Synthesis of 3'-amino-3'-deoxyguanosine and 3'-amino-3'-deoxyxyloguanosine monophosphate hepdirect prodrugs from guanosine

Article abstract of DOI:10.1080/15257770903307151

The synthesis of 3'-amino-3'-deoxyguanosine and 3'-amino-3'- deoxyxyloguanosine monophosphate HepDirect prodrugs from guanosine is reported. Initial incorporation of N,N-dibenzylformamidino protection of the C2-amino of guanosine masked the reactivity of

Full text of DOI:10.1080/15257770903307151

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Synthesis of 3′-Amino-3′-
Deoxyguanosine and 3′-Amino-3′-
Deoxyxyloguanosine Monophosphate
Hepdirect Prodrugs from Guanosine
Brett C. Bookser a , Nicholas B. Raffaele a , K. Raja Reddy a , Kevin
Fan a , Wenjian Huang a & Mark D. Erion a
a Metabasis Therapeutics, Inc., La Jolla, California, USA
Version of record first published: 20 Oct 2009.
To cite this article: Brett C. Bookser , Nicholas B. Raffaele , K. Raja Reddy , Kevin Fan , Wenjian
Huang & Mark D. Erion (2009): Synthesis of 3′-Amino-3′-Deoxyguanosine and 3′-Amino-3′-
Deoxyxyloguanosine Monophosphate Hepdirect Prodrugs from Guanosine, Nucleosides, Nucleotides
and Nucleic Acids, 28:10, 969-986
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Nucleosides, Nucleotides and Nucleic Acids, 28:969–986, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903307151
SYNTHESIS OF 3ꢀ-AMINO-3ꢀ-DEOXYGUANOSINE AND
3ꢀ-AMINO-3ꢀ-DEOXYXYLOGUANOSINE MONOPHOSPHATE
HEPDIRECT PRODRUGS FROM GUANOSINE
Brett C. Bookser, Nicholas B. Raffaele, K. Raja Reddy, Kevin Fan,
Wenjian Huang, and Mark D. Erion
Metabasis Therapeutics, Inc., La Jolla, California, USA
The synthesis of 3ꢁ-amino-3ꢁ-deoxyguanosine and 3ꢁ-amino-3ꢁ-deoxyxyloguanosine monophos-
2
phate HepDirect prodrugs from guanosine is reported. Initial incorporation of N,N-
dibenzylformamidino protection of the C2-amino of guanosine masked the reactivity of that group
and simplified purification of subsequent analogues. The first key intermediate, 9-(2,5-bis-O-tert-
butyldimethylsilyl-β-D-ribofuranosyl)-2-N-(N,N-dibenzylformamidino)guanine (3a), was prepared
in 60% yield after recycling of the undesired 3ꢁ,5ꢁ-bis-O-protected byproduct (4a) by simple
equilibration in methanol to a mixture of the two bis-O-protected compounds. Thus, protected, the
3ꢁ-position was manipulated to form the 3ꢁ-deoxyribo- or 3ꢁ-deoxyxylo-3ꢁ-azido derivatives (9 or 16,
respectively). Further selective manipulations provided the cis-5ꢁ-monophosphate (3-chlorophenyl)-
1,3-propanyl diester prodrugs (HepDirect prodrugs), 15 and 21. These HepDirect prodrugs were
demonstrated to activate to their respective NTPs in rat hepatocytes.
Keywords HepDirect prodrugs; 3ꢁ-amino-3ꢁ-deoxyguanosines; NTPs
INTRODUCTION
Early research into the synthesis of 3ꢁ-amino-nucleosides was driven
by the discovery of 9-(3-deoxy-3-((4-methoxy-l-phenylalanyl)amino)-β-d-
ribofuranosyl)-6-(dimethylamino)purine, commonly known as puromycin,
Received 8 July 2009; accepted 2 September 2009.
Supporting Information Available: 1H NMR for all new compounds.
Address correspondence to Brett Bookser, Helicon Therapeutics Inc., 7473 Lusk Blvd., San Diego,
CA 92121, USA. E-mail: brett bookser@mac.com
969

970
B. C. Bookser et al.
and its antibiotic properties.[1] Further research elaborated the series
with analogues having antibacterial, anticancer, and other properties of
potential medicinal benefit.[2] Later, 3ꢁ-azido-3ꢁ-deoxynucleosides, as ex-
emplified by 3ꢁ-azido-3ꢁ-deoxythymidine or AZT, propelled research in
antiviral applications on into modern times.[3] More recently groups
have been interested in the synthesis of 3ꢁ-amino-3ꢁ-deoxynucleosides and
their derivatives as ligands for purine receptors for a variety of dis-
ease applications.[4–5] Still other work has focused particularly on the
preparation of 3ꢁ-azido-3ꢁ-deoxyguanosine, 3ꢁ-azido-3ꢁ-deoxyxyloguanosine,
3ꢁ-amino-3ꢁ-deoxyguanosine, their 2ꢁ,3ꢁ-dideoxy analogues and their 5ꢁ-
phosphorylated derivatives for use as antivirals,[6] anticancer agents,[7] for
the preparation of backbone modified oligonucleotides,[8] and as bioor-
ganic tool molecules.[9]
Our interest in 3ꢁ-deoxy-3ꢁ-modified guanosine analogues originates
in their potential hepatitis C antiviral applications. Among the natu-
ral nucleotides, ribose modified guanosine 5ꢁ-triphosphate (GTP) ana-
logues are the most potent RdRp inhibitors.[10] When considering the
reported structural model of binding of known 2ꢁ-C-methyl-nucleoside
triphosphate inhibitors to RdRp, the 3ꢁ-position of the inhibitor NTP
is exposed to the solvent surface.[11] Thus, producing a series of 3ꢁ-
deoxyguanosines derivatized at the 3ꢁ-position in order to search for adven-
titious binding elements was chosen for exploration as HCV inhibitor NTP
precursors.
3ꢁ-Deoxy-3ꢁ-modified nucleoside analogues derive their activity when
converted to their respective nucleoside 5ꢁ-triphosphates (NTPs), which
act as polymerase inhibitors or chain terminators to growing viral DNA
or RNA.[12] However, if the nucleoside is an inefficient substrate for the
intracellular kinase cascade required for conversion to NTP, it will be in-
effective as an antiviral agent. The cis-5ꢁ-monophosphate (3-chlorophenyl)-
1,3-propanyl diester prodrug (HepDirect prodrug)[13] has been developed
as part of a kinase-bypass strategy to deliver NMP molecules specifically
to hepatocytes, which is especially relevant in the search for anti-HCV
drugs. Hence, in order to target nucleosides which can be converted to
the active NTP selectively in the target tissue (i.e., the liver), our synthetic
strategy required the incorporation of this phosphate triester group at the
5ꢁ-position in addition to manipulation of the 3ꢁ-site. As a consequence, mild
and selective reactions were employed to differentiate the two positions and
prepare the desired derivatives directly from guanosine.
The identification of potential HCV inhibitors is restricted by the
limited number of in vitro assays available for this target. The most direct
path involves first testing for inhibition of RdRp by the NTP, followed by
confirmation by testing the nucleoside in the cellular replicon model of
HCV inhibition. It is possible for the NTP to be a good RdRp inhibitor
but its nucleoside to be inactive in the replicon model because of a lack

3ꢁ-Amino-3ꢁ-deoxyguanosine NMP HepDirect Prodrugs
971
of cellular penetration or the previously mentioned inefficient conversion
to NTP by the cellular kinases. Alternatively, undesired metabolism of the
nucleoside within the replicon model Huh-7 cell may further diminish
potential activity.[14] Hence application of the NMP-SATE prodrug strategy
was found useful for establishing promising nucleosides as active in this
important assay.[14] While HepDirect prodrugs have been validated as
clinical candidates where SATE prodrugs have not,[13b] the HepDirect
prodrugs cannot be tested in the replicon assay because Huh-7 cells lack
sufficient CYP3A4 to metabolize this prodrug to the NMP. Moreover, this
report only describes the preparation of HepDirect prodrugs. An assessment
of the activation of these HepDirect prodrugs to their NTP derivatives in
hepatocytes is also reported. It is envisioned that the chemistry would apply
equally well to the preparation of their respective SATE prodrugs making
them available for replicon testing.
CHEMISTRY
For guanosine, amino- and azido-ribose modified derivatives have been
prepared by one of the following methods: (1) detailed synthetic manipu-
lation of guanosine;[6a,b,8,9b,c] (2) glycosylation of an acylated guanine with
a previously elaborated ribose molecule;[6c,7a,9a] and (3) transglycosylation
of an acylated guanine with an available ribose-modified nucleoside.[7b]
For the current work, the first approach was taken and due to the re-
activity of the 5ꢁ-phosphate triester prodrug, that is, HepDirect prodrug
group, strong hydrolytic, hydrogenolytic, and nucleophilic reactions were
avoided. Hence, the azide group was used to introduce the primary amino
group. The reaction sequence begins with the previously reported N2-
N ,N-dibenzylformamidino-protected guanosine 2[15] (Scheme 1). This hy-
drophobic N2-protecting group served to render the extremely polar guano-
sine nucleoside and derivatives into more chromatographically manageable
forms as well as remove the C2-NH2 active hydrogens as a source of potential
side reactions during subsequent reactions. A recent report[9c] and our
own experience, have determined that because of purification difficulties
it is not practical to prepare the desired 2ꢁ,5ꢁ-bis-O-t-butyldimethylsilyl-
protected (O-TBS-protected) guanosine analogue from a similar N2-N ,N-
dimethylformamidino-protected derivative.
The protection of nucleoside 2ꢁ- and 5ꢁ-hydroxy groups with TBS groups,
although well precedented[16] is known to proceed with only marginal se-
lectivity for guanosine analogues.[16b,c] After standard reaction conditions,
the mixture of 2ꢁ,5ꢁ-bis-O-TBS product 3a and 3ꢁ,5ꢁ-bis-O-TBS-product 4a
obtained was separated using standard silica gel chromatography. Con-
sistent with literature precedent,[1i,16b,17] it was observed that 3a and 4a
could be equilibrated by simple mixing in methanol at room temperature.

972
B. C. Bookser et al.
SCHEME 1 a) HC(OMe)2, Bn2NH, ACN, 80◦C (70%, see ref.[13]); b) TBS-Cl, imidazole, DMF (60% 3a
after recycling).
Since this was a concern with regards to structural integrity in the next
reaction, the isomerization was investigated in various solvents with the
results presented in Table 1. In methanol, equilibration of 3a to 4a was
complete after 22 hours at 22◦C or 1 hour at 80◦C to provide a 3a/4a
mixture of 58:42 or 54:46, respectively (entries 2 and 3). Confirmation of
the equilibration was demonstrated by isomerizing pure 4a into a 52:48
3a/4a mixture (methanol, 1 hour, 80◦C, entry 11). Evaluation of other
solvents revealed that isomerization occurred in DMSO (1 hour, 80◦C),
acetonitrile (1 hour, 80◦C), pyridine (17 hours, 80◦C), and THF (17 hours,
80◦C), (entries 5–8). However, the isomer 3a was stable in DMSO (22 hours,
22◦C), toluene (17 hours, 80◦C), and dichloromethane 17 hours, 22◦C
(entries 4, 9, and 10) thus indicating these solvents are compatible with
our planned chemistry. In fact, all aprotic solvents tested at 22◦C did not
induce significant isomerization of 3a to 4a (not listed). The equilibration
was further demonstrated to be general for nucleosides since adenosine and
uridine derivatives 3b, 3c, and 4c also were converted to isomeric mixtures
in methanol (1 hour, 80◦C), (entries 12–14). The equilibration process
(methanol, 22 hours, 22◦C) was used to advantage by recycling unwanted
isomer 4a obtained during the preparation of 3a into a mixture of 3a and 4a.
Performing the equilibration/chromatography recycling procedure three
times resulted in a 60% overall yield of 3a from 2 on a practical research
scale (43 g 3a prepared).
Scheme 2 describes how a double inversion of the 3ꢁ-position was
applied to the synthesis of the 3ꢁ-amino-3ꢁ-deoxyribofuranosyl analogue 15.
Certain reactions within this approach, or variations thereof, have been
applied to functionalization of the 2ꢁ- and 3ꢁ-positions of analogues of
guanine,[6a,b,9b,18] 2,6-diaminopurine,[18a,19] and adenine[1i,5,9b,18b] based

3ꢁ-Amino-3ꢁ-deoxyguanosine NMP HepDirect Prodrugs
973
TABLE 1 Solvent and temperature effect on 2ꢁ- to 3ꢁ-O-TBS isomerizationa
2ꢁ,5ꢁ-bis-O-
3ꢁ,5ꢁ-bis-O-
TBS
TBS
Entry
Cmpd
Solvent
Time
Temp
(%Y)a
(%Y)a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
4a
3b
3c
4c
MeOH
MeOH
MeOH
DMSO
DMSO
CH3CN
Pyridine
THF
toluene
CH2Cl2
MeOH
MeOH
MeOH
MeOH
4 h
22 h
1 h
22 h
1 h
22 C
22◦C
80◦C
22◦C
80◦C
80◦C
80◦C
80◦C
80◦C
22◦C
80◦C
80◦C
80◦C
80◦C
3a (75)
3a (58)
3a (54)
3a (99)
3a (57)
3a (65)
3a (57)
3a (72)
3a (>99)
3a (>99)
3a (52)
3b (51)
3c (62)
3c (62)
4a (25)
4a (42)
4a (46)
4a (1)
4a (43)
4a (35)
4a (43)
4a (28)
4a (<1)
4a (<1)
4a (48)
4b (49)
4c (38)
4c (38)
1 h
17 h
17 h
17 h
17 h
1 h
1 h
1 h
1 h
aPercentage of yield calculated from the mixture by HPLC peak area with compound retention times
verified by comparison with authentic samples.
nucleosides. Preparation of triflate 5, inversion with cesium acetate to 6
and methanolysis to the xylofuranosyl derivative 7 all proceeded satisfac-
torily. However, formation of triflate 8 was sensitive to time, temperature
and reagents. This is in contrast to the preparation of close analogues:
ribofuranosyl triflate 5 and a reported xyloadenosine 3ꢁ-O-triflate.[18b] An
optimal yield of 31% of 8 was realized only when trifluoromethanesulfonic
anhydride was used and the reaction was processed with starting material re-
maining in the reaction mixture (29% 7 recovered). The low yield is perhaps
not surprising since previous reports have described a low yield of triflate
from an arabinoguanosine analogue,[18c] as well as the formation of guanine
nucleoside O6-triflate as a product depending on reaction conditions.[18a,b]
Alternate approaches have been applied for the inversion of the 3ꢁ-position
of xyloguanosine analogues,[6a,b,9b] but this method was satisfactory for our
purposes.[20] Displacement of the triflate in 8 with lithium azide to form
9 followed by selective 5ꢁ-O-desilylation provided 10. Deprotonation of the
5ꢁ-hydroxy group with t-BuMgCl followed by phosphorylation with reagent
11[13a,13f,21] provided the HepDirect prodrug 12 in 93% yield. Sequential

974
B. C. Bookser et al.
SCHEME 2 Synthesis of ribofuranosyl analogue. a) TfCl, DMAP, DCM (80%); b) AcOH, Cs2CO3, DMF
(67%); c) MeONa, MeOH (84%); d) Tf2O, DMAP, DCM (31%, and 29% recovered 7); e) LiN3, DMF
(93%); f) 70% TFA in water (68%); g) t-BuMgCl, THF; 11 (93%); h) Et4NF, DMF (85%); i) 50% TFA in
water (107%); j) PPh3, NH3, MeOH, pyridine (95%).
deprotection of the TBS and amidine protecting groups provided azide
14 in high yield. Application of the Staudinger reduction[22] to the azide
provided the target amine 15 in 95% yield. Overall, from guanosine (1), the
preparation of ribo-analogue 15 required 12 steps, with an average yield of
77% for each step.
Preparation of the 3ꢁ-amino-3ꢁ-deoxyxylofuranosyl analogue 21 followed
a similar route and is described in Scheme 3. Displacement of the triflate
functionality in 5 with lithium azide produced xylo-analogue 16 which
was selectively deprotected to 17. Synthesis of the phosphate prodrug 18
followed by simultaneous deprotection of the amidine and TBS groups
with 50% trifluoroacetic acid in water provided the xylo-azide 19. Unlike
compound 14, the Staudinger reduction of azide 19 was best performed
stepwise with isolation of the intermediate triphenylphosphoranylidene 20
prior to ammoniolysis to the target amine 21. Overall, from guanosine (1),

3ꢁ-Amino-3ꢁ-deoxyguanosine NMP HepDirect Prodrugs
975
SCHEME 3 Synthesis of xylofuranosyl analogue. a) LiN3, DMF (100%); b) 70% TFA in water (75%); c)
t-BuMgCl, THF; 11 (65%); d) 50% TFA in water (77%); e) PPh3, pyridine (72%); f) NH4OH, MeOH
(87%).
the preparation of xylo-analogue 21 required 9 steps, with an average yield
of 76% for each step.
These HepDirect prodrugs were evaluated for activation to derivative
NTPs in the reported rat hepatocyte model.[23] The ribo-azide and -amine
14 and 15 at 500 µM produced 64.8 and 7.5 nmol/g NTP, respectively,
after a 2 hours incubation. The xylo-azide and -amine 19 and 21 at 250
µM produced 58.1 and 36.1 nmol/g NTP, respectively, after a 2 hours
incubation. Since the parent compounds 14, 15, 19, and 21 are activated
in rat hepatocytes to their respective NTPs, it is likely their planned amine
derivatives will also activate.
These routes provide both the ribo- and xylo-epimers of 3ꢁ-amino-3ꢁ-
deoxyguanosine as well as corresponding azido-analogues. Liver targeting
of their potentially bioactive NTPs is possible since they may be derived
from in vivo metabolic transformations of the installed HepDirect prodrug
of the 5ꢁ-phosphates. Further extensions of this methodology are envisioned
which involve the generation of additional analogues through standard

976
B. C. Bookser et al.
amine derivatization reactions, that have been reported previously for
other nucleoside systems.[1h-i,4a,c,24] This synthesis demonstrated that the
traditionally difficult guanosine nucleoside system can be manipulated
selectively in the 2ꢁ-, 3ꢁ-, and 5ꢁ-O- as well as the N2-positions with appropri-
ate protection/deprotection strategies to produce 3ꢁ-deoxy-GMP prodrugs
ready for 3ꢁ-amino derivatization. To study this series in the replicon cellular
model of HCV inhibition, another GMP prodrug such as SATE[14] would
be necessary and this synthesis should readily extend to the preparation of
those analogues.
EXPERIMENTAL
Glassware for moisture-sensitive reactions was flame-dried and cooled
to room temperature in a desiccator. All reactions were carried out under
an atmosphere of nitrogen. For the reactions, room temperature was 22◦C.
´
˚
Anhydrous solvents were purchased and stored over 4A molecular sieves.
Flash chromatography was performed on 230–400 mesh EM Science silica
1
gel 60. H NMR were obtained in DMSO-d6 at 200 MHz and spectra
were recorded in units δ with CD2HS(O)CD3 (δ 2.504) as the reference
line internal standard. Certain OH and NH proton chemical shifts were
confirmed by D2O exchange as indicated. Analytical HPLC was conducted
with three serially connected Chromolith SpeedRODs RP-18e, 100 × 4.6
mm. Solvent A = HPLC grade acetonitrile; Solvent B = 20 mM ammonium
phosphate buffer (pH 6.1, 0.018 M NH4H2PO4/0.002 M (NH4)2HPO4) with
5% acetonitrile. The UV detector was set to 255 nm and the flow rate was
4 mL/minutes. Gradient program: minutes (%B), 0 (100), 10 (60), 10.1
(100), 12 (100). Mass spectral data were obtained by an LCMS operating in
the positive ion mode. C, H, N microanalyses were performed by Robertson
Microlit Laboratories, Inc. (Madison, NJ, USA).
9-(2,5-Bis-O-tert-butyldimethylsilyl-β-D-ribofuranosyl)-2-N-(N,N-
dibenzylformamidino)guanine
(3a).
A
mixture
of
2-N -(N,N-
dibenzylformamidino)-9-(β-d-ribofuranosyl)guanine[15] (2) (49.08 g,
100.1 mmol), t-butylchlorodimethylsilane (45.25 g, 300.2 mmol), and
imidazole (23.85 g, 350.2 mmol) in 800 mL DMF was stirred at room
temperature for 6 hours. Then it was diluted with 600 mL of EtOAc and
extracted with 500 mL water, 500 mL brine, dried (MgSO4) and evaporated
to provide 120 g of residue. This was subjected to chromatography through
a 1200 g SiO2 column eluting with 3:2:1 EtOAc/hexanes/CH2Cl2 to provide
1
the 7.73 g of the title compound (3a) as an amorphous solid: H NMR
(200 MHz, DMSO-d6) δ −0.13 (s, 3H), −0.04 (s, 3H), 0.08 (s, 3H), 0.10 (s,
3H), 0.76 (s, 9H), 0.90 (s, 9H), 3.8–4.2 (m, 4H), 4.41 (t, 1H, J = 6 Hz),
4.58–4.63 (m, 5H), 5.16 (d, 1H, J = 6 Hz, 3ꢁ-OH, D2O exchanged), 5.90
(d, 1H, J = 5 Hz), 7.26–7.41 (m, 10H), 8.04 (s, 1H), 8.95 (s, 1H), 11.60

3ꢁ-Amino-3ꢁ-deoxyguanosine NMP HepDirect Prodrugs
977
(s, 1H, NH, D2O exchanged). Anal. Calcd for C37H54N6O5Si2 (719.1):%C,
61.81;%H, 7.57;%N, 11.69. Found:%C, 62.18;%H, 7.88;%N, 11.63. Further
elution with 10:3:3 EtOAc/hexanes/CH2Cl2 eluted a mixture of the title
compound (3a) and 9-(3,5-bis-O-tert-butyldimethylsilyl-β-d-ribofuranosyl)-
2-N -(N ,N -dibenzylformamidino)guanine (4a). Finally elution with 12:1:3
EtOAc/hexanes/CH2Cl2 provided 5.0 g compound 4a as an amorphous
1
solid: H NMR (200 MHz, DMSO-d6) δ 0.02 (s, 3H), 0.03 (s, 3H), 0.12 (s,
6H), 0.86 (s, 9H), 0.89 (s, 9H), 3.3–3.9 (m, 4H), 4.27 (m, 1H), 4.5–4.7
(m, 5H), 5.46 (d, 1H, J = 6 Hz, 2ꢁ-OH, D2O exchanged), 5.83 (d, 1H, J
= 6 Hz), 7.3–7.4 (m, 10H), 8.06 (s, 1H), 8.96 (s, 1H), 11.60 (s, 1H, NH,
D2O exchanged). Anal. Calcd for C37H54N6O5Si2•0.5H2O (728.1):%C,
61.04;%H, 7.61;%N, 11.54. Found:%C, 61.05;%H, 7.89;%N, 11.45. The
impure fractions were combined and rechromatographed in the same
manner to provide an additional 8.12 g of title compound (3a). Impure
fractions and isolated isomer 4a were combined and stirred in methanol for
16 hours at room temperature. This mixture was evaporated and subjected
to chromatographic purification as before to provide an additional 7.93 g of
title compound (3a). The remaining 3a/4a mixture isolated was subjected
to this isomerization/chromatographic purification process 2 times further
to provide an additional 19.36 g of title compound. Total yield of the title
compound (3a) was 43.14 g (60%).
Studies on the Isomerization of 9-(2,5-Bis-O-tert-butyldimethylsilyl-β-
D-ribofuranosyl)-2-N-(N,N-dibenzylformamidino)guanine (3a) into 9-(3,
5-Bis-O-tert-butyldimethylsilyl-β-D-ribofuranosyl)-2-N-(N,N-dibenzylforma-
midino)guanine (4a). A solution of compound 3a (20 mg, 0.028 mmol) in
0.4 mL of the specified solvent was stirred in a sealed vial at the specified
temperature. After the specified time, 0.020 mL was removed and diluted
with 1.5 mL of methanol and evaluated by HPLC to determine the isomeric
ratio. The same method was used to study the equilibration of 4a to 3a; 3b
to 4b; 3c to 4c; and 4c to 3c.
9-(2,5-Bis-O-tert-butyldimethylsilyl-3-O-trifluoromethylsulfonyl-β-D-
ribofuranosyl)-2-N-(N,N-dibenzylformamidino)guanine (5). To a mixture
of compound 3a (25 g, 34.8 mmol) and DMAP (12.7 g, 104.3 mmol)
in 350 mL CH2Cl2 at 0◦C was added slowly over a period of 30 minutes
trifluoromethanesulfonylchloride (4.1 mL, 38.3 mmol) and stirring
continued at 0◦C for 30 minutes. Then the mixture was partitioned
with water, the organic layer separated, washed with water, brine, dried
(MgSO4) and evaporated. The residue was dissolved in 60 mL of EtOAc,
diluted with 20 mL of CH2Cl2 and 20 mL of hexanes and subjected to
chromatography on a column containing 400 g SiO2 and eluted with 10:3:3
EtOAc/hexanes/CH2Cl2 to provide 23.7 g (80%) of the title compound
1
(5) as an amorphous white solid: H NMR (200 MHz, DMSO-d6) δ −0.41
(s, 3H), −0.02 (s, 3H), 0.03 (s, 3H), 0.10 (s, 3H), 0.73 (s, 9H), 0.84 (s, 9H),
3.8–4.0 (m, 2H), 4.36 (t, 1H, J = 6 Hz), 4.5–4.7 (m, 4H), 5.3–5.4 (m, 1H),

978
B. C. Bookser et al.
5.51 (d, 1H, J = 5 Hz), 5.94 (d, 1H, J = 8 Hz), 7.27–7.41 (m, 10H), 8.08 (s,
1H), 8.96 (s, 1H), 11.68 (s, 1H).
9-(3-O-Acetyl-2,5-bis-O-tert-butyldimethylsilyl-D-xylofuranosyl)-2-N-(N,N-
dibenzylformamidino)guanine (6). A mixture of acetic acid (3.2 mL, 56.4
mmol) and Cs2CO3 (18.4 g, 56.4 mmol) in 140 mL DMF was heated
to 80◦C for 1 hour, cooled to 0◦C and then compound 5 (24.0 g, 28.2
mmol) as a solution in 140 mL DMF cooled to 0◦C was added via cannula
needle. The mixture was stirred at 0◦C for 1 hour and then diluted with
EtOAc and washed with water, brine, dried (MgSO4) and evaporated. The
residue was subjected to chromatography on SiO2 and eluted with 3:2:1
EtOAc/hexanes/CH2Cl2 followed by 10:3:3 to provide 14.38 g (67%) of
1
the title compound (6) as an amorphous white solid: H NMR (200 MHz,
DMSO-d6) δ −0.12 (s, 3H), 0.04 (s, 3H), 0.07 (s, 6H), 0.77 (s, 9H), 0.87 (s,
9H), 2.01 (s, 3H), 3.83 (dd, 1H, J = 9, 5 Hz), 3.92 (dd, 1H, J = 9, 5 Hz),
4.37 (dd, 1H, J = 6, 2 Hz), 4.56 (s, 2H), 4.6–4.7 (m, 3H), 5.21 (dd, 1H, J
= 5, 2 Hz), 5.93 (d, 1H, J = 4 Hz), 7.2–7.4 (m, 10H), 7.98 (s, 1H), 8.98 (s,
1H), 11.64 (s, 1H). Anal. Calcd for C39H56N6O6Si2 (761.1):%C, 61.55;%H,
7.42;%N, 11.04. Found:%C, 61.32;%H, 7.74;%N, 11.20.
9-(2,5-Bis-O-tert-butyldimethylsilyl-β-D-xylofuranosyl)-2-N-(N,N-
dibenzylformamidino)guanine (7). A 0.5 M solution of sodium methoxide
in methanol was prepared by dissolving sodium metal (1.20 g, 50 mmol) in
100 mL of methanol. To a solution of compound 6 (14.38 g, 18.9 mmol) in
100 mL methanol cooled to 0◦C was added 75 mL of the 0.5 M solution of
sodium methoxide (37.5 mmol) and the resulting mixture stirred at 0◦C
for 30 minutes and then poured onto 400 mL of 0.2 N aqueous NH4Cl.
The resulting white solid was collected by filtration, dissolved in 400 mL
EtOAc, washed with brine, dried (MgSO4), and evaporated to provide 11.44
1
g (84%) of the title compound (7) as an amorphous white solid: H NMR
(200 MHz, DMSO-d6) δ −0.02 (s, 3H), 0.02 (s, 3H), 0.04 (s, 6H), 0.80 (s,
9H), 0.87 (s, 9H), 3.8–4.3 (m, 4H), 4.55 (s, 2H), 4.62 (s, 2H), 5.71 (d, 1H, J
= 4 Hz, 3ꢁ-OH, D2O exchanged), 5.87 (d, 1H, J = 2 Hz), 7.2–7.4 (m, 10H),
7.96 (s, 1H), 8.99 (s, 1H), 11.61 (s, 1H, NH, D2O exchanged). Anal. Calcd
for C37H54N6O5Si2 (719.1):%C, 61.81;%H, 7.57;%N, 11.69. Found:%C,
61.41;%H, 7.98;%N, 11.63.
9-(2,5-Bis-O-tert-butyldimethylsilyl-3-O-trifluoromethylsulfonyl-β-D-
xylofuranosyl)-2-N-(N,N-dibenzylformamidino)guanine (8). Trifluorome-
thanesulfonic anhydride (1.30 mL, 7.65 mmol), was added to a solution
of compound 7 (5.00 g, 6.95 mmol) and DMAP (2.55 g, 20.9 mmol) in 70
mL CH2Cl2 at −40◦C and then the solution was stirred at 0◦C for 1 hour.
It was diluted with CH2Cl2 and washed with aqueous 2.5% acetic acid,
water, aqueous saturated NaHCO3, brine, dried (MgSO4), and evaporated
to provide 6 g of crude product. This was adsorbed onto 18 g of SiO2 from
CH2Cl2 and subjected to chromatography through a column of 200 g of
SiO2 eluting with 3:2:1 EtOAc/hexane/CH2Cl2 to provide 1.7 g (31%)

3ꢁ-Amino-3ꢁ-deoxyguanosine NMP HepDirect Prodrugs
979
1
of the title compound (8) as an amorphous solid: H NMR (200 MHz,
DMSO-d6) δ -0.22 (s, 3H), -0.13 (s, 3H), 0.11 (s, 6H), 0.73 (s, 9H), 0.91 (s,
9H), 3.8–4.0 (m, 2H), 4.32 (m, 1H), 4.4–4.7 (m, 4H), 4.86 (dd, 2H, J = 8,
4 Hz), 5.92 (d, 1H, J = 8 Hz), 7.2–7.4 (m, 10H), 7.93 (s, 1H), 8.99 (s, 1H),
11.70 (s, 1H). Anal. Calcd for C38H53F3N6O7SSi2 (851.1):%C, 53.63;%H,
6.28;%N, 9.87. Found:%C, 53.27;%H, 6.51;%N, 9.95. Continued elution
with 12:1:3 EtOAc/hexane/CH2Cl2 provided 1.45 g (29%) of recovered
starting material (7).
9-(3-Azido-2,5-bis-O-tert-butyldimethylsilyl-3-deoxy-β-D-ribofuranosyl)-
2-N-(N,N-dibenzylformamidino)guanine (9). A mixture of compound 8
(4.40 g, 5.17 mmol) and LiN3 (791 mg, 15.5 mmol) in 52 mL of DMF was
stirred at 0◦C for 3 hours and then diluted with EtOAc and washed with
water, brine, dried (MgSO4), and evaporated. The residue was subjected to
chromatography eluting with 3:2:1 EtOAc/hexane/CH2Cl2 to provide 3.56
1
g (93%) of the title compound (9) as an amorphous solid: H NMR (200
MHz, DMSO-d6) δ -0.20 (s, 3H), 0.01 (s, 3H), 0.08 (s, 6H), 0.78 (s, 9H),
0.89 (s, 9H), 3.8–4.1 (m, 3H), 4.32 (t, 1H, J = 5 Hz), 4.59 (s, 2H), 4.63
(s, 2H), 4.87 (t, 1H, J = 5 Hz), 5.89 (d, 1H, J = 5 Hz), 7.2–7.4 (m, 10H),
8.02 (s, 1H), 8.97 (s, 1H), 11.61 (s, 1H). Anal. Calcd for C37H53N9O4Si2
(744.1):%C, 59.73;%H, 7.18;%N, 16.94. Found:%C, 59.35;%H, 7.46;%N,
16.48.
9-(3-Azido-2-O-tert-butyldimethylsilyl-3-deoxy-β-D-ribofuranosyl)-2-N-
(N,N-dibenzylformamidino)guanine (10). A solution of compound 9 (2.65
g, 3.56 mmol) and 15 mL of 70% TFA in water was stirred for 45 minutes at
room temperatur. Then it was poured onto a mixture of 250 mL saturated
aqueous NaHCO3 and 250 mL EtOAc and stirred vigorously for 15 minutes
at room temperature. The organic layer was separated and washed with
saturated aqueous NaHCO3, brine, dried (MgSO4) and evaporated. The
residue was subjected to chromatography eluting with 10:1 EtOAc/hexane
and then EtOAc to provide 1.53 g (68%) of the title compound (10) as an
1
amorphous solid: H NMR (200 MHz, DMSO-d6) δ -0.22 (s, 3H), -0.03 (s,
3H), 0.77 (s, 9H), 3.5–3.8 (m, 2H), 3.9–4.0 (m, 1H), 4.38 (dd, 1H, J = 5,
4 Hz), 4.57 (s, 2H), 4.62 (s, 2H), 5.04 (t, 1H, J = 5 Hz), 5.35 (t, 1H, J = 6
Hz, 5ꢁ-OH, D2O exchanged), 5.86 (d, 1H, J = 6 Hz), 7.2–7.4 (m, 10H), 8.13
(s, 1H), 8.94 (s, 1H), 11.64 (s, 1H, NH, D2O exchanged). Anal. Calcd for
C31H39N9O4Si·H2O (647.8):%C, 57.48;%H, 6.38;%N, 19.46. Found:%C,
58.10;%H, 6.54;%N, 18.83.
9-(3-Azido-2-O-tert-butyldimethylsilyl-cis-5-O-(4-(3-chlorophenyl)-1,3-
dioxa-2-oxophosphorinan-2-yl)-3-deoxy-β-D-ribofuranosyl)-2-N-(N,N-
dibenzylformamidino)guanine (12). To a solution of compound 10 (1.65 g,
2.62 mmol) in 52 mL of THF at −40◦C was added 6.3 mL of a 1 M solution
of t-BuMgCl in THF (6.29 mmol) and the mixture stirred for 15 minutes
at −40◦C. Then trans-4-(3-chlorophenyl)-2-(4-nitrophenoxy)-2-oxo-1,3,2-
dioxaphosphorinane (11)[13a] (1.45 g, 3.93 mmol) was added as a solid

980
B. C. Bookser et al.
and the mixture stirred at 0◦C for 1 hour. Then it was stirred at room
temperature for 16 hours, diluted with 1 M aqueous NH4Cl and extracted
into EtOAc. The organic layer was washed with brine, dried (MgSO4), and
evaporated. The residue was dissolved in EtOAc and loaded onto a column
of SiO2 and eluted with EtOAc and then 100:1 EtOAc/methanol to provide
1
2.1 g (93%) of the title compound (12) as an amorphous solid: H NMR
(200 MHz, DMSO-d6) δ −0.23 and −0.21 (s each, 3H),21 −0.03 and −0.01
(s each, 3H), 0.76 and 0.77 (s each, 9H), 2.0–2.2 (m, 2H), 4.1–4.2 (m, 1H),
4.2–4.6 (m, 7H), 5.11 (q, 1H, J = 6 Hz), 5.6–5.8 (m, 1H), 5.91 (d, 1H, J =
6 Hz), 7.2–7.5 (m, 14H), 8.04 (s, 1H), 8.96 (s, 1H), 11.62 and 11.63 (s each,
1H). Anal. Calcd for C40H47ClN9O7PSi (860.4):%C, 55.84;%H, 5.51;%N,
14.65. Found:%C, 55.69;%H, 5.62;%N, 14.32.
9-(3-Azido-cis-5-O-(4-(3-chlorophenyl)-1,3-dioxa-2-oxophosphorinan-2-
yl)-3-deoxy-β-D-ribofuranosyl)-2-N-(N,N-dibenzylformamidino)guanine
(13).
A
mixture of compound 12 (1.00 g, 1.16 mmol) and
tetraethylammonium fluoride in 12 mL of DMF was stirred for 2 hours
at room temperature. Then it was diluted with EtOAc and washed with
water, brine, dried (MgSO4), and evaporated. The residue was adsorbed
to SiO2 from a solution in CH2Cl2/methanol and then subjected to
chromatography on SiO2 eluting with 20:1 CH2Cl2/methanol followed by
10:1 to provide 733 mg (85%) of the title compound (13) as an amorphous
1
solid: H NMR (200 MHz, DMSO-d6) δ 2.0–2.2 (m, 2H), 4.12 (q, 1H, J =
4 Hz), 4.2–4.7 (m, 6H), 4.9–5.1 (m, 1H), 5.6–5.8 (m, 1H), 5.89 (d, 1H, J
= 6 Hz), 6.39 (br s, 1H, 2ꢁ-OH, D2O exchanged), 7.2–7.5 (m, 14H), 8.05
(s, 1H), 9.01 (s, 1H), 11.60 (s, 1H, NH, D2O exchanged). Anal. Calcd
for C34H33ClN9O7P (746.1):%C, 54.73;%H, 4.46;%N, 16.90. Found:%C,
54.48;%H, 4.67;%N, 16.93.
9-(3-Azido-cis-5-O-(4-(3-chlorophenyl)-1,3-dioxa-2-oxophosphorinan-2-
yl)-3-deoxy-β-D-ribofuranosyl)guanine (14). A mixture of compound 13
(733 mg, 0.98 mmol) in 10 mL of 50% aqueous TFA was stirred at room
temperature for 16 hours and then carefully added in a dropwise manner
to 75 mL of saturated aqueous NaHCO3 with rapid stirring. The resulting
white solid was collected and dried at (room temperature) rt/0.1 mm
vacuum to provide 566 mg (107%) of the title compound (14) as a white
amorphous solid: 1H NMR (200 MHz, DMSO-d6) δ 2.1–2.3 (m, 2H), 4.0–4.1
(m, 1H), 4.2–4.6 (m, 4H), 4.8–4.9 (m, 1H), 5.6–5.7 (m, 1H), 5.74 (d, 1H,
J = 5 Hz), 6.33 (d, 1H, J = 5 Hz, 2ꢁ-OH, D2O exchanged), 6.55 (br s, 2H,
NH2, D2O exchanged), 7.2–7.5 (m, 4H), 7.86 and 7.87 (s each, 1H), 10.70
(s, 1H, NH, D2O exchanged); MS calcd for C19H20ClN8O7P: M+1 = 539.1;
M+1 found: 539.7.
9-(3-Amino-cis-5-O-(4-(3-chlorophenyl)-1,3-dioxa-2-oxophosphorinan-
2-yl)-3-deoxy-β-D-ribofuranosyl)guanine (15). A mixture of compound 14
(303 mg, 0.56 mmol) triphenylphosphine (438 mg, 1.67 mmol), 2.5 mL of
6 M NH3 in methanol and 2.5 mL of pyridine was stirred at 50◦C for 1.5

3ꢁ-Amino-3ꢁ-deoxyguanosine NMP HepDirect Prodrugs
981
hours. The solvent was evaporated and the residue suspended in EtOAc.
An equal volume of ether was added and the resulting white solid was
collected by filtration and dried at rt/0.1 mm vacuum to provide 265 mg
1
(95%) of the title compound (15) as an amorphous white solid: H NMR
(200 MHz, DMSO-d6) δ 2.0–2.2 (m, 2H), 3.4–3.5 (m, 1H), 3.8–3.9 (m,
1H), 4.1–4.6 (m, 5H), 5.6–5.7 (m, 1H), 5.77 (d, 1H, J = 1 Hz), 6.48 (br s,
2H, NH2, D2O exchanged), 7.2–7.5 (m, 4H), 7.79 and 7.80 (s each, 1H);
MS calcd for C19H22ClN6O7P: M+1 = 513.1; M+1 found: 513.2. Anal.
Calcd for C19H22ClN6O7P•1.5H2O (539.9):%C, 42.27;%H, 4.67;%N, 15.57.
Found:%C, 41.90;%H, 4.49;%N, 16.01.
9-(3-Azido-2,5-bis-O-tert-butyldimethylsilyl-3-deoxy-β-D-xylofuranosyl)-2-
N-(N,N-dibenzylformamidino)guanine (16). A mixture of 5 (6.36 g, 7.47
mmol), lithium azide (1.90 g, 37.4 mmol), and DMF (100 mL) was stirred
at room temperature for 3 hours. The reaction mixture was partitioned
between ethyl acetate (300 mL) and saturated NaHCO3 (150 mL). The
layers were separated and the organic layer was washed with saturated
NaHCO3, dried (MgSO4), and evaporated. The residue was subjected to
chromatography (SiO2, 10:3:3 EtOAc/Hexane/CH2Cl2) which provided
1
5.78 g (100%) of the title compound (16) as a light yellow foam: H NMR
(200 MHz, DMSO-d6) δ -0.30 (s, 3H), −0.05 (s, 3H), 0.08 (d, 6H, J = 3 Hz),
0.75 (s, 9H), 0.89 (s, 9H), 3.8–3.9 (m, 2H), 4.3–4.4 (m, 1H), 4.4–4.8 (m,
7H), 5.84 (d, 1H, J = 6 Hz), 7.2–7.5 (m, 10H), 8.02 (s, 1H), 8.98 (s, 1H),
11.63 (s, 1H); MS calcd for C37H53N9O4Si2: M+1 = 744.4. M+1 found:
745.1. Anal. calcd. for C37H53N9O4Si2 (744.06): C, 59.73; H, 7.18; N, 16.94.
Found: C, 59.83; H, 7.19; N, 16.38.
9-(3-Azido-2-O-tert-butyldimethylsilyl-3-deoxy-β-D-xylofuranosyl)-2-N-
(N,N-dibenzylformamidino)guanine (17). A solution of 16 (4.99 g, 6.70
mmol) in 75 mL of 70% trifluoroacetic acid/water was stirred at room
temperature for 10 minutes. The reaction mixture was poured into a
beaker containing saturated NaHCO3 (300 mL) and ethyl acetate (300 mL)
with stirring. The layers were separated and the organic layer was washed
with saturated NaHCO3 (3×300 mL), dried (MgSO4), and evaporated.
The residue was subjected to chromatography (SiO2, 20:1 EtOAc/MeOH)
which provided 3.16 g (75%) of the title compound (17) as an amorphous
1
white solid: H NMR (200 MHz, DMSO-d6) δ -0.26 (s, 3H), -0.01 (s, 3H),
0.77 (s, 9H), 3.6–3.8 (m, 2H), 4.2–4.3 (m, 1H), 4.4–4.5 (m, 1H), 4.56 (s,
2H), 4.62 (s, 2H), 4.8–4.9 (m, 1H), 5.19 (m, 2H), 5.83 (d, 1H, J = 6 Hz),
7.2–7.5 (m, 10H), 8.10 (s, 1H), 8.97 (s, 1H), 11.66 (s, 1H); MS calcd for
C31H39N9O4Si: M+1 = 630.3. M+1 found: 630.3.
9-(3-Azido-2-O-tert-butyldimethylsilyl-cis-5-O-(4-(3-chlorophenyl)-1,3-
dioxa-2-oxophosphorinan-2-yl)-3-deoxy-β-D-xylofuranosyl)-2-N-(N,N-
dibenzylformamidino)guanine (18). A solution of 17 (1.23 g, 1.95 mmol) in
THF (100 mL) was cooled to −40◦C and a 1 M solution of t-butylmagnesium
chloride (in THF) (4.68 mL, 4.68 mmol) was added dropwise over 10

982
B. C. Bookser et al.
minutes. The reaction mixture was stirred for an additional 15 minutes.
at −40◦C. Then trans-4-(3-chlorophenyl)-2-(4-nitrophenoxy)-2-oxo-1,3,2-
dioxaphosphorinane (11) (1.08 g, 2.93 mmol) was added as a solid and
the mixture stirred at −40◦C for 15 minutes. The reaction mixture was
warmed to room temperature and stirred for 16 hours, diluted with EtOAc
(200 mL), washed with 2 N NH4Cl (200 mL), saturated NaHCO3 (100
mL), brine (100 mL), dried (MgSO4), and evaporated. The residue was
subjected to chromatography (SiO2, gradient of 2–5% MeOH/CH2Cl2)
which provided 1.09 g (65%) of the title compound (18) as an amorphous
light yellow solid: 1H NMR (200 MHz, DMSO-d6) δ −0.26 (s, 3H), −0.02 (s,
3H), 0.75 (s, 9H), 2.1–2.3 (m, 2H), 3.3–3.5 (m, 2H), 4.3–4.5 (m, 2H), 4.57
(s, 2H), 4.5–4.6 (m, 3H), 4.8–5.0 (m, 1H), 4.6–4.7 (m, 1H), 5.89 (d, 1H, J
= 6 Hz), 7.2–7.5 (m, 14H), 8.06 (s, 1H), 8.97 (s, 1H), 11.67 (s, 1H).
9-(3-Azido-cis-5-O-(4-(3-chlorophenyl)-1,3-dioxa-2-oxophosphorinan-2-
yl)-3-deoxy-β-D-xylofuranosyl)guanine (19). A solution of 18 (763 mg, 0.877
mmol) in a 1:1 mixture of trifluoroacetic acid/water (5 mL) was stirred at
room temperature for 16 hours. The reaction mixture was concentrated
and azeotroped with MeOH and acetonitrile. The residue was adsorbed
onto SiO2 and subjected to chromatography (SiO2, gradient of 5–10%
MeOH/CH2Cl2) which provided 370 mg (77%) of the title compound (19)
as an amorphous white solid.: 1H NMR (200 MHz, DMSO-d6) δ 2.1–2.3 (m,
2H), 3.4–3.5 (m, 2H), 4.31 (d, 2H, J = 6 Hz), 4.4–4.6 (m, 2H), 4.6–4.7
(m, 1H), 5.67 (d, 1H, J = 6 Hz), 5.6–5.7 (m, 1H), 6.34 (d, 1H, J = 6 Hz),
6.56 (br s, 2H), 7.3–7.5 (m, 4H), 7.87 (s, 1H), 10.72 (s, 1H); MS calcd for
C19H20ClN8O7P M+1 = 539.1. M+1 found: 539.0.
9-(cis-5-O-(4-(3-Chlorophenyl)-1,3-dioxa-2-oxophosphorinan-2-yl)-3-
deoxy-3-(triphenylphosphoranylidene)amino-β-D-xylofuranosyl)guanine
(20). To a solution of 19 (458 mg, 0.850 mmol) in pyridine (20 mL)
was added triphenylphosphine (668 mg, 2.55 mmol) and the solution
was stirred at room temperature for 1 hour. The reaction mixture was
concentrated, adsorbed onto SiO2 and subjected to chromatography (SiO2,
3:10:0.05 MeOH/CH2Cl2/conc. NH4OH) to provide 470 mg (72%) of the
1
title compound (20) as an amorphous white solid: H NMR (200 MHz,
DMSO-d6) δ 2.0–2.2 (m, 2H), 3.7–3.9 (m, 2H), 4.2–4.3 (m, 2H), 4.3–4.4 (m,
1H), 4.4–4.6 (m, 1H), 4.8–5.0 (m, 1H), 5.55 (d, 1H, J = 8 Hz), 5.6–5.8 (m,
1H), 6.2–6.3 (m, 1H), 6.45 (br s, 2H), 7.2–7.5 (m, 4H), 7.6–8.0 (m, 16H),
10.80 (s, 1H); MS calcd for C37H35ClN6O7P2: M+1 = 773.2. M+1 found:
773.5.
9-(3-Amino-cis-5-O-(4-(3-chlorophenyl)-1,3-dioxa-2-oxophosphorinan-2-
yl)-β-D-xylofuranosyl)guanine (21). A solution of 20 (335 mg, 0.443 mmol)
in a 1:1 mixture of concentrated NH4OH in MeOH (100 mL) was stirred
at room temperature for 8 hours. The reaction mixture was concentrated,
adsorbed onto SiO2, and subjected to chromatography (SiO2, 3:10:0.05
MeOH/CH2Cl2/conc. NH4OH) to provide 194 mg (87%) of the title

3ꢁ-Amino-3ꢁ-deoxyguanosine NMP HepDirect Prodrugs
983
compound (21) as a amorphous white solid: 1H NMR (200 MHz, DMSO-d6)
δ 2.1–2.3 (m, 2H), 3.4–3.5 (m, 2H), 4.1–4.2 (m, 2H), 4.2–4.4 (m, 3H),
5.61 (d, 1H, J = 5 Hz), 5.69 (d, 1H, J = 12 Hz), 5.80 (d, 1H, J = 5 Hz),
6.49 (br s, 2H), 7.3–7.6 (m, 4H), 7.98 (s, 1H), 10.66 (br s, 1H); MS calcd
for C19H22ClN6O7P: M+1 = 513.1; M+1 found: 513.3. Anal. Calcd. for
C19H22ClN6O7P•0.25H2O (517.4): C, 44.38; H, 4.38; N, 16.24. Found: C,
44.09; H, 5.13; N, 15.38.
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