Synthesis of methylenebis (Phosphonate) analogues of 2-, 4-, and 6-pyridones of nicotinamide adenine dinucleotide

Article abstract of DOI:10.1080/15257770.2011.575909

The synthesis of metabolically stable methylenebis(phosphonate) analogues of 2-, 4-, and 6-pyridones of nicotinamide adenine dinucleotide (NAD) is reported. In contrast to natural pyrophosphates, these NAD analogues are able to penetrate the cell membrane and can be used as probes in cellular assays. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.575909

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Synthesis of Methylenebis(Phosphonate)
Analogues of 2-, 4-, and 6-Pyridones of
Nicotinamide Adenine Dinucleotide
Dr Krzysztof Felczak a & Krzysztof W. Pankiewicz a
a Center for Drug Design , University of Minnesota , Minneapolis ,
Minnesota , USA
Published online: 02 Sep 2011.
To cite this article: Dr Krzysztof Felczak & Krzysztof W. Pankiewicz (2011) Synthesis
of Methylenebis(Phosphonate) Analogues of 2-, 4-, and 6-Pyridones of Nicotinamide
Adenine Dinucleotide, Nucleosides, Nucleotides and Nucleic Acids, 30:7-8, 512-523, DOI:
10.1080/15257770.2011.575909
To link to this article: http://dx.doi.org/10.1080/15257770.2011.575909
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Nucleosides, Nucleotides and Nucleic Acids, 30:512–523, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.575909
SYNTHESIS OF METHYLENEBIS(PHOSPHONATE)
ANALOGUES OF 2-, 4-, AND 6-PYRIDONES OF NICOTINAMIDE
ADENINE DINUCLEOTIDE
Krzysztof Felczak and Krzysztof W. Pankiewicz
Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA
The synthesis of metabolically stable methylenebis(phosphonate) analogues of 2-, 4-, and 6-
2
pyridones of nicotinamide adenine dinucleotide (NAD) is reported. In contrast to natural pyrophos-
phates, these NAD analogues are able to penetrate the cell membrane and can be used as probes in
cellular assays.
Keywords NAD analogues; IMPDH; NAD kinase
In recent years, intensive studies of nicotinamide adenine dinucleotide
(NAD) dependent biological processes have a revealed a variety of functions
of this molecule in nature and generated broad interest in evaluation of its
potential in biology and medicine. Metabolic pathways leading to the biosyn-
thesis and biodegradation of nicotinamide adenine dinucleotides, NAD and
NADP, are under detailed investigation; the focus is on understanding their
importance for the well-being of living organisms. The search for NAD-based
potential therapeutics is now a fast growing field with numerous drug can-
didates in clinical studies.[1]
In addition to its role as a co-enzyme in cellular redox reaction, NAD also
serves as a substrate for a number of NAD-utilizing enzymes, such as PARP
[poly(ADP-ribose) polymerase] and sirtuins (NAD-dependent deacetylases),
that release a significant amount of nicotinamide. Nicotinamide is recycled
in the cell into NAD or is excreted in urine (almost exclusively) as N-methyl-
nicotinamide. At the end of the 1970s, the 4-pyridone-3-carboxamide ri-
bonucleoside (1, Figure 1) had been isolated from the urine of patients with
chronic myelogenous leukemia (CML) and proposed as a potential marker
of progression of this disease.[2]
Received 14 March 2011; accepted 24 March 2011.
Address correspondence to Krzysztof W. Pankiewicz, Center for Drug Design, University of Min-
nesota, 516 Delaware St. SE, Minneapolis, MN 55455, USA. E-mail: panki001@umn.edu
512

Methylenebis(Phosphonate) Analogues
513
FIGURE 1 4-Pyridone-3-carboxamide riboside, its 5ꢁ-triphosphate, and 4-pyridone NAD(P) analogues.
It has been suggested that nucleoside 1 was likely formed due to oxida-
tion of NAD at the 4-position of the pyridine moiety to give the 4-pyridone
derivative of NAD 4. Subsequent cleavage of 4 by cellular phosphodiesterases
to the corresponding 5ꢁ-monophosphates and dephosphorylation of the
monophosphate 2 afforded 1.[3] Recently, a high concentration (compa-
rable to that of ATP) of 4-pyridone-3-carboxamide-1-β-D-ribonucleoside 5ꢁ-
triphosphate (3) has been reported in erythrocytes of patients with chronic
renal failure (CRF).[4] It was also found that mammalian adrenodoxin reduc-
tase, involved in steroid and vitamin D biosynthesis, oxidized NADP to give
the pyridone 6.[5,6] The same compound has been reported to be formed
as a result of incubation of Mycobacterium tuberculosis oxidoreductase FprA
with NADP.[7] However, it is not known whether 4-pyridone-NADP has any
physiological role.
The synthesis of 2-pyridone-3- and -5-carboxamide ribonucleosides 8 and
11, respectively (Figure 2), have been earlier reported by the authors[8] and
others.[9,10,11] The corresponding 2- and 6-pyridone-NAD analogues (9 and
12, respectively) were also prepared by enzymatic and chemical methods.[12]
As with 4-pyridone-NAD 4, these compounds might be of interest as potential
donors of the pyridone bases because accumulation of modified pyridone
bases in urine of CRF patients is well documented.[13]
Natural pyrophosphate analogues of 2-, 4-, and 6-pyridone-NAD (9, 4,
and 12, X = O) are metabolically unstable, cannot penetrate the cell mem-
brane, and therefore have not been evaluated in cellular assays. In contrast,
FIGURE 2 2-Pyridone-3-, and 5-carboxamide ribosides and their corresponding NAD analogues.

514
K. Felczak and K. W. Pankiewicz
structurally close methylenebis(phosphonate) analogues of NAD (e.g., ben-
zamide adenine dinucleotide analogue[14]) are resistant to metabolic degra-
dation and are able to enter the cell. Thus, we report herein the synthesis
of bis(phosphonate) analogues of 2-, 4-, and 6-pyridone-NAD (10, 5, and 13,
respectively) for evaluation in cellular assays and as potential starting mate-
rials for further modification, i.e. phosphorylation. A conversion of 5 to 7
may afford the potent and selective inhibitor of the mycobacterium FprA
enzyme.
For synthesis of title compounds we used our standard proce-
dure of N,N-disopropylcarbodiimide (DIC) promoted coupling of 2ꢁ, 3ꢁ-
O-isopropylidene protected pyridone-carboxamide riboside with 2ꢁ3ꢁ-O-
isopropylidene-adenosin-5ꢁ-yl-methylenebis(phosphonate) (14).[15,16]
SCHEME 1 Synthesis of 4-pyridone-NAD analogue.
To our surprise, when the isopropylidene protected 4-pyridone-3-
carboxamide nucleoside 15 was used as the starting material (Scheme 1),
a mixture of the desired methylenebis-(phosphonate) 4-pyridone-NAD ana-
logue 5 and 4-pyridone-3-nitrile derivative 18 was obtained, which could
not be separated on a preparative high performance liquid chromatography
(HPLC) column. Our earlier application of the identical coupling procedure
for the synthesis of methylenebis(phosphonate) analogue of benzamide ade-
nine dinucleotide (BAD) or tiazofurin adenine dinucleotide (TAD, Figure 3)
afforded the desired compounds in high yield and no formation of the cor-
responding 3-nitrile derivatives was observed.
Although dehydration of carboxyamides to nitriles in the presence of
DIC is a known reaction, apparently pyridone carboxyamides are more
sensitive to such dehydration than the aromatic benzamide ring of ben-
zamide riboside or thiazole-4-carboxamido group of tiazofurin. Indeed, the
FIGURE 3 Methylenebis(phosphonate) analogues of BAD and TAD.

Methylenebis(Phosphonate) Analogues
515
similar DIC coupling of the 2ꢁ, 3ꢁ-O-isopropylidene protected 2-pyridone-
5-carboxamide nucleoside 19 also afforded a mixture of the desired 6-
pyridone-NAD 13 and the corresponding nitrile derivative 22 in the ratio
of 1:2 (Scheme 2). Fortunately, these compounds were separable on HPLC
column and the desired 6-pyridone-NAD 13 was eluted first, followed by the
nitrile derivative 22.
SCHEME 2 Synthesis of 6-pyridone-NAD analogue.
Obviously, DIC coupling of adenosine-5ꢁ-methylenebis(phosphonate) 14
with pyridone-3-carboxylic esters ribosides instead of the carboxyamido ribo-
side derivatives would not result in dehydration. In addition, a conversion of
esters to amides by treatment with ammonia should be a simple and efficient
reaction.
As shown in Scheme 3, we modified our coupling procedure and pre-
pared 4-pyridone NAD 5 using methyl 2ꢁ, 3ꢁ-O-isopropylidene-4-pyridone-
3-carboxylate ribonucleoside 25 as the starting material for DIC coupling
with adenosine-5ꢁ-methylenebis(phosphonate) 14. Compound 25 was syn-
thesized from unreported methyl 4-pyridone-3-carboxylate ribonucleoside
24, which we prepared by Vorbrugen coupling of 4-pyridone-3-carboxylic
acid with 1, 2, 3, 5,-tetra-O-acetyl-β-D-ribofuranose to give 23 and its further
deacetylation followed by conversion to the methyl ester 24.
SCHEME 3 Synthesis of 4-pyridone-NAD analogue 5 from methyl ester of 4-pyridone-3-carboxylic acid
riboside.
The protected methylenebis(phosphonate) analogue 26 was obtained
in good yield. After conversion of 26 into the corresponding carboxyamido
derivative by treatment with methanolic ammonia, the isopropylidene pro-
tecting groups were removed with formic acid.

516
K. Felczak and K. W. Pankiewicz
In the same manner, coupling of methyl 2ꢁ, 3ꢁ-O-isopropylidene-
2-pyridone-3-carboxylate
ribonucleoside
27
with
adenosine-5ꢁ-
methylenebis(phosphonate) 14 followed by treatment with diazometane
and deprotection afforded a good yield of the desired 2-pyridone-NAD 10
(Scheme 4).
SCHEME 4 Synthesis of 2-pyridone-NAD analogue.
We evaluated our pyridone-NAD analogues as potential inhibitors of
human and mycobacterium IMP-dehydrogenase and NAD kinase. None
showed inhibition of IMPDH. Interestingly, 6-pyridone 13 inhibited both
human and mycobacterium NAD kinase equally (50% at the concentration
of 0.5 mM) but 2-pyridone 10 was found to be 8-fold more potent against
the human enzyme (80%) than Mycobacterium tuberculosis NAD kinase (9%)
at the same concentration.
It was recently found that 4-pyridone-3-carboxamide-adenine dinu-
cleotide phosphate 6 is not only a good ligand and a competitive inhibitor
of Mycobacterium tuberculosis oxidoreductase FprA (Ki = 1.2 µM) but also
inhibits Toxoplasma gondi FNR enzyme (Ki = 30 µM).[6] Thus, our synthetic
bis(phosphonate) analogue 5 when converted into the 2ꢁ-monophosphate
prodrug could be of therapeutic interest.
EXPERIMENTAL
General Methods
All commercial reagents (Sigma-Aldrich, St. Louis, MO, USA; Afla Aesar,
War Hill, MA, USA) were used as provided unless otherwise indicated. An
anhydrous solvent dispensing system (J. C. Meyer, Laguna Beach, CA, USA)
using two packed columns of neutral alumina was used for drying THF, Et2O,
and CH2Cl2,; two packed columns of molecular sieves were used to dry DMF.
Solvents were dispensed under argon. Analytical HPLC was performed on a
Varian (Lake Forest, CA, USA) Microsorb column (C18, 5 µ, 4.6 × 250 mm)
with a flow rate of 0.5 mL/min whereas preparative HPLC was performed on
a Varian Dynamax column (C18, 8 µ, 41.4 × 250 mm) with a flow rate of 40
mL/min. An isocratic or linear gradient of 0.1 M triethylammonium bicar-
bonate (TEAB) and aqueous MeCN (70%) were used. Flash chromatography
was performed using Teledyne ISCO CombiFlash Rf equipped with Teledyne

Methylenebis(Phosphonate) Analogues
517
ISCO RediSep Rf flash column silica cartridges (www.isco.com/combiflash)
with the indicated solvent system. Nuclear magnetic resonance (NMR) spec-
tra were recorded on a Varian 600 MHz with Me4Si, DDS, or signals from
residual solvent as the internal standard for 1H and external H3PO4 for 31P.
Chemical shifts are reported in ppm, and signals are described as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), brs (broad singlet), and
dd (double doublet). Values given for coupling constants are first order.
High-resolution mass spectra were recorded on an Agilent TOF II TOF/MS
instrument equipped with either an ESI or APCI interface. All reactions
were performed under an inert atmosphere of dry Ar in oven dried (150◦C)
glassware. The purity of the compounds was ≥95%, and it was determined
by HPLC using the above-mentioned analytical column.
4-Pyridone-3-(carboxylic acid)-1-(2, 3, 5-tri-O-acetyl
β-D-ribofuranoside) (23)
To the suspension of 4-pyridone-3-carboxylic acid[17] (1.39 g, 10 mmol)
and TAR (3.64 g, 11.28 mmol) in dry CH3CN (25 mL) BSTFA (6.5 ml,
25.7 mmol) was added. The mixture was stirred at room temperature for
1.5 hours. Excess silylating reagent was removed and the residue was dis-
solved in dry CH3CN (25 mL). The clear solution was cooled and TMSOTfl
(2.1 mL, 11.5 mmol) was added dropwise. The mixture was stirred at room
temperature for 6 hours, cooled in an ice, and sat. aq. NaHCO3 was added
dropwise to pH 5–6. Solvents were evaporated and the residue was dissolved
in water. 1 N HCL was added to adjust pH to 1–2, and the mixture was ex-
tracted with CH2Cl2. Organic fractions were combined, washed with water,
dried over MgSO4, and concentrated to give thick oil, which was purified on
silica gel flash column chromatography using CH2Cl2:MeOH (9:1). Crude
product was crystallized from EtOH to give 23 (3.8 g, 96%). 1H NMR (CDCl3)
δ 15.19 (s, 1H), 8.79 (d, J = 2.17 Hz, 1H), 7.78 (dd, J = 2.24, 7.72 Hz, 2H),
6.77 (d, J = 7.53 Hz, 1H), 5.62 (d, J = 5.07 Hz, 1H), 5.29 (m, 1H), 5.24 (m,
1H), 4.54 (m, 1H), 4.49 (dd, J = 2.40, 12.85, 1H), 4.39 (dd, J = 2.11, 12.85,
1H), 2.26 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H). HRMS C17H18NO10 396.0936
(M-H)−, found 396.0921.
Methyl 4-pyridone-3-carboxylate-1-β-D-ribofuranoside (24)
Compound 23 was suspended in methanolic ammonia (20 mL of 7 M
NH3), stirred at room temperature for 48 hours, evaporated, dissolved
in water, and passed through a column of Dowex 50 W(H+). The elu-
ents were evaporated, the residue co-evaporated with toluene, dissolved
in MeOH, cooled in an ice bath, and treated with TMS-CH2N2 until
small excess of TMSCH2N2 was present. When the reaction was com-
pleted, AcOH (2 mL) was added and the mixture was stirred at room

518
K. Felczak and K. W. Pankiewicz
temperature for 15 minutes, evaporated and co-evaporated with toluene,
EtOH, and finally purified by flash chromatography on RediSep using
1
CHCl3:MeOH (8 : 2) as eluent to give 24 (534 mg 62%). H NMR
(D2O) δ 8.71 (d, J = 2.25 Hz, 1H), 7.91 (dd, J = 2.25 Hz, J = 7.64
Hz, 1H), 6.55 (d, J = 7.64 Hz, 1H), 5.52 (d, J = 5.26 Hz, 1H), 4.22 (t,
J = 5.18 Hz, 1H), 4.17 (t, J = 4.64 Hz, 1H), 4.12 (m, 1H), 3.80 (dd,
J = 2.96 Hz, J = 12.84 Hz, 1H), 3.74 (s, 3H), 3.83 (dd, J = 3.96 Hz, J =
12.84 Hz, 1H). HRMS calcd for C12H16NO7 286.0921 (M + H)+, found
286.0930.
Methyl 4-pyridone-3-carboxylate -1-(2,
3-O-isopropylidene-β-D-ribofuranoside) (25)
Dimethoxypropane (1 mL, 8.5 mmol) was added to the suspension of 24
(485 mg, 1.7 mmol) in acetone (10 mL), followed by p-TsOH monohydrate
(300 mg, 1.7 mmol). The mixture was stirred at room temperature for 3
hours, excess of sat. aq. NaHCO3 was added and the mixture was diluted
with water and extracted with CH2Cl2. Extracts were combined, dried over
MgSO4, filtered, and dried in vaccuo to give 25 as a white foam (233 mg,
42%). 1H NMR (CDCl3) δ 8.39 (d, J = 2.30 Hz, 1H), 7.84 (dd, J = 2.38 Hz, J =
7.82 Hz, 1H), 6.35 (d, J = 7.67 Hz, 1H), 5.40 (d, J = 3.36 Hz, 1H), 4.90 (dd,
J = 1.23 Hz, J = 4.70 Hz, 1H), 4.69 (dd, J = 3.33 Hz, J = 5.85 Hz, 1H), 4.57
(bs, 1H), 4.47 (m, 1H), 3.98 (dd, J = 1.76 Hz, J = 11.76 Hz, 1H), 3.84 (dd,
J = 1.87 Hz, J = 11.76 Hz, 1H), 3.79 (s, 3H). HRMS calcd for C15H20NO7
326.1234 (M + H)+, found 336.1242.
4-Pyridone-3-carboxamide -1-β-D-ribofuranoside (1)
Compound 23 (3.8 g, 9.6 mmol) was dissolved in 100 mL of MeOH,
cooled in an ice bath, and TMS-CH2N2 (2M in diethyl ether) was slowly
added until the reaction was complete and slight excess of TMS-CH2N2
was present. The mixture was stirred for 15 minutes, 1.5 mL of AcOH was
added, and solvents were evaporated and coevaporated with toluene to leave
methyl-4-pyridone-3-carboxylate-1-(2, 3, 5, -tri-O-acetyl-β-D-ribofuranoside)
as a thick oil, which was used directly in the next step. The crude ester
was dissolved 7 M ammonia in MeOH (90 mL) in a pressure vessel. The
mixture was stirred for 3 days at room temperature, evaporated, and the
residue was purified by flash chromatography on silica gel with CH2Cl2 and
CH2Cl2:MeOH (95:5 and next 8:2) as eluents. A crystallization from MeOH
1
gave 1, which was washed with acetone and dried (0.875 g, 34%). H NMR
(D2O) δ 8.68 (d, J = 2.15 Hz, 1H), 7.93 (dd, J = 2.15 Hz, J = 7.65 Hz, 1H),
6.60 (d, J = 7.71 Hz, 1H), 5.54 (d, J = 5.57 Hz, 1H), 4.22 (t, J = 5.40 Hz, 1H),
4.17 (m, 1H), 4.13 (m, 1H), 3.79 (dd, J = 3.05 Hz, 12.82 Hz, 1H), 3.71 (dd,
J = 4.05 Hz, 12.82 Hz, 1H). 13C NMR (D2O) δ 61.1, 70.2, 75.8, 86.2, 97.2,

Methylenebis(Phosphonate) Analogues
519
118.5, 120.6, 139.0, 143.0, 168.0, 179.4. HRMS C11H15N2O6 271.0925 (M +
H)+, found 271.0936.
4-Pyridone-3-carboxyamide -1-(2, 3-O-isopropylidene
β-D-ribofuranoside) (15)
p-TsOH (190 mg, 1 mmol) was added to the suspension of nucleoside
1 (0.27 g, 1 mmol) in 5 mL of acetone, followed by dimethoxypropane
(0.6 mL, 5 mmol). The mixture was stirred at room temperature overnight.
Aq. ammonia (2 mL) was added and the whole mixture was evaporated,
diluted with water, extracted with AcOEt; extracts were dried with MgSO4,
filtered, and evaporated to leave a colorless foam, which was purified by
preparative TLC on silica gel with CHCl3:MeOH 9:1 to give 15 (205 mg,
66%) 1H NMR (CDCl3) δ 9.87 (brs, 1H), 8.82 (d, J = 2.25 Hz, 1H), 7.74 (dd,
J = 2.25 Hz, J = 7.64 Hz, 1H), 6.58 (d, J = 7.60 Hz, 1H), 5.75 (brs, 2H), 5.51
(m, 1H), 4.94 (m, 1H), 4.72 (m, 1H), 4.49 (m, 1H), 4.04 (dd, J = 1.90 Hz, J
= 12.20 Hz, 1H), 3.89 (dd, J = 3.55 Hz, J = 12.20 Hz, 1H), 1.61 (s, 3H), 1.36
(s, 3H). HRMS C14H19N2O6 311.1238 (M + H)+, found 311.1230.
4-Pyridone-NAD (5)
To the solution of 14 (88 mg, 0.13 mmol) in dry pyridine (0.7 mL), DIC
(80 µL, 0.52 mmol) was added, the mixture was stirred overnight at room
temperature., compound 25 (43 mg, 0.13 mmol) was added, and stirring
was continued for 70 hours at 65◦C. After cooling to room temperature, a
mixture of water (0.2 mL) and TEA (0.1 mL) was added, and the reaction
was heated for 6 hours at 65◦C. When the reaction was complete, the mixture
was evaporated and co-evaporated with EtOH, the residue was dissolved in
methanolic ammonia (7 M) and stirred at room temperature for 3 days, evap-
orated and the crude intermediate 26 was dissolved in 50% HCOOH (5 mL)
and stirred overnight at room temperature. Solvents were evaporated, and
the residue was co-evaporated with a mixture of EtOH and water. The crude
product was purified by preparative HPLC with 70% MeCN/0.1 M TEAB
(2-100 linear gradient). Fractions containing product were evaporated, the
residue was dissolved in water, and then passed through a column of Dowex
50 WX8-200 (Na+ form). After elution with water, ultraviolet (UV) active
fractions were collected, and lyophilized to give 5 (sodium salt) as a white
powder (32 mg, 20%). 1H NMR (D2O) δ 8.35 (d, J = 2.22 Hz, 1H), 8.21 (s,
1H), 8.02 (s, 1H),7.86 (dd, J = 2.22 Hz, J = 7.60 Hz, 1H), 6.39 (d, J = 7.65
Hz, 1H), 5.89 (d, J = 5.20 Hz, 1H), 5.39 (d, J = 5.97 Hz, 1H), 4.55 (t, J =
5.14 Hz, 1H), 4.36 (t, J = 4.82 Hz, 1H), 4.27 (m, 1H), 4.22 (m, 4H), 4.05 (m,
2H), 2.13 (t, J = 19.91 Hz, 2H). 31P NMR (D2O) δ 18.20 (d, J = 10.45 Hz),
18.18 (d, J = 10.45 Hz). HRMS C22H28N7O14P2 676.1175 (M-H)−, found
676.1134.

520
K. Felczak and K. W. Pankiewicz
The DIC promoted coupling starting from 15 afforded an inseparable
mixture of 2ꢁ, 3ꢁ-O-isopropylidene protected nitrile derivative 16 and car-
boxyamido analogue 17. Repeated attempts for HPLC separation of com-
pound 18 and 5 after deisopropylidenetion were also unsuccessful.
2-Pyridone-5-carboxyamide-1-(2, 3-O-isopropylidene
β-D-ribofuranoside) (19)
To the suspension of the 2-pyridone-5-carboxyamide-1-β-D-
ribofuranoside[8] (0.27 g, 1 mmol) in 5 mL of acetone, p-TsOH (190
mg, 1 mmol) was added, followed by dimethoxypropane (0.6 mL, 5 mmol).
The mixture was stirred at room temperature overnight. To the clear
solution aq. ammonia was added and the whole mixture evaporated, diluted
with water, and extracted with AcOEt; extracts were dried over MgSO4
and evaporated to leave a crude product, which was purified by flash
chromatography on silica gel with CHCl3:MeOH 9:1. Fractions containing
the desired product were combined, and concentrated in vacuo to give 19
as an oil (250 mg, 81%). 1H NMR (CDCl3) δ 8.38 (d, J = 2.18 Hz, 1H), 7.31
(dd, J = 9.40 Hz, J = 2.18 Hz, 1H), 6.56 (d, J = 9.40 Hz, 1H), 5.83 (d, J =
1.90 Hz, 1H), 5.74 (brs, 2H), 5.07 (m, 1H), 5.00 (m, 1H), 4.42 (m, 1H),
4.01 (dd, J = 1.80 Hz, J = 12.35 Hz, 1H), 3.82 (dd, J = 3.60 Hz, J = 12.35
Hz, 1H), 1.60 (s, 3H), 1.36 (s, 3H). HRMS calcd for C14H19N2O6 311.1238
(M+H)+, found 311.1249.
6-Pyridone-NAD (13)
2ꢁ,3ꢁ-O-Isopropylidene-adenosin-5ꢁ-yl-methylenebis(phosphonate) (14,
88 mg, 0.13 mmol) and 19 (40.3 mg, 0.13 mmol) were dissolved in dry
pyridine (0.7 mL), and to the resulting solution, DIC (80 µL, 0.52 mmol)
was added. The resulting mixture was stirred at room temperature for 1 hour
and then at 65◦C for 69 hours, cooled to room temperature, treated with a
mixture of H2O and TEA (200 µL and 100 µL, respectively) and heated at
65◦C for 6 hours. The mixture was evaporated and co-evaporated with EtOH.
The residue was dissolved in 80% TFA, stirred vigorously for 3 hours at 0◦C,
evaporated, co-evaporated with EtOH, and dissolved in a small amount of
MeOH. The crude mixture was purified by preparative HPLC with 70%
MeCN/0.01M TEAB (5–15 linear gradient) to give two main products. Frac-
tions containing the major product were pooled, evaporated to dryness,
co-evaporated with H2O and EtOH, dissolved in a small amount of H2O,
and passed through a small column of Dowex 50 WX8-200 (Na+), combined
and lyophilized to give 22 as a white powder (20 mg, 22%). 1H NMR (D2O)
δ 8.33 (s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.40 (d, J = 9.29 Hz, 1H), 6.32
(d, J = 9.42 Hz, 1H), 5.86 (d, J = 5.46 Hz, 1H), 5.62 (s, 1H), 4.42 (t, J =
5.48 Hz, 1H), 4.35 (t, J = 4.92 Hz, 1H), 4.21 (m, 1H), 4.14 (m, 2H), 4.05
(m, 4H), 3.97 (m, 1H), 2.14 (t, J = 19.95 Hz, 2H). 31P NMR (D2O) δ 18.32

Methylenebis(Phosphonate) Analogues
521
(d, J = 10.49 Hz), 18.22 (d, J = 10.49 Hz). HRMS calcd for C22H26N7O13P2
658.1069 (M-H)−, found 686.1084. Fractions containing the minor product
were pooled, evaporated to dryness, co-evaporated with H2O and EtOH, and
converted into the Na+ salt as described above to give 13 as a white powder
(10 mg, 11%). 1H NMR (D2O) δ 8.27 (s, 1H), 8.10 (s, 1H), 8.02 (s, 1H), 7.62
(d, J = 9.05 Hz, 1H), 6.34 (d, J = 9.60 Hz, 1H), 5.82 (d, J = 5.82 Hz, 1H),
5.75 (d, J = 2.48 Hz, 1H), 4.50 (t, J = 5.75 Hz, 1H), 4.31 (t, J = 4.82 Hz, 1H),
4.16 (m, 2H), 4.07 (m, 6H), 4.02 (m, 1H), 2.12 (t, J = 19.95 Hz, 2H). 31P
NMR (D2O) δ 18.39 (d, J = 10.84 Hz), 18.18 (d, J = 10.84 Hz). HRMS calcd
for C22H28N7O14P2 676.1175 (M-H)−, found 676.1105.
Methyl 2-pyridone-3-carboxylate -1-(2,3-O-isopropylidene
β-D-ribofuranoside) (27)
2-Pyridone-3-(carboxylic acid)-1-(β-D-ribofuranoside)[8] (1.2 g, 4.5
mmol) was suspended in MeOH (50 mL) and cooled in an ice bath and
TMS-CH2N2 (3.37 mL, 6.75 mmol) was added until small excess of reagent
was present. The mixture was stirred at room temperature until the reaction
was complete. Solvents were evaporated, and the residue dried and used
in the next step without further purification. Dried crude methyl ester was
suspended in acetone (30 mL) and to this suspension dimethoxypropane
(3 mL) was added followed by p-toluenesulphonic acid monohydrate (860
mg, 4.5 mmol). The mixture was stirred at room temperature for 4 hours,
neutralized with NaHCO3, concentrated, diluted with water, and extracted
with EtOAc. Extracts were combined, washed with H2O, dried over MgSO4,
and finally concentrated to leave semisolid 27 (0.5g, 34%). 1H NMR (CDCl3)
δ 8.18 (dd, J = 2.20 Hz, J = 7.10 Hz, 1H), 7.69 (dd, J = 2.10 Hz, J = 6.76 Hz,
1H), 6.30 (t, J = 7.17 Hz, 1H), 5.68 (d, J = 2.23 Hz, 1H), 5.18 (dd, J = 2.28
Hz, J = 6.40 Hz, 1H), 5.09 (dd, J = 3.93 Hz, J = 6.40 Hz, 1H),4.71 (brs, 1H),
4.33 (m, 1H), 3.93 (dd, J = 2.52 Hz, J = 12.20 Hz, 1H), 3.87 (s, 3H), 3.83
(dd, J = 3.82 Hz, J = 12.20 Hz, 1H), 1.57 (s, 3H), 1.33 (s, 3H). HRMS calcd
for C15H20NO7 326.1234 (M + H)+, found 326.1246.
2-Pyridone-NAD (10)
Compound 14 (88 mg, 0.13 mmol) was dissolved in dry pyridine (0.7
mL) and DIC (80 µL, 0.52 mmol) was added. To this mixture compound
27 (43 mg, 0.13 mmol) was added and the reaction mixture was heated for
69 hours at 65◦C. After the reaction was completed H2O (200 µL) was added
and the mixture was heated for 6 hours. The mixture was evaporated and co-
evaporated with EtOH, the residue was dissolved in 7M NH3 in MeOH, and
stirred at room temperature for 3 days. The residue was dissolved in a mixture
of HCOOH and water 1:1 (10 mL), and stirred overnight at room tempera-
ture, evaporated and purified by preparative HPLC with 70% MeCN/0.01M

522
K. Felczak and K. W. Pankiewicz
TEAB (5–15 linear gradient). Fractions containing the product were col-
lected, evaporated to dryness, co-evaporated with H2O and EtOH, dissolved
in a small amount of H2O, and passed through a small column of Dowex 50
WX8-200 (Na+). Fractions containing the desired product were combined
and lyophilized to give 10 as a white powder (20 mg, 23%). 1H NMR (D2O)
δ 8.16 (s, 1H), 7.99 (s, 1H), 7.90 (m, 2H), 6.29 (t, J = 7.04 Hz, 1H), 5.78
(m, 2H), 4.33 (t, J = 4.62 Hz, 1H), 4.19 (t, J = 5.05 Hz, 1H), 4.14 (m, 2H),
4.08 (m, 2H), 3.97 (m, 3H), 3.91 (m, 1H), 2.10 (t, J = 20.04 Hz, 2H). 31P
NMR (D2O) δ 18.08 (d, J = 11.06 Hz), 18.06 (d, J = 11.06 Hz). HRMS
C22H28N7O14P2 676.1175 (M-H)−, found 676.1112.
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