in vitro inhibition of the measles virus by novel ring-expanded ('fat') nucleoside analogues containing the imidazo[4,5-e]diazepine ring system.

Article abstract of DOI:10.1016/S0960-894X(02)00762-X

The synthesis and in vitro anti-measles virus (anti-MV) activity of a class of ring-expanded ('fat') nucleoside analogues (1-4) containing the title heterocyclic ring system are reported. The target compounds were synthesized by base-catalyzed condensations of 4,5-dicarboxylic acid esters of the appropriately substituted imidazole-1-ribosides with suitably substituted guanidine derivatives. Compounds were screened for anti-MV activity in African green monkey kidney cells (CV-1), employing ribavirin as the control standard. While the parent compound 1 itself failed to show any significant antiviral activity against MV, its analogues containing hydrophobic substituents at the 2-position (2) or the 6-position (4) showed promising antiviral activity at submicromolar or micromolar concentration levels with no apparent toxicity to the host cell line. Both compounds showed higher anti-MV activity than the control drug ribavirin.

Full text of DOI:10.1016/S0960-894X(02)00762-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3391–3394
In Vitro Inhibition of the Measles Virus by Novel
Ring-Expanded (‘Fat’) Nucleoside Analogues Containing
the Imidazo[4,5-e][1,3]diazepine Ring System
Ning Zhang,a Huan-Ming Chen,a Ramesh Sood,b Kishna Kalicharran,b Ali I. Fattom,b
Robert B. Naso,b Dale L. Barnard,c Robert W. Sidwellc and Ramachandra S. Hosmanea,*
aLaboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland, Baltimore County,
1000 Hilltop Circle Baltimore, MD 21250, USA
bW. W. Karakawa Microbial Pathogenesis Laboratory, Nabi, 12280 Wilkins Avenue, Rockville, MD 20852, USA
cInstitute for Antiviral Research, Utah State University, Logan, UT 84322, USA
Received 28 July 2002; accepted 29 August 2002
Abstract—The synthesis and in vitro anti-measles virus (anti-MV) activity of a class of ring-expanded (‘fat’) nucleoside analogues
(1–4) containing the title heterocyclic ring system are reported. The target compounds were synthesized by base-catalyzed con-
densations of 4,5-dicarboxylic acid esters of the appropriately substituted imidazole-1-ribosides with suitably substituted guanidine
derivatives. Compounds were screened for anti-MV activity in African green monkey kidney cells (CV-1), employing ribavirin as
the control standard. While the parent compound 1 itself failed to show any significant antiviral activity against MV, its analogues
containing hydrophobic substituents at the 2-position (2) or the 6-position (4) showed promising antiviral activity at submicromolar
or micromolar concentration levels with no apparent toxicity to the host cell line. Both compounds showed higher anti-MV activity
than the control drug ribavirin.
# 2002 Elsevier Science Ltd. All rights reserved.
With over one million child deaths per year, measles
virus (MV) is a major human pathogen, and ranks 8th
as the cause of death worldwide, especially in the devel-
oping countries.1 Despite large vaccination campaigns,
MV is still resisting eradication, and there is no avail-
able therapeutic treatment. The MV infection causes a
respiratory disease which is, more often than not, con-
trolled solely by the immune response. However, MV
infection can lead to a severe immunosuppression that is
responsible for additional opportunistic infections.2
Furthermore, in certain cases, MV establishes persistent
infection of the brain leading to neurological compli-
cations.3 MV is an enveloped, negative, single-stranded
RNA virus belonging to the Paramyxoviridae family.4
The intricate transcription and genome replication
mechanisms of MV, coupled with the fine tuning of
expression of its gene products, make MV one of the
most sophistcated and clinically attractive viruses to
study the life cycle as well as structural and biochemical
basis of viral gene expression. The discovery of potent,
new MV inhibitors may pave the way to novel approa-
ches to combat the viral epidemic. Such inhibitors may
also facilitate the in-depth structural and molecuar bio-
logical investigations that could further assist in studies
of other similar human pathogens such as the Ebola
virus. We report here a novel class of selective anti-MV
agents (1–4) that can be considered as ring-expanded
(‘fat’) analogues of purine nucleosides, and contain the
title 5:7-fused imidazo[4,5-e][1,3]diazepine ring system.
*Corresponding author. Tel.:+1-410-455-2520; fax:+1-410-455-1148;
e-mail: hosmane@umbc.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00762-X

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N. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3391–3394
Scheme 1.
The general procedure for the synthesis (see Scheme 1)
of the target nucleosides involved glycosylation of the
appropriate 2-substituted imidazole analogues contain-
ing carboxylic acid ester groups at the 4- and 5-posi-
tions, followed by condensation of the resulting
imidazole nucleosides with the appropriately substituted
guanidine derivatives. We have already reported the
synthesis of the parent nucleoside 1.5 The synthesis of
the necessary 2-phenylimidazole precursor 5b was
accomplished by esterification of the corresponding
2-phenyl-4,5-imidazole- dicarboxylic acid6ꢀ9 with anhy-
drous methanolic hydrogen chloride. The diacid, in
turn, was prepared by one of the following two known
methods, including (a) nitration of d-tartaric acid, fol-
lowed by ring-closure of the resulting dinitrate by treat-
ment with benzaldehyde and ammonium hydroxide,6,7
or (b) basic hydrolysis (6 N NaOH) of 2-phenyl-4,5-
dicyanoimidazole. The latter was synthesized either by
ring-closure of N-benzylidenediaminomaleonitrile with
N-chlorosuccinimide, and nicotinamide in dimethyl-
formamide8 or by diazotization of 2-amino-4,5-dicyano-
imidazole with sodium nitrite and hydrochloric acid,
followed by replacement of the resulting diazo func-
tionality by phenyl group by heating with benzene at
reflux.9 The overall product yield by either of the two
methods, a or b, was nearly identical although method b
with 2-amino-4-cyanoimidazole as the starting material
was our method of choice because of its simplicity,
convenience, and ease of preparation. Nucleosides 3 and
4 were synthesized by condensation of 6a with methyl-
and ethylguanidine, liberated from the respective com-
mercially available hydrochloride and sulfate salts by
treatment with sodium methoxide in methanol. All final
products and intermediates were fully characterized by
NMR and mass spectral data, coupled with elemental
microanalyses.10
Antiviral efficacy of compounds 1–5 was assessed in
vitro by two different methods, including the Cyto-
pathic Effect (CPE) inhibition assay and Neutral Red
(NR) uptake assay.11 African green monkey kidney cells
(CV-1) were employed for both assays, using ribavirin
as a positive control drug. Ribavirin, a broad spectrum
antiviral nucleoside analogue containing a triazole moi-
ety, is known to mimic guanosine.12 The EC50 and IC50
values, along with a selective index (SI) for each com-
pound, are collected in Table 1.
Table 1. Anti-measles virus activity of compounds 1–4 in vitro (CV-1
cell)
Compd
Assay used
EC50 (mM) IC50 (mM)
SI
(IC50/EC50
)
1
2
3
4
CPE inhibition
Neutral red
>100
>100
>100
>100
>0
>0
CPE inhibition
Neutral red
10
0.5
>100
>100
>10
>200
CPE inhibition
Neutral red
>100
>100
>100
>100
>0
>0
CPE inhibition
Neutral red
2
1.2
>100
>100
>50
>83
Ribavirin CPE inhibition
(Control) Neutral red
20
16
1160
612
58
38

N. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3391–3394
3393
While the parent compound 1 failed to show any sig-
nificant anti-MV activity, data from Table 1 suggest
that the introduction of the hydrophobic phenyl group
at position-2 of the heterocyclic ring, as in 2, results in
antiviral activity. Antiviral activity also comes from
introduction of a hydrophobic substituent bulkier than
methyl group at position-6 as in compound 4. These
initial findings suggest that a few more analogues of 1,
containing substituents with increasing hydrophilicity at
position-2 and/or position-6 need to be synthesized and
screened against the measles virus in order to fully
explore the structure–activity relationship (SAR) and
the antiviral potential of these novel class of ring-
expanded nucleoside analogues. Such an endeavor is
currently in progress.
10. General procedure for preparation of 6 by ribosylation of 5: A
solution of the appropriately substituted methyl 4,5-imidazole-
dicarboxylate13 (5) (10 mmol) and 1-O-acetyl-2,3,5-tri-O-benzyl-
b,-d-ribofuranose (5.04 g, 10 mmol) in dry acetonitrile (50
mL) was placed into a flame-dried 100-mL round-bottom
flask. The solution was stirred in an ice bath for 10 min. Then,
1,1,1,3,3,3-hexamethyldisilazane (HMDS) (7 mL, 33 mmol),
chlorotrimethylsilane (TMSCl) (4.5 mL, 36 mmol) and tri-
fluoromethanesulfonic acid (TFMSA) (3 mL, 36 mmol) were
consecutively added to the above solution. The resulting solu-
tion was stirred in an ice-bath for 1 h. The reaction was com-
plete as shown by TLC analysis [silica gel plates, chloroform/
methanol (30:1)]. The reaction mixture was evaporated to
dryness in vacuo. The resulting residue was dissolved in
chloroform, and washed with saturated aqueous sodium
bicarbonate and water respectively. After drying over anhy-
drous Na2SO4 and filtering, the chloroform solution was eva-
porated to dryness in vacuo to obtain the product 6 either as a
foam or solid, which was recrystallized from the appropriate
solvent or purified by silica gel flash chromatography to give
the pure product 6. The solvent of recrystallization and/or
eluting solvent for column or prep-TLC chromatography,
along with physical, spectral, and analytical data for 6 are
given below:
Conclusion
Ring-expanded (‘fat’) nucleoside analogues represent a
new class of compounds that show promising anti-
measles virus activity at submicromolar or micromolar
concentration levels with no apparent toxicity to the
host cell line. Both compounds showed higher anti-MV
activity than the control drug ribavirin. Nucleosides 2
and 4 are promising candidates for further extensive
structure–activity relationship (SAR) studies and drug
development
Methyl 2-phenyl-1-(2,3,5-tri-O-benzoyl-ꢀ-D-ribofuranosyl)-
4,5-imidazoledicarboxylate (6b): Foam, recrystallized from
methanol, colorless crystals (6.0 g, 85%), Rf 0.74 (chloroform-
methanol (30:1)), mp 139–140 ꢁC, H NMR (CDCl3) d, 7.24–
1
8.06 (m, 20H, ArH), 6.32 (dd, 1H, J=5.7 and 6.9 Hz, 20-H),
6.22 (d, 1H, J=5.7 Hz, 10-H), 5.82 (dd, 1H, J=6.3 and 6.9 Hz,
30-H), 4.71 (dd, 1H, J=3.6 and 11.7 Hz, 50-H), 4.58 (dd, 1H,
J=6.9 and 11.7 Hz, 50-H), 4.52 (m, 1H, 40-H), 3.96 (s, 3H,
OMe), 3.93 (s, 3H, OMe), 13C NMR (CDCl3) d, 52.32 (CH3),
53.18 (CH3), 63.69 (50-C), 70.22 (30-C), 74.12 (20-C), 79.96 (40-
C), 89.54 (10-C), 126.22, 126.22, 128.43, 128.43, 128.29 (Ph-30,
50, 20, 60, 40), 128.34, 128.34, 128.39, 128.39, 128.55, 128.55
(Ph-m), 129.68, 129.68, 129.68, 129.73, 129.73, 129.73, 129.84,
129.84, 129.84 (Ph-o or p), 130.29, 130.29, 130.37 (imidazole),
133.22, 133.46, 133.66 (Ph-C1), 150.69 (Ph-10), 161.54 (C¼O),
162.56 (C¼O), 164.70, 164.83, 166.12 (PhC¼O), Anal. calcd
for C39H32N2O11: C, 66.47, H, 4.58, N, 3.96. Found: C, 66.45,
H, 4.66, N, 3.96.
Acknowledgements
The research was supported by grants (#RO1 CA 71079
and #9RO1 AI55452) from the National Institutes of
Health. The project was also funded in part by an award
(#2001.10) from the Maryland Industrial Partnerships
(MIPS) program, which was co-funded by Nabi
Biopharmaceuticals, Inc. We gratefully acknowledge
Dr. Christopher Tseng, the Program Officer of Antiviral
Research and Antimicrobial Chemistry of the Virology
Branch of the National Institute of Allergy and Infec-
tious Diseases (NIAID), Bethesda, Maryland, for his
support and encouragement throughout the course of
this work.
General procedure for preparation of nucleosides 2–4 by con-
densation of 6 with guanidine: Guanidine hydrochloride (0.38 g,
4 mmol) was added to 4 mL of 2.3 M sodium methoxide
solution resulting from sodium (0.75 g) dissolved in 15 mL of
absolute methanol. The mixture was stirred in an ice-bath for
30 min. The precipitated sodium chloride was removed and
the filtrate was poured into a solution of appropriately sub-
stituted methyl 1-(2,3,5-tri-O-benzoyl-b,-d-ribofuranosyl)-4,5-
imidazoledicarboxylate (6) (1 mmol) in 20 mL of absolute
methanol. The mixture was stirred at room temperature for
24–48 h, until when TLC showed the completion of reaction.
The reaction mixture was filtered if necessary. The clear filtrate
was treated with a solution of methanol saturated with HCl
gas to pH 6.5–7.0. The resulting precipitate was filtered and
washed with water and methanol to give the product. The
solvent of recrystallization and/or eluting solvent for column
or prep-TLC chromatography, along with physical, spectral,
and analytical data for 2–4 are given below: 6-Amino-4,5-
dihydro-8H-2-phenyl-1-(ꢀ-D-ribofuranosyl)imidazo[4,5-e][1,3] dia-
zepine-4,8-dione (2): Yield 85%, an analytical sample was pre-
pared by preparative TLC on silica gel plate, using a mixture
of acetonitrile–water (4:1) as a developing solvent, Rf 0.26
[chloroform-methanol-30% ammoniun hydroxide (2:1:0.3)),
References and Notes
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mp >250 ꢁC, H NMR (DMSO-d6) d, 10.71 (br s, 1H, NH,
1
exchangeable with D2O), 7.75–7.47 (m, 5H, Ph), 7.47 (br s,
1H, NH, exchangeable with D2O), 6.53 (brs, 1H, NH,
exchangeable with D2O), 5.84 (d, 1H, J=5.7 Hz, 10-H), 5.33

3394
N. Zhang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3391–3394
(d, 1H, J=6.3 Hz, OH, exchangeable with D2O), 4.96 (d, 1H,
J=6.3 Hz, OH, exchangeable with D2O), 4.72 (t, 1H, J=5.4
Hz, OH, exchangeable with D2O), 4.44 (q, 1H, J=6.0 Hz, 20-
H, it became t after D2O exchange), 3.70 (dd, 1H, J=11.7 and
5.7 Hz, 50-H), 3.62 (dd, 1H, J=11.7 and 6.3 Hz, 50-H), 3.52–
3.36 (m, 2H, 30,40-H), 13C NMR (DMSO-d6) d, 61.55 (C-50),
69.02 (C-30), 72.14 (C-20), 85.19 (C-40), 90.48 (C-10), 128.12,
128.12 (Ph-30,50), 129.88, 129.88 (Ph-20,60), 129.40 (Ph-40),
130.50, 131.16 (C-3a,8a), 140.75 (C-2), 149.92 (Ph-10), 161.81,
162.54 (C-4,8), 166.76 (C-6), HRMS (FAB): Calcd. for
C17H18N5O6 388.1257, found 388.1276, Anal. calcd for
C17H17N5O.6CH3CN.2/3H2O: C, 51.82, H, 4.88, N, 19.08.
Found: C, 51.66, H, 4.43, N, 19.04.
4,5-Dihydro-8H-2-(N-methyl)amino-1-(ꢀ,-D-ribofuranosyl)-
imidazo[4,5-e][1,3]diazepine-4,8-dione (3): Yield 93%, Mp>
210 ꢁC (dec.), 1H NMR (DMSO-d6): d, 10.63 (brs, 1H, NH,
exchangeable with D2O), 8.54 (s, 1H, imidazole), 7.01 (brs,
1H, NH, exchangeable with D2O), 6.36 (d, J=2.6 Hz, 1H,
10-H), 5.47 (d, J=5.1 Hz, 1H, OH, exchangeable with D2O),
5.16 (t, J=4.9 Hz, 1H, OH, exchangeable with D2O), 5.07 (d,
J=5.5 Hz, 1H, OH, exchangeable with D2O), 4.06 (m, 2H, 20-
and 30-H), 3.90 (m, 1H, 40-H), 3.73 (dd, J1=12.1 Hz, J2=4.0
Hz, 1H, 50-H1), 3.58 (dd, J1=11.7 Hz, J2=4.0 Hz, 1H, 50-H2),
2.75 (d, J=4.0 Hz, 3H, CH3), Anal. calcd for
C12H15N5O6AH2O: C, 41.98, H, 4.99, N, 20.40 Found: C,
42.30, H, 4.79, N, 20.35
4,5-Dihydro-8H-2-(N-ethyl)amino-1-(ꢀ,-D -ribofuranosyl)
imidazo[4,5-e][1,3]diazepine-4,8-dione (4): Yield 90%, Mp>
219 ꢁC (dec.), 1H NMR (DMSO-d6) d, 10.48 (brs, 1H, NH,
exchangeable with D2O), 8.55 (s, 1H, imidazole), 7.07 (brs,
1H, NH, exchangeable with D2O), 6.36 (d, J=2.9 Hz, 1H,
10-H), 5.50 (d, J=4.8 Hz, 1H, OH, exchangeable with D2O),
5.18 (t, J=4.8 Hz, 1H, OH, exchangeable with D2O), 5.08 (d,
J=5.1 Hz, 1H, OH, exchangeable with D2O), 4.06 (m, 2H, 20-
and 30-H), 3.90 (m, 1H, 40-H), 3.73 (dd, J1=11.7 Hz, J2=4.4
Hz, 1H, 50-H1), 3.58 (dd, J1=11.9 Hz, J2=4.2 Hz, 1H, 50-H2),
3.24 (m, 2H, CH2), 2.75 (t, J=7 .1 Hz, 3H, CH3), Anal. calcd
for C13H17N5O6: C, 46.02, H, 5.05, N, 20.64. Found: C, 45.59,
H, 5.14, N, 20.43.
11. Barnard, D. L.; Hill, C. L.; Gage, T.; Matheson, J. E.;
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