Intracellular metabolism and potential cardiotoxicity of a β-D-2’-C-methyl-2,6-diaminopurine ribonucleoside phosphoramidate that inhibits hepatitis C virus replication

Article abstract of DOI:10.1080/15257770.2019.1671594

β-D-2’-C-Methyl-2,6-diaminopurine ribonucleoside (2’-C-Me-DAPN) phosphoramidate prodrug (DAPN-PD) is a selective hepatitis C virus inhibitor that is metabolized intracellularly into two active metabolites: 2’-C-Methyl-DAPN triphosphate (2’-C-Me-DAPN-TP) a

Full text of DOI:10.1080/15257770.2019.1671594

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Intracellular metabolism and potential
cardiotoxicity of a β-D-2’-C-methyl-2,6-
diaminopurine ribonucleoside phosphoramidate
that inhibits hepatitis C virus replication
Sijia Tao, Longhu Zhou, Hongwang Zhang, Shaoman Zhou, Sheida Amiralaei,
Jadd Shelton, Maryam Ehteshami, Yong Jiang, Franck Amblard, Steven J.
Coats & Raymond F. Schinazi
To cite this article: Sijia Tao, Longhu Zhou, Hongwang Zhang, Shaoman Zhou, Sheida Amiralaei,
Jadd Shelton, Maryam Ehteshami, Yong Jiang, Franck Amblard, Steven J. Coats & Raymond F.
Schinazi (2019): Intracellular metabolism and potential cardiotoxicity of a β-D-2’-C-methyl-2,6-
diaminopurine ribonucleoside phosphoramidate that inhibits hepatitis C virus replication,
Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2019.1671594
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2019.1671594
Intracellular metabolism and potential cardiotoxicity of
a b-D-2’-C-methyl-2,6-diaminopurine ribonucleoside
phosphoramidate that inhibits hepatitis C virus
replication
Sijia Tao, Longhu Zhou, Hongwang Zhang, Shaoman Zhou, Sheida Amiralaei,
Jadd Shelton, Maryam Ehteshami, Yong Jiang, Franck Amblard, Steven J.
Coats, and Raymond F. Schinazi
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics,
Emory University School of Medicine, Atlanta, GA, USA
ABSTRACT
ARTICLE HISTORY
Received 2 August 2019
Accepted 18 September 2019
b-D-2’-C-Methyl-2,6-diaminopurine ribonucleoside (2’-C-Me-
DAPN) phosphoramidate prodrug (DAPN-PD) is a selective
hepatitis C virus inhibitor that is metabolized intracellularly
into two active metabolites: 2’-C-Methyl-DAPN triphosphate
(2’-C-Me-DAPN-TP) and 2’-C-methyl-guanosine 5’-triphosphate
(2’-C-Me-GTP). BMS-986094 and IDX-184 are also bioconverted
to 2’-C-Me-GTP. A phase IIb clinical trial with BMS-986094 was
abruptly halted due to adverse cardiac and renal effects.
Herein, we developed an efficient large scale synthesis of
DAPN-PD and determined intracellular pharmacology of
DAPN-PD in comparison with BMS-986094 and IDX-184, versus
Huh-7, HepG2 and interspecies primary hepatocytes and
human cardiomyocytes. Imaging data of drug treated human
cardiomyocytes was found to be most useful in determining
toxicity potential as no obvious beating rate change was
observed for IDX-184 up to 50 mM up at 48 h. However, with
BMS-986094 and DAPN-PD at 10 mM changes to both beat
rate and rhythm were noted.
KEYWORDS
Nucleoside prodrug;
hepatitis C virus;
intracellular metabolism;
hepatocytes; human
cardiomyocytes;
cardiotoxicity
Introduction
The nucleoside analog antiviral inhibitor (NI), sofosbuvir,[1] revolutionized
the treatment of hepatitis C virus (HCV) infection and established NIs as
an important class of anti-HCV agents due to their pan-genotypic potential
and high barrier to the selection of resistant virus.[2] Despite this resound-
ing success there have been many clinical failures with this class of anti-
HCV agents,[3,4] most notably the failure of BMS-986094 (formerly known
as INX-189 from Inhibitex, Inc., purchased by Bristol-Myers Squibb,
Inc.)[5,6] which was halted in phase IIb clinical trials due to cardiac and
CONTACT Raymond F. Schinazi
rschina@emory.edu
Department of Pediatrics, Emory University School of
Medicine, E420, 1760 Haygood drive, Atlanta, GA 30322.
ß 2019 Taylor & Francis Group, LLC

2
S. TAO ET AL.
O
N
NH2
N
OMe
N
N
N
N
N
N
N
NH
NH2
O
P
O
O
O
P
O
O
HO
S
O
O
NH2
NH2
N
N
O
N
H
P
N
H
O
O
O
Me
OH
O
Me
OH
Me
OH
O
O
HN
O
O
O
HO
HO
HO
O
DAPN-PD
BMS-986094 (INX-189)
IDX-184
NH2
O
OMe
N
N
N
N
N
NH
NH2
O
P
O
P
O
P
OH
O
P
O
P
O
P
OH
O
P
O
P
O
P
OH
O
O
O
N
N
O
Me
OH
N
O
Me
OH
NH2
NH2
N
N
N
HO
O
O
HO
O
O
HO
O
O
O
Me
OH
OH OH
OH OH
OH OH
HO
HO
HO
2'-C-Me-GTP
2'-C-Me-6-OMe-GTP
2'-C-Me-DAPN-TP
Figure 1. Structures of DAPN-PD, IDX-184 and BMS-986094. DAPN-PD was metabolized intracel-
lularly into two nucleoside 5’-triphosphates (NI-TP): 2’-C-Me-DAPN-TP and 2’-C-Me-GTP. IDX-184
generated one NI-TP: 2’-C-Me-GTP. BMS-986094 was metabolized into two NI-TPs: 2’-C-Me-GTP
and 2’-C-Me-6-OMe-GTP.
renal adverse effects (Figure 1).[7] In this study, a patient receiving BMS-
986094 for 40 days presented with rapidly progressive heart failure charac-
terized by pronounced electrocardiographic changes and decreased
ventricular ejection fraction accompanied by acute renal failure. Of the 34
patients treated with BMS-986094 in this phase IIb study, 14 had some evi-
dence of cardiac dysfunction and there was one death.
BMS-986094 is metabolized intracellularly to 2’-C-Me-GTP which is the
actual HCV NS5B inhibitor (Figure 1) Mitochondrial toxicity has been
associated with long-term use of some 2’-deoxynucleoside analogs,[8,9] espe-
cially in treatment of HIV and HBV, where the active 5’-triphosphorylated
nucleoside metabolites can also serve as substrates for the mitochondrial
DNA polymerase gamma. In the case of BMS-986094 the observed toxicity
has been attributed to efficient incorporation of 2’-C-Me-GTP by mito-
chondrial RNA polymerase (POLRMT) resulting in mitochondrial tox-
icity[10,11] although some studies have come to a different conclusion.[12,13]
However, incorporation by POLRMT, mitochondrial toxicity and increased
lactic acid levels in HepG2 cells have been reported by other groups[14,15]
including our own work.[16,17] Additionally, BMS-986094’s (but nþot its
ꢀ
metabolites) blocking of the human ether-a-go-go-related (hERG) K ion
channel was predicted computationally and was subsequently confirmed in
two different biological assays.[18] A study done by BMS looking at the
metabolism of BMS-986094 in human hepatocytes, human cardiomyocytes
and ultimately studying various monkey tissues three weeks post a three-
week dosing (week 6) found high levels of 2’-C-Me-GTP in the heart and
kidney (2,610–4,280 ng/mL). They concluded “Persistent high concentra-
tions of the active triphosphorylated metabolite, INX-09114 (2’-C-Me-

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
GTP), in monkey heart and kidney appeared to correlate with toxicities in
these tissues.”[19]
The FDA placed a clinical hold on IDX-184 (Idenix, Inc).[20] which was
also undergoing phase IIb clinical studies at the time, as the same active
metabolite, 2’-C-Me-GTP, was produced (Figure 1)[21,22] At the same time
our group was in late stage preclinical studies of a novel anti-HCV NI,
DAPN-PD,[23] that metabolized intracellularly into two active metabolites:
2’-C-Me-DAPN-TP and 2’-C-Me-GTP, (Figure 1)[15,16] Unlike the broad
toxicity reported with BMS-986094,[24] we had observed no in vitro cyto-
toxicity in various cell lines up to 100 lM and more importantly no mito-
chondrial toxicity or increase in lactic acid levels up to 50 lM in HepG2
cells.[15,16] As we initiated animal studies we thought it worthy to compare
the metabolism profile of our drug, DAPN-PD, with BMS-986094 and
IDX-184 to determine intercellular delivery of prodrug, triphosphate(s) and
various other metabolites versus liver cell lines, various species of primary
hepatocytes and human cardiomyocytes. We also visually recorded cardio-
myocytes up to 48 h after exposure to each drug to determine the contrac-
tion and rhythm and overall morphology. Our goal was to predict if
DAPN-PD and IDX-184 would have a similar in vivo toxicity profile to
that found with BMS-986094.
Materials and methods
Nucleoside analog compounds
The medicinal chemistry synthesis and characterization of DAPN-PD was
described elsewhere,[15] and the process route is described below. 2’-C-Me-
DAPN, 2’-C-Me-DAPN-monophosphate (-MP) 2’-C-Me-DAPN-TP, 2’-C-
Me-G, 2’-C-Me-GMP, 2’-C-Me-GTP, BMS-986094 and IDX-184 were
synthesized in house with a purity higher than 98%, as determined by
HPLC-UV analysis (as an approximate 1:1 mixture of phosphorous diaster-
eomers for DAPN-PD, BMS-9860945 and IDX-18425[25]). ddATP was pur-
chased from Sigma-Aldrich (St. Louis, MO). All other reagents were the
highest quality available from Thermo Fisher Scientific (Waltham, MA).
Cellular pharmacology studies in Huh-7 and HepG2 cells
The human hepatocellular carcinoma cell lines (Huh-7 and HepG2) were
seeded at 1 ꢀ 106 per well in 12-well plates and incubated in a cell culture
incubator at 37 ꢁC with a humidified atmosphere of 5% CO2. Adherent
cells were subsequently exposed to 50 mM of DAPN-PD, IDX-184 or BMS-
986094. At 4 h, drug-containing medium was removed and cells were
washed twice with ice-cold phosphate buffered saline (PBS). Cells were

4
S. TAO ET AL.
resuspended in 70% ice-cold methanol containing 20 nM ddATP overnight
at ꢂ20 ꢁC. The supernatants were then dried under a flow of air and dried
samples stored at ꢂ20 ꢁC until analyzed by LC-MS/MS.
Cellular pharmacology studies in primary mouse, rat, dog, monkey and
human hepatocytes
Cryopreserved primary mouse, rat, dog, monkey and human hepatocytes
and medium were purchased from Life Technologies (Grand Island, NY).
After thawing in a 37 ꢁC water bath for approximately 2 min, the cell sus-
pension was immediately transferred to 48 mL pre-warmed cryopreserved
hepatocyte recovery medium (for human) or Williams’ medium E and hep-
atocyte plating supplement pack (for animal) and then centrifuged at room
temperature (human hepatocytes: 100 ꢀ g for 10 min, dog and monkey hep-
atocytes: 65 ꢀ g for 4 min, mouse and rat hepatocytes: 55 ꢀ g for 3 min,
respectively) according to the supplier’s protocol. The resulting pellet was
resuspended in pre-warmed incubation medium and cell viability and yield
determined by Trypan blue exclusion dye. Cells were suspended at a target
density of approximately 0.5 ꢀ 106 (mouse) or 1.0 ꢀ 106 cells/mL (rat, dog,
monkey and human), respectively, for cellular pharmacology studies. Cell
suspension (1 mL/well of 12-well plates) was incubated at 37 ꢁC with a
humidified atmosphere of 5% CO2 with DAPN-PD, IDX-184 or BMS-
986094 at a final concentration of 50 mM. At 4 h, the extracellular medium
was removed by centrifugation and cell pellets were washed with ice-cold
PBS twice in order to remove any residual medium. Cells were resuspended
in 70% ice-cold methanol containing 20 nM ddATP overnight at ꢂ20 ꢁC.
Supernatants were dried under a flow of air and dried samples stored at
ꢂ20 ꢁC until LC-MS/MS analysis.
Cellular accumulation of DAPN-PD, BMS-986094 and IDX-184 in human
cardiomyocytes
Freshly thawed cryopreserved human cardiomyocytes (Cellular Dynamics
International, Madison, WI) were seeded in 12-well collagen coated plates
with iCell plating medium (Cellular Dynamics International, Madison, WI)
at a density of 480,000 (plating efficiency 53%) and incubated in a cell cul-
ture incubator at 37 ꢁC, 7% CO2. After 48 h, non-adhered cardiomyocytes
and debris were removed by rinsing twice with iCell maintenance medium
and the attached cells were incubated for an additional of 8 days in main-
tenance medium, with fresh medium replacement every other day. At day
10, when the cells beat spontaneously and had reached a stable beating
rate, 10 mM of DAPN-PD, IDX-184, BMS-986094 and sofosbuvir (negative

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
control) were added to each well respectively. At 4 h and 8 h after incuba-
tion, extracellular medium was removed and the cell layer washed with
1 mL of ice-cold PBS three times to remove any residual drug-containing
medium. Cells were resuspended in 70% methanol containing 20 nM
ddATP overnight at ꢂ20 ꢁC. The supernatants were dried under a flow of
air and dried samples stored at ꢂ20 ꢁC until LC-MS/MS analysis.
Quantification of intracellular metabolites by LC-MS/MS
A rapid ion-pairing high performance liquid chromatography coupled with
electrospray tandem mass spectrometry (LC-MS/MS) method[26] was
adapted to quantify the intracellular metabolites. Prior to analysis, each
sample was reconstituted in 200 mL mobile phase. An Ultimate 3000 HPLC
system (Thermo Fisher Scientific, Waltham, MA) was used for separation.
The processed samples were injected on a Hypersil GOLD column
(100 ꢀ 1 mm) with a 3 mm particle size (Thermo Fisher Scientific, Waltham,
MA). An ion-pair method (Mobile phase A, consisted of 2 mM ammonium
phosphate and 3 mM hexylamine, and mobile phase B, acetonitrile) was
used for a gradient elution. Mobile phase B was increased from 5 to 80%
over 13 min, kept at 80% for 4 min, and returned to initial conditions with-
out ramp. The flow rate was maintained at 50 mL/min. Mass spectrometric
detection was performed on a TSQ Quantum Ultra triple quadrupole mass
spectrometer equipped with electrospray ionization source (Thermo Fisher
Scientific, Waltham, MA). Positive SRM detection mode was used with a
spray voltage of 3.2 KV, sheath gas at 55 (arbitrary units), ion sweep gas at
0.3 (arbitrary units), auxiliary gas at 5 (arbitrary units), and a capillary tem-
perature of 380 ꢁC. The collision cell pressure was maintained at 1.5
mTorr. Xcalibur 2.0 was used to perform data analyses. The calibration
curves were generated from standards of the three prodrugs, parent
nucleosides, 2’-C-Me-DAPN-MP, 2’-C-Me-DAPN-TP, 2’-C-Me-GMP and
2’-C-Me-GTP using extracts from untreated cells. The lower limit of quan-
tification for all analytes ranged from 0.5 to 1 nM.
Recording human cardiomyocyte beating patterns
Human cardiomyocytes were thawed and cultured as described above. Ten
days post-plating when all cells demonstrated regular synchronous beating,
cardiomyocytes were then exposed to 2, 10 and 50 mM of DAPN-PD, BMS-
986094, IDX-184 or sofosbuvir (control) in duplicate. Cultures were imaged
using a PerkinElmer spinning disc confocal microscope (PerkinElmer,
Waltham, MA) and 60 second videos were recorded at 6 fps using a
Hamamatsu Flash 4.0 sCMOS camera and Velocity Software at 2, 4, 8, 12,

6
S. TAO ET AL.
Scheme 1. Synthesis of pentafluoro phosphoryl ester 4. Reagents and conditions: (a) (i) iPrOH,
cat. H2SO4, rt, 18 h; (ii) NaHCO3 90%; (b) POCl3, t-BuOMe, rt 2 h; (c) Et3N, t-BuOMe, DCM, 2 h, rt;
(d) Et3N, DCM, 5 oC, to rt, 2 h, 25% (2 steps).
24, and 48 h after drug exposure. Contractility of human cardiomyocytes
was analyzed by manual counting to determine contraction rate
and rhythm.
Results and discussion
Chemistry
With DAPN-PD progressing into animal studies we required kg quantities
which necessitated reworking the synthesis versus what was developed for
gram scale preparation.[16] Hydrolysis of 3 kg of dihydrocoumarin, 1, in
anhydrous isopropanol was found to proceed in comparable yield and pur-
ity at room temperature in 18 h versus the small scale reaction which was
done at reflux (Scheme 1) The intermediate phenyl dichlorophosphate, 2,
was formed at ꢂ10 ꢁC then 2 h toward room temperature whereas the
small-scale reaction was done ꢂ70 ꢁC then 18 h at room temperature.
The solvent was also switched from ethyl ether to methyl t-butyl ether. The
same adjustments to the conditions for the L-alanine isopropyl ester add-
ition and reaction time provided intermediate 3, which was deemed too
reactive to handle efficiently on large scale, hence, reaction with pentafluor-
ophenol in the presence of triethylamine gave 4 as a stable solid with a 54/
46 diastereomeric ratio at the phosphorous center (Scheme 1).
For the large-scale synthesis of DAPN-PD the medicinal chemistry devel-
oped Boc protection/deprotection route.[16] was deemed to have too many
steps that required purification for efficient scaleup. Ultimately a route was
developed that relied on a transient intermediate 6-diazo purine nucleoside
(Scheme 2)[27,28]
The 6-chloro-2-aminopurine tribenzoylated nucleoside 6 was synthesized
from known sugar 5[29] and 6-chloro-2-aminopurine in the presence of

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Scheme 2. Synthesis of DAPN-PD. Reagents and conditions: (a) DBU, TMSOTf, CH3CN, 65 ꢁC,
3 h, 79.5%; (b) NH3/MeOH, rt, 12 h, 69.9%; (c) 4, THF, t-BuMgCl, 0 ꢁC to rt, 6 h, 80%; (d) NaN3,
(Bu)4NI, DMF, 80 ꢁC, 18 h; (e) H2, 20% Pd(OH)2, i-PrOH, rt, 52% (two steps).
DBU with addition of TMSOTf at 0 ꢁC as opposed to ꢂ40 ꢁC used with the
small-scale route. The unprotected 6-chloro-2-aminopurine nucleoside 6
was obtained by debenzylation with ammonia in methanol. Introduction of
the prodrug moiety was accomplished with 950 grams of 6 and 2.56 kilo-
grams of 4 by the addition of t-BuMgCl at 0 ꢁC. Stirring was continued for
6 h during which time the temperature was maintained below 30 ꢁC. The 6-
azido intermediate 8 was prepared from 1.68 kilograms of 7 by reaction
with sodium azide in the presence of tetrabutylammonium iodide. After
the reaction was complete the excess sodium azide was removed by water
extraction and the dried organic layer was stirred with charcoal and filtered
through celite. Reduction of the 6-azido intermediate 8 was accomplished
with 20% palladium hydroxide on carbon in the presence of hydrogen pro-
viding 847 grams of DAPN-PD.
Anti-HCV activity and cytotoxicity profile of DAPN-PD, IDX-184 and
BMS-986094
DAPN-PD, IDX-184 and BMS-986094 were evaluated for inhibition of
HCV genotype 1 b RNA replication in Huh-7 cells using a subgenomic
HCV replicon system.[30] Cytotoxicity in Huh-7 cells was determined sim-
ultaneously by extraction and amplification of both HCV RNA and cellular
ribosomal RNA (rRNA).[31] In addition, cytotoxicity was determined in pri-
mary human peripheral blood mononuclear (PBM) cells, human lympho-
blastoid cells (CEM), and African Green monkey Vero cells.[32,33] The
results are summarized in Table 1. While DAPN-PD and IDX-184 both
display sub-micromolar EC50’s in our HCV replicon system, BMS-986094

8
S. TAO ET AL.
Table 1. HCV replicon and cytotoxicity data for DAPN-PD, IDX-184 and BMS-986094.
Replicon Huh-7 (mM)
Cytotoxicity (CC50, mM)
Compounds
EC50 EC90
Huh-7
PBM
Vero
CEM
DAPN-PD
BMS-986094
IDX-184
0.7
0.02
0.3
2.5
0.04
0.9
>10
0.8
>10
>100
4.7
>100
>100
14
>100
>100
8.7
>100
Figure 2. Total levels of NI-TP metabolites of DAPN-PD, IDX-184 and BMS-986094 in Huh-7 and
HepG2 cells. 50 mM of each compound was incubated with Huh-7 or HepG2 cells for 4 h at
37 ꢁC. Values represent the mean of three replicates. For DAPN-PD, total NI-TP is 2’-C-Me-DAPN-
TP and 2’-C-Me-GTP. For IDX-184, NI-TP is 2’-C-Me-GTP. For BMS-986094, total NI-TP is 2’-C-Me-
GTP and 2’-C-Me-6-OMe-GTP.
has an impressive EC50 of 20 nM. However, as reported early on by
Idenix,[21] BMS-986094 displays concerning toxicity versus multiple
cell lines.
Intracellular metabolism of DAPN-PD, IDX-184 and BMS-986094 in
Huh-7 and HepG2 cells. Cellular uptake of DAPN-PD, IDX-184 and BMS-
986094 were evaluated side-by-side in both Huh-7 and HepG2 cells to
compare their intracellular metabolism. In both Huh-7 and HepG2 cell
lines, DAPN-PD generated two distinct 5’-triphosphorylated nucleoside
metabolites, 2’-C-Me-DAPN-TP and 2’-C-Me-GTP, with comparable total
NI-TP levels to IDX-184. The major NI-TP metabolized from BMS-986094
was 2’-C-Me-GTP (240 and 350 pmol/million cells in Huh-7 and HepG2
cells, respectively) which was 94- and 88-fold higher than the total NI-TP
generated from DAPN-PD, and 77- and 170-fold higher than that gener-
ated from IDX-184. Interestingly, for all three prodrugs, their respective
metabolite profiles were similar in both cell lines (Figure 2 and Table 2).
As shown in Table 2, BMS-986094 also delivered very high level of the
parent prodrug relative to DAPN-PD and IDX-184. In fact, the level

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Table 2. Comparison of DAPN-PD, IDX-184 and BMS-986094 intracellular metabolism in Huh-7
and HepG2 cells.
Intracellular concentration of prodrugs and metabolites (pmol/106 cells SD)b
Compounda Cell type
Prodrugs
170
390 33
Nc
47
27
NA
NA
220 18
220
2’-C-Me-G
NTPc
2’-C-Me-GTP
DAPN-PD
Huh-7
HepG2
Huh-7
HepG2
4
3
2
7.5 1.5
90 12
5.6 1.6
0.55 0.11
1.2 0.01
NA
NA
0.32 0.04
0.57 0.10
2.0 0.1
2.8 0.4
3.1 0.3
2.1 0.4
240 13
IDX-184
11
21
1
1
20
1
BMS-986094 Huh-7
HepG2
2,900 358
2,400
640 36
4,900 108
9
1
350 7
aCells were incubated with each compound for 4 h at 50 mM.
bValues represent the mean SD of three replicates.
cFor DAPN-PD, N is 2’-C-Me-DAPN; for BMS-986094, N is 2’-C-Me-6-OMe-G.
NA is not applicable.
detected for BMS-986094 were 260- and 110-times the levels observed for
IDX-184 and 17- and 6-times the levels observed for DAPN-PD in Huh-7
and HepG2 cells, respectively. Also, the amount of 2’-C-Me-6-OMe-GTP
generated from BMS-986094 is not significant relative to the levels of 2’-C-
Me-GTP that were produced but in the case of DAPN-PD the levels of
DAPN-TP are significant relative to the levels of 2’-C-Me-GTP produced.
Based on the studies outlined above, the high levels of intracellular BMS-
986094 would lead to blocking of the hERG potassium ion channel while
high levels of 2’-C-Me-GTP would result in incorporation by POLRMT
leading to mitochondrial toxicity and increased lactic acid levels.
NA is not applicable.
Intracellular metabolism of DAPN-PD, IDX-184 and BMS-986094 in mouse,
rat, dog, monkey and human hepatocytes
The intracellular metabolism studies were conducted in primary hepato-
cytes from mouse, rat, dog, monkey and human. Accordingly, each primary
cell culture was incubated with DAPN-PD, IDX-184 or BMS-986094
(50 mM, 4 h) and intracellular metabolites and intact prodrugs were readily
detected form cell extracts by LC-MS/MS (Table 3). Surprisingly, BMS-
986094 give the highest total TP concentration only in dog hepatocytes
where IDX-184 and DAPN-PD produced their lowest levels among the five
species tested. In all five species evaluated, DAPN-PD was metabolized into
two nucleoside triphosphates: 2’-C-Me-DAPN-TP and 2’-C-Me-GTP but
the ratio varied greatly depending on the species while the level of 2’-C-
Me-6-OMe-GTP produced from BMS-986094 was quite low across all spe-
cies and undetectable in mouse hepatocytes. The variation in NI-TP levels
in different species and variation in the ratios of 2’-C-Me-DAPN-TP versus
2’-C-Me-GTP observed with DAPN-PD suggest that the interspecies differ-
ences may be attributed to the amount or efficiency of enzyme(s) respon-
sible for the deamination of DAPN-PD or one of its metabolites.[34] In

10
S. TAO ET AL.
Table 3. Comparison of DAPN-PD, IDX-184 and BMS-986094 intracellular metabolism in
mouse, rat, dog, monkey and human primary hepatocytes.
Intracellular concentration prodrugs and metabolites (pmol/106 cells SD)b
Compounda Hepatocyte species
Prodrugs
13
0.90 0.50
Nc
2’-C-Me-G
NTPc
2’-C-Me-GTP
DAPN-PD
Mouse
Rat
1
1,300 70
310 67
BLOQ
72 47
130
4
48
45
4
5
99 45
Dog
6.3 1.5
113
4
11
6.8 4.5
200 13
1,300 137
320 155
4
14
180
550 16
NA
1
6
2.5 0.2
Monkey
Human
Mouse
Rat
160
70
220
4
6
3
440 43
2,100 744
NA
13
23
1
2
IDX-184
1,300 29
780 308
1,100 471
NA
NA
Dog
Monkey
Human
110
75
160 18
880 135
52
2,200 68
150 19
4,100 569
2
3
NA
NA
NA
190 19
230
8
NA
NA
NA
BLOQ
0.60 0.10
0.60 0.04
6.2 2.4
2.7 0.3
150
660
200 18
320 37
250 45
310 17
5
3
810 160
2,500 181
440 53
210 48
1,800 163
510 32
BMS-986094 Mouse
Rat
Dog
Monkey
Human
14
3
130 13
200 10
960 13
87
240 13
3
9,500 431
aCells were incubated with each compound for 4 h at 50 mM.
bValues represent the mean SD of three replicates.
cFor DAPN-PD, N is 2’-C-Me-DAPN; for BMS-986094, N is 2’-C-Me-6-OMe-G.
BLOQ, below limit of quantification. NA, not applicable.
primary human hepatocytes, 2’-C-Me-DAPN-TP was the major metabolite
produced by DAPN-PD, whereas 2’-C-Me-GTP was the primary metabolite
observed with BMS-986094 and IDX-184. Additionally, the amount of 2’-
C-Me-DAPN-TP produced by DAPN-PD (550 pmol/million cells) was
markedly higher than the level of 2’-C-Me-GTP from BMS-986094
(240 pmol/million cells) or IDX-184 (200 pmol/million cells). Human hepa-
tocytes showed the highest phosphorylation efficiency for DAPN-PD, fol-
lowed by monkey, mouse and rat. In contrast, BMS-986094 had similar
metabolism profiles in mouse, rat, dog and human hepatocytes.
For IDX-184, the mouse, rat and monkey hepatocytes produced higher 2’-C-
Me-GTP levels than detected in dog and human hepatocytes and also produced
significantly higher amounts of TP relative to the two other drugs (Figure 3).
Cellular accumulation of DAPN-PD, IDX-184 and BMS-986094 in human
cardiomyocytes
As BMS-986094 was reported to have cardiotoxicity in humans,[6] we
thought it was important to determine if NI-TP levels could be correlated
with potential toxicity in human cardiomyocytes[18] with each of the three
drugs. We initially dosed cardiomyocytes at 50 mM as we did for hepato-
cytes, Huh-7 and HepG2 cells, however, BMS-986094 treated cardiomyo-
cytes stopped beating within 2 h after drug administration, indicating an
acute cardiac effect. Therefore, we reduced the maximum concentration to
10 mM for this cellular pharmacology study. Using a similar approach, the

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Figure 3. Total levels of active NI-TP metabolite of DAPN-PD, IDX-184 and BMS-986094 in inter-
species primary hepatocytes. 50 mM of each compound was incubated with each species of
hepatocytes for 4 h at 37 ꢁC. Values represent the mean of three replicates. NI-TP: nucleoside
5’-triphosphate. For DAPN-PD, total NI-TP is 2’-C-Me-DAPN-TP and 2’-C-Me-GTP. For IDX-184, NI-
TP is 2’-C-Me-GTP. For BMS-986094, total NI-TP is 2’-C-Me-GTP and 2’-C-Me-6-OMe-GTP.
Table 4. Comparison of DAPN-PD, IDX-184, BMS-986094 and sofosbuvir intracellular metabol-
ism in human cardiomyocytes.
Intracellular concentration of the metabolites and prodrugs in human
cardiomyocytes (pmol/106 cells SD)b
Compoundsa
DAPN-PD
Time (h)
Prodrugs
Nc
2’-C-Me-G
NTPc
2’-C-Me-GTP
4
8
4
8
4
8
4
8
70
51
21
3
1
1
6.2 0.1
4.6 0.5
NA
8.0 2.2
9.1 0.7
3.6 0.7
4.3 0.3
650 74
440 41
NA
18
16
NA
NA
3
2
28
43
2
1
IDX-184
2.9 0.7
5.4 0.3
410 42
540 29
NA
9.8 1.4
800 118
810 80
NA
BMS-986094
16
11
1
1
BLOQ
BLOQ
Sofosbuvir
(control)
56
42
3
4
BLOQ
BLOQ
24
64
1
1
NA
NA
aCells were incubated with each compound for 4 and 8 h at 10 mM.
bValues represent the mean SD of three replicates.
cFor DAPN-PD, N is 2’-C-Me-DAPN; for BMS-986094, N is 2’-C-Me-6-OMe-G; for sofosbuvir, N is 2’-F-2’-C-Me-U.
BLOQ, below limit of quantification. NA, not applicable.
intracellular metabolism of DAPN-PD, IDX-184 and BMS-986094 were
assessed in human cardiomyocytes.[35] exposed to 10 mM concentrations of
these prodrugs for 4 and 8 h. Intracellular metabolites and intact prodrug
levels were detected in cell extracts by LC-MS/MS (Table 4).
In human cardiomyocytes, sofosbuvir produced 24 and 65 pmol/million
cells of 2’-F-2’-C-Me-UTP at 4 h and 8 h respectively, while BMS-986094
produced a much higher level of 2’-C-Me-GTP (410 and 540 pmol/million
cells at 4 h and 8 h). The total NI-TP levels for BMS-986094 were signifi-
cantly higher than levels detected with DAPN-PD and IDX-184 at 4 h (9-
and 138-fold, respectively) and at 8 h (9- and 100-fold, respectively) (Figure
4). Furthermore, the level of intact BMS-986094 was also significantly

12
S. TAO ET AL.
Figure 4. Intracellular accumulation of total NI-TP metabolite and intact prodrug of DAPN-PD,
IDX-184, BMS-986094 and sofosbuvir at 4 and 8 h in human cardiomyocytes. (A) Intracellular
level of total NI-TP metabolite(s) of DAPN-PD, IDX-184, BMS-986094 and sofosbuvir and (B)
Intracellular level of intact prodrug of DAPN-PD, IDX-184, BMS-986094 and sofosbuvir at 4 h
and 8 h in human cardiomyocytes at (10 mM, 37 ꢁC). Values represent the mean of three repli-
cates. NI-TP: nucleoside 5’-triphosphate. For DAPN-PD, total NI-TP is 2’-C-Me-DAPN-TP and 2’-C-
Me-GTP. For IDX-184, NI-TP is 2’-C-Me-GTP. For BMS-986094, total NI-TP is 2’-C-Me-GTP and 2’-
C-Me-6-OMe-GTP. For sofosbuvir, NI-TP is 2’-F-2’-C-Me-UTP.
higher than levels of DAPN-PD and IDX-184 at 4 h (11- and 37-fold,
respectively) and at 8 h (15- and 82-fold, respectively). BMS-986094 pro-
duced the highest level of 2’-C-Me-GTP in human cardiomyocytes of any
cell line tested despite the fact that the incubations were done 5-fold less
concentrated. Interestingly, the levels in human cardiomyocytes for 2’-C-
Me-GTP from IDX-184 were among the lowest observed in any cell line
even when the 5-fold reduction in incubation concentration is considered.
Imaging of human cardiomyocytes after exposure to DAPN-PD, BMS-986094,
and IDX-184
To assess potential acute cardiotoxicity of DAPN-PD, BMS-986094 and
IDX-184 and determine if there is a correlate between 2’-C-Me-GTP levels
and contraction rate and rhythm, we recorded the cardiomyocytes beating
up to 48 h after treatment (2, 10 and 50 mM) each drug to observe the con-
traction and rhythm. For the control (sofosbuvir), no apparent change in
beating rate or rhythm at all three concentrations up to 48 h was observed
(Table 5). For BMS-986094, no clear change in beating rate or rhythm was
noted at 2 mM up to 48 h; however, at 10 mM, higher beating rates were
observed starting from 12 h compared with untreated cells. The higher
beating rate was more obvious at 24 and 48 h where the blank control and
SOF control beat rates have slowed notably. At 50 mM the cardiomyocytes
stopped beating within 2 h, resumed briefly at 12 and 24 h, but with very
unstable rhythm. For IDX-184, no apparent change in beating was observed

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
Table 5. Beating rate (beats/minute) of human cardiomyocytes after exposure to DAPN-PD,
IDX-184, BMS-986094 and sofosbuvir.
Compound
DAPN-PD
Conc. (mM)
2 h
4 h
34.0
23.5 0.7
21.0
31.5 0.7
30.0
8 h
37.0
26.0 1.4
22.0
37.0 1.4
34.0
12 h
36.0
25.5 0.7
21.0
36.0 1.4
34.0 1.4
34.5 0.7
24 h
48 h
2
33.5 0.7
24.5 0.7
21.5 0.7
32.0
30.0 1.4
33.0
31.0
31.0
0
36.5 2.1
34.0 1.4
32.5 0.7
32.5 1.7
0
0
0
34.0 1.4
25.5 0.7
27.5 0.7
25.5 0.7
17.0 0
24.0 1.4
25.0 1.4
27.5 0.7
26.5 0.7
35.5 2.1
10
50
2
10
50
2
10
50
2
10
50
0
0
0
0
19.0
0
IDX-184
0
32.5 0.7
31.0 1.4
34.5 4.9
32.5 0.7
34.5 0.7
0
0
0
0
0
0
31.5 0.7
30.5 0.7
34.5 0.7
34.5 0.7
BMS-986094
Sofosbuvir (control)
Blank control
36.0
36.0
32.0
0
0
31.0
0
0
0
35.0
0
0
0
34.0
33.0
30.0
0
0
0
0 0
35.0 1.4
33.0 1.4
38.5 0.7
35.0 1.4
34.5 0.7
34.2 1.6
36.5 0.7
33.5 0.7
33.0 1.4
34.2 3.3
25.5 0.7
23.0
0
32.0
0
29.5 0.7
28.6 1.5
23.5 0.7
23.6 1.9
31.8 0.8
up to 48 h at all three concentrations. And for DAPN-PD, we did not
observe beating changes at 2 mM. However, at 10 and 50 mM, the beating
rate dropped within 2 h then remained at a reduced rate out to 48 h. The
rhythm was also altered and irregular at these two higher concentrations.
Cardiomyocytes are non-dividing muscle cells with high-energy require-
ments for normal function which makes them more susceptible to mito-
chondrial toxicity.[36–38] The high level of the intact BMS-986094 prodrug
at 4 h and equal amounts at 8 h demonstrate a rapid saturation of the car-
diomyocytes and would likely maintain high levels of 2’-C-Me-GTP much
longer than either DAPN-PD (16 times lower at 8 h) or IDX-184 (83 times
lower at 8 h).[39] It is also worth highlighting that at 10 mM BMS-986094
caused a slight increase in beat rate while DAPN-PD at 10 mM caused the
beat rate to decrease indicating that the toxic effects of DAPN-PD may be
wholly or partially derived from different mechanisms.
Conclusions
As earlier studies[14,18] concluded, the extremely high levels of 2’-C-Me-
GTP in cardiomyocytes generated by incubation with BMS-986094 coupled
with its incorporation by human mitochondrial RNA polymerase
(POLRMT)[9,10,15] appears to play a major role in the clinically observed
adverse cardiac effects. Our earlier work[15] showed DAPN-TP to have a
similar incorporation level with POLRMT as 2’-C-Me-GTP, but its total
NI-TP level in cardiomyocytes was 9 times lower than that from BMS-
986094 after 4 and 8 h incubation. Thus, their similar level of toxicity
observed upon visual observation of beat rate and rhythm of dosed cardio-
myocytes would appear to result from a mechanism beyond or in combin-
ation with inhibition of POLRMT. Interestingly, no apparent changes to
beat rate or rhythm were observed for IDX-184 treated cardiomyocytes,
indicating that there may be a therapeutic window for IDX-184 in further

14
S. TAO ET AL.
human studies. In this study, we found cardiomyocyte imaging as a tool to
predict cardiotoxicity, to provide the most valuable technique to rapidly
predict cardiotoxicity prior to animal and eventually human studies.
Several subsequent animal studies in both rats and dogs were performed
with DAPN-PD to determine the validity of our in vitro results and estab-
lish a potential therapeutic window. Single dose escalation studies set the
maximum tolerated dose at a promising 1,000 mg/kg for both rats and
dogs. However, seven day repeat dosing in both dogs and rats set the no-
observed-adverse-effect-level (NOAEL) at <40 mg/kg/day with higher doses
having adverse effects on multiple organs and tissues including cardiac tis-
sue and function. Having determined a therapeutic windows of close to
ten, DAPN-PD preclinical development was abandoned.
Experimental protocols
Chemistry
General information
Reagents were purchased from commercial sources. Unless noted otherwise,
the materials used in the examples were obtained from readily available
commercial suppliers or synthesized by standard methods known to one
skilled in the art of chemical synthesis. Signal multiplicities are represented
by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadru-
plet), br (broad), bs (broad singlet), m (multiplet). All J-values are in Hz.
Isopropyl 3-(2-(((((S)-1-isopropoxy-1-oxopropan-2-
yl)amino)(perfluorophenoxy)phosphoryl)oxy)phenyl)propanoate (4)
A dry, N2 flushed, 100 L reactor was charged with dihydrocoumarin (3 kg,
20.2 mol) isopropyl alcohol (30.0 L) and H2SO4 (0.09 kg, 0.92 mol). The
mixture was stirred at room temperature for 12 hours and then the pH of
the mixture was adjusted to 6.5 ꢃ 7.0 by addition of NaHCO3 (6.21 kg,
73.9 mol). The resulting mixture was filtered through a pad of celite and
the solvent was removed under reduced pressure. To the residue was added
dichloromethane (6 L) and the mixture was dried over anhydrous Na2SO4
(3 kg). The mixture was filtered through a pad of silica gel, washed with
dichloromethane (18 L) and evaporated under reduced pressure to afford
3.8 kg (90.4%) of crude isopropyl ester which was used directly in the next
1
step. H-NMR (400 MHz, CDCl3) d 7.44 (s, 1 H), 7.09-7.15 (m, 2 H), 6.85-
6.90 (m, 2 H), 5.03 (m, 1 H), 2.92 (t, J ¼ 6.8 Hz, 2 H), 2.72 (t, J ¼ 6.8 Hz,
2 H), 1.23 (d, J ¼ 6.0 Hz, 6 H); LRMS Calcd for C12H17O3 (M þ 1)þ 209.12,
found 209.20.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
To a stirred solution of the above isopropyl ester (3.8 kg) in t-butyl
methyl ether (38 L) was added POCl3 (2.8 kg, 11.1 mol). The mixture was
cooled to ꢂ10 ꢁC and triethylamine (2.4 kg, 23.7 mol) was added dropwise.
After complete addition, the resulting mixture was allowed to warm to a
temperature between 15 and 30 ꢁC and stirred for an additional hour. The
mixture was cooled to ꢂ10 ꢁC and a solution of isopropyl L-alaninate
hydrochloride (3.06 kg, 18.3 mol) and triethylamine (3.7 kg, 36.6 mol) in
dichloromethane (14.3 L) was added dropwise to the reaction mixture and
the internal temperature was raised to ambient temperature. After the reac-
tion was complete, the mixture was filtered to remove the remaining salts
followed by evaporation of the filtrate. To the resulting residue was added
dichloromethane (26.6 L) and the mixture was cooled down to between 0
and 5 ꢁC. Pentafluorophenol (3.36 kg, 18.3 mol) and triethylamine (1.85 kg,
18.3 mol) in dichloromethane (3.8 L) were added dropwise to the reaction
mixture over 1 to 2 hours while maintaining the temperature between 15
and 30 ꢁC. After one additional hour, the mixture was cooled to a tempera-
ture between 0 and 5 ꢁC and 1 N HCl (30.4 L) was added and the layers
were separated. The organic layer was further washed with saturated
NaHCO3 (38 L X 2), brine (38 L X 2) and dried over anhydrous Na2SO4,
filtered and evaporated under reduced pressure to afford 4 (2.6 kg, 25.1%)
1
(97.2% purity, PR/PS ratio ¼ 54/46). H-NMR (Acetone-d6, 400 MHz) d
7.40-6.77 (m, 3 H), 5.04-4.93 (m, 2 H), 4.12-3.84 (m, 3 H), 3.05-2.80 (m,
2 H), 2.78-2.42 (m, 2 H), 1.45-1.28 (m, 3 H), 1.25-1.14 (m, 12 H).
(2R,3R,4R,5R)-2-(2-Amino-6-chloro-9H-purin-9-yl)-5-(hydroxymethyl)-3-
methyltetrahydrofuran-3,4-diol (6)
To a dry, N2 flushed, 100 L glass-lined reactor precooled (ꢂ10 ꢁC) contain-
ing a solution of 5 (4.0 kg, 6.89 mol), 2-amino-6-chloropurine (1.29 kg,
7.58 mol), and 1,8-diazabicycl[5.4.0]undec-7-ene (DBU) (3.15 kg, 20.67 mol)
in anhydrous acetonitrile (40 L) was added trimethylsilyl triflate (6.13 kg,
6.88 mmol) dropwise. After stirring at 0 ꢁC for 30 minutes, the reaction
mixture was heated at 65 ꢁC with stirring for an additional 3 h, allowed to
come to room temperature, then diluted with dichloromethane (40.0 L).
The resulting solution was washed with saturated aqueous sodium bicar-
bonate (20 L), filtered through a pad of celite and the layers were separated.
The organic phase was dried over anhydrous sodium sulfate (4.0 kg), fil-
tered and the volatiles were removed under reduced pressure. To the
resulting residue was added isopropyl alcohol (20.0 L) and the resulting
slurry was stirred at 50 ꢁC until all solids had dissolved. The homogeneous
solution was cooled to room temperature with stirring, after 2 hours the
resulting solid was collected by filtration and dried under vacuum. The

16
S. TAO ET AL.
tribenzoylated nucleoside (3.44 kg, 79.5%) was obtained and determined to
1
be 98.6% pure by HPLC analysis. H-NMR (CD3OD, 400 MHz) d 8.56 (s,
1 H), 6.01 (s, 1 H), 4.20 (d, J ¼ 8.8 Hz, 1 H), 4.07-3.98 (m, 2 H), 3.85 (dd,
J ¼ 12.6 Hz, J ¼ 3.0 Hz, 1 H), 0.97 (s, 3 H). 13C NMR (CD3OD, 100 MHz) d
20.2, 61.0, 73.4, 80.3, 84.3, 92.7, 124.9, 142.6, 151.7, 154.7, 161.6; LRMS
Calcd for C11H15ClN5O4 (M þ 1)þ 316.08, found 316.21.
To a dry, N2 flushed, 100 L glass-lined reactor at room temperature con-
taining a solution of tribenzoylated nucleoside (1.1 g, 1.66 mmol) from
above was treated with a saturated solution of ammonia in methanol
(66.0 L). The reaction mixture was stirred at room temperature for 12 h to
reach completion. After removal of the solvent under reduced pressure, to
the residue was added methanol (8.25 L) and the resulting mixture was
heated at 50 ꢁC with stirring for 30 minutes. After cooling to room tem-
perature, dichloromethane (33.0 L) was added and stirring was continued
for 2 h. The resulting solid was collected by filtration and dried under vac-
uum for 12 h. Compound 6 was obtained (1.16 kg, 69.9%) and determined
1
to be 97.4% pure by HPLC analysis. H-NMR (400 MHz, DMSO-d6): 8.01
(s, 1 H), 6.73 (s, 2 H), 5.81 (s, 2 H), 5.76 (d, J ¼ 2.8 Hz, 1 H), 5.18-5.22 (t,
J ¼ 6.8 Hz), 5.05 (s, 1 H), 0.78 (s, 3 H), 3.98-4.02 (m, 1 H), 3.78-3.84 (m,
2 H), 3.63-3.67 (m, 1 H). LC/MS calcd. for C11H16N6O4 (M þ 1)þ 296.12,
observed: 297.32.
Isopropyl 3-(2-(((((2 R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-3,4-
dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-isopropoxy-1-
oxopropan-2-yl)amino)phosphoryl)oxy)phenyl)propanoate (7)
A dry, N2 flushed, 100 L reactor was charged with 6 (0.95 kg, 3.0 mol), 4
(2.56 kg, 4.5 mol) and tetrahydrofuran (14.3 L) then the mixture was cooled
to 0 ꢁC. Next, t-BuMgCl (4.51 L, 9.02 mol, 2 M solution in tetrahydrofuran)
was added while maintaining the internal temperature below 30 ꢁC. After
complete addition, stirring was continued for 6 hours at ambient tempera-
ture. The mixture was cooled to between 0 and 5 ꢁC and NH4Cl (9.5 L,
5 wt% aq.) was added and the organic solvent was removed under reduced
pressure. To the remaining aqueous layer was added 9.5 L of ethyl acetate
and the layers were separated. The organic layer was washed with saturated
NaHCO3 (9.5 L X 2), brine (9.5 L X 2) and dried over anhydrous Na2SO4
(3.8 kg), filtered and evaporated under reduced pressure. The residue was
purified by silica-gel column chromatography (dichloromethane/methanol
¼ 30/1 to 10/1, v/v) to afford 7 (1.68 kg, 80% yield) (91.7% purity, Rp/Sp
1
ratio ¼ 51/49). HNMR (400 MHz, CD3OD) d 8.16 (s, 1 H), 8.17 (s, 1 H),
7.37–7.04 (m, 4 H), 5.99 (s, 1 H), 5.97 4.97–4.91 (m, 2 H), 4.60–4.49 (m,
2 H), 4.32–4.18 (m, 2 H), 3.95–3.90 (m, 1 H), 2.99–2.96 (m, 2 H), 2.61–2.57

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
(m, 2 H), 1.34–1.31 (m, 3 H), 1.21–1.15 (m, 12 H), 1.00–0.99 (m, 3 H);
31PNMR (162 MHz, CD3OD)
d
3.80, 3.72; LRMS calcd for
C29H40ClN6O10P (M þ H)þ 699.23, found 699.40.
Isopropyl 3-(2-(((((2 R,3R,4R,5R)-5-(2,6-diamino-9H-purin-9-yl)-3,4-dihydroxy-
4-methyltetrahydrofuran-2-yl)methoxy)(((S)-1-isopropoxy-1-oxopropan-2-
yl)amino)phosphoryl)oxy)phenyl)propanoate (9)
A dry, N2 flushed, 100 L reactor was charged with 7 (1.68 kg, 2.4 mol),
NaN3 (0.23 kg, 3.5 mol), tetrabutylammonium iodide (0.44 kg, 1.2 mol),
dimethylformamide (8.4 L) and the mixture was heated to between 65 and
80 ꢁC for 18 hours. The mixture was allowed to cool to room temperature
and BuBr (0.42 kg, 3.1 mol) was added. After stirring for 1 hour, the solvent
was removed under reduced pressure.
To the resulting mixture was added ethyl acetate (58.8 L) and aqueous
NaCl (42 L, 10 wt% aqueous) and the layers were separated. To the sepa-
rated organic layer was added 0.6 kg of Na2SO4 and 0.4 kg of decolorizing
charcoal. The resulting mixture was then stirred for an hour, filtered
through a pad of celite and the filtrate was evaporated under reduced pres-
sure. To the residue were added isopropyl alcohol (16.8 L), 20% Pd(OH)2
on carbon (0.34 kg) and hydrogen gas was charged (1.0 to 1.5 kgf/cm2)
with stirring for 11 hours at room temperature. The mixture was filtered
through a pad celite and the filtrate was concentrated under reduced pres-
sure. Ethyl acetate (25 L) and NaCl (17 L, 10 wt% aqueous) were added to
the above residue and the layers were separated. The separated organic
layer was washed with brine and dried over anhydrous Na2SO4 (0.6 kg), fil-
tered and evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (ethyl acetate/methanol, 30/1 to 10/1, v/
v) to afford 9 (847 g, 52% over two steps) (99.0% purity, Rp/Sp ratio ¼
51:49). The two diasteroisomers were separated on small scale using silica
gel column chromatography with a gradient of 0 to 10% MeOH in CH2Cl2.
1
14c-Sp: HNMR (400 MHz, CD3OD) d 7.86 (s, 1 H), 7.37 (d, J ¼ 8.4 Hz,
1 H), 7.25 (d, J ¼ 7.2 Hz, 1 H), 7.17 (dt, J ¼ 8.0 Hz, J ¼ 1.6 Hz, 1 H), 7.08 (t,
J ¼ 7.2 Hz, 1 H), 5.94 (s, 1 H), 5.05-4.84 (m, 2 H), 4.62-4.46 (m, 2 H), 4.22
(s, 2 H), 3.95-3.91 (m, 1 H), 2.99 (t, J ¼ 8.0 Hz, 2 H), 2.60 (t, J ¼ 8.0 Hz,
2 H), 1.33 (d, J ¼ 7.2 Hz, 3 H), 1.20 (d, J ¼ 6.4 Hz, 3 H), 1.19 (d, J ¼ 6.0 Hz,
3 H), 1.15 (d, J ¼ 6.0 Hz, 3 H), 1.14 (t, J ¼ 6.4 Hz, 3 H), 0.96 (s, 3 H);
31PNMR (162 MHz, CD3OD) d 5.08; LRMS calcd for C29H43N7O10P
1
(M þ H)þ 680.28, found 680.12. 14c-Rp: HNMR (400 MHz, CD3OD) d
8.03 (s, 1 H), 7.38 (d, J ¼ 8.0 Hz, 1 H), 7.26 (d, J ¼ 7.6 Hz, 1 H), 7.18 (dt,
J ¼ 7.6 Hz, J ¼ 1.6 Hz, 1 H), 7.10 (t, J ¼ 7.2 Hz, 1 H), 5.91 (s, 1 H), 4.95-4.79
(m, 2 H), 4.53-4.46 (m, 2 H), 4.18 (s, 2 H), 3.95-3.91 (m, 1 H), 2.99 (t,

18
S. TAO ET AL.
J ¼ 8.0 Hz, 2 H), 2.61 (t, J ¼ 8.0 Hz, 2 H), 1.35 (d, J ¼ 7.2 Hz, 3 H), 1.19-1.16
(m, 12 H), 0.99 (s, 3 H); 31PNMR (162 MHz, CD3OD) d 5.02; LRMS calcd
for C29H43N7O10P (M þ H)þ 680.28, found 680.14.
Acknowledgments
This paper is dedicated to our friend and colleague Professor Akira Matsuda’s in celebra-
tion of his 70th birthday.
Funding
This work was supported in part by CFAR (Center for AIDS Research, Emory University)
grant NIH5P30-AI-50409 (to RFS).
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