Antiproliferative and antibacterial activity of some glutarimide derivatives

Article abstract of DOI:10.3109/14756366.2015.1070844

Antiproliferative and antibacterial activities of nine glutarimide derivatives (1–9) were reported. Cytotoxicity of compounds was tested toward three human cancer cell lines, HeLa, K562 and MDA-MB-453 by MTT assay. Compound 7 (2-benzyl-2-azaspiro[5.11]heptadecane-1,3,7-trione), containing 12-membered ketone ring, was found to be the most potent toward all tested cell lines (IC₅₀ = 9–27 μM). Preliminary screening of antibacterial activity by a disk diffusion method showed that Gram-positive bacteria were more susceptible to the tested compounds than Gram-negative bacteria. Minimum inhibitory concentration (MIC) determined by a broth microdilution method confirmed that compounds 1, 2, 4, 6–8 and 9 inhibited the growth of all tested Gram-positive and some of the Gram-negative bacteria. The best antibacterial potential was achieved with compound 9 (ethyl 4-(1-benzyl-2,6-dioxopiperidin-3-yl)butanoate) against Bacillus cereus (MIC 0.625 mg/mL; 1.97 × 10^?3mol/L). Distinction between more and less active/inactive compounds was assessed from the pharmacophoric patterns obtained by molecular interaction fields.

Full text of DOI:10.3109/14756366.2015.1070844

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1070844
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RESEARCH ARTICLE
Antiproliferative and antibacterial activity of some glutarimide
derivatives
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Jelena B. Popovic-Djordjevic , Anita S. Klaus , Zeljko S. Zizak , Ivana Z. Matic , and Branko J. Drakulic
1Department of Chemistry and Biochemistry and 2Department for Industrial Microbiology, Faculty of Agriculture, University of Belgrade, Belgrade,
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4
Serbia, Institute of Oncology and Radiology of Serbia, Belgrade, Serbia, and Department of Chemistry, Institute of Chemistry, Technology and
Metallurgy, University of Belgrade, Belgrade, Serbia
Abstract
Keywords
Antiproliferative and antibacterial activities of nine glutarimide derivatives (1–9) were reported.
Cytotoxicity of compounds was tested toward three human cancer cell lines, HeLa, K562 and
MDA-MB-453 by MTT assay. Compound 7 (2-benzyl-2-azaspiro[5.11]heptadecane-1,3,7-trione),
containing 12-membered ketone ring, was found to be the most potent toward all tested cell
lines (IC50 ¼ 9–27 mM). Preliminary screening of antibacterial activity by a disk diffusion method
showed that Gram-positive bacteria were more susceptible to the tested compounds
than Gram-negative bacteria. Minimum inhibitory concentration (MIC) determined by a broth
microdilution method confirmed that compounds 1, 2, 4, 6–8 and 9 inhibited the growth of all
tested Gram-positive and some of the Gram-negative bacteria. The best antibacterial potential
was achieved with compound 9 (ethyl 4-(1-benzyl-2,6-dioxopiperidin-3-yl)butanoate) against
Bacillus cereus (MIC 0.625 mg/mL; 1.97 ꢀ 10ꢁ3 mol/L). Distinction between more and less active/
inactive compounds was assessed from the pharmacophoric patterns obtained by molecular
interaction fields.
Antitumor agents, heterocycles,
structure–activity analysis
History
Received 7 May 2015
Revised 25 June 2015
Accepted 26 June 2015
Published online 6 August 2015
Introduction
In 1990s it was discovered that thalidomide, a well-known
synthetic glutarimide derivative, has anti-inflammatory and
antiangiogenic properties. It was approved as a drug for the
treatment of certain cancers (newly diagnosed multiple myeloma)
and for complication arised from leprosy. Later on, analogs of
thalidomide with increased potency, 3-amino-thalidomid (poma-
lidomid, Pomalyst) and a-(3-aminophthalimido) glutarimide
(lenalidomid, Revlimid) have been developed20,21. Lenalidomid
has been used for the treatment of multiple myeloma, while
pomalidomid has been recently approved by FDA for the
treatment of relapsed and refractory multiple myeloma22.
Some estrone derivatives with the D-ring replaced with the
glutarimide moiety showed potent inhibition of steroid sulfatase,
an enzyme involved in the pathway of the development of
hormone-dependent breast tumors23; while aminoglutethimide,
the non-steroidal aromatase inhibitor, is in use for the treatment of
hormone-sensitive metastatic breast cancer24,25. In the past
decade, antitumor activity in vitro of mitonafide26, amonafide27
and naphthalimide28,29 derivatives was intensively examined.
Upon detail analysis of bioactive compounds that comprise
glutarimide moiety in their structure, as is briefly outlined in
previous paragraphs, we concluded that data on antiproliferative
and on antibacterial activity of compounds 1–9 (Figure 1) cannot
be found in literature. Those compounds have been prepared in our
group to demonstrate novel synthetic approach of glutarimide ring
closure reaction30. All compounds have a common N-substituted
glutarimide moiety in their structure, and bear structurally diverse
substituents in positions 3 and/or 4 of glutarimide ring. We have
tested antiproliferative and antibacterial activity of compounds
1–9 in vitro, and hereby report on the results obtained.
Both naturally occurring and synthetic cyclic imides, especially
five- and six-membered systems, are an important group of
bioactive molecules. They exhibit widespread pharmacological
effects, including antitumor1–4, anti-inflammatory5, immunomo-
dulatory, antiangiogenic and anxiolytic6–8
.
Isolation and examination of pharmacologically active natural
glutarimides started in 1960s. Initially, cycloheximide9 and
streptimidone10–12 were examined as antibiotics, but later it
was found that they acted as very potent cytotoxic agents13,14
.
The structurally related streptimidone derivative, 9-methylstrepti-
midone, exerts a significant inhibitory activity toward nuclear
factor-kB (N-kB)15. N-kB is involved in cancer and inflamma-
tions. Alkaloids (+)-sesbanimide A and (ꢁ)-sesbanimide B were
isolated from the seeds of the leguminous plant Sesbania
drummondii1,16
. Ethanol extracts of S. drummondii seeds
showed significant inhibitory activity against P388 murine
leukemia model in mice (in vivo)17–19. Structurally related natural
product lactimidomycin (LTM), 12-membered unsaturated
macrolide antibiotic that comprise biosynthetically rare glutar-
imide side chain, produced by Streptomyces amphibiosporus
R310-104 (ATCC 53964), display strong cytotoxicity against a
number of human tumor cell lines in vitro, in vivo antitumor
activity in mice model and potent antifungal activity2.
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Address for correspondence: Jelena B. Popovic-Djordjevic, Department
of Chemistry and Biochemistry, Faculty of Agriculture, University of
Belgrade, Nemanjina 6, 11080 Belgrade, Serbia. E-mail: jelenadj@
agrif.bg.ac.rs

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J. B. Popovic-Djordjevic et al.
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J Enzyme Inhib Med Chem, Early Online: 1–9
Figure 1. Structures of glutarimide deriva-
tives 1–9.
Methods
the wells. Absorbance was measured at 570 nm the next day.
To achieve cell survival (S%), absorbance at 570 nm of a sample
with cells grown in the presence of various concentrations of
Antiproliferative activity
Stock solutions of investigated compounds were prepared in compounds tested was divided with absorbance of control sample
a nutrient medium (RPMI-1640) supplemented with 3 mM (the absorbance of cells grown in nutrient medium only).
L-glutamine, 100 mg/mL streptomycin, 100 IU/mL penicillin, Absorbance of blank was always subtracted from absorbance of
10% heat inactivated fetal bovine serum (FBS) and 25 mM a corresponding sample with cells. All experimentally obtained
Hepes, adjusted to pH 7.2 by bicarbonate solution. RPMI-1640, IC50 data were means of three measurements done in triplicate.
FBS, Hepes and L-glutamine were products of Sigma Chemical
Co., St. Louis, MO. Human cervix adenocarcinoma HeLa, breast
The antibacterial activity testing
carcinoma MDA-MB-453 and normal lung fibroblast MRC-5
cells were cultured as monolayers in the nutrient medium, while The antibacterial activity of compounds 1, 2, 4–8 and 9 was
human myelogenous leukemia K562 cells were maintained as determined against four Gram-positive (Staphylococcus aureus,
suspension culture. The cells were grown at 37 ꢂC in 5% CO2 and Enterococcus faecalis, Bacillus cereus, Listeria monocytogenes)
humidified air atmosphere.
and seven Gram-negative bacterial species [Pseudomonas
HeLa (2000 cells per well), MDA-MB-453 (3000 c/w) and aeruginosa, Escherichia coli, Salmonella enteritidis, Proteus
MRC-5 (5000 c/w) cells were seeded into 96-well microtiter hauseri, Shigella sonnei, Yersinia enterocolitica, E. coli
plates and 20 h later, after the cell adherence, five different (O157:H7)]. Selected bacterial strains originated from ATCC
concentrations of investigated compounds were added to the (American Type Culture Collection, Rockville, MD). These
wells. Final concentrations were in the range from 200 to microorganisms were chosen for the bioassay as the well-known
12.5 mM. Only nutrient medium was added to the cells in the food spoilage and pathogenic bacteria. Each species was main-
control wells. Investigated compound was added to a suspension tained on Mueller–Hinton agar (MHA), which was also used to
of leukemia K562 cells (5000 cells/ well) 2 h after cell seeding, confirm the absence of contamination and the validity of the
in the same final concentrations applied to HeLa, MDA-MB-453 inocula. Before testing, each species was recovered by sub-
and MRC-5 cells. All experiments were done in triplicate. culturing in Mueller–Hinton broth (MHB), aerobically, for 24 h,
Nutrient medium with corresponding concentrations of com- at 37 ꢂC. Working concentrations of approximately 105–106 cfu/
pound, but void of cells, was used as blank.
mL, used for antibacterial activity assays, were prepared by
Cell survival was determined by MTT test according to the proper dilution of culture in microbiological medium.
method of Mosmann and modified by Ohno and Abe, 72 h after Compounds were dissolved in DMSO (2%) to prepare stock
addition of the compounds31,32. Briefly, 20 mL of MTT solution solutions at a concentration of 40 mg/mL, sterilized by filtration
¨
(5 mg/mL in phosphate buffered saline) was added to each well. through a 0.22-mm membrane filter (Sartorius AG – Gottingen,
Samples were incubated for further 4 h at 37 ꢂC in humidified Germany) according to Tepe et al. and further diluted in MHB to
atmosphere with 5% CO2. Then, 100 mL of 10% SDS was added to a working solutions. DMSO was chosen as a non-toxic solvent33.

DOI: 10.3109/14756366.2015.1070844
Activities of glutarimide derivatives
3
Disk diffusion assay was performed using a slightly modified surface area, volume and virtual log P, were calculated in
CLSI34. Each bacterial culture (approximately 105–106 cfu/mL) VegaZZ, using 1.4 A probe . Molecular interaction fields (MIF)
˚
43
was added (0.1 mL) to Petri dishes (90 mm) containing MHA around molecules were calculated by a GRID method, as applied
44
(20 mL). Three sterile blank paper disks (6 mm in diameter, in Pentacle program, using grid resolution of 0.4 A . Hydrogen-
˚
Susceptibility Test Discs, SD 067-5CT, HiMedia, Mumbai, India) bond donor (N1), hydrogen-bond acceptor (O), hydrophobic
were placed on the surface of each agar plate and inoculated with (DRY) and shape (TIP) probes were used. AMANDA algorithm
10 mL of the compound (20 mg/mL). After 2 h at 25 ꢂC, the plates were used for the extraction of hot spots (nodes) from the obtained
were incubated aerobically, for 24 h at 37 ꢂC. After incubation MIFs (discretization); the distances and relative position of the
period, inhibition zone (mm) was measured including the initial nodes were described by maximum auto- and cross-correlation
diameter of the disk. Tests were performed in triplicate and the (MACC2) (encoding). For more exhaustive description of
results were analyzed for statistical significance. The plates with applied methodology see original reference45. Auto- and cross-
MHA were sterility controls. Negative controls were disks correlograms, obtained by the Pentacle program are depicted
impregnated with DMSO. As positive controls disks (Sigma- in matrix-like representation, named as heatmap. Values of
Aldrich GmbH, Steinheim, Germany) with gentamicin (30 mg) variables are color-coded from red (low value) to blue (high
and tetracycline (30 mg) were used.
value). For color code in heatmap, depicted in Figure 3, see
Broth microdilution method was employed to determine on-line version of the article. Correlograms encode molecular
minimum inhibitory concentrations (MICs)34,35. Concentrations descriptors grounded on two-point pharmacophoric pattern,
of compound ranged from 10.0 to 0.048 mg/mL. Test bacterial which represent the local minima of two probes (nodes) around
culture (50 mL) in a MHB was added to the wells of a sterile 96- molecule, including distance between those nodes. All blocks of
well microtiter plate (Sarstedt, Numbrecht, Germany) already correlograms (DRY-DRY, O-O, N1-N1, TIP-TIP, DRY-O, DRY-
containing 50 mL of twofold serially diluted compound in MHB. N1, DRY-TIP, O-N1, O-TIP, N1-TIP) were considered during
The final volume in each well was 100 mL. The microplates were analysis.
prepared in triplicate and incubated aerobically, for 24 h at
37 ꢂC. Wells with MHB was used as a sterility control, while
negative controls were wells with tested compound in 50 mL of
Chemistry
MHB, but void of bacteria. Positive controls were wells with a Chemicals and solvents were purchased from Merck (Darmstadt,
bacterial suspension in 50 mL of MHB and wells with a bacterial Germany), Sigma-Aldrich and Fluka (Basel, Switzerland). All
suspension in a MHB with DMSO, in amounts corresponding to reagents were of analytically pure. All solvents were dried by
the highest quantity present in the broth microdilution assay (to standard methods and distilled before use. The sodium hydride
prove that DMSO had no inhibition effect on the bacterial was used as a 60% dispersion in mineral oil. 18-Crown-6 ether
growth). A microplate shaker (Lab Companion, VM-96B, Seoul, was prepared according to a literature procedure46. Reactions
South Korea) was used for mixing the content of each well at were monitored on silica gel precoated TLC plates, HF254
900 rpm for 1 min prior to incubation in the cultivation (Merck). The dry-flash chromatography on silica gel (12–16 m,
conditions described above. To indicate cellular respiration ICN Pharmaceuticals, Costa Mesa, CA) was used to purify the
2,3,5-triphenyltetrazolium chloride (TTC) (Aldrich Chemical reaction products. Anhydrous reactions were carried out in oven-
Company Inc., Sigma-Aldrich, St. Louis, MO) was added to the dried glassware in extra pure argon atmosphere. 1H and 13C
culture medium. The final concentration of TTC after inocula- NMR spectra were recorded on a Bruker Avance 500 (Bruker
tion was 0.05%. Viable microorganisms enzymatically reduced BioSpin GmbH, Karlsruhe, Germany), or Varian (Palo Alto, CA)
white TTC to a pink TPF (1,3,5-triphenylformazan). The MIC Gemini 2000 instruments on 500/125 or 200/50 MHz, in CDCl3
was defined as the lowest sample concentration that prevented with TMS as an internal reference. ESI-MS spectra were
this change and exhibited complete inhibition of bacterial recorded on Agilent Technologies (Santa Clara, CA) 6210-1210
growth.
TOF-LC-ESI-HR/MS instrument in positive mode. Integrity and
All measurements were done in triplicate and data were purity of all compounds used for biological tests are routinely
expressed as mean standard deviation. The experimental data checked by NMR and ESI/HR-MS.
were subjected to an one-way analysis of variance (ANOVA) and
Fisher’s LSD was calculated to detect significant difference
General procedure for the synthesis of compounds 1 and 2
(p ꢃ 0.05) between the mean values.
1-Benzyl-piperidine-2,6-dione (1, C12H13NO2). Toluene (20 mL)
and sodium hydride (2.9 g, 72 mmol) were placed in a two-necked
flask equipped with reflux condenser and dropping funnel. A
Molecular modeling
Initial 3D structures of compounds 1–9 were generated from
SMILES notation in CORINA assuming R stereochemistry for all
stereogenic centers, except for C2 of the phenethyl moiety of
compound 636,37. The S stereochemistry was ascribed to this
stereogenic center, on the ground of experimental data30. Initial
structures were imported in VegaZZ38. Up to 20 conformations,
representing local energy minima, were obtained by conform-
ational search on the molecular mechanics level (MMFF94s force
field), using Boltzmann jump algorithm in AMMP39,40. Each
conformation of each compound was minimized by the semi-
empirical molecular orbital PM6 method, using implicit solvation
in water (COSMO) to root mean square gradient of 0.01; by
MOPAC201241,42. Conformation of each compound that had the
lowest heat of formation (implying the most stable one) was
chosen for further modeling. 3D-dependent whole-molecular
properties of compounds, surface area, polar surface area, apolar
solution of 4-benzylcarbamoyl-butyric acid methyl ester 1 c
(5.0 g, 18 mmol) in toluene (ꢄ30 ml) was added and mixture
was refluxed for 3 h. After cooling at room temperature the
mixture was filtered and the filtrate was evaporated under reduced
1
pressure to give 65 % of compound 1 (C12H13NO2). H NMR
(200 MHz, CDCl3): ꢀ ¼ 7.36–7.24 (m, 5H, Ar–H), 4.94 (s, 2H;
CH2), 2.65 (t, J ¼ 6.5 Hz, 4H, CH2), 1.98–1.88 (m, 2H, CH2) ppm;
13C NMR(50 MHz, CDCl3): ꢀ ¼ 172.5 (CCOimide), [137.3, 128.8,
128.4, 127.4 (CAr)], 42.6 (CCH2), 32.9 (CCH2), 17.0 (CCH) ppm;
HR-MS (ESI, m/z): calcd. for C12H13NO2 [M + NH4]+: 221.1284;
found: 221.1278.
8-Benzyl-8-aza-spiro[4.5]decane-7,9-dione (2, C16H19NO2).
1
Prepared from methyl ester 2e. Yield 75%; H NMR (200 MHz,
CDCl3): ꢀ ¼ 7.37–7.24 (m, 5H, Ar–H), 4.94 (s, 2H, CH2), 2.60
(s, 4H, CH2), 1.72–1.65 (m, 4H, CH2), 1.50–1.44 (m, 4H, CH2)
ppm; 13C NMR (50 MHz, CDCl3): ꢀ ¼ 172.1(CCOimide), [137.2,

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J. B. Popovic-Djordjevic et al.
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J Enzyme Inhib Med Chem, Early Online: 1–9
128.5, 128.3, 127.3 (CAr)], 44.7 (CCH2), 42.6 (CCH2), 39.4 (C),
2-Benzyl-2-azaspiro[5.7]tridecane-1,3,7-trione (8, C19H23NO3).
1
37.4 (CCH2), 24.1 (CCH2) ppm; HR-MS (ESI, m/z): calcd. for Yield 53%; H NMR (200 MHz, CDCl3): ꢀ ¼ 7.28–7.23 (m, 5H,
C16H19NO2 [M + H]+: 258.1489; found: 258.1480.
Ar–H), 4.91 (s, 2H, CH2), 3.19–2.40 (m, 6H, CH2), 2.25 (ddd,
J ¼ 12.3 Hz, J ¼ 6.2 Hz, J ¼ 3.4 Hz, 1H, CH2), 1.88–1.53 (m, 9H,
CH2) ppm; 13C NMR (50 MHz, CDCl3): ꢀ ¼ 213.3 (CCOketo),
172.3 (CCOimide), 171.6 (CCOimide), [137.0, 128.4, 128.2, 127.3
(CAr)], 57.9 (C), 43.1 (CCH2), 38.3 (CCH2), 31.4 (CCH2), 30.0
(CCH2), 29.5 (CCH2), 25.7 (CCH2), 23.9 (CCH2), 23.8 (CCH2),
22.5 (CCH2) ppm; HR-MS (ESI, m/z): calcd. for C19H23NO3
[M + H]+: 314.1751, found: 314.1738.
General procedure described in our previous paper was used for
preparation of compounds 3–9
2,6-Dioxo-1-phenethylpiperidine-3-carbonitrile (3, C14H14N2O2).
1
Yield 65%; H NMR (200 MHz, CDCl3): ꢀ ¼ 7.34–7.19 (m, 5H,
Ar–H), 4.05 (splitted t, J ¼ 7.0 Hz; J ¼ 2.1 Hz, 2H, CH2), 3.71 (dd,
J ¼ 9.3 Hz, J ¼ 5.3 Hz, 1H, CH), 2.95–2.81 (m, 3H, CH2), 2.72–
2.56 (m, 1H, CH2), 2.36–2.17 (m, 2H, CH2) ppm; 13C NMR
(50 MHz, CDCl3,): ꢀ ¼ 169.6 (CCOimide), [137.7, 129.0, 128.5,
126.7 (CAr)], 115.1 (CCN), 41.6 (CCH2), 35.7 (CCH2), 33.6
(CCH), 30.7 (CCH2), 21.5 (CCH2) ppm; HR-MS (ESI, m/z):
calcd. for C14H14N2O2 [M + NH4]+: 260.1393, found: 260.1389.30
Tert-pentyl-2,6-dioxo-1-phenethylpiperidine-3-carboxylate (4,
Ethyl
4-(1-benzyl-2,6-dioxopiperidin-3-yl)butanoate
(9,
C18H23NO4). Yield 42%; 1H NMR (200 MHz, CDCl3):
ꢀ ¼ 7.37–7.22 (m, 5H, Ar–H), 4.94 (s, 2H, CH2), 4.13 (q,
J ¼ 7.1 Hz, 2H, CH2), 2.82 (dt, J ¼ 17.6 Hz, J ¼ 4.9 Hz, 1H, CH2),
2.64 (dd, J ¼ 11.1 Hz, J ¼ 5.3 Hz, 1H, CH2), 2.54–2.42 (m, 1H,
CH), 2.34 (t, J ¼ 7.2 Hz, 2H, CH2), 2.12–1.90 (m, 2H, CH2),
1.82–1.56 (m, 4H, CH2), 1.25 (t, J ¼ 7.1 Hz, 3H, CH3) ppm; 13C
NMR (50 MHz, CDCl3): ꢀ ¼ 174.5 (CCOimide), 173.2 (CCOester),
172.2 (CCOimide), [137.3, 128.5, 128.3, 127.3 (CAr)], 60.3
(CCH2), 42.9 (CCH2), 41.9 (CCH2), 33.9 (CCH2), 31.9 (CCH2),
29.6 (CCH2), 22.2 (CCH2), 22.0 (CCH2), 14.1 (CCH3) ppm;
HR-MS (ESI, m/z): calcd. for C18H23NO4 [M + H]+: 318.1700,
found: 318.1691.
1
C19H25NO4). Yield 70%; H NMR (200 MHz, CDCl3) ꢀ ¼ 7.30–
7.21 (m, 5H, Ar–H), 4.04–3.96 (m, 2H, CH2), 3.57–3.51 (m, 1H,
CH), 2.86–2.78 (m, 2H, CH2), 2.67 (dt, J ¼ 11.6 Hz, J ¼ 5.8 Hz,
2H, CH2), 2.25–2.07 (m, 2H, CH2), 1.80 (q, J ¼ 7.5 Hz, 2H, CH2),
1.46 (s, 6H, CH3), 0.90 (t, J ¼ 7.5 Hz, 3H, CH3) ppm; 13C NMR
(50 MHz, CDCl3): ꢀ ¼ 171.3 (CCOimide), 168.8 (CCOimide), 167.7
(CCOester), [138.4, 128.9, 126.4 (CAr)], 85.6 (C), 50.0 (CCH),
41.1 (CCH2), 33.8 (CCH2), 33.3 (CCH2), 30.8 (CCH2), 30.2
(CCH2), 25.3 (CCH3), 20.7 (CCH2), 8.1 (CCH3) ppm; HR-MS
(ESI, m/z): calcd. for C19H25NO4 [M + H]+: 332.1856, found:
332.1841.
8-Oxaspiro[4.5]decane-7,9-dione (2c) was prepared according to
a modified literature procedure47.
7,9-Dioxo-8-azaspiro[4.5]decane-6,10-dicarbonitrile (2a) and
2,20-cyclopentane-1,1-diyldi acetic acid (2b) were prepared by a
known literature procedure48. Methyl 5-chloro-5-oxopentanoate
(1b) and methyl [1-(2-chloro-2-oxoethyl)cyclopentyl] acetate (2d)
Tert-pentyl-1-benzyl-4-methyl-2,6-dioxopiperidine-3-carboxyl-
1
ate (5, C19H25NO4). Yield 46.6%; H NMR (200 MHz, CDCl3):
were prepared by
procedure49.
a modification of a known literature
ꢀ ¼ 7.38–7.24 (m, 5H, Ar–H), 5.04–4.88 (m, 2H, CH2), 3.59
(d, J ¼ 4.9 Hz, 0.13 H, CH), 3.21 (d, J ¼ 9.0 Hz, 0.59 H, CH), 2.83
(dd, J ¼ 16.5, J ¼ 3.9 Hz, 1 H, CH), 2.74–2.68 (m, 0.41 H, CH2),
2.60–2.46 (m, 1H, CH2), 2.43–2.29 (m, 1H, CH2), 1.76
(dt, J ¼ 9.4 Hz, J ¼ 4.8 Hz, 2H, CH2), 1.44 (s, 5H, CH3), 1.36
(d, J ¼ 3.5 Hz, 1H, CH3), 1.14–1.07 (m, 3H, CH3), 0.91–0.75 (m,
3H, CH3) ppm; 13C NMR (50 MHz, CDCl3): ꢀ ¼ 170.9
(CCOimide), 169.1(CCOimide), 167.4 (CCOester), [136.9, 128.7,
128.4, 127.5 (CAr)], 85.4 (C), 57.9 (CCH), 43.0 (CCH2), 38.6
(CCH2), 33.4 (CCH2), 27.7 (CCH), 25.3 (CCH3), 25.2 (CCH2),
19.1 (CCH3), 8.1 (CCH3) ppm; HR-MS (ESI, m/z): calcd. for
C19H25NO4 [M + NH4]+: 349.2122, found: 349.2128.
Methyl 5-(benzylamino)-5-oxopentanoate (1c) and methyl 2-(1-(2-
(benzylamino)-2-oxoethyl) cyclopentyl) acetate (2e) were pre-
pared according to a modified literature procedure50.
Methyl 2-methylbutan-2-yl propanedioate (3b) was prepared by a
literature procedure51. Methyl 2-(phenylsulfonyl)acetate (3c) was
prepared by a literature procedure52.
Acrylamides (4a, 4c, 4d) and (E)-N-benzylbut-2-enamide (4b)
were prepared according to a modified literature procedure50.
ꢁ-Ketoesters (5a–c) were prepared by a modification of a
literature procedure53.
1-((R)-1-Phenylethyl)-3-(phenylsulfonyl)piperidine-2,6-dione
(6, C19H19NO4S). Yield 68%; 1H NMR (200 MHz, CDCl3):
ꢀ ¼ 7.90–7.26 (m, 10H, Ar–H), 6.19–6.00 (m, 1H, CH), 4.13–
3.942 (m, 1H, CH), 3.44–3.14 (m, 1H, CH2), 2.92–2.71 (m, 2H,
Results and discussion
Chemistry
Glutarimide derivatives, depicted in Figure 1, were synthesized
according to previously reported procedures of our30 or other
research groups. Compounds 1 and 2 were prepared according to
a modified literature procedure. Cyclization of amido-esters,
derived from corresponding glutaric acid anhydrides, in presence
of the base (NaH), was carried out by reflux in toluene (ꢄ3 h),
yielding glutarimides 1 and 2 (Scheme 1). Amido-esters 1c and 2e
were prepared from 1a and 2c, respectively, by a modification of
CH2), 2.45–2.25 (m, 1H, CH2), 1.77–1.71 (m, 3H, CH3) ppm; 13
C
NMR (50 MHz, CDCl3): ꢀ ¼ 171.1 (CCOimide), 170.8 (CCOimide),
[139.6, 134.5, 134.4, 129.2, 129.0, 128.1, 128.0, 127.7, 127.0,
126.8 (CAr)], 66.2 (CCH), 50.3 (CCH), 50.1 (CCH), 29.8 (CCH2),
29.4 (CCH2), 17.6 (CCH3), 17.4, (CCH3), 15.8 (CCH2) ppm; HR-
MS (ESI, m/z): calcd. for C19H19NO4S [M + H]+: 358.1099,
found: 358.1108.
standard methods47,49,50
.
2-Benzyl-2-azaspiro[5.11]heptadecane-1,3,7-trione
(7,
C23H31NO3). Yield 42.1%; 1H NMR (500 MHz, CDCl3):
ꢀ ¼ 7.34–7.23 (m, 5H, Ar–H), 4.95 (ABq, 2H, CH2,
J ¼ 14.6 Hz), 3.23–3.17 (ddd, 1H, CH2, J ¼ 7.6 Hz, J ¼ 5.3 Hz,
J ¼ 1.0 Hz), 2.68–2.56 (m, 2H, CH2), 2.59–2.53 (m, 2H, CH2),
2.00–1.95 (m, 1H, CH2), 1.60–1.47 (m, 4H, CH2), 1.33–1.19 (m,
15H, CH2), 0.96–0.89 (m, 1H, CH2) ppm; 13C NMR (125 MHz,
CDCl3): ꢀ ¼ 204.8 (CCOketo), 172.7 (CCOimide), 171.9
(CCOimide), [136.9, 128.8, 128.4, 127.5 (CAr)], 60.0 (CCH),
43.4 (CCH2), 34.7 (CCH2), 33.9 (CCH2), 29.9 (CCH2), 26.3
(CCH2), 26.2 (CCH2), 23.3 (CCH2), 23.1 (CCH2), 22.0 (CCH2),
21.7 (CCH2), 18.8 (CCH2) ppm; HR-MS (ESI, m/z): calcd. for
C23H31NO3 [M + H]+: 370.2377, found: 370.2371.
Compounds 3–9 were synthesized by tandem process described
in our previous paper30. The process involved a base-catalyzed
Michael addition of active methylene compounds to secondary
acrylamides or crotonamides, followed by intramolecular
N-acylation of the carboxamido group. Synthesis of derivatives
3–6 was performed by reacting methyl l,2-cyanoacetate (3a),
methyl t-pentyl malonate (3b) and methyl 2-(phenylsulfonyl)acet-
ate (3c) with N-substituted acryl- and crotonamides (4a–c), under
the reaction conditions (Scheme 2A). Yields of products were
42–72%. In the reaction of ꢁ-keto esters, comprising 5, 8 and 12
member rings, 5a–c, with N-benzyl acrylamide, imides 9, 8 and 7,
respectively, were obtained (Scheme 2B).

DOI: 10.3109/14756366.2015.1070844
Activities of glutarimide derivatives
5
Scheme 1. Synthetic path to obtain com-
pounds 1 and 2.
Scheme 2. Synthetic paths to obtain com-
pounds (A) 3–6; and (B) 7–9.

´ ´
J. B. Popovic-Djordjevic et al.
6
J Enzyme Inhib Med Chem, Early Online: 1–9
Antiproliferative activity
72 h of exposure to compounds31,32. IC50 values (Table 1) are
shown in molar concentrations, as the mean SD, determined
from three independent measurements.
The cytotoxicity of compounds 1–9 was tested toward selected
human cancer cell lines: cervix adenocarcinoma HeLa, human
myelogenous leukemia K562 and human breast carcinoma MDA-
MB-453 cells. Cell survival was determined by MTT test, after
IC50 was defined as the concentration of the compound
inhibiting cell survival by 50%, compared with a vehicle-treated
control cells. The most potent compound, 7, was also tested
toward MRC-5, normal lung fibroblast cells. Compounds 2, 4, 5–
8 and 9 exerted dose-dependent cytotoxicity toward malignant
cells, while compound 7 appeared as the most active. Compound
7 exerted humble selectivity, with IC50 toward normal cells
ꢄ37 mM. The decrease in survival of target cells induced by
compound 7 is shown in Figure 2.
Table 1. Concentrations of compounds 1–9 that induced 50% decrease in
cell survival (IC50).
IC50 (mM)
Compound
HeLa
4200
150.2 2.0 103.9 4.4
K562
4200
MDA-MB-453
MRC5
1
2
3
4
5
6
7
8
9
4200
167.8 3.8
4200
–
–
–
–
Antibacterial activity
4200
119.3 1.3
108.4 8.1
4200
96.6 2.5
81.1 0.8
Antibacterial activity of compounds 1, 2, 4–8 and 9 was examined
against selected foodborne pathogenic bacteria, Gram-positive
species S. aureus (ATCC 25923), E. faecalis (ATCC 29212), B.
cereus (ATCC 10876), L. monocytogenes (ATCC 19115) and
Gram-negative species P. aeruginosa (ATCC 27853), E. coli
(ATCC 25922), S. enteritidis (ATCC 13076), P. hauseri (ATCC
13315), S. sonnei (ATCC 29930), Y. enterocolitica (ATCC
27729), E. coli O157:H7 (ATCC 12900). A preliminary screen-
ing, done by a disc diffusion method, indicated the ability of
bacteria to produce visible growth in the presence of compounds
1, 2, 4–8 and 9. In most cases, Gram-negative bacteria were more
resistant to the tested compounds than Gram-positive bacteria
(Table 2). The diameters of the inhibition zones, determined by a
disk diffusion method, ranged from 6.4 to 14.6 mm, in the
presence of 200 mg of compounds tested.
150.9 0.2
135.7 0.2
ꢄ200
–
154.3 6.9 147.1 8.7
26.8 4.7 8.98 0.64
146.7 5.9 150.0 0.1
186.5 1.2 193.6 6.4
–
36.73 0.65
27.36 0.20
138.1 3.5
195.2 4.8
–
–
The Gram-positive bacteria B. cereus were the most sensitive
to compounds 4 and 9 (14.6 and 14.2 mm, respectively). Such
activity was comparable to the effect of commercial antibiotic
gentamicin (14.8 mm). Also, there was no statistically significant
difference between the susceptibility of B. cereus on commercial
antibiotic tetracycline (12.4 mm) and compounds 2, 7 and 8 (12.1,
12.9 and 12.4 mm, respectively). Among Gram-negative bacteria,
S. sonnei was the most sensitive to compounds 2, 4 and 6 (11.5,
10.4 and 10.2 mm, respectively), comparable to antibiotic tetra-
cycline (11.4 mm). Other compounds did not show significant
antimicrobial activity against tested bacteria in the applied
concentration. Compound 5 did not show any antimicrobial
effect on tested Gram-positive and Gram-negative bacteria.
Due to the fact that the disk diffusion method is not entirely
reliable in determining the antimicrobial properties, the broth
microdilution method, as a rapid and quantitative method
Figure 2. Dose–response curves for the cytotoxicity of compound 7
toward HeLa, K562, MDA-MB-453 and MRC-5 cells. Percentage
of viable cells (S%) was plotted against various concentrations of
compound 7.
Table 2. Antibacterial activity of compounds 1, 2, 4, 6–8 and 9, determined by the disk diffusion method.
Diameter of inhibition zone (mm) including the initial diameter of the disk (6 mm)*
Bacterial strain
1
2
4
6
7
8
9
Gy
Tz
S. aureus
E. faecalis
B. cereus
L. monocytogenes
P. aeruginosa
E. coli
S. enteritidis
P. hauseri
S. sonnei
Y. enterocolitica
E. coli(0157:H7)
8.2 0.2f
10.1 0.5c
9.3 0.0c
9.2 0.3e
–x
11.3 0.4d 13.3 0.4c
10.8 0.2c 9.3 0.2d
8.5 0.2f
8.1 0.3e
9.6 0.5c
8.1 0.1f
7.4 0.4c
8.0 0.6c
7.1 0.2e
8.6 0.6c
10.2 0.2c
7.4 0.3d
7.8 0.1c
12.6 0.2c
12.9 0.1c
9.8 0.2eô 18.2 0.8b 29.9 0.5a
9.4 0.5d 10.2 0.4c
10.3 0.2c 12.1 0.7b 15.8 0.5a
14.8 0.2a 12.4 0.3b
12.6 0.7b 15.2 0.4a
12.1 0.9b 14.6 0.3a
12.9 0.0b 12.4 0.2b 14.2 0.1a
10.1 0.7d 10.5 0.5d 11.6 0.4c
12.1 0.2b
8.3 0.5f
7.3 0.3c
8.2 0.5c
9.1 0.1c
6.9 0.3d
9.6 0.4b
7.1 0.3c
7.0 0.2c
7.7 0.1c
–
7.5 0.5c
7.9 0.3c
6.8 0.6e
–
21.3 0.4a
22.9 0.8a
25.3 0.4a
25.6 0.5a
16.6 0.4a
30.9 0.4a
9.1 0.2b
11.4 0.4b
22.5 0.6b
24.8 0.3b
11.4 0.4b
26.8 0.8b
8.3 0.5c
7.9 0.3d
–
–
–
–
8.3 0.6d
–
6.4 0.7d
9.8 0.1d
7.7 0.2d
–
6.8 0.3d
9.9 0.4d
7.9 0.3d
–
9.7 0.6d 11.5 0.8b 10.4 0.3c
8.7 0.0e
7.1 0.4d
–
8.9 0.0c
8.1 0.7c
8.4 0.6c
–
8.6 0.2c
7.6 0.3c
21.8 0.6b 33.7 0.1a
*Data are expressed as mean standard deviation (n ¼ 3).
yG-gentamicin.
zT-tetracycline.
ôWithin the same row, means followed by different letters are significantly different at p50.05 (ANOVA, Fisher’s LSD).
xInhibition zone not achieved.

DOI: 10.3109/14756366.2015.1070844
Activities of glutarimide derivatives
7
Figure 3. Heatmap depiction of descriptors (auto- and cross-correlo-
grams), calculated by AMANDA algorithm, for compounds 1–9.
for determining of MIC, based on the color change caused by
the enzymatic activity of viable microorganisms, was applied
(Table 3). Compounds 1, 2, 4, 6–8 and 9 inhibited the growth of
all tested Gram-positive and some of the Gram-negative bacteria.
Achieved MICs were in the range of 0.625–10.0 mg/mL
(1.97 ꢀ 10ꢁ3– 4.92 ꢀ 10ꢁ2 mol/L).
The highest antibacterial potential was reached with compound
9 against B. cereus (MIC was 0.625 mg/mL ¼ 1.97 ꢀ 10ꢁ3 mol/L).
MICs against Gram-negative bacteria exceeded 10.0 mg/mL, for
the majority of compounds, including compound 5, proved to be
inactive in concentrations up to 10.0 mg/mL against all bacterial
strains tested. Exceptions were compound 4, whose antibacterial
activity ranged from 5.0 to 10.0 mg/mL against all tested Gram-
negative bacteria and compounds 2, 6 and 7, with MICs of
10.0 mg/mL (3.89 ꢀ 10ꢁ2, 2.80 ꢀ 10ꢁ2 and 2.71 ꢀ 10ꢁ2 mol/L,
respectively) against S. sonnei.
Structure–activity relationship
Although exerting humble potency, derivatives 9 and 4 appeared
most potent against bacteria, while derivative 7 was the one with
fair potency toward human tumor cells. In order to rationalize
structural features associated with the most potent compounds we
calculated molecular descriptors derived from the 3D structures of
compounds. For modeling studies structures of all compounds
(1–9) were prepared as described in our previous paper54, see
section ‘‘Methods’’. Due to low number of compounds tested
against bacteria, or toward human tumor cells, and limited
potencies, we did not build novel quantitative structure–activity
models, but envisaged to use molecular properties and to use
previously built model to draw conclusions on structural charac-
teristics needed for significant potency. To predict potency of
compound 7 toward K562 cells, we projected structures of
compounds 2, 7 and 8 (all spirocyclic compounds in our dataset)
on model previously derived for antiproliferative activity data of
glutarimide derivatives toward K562 cells, collected from
National Cancer Institute (NCI) repository54. Unfortunately, our
prediction failed. Compounds 2 and 7 were predicted as more
potent comparing to compound 8 (data not shown). There are
several possible reasons for the poor prediction. First, in the

´ ´
J. B. Popovic-Djordjevic et al.
8
J Enzyme Inhib Med Chem, Early Online: 1–9
model of antiproliferative activity of NCI glutarimides toward 4, most probably because ester keto group, as a HBA, was
K562 cells, potency data span range from 4 to 8.6 (p(GI50) hindered with branched alkyl moiety. All variables described for
values). Approximating equality between p(IC50) and p(GI50) compounds 9 and 4 are depicted in Figures S20 and S21
values, which for sure is not the most stringent criteria; (Supplemental Information). So, along with overall large molecu-
compounds 2 and 7, with p(IC50) of 3.98 and 3.82, respectively, lar volumes, due to alkyl chains bound to position 3 of
are out of the range of potencies that NCI model covers. Next, glutarimide moiety, compound 9, which exerted better antibac-
antiproliferative data of compounds tested by NCI were obtained terial activity than the rest of compounds, has HBA moiety in this
by Sulforhodamine B assay after 48 h of exposure of cells to alkyl chain and this HBA is not hindered by the vicinal parts of
compounds, while we used MTT assay and exposed cells to molecule.
compounds during 72 h. The most potent compounds in NCI set
comprise unsubstituted glutarimide nitrogen, while all compounds
in our set bear bulky substituent in this position. Finally, our
Conclusion
The cytotoxicity study of compounds 1–9 toward selected human
cancer cell lines showed that compound 7 was the most potent,
with IC50 values 8.98, 26.8 and 27.36 mM, toward K562, HeLa and
MDA-MB-453 cells, respectively. This compound exerted modest
selectivity toward normal control MRC-5 cells with IC50 ꢄ37 mM.
Preliminary screening of antibacterial activity by a disk diffusion
method showed that Gram-positive bacteria were more susceptible
to tested compounds than Gram-negative bacteria. Compounds 4
and 9 expressed the highest inhibition on growth of Gram-positive
bacteria B. cereus and it is comparable to antibiotic gentamicin.
S. sonnei (Gram-negative bacteria) was the most sensitive to the
compound 2 (comparable to antibiotic tetracycline). Using a
quantitative method for determination of MIC the highest
antibacterial potential was achieved with compound 9 against
B. cereus. Compound 5 did not show any antimicrobial effect on
tested Gram-positive and Gram-negative bacteria in any of applied
tests. Whole-molecular descriptors derived from 3D structures of
examined compounds and structure–activity model based on MIF,
calculated by the GRID method, rationalized structural features
associated with the most potent compounds. Compound with
bulky hydrophobic moiety distal from glutarimide ring exerted
best antiproliferative potency within studied set.
model of antiproliferative activity of NCI glutarimides was built
on the pharmacophoric similarity patterns (including spatial
positions of HBA, HBD, hydrophobic and shape features), but did
not include whole-molecule properties, as surfaces and volumes.
Next, we compared whole-molecular properties of compounds 1–
9 (surface area, polar surface area, apolar surface area, volume,
virtual log P; Supplemental Information, Table S1) derived from
the 3D structures of compounds. We concluded that derivative 7,
most potent toward human tumor cells tested, had the largest
molecular volume. This observation is in accordance with the fact
that compounds having largest molecular volumes are among
most potent in NCI glutarimide set (Figure 4a in reference54). To
obtain more data on structural features associated with most
potent compounds in our set, we calculated MIF, with hydrogen-
bond donor (N1), hydrogen-bond acceptor (O), hydrophobic
(DRY) and shape (TIP) probes around compounds 1–9 in program
Pentacle, and visually inspected interaction patterns, obtained by
two-point pharmacophoric features offered by AMANDA algo-
rithm45,55. Blocks of correlograms were depicted as a heatmap
(Figure 3). Features that separate the most potent compounds
from the rest appeared straightforwardly.
We observed TIP-TIP and DRY-TIP blocks of variables which
were broader for compounds 4, 7 and 9, comparing with the same
block of variables for the rest of compounds. Along with this, the
N1-TIP block of variables is significantly broader for compound
9, compared to other compounds. So, variables in those three
blocks, with the largest distances between nodes, made distinction
between compounds 4, 7 and 9 and the rest. Variables TIP-TIP-
We found that compound 7 exerts the highest antiproliferative
potency within studied set. This derivative will be a starting point
for further structural modifications in order to obtain more potent
compounds. As compound 7 exerts modest selectivity, the second
goal of structural modifications will be improvement of select-
ivity of new derivatives.
˚
230, encoding nodes of shape probe on distance of ꢄ17.1 A
(Supplemental Information, Figure S19a), and DRY-TIP-408,
encoding node of hydrophobic probe and the node of shape probe
Declaration of interest
The authors have no conflicts of interest. Authors acknowledge the
Ministry of Education, Science and Technological Development of the
Republic of Serbia for financial support (Grant Nos. 172032 and 175011).
˚
on distance of ꢄ17.4 A (Supplemental Information, Figure S19b),
clearly separated compound 7, most potent toward tumor cells
tested, from the rest. Along the largest molecular volume,
compound 7 comprises bulky cycloalkyl moiety distal from the
benzyl ring bound to glutarimide N. We observed similar
structural features for compounds 9 and 4, most active against
Gram-positive bacteria. Variables TIP-TIP-233 (nodes of the
References
1. Matsuda F, Terashima S. Total synthesis of natural (+)-sesbanimide
A and (ꢁ)-sesbanimide B. Tetrahedron 1988;44:4721–36.
2. Ju J, Rajski SR, Lim SK, et al. Lactimidomycin iso-migrastatin and
related glutarimide-containing 12-membered macrolides are extre-
mely potent inhibitors of cell migration. J Am Chem Soc 2009;131:
1370–1.
3. Schneider-Poetsch T, Ju J, Eyler DE, et al. Inhibition of eukaryotic
translation elongation by cycloheximide and lactimidomycin. Nat
Chem Biol 2010;6:209–17.
4. Sugawara K, Nishiyama Y, Toda S, et al. Lactimidomycin, a new
glutarimide group antibiotic. Production, isolation, structure and
biological activity. J Antibiot 1992;45:1433–41.
5. Machado AL, Lima LM, Araujo Jr JX, et al. Design, synthesis and
antiinflammatory activity of novel phthalimide derivatives, struc-
turally related to thalidomide. Bioorg Med Chem Lett 2005;15:
1169–72.
˚
shape probe on distance of ꢄ18.1 A) and DRY-TIP-410 (node of
˚
the hydrophobic probe on distance of ꢄ18.1 A from the node of
shape probe) encoded compound 9. Variables TIP-TIP-234 (nodes
˚
of the shape probe on distance of ꢄ18.4 A) and DRY-TIP-411
˚
(node of the hydrophobic probe on distance of ꢄ18.4 A from the
node of shape probe) encode compound 4. Along with those two
variables, comparable with variables associated with compound 7,
that described overall shape and spatial position of benzyl moiety
and branched alkyl chains attached to position 3 of glutarimide
ring in compounds 9 and 4; we observed additional variables
characteristic for compound 9. Variables N1-TIP-584 (HBD
associated with keto group in glutarimide ring, and alkyl moiety
of the ester, associated with the shape probe, on spatial distance of
˚
ꢄ17.1 A) and N1-N1-157 (HBDs associated with keto group in
6. Bartlett JB, Dredge K, Dalgleish AG. The evolution of thalidomide
and its IMiD derivatives as anticancer agents. Nat Rev Cancer 2004;
4:314–22.
glutarimide ring and with ester keto moiety) are typical for
compound 9. We did not observe similar variables for compound

DOI: 10.3109/14756366.2015.1070844
Activities of glutarimide derivatives
9
7. Wu YH, Rayburn JW, Allen LE, et al. Psychosedative agents. 2. 8- 31. Mosmann T. Rapid colorimetric assay for cellular growth and
(4-Substituted l-piperazinylalkyl)-8-azaspiro[4.5]decane-7,9-diones.
J Med Chem 1972;15:477–9.
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 1983;65:55–63.
8. Barrdell LB, Fitton A. Tandospirone. CNS Drugs 1996;5:147–52.
9. Obrig TG, Culp WJ, Mckeehan WL, Hardesty B. The mechanism by
which cycloheximide and related glutarimide antibiotics inhibit
peptide synthesis on reticulocyte ribosomes. J Biol Chem 1971;246:
174–81.
10. Kondo H, Oritani T, Kiyota H. Synthesis and antifungal activity of
the four stereoisomers of streptimidone, a glutarimide antibiotic
from Streptomyces rimosus forma paromomycinus. Eur J Org Chem
2000;2000:3459–62.
32. Ohno M, Abe T. Rapid colorimetric assay for the quantification of
leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). J Immunol
Methods 1991;145:199–203.
33. Tepe B, Daferera D, Sokmen A, et al. Antimicrobial and antioxidant
activities of the essential oil and various extracts of Salvia tomentosa
Miller (Lamiaceae). Food Chem 2005;90:333–40.
34. Clinical and Laboratory Standards Institute (CLSI). Performance
standards for antimicrobial susceptibility testing: 15th informational
supplement 2005. CLSI Document M100-S15. Pennsylvania: CLSI.
11. Frohardt RP, Dion HW, Jakubowski ZL, et al. Chemistry of
streptimidone, a new antibiotic. J Am Chem Soc 1959;81:5500–6.
12. Kim BS, Moon SS, Hwang BK. Isolation, antifungal activity, and
structure elucidation of the glutarimide antibiotic, streptimidone,
produced by Micromonospora coerulea. J Agric Food Chem 1999;
47:3372–80.
ˇ
ˇ
ˇ
35. Klancnik A, Piskernik S, Jersek B, Smole Mozina S. Evaluation of
diffusion and dilution methods to determine the antibacterial activity
of plant extracts. J Microbiol Methods 2010;81:121–6.
36. Sadowski J, Gasteiger J. From atoms and bonds to three-dimensional
atomic coordinates: automatic model builders. Chem Rev 1993;93:
2567–81.
13. Ha DKK, Lau WH. Effect of recombinant human tumor necrosis
factor on human nasopharyngeal carcinoma cell line in vitro. Cancer
Lett 1988;41:217–24.
14. Andres MI, Sanz P, Garfia A, et al. Induction of cell differentiation
and other in vitro effects of cycloheximide on neuroblastoma cells.
In Vitro Mol Toxicol 1977;10:319–28.
15. Ishikawa Y, Tachibana M, Matsui C, et al. Synthesis and biological
evaluation on novel analogs of 9-methylstreptimidone, an inhibitor
of NF-kB. Bioorg Med Chem Lett 2009;19:1726–8.
16. Powell RG, Smith Jr CR, Weisleder D. Sesbanimide A and related
tumor inhibitors from Sesbania drummondii: structure and chemis-
try. Phytochemistry 1984;23:2789–96.
17. Powell RG, Smith CR, Weisleder D, et al. Sesbanimide, a potent
antitumor substance from Sesbania drummondii seed. J Am Chem
Soc 1983;105:3739–41.
18. Powel RG, Smith Jr CR. An investigation of the antitumor activity
of Sesbania drummondii. J Nat Prod 1981;44:86–90.
19. Powel RG, Smith Jr CR, Madrigal RV. Antitumor activity of
Sesbania vesicaria, S. punicea, and S. drummondii seed extracts.
Planta Med 1976;30:1–8.
20. Armoiry X, Aulagner G, Facon T. Lenalidomide in the treatment of
multiple myeloma: a review. J Clin Pharm Ther 2008;33:219–26.
21. Lentzsch S, Rogers MS, LeBlanc R, et al. S-3-Amino-phthalimido-
glutarimide inhibits angiogenesis and growth of B-cell neoplasias in
mice. Cancer Res 2002;62:2300–5.
22. McCarthy PL, Owzar K, Hofmeister CC, et al. Lenalidomide after
stem-cell transplantation for multiple myeloma. N Engl J Med 2012;
366:1770–81.
23. Fischer DS, Woo LW, Mahon MF, et al. D-ring modified estrone
derivatives as novel potent inhibitors of steroid sulfatase. Bioorg
Med Chem 2003;11:1685–700.
24. Brueggemeier RW, Hackett JC, Diaz-Cruz ES. Aromatase inhibitors
in the treatment of breast cancer. Endocr Rev 2005;26:331–45.
25. Santen RJ, Brodie H, Simpson ER, et al. History of aromatase: saga
of an important biological mediator and therapeutic target. Endocr
Rev 2009;30:343–75.
26. Antonini I, Volpini R, Dal Ben D, et al. Design, synthesis, and
biological evaluation of new mitonafide derivatives as potential
antitumor drugs. Bioorg Med Chem 2008;16:8440–6.
27. Norton JT, Witschi MA, Luong L, et al. Synthesis and anticancer
activities of 6-amino amonafide derivatives. Anti-Cancer Drugs
2008;19:23–36.
37. Sadowski J, Gasteiger J, Klebe G. Comparison of automatic three-
dimensional model builders using 639 X-ray structures. J Chem
Inform Model 1994;34:1000–8.
38. Pedretti A, Villa L,Vistoli G. VEGA – an open platform to develop
chemo-bio-informatics applications, using plug-in architecture and
script programming. J Comput-Aided Mol Des 2004;18:167–73.
39. Halgren TA. MMFF VI. MMFF94s option for energy minimization
studies. J Comput Chem 1999;20:720–9.
40. Harrison RW. Stiffness and energy conservation in molecular
dynamics: an improved integrator. J Comput Chem 1993;14:
1112–22.
41. Stewart JJP. Optimization of parameters for semiempirical methods
V: modification of NDDO approximations and application to 70
elements. J Mol Model 2007;13:1173–213.
42. Stewart JJP. MOPAC: a semiempirical molecular orbital program. J
Comput-Aided Mol Des 1990;4:1–103.
43. Gaillard P, Carrupt PA, Testa B, Boudon A. Molecular lipophilicity
potential, a tool in 3D QSAR: method and applications. J Comput-
Aided Mol Des 1994;8:83–96.
44. Goodford PJ. A Computational procedure for determining energet-
ically favorable binding sites on biologically important macromol-
ecules. J Med Chem 1985;28:849–57.
´
´
´
45. Duran A, Martınez GC, Pastor M. Development and validation of
AMANDA, a new algorithm for selecting highly relevant regions in
molecular interaction fields. J Chem Inform Model 2008;48:
1813–23. Pentacle 1.0.6. http://www.moldiscovery.com/software/
pentacle/
46. Gokel GW, Cram DJ, Liotta CL, et al. 18-Crown-6 (1,4,7,10,13,16-
¨
hexaoxacyclooctadecane). Org Syn 1988;VI:301–2.
47. Cason J. b-Methylglutaric anhydride. Org Syn 1963;IV:630–1.
48. Farmer HH, Rebjohn N. b-Ethyl-b-methylglutaric acid. Org Syn
1963;IV:441–2.
49. Cason J. b-Carbomethoxypropionyl chloride. Org Syn 1955;III:
169–70.
50. Vogel AI. Vogel’s text book of practical organic chemistry. 5th ed.
Harlow: Longman Group, UK Limited; 1989.
51. Strube RE. Ethyl tert-butyl malonate. Org Syn 1963;IV:417–18.
52. Paquet LA, ed. Encyclopedia of reagents for organic synthesis. Vol.
7. Pennsylvania State University, John-Wiley; 1995. Available from:
http://eu.wiley.com/WileyCDA/WileyTitle/productCd-0470017546.
html [last accessed 21 Jul 2015].
53. Krapcho AP, Diamanti J, Cayen C, Bingham R. 2-
Carbethoxycyclooctanone. Org Syn 1973;V:198–9.
28. Wu A, Xu Y, Qian X, et al. Novel naphthalimide derivatives as
potential apoptosis-inducing agents: design, synthesis and biological
evaluation. Eur J Med Chem 2009;44:4674–80.
29. Machado KE, Navakoski de Oliveira K, Santos-Bubniak L, et al.
Evaluation of apoptotic effect of cyclic imide derivatives on murine
B16F10 melanoma cells. Bioorg Med Chem 2011;19:6285–91.
´
´
ˇ
´
´
´
´
54. Popovic-Djordjevic JB, Dosen-Micovic LjI, Juranic IO, Drakulic BJ.
Antiproliferative activity of NCI-DTP glutarimide derivatives: an
alignment independent 3D QSAR study. J Serbian Chem Soc 2010;
75:1167–79.
´
´
´
´
55. Cruciani G, ed. Methods and principles in medicinal chemistry,
molecular interaction fields: applications in drug discovery and
ADME prediction. Weinheim: Wiley-VCH; 2006.
30. Popovic-Ðordevic JB, Ivanovic MD, Kiricojevic VD. A novel
tandem process leading to functionalized glutarimides. Tetrahedron
Lett 2005;46:2611–14.
Supplementary materials available online