The practical synthesis of a methylenebisphosphonate analogue of benzamide adenine dinucleotide: Inhibition of human inosine monophosphate dehydrogenase (type I and II)

Article abstract of DOI:10.1021/jm960641y

β-Methylene-BAD (8), a nonhydrolyzable analogue of benzamide adenine dinucleotide (BAD), was synthesized as potential inhibitor of human inosine monophosphate dehydrogenase (IMPDH). Treatment of 2',3'O- isopropylideneadenosine 5'-methylenebisphosphonate (15) with DCC afforded P¹,P⁴-bis(2',3'-O-isopropylideneadenosine) 5'-P¹,P²:P³,p⁴- dimethylenetetrakisphosphonate (17). This compound was further converted with DCC to an active intermediate 18 which upon reaction with 3.(2',3'-O- isopropylidene-β-D-ribofuranosyl)benzamide (19) gave, after hydrolysis and deisopropylidenation, the desired β-methylene-BAD (8) in'95% yield. In a similar manner, treatment of 18 with 2',3'-O-isopropylidenetiazofurin (21) followed by hydrolysis and aleprotection afforded β-methylene-TAD (5) in 91% yield. Compound 8 (IC₅₀ = 0.665 μM) was found to be a 6-8 times less potent inhibitor of IMPDH than 5 (IC₅₀ = 0.107 μM) and was almost equally potent against IMPDH type I and type II. Although TAD and β-methylene-TAD were bound by LADH with the same affinity, the binding affinity of 8 toward LADH (K(i) = 333 μM) was found to be 50-fold lower than that of the parent pyrophosphate 7 (K(i) = 6.3 μM).

Full text of DOI:10.1021/jm960641y

J . Med. Chem. 1997, 40, 1287-1291
1287
Notes
Th e P r a ctica l Syn th esis of a Meth ylen ebisp h osp h on a te An a logu e of Ben za m id e
Ad en in e Din u cleotid e: In h ibition of Hu m a n In osin e Mon op h osp h a te
Deh yd r ogen a se (Typ e I a n d II)1
Krzysztof W. Pankiewicz,*,† Krystyna Lesiak,† Andrzej Zatorski,‡ Barry M. Goldstein,§ Stephen F. Carr,
Marek Sochacki,| Alokes Majumdar,† Michael Seidman,† and Kyoichi A. Watanabe†
Codon Pharmaceuticals, Inc., 200 Perry Parkway, Gaithersburg, Maryland 20877, Laboratory of Organic Chemistry,
Sloan-Kettering Institute for Cancer Research, New York, New York 10021, Department of Biophysics, University of Rochester
Medical Center, Rochester, New York 14642, Department of Biochemistry, Roche Bioscience, Palo Alto, California 94304, and
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland
Received September 10, 1996X
â-Methylene-BAD (8), a nonhydrolyzable analogue of benzamide adenine dinucleotide (BAD),
was synthesized as potential inhibitor of human inosine monophosphate dehydrogenase
(IMPDH). Treatment of 2′,3′-O-isopropylideneadenosine 5′-methylenebisphosphonate (15) with
DCC afforded P1,P4-bis(2′,3′-O-isopropylideneadenosine) 5′-P1,P2:P3,P4-dimethylenetetrakispho-
sphonate (17). This compound was further converted with DCC to an active intermediate 18
which upon reaction with 3-(2′,3′-O-isopropylidene-â-D-ribofuranosyl)benzamide (19) gave, after
hydrolysis and deisopropylidenation, the desired â-methylene-BAD (8) in 95% yield. In a similar
manner, treatment of 18 with 2′,3′-O-isopropylidenetiazofurin (21) followed by hydrolysis and
deprotection afforded â-methylene-TAD (5) in 91% yield. Compound 8 (IC50 ) 0.665 µM) was
found to be a 6-8 times less potent inhibitor of IMPDH than 5 (IC50 ) 0.107 µM) and was
almost equally potent against IMPDH type I and type II. Although TAD and â-methylene-
TAD were bound by LADH with the same affinity, the binding affinity of 8 toward LADH (Ki
) 333 µM) was found to be 50-fold lower than that of the parent pyrophosphate 7 (Ki ) 6.3
µM).
In tr od u ction
II IMPDH is expected to provide significant therapeutic
advantage by reducing potential toxicity caused by
inhibition of the type I isoform.
Among cellular dehydrogenases, inosine monophos-
phate dehydrogenase (IMPDH), which catalyzes the
NAD-dependent conversion of inosine 5′-monophosphate
(IMP) to xanthosine 5′-monophosphate (XMP) is an
important target in cancer chemotherapy.2,3 The onco-
lytic C-nucleosides, tiazofurin4,5 (2-â-D-ribofuranosylthi-
azole-4-carboxamide, TR, 3, Chart 1) and benzamide
riboside6 (3-â-D-ribofuranosylbenzamide, BR, 6) are
potent inhibitors of IMPDH.7-10 These compounds
require a unique metabolic activation. Both 3 and 6 are
converted by cellular enzymes to the corresponding
analogues of nicotinamide adenine dinucleotide (NAD,
2), thiazole-4-carboxamide adenine dinucleotide11-13
(TAD, 4), and benzamide adenine dinucleotide14 (BAD,
7). TAD and BAD compete with the natural cofactor
NAD, but since they cannot participate in hydride
transfer, they act as competitive inhibitors of IMPDH.
It has been recently discovered that human IMPDH
exists as two isoforms, types I and II.15 Type I is
constitutively expressed and is the dominant isoform
in normal cells, while type II is selectively up-regulated
in neoplastic and replicating cells and emerges as the
major species.16 Thus, the selective inhibition of type
The idea to use analogues of NAD in order to
selectively inhibit some cellular dehydrogenases emerged
long time ago but it has been criticized since its
inception. For example, it was emphasized that numer-
ous cellular dehydrogenases depend on NAD as the
cofactor, and since the cofactor binding domain in all of
these proteins was conserved, a sufficiently selective
inhibition of individual cellular dehydrogenases could
not be achieved. Contrary to such criticism, potent and
selective inhibition of 6-phosphogluconate dehydroge-
nase by 6-amino-NAD (10, Ki ) 0.13 µM) was reported
as early as in 1971.17 Later, the synthesis of TAD18 (4)
and its methylenebisphosphonate analogue, â-methyl-
ene-TAD19 (5) was published. Both 4 and 5 showed
potent and selective inhibitory activity against IMPDH
with Ki in the range of 0.1 µM. These compounds did
not inhibit alcohol, lactate, glutamate, and malate
dehydrogenases at concentrations up to 200 µM. Re-
cently, we prepared C-nucleoside analogues of the
natural cofactor C-NAD (12) and C-PAD (14)20 and
found that C-NAD, but not C-PAD, was an extremely
potent and specific inhibitor of horse liver alcohol
dehydrogenase (LADH) with Ki ) 1-2 nM.21,22
* Author for correspondence and reprints (phone 301-527-2063, FAX
301-208-6997).
In spite of the promising initial results, progress
toward biomedical utilization of TAD and BAD has been
hampered due mainly to the lack of efficient synthetic
procedures for these compounds. Recently, we devel-
oped a modified procedure for imidazolide activation of
† Codon Pharmaceuticals, Inc.
‡ Sloan-Kettering Institute for Cancer Research.
§ University of Rochester Medical Center.
Roche Bioscience.
| Polish Academy of Sciences.
X Abstract published in Advance ACS Abstracts, March 1, 1997.
S0022-2623(96)00641-3 CCC: $14.00 © 1997 American Chemical Society

1288 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Notes
Ch a r t 1
18, showing numerous new phosphorus resonances in
the 31P NMR spectrum. Intermediate 18 may consist
of compound(s) resulting from further DCC-promoted
dehydration of 17 to monocyclic, bicyclic, and/or mac-
rocyclic derivatives of 17. Compound(s) 18 cannot be
isolated due to its (their) susceptibility to hydrolysis.
Multisignal resonances in 31P NMR either are due to
polyphosphate composition of 18 or represent a mixture
of diastereomers formed during cyclization of 17 (phos-
phorus atoms of 18 become chiral upon dehydration).
Whatever the structure of 18 is, when nucleoside 19 was
added at this stage of reaction, the number of phospho-
rus resonances was reduced to two broad singlets, and
hydrolysis with water afforded the desired dinucleotide
20 in 95% yield. Similarly, addition of 2′,3′-O-isopro-
pylidenetiazofurin (21) to intermediate 18 gave after
hydrolysis the protected â-methylene-TAD (22) in excel-
lent yield. Deisopropylidenation of 20 and 22 with
Dowex 50/H+ afforded compounds 8 and 5, respectively.
This procedure is amenable to gram-scale synthesis of
compounds such as 5 and 8; the only limitation is the
availability of the corresponding starting nucleosides.
Biologica l Resu lts
We examined the inhibitory effects of 8 and 5 against
IMPDH type I and type II. The IC50’s of these com-
pounds for each isoform were measured as reported
previously.24 â-Methylene-TAD was equally potent
against IMPDH type I (IC50 ) 0.107 µM) and type II
(IC50 ) 0.109 µM), showing an inhibitory potency
identical to that of TAD.19 â-Methylene-BAD was found
to be 6-8 times less potent than â-methylene-TAD and
slightly less potent against type II (IC50 ) 0.948 µM)
than type I IMPDH (IC50 ) 0.665 µM). These are
similar relative potencies to those reported for BAD
versus TAD.24 Thus, both â-methylene-TAD and â-me-
thylene-BAD do not exhibit any significant selectivity
against IMPDH type II.
We also examined inhibition of LADH by both BAD
and â-methylene-BAD. The pattern of inhibition with
respect to NAD is competitive in both cases. Measured
Ki’s for inhibition of LADH with NAD as the variable
substrate are 6.3 ( 1.43 µM for BAD and 333 ( 62 µM
for â-methylene-BAD. Thus, replacement of the pyro-
phosphate oxygen with a -CH2- linkage results in a 50-
fold decrease in binding affinity, comparable to a ca. 2.4
kcal/mol difference in free energy. LADH follows an
ordered sequential mechanism, the binding of cofactor
preceding that of substrate.27 The observed pattern of
inhibition is consistent with binding of the inhibitor at
the normal cofactor site.28 The pyrophosphate oxygen
in NAD does not participate in any specific intermo-
lecular interactions in either open or closed complexes
with LADH.22,27 Thus, the observed difference in Ki’s
is surprising. However, adjacent phosphorus oxygens
do participate in a number of polar interactions.27
Subtle steric and/or electronic effects of the methylene
substitution may perturb these interactions,29 resulting
in the weaker binding of â-methylene-BAD. Crystal-
lographic studies of the inhibitor-LADH complexes are
underway to address this question.
nucleoside 5′-monophosphates for the efficient synthesis
of dinucleoside pyrophosphates.23 Using this method,
we prepared BAD24 in yields over 90%. In this paper
we report an efficient synthesis of a methylenebispho-
sphonate analogue of BAD (8), which could be prepared
in yields over 90% by our new method of DCC activation
of adenosine 5′-methylenebisphosphonate. We also
report inhibitory effects of a newly synthesized 8 against
the two isoforms of human IMPDH. In addition, inhibi-
tion of horse liver alcohol dehydrogenase (LADH) by
both BAD (7) and â-methylene BAD (8) was examined.
Inhibition of cell growth by TAD, BAD, and their
methylenebisphosphonate analogues was also studied.
The new compound was tested as potential inducer of
differentiation in human K562 erythroid leukemia cells.
Ch em ica l Syn th esis
We report herein an efficient method of dicyclohexy-
lcarbodiimide (DCC) activation of the adenosine 5′-
methylenebisphosphonate25 (15, Scheme 1) for coupling
with another nucleoside. It has been generally accepted
that DCC forms an amidine intermediate (such as 16)
which is subsequently displaced by the 5′-OH group of
the suitably protected nucleoside to give the desired
product 20. We found, however, that upon admixture
of 3-4 equiv of DCC to the pyridine solution of 15
and
3-(2,3-O-isopropylidene-â-D-ribofuranosyl)benz-
amide6 (19), the early product of this reaction was the
P1,P2,P3,P4-bismethylene analogue of adenosine tetra-
phosphate (17, Ap4A),26 which could be isolated in good
yield. In the absence of 19, addition of DCC to the
pyridine solution of 15 or to the solution of isolated Ap4A
analogue 17 resulted in formation of an intermediate
Analysis of the influence of the compounds on growth
of K562 cells showed that the methylene derivatives of
BAD and TAD were active as inhibitors of K562 cell
growth, although concentrations higher than TR or TAD

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1289
Sch em e 1
Ta ble 1. Inhibition of Growth of K562 Cells by
â-Methylene-BAD as Compared with Inhibitory Activity of TR,
TAD, and â-Methylene-TAD
cell. Instead, it seems probable that TAD is active itself.
This implies that it is taken up by the cells despite the
pyrophosphate linkage. The lower activity of the me-
thylene BAD derivative is in accord with the reduced
activity against IMPDH.
growth
compound
IC50 (µM)
(% of untreated control)
TR
TAD
3
3.7
60
18
90
80
90
90
Exp er im en ta l Section
â-methylene- BAD
â-methylene-TAD
Gen er a l Meth od s. HPLC was performed on a Dynamax-
60A C18-83-221-C column with flow rate of 5 mL/min or
Dynamax-300A C18-83-243-C column with a flow rate of 20
mL/min of 0.1 M Et3N H2CO3 (TEAB) followed by a linear
gradient of 0.1 M TEAB-aqueous MeCN (70%). Elemental
analyses were performed by M-H-W Laboratories, Phoenix, AZ.
Nuclear magnetic resonance spectra were recorded on a J EOL
Eclipse 270 and Bruker-AMX-400 spectrometer with Me4Si or
DDS as the internal standard for 1H and external H3PO4 for
31P. Chemical shifts are reported in ppm (δ), and signals are
described as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), bs (broad singlet), and dd (double doublet). Values
given for coupling constants are first order. Negative ion mass
spectra were recorded using Finnigan TSQ instrument equipped
with electronspray ion source.
Ta ble 2. Differentiation of K562 Cells by â-Methylene-BAD,
TR, TAD, and â-Methylene-TAD without and with
Coadministration of Guanosine
% benzidine positive cells
compound
concn (µM)
-Gua
+Gua
TR
3.8
19
7.5
15
60
120
15
36
80
50
70
45
75
34
85
12
40
15
30
15
17
23
30
TAD
â-methylene-BAD
â-methylene-TAD
30
P 1,P 4-Bis(2′,3′-O-isopr opyliden eaden osin e) 5′-P 1,P 2:P 3,P 4-
d im et h ylen et et r a k isp h osp h on a t e (17). 2′,3′-O-Isopro-
pylideneadenosine 5′-methylenebisphosphonate (62 mg, 0.11
mmol) as monotriethylammonium salt was dissolved in pyri-
dine (0.5 mL) containing DCC (25 mg, 0.12 mmol), and the
mixture was stirred at room temperature for 1 h and concen-
trated in vacuo. The residue was dissolved in 0.1 M Et3N H2-
CO3 (TEAB) and chromathographed on HPLC to give 17 (36
mg, 59%) as the ditriethylammonium salt (after lyo-
philization): 1H NMR (D2O) δ 1.22-1.26 (two t, 18H, Et3N),
1.44 (s, 6H, iPr), 1.67 (s, 6H, iPr), 2.31-2.41 (m, 4H, two
PCH2P), 3.02-3.16 (two q, 12H, Et3N), 4.05, 4.08 (dt, 4H, H5′,
J 4′,5′ ) 4.2 Hz, J 5′,5′′ ) 11.4 Hz, J 5′,P ) 4.2 Hz), 4.12-4.17 (m,
were required (Table 1). Coadministration of guanosine
blocked the inhibition of cell growth by all compounds,
indicating that IMPDH was the target of both methyl-
ene derivatives.
Cell differentiation was monitored by analysis of
hemoglobin expression. Hemoglobin was induced by all
compounds in the same concentration ranges as the
IC50. Guanosine partially blocked the production of
benzidine positive cells (Table 2).
These results indicate that the methylene derivatives
are not as active as the TR or the dinucleotide derivative
of TR, TAD. Since TAD shows activity at concentrations
similar to TR, it is unlikely that the activity of TAD is
due to breakdown and recycling of the TR moiety in the
4H, H5′′), 4.58-4.59 (m, 2H, H4′), 5.23 (dd, 2H, H3′, J 2′,3′
)
6.1 Hz, J 3′,4′ ) 1.9 Hz), 5.36 (dd, 2H, H2′, J 1′,2′ ) 3.7 Hz), 6.14
(d, 2H, H1′), 8.11, 8,41 (two 2H singlets, H2, H8); 31P NMR
(D2O) δ 10.51 (P2, P3), 21.35 (P1, P4); MS (ES) m/ z 911 (M -
H)-, 455 (M - 2H)2-
.

1290 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Notes
P 1-(3-â-D-Ribofu r a n os-5-ylben za m id e) P 2-Ad en osin e-5′-
(Meth ylen ebisp h osp h on a te) (8, â-Meth ylen e-BAD). (A)
Syn th esis fr om 15. 2′,3′-O-Isopropylideneadenosine 5′-me-
thylenebisphosphonate (62 mg, 0.11 mmol) as the monotri-
ethylammonium salt was dissolved in pyridine (1.5 mL)
containing DCC (49 mg, 0.24 mmol), and the mixture was
stirred at room temperature for 20 h. 31P NMR analysis
showed disappearance of resonance signals of 15 (δ 15.46,
singlet) and formation of an intermediate 18 with character-
istic multisignal resonances. At this time nucleoside 19 (32
mg, 0.11 mmol) was added and the reaction mixture was kept
at 65 °C (2 days) until the 31P spectrum of the reaction showed
the formation of a simple AB system (δ ) 16.90). Then water
was added, and the mixture was stirred at room temperature
for 3 h and concentrated in vacuo. The residue was chromato-
graphed on HPLC column to give P1-[3-(2,3-O-isopropylidene-
â-D-ribofuranos-5-yl)benzamide] P2-(2′,3′-O-isopropylidenead-
enosine) 5′-methyelenebisphosphonate (20) as the ditriethyl-
ammoniun salt (102 mg): 1H NMR (D2O) δ 1.27-1.31 (t, 18H,
Et3N), 1.40 (s, 3H, iPr), 1.46 (s, 3H, iPr), 1.64 (s, 3H, iPr), 1.68
(s, 3H, iPr), 2.05-2.20 (m, 2H, PCH2P), 3.21 (q, 12H, Et3N),
4.09-4.12 [m, 4H, H5′,H5′′(B) and (A)], 4.26-4.28 [m, 1H, H4′-
(B)], 4.59-4.64 [m, 2H, H4′(A), H2′(B)], 4.80 [1H, H1′(B)], 4.88
[dd, 1H, H3′(B), J 1′,2′ ) 5.7 Hz, J 2′,3′ ) 6.5 Hz), 5.20 [m, 1H,
H3′(A)], 5.30 [dd, 1H, H2′(A), J 1′,2′ ) 3.0 Hz, J 2′,3′ ) 6.5 Hz],
6.12 [d, 1H, H1′(A)], 7.39 [pseudo t, 1H, H5(B)], 7.46 [d, 1H,
H4(B), J 4,5 ) 7.8 Hz], 7.64 [d, 1H, H6(B), J 5,6 ) 7.8 Hz], 7.67
(s, 1H, H2(B)], 8.15, 8.40 [two 1H singlets, H2(A), H8(A)].
Compound 20 was treated with Dowex 50-X8/H+ in water and
purified by passing through the column of Dowex 50-X8/H+ to
give â-methylene-BAD (8, 69 mg, 95%): 1H NMR (D2O) δ 2.27
(pseudo t, 2H, PCH2P), 4.05 [dd, 1H, H2′(B), J 1′,2′ ) 7.0 Hz,
J 2′,3′ ) 5.1 Hz], 4.17-4.20 [m, 4H, H5′,5′′(A) and (B)], 4.25-
4.27 [m, 2H, H3′(B), H4′(B)], 4.37 [pseudo t, 1H, H3′(A)], 4.66
[pseudo t, H2′(A)], 4.80 [d, 1H, H1′(B)], 6.06 [d, 1H, H1′(A),
J 1′,2′ ) 4.9 Hz], 7.41 [pseudo t, 1H, H5(B)], 7.56 [d, 1H, H4(B),
J 4,5 ) 7.8 Hz], 7.63 [d, 1H, H6(B), J 5,6 ) 7.8 Hz], 7.69 (s, 1H,
H2(B)], 8.32, 8.57 [two 1H singlets, H2(A), H8(A)], 31P NMR
(D2O) δ 20.94 (s); MS (ES) m/ z 659 (M - H)-.
(B) Syn th esis fr om 17. Compound 17 (30 mg, 0.027 mmol)
as the ditriethylammonium salt was dissolved in pyridine (0.5
mL) containing DCC (20 mg, 0.1 mmol), and the mixture was
stirred at room temperature for 3 h. 31P NMR analysis showed
the disappearance of resonance signals of 17 with simulta-
neous formation of intermediate 18 with characteristic mul-
tisignal resonances. At this time nucleoside 19 (19 mg, 0.06
mmol) was added, and the reaction mixture was kept at 65 °C
(2 days) until the 31P spectrum of the reaction showed the
formation of the AB system (δ ) 16.90). Water was added,
and the mixture was stirred at room temperature for 3 h and
then concentrated in vacuo. The residue was chromato-
graphed on HPLC column to give 20 (21 mg, 91%) as the
ditriethylammoniun salt. The analytical data of this com-
pound was identical to that of 20 reported above.
P 1-Tia zofu r in P 2-Ad en osin e-5′-m eth ylen ebisp h osp h o-
n a te (â-Meth ylen e-TAD, 5). 2′,3′-O-Isopropylideneadenosine
5′-methylenebisphosphonate (620 mg, 1.1 mmol) as the monot-
riethylammonium salt was dissolved in pyridine (30 mL)
containing DCC (500 mg, 2.5 mmol), and the mixture was
stirred at room temperature for 20 h. 31P NMR analysis
showed conversion of 15 (δ ) 15.46, singlet) into intermediate
18 with characteristic multisignal resonances. At this time
nucleoside 21 (330 mg, 1.1 mmol) was added, and the reaction
mixture was kept at 65 °C (2 days) until the 31P spectrum
became the simple AB system (δ ) 17.80). The reaction was
quenched by addition of water, and the mixture was stirred
H4′ of adenosine), 4.89-4.95 (2H, m, H2′, 3′ of tiazofurin), 5.09
(1H, d, H1′ of tiazofurin, J 1′2′ ) 3.4 Hz), 5.22 (1H, dd, H3′, J 2′,3′
) 6.2 Hz, J 3′,4′ ) 2.2 Hz), 5.36 (1H, dd, H2′, J 1′,2′ ) 3.5 Hz),
6.18 (1H, d, H1′), 8.06, 8.14, and 8.42 (three 1H singlets, H2,
H8 adenosine and H5 tiazofurin). The rest of the residue was
dissolved in water (100 mL), treated with Dowex50/H+, chro-
matographed on the column of Dowex50/H+, and concentrated
in vacuo. Final purification on HPLC afforded 5 (870 mg,
1
91%). The H NMR spectrum of 5 was identical with that of
authentic sample.
In h ibition of LADH. Horse liver ADH was obtained in
crystalline form from Boehringer Mannheim Biochemicals,
Indianapolis, IN. â-Nicotinamide adenine dinucleotide in
crystalline free acid form was also obtained from Boehringer.
BAD was prepared as reported earlier,24 and â-methylene-BAD
was synthesized as described above.
LADH in crystalline suspension was spun down to a pellet
and dissolved in 100 mM Tris-HCl buffer containing 100 mM
KCl adjusted to pH 8 at 23 °C. Kinetic constants of the
inhibitors with respect to NAD were obtained by monitoring
spectrophotometrically the rate of production of NADH during
the reaction. The course of the reaction was followed by
measuring the changing absorbance of the reaction mixture
at 340 nm, the absorbance peak for NADH, using Beckman
DU-65 spectrophotometer, and an extinction coefficient of 6.22
A mM-1 cm-1 for the reduced cofactor.30 Substrate, cofactor,
and inhibitor were combined and equilibrated at 23 °C for 5
min. The reaction was initiated by the introduction of 10 µL
of enzyme solution on a mixing plunger, and NADH production
was monitored for between 2 and 4 min, depending on the
activity of the protein solution.
Initial velocities were measured at four NAD concentration
(25, 50, 100, and 200 µM) at the fixed saturating substrate
concentration (1.2 µM). Velocities were measured in the
absence of inhibitor and in presence of multiple concentrations
of BAD and â-methylene-BAD (5-40 µM and 120-300 µM,
respectively). Values of inhibition constants and patterns of
inhibition were obtained as described by us earlier23 by direct
least-squares fits to the nonreciprocal forms of the Michaelis-
Menten rate equations.31
Cells, Gr ow th , a n d Differ en tia tion Assa ys. Human
erythroleukemia K562 cells were grown in RPMI 1640 medium
supplemented with 10% heat-inactivated calf serum, glutamine,
and penicillin/streptomycin. Growth assays were performed
by suspending cells at a concentration of 5 × 103 mL in a final
volume of 200 in a 96-well microtiter plate. The chemicals,
with or without added guanosine (30 µM), were added at the
start of the assay. After 5 days of incubation, 100 µL of
medium was removed from each well and 10 µL of MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma]
was added, and the mixture incubated at 37 °C for 3 h.32 The
insoluble formazan was then dissolved by addition of 150 µL
of 2-propanol/0.04 mM HCl. The plates were then read in a
plate reader at 550 nm. All points were done in triplicate.
The concentration required for 50% inhibition of growth (IC50
)
was calculated using an Excel program (Microsoft). The
results were averaged from four experiments.
The expression of hemoglobin was measured by benzidine
staining.33 Cells in 2 mL were incubated for 5 days in the
presence of compounds. An equal volume of cells and freshly
prepared staining solution (0.1% benzidine in 0.25% hydrogen
peroxide) were mixed and incubated at room temperature for
5 min. A total of 300-400 cells were counted to determine
the percentage of benzidine positive cells. The experiments
were done in duplicate, and the reported results are the
average of three experiments.
at room temperature for 3 h and concentrated in vacuo.
A
small amount of the residue (50 mg) was chromatographed
on an HPLC column to give the analytical sample of P1-(2′,3′-
O-isopropylidenetiazofurine P2-(2′,3′-O-isopropylideneadenos-
ine) 5′-methylenebisphosphonate (22): 1H NMR (D2O) δ 1.26
(18H, t, Et3NH+), 1.38, 1.45, 1.60 and 1.67 (3H each, Me-
isopropylidene of tiazofurin and adenosine), 2.07 (2H, t,
PCH2P, J P-H ) 20.1 Hz), 3.19 (12H, q, Et3NH+), 3.88-3.98
(2H, m, H5′,5′′ of tiazofurin), 4.07-4.12 (2H, H5′,5′′ of adenos-
ine), 4.35-4.38 (1H, m, H4′ of tiazofurin), 4.59-4.62 (1H, m,
Refer en ces
(1) NAD analogues 10. For part 9, see: Pankiewicz, K. W.; Zatorski,
A.; Watanabe, K. A. NAD Analogues as Potential Anticancer
Agents: Conformational Restrictions as Basis for Selectivity.
Acta Biochim. Pol. 1996, 43, 183-194.
(2) J ackson, R. C.; Weber, G.; Morris, H. P. IMP Dehydrogenase:
An Enzyme Linked with Proliferation and Malignancy. Nature
1975, 256, 331-333.

Notes
(3) Robins, R. K. Nucleoside and Nucleotide Inhibitors of Inosine
Monophosphate Dehydrogenase (IMP) as Potential Antitumor
Inhibitors. Nucleosides Nucleotides 1982, 1, 35-44.
(4) Fuertes, M.; Garzia-Lopez, T.; Garzia-Munoz, G.; Stud, M.
Synthesis of C-Glycosyl Thiazoles, J . Org. Chem. 1976, 41,
4074-4077.
(5) Srivastava, P. C.; Pickering, M. V.; Allen, L. B.; Streeter, D. G.;
Campbell, M. T.; Witkowski, J . T.; Sidwell, R. W.; Robins, R. K.
Synthesis and Antiviral Activity of Certain Thiazole C-Nucleo-
sides. J . Med. Chem. 1977, 20, 256-260.
(6) Krohn, K.; Heins, H.; Wielckens, K. Synthesis and Cytotoxic
Activity of C-Glycosidic Nicotinamide Riboside Analogues. J .
Med. Chem. 1992, 35, 511-517.
(7) Robins, R. K.; Finch, R. A.; Avery, T. L. Nucleoside and
Nucleotide Modulation of Oncogenic Expression: A New Ap-
proach to Cancer Chemotherapy. In Anticancer Drug Discovery
and Development: Natural Products and New Molecular Models;
Valeriote, F. A., Corbett, T. H., Baker, L. H., Eds.; Kluwer
Academic Publisher: Boston, 1994; pp 149-182.
(8) J ayaram, H. N.; Lapis, E.; Tricot, G.; Kneebone, P.; Paulik, E.;
Zhen, W.; Engeler, G. P.; Hoffman, R.; Weber, G. Clinical
Pharmacokinetic Study of Tiazofurin Administered as 1-Hour
Infusion. Int. J . Cancer 1992, 51, 182-188.
(9) J ayaram, H. N.; Gharehbaghi, K.; J ayaram, N. H.; Rieser, J .;
Krohn, K.; Paull, K. D. Cytotoxicity of a New IPM Dehydroge-
nase Inhibitor, Benzamide Riboside, to Human Myelogenous
Leukemia K562 Cells. Biochem. Biophys. Res. Commun. 1992,
186, 1600-1606.
(10) Gharehbaghi, K.; Sreenath, A.; Hao, Z.; Paull, K. D.; Szekeres,
T.; Cooney, D. A.; Krohn, K.; J ayaram, H. N. Comparison of
Biochemical Parameters of Benzamide Riboside, a New Inhibitor
of IMP Dehydrogenase, with Tiazofurin and Selenazofurin.
Biochem. Pharmacol. 1994, 48, 1413-1419.
(11) Fridland, A.; Conelly, M. C.; Robbins, T. J . Tiazofurin Metabo-
lism in Human Lymphoblastoids Cells: Evidence for Phospho-
rylation by Adenosine Kinase and 5′-Nucleotidase. Cancer Res.
1986, 46, 532-537.
(12) Cooney, D. A.; J ayaram, H. N.; Glazer, R. A.; Kelly, J . A.;
Marquez, V. E.; Gebeyehu, G.; van Cott, A. C.; Zwelling, L. A.;
J ohns, D. G. Studies on the Mechanism of Action of Tiazofurin
Metabolism to an Analog of NAD with Potent IMP Dehydroge-
nase- Inhibitory Activity. Adv. Enzyme Regul. 1983, 21, 271-
303.
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1291
(19) Marquez, V. E.; Tseng, C. K. H.; Gebeyehu, G.; Cooney, D. A.;
Ahluwalia, G. S.; Kelly, J . A.; Dalal, M.; Fuller, R. W.; Wilson,
Y. A.; J ohns, D. G. Thiazole-4-carboxamide Adenine Dinucleotide
(TAD). Analogues Stable to Phosphodiesterase Hydrolysis. J .
Med. Chem. 1986, 29, 1726-1731.
(20) Pankiewicz, K. W.; Zeidler, J .; Ciszewski, L. A.; Bell, J . E.;
Goldstein, B. M.; J ayaram, H. N.; Watanabe, K. A. Synthesis of
Isosteric Analogues of Nicotinamide Adenine Dinucleotide Con-
taining C-Nucleotide of Nicotinamide and Picolinamide. J . Med.
Chem. 1993, 36, 1855-1859.
(21) Goldstein, B. M.; Li, H.; Bell, J . E.; Zeidler, J .; Pankiewicz, K.
W.; Watanabe, K. A. CNAD: A Potent and Specific Inhibitor of
Alcohol Dehydrogenase. J . Med. Chem. 1994, 37, 392-399.
(22) Li, H.; Hallows, W. H.; Punzi, J . S.; Pankiewicz, K. W.; Wa-
tanabe, K. A. Goldstein, B. M. Crystallographic Studies of
Isosteric NAD Analogues Bound to Alcohol Dehydrogenase:
Specificity and Substrate Binding in Two Ternary Complexes.
Biochemistry 1994, 33, 11734-11744.
(23) Zatorski, A.; Goldstein, B. M.; Colby, T. D.; J ones, J . P.;
Pankiewicz, K. W. Potent Inhibitors of Human Inosine Mono-
phosphate Dehydrogenase Type II. Fluorine-Substituted Ana-
logues of Thiazole-4-carboxamide Adenine Dinucleotide. J .
Med.Chem. 1995, 38, 1098-1105.
(24) Zatorski, A.; Watanabe, K. A.; Carr, S. F.; Goldstein, B. M.;
Pankiewicz, K. W. The Chemical Synthesis of Benzamide
Adenine Dinucleotide: Inhibition of Inosine Monophosphate
Dehydrogenase (Type I and II). J . Med. Chem. 1996, 39, 2422-
2426.
(25) Davisson, V. J .; Davis, D. R.; Dixit, V. M.; Poulter C. D. Synthesis
of Nucleotide 5′-Diphosphates from 5′-O-Tosyl Nucleosides. J .
Org. Chem. 1987, 52, 1794-1801.
(26) Blackburn, G. M.; Guo, M.-J .; McLennan, A. G. Synthetic
A
Structural Analogues of Dinucleoside Polyphosphates. In Ap4
and Other Dinucleoside Polyphosphates; McLennan, A. G., Ed.;
CRC Press, Inc.: Boca Raton, 1994; pp 313-314.
(27) Eklund, H.; Branden, C. I. In Biological Macromolecules and
Assemblies. Vol. 3. Active Sites of Enzymes; J urnak, F. A.,
McPherson, A., Eds.; J ohn Wiley and Sons: New York, 1987;
Chapter 2.
(28) Segel, I. H. Enzyme Kinetics. Behavior and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems; J ohn Wiley and
Sons: New York, 1975; pp 560-590.
(29) Blackburn, G. M.; Eckstein, F.; Kent, D. E.; Perree, T. D. Isopolar
vs. Isosteric Phosphonate Analogues of Nucleotides. Nucleosides
Nucleotides 1985, 4, 165-167.
(30) Kaplan, N. O. The Pyridine Coenzymes. In The Enzymes, Vol.
III, 2nd ed.; Boyer, P. D., Lardy, H., Myrbak, K., Eds.; Academic
Press: New York, 1960; Vol. III, pp 105-169.
(31) Cleland, W. W. Statistical Analysis of Enzyme Kinetics Data.
Methods Enzymol. 1979, 63, 103-139.
(32) Heo, D. S.; Park, J . G.; Hata, K.; Day, R.; Herberman, R. B.;
Whiteside, T. L. Evaluation of tetrazolium based semiautomatic
colorimetric assay for measurement of human antitumor cyto-
toxicity. Cancer Res. 1990, 50, 3681-3690.
(13) Yamada, Y.; Natsumeda, Y.; Weber, G. Action of Active Metabo-
lites of Tiazofurin and Ribavirin on Purified IMP Dehydroge-
nase. Biochemistry 1988, 27, 2193-2196.
(14) Gharehbaghi, K.; Paull, K. D.; Kelley, J . A.; Barchi, J . J ., J r.;
Marquez, V. E.; Cooney, D. A.; Monks, A.; Scudiero, D.; Krohn,
K.; J ayaram, H. N. Cytotoxicity and Characterization of an
Active Metabolite of Benzamide Riboside, a Novel Inhibitor of
IMP Dehydrogenase. Int. J . Cancer 1994, 56, 892-899.
(15) Carr, S. F.; Papp, E.; Wu, J . C.; Natsumeda, Y. Characterization
of Human Type I and Type II IMP Dehydrogenase. J . Biol.
Chem. 1993, 268, 27286-27290.
(16) Nagai, M.; Natsumeda, Y.; Weber, G. Proliferation-Linked
Regulation of Type II 5′-Monophosphate Dehydrogenase Mes-
senger RNA Expression in Human Leukemias. Cancer Res.
1991, 52, 3886-3890.
(17) Herken, H. Antimetabolic Action of 6-Aminonicotinamide on the
pentose pathway in the brain. In Mechanism of Toxicity;
Aldridge, W. N., Ed.; Mc Millan: London, 1971; pp 189-203.
(18) Gebeyehu, G.; Marquez, V. E.; Kelly, J . A.; Cooney, D. A.;
J ayaram, H. N.; J ohns, D. G. Synthesis of Thiazole-4-carboxa-
mide Adenine Dinucleotide. J . Med. Chem. 1983, 26, 922-925.
(33) Orkin, S. H.; Harosi, F. I.; Leder, P. Differentiation in erythro-
leukemia cells and their somatic hybrids. Proc. Natl. Acad. Sci.
U.S.A. 1978, 72, 98-102.
J M960641Y