2′-position are quite different due to their
close proximity to other neighboring groups. This molec-
ular modeling supports assignment of isomers I and II
29
corresponding to Rp and Sp, respectively.
-
weight as 446.2 (M ). From these model experiments and
(
28) Thatcher, G. R. J.; Campbell, A. S. J. Org. Chem. 1993, 58,
272.
29) Please see the 3-D structures for Rp-24a and Sp-24a from
molecular modeling in the Supporting Information. Also refer to ref
due to the partial overlap between peaks 1 and 3 in
Figure 5, we concluded that the oxathiaphospholane ring-
opening process catalyzed by DBU in 22 did not cause
significant racemization in the resulting nucleoside amino
acid boranophosphoramidate 24.
2
(
2
5 for molecular modeling. The result from CHEM3D ULTRA was
preliminary, and further calculations are needed to confirm the
conclusions.
J. Org. Chem, Vol. 70, No. 6, 2005 2179

Li and Shaw
such as 24a were contaminated with DPCP because both
boranophosphoramidates 24 and the DPCP hydrolysis
products were highly soluble in water. Further purifica-
tion via silica gel chromatography did not resolve the
problem. Hence, the relatively lengthy procedure and
difficulties in purifying the final product limited the
application of H-phosphonate chemistry in preparation
of boranophosphoramidates and other relating phosphate
analogues.
Synthesis of nucleoside amino acid boranophosphora-
midate was better accomplished via a 1,3,2-oxathiaphos-
pholane approach. The key intermediate oxathiaphos-
pholane-borane complex 22 was obtained through
phosphitylation of an amino acid methyl ester with
2
-chloro-1,3,2-oxathiaphospholane followed by boronating
with borane-dimethyl sulfide. Comparative studies on
different boronating reagents and the optimal conditions
for DBU-catalyzed ring-opening condensation were car-
ried out. The reaction of DBU-assisted nucleophilic attack
of nucleoside on the phosphorus atom of the oxathia-
phospholane ring in 22 furnished an approach to various
boranophosphoramidate analogues 24a-h and 9 in good
yields. Hence, the borane complex 22 represents a new
intermediate for condensation reaction with various
nucleosides. The oxathiaphospholane strategy is of par-
ticular interest since it provides a facile and efficient
route to prepare biologically important analogues of
normal phosphoramidates: boranophosphoramidates 24.
The two diastereomers of boranophosphoramidates 24a-f
were successfully separated by RP-HPLC. The structures
for all of the final compounds 24a-g and 9 were
1
confirmed by spectroscopic methods including 2D- H
NMR. We also confirmed by HPLC and mass analyses
that the presence of DBU in the oxathiaphospholane ring-
opening reactions did not induce noticeable racemization
of boranophosphorylated derivatives of amino acids. It
is worthwhile to emphasize that due to the mildness of
the reaction conditions and the easily accessible phos-
phitylating reagent 20, the synthetic protocol developed
here can be considered as a general method for the
preparation of molecules with the presence of both P-N
and P-B bonds.
FIGURE 5. Separation profiles by RP-HPLC for diastereo-
mers of (A) 2(S)-[(3′-O-acetylthymidin-5′-yl)boranophosphor-
ylamino]proprionic acid methyl ester 24a, (B) 2(R)-[(3′-O-
acetylthymidin-5′-yl)boranophosphorylamino]proprionic acid
methyl ester 24g, and (C) 2(R,S)-[(3′-O-acetylthymidin-5′-yl)-
boranophosphorylamino]proprionic acid methyl ester 24h. The
separation was carried out on the Waters Delta-Pak C18
column with gradient 5-40% acetonitrile in 100 mM TEAB
(
pH 8.0) over 60 min at a flow rate of 1.0 mL/min. The
absorbance was monitored at λ ) 260 nm. Peak 1: isomer I of
compound 24a. Peak 2: isomer II of compound 24a. Peak 3:
diastereomer mixtures of compound 24g.
In summary, we developed two new strategies for the
introduction of a boranophosphate moiety into amino
acids to obtain nucleoside amino acid boranophosphora-
midates 24. On the basis of an “adjacent”-type mecha-
nism, the tentative assignment of configurations for both
diastereomers was carried out through molecular model-
ing and proton spectra in which the results were all in
accordance with each other. The two diastereomers of
nucleoside amino acid boranophosphoramidates are ex-
pected to have different substrate properties toward
phosphoramidases and should be useful for investigating
the roles of phosphate and metal ions in biological
processes to elucidate the stereochemical and metal
requirements of the enzymatic reactions involving phos-
phoramidases. Moreover, due to the presence of a borane
group,10 the boranophosphoramidates are expected to
have increased lipophilicity relative to their parent
phosphoramidate compounds, which would facilitate the
Conclusions
We developed two strategies for the synthesis of
nucleoside boranophosphoramidates conjugated with
amino acids. One strategy involved H-phosphonate chem-
istry in which the key intermediate, silylated phosphora-
midite diester 6, was obtained through a series of reaction
steps starting from nucleoside H-phosphonate in the
presence of condensing reagent DPCP. Subjecting the key
intermediate 6 to boronation followed by hydrolysis with
water resulted in the formation of nucleoside amino acid
boranophosphoramidate 9. Different condensing reagents
were examined, and DPCP appeared to be the best choice.
Due to the presence of a DMT group in the boranophos-
phoramidate 9, the condensing reagent DPCP could be
separated from the product 9 by aqueous extraction.
However, if the molecule lacked a hydrophobic group like
DMT, final product boranophosphoramidate compounds
1
,12
delivery of prodrugs containing antiviral nucleosides.
All of these advantages in combination with the potential
2180 J. Org. Chem., Vol. 70, No. 6, 2005

Nucleoside Boranophosphoramidate Prodrugs
utility as a carrier of 10B in BNCT30 make the nucleoside
amino acid boranophosphoramidate a useful compound
and biochemical tool in antiviral drug research.
3.15 (m, 4H, H-5′ and CH
2
); 2.99 (q, J ) 7.2 Hz, 6H, CH
TEAH ); 1.86 (m, 2H, H-2′); 1.32 (s, 3H, CH -5); 1.29 (t, J )
from TEAH ); (+)0.78 to (-)0.17 (br, 3H, BH ).
Cl): δ 93.5 (m, 1P). UV (H O): Isomer I λmax
13.6, 272.3 nm; Isomer II λmax 212.4, 272.3 nm. FAB-MS m/z
2
from
+
3
+
7
.2 Hz, 9H, CH
3
3
3
1
P NMR (CD
3
2
2
-
11
Experimental Section
821.3 (M ). HRMS: found, m/z 924.4498 (for B); calcd for
+
+
49 5 10
C H64BN O P [for (M + TEA + 2H) ], 924.4484.
2
-Chloro-1,3,2-oxathiaphospholane19a and 2-chloro-5,5-di-
Synthesis of Nucleoside Amino Acid Boranophospho-
3
1
methyl-2-oxo-1,3,2-dioxaphosphinane (NEP-Cl) were syn-
thesized according to the reported procedure. 2,4,6-Trichlo-
rophenol was recrystallized from benzene and ethanol. All
solvents were freshly distilled under argon from calcium
hydride. All boronating reagents except borane-dimethyl
sulfide complex, diisopropylethylamine (DIPEA), and 1,8-
diazabicyclo[5.4.0]-undec-7-ene (DBU) were dried by 4 Å
molecular sieves overnight. Nucleoside H-phosphonate, nu-
cleosides, and amino acid methyl ester hydrochlorides were
ramidates via an Oxathiaphospholane Approach: Gen-
eral Procedure. Amino acid methyl ester hydrochloride 19
(
0.5 mmol, 1.0 equiv) and DIPEA (1.0 mmol, 2.0 equiv) were
dissolved in methylene chloride. When the solution became
clear, 2-chloro-1,3,2-oxathiaphospholane 20 (0.55 mmol, 1.1
equiv) was added. After 30 min borane-dimethyl sulfide
complex (2.5 mmol, 5.0 equiv) was added, and the reaction
mixture was stirred for another 30 min. A freshly prepared
solution of nucleoside (0.4 mmol, 0.8 equiv) and DBU (1.5-5
equiv, see Table 3) in acetonitrile was injected into the reaction
vessel via syringe. The reaction mixtures were then heated to
dried overnight under vacuum in the presence of P
to use. Triethylammonium bicarbonate was prepared from
triethylamine, H O, and CO . All the reactions were carried
2 5
O prior
2
2
4
0-60 °C for 1-5 h (see Table 3). When the reaction was
out under an argon atmosphere unless otherwise stated. All
of the final products were in the triethylammonium salt forms.
completed, the reaction mixtures were treated with triethyl-
amine/water (1:1, v/v) for 15 min. Solvent was removed under
vacuum, and the residue was coevaporated with ethanol (15
mL) three times before being loaded to the silica gel. Flash
chromatography using a stepwise gradient of methanol (3-
15%) in chloroform containing 0.5% TEA gave the desired
product as an amorphous solid.
1
1
Proton assignment was based on the 2D H- H COSY.
Synthesis of 5′-O-Dimethoxytritylthymidin-3′-yl L-
Tryptophanyl-H-phosphonamidate 7 via H-Phosphonate
Chemistry. 5′-O-Dimethoxytritylthymidine 3′-H-phosphonate
+
1
(
(TEAH salt, 0.55 mmol, 1 equiv) and 2,4,6-trichlorophenol
0.60 mmol, 1.1 equiv) were rendered anhydrous by repeated
Reverse-Phase HPLC Separation of Diastereomers of
Nucleoside Amino Acid Boranophosphoramidates. The
separation of diastereomers of each nucleoside amino acid
boranophosphoramidate was carried out by ion-pairing chro-
matography on a semipreparative reverse-phase column (Wa-
ters Delta-Pak C18, 15 µm, 100 Å, 25 × 100 mm, with
Z-module) at a flow rate of 8 mL/min using a gradient or
isocratic elution [100 mM triethylammonium bicarbonate
(TEAB) buffer, pH ) 8.0, and acetonitrile, see Table 4].
Fractions containing the same isomer (similar retention time)
were combined, and the solvent was removed under reduced
pressure. The excess amount of TEAB and acetonitrile was
removed by repeated lyophilization with deionized water. The
retention time and ratio for different diastereomers of nucleo-
side amino acid boranophosphoramidates 24a-f are sum-
marized in Table 4.
coevaporation with added pyridine, dissolved in methylene
chloride, and condensing reagent was added. When the
3
2
condensation was completed, chlorotrimethylsilane (1.65
mmol, 3 equiv) was injected into the reaction vessel and
immediately followed by the addition of triethylamine (3.3
mmol, 6 equiv). After 5 min L-tryptophan methyl ester
hydrochloride (0.44 mmol, 0.8 equiv) dissolved in anhydrous
triethylamine (1.1 mmol, 2 equiv) was injected into the reaction
vessel. After stirring for 2 h the aminolysis reaction mixture
containing silylated phosphoramidite diester 6 was diluted
with methylene chloride (30 mL) and washed repeatedly with
0
.5 M NaHCO
Na SO ; the solvent was removed under vacuum; and the
residue was coevaporated with a toluene-acetonitrile mixture
1:1, v/v). Crude product 7 was obtained as an off-white foam.
3
(5 × 30 mL). The organic layer was dried over
2
4
(
3
1
3
P NMR (CDCl ): δ 15.12, 15.05. HRMS found, m/ z 809.2943
1
1
+
+
(
46 4 10
for B); calcd for C43H N O P (MH ), 809.2952. For the
2(S)-[(3′-O-Acetylthymidin-5′-yl)boranophosphorylami-
equivalency of different condensing reagents and the corre-
sponding yield for crude product 7, please refer to Table 1.
Synthesis of 2(S)-[(5′-O-Dimethoxytritylthymidin-3′-
yl)boranophosphorylamino]-3-(3-indolyl)proprionic Acid
Methyl Ester 9 via H-Phosphonate Chemistry. Silylated
phosphoramidite diester 6 was obtained as described above
when 1.0 equiv of DPCP was used as a condensing reagent.
The reaction mixture was treated with borane-dimethyl
sulfide (5.5 mmol, 10 equiv) for 45 min followed by addition of
water (5 mL). The reaction mixture was stirred for another
no]proprionic Acid Methyl Ester (24a). The diastereomeric
mixture of compound 24a was prepared in 45% yield (99 mg)
following the general procedure by using L-alanine methyl
ester (HCl salt), 3′-O-acetylthymidine, and 3 mol equiv of DBU
at 40 °C for 2 h.
Isomer I (tentatively assigned as Rp isomer). 1H NMR
(
(
)
)
3
2
D O): δ 7.59 (s, 1H, H-6), 6.18 (t, J ) 6.8 Hz, 1H, H-1′), 5.17
m, 1H, H-3′), 4.17 (m, 1H, H-4′), 3.82 (m, 2H, H-5′), 3.70 (t, J
7.6 Hz, 1H, CH), 3.54 (s, 3H, CH
3
from CO
from TEAH ), 2.29 (m, 2H, H-2′), 1.97 (s,
from OAc), 1.79 (s, 3H, CH -5), 1.14 (d, J ) 7.6 Hz,
), 1.11 (t, J ) 7.2 Hz, 9H, CH
2
Me), 3.03 (q, J
+
7.2 Hz, 6H, CH
2
3
0 min and then concentrated in a vacuum. The residue was
dissolved in methylene chloride (30 mL) and extracted with
.5 M NaHCO
(3 × 20 mL). The organic phase was dried over
Na SO
H, CH
H, CH
3
3
+
3
3
3
from TEAH ), (+)0.90
3 2
). P NMR (D O): δ 90.7 (m, 1P). UV
0
3
31
to (-)0.35 (br, 3H, BH
O): max 266.4 nm. FAB-MS: m/z 446.3 (M ). HRMS:
3 9
found, m/z 446.1501 (for B); calcd for C16 26BN O P [for (M
2
4
and then evaporated to dryness. The residue was
+
(
H
2
λ
chromatographed on a silica gel column using a stepwise
11
+
H
gradient of methanol (0-5%) in chloroform containing 0.5%
+
-
2e) ], 446.1500.
Isomer II (tentatively assigned as Sp isomer). 1H NMR
O): δ 7.61 (s, 1H, H-6), 6.17 (dd, J ) 5.6, 6.0 Hz, 1H, H-1′),
.16 (m, 1H, H-3′), 4.20 (m, 1H, H-4′), 3.82 (m, 2H, H-5′), 3.76
from CO Me), 3.03
from TEAH ), 2.32 (m, 1H, H-2′), 2.21
m, 1H, H-2′), 1.97 (s, 3H, CH from OAc), 1.79 (s, 3H, CH -5),
.16 (d, J ) 7.2 Hz, 3H, CH ), 1.11 (t, J ) 7.2 Hz, 9H, CH
1
TEA to afford 9 as a foam (130 mg, 32% yield). H NMR (CD
3
-
Cl): δ 7.57-7.50, 7.37-6.98, 6.81-6.79 (m, 19H, ArH and H-6);
(
D
2
6
.36, 6.21 (2t, two P-isomers, J ) 6.0 Hz, 1H, H-1′); 4.98, 4.75
5
(
2m, two P-isomers, 1H, H-3′); 4.24 (m, 1H, H-4′); 4.15, 4.12
2m, two P-isomers, 1H, CH); 3.74 (s, 6H, OMe from DMT);
(
(
(
t, J ) 7.6 Hz, 1H, CH), 3.54 (s, 3H, CH
3
2
(
+
q, J ) 7.2 Hz, 6H, CH
2
3
2
.56, 3.44 (2s, two P-isomer, 3H, OMe from CO Me); 3.40-
3
3
1
3
3
(
(
(
30) Hawthorne, M. F. Angew. Chem., Int. Ed. Engl. 1993, 32, 950.
+
31
from TEAH ), (+)0.60 to (-)0.20 (br, 3H, BH
3
). P NMR
31) McConnell, R. L.; Coover, H. W. J. Org. Chem. 1959, 24, 630.
32) A sample of reaction mixture (10 µL) was quenched with dry
(
D
2
O): δ 91.6 (m, 1P). UV (H
2
O): λmax 266.4 nm. FAB-MS: m/z
+
11
4
46.3 (M ). HRMS: found, m/z 446.1501 (for B); calcd for
methanol (20 µL) prior to analysis to convert a hydrolytically unstable
aryl H-phosphonate 3 into the stable methyl ester.
+
+
C
16
H
26BN
3
O
9
P
[for (M - 2e) ], 446.1500.
J. Org. Chem, Vol. 70, No. 6, 2005 2181

Li and Shaw
2
(S)-[(3′-O-Acetylthymidin-5′-yl)boranophosphorylami-
ring), 6.83 (t, J ) 7.6 Hz, 1H, H-5 on indolyl ring), 5.91 (t, J
) 6.8 Hz, 1H, H-1′), 4.10 (m, 1H, H-3′), 3.79 (m, 2H, H-4′ and
no]-3-(3-indolyl)proprionic Acid Methyl Ester (24b). The
diastereomeric mixture of compound 24b was prepared in 40%
yield (106 mg) following the general procedure by using
L-tryptophan methyl ester (HCl salt), 3′-O-acetylthymidine,
and 3 mol equiv of DBU at 60 °C for 2 h.
CH), 3.58 (m, 1H, H-5′), 3.51 (s, 3H, CH
(m, 1H, H-5′), 3.01 (q, J ) 7.2 Hz, 6H, CH
(m, 1H, CH ), 2.87 (m, 1H, CH ), 2.12 (m, 1H, H-2′), 2.00 (m,
1H, H-2′), 1.58 (s, 3H, CH -5), 1.10 (t, J ) 7.2 Hz, 9H, CH
3
from CO
2
Me), 3.45
+
2
from TEAH ), 2.99
2
2
3
3
Isomer I (tentatively assigned as Rp isomer). 1H NMR
+
31
from TEAH ), (+)0.50 to (-)0.40 (br, 3H, BH
(D O): δ 91.7 (m, 1P). UV (H O): λmax 215.9, 269.9 nm. FAB-
MS m/z 544.1 (M ). HRMS: found, m/z 544.1882 (for B); calcd
H28BN O P [for (M - 2e) ], 544.1881.
7 7
Isomer II (tentatively assigned as Sp isomer). 1H NMR
O): δ 7.46 (d, J ) 7.6 Hz, 1H, H-4 on indolyl ring), 7.20 (d,
J ) 8.0 Hz, 1H, H-7 on indolyl ring), 7.16 (s, 1H, H-6), 7.02 (s,
H, H-2 on indolyl ring), 6.98 (dd, J ) 7.2, 7.6 Hz, 1H, H-6 on
indolyl ring), 6.90 (dd, J ) 7.6, 8.0 Hz, 1H, H-5 on indolyl ring),
3
). P NMR
(
D
2
O): δ 7.37 (d, J ) 8.0 Hz, 1H, H-4 on indolyl ring), 7.22 (s,
2
2
-
11
1
H, H-6), 7.21 (d, J ) 7.6 Hz, 1H, H-7 on indolyl ring), 7.01 (s,
H, H-2 on indolyl ring), 6.96 (dd, J ) 7.2, 8.0 Hz, 1H, H-6 on
+
+
1
for C22
indolyl ring), 6.81 (dd, J ) 7.2, 7.6 Hz, 1H, H-5 on indolyl ring),
6
.03 (dd, J ) 6.0, 6.0 Hz, 1H, H-1′), 5.01 (m, 1H, H-3′), 3.95
(
D
2
(
m, 1H, H-4′), 3.82 (q, J ) 8.8 Hz, 1H, CH) 3.66 (m, 1H, H-5′),
3
.50 (s, 3H, CH
.2 Hz, 6H, CH
3
from CO
from TEAH ), 2.97, 2.89 (2m, 2H, CH
from OAc), 1.90 (m, 1H, H-2′),
2
Me), 3.37 (m, 1H, H-5′), 2.99 (q, J )
1
+
7
2
2
), 2.07
(
m, 1H, H-2′), 1.94 (s, 3H, CH
.56 (s, 3H, CH -5), 1.09 (t, J ) 7.2 Hz, 9H, CH
+)0.80 to (-)0.40 (br, 3H, BH
P). UV (H O): λmax 220.6, 269.9 nm. FAB-MS: m/z 561.1 (M ).
3
5
3
.85 (t, J ) 6.4 Hz, 1H, H-1′), 4.01 (m, 2H, H-3′ and H-4′),
.86 (m, 1H, CH), 3.60 (m, 1H, H-5′), 3.55 (s, 3H, CH from
Me), 3.53 (m, 1H, H-5′), 3.05 (m, 1H, CH ), 3.02 (q, J )
from TEAH ), 2.86 (m, 1H, CH ), 1.96 (m,
H, H-2′), 1.61 (m, 1H, H-2′), 1.57 (s, 3H, CH -5), 1.10 (t, J )
from TEAH ), (+)0.52 to (-)0.18 (br, 3H, BH ).
P NMR (D O): δ 91.5 (m, 1P). UV (H O): λ 211.2, 269.9
+
1
3
3
from TEAH ),
3 2
). P NMR (D O): δ 91.5 (m,
3
3
1
(
CO
2
2
-
1
2
+
7
1
7
.2 Hz, 6H, CH
2
2
11
+
HRMS: found, m/z 561.1896 (for B); calcd for C24
4 9
H31BN O P
3
+
[
for (M - 2e) ], 561.1922.
Isomer II (tentatively assigned as Sp isomer). 1H NMR
O): δ 7.45 (d, J ) 7.6 Hz, 1H, H-4 on indolyl ring), 7.19
m, 2H, H-7 on indolyl ring and H-6), 7.01 (s, 1H, H-2 on indolyl
+
.2 Hz, 9H, CH
3
3
31
2
2
max
(
(
D
2
-
nm. FAB-MS: m/z 544.1 (M ). HRMS: found, m/z 544.1877
11
+
+
(
for B); calcd for C22
7 7
H28BN O P [for (M - 2e) ], 544.1881.
ring), 6.96 (dd, J ) 7.2, 7.6 Hz, 1H, H-6 on indolyl ring), 6.88
dd, J ) 7.2, 7.6 Hz, 1H, H-5 on indolyl ring), 5.96 (t, J ) 7.2
Hz, 1H, H-1′), 4.95 (m, 1H, H-3′), 4.02 (m, 2H, H-4′ and CH)
.61 (m, 2H, H-5′), 3.54 (s, 3H, CH
.2 Hz, 6H, CH
2(S)-[(2′,3′-Dideoxy-2′,3′-didehydrothymidin-5′-yl)bora-
(
nophosphorylamino]proprionic Acid Methyl Ester (24e).
The diastereomeric mixture of compound 24e was prepared
in 40% yield (78 mg) following the general procedure by using
L-alanine methyl ester (HCl salt), d4T, and 1.5 mol equiv of
DBU at 40 °C for 1 h.
3
3
from CO
from TEAH ), 2.98, 2.86 (2m, 2H, CH
3
from OAc), 1.57 (m, 4H, H-2′ and CH -
2
Me), 3.01 (q, J )
+
7
2
2
), 1.95
(
m, 4H, H-2′ and CH
), 1.10 (t, J ) 7.2 Hz, 9H, CH
3
+
5
3
from TEAH ), (+)0.60 to
). P NMR (D O): δ 91.3 (m, 1P). UV
λmax 219.4, 269.9 nm. FAB-MS: m/z 561.1 (M ).
Isomer I (tentatively assigned as Rp isomer). 1H NMR
(D O): δ 7.27 (s, 1H, H-6), 6.70 (s, 1H, H-1′), 6.29 (d, J ) 6.0
2
3
1
(
(
-)0.40 (br, 3H, BH
O):
3
2
-
H
2
11
+
Hz, 1H, H-2′), 5.78 (d, J ) 6.0 Hz, 1H, H-3′), 4.87 (m, 1H, H-4′),
3.92 (m, 1H, H-5′), 3.65 (m, 1H, H-5′), 3.48 (s, 3H, CH from
4 9
HRMS: found, m/z 561.1915 (for B), calcd for C24H31BN O P
+
3
[
for (M - 2e) ], 561.1922.
(S)-[(3′-Deoxy-3′-azidothymidin-5′-yl)boranophospho-
CO
CH
2
Me), 3.31 (q, J ) 7.2 Hz, 1H, CH), 3.00 (q, J ) 7.2 Hz, 6H,
2
+
2
from TEAH ), 1.72 (s, 3H, CH
3
-5), 1.10 (t, J ) 7.2 Hz,
), (+)0.40-
O): δ 92.1 (m, 1P). UV
rylamino]proprionic Acid Methyl Ester (24c). The dia-
stereomeric mixture of compound 24c was prepared in 38%
yield (80 mg) following the general procedure by using L-
alanine methyl ester (HCl salt), AZT, and 5 mol equiv of DBU
at 45 °C for 5 h.
+
9
H, CH
3
from TEAH ), 1.03 (d, J ) 7.2 Hz, 3H, CH
3
3
1
(
(
-)0.42 (br, 3H, BH
O):
3
). P NMR (D
2
+
H
2
λ
max 214.7, 266.4 nm. FAB-MS: m/z 386.2 (M ).
11
+
3 7
HRMS: found, m/z 386.1283 (for B); calcd for C14H22BN O P
+
[
for (M - 2e) ], 386.1288.
Isomer II (tentatively assigned as Sp isomer). 1H NMR
O): δ 7.39 (s, 1H, H-6), 6.75 (s, 1H, H-1′), 6.28 (d, J ) 6.0
Isomer I (tentatively assigned as Rp isomer). 1H NMR
(
(
2
D O): δ 7.50 (s, 1H, H-6), 6.10 (t, J ) 6.8 Hz, 1H, H-1′), 4.32
(
D
2
dt, J ) 4.8, 5.6 Hz, 1H, H-3′), 3.97 (m, 1H, H-4′), 3.84 (m, 1H,
Hz, 1H, H-2′), 5.77 (d, J ) 6.0 Hz, 1H, H-3′), 4.88 (m, 1H, H-4′),
.75 (m, 2H, H-5′), 3.66 (m, 1H, CH), 3.52 (s, 3H, CH from
from TEAH ), 1.73 (s,
H-5′), 3.80 (m, 1H, H-5′), 3.63 (q, J ) 6.4 Hz, 1H, CH), 3.53 (s,
H, CH from CO Me), 3.03 (q, J ) 7.2 Hz, 6H, CH from
TEAH ), 2.34 (t, J ) 6.4 Hz, 2H, H-2′), 1.78 (s, 3H, CH -5),
3
3
3
3
2
2
+
+
CO
2
Me), 3.01 (q, J ) 7.2 Hz, 6H, CH
3
-5), 1.10 (t, J ) 7.2 Hz, 9H, CH from TEAH ), 1.09
2
3
+
+
3H, CH
3
1
.11 (t, J ) 7.2 Hz, 9H, CH
3
from TEAH ), 1.07 (d, J ) 6.4
3
1
3
1
(d, J ) 7.2 Hz, 3H, CH ), (+)0.42 to (-)0.42 (br, 3H, BH ).
P
Hz, 3H, CH
3
), (+)0.82 to (-)0.30 (br, 3H, BH
3
). P NMR
3
3
NMR (D
2
O): δ 91.3 (m, 1P). UV (H
2
O): λmax 215.9, 266.4 nm.
(
D
2
O): δ 90.9 (m, 1P). UV (H
2
O): λmax 267.6 nm. FAB-MS m/z
-
-
11
FAB-MS: m/z 386.1 (M ). HRMS: found, m/z 386.1273 (for
4
29.2 (M ). HRMS: found, m/z 429.1473 (for B); calcd for
1
1
-
+
+
14 22 3 7
B); calcd for C H BN O P , 386.1288.
C
14
H
23BN
6
7
O P
[for (M - 2e) ], 429.1459.
Isomer II (tentatively assigned as Sp isomer). 1H NMR
O): δ 7.56 (s, 1H, H-6), 6.08 (t, J ) 6.8 Hz, 1H, H-1′), 4.29
m, 1H, H-3′), 4.02 (m, 1H, H-4′), 3.83 (m, 2H, H-5′), 3.76 (q,
from CO Me), 3.03 (q,
from TEAH ), 2.32 (m, 2H, H-2′), 1.78 (s,
-5), 1.15 (d, J ) 7.2 Hz, 3H, CH ), 1.11 (t, J ) 7.2 Hz,
2(S)-[(2′,3′-Dideoxy-2′,3′-didehydrothymidin-5′-yl)bora-
(
(
D
2
nophosphorylamino]-3-(3-indolyl)proprionic Acid Meth-
yl Ester (24f). The diastereomeric mixture of compound 24f
was prepared in 38% yield (91 mg) following the general
procedure by using L-tryptophan methyl ester (HCl salt), d4T,
and 1.5 mol equiv of DBU at 60 °C for 2 h.
J ) 7.2 Hz, 1H, CH), 3.55 (s, 3H, CH
3
2
+
J ) 7.2 Hz, 6H, CH
2
3
9
H, CH
H, CH
3
3
+
31
Isomer I (tentatively assigned as Rp isomer). 1H NMR
3
from TEAH ), (+)0.54 to (-)0.32 (br, 3H, BH
O): δ 91.9 (m, 1P). UV (H O): λmax 267.6 nm. FAB-
3
).
P
NMR (D
MS: m/z 429.1 (M ). HRMS: found, m/z 429.1450 (for B);
calcd for C14
2
2
(D O): δ 7.37 (d, J ) 8.0 Hz, 1H, H-4 on indolyl ring), 7.27 (d,
2
-
11
J ) 8.2 Hz, 1H, H-7 on indolyl ring), 7.17 (s, 1H, H-6), 7.03
-
6 7
H23BN O P , 429.1459.
(
dd, J ) 6.8, 8.0 Hz, 1H, H-6 on indolyl ring), 7.01 (s, 1H, H-2
2(S)-[(3′-Deoxy-3′-azidothymidin-5′-yl)boranophospho-
on indolyl ring), 6.91 (dd, J ) 6.8, 8.0 Hz, 1H, H-5 on indolyl
ring), 6.68 (s, 1H, H-1′), 6.17 (d, J ) 6.0 Hz, 1H, H-2′), 5.67 (d,
J ) 6.0 Hz, 1H, H-3′), 4.74 (m, 1H, H-4′), 3.65 (m, 3H, CH and
H-5′), 3.39 (s, 3H, CH from CO Me), 2.94 (q, J ) 7.2 Hz, 6H,
rylamino]-3-(3-indolyl)proprionic Acid Methyl Ester (24d).
The diastereomeric mixture of compound 24d was prepared
in 35% yield (90 mg) following the general procedure by using
L-tryptophan methyl ester (HCl salt), AZT, and 5 mol equiv of
DBU at 60 °C for 8 h.
3
2
+
CH from TEAH ), 2.91 (d, J ) 5.2 Hz, 2H, CH ), 1.64 (s, 3H,
2
2
+
CH -5), 1.07 (t, J ) 7.2 Hz, 9H, CH from TEAH ), (+)0.50 to
3
3
Isomer I (tentatively assigned as Rp isomer). 1H NMR
31
3 2
(-)0.42 (br, 3H, BH ). P NMR (D O): δ 92.1 (m, 1P). UV
-
(
D
2
O): δ 7.38 (d, J ) 7.6 Hz, 1H, H-4 on indolyl ring), 7.23 (d,
2
(H O): λmax 224.1, 269.9 nm. FAB-MS m/z 501.1 (M ). HRMS:
1
1
+
J ) 8.4 Hz, 1H, H-7 on indolyl ring), 7.18 (s, 1H, H-6), 7.01 (s,
H, H-2 on indolyl ring), 6.97 (t, J ) 7.6 Hz, 1H, H-6 on indolyl
found, m/z 501.1712 (for B); calcd for C22
H
27BN
4
O
7
P [for (M
+
1
- 2e) ], 501.1710.
2182 J. Org. Chem., Vol. 70, No. 6, 2005

Nucleoside Boranophosphoramidate Prodrugs
Isomer II (tentatively assigned as Sp isomer). 1H NMR
eomeric mixture of compound 24h was prepared in 37% yield
(81 mg) following the general procedure by using racemic D,L-
alanine methyl ester (HCl salt), 3′-O-acetylthymidine, and 3
mol equiv of DBU at 40 °C for 2 h.
2
(D O): δ 7.41 (d, J ) 8.0 Hz, 1H, H-4 on indolyl ring), 7.26 (d,
J ) 7.6 Hz, 1H, H-7 on indolyl ring), 7.21 (s, 1H, H-6), 7.02
dd, J ) 7.2, 8.0 Hz, 1H, H-6 on indolyl ring), 6.99 (s, 1H, H-2
(
on indolyl ring), 6.93 (dd, J ) 7.2, 7.6 Hz, 1H, H-5 on indolyl
ring), 6.66 (s, 1H, H-1′), 6.07 (d, J ) 5.6 Hz, 1H, H-2′), 5.60 (d,
J ) 5.6 Hz, 1H, H-3′), 4.69 (m, 1H, H-4′), 3.97 (dd, J ) 6.8, 7.2
Diastereomer Mixture. UV (H
2
O): λmax 266.4 nm. FAB-MS:
+
11
m/z 446.1 (M ). HRMS: found, m/z 446.1510 (for B); calcd
+
+
3 9
for C16H26BN O P [for (M - 2e) ], 446.1500.
3 2
Hz, 1H, CH), 3.59 (m, 2H, H-5′), 3.43 (s, 3H, CH from CO -
+
Me), 2.95 (q, J ) 7.2 Hz, 6H, CH
.8 Hz, 2H, CH ), 1.57 (s, 3H, CH
2
from TEAH ), 2.93 (d, J )
2(S)-[(5′-O-Dimethoxytritylthymidin-3′-yl)boranophos-
phorylamino]-3-(3-indolyl)proprionic Acid Methyl Ester
(9). The diastereomeric mixture of compound 9 was prepared
in 36% yield (133 mg) following the general procedure by using
L-tryptophan methyl ester (HCl salt), 5′-O-DMT-thymidine,
and 5 mol equiv of DBU at 50 °C for 5 h.
6
2
3
-5), 1.07 (t, J ) 7.2 Hz, 9H,
+
31
CH
3
from TEAH ), (+)0.57 to (-)0.26 (br, 3H, BH
3
). P NMR
(
D
2
O): δ 90.5 (m, 1P). UV (H
2
O): λmax 222.9, 269.9 nm. FAB-
-
11
MS: m/z 501.2 (M ). HRMS: found, m/z 501.1721 (for B);
+
+
calcd for C22
2
4 7
H27BN O P [for (M - 2e) ], 501.1710.
(R)-[(3′-O-Acetylthymidin-5′-yl)boranophosphorylami-
1H NMR and 31P NMR data were the same as that of
compound 9 prepared earlier via H-phosphonate chemistry.
no]proprionic Acid Methyl Ester (24g). The diastereomeric
mixture of compound 24g was prepared in 38% yield (83 mg)
following the general procedure by using D-alanine methyl
ester (HCl salt), 3′-O-acetylthymidine, and 3 mol equiv of DBU
Acknowledgment. This work was supported by an
NIH grant (R01 AI52061) to B.R.S. and a Paul M. Gross
fellowship from Duke University to P.L. We thank Mr.
Michael Goldsmith for helpful discussion about molec-
ular modeling and calculations.
at 40 °C for 2 h.
1
Diastereomer Mixture. H NMR (D
2
O): δ 7.63, 7.57 (2s, 2
isomers, 1H, H-6), 6.20 (t, J ) 6.8 Hz, 1H, H-1′), 5.18 (m, 1H,
H-3′), 4.15 (m, 1H, H-4′), 3.85-3.68 (m, 3H, CH and H-5′), 3.49
(
s, 3H, CH
3
from CO
TEAH ), 2.21 (m, 2H, H-2′), 1.97 (s, 3H, CH
s, 3H, CH -5), 1.15 (d, J ) 7.2 Hz, 3H, CH
Hz, 9H, CH
2
Me), 3.03 (q, J ) 7.2 Hz, 6H, CH
from OAc), 1.80
), 1.11 (t, J ) 7.2
from TEAH ), (+)0.42 to (-)0.20 (br, 3H, BH ).
O): δ 90.9 (m, 1P). UV (H O): λmax 266.4 nm.
FAB-MS: m/z 446.1 (M ). HRMS: found, m/z 446.1514 (for
2
from
+
3
(
3
3
Supporting Information Available: 1D- H NMR, 31P
1
+
1
3
3
NMR, and selected 2D- H NMR spectra for compounds 9 and
31
P NMR (D
2
2
2
4a-g; HPLC profiles for compounds 24a-f; molecular mod-
+
eling for compound 24a. This material is available free of
11
+
+
B); calcd for C16
(R,S)-[(3′-O-Acetylthymidin-5′-yl)boranophosphoryl-
amino]proprionic Acid Methyl Ester (24h). The diaster-
3 9
H26BN O P [for (M - 2e) ], 446.1500.
charge via the Internet at http://pubs.acs.org.
2
JO0481248
J. Org. Chem, Vol. 70, No. 6, 2005 2183