Ribose-modified mizoribine analogues: Synthesis and biological evaluation

Article abstract of DOI:10.1080/15257770500334673

□ Synthesis, conformational analysis and antitumor evaluation of 2′- and 3′-C-methyl analogues of mizoribine (bredinine, 4-carbamoyl-1-β-D-ribofuranosylimidazole-5-olate) are reported. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770500334673

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Ribose-modified Mizoribine Analogues: Synthesis and
Biological Evaluation
Palmarisa Franchetti a , Michela Pasqualini a , Loredana Cappellacci a , Riccardo Petrelli a
Patrizia Vita a , Mario Grifantini a & Hiremagalur N. Jayaram b
,
a Dipartimento di Scienze Chimiche , Università di Camerino , Camerino, Italy
b Department of Biochemistry and Molecular Biology , Indiana University School of Medicine ,
Indianapolis, IN, U.S.A.
Published online: 16 Aug 2006.
To cite this article: Palmarisa Franchetti , Michela Pasqualini , Loredana Cappellacci , Riccardo Petrelli , Patrizia Vita ,
Mario Grifantini & Hiremagalur N. Jayaram (2005) Ribose-modified Mizoribine Analogues: Synthesis and Biological Evaluation,
Nucleosides, Nucleotides and Nucleic Acids, 24:10-12, 2023-2027, DOI: 10.1080/15257770500334673
To link to this article: http://dx.doi.org/10.1080/15257770500334673
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Nucleosides, Nucleotides, and Nucleic Acids, 24:2023–2027, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500334673
RIBOSE-MODIFIED MIZORIBINE ANALOGUES:
Synthesis and Biological Evaluation
Palmarisa Franchetti, Michela Pasqualini, Loredana Cappellacci, Riccardo
ᮀ
Petrelli, Patrizia Vita, and Mario Grifantini
Dipartimento di Scienze Chimiche,
Universita` di Camerino, Camerino, Italy
ᮀ
Hiremagalur N. Jayaram
Department of Biochemistry and Molecular Biology,
Indiana University School of Medicine, Indianapolis, IN, U.S.A.
Synthesis, conformational analysis and antitumor evaluation of 2ꢀ- and 3ꢀ-C-methyl
ᮀ
analogues of mizoribine (bredinine, 4-carbamoyl-1- -D-ribofuranosylimidazole-5-olate) are reported.
Keywords Mizoribine analogues; IMPDH inhibitors; Antitumor agents
Mizoribine (bredinine, 4-carbamoyl-1- -D-ribofuranosylimidazolium-5-
olate, MZR), an imidazole nucleoside isolated from Eupenicillium brefel-
dianum,[1] is clinically used as immunosuppressive agent to inhibit rejection
reactions after renal transplantation and in the treatment of lupus nephritis
and rheumatoid arthritis.[2] Mizoribine also displayed tumor cells growth
inhibition[3] and antiviral activity.[4] The biological activities of mizoribine
are due to the ability of its metabolite mizoribine 5ꢁ-monophosphate (MZR-
5ꢁ-P) to inhibit IMP dehydrogenase (IMPDH) and guanosine 5ꢁ-monophos-
phate synthetase (GMP-synthetase).[2] Both enzymes are required for the
de novo biosynthesis of GMP starting from IMP. Thus, MZR-5ꢁ-P almost
completely inhibits guanine nucleotide synthesis, which may explain one
of the significant immunosuppressive roles of mizoribine, inhibitory effects
for T cells and B cells proliferation.
Recently, it was ascertained that MZR-5ꢁ-P behaves as a transition-state
analogue inhibitor able to bind the active site of the enzyme adopting the
Dedicated to the Memory of Dr. John A. Montgomery.
Received 28 January 2005; accepted 10 May 2005.
Address correspondence to Mario Grifantini, Dipartimento di Scienze Chimiche, Universita` di
Camerino, Via S. Agostino 1, 62032 Camerino, Italy. Fax: 39-0737-637345; E-mail: mario.grifantini@
unicam.it
2023

2024
P.Franchetti et al.
C3ꢁ-endo ribosyl pucker as the substrate xantosine monophosphate (XMP).[5]
In this paper, we report on the synthesis and the biological evaluation of mi-
zoribine analogues in which the ribose moiety was modified by substitution
of the hydrogen atoms in 2ꢁ- or 3ꢁ-position with a methyl group (2ꢁ-Me-MZR
and 3ꢁ-Me-MZR). These modifications are able to stabilize the ribose con-
formation into the C3ꢁ-endo and C2ꢁ-endo forms, respectively.[6]
CHEMISTRY
The 2ꢁ-C-methyl analogue of mizoribine (2ꢁ-Me-MZR, 1) was synthesized
as reported in Scheme 1. Glycosylation of silylated derivative of 3-(4-
nitrobenzyl)-5-carbamoylimidazolium-4-olate (3)[7] with 1-bromo-2,3,5-tri-
O-benzoyl-2-C-methyl- -D-ribofuranose (4) in the presence of HgO and
HgBr2 gave the nucleoside derivative 5. The deprotection of nucleobase
SCHEME 1 i) HMDS, (NH4)2SO4 a., xylene a.; ii) HgO, HgBr2, ClCH2CH2Cl a.; iii) HCOONH4,
10% Pd/C, MeOH a.; iv) MeONa, MeOH a.; v) Dowex 50W-X8, H form.

Ribose-modified Mizoribine Analogues
2025
of 5 and the debenzoylation gave the desired product 4-carbamoyl-1-(2-C-
methyl- -D-ribofuranosyl)imidazolium-5-olate (1).
The 3ꢁ-C-methyl analogue of mizoribine (3ꢁ-Me-MZR, 2) was obtained as
reported in Scheme 2. Condensation of 3 with 1,2,3-tri-O-acetyl-5-O-benzoyl-
3-C-methyl-D-ribofuranose (6)[8] in the presence of TMSOTf and stannic
chloride gave the nucleoside 7. Removal of the nitrobenzyl group and de-
blocking of 8 under basic conditions gave the desired product 4-carbamoyl-
1-(3-C-methyl- -D-ribofuranosyl)imidazolium-5-olate (2) in high yield. The
same procedure, starting from 3 and 1,2,3,5-tetra-O-acetyl- -D-ribofuranose,
was followed to obtain mizoribine,[7] which was used as reference product
for biological assays. Structural assignments for compounds 1 and 2 were
based on elemental analysis and 1H NMR and mass spectrometric data.
Proton nuclear magnetic resonance (1H NMR) data and nuclear Over-
hauser enhancement (NOE) experiments were employed to determine the
predominant conformation of mizoribine and its 2ꢁ- and 3ꢁ-C-methyl ana-
logues in solution. NOE effects in compounds were determined in D2O.
The H-2ꢁ enhancement when H-2 proton in MZR and 3ꢁ-Me-MZR was irra-
diated (2.6 and 4.5%, respectively) supports a spatial arrangement where
H-2 of nucleobase and H-2ꢁ are proximate, as would be the case in the syn
conformers. The irradiation of H-2 proton in 2ꢁ-Me-MZR showed the H-3ꢁ
enhancement (3.6%) in the ribose ring supporting a spatial arrangement
where H-2 and H-3ꢁ protons are proximate, and therefore this nucleoside
SCHEME 2 i) HMDS, (NH4)2SO4 a., xylene a.; ii) TMSOTf, SnCl4, ClCH2CH2Cl a.; iii) HCOONH4,
10% Pd/C, MeOH a.; iv) MeONa, MeOH a.; v) Dowex 50W-X8, H form.

2026
P.Franchetti et al.
prefers the anti conformation. Further information concerning the solu-
tion conformation was obtained by the coupling constant values. The 3ꢁ-C
ꢀ
ꢀ
substitution precludes getting information from the J3 4 value leaving only
the J1 2 as a clue to the sugar puckering. It was found that in 3ꢁ-Me-MZR
ꢀ
ꢀ
ꢀ
ꢀ
the J1 2 value was 8.2 Hz, indicating that this nucleoside is predominantly
S-puckered (C2ꢁ-endo).
It was impossible to establish the pucker conformation of the sugar
of 2ꢁ-Me-MZR on the basis of J3 4 owing the overlapping of H-3ꢁ and
ꢀ
H-4ꢁ signals. However, it is well known that 2ꢁ-C-methyl-ribonucleosides are
predominantly N-puckered (C3ꢁ-endo).[6]
BIOLOGICAL EVALUATION
Mizoribine and its 2ꢁ- and 3ꢁ-C-methyl derivatives 1 and 2 were evaluated
for their ability to inhibit the replication of human myelogenous leukemia
K562. K562 cells (1 × 105 cells/ml) were cultured in 0.1 ml volume in
96 well plates. Twenty-four hours later, cells were incubated with various
concentrations of agents in 3 ml volume in triplicates for 72 h and the cell
proliferation was determined by using MTS assay. Surprisingly, mizoribine
and its analogues 1 and 2 proved to be inactive against this type of tumor
cells up to 100 M concentration. The inactivity of these nucleosides
might be due to their inability to be converted into the corresponding 5ꢁ-
monophosphate in K562 cells. Further experiments are underway in order
to check this hypothesis.
ACKNOWLEDGMENTS
The research was supported by the Italian MIUR (PRIN2002), and
by the Project Development Program, Research and Sponsored Programs,
Indiana University, Indianapolis.
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2027
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