Synthesis and spectroscopic characterization of cyclobutyl hydantoins

Article abstract of DOI:10.1016/j.molstruc.2018.06.007

The hydantoin moiety has proved to be an important pharmacophore that confers a wide range of biological properties to different derivatives. Thus, synthetic methods have been developed to obtain such molecules. Herein, we describe the heterocyclization process to obtain imidazolidine-2,4-diones (hydantoin compounds) from methylcyclobutyl ketones and cyclobutanones derived from (?)-(1S)-α-pinene and (?)-(1S)-verbenone through the Bucherer-Berg reaction. The methylcyclobutyl hydantoins and the spirohydantoin obtained were fully characterized, determining their absolute stereochemistry by nuclear magnetic resonance experiments and theoretical calculations.

Full text of DOI:10.1016/j.molstruc.2018.06.007

Accepted Manuscript
Synthesis and spectroscopic characterization of cyclobutyl hydantoins
Juan Caturelli, M. Florencia Martini, Lucas Fabian, Graciela Y. Moltrasio, Albertina
G. Moglioni
PII:
S0022-2860(18)30708-7
10.1016/j.molstruc.2018.06.007
MOLSTR 25294
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
13 March 2018
01 June 2018
Please cite this article as: Juan Caturelli, M. Florencia Martini, Lucas Fabian, Graciela Y. Moltrasio,
Albertina G. Moglioni, Synthesis and spectroscopic characterization of cyclobutyl hydantoins,
Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.06.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis and spectroscopic characterization of cyclobutyl hydantoins
Juan Caturellia, M. Florencia Martinia,b, Lucas Fabiana,b, Graciela Y. Moltrasioc and
Albertina G. Moglionia,b,*
a Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Cátedra de Química Medicinal, Junín 956 PP (1113), Buenos
Aires, Argentina.
b CONICET-Universidad de Buenos Aires. Instituto de la Química y Metabolismo del Fármaco (IQUIMEFA), Junín 956 SS (1113)
Buenos Aires, Argentina.
c Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Orgánica, Junín 956 3er floor, Buenos
Aires, Argentina.
* Corresponding author: Departamento de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Instituto de
la Química y Metabolismo del Fármaco (IQUIMEFA), Junín 956, (1113) Buenos Aires, Argentina.
E-mail addresses: amoglio@ffyb.uba.ar
Abstract
The hydantoin moiety has proved to be an important pharmacophore that confers a wide
range of biological properties to different derivatives. Thus, synthetic methods have been
developed to obtain such molecules. Herein, we describe the heterocyclization process to
obtain imidazolidine-2,4-diones (hydantoin compounds) from methylcyclobutyl ketones and
cyclobutanones derived from (-)-(1S)--pinene and (-)-(1S)-verbenone through the
Bucherer-Berg reaction. The methylcyclobutyl hydantoins and the spirohydantoin obtained
were fully characterized, determining their absolute stereochemistry by nuclear magnetic
resonance experiments and theoretical calculations.
Keywords: methylcyclobutyl ketones, cyclobutanones, Bucherer-Berg reaction, hydantoins,
nuclear magnetic resonance
1

ACCEPTED MANUSCRIPT
Introduction
Hydantoins and their derivatives constitute a group of privileged structures [1] displaying a wide
range of biological activities [2]. These properties have encouraged their use in fragment-based
drug design processes [3]. These procedures have led to the discovery of novel entities with
therapeutic utility (Figure 1) [4].
Figure 1. Some therapeutically useful hydantoins
The hydantoin core is present in some natural products, for example (+)-hydantocidin [2], which is
a spironucleoside structure with herbicidal activity. This compound can be isolated from
Streptomyces hygroscopicus, growing in Japan. The hydantoin moiety is also present in many
alkaloids present in sponges and corals, such as aplysinopsins [2], which are known to have
cytotoxic properties (Figure 2).
Figure 2. Natural products bearing the hydantoin moiety
One of the methods of choice for the synthesis of 5,5-disubstituted hydantoins is a multicomponent
reaction proposed by Bucherer and Berg (BB) in 1934 [5]. This reaction involves the
heterocyclization of either an aldehyde or a ketone with ammonium carbonate and potassium
cyanide under strong basic conditions (Scheme 1). Other methods of synthesis, as well as the
reactivity of this group of heterocyclic compounds, have been reviewed by Meusel and Gutschow
in 2004 and by Colacino et al, in 2017 [6].The behavior of these compounds in solid phase has
been recently described by Nogueira et al (2017) [7].
2

ACCEPTED MANUSCRIPT
Scheme 1. General procedure to obtain hydantoins from carbonyl compounds
In this work we describe the synthesis and characterization of two hydantoine classes developed in
our laboratory, namely, class I hydantoins, obtained from cyclobutanones, and class II hydantoins
obtained from methylcyclobutyl ketones. The synthesis of these compounds was achieved through
the BB reaction on homochiral ketones derived from terpenes(Figure 3).
O
O
NH
*
O
HN
HN
NH
O
*
O
O
MeO
n
MeO
n
type II
type I
Figure 3. General structure for the hydantoins synthesized, with n = 0 or 1.
Results and Discussion
The cyclobutyl precursors were prepared through the cleavage of commercially available bicyclic
enantiomerically enriched (-)-(1S)--pinene and (-)-(1S)-verbenone, according the pathway
showed in the Scheme 2.
Scheme 2. Synthesis of ketones 1-4 and 10. Reagents and conditions: i) NaIO4, RuCl3,
H2O/CH3CN/CCl4, rt; ii) CH2N2, Et2O, 50 ºC; iii) m-CPBA, CH2Cl2, rt; iv) K2CO3, H2O/MeOH/THF, rt; v)
DMP, CH2Cl2, rt; vi) (CH2OH)2, PPTS, PhH, reflux; vii) LiBH4, THF, rt; viii) PPTS, (CH3)2CO, 56 ºC.
3

ACCEPTED MANUSCRIPT
The synthetic pathways outlined in Scheme 2 allowed obtaining methylcyclobutyl ketones 1 [8] and
3 [8] and cyclobutanones 2 [9] and 4 [9] through the ruthenium-catalysed oxidative cleavage of the
double bond present in (-)-(1S)--pinene and (-)-(1S)-verbenone.
Ketones 1-4 were then treated with ammonium carbonate and potassium cyanide, under BB
conditions, to form hydantoins 5-8 (Scheme 3).
O
O
NH
*
O
O
HN
HN
O
NH
i
i
*
O
R
R
R
R
7, 8, 11
1, 3, 10
2, 4
5, 6
1, 2, 5, 7 R = CH2CO2CH3
3, 4, 6, 8 R = CO2CH3
10, 11 R = CH2OH
Scheme 3. Synthesis of hydantoins 5-8 and 11.Reagents
and conditions: i) (NH4)2CO3, KCN, NH4Cl, H2O/EtOH, 60ºC.
Under BB conditions, to obtain class I hydantoins, cyclobutanones 2 and 4 were extremely
reactive, obtaining only one hydantoin compound in the case of cyclobutanone 2. The expected
hydantoine 6 could not be obtained. Considering that the starting material has one stereogenic
center, with a second one being generated during the reaction; only two diastereoisomeric
products were possible. Only compound 5 was identified as one hydantoinic product, which could
be isolated by preparative thin layer chromatography from the complex reaction mixture containing
ketone 2. The NMR (Cl3CD) spectra analysis of compound 5 is shown in Table 1.
The NOESY and nOe experiments carried out with compound 5 in DMSO-d6, a solvent that, in this
case, allowed a greater separation of the signals corresponding to the methyl groups,
demonstrated that the hydrogen atom signals corresponding to the methyl group appearing at a
higher field (δ 0.96 ppm) presented a nOe effect with the methine hydrogen atom at δ 2.48 ppm.
On the other hand, the hydrogen atoms corresponding to the side chain methylene group
presented a nOe effect with the methyl group at δ 0.99 ppm, and with the endocyclic methylene
hydrogen atom (3'-Ha). These phenomena allowed concluding that these three atomic groups are
on the same face of the ring (Figure 4). Although different NMR experiments allowed assigning
each hydrogen and carbon atoms of compound 5, the configuration of the spiro-carbon could not
be determined.
4

ACCEPTED MANUSCRIPT
Table 1. Chemical shifts (ppm) and hetero- and homonuclear correlations for compound 5 determined
inCl3CD.
H
O
N
MeO
3
2
O
10'
8'
6'
NH
3'
1
2'
O
4'
1'
9'
δ 13
C
δ 1H
Position
δ 1H
(HSQC)
172.4
34.7
(HMBC)
1
2
-
3.69 and 2.35
Ha/b 2.35 (dd, J= 7.8, 15.5 Hz, 1H)
Ha/b 2.42 (dd, J= 7.6, 15.4 Hz, 1H)
3
3.69 (s, 3H)
51.7
46.9
34.3
35.0
1'
2'
3'
-
2.35; 2.65
2.80 (ddd, J= 7.8, 8.0, 8.0 Hz, 1H)
2.35; 2.42; 1.98 and 1.15
Ha/b2.65 (dd, J= 8.5, 11.4 Hz, 1H)
Ha/b 1.98 (dd, J= 11.2, 11.2 Hz, 1H)
4'
5'
-
66.0
-
1.15
5.76 (br, 1H)
-
6'
155.4
-
7'
7.51 (br, 1H)
-
8'
175.2
22.6
19.0
9'
1.16 (s, 3H)
1.15 (s, 3H)
10'
2.30
2.37
1.88
H
H
H
O
H
MeO
N
0.99
NH
O
H
2.48
0.96
O
Figure 4. nOe effect observed for compound 5
Chemical shifts in 1HNMR spectra were determined in DMSO-d6.
Given that only one diastereomer of compound 5 was obtained, both possible stereoisomers were
considered for structural assignment. This was carried out, taking into account that the closure of
the hydantoin ring can generate the compound 5a, with the nitrogen atom located in relation syn to
the chain in position 2’ of the original ring, and its diastereomer 5b of anti ratio (Figure 5).
5

ACCEPTED MANUSCRIPT
O
H
MeO
O

MeO

2
O
N
1

2'
4'(R)
NH
(S)
(R)
NH
(R)
O
N
H
O
O
5b
5a
Figure 5. Possible stereoisomers of compound 5
In order to determine the absolute configuration of the new chiral center, computational chemistry
methods, such as DP4 and ANN were employed to distinguish both diastereoisomers, allowing the
assignment of one of them to the compound obtained. Possible structures were subjected to
conformational searching using the “conformational search” module of the Hyperchem 8.0.92
software [10]. These conformational searches were performed starting with the structures
previously optimized with the MM+ force field corresponding to each diastereoisomer. For the α
and β angles (Figure 5), the rotation was studied between 0° and ± 180°, rotating every 10°, and
allowing the simultaneous folding of the cyclobutanic ring.
As described by Pawar et al., the γ angle was fixed at 180° (conformation Z) [11]. The
convergence criterion in each case was defined at 1000 cycles or a gradient less than 0.01
kcal/mol, leaving the other parameters unchanged. At least 10 conformers for each
diastereoisomer, whose energy values were  5kcal/mol in relation to the lowest energy conformer,
were selected. These 20 conformers were then re-optimized using the density functional theory
(DFT) module///tool of the Gaussian 03 software [12], using the Becke, 3-parameter, Lee Yang-
Parr (B3LYP) functional combined with the 6-31+g(d,p) base. Those conformers displaying energy
values within 2kcal/mol in relation to the lowest energy conformer were selected (Table 2).
Table 2. Relative energy values obtained by the quantum mechanical method for conformers, calculated
with DFT B3LYP/6-31+g(d,p).
5a (2'R, 4'R)
5b (2'R, 4'S)
relative energy (kcal/mol)
0.171445
Conformer relative energy (kcal/mol)
1
2
0.863699
0.799112
0.000000
0.000069
2.357174
2.356911
0.375184
0.375184
0.960933
0.859761
0.173021
3
8.017752
4
8.150602
5
8.021953
6
0.174333
7
0.171971
8
0.172496
9
0.000000
10
0.000263
6

ACCEPTED MANUSCRIPT
Finally, the Gauge Independent Atomic Orbitals (GIAO) method [13] was applied for each selected
conformer, using the mPW1PW91 functional to obtain the values of the shielding constant (σ) for
each atom. This set of values was subjected to weighting through the Boltzmann analysis on all
selected conformers. The DP4 method (http://www-jmg.ch.cam.ac.uk/tools/nmr/DP4/), developed
by Smith and Goodman (2010) [14] was applied to these data. Taking together the chemical shifts
1
of H and 13C atoms (data not shown), the highest probability value obtained was 99.7% for the
stereoisomer 5a, with the absolute configuration 2'R,4'R. This configuration was assigned to
compound 5.
1
These values were also loaded, together with the experimental chemical shifts of the 13C and H
atoms, in the spread sheet containing the neural network provided by Zanardi et al. [14b]. The
correlation analysis performed between the experimental and the calculated data for the2'R,4'R
isomer (5a) and isomer 2'R,4'S (5b) indicated that the structure 5a was the correct one in the ANN-
TMSvac, in accordance with the results obtained applying the methodology previously described
by Goodman et al. [14a]. Therefore, we decided to apply DP4 and CP3, for the rest of the
compounds described in this work.
The absolute configuration of the newly formed stereogenic center in compound 5 can be
explained on the basis of the mechanistic considerations raised by other authors [5]. In the
intermediate A, the hydrogen bond formation between the amino group and the carbonyl group of
the ester in the 1,3-cis relative position, would further favor this reaction pathway (Scheme 4). The
presence of the methyl ester chain is a determining factor in the course of the reaction, as we have
previously observed in other heterocyclization reactions[15]. Besides, such methyl ester group is
responsible for the observed diastereoselectivity.
NH
O
MeO
O
N
O
CN
NH2
NH2
C
O
HN
O
CN
NH
C N
MeO
O
MeO
O
MeO
O
MeO
O
A
B
O
O
O
H
HN
O
O
N
HN
HN
O
NH
O
O
NH
NH
O
O
O
O
NH
MeO
MeO
MeO
MeO
5a
5b
Scheme 4. Possible reaction pathway to obtain spirohydantoins 5a and 5b
7

ACCEPTED MANUSCRIPT
From ketones 1 and 3, a complex mixture of products was obtained. Taking into account that a
new chiral center was generated during the reaction, and the potential isomerization from a cis to a
trans configuration in the starting materials [8], we decided to determine whether the stereogenic
centers had been modified during the heterocyclization reaction. To that end, a complete
determination of the conformation of all diastereoisomers was determined by mono- and bi-
dimensional NMR methodologies (HSQC, HMBC and NOESY).
The analysis of the complex reaction mixture resulting from ketone 1 shows a main product
together with other compounds, including saponification derivatives of the methyl ester group.
Mono- and bi-dimensional NMR spectra allowed determining that the main product, isolated from
the reaction mixture, corresponded to hydantoin 7. The complete assignments of compound 7 are
presented in Table 3. The nOe-effects observed in the NOESY spectrum of this compound (see
figure in Table 3) allowed concluding that the substituents at the 1,3-positions in the cyclobutane
ring retained the cis relative configuration. However, the configuration assignment of the new
stereocenter (C5) created in the hydantoin ring could not be determined. In order to assign such
configuration, the conformational minima for each of the possible diastereoisomers of compound 7
(7a and 7b) were calculated. For this purpose, a geometry optimization was performed to obtain
the σ values for each H and C atom. The chemical shifts of H atoms for 7a and 7b are shown in
Table 3.
8

ACCEPTED MANUSCRIPT
Table 3. Chemical shifts (ppm) assigned from hetero- and homonuclear correlations spectra for compound
7a and 7b (COSY and HSQC spectra) and calculated chemical shifts for H of compound 7
O
4
O
2
O
2
NH
NH
HN
1'
HN
2
MeO
7'
4
4
5'
HN 5
9'
4'
O
MeO
7'
NH
6
O
O
4'
5
5
5'
9'
4'
3'
6'
1'
6
O
3'
H
H
5'
2'
1'
MeO
7'
6
O
3'
6'
8'
2'
9'
8'
2'
1'R,3'R,5R
H
1'R,3'R,5S
O
6'
8'
7a
7
7b
7a
δ1Hcalc.
7b
δ1Hcalc.
Position
δ1Hexp.
δ13Cexp.
6'
5'
-
173.1
34.7
2.27 (m, 1H)
2.18
2.29
2.11
2.47
2.34 (ddd, J= 7.7, 7.8,
8.2 Hz, 1H)
7'
1'
2'
3'
3.67 (s, 3H)
3.71
1.89
3.62
2.30
51.5
38.4
42.7
48.3
2.27 (m, 1H)
-
2.32 (m, 1H)
2.25
2.13
2.13
1.22
1.63 (dd, J= 10.8, 10.9
4'
24.3
Hz, 1H)
1.92 (m, 1H)
2.29
2.32
8'
9'
1
1.22 (s, 3H)
1.15
1.20
30.9
17.1
-
1.08 (s, 3H)
0.94
0.90
8.68
-
-
2
-
-
-
157.0
-
3
6.18
-
-
4
-
-
-
-
-
176.9
64.7
22.4
5
-
6
1.44 (s, 3H)
1.25
1.12
The energy difference between both diastereoisomers of 7 is small (ΔE=1.99 kcal/mol), being the
stereoisomer of configuration 1'R,3’R,5S (7a) slightly more stable. Taking into account that only
one of the two posible diastereoisomers of compound 7was characterized, and that such structure
could correspond to one of the two stereoisomers calculated, the DP4 method applied. The same
procedure was followed for compound 5. In this case, the highest probability value (93.9%),
1
obtained with data calculated for H atoms, was assigned to stereoisomer 7b, with the absolute
configuration 1'R,3'R,5R. Then, the structure 7b was assigned to the isolated compound 7.The
DP4 method applied to 1H and 13C chemical shifts (data not shown) supported this assignment.
9

ACCEPTED MANUSCRIPT
From ketone 3, a mixture of diastereoisomers could be isolated by preparative TLC. The bi-
dimensional HMBC and HSQC NMR spectra of compounds 8, allowed determining the position of
atoms in 8a and 8b. Table 4 shows the complete assignment of each stereoisomer. The nOe
effects observed for compounds 8a and 8b (Figure 6) indicated that the cis relative configuration
present in the starting material was maintained.
O
2.25
H
H
2.26
N
O
O
H
O
O
NH
O
1.11
HN
H
1.02
MeO
2.72
NH
2.41
MeO
2.75
H
H
H
2.44
1.35
1.25
8b
8a
Figure 6. nOe effect observed for compounds 8a and 8b
In order to define the configuration of the new chiral center generated in each one of the
diastereoisomers of compound 8, the conformational minima were calculated as performed for
compound 5. The calculated energy difference between both diastereoisomers is small
(ΔE=1.80kcal/mol) with a slight difference in favor of the 1'R,3'S,5S configuration.
Considering that the two possible diastereoisomers of 8 were characterized and that each structure
may correspond to one of the two stereoisomers calculated, the CP3 method (http://www-
jmg.ch.cam.ac.uk/tools/nmr/CP3.html) was applied as described by Smith and Goodman [14].By
the CP3 method a 100% probability was obtained between the 8a isomer values and the
calculated values for the 1'R,3'S,5R absolute configuration and between the 8b isomer values and
the calculated values for the 1'R,3'S,5S absolute configuration. No changes in this probability value
1
were obtained when the CP3 method was applied to the combination of H and13C chemical shift
values (data not shown).
10

ACCEPTED MANUSCRIPT
Table 4.Chemical shifts (ppm) and hetero- and homonuclear correlations for compounds 8a and 8b and
calculated chemical shifts (ppm) for 1H of compounds 8
O
2
NH
O
HN
HN
4
NH
O
O
5
O
5
6'
6'
3'
5'
1'R,3'S,5R
O
MeO
8'
MeO
8'
7'
6
6
4'
3'
1'
1'
2'
5'
1'R,3'S,5S
δ 1Hcalc.
δ 1Hcalc.
8a
8b
δ 13Cexp.
δ 13Cexp.
δ 1H exp.
δ 1H exp.
Position
for 1'R,3'S,5S
for 1'R,3'S,5R
(HSQC)
45.5
(HSQC)
46.1
1'
2'
3'
2.72 (m, 1H)a
-
2.75 (m, 1H)a
-
2.58
-
2.54
-
44.7
44.3
2.41 (m, 1H)a
Ha/b1.85 (ddd, J= 8.3,
8.7, 11.1 Hz, 1H)
Ha/b2.26 (m, 1H)a
1.35(s, 3H)
1.11 (s, 3H)
-
47.5
2.44 (m, 1H)a
46.1
2.36
2.48
1.64
Ha/b2.15 (ca, 1H)
4'
19.7
20.5
2.24
Ha/b2.25 (m, 1H)a
1.25(s, 3H)
1.02 (s, 3H)
-
2.07
1.26
0.91
-
2.15
1.31
1.09
-
5'
6'
7’
8'
1
31.3
17.8
172.4
51.6
-
31.3
17.3
173.1
51.4
-
3.68 (s, 3H)
6.86
3.69 (s, 3H)
6.28
3.75
-
3.70
-
156.3/15
6.0
-
2
-
-
156.3/156.0
-
3
4
5
6
8.68/8.80
-
8.68/8.80
-
-
-
-
176.8
64.9
22.4
-
176.4
64.3
24.3
-
-
-
-
-
-
1.38(s, 3H)
1.25
1.45(s, 3H)
1.17
aThese values were observed overlapped on the spectrum. An approximate assignment of signals was done according to the best
adjustment with respect to the calculated values. ca: complex absorption.
Taking into account that the hydantoinic compounds obtained could be useful intermediates to
prepare nucleoside analogs, the direct reduction of the ester group present in compounds 7 and 8,
to the hydroxymethyl group was performed. Even though lithium aluminum hydride is known to
render a complex mixture of reduced heterocycles, even generating the diamine [16], this
compound was employed, since under the mild experimental conditions employed in this work, it
proved to render compound 9 [8]. To date, the reduction of hydantoinic compounds using this
reagent has not been reported.
The complex mixture obtained lacked the 3'-carbinol hydantoins, thus it was concluded that this
reducing agent also affects the heterocycle, rendering a wide range of products that are in different
11

ACCEPTED MANUSCRIPT
states of reduction. Therefore, compound 10 [15] was synthetized through a three-step process
obtaining 47% global yield. The application of BB´s conditions reaction on compound 10 rendered
two hydantoic products (11a and 11b). This behavior was similar to that obtained when the BB’s
reaction is applied to its parent compound 6. The isolation of 11a and 11b was not possible by any
chromatographic method, including HPLC. Therefore, a complete assignment of this mixture was
attempted through mono- and bidimentional NMR studies. NOESY experiments confirmed that the
1,3-cis relative configuration remained unaffected, confirming that the products were a
diastereoisomeric 1:1 mixture of hydantoins (Figure 7).
The HMBC and HSQC spectra, allowed determining the position of atoms in each of these
molecules. Table 5 shows the complete assignment for each diastereoisomer of 11.
O
H
N
O
H
O
H
NH
O
HN
H
HO
NH
HO
H
H
H
11a
11b
Figure 7. nOe effect observed for compounds 11a and 11b
In order to determine the configuration of both diastereoisomers of 11 (a and b), the conformational
minima were calculated, as previously described for compounds 5 and 6. Table 5 also shows the δ
values calculated by GIAO for 1H and 13C atoms of 11a and 11b. The calculated energy difference
between both diastereoisomers was notsignificant (ΔE=0.06 kcal/mol), with a slight difference in
favor of the 1'R,3'S,5S configuration. This result is in line with those obtained experimentally.
Given that the two possible diastereoisomers of 11 were characterized, and knowing that each
structure could correspond to one of the calculated stereoisomers, the CP3 method was applied.
1
However, the calculation of probabilities employing the H and 13C chemical shifts independently
led to contradictory results. An approximate assignment was attained by the combination of both
values, obtaining a probability value of 98.8% between the 11a isomer values and the calculated
values for the 1'R,3'S,5S absolute configuration, and between the 11b isomer values and the
calculated values for the 1'R,3'S,5R absolute configuration.
12

ACCEPTED MANUSCRIPT
Table 5. Experimental and calculated chemical shifts (ppm), and hetero- and homonuclear correlations for
compounds 11a and 11b
O
O
2
NH
2
HN
4
4'
4
NH
O
HN
4'
5
5
5'
5'
7'
O
7'
HO
8'
HO
8'
6
6
3'
6'
1'
3'
6'
1'R,3'S,5S
1'
2'
1'R,3'S,5R
11a
11b
1’R,3’S,5R
1’R,3’S,5S
Position
δ 1H
δ 13
C
δ 1H
δ 13
C
δ 13C calc. δ 1Hcalc. δ 13Ccalc.
δ 1Hcalc.
(HSQC)
-
(HSQC)
-
1
2
*
-
*
-
-
-
-
156.9/15
7.3
156.9/1
57.3
-
151.7
-
150.4
3
4
*
-
-
-
*
-
-
-
-
-
-
178.7
63.3
22.8
48.5
41.7
43.7
21.5
178.9
63.4
24.2
46.2
41.0
44.6
22.5
173.2
69.9
27.4
50.0
48.3
49.2
28.8
174.6
68.7
21.8
55.6
47.5
47.7
30.3
5
-
-
6
1.18 (s, 3H)
2.02 (m, 1H)a
-
1.10 (s, 3H)
2.04 (m, 1H)a
-
1.57
2.39
-
1.57
2.33
-
1’
2’
3'
4’
1.84 (m, 1H)a
Ha/b1.48 (m, 1H)a
Ha/b1.57 (m, 1H)
Ha/b3.26 (m, 1H)
Ha/b3.33 (m, 1H)
1.11 (s, 3H)
1.86 (m, 1H)a
Ha/b1.50 (m, 1H)a
Ha/b1.83 (m, 1H)a
Ha/b3.34 (m, 1H)
Ha/b3.50 (m, 1H)
0.98 (s, 3H)
2.69
2.03
2.27
2.81
5'
6'
7’
8’
61.7
61.9
68.3
3.84
4.00
1.43
1.33
67.6
3.90
4.10
1.49
1.24
32.6
16.7
31.9
15.6
33.6
20.0
34.5
19.6
0.97 (s, 3H)
0.93 (s, 3H)
aValues were observed overlapped on the spectrum. An approximate assignment was done, according to the best adjustment with
respect to the calculated values.*Since the spectrum was performed in D2O,the assignment was not possible.
Since compounds 11 are nucleoside analogs, and their isolation was not possible, a preliminary
screening of their biological activity was undertaken. The antiproliferative activity of these
compounds was evaluated to guide future studies evaluating either the antiviral or the anticancer
activity. The MTS reduction assay was performed to evaluate the inhibitory activity of 11 against
U937human promyelocytic leukemia cells to find that the mixture of 11 did not display any
antiproliferative activity between 4.40 and 100 M.
13

ACCEPTED MANUSCRIPT
Conclusions
• The reactivity of enantiopure methylcyclobutyl ketones and cyclobutanones was studied through
the BB reaction. Complex mixtures of products were obtained in all cases, including decomposition
ones.
• Product 5 (class I hydantoin) was obtained as one of the two possible diastereoisomers. Its
configuration could be explained on the basis of the influence of the ester group on the chiral
center present on the starting material 2.
• An exhaustive structural analysis of the new compounds 7, 8 and 11(class II hydantoins),
employing mono- and bi-dimensional NMR spectroscopy methods, allowed confirming the
retention of the 1,3-cis relative stereochemistry of the cyclobutanic precursors.
• The use of mono- and bi-dimentional NMR methods, together with theoretical calculations, was
useful to assign the absolute configuration of compounds 5, 7, 8 and 11.
Experimental Section
General
Reagents were purchased from Sigma-Aldrich, and used without further purification. Solvents were
distilled before use by standard purification methods [17,18]. (-)-(1S)-α-pinene (98 %) and (-)-
(1S)-verbenone (94%), were purchased from Acros Organics and employed without purification.
Thin layer chromatography (TLC) and preparative TLC were performed on Silica Gel F254 (Merck).
The visualization reagents employed were 1.6% KMnO4 in H2SO4 conc.; 2.2% CH3OC6H4CHO in
EtOH or iodine. Column chromatography was performed on Silica Gel 60 240-400 mesh (Merck)
with the solvent mixture indicated in each case. Melting points were measured in a Thomas-
1
Hoover equipment. H and13C NMR spectra, homo- and heteronuclear correlation spectroscopy
and NOESY experiments were recorded in a Bruker 300 MHz, Bruker 500 MHz or Bruker
UltraShield 600 MHz spectrometers. Unless otherwise specified, all experiments were performed
at ambient temperature in the following solvents: CDCl3, DMSO-d6 or D2O as stated in each case.
Chemical shifts are expressed in ppm (relative to the solvent). Coupling constants (J) are in Hz.
FT-IR spectra were recorded as a film from acetone with a Nicolet 380 Thermo spectrometer. High
resolution mass spectra (HRMS) were determined in a Bruker micrOTOF-Q II™ spectrometer.
Conformational searches were performed using MM+ force field with the Hyperchem program [10].
All conformers within 5 kcal/mol from the minimal energy one, were re-optimized using B3LYP/6-
31+g (d,p) level with Gaussian G03 program package [12]. Conformers, within 2kcal/mol from the
global minimum, were used in NMR calculations. A single point using mPW1PW91/6-31+g(d,p)
functional and the GIAO method [13] were employed to calculate the shielding constants ().The
tetramethylsilane (TMS) molecule has also optimized to determine the chemical shifts as a function
14

ACCEPTED MANUSCRIPT
of this reference compound. These values, showing a Boltzmann distribution, were analyzed
through different methods, as appropriate: CP3, DP4 or ANN-TMSvac [14].
Procedures and analytical data
Synthesis of hydantoins. General procedure.
To a solution of ketone (1 equiv) in a 1:1 EtOH/H2O mixture (26 mL), (NH4)2CO3 (1.83 g, 22.07
mmol, 10 equiv) and NH4Cl (410 mg, 7.67 mmol, 3.6 equiv) were added followed by, KCN (560
mg, 8.63 mmol, 4 equiv) after 15 min. The solution was stirred at 60 °C for 72 h. The solvent was
reduced in vacuo, and the solid obtained was filtered and washed with cold water. The product was
purified by preparative TLC, eluting with either PhCH3/EtOH 6:1 or hexane/EtOAc/MeOH 7:2:1 as
stated in each case.
Methyl (2'R,4'R)-(1',1'-dimethyl-6',8'-dioxo-5',7'-diaza-spiro[3.4]oct-2'-yl)-acetate 5
Compound 5 was obtained as a white solid and purified by preparative TLC (PhCH3/EtOH 6:1, Rf:
0.31) (101 mg, 20%) mp: 88-99 °C (decomposition), IR max (cm-1) (film) 3292, 2970, 2931,
1
1720, 1367, 1205. H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3): see Table 1.
HRMS: Calcd for C11H16N2NaO4 [M+Na]+ 263.10078. Found 263.10152.
(5RS)-(1'R,3'R)-5-(3'-methylacetate-2',2'-dimethylcyclobutyl)-imidazolidin-2,4-dione 7
Compound 7 was obtained as a white solid. The purification of each diastereoisomer by
preparative TLC (hexane/EtOAc/MeOH 7:2:1) was not successful. Therefore, they were analyzed
as a mixture (425 mg, 52%) mp: 219-220 °C IR max (cm-1) (film) (as a 4:1 diastereoisomeric
1
mixture) 3261, 3055, 3041, 2962, 2873, 1753, 1727, 1431, 1265, 1171, 771. H NMR (600 MHz,
CDCl3) and 13C NMR (150 MHz, CDCl3): see Table 3. HRMS: Calcd for C13H20N2O4Na [M+Na]+
291.1321. Found 291.1334.
(5RS)-(3S,3'R)-5-(3'-methoxycarbonyl-2',2'-dimethylcyclobutyl)-5-methyl-imidazolidin-2,4-dione 8
Compound 8 was obtained as a white solid. The purification of each diastereoisomer by
preparative TLC (hexane/EtOAc/MeOH7:2:1) was not successful. Therefore, they were analyzed
as a mixture (229 mg, 44%) IR max (cm-1) (film) (as a 1:1 diastereoisomeric mixture) 2958, 2924,
1724, 1464, 1456, 1375, 1230, 1203.1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3):
see Table 4. HRMS: Calcd for C12H18N2O4Na [M+Na]+ 277.1164. Found 277.1165.
(5RS)-(1'R,3'S)-5-(3'-hydroxymethyl-2',2'-dimethyl-cyclobutyl)-5-methyl-imidazolidin-2,4dione 11
Compound 11 was obtained as a white solid. The purification of each diastereoisomer by
preparative TLC (hexane/EtOAc/MeOH 7:2:1) was not successful. Therefore, they were analyzed
as a mixture (133 mg, 45%) IR max (cm-1) (film) (as a mixture) 3294, 2954, 2929, 2877, 2748,
15

ACCEPTED MANUSCRIPT
1759, 1410, 1367. 1H NMR (600 MHz, D2O) and 13C NMR (150 MHz, D2O): see Table 5. HRMS:
Calcd for C12H18N2O4Na [M-H]- 225.12392. Found 225.12444.
Reduction using LiBH4. General procedure
To a solution of hydantoin (1 equiv) in dry THF (9.6 mL), a 2M solution of LiBH4 in THF was added
dropwise (9.60mmol, 3.4 equiv). The solution was kept under a nitrogen atmosphere at reflux
temperature with stirring for 24 h. The mixture was then diluted with MeOH (3 mL) and H2O (1 mL).
The solvent was reduced, and the resulting solution was extracted with EtOAc (4x30 mL). The
organic phase was separated, washed with brine and dried over Na2SO4until dryness.
Cell proliferation assay
The in vitro cell proliferation was determined by a colorimetric assay (CellTiter 96 AQueous Non-
Radioactive Cell Proliferation Assay, Promega, USA) according to the manufacturer’s instructions.
For the MTS assay, U937 human promyelocytic leukemia cells (American Type Culture Collection,
Rockville, MD, USA) growing in exponential phase (2.0x106cells/mL) were seeded in a 96-well
plate and incubated in an atmosphere of 5% CO2 at 37°C. Cells were incubated with serial
dilutions of the mixture of compounds 11 (4.4μM to 100 μM), 0.05% (v/v) DMSO (vehicle control
group) or 400 μM db-AMPc (positive inhibition control). After incubation for 48h, 20 μl of MTS were
added to each well and further incubated for 2h at 37°C. The absorbance was measured at 490
nm using the FlexStation 3 microplate reader (Molecular Devices Inc., USA). Assays were carried
out in triplicate and at least three independent experiments were conducted.
Acknowledgements
This work was financially supported by grant PICT 2012-0032 from Agencia Nacional de
Promoción Científica y Tecnológica, grant B033 from Universidad de Buenos Aires and grant PIP
11220120100074CO from Consejo Nacional de Investigaciones Científicas y Tecnológicas
(Argentina).
References:
[1] P. J. Hajduk and J. Greer, Nat. Rev. Drug Discov. 6 (2007) 211-219.
[2] a) M. Nakajima, K. Itoi, Y. Takamatsu, H. Okazaki, T. Kinoshita, M. Shindou, K. Kawakubo, T. Honma,
M. Toujigamori, T. Haneishi, J. Antibiot. 44 (1991) 293-300. b) M. S.Luer, Neurol. Res. 20 1998 178-
182, c) D. Bialonska and J.K. Zjawiony, Marine Drugs 7 (2009) 166-183.
[3] H. Prevet, M. Flipo, P. Roussel, B. Deprez and N. Willand, Tetrahedron Lett.57 (2016) 2888-2894.
[4] a) Y. Chen, L. Su, X. Yang, W. Pan and H. Fang, Tetrahedron 71 (2015) 9234-9239; b) M. A. Khanfar,
B. A. Asal, M. Mudit, A. Kaddoumi and K. A. El Sayed, Bioorg. Med. Chem.17(2009) 6032; c) M.
Jansen, H. Potschka, C. Brandt, W. Löscher and G. Dannhardt, J. Med. Chem. 46 (2003) 64-73.
16

ACCEPTED MANUSCRIPT
[5] J. T. Edward and C. Jitrangsri, Can. J. Chem. 53 (1975) 3339-3350.
[6] a) M. Meusel and M. Gütschow, Org. Prep. Proced. Int. 36 (2004) 391-443; b) L. Konnert, F. Lamaty, J.
Martinez and E. Colacino, Chem. Rev.117 (2017) 13757−13809.
[7] a) B. A. Nogueira, G. O. Ildiz, J. Canotilho, M. E. S. Eusébio, M. S. C. Henriques, J. A. Paixão and R.
Fausto, J. Phys. Chem. A 121(2017)5267-5279; b) B. A. Nogueira, G. O. Ildiz, M. S. C. Henriques, J. A.
Paixão and R. Fausto, J. Mol. Struct. 1148 (2017) 111-118.
[8] A. G. Moglioni, E. García-Expósito, G. P. Aguado, T. Parella, V. Branchadell, G. Y. Moltrasio and R. M.
Ortuño, J. Org. Chem. 65 (2000) 3934-3940.
[9] J. A. Caturelli Kuran and A. Moglioni, Synthetic Commun. 44 (2014) 2393-2400.
[10] HyperChem® Professional 7.51, Hypercube, Inc. 1115NW 4th Street, Gainesville, FL 32601, US.
[11] D. M. Pawar, A. A. Khalil, D. R. Hooks, K. Collins, T. Elliott, J. Stafford, L. Smith and E. A. Noe, J. Am.
Chem. Soc. 120 (1998) 2108-2112.
[12] Gaussian 03, Revision C.02, M. J. Frisch, G. W Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J.
R. Cheeseman, J. A. Montgomery Jr., T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M.
Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H.
Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y.
Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A.
D.Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V.
Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I.
Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M.
Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez and J. A. Pople,
Gaussian, Inc., Wallingford CT, 2004.
[13] K. Wolinski, J. F. Hilton and P. Pulay, J. Am. Chem. Soc. 112 (1990) 8251-8260.
[14] a) S. G. Smith and J. M. Goodman, J. Org. Chem. 74 (2009) 4597-4607, b) S. G. Smith and J. M.
Goodman, J. Am. Chem. Soc. 132 (2010)12946-12959, c) A. M. Sarotti, Org. Biomol. Chem.11(2013)
4847-4859
[15] G.P. Sarmiento, P.D. Rouge, L. Fabian, D. Vega, R. M. Ortuño, G. Moltrasio and A. G.
Moglioni,Tetrahedron Asymmetry 22 (2011) 1924-1929
[16] S. Cortes and H. Kohn, J. Org. Chem. 48 (1983) 2246-2254.
[17] B. S. Furniss, A.J. Hannaford, P.W.G. Smith and A.R. Tatchell, Vogel’s Textbook of Practical Organic
Chemistry; fifth ed.; Longman, England,1989.
[18] W. L. F. Armarego, D.D. Perrin, Purification of Laboratory Chemicals; fourthed ed., Butterworth-
Heinemann, 1997.
17

ACCEPTED MANUSCRIPT
Highlights
A new series of hydantoin compounds derived from cyclobutyl ketones and
cyclobutanone were synthesized.
Class I and class II were fully characterized by mono and bi-dimensional
NMR spectroscopy.
Theoretical calculations were assayed for stereochemical assignment of the
hydantoin compounds.