Synthesis, structural characterization, DFT calculations and antiproliferative evaluation of novel spirohydantoin derivatives containing a substituted benzyl moiety

Article abstract of DOI:10.1016/j.molstruc.2018.11.071

Two series of cycloalkanespiro-5-hydantoins, namely cyclohexanespiro-5-hydantoins and cycloheptanespiro-5-hydantoins with a 4-substituted benzyl or a 2-(4-substituted phenyl)-2-oxoethyl group at N3 position, were synthesized and their effects on proliferation of human colon (HCT-116), leukemia (K562) and breast (MDA-MB-231) cancer cell lines were tested. For comparison, we also described the 5,5-diphenylhydantoin analogues. The structural features of the investigated compounds were characterized by elemental analysis, FT-IR, UV–Vis, ¹H and ¹³C NMR spectroscopy and X-ray crystallography. Regarding their structure–activity relationships, it was shown that the substitution on the benzyl moiety with the methoxy, chloro or bromo group potentiated the antiproliferative activity relative to the parent compounds, while an increase in the size of the cycloalkyl group resulted mostly in a decrease of the antiproliferative activity. The single crystal X-ray analysis revealed the existence of dimers and chains formed by the N–H?O hydrogen bonds. The analysis of the molecular descriptors of Lipinski demonstrated that all investigated compounds obeyed the rule of five. To further understand their geometry and electronic structure, DFT calculations with B3LYP method using 6-311++G(d,p) basic set were performed. In this context, the UV–Vis spectra of the investigated compounds were analyzed in detail, whereby the predicted absorption spectra from DFT calculation matched the experimentally obtained ones, with a good correlation. The interesting physico-chemical and pharmacologically relevant properties of the investigated compounds warrant their further investigation.

Full text of DOI:10.1016/j.molstruc.2018.11.071

Accepted Manuscript
Synthesis, structural characterization, DFT calculations and antiproliferative
evaluation of novel spirohydantoin derivatives containing a substituted benzyl moiety
Anita M. Lazić, Lidija D. Radovanović, Bojan Đ. Božić, Biljana Đ. Božić Nedeljković,
Vesna D. Vitnik, Željko J. Vitnik, Jelena R. Rogan, Nataša V. Valentić, Gordana S.
Ušćumlić, Nemanja P. Trišović
PII:
S0022-2860(18)31384-X
DOI:
https://doi.org/10.1016/j.molstruc.2018.11.071
MOLSTR 25900
Reference:
To appear in: Journal of Molecular Structure
Received Date: 6 August 2018
Revised Date: 19 November 2018
Accepted Date: 22 November 2018
Please cite this article as: A.M. Lazić, L.D. Radovanović, Bojan.Đ. Božić, Biljana.Đ. Božić Nedeljković,
V.D. Vitnik, Ž.J. Vitnik, J.R. Rogan, Nataš.V. Valentić, G.S. Ušćumlić, N.P. Trišović, Synthesis,
structural characterization, DFT calculations and antiproliferative evaluation of novel spirohydantoin
derivatives containing a substituted benzyl moiety, Journal of Molecular Structure (2018), doi: https://
doi.org/10.1016/j.molstruc.2018.11.071.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, structural characterization, DFT calculations and antiproliferative
evaluation of novel spirohydantoin derivatives containing a substituted benzyl
moiety
Anita M. Lazića, Lidija D. Radovanovića, Bojan Đ. Božićb, Biljana Đ. Božić Nedeljkovićb,
Vesna D. Vitnikc, Željko J. Vitnikc, Jelena R. Rogand, Nataša V. Valentićd, Gordana S.
Ušćumlićd, Nemanja P. Trišović*,d
aInnovation Center, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade,
Serbia
bFaculty of Biology, University of Belgrade, Belgrade, Serbia
cDepartment of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of
Belgrade, Belgrade, Serbia
dFaculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia
*Corresponding author. Tel.: +381113303869; fax: +381113370387.
E-mail address: ntrisovic@tmf.bg.ac.rs
1

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Abstract
Two series of cycloalkanespiro-5-hydantoins, namely cyclohexanespiro-5-hydantoins and
cycloheptanespiro-5-hydantoins with a 4-substituted benzyl or a 2-(4-substituted phenyl)-2-
oxoethyl group at N3 position, were synthesized and their effects on proliferation of human
colon (HCT-116), leukemia (K562) and breast (MDA-MB-231) cancer cell lines were tested.
For comparison, we also described the 5,5-diphenylhydantoin analogues. The structural
features of the investigated compounds were characterized by elemental analysis, FT-IR, UV-
1
Vis, H and 13C NMR spectroscopy and X-ray crystallography. Regarding their structure–
activity relationships, it was shown that the substitution on the benzyl moiety with the
methoxy, chloro or bromo group potentiated the antiproliferative activity relative to the
parent compounds, while an increase in the size of the cycloalkyl group resulted mostly in a
decrease of the antiproliferative activity. The single crystal X-ray analysis revealed the
existence of dimers and chains formed by the N–HꢀꢀꢀO hydrogen bonds. The analysis of the
molecular descriptors of Lipinski demonstrated that all investigated compounds obeyed the
rule of five. To further understand their geometry and electronic structure, DFT calculations
with B3LYP method using 6-311++G(d,p) basic set were performed. In this context, the UV-
Vis spectra of the investigated compounds were analyzed in detail, whereby the predicted
absorption spectra from DFT calculation matched the experimentally obtained ones, with a
good correlation. The interesting physico-chemical and pharmacologically relevant properties
of the investigated compounds warrant their further investigation.
Keywords: Spirohydantoin; Antiproliferative activity; X-ray structure determination; DFT
calculation.
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1. Introduction
A number of reasons have been put forward to promote the pharmacological applications of
derivatives of hydantoin (imidazolidine-2,4-dione) [1]. These compounds are mostly small
molecules, lipophilic and easily penetrate biological membranes. When properly substituted,
derivatives of hydantoin exhibit various pharmacological activities, e.g. anticonvulsant [2]
antiarrhythmic [3], antiviral [4], anti-inflammatory [5] and anti-HIV activity [6]. A
representative example of a hydantoin-based drug is phenytoin (5,5-diphenylhydantoin,
Dilantin), which is widely used in the control of the grand mal types of epilepsy and cardiac
arrhythmias [7].
Due to their ability to interfere with multiple signaling pathways influencing cancer
metastasis, derivatives of hydantoin have been recognized as promising therapeutic agents in
the cancer treatment. The non-steroidal antiandrogen nilutamide (3-(4-nitro-3-
trifluoromethylphenyl)-5,5-dimethylhydantoin, Nilandron), is used in combination with
surgical castration for the treatment of metastatic prostate cancer [8]. Its ferrocenyl-aryl-
hydantoin derivatives have been shown to retain a modest affinity for the androgen receptor,
while the N-substituted complexes show a weak or moderate antiproliferative effect on
hormone-dependent and -independent prostate cancer cells [9]. A linkage of aryl hydantoin-
based antiandrogen through a short polyethylene glycol linker to genistein results in a
conjugate which derives its antiproliferative activity against prostate cancer cell lines in a
similar manner [10].
The 5-benzylidene hydantoin core has also been recognized as a promising scaffold to
develop new antiproliferative compounds. Carmi et al. have reported that 5-arylidene
hydantoins inhibit the EGFR kinase and exhibited an antiproliferative action on A431 human
epidermoid carcinoma cells [11]. Regarding the mechanism of action, their growth-inhibitory
effects in the lung (A549) cancer cell line has been associated with an accumulation of the
cells in the S phase of the cell cycle and induction of genomic DNA damage [12]. Z-5-(4-
Hydroxyphenyl)methylene hydantoin, a marine natural product, and its derivatives enhance
tight junction formation and exhibit anti-invasive and anti-migratory activities in vitro against
metastatic prostate cancer cells and inhibit tumor growth in distant organs in mouse models
[13]. The corresponding structure–activity (SAR) studies have revealed the importance of
size and lipophilic parameters, whereby log P and molecular volume are the most influential
descriptors [14]. Jiang and Zeng have synthesized a series of structurally related hydantoin
derivatives and tested for the antitumor activity against HepG2 cancer cells [15].
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Spiromustine
(3-(2-(bis(2-chloroethyl)amino)ethyl)-1,3-diazaspiro[4.5]decane-2,4-
dione) has been developed to cross the blood-brain barrier; thus it has been reported to
preferentially localize in brain tumors relative to normal brain tissue [16]. Several diversely
substituted diazaspiro hydantoins show growth inhibitory effects against human breast (MCF-
7), hepatocellular (HepG-2), cervix (HeLa) and colon (HT-29) cancer cell lines [17]. In this
case, substitution at N3 position in the hydantoin ring appears to have a key role in the
antiproliferative activity, i.e., compounds bearing the aryl groups show better inhibition
relative to those with the alkyl substituents. When additionally investigating the inhibition of
the leukemia (K562) cell proliferation, Kavitha et al. have reported that the most promising
candidate among these compounds induces cell apoptosis through mitochondrial pathway
following cell cycle arrest [18]. Hydantoin derivatives of the dihydrothieno[2,3-b]naphtho-
4,9-dione system with a distal amine moiety exhibit a similar or greater cytotoxic potency
than doxorubicin, a standard chemotherapy drug, against the human breast (MCF-7) and
colon (SW 620) cancer cell lines [19]. Their cytotoxicity might be ascribed, among others, to
an electrostatic interaction of the positively charged ammonium cation with a negatively
charged binding site. Spiro bisheterocycles containing the hydantoin moiety have been shown
to promote apoptosis of breast (MCF-7 and MDA-MB-23) cancer cell lines via p53-
dependent and -independent pathways [20].
Alanazi et al. have reported that 5,5-diphenylhydantoins with an alkyl, aryl or
phenacyl group at N3 position possess selective activity against the renal (A498 and UO31)
cancer cell lines [21]. The introduction of an additional piperydinyl unit has resulted in a
compound which shows the strong activity against the melanoma (MDA-MB-435) and breast
(MCF-7) cancer cell lines. The docking study has revealed that the hydantoin moiety binds to
a narrow hydrophobic pocket in the enzyme through formation of the hydrogen bonds, while
the π–π and π–cation interactions between the compound and the binding site have also been
observed. In this context, we have shown that 3-(4-substituted benzyl)-5,5-diphenyl- and 3-
(4-substituted benzyl)-5-ethyl-5-phenylhydantoins exhibit the superior antiproliferative
activity against breast (MDA-MB-231) cancer cell line than against colon (HCT-116) cancer
cell line, whereby compounds bearing two phenyl groups at C5 position possess higher
potencies than those with one phenyl group [22].
In the present study, two series of cycloalkanespiro-5-hydantoins bearing a
4-substituted benzyl or a 2-(4-substituted phenyl)-2-oxoethyl group at N3 position were
synthesized (Figure 1, Series 1 and 2) and their effects on proliferation of human colon
(HCT-116), leukemia (K562) and breast (MDA-MB-231) cancer cell lines were tested. A
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focus was thus placed on the overall effects of the structural modifications at N3 and C5
positions of the hydantoin ring on the observed activity. In this context, we described the
antiproliferative activities of several key compounds from our previous study [22] and the
study of Alanazi [21], 3-(4-substituted benzyl)-5,5-diphenylhydantoins and 3-(2-(4-
substituted phenyl)-2-oxoethyl)-5,5-diphenylhydantoins (Fig. 1, Series 3), against human
colon (HCT-116) and breast (MDA-MB-231) cancer cell lines and additionally determined
their antiproliferative activities against leukemia (K562) cancer cell line in this study. To
understand the geometry and electronic structure of investigated hydantoin derivatives, DFT
calculations with B3LYP method using 6-311++G(d,p) basic set were performed. Coupled
with the single crystal X-ray analysis of the representative compounds (1c and 2a), the
presented study provided basis for understanding of a structure–activity relationship of the
investigated compounds, thus enabling development of new synthetic spirohydantoins for the
cancer treatment.
<Figure 1>
2. Experimental section
2.1. Chemistry
1
The H and 13C NMR spectral measurements were performed on a Bruker AC 250
1
13
spectrometer at 200 MHz for the H NMR and 50 MHz for the C NMR spectra or on a
1
Bruker 300 spectrometer at 400 MHz for the H NMR and 100 MHz for the 13C NMR
spectra. The spectra were recorded at room temperature in DMSO-d6. The chemical shifts
1
were expressed in ppm values referenced to TMS (δH = 0 ppm) in H NMR spectra, and the
13
residual solvent signal (δC = 39.5 ppm) in C NMR spectra. FT-IR spectra were recorded
using a Bomem MB series 100 spectrophotometer (υmax are given in cm-1) in the form of KBr
pellets. The UV-Vis spectra were measured with a Shimadzu 1700 spectrophotometer. The
elemental analyses of the investigated compounds were carried out by standard analytical
micromethods using an Elemental Vario EL III microanalyzer.
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2.1.1. General procedure for the preparation of 1a−1g and 2a−2g
The compounds of series 1 and 2 were obtained following the synthetic protocol shown in
Scheme 1. 1,3-Diazaspiro[4.5]decane-2,4-dione and 1,3-diazaspiro[4.6]undecane-2,4-dione
were synthesized according to the modified procedure of Bucherer and Lieb [23]. The
compounds 1a−1i and 2a−2i were prepared using a modified procedure described previously
[24]. 1,3-Diazaspiro[4.5]decane-2,4-dione or 1,3-diazaspiro[4.6]undecane-2,4-dione (0.01
mol) and potassium carbonate (0.03 mol) were dissolved in DMF (60 cm3). After half an
hour, 4-substituted benzyl halide (0.011 mol) was added in the solution. The reaction mixture
was heated at 80 °C for three days. The reaction mixture was poured into three times the
volume of water and extracted with ethyl acetate. The organic layer was washed with 5 %
sodium hydroxide and water and dried over anhydrous magnesium sulphate. The residual
solvent was removed by distillation and the crude product was purified by recrystallization
from ethanol.
<Scheme 1>
2.1.2. General procedure for the preparation of 1h, 1i, 2h, 2i, 3h, 3i
The compounds 1h, 1i, 2h, 2i, 3h and 3i were prepared according to the procedure given in
[21] (Scheme 2). A mixture of the appropriate hydantoin (cycloalkanespiro-5-hydantoin or
5,5-diphenylhydantoin) (0.01 mol) and potassium carbonate (0.01 mol) was stirred in
acetone (60 cm3) at room temperature for half an hour. A solution of 1-(4-substituted
phenyl)-2-chloroethanone (0.011 mol) in acetone was added and the reaction mixture was
stirred for 24 hours at room temperature. The obtained solid was filtered and recrystallized
from ethanol.
<Scheme 2>
3-Benzyl-1,3-diazaspiro[4.5]decane-2,4-dione (1a)
Yield 73%; White solid; mp 151−153 °C; FT-IR (KBr pill, ν cm-1) 3323 (NH), 1771, 1708
1
(C=O); H NMR (200 MHz, DMSO−d6): δ /ppm = 8.81 (s, 1H, NH), 7.33−7.19 (m, 5H,
13
−C6H5−), 4.52 (s, 2Н, –CH2–), 1.68−1.26 (m, 10H, −C6H10−); C NMR (50 MHz, DMSO
d6): δ/ppm = 176.8, 155.9, 137.2, 128.8, 127.6, 127.2, 61.3, 41.0, 38.5, 24.6, 21.0; Elemental
anal. calc. (%) for C15H18N2O2: C, 69.74; H, 7.02; N, 10.84; found: C, 69.70; H, 7.05; N,
10.80.
̶
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3-(4-Меthylbenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1b)
Yield 56%; White solid; mp 211−213 °C; FT-IR (KBr pill, ν cm–1) 3220 (NH), 1771, 1704
(C=O); 1H NMR (200 MHz, DMSO
−C6H4−), 7.08 (d, J = 8 Hz, 2H, −C6H4−), 4.46 (s, 2H, −CH2−), 2.26 (s, 3H, −CH3),
1.66−1.25 (m, 10H, −C6H10−); 13C NMR (50 MHz, DMSO
d6): δ/ppm = 176.9 155.9, 136.7,
̶ d6): δ/ppm = 8.79 (s, 1H, NH), 7.13 (d, J = 10 Hz, 2H,
̶
134.2, 129.4, 127.3, 61.3, 40.8, 33.5, 24.4, 21.0, 20.9 (2C); Elemental anal. calc. (%) for
C16H20N2O2: C, 70.56; H ,7.40; N, 10.29; found: C, 70.62; H, 7.42; N, 10.34.
3-(4-Methoxybenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1c)
Yield 58%; White solid; mp 162−165 °C; FT-IR (KBr pill, ν cm–1): 3295 (NH), 1773, 1696
1
(C=O); H NMR (200 MHz, DMSO−d6): δ/ppm = 8.77 (s, 1H, NH), 7.17 (d, 2H, J = 8 Hz,
−C6H4−), 6.89 (d, 2H, J = 8 Hz, −C6H4−), 4.46 (s, 2H, –CH2–), 3.73 (s, 3H, −CH3),
1.62−1.26 (m, 10H, −C6H10−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 176.8, 158.8,
155.9, 129.2, 128.8, 114.1, 61.2, 55.2, 40.5, 33.5, 24.6, 21.00; Elemental anal. calc. (%) for
C16H20N2O3: C, 66.65; H, 6.99; N, 9.72; found: C, 66.76; H, 7.04; N, 9.65.
3-(4-Chlorobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1d)
Yield 66%; White solid mp 185−186° °C; FT-IR (KBr pill, ν cm–1): 3232 (NH), 1773, 1710
(C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.83 (s, 1H, NH), 7.40 (d, J = 8 Hz, 2H,
−C6H4−), 7.24 (d, J = 8 Hz, 2H, −C6H4−), 4.52 (s, 2H, −CH2−), 1.68−1.03 (m, 10H,
−C6H10−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 176.8, 155.7, 136.1, 132.2, 129.3,
128.8, 61.3, 40.4, 33.5, 24.6, 21.0; Elemental anal. calc. (%) for C15H17N2O2Cl: C, 61.54; H,
5.85; N, 9.57; found: C, 61.57; H, 5.89; N, 9.52.
3-(4-Bromobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1e)
Yield 70%; White solid; mp 193−194 °C; FT-IR (KBr pill, ν cm–1): 3216 (NH), 1768, 1709
(C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.83 (s, 1H, NH), 7.53 (d, J = 10 Hz, 2H,
−C6H4−), 7.18 (d, J = 8 Hz, 2H, −C6H4−), 4.50 (s, 2H, −CH2−), 1.67−1.03 (m, 10H,
−C6H10−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 176.8, 155.7, 136.6, 131.7, 129.6,
120.7, 61.3, 40.5, 33.5, 24.6, 21.0; Elemental anal. calc. (%) for C15H17N2O2Br : C, 53.43; H,
5.08; N, 8.31; found: C, 53.48; H, 5.02; N, 8.35.
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3-(4-Cyanobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1f)
Yield 52%; White solid; mp 179−182 °C; FT-IR (KBr pill, ν cm–1): 3243 (NH), 2231 (CN),
1771, 1714 (C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.88 (s, 1H, NH), 7.81 (d, 2H,
J = 8 Hz, −C6H4−), 7.40 (d, 2H, J = 8 Hz, −C6H4−), 4.62 (s, 2H, –CH2–), 1.70−1.03 (m, 10H,
−C6H10−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 176.9, 155.6, 142.7, 132.8, 128.1,
118.9, 110.4, 61.5, 40.7, 33.5, 24.5, 21.0; Elemental anal. calc. (%) for C16H17N3O2: C,
67.83; H, 6.05; N, 14.83; found: C, 67.90; H, 6.05; N, 14.83.
3-(4-Nitrobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1g)
Yield 74%; Yellowish solid; mp 185−188 °C; FT-IR (KBr pill, ν cm–1): 3235 (NH), 1772,
1708 (C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.90 (s, 1H, NH), 8.21 (d, 2H, J = 10
Hz, −C6H4−), 7.49 (d, 2H, J = 8 Hz, −C6H4−), 4.68 (s, 2H, −CH2–), 1.70−1.03 (m, 10H,
−C6H10−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 176.9, 155.6, 147.0, 144.8, 128.4,
124.0, 61.5, 40.6, 33.4, 24.5, 21.0; Elemental anal. calc. (%) for C15H17N3O4: C, 59.40; H,
5.65; N, 13.85; found: C, 59.32; H, 5.60; N, 13.94.
3-(2-(4-Fluorophenyl)-2-oxoethyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1h)
Yield 40%; White solid; mp 177−180 °C; FT-IR (KBr pill, ν cm–1): 3301 (NH), 1765, 1716,
1
1701 (C=O); H NMR (200 MHz, DMSO−d6): δ/ppm = 8.85 (s, 1H, NH), 8.13 (q, 2H, J =
3.2 Hz, −C6H4−), 7.40(t, 2H, J = 9 Hz, −C6H4−), 4.92 (s, 2H, –CH2–), 1.79−1.28 (m, 10H,
13
−C6H10−); C NMR (50 MHz, DMSO−d6): δ/ppm = 191.3, 177.1, 165.8 (d, J = 251.5 Hz),
155.9, 131.5 (d, J = 9.5 Hz), 131.1 (d, J = 2.5 Hz), 116.4 (d, J = 22 Hz), 61.7, 44.4, 33.6,
24.6, 21.1; Elemental anal. calc. (%) for C16H17FN2O3: C, 63.15; H, 5.63; N, 9.21; found: C,
63.04; H, 5.60; N, 9.16.
3-(2-(4-Chlorophenyl)-2-oxoethyl)-1,3-diazaspiro[4.5]decane-2,4-dione (1i)
Yield 45%; White solid; mp 248−250 °C; FT-IR (KBr pill, ν cm–1): 3109 (NH), 1777, 1708,
1697 (C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.85 (s, 1H, NH), 8.05 (d, 2H, J = 8
Hz, −C6H4−), 7.64 (d, 2H, J = 8 Hz, −C6H4−), 4.92 (s, 2H, –CH2–), 1.79−1.29 (m, 10H,
−C6H10−); 13C NMR (50 MHz, DMSO−d6): δ/ppm) = 191.8, 177.0, 155.6, 139.4, 133.0,
130.3, 129.4, 61.7, 44.4, 33.6, 24.6, 21.0; Elemental anal. calc. (%) for C16H17N2O3Cl: C,
59.91; H, 5.34; N, 8.73; found: C, 59.87; H, 5.31; N, 8.69.
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3-Benzyl-1,3-diazaspiro[4.6]undecane-2,4-dione (2a)
Yield 63%; White solid; mp 117−118 °С; FT-IR (KBr pill, ν cm–1): 3233 (NH), 1771, 1703
1
(C=O); H NMR (200 MHz, DMSO−d6): δ/ppm = 8.66 (s, 1H, NH), 7.37−7.18 (m, 5H,
−C6H5−), 4.51 (s, 2H, –CH2–), 1.86−1.56 (m, 12H, −C7H12−); 13C NMR (50 MHz,
DMSO−d6): δ/ppm = 178.0, 155.7, 137.2, 128.8, 127.6, 127.2, 63.9, 41.0, 37.2, 28.9, 22.3;
Elemental anal. calc. (%) for C16H20N2O2: C, 70.56; H, 7.40; N, 10.29; found: C, 70.52; H,
7.44; N, 10.26.
3-(4-Меthylbenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (2b)
Yield 56%; White solid; mp 172 ̶
175 °С; FT-IR (KBr pill, ν cm–1): 3238 (NH), 1769, 1702
1
(C=O); H NMR (200 MHz, DMSO−d6): δ/ppm = 8.64 (s, 1H, NH), 7.16 (d, 2H, J = 8 Hz,
−C6H4−), 7.10 (d, 2H, J = 8 Hz, −C6H4−), 4.46 (s, 2H, −CH2–), 2.27 (s, 3H, −CH3),
1.81−1.59 (m, 12H, −C7H12−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 178.0, 155.7,
136.7, 134.2, 129.3, 127.3, 63.8, 40.8, 37.1, 28.9, 22.2, 20.8; Elemental anal. calc. (%) for
C17H22N2O2: C, 71.30; H, 7.74; N, 9.78; found: C, 70.28; H, 7.80; N, 9.75.
3-(4-Methoxybenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (2c)
Yield 53%; White solid; mp 132−134 °С; FT-IR (KBr pill, ν cm–1): 3259 (NH), 1767, 1706
1
(C=O); H NMR (200 MHz, DMSO−d6): δ/ppm = 8.61 (s, 1H, NH), 7.15 (d, 2H, J = 8 Hz,
−C6H4−), 6.88 (d, 2H, J = 8 Hz, −C6H4−), 4.42 (s, 2H, −CH2−), 3.73 (s, 3H, −CH3),
1.84−1.57 (m, 12H, −C7H12−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 178.0, 158.8,
155.8, 129.2, 128.9, 114.2, 63.8, 55.3, 40.5, 37.15, 28.9, 22.3; Elemental anal. calc. (%) for
C17H22N2O3: C, 67.53; H, 7.33; N, 9.26; found: C, 67.50; H, 7.37; N, 9.23.
3-(4-Chlorobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (2d)
Yield 65%; White solid; mp 170−173 °С; FT-IR (KBr pill, ν cm–1): 3231 (NH), 1769, 1721
(C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.68 (s, 1H, NH), 7.39 (d, J = 8.4 Hz, 2H,
−C6H4−), 7.23 (d, J = 8.6 Hz, 2H, −C6H4−), 4.49 (s, 2H, −CH2−), 1.85−1.55 (m, 12H,
−C7H12−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 178.0, 155.6, 136.2, 132.2, 129.3,
128.8, 63.9, 40.4, 37.1, 28.9, 22.3; Elemental anal. calc. (%) for C16H19N2O2Cl: C, 62.64; H,
6.24; N, 9.13; found: C, 62.59; H, 6.27; N, 9.10.
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3-(4-Bromobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (2e)
Yield 67%; White solid; mp 178−180 °С; FT-IR (KBr pill, ν cm–1): 3232 (NH), 1770, 1720
(C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.68 (s, 1H, NH), 7.52 (d, J = 8.4 Hz, 2H,
−C6H4−), 7.17 (d, J = 8.4 Hz, 2H, −C6H4−), 4.47 (s, 2H, −CH2−), 1.85−1.55 (m, 12H),
−C7H12−); 13C NMR (50 MHz, DMSO−d6): δ/ppm: 178.0, 155.6, 136.6, 131.7, 129.7, 120.7,
63.9, 40.5, 37.1, 28.9, 22.3; Elemental anal. calc. (%) for C16H19N2O2Br : C, 54.71; H, 5.45;
N, 7.98; found: C, 54.68; H, 5.48; N, 8.03.
3-(4-Cyanobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (2f)
Yield 53%; White solid; mp 171−173 °С; FT-IR (KBr pill, ν cm–1): 3239 (NH), 1772, 1720
(C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.73 (s, 1H, NH), 7.81 (d, 2H, J = 8 Hz,
−C6H4−), 7.38 (d, 2H, J = 10 Hz, −C6H4−), 4.59 (s, 2H, −CH2–), 1.85−1.56 (m, 12H,
−C7H12−); 13C NMR (50 MHz, DMSO−d6): δ/ppm: 178.0, 155.5, 142.8, 132.9, 128.2, 118.9,
110.5, 64.1, 40.8, 37.1, 28.9, 22.3; Elemental anal. calc. (%) for C17H19N3O2: C, 68.67; H,
6.44; N, 14.13; found: C, 68.64; H, 6.49; N, 14.09.
3-(4-Nitrobenzyl)-1,3-diazaspiro[4.5]decane-2,4-dione (2g)
Yield 73%; Yellow solid; mp 166−169 °С; FT-IR (KBr pill, ν cm–1): 3229 (NH), 1772, 1710
(C=O); 1H NMR (200 MHz, DMSO−d6): δ/ppm = 8.75 (s, 1H, NH), 8.20 (d, 2H, J = 10 Hz,
−C6H4−), 7.47 (d, 2H, J = 8 Hz, −C6H4−), 4.64 (s, 2H, –CH2–), 1.86−1.56 (m, 12H,
−C7H12−); 13C NMR (50 MHz, DMSO−d6): δ/ppm = 178.0, 155.5, 147.1, 144.8, 128.5,
124.1, 64.1, 40.6, 37.1, 28.9, 22.3; Elemental anal. calc. (%) for C16H19N3O4: C, 60.56; H,
6.03; N, 13.24; found: C, 60.52; H, 6.08; N, 13.20.
3-(2-(4-Fluorophenyl)-2-oxoethyl)-1,3-diazaspiro[4.6]undecane-2,4-dione (2h)
Yield 43%; White solid; mp 140−142 °C; FT-IR (KBr pill, ν cm–1): 3287 (NH), 1769, 1714,
1
1700 (C=O); H NMR (400 MHz, DMSO−d6): δ/ppm = 8.73 (s, 1H, NH), 8.14 (q, 2H, J =
2.8 Hz, −C6H4−), 7.42 (t, 2H, J = 8.8 Hz, −C6H4−), 4.92 (s, 2H, –CH2–), 1.95−1.59 (m, 12H,
13
−C7H12−); C NMR (100 MHz, DMSO−d6): δ/ppm = 191.5, 178.4, 166.0 (d, J = 255 Hz),
155.7, 131.7 (d, J = 9.0 Hz), 131.4 (d, J = 2.8 Hz), 116.6 (d, J = 22 Hz), 64.5, 37.4, 29.2,
22.2. Elemental anal. calc. (%) for C17H19N2O3F: C, 64.14; H, 6.02; N, 8.80; found: C, 64.10;
H, 6.06; N, 8.77.
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3-(2-(4-Chlorophenyl)-2-oxoethyl)-1,3-diazaspiro[4.6]undecane-2,4-dione (2i)
Yield 47%; White solid; mp 206−208 °C; FT-IR (KBr pill, ν cm–1): 3231 (NH), 1775, 1714,
1700 (C=O); 1H NMR (400 MHz, DMSO−d6): δ/ppm = 8.72 (s, 1H, NH), 8.17
−C6H4−), 7.46 7.37 (m, 2H, −C6H4−), 4.91 (s, 2H, –CH2–), 1.96−1.59 (m, 12H, −C7H12−);
̶ 8.10 (m, 2H,
̶
13C NMR (100 MHz, DMSO−d6): δ/ppm = 192.2, 178.4, 155.8, 139.3, 133.2, 130.5, 129.6,
64.5, 44.7, 37.4, 28.9, 22.5. Elemental anal. calc. (%) for C17H19N2O3Cl: C, 60.99; H, 5.72;
N, 8.37; found: C, 60.90; H, 5.76; N, 8.33.
3-(2-(4-Fluorophenyl)-2-oxoethyl-5,5-diphenylimidazolidine-2,4-dione (3h)
Yield 45%; White solid; mp 244−245 °C; FT-IR (KBr pill, ν cm–1): 3233 (NH), 1780, 1704,
1
1690 (C=O); H NMR (400 MHz, DMSO−d6): δ/ppm = 9.80 (s, 1H, NH), 8.16 (q, 2H, J =
3.2 Hz, −C6H4−), 7.47−7.37 (m, 12H, 2×C6H5 and −C6H4− ), 5.07 (s, 2H, −CH2−); 13C NMR
(100 MHz, DMSO−d6): δ/ppm = 191.3, 173.9, 166.1 (d, J = 252 Hz), 155.3, 140.0, 131.8 (d,
J = 9.6 Hz), 131.2 (d, J = 2.8 Hz), 129.0, 128.7 , 127.4, 116.6 (d, J = 22 Hz), 70.2, 45.3.
Elemental anal. calc. (%) for C23H17N2O3F: C, 71.13; H, 4.41; N, 7.21; found: C, 71.09; H,
4.46; N, 7.18.
3-(2-(4-Chlorophenyl)-2-oxoethyl-diphenylimidazolidine-2,4-dione (3i)
Yield 43%; White solid; mp 241−242 °C; FT-IR (KBr pill, ν cm–1): 3219 (NH), 1780, 1705,
1
1692 (C=O); H NMR (400 MHz, DMSO−d6): δ/ppm = 9.83 (s, 1H, NH), 8.13 (d, 2H, J =
8.4 Hz, −C6H4−), 7.71 (d, 2H, J = 8.4 Hz, −C6H4−), 7.49−7.42 (m, 10H, 2 × C6H5), 5.13 (s,
13
2H, −CH2−); C NMR (100 MHz, DMSO−d6): δ/ppm = 191.9, 173.8, 155.3, 140.0, 139.7,
133.2, 130.6 , 129.6, 129.0, 128.7, 127.4, 127.1, 126.6, 70.2, 45.4; Elemental anal. calc. (%)
for C23H17N2O3Cl: C, 68.23; H, 4.23; N, 6.92; found: C, 68.20; H, 4.26; N, 6.89.
2.2. Biological characterization
2.2.1. Compounds and solutions
The RPMI 1,640 cell culture medium, fetal bovine serum (FBS), and MTT were purchased
from Sigma Chemical Company, USA. MTT was dissolved (5 mg cm-3) in phosphate
buffered saline (pH 7.2) and filtered (0.22 lm) before use.
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2.2.2. Cells
Normal rat peritoneal macrophages and tumor cell lines including human colon cancer (HCT-
116), leukemia (K562) and human breast cancer (MDA-MB-231) cell lines were maintained
in a culture using a nutrient medium RPMI 1640 with 10 % FBS and antibiotics.
2.2.3. Treatment of peritoneal macrophages for evaluation of cytotoxic effect
The biocompatibility testing of the newly synthesized compounds is the first step in their
biological characterization. The rat peritoneal macrophages were used for the evaluation of
biocompatibility. Rat peritoneal macrophages were seeded in 96-well flat-bottomed microtiter
plate and cultivated in 0.1 cm3 of full culture media during 24 h. After that, the investigated
compounds were added to cells in the final concentrations (0.01, 0.1, 1, 10, and 100 ꢁmol dm-
3), except in the control wells, where only the medium was added. Thusly prepared cell
cultures were incubated during an additional 24 h. The stock solutions of the compounds were
prepared in DMSO and dissolved in the corresponding medium to the required working
concentrations. The effects of the investigated compounds on the viability/proliferation of
peritoneal macrophages was determined 24 hours later by the MTT test [25], modified by
Ohno and Abe [26]. Briefly, 0.02 cm3 of MTT dye (5 mg cm-3) was added to each well. After
incubation for further 3 h, 0.1 cm3 of 10 % sodium dodecyl sulfate was added to extract the
insoluble product formazan resulting from conversion of the MTT dye by viable cells. The
number of viable cells in each well is proportional to the intensity of the absorbance of light,
which was then read in an ELISA plate reader at 570 nm.
2.2.4. Treatment of cell lines for antiproliferative in vitro screening
The target cells HCT–116 (10000 cells per well), K562 (100000 cells per well) and MDA–
MB–231 (100000 cells per well) were seeded in triplicate into a wells of a 96-well, flat-
bottomed microtiter plate in 0.1 cm3 culture medium. Twenty-four hours later, after the cell
adaptation and adherence for both cell lines, 0.1 cm3 of the investigated compound was
added to cells in the final concentration (0.01, 0.1, 1, 10 and 100 ꢁmol dm-3), except in the
control wells, where only culture medium was added to the cells and was incubated during
additional 24 h. The effect of the investigated compounds on cancer cell survival was
determined after 24 h by the MTT test described in the previous section. The antiproliferative
effect of the compounds was expressed as a percentage of inhibition of proliferation of non-
treated cell. It was calculated as 100% minus the ratio between the absorbance of each dose
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of the compounds.
2.3. X-ray structure determination
Single-crystal X-ray diffraction data were collected at room temperature (295 K) on an
Oxford Gemini S diffractometer equipped with CCD detector using monochromatized Mo Kα
radiation (λ = 0.71073 Å). Intensities were corrected for absorption using the multi-scan
method. Both structures were solved by direct methods (SIR92) [27] and refined on F2 by
full-matrix least-squares using the programs SHELXL-97 (1c) [28], SHELXL 2018-3 (2a)
[29] and WinGX [30]. All non-hydrogen atoms were refined anisotropically. The positions of
H atoms connected to C atoms in 1c and to C and N atoms in 2a were calculated on
geometric criteria and refined using the riding model with Uiso = 1.2Ueq(C,N). The hydrogen
atom bonded to N1 in 1c was found in ꢂF maps and added to the structural models before the
final cycle of refinement with the fixed positional and atomic displacement parameters. The
selected crystal data and refinement results for 1c and 2a are listed in Table 1. The
crystallographic data for 1c and 2a were deposited at the Cambridge Crystallographic Data
Centre with CCDC reference number 1851483 and 1851484.
<Table 1>
2.4. Computational details
All DFT calculations were performed using the Gaussian 09 program package [31] with
B3LYP method [33] and 6-311++G(d,p) basis set. The default convergence criteria were
used without any constraint on the geometry. The stability of the optimized geometry was
confirmed by frequency calculations, which gave real values for all frequencies. The solvent
effect was introduced by the Conductor Polarizable Continuum Model (CPCM) [33].
UV absorption energies of these compounds were calculated by TD-DFT B3LYP
method in ethanol as solvent. The frontier molecular orbital energies and energy gap of the
investigated compounds are also calculated at the same level of DFT theory. The GaussView
5.0 graphical interface was used to visualize molecular orbitals [34].
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3. Results and Discussion
3.1 Antiproliferative evaluation
The antiproliferative potential of the investigated compounds was evaluated against HCT-
116, K562 and MDA-MB-231 cancer cell lines. The cytotoxicity assays against tumor cell
lines were performed in vitro with various concentrations of compounds [35]. The results for
each compound were reported as the percentage of inhibition of treated cells prolifertion
compared to the non-treated control cells. First of all, the in vitro screening data have
revealed that all investigated compounds are non-toxic to normal cells. On the other side, all
investigated compounds demonstrate a statistically significant antiproliferative potential
against to different tumor cells in the investigated concentration range.
The investigated compounds exhibit mild to moderate inhibitory effects against
proliferation of all three investigated tumor cell lines at concentration of 100 ꢁmol dm-3
(Figs. 2–4 and Tables S1–S5 (Electronic Supplementary Information)). Some general
conclusions concerning structure–activity relationships of the investigated compounds
cannot be simply drawn from the data shown in Figs. 2–4. When the substituent X is the
same, the increase in the size of the cycloalkyl group mostly results in decrease of the
antiproliferative activity. Exceptions from this trend were largely observed with inhibition of
HCT-116 cell proliferation. The parent compounds 1a and 2a appear to be the least active
ones, while the substitution on the benzyl moiety with the methoxy, chloro or bromo group
potentiates the antiproliferative activity. Regarding the inhibition of HCT-116 and K562
tumor cell proliferation, the introduction of the methyl group does not produce an improved
antiproliferative activity relative to the parent compounds. Based on this, we may postulate
that, in these cases, effects of the electron-rich substituent X (attraction or repulsion) may
cause either a tighter interaction or a loosening of the contacts to the amino acid residues in
the binding pocket of the protein involved in mediating their antiproliferative action. These
impacts alter its function, thus resulting in the increased antiproliferative activity relative to
the parent compounds. The replacement of the bridging methylene group with the oxoethyl
group can result in compounds with a lower antiproliferative activity. However, some
exceptions were identified when analysing the inhibition of K562 cancer cell proliferation of
cycloalkanespiro-5-hydantoins.
<Figure 2>
<Figure 3>
<Figure 4>
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3.2. Crystal structures of compounds 1c and 2a
Compounds 1c and 2a crystallize in monoclinic systems, but in different space groups: P21/c
for 1c and P21/n for 2a. The asymmetric units of compounds together with atomic numbering
scheme are presented in Fig. 5. The hydantoin and cyclohexane (C5–C10) rings in 1c are
almost perpendicular, with dihedral angle of 89.5(1)°. The analogous angle for the
cycloheptane (C5–C11) ring in 2a is similar and amounts 89.1(1)°. Both cyclohexane and
cycloheptane rings adopt a distorted chair conformation and the maximal deviations from
their corresponding planes are: 0.256(2) Å for C9 atom in 1c and 0.382(2) for C7 atom in 2a.
The hydantoin ring in 1c is more planar than the same ring in 2a, where maximal deviations
are 0.0148(1) Å for N3 and 0.0181(7) Å for C4. The selected bond lengths, angles and
torsion angles for both compounds are listed in Table 2. The values are similar with
analogous bond lengths, angles and torsion angles found in related hydantoin derivatives [22,
36, 37].
<Figure 5>
The molecules of 1c are linked by strong intermolecular N–H···O hydrogen bonds
(Table 3) between N1 atom, which belongs to the hydantoin ring of one molecule and O2
atom from the hydantoin ring of an adjacent molecule, permitting the formation of infinite
pseudo-chains along [010] direction (Fig. 6). The formation of supramolecular chains is not
unusual for these compounds and the chains have been found in crystal packing of similar
hydantoin derivatives [22]. The neighboring chains are further stabilized by weak C–H···π
interactions (H···Cg distance is 2.860 Å) between H18 atom from the metoxy group and the
adjacent phenyl ring (Fig. 7) and together with numerous C–H···O contacts (Table 3)
construct a three-dimensional network.
By intermolecular N–H···O hydrogen bonds (Table 3 and Fig. 8), the molecules of 2a
are connected into centrosymmetric dimmers parallel to the b-axis. The formation of the
centrosymmetric dimmers are not rare for this type of compounds and they are found in
similar systems [22, 36, 37]. The crystal packing of 2a is stabilized by the C–H···O
interactions (Table 3), while additional weak C–H···π interactions (H···Cg distance is 2.940
Å) between H16 atom from one phenyl ring and the π-system of the adjacent phenyl ring
(Fig. 9) allow the formation of a three-dimensional structure.
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<Table 2>
<
Table 3>
<Figure 6>
<Figure 7>
<Figure 8>
<Figure 9>
Both introduction of different substituents X and change in the size of the cycloalkyl
group produce a variety in their crystal structures. Despite N–H···O hydrogen bonds
dominate, weak interactions promote either stabilization of the crystal structure or its
alteration. Regarding compounds 1d and 1e, we have previously reported that halogen
bonding (X···O) interactions form a supramolecular pseudo-hexagonal network [37]. On the
other side, compounds 2d and 2e build a different crystal packing based on the X···π
interactions. It is clear that tailoring the crystal structure of the investigated compounds
through the proposed structural changes should be taken into account when considering their
biopharmaceutical aspects, especially those related to their low aqueous solubility. Poorly
soluble compounds can provide a risk of low bioavailability with consequences for safety
and efficacy. However, a comprehensive knowledge of drugs at the molecular level is
required to determine the appropriate approach to improving solubility and dissolution rate.
3.3. Molecular geometry
By applying the DFT method at B3LYP/6-311++G(d,p) level of theory, the optimized
geometry parameters for all investigated cycloalkanespiro-5-hydantoines were determined.
The most significant geometric parameters (bond lengths, bond angles and torsion angles)
are listed in Tables S6 and S7 (Electronic Supplementary Information).
According to the DFT calculations, the cycloheptanespiro-5-hydantoine derivatives
exist in two conformational forms relative to each other as 81:19. The first form is presented
in Fig. 10, while in the other form the cycloheptane rings occupies a stable conformation of
the distorted chair (Fig. S1, Electronic Supplementary Information). The difference in energy
between these two conformations is 0.868 kcal mol-1.
<Figure 10>
As expected, the heterocyclic ring is affected by a π-conjugation of the amide type
16

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which is evident from the N–C bond lengths (N1–C2, N1–C5, N3–C2 and N3–C4, Table S6
and S7, Electronic Supplementary Information) [38]. The substituent effect is reflected on
small changes of length of bonds bridging the hydantoin and phenyl rings. An increase of the
electron accepting ability of the substituent X is associated with a shortening of the N–C11
bond in the compounds of series 1 and the N–C12 bond in the compounds of series 2,
contrary to the case of the electron donating substituents, where a slight lengthening is
observed. On the other side, the C11–C12 bond in the compounds of series 1 and the C12–
C13 bond in the compounds of series 2 lengthen along with an increase of the electron
accepting ability of the substituent X when compared to the parent compounds. The electron
donating substituents have an opposite effect. The planes defined by the hydantoin and
cycloalkyl rings are approximately perpendicular to each other. The situation is similar
regarding the relative orientation of the hydantoin and phenyl rings (Table 2).
When comparing the crystallographically determined structure and structure
calculated by the DFT methods, it can be seen that the main differences result from the
different bond lengths and bond angles in the cycloalkane ring (the difference ranges from
0.012 to 0.033 A). This can be attributed to so-called packing effects, which distort the
structures. Analogously, a notable difference in the length of the N1–C2 and N1–C5 bonds
can be ascribed to the involvement of the N1 atom in various intermolecular interactions,
primarily hydrogen bonding, which is not present in the isolated state, i.e. in the DFT
calculations. Furthermore, the differences found in the relative orientation of the hydantoin
and lipophilic cycloalkyl and phenyl groups in the crystal and as an isolated molecule
indicate that these molecules can easily change their conformation upon binding to a protein.
3.5. Experimental and theoretical study of electronic absorption spectra
The absorption spectra of the investigated compounds retain essentially the character of those
of 3-(4-substituted benzyl)-1,3-diazaspiro[4.4]nonane-2,4-diones [36]. Only the absorption
spectra of the compounds of series 2 in ethanol are given here (Fig. 11 and Table 4), because
both series of compounds (1 and 2) exhibit the same trends in λmax as a function of the nature
of the substituent X and no significant solvatochromic shift was observed. Their main feature
is an intense absorption band with a shoulder on the low-energy side in the region of 207–
266 nm and a broad, weak absorption band in the region of 274–281 nm.
<Figure 11>
17

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<Table 4>
When the donating or accepting ability of the substituent X increases, a red shift of
the absorption maxima is observed. It is evident that, because of the C,H-hyperconjugation of
the bridging CH2 group, the chromophoric system involves both hydantoin and aryl moieties.
To understand the shift of this band under the influence of the substituent X, the data from
Table 4 were plotted as a function of the Hammett substituent constant (Fig. 12).
<Figure 12>
There is a split in the Hammett plot at the parent compound, whereby the electron-
donating effect is somehow stronger than electron accepting effect (ρ = +12.4 vs. ρ = –10.8).
When the substituent X is a resonance electron-accepting substituent (NO2 and CN), the data
fall distinctly off the linear relationship, as expected.
The UV-Vis absorption spectra for representative compounds were calculated in
relation to their optimized geometry by applying the B3LYP method with 6-311++G(d,p)
basic set (Fig. 13, Table S8). Solvent effects were determined using the TD-DFT method.
In the compound 2c with the electron-donating methoxy group, a closer look into the
two major transitions (HOMO→LUMO+2 and HOMO→LUMO+4) based on the shape of
the molecular orbital in Fig. 13a suggests a typical π→π* character. When the substituent X
is the electron-accepting NO2 group (compound 2g), a higher amount of the orbital
distribution is observed on the aryl moiety in the LUMO orbital. Thus, charge may transfer in
the HOMO–2→LUMO and HOMO–1→LUMO transitions resulting in the observed
absorption bands (Fig. 13b).
<Figure 13>
The orbital distributions of their HOMOs and LUMOs are presented in Fig. 14. The
HOMO energy describes the region of a molecule, which can donate electrons during the
formation of complexes with proteins involved in mediating its biological effects. On the
other hand, the LUMO energy refers to its ability to accept the electrons from proteins [39].
In the case of the parent compounds (1a and 2a) or when X is an electron-donating
substituent, the HOMOs mainly involve contributions of π-orbitals of the aryl moiety, while
the LUMOs are spread across the entire molecule. The electron-accepting substituent X has
18

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the opposite effect: the HOMOs are distributed over the entire molecule, while the LUMOs
are shifted towards the aryl moiety. In the case of the compounds with the bridging oxoethyl
group, the HOMOs extend mainly over the hydantoin ring, while the LUMOs are mainly
composed of those π orbital from the aryl moiety. The HOMO–LUMO energy gaps are in
the range from 4.4 to 6.3 eV (Table 5). The larger HOMO–LUMO energy gaps of the
investigated compounds refer to their higher kinetic stability and lower chemical reactivity.
In comparison with the parent compounds, the electron-donating substituents X elevate both
HOMO and LUMO energy levels simultaneously. Oppositely, the HOMO and LUMO
energy levels are lower than that of the parent compounds, when X is a resonance electron-
accepting substituent (NO2 and CN). With enlarging the cycloalkane ring, the energy gap
remains almost unchanged.
<Figure 14>
<Table 5>
3.6 ’Rule of five’ properties
The Lipinski rule of five is widely used to estimate drug-like properties [40]. According to
this rule, compounds with good permeability have log P < 5, molecular weight < 500 g mol-
1, the number of hydrogen bond acceptors < 10 and the number of hydrogen bond donors <
5.
The analysis of molecular descriptors shows that the investigated compounds obey
rule of five (Table 5). When compared to the phenytoin derivatives [22], the physico-
chemical properties of the investigated compounds are moving towards somewhat lower
molecular weight (258–335), and coincidentally also somewhat lower lipophilicity (2.6–3.8).
As expected, lipophilicity increases with the size of the cycloalkyl group. With the exception
of the methoxy group, the introduction of the substituent X results in the increase of
lipophilicity relative to the parent compounds (1a and 2a). This will lead to their higher
partitioning into the lipophilic phase of a biological membrane or lipophilic domains of a
protein. Furthermore, a higher local concentration of the investigated compounds can be
expected near a binding site, but it does not necessarily indicate its higher biological activity.
On the other side, lipophilicity decreases when the bridging methyl group is replaced with
the ethyloxo group. The investigated compounds contain both proton acceptor and proton
donor groups, which indicates their high capacity for hydrogen bonding, especially toward
proton donor of the binding site. The number of rotatable bonds in the investigated
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compounds is almost constant and low, thus indicating that large changes of their
conformation upon binding to a binding site are not expected. An increase in polar surface
area (PSA) of the investigated compounds arises from both introduction of the substituent X
and replacement of the bridging methyl group with the ethyloxo group. Generally, relatively
high values of PSA may result in worsening of their absorption.
<Table 6>
4. Conclusion
In the present paper, we reported the synthesis and antiproliferative evaluation of two series
cycloalkanespiro-5-hydantoins with a 4-substituted benzyl or a 2-(4-substituted phenyl)-2-
oxoethyl group at N3 position. For comparison, we also described the 5,5-diphenylhydantoin
analogues. The investigated compounds exhibited mild to moderate effects against
proliferation of human colon (HCT-116), leukemia (K562) and breast (MDA-MB-231)
cancer cell lines at concentration of 100 ꢁmol dm-3, while they were non-toxic to normal
cells. Some interesting structure–activity findings were revealed. The substitution on the
benzyl moiety with the methoxy, chloro or bromo group potentiated the antiproliferative
activity relative to the parent compounds, while an increase in the size of the cycloalkyl
group resulted in a decrease in the antiproliferative activity in most cases. The replacement
of the bridging methylene group with the oxoethyl group can result in compounds with a
lower antiproliferative activity. The investigated compounds were compatible with the ’rule
of five’ indicating that they have satisfactory pharmacokinetic properties and bioavailability.
Regarding the crystallographic analysis, the investigated compounds adopted
different packing arrangements, either hydrogen bonded dimeric units or one-dimensional
pseudo-chains. Controlling the crystal structure of the investigated compounds through the
proposed structural changes should be taken into account when considering their
biopharmaceutical aspects, especially those related to their low aqueous solubility. The
theoretical calculations reproduced the experimental data well, thus describing the geometry
and electronic structures accurately and providing detailed spectroscopic results.
We find that the presented results shed light on the strategy for development of new
cycloalkanespiro-5-hydantoins
antiproliferative activities.
with
specific
physico-chemical
properties
and
20

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Acknowledgement
The authors are grateful to the Ministry of Education, Science and Technological
Development of the Republic of Serbia for financial support (Projects No. 172013, 172035,
173052 and III45007).
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Figure Captions
Fig. 1. Chemical structures of the investigated 1,3-diazaspiro[4.5]decane-2,4-diones (Series
1, labeled as 1a–1i), 1,3-diazaspiro[4.6]undecane-2,4-diones (Series 2, labeled as 2a–2i), and
5,5-diphenylhydantoins (Series 3, labeled as 3a–3i).
Fig. 2. The effect of he investigated compounds against proliferation of the HCT–116 cell
line at concentration 100 ꢁmol dm-3. Data for the compounds 3a–3e were taken from our
previous paper [22].
Fig. 3. The effect of the investigated compounds against proliferation of the K562 cell line at
concentration 100 ꢁmol dm-3.
Fig. 4. The effect of the investigated compounds against proliferation of the MDA–MB–231
cell line at concentration 100 ꢁmol dm-3. Data for the compounds 3a–3e are taken from our
previous paper [22].
Fig. 5. The asymmetric units of 1c and 2a. Displacement ellipsoids are drawn at 30%
probability level and hydrogen atoms are shown as small spheres of arbitrary radii.
Fig. 6. The polymeric pseudo-chain of 1c along the b-axis formed by hydrogen bonds
(dashed lines). The hydrogen atoms are omitted for clarity.
Fig. 7. C–H···π interactions (dot lines) between adjacent chains in 1c.
Fig. 8. The centrosymmetric dimer of 2a. Symmetry code, (i): –x+1, –y, –z.
Fig. 9. The crystal packing diagram of 2a in the almost ac-plane. Hydrogen bonds and C–
H···π interactions are presented with dash and dot lines, respectively.
Fig. 10. The most stable conformations of compounds 1a and 2a.
Fig. 11. The absorption spectra of the compounds 2a–2g at concentration ~1.0×10–5
mol dm-3 in ethanol.
Fig. 12. Relationships between νmax and σp for the investigated 3-(4-substituted benzyl)-1,3-
diazaspiro[4.6]undecane-2,4-diones (series 2).
Fig. 13. Experimental (dashed line) and simulated (full line) UV-Vis absorption spectra of
the compounds 2c (a) and 2g (b) in ethanol.
Fig. 14. The molecular orbitals and energy gaps between HOMO and LUMO of compounds
2a, 2c, 2g and 2i in the gas phase calculations.
Scheme 1. Synthesis of the investigated cyclohexanespiro-5-hydantoine derivatives (1a–1g)
and cycloheptanespiro-5-hydantoine derivatives (2a–2g).
Scheme 2. Synthesis of the investigated cycloalkanespiro-5-hydantoine derivatives (1h, 1i,
2h, 2i) and 5,5-diphenylhydantoine derivatives (3h, 3i).
26

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Table 1. Crystal data and structure refinements for 1c and 2a.
Compound
Formula
1c
C16H20N2O3
288.34
0.42×0.34×0.05
Monoclinic
P21/c
2a
C16H20N2O2
272.34
0.87×0.31×0.10
Monoclinic
P21/n
Formula weight (g mol-1)
Crystal size (mm3)
Crystal system
Space group
a (Å)
13.681(3)
11.474(2)
10.089(2)
110.76(3)
1480.9(5)
4
12.783(3)
6.2750(13)
18.635(4)
105.94(3)
1437.3(5)
4
b (Å)
c (Å)
β (°)
V (Å3)
Z
F(000)
616
0.090
1.293
584
0.084
1.259
ꢀ (mm-1)
ρc (g cm-3)
θ range (º)
3.18–25.35
–14→16
–7→13
3.32–26.02
–15→15
–7→7
–23→23
0.0603ii
1.067
Index ranges,
h, k, l
–11→12
R indices (all data)
Goodness-of-fit
Rint
0.0723i
1.068
0.0221
0.133, –0.193
0.0227
0.292, –0.321
ꢀρmax, ꢀρmin (e Å-3)
i w = 1 / [s2(Fo 2) + (0.0392P)2 + 0.1715P] where P = (Fo 2+ 2Fc2)/3;
2
2
ii w = 1 / [s2(Fo ) + (0.1036P)2 + 0.3109P] where P = (Fo + 2Fc2)/3.

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Table 2. The experimental and calculated values of selected bond lengths, bond angles and
torsion angles for compounds 1c and 2a.
1c
(calc.)
2a A
2a B
Parameter
1c (exp.)
Parameter
2a (exp.)
(calc.)i
(calc.)i
Bond lengths (Å)
N1−C2
N1−C5
Bond length (Å)
N1−C2
N1−C5
1.367
1.467
1.212
1.413
1.376
1.212
1.543
1.545
1.548
1.538
1.538
1.543
1.540
1.538
1.466
1.516
1.399
1.398
1.393
1.395
1.394
1.394
1.342(2)
1.460(2)
1.215(2)
1.405(2)
1.358(2)
1.220(2)
1.516(2)
1.531(2)
1.525(3)
1.519(3)
1.517(3)
1.515(2)
1.518(3)
1.464(2)
1.502(2)
1.376(3)
1.376(3)
1.375(2)
1.378(3)
1.373(3)
1.388(3)
1.367
1.466
1.212
1.413
1.375
1.212
1.539
1.543
1.546
1.534
1.535
1.535
1.535
1.468
1.513
1.394
1.402
1.396
1.397
1.400
1.387
1.337(2)
1.455(2)
1.224(2)
1.391(2)
1.369(2)
1.206(2)
1.526(2)
1.539(3)
1.527(3)
1.506(3)
1.508(5)
1.482(4)
1.510(3)
1.520(3)
1.447(2)
1.509(3)
1.377(3)
1.379(3)
1.379(3)
1.367(3)
1.372(3)
1.389(3)
1.366
1.468
1.212
1.413
1.376
1.212
1.543
1.545
1.553
1.539
1.538
1.534
1.538
1.538
1.466
1.516
1.399
1.398
1.393
1.395
1.394
1.394
C2−O1
C2−O1
C2−N3
C2−N3
N3−C4
N3−C4
C4−O2
C4−C5
C5−C6
C5−C10
C6−C7
C4−O2
C4−C5
C5−C6
C5−C11
C6−C7
C7−C8
C7−C8
C8−C9
C8−C9
C9−C10
N3−C11
C11−C12
C12−C13
C12−C17
C13−C14
C14−C15
C15−C16
C16−C17
C9−C10
C10−C11
N3−C12
C12−C13
C13−C14
C13−C18
C14−C15
C15−C16
C16−C17
C17−C18
Bond angles (°)
N1−C5−C4
N1−C5−C11
C4−C5−C11
N1−C5−C6
C4−C5−C6
C6−C5−C11
Torsion angles (°)
C4−N3−C12−C13
N3−C12−C13−C14
N1−C5−C6−C7
Bond angles (°)
N1−C5−C4
100.4
111.4
108.7
112.2
108.0
114.9
100.6(1)
112.5(1)
111.7(2)
112.1(1)
109.1(1)
110.4(1)
100.7
112.5
110.0
112.4
110.3
110.6
100.6(1)
111.2(2)
109.0(1)
111.7(2)
108.0(2)
115.4(2)
100.3
109.7
109.5
112.3
108.8
115.1
N1−C5−C10
C4−C5−C10
N1−C5−C6
C4−C5−C6
C6−C5−C10
Torsion angles (°)
C4−N3−C11−C12
N3−C11−C12−C13
N1−C5−C6−C7
C4−C5−C6−C7
N3−C4−C5-C10
C2−N3−C11−C12
−86.0
−84.9
76.0
81.3(2)
–99.3(2)
71.7(2)
91.8
–88.2
72.8
–84.7(2)
–11.3(2)
65.9(2)
−88.0
−85.2
165.4
–85.4
122.8
90.6
−174.2
120.2
92.2
–177.6(2)
120.7(2)
−102.3(2)
–175.8 C4−C5−C6−C7
175.6(2)
119.1(2)
101.0(2)
121.7
–88.1
N3−C4−C5−C11
C2−N3−C12−C13
iRegarding the optimized conformations, the DFT calculations reveal two energetically close forms of 2a (Fig. S1,
Electronic Supplementary Information).