Synthesis and antiviral activities of synthetic glutarimide derivatives

Article abstract of DOI:10.1248/cpb.58.1436

A series of novel glutarimide compounds were synthesized and their antiviral activities were evaluated. The compounds displaying the strongest antiviral activities included 5, 6f, 7e and 9 against coxsackievirus B3 (Cox B3), 10 and 6f against influenza vi

Full text of DOI:10.1248/cpb.58.1436

1436
Regular Article
Chem. Pharm. Bull. 58(11) 1436—1441 (2010)
Vol. 58, No. 11
Synthesis and Antiviral Activities of Synthetic Glutarimide Derivatives
Xing-yue JI, Zhao-jin ZHONG, Si-tu XUE, Shuai MENG, Wei-ying HE, Rong-mei GAO,
Yu-huan LI,* and Zhuo-rong LI*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College; Beijing
100050, People’s Republic of China.
Received April 6, 2010; accepted August 2, 2010; published online August 4, 2010
A series of novel glutarimide compounds were synthesized and their antiviral activities were evaluated. The
compounds displaying the strongest antiviral activities included 5, 6f, 7e and 9 against coxsackievirus B3 (Cox
B3), 10 and 6f against influenza virus A (influenza A) and 7a against herpes simplex virus 2 (HSV-2). However,
most of the synthetic glutarimides showed comparatively much weaker activity against influenza A, Cox B3 and
HSV-2 than the natural glutarimide compounds tested. Based on the results, it seemed likely that a conjugated
system at the b-substituted moiety provides stronger antiviral activity.
Key words glutarimide derivative; synthesis; antiviral activity; structure–activity relationship
Glutarimide antibiotics, separated from different Strepto- ocarditis, and EV71 infectious disease in children became an
mycete fermentations, include more than 20 kinds of natural important public health problem recently in China. In pres-
products. Most of those compounds possess a b-substituted ent, there are no effective drugs against these infections in
glutarimide moiety in the structure, such as 9-methylstrepti- clinic. Although the application of CHX was limited because
midones,1) cycloheximide (CHX)2,3) and migrastatin4,5) (Fig. of its high toxicity,17—19) it is still a good leading compound
1). Glutarimide antibiotics have been extensively investigated for synthesizing and evaluating novel antiviral agents with
because of their excellent and diverse spectrum of biological broad spectrum activities.
activities.6—9) Most of the natural glutarimides possess anti-
fungal, antibacterial, antiprotozoal, antiviral, antitumor and analogs with glutarimide moiety in their structures, and stud-
immuno suppression activities. ied their antiviral activities and toxicity. Additionally, the
In view of this, we designed and synthesized a series of
CHX, which is a well-known inhibitor of protein synthe- structure–activity relationships (SAR) of the synthesized glu-
sis, shows strong inhibitory activities against plant fungi, tarimide compounds were also investigated.
influenza virus, reovirus and arbovirus.10—12) Streptimidone
Chemistry (2,6-Dioxopiperidin-4-yl)acetaldehyde 5, a
inhibits the development of plant diseases such as phytoph- common intermediate for the synthesis of glutarimide com-
thora blight on pepper, gray mold on cucumber leaves, and pounds, was synthesized according to Fig. 2.7,20)
leaf blast on rice leaves.13) 9-Methylstreptimidone was iso-
As shown in Fig. 2, diethyl b-oxo-glutarate was chosen as
lated from Streptomyces sp., it is responsible for the high the starting material in our research. It was reacted with
antiyeast and antifungal activity of the Streptomycete fer- cyanoacetic acid under the conditions of Cope condensation
mentation, and its antifungal activity is compared with that to afford diethyl b-cyanomethylideneglutarate 1, which was
of streptimidone.1) Migrastatin was isolated from a cultured catalytically hydrogenated in ethanol using 5% Pd/SrCO3 as
broth of Streptomyces sp. as an inhibitor of tumor cell migra- catalyst to yield diethyl b-cyanomethylglutarate 2. Then
tion.4) The 14-membered lactone core in the structure of compound 2 was heated under reflux with moderately strong
migrastatin is the essential part for its inhibitory activity of hydrochloric acid and the resulting homogeneous solution
tumor metastasis.5) Naramycin B is a natural occurring was evaporated to small bulk which was heated to 235 °C
stereoisomer of CHX, and it is active against microorganisms until a clear quiescent liquid was obtained. The melt crystal-
sensitive to CHX.14) In our previous research, it was demon- lized on cooling and recrystallized from methanol to give an
strated that CHX and streptimidones possess potential excellent yield of 3. Treated 3 with SOCl2 under reflux for
inhibitory activities against DNA and RNA viruses, such as 30 min, on cooling, compound 4 was crystallized out as a
human immunodeficiency virus-1 (HIV-1), hepatitis B virus pale yellow solid. Before the Rosenmund reduction, com-
(HBV), herpes simplex virus (HSV), coxsackie B viruses
(Cox B3, Cox B6), human enterovirus 71 (EV71) and
influenza virus.15,16) Especially, coxsackie B viruses are con-
sidered to have an important role in the etiology of viral my-
Fig. 2. The Synthesis of the Common Intermediate 3-Glutarimidylac-
etaldehyde
Reagents and conditions: (i) cyanoacetic acid, b-anline, HOAc, dry benzene, reflux;
(ii) 5% Pd/SrCO3, EtOH, H2, 1 atm; (iii) 15% aq HCl, reflux 30 min; (iv) 235 °C, 2 h,
Fig. 1. The Structures of Naturally Occurring Glutarimide Compounds
(v) SOCl2, reflux 0.5 h; (vi) 10% Pd/BaSO4, dry toluene, H2, 1 atm, reflux, 3 h.
∗ To whom correspondence should be addressed. e-mail: yuhuanlibj@126.com; l-z-r@263.net
© 2010 Pharmaceutical Society of Japan

November 2010
1437
Fig. 4. The Stereo Configurations of Compounds 6b, 6d and 6e
Fig. 3. The Synthesis of Glutarimide Derivatives
Reagents and conditions (i): LDA, tetramethylethylenediamine (TMEDA), THF,
Ϫ70 °C, 0.5 h, (ii): BF3·Et2O, CH2Cl2, reflux, 2 h.
Fig. 5. A Proposed Mechanism for the Formation of anti Isomer
pound 4 must be recrystallized once from dry toluene and the
desired key intermediate (2,6-dioxopiperidin-4-yl)acetalde-
hyde 5 was obtained under the conditions of Rosenmund
reduction. Compounds 1—5 are known structures,7,20) but
1
only the H-MNR spectrum data of compounds 1 and 2 was
Fig. 6. The Configurations of 7b, 7d and 7e
reported.21,22) We report here for the first time the H-NMR
1
spectrum data of compounds 3 and 5.
As shown in Fig. 3, a series of synthetic glutarimide deriv- products 6b and 6d were identified as the anti isomers. Com-
atives were obtained by condensing compound 5 with differ- pounds 6b and 6d showed 8-H signals at around 3.8 ppm in
1
ent types of ketones, to yield novel compounds 6b, 7b, 6c, the H-NMR spectrum which was consistent with that of
7c, 6f, and 7f. Not all the alkalis used in the common aldol naramycin B. The configurations of the compounds men-
condensation, such as NaOH, NaH and NaOEt, were suitable tioned above can also be supported by the mechanism
for the aldol reaction because the glutarimide moiety is sen- depicted in Fig. 5.
sitive to base.3,23—25) Johnson et al.,3) and Egawa et al.20)
In this work, the configuration of the dehydration com-
1
reported that this type of condensation can be carried out pounds was first determined by H-NMR and nuclear Over-
under the conditions of the Nielsen condensation, but the hauser effect spectroscopy (NOESY) experiments. The con-
yield was disappointing (4—12%). In this paper, we em- figuration of the double bond in the dehydration products 7a,
ployed the method of Kudo et al.26) with a small modifica- 7c and 7f are the E configurations which were confirmed by
tion: Lithium diisopropylamide (LDA) was used as the alkali the coupling constant (15.6, 16.0, 15.0 Hz) between the
for enolizing the ketone. In addition, 2 eq of LDA was used alkene double bond hydrogen. The configuration of com-
because of the acidity of (2,6-dioxopiperidin-4-yl)acetalde- pounds 7b, 7d and 7e was determined by NOESY experi-
hyde. Consequently, 60% was achieved. The resulting com- ments.
pounds were dehydrated easily by treatment with BF3·Et2O
in dichloromethane to afford the dehydration products.
Using 7b (Fig. 6) as an example, irradiation of 6-H
enhanced the intensity of 7-H, and no enhancement of inten-
Didemethylcycloheximide (compound 6e) was first syn- sity of 4-H was observed. Moreover, irradiation of 7-H en-
thesized by Kondo et al.,27) and its stereo configuration was hanced the intensity of 4-H. Such data indicated that the con-
ascertained as an anti-configuration based on the data that the figuration of the 7b is E configuration, similar to 7d and 7e.
1
7-H signals of 6e were at 3.8 ppm in H-NMR spectrum,
Compound 9, a known natural antibiotic Actiphenol,
which was in accordance with the data for naramycin B (Fig. was synthesized according to a literature method.28) Its ana-
1), while the 7-H signals of the syn isomer was at about log 10 was first synthesized according to Fig. 7. 2,4-Di-
4.2 ppm, which was in accordance with the data for natural methylphenyl(2,6-dioxopiperidin-4-yl)acetate 8 can be ob-
cycloheximide (Fig. 1).26)
tained by mixing (2,6-dioxopiperidin-4-yl)acetyl chloride 4
As shown in Fig. 4, the configurations of the condensation and 2,4-dimethylphenol in dry pyridine and refluxed for 2 h.

1438
Vol. 58, No. 11
Fig. 7. Synthesis Route for Actiphenol and Its Hydrolyzed Derivatives
Reagents and conditions: (i): dry pyridine, reflux 2 h (ii): anhydrous AlCl3, 155 °C, (iii): ZnBH4/DME, room temperature, 2 h
Table 1. The Antiviral Activities of the Synthesized Glutarimide Derivatives
Cox B3
Influenza virus A
HSV-2
Compounds
TC50
mg/ml
IC50
mg/ml
TC50
mg/ml
IC50
mg/ml
TC50
mg/ml
IC50
mg/ml
SI
SI
SI
5
9
10
6a
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
7f
46.22
80.12
38.52
17.25
14.37
Ͼ18.52
129.34
Ͼ2.06
Ͼ166.67
Ͼ6.67
55.56
Ͼ0.69
Ͼ166.67
Ͼ18.52
Ͼ166.67
18.52
3.33
2.68
5.58
—
Ͼ3.87
—
—
—
4.33
—
—
Ͼ500
122.8
80.1
Ͼ500
288.7
Ͼ500
Ͼ500
Ͼ500
96.2
Ͼ500
240.4
Ͼ500
288.7
309.2
96.2
Ͼ200
34.4
—
200
71.59
38.52
123.68
Ͼ18.52
Ͼ18.52
Ͼ166.67
2.06
Ͼ18.52
Ͼ6.67
43.11
Ͼ0.69
Ͼ388.03
Ͼ18.52
Ͼ166.6
Ͼ6.67
Ͼ18.52
Ͼ0.69
1.63
1.62
—
—
—
3.00
—
—
5.58
—
—
—
—
—
—
—
205.0
26.5
/
3.57
9.00
—
—
—
Ͼ1.29
—
2.2
—
—
8.90
Ͼ500
4.28
Ͼ500
32.08
240.37
1.19
Ͼ500
32.08
500
Ͼ167.0
Ͼ167.0
Ͼ500
388.0
Ͼ167.0
43.11
Ͼ500
Ͼ55.6
Ͼ500
166.7
18.5
Ͼ500
6.17
96.23
32.08
240.37
1.71
Ͼ500
96.23
Ͼ500
32.08
96.23
3.56
334.1
83.99
/
—
—
—
1.7
32.08
96.23
1.89
21.3
83.99
/
1.73
28.90
—
236.7
110.5
/
16.7
3.00
535
3.7
4344.8
600.1
/
Ͼ0.23
0.09
0.76
32.1
0.50
1.6
0.29
2.13
/
CHX
S632A2
Oseltamivir
Ribavirin
Aciclovir
267.50
5.77
1260
1278
/
3.17
/
/
/
2000
271.4
/
7.37
/
/
/
/
500
4.84
103.3
Actiphenol 9 was obtained using the Fries rearrangement. inactive. However, compound 9 and 10 showed low activity
The system of NaBH4/MeOH failed to reduce the carbonyl against Cox B3 virus and influenza virus A respectively.
of Actiphenol 9 because of its sensitivity to base. Therefore, Therefore, it is possible that a conjugated system at the b-
ZnBH4/1,2-dimethoxyethane (DME) was explored to reduce substituted moiety favors the antiviral activities. Compounds
the carbonyl to give compound 10 in good yield. ZnBH4/ 6a, 6b, 6e, 6d were also inactive against the viral panel how-
DME was prepared by adding NaBH4/DME to the suspen- ever cycloheximide possessed potent antiviral activities. It
sion of anhydrous ZnCl2 in DME, and the mixture was can be concluded that the syn isomer rather than the anti iso-
heated under reflux for 3 h, and then was filtered to remove mer of the aldol condensation compounds was necessary for
the insoluble solid.
antiviral activity, but it is also possible that it is the methyl
group at the a position of the ketonic carbonyl of cyclohex-
imide that dominated the antiviral activity.
Results and Discussion
The antiviral activities of the synthesized glutarimide com-
Among the dehydration compounds, compound 7e showed
pounds against Cox B3, HSV-2 (SAV strain) and influenza A certain activity against the Cox B3 virus, but it still presented
(A/Jifang/15/90, H1N1) were evaluated and the results are high toxicity to the Vero cell (IC50ϭ32.08 mg/ml). It is wor-
summarized in Table 1. The activities against Cox B3, and thy of our attention that the dehydration compound 7a showed
HSV-2 virus were tested by using African green monkey kid- potent activity against HSV-2 virus, whose IC50 value (2.06
ney cells (Vero cell) as the virus host, and the anti-influenza mg/ml) was even lower than that of S632A2 and Aciclovir.
A activity was determined in Madin-Darby Canine Kidney Consequently, it can be concluded that the dehydration com-
(MDCK).
pounds which contain an a, b unsaturated ketone at the b-
As shown in Table 1, compound 6f was active against Cox substituted moiety favor more potent antiviral activities, es-
B3, and influenza virus A. Although its IC50 was much pecially 7a, which contains a saturated branched-chain at the
higher than that of S632A2, it presented much lower toxicity ketone moiety. However, most of the dehydration compounds
to MCDK in comparison with S632A2. Compound 6c, how- showed high toxicity to the Vero cell.
ever, showed no activities against any virus, although it pos-
As shown in the Table 1, the same glutarimide compound
sessed a structure similar to 6f except for the additional shows antiviral activities against different kinds of viruses,
methyl group at the benzylic position. Consequently, it can such as 6f, CHX and S632A2. It is well known that CHX is a
be concluded that the methyl group at the benzylic position is inhibitor of protein synthesis, and inhibits virus replication
imperative to the antiviral activities. Compound 6a was also mostly by directly inhibiting virus protein synthesis and/or

November 2010
1439
(2,6-Dioxopiperidin-4-yl)acetic Acid (3)
A
mixture of diethyl b-
inhibiting host cell protein synthesis indirectly interfere virus
replication. And it is deduced that the glutarimide derivatives
synthesized in this paper share the same mechanism. Conse-
quently, there is no doubt that the glutarimide antibiotics
present broad spectrum antiviral activities. However, the pre-
cise molecule mechanism of glutarimide compounds against
virus replication needs to be illustrated.
cyanomethylglutarate (15 g, 0.07 mol) and 15% HCl (50 ml) solution was
heated under reflux for 30 min to give a homogenous solution, and the reac-
tion mixture was concentrated in vacuo to afford a pale yellow oil which was
then heated to 235 °C until a clear quiescent liquid was obtained. The liquid
was cooled and the product was crystallized out, after recrystallization from
absolute MeOH, the title compound was obtained (9.4 g, yield 78%) as
1
white solid. mp 172—173 °C. H-NMR (DMSO-d6) d: 2.29 (3H, m), 2.38
(2H, m), 2.53 (2H, dd, Jϭ3.2, 16 Hz), 10.71 (1H, s), 12.29 (1H, s).
(2,6-Dioxopiperidin-4-yl)acetyl Chloride (4) A slurry of the acid
(8.6 g, 0.05 mol) and thionyl chloride (40 ml) was heated under reflux for
25 min. The orange reaction mixture was cooled and the crystalline solid
that precipitated was filtered rapidly, washed with dry toluene. A colorless
prism was obtained after recrystallization from dry toluene (yield 70%). mp
130—132 °C.
Conclusion
In summary, a series of novel glutarimide compounds were
synthesized and the configuration of the aldol condensation
products and the dehydration products was ascertained along
with their antiviral activities. Some compounds presented po-
tent antiviral activity, especially 6f. Although its IC50 value
(2,6-Dioxopiperidin-4-yl)acetaldehyde (5) To a flask with a gas inlet
tube, stirrer and condenser were added dry toluene (150 ml), once-recrystal-
was higher than S632A2, it was less toxic to the MDCK. The lized (2,6-dioxopiperidin-4-yl)acetyl chloride (3.4 g, 0.018 mol) and 10%
palladiumon–barium sulfate catalyst (0.3 g). The mixture was heated under
reflux and hydrogen was passed through the system. The hydrochloric acid
formed was neutralized with KOH (0.94 g, 0.016 mol) in water (10 ml).
When the solution of KOH became neutral, the reaction was stopped and the
methyl group at the benzylic position was imperative to the
antiviral activity; accordingly, we can deduce that the methyl
at a position of the ketonic carbonyl of S632A2 may also be
essential to the antiviral activity. A conjugated system in the
b-substituted moiety particularly at the b position of the ke-
tonic carbonyl also favored the antiviral activity. Because
compounds 6b, 6d and 6e showed no antiviral activity, it can
be deduced that the syn isomer rather than the anti isomer
was important to the antiviral activity. However, just like the
importance of the methyl to the antiviral activity of 6f and
S632A2, it is possible that the methyl at the a position of the
ketonic carbonyl of CHX also determined the antiviral activ-
ity. Meanwhile, the dehydration compounds which contained
an a, b unsaturated ketone at the b-substituted moiety
favored the antiviral activities, in particular 7a. However,
most of the dehydration compounds showed high toxicity to
the Vero cell. Therefore, our future work will be focused on
the synthesis and evaluation of the antiviral activities of the
hot toluene solution was filtered free of catalyst. On cooling, long colorless
needles of the aldehyde were deposited. These were removed and a second
crop was obtained by concentrating the filtrate. In total, 1.8 g (yield 64%) of
the aldehyde was obtained. mp 121—123 °C. 1H-NMR (CDCl3) d: 2.38 (2H,
dd, Jϭ10.8, 18 Hz), 2.61 (2H, d, Jϭ6.0 Hz), 2.71 (3H, m), 7.92 (1H, br),
9.78 (1H, s).
4-(2-Hydroxy-5-methyl-4-oxohexyl)piperidin-2,6-dione (6a) To a so-
lution of 2 N LDA in hexane (3 ml) in dry THF was added 3-methylbutan-2-
one (516 mg, 6 mmol) and tetramethylethylenediamine (TMEDA) (697 mg,
6 mmol) at Ϫ70 °C under N2 atmosphere. The mixture was stirred 30 min at
this temperature, then a solution of (2,6-dioxopiperidin-4-yl)acetaldehyde
(465 mg, 3 mmol) in dry THF (10 ml) was added at Ϫ70 °C, and stirring was
continued for another 30 min. The reaction was quenched with 10% of
HOAc aqueous solution (30 ml), and extracted with CH2C12 (3ϫ10 ml). The
extract was washed with aq. NaHCO3 and brine, and dried over anhydrous
MgSO4. Evaporation of the solvent left a pale yellow oil, which was tritu-
rated with petroleum ether to wash away the unreacted ketone, and the
residue was recrystallized form CH2Cl2/petroleum ether to give a white solid
1
syn isomer of the aldol condensation and the dehydration of the title compound (yield 60%). mp 94—96 °C H-NMR (DMSO-d6) d:
0.97 (6H, d, Jϭ6.8 Hz), 1.33 (2H, m), 2.24 (3H, m), 2.45 (2H, m), 2.58 (3H,
compounds, in order to further investigate the SAR of the
m), 3.94 (1H, m), 4.67 (1H, d, Jϭ5.6 Hz), 10.64 (1H, s). Electrospray ioniza-
glutarimide antibiotics.
tion (ESI)-MS m/z: 242.13911 [MϩH]ϩ (Calcd for C12H20NO4: 242.13923).
(E)-4-(5-Methyl-4-oxohex-2-enyl)piperidin-2,6-dione (7a) A solution
Experimental
of 6a (200 mg, 0.83 mmol) and 46% BF3·Et2O (1 ml) in CH2Cl2 (15 ml) was
Chemistry All reagents and solvent were used without purification heated under reflux for 2 h. The reaction mixture was poured into saturated
except for the benzene and toluene which were dried with sodium before aq. NaHCO3 (20 ml), the organic layer was separated and the aqueous layer
usage, and tetrahydrofuran (THF), pyridine which were redistilled from was extracted with CH2Cl2 (2ϫ10 ml). The organic layer was combined and
sodium and benzophenone. 1H-NMR spectra were recorded in CDCl3 or
washed with saturated aqueous solution of NaHCO3, water, and brine suc-
DMSO-d6 on a Varian Inova 400/500 MHz spectrometer (Varian, San Fran- cessively. Dried over anhydrous MgSO4, and removed of the solvent to give
cisco, CA, U.S.A.). Chemical shift was reported in parts per million relative a pale yellow oil which was recrystallized from CH2Cl2/petroleum ether to
1
to tetramethylsilane as the internal standard. Melting points were determined afford the title compound as a white solid (yield 78%). mp 62—64 °C. H-
with a X6 microscope melting point apparatus and were uncorrected.
Diethyl b-Cyanomethylideneglutarate (1) A mixture of diethyl b-oxo- (1H, d, Jϭ15.6 Hz), 6.75 (1H, m), 7.80 (1H, s). Electron ionization (EI)-MS
NMR (CDCl3) d: 1.12 (6H, d, Jϭ6.8 Hz), 2.32 (5H, m), 2.74 (3H, m), 6.26
glutarate (50 g, 0.25 mol), cyanoacetic acid (23.3 g, 0.27 mol), b-alanine m/z: 223.1198 [M]ϩ (Calcd for C12H17NO3: 223.1208).
(1.74 g) and glac HOAc (7.5 ml) in dry benzene (50 ml) were heated under
4-[2-Hydroxy-2-(2-oxocyclopentyl)ethyl]piperidin-2,6-dione (6b) By
reflux and H2O was liberated. After 10 h, b-alanine (0.75 g) and glc HOAc a similar method to 6a, compound 6b was synthesized (yield 56%) as a
1
(2.5 ml) were added, and the reaction mixture was heated under reflux until white solid. mp 120—122 °C. H-NMR (DMSO-d6) d: 1.15 (1H, m), 1.62
no more H2O was separated out (ca. 18 h). The mixture was cooled and (3H, m), 1.76 (1H, m), 1.88 (2H, m), 2.16 (5H, m), 2.51 (2H, m), 3.79 (1H,
EtOAc (50 ml) was added, the organic layer was washed successively with m), 4.77 (1H, d, Jϭ5.2 Hz), 10.63 (1H, s). ESI-MS m/z: 240.12557 [MϩH]ϩ
water, a saturated solution of NaHCO3, water and brine. The organic layer (Calcd for C12H18NO4: 240.12358).
was separated out and dried over anhydrous MgSO4. The solvent was
removed and the residue was purified by column chromatography on silica similar method to 7a, compound 7b was obtained (yield 76%) as a white
gel to give the title compound (41.8 g, yield 75%) as a colorless oil. 1H-
solid. mp 90—92 °C. 1H-NMR (CDCl3) d: 0.85 (1H, m), 1.97 (2H, m), 2.26
NMR (CDCl3) d: 1.26 (6H, t, Jϭ6.8 Hz), 3.36 (2H, s), 3.6 (2H, s), 4.15 (4H, (2H, t, Jϭ6.8 Hz), 2.35 (4H, m), 2.57 (2H, t, Jϭ6.8 Hz), 2.72 (2H, d, Jϭ13.2
q, Jϭ6.8 Hz), 5.50 (1H, s).
Hz), 6.45 (1H, t, Jϭ6.8 Hz), 7.83 (1H, s). EI-MS m/z: 221.1035 [M]ϩ
(E)-4-[2-(2-Oxocyclopentylidene)ethyl]piperidin-2,6-dione (7b) By a
Diethyl b-Cyanomethylglutarate (2) Catalytic hydrogenation of (Calcd for C12H15NO3: 221.1052).
diethyl b-cyanomethylideneglutarate (40 g, 0.18 mol) in absolute EtOH (300
4-(2-Hydroxy-4-oxo-5-o-tolylpentyl)piperidin-2,6-dione (6c) By
a
ml) was carried out at atmospheric pressure using 5% Pd/SrCO3 (4.0 g) as
similar method to 6a, compound 6c was synthesized (yield 54%) as a white
the catalyst for 24 h. The catalyst was filtered away, and the filtrate was con- solid. mp 65—66 °C. 1H-NMR (DMSO-d6) d: 1.28 (3H, m), 2.06 (1H, d, Jϭ
centrated to give a colorless oil which was used directly in the next step 6.8 Hz), 2.12 (3H, s), 2.26 (5H, m), 3.79 (2H, s), 3.93 (1H, m), 4.82 (1H, d,
(yield 98%). 1H-NMR (CDCl3) d: 1.26 (6H, t, Jϭ6.8 Hz), 2.49 (4H, m),
2.69 (3H, m), 4.15 (4H, q, Jϭ6.8 Hz).
Jϭ5.6 Hz), 7.10 (4H, m), 10.64 (1H, s). ESI-MS m/z: 304.15541 [MϩH]ϩ
(Calcd for C17H22NO4: 304.15488).

1440
(E)-4-(4-Oxo-5-o-tolylpent-2-enyl)piperidin-2,6-dione (7c) By a simi-
lar method to 7a, compound 7c was obtained (yield 78%) as a white solid.
mp 152—154 °C. 1H-NMR (CDCl3) d: 2.22 (3H, s), 2.28 (5H, m), 2.70 (2H,
d, Jϭ17.0 Hz), 3.82 (2H, s), 6.21 (1H, d, Jϭ16.0 Hz), 6.78 (1H, m), 7.11
(1H, d, Jϭ6.5 Hz), 7.18 (3H, m), 7.73 (1H, s). EI-MS m/z: 285.1351 [M]ϩ
(Calcd for C17H19NO3: 285.1365).
Vol. 58, No. 11
added to adjust the pH to 2. The mixture was extracted with CH2Cl2 (3ϫ15
ml), the organic layer was washed with a saturated aqueous solution of
NaHCO3, water, and brine successively, and dried over anhydrous MgSO4.
The solvent was removed to afford a white solid which was purified by col-
umn chromatography over silica gel to give the title compound (yield 78%).
1
mp 166—168 °C. H-NMR (DMSO-d6) d: 1.55 (2H, m), 2.09 (3H, s), 2.14
4-[2-Hydroxy-2-(5-methyl-2-oxocyclohexyl)ethyl]piperidin-2,6-dione
(6d) By a similar method to 6a, compound 6d was synthesized (yield
(3H, s), 2.26 (3H, m), 2.58 (2H, m), 4.87 (1H, m), 5.55 (1H, d, Jϭ4.4 Hz),
6.74 (1H, s), 6.84 (1H, s), 8.27 (1H, s), 10.64 (1H, s). EI-MS m/z: 277.1301
[M]ϩ (Calcd for C15H19NO4: 277.1314).
1
55%) as a white solid. mp 125—127 °C. H-NMR (DMSO-d6) d: 0.93 (3H,
d, Jϭ6.8 Hz), 1.36 (4H, m), 1.75 (1H, m), 1.90 (1H, m), 2.09 (2H, m), 2.22
(4H, m), 2.62 (3H, m), 3.82 (1H, m), 4.72 (1H, d, Jϭ6.8 Hz), 10.65 (1H, s).
ESI-MS m/z: 268.15707 [MϩH]ϩ (Calcd for C14H22NO4: 268.15488).
(E)-4-[2-(5-Methyl-2-oxocyclohexylidene)ethyl]piperidin-2,6-dione
(7d) By a similar method to 7a, compound 7d was obtained (yield 78%) as
a white solid. mp 125—127 °C. 1H-NMR (CDCl3) d: 1.07 (3H, d, Jϭ6.4
Hz), 1.53 (1H, m), 1.93 (3H, m), 2.17 (2H, m), 2.36 (4H, m), 2.64 (4H, m),
6.49 (1H, t, Jϭ7.6 Hz), 7.78 (1H, s). EI-MS m/z: 249.1348 [M]ϩ (Calcd for
C14H19NO3: 249.1365).
Antiviral Assays African green monkey kidney cells (Vero), Madin-
Darby Canine Kidney (MDCK), Coxsackie viruses (Cox B3 Nancy strain),
influenza A (A/Jifang/15/90, H1N1) and HSV-2 (SAV strain) were all from
the Institute of Virology, Chinese Academy of Preventive Medicine.
Cytotoxicity Determination. Cytotoxicity Assay The cytotoxicity of
compounds in the presence of Vero and MDCK cells were monitored by cy-
topathic effect (CPE). Vero and MDCK cells (2.5ϫ104/well) were plated
into a 96-well plate. A total of 24 h later, the monolayer cells were incubated
in the presence of various concentrations of test compounds. After 48 h of
culture at 37 °C and 5% CO2 in a carbon–dioxide incubator, the cells were
monitored by CPE. Median toxic concentration (TC50) was calculated by
Reed and Muench analyses.
4-[2-Hydroxy-2-(2-oxocyclohexyl)ethyl]piperidin-2,6-dione (6e) By a
similar method to 6a, compound 6e was synthesized (yield 61%) as a white
1
solid. mp 93—95 °C. H-NMR (DMSO-d6) d: 1.29 (2H, t, Jϭ6.0 Hz), 1.44
(1H, m), 1.59 (2H, m), 1.81 (2H, m), 1.97 (1H, m), 2.27 (5H, m,), 2.38 (1H,
m), 2.56 (2H, m), 3.87 (1H, m), 4.51 (1H, d, Jϭ5.6 Hz), 10.63 (1H, s). ESI-
MS m/z: 254.14052 [MϩH]ϩ (Calcd for C13H20NO4: 254.13923).
Anti Coxsackie B3 and HSV-2 Activity Assay Confluent Vero cells
grown in 96-well microplates were infected respectively with 100 median
tissue culture infective dose (100TCID50) HSV-2 or Cox B3. After 1 h ad-
sorption at 37 °C, the monolayers were washed by phosphate buffered saline
(PBS) and incubated at 37 °C in the maintenance medium (MEM plus 2%
fetal bovine serum (FBS)) with or without different concentrations of test
compounds. Viral cytopathic effect (CPE) was observed when the viral con-
trol group reached 4 and the antiviral activity of tested compounds was de-
termined by the Reed and Muench analyses.
(E)-4-[2-(2-Oxocyclohexylidene)ethyl]piperidin-2,6-dione (7e) By a
similar method to 7a, compound 7e was obtained (yield 82%) as a white
1
solid. mp 152—154 °C. H-NMR (CDCl3) d: 1.78 (2H, m), 1.88 (2H, m),
2.22 (2H, t, Jϭ6.4 Hz), 2.33 (3H, m), 2.45 (4H, m), 2.73 (2H, m), 6.50 (1H,
t, Jϭ7.6 Hz), 7.77 (1H, s). EI-MS m/z: 235.1200 [M]ϩ (Calcd for C13H17NO3:
235.1208).
4-(2-Hydroxy-4-oxo-5-o-tolylhexyl)piperidin-2,6-dione (6f) By a sim-
ilar method to 6a, compound 6f was synthesized (yield 63%) as a white
solid. mp 121—123 °C. 1H-NMR (DMSO-d6) d: 1.20 (3H, d, Jϭ7.0 Hz),
1.26 (2H, m), 2.16 (4H, m), 2.31 (3H, s), 2.35 (1H, d, Jϭ7.0 Hz), 2.44 (2H,
m,), 3.84 (1H, br), 4.01 (1H, q, Jϭ7.0 Hz), 4.65 (1H, d, Jϭ5.5 Hz), 6.97
(1H, d, Jϭ6.5 Hz), 7.16 (2H, m), 7.19 (1H, d, Jϭ5.5 Hz), 10.62 (1H, s). ESI-
MS m/z: 318.17107 [MϩH]ϩ (Calcd for C18H24NO4: 318.17053).
Anti-influenza A Assays Confluent MDCK cells grown in 96-well mi-
croplates were infected 100TCID50 with influenza A. After 2 h of adsorption
at 37 °C, the monolayers were washed by PBS and incubated at 37 °C in the
maintenance medium with or without different concentrations of test com-
pounds. Viral cytopathic effect (CPE) was observed when the viral control
group reached 4 and the antiviral activity of glutarimide derivatives was de-
termined by the Reed and Muench analyses.
(E)-4-(4-Oxo-5-o-tolylhex-2-enyl)piperidin-2,6-dione (7f) By a simi-
lar method to 7a, compound 7f was obtained (yield 81%) as a white solid.
mp 149—151 °C. 1H-NMR (CDCl3) d: 1.41 (3H, d, Jϭ6.5 Hz), 2.17 (5H,
m), 2.44 (3H, s), 2.60 (2H, d, Jϭ16.0 Hz), 4.02 (1H, q, Jϭ6.5 Hz), 6.04 (1H,
d, Jϭ15.0 Hz), 6.68 (1H, m), 7.02 (1H, t, Jϭ5.0 Hz), 7.22 (3H, m), 7.71
(1H, s). EI-MS m/z: 300.16142 [MϩH]ϩ (Calcd for C18H22NO3: 300.15997).
2,4-Dimethylphenyl(2,6-dioxopiperidin-4-yl)acetate (8) The acid
chloride 4 (from 5 g, of 3) was dissolved in dry pyridine (35 ml) and 2,4-di-
methylphenol (5 g, 0.041 mol) added in one portion. The mixture was heated
at 90 °C for 2 h, and then poured into a mixture of water (250 ml) and meth-
ylene chloride (100 ml). Some unwanted black material was separated at this
stage by filtration. The organic layer in the filtrate was washed with dilute
hydrochloric acid, then water, and dried over anhydrous magnesium sulfate.
Removed of the solvent to give a white solid followed by recrystallization
from methylene chloride–ether to gave the title compound (yield 80%), mp
Acknowledgements This work was supported by the National Natural
Science Foundation of China (Grant no. 30772600) and National S&T Major
Special Project on Major New Drug Innovation (Item Number: 2009ZX09103-
135).
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