Supporting Material
Synthesis, characterization and antimicrobial evaluation of a small library of ferrocenecontaining acetoacetates and phenyl analogs - the discovery of a potent anticandidal agent
Niko S. Radulovića*, Marko Z. Mladenovića, Zorica Radić-Stojanovićb, Goran A.
Bogdanovićc, Dragana Stevanovićd and Rastko D. Vukićevićd
Spectral characterization;
Table S1 1H and 13C NMR spectroscopic data (400 and 100 MHz, respectively) for
compound 3b, 3f, 3d, 4d;
Table S2 NOESY spectroscopic data for compound 3b, 3f, 3d, 4d;
Table S3 Crystallographic data for 3f;
Table S4 Selected bond lengths (Å) and bond angles (°) for crystal structure of 3f;
Figure S1 a) 1H-NMR spectrum of compound 3b in CDCl3 with (1) no shift agent, (2) 4.2%,
(3) 8.4%, (4) 12.6%, and (5) 16.8% of Eu(fod)3 (molar ration of the shift agent and 3b); b)
Expanded section (3.70-4.30 ppm) of spectra under a);
Figure S2 a) 1H-NMR spectrum of compound 3d in CDCl3 with (1) no shift agent added, (2)
4.2%, (3) 8.4% and (4) 16.8% of Eu(fod)3 (molar ration of the shift agent and 3d); b)
Expanded sections (3.6-4.7, 5.1-5.4 and 5.6-6.3 ppm) of spectra under a);
Figure S3 Proposed structure of complexes of Eu(fod)3 shift regent and compounds 3b and
3d with R = methyl and allyl group, respectively;
Figure S4 MS fragmentation of benzyl or ferrocenylmethyl methyl acetoacetates (1) and the
fragmentation observed only for compounds with the benzyl moiety (2, 3)
Figure S5 The projection of molecule 3f illustrating the eclipsed conformation of Cp rings;
Figure S6 The weak H-bond C6−H6b…O1i [symmetry code: (i) −x−1,−y, −z] connects the
molecules in centrosymmetric dimer;
Figure S7 The Fc-Fc interaction based on electrostatic complementarity between two Fc units
[6]. The Fc-Fc dimers are shown in two orthogonal projections;
Figure S8 The chain (in two projections) formed by the C6−H6b…O1 hydrogen bonds
(Figure S6) and Fc-Fc interactions (Figure S7)
Figure S9 The unsubstituted Cp ring participates in C−H…π interactions as π-acceptor of
C10 methyl groups. Dotted blue lines label the C…H contacts with the distance shorter than
2.9 Å;
Figure S10 Cathodic and anodic peak currents of 3d obtained at different scan rates in
acetonitrile.
Spectral characterization
The synthesized compounds, 3a-h, 4a-h, 6 and 7, have been fully characterized by standard
spectroscopic techniques (IR, MS, 1H and 13C NMR). All spectral data were fully consistent
with the proposed structures and the data for known compounds 4a [1], 4b [2], 4h [3] and 6
[3] (these compounds have been already reported together with their spectral data). Although
compound 7 was described in the literature [4], this is the first report on its spectral properties.
The main common feature of the IR spectra of all of the new methyl acetoacetates was a
strong band at 1705–1715 for 3a-h or at 1710–1715 cm-1 for 4a-h, 6 and 7 which could be
attributed to the ketone carbonyl group (C=O) stretching vibrations. Another prominent band
was a sharp, medium intensity absorption of another C=O (from COOMe moiety) at 1738–
1751 cm-1.
UV spectra of all ferrocenyl substituted methyl acetoacetates (3a-h) showed two absorption
maxima at approximately the same wavelength - 205 and 438 nm. The phenyl counterparts
(4a-h) had one absorption maximum at ca. 195 nm.
With few exceptions, the H and C atom resonances from the NMR spectra (at 200 and 50
MHz, respectively) of these compounds could be assigned on the basis of chemical-shift
theory, signal intensities and multiplicities, substituent effects, and by comparison with
literature data for known methyl acetoacetates (4a,b,h and 6). Thus, in the 1H NMR spectra of
3a-h, signals of the unsubstituted cyclopentadienyl (Cp) ring protons appeared at almost the
same position in the spectra (a broad singlet at 4.17–4.11 ppm). Interestingly, compounds 3ah showed different patterns of proton multiplets from the substituted ring of the ferrocenyl
groups. Probably caused by the differing anisotropic effect of the carbonyl groups determined
by conformational constraints, 1H NMR spectrum for compound 3a contained only a broad
signal at 4.06–4.01 ppm (corresponding to 4H), whereas compounds 3f and 3g contained
three multiplets (4.07–4.02 (2H), 4.01–3.96 (1H) and 3.96–3.91 (1H)) and the remaining
compounds had two multiplets (4.09–4.00 (3H) and 4.01–3.92 (1H)). On the other hand,
protons of the phenyl rings in the series 4a-h had almost the same chemical shift (signals in
the range 7.32–6.90 ppm). All synthesized compounds displayed singlets at 3.73–3.65 ppm
(3H, COOCH3) and 2.18–1.93 ppm (3H, CH3CO), as well as a specific AB second order
multiplet signal 3.12–2.78 (-CH2Fc) and 3.32–2.99 (-CH2Ph) ppm, respectively for 3c-f and
4d-g.
The off-resonance H atom decoupled 13C-NMR spectra of all synthesized compounds showed
the expected number of peaks in the aliphatic, aromatic and/or ferrocenyl and C=O regions
and these spectra also corroborated the structure of all synthesized ferrocenyl, phenyl and
dialkyl substituted methyl acetoacetates.
In order to resolve overlapping peaks in the proton NMR of 3b,d,f and 4b we decided to
record and reanalyze their 1H and 13C spectra at 400 and 100 MHz (Table S1), respectively.
Additionally, 2D NMR experiments (NOESY (Table S2), HSQC, DEPT, 1H–1H COSY and
HMBC) were conducted to corroborate the previously proposed assignment of peaks.
Moreover, alongside to the confirmation of peak assignments, in an attempt to resolve signals
of the substituted cyclopentadienyl ring (2’-5’) in 3b and the two signals at C2’’’ and C3’’’ in
3d, 1H NMR spectra of 3b and 3d were also recorded at 400 MHz with the addition of
increasing amounts of a lanthanide shift reagent (Eu(fod)3). As can be seen from Figure S1
and S2, the values of the chemical shifts observed were dependent on the amount of added
Eu(fod)3. The changes in chemical shift values can be explained by complexation of 3b,d
with Eu(fod)3. (The induced shifts arise from dipolar (pseudocontact) interaction between the
shift reagent and the substrate [5].)
Both compounds contain two potential coordination sites; unshared electron pairs at two
oxygens of the acetoacetyl moiety (Figure S3). The mode of complexation suggested in
Figure S3 is substantiated by significant changes observed for the chemical shifts of protons
that were in close proximity to the carbonyl groups and only negligible changes for the
remote protons of the unsubstituted Cp ring. Increasing Eu(fod)3 relative amount caused a
shift of the substituted Cp ring protons (H-3’, 4’ and 2’ or 5’) towards lower field. The
multiplet signals of 3b at ca. 4.02 and 4.05 ppm gradually shifted to a single low-field
multiplet at 4.28 ppm (after the addition of 12 mg of Eu(fod)3) in agreement with their
assignation as H-2’, 5’, whereas the signals of H-3’ and 4’, originally at ca. 4.07 ppm, shifted
much more slowly and finally overlapped with the protons from the unsubstituted Cp ring at
4.16 ppm (with 12 mg of the shift agent added).
Likewise, incremental addition of Eu(fod)3 resulted in a simplification of the 1H NMR
spectrum for 3d in CDCl3. The second-order spin system (multiplets at 5.12 and 5.15 ppm)
corresponding to protons of the allyl moiety (H-2’’’ and 3’’’) was transformed into a firstorder spin system (6.15 (doublet of doublet of triplets, J = 17.3, 10.1, 7.3 Hz, 1H) for H-2’’’,
5.38 (doublet of doublets, J = 17.3, 1.6 Hz, 1H) for trans-H-3’’’ and 5.27 (doublet of
doublets, J = 10.1, 1.6 Hz, 1H) for cis-H-3’’’ protons in the spectrum of 3d with the addition
of 12 mg of Eu(fod)3) even with a low (4.2-16.8%) molar ratio of Eu(fod)3 to 3d.
Their electron impact (EI) mass spectra clearly revealed fragmentations taking place in two
segments of the molecules, i.e., the alkyl substituted methyl acetoacetate and
ferrocenylmethyl units (Figure S4). All of the molecular ion peaks [M+.] for compounds 3a-h
were also the base peaks. All mass spectra contained high intensity fragments at m/z 43
[CH3CO]+, 56 [Fe]+, 121 [C5H5Fe]+ and 199 [C5H5FeC5H4CH2]+. Ions formed by the loss of a
Cp fragment, [M - C5H5]+, are considered a characteristic of compounds possessing a
ferrocenyl unit. Strangely, MS of our compounds contained no such ions, but some of them
exhibited fragments resulting from a simultaneous loss of cyclopentadiene and
CH3COC(R)COOCH3 fragments resulting in the detection of a quasi benzene-iron complex
[FeC6H6]+.
Even one of the mentioned most intense ions at m/z 199 could be represented as an iron
sandwich complex with Cp and benzene ligands, i.e. [C5H5FeC6H6]+. The analogous ion in the
benzyl series, the tropylium ion (m/z 91), was the most intensive fragment of their mass
spectra, except for 4a and 4f. However, some of the most prominent ions detected for all
compounds from the benzyl series, formed by the same bond fission (either a loss of an acetyl
group or a COOMe group) giving fragments, for example for 4a, at m/z 147 and 163, were not
detected at all in the ferrocene series.
Table S1 1H and 13C NMR spectroscopic data (400 and 100 MHz, respectively) for compound 3b, 3f, 3d, 4d
3b
1
H
3f
13
C
1
H
3d
13
C
1
H
4d
13
C
1
H
13
C
1
2.12 (s, 3H)
26.5
2.06 (s, 3H)
27.3
2.08 (s, 3H)
27.5
2.13 (s, 3H)
27.5
2
/
205.4
/
205.1
/
204.5
/
204.2
3
/
61.1
/
65.0
/
65.1
/
64.9
4
/
173.0
/
172.4
/
172.1
/
171.1
5
3.72 (s, 3H)
52.2
3.70 (s, 3H)
52.0
3.72 (s, 3H)
52.1
3.72 (s, 3H)
52.1
3.04 (d, J = 14.1 Hz,
6
1H), 2.87 (d, J = 14.1
3.02 (d, J = 14.5 Hz,
36.0
Hz, 1H)
1H), 2.96 (d, J = 14.5
3.02 (d, J = 14.5 Hz,
33.0
Hz, 1H)
1H), 2.96 (d, J = 14.5
3.26 (d, J = 14.1
33.0
Hz, 1H)
Hz, 1H), 3.17 (d, J
37.8
= 14.1 Hz, 1H)
7.10 (d, J = 6.5 Hz,
2’ and 6’
/
/
/
/
/
/
129.9
2H)
1’
3’, 5’, 4’
/
/
82.4
/
4.02 (doublet of
2’ or 5’
/
/
/
3.97 (pseudo quartet,
69.6
triplets, J = 2.5, 1.4
82.3
/
/
/
/
136.2
7.32-7.17
126.9
(overlapping peaks,
,
3H)
128.3
/
/
4.00 (doublet of
69.5
J = 1.8 Hz, 1H)
82.0
69.5
doublets, J = 3.1, 1.7
Hz, 1H)
Hz, 1H)
4.05 (doublet of
4.01 (pseudo quartet,
2’ or 5’
/
/
69.7
doublets, J = 3.1, 1.7
69.7
/
/
/
/
/
/
J = 1.8 Hz, 1H)
Hz, 1H)
3’, 4’ (and
4.07–4.02
67.8,
4.08 (doublet of
4.05 (pseudo triplet, J
2’ or 5’ for
(overlapping peaks,
67.9,
3H)
67.9
70.0
4.11 (broad singlet,
1’’-5’’
68.0
Hz, 2H)
4.13 (broad singlet,
68.7
5H)
67.9,
doublets, J = 3.1, 1.7
= 1.8 Hz, 2H)
3b)
67.8,
4.13 (broad singlet,
68.7
5H)
68.7
5H)
2.55 (doublet of
2.49 (broad doublet, J
1’’’
1.27 (s, 3H)
19.2
2.61 (broad doublet,
30.5
triplets, J = 7.3, 1.2
36.3
= 7.0 Hz, 2H)
36.2
J = 7.2 Hz, 2H)
Hz, 2H)
4.94 (triplet of septet,
2’’’
/
/
117.7
5.64 (m, 1H)
132.5
5.78 (m, 1H)
132.3
J = 7.0, 1.4 Hz, 1H)
cis-H 5.14 (m, 1H),
cis-H 5.12 (m, 1H),
3’’’
/
/
/
135.3
119.0
trans-H 5.18 (broad
119.2
trans-H 5.15 (m, 1H)
singlet, 1H)
4’’’(cis to
1.61 (broad doublet, J
/
CH2)
/
18.1
= 1.4 Hz 3H)
/
/
/
/
5’’’(trans to
1.74 (pseudo quartet,
/
CH2)
/
26.0
J = 1.4 Hz, 3H)
/
/
Table S2 NOESY spectroscopic data for compound 3b, 3f, 3d, 4d
H-1
H-5
3b
3f
3d
4d
H-1’’’, H-5, H-6, H-2’, H-5’
H-5, H-6, H-2’ or H-5’ (δ =
H-5, H-6, H-2’ or H-5’ (δ =
H-5, H-6, H-2’ or H-6’,
4.01), H-1’’’, H-2’’’, H-5’’’
4.05), H-1’’’, H-2’’’, H-3’’’
H-1’’’, H-2’’’, H-3’’’
H-1, H-6, H-1’’’, H-4’’’, H-
H-1, H-6, H-2’ or H-5’ (δ =
H-1, H-6, H-2’ or H-6’,
5’’’
4.05), H-1’’’, H-2’’’
H-3’ and/or H-4’, H-5’,
H-1, H-6, H-1’’’
H-1’’’, H-2’’’, H-3’’’(cis)
H-6
H-1, H-5, H-1’’’, H-2’, H-5’
H-1, H-5, H-2’, H-5’, H-1’’’,
H-1, H-5, H-2’, H-5’, H-
H-1, H-5, H-2’ or H-6’,
H-2’’’, H-4’’’, H-5’’’
1’’’, H-2’’’, H-3’’’(-trans)
H-3’ and/or H-4’, H-5’,
H-1’’’, H-2’’’, H-3’’’(cis)
H-2’ or H-5’ (at the lower
H-1, H-6, H-1’’’
H-6, H-1’’’
H-6, H-1’’’
/
/
H-1, H-6, H-1’’’, H-2’’’, H-
H-1, H-6, H-1’’’, H-2’’’
/
/
H-1, H-5, H-6, H-1’’’, H-
chemical shift)
H-2’ or H-5’ (at the higher
4’’’, H-5’’’
chemical shift)
H-2’ or H-6’
/
/
2’’’, H-3’’’ (-cis)
H-3’ and/or H-4’, H-5’
/
/
/
H-5, H-6, H-1’’’
H-3’, H-4’ (and for 3b H-2’ or
H-1, H-6, H-1’’’
/
/
/
H-1’’-H-5’’
/
/
/
/
H-1’’’
H-1, H-5, H-6, H-2’, H-5’
H-1, H-5, H-6, H-2’, H-5’, H-
H-1, H-5, H-6, H-2’’’, H-
H-1, H-5, H-6, H-2’ or
2’’’, H-4’’’, H-5’’’
3’’’, H-2’, H-5’
H-6’, H-3’ and/or H-4’,
H-5’)
H-5’, H-2’’’, H-3’’’
H-2’’’
/
H-1, H-6, H-2’ or H-5’ (δ =
H-1, H-5, H-6, H-2’ or H-5’
H-1, H-5, H-6, H-2’ or
4.01), H-1’’’, H-5’’’
(δ = 4.05), H-1’’’, H-3’’’(-
H-6’, H-1’’’, H-3’’’(-cis)
cis)
H-3’’’
/
/
H-3’’’ (-cis): H-1, H-1’’’, H-
H-3’’’ (-cis): H-1, H-5,
2’’’. H-3’’’ (-trans): H-1, H-
H-6, H-2’ or H-6’, H-
1’’’, H-6
1’’’, H-2’’’. H-3’’’ (trans): H-1, H-1’’’
H-4’’’
/
H-5, H-6, H-2’ or H-5’ (δ =
/
/
/
/
4.01), H-1’’’
H-5’’’
/
H-1, H-5, H-6, H-2’ or H-5’
(δ = 4.01), H-1’’’, H-2’’’
Table S3 Crystallographic data for 3f
Empirical formula
C21 H26 O3 Fe
Formula weight
382.27
Color, crystal shape
Orange, prism
Crystal size (mm3)
0.20 x 0.16 x 0.13
Temperature (K)
293(2)
Wavelength (Å)
1.5418
Crystal system
Monoclinic
Space group
P21/a
Unit cell dimensions
a (Å)
9.6899(4)
b (Å)
19.8839(7)
c (Å)
10.6516(4)
α (°)
90
β (°)
110.053(4)
γ (°)
90
V (Å3)
1927.86(13)
Z
4
Dcalc (Mg/m3)
1.317
μ (mm-1)
6.394
θ range for data collection (°)
4.42 to 72.10
Reflections collected
6819
Independent reflections, Rint
3711, 0.0293
Completeness (%) to θ = 67°
99.9
Refinement method
Full-matrix least-squares on F2
Data / restraints / parameters
3711 / 0 / 230
Goodness-of-fit on F2
1.056
Final R1/wR2 indices [I >2σ(I)]
0.0451 / 0.1116
Final R1/wR2 indices [I >2σ(I)]
0.0560 / 0.1201
Largest diff. peak and hole (eÅ-3)
0.347 /−0.577
Table S4 Selected bond lengths (Å) and bond angles (°) for crystal
structure of 3f
O1−C1
1.186(3)
C1−O2−C8
115.6(2)
O2−C1
1.333(3)
O1−C1−O2
124.1(2)
O2−C8
1.443(3)
O1−C1−C2
125.8(2)
O3−C9
1.200(3)
O2−C1−C2
110.04(19)
C1−C2
1.529(3)
C1−C2−C9
109.24(19)
C2−C9
1.539(3)
C1−C2−C11
108.75(19)
C2−C11
1.548(3)
C9−C2−C11
109.32(18)
C2−C3
1.550(3)
C1−C2−C3
107.78(19)
C3−C4
1.501(3)
C9−C2−C3
108.9(2)
C4−C5
1.319(4)
C11−C2−C3
112.82(19)
C5−C6
1.494(4)
C4−C3−C2
113.9(2)
C5−C7
1.510(4)
C5−C4−C3
126.6(3)
C9−C10
1.503(4)
C4−C5−C6
125.1(3)
C11−C12
1.501(3)
C4−C5−C7
120.4(3)
C6−C5−C7
114.5(3)
O3−C9−C10
121.0(2)
O3−C9−C2
121.6(2)
C10−C9−C2
117.3(2)
C12−C11−C2
114.57(19)
Figure S1 a) 1H-NMR spectrum of compound 3b in CDCl3 with (1) no shift agent, (2) 4.2%,
(3) 8.4%, (4) 12.6%, and (5) 16.8% of Eu(fod)3 (molar ration of the shift agent and 3b)
Figure S1 b) Expanded section (3.70-4.30 ppm) of spectra under a)
Figure S2 a) 1H-NMR spectrum of compound 3d in CDCl3 with (1) no shift agent added, (2)
4.2%, (3) 8.4% and (4) 16.8% of Eu(fod)3 (molar ration of the shift agent and 3d)
Figure S2 b) Expanded sections (3.6-4.7, 5.1-5.4 and 5.6-6.3 ppm) of spectra under a)
Figure S3 Proposed structure of complexes of Eu(fod)3 shift regent and compounds 3b and
3d with R = methyl and allyl group, respectively
Figure S4 MS fragmentation of benzyl or ferrocenylmethyl methyl acetoacetates (1) and the
fragmentation observed only for compounds with the benzyl moiety (2, 3)
Figure S5 The projection of molecule 3f illustrating the eclipsed conformation of Cp rings
Figure S6 The weak H-bond C6−H6b…O1i [symmetry code: (i) −x−1,−y, −z] connects the
molecules in centrosymmetric dimer
Figure S7 The Fc-Fc interaction based on electrostatic complementarity between two Fc units
[6]. The Fc-Fc dimers are shown in two orthogonal projections
Figure S8 The chain (in two projections) formed by the C6−H6b…O1 hydrogen bonds
(Figure S6) and Fc-Fc interactions (Figure S7)
Figure S9 The unsubstituted Cp ring participates in C−H…π interactions as π-acceptor of
C10 methyl groups. Dotted blue lines label the C…H contacts with the distance shorter than
2.9 Å
Figure S10 Cathodic and anodic peak currents of 3d obtained at different scan rates in
acetonitrile
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