Synthesis of a novel carbocyclic analog of bredinin

Article abstract of DOI:10.3390/molecules180911576

The natural nucleoside antibiotic, bredinin, exhibits antiviral and other biological activities. While various nucleosides related to bredinin have been synthesized, its carbocyclic analog has remained unknown. Synthesis of this heretofore unknown analog of bredinin is described. The key precursor, (3aS,4R,6R,6aR)-6-((methoxymethoxy) methyl)-2,2-dimethyltetrahydro-3aH- cyclopenta[d][1,3]dioxol-4-amine (5), was prepared from the commercially available compound, (1R,4S)-2-azabicyclo[2.2.1] hept-5- en-3-one (4). Our initial approach used intermediate 6, derived in three transformations from 5, for the key photolytic step to produce the desired ring-opened precursor to the target compound. This photochemical transformation was unsuccessful. However, an appropriately protected and related precursor was synthesized from 5 through the following side-chain functional group transformations: elaboration of the amino group through malonyl ester formation, oximation at the central carbon, conversion of ester to amide and catalytic reduction of the oxime group. This precursor, on treatment with triethylorthoformate and catalytic acetic acid in ethanol, underwent cyclization to produce the desired 4-carbamoyl-imidazolium-5- olate ring. Deprotection of the latter product proceeded smoothly to give the carbocyclic analog of bredinin. This target molecule exhibits antiviral activity, albeit low, against a number of RNA viruses. Further biological evaluations are in progress.

Full text of DOI:10.3390/molecules180911576

Molecules 2013, 18, 11576-11585; doi:10.3390/molecules180911576
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of a Novel Carbocyclic Analog of Bredinin
Vasu Nair * and Fan Zhang
Center for Drug Discovery and College of Pharmacy, University of Georgia, Athens, GA 30602, USA
*
Author to whom correspondence should be addressed; E-Mail: vnair@rx.uga.edu or vnair@uga.edu;
Tel.: +1-706-542-6293; Fax: +1-706-583-8283.
Received: 23 August 2013; in revised form: 5 September 2013 / Accepted: 10 September 2013 /
Published: 17 September 2013
Abstract: The natural nucleoside antibiotic, bredinin, exhibits antiviral and other
biological activities. While various nucleosides related to bredinin have been synthesized,
its carbocyclic analog has remained unknown. Synthesis of this heretofore unknown
analog of bredinin is described. The key precursor, (3aS,4R,6R,6aR)-6-((methoxy-
methoxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-amine (5), was
prepared from the commercially available compound, (1R,4S)-2-azabicyclo[2.2.1] hept-5-
en-3-one (4). Our initial approach used intermediate 6, derived in three transformations
from 5, for the key photolytic step to produce the desired ring-opened precursor to the
target compound. This photochemical transformation was unsuccessful. However, an
appropriately protected and related precursor was synthesized from 5 through the following
side-chain functional group transformations: elaboration of the amino group through
malonyl ester formation, oximation at the central carbon, conversion of ester to amide
and catalytic reduction of the oxime group. This precursor, on treatment with
triethylorthoformate and catalytic acetic acid in ethanol, underwent cyclization to produce
the desired 4-carbamoyl-imidazolium-5-olate ring. Deprotection of the latter product
proceeded smoothly to give the carbocyclic analog of bredinin. This target molecule
exhibits antiviral activity, albeit low, against a number of RNA viruses. Further biological
evaluations are in progress.
Keywords: bredinin; carbocyclic analog; synthesis; intramolecular cyclization;
antiviral activity

Molecules 2013, 18
. Introduction
Bredinin (1; zwitterion form 2, Figure 1) is an imidazole nucleoside antibiotic, which was originally
11577
1
isolated from the culture filtrates of Eupenicillium brefeldianum [1]. This natural nucleoside, also
called mizoribine, exhibits broad-spectrum antiviral activity, including activity against three strains of
respiratory syncytial virus (RSV), one strain each of Flu A and B, parainfluenza virus types 2 and 3,
mumps virus, and the measles virus (MLSV) [2]. EC50 values in the submicromolar range were
observed with some of these viruses. This natural compound also exhibits a synergistic effect against
the bovine viral diarrhea virus (BVDV) in combination with IFN-α. The EC50 against BVDV in
MDBK cells for a 10:1 combination of IFN-α and bredinin was 0.66 μM, whereas bredinin alone
showed an EC50 of 5.3 μM [3]. Bredinin has also been found to have activity against the hepatitis C
virus [4,5].
Figure 1. Structure of bredinin 1, its zwitterionic form 2 [6] and its carbocyclic analog 3.
This compound also shows activity against the vaccinia virus [1] and the human cytomegalovirus,
HCMV [7]. It has been approved in Japan for use in the prevention of organ allograft rejection, and in
the treatment of rheumatoid arthritis, primary nephrosis, lupus nephritis, dermatomyositis and autoimmune
dermatoses [8]. It is also clinically used as an immunosuppressant in renal transplantation [9,10] and
for autoimmune diseases such as rheumatism [11].
i
Bredinin is one of the most potent known inhibitors of IMPDH (K 0.4 nM E. coli IMPDH and
4
.0 nM for human type II IMPDH). Its active metabolite, bredinin 5'-monophosphate, is thought to
mimic the transition state of IMPDH catalysis [12]. Thus, the mechanism of its antiviral activity
appears to be associated with inhibition of IMPDH by bredinin monophosphate [12,13] (Scheme 1).
Consistent with this suggestion is the observation that the activities of bredinin against viruses can be
reversed by guanosine and guanosine monophosphate [2].
Because of the significant biological activity exhibited by bredinin, we sought to develop synthetic
routes to carbocyclic analogs of this compound that would be of potential interest in the RNA antiviral
drug discovery area. However, an issue with the structure of bredinin is that it exists in the charged
zwitterionic form 2 and this has been confirmed by X-ray crystallographic studies [6,14]. The
zwitterionic structure of the heterocyclic ring of bredinin has produced significant limitations in the
synthesis of new analogs and homologs of this compound. In this report, we provide a methodology
for the synthesis of 1-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-5-hydroxy-1H-
imidazole-4-carboxamide (3), the heretofore unknown carbocyclic analog of bredinin. The

Molecules 2013, 18
11578
methodology described should have generality for the synthesis of other carbocyclic analogs and
derivatives of this biologically interesting compound [15–19].
Scheme 1. Transition state in the conversion of IMP to XMP catalyzed by IMPDH [12].
2
. Results and Discussion
In planning the synthesis of 3 and from the point of view of selectivity, we considered it preferable
for this carbocyclic nucleoside to be synthesized through a linear rather than a convergent approach in
order to avoid side products resulting from alkylation of the nitrogens of imidazole base and to circumvent
possible complications arising from the presence of the hydroxyl group of the imidazole base.
We initiated our synthetic effort (Scheme 2) with the commercially available bicyclic lactam,
(
1R,4S)-2-azabicyclo[2.2.1] hept-5-en-3-one (4). This chiral building block was used because of its
versatility in the synthesis of other carbocyclic nucleosides [20]. Synthesis of the key intermediate,
3aS,4R,6R,6aR)-6-((methoxymethoxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclo-penta[d][1,3]dioxol-4-
(
amine (5) from 4 involved the following transformations: hydroxylation of 4 through treatment with
osmium tetroxide in the presence of N-methylmorpholine-N-oxide (NMO), which resulted in
hydroxylation from the α-face, as previously reported [16,21–23]; protection of the diol and the lactam
NH groups; reductive cleavage of the lactam and protection of the resulting alcohol; and selective
aqueous hydrolysis of the NH protecting group [16]. It is relevant to note that the use of
tert-butyldimethylsilyl ether (TBDMS) for protection of the 5'-hydroxyl group proved less successful,
as the eventual deprotection step became problematic because of the poor water solubility of the
TBDMS-protected compound.

Molecules 2013, 18
Scheme 2. The photochemical approach to the carbocylic analog of bredinin.
11579
O
MOMO
NH2
NH
3 steps
(g) - (i)
7
steps
(
a) - (f)
O
O
4
5
O
O
O
H N
2
N
N
NH2
NH2
NH2
HN
AcO
AcO
3
(
j)
AcO
OAc
AcO
OAc
6
7
Reagents and conditions: (a) OsO
4 2
, NMO, H O:t-BuOH (1:1.5), 70 °C, 30 min, 94%; (b) 2,2-DMP, TsOH,
DMF, r.t., 20 h, 84%; (c) Boc O, DMAP, CH CN, r.t., 15 h, 98%; (d) NaBH , CH OH, 0 °C, 2 h;
2
3
4
3
(
e) ClCH OCH , (iPr) NC H , CH Cl , r.t., 5 h; (f) water, reflux, 6 h, 94%; (g) EtO-CH=NCH(CN)CONH ,
2 3 2 2 5 2 2 2
CH CN, 90 °C, 15 min, 63%; (h) 1N HCl, reflux, 1 h, 78%; (i) Ac O, (C H ) N, DMAP, CH CN, r.t.,1 h,
3
2
2
5 3
3
6
5%; (j) 0.1N HCl, 250 nm or 300 nm UV, r.t., 1 h.
Based on previous literature methodology on the synthesis of bredinin [24,25], we considered
intermediate 7 (Scheme 1) to be an appropriate precursor to prepare our target compound 3, as
aminomalonamide derivatives of riboside were reported to cyclize to 5-hydroxyimidazole compounds
through treatment with triethyl orthoformate. Thus, in order to synthesize 7, compound 5 was first
treated with ethyl-N-(carbamoylcyanomethyl)formimidate followed by deprotection of the MOM
group and acetylation to produce key precursor 6 (31% yield for three steps). However, the photochemical
reaction of 6 with ultraviolet light at λmax 254 nm or 300 nm produced an intractable mixture of
products, from which compound 7 could not be detected or isolated. Interestingly, in this photolysis
step, carbocyclic intermediate 6 behaved totally differently compared to its ribosyl counterpart [24].
Failure of the photolytic ring opening of imidazole 6 prompted us to investigate alternative
pathways [26] to produce intermediate 7 or its derivatives, which could be cyclized to the target
compound 3. The successful strategy that we examined (Scheme 3) was through the synthesis of the
malondiamide intermediate 11. Although this pathway was somewhat longer than the photolytic
pathway of Scheme 1, we chose to use this more viable approach to prepare intermediate 11, in order
to circumvent the decomposition problem of the precursor that had been observed during ultraviolet
irradiation in the photolysis approach. In this thermal approach, the protected cyclopentylamine 5 was
first treated with ethyl 3-chloro-3-oxopropionate in triethylamine to give the amide 8 (73%).
Modification of the malonate side chain of 8 by treatment with aqueous sodium nitrite in acetic acid
produced the oxime 9 (46%). On treatment with methanolic ammonia at 0 °C for 12 h, oxime 9 was
almost quantitatively converted to the malonamide 10. Reduction of 10 with 10% platinum on carbon
gave the desired amine precursor 11 in 77% purified yield.

Molecules 2013, 18
11580
Most of the reported conditions for cyclisation to produce the imidazole ring in the reported
synthesis of bredinin involved heating the malonamide intermediates with orthoformate or
formimidate derivatives in DMF [24,27]. These procedures were completely unsuccessful with
intermediate 11. However, cyclization to the 5-hydroxyimidazole ring did occur effectively when
malonamide 11 was heated at 90 °C with triethyl orthoformate in the presence of catalytic amounts of
acetic acid in ethanol. Purification by HPLC afforded the protected carbocyclic bredinin 12 as a pure
product in 55% yield. Deprotection of 12 in methanol under reflux conditions in the presence of 1N
HCl gave the target molecule 3 in 54% isolated yield, after HPLC purification (Scheme 3).
Scheme 3. Successful route for the synthesis of a carbocyclic analog of bredinin.
Reagents and conditions: (a) ClCOCH
2 2 2 5 2 5 3 3
CO C H , (C H ) N, CH CN, 0 °C, 30 min, then r.t., 2 h, 73.3%;
(
b) NaNO , CH CO H, THF, r.t., 40 h, 46%; (c) NH , CH OH, 0 °C, 12 h, 99.1%; (d) H , 10% Pt/C, CH OH,
2
3
2
3
3
2
3
r.t., 0.5 h, 77%; (e) HC(OC H ) , catalytic CH CO H, C H OH, 90 °C, 1.5 h, 55%; (f) 1 N HCl, CH OH,
2
5 3
3
2
2
5
3
reflux, 1h, 53.6%.
Compound 3 was evaluated for in vitro antiviral activity against a number of RNA viruses [28].
However, only low activity was seen with West Nile virus (New York), Rift Valley Fever virus
(
MP-12), FluA (H1N1) and FluA (H5N1), and Tacaribe virus (TRVL). Further antiviral studies against
other RNA viruses are in progress and those results will be reported elsewhere when the studies
are completed.

Molecules 2013, 18
11581
3. Experimental
3.1. General Procedures
All commercial chemicals and solvents used in the synthetic steps were purchased from Aldrich or
Fisher Scientific and used as received. Reactions were monitored by thin layer chromatography (TLC)
using appropriate developing solvents and pre-coated silica gel plates (UV 254 nm) purchased from
Merck and Co. Each synthetic reaction was performed under an inert atmosphere of dry nitrogen
1
13
unless stated otherwise. H-NMR and C-NMR spectra were recorded on a Varian Unity Inova
00 MHz or a Varian Mercury Plus 400 MHz NMR spectrometer. Chemical shifts are reported in δ
5
(
(
ppm) relative to tetramethylsilane (TMS) as internal standard and multiplicities are given as singlet
s), doublet (d), quartet (q), multiplet (m) and broad singlet (br s). High resolution mass spectral data
were obtained on a Q-TOF Ion Mobility mass spectrometer. Ultraviolet spectra were recorded on
Varian Cary Model 3 spectrophotometer. HPLC purifications were performed on an analytical Delta
Pak C column using a Beckman Coulter Gold 127 HPLC system with a Gold 166 UV analytical
1
8
detector. The data were collected and processed using a Gold software package.
(
1S,2R,3R,5R)-3-(acetoxymethyl)-5-(5-amino-4-carbamoyl-1H-imidazol-1-yl)cyclopentane-
1,2-diyl
diacetate (6). To a solution of ethyl N-[ethoxycarbonyl (cyano)methyl] formimidate (0.516 g, 2.80 mmol)
in acetonitrile (5 mL) was added a solution of amine 5 [16,23] (0.6 g, 1.81 mmol) in acetonitrile (10 mL)
and the mixture was refluxed at 90 °C for 15 min. The reaction mixture was concentrated to dryness,
and the residue was purified by silica gel column chromatography using chloroform/methanol (98:2)
1
as eluent to give the imidazole intermediate (0.423 g, 63.3%). H-NMR (500 MHz, CDCl
3
) δ 7.00 (s,
1
4
1
1
H), 6.48 (bs, 1H), 5.60 (brs, 2H), 4.69–4.67 (m, 2H), 4.60–4.58 (m, 1H), 4.43 (t, J = 7.0 Hz, 1H),
.26–4.21 (m, 1H), 3.73–3.71 (m, 1H), 3.67–3.64 (m, 1H), 3.40 (s, 3H) 3.34 (m, 1H), 2.55–2.50 (m,
13
H), 2.47–2.42 (m, 1H), 2.26–2.19 (m, 1H), 1.61 (s, 3H), 1.31 (s, 3H). C-NMR (125 MHz, CDCl
3
) δ
67.1, 150.0, 144.1, 126.4, 114.6, 113.6, 96.7, 85.5, 81.4, 67.9, 60.6, 55.5, 43.2, 31.6, 27.3, 24.9. This
product (0.1 g, 0.29 mmol) was deprotected by heating at 90 °C in 1N hydrochloric acid (10 mL) for 1 h.
After cooling to room temperature, the reaction mixture was neutralized with a saturated solution of
sodium bicarbonate (50 mL). The solvent was removed under reduced pressure, and the residue was
1
purified by HPLC (C18 column) to give the deprotected product (0.058 g, 78.0%). H NMR (500 MHz,
D
2
O) δ 7.35 (s, 1H), 4.26–4.21 (m, 1H), 4.12–4.09 (m, 1H), 3.89–3.87 (m, 1H), 3.55 (d, J = 6.0 Hz,
H), 2.35–2.29 (m, 1H), 2.12–2.09 (m, 1H), 1.51–1.45 (m, 1H). Acetic anhydride (0.094 mL, 1.0 mmol)
was added dropwise to a mixture of the deprotected compound (0.052 g, 0.2 mmol), triethylamine
0.27 mL, 2.0 mmol) and DMAP (2 mg, 0.0164 mmol) in acetonitrile (5 mL). The reaction mixture
2
(
was stirred for 1 h at room temperature and MeOH (0.5 mL) was added to decompose the excess acetic
anhydride. The solution was concentrated under vacuum and the residue was purified by silica gel
1
column chromatography to give 6 (0.050 g, 65.1%). H-NMR (500 MHz, CDCl ) δ 7.16 (s, 1H), 6.61
3
(
brs, 1H), 5.37 (brs, 1H), 5.23 (s, 2H), 5.20-5.18 (m, 1H), 5.16–5.13 (m, 1H), 4.50–4.45 (m, 1H),
4
1
7
.28–4.24 (m, 1H), 4.19–4.14 (m, 1H), 2.63–2.53 (m, 2H), 2.12 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H),
1
3
.95–1.90 (m, 1H). C-NMR (125 MHz, CDCl
3
) δ 170.8, 170.1, 169.9, 166.9, 143.1, 126.9, 114.5,
6.3, 71.8, 63.9, 55.4, 40.0, 28.0, 20.9, 20.8, 20.7.

Molecules 2013, 18
11582
Ethyl 3-((3aS,4R,6R,6aR)-6-((methoxymethoxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]-
dioxol-4-yl-amino)-3-oxopropanoate (8). To a solution of 5 (0.349 g, 1.50 mmol), triethylamine (0. 607 g,
6
0
.00 mmol) in acetonitrile (5 mL) was added ethyl 3-chloro-3-oxopropionate (0.248 g, 1.65 mmol) at
°C. This reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 2 h. The
solvent was removed under reduced pressure and the residue was purified by silica gel column
1
chromatography using chloroform/methanol (99:1) to give compound 8 (0.38 g, 73.3%). H-NMR (500
MHz, CDCl ) δ 7.64 (d, J = 8.0 Hz, 1H), 4.69 (d, J = 7.0 Hz, 1H), 4.67 (d, J = 7.0 Hz, 1H), 4.52–4.50 (m,
3
1H), 4.41–4.39 (m, 1H), 4.37–4.36 (m, 1H), 4.17 (q, J = 7.0 Hz, 2H), 3.66–3.64 (m, 1H), 3.56–3.53 (m,
1
H), 3.37 (s, 3H), 3.27 (s, 2H), 2.51–2.47 (m, 1H), 2.40–2.38 (m, 1H), 1.53–1.49 (m, 1H), 1.47
1
3
(
s, 3H), 1.27 (t, J = 7.0 Hz, 3H), 1.27 (s, 3H); C-NMR (125 MHz, CDCl ) δ 169.4, 164.4, 111.5,
3
9
6.6, 86.7, 83.5, 69.3, 61.5, 55.7, 55.4, 45.1, 41.5, 33.4, 27.1, 24.7, 14.0; HRMS (ESI) calcd for
+
C
16
H28NO
7
:[M+H] = 346.1866, found 346.1867.
Ethyl 2-(hydroxyimino)-3-((3aS,4R,6R,6aR)-6-((methoxymethoxy)methyl)-2,2-dimethyltetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-ylamino)-3-oxopropanoate (9). A solution of sodium nitrite (1.1 g, 16 mmol)
in H O (1 mL) was added to compound 8 (0.360 g, 1.04 mmol) in acetic acid (4 mL) and
2
tetrahydrofuran (4 mL) and the reaction mixture was stirred at room temperature for 40 h and then
2 4
extracted with ethyl acetate (2 × 20 mL). The combined organic extracts were dried (Na SO ) and
concentrated under vacuum to give a yellow oil which was purified by silica gel column chromatography
1
using CHCl
3
/CH
3
OH (98:2) to give 9 (0.18 g, 46%). H-NMR (500 MHz, CDCl
3
) δ 10.3 (brs, 1H),
7
.96 (d, J = 9.0 Hz, 1H), 4.75–7.72 (m, 2H), 4.60–4.58 (m, 1H), 4.49–4.45 (m, 2H), 4.41 (q, J = 7.0 Hz,
H), 3.76–3.73 (m, 1H), 3.62–3.59 (m, 1H), 3.41 (s, 3H), 2.64–2.60 (m, 1H), 2.47–2.44 (m, 1H), 1.59–1.56
2
1
3
(
m, 1H), 1.50 (s, 3H), 1.39 (t, J =7.0 Hz, 3H), 1.33 (s, 3H). C-NMR (125 MHz, CDCl
3
) δ 161.4,
1
59.2, 145.9,111.1, 96.5, 86.8, 84.1, 69.7, 62.4, 55.6, 55.0, 45.6, 33.2 ,27.0, 24.6,14.0; HRMS (ESI)
+
27 2 8
calcd for C16H N O [M+H] = 375.1767, found 375.1761.
1
2
-(Hydroxyimino)-N -((3aS,4R,6R,6aR)-6-((methoxymethoxy)methyl)-2,2-dimethyl-tetrahydro-3aH-
cyclopenta[d][1,3]dioxol-4-yl)malonamide (10). Compound 9 (0.246 g, 0.66 mmol), dissolved in a
saturated solution of ammonia in methanol (15 mL), was stirred for 12 h at 0 °C. After the reaction
was complete, the mixture was concentrated under reduced pressure. The residue was purified by silica
1
gel column chromatography using CHCl
3
/CH
3
OH (99:1) to give compound 10 (0.23 g, 99.1%). H-NMR
(500 MHz, CDCl
3
) δ 9.70 (s, 1H), 8.60 (d, J = 9.0 Hz, 1H), 6.00 (brs, 1H), 4.78–4.76 (m, 2H),
4
2
.62–4.61 (m, 1H), 4.52–4.49 (m, 1H), 4.42–4.40 (m, 1H), 3.77 (m, 1H), 3.74 (m, 1H), 3.41 (s, 3H),
1
3
.72–2.65 (m, 1H), 2.51–2.48 (m, 1H), 1.60–1.57 (m, 1H), 1.52 (s, 3H), 1.33 (s, 3H). C-NMR (125
MHz, CDCl
3
) δ 165.1, 162.8, 137.1, 111.8, 96.5, 86.7, 83.7, 68.9, 55.9, 55.4, 44.8, 33.3, 27.1, 24.7;
+
HRMS (ESI) calcd for C14
H
24
N
3
O
7
[M+H] = 346.1614, found 346.1613.
1
2
-Amino-N -((3aS,4R,6R,6aR)-6-((methoxymethoxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta-
[
d][1,3]dioxol-4-yl)malonamide (11). A mixture of compound 10 (0.110 g, 0.32 mmol) and 10% Pt/C
20 mg) in methanol (30 mL) was stirred under 14 psi pressure of hydrogen at room temperature for
.5 h. The catalyst was then filtered off using a bed of celite and the filtrate was evaporated under
reduced pressure. The residue was purified by silica gel column chromatography using CHCl /CH OH
(
0
3
3

Molecules 2013, 18
11583
1
(
97:3) to give 11 (0.082 g, 77.3%). H-NMR (500 MHz, CDCl ) δ 8.5–8.47 (m, 1H), 7.7–7.73 (m, 1H),
3
5.75 (brs, 1H), 4.75–4.69 (m, 2H), 4.5–4.50 (m, 1H), 4.4–4.34 (m, 2H), 4.16 (brs, 1H), 3.7–3.67 (m,
1
H), 3.5–3.55 (m, 1H), 3.40 (s, 3H), 2.57–2.43 (m, 4H), 1.5–1.52 (m, 1H), 1.51 (s, 3H), 1.32 (s, 3H).
1
3
C-NMR (125 MHz, CDCl
3
) δ 171.2, 169.4, 111.5, 96.6, 87.0, 83.8, 69.3, 57.0, 55.8, 55.4, 45.2, 33.5,
+
2
7.1, 24.7; HRMS (ESI) calcd for C14
H
26
N
3
O
6
[M+H] = 332.1822, found 332.1801.
5
-Hydroxy-1-((3aS,4R,6R,6aR)-6-((methoxymethoxy)methyl)-2,2-dimethyltetrahydro-3aH-cyclopenta-
[d][1,3]dioxol-4-yl)-1H-imidazole-4-carboxamide (12). To the solution of 11 (0.082 g, 0.25 mmol) in
anhydrous ethanol (8 mL) was added triethyl orthoformate (8 mL) and acetic acid (0.1 mL) and the
mixture was heated at 90 °C under argon for 1.5 h. After the reaction was complete, the solvent was
removed under reduced pressure and the residue was purified by HPLC (C column) to give 12 (0.047 g,
1
8
1
5
4
1
1
5%). H-NMR (500 MHz, DMSO-d
6
) δ 8.25 (s, 1H), 7.13 (brs, 1H), 6.66 (brs, 1H), 4.89–4.86 (m, 1H),
.60 (s, 2H), 4.50–4.45 (m, 2H), 3.54–3.51 (m, 2H), 3.28 (s, 3H), 2.29–2.25 (m, 1H), 2.21–2.18 (m,
1
3
H), 2.01–1.96 (m, 1H), 1.46 (s, 3H), 1.24 (s, 3H); C-NMR ( 125 MHz, CD OD) δ 163.1, 155.7,
3
25.1, 113.4, 100.1, 96.2, 83.4, 81.6, 68.3, 59.5, 54.2, 43.9, 33.6, 26.4, 24.1; UV λmax (MeOH) 240 nm
+
(
ε 4100), 284 nm (ε 7900); HRMS (ESI) calcd for C H N O [M+H] = 342.1665, found 342.1667.
1
5
24
3
6
1
-((1R,2S,3R,4R)-2,3-Dihydroxy-4-(hydroxymethyl)cyclopentyl)-5-hydroxy-1H-imidazole-4-carbox-
amide (3). Compound 12 (37 mg, 0.11 mmol) in methanol (8 mL) and 1N HCl (1 mL) was heated at
5 °C for 1 h. After cooling to room temperature, the reaction mixture was treated with saturated
5
sodium bicarbonate to adjust its pH to 6. The solvent was then removed under reduced pressure and
the residue was purified by HPLC using a Delta Pak C18 (15 µm, 10 Å) column with the eluting
solvent system being water /acetonitrile (acetonitrile from 0 to 100%). The flow rate was 5 mL/min
and the retention time was 55.8 min (254 nm detection). After lyophilization, 14.9 mg of pure title
1
compound 3 was obtained (53.6% yield). H-NMR (500 MHz, D
2
O) δ 8.16 (s, 1H), 4.51 (unresolved
dd, 1H), 4.32 (m, 1H), 4.00 (m, 1H), 3.67–3.61 (m, 2H), 2.44–2.38 (m, 1H), 2.23-2.19 (m, 1H),
1
3
1
3
.61–1.55 (m, 1H). C-NMR (125 MHz, CD OD) δ 163.3, 156.2, 125.3, 100.3, 75.2, 72.2, 63.0, 58.3,
4
5.3, 28.6; UV λmax (MeOH) 240 nm (ε 4,200), 283 nm (ε 8,600); HRMS calcd for C10
H
16
N
3
O
5
+
[M+H] = 258.1090, found 258.1104.
3.2. Antiviral Activity Assays
The RNA antiviral activity protocols involved the following assays: virus yield reduction, neutral
red and visual (http://www.usu.edu/iar/In_vitro_assays.html).
4
. Conclusions
Synthesis of compound 3, the first carbocyclic analog of bredinin, is reported. The methodology
developed is general and can be applied to the synthesis of other carbocyclic analogs of this
biologically-active natural nucleoside. The antiviral evaluation revealed that the compound shows low
activity against some RNA viruses. Further antiviral studies are in progress.

Molecules 2013, 18
11584
Acknowledgments
This research was supported by the National Institute of Allergy and Infectious Diseases of the
National Institutes of Health, USA, through Research Award U19 AI 056540. We thank the Institute of
Antiviral Research, Utah State University, Logan, Utah, USA for the antiviral data. The authors also
thank members of our UGA research group for helpful suggestions during the preparation of
this manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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