Expedient total synthesis of triciribine and its prodrugs

Article abstract of DOI:10.1080/00397911.2010.524342

Triciribine (TCN, 1) and its monophosphate (TCNP, 2) are ricyclic nucleotide derivatives that have potential antineoplastic activity. Triciribine inhibits the phosphorylation, activation, and signaling of Akt-1, -2, and -3, which may result in the inhibition of Akt-expressing tumor cell proliferation. Both TCN and TCNP have very low bioavailability, and the development of both drugs as intravenous (IV) treatments was halted because of the toxicity induced by the high doses needed for their use as general cytotoxic agents. This publication describes an expedient and straightforward total synthesis of amino acid prodrugs (3, 4) of TCN and TCNP. In our study, both the prodrugs significant improved the plasma exposure of the parent drugs and the prodrugs.Copyright TSRL Inc.

Full text of DOI:10.1080/00397911.2010.524342

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Expedient Total Synthesis of Triciribine
and Its Prodrugs
Wei Shen a , Jae-Seung Kim a & John Hilfinger a
a TSRL Inc. , Ann Arbor , Michigan , USA
Accepted author version posted online: 26 Jul 2011.Published
online: 06 Oct 2011.
To cite this article: Wei Shen , Jae-Seung Kim & John Hilfinger (2012) Expedient Total Synthesis
of Triciribine and Its Prodrugs, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 42:3, 358-374, DOI: 10.1080/00397911.2010.524342
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Synthetic Communications1, 42: 358–374, 2012
Copyright # TSRL Inc.
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2010.524342
EXPEDIENT TOTAL SYNTHESIS OF TRICIRIBINE
AND ITS PRODRUGS
Wei Shen, Jae-Seung Kim, and John Hilfinger
TSRL Inc., Ann Arbor, Michigan, USA
GRAPHICAL ABSTRACT
Abstract Triciribine (TCN, 1) and its monophosphate (TCNP, 2) are tricyclic nucleotide
derivatives that have potential antineoplastic activity. Triciribine inhibits the phosphoryla-
tion, activation, and signaling of Akt-1, -2, and -3, which may result in the inhibition of
Akt-expressing tumor cell proliferation. Both TCN and TCNP have very low bioavailabil-
ity, and the development of both drugs as intravenous (IV) treatments was halted because
of the toxicity induced by the high doses needed for their use as general cytotoxic agents.
This publication describes an expedient and straightforward total synthesis of amino acid
prodrugs (3, 4) of TCN and TCNP. In our study, both the prodrugs significant improved
the plasma exposure of the parent drugs and the prodrugs.
Keywords Amino acid prodrugs; antitumor; bioavailability; phosphoramidate prodrug;
triciribine; triciribine monophosphate
INTRODUCTION
Triciribine (TCN, NSC-154020, compound 1, Fig. 1) is a tricyclic nucleotide
derivative first synthesized by Schram and Townsend in 1971.[1] The compound
showed promise as an anticancer agent but had very poor solubility. In response
Received August 6, 2010.
Address correspondence to Wei Shen, TSRL Inc., 540 Avis Drive, Suite A, Ann Arbor, MI 48108,
USA. E-mail: wshen@tsrlinc.com; weiweishen@yahoo.com
358

TOTAL SYNTHESIS OF TRICIRIBINE PRODRUGS
359
Figure 1. TCN, TCNP, and their prodrugs.
to the poor solubility, they synthesized the 50-monophosphate of triciribine (TCNP,
compound 2, Fig. 1), which was found to be highly soluble. TCN was the most
potent AKT inhibitor among 1992 compound library from the National Cancer
Institute (NCI) Diversity Set.[2] AKT, also named protein kinase B, a serine=
threonine-specific protein kinase, is a member of the PI3K=Akt=mTOR pathway
involved in many key cellular processes that are overexpressed in a variety of
cancers. Ectopic expression of AKT, especially constitutively active AKT, induces
cell survival and malignant transformation, whereas inhibition of AKT activity
stimulates apoptosis in a range of mammalian cells.[3–6] Furthermore, activation of
AKT has been shown to associate with tumor invasiveness and chemoresistance.[7]
These observations establish AKT as an attractive target for cancer therapy. In a
screen using the NCI diversity set, TCNP was found to suppress growth and induce
apoptosis of cancer cells overexpressing AKT.[2] Furthermore, it was shown to
directly inhibit phosphorylation and consequent activation of all three isoforms of
AKT.[2] The water-soluble derivative, TCNP, does not inhibit upstream activators
of AKT such as PI3K and PDK1, nor does it inhibit other Ser=Thr kinases, making
it highly selective and specific for AKT. Consequently, TCNP attenuates the
phosphorylation of AKT’s downstream target enzymes (substrates). In a xenograft
model in nude mice, TCNP treatment significantly inhibited the growth of three
cancer cell lines overexpressing AKT, while it had little effect on cancer cell lines
expressing low levels of AKT.[2] These results were the first to show that TCNP
selectively inhibits the growth of tumors that overexpress AKT.[2]
Neither TCN nor TCNP are orally bioavailable. However, because of the high
solubility of TCNP, this compound was suitable for intravenous (IV) formulation
and was therefore tested in the late 1980s and early 90s as a general cytotoxic agent
for cancer treatment in phase I and phase II clinical trials by the NCI under IND
18,905.[8–11] In a phase I trial of 33 evaluable patients, using a 5-day continuous
infusion schedule every 42 days, modest activity was observed by improvement in
metastasis in a patient with papillary thyroid cancer, disease stabilization in a patient
with mesothelioma, and mixed responses in three patients with sarcoma, colorectal
cancer, and tonsillar carcinoma, respectively. The investigators noted cumulative
toxicity at the 30 mg=m2=day cohort with hyperglycemia, myelosuppression, throm-
bocytopenia, elevated pancreatic enzymes, and hepatotoxicity. Therefore, they
recommended a dose of 20 mg=m2=day for phase II.[10] In a phase II study of 24

360
W. SHEN, J.-S. KIM, AND J. HILFINGER
patients with advanced cervical squamous cell carcinoma, the same 5-day continuous
infusion schedule was given at a starting dose of 35 mg=m2=day. Two objective
responses were seen: one was a complete response lasting longer than 19 months,
and the second was a partial response lasting longer than 5 months. Only one subject
developed grade 4 toxicity.[12] Other clinical breast cancer and colorectal cancer
studies did not show significant activity following low levels of the drug and were
toxic at higher doses.[13–16] The development of TCNP was halted at that time
because of the toxicity induced by the high doses needed for its use as a general
cytotoxic agent together with its mild efficacy.
The prodrug approach for improvement of oral bioavailability has been highly
effective as demonstrated by the success of antiviral prodrugs such as adefovir
dipivoxil,[17] famciclovir,[18] tenofovir disoproxil,[19] valacyclovir,[20] and valganciclovir.[21]
These prodrugs exhibited 4–10-fold great oral absorption than their parent drug.
Interestingly, the amino acid prodrugs valacyclovir and valganciclovir are actively trans-
ported by oligopeptide transporters, which are highly expressed in the gastrointestinal
tract.[22–25] A prodrug strategy for TCN=TCNP could bolster the intestinal permeability
of the drug to maintain the effectiveness of the parent drugs at lower dosage, which can
overcome the toxicity induced by the high dose needed during IV treatment. Based
on these rationales, prodrugs of TCN and TCNP were synthesized and tested for
bioavailability enhancement. In this article, we report the synthesis and bioavailability
studies of the prodrugs of TCN.
CHEMISTRY
The purpose of developing orally available prodrugs of TCN is to accomplish
lower effective dosage, orally available drug delivery, greater flexibility in dose sched-
uling, and significantly improved patient compliance. The drug should have good
permeation across intestinal membranes, relatively good stability in the gastrointes-
tinal tract, and efficiency in the chemical and enzymatic conversion to parent drugs
in plasma or intracell. In this case, the parent drugs are TCN and=or TCNP. Two
prodrugs were designed based on the mentioned criteria (compounds 3 and 4,
Fig. 1). Compound 1 is a 50-O-L-valine methyl ester of phosphoramidate of TCN
and compound 2 is 50-O-L-valyl ester of TCN. L-Valine was chosen in our explo-
ration because it is a good substrate of oligopeptide transporters such as hPEPT1
that are highly expressed in the gastrointestinal tract.[26]
During the synthesis of the prodrugs of TCN, a big challenge was lack of
efficient synthetic route to TCN itself. Triciribine was originally synthesized from
the naturally occurring antibiotic toyocamycin.[1] The toyocamycin is not commer-
cially available anymore. Another method started from tetracyanoethylene via
4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine as key intermediate through 10
steps to obtain triciribine at around 10% total yield.[27–29] The reproducibility of
the latter method is very difficult in our laboratory. The major barrier was the
difficulty in preparation of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine from
tetracyanoethylene. A significant amount of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]
pyrimidine was lost during a tedious workup procedure. To prepare gram or even
kilogram amounts of triciribine, we developed a different procedure to avoid using
4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine as an intermediate (Scheme 1).

TOTAL SYNTHESIS OF TRICIRIBINE PRODRUGS
361
Scheme 1. New method for the synthesis of triciribine.
In this novel synthetic route, we chose 6-chloro-7-iodo-7-deazapurine (also called
4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 5) as starting material. It is commercially
available from several vendors. Glycosylation product (6) of 6-chloro-7-iodo-7-
deazapurine was obtained with 80% yield by first silylating compound 5 with
N,O-bis(trimethylsilyl) acetamide (BSA), then treating it with 1-O-acetyl-2,3,5-tri-O-
benzoyl-b-D-ribofuranose, followed by trimethylsilyl trifluoromethanesulfonate
(TMSOTf) under argon at 80ꢀC. The key step of the new method was to selectively
introduce the cyano group at the 7-position of the deazapurine ring of compound
6. Transition-metal-catalyzed cyanation of aryl halides is a common and useful trans-
formation in organic synthesis. The reaction goes smoothly with iodide and bromide
at mild condition while more harsh conditions are needed to make reaction on choride
occur.[30,31] When palladium complexes were applied as catalyst, the reaction of
aryl-chloride cyanation was very slow.[32–34] The difference in reactivity between iodide
and chloride is sufficient to permit preferential cyanation of the iodide in the presence
of both halogens.[35] In our study, the cyanation was carried out smoothly with 83%
yield by treatment with tributyltin cyanide and palladium tetraphenylphosphine in
anhydrous tetrahydrofuran (THF) to form 4-chloro-5-cyno-7-[(2,3,5-tri-O-benzoyl)-
b-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (7). Treatment of compound (7)
with methylhydrazine in ethanol at room temperature for 1 h afforded
4-(1-methylhydazino)-5-cyano-7-[(2,3,5-tri-O-benzoyl)-b-D-ribofuranosyl]-7H-pyrrolo
[2,3-d]pyrimidine (8) in 73% yield. Deprotection of compound 8 and subsequent ring

362
W. SHEN, J.-S. KIM, AND J. HILFINGER
Scheme 2. Selective protection of 20 and 30 dihydroxyl groups of triciribine. (v) DMPA, TBDMSCI,
levulinic acid, DCC; and (vi) TBAF, glacial acetic acid.
closure were accomplished by following the existing method[8] to obtain triciribine in
78% yield (38% yield from compound 5).
During the synthesis of the TCN prodrugs, the 20,30-dihydroxyl groups of TCN
were selectively protected by dilevulinate ester groups with two steps as shown in
Scheme 2[36] so that potential side reactions can be avoided and the solubility of
the intermediates in organic solvent can be improved. The levulinate group can
survive the synthetic conditions for the prodrugs and can be easily removed by
treatment with 1 ml of 2 M hydrazine hydrate in pyridine–acetic acid buffer for
10 min.[36] In addition, the levulinate is less prone to migration between adjacent
hydroxyl groups on the sugar residue than other ester protection group such as
benzoate and acetate.[36] Importantly, the exocyclic amine of the tricyclic base moiety
was not levulinated as long as the reaction time was limited to 2 to 3 h.[36] The
Scheme 3. Synthesis of the prodrug of TCN and TCNP. (vii) Methylchlorophenylphosphoryl P-N-L-
valine, N-methylimidazolo; (vii) hydrazine hydrate, pyridine–acetic acid buffer; (ix) Box-valine, DMAP,
DCC; and (x) TFA, DCM.

TOTAL SYNTHESIS OF TRICIRIBINE PRODRUGS
363
protected TCN was coupled with N-Boc-L-valine to form the fully protected 50-O-
valyl ester of TCN. Fully protected 50-O-[(valine methyl ester) phosphoramidate]
of TCN was constructed by reaction of protected TCN with phenyl (valine methyl
ester) phosphochloridate 11 that was prepared based on the existing method.[37]
The final prodrugs were obtained after removal of the levulinate group by hydrazine
and=or removal of Boc group by trifluoroacetic acid (TFA). The entire synthetic
route was illustrated in Scheme 3.
RESULTS AND DISCUSSION
Prodrug Stability
The stability of the prodrugs was assessed in 100 mM phosphate buffer, pH 6.5,
and rat liver homogenates at 37ꢀC. For the buffer stability, timed aliquots were taken
at 0, 1, 2, and 3 h and mixed with cold 10% trichloroacetic acid (TCA) in preparation
for analysis. For the liver homogenates, samples were taken at 0, 5, 10, 20, 30, and
90min, mixed with cold 10% TCA, and then centrifuged for 10min to remove preci-
pitated liver homogenate protein. All samples were analyzed by liquid chromato-
graphy–tandem mass spectrometry (LC=MS=MS (using an assay developed at
TSRL. Briefly, 10mL aliquots of sample were separated on a C18, 2.2 mm ꢁ 10cm col-
umn (Higgins Analytical), at a flow rate of 0.2 ml=min over a run time of 3 min. The
mobile phase for the separation consisted of an isocratic gradient using the following
mobile phases: (A) 70% formic acid in water to (B) 30% acetonitrile. The MS=MS
detector was run under MRM positive acquisition mode, with a cone voltage of
30 V and collision energy of 5 V with a collision gas pressure of 1 ꢁ 10ꢂ3 mbar.
According to our study (Table 1), all the compounds were stable in phosphate
buffer at pH 6.5. The 50-O-valyl ester prodrug of TCN broke down rapidly to
the parent TCN in liver homogenates, with a t1=2 equal to 12 min. In contrast the
TCN, TCNP and phosphoramidate prodrugs were stable over a 90 min time course
in liver homogenates.
Prodrug Absorption
The oral absorptions of TCN and TCNP were negligible (U.S. patent US2008=
058862). This was tested by direct injection of the TCN or TCNP into the duodenum
of rats and measuring the resulting systemic plasma levels of the drugs. In this
system, male albino Sprague–Dawley rats, 9–10 weeks old and weighing
250–350 g, were fasted for 18 h with free access to water. The rats were anesthetized
with 2–5% isofluorane. To withdraw timed plasma samples, a catheter was placed in
Table 1. Stability of TCN, TCNP, and their prodrugs in phosphate buffer and liver homogenates
Compound
Phosphate buffer (pH 6.5)
Liver homogenates
TCN
TCNP
Stable
Stable
Stable
Stable
Stable
Stable
12 min
Stable
50 O-Valyl triciribine
50 Valyl phosphoramidate prodrug

364
W. SHEN, J.-S. KIM, AND J. HILFINGER
the jugular vein. The abdomen was opened by a 4 to 5-cm midline incision and the
duodenal segment was located. Three mg (0.5 ml of a 6 mg=ml drug solution of
TCNP or suspension of TCN) were injected directly into the duodenal segment,
the intestine was placed back into the abdominal cavity, and the incision was covered
with gauze. Plasma samples (ꢃ0.5 ml) were withdrawn over a 4-h period, and the
systemic plasma concentrations of the injected prodrug and=or parent compounds
were determined simultaneously using an LC=MS=MS method. In this system,
the plasma levels of both TCN and TCNP were minimal; in the case of TCN,
measurable levels of drug were below the detection limit of the drug (<1 ng=ml).
For TCNP, the resulting Cmax in plasma was only 2.2 ng=ml (Fig. 2A and Table 2).
We then tested the intestinal absorption of the two prodrugs in the same
system. In contrast, the prodrug compounds (Fig. 2B and C) showed much greater
exposure levels in plasma compared to the two parent compounds, TCNP and TCN.
Dosing with the two prodrugs resulted in more complex Cp ꢁ time curves, because
all of the prodrugs were hydrolyzed in liver, at least in part, to their parent com-
pound. The 50-O-valyl triciribine had a peak concentration of 53.4 ng=ml and also
showed low levels of TCN (5.2 ng=ml). The 50-O-valyl phosphoramidate triciribine
prodrug showed the greatest absorption, and, in this case, the major detectable
compound was the parent compound, TCNP, which had a Cmax of 173.6 ng=ml.
Figure 2. Plasma concentration versus time curves for TCN, TCNP, and their prodrugs. (Figure is
provided in color online.)

TOTAL SYNTHESIS OF TRICIRIBINE PRODRUGS
365
Table 2. PK parameters of Cmax and AUC after duodenal administration of TCN, TCNP, and their
respective prodrugs (Cmax and AUC values were determined from the Cp ꢁ time curves shown in Fig. 2)
Fold
enhancement
Fold
enhancement
Compound
TCN
TCNP
Prodrug
C
max from oral dosing for TCN, TCNP, s0-O-valyl ester of TCN, and 50-O-
[(valine methyl ester) phosphoramidate] of TCN (ng=ml)
TCN
BDL
—
7.2
—
—
—
2.2
—
—
na
78
—
—
TCNP
50-O-Valyl triciribine
50-O-Valyl phosphoramidate
TCN
Significant
Significant
BDL
173.6
52.2
5.3
13.4
Dose norm. AUC0–4 for TCN, TCNP, s0-O-valyl ester of TCN, and 50-O-
[(valine methyl ester) phosphoramidate] of TCN (ng=ml) ꢄ h
TCN
TCNP
BDL
—
—
—
—
4.4 ꢅ 0.9
BDL
—
—
—
—
50-O-Valyl triciribine
21.5 ꢅ 4.8 Significant
Na
139
161 ꢅ 33.7
17.4 ꢅ 2.5
50-O-Valyl phosphoramidate 47.7 ꢅ 2.2 Significant
615 ꢅ 11.4
TCN
Total quantification of drug exposure of TCN containing compounds (AUC0–4hr)
also showed that the phosphoramidate-linked prodrug had the greatest potential
for oral absorption. These results are summarized in Table 2. As can be seen in
the data tables, both the TCN prodrugs yielded significant increases in circulating
levels of TCN, TCNP, or both as well as high levels of circulating prodrug. With
regard to Cmax, L-val ester prodrug yielded the greatest peak concentrations of pro-
drug, while the valyl phosphoramidate prodrug showed the greatest level of TCNP.
More importantly, the total exposure of active TCN and=or TCNP components in
plasma was significantly increased for both prodrugs when compared to either
TCN or TCNP. This can be seen in the AUC portion of Table 2 and is illustrated
graphically in Fig. 3. Thus, the L-valyl ester prodrug yielded significant increase in
circulating TCN levels compared with both TCN and TCNP dosing. Similarly, the
Figure 3. Intestinal dosing of TCN and TCNP prodrugs shows enhanced levels of circulating prodrug and
parents compared with TCN and TCNP along. The AUC0–4 hr of TCN (red bars), TCNP (blue bars), and
prodrug (green bars) after intestinal dosing of the prodrugs at a level of 10 mg=kg is compared to AUC
levels after TCN and TCNP dosing. The data are taken from Table 1. (Figure is provided in color online.)

366
W. SHEN, J.-S. KIM, AND J. HILFINGER
(L-valine methyl ester) phosphoramidate prodrugs had very significant increases in
TCN levels and 139-fold increase in TCNP levels, which indicated that both TCN
and TCNP can be metabolites of the phsophoramidate after passing through the
liver. Additionally, both compounds yielded significant levels of circulating prodrugs
in the plasma, which may serve as an additional depot of active compounds after
entering the caner cell. Taken together, these data strongly support our hypothesis
that the amino acid prodrugs of TCN or TCNP can significantly boost the oral avail-
ability of active compounds (TCN and TCNP).
EXPERIMENTAL
6-Chloro-7-iodo-7-deazapurine was purchased from Sigma-Aldrich Co. All
reagents employed were of American Chemical Society (ACS) grade or finer and
were used without further purification unless otherwise stated. All chemicals were
obtained from Sigma-Aldrich Co. For anhydrous reactions, solvents were dried
according to Vogel’s Textbook of Practical Organic Chemistry.[38] Removal of
solvent was performed under reduced pressure using a Buchi rotary evaporator,
¨
followed by evacuation (<0.1 mm Hg) to constant sample weight. Deionized water
was obtained from a Milli-Q reagent water system (Millipore Co., Milford, MA).
All reactions were carried out under an argon atmosphere in dried glassware. All
reactions and fractions from column chromatography were monitored by thin-layer
chromatography (TLC) using plates with an ultraviolet (UV) fluorescent indicator
(normal SiO2, Merck 60 F254). One or more of the following methods were used
for visualization; UV absorption by fluorescence quenching and iodine staining.
Flash chromatography was performed according to the method of Still[39] using
Merck type 60, 230 to 400-mesh silica gel. Fast atom bombardment mass spectra
(FAB-MS) were recorded on a Quattro II mass spectrometer. Nuclear magnetic reson-
ance (NMR) spectra were obtained on a Bruker 400 spectrometer. 1H NMR chemical
shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane
(TMS) using the residual proton resonance of solvents as reference: D2O d 4.8 and
1
dimethylsulfoxide (DMSO-d6) d 2.50. H NMR data are reported in the following
order: number of protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet,
qn, quintet; and m, multiplet), and coupling constant (J) in hertz (Hz). When appro-
priate, the multiplicity is preceded by br, indicating that the signal was broad. Yields
refer to isolated yields of compounds estimated to be >95% pure as determined by
1H NMR and analytical high-performance liquid chromatography (HPLC).
4-Chloro-5-iodo-7-[(2,3,5-tri-O-benzoyl)-b-D-ribofuranosyl]-7H-
pyrrolo[2,3-d]pyrimidine (6)
N,O-Bis(trimethylsilyl) acetamide (BSA, 4.8 mL, 20 mmol) was added to a
stirred suspension of 6-chloro-7-iodo-7-deazapurine (5) (5 g, 18 mmol) in dry aceto-
nitrile (122 mL) under an argon enviroment. After stirring at room temperature
for 20 min, 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose (10 g, 20 mmol) was
added, followed by the addition of trimethylsilyl trifluoromethanesulfonate
(3.5 mL, 19 mmol). The reaction mixture was stirred at room temperature for
30 min, after which the flask was transferred to a preheated oil bath at 80 ꢀC. After

TOTAL SYNTHESIS OF TRICIRIBINE PRODRUGS
367
stirring for 2 h at 80 ꢀC, the reaction mixture was cooled to room temperature and
diluted with ethyl acetate (200mL). The organic phase was sequentially washed with
aqueous saturated NaHCO3 and brine, dried with anhydrous Na2SO4, and concen-
trated to provide the crude product, which was purified by flash column chromato-
graphy (cyclohexane = ethyl acetate 3=1, v=v) to provide 6 as a pale yellow foam
1
(10 g, 80%). H NMR (DMSO-d6) d: 8.61 (s, 1H, proton at 2 position of pyrimidine
ring), 8.31 (s, 1H, proton at 6 position of pyrimidine), 8.09–7.94 (m, 4H, proton at
benzoates), 7.85–7.88 (m, 2H, proton at benzoates), 7.72–7.60 (m, 3H, proton at benzo-
ates), 7.55–7.44 (m, 6H, proton at benzoates), 6.73–6.71 (d, 1H, J 4.93 Hz, 10 proton of
ribofuranose); 6.32–6.29 (t, 1H, J 6.21 & 5.15 Hz), 6.17–6.14 (t, 1H, J 6.00 & 5.79 Hz),
4.90–4.79 (m, 2H), and 4.71–4.67 (m, 1H); 20, 30, 40, and 50 protons of ribofuranose.
Mass spectrum: calculated for: C32H23ClIN3O7 (723.90). FAB-MS: m=z 723.86.
Analytical calculation for C32H23ClIN3O7: C, 53.09; H, 3.20; N, 5.80, Found: C,
53.12; H, 3.16; N, 5.74.
4-Chloro-5-cyano-7-[(2,3,5-tri-O-benzoyl)-b-D-ribofuranosyl]-7H-
pyrrolo[2,3-d]pyrimidine (7)
To a deaerated solution of 4-chloro-5-iodo-7-[(2,3,5-tri-O-benzoyl)-b-D-
ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (6) (5 g, 6.9 mmol) in 25 ml of anhydrous
DMF were added 2.4 g (7.6 mmol) tributyltin cyanide and 0.40 g (0.34 mmol)
palladium tetraphenylphosphine. After dearaton for half an hour, the mixture was
stirred at 95 ꢀC under protection of argon gas for 6 h. Solvent and volatile compo-
nent were removed under reduced pressure, and the residue was dissolved in
100 ml ethyl acetate and washed with 100 ml ammonium chloride saturated aqueous
solution. After separation of the ethyl acetate layer, the aqueous layer was washed
three times with 30 ml ethyl acetate. The combined ethyl acetate solution was washed
with 50 ml brine and dried with anhydrous sodium sulfate. After filtration, the ethyl
acetated solution was concentrated, and the residue was purified by flash chromato-
graphy with hexane=ethyl acetate (3:2) to afford 3.1 g compound 7 as a yellow oil
(72% yield). 1H NMR (DMSO-d6) d: 8.535 (s, 1H, proton at 2 position of pyrimidine
ring), 8.175 (s, 1H, proton at 2 position of pyrimidine), 8.017–8.086 (m, 4H, proton
at benzoates), 7.871–7.894 (m, 2H, proton at benzoates), 7.341–7.661 (m, 9H, proton
at benzoates), 6.642–6.668 (d, 1H, J 5.38 Hz, 10 proton of ribofuranose), 6.195–6.167
(t, 1H, J 5.82 & 5.66 Hz), 6.114–6.088 (t, 1H, J 5.30 & 6.02 Hz), and 4.976–4.702
(m, 3H): 20, 30, 40, and 50 protons of ribofuranose Mass spectrum: calculated for:
C33H23ClN4O7 (623.01). FAB-MS: m=z 623.28. Analytical calculation for C33H23
ClN4O7: C, 63.62; H, 3.72; N, 8.99, Found: C, 63.54; H, 4.00; N, 9.05.
4-(1-Methylhydrazino)-5-cyano-7-[(2,3,5-tri-O-benzoyl)-b-D-
ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (8)
To a solution of 4-chloro-5-cyano-7-[(2,3,5-tri-O-benzoyl)-b-D-ribofuranosyl]-
7H-pyrrolo[2,3-d]pyrimidine (7) (2 g, 3.2 mmol) in 50 ml of 1:1 volume mixture of
anhydrous chloroform and anhydrous ethanol was added 0.3 ml methylhydrazine
(0.26 g, 5.7 mmol) and stirred under protection of argon gas for 3.5 h. Another
0.3 ml of methylhydrazine was added and the reaction was stirred for another 1.5 h.

368
W. SHEN, J.-S. KIM, AND J. HILFINGER
Solvent and volatile component were removed under reduced pressure, and the
residue was purified by flash chromatography with hexane=ethyl acetate (1:1 and
1:2) to afford 1.9 g compound 8 as an pale yellow oil (93% yield). 1H NMR
(DMSO-d6) d: 8.100 (s, 1H, proton at 2 position of pyrimidine ring), 7.828 (s, 1H,
proton at 6 position of pyrimidine), 8.040–8.017 (m, 2H, proton at benzoates),
7.947–7.923 (m, 2H, proton at benzoates), 7.858–7.828 (m, 2H, proton at benzoates),
7.705–7.607 (m, 3H, proton at benzoates), 7.560–7.411 (m, 6H, proton at benzoates),
6.629–6.615 (d, 1H, J 5.46 Hz, 10 proton of ribofuranose), 6.272–6.243 (t, 1H, J 5.82 &
5.66 Hz), 6.112–6.084 (t, 1H, J 5.30 & 6.02 Hz), and 4.808–4.650 (m, 3H): 20, 30, 40, and
50 protons of ribofuranose; 4.938 (s, 2H, 2 proton at hydrazine), 3.264 (s, 3H, methyl
group of methylhydrazine). Mass spectrum: calculated for C33H28IN5O7 (733.51).
FAB-MS: m=z 734.07. Analytical calculation for C33H28IN5O7: C, 54.04; H, 3.85;
N, 9.55, Found: C, 54.06; H, 4.08; N, 9.50.
4-Methyl-6-amino-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-d]pyrimidino
[4,5-c]pyridazine (triciribine) (1, TCN or triciribine) (Modified
from Existing Method)
4-(1-Methylhydrazino)-5-cyano-7-[(2,3,5-tri-O-benzoyl)-b-D-ribofuranosyl]-7H-
pyrrolo[2,3-d]pyrimidine (8) (1.0 g, 1.6 mmol) and sodium methoxide (0.38 g,
7.1 mmol) were suspended in methanol anhydrous (100 mL) and stirred at 20 ꢀC with
protection of argon. After 1 h, the reaction mixture was heated at reflux temperature
with protection of argon for 18 h. The solvent and volatile component were removed
under reduced pressure, and the yellow residue was purified by recrstallization from
methanol and water to obtain 0.37g (74%) of pure TCN.
1H NMR (DMSO-d6) d 8.03 (s, 1H, proton at 2 position of hetero-tricyclic
ring), 7.07 (s, 1H, proton at 7 position of hetero-tricyclic ring), 6.25 (s, 2H, NH2),
5.82 to 5.80 (d, 1H, J 6.54 Hz, 10 proton of ribofuranose), 5.60–5.57 (q, 1H, OH),
5.39–5.37 (d, 1H, J 6.63 Hz, OH), 5.18–5.17 (d, 1H, J 4.50 Hz, OH), 4.51–4.47
(m, 1H), 4.12–4.08 (m, 1H), 3.98–3.95 (m, 1H), 3.64–3.50 (m, 2H), 20, 30, 40, and 50
protons of ribofuranose, 3.40 (s, 3H, NCH3). Mass spectrum: calculated for
C13H16N6O4 (320.12). FAB-MS: m=z 321.27.
Analytical calculation for C13H16N6O4 ꢄ MeOH: C, 47.72; H, 5.68; N, 23.86.
Found: C, 47.80; H, 5.53; N, 23.75.
4-Methyl-6-amino-8-[5’-O-(tert-butyldimethylsilyl)-2’,3’-O-
dilevylinyl-b-D-ribofuranosyl]pyrrolo[4,3,2-d]pyrimidino[4,5-
c]pyridazine (9)
The described procedure[36] was modified. To a stirred suspension of 1.0 g
(3.12 mmol) of dry TCN (1) in 15 mL of anhydrous N,N-dimethylformamide
was added 0.91 g (7.5 mmol) of DMPA, followed by 0.56 g (3.74 mmol) of tert-
butyldimethylchlorosilane. The mixture was stirred with protection of argon at room
temperature for 20 h, at the end of which time TLC (9:1 DCM=MeOH) indicated
that the starting material disappeared. A solution of levulinic acid (1.98 g, 17 mmol)
in 10 ml of anhydrous ethyl acetate was added followed by solution of N,N0-dicyclo-
hexylcarbodiimide (DCC) (6.7 g, 32.6 mmol) in 10 mL anhydrous ethyl acetate. The

TOTAL SYNTHESIS OF TRICIRIBINE PRODRUGS
369
reaction mixture was stirred under protection of argon at room temperature for 1.5 h.
TLC (9:1 DCM=MeOH) indicated the reaction was completed. Anhydrous EtOH
(4mL) was added to consume all unreacted levulinic acid. After an additional
30min, the precipitate (DCU) was filtered off, and the solvent was removed from
filtrate in vacuo. The residue was redissolved in 50ml ethyl acetate and extracted with
saturated ammonium chloride aqueous solution (40 mL) once, water (40 mL) twice, and
brine (40ml) once. The organic layer was dried with anhydrous Na2SO4 before being
evaporated to dryness. The resulted syrup was further purified by silica flash chroma-
tography (4% MeOH in DCM as eluent) to obtain 1.56g pure product (9) (80%).
1H NMR (DMSO-d6) d 0.037 (6H, 2 s, SiMe2), 0.846 (9H, s, t-butylSi); levuli-
nate: 2.047 (3H, s CH3), 2.134 (3H, s, CH3), 2.457–2.473 (2H, t, CH2), 2.572–2.752
(4H, m, two CH2), 2.769–2.784 (2H, m, CH2); : ribofuranose: 3.781–4.183 (3H, m),
3
0
5.495–5.519 (1H, m), 5.808–5.837 (1H, m), 6.156 (1H, d, J ¼ 5.96 Hz, H1 );
hetero-tricyclic ring: 3.40 (s, 3H, NCH3), 6.224 (s, 2H, NH2), 7.014 (s, 1H, proton
at 7 position), 8.066 (s, 1H, proton at 2 position).
Mass spectrum: calculated for C29H42N6O8Si (630.76). FAB-MS: m=z 631.41
Analytical calculation for C29H42N6O8Si: C, 55.22; H, 6.71; N, 13.32. Found:
C, 55.28; H, 6.68; N, 13.45.
4-Methyl-6-amino-8-[2’,3-O’-dilevylinyl-b-
D-ribofuranosyl]pyrrolo[4,3,2-d]pyrimidino[4,5-c]pyridazine (10)
One gram (1.59 mmol) of 9, which had a yellow foamy appearance, was
dissolved in a solution of 1 M TBAF in THF (7.2 mL) containing glacial acetic acid
(0.67 mL). The reaction mixture was stirred under argon at room temperature for
1.5 h and was then directly filtered through a silica gel plug (4 ꢁ 5 cm) with THF
as eluant. The collected fractions were evaporated to dryness in vacuo. The residue
was redissolved in DCM and purified by silica-gel flash chromatography with 7%
methanol in DCM as eluant to obtain 0.61 g (75% yield) of a gumlike product, which
was crystallized from ethyl acetate as a pale yellow solid (10).
1H NMR (DMSO-d6): levulinate: 2.010 (3H, s CH3), 2.143 (3H, s, CH3),
2.501–2.512 (2H, t, CH2), 2.601–2.617 (2H, m, CH2), 2.682–2.698 (2H, m, CH2),
3.154–3.170 (2H, m, CH2); ribofuranose: 3.674–3.896 (2H, m), 3.961–4.057 (1H,
3
3
m), 5.035–5.127 (1H, t, 50OH, J ¼ 5.48), 5.409–5.736 (2H, m), 6.137 (1H, d, J ¼
5.84 Hz, H10); hetero-tricyclic ring: 3.38 (s, 3H, NCH3), 6.215 (s, 2H, NH2), 7.010
(s, 1H, proton at 7 position), 7.973 (s, 1H, proton at 2 position).
Mass spectrum: calculated for: C23H28N6O8 (516.5). FAB-MS: m=z 517.96.
Analytical calculation for C23H28N6O8: C, 53.48; H, 5.46; N, 16.27. Found: C,
53.45; H, 5.61; N, 16.18.
4-Methyl-6-amino-8-[5’-O-(methylphenylphosphoryl P!N-L-valine)-
2’,3’-O-dilevylinyl-b-D-ribofuranosyl]pyrrolo[4,3,2-d]pyrimidino
[4,5-c]pyridazine (12)
Methylchlorophenylphosphoryl P!N-L-valine (11)[37] (1.77 g, 5.8 mmol) in
5 ml anhydrous THF was added into a flask containing 0.6 g 10 (1.2 mmol) in
15 ml THF, followed by addition of 1 ml (11 mmol) N-methylimidazole. The reaction

370
W. SHEN, J.-S. KIM, AND J. HILFINGER
was stirred for 5 h at room temperature. The resultant mixture was concentrated
under vacuum. The residue was absorbed in 2 g of silica gel and subjected to silica-gel
flash chromatograhph (4% of MeOH in DCM) to obtain 0.62 g 12 (68%).
1H NMR (DMSO-d6) d valine methyl ester: 0.733–0.823 (6H, m, two CH3),
1.871–1.954 (1H, m, CH), 3.462–3.540 (1H, m, CH), 3.541–3.575 (3H, 2s, OCH3),
5.779–5.935 (1H, m, NH of valine); levulinate: 2.041 (3H, s CH3), 2.227 (3H, s,
CH3), 2.583–2.742 (4H, m, 2ꢁCH2), 2.846–2.901 (2H, m, CH2), 3.301–3.386 (2H,
m, CH2); ribofuranose: 3.953–4.063 (1H, m), 4.102–4.179 (2H, m), 4.836–5.062
(2H, m), 6.312 (1H, d, H10); C6H5: 7.126–7.178 (3H, m), 7.307–7.367 (2H, m);
hetero-tricyclic ring: 3.41 (s, 3H, NCH3), 6.917 (2H, s, NH2), 7.254 (s, 1H, proton
at 7 position), 8.137 (s, 1H, proton at 2 position). Mass spectrum: calculated for:
C35H44N7O12P (785.74). FAB-MS: m=z 786.38. Analytical calculation for C35H44
N7O12P: C, 53.50; H, 5.64; N, 12.48. Found: C, 53.68; H, 5.81; N, 12.39.
4-Methyl-6-amino-8-[5’-O-(methylphenylphosphoryl P!N-L-valine)-
b-D-ribofuranosyl]pyrrolo[4,3,2-d]pyrimidino[4,5-c]pyridazine (3)
Compound 12 (0.5 g, 0.63 mmol) was dissolved in 1 ml pyridine. Hydrazine
hydrate (1.25 mL, 2 M) in pyridine–acetic acid buffer was added. The reaction mix-
ture was stirred at room temperature for 15 min, after which time the mixture was
directly loaded on top of a prepared silica-gel column and eluent with 15% MeOH
in DCM to obtain the final product (10). Preparative HPLC (gradient, 0 to 35%
MeOH in water during 35 min) was used to obtain 0.31 g pure products 3 (83%).
1H NMR (DMSO-d6) d valine methyl ester: 0.713–0.821 (6H, m, two CH3),
1.879–1.895 (1H, m, CH), 3.458–3.519 (1H, m, CH), 3.538–3.570 (3H, 2s, OCH3),
5.876–5.922 (1H, m, NH of valine); ribofuranose: 4.132–4.188 (3H, m), 4.238–
4.276 (2H, m), 5.4–5.6 (2H, broadband 2OH), 5.936 (1H, d, H10); C6H5: 7.144–
7.177 (3H, m), 7.305–7.358 (2H, m); hetero-tricyclic ring: 3.455 (s, 3H, NCH3),
6.4–6.6 (2H, broadband, NH2), 7.219 (s, 1H, proton at 7 position), 8.135 (s, 1H, pro-
ton at 2 position). Mass spectrum: calculated for C25H32N7O8P (589.54). FAB-MS:
m=z 590.75. Analytical calculation for C25H32N7O8P: C, 50.93; H, 5.47; N, 16.63.
Found: C, 51.05; H, 5.64; N, 16.54.
4-Methyl-6-amino-8-[2’,3’-O-dilevylinyl-5’-O-Na-Boc-L-valyl-b-
D-ribofuranosyl]pyrrolo[4,3,2-d]pyrimidino[4,5-c]pyridazine (13)
aN-Boc-L-valine, (218 mg, 1 mmol) 15 mg (0.12 mmol) of DMAP, and 260 mg
(0.5 mmol) of compound 10 was dried in a 250-ml, single-necked, round-bottom
flask under high vacuum overnight. Anhydrous DMF (5 ml) was added. After stirred
for 5 min at 0 ꢀC, 208 mg (1.00 mmol) of DCC in 5 ml DMF were added dropwise at
0 ꢀC. After 4 h, TLC indicated completion of the reaction. Anhydrous ethanol (5 ml)
was added, and the reaction mixture was stirred overnight. DMF was removed under
high vacuum by rotavapor at 40 ꢀC. The residue was dissolved in 50 ml ethyl acetate.
The insoluble white precipitate was removed by filtration. The filtrate was consecu-
tively washed with 20 ml saturated ammonium chloride, 20 ml water, and 20 ml
brine. After drying with anhydrous sodium sulfate, the solvent was removed in
vacuo. The residue was dissolved in a minimum amount of dichloromethane and

TOTAL SYNTHESIS OF TRICIRIBINE PRODRUGS
371
absorbed in 2 g of silica gel by removal of dichloromethane with aid of rotavopor.
The mixture was loaded on top of a 30 g silica-gel column. Flash chromatography
with solvent of 93:7 (v=v) dichloromethane and methanol led to 260 mg compound
13 (72%).
1H NMR (DMSO-d6): d valyl: 0.899–0.922 (6H, m, 2ꢁCH3,), 1.413 (9H, s, Boc),
2.213–2.274 (1H, m, CH), 3.923–3.951 (1H, m, CH), 7.328 (1H, d, J ¼ 5.0, NH), levu-
linate: 2.053 (3H, s CH3), 2.171 (3H, s, CH3), 2.457–2.473 (2H, t, CH2), 2.489–2.494
(2H, m, CH2), 2.498–2.507 (2H, m, CH2), 2.691–2.749 (2H, m, CH2); ribofuranose
ring: 3.781–3.883 (1H, m), 4.037–4.153 (2H, m), 4.910–5.127 (2H, m), 6.150 (1H, d,
2J ¼ 5.96 Hz, H10); hetero-tricyclic ring : 3.412 (s, 3H, NCH3), 6.228 (s, 2H, NH2),
7.020 (s, 1H, proton at 7 position), 8.104 (s, 1H, proton at 2 position). Mass spectrum:
calculated for C33H45N7O11: 715.75. FAB-MS: m=z 716.53 (M þ 1). Analyticla calcula-
tions for C33H45N7O11: C, 55.38; H, 6.34; N, 13.70. Found: C, 55.42; H, 6.19; N, 13.66.
4-Methyl-6-amino-8-[5’-O-L-valyl-b-D-ribofuranosyl]pyrrolo[4,3,2-
d]pyrimidino[4,5-c]pyridazine (4)
Compound 13 (200 mg) was dissolved in 2 ml pyridine. (Hydrazine hydrate 1 ml,
2 M) in pyridine–acetic acid buffer was added. After stirring the solution for 10 min at
room temperature, the reactant disappeared and a slow migrating fraction was
detected by TLC (8:2 dichloromethane=methanol). The excess hydrazine was elimi-
nated through reaction with pentane-2,4-dione for another 10 min. After evaporating
the volatile components, the residue was dissolved in EtOAc and washed with 20 ml
saturated ammonium chloride, water, and brine. The organic layer was dried over
anhydrous Na2SO4, filtered, and evaporated to dryness. The residue was dissolved
in a minimum amount of dichloromethane and absorbed in 2 g of silica gel by removal
of dichloromethane with aid of rotavopor. The mixture was loaded on top of a 30 g
silica-gel column. Flash chromatography with solvent of 85:15 (v=v) dichloromethane
and methanol led to 138 mg compound 4 that still contained a trace of impurity.
Without further purification, 6 ml of 1:1 TFA and dichloromethane were added to
the residue and stirred for 4 h. Solvent was removed in vacuo. The residue was washed
with cold diethyl ether. The resulting white solid was purified by reverse-phase pre-
parative HPLC to obtain 100 mg compound 4 (85% from compound 13). HPLC con-
dition: gradient, 0 to 85% methanol in water containing 0.02% TFA within 30 min.
1H NMR (D2O): d valyl: 0.962–1.012 (6H, m, 2ꢁCH3,), 2.299–2.482 (1H, m,
2
CH), 4.025–4.088 (1H, d, CH, J ¼ 4.75 Hz) ribofuranose ring: 3.782–3.802 (2H,
m), 4.366–4.378 (1H, m), 4.796–4.827 (1H, m), 5.434–5.453 (1H, m), 6.024 (1H, d,
J ¼ 7.22 Hz, H10); hetero-tricyclic ring: 3.482 (s, 3H, NCH3), 7.376 (s, 1H, proton
at 7 position), 8.039 (s, 1H, proton at 2 position). Mass spectrum: calculated for
C18H25N7O5: 419.44. FAB-MS: m=z 420.50(M þ 1). Analytical calculations for
C18H25N7O52TFA: C, 40.81; H, 4.20; N, 15.14. Found: C, 40.65; H, 4.17; N, 15.23.
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