"Abbreviated" NAD+analogues containing a phosphonate function

Article abstract of DOI:10.1135/cccc19961538

"Abbreviated" NAD⁺ analogues with anionic phosphonate function as a part of the link between the adenine and nicotinamide moieties, 9-(2-phosphonomethoxyethyl)adenine 2-(3-carbamoylpyridinium)ethyl ester (1a), (R)- and (S)-9-(2-phosphonomethoxypropyl)adenine 2-(3-carbamoylpyridinium)ethyl ester (1b and 1c), (RS)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine 2-(3-carbamoylpyridinium)ethyl ester (1d), and (S)- and (R)-1-[3-(adenin-9-yl)-2-phosphonatomethoxypropyl]-3-carbamoylpyridinium (2a and 2b), were prepared by multistep syntheses using the Zincke reaction in the last step.

Full text of DOI:10.1135/cccc19961538

1538
Hockova, Masojidkova, Holy:
“ABBREVIATED” NAD+ ANALOGUES CONTAINING
A PHOSPHONATE FUNCTION
1
2
Dana HOCKOVA , Milena MASOJIDKOVA and Antonin HOLY
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic; e-mail: 1 lasice@uochb.cas.cz, 2 saman@uochb.cas.cz
Received April 18, 1996
Accepted July 12, 1996
Dedicated to the memory of Dr Zdenek Arnold.
“Abbreviated” NAD+ analogues with anionic phosphonate function as a part of the link between the
adenine and nicotinamide moieties, 9-(2-phosphonomethoxyethyl)adenine 2-(3-carbamoylpyridi-
nium)ethyl ester (1a), (R)- and (S)-9-(2-phosphonomethoxypropyl)adenine 2-(3-carbamoylpyridi-
nium)ethyl ester (1b and 1c), (RS)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine 2-(3-carbamoyl-
pyridinium)ethyl ester (1d), and (S)- and (R)-1-[3-(adenin-9-yl)-2-phosphonatomethoxypropyl]-3-car-
bamoylpyridinium (2a and 2b), were prepared by multistep syntheses using the Zincke reaction in the
last step.
Key words: NAD+ analogues; Phosphonate nucleotide analogues; Zincke reaction.
Nicotinamide adenine dinucleotide (NAD) and related nicotinamide adenine dinucleo-
tide phosphate are involved in many enzymatic redox reactions catalyzed by dehy-
drogenases and reductases. The coenzymatic activity of NAD depends not only on the
nicotinamide moiety (that is during the redox reaction reduced to its 1,4-dihydro form
and vice versa) but also on the rest of the molecule. This “nonfunctional” part that
remains chemically unaltered, can induce significant conformational changes and is
important for the interaction with the enzyme. A great number of NAD analogues with
modified various parts of the molecule has been prepared to investigate the structure–
activity relationship. Considering all the metabolic roles of NAD, its analogues may
have very interesting biological properties1.
We studied such modification of the coenzyme, where the adenine and the nicotin-
amide moieties are preserved, but the ribosediphosphoribose link is replaced by an
acyclic chain2,3. Functionalized alkyl chains were chosen as linking elements because
of their flexibility and greater stability compared to ribose.
In this paper we report on compounds containing, contrary to the previous analo-
gues2, an anionic phosphonate function that either compensates the positive charge of
the carbamoylpyridinium moiety (compounds 1a–1d) or even changes the total charge
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

NAD+ Analogues
1539
of the molecule to a negative one (compounds 2a and 2b), to some extent similarly as
in the NAD+ molecule. As parent structures for the design of such NAD+ analogues we
have chosen the acyclic phosphonate nucleotide analogues 9-(2-phosphonomethoxy-
ethyl)adenine (PMEA, 3a), 9-(2-phosphonomethoxypropyl)adenine (PMPA, 3b, 3c)
and 9-(3-hydroxy-2-phosphonomethoxypropyl)adenine (HPMPA), that exhibit mani-
fold biological effects4,5
.
Compounds of the first group (1a–1d) (Scheme 1) contain a phosphonomethyl mono-
ester function as a part of the acyclic chain joining the adenine and nicotinamide
moieties. Starting with the corresponding phosphonates 3a–3c (PMEA, ref.6, (R)- or
(S)-PMPA, ref.7) and 3′-O-benzyl HPMPA (3d), prepared by alkylation of adenine with
diisopropyl {[1-(benzyloxy)methyl]-2-(p-toluenesulfonyloxy)}ethoxymethylphospho-
nate8 followed by deprotection of the phosphonate group, the 2-azidoethyl monoesters
NH
NH
2
2
N
N
N
N
a
N
N
N
N
R
R
*
*
O
O
P(O)(OH)
O
P
OCH CH N
2 2 3
2
OH
3
4
b
NH
N
2
N
N
c
N
R
*
O
O
P
OCH CH NH
2 2 2
OH
5
NH
N
2
O
4"
N
N
N
8
R
H
R
H
1,5
3,4
5"
6"
NH
2
2
a
b
c
d
a
b
c
d
2"
9’
R
N
1’
*
(R)-CH
(R)-CH
3
(S)-CH
3
3
O
2’
8’
(S)-CH
3
O
P
O
7’
CH OH
CH OCH C H
2 2 6 5
2
4’
O
1
a) 1. (Cl CO) CO, 2.HOCH CH N ; b) Pd-C/H ;
3
2
2
2
2 3
c) DBU/3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1540
Hockova, Masojidkova, Holy:
4a–4d were prepared by the standard method using triphosgene/dimethylformamide for
activation9. Reduction of the azido group (and simultaneous cleavage of the benzyl
group in the derivative 4d) over palladium on charcoal afforded the aminoethyl esters
5a–5d. These amino derivatives were converted into the NAD+ analogues 1a–1d by
reaction with 3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride in absolute methanol
(Zincke reaction)10. Since in the presence of the acid group the amino function is proto-
nated, the Zincke reaction, based on nucleophilic attack by amine at the position 2 of the
activated pyridinium salt, had to be carried out in the presence of a strong non-nucleo-
philic amine base, e.g. 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
In the stereoisomeric compounds 2a and 2b (Scheme 2) the acyclic chain is function-
alized with a free phosphonomethoxy moiety. The first step in the synthesis of the
(R)-enantiomer 2b was the alkylation of adenine by diisopropyl (S)-[1-azidomethyl-2-
(p-toluenesulfonyloxy)]ethoxymethylphosphonate11 in the presence of cesium carbonate to
obtain (R)-azido derivative 6b. The (S)-enantiomer 6a was prepared from diisopropyl
(S)-HPMPA12 via its O-tosyl derivative that was converted to the azido compound by
NH
NH
N
NH
2
OCH PO(OiPr)
2
2
2
2
TsO
N
3
N
N
N
N
N
a
N
N
N
b
NH
N
N
*
O
P
OH
O
P
N
3
iPrO
O
iPrO
O
OiPr
OiPr
6
Configuration
Configuration
2,6
7
(S)
(R)
(R)
(S)
a
b
a
b
c
NH
NH
N
2
2
N
N
N
N
d
N
8
2
N
O
N
2"
5’
1’
*
*
O
NH
O
P
N
NH
2
2
2’
4’
HO
P
O
4"
HO
O
6"
5"
OH
O
7
2
a) 1. TsCl/Py, 2. NaN /DMF; b) CsCO /DMF; c) 1. Pd-C/H , 2. Me SiBr/CH CN;
3
3
2
3
3
d) DBU/3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride
SCHEME 2
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

NAD+ Analogues
1541
reaction with sodium azide. After reduction of 6a and 6b over palladium on charcoal,
the protecting isopropyl ester groups were removed by action of bromotrimethylsilane
to form amino analogues of HPMPA 7a and 7b. The last step of the reaction sequence
was the Zincke reaction of derivatives 7a and 7b that under the above-mentioned con-
ditions gave the desired compounds 2a and 2b.
The structures of the resulting NAD+ analogues 1a–1d, 2a and 2b were confirmed by
1H NMR (Table I) and 13C NMR spectra (Table II). High resolution mass spectrometry
was used to determine the molecular formulas instead of microanalyses, because of the
extremely hygroscopic character of the compounds.
The cytostatic activity was tested on L-1210 mouse leukemia cells. Neither of the
compounds exhibited significant cytostatic activity and neither was cytotoxic. In vitro
activities against DNA viruses and retroviruses were determined at the Rega Institute
for Medical Research (Professor E. De Clercq, Head), Catholic University Leuven, Bel-
gium; these results, together with in vitro enzyme studies, will be published in detail
and compared with other types of NAD+ analogues in a separate communication.
EXPERIMENTAL
Unless stated otherwise, solvents were evaporated at 40 °C/2 kPa and compounds were dried at 2 kPa
over P2O5. Melting points were determined on a Kofler block and are uncorrected. Analytical TLC
were performed on Silufol UV254 plates (Kavalier Votice, Czech Republic). Preparative TLC were
carried out on 40 × 17 × 0.4 cm loose layer plates of silica gel containing UV indicator. Paper elec-
trophoresis was carried out on a Whatman No. 3 MM paper at 40 V/cm for 1 h in 0.05 M triethylam-
monium hydrogen carbonate (TEAB) at pH 7.5; the electrophoretical mobilities are referenced to
uridine 3′-phosphate. NMR spectra were measured on a Varian Unity 500 spectrometer (500 MHz for
1H and 125.7 MHz for 13C NMR) in hexadeuteriodimethyl sulfoxide referenced to the solvent signals
(2.5 ppm for 1H and 39.7 ppm for 13C NMR), or in deuterium oxide containing sodium deuteroxide
with sodium 3-(trimethylsilyl)propanesulfonate as an internal standard for 1H NMR and dioxane as
an external standard for 13C NMR (δ(dioxane) 66.86 ppm). Mass spectra were measured on a ZAB-EQ
(VG Analytical) spectrometer using the FAB technique (ionization by Xe, accelerating voltage 8 kV,
glycerol matrix). Dimethylformamide was distilled from P2O5 and stored over molecular sieves (4A).
The cytostatic assays were performed in the laboratory of Dr I. Votruba at this Institute.
9-(3-Benzyloxy-2-phosphonomethoxypropyl)adenine (3d)
A stirred mixture of adenine (1.0 g, 7.4 mmol), cesium carbonate (1.2 g, 3.7 mmol) and dimethylform-
amide (10 ml) was heated at 120 °C for 1 h. After addition of diisopropyl {[1-(benzyloxy)methyl]-2-(p-toluene-
sulfonyloxy)}ethoxymethylphosphonate8 (2.0 g, 3.9 mmol) in dimethylformamide (15 ml), the heating
at 120 °C was continued for 4 h. The reaction mixture was taken down, and the product of alkylation
was isolated by preparative thin-layer chromatography on silica gel (15% of methanol in chloroform).
Then acetonitrile (60 ml) and bromotrimethylsilane (5 ml) were added to the dried residue and the
mixture was stirred at room temperature for 24 h. The solvent was evaporated, the residue was codistilled
with acetonitrile, dissolved in water and made alkaline with triethylamine. After evaporation in vacuo
the residue was deionized on Dowex 50X8 (H+ form, 70 ml). The product-containing fraction was
evaporated and applied onto a column of Dowex 1 (acetate form, 50 ml). The column was washed
with water and 0.25 M acetic acid (0.5 l), then the Dowex was stirred with hot 5 M acetic acid and
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1542
Hockova, Masojidkova, Holy:
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

NAD+ Analogues
1543
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1544
Hockova, Masojidkova, Holy:
filtered off. The filtrate was evaporated and the residue codistilled with water to obtain the pure com-
pound 3d (1.05 g, 68%), m.p. 240–243 °C. 1H NMR spectrum (D2O): 8.09 s, 1 H (H-2); 8.18 s, 1 H
(H-8); 7.32–7.24 m, 3 H and 7.1–6.9 m, 2 H (arom. H); 4.43 dd, 1 H, J(1′a,2′) = 4.4, J(gem) = 14.7
(Ha-1′); 4.39 dd, 1 H, J(1′b,2′) = 5.6, J(gem) = 14.7 (Hb-1′); 4.34 d, 1 H, J(gem) = 11.5 (CH2Ph); 3.95 m,
1 H (H-2′); 3.66 dd, 1 H, J(P,CHa) = 9.5, J(gem) = 12.0 (Ha-4′); 3.63 dd, 1 H, J(P,CHb) = 9.8,
J(gem) = 12.0 (Hb-4′); 3.61 dd, 1 H, J(5′a,2′) = 4.5, J(gem) = 11.0 (Ha-5′); 3.48 dd, 1 H, J(5′b,2′) =
3.9, J(gem) = 11.0 (Hb-5′). Mass spectrum (FAB), m/z (rel.%): 394 (80) [M + H]+. For C16H20N5O5P
(393.3) calculated: 48.86% C, 5.13% H, 17.80% N, 7.87% P; found: 48.53% C, 5.07% H, 17.69% N,
8.25% P.
TABLE II
13C NMR spectra (δ, ppm) of compounds 1 and 2
Atom
1aa
1bb
1cb
1da
2ab
2ba
C-2 150.80
C-4 148.79
C-5 118.11
C-6 151.16
C-8 143.01
152.01
148.81
117.60
154.98
142.96
47.50
151.84
148.42
117.28
154.75
142.71
47.45
152.54
149.89
118.63
156.12
141.79
44.05
152.35
148.60
117.67
155.18
142.80
43.10
152.69
149.96
118.54
156.19
142.98
43.06
C-1′
C-2′
43.25
70.26 d
75.44 d
75.37 d
80.59 d
77.55 d
76.90 d
J(P,C) = 11.7 J(P,C) = 11.8 J(P,C) = 11.7 J(P,C) = 10.0 J(P,C) = 11.7 J(P,C) = 6.3
64.90 d 62.71 d 62.67 d 66.40 d 66.61 d 68.28 d
J(P,C) = 160.2 J(P,C) = 160.2 J(P,C) = 161.1 J(P,C) = 156.3 J(P,C) = 155.3 J(P,C) = 150.5
C-4′
C-5′
C-7′
–
–
–
–
62.04
61.76
61.52 d
J(P,C) = 4.0
62.28 dc
J(P,C) = 5.9
62.01 dc
J(P,C) = 5.9
62.26 d
J(P,C) = 4.0
62.33
–
–
–
–
–
–
63.53 d
J(P,C) = 6.0
62.03 d
J(P,C) = 6.0
62.80 d
J(P,C) = 5.9
C-8′
C-9′
–
15.51
15.49 d
60.75
J(P,C) = 3.1
C-2′′ 147.20
C-3′′ 133.87
C-4′′ 146.16
C-5′′ 127.88
C-6′′ 144.09
C=O 162.96
146.63
133.18
144.30c
127.89
144.04c
165.03
146.52
133.18
144.22c
127.84
143.94c
164.88
146.93
134.11
145.20
127.79
143.94
163.82
147.14
133.24
144.56
127.80
144.38
165.36
147.43
134.32
145.89
127.56
144.68
163.79
a
b
c
(CD3)2SO or D2O. Signals may be reversed.
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NAD+ Analogues
1545
Synthesis of Azidoethyl Phosphonates 4. General Procedure
Triphosgene (3.12 g, 10.5 mmol) was slowly added at room temperature to a mixture of compound 3
(3.5 mmol) and dimethylformamide (100 ml). After stirring for 1 h at room temperature, 2-azido-
ethanol (10 ml) was added and the mixture was allowed to stand for 20 h. The solution was neu-
tralized with TEAB and then concentrated ammonia (30 ml) was added. After standing overnight and
filtration, the solvent was evaporated, the residue was codistilled with water and applied onto a Dowex
50X8 column (H+ form, 100 ml). The column was washed with water and the product was eluted
with aqueous ammonia (1 : 20). The fraction containing the product was evaporated and applied on
a column of Dowex 1 (acetate form, 50 ml). Elution with a gradient of 0–0.5 M acetic acid afforded
the compound 4.
9-(2-Phosphonomethoxyethyl)adenine 2-azidoethyl ester (4a): Yield 1.02 g (85%), m.p. 183–185 °C.
1H NMR spectrum ((CD3)2SO): 8.185 s, 1 H (H-2); 8.18 s, 1 H (H-8); 7.73 brs, 2 H (NH2); 4.34 t,
2 H, J(1′,2′) = 5.1 (H-1′); 3.94 dt, 2 H, J(7′, 8′) = 4.9, J(POCH) = 7.3 (H-7′); 3.89 t, 2 H, J(2′,1′) =
5.1 (H-2′); 3.70 d, 2 H, J(P,CH) = 8.3 (H-4′); 3.38 t, 2 H, J(8′,7′) = 4.9 (H-8′). Mass spectrum
(FAB), m/z (rel.%): 343 (100) [M + H]+. For C10H15N8O4P . 1/3 H2O (348.3) calculated: 34.48% C,
4.53% H, 32.17% N, 8.89% P; found: 34.87% C, 4.65% H, 32.00% N, 8.50% P.
(R)-9-(2-Phosphonomethoxypropyl)adenine 2-azidoethyl ester (4b): Yield 1.13 g (90%), m.p. 136–
1
138 °C. H NMR spectrum ((CD3)2SO): 8.35 s, 1 H (H-2); 8.37 s, 1 H (H-8); 7.68 brs, 2 H (NH2);
4.45 dd, 1 H, J(1′a,2′) = 3.0, J(gem) = 14.9 (Ha-1′); 4.29 dd, 1 H, J(1′b,2′) = 7.6, J(gem) = 14.9
(Hb-1′); 4.00 m, 1 H (H-2′); 3.79–3.85 m, 2 H, (H-7′); 3.75 dd, 1 H, J(P,CHa) = 9.1, J(gem) = 13.4
(Ha-4′); 3.53 dd, 1 H, J(P,CHb) = 9.8, J(gem) = 13.4 (Hb-4′); 3.35 m, 1 H and 3.30 m, 1 H (H-8′);
1.25 d, 3 H, J(9′, 2′) = 6.3 (H-9′). Mass spectrum (FAB), m/z (rel.%): 357 (100) [M + H]+.
(S)-9-(2-Phosphonomethoxypropyl)adenine 2-azidoethyl ester (4c): Yield 1.20 g (96%), m.p. 137–
1
138 °C. H NMR spectrum ((CD3)2SO): 8.37 s, 2 H (H-2 and H-8); 7.70 brs, 2 H (NH2); 4.46 dd, 1 H,
J(1′a,2′) = 2.9, J(gem) = 14.7 (Ha-1′); 4.29 dd, 1 H, J(1′b,2′) = 7.8, J(gem) = 14.7 (Hb-1′); 4.01 m,
1 H (H-2′); 3.77-3.86 m, 2 H, (H-7′); 3.76 dd, 1 H, J(P,CHa) = 9.0, J(gem) = 13.4 (Ha-4′); 3.50 dd,
1 H, J(P,CHb) = 9.8, J(gem) = 13.4 (Hb-3′); 3.35 m, 1 H and 3.30 m, 1 H (H-5′); 1.24 d, 3 H,
J(6′,2′) = 6.3 (H-6′). Mass spectrum (FAB), m/z (rel.%): 357 (100) [M + H]+.
(RS)-9-(3-Hydroxy-2-phosphonomethoxypropyl)adenine 2-azidoethyl ester (4d): Yield 1.26 g
(77%), m.p. >250 °C. 1H NMR spectrum ((CD3)2SO): 8.23 s, 1 H (H-2); 8.13 s, 1 H (H-8); 7.36–
7.20 m, 5 H (arom. H); 4.46 s, 2 H (CH2Ph); 4.38 dd, 1 H, J(1′a,2′) = 3.9, J(gem) = 14.2 (Ha-1′); 4.25 dd,
1 H, J(1′b,2′) = 6.3, J(gem) = 14.2 (Hb-1′); 3.96 m, 1 H (H-2′); 3.79 m, 2 H, (H-7′); 3.58 m, 2 H
(H-8′); 3.50–3.30 m, 4 H (H-4′) and (H-9′). Mass spectrum (FAB), m/z (rel.%): 463 (40) [M + H]+.
For C18H23N8O5P (462.4) calculated: 46.75% C, 5.01% H, 24.24% N, 6.70% P; found: 46.51% C,
5.26% H, 23.97% N, 6.50% P.
Synthesis of Aminoethyl Phosphonates 5. General Procedure
The corresponding compound 4 (2 mmol) was hydrogenated in methanol (70 ml) over 10% palla-
dium on charcoal (0.6 g) with stirring for 24 h at room temperature. The mixture was filtered through
a Celite pad. The catalyst was washed with hot methanol and hot water. The filtrate was evaporated
and compounds 5a–5c were obtained as colourless foams, 5d after crystallization from aqueous ethanol
as a solid.
1
9-(2-Phosphonomethoxyethyl)adenine 2-aminoethyl ester (5a): Yield 0.57 g (90%). H NMR spec-
trum ((CD3)2SO): 8.18 s, 1 H (H-2); 8.13 s, 1 H (H-8); 7.19 brs, 2 H (NH2); 4.29 t, 2 H, J(1′,2′) =
5.2 (H-1′); 3.84 m, 2 H (H-7′); 3.82 t, 2 H, J(2′,1′) = 5.2 (H-2′); 3.50 brs, 2 H (NH′2); 3.47 d, 2 H,
J(P,CH) = 8.3 (H-4′); 2.85 t, 2 H, J(7′,8′) = 5.0 (H-8′). Mass spectrum (FAB), m/z (rel.%): 317 (100)
[M + H]+.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1546
Hockova, Masojidkova, Holy:
1
(R)-9-(2-Phosphonomethoxypropyl)adenine 2-aminoethyl ester (5b): Yield 0.41 g (62%). H NMR
spectrum (D2O): 8.23 s, 1 H (H-2); 8.20, 1 H (H-8); 4.38 dd, 1 H, J(1′a,2′) = 3.2, J(gem) = 14.9 (Ha-1′);
4.24 dd, 1 H, J(1′b,2′) = 7.6, J(gem) = 14.9 (Hb-1′); 4.05–3.97 m, 3 H (H-2′ and H-7′); 3.75 dd,
1 H, J(P,CHa) = 9.3, J(gem) = 13.4 (Ha-4′); 3.58 dd, 1 H, J(P,CHb) = 9.8, J(gem) = 13.4
(Hb-4′); 3.06 t, 2 H, J(7′,8′) = 5.1 (H-8′); 1.23 d, 3 H, J(9′,2′) = 6.3 (H-9′). Mass spectrum (FAB), m/z (rel.%): 331 (60)
[M + H]+.
(S)-9-(2-Phosphonomethoxypropyl)adenine 2-aminoethyl ester (5c): Yield 0.49 g (75%). 1H NMR
spectrum (D2O): 8.21 s, 1 H (H-2); 8.18, 1 H (H-8); 7.70 brs, 2 H (NH2); 4.36 dd, 1 H, J(1′a,2′) = 3.1,
J(gem) = 14.7 (Ha-1′); 4.22 dd, 1 H, J(1′b,2′) = 7.8, J(gem) = 14.7 (Hb-1′); 3.98 m, 1 H (H-2′); 3.72 dd, 1 H,
J(P,CHa) = 9.0, J(gem) = 13.4 (Ha-4′); 3.56 q, 2 H, J(7′,8′) = 5.2, J(POCH) = 6.6 (H-7′); 3.46 dd, 1 H,
J(P,CHb) = 9.8, J(gem) = 13.4 (Hb-4′); 2.57 t, 2 H, J(7′,8′) = 5.2 (H-8′); 1.23 d, 3 H, J(9′,2′) = 6.3
(H-9′). Mass spectrum (FAB), m/z (rel.%): 331 (100) [M + H]+.
(RS)-9-(3-Hydroxy-2-phosphonomethoxypropyl)adenine 2-aminoethyl ester (5d): Yield 0.37 g
1
(53%), m.p. 225–227 °C. H NMR spectrum (D2O): 8.17 s, 1 H (H-2); 8.16, 1 H (H-8); 4.41 dd, 1 H,
J(1′a,2′) = 4.0, J(gem) = 14.9 (Ha-1′); 4.35 dd, 1 H, J(1′b,2′) = 7.6, J(gem) = 14.9 (Hb-1′); 3.89 m,
1 H (H-2′); 3.85 m, 3 H (Ha-9′ and H-7′); 3.76 dd, 1 H, J(P,CHa) = 9.3, J(gem) = 13.4 (Ha-4′); 3.59 dd,
1 H, J(9′b,2′) = 4.3, J(gem) = 12.5 (Hb-9′); 3.53 dd, 1 H, J(P,CHb) = 9.8, J(gem) = 13.4 (Hb-4′);
3.10 t, 2 H, J(7′,8′) = 5.0 (H-8′). Mass spectrum (FAB), m/z (rel.%): 347 (30) [M + H]+. For
C11H19N6O5P . 1.5 H2O (373.3) calculated: 35.39% C, 5.94% H, 22.51% N; found: 35.06% C, 5.68% H,
22.54% N.
Diisopropyl (S)-9-(3-Azido-2-phosphonomethoxypropyl)adenine (6a)
p-Toluenesulfonyl chloride (1.48 g, 7.8 mmol) and 4-dimethylaminopyridine (10 mg) were added at
–10 °C to a stirred solution of diisopropyl (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine (1.5 g,
3.9 mmol) in pyridine (30 ml). After standing at room temperature overnight, water (10 ml) was
added and the mixture was taken down. The residue was taken up in chloroform (100 ml), washed
with water and dried over magnesium sulfate. After filtration, the solvent was evaporated and sodium
azide (1.4 g, 22 mmol) and dimethylformamide (30 ml) were added. The mixture was stirred for 4 h
at 100 °C, filtered while hot and the filtrate taken down. The residue was codistilled with toluene and
the product was purified by preparative thin-layer chromatography. Yield 0.8 g (50%) of compound 6a,
1
m.p. 110–112 °C. H NMR spectrum ((CD3)2SO): 8.14 s, 1 H (H-2); 8.08, 1 H (H-8); 7.22 brs, 2 H
(NH2); 4.53 dsept, 1 H and 4.48 dsept, 1 H, J(CH,CH3) = 6.1, J(P,OCH) = 7.6 (2 × POCH); 4.35 dd,
1 H, J(1′a,2′) = 4.2, J(gem) = 14.6 (Ha-1′); 4.29 dd, 1 H, J(1′b,2′) = 6.6, J(gem) = 14.6 (Hb-1′); 4.04 m,
1 H (H-2′); 3.89 dd, 1 H, J(P,CHa) = 9.0, J(gem) = 13.7 (Ha-4′); 3.85 dd, 1 H, J(P,CHb) = 9.5,
J(gem) = 13.7 (Hb-4′); 3.65 dd, 1 H, J(5′a,2′) = 4.2, J(gem) = 13.4 (Ha-5′); 3.30 dd, 1 H, J(5′b,2′) = 5.4,
J(gem) = 13.4 (Hb-5′); 1.21 d, 3 H, 1.18 d, 3 H, 1.17 d, 3 H and 1.13 d, 3 H, J(CH3,CH) = 6.1 (6 × CH3).
Mass spectrum (FAB), m/z (rel.%): 413 (100) [M + H]+. For C15H25N8O4P (412.4) calculated: 43.68% C,
6.11% H, 27.18% N; found: 43.72% C, 5.88% H, 26.61% N.
Diisopropyl (R)-9-(3-Azido-2-phosphonomethoxypropyl)adenine (6b)
A stirred mixture of adenine (1.3 g, 9.6 mmol), cesium carbonate (1.6 g, 4.9 mmol) and dry di-
methylformamide (40 ml) was heated at 120 °C for 1 h. After addition of diisopropyl (S)-[1-azido-
methyl-(2-p-toluenesulfonyloxy)]ethoxymethylphosphonate11 (4.4 g, 9.8 mmol) in dimethylformamide
(20 ml), the heating at 120 °C was continued for 6 h. The reaction mixture was taken down and the
product 6b (2.0 g, 50%) was obtained by chromatography on a column of silica gel (150 ml, 2% of
1
methanol in chloroform), m.p. 107–110 °C. H NMR spectrum ((CD3)2SO): 8.15 s, 1 H (H-2); 8.10,
1 H (H-8); 7.30 brs, 2 H (NH2); 4.53 dsept, 2 H, J(CH,CH3) = 6.1, J(P,OCH) = 7.6 (2 × POCH);
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

NAD+ Analogues
1547
4.36 dd, 1 H, J(1′a,2′) = 4.2, J(gem) = 14.6 (Ha-1′); 4.29 dd, 1 H, J(1′b,2′) = 6.6, J(gem) = 14.6
(Hb-1′); 4.04 m, 1 H (H-2′); 3.89 dd, 1 H, J(P,CHa) = 9.0, J(gem) = 13.9 (Ha-4′); 3.85 dd, 1 H,
J(P,CHb) = 9.5, J(gem) = 13.9 (Hb-4′); 3.65 dd, 1 H, J(5′a,2′) = 4.1, J(gem) = 13.4 (Ha-5′); 3.30 dd,
1 H, J(5′b,2′) = 5.1, J(gem) = 13.4 (Hb-5′); 1.21 d, 3 H, 1.18 d, 3 H, 1.16 d, 3 H and 1.12 d, 3 H,
J(CH3,CH) = 6.1 (6 × CH3). Mass spectrum (FAB), m/z (rel.%): 413 (80) [M + H]+. For
C15H25N8O4P (412.4) calculated: 43.68% C, 6.11% H, 27.18% N; found: 43.52% C, 5.99% H,
26.93% N.
(R)-9-(3-Amino-2-phosphonomethoxypropyl)adenine (7a)
and (S)-9-(3-Amino-2-phosphonomethoxypropyl)adenine (7b)
Compound 6a (0.82 g, 2 mmol) was hydrogenated in methanol (70 ml) over 10% palladium on char-
coal (0.6 g) with stirring for 24 h at room temperature. The mixture was filtered through a pad of
Celite. The catalyst was washed with hot methanol and hot water (100 ml each). The filtrate was
evaporated and the residue was dried in vacuo and codistilled with acetonitrile. Acetonitrile (20 ml)
and bromotrimethylsilane (2 ml) were added and the mixture was stirred at room temperature for 24 h.
The solvent was evaporated, the residue was codistilled with acetonitrile, dissolved in water and
made alkaline with triethylamine. After evaporation in vacuo the residue was deionized on Dowex
50X8 (H+ form, 70 ml). The product-containing fraction was evaporated and applied on a column of
Dowex 1 (acetate form, 50 ml). Elution with a linear gradient of acetic acid (0–0.5 M, 1 l each) and
1
evaporation afforded compound 7a (0.4 g, 66%). H NMR spectrum (D2O): 8.23 s, 1 H (H-2); 8.21,
1 H (H-8); 7.30 brs, 2 H (NH2); 4.57 dd, 1 H, J(1′a,2′) = 4.1, J(gem) = 15.1 (Ha-1′); 4.43 dd, 1 H,
J(1′b,2′) = 5.4, J(gem) = 15.1 (Hb-1′); 4.16 m, 1 H (H-2′); 3.77 dd, 1 H, J(P,CHa) = 9.5, J(gem) = 12.7
(Ha-4′); 3.56 dd, 1 H, J(P,CHb) = 9.5, J(gem) = 12.7 (Hb-4′); 3.28 dd, 1 H, J(5′a,2′) = 2.9, J(gem) = 13.4
(Ha-5′); 2.86 dd, 1 H, J(5′b,2′) = 9.8, J(gem) = 13.4 (Hb-5′). Mass spectrum (FAB), m/z (rel.%): 303
(100) [M + H]+.
1
Compound 7b was obtained by the same procedure; yield 0.3 g (50%). H NMR spectrum (D2O):
8.24 s, 1 H (H-2); 8.22, 1 H (H-8); 7.32 brs, 2 H (NH2); 4.58 dd, 1 H, J(1′a,2′) = 4.2, J(gem) = 15.1
(Ha-1′); 4.43 dd, 1 H, J(1′b,2′) = 5.4, J(gem) = 15.1 (Hb-1′); 4.18 m, 1 H (H-2′); 3.76 dd, 1 H,
J(P,CHa) = 9.4, J(gem) = 12.7 (Ha-4′); 3.58 dd, 1 H, J(P,CHb) = 9.5, J(gem) = 12.7 (Hb-4′); 3.29 dd,
1 H, J(5′a,2′) = 2.9, J(gem) = 13.4 (Ha-5′); 2.86 dd, 1 H, J(5′b,2′) = 9.8, J(gem) = 13.4 (Hb-5′).
Mass spectrum (FAB), m/z (rel.%): 303 (100) [M + H]+.
NAD+ Analogues 1 and 2. General Procedure
DBU was added dropwise to a suspension of compound 5 or 7 (1 mmol) in dry methanol (20 ml)
until the pH reached 7.5. Then Zincke salt3 (3-carbamoyl-1-(2,4-dinitrophenyl)pyridinium chloride,
0.34 g, 1.05 mmol) was added and the mixture was stirred for 5 h. The crude product was precipi-
tated with ether (50 ml) and filtered off. The precipitate was dissolved in water (20 ml) and washed
with ether (10 × 20 ml). In the case of 1a, 2a and 2b, water was evaporated, the residue was dis-
solved in methanol and the product was precipitated by addition of ether. In the other cases, the
aqueous solution of 1b or 1c was applied onto a column of Amberlite (H+, 20 ml). After washing
with water the resin was mixed with water (100 ml), the mixture adjusted to pH 7.5 with ammonia
and the suspension filtered. After evaporation of water, the residue was dissolved in methanol and
the product was precipitated by addition of ether.
9-(2-Phosphonomethoxyethyl)adenine 2-(3-carbamoylpyridinium)ethyl ester (1a): Yield 0.30 g
(68%). Mass spectrum (FAB), m/z (rel.%): 422 (20) [M + H]+. For C16H20N7O5P . 3 H2O (475.4)
calculated: 40.42% C, 5.51% H, 20.62% N, 6.52% P; found: 40.42% C, 5.72% H, 20.43% N, 6.13% P.
Exact mass (FAB HRMS) for C16H21N7O5P calculated: 422.1342; found: 422.1295.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1548
Hockova, Masojidkova, Holy:
(R)-9-(2-Phosphonomethoxypropyl)adenine 2-(3-carbamoylpyridinium)ethyl ester (1b): Yield 0.31 g
(71%). Mass spectrum (FAB), m/z (rel.%): 436 (20) [M + H]+. For C17H22N7O5P . 2.5 H2O (480.4)
calculated: 42.50% C, 5.67% H, 20.41% N; found: 42.24% C, 5.65% H, 20.13% N. Exact mass
(FAB HRMS) for C17H23N7O5P calculated: 436.1498; found: 436.1493.
(S)-9-(2-Phosphonomethoxypropyl)adenine 2-(3-carbamoylpyridinium)ethyl ester (1c): Yield 0.22 g
(50%). Mass spectrum (FAB), m/z (rel.%): 436 (40) [M + H]+. For C17H22N7O5P . 2.5 H2O (480.4)
calculated: 42.50% C, 5.67% H, 20.41% N; found: 42.35% C, 5.79% H, 20.03% N. Exact mass
(FAB HRMS) for C17H23N7O5P calculated: 436.1498; found: 436.1606.
(RS)-9-(3-Hydroxy-2-phosphonomethoxypropyl)adenine 2-(3-carbamoylpyridinium)ethyl ester
(1d): Yield 0.32 g (70%). Mass spectrum (FAB), m/z (rel.%): 452 (40) [M + H]+. Exact mass (FAB
HRMS) for C17H23N7O6P calculated: 452.1447; found: 452.1299.
(S)-1-[3-(Adenin-9-yl)-2-phosphonatomethoxypropyl]-3-carbamoylpyridinium (2a): Yield 0.31 g
(76%). Mass spectrum (FAB), m/z (rel.%): 408 (60) [M + H]+. Exact mass (FAB HRMS) for
C15H19N7O5P calculated: 408.1185; found: 408.1147.
(R)-1-[3-(Adenin-9-yl)-2-phosphonatomethoxypropyl]-3-carbamoylpyridinium (2b): Yield 0.25 g
(61%). Mass spectrum (FAB), m/z (rel.%): 408 (90) [M + H]+. Exact mass (FAB HRMS) for
C15H19N7O5P calculated: 408.1185; found: 408.1241.
1
For H and 13C NMR spectra of compounds 1 and 2 see Tables I and II.
REFERENCES
1. Dolphin D., Poulson R., Avramovic O. (Eds): Pyridine Nucleotide Coenzymes, Part A, Vol. II.
Wiley, New York 1987.
2. Hockova D., Votavova H., Holy A.: Tetrahedron Asymm. 6, 2375 (1995).
3. Juricova K., Smrckova S., Holy A.: Collect. Czech. Chem. Commun. 60, 237 (1995).
4. Holy A., Dvorakova H., Jindrich J. in: Antibiotics and Antiviral Compounds (K. Krohn, H. A.
Kirst and H. Maag, Eds), p. 455. VCH, Weinheim 1993.
5. Holy A. in: Advances in Antiviral Drug Design (E. De Clercq, Ed.), Vol. 1, p. 178. JAI Press
Inc., Greenwich, Connecticut 1993.
6. Holy A., Rosenberg I.: Collect. Czech. Chem. Commun. 52, 2801 (1987).
7. Holy A., Dvorakova H., Masojidkova M.: Collect. Czech. Chem. Commun. 60, 1390 (1995).
8. Hocek M., Masojidkova M., Holy A., Andrei G., Snoeck R., Balzarini J., De Clercq E.: Collect.
Czech. Chem. Commun. 61, 1525 (1996).
9. Alexander P., Holy A., Masojidkova M.: Collect. Czech. Chem. Commun. 59, 1853 (1994).
10. Zincke T., Wuerker W.: Justus Liebigs Ann. Chem. 341, 365 (1905).
11. Holy A., Dvorakova H.: Nucleosides Nucleotides 14, 695 (1993).
12. Holy A.: Collect. Czech. Chem. Commun. 58, 649 (1993).
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