New thalidomide-resembling dicarboximides target ABC50 protein and show antileukemic and immunomodulatory activities

Article abstract of DOI:10.3390/biom9090446

We identified novel dicarboximides that were selectively cytotoxic towards human leukemia cells. Using chemical and biological methods, we characterized the biological activity, identified cellular protein targets and defined the mechanism of action of th

Full text of DOI:10.3390/biom9090446

biomolecules
Article
New Thalidomide-Resembling Dicarboximides
Target ABC50 Protein and Show Antileukemic and
Immunomodulatory Activities
Marcin Cies´lak 1,
*
, Julia Kaz´mierczak-Baran´ska 1, Karolina Królewska-Golin´ska 1,
Mariola Napiórkowska 2 , Iga Stukan 3, Urszula Wojda 3 and Barbara Nawrot 1
1
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 112 Sienkiewicza Str.,
90-363 Lodz, Poland
2
Department of Medical Chemistry, Medical University of Warsaw, 3 Oczki Str., 02-007 Warsaw, Poland
Laboratory of Preclinical Testing of Higher Standard, Nencki Institute of Experimental Biology of Polish
3
Academy of Sciences, 02-093 Warsaw, Poland
*
Correspondence: marcin@cbmm.lodz.pl; Tel.: +48-42-6803-230
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 31 July 2019; Accepted: 2 September 2019; Published: 4 September 2019
Abstract: We identified novel dicarboximides that were selectively cytotoxic towards human leukemia
cells. Using chemical and biological methods, we characterized the biological activity, identified
cellular protein targets and defined the mechanism of action of the test dicarboximides. The reported
IC50 values (concentration required to reduce cell survival fraction to 50% of control) of selected
dicarboximides were similar or lower than IC50 of registered anticancer drugs, for example cytarabine,
sorafenib, irinotecan. Test compounds induced apoptosis in chronic myelogenous (K562) and
acute lymphoblastic (MOLT-4) leukemia cells by activation of receptor and mitochondrial apoptotic
pathways and increased the expression of proapoptotic genes (BAX, NOXA, HTRA2, TNFRSF10B,
ESRRBL1). Selected dicarboximides displayed immunomodulatory activity and downregulated
IKZF1 and IKZF3 transcription factors in K562 and MOLT-4 leukemia cells. ATP-binding cassette
protein 50 (ABC50) was identified as a target for dicarboximides. Cancer cells with knocked down
ABC50 showed increased resistance to dicarboximides. Based on the structure of dicarboximides and
thalidomide, novel proteolysis-targeting chimeras (PROTACs) were synthesized and used as tools to
downregulate ABC50 in leukemia cells.
Keywords: apoptosis; dicarboximides; anticancer compounds; cytotoxicity; leukemia; ABC50
(ABCf1); protac (proteolysis targeting chimera)
1. Introduction
Cancer diseases with quickly rising mortality rates constitute nowadays the major problem of
clinical medicine. There is an ongoing need for new anticancer drugs with improved pharmacological
profile. While the design of new drugs would be facilitated by development of more detailed knowledge
of genetic and molecular mechanisms of cancer pathogenesis, many of them are derivatives of already
known therapeutics. Effective anticancer therapy should not only inhibit proliferation of cancer cells
but also induce their apoptosis. Apoptosis, or programmed cell death, is a metabolic process used
by an organism for removal of harmful, unnecessary or damaged cells in a controlled way. A proper
balance between cell proliferation and apoptosis maintains the optimal numbers and types of cells
during development as well as in mature tissues. Cancer cells can escape these maintenance processes,
so there is a growing demand for methods to get them back on track of controlled apoptotic elimination.
In principle, there are two pathways leading to apoptosis: (1) receptor mediated (extrinsic, utilizing
extracellular signals sensed by death receptors), and (2) mitochondrial (intrinsic). Sometimes these
Biomolecules 2019, 9, 446; doi:10.3390/biom9090446
www.mdpi.com/journal/biomolecules

Biomolecules 2019, 9, 446
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pathways overlap leading to the amplification of proapoptotic signals [
1–4
]. Key enzymes involved in
apoptosis are cysteine proteases called caspases, which operate either as initiators (caspases 8, 9, 10) or
effectors (caspases 3, 6 and 7). The latter are responsible for cell disintegration during the final stage of
apoptosis [5,6]. Necrosis, in contrast to apoptosis, is an unregulated premature cell death upon an
injury, detrimental to cells.
Numerous reports show that dicarboximides exhibit the anti-neoplastic activity. The first
clinical trials of naphthalimide derivatives (amonafide and mitonafide) as antineoplastic compounds
were carried out in the 1980s [7,8]. Although in the phases II and III these compounds showed
anticancer activity in leukemias and solid tumors, they were abandoned because of serious side
effects. The cytotoxicity of these compounds resulted mainly from intercalation into DNA and
inhibition of topoisomerase II activity [
indolomaleimide derivatives, which exert anticancer activity by intercalation to DNA, induction of
apoptosis, cell-cycle arrest and inhibition of protein kinase C [11 12]. Thus, dicarboximides have
9,10]. Another interesting class of compounds consists of
,
interesting biological properties and may affect a variety of biological processes, with undisputable
potential as anticancer drugs.
Thalidomide and its derivatives lenalidomide and pomalidomide are probably the most recognized
representatives of dicarboximides. They are collectively known as immunomodulatory drugs (IMiDs)
and display a wide range of biological activities including inhibition of angiogenesis, induction of
oxidative stress, cytotoxicity and immunomodulation. IMiDs are currently used for treatment of
hematological malignancies, particularly multiple myeloma [13]. From chemical perspective these
compounds are composed of phthalimide and glutarimide moieties. One of the possible mechanisms
of action of IMiDs as anti-leukemia (particularly anti-myeloma) agents has been identified and is
based on selective targeting of cellular proteins for ubiquitination by cullin-4 RING E3 ligase (CRL4).
Ubiquitinated proteins are subsequently degraded by a proteasome. It was demonstrated that IMiDs
bind cereblon (CRBN), a protein that is a part of the CRL4 complex [14,15]. Binding of IMiDs to CRBN
results in enhanced ubiquitination and degradation of two zinc finger transcription factors: IKZF1
(Ikaros family zinc finger protein 1, Ikaros) and IKZF3 (Ikaros family zinc finger protein 3, Aiolos).
These transcription factors are necessary for myeloma cells survival thus their degradation is lethal
for myeloma cells. Interestingly, IMiDs-driven downregulation of IKZF1/IKZF3 is rather non-toxic to
T-cells or T-cell leukemias but results in increased expression of interleukin 2 (IL-2) and decreased
expression of tumor necrosis factor alpha (TNF-α) in monocytes [16,17].
IKZF1 and IKZF3 are indispensable for development of lymphoid tissue, particularly T- and
B-lymphocytes and natural killer (NK) cells. IKZF1 is expressed early during the formation of
lymphoid system while IKZF3 is expressed in the middle and late stages of T- and B-cell maturation.
Interestingly, Ikaros is necessary for pluripotent stem cells differentiation not only to lymphoid but also
myeloid/erythroid lineages. Studies in mice showed that deletion of DNA binding domain of Ikaros
resulted in generation of abnormal T-cells and was lethal to T-cell leukemias [18]. These data suggest
that downregulation of Ikaros and/or Aiolos either by low molecular weight organic compounds or by
genetic modifications can have cytotoxic effects on hematological malignancies including T-cell and
myeloid/erythroid leukemias.
In the present study we investigated pro-apoptotic properties and the mechanism of cytotoxicity
of new derivatives of dicarboximides. These compounds were earlier found to be remarkably cytotoxic
and selective towards human leukemia cells (K562, HL-60), and non-toxic to adherent cancer cells
(HeLa, human cervix carcinoma) and to primary endothelial cells (HUVEC, human umbilical vein
endothelial cells) [19,20]. We identified cellular proteins targeted by these compounds and investigated
their immunomodulatory activity.
The complete list of dicarboximides, biological tests and cell lines is included in the supplementary
material (Table S1).

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2. Materials and Methods
2.1. Chemistry
All solvents, reagents and chemicals used in these studies were purchased from Aldrich Chemical
and Merck AG. Melting points were determined with Electrothermal 9100 capillary apparatus and
were uncorrected. The nuclear magnetic resonance spectra were recorded in deuterated D6 dimethyl
sulfoxide (DMSO-d6) or CDCl3 on VMNRS300 operating at 300 MHz (1H nuclear magnetic resonance
(NMR)) and 75 MHz (13C-NMR). Chemical shifts (
δ) are expressed in parts per million relatives to
tetramethylsilane used as the internal reference. Coupling constants (J) values are given in hertz
(Hz) and spin multiples are given as s (singlet), d (dublet), t (triplet), m (multiplet). Mass spectral
ESI (Electrospray Ionization) measurements were carried out on a MicrOTOF II (Bruker, Bremen,
Germany) instrument with TOF detector. The spectra were obtained in the positive ion mode. Flash
chromatography was performed on Kieselgel (Merck, Darmstadt, Germany) 0.05–0.2 mm reinst
(70–325 mesh ASTM) silica gel using chloroform as eluent. Progress of the reactions described in
experimental section was monitored by TLC (Thin Layer Chromatography) on silica gel (plates with
fluorescent indicator 254 nm, layer thickness 0.2 mm, Kieselgel G (Merck, Darmstadt, Germany), using
chloroform-methanol as an eluent system at the v/v ratio of 9:1 or 9.5:0.5.
Dicarboximides
1–6 (Table 1) were synthesized according to previously reported procedures [20,21].
Compounds 6a, 6b, 7–9 were synthesized as described below.
Table 1. Chemical structure and the numbering of dicarboximides.
Structure * Compound Structure *
Compound
1
H C
C2H5
3
O
CH3
CH3
C H
2
5
O
N
CH
3
C6H5
C6H5
N
H C
5
6
6
O
N
4
5
O
N
H C
5
O
O
C2H5
C H
2
5
CH
CH
C2H5
3
O
C2H5
C2H5
C H
2
5
O
HO
NH
H5C6
H5C6
N
H C
5
6
3
O
N
2
3
O
N
H C
5
6
O
O
C2H5
C H
2
5
C6H5
C2H5
O
O
CH3
H5C6
H5C6
H5C6
H5C6
N
N
N
CH3
O
N
6
O
O
C6H5
C2H5
H
N
C6H5
O
HO
NH2
S
O
H5C6
H5C6
N
H
C6H5
N
O
6a
6b
HO
NH
H5C6
O
N
O
H
5C6
O
C6H5
C6H5

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Table 1. Cont.
Compound
Compound
Structure *
Structure *
C2H5
CH3
O
O
H5C6
H5C6
H5C6
O
N
O
O
N
O
7
9
8
N
H
N
H
H5C6
O
O
O
O
CH3
C2H5
C6H5
O
H5C6
H5C6
O
N
O
N
H
O
O
C6H5
* all amine derivatives of dicarboximides were used as hydrochlorides.
2.1.1. Synthesis of 1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (D)
The starting imide was prepared by the methods described earlier [20]. A mixture of
D
1,2,3,4-tetraphenylcyclopenta-1,3-diene (0.02 mol) and maleimide (0.022 mol) was heated for 6 h
in 20 mL of benzene. When the reaction was completed the solvent was evaporated under reduced
pressure and the solid residue was crystallized from benzene.
Molecular weight (M.W.) = 467.5571; C33H25NO2; Yield: 81%, white powder, m. p. 246.5–246.9 ◦C
(from benzene); 1H NMR (300 MHz, DMSO-d6+ TMS (tetramethylsilane),
δ/ppm): 11.44 (1H, s, NH),
7.74 (4H, d, J = 7.2, H-aromatic), 7.24 (6H, m, H-aromatic), 6.88 (6H, m, H-aromatic), 6.60 (4H, m,
H-aromatic), 4.20 (2H, s, C2-H, C6-H), 3.23 (1H, d, J = 8.7, C10-H), 2.23 (1H, d, J = 8.4, C10-H); 13C-NMR
(DMSO, 75 MHz) δ
(ppm): 178.0 (2C-3, 5), 145.0 (2C-11, 110), 139.8 (2C-15, 150), 134.5 (2C-8, 9), 129.6
(4C-13, 130), 129.1 (4C-16, 160), 127.7 (4C-12, 120), 127.1 (4C-17, 170), 126.6 (2C-18, 180), 126.4 (2C-14, 140),
65.1 (C-10), 62.9 (2C-1, 7), 52.9 (2C-2, 6). High Resolution Mass Spectrometry (HRMS, m/z): calculated
value for [M + Na]+ 100% = 490.1778; found 100% = 490.1777, 35.6% = 491.1817, 6.6% = 492.1856.
2.1.2. Synthesis of 4-[-3-(amino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-
ene-3,5-dione (6a)
The amino alkanol derivative 6a was obtained in two-step reaction (Scheme 1) according to the
method described previously [20].
Step I: the mixture of 1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (
D) (0.01 mol)
with 1-chloro-2,3-epoxypropane (50 mL), in the presence of an anhydrous K2CO3 (0.01 mol) was
stirred on a magnetic stirrer under reflux with a tube with CaCl2. The reaction was carried out at
room temperature, for 15 h. When the reaction was completed (TLC control) the inorganic precipitate
was filtered off and the filtrate was concentrated. The obtained oily product was purified by column
chromatography on silica gel (eluent: chloroform and chloroform: methanol; 50:0.2, v/v) to deliver
pure derivative D1.
Step II: to the solution of 4-(oxirane-2-ylmethyl)-1,7,8,9-tetraphenyl-4 azatricyclo[5.2.1.02,6]dec-8-
ene-3,5-dione (D1) (0.001 mol) in a mixture of methanol/water (10:1 v/v) ammonium hydroxide
(concentrated, 5 mL) was added. Reactions were carried out at room temperature on a magnetic stirrer
under reflux for 72 hours. When the reaction was completed (TLC control), the excess of the solvent
was evaporated, and the crude product was purified by column chromatography on silica gel (eluent:
chloroform or chloroform: methanol; 50:0.2, 50:0.5, v/v).

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O
NH
O
O
O
NH3(aq)
Cl
O
+
CH3OH/H2O
N
N
NH2
O
O
HO
O
D
D1
6a
DMAP
DCC
HOBT
DMF
biotin
O
O
NH
HN
N
O
NH
S
HO
O
6b
Scheme 1. Synthesis of derivative 6a and biotinylated derivative 6b.
4-(oxiran-2-ylmethyl)-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (D1)
M.W. = 523.6203; C36H29NO3; Yield: 52%; white powder, m.p. 229–230 ◦C (from MeOH); 1H-NMR
(300MHz, CDCl3, /ppm): 7.70 (4H, m, H-aromatic), 7.28 (6H, m, H-aromatic), 6.93 (6H, m, H-aromatic),
δ
6.55 (4H, m, H-aromatic), 4.18 (2H, s, C2-H, C6-H), 3.78 (1H, m, C19-H), 3.46 (1H, m, C19-H), 3.16 (1H,
d, J = 9.0, C10-H), 3.07 (1H, m, C20-H), 2.64 (2H, m, C21-H), 2.34 (1H, d, J = 9.0, C10-H); 13C NMR
(CDCl3, 75 MHz) δ
(ppm): 176.4 (2C-3, 5), 145.1 (2C-11, 110), 139.7 (2C-15, 150), 134.2 (2C-8, 9), 129.4
(4C-13, 130), 129.1 (4C-16, 160), 127.8 (4C-12, 120), 127.2 (4C-17, 170), 126.7 (2C-18, 180), 126.5 (2C-14, 140),
65.2 (C-10), 63.0 (2C-1, 7), 52.1 (2C-2, 6), 51.8 (C-20), 48.2 (C-19), 45.3 (C-21); HRMS (m/z): calculated
value for [M + Na]+ 100% = 546.2410; found 100% = 546.2901, 10% = 547.3011.
4-[-3-(amino)-2-hydroxypropyl]-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (6a)
M.W. = 540.6508 (free amine); C36H32N2O3; Yield: 34%; oil; 1H-NMR (300 MHz, CDCl3, δ/ppm): 7.70
(4H, m, H-aromatic), 7.25 (5H, m, H-aromatic), 6.89 (7H, m, H-aromatic), 6.56 (4H, m, H-aromatic), 4.17
(2H, m, C2-H, C6-H), 3.80 (1H, m, C20-H), 3.57 (2H, m, C19-H), 3.16 (1H, m, C10-H), 2.78 (1H, m, C21-H),
2.62 (1H, m, C21-H), 2.34 (1H, m, C10-H); 13C NMR (CDCl3, 75 MHz)
δ (ppm): 177.1 (C-3/R/S-20),
177.0 (C-5/R/S-20), 145.2 (C-11/S-20), 145.1 (C-11/R-20), 144.9 (C-110/S-20), 144.9 (C-110/R-20), 139.3
(C-15/S-20), 139.3 (C-15/R-20), 139.2 (C-150/S-20), 139.2 (C-150/R-20), 134.0 (C-8/S/R-20), 133.9 (C-9/S-20),
133.8 (C-9/R-20), 129.9 (2C-13/S-20), 129.9 (2C-13/R-20), 129.8 (2C-130/S-20), 129.8 (2C-130/R-20), 129.1
(4C-160, 16), 128.0 (4C-120, 12), 127.4 (2C-170), 127.3 (2C-17), 127.1 (C-18), 127.0 (C-180), 126.8 (2C-14,
140), 69.4 (C-20), 65.9 (C-10/R-20), 65.8 (C-10/S-20), 63.6 (C-7/S-20), 63.6 (C-7/R-20), 63.6 (C-1/S-20), 63.5
(C-1/R-20), 52.2 (C-6/S-20), 52.2 (C-6/R-20), 49.5 (C-2), 44.4 (C-21), 42.8 (C-19); HRMS (m/z): calculated
value for [M + H]+ 100% = 541.2486; found 100% = 541.2486, 53.4% = 542.2501, 10% = 543.2530.
2.1.3. Synthesis of Biotinylated Derivative 6b
To a mixture of 0.795 mmol (430 mg) of derivative 6a, dicyclohexylcarbodiimide (DCC, 164 mg,
0.795 mmol, N,N-dimethylaminopyridine (DMAP, 9.71 mg, 0.0795 mmol) and hydroxybenzotriazole
(HOBT, 10.7 mg, 0.0795 mmol), dissolved in N,N-dimethylformamide (DMF, 20 mL), a solution of
0.795 mmol of biotin (194 mg) in DMF (12 mL) was slowly added dropwise and the reaction mixture
was stirred for 24 h at room temperature. Then the solvent was removed under reduced pressure.

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The reaction mixture was poured at the silica gel column and the desired product was eluted with the
gradient of methanol (0 to 10%) in chloroform. The numbering of atoms of derivative 6b used in the
description of nuclear magnetic spectra is available in supplementary material (Figure S1).
N-[2-hydroxy-3-(1,7,8,9-tetraphenyl)-3,5-dioxo-4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl)propyl]-5-(2-oxo
hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (6b)
M.W. = 766.3188; C46H46N4O5S; Yield: 54,5%; oil; 1H-NMR (300 MHz, CDCl3,
δ/ppm): 7.66 (4H, m,
H-aromatic), 7.26 (4H, m, H-aromatic), 7.14 (2H, m, H-aromatic), 6.94 (2H, m, H-aromatic), 6.84 (4H,
m, H-aromatic), 6.52 (4H, m, H-aromatic), 6.31 (1H, m, NH-biot), 5.01 (1H, m, NH-biot), 4.34 (1H, m,
C28-H), 4.16 (3H, m, C29-H, C19-H), 4.00 (1H, m, C20-H), 3.52 (3H, m, C27-H, C30-H), 3.32 (1H, m,
C2-H), 3.06 (2H, m, C21-H), 2.73 (1H, m, C10-H), 2.54 (1H, m, C6-H), 2.30 (1H, m, C10-H), 2.17 (2H, m,
C23-H), 1.61 (4H, m, C24-H, C26-H), 1.39 (2H, m, C25-H). 13C NMR (CDCl3, 75 MHz)
δ (ppm): 177.2
(C-3/R/S-20), 177.2 (C-5/R-20), 177.1 (C-5/S-20), 174.3 (C-22/S-20), 174.2 (C-22/R-20), 164.8 (C-31/S-20),
164.7 (C-31/R-20), 145.3 (C-11/S-20), 145.2 (C-11/R-20), 144.9 (C-110/S-20), 144.7 (C-110/R-20), 139.3
(C-15/S-20), 139.3 (C-15/R-20), 139.2 (C-150/S-20), 139.2 (C-150/R-20), 134.1 (C-8/S-20), 134.0 (C-8/R-20),
133.9 (C-9/S-20), 133.8 (C-9/R-20), 129.9 (2C-13/S-20), 129.9 (2C-13/R-20), 129.8 (2C-130/S-20), 129.8
(2C-130/R-20), 129.1 (2C-16), 129.0 (2C-160), 128.0 (4C-120, 12), 127.3 (4C-170, 17), 127.1 (C-18), 127.0
(C-180), 126.7 (C-14), 126.7 (C-140), 77.2 (C-20), 68.1 (C-10/S-20), 67.7 (C-10/R-20), 63.5 (C-7/S-20), 63.4
(C-7/R-20), 63.4 (C-1/S-20), 63.3 (C-1/R-20), 61.5 (C-28/S-20), 61.4 (C-28/R-20), 60.3 (C-29), 55.7 (C-27/S-20),
55.6 (C-27/R-20), 52.3 (C-6/S-20), 52.2 (C-6/R-20), 51.9 (C-2/S-20), 51.8 (C-2/R-20), 43.0 (C-21), 42.8 (C-19),
40.3 (C-30), 36.0 (C-23/S-20), 35.8 (C-23/R-20), 28.2 (C-25/S-20), 28.0 (C-25/R-20), 27.9 (C-26/S-20), 27.7
(C-26/R-20), 25.9 (C-24/S-20), 25.5 (C-24/R-20); HRMS (m/z): calculated value for [M + H]+ 100% =
767.3261; found 100% = 767.3260;
2.1.4. Synthesis of Derivatives 7–9
The general synthesis of derivatives 7–9 is shown on Scheme 2.
O
O
O
C H
H C
C H
H C
6
5
5
6
H C
2
5
5
2
CH
3
3
H C
C H
6 5
C H
H C
C H
H C
6
5
5
6
5
6
6
5
5
6
2
A
B
C
L-glutamine;
R
R
O
R
R
A or B or C
O
carbonyl diimidazole;
DMSO
H C
5
6
H C
5
6
+
O
O
N
O
benzene
O
O
O
O
N
H
H C
5
6
H C
5
6
O
O
O
7: R = -CH3
8: R = -C2H5
9: R = -C6H5
A1: R = -CH3
B1: R = -C2H5
C1: R = -C6H5
Scheme 2. Synthesis of derivatives 7–9.
Synthesis of Anhydrides A1, B1 and C1
An appropriate cyclopentadienone:

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2,5-dimethyl-3,4-diphenylcyclopentadienone as a dimer (0.002 mol of dimer)
2,5-diethyl-3,4-diphenylcyclopenta-2,4-dienone (0.007 mol)
1,2,3,4-tetraphenylcyclopenta-1,3-dienone (0.007 mol)
was dissolved in benzene (50 mL). Next, maleic anhydride (0.008 mol) was added to the solution.
The reaction mixture was refluxed for 48 h. When the reaction was complete (TLC control) the mixture
was left at the room temperature for 12 h, obtaining solid product. The obtained crude product was
crystallized from benzene to give the white solid. Finally, product was filtered and dried.
1,7-dimethyl-8,9-diphenyl-4-oxatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (A1)
M.W. = 358.1205; C23H18O4; Yield: 60%; white powder, m.p. 188–190 ◦C; 1H-NMR (300 MHz, CDCl3,
δ
/ppm): 11.10 (1H, s, -NH), 7.19 (6H, m, H-aromatic), 6.97 (4H, m, H-aromatic), 3.49 (2H, s, C2-H,
C6-H), 1.58(6H, s, -CH3); 13C NMR (CDCl3, 75 MHz)
δ (ppm): 197.5 (C-10), 169.6 (2C-3, 5), 142.5 (2C-8,
9), 132.0 (2C-12, 120), 129.5 (4C-14, 140), 128.2 (2C-15, 150), 128.1 (4C-13, 130), 56.5 (2C-1, 7), 49.0 (2C-2, 6),
11.8 (2C-11, 110); HRMS (m/z): calculated value for [M + Na]+ 100% = 381.1097; found 100% = 381.1097,
25.3% = 382.1134.
1,7-diethyl-8,9-diphenyl-4-oxatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (B1)
◦
1
M.W. = 386.4397; C25H22O4; Yield: 75%; white powder, m.p. 146–147 C; H NMR (300 MHz,
DMSO-d6+ TMS, /ppm): 7.20 (6H, m, H-aromatic), 6.97 (4H, m, H-aromatic), 4.02 (2H, s, C2-H, C6-H),
δ
2.09 ( 2H, m, -CH2-), 1.88 ( 2H, m, -CH2-), 0.87 (6H, t, J = 7.5, -CH3); 13C NMR (DMSO, 75 MHz)
δ
(ppm): 197.0 (C-10), 171.3 (2C-3, 5), 142.4 (2C-8, 9), 133.0 (2C-13, 130), 129.0 (4C-15, 150), 128.1 (2C-16,
160), 127.7 (4C-14, 140), 59.3 (2C-1, 7), 45.5 (2C-2, 6), 18.5 (2C-11, 110) 8.8 (2C-12, 120); HRMS (m/z):
calculated value for [M + Na]+ 100% = 409.1410; found 100% = 409.1410, 28.3% = 410.1437.
1,7,8,9-tetraphenyl-4-oxatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (C1)
M.W. = 482.5253; C33H22O4; Yield: 65%; white powder, m.p. 216–218 ◦C; 1H-NMR (300 MHz, CDCl3,
δ
/ppm): 7.64 (2H, m, H-aromatic), 7.40 (4H, m, H-aromatic), 7.24 (2H, m, H-aromatic), 6.93 (2H, m,
H-aromatic), 6.91 (4H, m, H-aromatic), 6.74 (4H, m, H-aromatic), 4.46 (2H, s, C2-H, C6-H); 13C NMR
(CDCl3, 75 MHz)
δ
(ppm): 194.1 (C-10), 169.1 (2C-3, 5), 142.3 (2C-8, 9), 132.0 (4C-11, 110, 15, 150), 131.5
(4C-16, 160), 130.1 (4C-17, 170), 130.0 (4C-13, 130), 128.4 (2C-18, 180), 128.2 (4C-12, 120), 127.9 (2C-14,
140), 65.0 (2C-1, 7), 46.6 (2C-2, 6); HRMS (m/z): calculated value for [M + Na]+ 100% = 505.1410; found
100% = 505.1410, 35.5% = 506.1431.
Synthesis of Derivatives 7–9
l-glutamine (0.0056 mol) was suspended in DMSO (10 mL) at room temperature. Next,
an ◦appropriate anhydride (A1, B1, C1) was added and the solution was stirred at temperature
80 C. When reaction was finished (TLC monitoring, developing system: chloroform: methanol
9:1 v/v) the mixture was cooled to 20 ◦C, filtered and poured into a round-bottom flask containing
carbonyl-diimidazole (0.006 mol) in DMSO (6 mL) at 20 ◦C [22]. The resulting solution was heated to
85–90 ◦C and stirred at this temperature to the end of the reaction (TLC monitoring, developing system:
chloroform: methanol 9:1 v/v). Then the solution was poured into cold water (about 5 ◦C) and the
mixture was stirred at room temperature for 24 h. The precipitated solid was filtered, crystalized from
methanol: water (1:4 v/v) system and dried overnight to give crystalline white product. In the biological
experiments the derivatives
7–9 were used as mixtures of diastereoisomers. The numbering of atoms
of compounds used in the description of nuclear magnetic spectra is available in supplementary
7–9
material (Figure S2).
4-(2,6-dioxopiperidin-3-yl)-1,7-dimethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (7)
◦
1
M.W. = 468.3467; C28H24N2O5; Yield: 30%; white powder, m.p. 217–218 C; H-NMR (300 MHz,
DMSO-d6+ TMS, /ppm): 11.10 (1H, s, -NH), 7.19 (6H, m, H-aromatic), 7.01 (2H, m, H-aromatic), 6.91
δ

Biomolecules 2019, 9, 446
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(2H, m, H-aromatic), 5.01 (1H, m, C30-H), 3.64 (2H, m, C2-H, C6-H), 2.85 (1 H, m, C50-H), 2.56 (1 H,
m, C50-H), 2.38 (1H, m, C40-H), 1.75 (1H, m, C40-H), 1.43 (3H, s, -CH3), 1.38 (3H, s, -CH3); 13C NMR
(DMSO, 75 MHz) δ
(ppm): 198.5 (C-10), 175.1 (2C-3, 5), 172.4 (C-60), 168.6 (C-20), 141.4 (C-8), 140.9 (C-9),
133.0 (2C-12, 120), 129.4 (4C-14, 140), 127.9 (2C-15, 150), 127.4 (4C-13, 130), 79.17 (C-30), 55.7 (2C-1, 7),
49.5 (2C-2, 6), 30.6 (C-50), 21.4 (C-40), 11.9 (2C-11, 110); HRMS (m/z): calculated value for [M + Na]+
100% = 491.1577; found 100% = 491.1576, 35% = 492.1584.
4-(2,6-dioxopiperidin-3-yl)-1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (8)
◦
1
M.W. = 496.1997; C30H28N2O5; Yield: 40%; white powder, m.p. 232–234 C; H-NMR (300 MHz,
DMSO-d6+ TMS, δ/ppm): 11.12 (1H, s, -NH), 7.17 (6H, m, H-aromatic), 7.00 (2H, m, H-aromatic), 6.92
(2H, m, H-aromatic), 5.04 (1H, m, C30-H), 3.78 (2H, m, C2-H, C6-H), 2.84 (1 H, m, C50-H), 2.80 (1 H, m,
C50-H), 2.50 (1H, m, C40-H), 2.05 (2H, m, -CH2-), 1.87 (2H, m, -CH2-), 1.71 ( 1H, m, C40-H), 0.88 (6H, t,
J = 6.9 Hz, -CH3); 13C NMR (DMSO, 75 MHz)
δ
(ppm): 198.1(C-10), 175.5 (2C-3, 5), 172.4 (C-60), 168.6
(C-20), 141.8 (2C-8, 9), 133.4 (2C-13, 130), 129.2 (4C-15, 150), 127.1 (2C-16, 160), 127.4 (4C-14, 140), 59.3
(2C-1, 7), 49.6 (C-30), 43.5 (2C-2, 6), 30.6 (C-50), 21.4 (C-40), 19.0 (2C-11, 110), 9.0 (2C-12, 120); HRMS
(m/z): calculated value for [M + Na]+ 100% = 519.1890; found 100% = 519.1891, 30% = 520.1899.
4-(2,6-dioxopiperidin-3-yl)-1,7,8,9-tetraphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione (9)
◦
1
M.W. = 592.1998; C38H28N2O5; Yield: 30%; white powder, m.p. 230–232 C; H-NMR (300 MHz,
DMSO-d6+ TMS, /ppm): 11.16 (1H, s, -NH), 7.69 (4H, m, H-aromatic), 7.36 (6H, m, H-aromatic), 6.91
(6H, m, H-aromatic), 6.78 (4H, m, H-aromatic), 5.16 (1H, m, C30-H), 4.60 (2H, m, C2-H, C6-H), 2.85 (1 H,
m, C50-H), 2.81 (1 H, m, C50-H), 2.50 (1H, m, C40-H), 1.74 (1H, m, C40-H); 13C NMR (DMSO, 75 MHz)
δ
δ
(ppm): 194.8 (C-10), 174.6 (2C-3, 5), 172.4 (C-60), 168.5 (C-20), 141.6 (2C-8, 9), 132.9 (4C-11, 110,15,150),
132.7 (4C-16, 160), 130.5 (4C-17, 170), 129.8 (4C-13, 130), 127.7 (2C-18, 180), 127.4 (4C-12, 120), 127.3 (2C-14,
140), 79.16 (C-30), 65.2 (2C-1, 7), 50.0 (2C-2, 6) 30.5 (C-50), 21.3 (C-40); HRMS (m/z): calculated value for
[M + Na]+ 100% = 615.1890; found 100% = 615.1890, 30% = 616.1909, 6.25% = 617.1952.
2.2. Cell Culturing and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Cytotoxicity Assay
Human umbilical vein endothelial cells (purchased from Life Technologies, Carlsbad, CA,
USA) were cultured according to the manufacturer’s instructions in Medium 200 supplemented
with Low Serum Growth Supplement. 1
×
104 cells were seeded on each well on a 96-well plate
(Nunc, Roskilde, Denmark). The HeLa (human cervix carcinoma, cat. #93021013), CFPAC (human
pancreatic adenocarcinoma, cat. #91112501), K562 (chronic myelogenous leukemia, cat. #89121407),
HL-60 (acute myelogenous leukemia, cat. #98070106), and MOLT-4 (Human acute T lymphoblastic
leukemia, cat. #85011413) cells were purchased from European Collection of Authenticated Cell
Cultures (ECACC). They were cultured in an RPMI 1640 medium supplemented with antibiotics
(streptomycin, penicillin) and 10% fetal calf serum (20% for HL60), in a 5% CO2-95% air atmosphere.
7
×
103 cells were seeded on each well on a 96-well plate (Nunc). 24 hours later the cells were exposed
to the test compounds. The aliquots of stock solutions of the test compounds (freshly prepared
in DMSO) were added to the cell cultures to yield final concentrations of 1, 10−1, 10−2, 10−3, 10−4
and 10−5 mM. The concentration of DMSO in the cell culture medium was 1%. The IC50 value (the
concentration of a test compound required to reduce the cell survival fraction to 50% of the control)
was calculated from a dose-response curve and used as a measure of cellular sensitivity to a given
,
treatment. The IC50 values are given as means (
±
standard deviation (SD)) from 2 independent
experiments with 4 technical replicates. The cytotoxicity of all compounds was determined by the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma, St. Louis, MO, USA).
Briefly, after 24 h or 48 h of incubation with a given compound, the cells were treated with the MTT
reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved in 20% SDS and
50% DMF at pH 4.7 and VIS light absorption was measured at 570 and 650 nm on a microplate reader

Biomolecules 2019, 9, 446
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FLUOstar Omega (BMG Labtech, Offenburg, Germany). As a control (100% viability), cells grown in
the presence of 1% DMSO were used.
2.3. Activation of Caspase-3/7 and Caspase-8/9 Determined by Fluorescent and Luminescent Assays
K562 and MOLT-4 cells were cultured in a RPMI 1640 medium supplemented with antibiotics
and 10% fetal bovine serum in a 5% CO2 atmosphere at 37 ◦C. 20
×
103 cells were seeded on each well
on a 96-well plate. After 24 h the cells were exposed to the test compounds at concentration of 5
IC50 for another 18 h (caspase 3 and 7), or 3 h and 6 h (caspase 8 and 9). The cells were also exposed
to 1% DMSO (control) or 1 M staurosporine (a strong inducer of cell apoptosis, Sigma, St. Louis,
×
µ
MO). The induction of cell apoptosis was analyzed by Apo-ONE® Homogeneous Caspase-3/7 Assay
(Promega, Madison, WI, USA). After 18 h of incubation with the test compounds, the cells were
treated with the caspase-3/7 reagent according to manufacturer’s instructions and incubated for an
additional 1.5 hours at room temperature. The fluorescence in each well was measured in triplicate
(excitation at 485 nm, emission measured at 520 nm) using a microplate reader FLUOStar Omega
(BMG-Labtech, Offenburg, Germany). For normalization of the data, the caspase activity in the control
cells (exposed to 1% DMSO) was taken as 1.0. The apoptosis pathway was identified by measuring
the activity of caspases 8 and 9 using Caspase-Glo 8 Assay and Caspase-Glo 9 Assay kits (Promega,
Madison, WI, USA). After incubation of MOLT-4 and K562 cells with the test compounds for 3 h and 6 h,
respectively, the cells were treated with the pro-luminescent substrates according to the manufacturer’s
instructions and incubated for an additional 40 min at room temperature. Luminescence was measured
(in triplicate) using a microplate reader FLUOStar Omega. For normalization of the data, the caspase
activity in the control cells (exposed to 1% DMSO) was taken as 1.0.
2.4. Cleavage of Caspase 3/8/9 and Poly(ADP-Ribose)Polymerase (PARP)
0.15
×
106 cells/ml were seeded on a 6-well plate (Nunclon, Thermo Fisher Scientific, Waltham,
MA, USA). Following 24 hour preincubation the cells were treated with either the compound or 1%
DMSO (control) for 24 or 48 hours. Cells were lysed using RIPA Buffer (Sigma) supplemented with
Complete Protease Inhibitor Coctail (Roche, Basel, Switzerland) and PhosSTOP (Roche). Samples
containing 20
µ
g protein were resuspended in 4 Laemmli’s Buffer (Biorad, Hercules, CA, USA),
×
separated on 10% acrylamide gel (TGX Stain-Free Gel Kit, Biorad) and blotted onto nitrocellulose
membrane (Panreac Applichem ITW, Darmstadt, Germany). Blots were blocked in 5% non-fat dry
milk in TBST (1% (v/v) Tween-20, 140 mM NaCl, 25 mM Trizma, pH = 7.4 adjusted with HCl)
for 1 h and incubated overnight with the antibodies against caspase-9 (9502), caspase-8 (9746),
caspase-3 (9662), caspase-3 cleaved (9661) and β-actin (3700), all from Cell Signaling Technology, and
antibody against poly(ADP-ribose)polymerase (PARP, 551025) from BD Pharmingen. Membranes were
incubated with appropriate species-specific secondary antibodies conjugated with HRP (Cell Signaling
Technology, Danvers, MA, USA). Blots were developed using ECL substrate (Biorad, Hercules, CA,
USA). The experiment was performed in at least three independent biological replicates.
2.5. Annexin V/Propidium Iodide (PI) Flow Cytometry
The phosphatidylserine on the extracellular side of cell membranes was quantified using an
FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) according to the
manufacturer’s instruction. 106 cells were seeded on a 6-well plate (Nunc). 24 h later the cells were
exposed to the test compounds at concentration of 5
cells). The control cells were exposed to 1% DMSO or 1
were centrifuged at 700 rpm (120 g) for 5 min, washed twice with cold PBS and then resuspended in
1x binding buffer to a density of 106 cells/ml. 100 µL of the resuspended cells was stained in the dark
with 5 l of Annexin V-fluoroscein isothiocyanate (FITC) solution and 10 L propidium iodide (PI)
solution for 15 min at room temperature. The samples were then diluted with 400 L of a binding
buffer and within 1 h analyzed with a flow cytometer (BD FACSCaliburTM, BD Biosciences, San Jose,
×
IC50 for another 6 h (K562 cells) or 3 h (MOLT-4
µM staurosporine. After treatment the cells
×
µ
µ
µ

Biomolecules 2019, 9, 446
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CA, USA). After treatment, four populations of cells were assessed: (1) viable (neither apoptotic nor
necrotic, Annexin V-FITC and PI negative), (2) apoptotic (Annexin V-FITC positive and PI negative),
(3) late apoptotic (Annexin V-FITC and PI positive), and (4) necrotic (Annexin V-FITC negative and
PI positive).
2.6. Annexin V/7-Aminoactinomycin D (7-AAD) Flow Cytometry
0.15
MA, USA). Following 24 h preincubation the cells were treated with either the compound or 1%
DMSO (control) for 24 or 48 h. After indicated time, the cells were washed with cold PBS (w/o Mg2+
×
106 cells/ml were seeded on a 6-well plate (Nunclon, Thermo Fisher Scientific, Waltham,
,
Ca2+), stained with Annexin V (FITC Annexin V Apoptosis Detection Kit with 7-aminoactinomycin
D (7-AAD), Biolegend, San Diego, CA, USA) according to manufacturer instructions and analysed
using BD FACSCalibur flow cytometer. Obtained Flow Cytometry Standard files were analyzed using
BD FACSDiva Software (FACSDiva Version 6.1.3, Becton Dickinson Biosciences, San Jose, CA, USA).
The experiment was performed three times in at least two technical replicates.
2.7. Real-Time Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
K562 tumor cells were seeded on a 6-well plate (Nunc, Roskilde, Denmark) in an amount of
3
×
106 cells/well and were exposed to the test compounds at concentration of 5
×
IC50 for 18 h.
The control cells were exposed to 1% DMSO or 1
µM staurosporine. The total RNA pool was isolated
from the cells lysates using TriPure Isolation Reagent (Roche, Basel, Switzerland) according to the
manufacturer’s instruction. Purity and integrity of RNA was checked spectrophotometrically with a
NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent
2100 Bioanalyzer instrument. All RNA samples used for microarray studies had RIN values
Reverse transcription and polymerase chain reaction (RT-PCR) amplification reactions were performed
in one step using a LightCycler 1.0 Instrument (Roche) and LightCycler RNA Amplification Kit
SYBR Green I (Roche). Each sample contained 250 ng of total RNA, 2 mM MgCl2, 2 l of 5 SYBR
Green, 0.5 M of forward and reverse primers, and 0.2 l of the enzyme mix (total sample volume of
10 l). The qRT-PCR reactions were optimized for each studied gene and performed according to a
≥
9.
®
µ
×
µ
µ
µ
general protocol: a reverse transcription reaction (RT) for 10 min. at 55 ◦C, and a denaturation step for
30 s at 95 ◦C. The three steps of PCR quantification reaction included: I—denaturation (0 s at 95 ◦C),
II—annealing (10 s at 60 ◦C), III—product extension (8 s at 72 ◦C), 45 cycles in total. Subsequent melting
experiments were performed by quick denaturation at 95 ◦C, annealing over 10 s at 65 ◦C and heating
up to 95 ◦C at 0.1 ◦C/s. The BAX, PMAIP1, HTRA2, TNFRSF 10B, and ESRRBL1 mRNA levels were
normalized against a reference GAPDH mRNA. Changes in mRNA expression invoked by the tested
compounds were calculated using ∆∆Ct method (calibrator- RNA isolated with K562 cells exposed
to 1% DMSO, averaged values
±
standard deviation from 4 experiments are given). The following
primers (sequences given in 50 to 30 direction) were used for PCR reactions: BAX Fwd: AAG GTG
CCG GAA CTG ATC AG, Rev: GCG TCC CAA AGT AGG AGA GG, (product size 121 bp); PMAIP
Fwd: GCT CAG GAA CCT GAC TGC AT, Rev: GCA CCC ATG AAT GCA CCT TC, (product size
138 bp); HTRA2 Fwd: TCC CTA TCT CGA ACG GCT CA, Rev: TGA ATC CTC AGC GTT GCG AT
(product size 175 bp); TNFRSF10B Fwd: GAA GTT GGG CCT CAT GGA CA, Rev: GGT GTG GAC
AGA GGC ATC TC (product size 127 bp); ESRRBL1 Fwd: CAG CTG AGT GAG GCA AAG GA, Rev:
GGA GCA CCA TCA GTC ATG CT (product size 146 bp); GAPDH Fwd: CAT CAT CTC TGC CCC
CTC TC, Rev: CTG TTG AAA CCA TAG CAC CT (product size 159 bp).
2.8. The Effect of Thalidomide, Lenalidomide and Test Dicarboximides on the IKZF1 and IKZF3 Level in
Leukemia Cells
K562 or MOLT4 cells were seeded on 6-well plate (2
containing 10% FBS. MOLT4 cells were exposed to 1% DMSO (vehicle control), test compounds
(2 M), (5 M), (10 M). Similarly, K562 cells were exposed to 1% DMSO, test compounds
×
106 cells/well) in a RPMI 1640 medium
6
6
µ
3
µ
5
µ

Biomolecules 2019, 9, 446
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(1
µ
M),
3
(1
µ
M),
5
(1
µ
M) for 48 h. Cells treated with 100
µ
M thalidomide or 10
µ
M lenalidomide
served as positive controls. After incubation, cells were centrifuged (600 rpm, 6 min, 24 ◦C), washed
once with PBS and lysed in 20 mM Tris (pH 7.2) containing 1% Triton X-100 supplemented with
Complete®protease inhibitors cocktail (Roche). The levels of IKZF1 and IKZF3 were assessed by
immunoblotting (IB).
2.9. IKZF1, IKZF3 and ABC50 Immunoblotting
Cells were lysed in TBS buffer containing 1% Triton X-100 and Complete®protease inhibitor
cocktail (Roche) for 15 min on ice with occasional vortexing. Insoluble material was centrifuged
(12,000 rpm, 15 min, 4 ◦C) and supernatants were collected. Total protein concentration was measured
with DC Protein Assay (Bio-Rad, Hercules, CA, USA). Cell lysates containing 30
µg of total protein
were mixed with Laemli buffer supplemented with SDS and
β-mercaptoethanol, denatured (10 min,
◦
95 C) and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
electrophoresis (4% stacking and 10% resolving gels). Resolved proteins were electrotransferred
(semi-dry, 90 min, 1.5 mA/cm2) to a 0.45
µm nitrocellulose membrane (Thermo Scientific, Waltham,
MA, USA). Membranes were blocked for 2 hours in TBST buffer (20 mM Tris-HCl pH 7.5, 0.9%
NaCl, 0.1% Tween 20) containing 5% BSA (BioShop, Burlington, ON, Canada) and probed with
primary antibodies (overnight at 4 ◦C) diluted in TBST buffer with 0.5% BSA. Tubulin served as a
loading control for IB experiments. Anti IKZF3 (rabbit monoclonal, dilution 1:500), anti IKZF1 (rabbit
polyclonal, dilution 1:500) were from Abcam. Anti IRF4 (rabbit polyclonal, dilution 1:500) were
from Cell Signaling Technology. Anti α-tubulin (mouse monoclonal IgG, 1:1000) was from Sigma
Aldrich. Anti ABC50 antibodies (mouse monoclonal ABC5H06, dilution 1:100) was from Abcam. After
extensive washing with TBST buffer, membranes were probed with secondary goat anti-rabbit or goat
anti-mouse HRP-conjugated IgG (1:5000 in TBST, Santa Cruz Biotech, Dallas, TX, USA) for 45 min at
room temperature. Membranes were washed in TBST buffer and the chemiluminescent signal was
developed with ECL Plus Western Blotting Substrate (Thermo Scientific) and visualized in Syngene
G:Box detection system. Protein bands intensities were analysed using Image Quant (version 5.0, GE
Healthcare Life Sciences, Pittsburgh, PA, USA).
2.10. Identification of Cellular Proteins Targeted by Dicarboximides—Pull-Down Assay
300 nmoles of biotinylated 6b was added to 0.6 mL of the high capacity streptavidin agarose
resin (Thermo Scientific, Waltham, MA, USA) equilibrated in 50% DMSO and incubated for 2 h (room
temperature, gently mixing, total volume 0.7 mL). The K562 cell lysate was prepared in the buffer
containing 0.5% NP-40 and supplemented with protease inhibitors coctail (Roche). Next, the beads
with immobilized 6b were added to the K562 cell lysate (9 mg of total protein) and incubated for 2 h
at 4 ◦C with gently mixing. As a control, K562 cell lysate incubated with the streptavidin agarose
beads was used. After incubation the beads were centrifuged (2000 rpm, 2 min, 4 ◦C) and washed
6 times with 5 mL of lysis buffer. After the last wash, the beads were resuspended in Laemli buffer and
denatured 10 min at 95 ◦C. Released proteins (40
µL/well) were separated by SDS-PAGE (4% stacking,
8% resolving gel) and silver stained with Pierce Silver Stain Kit (Thermo Scientific, Waltham, MA, USA).
Bands of interest were cut out of the gel and digested with trypsin. Tryptic peptides were separated
by liquid chromatography (LC) and analyzed by LC-MS-MS/MS with Orbitrap mass spectrometer
(Thermo). The sequences of the MS/MS spectra were identified by correlation with the human protein
sequence database (Sprot 2016_11, H.) using MASCOT software (version 2.6.2, Matrix Science Inc.,
Boston, MA, USA) (http://www.matrixscience.com/). The significance threshold was p < 0.05 and ions
score or expect cut-off was 22.
2.11. Analysis of ABC50 Protein: Cell Culture, siRNA, Transfections
HeLa cells were transfected with a siRNA with the aid of Lipofectamine 3000 (Invitrogen, Carlsbas,
CA, USA) according to manufacturer’s instructions. The complexes of Lipofectamine 3000 with

Biomolecules 2019, 9, 446
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RNA were prepared in Opti-MEM Reduced-Serum Medium (Gibco, Waltham, MA, USA). The ratio
of Lipofectamine3000/nucleic acid (v/w) was 2/1. The final concentration of the siRNA was 100 nM
for single siRNA transfections. Unless otherwise stated, cells transfected with a given siRNA were
analyzed 48 h post-transfection. siRNAs for ABC50 (NM_001090) were designed with the aid of
the siDESIGN Center (www.dharmacon.com), synthesized in-house (the Department of Bioorganic
Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences (CMMS
PAS) and purified by high-performance liquid chromatography (HPLC). The silencing efficiency of
siRNAs was determined by western blotting of ABC50 protein in transfected HeLa cells. The following
siRNA duplexes (S—sense strand, AS—antisense strand) were used:
si166 (S) 50 CAG ACA AAG UGG UGA AGA ATT/(AS) 50 UUC UUC ACC ACU UUG UCU GTT
si1612 (S) 50 AUG ACC AGG GCU UCU UGG ATT/(AS) 50 UCC AAG AAG CCC UGG UCA UTT
si1730 (S) 50 AGA ACU GCU GAA ACA GUA UTT/(AS) 50 AUA CUG UUU CAG CAG UUC UTT
Control siRNA (siCTL): (S) 50ACA UGA AGC AGC ACG ACU UTT/(AS) 50AAG UCG UGC UGC
UUC AUG UTT
2.12. Statistical Analysis
Statistical analysis was performed with STATISTICA 10 (StatSoft, Round Rock, TX, USA) and
Microsoft Excel (Excel 2010, Microsoft, Redmond, WA, USA). Routinely the t-test was used, while the
non-parametric Mann–Whitney U test was applied for the data not fitting the normal distribution.
P values below 0.05 were considered as statistically significant. For majority of presented experiments
the results are given as a mean (
least in triplicate.
±
SD) from two or three independent experiments, each carried out at
3. Results
3.1. Dicarboximides Are Cytotoxic toward Leukemia Cells
We have earlier shown [20] that dicarboximides 15 were highly cytotoxic towards human leukemia
cells K562 and HL-60. Interestingly, these compounds were neither cytotoxic to adherent cancer cells
(HeLa) nor to primary endothelial cells (HUVEC). Since K562 and HL-60 cells represent the chronic
myeloid leukemia (CML) and acute myeloid leukemia (AML) type, respectively, we extended our
studies to human MOLT-4 cells belonging to acute lymphoblastic leukemia (ALL). We also used
the adherent human pancreatic cancer cell line CFPAC as the representative of the solid cancers.
Please note, that in the biological tests described below, all dicarboximides were used as mixtures
of diastereomers. Cytotoxicity of the test compounds
determined using the MTT test. Compounds displayed significant cytotoxicity toward MOLT-4
and were non-toxic to adherent CFPAC cells, except of compound (Table 2). These results support our
previous observations that some dicarboximides demonstrate selective cytotoxicity against leukemia
cells, particularly compounds and 20]. Compounds and were much more cytotoxic toward
leukemia cells (IC50 values 1–20 M) than cytarabine (IC50 300 M) and showed similar cytotoxicity as
1–6 towards MOLT-4 and CFPAC cells was
1–6
6
3
5
[
3,
5
6
µ
µ
sorafenib and irinotecan (Table 2). Bortezomib and doxorubicin were highly cytotoxic to both cancer
and normal cells. Although test dicarboximides were less potent than bortezomib or doxorubicin, their
therapeutic indexes (Table 2) suggest that they might be safer in use, with less potential side effects.
In this respect, test dicarboximides could be superior over standard anticancer drugs.

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Table 2. Cytotoxicity of test compounds, cytarabine, bortezomib, sorafenib, irinotecan and doxorubicin
in different cell lines after 48 h. IC50 mean values [µM] ± standard deviation (SD) are shown.
Compound
HeLa
>1000 *
0.05 ± 0.01 *
20.0 ± 2.6 *
200 ± 11 *
0.50 ± 0.01 *
>100 **
K562
300 ± 19 *
0.04 ± 0.01 *
3.0 ± 0.3 *
10.0 ± 0.9 *
0.15 ± 0.01 *
10 ± 0.59 **
4.5 ± 0.29 **
2.0 ± 0.31 **
8 ± 0.53 **
1.0 ± 0.05 **
1.0 ± 0.3 **
12 ± 1.5
HL-60
300 ± 18 *
0.3 ± 0.1 *
15.0 ± 0.6 *
70.0 ± 2.5 *
0.15 ± 0.03 *
>100 **
>100 **
>100 **
>100 **
2.0 ± 0.05 **
5.0 ± 0.24 **
nd
HUVEC
>1000 *
<0.01 *
20.0 ± 4.1 *
30.0 ± 3.7 *
0.10 ± 0.03 *
>100 **
>100 **
>100 **
>100 **
>100 **
>100 **
>100
MOLT-4
nd
CFPAC
nd
TI ***
3.3
0.25
6.7
3
cytarabine
bortezomib
nd
nd
sorafenib
nd
nd
irinotecan
nd
nd
doxorubicin
nd
nd
0.67
10
1
2
20.0 ± 2.7
30.0 ± 2.3
9.0 ± 0.6
30.0 ± 2.0
20.0 ± 2.3
4.0 ± 0.2
5 ± 0.59
1.5 ± 0.06
150
>1000
>1000
>1000
>1000
>1000
40.0 ± 3.1
nd
>100 **
22
3
>100 **
50
4
>100 **
12.5
100
100
83
5
>100 **
6
1.0 ± 0.2 **
20 ± 1.9
64 ± 10
6a
6b
7
7 ± 1.2
nd
3.8 ± 0.22
200
nd
0.5
1.2
2.6
nd
180
170
nd
nd
8
>200
25
nd
65
18
nd
9
>200
>200
nd
>200
>200
nd
nd—not determined, * taken from [23], ** taken from [20], *** TI was calculated as a ratio of IC50 (concentration
required to reduce cell survival fraction to 50% of control) for HUVEC and K562.
3.2. Dicarboximides Induce Apoptosis in Leukemia Cells
We have previously reported that compounds
3 and 6 induce apoptosis in K562 (CML) leukemia
cells [20]. Here we show that dicarboximides are significantly cytotoxic for MOLT-4 (ALL) cells
1–6
(Table 2). This finding prompted us to investigate further whether these compounds induce apoptosis
in the MOLT-4 cells. In the below apoptosis studies (i.e., caspase 3/7/8/9 activation or cleavage assays,
annexin V/PI staining) dicarboximides were used at the arbitrarily chosen concentration of 5 IC50.
×
Apoptosis is characterized by strong activation of caspase 3 and 7 (caspase 3/7) which in turn cleave
several key cellular proteins leading to cell death. Some of these cleaved proteins serve as markers of
apoptosis, for example PARP. Another hallmark of apoptosis is a proteolysis of pro-caspase 3 yielding
a 17 kDa protein fragment. Activity of caspase 3/7 can also be measured directly using profluorescent
DEVD peptide substrate. As shown on Figure 1a, incubation of MOLT-4 cells with dicarboximides
3
,
5
and
6
led to a strong activation of caspase 3/7 suggesting that these compounds show a strong
induced apoptosis in
pro-apoptotic activity. Using different assays, we demonstrated that compound
5
K562 cells. This is evidenced by the presence of cleavage fragments of PARP (85 kDa) and caspase 3
(17 kDa) (Figure 1b).

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10
9
8
7
6
5
4
3
2
1
0
(a)
(b)
Figure 1. Test dicarboximides activate caspase 3 and 7 in leukemia cells.
in MOLT-4 cells treated with dicarboximides or staurosporine (positive control) for 18 h. The test
compounds were used at the indicated concentrations (5 IC50). The results are normalized to the
sample containing MOLT-4 cells exposed to 1% DMSO. Means SD are shown. Abbreviations:
MOLT-4—untreated cells. ST—staurosporine. ( ) Caspase 3 and poly(ADP-ribose)polymerase (PARP)
cleavage in K562 cells treated with dicarboximide for 24 h. Cells incubated with 1% DMSO were
(a) Activity of caspase 3/7
×
±
b
5
used as a control (0). After indicated time, lysates were collected and immunoblotted for caspase-3 and
PARP cleavage. The experiment was performed in at least three independent biological replicates and
representative Western blot results are shown.
To confirm the ability of compounds 3, 5 and 6 to induce apoptosis in leukemia cells, we tested
phosphatidylserine (PS) distribution in K562 and MOLT-4 cells using fluorescently labelled annexin V.
In the non-apoptotic cells, PS is present only in the inner (cytoplasmic) leaflet of the cell membrane.
During apoptosis PS undergoes translocation to the outer leaflet giving a signal for phagocytosis.

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The cells were treated with
3
,
5
and
6
(at the concentrations of 5
×
IC50) or 1
µ
M staurosporine (positive
control) and 1% DMSO (negative control). Necrotic cells were visualized with propidium iodide (PI) or
7-aminoactinomycin D (7-AAD). As shown in Figure 2a, dicarboximides and induced apoptosis
in K562 cells (please note the different incubation times for compounds and ). Staurosporine
(positive control) induced apoptosis in only about 25% of K562 cells including both early (LR) and
late (UR) apoptotic cells. In the presence of and we observed more than 90% of apoptotic cells,
while compound induced apoptosis in about 44% of K562 cells. Similar results were obtained in
3
,
5
6
3,
6
5
5
6
3
MOLT-4 cells. In the preliminary experiments we found that MOLT-4 cells were more sensitive to
the apoptosis-inducing factors therefore, only 3h incubation with staurosporine or test compounds
was applied. The results shown in Figure 2b indicate that compounds
3, 5 and 6 induced massive
apoptosis in about 76%, 78% and 99% of MOLT-4 cells, respectively and the late apoptotic phase was
prevailing. Altogether, these data further confirm that compounds
extensive apoptosis in K562 and MOLT-4 cells.
3, 5 and 6 induce efficient and
(a)
Figure 2. Cont.

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(b)
Figure 2. Flow cytometry analysis of apoptosis in leukemia cells induced by the test compounds or
staurosporine. Apoptosis was determined by Annexin V Fluoroscein Isothiocyanate (FITC) Apoptosis
Detection Kit I, (BD Pharmingen). The test compound and time of incubation with cells is given
above each scatterplot.
(a) scatterplots of K562 cells stained with Annexin V FITC and PI (or
7-AAD, FITC Annexin V Apoptosis Detection Kit with 7-AAD, BioLegend). Abbreviations: K562
+ DMSO—cells treated with 1% DMSO; ST—staurosporine. Tested compounds were used at the
following concentrations:
3 (10 µM), 6 (5 µM), 5 (5 µM); staurosporine was used at 1µM concentration;
(
b) scatterplots of MOLT-4 cells stained with Annexin V FITC and PI. Abbreviations: MOLT-4 +
DMSO—cells treated with 1% DMSO. Tested compounds were used at the following concentrations:
3 (45 µM), 6 (20 µM), 5 (100 µM).
3.3. Dicarboximides Activate Apoptosis via Receptor and Mitochondrial Pathways
To identify which of the apoptotic pathways: receptor or mitochondrial is activated by
dicarboximides, we measured the activity of caspase 8 and 9 (caspases 8/9) in leukemia cells exposed
to compounds 3, 5 and 6. In non-apoptotic cells, caspases 8/9 exist as inactive pro-enzymes which
are cleaved upon initiation of apoptosis, yielding enzymatically active caspases. Activated (cleaved)
caspases 8 and 9 are markers of the receptor and mitochondrial apoptotic pathway, respectively.
Figure 3 clearly indicates that caspases 8/9 were strongly activated in K562 treated with
MOLT-4 cells treated with and . Treatment of K562 cells with or resulted in more than 2-fold
increase of activity of caspases 8/9 compared to the control (Figure 3a). Even stronger activation of
caspases 8/9 (more than 10-fold) was observed in MOLT-4 cells exposed to or (Figure 3b). Incubation
of K562 cells with compound also led to the activation of caspases 8/9. Cleaved (i.e., activated)
forms of caspases 8/9 were detected in K562 cells after 48 h incubation with , while after 24 h we
3, 5, 6 and in
3
6
3
6
a
3
6
5
5
observed cleaved form of caspase 9 (Figure 3c). These results indicate that compounds
induce apoptosis in leukemia cells by both the receptor and mitochondrial pathways.
3, 5 and 6

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6
5
4
3
2
1
0
caspase 8
caspase 9
(a)
25
caspase 8
caspase 9
20
15
10
5
0
(b)
Figure 3. Cont.

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(c)
Figure 3. Activation of caspase 8 and caspase 9 in leukemia cells treated with compounds
K562 ( ) or MOLT-4 cells ( ) were treated with or staurosporine (ST) for 6 or 3 hours, respectively.
Activity of caspases in cells exposed to 1% DMSO was normalized to 1.0. Means +/ SD are shown.
) Western blot analysis of cleavage of caspase 8 and caspase 9 in K562 cells incubated with (5 M)
3, 5 and 6.
a
b
3, 6
−
(
c
5
µ
for 24 and 48 hours. Cells incubated with 1% DMSO were used as a control. After indicated time,
lysates were collected and immunoblotted for cleavage of caspase 8 and caspase 9. The experiment was
performed in at least three independent biological replicates and representative Western blot results
are shown.
3.4. Dicarboximides Change the Expression Profile of Genes Involved in Apoptosis
Furthermore, we assessed the expression of five apoptosis-related genes using real-time RT-qPCR.
The selected genes encode the following proteins: BAX (BCL-2-associated X protein), PMAIP1 (NOXA)
(phorbol-12-myristate-13-acetate-induced protein 1), HTRA2 (OMI) (high temperature requirement
A2 serine protease), TNFRSF 10B (DR5) (tumor necrosis factor receptor superfamily, member 10B),
and ESRRBL1 (IFT57) (intraflagellar transport 57 homolog (Chlamydomonas)). As shown in Figure 4
dicarboximides 3 and 6 significantly induced the expression of mRNAs encoding proteins involved
in both the receptor (TNFRSF 10B, ESRRBL1) and mitochondrial (BAX, PMAIP1, HTRA2) apoptotic
pathways. The concentration of dicarboximides was kept the same as in the apoptosis activation
studies i.e., 5
were similar to staurosporine. These data corroborate with our results demonstrating that test
dicarboximides activated caspase 8/9 leading to apoptosis via receptor and mitochondrial pathways.
×
IC50. The expression profiles of analyzed mRNAs obtained upon the use of 3 and
6

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Figure 4. Real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of
relative mRNAs levels in K562 cells after 18 h of incubation with staurosporine (1 M, positive control)
and tested compounds (10 M) and (5 M). Levels of analyzed mRNAs in K562 cells treated with
µ
3
µ
6
µ
1% DMSO (reference sample) were expressed as 1.0. Statistically significant changes are indicated with
asterisks (p < 0.05, Mann-Whitney U test).
3.5. Identification of Cellular Proteins Targeted by Dicarboximides—Pull-Down Assay
To identify cellular targets for the test dicarboximides, we have synthesized a biotinylated
derivative of 6a. Compound 6a exhibited significant cytotoxicity toward leukemia cells and was
chosen for biotinylation due to the presence of hydroxyl and amine groups (Table 2). The biotinylated
derivative 6b retained cytotoxicity toward K562 and MOLT-4 cells with IC50 of 7 and 1.5 µM, respectively
(Table 2). Next, 6b was immobilized on streptavidin-agarose beads and incubated with the lysate of
K562 cells. Bound cellular proteins were pulled-down, separated by SDS-PAGE and visualized by
silver staining. We have observed six protein bands located between 250 and 75 kDa that were uniquely
present or significantly enhanced in a pull-down material with 6b (Figure 5a). These bands were
excised, and their protein content was identified by mass spectrometry (Supplementary materials).
In five out of six bands analyzed, the ABC50 protein was identified and significantly enriched (Table 3).
Western blot analysis of pull-down material with immobilized 6b confirmed the presence of ABC50
(Figure 5b). Therefore, we focused on the ABC50 protein as a target for the test dicarboximides.

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(a)
(b)
Figure 5.
alone or incubated with 6b. The gel was silver stained. M—protein marker lane in kDa. Numbers
on the right correspond to band number entries in Table 3 (below). ( ) Western blot identification of
(a) SDS-PAGE of the streptavidin-agarose pull down samples obtained from lysates of K562
b
ABC50 in a material pulled-down from lysates of K562 cells. Mouse monoclonal anti ABC50 antibodies
were used. Pull-down was performed in the presence (+) or in the absence (−) of compound 6b.

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Table 3. Identification of cellular proteins pulled-down with 6b.
Exponentially
No. of
Significant Significant
Matches
No. of
Band
Mass
(Da)
Modified Protein
Abundance Index
(emPAI)
Database
No.
Entry
Score
2693
289
Description
Sequences
1
2
Sprot
Sprot
Q9Y490
Q8NE71
271,766
96,323
72
59
18,629
Talin-1
ATP-binding
cassette sub-family
F member 1 (ABCF1
or ABC50)
8
8
0.42
3
4
5
6
Sprot
Sprot
Sprot
Sprot
Q8NE71
Q8NE71
Q8NE71
Q8NE71
565
1923
651
96,323
96,323
96,323
96,323
15
43
18
12
14
29
16
11
0.85
32,905
42,767
0.62
ABC50
ABC50
ABC50
ABC50
495
3.6. ABC50 Knockdown Abolishes HeLa Sensitivity to Dicarboximide 6
Results of the pull-down experiments strongly suggested that ABC50 might be a cellular target
for test dicarboximides. To confirm the role of this protein in dicarboximides-induced cytotoxicity,
we investigated whether siRNA-mediated downregulation of ABC50 renders the cells less sensitive
to 6. First, we tried to deliver siRNA to K562 or MOLT-4 cells either by transfection or nucleofection
(electroporation). Despite several attempts, we were not able to efficiently downregulate the expression
of ABC50 in K562 or MOLT-4 cells, probably due to inefficient delivery of siRNA. Therefore, we
switched to HeLa cells which are more prone to transfection and they are also sensitive to compound
6
(Table 2). We identified siRNA designated as si166, that efficiently downregulated ABC50 protein in
HeLa cells (Figure 6a). Subsequently, Hela cells were transfected with si166 and 24h later were treated
with increased concentrations of
cells was measured with an MTT assay. As shown in Figure 6b, HeLa cells with downregulated ABC50
were less sensitive to when compared with control cells, i.e., transfected with control siRNA (siCTL).
In the presence of at the concentration of up to 2.5 M the viability of HeLa cells with knocked-down
ABC50 (HeLa kdABC) was not affected, while the viability of cells with normal levels of ABC50 was
significantly lower. Only at the highest concentration of (i.e., 5 M) the viability of HeLa kdABC
6
(i.e., 0.5, 1, 2.5 and 5
×
IC50). After additional 48 h the viability of
6
6
µ
6
µ
was reduced about 20%. These data confirm that observed cytotoxicity of dicarboximides is likely the
result of targeting the ABC50 protein.
(a)
Figure 6. Cont.

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140
120
100
80
siCTRL
si166
60
40
20
0
0
0.5
1
2.5
5
concentration of 6 [µM]
(b)
Figure 6. (a) Downregulation of ABC50 by siRNA. HeLa cells were transfected with a 100 nM of
siRNA. 48 h posttransfection ABC50 levels were determined by Western blotting. ABC50 level in cells
transfected with control siRNA (siCTRL) was used as a reference. Tubulin served as a loading control.
HeLa—non-transfected cells. (b) siRNA-mediated knockdown of ABC50 impairs cytotoxicity of 6 in
HeLa cells. Cells were transfected with the indicated siRNAs and treated with increased concentrations
of 6. Cells incubated with 1% DMSO were used as a control (0).
3.7. Dicarboximides Reduce the Level of IKZF1/3 Transcription Factors in Leukemia Cells
Thalidomide and its derivatives (lenalidomide and pomalidomide) belong to the class of organic
compounds known as phthalimides and display immunosuppressive and anti-angiogenic activity.
Thalidomide binds to the cereblon protein and modifies the function of the E3 ubiquitin ligase leading
to downregulation of the transcription factors: Ikaros family zinc finger protein-1 (IKZF1, Ikaros) and
3 (IKZF3, Aiolos). Similarities in chemical structure between the test dicarboximides and thalidomide,
i.e., the presence of phthalimide-like moiety, allowed us to hypothesize that the mechanism of action
might be similar. We examined whether incubation of K562 and MOLT-4 cells with
any effect on the level of IKZF1 and IKZF3. Dicarboximides were evaluated at the concentrations of
IC50 (K562) or 0.5 IC50 (MOLT-4), to limit the cytotoxic effect. As shown in Figure 7, we observed a
significant diminution of IKZF1 and IKZF3 in the leukemia cells treated with compounds and . This
effect was similar to lenalidomide or thalidomide used as reference compounds. Incubation of cells
with compound led to a modest reduction of IKZF1 and IKZF3. These results support our hypothesis
3, 5 and 6 had
×
5
6
3
that the biological activity of the test dicarboximides is similar to thalidomide, i.e., they modulate the
level of IKZF1 and IKZF3 in leukemia cells.

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(a)
(b)
Figure 7. Dicarboximides downregulate the levels of IKZF1 and IKZF3 transcription factors in leukemia
cells. ( ) K562 cells treated with lenalidomide (10 M), thalidomide (100 M) and dicarboximides
(1 M), (1 M) or (1 M) for 48 h. ( ) MOLT4 cells treated with lenalidomide (10 M), thalidomide
(100 M) and dicarboximides (5 M), (10 M) or (2 M) for 48 h. Representative blots from at
least 3 independent experiments are shown.
a
µ
µ
3
µ
5
µ
6
µ
b
µ
µ
3
µ
5
µ
6
µ
3.8. Glutarimide Derivatives of Dicarboximides as Modulators of ABC50 in Cancer
Cells—Proteolysis-Targeting Chimeras (PROTACs) Approach
ABC50 was identified as the target of the test dicarboximides in the pull-down experiments.
Therefore, we examined whether dicarboximides could be used as proteolysis-targeting chimeras
(PROTACs) to downregulate the ABC50 protein in leukemia cells. We synthesized the chimeras of
3, 5
and in which alkylamine chain was replaced with glutarimide, while the phthalimide-like portion
6

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remained unchanged or slightly modified (as in the case of
6
, where the methylene bridge in the
phthalimide-like ring was replaced by ketone bridge in
9). These chimeras are shown in Table 1 as 7, 8
and . A glutarimid ring endows the chimeras with the structural basis to bind cereblon protein, a part
9
of E3 ubiquitin ligase complex. The phthalimide-like portion of chimeras would bind ABC50 protein
thus facilitating its ubiquitination by E3 ubiquitin ligase and subsequent degradation in a proteasome.
It must be noted that
incubated with chimeras
ABC50 was determined by Western blotting. The concentrations of
to minimize the cytotoxic effect. Figure 8a shows the level of ABC50 was significantly reduced by
chimeras and in K562 cells. A weaker anti-ABC50 effect was observed in HeLa cells (Figure 8b).
Compounds and significantly downregulated ABC50, while didn’t have such pronounced effect.
7,
8
and
9
were evaluated as mixtures of enantiomers. K562 and HeLa cells were
M), (15 M) and (30 M) for 96 h and subsequently the level of
and were below their IC50
7
(30
µ
8
µ
9
µ
7,
8
9
7,
8
9
7
8
9
These results demonstrate that test dicarboximides may be used as PROTACs for the inhibition of
ABC50 in cancer cells.
(a)
(b)
Figure 8. The effect of dicarboximide-based proteolysis-targeting chimeras (PROTACs) on the ABC50
protein in the (
was detected by Western blotting. Compounds
DMSO 1% (control). Representative blots from at least 3 independent experiments are shown.
a
) K562 or (
b
) HeLa cells. Cells were incubated with compounds for 96 h and the ABC50
7
and were used at 30 M, was used at 15 M,
9
µ
8
µ
4. Discussion
We have earlier shown [20] that the compounds
1–6 (enlisted in Table 1) were selectively
cytotoxic towards adherent (HeLa) or suspension (K562, HL-60) cancer cells, being non-toxic towards
normal HUVEC cells. In the present studies we aimed to characterize the pathway of a cell death
(necrosis vs apoptosis) and to identify mechanism of activity and cellular protein target(s) of the test
dicarboximides. Due to structural similarities between test dicarboximides and thalidomide (presence
of the phthalimide-like ring), we also evaluated their immunomodulatory activity, i.e., the effect on
IKZF1 and IKZF3.
We determined cytotoxicity of the screened compounds towards two additional cancer cell lines,
pancreatic cancer CFPAC and leukemia MOLT-4. The corresponding IC50 values were in the range of
1.5–40
µM (Table 2). Interestingly, compounds
1–6 were not cytotoxic towards normal HUVEC cells,
and except for
6
, towards adherent HeLa and CFPAC cells. This indicates that the test dicarboximides
show, to some degree, selective cytotoxicity for leukemia cells. We also compared the cytotoxicity of the
screened dicarboximides with reference drugs: cytarabine, bortezomib and doxorubicin (anti-leukemia
drugs), sorafenib (used in the therapy of liver and kidney cancer) and irinotecan (colon cancer).
The results summarized in Table 2 indicate that the IC50 values of dicarboximides in the leukemia cells
are significantly lower than cytarabine and similar to sorafenib or irinotecan. However, it must be
emphasized that the test compounds are virtually non-toxic towards primary endothelial cells, while

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the reference drugs (except for cytarabine) show potent cytotoxicity against these cells. Therefore, the
test dicarboximides might be clinically more beneficial than the standard anticancer drugs.
Cytotoxicity of
3
,
5
and
6
towards K562 and MOLT-4 cells correlated with increased activity
and exert
of caspases 3 and 7 (Figure 1) and enhanced Annexin-V staining (Figure 2). Thus,
3
,
5
6
cytotoxicity towards K562 and MOLT-4 cells by inducing massive and effective apoptosis. Moreover,
these compounds increased the activity of caspases 8 and 9 (Figure 3) in K562 and MOLT-4 cells,
indicating activation of both, receptor and mitochondrial apoptotic pathways. This conclusion is
further supported by the results of expression of several pro-apoptotic genes. We have found that
3 and
6
significantly upregulated the expression of genes involved in the receptor (TNFRSF 10B, ESRRBL1)
and in the mitochondrial (BAX, PMAIP1, HTRA2) pathways of apoptosis (Figure 4). In this respect,
the test dicarboximides resemble other derivatives, for example, naphthalimides and maleimides.
It was demonstrated that naphthalimides and maleimides activated the mitochondrial apoptotic
pathway by changing the mitochondrial potential, increasing the level of the AIF and pro-apoptotic Bax
proteins, and decreasing the level of anti-apoptotic proteins Bcl-2 and survivin [10,24]. On the other
hand, maleimides activated the receptor apoptotic pathway by increasing the level of the membrane
Fas receptor in Jurkat cells [25]. The profiles of gene expression (Figure 4) were similar to control
experiment, where the cells were treated with staurosporine. Staurosporine is mainly involved in
the activation of mitochondrial pathway of apoptosis, but the receptor and the caspase-independent
pathways are not excluded [26,27].
In a pull-down assay followed by mass spectrometry analysis, ABC50 (or ABCF1) protein was
identified as a possible target of the test dicarboximides. For this experiment we used biotinylated
derivative 6b. Its cytotoxic properties were slightly diminished comparing the parent 6a. Biotinylation
may lead to a different pattern of hydrogen bonds the compound can form with biomolecules.
The reduced selectivity of 6b toward K562 cancer cells and increased trifold IC50 for MOLT-4 cells
can result from different steric hindrance of hydroxyl group and/or chemical “inactivation” (in terms
of hydrogen bonding) of the amine (-NH2) group when compared to the parental compound 6a
.
In addition, compound 6b may also interfere with important metabolic processes that depend on
biotin, a coenzyme for carboxylases. We also demonstrated that HeLa cells with knocked-down ABC50
showed increased resistance to the test dicarboximides, confirming the ABC50 as a target. ABC50
belongs to a family of ATP-binding cassette (ABC) proteins. Most ABC proteins are membrane-bound
ATP-dependent pumps. In contrast, ABC50 lacks a transmembrane domain and does not function
as a transmembrane transporter. This protein locates primarily in the cytoplasm and nucleoplasm
of cells, where it binds to eukaryotic initiation factor 2 (eiF2) and promotes interaction between
eiF2 and methionyl-tRNA. This suggests that ABC50 is involved in ribosome biogenesis [28] and
initiation and control of protein synthesis [29]. Lindqvist et al. demonstrated that inhibitors of protein
synthesis trigger the cell death [30]. It was also demonstrated that siRNA-driven depletion of ABC50
in HeLa cells led to the inhibition of total protein synthesis [29]. Thus, the observed cytotoxicity of
dicarboximides and their pro-apoptotic activity could result from binding to ABC50 and impairing
protein synthesis. Other studies indicate that ABC50 is an important regulator of innate immune
responses and autoimmune disorders. Lee et al. demonstrated that si-RNA mediated knockdown
of ABC50 in mouse embryonic fibroblasts (MEFs) resulted in a significant inhibition of synthesis
of CXCL10 in response to interferon-stimulatory DNA [31]. Other study indicated that TNF-alpha
strongly increased the expression of ABC50 mRNA in human synoviocytes [32]. Increased copy number
of ABC50 gene was also associated with increased risk of gout and autoimmune pancreatitis [33,34].
Having demonstrated that the test dicarboximides target ABC50 and based on their structural
similarity to IMIDs (i.e., thalidomide, lenalidomide), we hypothesized that dicarboximides might
inhibit the synthesis and/or induce proteasome-dependent degradation of immunomodulatory proteins.
Indeed, incubation of leukemia cells with the test dicarboximides resulted in downregulation of
transcription factors IKZF1 and IKZF3 (Figure 7), which are particularly important for development of
T- and B-lymphocytes. Among dicarboximides, compounds 1–5 differ in an amino alkyl substituent

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attached to the imide nitrogen atom. Unfortunately, we are unable to provide any rationale for changes
in their activity (especially in HL60 cells) upon change of that substituent. Although from the chemical
point of view, these differences are only minute, one cannot exclude that they are important in highly
specific interactions, which to us remain obscure. Compounds
cytotoxicity. While compounds and possess the same phthalimide-like moiety as compounds
and , their amino alkyl/hydroxyl-amino alkyl chains are the most extended. Compound contains
ethyl chain linked to piperidine residue via the nitrogen atom, while has an n-propanol chain linked
to isopropyl amine (secondary amine), and such a structure offers both—hydrophobic and hydrogen
bond interactions with the target biomolecule. On the other hand, compound has the most extended
phthalimide-like part, with the central ring substituted with 4 phenyl groups and with the methylene
bridge. Compound exhibits higher cytotoxicity than , while both compounds have the same amino
3, 5 and 6 show the most potent
3
5
1, 2
4
3
5
6
6
1
alkyl chain but differ in phthalimide-like portion, so, in principle, it might be possible than this
“extended” aromatic system may more efficiently interact with the target biomolecules by stacking
interactions or exert beneficial intercalating properties. Unfortunately, hydrolysis of plasmid DNA
with restriction endonuclease provided no evidence that such a process operates to any measurable
extent [20].
Moreover, we obtained novel PROTAC chimeras by extending the dicarboximides 3, 5 and 6 with
a glutarimide fragment of thalidomide. They target the ABC50 protein via the dicarboximide moiety
and recruit the E3 ubiquitin ligase by the glutarimide part. In cellular experiments, they proved to be
effective tools for presumably proteasome-dependent downregulation of ABC50 protein in cells.
Based on the obtained results we propose the putative mechanisms of action of dicarboximides
1–6 and 7–9, which are schematically depicted below.
5. Conclusions
In summary, we have identified novel dicarboximides that are cytotoxic for cancer cells and show
some preference toward leukemia cells. We also provided evidence that these compounds induced
leukemia cells death by activating receptor and mitochondrial apoptotic pathways. The molecular
mechanism of their cytotoxicity depends at least in part on ABC50 protein, which was identified

Biomolecules 2019, 9, 446
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as a target of dicarboximides in a pull-down assay. We also demonstrated that dicarboximides
could be successfully used as tools for targeted proteolysis of ABC50. Similar to thalidomide, the
test dicarboximides displayed immunomodulatory activity and decreased the levels of IKZF1 and
IKZF3 transcription factors. Thus, biological activity of the test dicarboximides makes them potential
candidates for being lead compounds for synthesis and biological evaluation of new anticancer agents.
6. Patents
The compounds described in this publication and their application are the subject of European
and Polish Patent Applications: EP13176421.9, EP13150611.5, P.400000, P.398193.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/9/446/s1,
Figure S1: The numbering of atoms of derivative 6b used in the description of nuclear magnetic spectra, Figure S2:
The numbering of atoms of compounds 7–9 used in the description of nuclear magnetic spectra, Table S1: Tests
and cell lines used to characterize the biological activity of dicarboximides.
Author Contributions: Conceptualization, M.C.; Data curation, M.C., J.K.-B., K.K.-G., M.N. and I.S.; Funding
acquisition, M.N., U.W. and B.N.; Investigation, M.C., J.K.-B., K.K.-G., M.N. and I.S.; Methodology, M.C., J.K.-B.,
K.K.-G., M.N. and I.S.; Project administration, M.C., U.W. and B.N.; Supervision, M.C., U.W. and B.N.; Visualization,
M.C., J.K.-B., K.K.-G., M.N. and I.S.; Writing—original draft, M.C. and J.K.-B.; Writing—review and editing, M.C.,
K.K.-G., M.N., I.S., U.W. and B.N.
Funding: This research was funded by: Ministry of Science and Higher Education, grant number
PBZ-MNiSW-07/I/2007 (B.N.), National Science Centre, grants numbers DEC-2012/05/N/NZ7/01174 (K.K.-G.),
UMO-2014/15/B/NZ7/00966 (U.W., B.N., M.N.) and by statutory funds of the CMMS PAS, Lodz and WMU, Warsaw.
Acknowledgments: The authors thank to: Piotr Guga for helpful discussions and critical reading of the manuscript;
Łukasz Pe˛czek for help with statistical analysis of real-time RT-PCR data; Milena Sobczak for preliminary synthesis
of 6b.
Conflicts of Interest: The authors declare no conflict of interest.
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