Ring-expanding rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-imidazol- 4-ones in synthesis of flutimide analogs

Article abstract of DOI:10.1016/j.tet.2014.04.013

The RNA-dependent transcriptase of influenza virus is an attractive antiviral target, still not addressed by any commercialized drugs. Flutimide, a fungal metabolite, comprising an unusual 2,6-diketopiperazine core has earlier been shown to inhibit the endonuclease activity of influenza transcriptase. In this paper we present a novel synthetic approach to 2,6-diketopiperazines, based on the rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-imidazol-4-ones and synthesis and anti-influenza evaluation of a series of novel flutimide analogs.

Full text of DOI:10.1016/j.tet.2014.04.013

Accepted Manuscript
Ring-expanding rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-imidazol-4-ones
in synthesis of flutimide analogs
Mikhail S. Baranov, Irina T. Fedyakina, Mikhail Yu. Shchelkanov, Ilia V. Yampolsky
PII:
S0040-4020(14)00506-7
10.1016/j.tet.2014.04.013
TET 25463
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 5 December 2013
Revised Date: 21 March 2014
Accepted Date: 7 April 2014
Please cite this article as: Baranov MS, Fedyakina IT, Shchelkanov MY, Yampolsky IV, Ring-expanding
rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-imidazol-4-ones in synthesis of flutimide analogs,
Tetrahedron (2014), doi: 10.1016/j.tet.2014.04.013.
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ACCEPTED MANUSCRIPT
Ring-expanding rearrangement of 2-acyl-5-arylidene-3,5-
dihydro-4H-imidazol-4-ones in synthesis of flutimide analogs
Mikhail S. Baranov a, Irina T. Fedyakina b, Mikhail Yu. Shchelkanov b,
Ilia V. Yampolskya,*
a Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10,
Moscow 117997, Russia
b D.I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, Gamaleya 16,
Moscow 123098, Russia
* Corresponding author. Mikhail S. Baranov, Tel: +7499- 7248122, Fax: +7495- 3307056, e-
mail: baranovmikes@gmail.com
Graphical abstract
Аbstract
The RNA-dependent transcriptase of influenza virus is an attractive antiviral target, still
not addressed by any commercialized drugs. Flutimide, a fungal metabolite, comprising an
unusual 2,6-diketopiperazine core has earlier been shown to inhibit the endonuclease activity of
influenza transcriptase. In this paper we present a novel synthetic approach to 2,6-
diketopiperazines, based on the rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-imidazol-4-
ones and synthesis and anti-influenza evaluation of a series of novel flutimide analogs.
Key words:
Influenza, flutimide, imidazolones, rearrangement.

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1. Introduction
The RNA-dependent transcriptase of influenza virus is an attractive antiviral target, still
not addressed by any commercialized drugs. A number of inhibitors for this enzymatic complex
have been proposed, including 4-substituted 2,4-dioxobutanoic acids1 and N-hydroxy amides and
imides.2 In 1995 Singh and colleagues reported isolation and structural characterization of a
fungal metabolite flutimide, that was shown to selectively inhibit the endonuclease ativity of the
influenza viral transcriptase at micromolar concentrations (IC50 ~ 3µ
M).3
Fig 1. Flutimide, a fungal metabolite possessing an anti-influenza activity
Flutimide was found to have an unusual structure, comprising a 2,6-diketopiperazine core
(Fig. 1.). There are only few examples of natural 3,4-dehydro-2,6-diketopiperazines, including
antibacterial agent Coelomycin and its derivatives.4 Several synthetic approaches to this
heterocyclic core have been reported.5
The total synthesis of flutimide was presented in 1995 by the Singh research group.6 Later,
in 2001 the same team described synthesis of flutimide structural analogs in order to find
compounds with improved activity.5a Several derivatives showed increased potencies compared
to flutimide (down to 0.8
provided low total yield.
µM), but the method of their synthesis included about 12 stages and
In this paper we present a novel synthetic approach to 2,6-diketopiperazines, based on the
rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-imidazol-4-ones and synthesis and anti-
influenza evaluation of a series of novel flutimide analogs.
2. Results and discussion
In the course of our investigations in the chemistry of chromophores of fluorescent
proteins we found that synthetic chromophore of the asFP595 chromoprotein from Anemonia
sulcata and its analogs easily transform into 2,6-diketopiperazines in acidified aqueous solutions
(Fig 2.).7

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Fig 2. Ring-expanding rearrangement of the red fluorescent proteins chromophores with
proposed mechanism. Alk = (CH2)2SMe : AsFP595 chromoprotein; Alk = (CH2)4NH2 : ZFP538
fluorescent protein.
The mechanism of this transformation presumably includes protonation of imidazole
nitrogen, nucleophilic ring opening upon addition of a water molecule and finally, recyclization
at the keto-group .
We found the reaction to proceed easily in polar protonic solvents at acid concentrations in
the broad range 0.1–5 M. The maximal yields were achieved using aqueous hydrochloric acid
diluted with ethanol to 0.5–1M. The conversion is complete upon several hours at room
temperature.
The reaction was found to be non-sensitive to the nature of aliphatic substituent in acyl
group (R') and little sensitive to the nature of a substituent at nitrogen atom (R). This allowed us
to synthesize a series of flutimide analogs with different substituents at positions 1 and 3
(Scheme 1).

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Scheme 1. Synthesis of flutimide analogs 4a-d using the ring-expanding rearrangement.
The 2-acylimidazolones 3 were obtained by oxidation of the corresponding 2-
alkylimidazolones 2 with selenium dioxide, earlier described by us.7a This synthetic step was
found to be incompatible with unprotected N-hydroxyimidazolones. Also, the acidic conditions
of 2,6-diketopiperazine ring formation excluded using acid-labile protecting groups at N-hydroxy
group. Therefore, we used N-methoxyimidazolones 2c,d and N-methylated imidazolones 2a,b.
We found that addition of water to the reaction mixture during the oxidation with SeO2 in
some cases led to direct formation of the rearrangement product. This effect is minute for N-
methylated analogs 2a,b whereas in case of N-methoxyimidazolones 2c,d a complete conversion
was achieved. The overall yields at the two stages (oxidation-recyclization) were 30–40% both
for consecutive and one-pot methods.
Since NMR and other spectral analyses of the rearrangement products provided only
indirect evidence of 2,6-diketopiperazine core formation, we performed an X-ray
crystallographic study of compound 4d (Fig 3)8
Fig 3. General view of compound 4d in representation of atoms via thermal ellipsoids at
50% probability level.
Deprotection of the O-methylated N-hydroxy functionality was completed by using boron
tribromide (Scheme 2). We found that demethylation of N-hydroxy group occurs faster than

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demethylation of phenolic oxygen, this allowed us to selectively deprotect the N-OMe moiety in
dimethoxy derivative 4e to obtain compound 5e.
Scheme 2. Synthesis of flutimide analogs.
Evaluation of antiviral activities and toxicities of compounds 4 and 5 was done on MDCK
(Madin-Darby canine kidney) cells, infected with Rimantadine-resistant influenza virus (H1N1
subtype). Among the compounds synthesized, 5e was identical to the compound 1c from the
paper by Singh et al,5a which provided reference to the earlier obtained data. Indeed, the in vivo
antiviral activity of compound 5e (IC50 3.0 ꢀM) was comparable with the earlier reported
transcriptase inhibitory activity of the same compound (0.8 ꢀM). Expectedly, the IC50 of 5e
increased upon transition from an enzyme-based assay to the cell-based assay.
Table 1
CC50, IC50 and therapeutic indices of compounds 4 and 5.
Therapeutic
a
Compound CC50 (µМ)
IC50b (µМ)
indexc
511
-
>>200
>>200
-
-
4a
4b
150
33
160
81
14
9.9
62
25
54
17
4.5
3.0
2.5
1.3
2.9
4.8
3.0
3.3
4c
4d
4e
5c
5d
5e
a 50% cytotoxic concentration (CC50; µМ);

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b 50% inhibitory concentration (IC50; µМ);
c 50% therapeutic index (IC50/CC50 ratio; non-dimensional).
The compounds with a free N-hydroxyl (5c-e) possessed the highest antiviral activities,
which is in agreement with the data by Singh et al (Table 1). However, the N-methoxy
derivatives 4c-e appeared to have noticeable activities, whereas the MOM-protected analogs
described in5a (IC50 25–62µM) did not inhibit the viral transcriptase(IC50 >200 µM). The N-
methylated derivatives 4a,b were found to be the least active. The compounds bearing isobutyl
substituent at the position 3 of diketopiperazine core (4d,e and 5d,e) were more active than their
3-methylated homologs.
Interestingly, the flutimide analogs tested possessed similar therapeutic indices (IC50/CC50
ratio), which indicates a link between their antiviral activity and toxicity.
3. Conclusion
In the present report a novel synthetic approach to substituted 2,6-diketopiperazines,
including analogs of antiviral candidate flutimide is described. The applicability of this approach
for synthesis of diverse 2,6-diketopiperazines is supported by availability of a broad range of
precursor imidazolones 2.9

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4. Experimental section
CAUTION: Selenium dioxide is extremely poisonous and proper care should be taken
when working with it.
4.1. General
Commercially available reagents were used without additional purification. For column
chromatography, E. Merck Kieselgel 60 was used. NMR spectra were recorded on a 700-MHz
Bruker Avance III NMR at 293 K. Chemical shifts are reported relative to residue peaks of
CDCl3 (7.27 ppm for 1H and 77.0 ppm for 13C) or DMSO-d6 (2.51 ppm for 1H and 39.5 ppm for
13C). Melting points were measured on a SMP 30 apparatus. IR spectra were obtained on Nicolet
iS10 FT-IR spectrometer (Thermo Fisher Scientific Inc. USA). Elemental analyses were
performed at the microanalysis facility of Nesmeyanov Intsitute of Organoelement Compounds
(Moscow, Russia). High-resolution mass spectra were obtained on an Agilent 6224 TOF LC/MS
System (Agilent Technologies, Santa Clara, CA, USA) equipped with a dual-nebulizer ESI
source. Data acquisition and analysis was performed by the MassHunter Workstation software
(Agilent Technologies, Santa Clara, CA, USA).
Purity of compounds (4a-e, 5c-e) used in biological test was analyzed by HPLC using
Waters Breeze Series liquid chromatograph with Diode-array detector 2998 (detection in range
210 nm to 800 nm). The Ascentis (Supelco, USA) RP-Amide column (150mm×4.6 mm, 3 µm
o
particle diameter) was operated at 35 C, flow rate 1.0 mL/min, mobile phase was mixture of
acetonitrile and water (1:1 v/v).
4.2. (Z)-4-(4-Acetoxybenzylidene)-oxazol-5(4H)-ones
4.2.1 (Z)-4-(4-Acetoxybenzylidene)-2-methylloxazol-5(4H)-one (1a) was synthesized as
reported previously.7a
4.2.2 (Z)-4-(4-Acetoxybenzylidene)-2-isopentyloxazol-5(4H)-one (1b). To the solution of 2-
(4-methylpentanamido)acetic acid (38.0 g, 0.22 mol) in dry THF (100 mL), DCC (45 g, 0.22
mol) in 100 mL of THF was added. The mixture was stirred for 24h, filtered and evaporated. 4-
acetoxy-benzaldehyde (32.8 g, 200 mmol), NaOAc (16.4 g, 200 mmol) and acetic anhydride
o
(150 mL) was added. The mixture was stirred at 35 C for 3 days, the acetic anhydride was
removed in vacuo and the product was purified by chromatography (EtOAc:hexane 1:1; Rf =
0.35). The title compound was obtained as an yellowish solid (31.1 g, 52%); mp 106–108 oC; 1H
NMR (CDCl3) δ 8.13 (d, J 8.8 Hz, 2H), 7.20 (d, J 8.8 Hz, 2H), 7.11 (s, 1H), 2.67 (t, J 7.5 Hz,
13
2H), 2.33 (s, 3H), 1.75–1.69 (m, 3H), 0.99 (d, J 6.2 Hz, 6H); C NMR (CDCl3) δ 21.1 (CH),
22.2 (2CH3), 27.5 (CH2), 27.7 (CH2), 33.9 (CH3), 122.1 (2CH), 129.9 (CH), 130.9, 132.6, 133.5
(2CH), 152. 7, 167.8, 168.9, 169.6; HRMS (ESI) calcd for C17H19NO4 ([M+H]+) 302.1387,
found 302.1379.

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4.3. (Z)-4-(4-Hydroxybenzylidene)-1H-imidazol-5(4H)-ones
4.3.1 (Z)-2-Ethyl-4-(4-hydroxybenzylidene)-1-methyl-1H-imidazol-5(4H)-one (2a) was
synthesized as reported previously.7a
4.3.1 (Z)-2-Isopentyl-4-(4-hydroxybenzylidene)-1-methyl-1H-imidazol-5(4H)-one (2b). In a
solution of 3.0g (10 mmol) of 1b in 50 mL of ethanol the solution of methylamine (wt 40%, 2.0
g, 26 mmol) was added. The mixture was stirred for 20 min, acidified, filtered and evaporated.
The crude product was dissolved in 20 mL of DMF and 330mg (1 mmol) of Cs2CO3 was added.
The mixture was refluxed for 5–10 min (observed by TLC – EtOH:CHCl3 1:10), evaporated,
dissolved in water (50 mL) and acidified by HCl to pH = 3. The mixture was extracted by EtOAc
(3 * 50 mL), washed by water (1 * 50 mL), brine, dried over anhydrous Na2SO4. The solvent
was evaporated and the product was purified by column chromatography (EtOH:CHCl3 1:15; Rf
= 0.45). Yellowish solid (1.62 g, 60%); mp 175–177 oC; 1H NMR (DMSO) δ 10.08 (s, 1H), 8.08
(d, J 8.9 Hz, 2H), 6.88 (s, 1H), 6.84 (d, J 8.9 Hz, 2H), 3.08 (s, 3H), 2.61 (t, J 7.8 Hz, 2H), 1.70–
13
1.65 (m, 1H), 1.62 (q, J 8.0 Hz, 2H), 0.95 (d, J 6.7 Hz, 6H); C NMR (DMSO) δ 22.2 (2CH3),
25.9 (CH2), 26.0 (CH), 27.3 (CH2), 33.6 (CH3), 115.7 (2CH), 125.4, 125.5 (CH), 134.1 (2CH),
136.2, 159.5, 164.9, 170.0; IR (neat) 3130 (w), 2950 (m), 2360 (m), 1670 (s), 1636 (s), 1580 (m),
1460 (m), 1235 (ws), 1160 (m), 820 (m) cm-1; Anal. Calcd for C16H20N2O2: C, 70.56; H, 7.40; N,
10.29. Found: C, 70.64; H, 7.63; N, 10.25. HRMS (ESI) calcd for C16H20N2O2 ([M+H]+)
273.1598 found 273.1605.
4.3.3 (Z)-2-Ethyl-4-(4-hydroxybenzylidene)-1-methoxy-1H-imidazol-5(4H)-one (2c). In a
suspension of 1a (3.5g (13 mmol)) in 50 mL of ethanol the solution of O-methylhydroxylamine
was added (the solution was prepared by dilution of amine hydrochloride (2.6g (31 mmol) and
1.1g (28 mmol) of NaOH in 50 mL of ethanol and 5 mL of water). The mixture was stirred for
20 min, acidified, filtered and evaporated. The crude product was dissolved in 20 mL of DMF
and 330mg (1 mmol) of Cs2CO3 was added. The mixture was refluxed for 5–10 min (observed
by TLC – EtOH:CHCl3 1:10 ; Rf = 0.65), evaporated, dissolved in water (50 mL) and acidified
by HCl to pH = 3. The mixture was extracted by EtOAc (3 * 50 mL), washed by water, brine,
dried over anhydrous Na2SO4. The solvent was evaporated and the product was purified by
column chromatography (EtOH:CHCl3 1:15; Rf = 0.50). The title compound was obtained as an
yellowish solid (1.12 g, 35%); mp 180–183 oC; 1H NMR (DMSO) δ 10.19 (s, 1H), 8.10 (d, J 8.8
Hz, 2H), 6.97 (s, 1H), 6.84 (d, J 8.8 Hz, 2H), 3.94 (s, 3H), 2.69 (q, J 7.3 Hz, 2H), 1.27 (t, J 7.3
Hz, 3H); 13C NMR (DMSO) δ 9.1 (CH3), 20.0 (CH2), 65.0 (CH3), 115.8 (2CH), 124.9, 127.7
(CH), 132.2, 134.7 (2CH), 160.1, 161.7, 164.3; IR (neat) 3130 (w), 2980 (m), 2360 (m), 1580
(m), 1556 (s), 1386 (m), 1290 (s), 1189 (ws), 1168 (s), 1135 (s), 1106 (s), 1082 (s), 953 (m), 881

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(m) cm-1; Anal. Calcd for C13H14N2O3: C, 63.40; H, 5.73; N, 11.38. Found: C, 63.44; H, 5.87; N,
11.38; HRMS (ESI) calcd for C13H14N2O3 ([M+H]+) 247.1077, found 247.1099.
4.3.4 (Z)-2-Isopentyl-4-(4-hydroxybenzylidene)-1-methoxy-1H-imidazol-5(4H)-on (2d). In
a solution of 1b (30.1g (100 mmol)) in 200 mL of ethanol the solution of O-
methylhydroxylamine was added (the solution was prepared by dilution of amine hydrochloride
[10.0 g (120 mmol) and 4.4 g (110 mmol) of NaOH in 150 mL of ethanol and 20 mL of water].
The mixture was stirred for 30 min, acidified, filtered and evaporated. The mixture was dissolved
in 750 mL of EtOAc, washed by water (1 * 150 mL), brine, dried over anhydrous Na2SO4 and
o
evaporated. The crude product was heated at 180 C for 5 minutes, cooled to room temperature
and dissolved in 500 mL of ethanol. The mixture was stirred with aqueous ammonia (25%, 15
mL) for 10 minutes. The solvent was evaporated (temperature below 40 oC) and the mixture was
dissolved in 750 mL of EtOAc, acidified, washed by water (2 * 150 mL), brine and dried over
anhydrous Na2SO4. The solvent was evaporated and the product was purified by column
chromatography (EtOAc:hexane 1:1; Rf = 0.35). Yellowish solid (10.7 g, 37%); mp 120–122 oC;
1H NMR (CDCl3) δ 8.05 (d, J 8.8 Hz, 2H), 7.12 (s, 1H), 7.00–6.90 (brs, 1H), 6.91 (d, J 8.8 Hz,
13
2H), 4.02 (s, 3H), 2.65 (t, J 7.9 Hz, 2H), 1.75–1.70 (m, 3H), 0.99 (d, J 6.3 Hz, 6H); C NMR
(CDCl3) δ 22.2 (2CH3), 25.4, 27.9, 34.4, 65.3 (CH3), 116.1 (2CH), 126.4, 129.8 (CH), 132.8,
134.9 (2CH), 158.8, 161.1, 165.8; IR (neat) 3145 (w), 2950 (m), 2361 (m), 1680 (s), 1638 (s),
1580 (m), 1510 (s), 1460 (m), 1230 (m), 1140 (m), 1103 (s), 1065 (m), 993 (s), 946 (s), 854 (m)
cm-1; Anal. Calcd for C16H20N2O3: C, 66.65; H, 6.99; N, 9.72. Found: C, 66.69; H, 6.84; N, 9.64;
HRMS (ESI) calcd for C16H20N2O3 ([M+H]+) 289.1547 found 289.1556.
4.4. General procedure of oxidation
To the solution of 2 (25 mmol) in dioxane (200 mL), the selenium dioxide (5.50 g, 50
mmol) and water (15 mL) was added. The mixture was refluxed for 15 minutes (monitored by
TLC (EtOH:CHCl3, alumina oxide)). The solvent was evaporated and the mixture was dissolved
in EtOAc (400 mL), washed by water (2 * 50 mL), brine, dried over anhydrous Na2SO4. The
solvent was evaporated and the products were purified by column chromatography.
4.4.1 Oxidation of 2a and 2b
In these reactions only ketone products 3 were formed:
(Z)-2-Acetyl-4-(4-hydroxybenzylidene)-1-methyl-1H-imidazol-5(4H)-one (3a). Described
previously.7a
(Z)-4-(4-Hydroxybenzylidene)-1-methyl-2-(3-methylbutanoyl)-1H-imidazol-5(4H)-one
o
1
(3b). Red solid (458 mg, 64%); mp 202–204 C; (EtOH:CHCl3 1:10; Rf = 0.40); H NMR
(DMSO) δ 10.60–10.50 ( brs, 1H), 8.20 (d, J 8.9 Hz, 2H), 7.37 (s, 1H), 6.91 (d, J 8.9 Hz, 2H),
3.29 (s, 3H), 3.00 (d, J 6.7 Hz, 2H), 2.25–2.10 (m, 1H), 0.98 (d, J 6.7 Hz, 6H); 13C NMR

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(DMSO) δ 22.4 (2CH3), 24.3, 28.3, 46.5 (CH3), 116.3 (2CH), 124.9, 134.2 (CH), 135.3, 135.7
(2CH), 153.5, 161.5, 169.6, 194.9 (CO); HRMS (ESI) calcd for C16H18N2O3 ([M+H]+) 287.1390
found 287.1408.
4.4.2 Oxidation of 2c and 2d
In these case formation of 3H-pyrazine-2,6-diones 4 were observed as major process:
(Z)-3-(4-Hydroxybenzylidene)-1-methoxy-5-methylpyrazine-2,6(1H,3H)-dione
(4c).
Orange solid (2,02 g, 31%); mp ~ 210 oC with decomposition; (EtOH:CHCl3 1:10; Rf = 0.35); 1H
NMR (DMSO) δ 10.51 (s, 1H), 8.20 (d, J 8.8 Hz, 2H), 7.63 (s, 1H), 6.88 (d, J 8.8 Hz, 2H), 3.84
13
(s, 3H), 2.37 (s, 3H); C NMR (DMSO) δ 20.7 (CH3), 6.3 (CH3), 116.1 (2CH), 125.1, 130.7,
136.5 (2CH), 140.4 (CH), 154.4, 154.5, 159.7, 161.5; IR (neat) 3330 (w), 1702 (ws), 1652 (ws),
1597 (ws), 1542 (ws), 1510 (m), 1370 (m), 1170 (m), 1145 (m), 1046 (s), 845 (m) cm-1; Anal.
Calcd for C13H12N2O4*0.33H2O: C, 58.64; H, 4.80; N, 10.52. Found: C, 58.68; H, 4.91; N,
10.52; HRMS (ESI) calcd for C13H12N2O4 ([M+H]+) 261.0870 found 261.0865.
(Z)-3-(4-Hydroxybenzylidene)-5-isobutyl-1-methoxypyrazine-2,6(1H,3H)-dione
(4d).
Orange solid (2.44 g, 33%); mp 205–208 oC; (EtOH:CHCl3 1:15; Rf = 0.45); 1H NMR (CDCl3) δ
8.20 (d, J 8.7 Hz, 2H), 7.82 (s, 1H), 6.95 (d, J 8.7 Hz, 2H), 5.90–5.85 (brs, 1H), 4.02 (s, 3H),
2.76 (d, J 7.2 Hz, 2H), 2.35–2.15 (m, 1H), 1.06 (d, J 6.5 Hz, 6H); 13C NMR (CDCl3) δ 22.6
(2CH3), 26.5 (CH), 41.9 (CH2), 64.2 (CH3), 116.2 (2CH), 126.9, 131.1, 136.9 (2CH), 142.9
(CH), 154.6, 158.9, 159.7, 160.3; IR (neat) 3260 (w), 2960 (m), 1702 (ws), 1658 (ws), 1603
(ws), 1538 (ws), 1505 (m), 1360 (m), 1250 (ws), 1210 (m), 1170 (m), 860 (m) cm-1; Anal. Calcd
for C16H18N2O4: C, 63.56; H, 6.00; N, 9.27. Found: C, 63.54; H, 5.94; N, 9.36; HRMS (ESI)
calcd for C16H18N2NaO4 ([M+Na]+) 325.1164 found 325.1153.
4.5. Recyclization in acidic conditions
The solution of 3a-b (1 mmol) in ethanol (20 mL) was cooled to 0 oC, and the concentrate
solution of HCl (36%, 3.5 mL) was added. The mixture was heated to room temperature and
stirred overnight (monitored by TLC (EtOH:CHCl3, alumina oxide)). The mixture was dissolved
in EtOAc (100 mL) and water (50 mL) and excess of NaHCO3 was added. The organic layer was
separated, washed by water (2 * 30 mL), brine and dried over anhydrous Na2SO4. The solvent
was evaporated and the product was purified by column chromatography.
4.5.1 (Z)-3-(4-Hydroxybenzylidene)-1,5-dimethylpyrazine-2,6(1H,3H)-dione (4a). Orange
solid (160 mg, 66%); mp ~200 oC with decomposition; (EtOH:CHCl3 1:10; Rf = 0.35); 1H NMR
(DMSO) δ 10.43 (s, 1H), 8.19 (d, J 8.8 Hz, 2H), 7.62 (s, 1H), 6.88 (d, J 8.8 Hz, 2H), 3.15 (s,
3H), 2.36 (s, 3H); 13C NMR (DMSO) δ 21.4 (CH3), 40.9 (CH3), 116.5 (2CH), 132.5, 136.7
(2CH), 140.3 (CH), 154.7, 158.1, 161.6, 163.2, 163.8; HRMS (ESI) calcd for C13H12N2O3
([M+H]+) 245.0921 found 245.0934.

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4.5.2 (Z)-3-(4-Hydroxybenzylidene)-5-isobutyl-1-methylpyrazine-2,6(1H,3H)-dione (4b).
o
Orange solid (163 mg, 57%); mp ~280 C with decomposition; (EtOH:CHCl3 1:10; Rf = 0.50);
1H NMR (DMSO) δ 10.44 (s, 1H), 8.19 (d, J 8.8 Hz, 2H), 7.62 (s, 1H), 6.87 (d, J 8.8 Hz, 2H),
2.61 (d, J 7.0 Hz, 2H), 2.25–2.20 (m, 1H), 0.98 (d, J 6.7 Hz, 6H); 13C NMR (DMSO) δ 22.4
(2CH3), 25.6 (CH2), 25.7 (CH2), 41.6 (CH3), 115.9 (2CH), 125.2, 130.1, 136.1 (2CH), 140.1
(CH), 155.9, 157.4, 161.1, 162.5; HRMS (ESI) calcd for C13H12N2O3 ([M+H]+) 287.1390 found
287.1377.
4.6. Demethylation by boron tribromide
o
Compound 4 (0.5 mmol) were dissolved in dry dichloromethane (3 mL), cooled to 0 C
and the solution of boron tribromide in dichloromethane (1M, 1 mL) was added. The mixture
was stayed at room temperature for 5 minutes and after that 50 mL of dichloromethane and 20
mL of water was added. The NaHCO3 was added to pH = 5. The organic layer was washed by
water (2 * 30 mL), brine and dried over Na2SO4. The solvent was evaporated (at the temperature
below 40 oC) and the product was purified by column chromatography.
4.6.1 (Z)-1-Hydroxy-3-(4-hydroxybenzylidene)-5-methylpyrazine-2,6(1H,3H)-dione (5c).
Orange solid (26 mg, 21%); mp ~210 oC with decomposition; (EtOH:CHCl3 1:10; Rf = 0.35); 1H
NMR (DMSO) δ 10.80 (s, 1H), 10.49 (s, 1H), 8.20 (d, J 8.8 Hz, 2H), 7.63 (s, 1H), 6.89 (d, J 8.8
Hz, 2H), 2.37 (s, 3H); 13C NMR (DMSO) δ 20.8 (CH3), 116.1 (2CH), 125.1, 130.7, 136.5 (2CH),
140.4 (CH), 154.4, 154.9, 160.5, 161.4; IR (neat) 3200 (w), 2940 (m), 2360 (m), 1640 (m), 1543
(s), 1460 (m), 1410 (ws), 1320 (m), 1260 (ws), 1050 (m), 970 (m), 840 (m) cm-1; HRMS (ESI)
calcd for C12H10N2O4 ([M+H]+) 247.0713 found 247.0719.
4.6.2 (Z)-1-Hydroxy-3-(4-hydroxybenzylidene)-5-isobutylpyrazine-2,6(1H,3H)-dione (5d).
o
Isolated as monohydrate. Orange solid (41 mg, 28%); mp ~220 C with decomposition;
1
(EtOH:CHCl3 1:10; Rf = 0.40); H NMR (DMSO) δ 10.80–10.70 (brs, 1H), 10.60–10.55 (brs,
1H), 8.21 (d, J 8.7 Hz, 2H), 7.65 (s, 1H), 6.88 (d, J 8.7 Hz, 2H), 2.62 (d, J 6.9 Hz, 2H), 2.22–
2.12 (m, 1H), 0.99 (d, J 6.5 Hz, 6H); 13C NMR (DMSO) δ 22.3 (2CH3), 25.9 (CH), 39.3 (CH2),
116.0 (2CH), 125.2, 130.6, 136.4 (2CH), 140.9 (CH), 154.9, 156.1, 160.3, 161.4; IR (neat) 3420
(w), 2950 (m), 2360 (m), 1684 (s), 1580 (ws), 1570 (ws), 1434 (s), 1317 (s), 1210 (m), 1190
(ws), 1120 (m), 990 (m), 858 (s), 760 (m) cm-1; Anal. Calcd for C15H16N2O4*H2O: C, 58.82; H,
5.92; N, 9.15. Found: C, 58.93; H, 5.81; N, 9.02; HRMS (ESI) calcd for C15H16N2O4 ([M+H]+)
289.1183 found 289.1187.
4.7. (Z)-1-Hydroxy-5-isobutyl-3-(4-methoxybenzylidene)pyrazine-2,6(1H,3H)-dione
(5e)
4.7.1 (Z)-5-Isobutyl-1-methoxy-3-(4-methoxybenzylidene)pyrazine-2,6(1H,3H)-dione (4e).
To the solution of 4d (302 mg, 1.0 mmol) in dry DMF (30 mL), the methyl iodide (560 mg, 4

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mmol) and NaHCO3 (340 mg, 4 mmol) was added. The mixture was stirred for 50 minutes
(monitored by TLC (EtOH:CHCl3 1:20), diluted with EtOAc (150 mL), washed by water (2 * 50
mL), brine and dried over Na2SO4. The solvent was evaporated and the product was purified by
o
column chromatography (CHCl3; Rf = 0.30). Yellowish solid (262 mg, 83%); mp 144–146 C;
1H NMR (CDCl3) δ 8.23 (d, J 8.8 Hz, 2H), 7.81 (s, 1H), 6.98 (d, J 8.8 Hz, 2H), 4.01 (s, 3H), 3.90
13
(s, 3H), 2.75 (d, J 7.1 Hz, 2H), 2.33–2.25 (m, 1H), 1.06 (d, J 6.6 Hz, 6H); C NMR (CDCl3) δ
22.6 (2CH3), 26.5 (CH), 41.9 (CH2), 55.5 (CH3), 64.1 (CH3), 114.6 (2CH), 126.8, 131.2, 136.5
(2CH), 142.8 (CH), 154.6, 156.8, 160.1, 163.0; IR (neat) 2940 (m), 2360 (m), 1715 (s), 1700
(ws), 1673 (s), 1590 (m), 1555 (s), 1440 (m), 1350 (m), 1160 (ws), 1030 (ws), 826 (s), 740 (m)
cm-1; Anal. Calcd for C17H20N2O4: C, 63.54; H, 6.37; N, 8.86. Found: C, 63.73; H, 6.14; N, 8.66;
HRMS (ESI) calcd for C17H20N2O4 ([M+H]+) 317.1496 found 317.1499.
4.7.2 (Z)-1-Hydroxy-5-isobutyl-3-(4-methoxybenzylidene)pyrazine-2,6(1H,3H)-dione (5e).
o
A solution of (4e) (160 mg, 0.5 mmol) in dry dichloromethane (3 mL) was cooled to 0 C
and the solution of boron tribromide in dichloromethane (1M, 0.7 mL) was added. The mixture
was stayed at room temperature for 5 minutes and after that 50 mL of dichloromethane and 20
mL of water was added. The NaHCO3 was added to pH = 5. The organic layer was washed by
water (2 * 30 mL), brine and dried over Na2SO4. The solvent was evaporated (at the temperature
below 40 oC) and the product was purified by column chromatography (CHCl3:EtOH 10:1; Rf =
o
o
1
0.40). Orange solid (95 mg, 63%); mp 157–159 C (lit 152–156 C5a); H NMR (CDCl3) δ 8.27
(d, J 7.6 Hz, 2H), 7.86 (s, 1H), 6.99 (d, J 7.6 Hz, 2H), 3.91 (s, 3H), 2.76 (d, J 3.3 Hz, 2H), 2.35–
2.22 (m, 1H), 1.05 (d, J 6.1 Hz, 6H); IR (neat) 3080 (w), 2960 (wm), 2360 (m), 1703 (s), 1647
(s), 1603 (s), 1560 (m), 1190 (ws) 1120 (m), 1010 (m), 985 (s), 830 (m) cm-1; HRMS (ESI) calcd
for C16H18N2O4 ([M+Na]+) 325.1159 found 325.1150.
4.8 Cytotoxity and antiviral activity
Cytotoxic effects of the compounds in the MDCK (Madin-Darby canine kidney) cell
culture were visually assessed after 48–72 hours of incubation with the compounds investigated.
Then the cell monolayer was washed and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide sodium salt (XTT) was added at 1 mg/mL. After 4 hours of
incubation the results were counted in an automatic colorimeter at wavelength of 450 nm and a
50% cytotoxic dose was calculated.
Studies of the antiviral activity of flutimide analogs and oseltamivir carboxylate at
1 mkg/mL as positive control were performed on 96-well plates with a monolayer of MDCK
cells inoculated by influenza virus A/IIV-Moscow/01/2009 (H1N1) pdm09 (full analogue of
prototype A/California/07/2009 (H1N1) pdm09 resistant for rimantadine).10 Simultaneously with
the infection of a monolayer the tested compounds were added at various concentrations. Panels

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were incubated for 24 hours at 37 °C and then quenched by fixing the cells with 80% acetone in
a phosphate buffer. An enzyme immunoassay technique was performed as described previously11
to assess concentration of viral proteins.

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Acknowledgements
The authors gratefully acknowledge support from the MCB program of the Russian
Academy of Sciences, the Russian Foundation for Basic Research Grant 14-03-31162-mol-a.
B.M.S. was supported by a stipend from the Program of the President of the Russian Federation.
Supplementary data
HPLC and IR data are available. Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.tet.

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References and notes
1.
Tomassini, J.; Selnick, H.; Davies, M. E.; Armstrong, M. E.; Baldwin, J.; Bourgeois, M.;
Hastings, J.; Hazuda, D.; Lewis, J.; McClements, W.; Ponticello, G.; Radzilowski, E.;
Smith, G.; Tebben, A.; Wolfe, A. Antimicrob. Agents Chemother. 1994, 38 (12), 2827–
2837.
2.
3.
4.
Parkes, R. E.; Ermert, P.; Fässler, J.; Ives, J.; Martin, J. A.; Merrett, J. H.; Obrecht, D.;
Williams, G.; Klumpp, K. J. Med. Chem. 2003, 46, 1153–1164.
Hensens, O. D., Goetz, M. A., Liesch, J. M., Zink, D. L., Raghoobar, S. L.; Helms, G. L.;
Singh S. B. Tetrahedron Lett. 1995, 2005–2008.
(a) Goetz, V. A.; Zhang, C.; Zink, D. L.; Arocho, V.; Vicente F. The Journal of Antibiotics
2010, 63, 512–518; (b) Chinworrungsee, V.; Kittakoop, P.; Saenboonrueng, J.;
Kongsaeree, P.; Thebtaranonth Y. J. Nat. Prod. 2006, 69, 1404–1410.
5.
(a) Singh S.B.; Tomassini J.E. J. Org. Chem. 2001, 66, 5504–5516; (b) Song, J.; Guo, C.;
Adele, A.; Yin, H.; Gong L. Chem. Eur. J. 2013, 10, 3319–3323; (c) DeLorbe, J.E.; Jabri,
S.Y.; Mennen, S.; Overman, L.E.; Zhang, F. J. Am. Chem. Soc. 2011, 133, 6549–6552; (d)
González-Vera, J.A.; García-López, T.; Herranz, R. J. Org. Chem. 2005, 70 (9), 3660–
3666; (e) Akhigbe, J.; Brückner, C. Eur. J. Org. Chem. 2013, 18, 3876–3884; (f) Suna, V.;
Haob, X.; Liua, S.; Haoa, H. Tetr. Lett. 2013, 54, 7, 692–694.
6.
7.
Singh S. B. Tetrahedron. Lett. 1995, 2009–2012.
(a) Yampolsky, I.V.; Remington, S.; Martynov, V.I.; Potapov, V.K.; Lukyanov, S.;
Lukyanov, K. A. Biochemistry 2005, 44, 5788–5793; (b) Yampolsky, I.V.; Balashova
N.A.; Lukyanov, K. A. Biochemistry 2009, 48, 8077–8082.
8.
Crystals of 4d (C16H18N2O4, M = 302.32) grown from methanol were colourless,
monoclinic, space group P 21/n. X-ray diffraction data were collected using a “Bruker
SMART APEX2” CCD diffractometer (λ(MoKα) = 0.71073Å, graphite monochromator)
at 100(2) K: a =5.9730(4), b = 16.6528(11), c = 14.6606(9) Å; β = 94.5571(14)°.
Intensities of 7850 reflections were measured and 3828 independent reflections [Rint
=
0.0445] were used in further refinement. Initially spherical atom refinements were
undertaken with SHELXTL PLUS 5.0 [Sheldrick G.M. Acta. Cryst. 2008, A64, 112–122.]
using the full-matrix least-squares method. All non-hydrogen atoms were allowed to have
an anisotropic thermal motion. The refinement converged to wR2 = 0.1059 and GOF =
1.005 for all independent reflections (R1 = 0.0432 was calculated against F for 2693
observed reflections with I > 2σ(I)). Atomic coordinates, bond lengths, angles, and thermal
parameters have been deposited at the Cambridge Crystallographic Data Center (CCDC)
with number 983835.

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9.
Baranov, M.S.; Lukyanov, K.A.; Yampolsky, I.V. RJBC. 2013, 3, 223–244.
10. Lvov, D. K.; Burtseva, E. I.; Prilipov, A. G.; Bazarova, M. V.; Kolobukhina, L. V.;
Merkulova, L. N. Vopr. Virusol. 2009, 54 (5), 10–14.
11. Leneva, I. A.; Fadeeva, N. I.; Fedjakina, I. T.; Gus'kova, T. A.; Hristova, M. L.; Sokolova,
M. V.; Kharitonenkov, I. G. Pharm. Chem. J. 1994, 28(9), 605–610.

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Supporting Information
Ring-expanding rearrangement of 2-acyl-5-arylidene-3,5-dihydro-4H-
imidazol-4-ones in synthesis of flutimide analogs
a,*
Mikhail S. Baranov a, Irina T. Fedyakina b, Mikhail Yu. Shchelkanov b, Ilia V. Yampolsky
a Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10,
Moscow 117997, Russia
b D.I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, Gamaleya 16, Moscow
123098, Russia
* Corresponding author. Mikhail S. Baranov, Tel: +7499- 7248122, Fax: +7495- 3307056, e-mail:
baranovmikes@gmail.com.
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