Synthesis of theophylline-based iridium(I) N-heterocyclic carbene complexes including fluorinated-thiophenolate ligands. Preliminary evaluation of their in vitro anticancer activity

Article abstract of DOI:10.1016/j.ica.2020.119588

A series of theophylline-based Ir(I) N-heterocyclic carbene complexes including fluorinated-thiolate ligands have been prepared and fully characterized. The molecular structures of the complexes [(NHC)Ir(SC₆F₅)(COD)] (3a) and [(NHC)I

Full text of DOI:10.1016/j.ica.2020.119588

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Research paper
Synthesis of Theophylline-Based Iridium(I) N-Heterocyclic Carbene Com‐
plexes Including Fluorinated-Thiophenolate ligands. Preliminary Evaluation
of Their In Vitro Anticancer Activity
Itzel Eslava-Gonzalez, Hugo Valdés, María Teresa Ramírez-Apan, Simón
Hernandez-Ortega, Miriam Rosario Zermeño-Ortega, Alcives Avila-Sorrosa,
David Morales-Morales
PII:
DOI:
Reference:
S0020-1693(19)32005-5
https://doi.org/10.1016/j.ica.2020.119588
ICA 119588
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Inorganica Chimica Acta
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Accepted Date:
20 December 2019
6 March 2020
8 March 2020
Please cite this article as: I. Eslava-Gonzalez, H. Valdés, M. Teresa Ramírez-Apan, S. Hernandez-Ortega, M.
Rosario Zermeño-Ortega, A. Avila-Sorrosa, D. Morales-Morales, Synthesis of Theophylline-Based Iridium(I) N-
Heterocyclic Carbene Complexes Including Fluorinated-Thiophenolate ligands. Preliminary Evaluation of Their
In Vitro Anticancer Activity, Inorganica Chimica Acta (2020), doi: https://doi.org/10.1016/j.ica.2020.119588
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Synthesis of Theophylline-Based Iridium(I) N-Heterocyclic Carbene
Complexes Including Fluorinated-Thiophenolate ligands.
Preliminary Evaluation of Their In Vitro Anticancer Activity.
aItzel Eslava-Gonzalez, aHugo Valdés*, aMaría Teresa Ramírez-Apan, aSimón Hernandez-
Ortega, bMiriam Rosario Zermeño-Ortega, cAlcives Avila-Sorrosa and aDavid Morales-
Morales*
aInstituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad
Universitaria, Ciudad de México, C.P. 04510, México. bUniversidad Autónoma de
Chihuahua, Facultad de Ciencias Químicas. Circuito Universitario, Campus Universitario
#2, Chihuahua, Chihuahua, C.P. 31125 México. cInstituto Politécnico Nacional, Escuela
Nacional de Ciencias Biológicas, Departamento de Química Orgánica. Carpio y Plan de
Ayala S/N, Colonia Santo Tomás, 11340 Ciudad de México, México
*Corresponding author. Tel.: +52 55 56224514; fax: +52 55 56162217.
E-mail address: damor@unam.mx (D. Morales-Morales)
Abstract
A series of theophylline-based Ir(I) N-heterocyclic carbene complexes including
fluorinated-thiolate ligands have been prepared and fully characterized. The molecular
structures of the complexes [(NHC)Ir(SC6F5)(COD)] (3a) and [(NHC)Ir(SC6F4H-4)(COD)]
(3b) were unambiguously determined by single crystal X-ray diffraction analysis. Both
compounds were isostructural, having the theophylline-imidazolylidene ligand coordinated
to the metal center and completing the coordination sphere with a 1,5-COD and
thiophenolate ligands. Interestingly, both complexes exhibit both inter- and intra-molecular
π–stacking interactions between the fluorinated ring of the thiolate and the theophylline-
based NHC ligand. Furthermore, for the series of Ir(I) NHC complexes preliminary in vitro
anticancer activity experiments were performed on six human cancer cell-lines, i.e. glia
cells of nervous central system (U-251), prostate (PC-3), leukemia (K-562), colon (HCT-
15), breast (MCF-7) and lung (SKLU-1). Being complex (3) the one showing the best
performance (compared to cisplatin) against PC-3 and SKLU-1 with IC50 values of 7.8 ±
0.4 μM and 10.7 ± 0.7 μM, respectively.
Keywords: N-heterocyclic carbene; Iridium complexes; Theophylline complex; Cancer;
Cytotoxic activity; Thiolate complexes; Xanthines.
1

1. Introduction
In the past two decades, the use of natural products as scaffolds for the preparation of
complexes has attracted much attention [1]. Several natural products can be easily isolated,
and their pharmaceutical applications have been well established.[1] Thus, the combination
of biological active compounds with a metal center can produce species with interesting
pharmaceutical applications. In this sense, xanthines are natural products that can be
extracted principally from cacao, coffee and tea. Among the best-known xanthines, we
have caffeine, theophylline and theobromine, whose structures are excellent scaffolds for
the preparation of N-heterocyclic carbene (NHC) ligands. In general, NHC ligands may
form very strong C-M bonds with almost any transition metal, avoiding NHC-metal bond
dissociation, a highly desirable feature for the design of catalysts and pharmaceutical
agents.
One of the first examples of xanthine-based NHC complexes was described in 1975
by Taube [2], since then few reports have been published [3]. These reports have focused in
their biological [4-10] and catalytic applications [11-13], as well as their preparation [14-
18] that often is not trivial. In 2014, Picquet and Casini described the antitumor activity of a
series of Au(I) xanthine-NHC complexes against human ovarian cancer cell line A2780/R,
which is resistant to cisplatin [6]. These compounds showed IC50 values in the micromolar
scale, being lower than that of cisplatin. Other xanthine-NHC complexes based on
transition metals such as Ag(I) [7], Pt(II) [19] and Pd(II) [10] have also been tested against
some cancer cell lines (Figure 1) showing promising results.
Recently, Iridium has been used for the design of antiproliferative agents, especially
when its oxidation state is +III [20-30]. However, there are some promising examples of
Ir(I) complexes for this purpose [31-33]. Metzler-Nolte and co-workers described the
2

cytotoxic activity of a series of complexes of the type [(NHC)IrCl(COD)], where NHC
corresponds to imidazolylidene and triazolylidene ligands [34-36]. These compounds
showed IC50 values in the micromolar scale, ranging from 10.3 ± 2.9 to 46.920 ± 0.085 μM
for MCF-7. Based on the aforementioned reasons, herein we reported the synthesis and
preliminary cytotoxic activity of a series of Ir(I) theophylline-NHC complexes including
fluorinated-thiophenolate ligands (Figure 1).
Figure 1. Cytotoxic xanthine-based complexes
2. Results and discussion
The preparation of 7-benzyl-1,3,9-trimethylxanthinium tetrafluoroborate (2) was
performed by a slight modification of a protocol described in the literature (Scheme 1)
[6,10]. Thus, theophylline was reacted with KOH, followed by the addition of 1 eq. of
benzyl bromide, affording the mono-benzylated theophylline compound (1) in 79 % yield.
The further reaction of this compound with [O(CH3)3]BF4 in CH2Cl2 afforded (2) in 80 %
1
yield. Spectroscopic analyses by H and 13C{1H} were in agreement with those reported
[10].
3

Scheme 1. Synthesis of the theophylline-azolium salt (2).
The coordination to iridium was achieved by reacting the imidazolium salt (2) with
K2CO3 and [IrCl(COD)]2 in THF (Scheme 2). The reaction was allowed to proceed for 24 h
at 50 ºC. After purification via chromatographic column, compound (3) was isolated as a
yellow solid in 41 % yield. With this complex on hand we were interested in exchanging
the chloride ligand by aromatic fluorinated thiolates. For this purpose, we reacted the Ir(I)
complex (3) with the corresponding thiolate lead salt [Pb(SArF)2] in acetone at room
temperature for 24 h (Scheme 2). The high insolubility in acetone of the PbCl2 produced,
drives the reaction to products, affording the desired complexes in good yields (Scheme 2).
Scheme 2. Preparation of Ir(I) theophylline-NHC complexes 3, 3a and 3b.
The new Ir(I) species were characterized by NMR spectroscopy and mass
spectrometry. For the series of complexes, the 1H NMR reveals the loss of symmetry of the
4

1,5-cyclooctadiene ligand due to coordination of the NHC to the metal. This loss of the
13
symmetry was also noted in the C{1H} NMR spectra, producing eight signals assignable
to the 1,5-cyclooctadiene moiety. The characteristic signal of the carbene carbon atom
appears in a similar chemical shift (δ 188 ppm) for the three complexes. Because of the
coordination of the thiolate ligand, the signals due to the olefinic carbons trans to the NHC
are shifted upfield, while the opposite effect was observed for the signals of the carbons
trans to the thiolate ligand. The mass spectra of the complexes provided further
information, producing clean spectra showing the molecular ions at 619.926 m/z for (3),
785.326 m/z for (3a) and 767.252 m/z for (3b) respectively. All these data, as well as
elemental analysis results are coherent with the proposed structures.
a)
b)
Figure 1. Molecular structure of a) (3a) and b) (3b). Hydrogen atoms are omitted for
clarity. Ellipsoids are at the 50 % probability level. Selected bond lengths (Å): (3a): Ir(1)-
C(2) 2.024(4), Ir(1)-C(20) 2.123(8), Ir(1)-C(21) 2.131(8), Ir(1)-C(24) 2.210(6), Ir(1)-C(25)
2.197(6), Ir(1)-S(1) 2.331(2). (3b): Ir(1)-C(2) 2.009(4), Ir(1)-C(27) 2.130(6), Ir(1)-C(26)
2.117(7), Ir(1)-C(30) 2.219(5), Ir(1)-C(31) 2.197(5), Ir(1)-S(1) 2.339(1). Selected Bond
Angles (º): (3a): C(2)-Ir(1)-S(1) 92.0(1), C(2)-Ir(1)-C(21) 94.1(2), C(2)-Ir(1)-C(20)
93.1(2), C(2)-Ir(1)-C(24) 163.6(2), C(2)-Ir(1)-C(25) 160.6(2), C(21)-Ir(1)-S(1) 162.5(2),
C(20)-Ir(1)-S(1) 157.8(2), Ir(1)-S(1)-C(28) 110.2(2). (3b): C(2)-Ir(1)-S(1) 91.0(1), C(2)-
Ir(1)-C(27) 94.2(2), C(2)-Ir(1)-C(26) 93.2(2), C(2)-Ir(1)-C(30) 166.5(2), C(2)-Ir(1)-C(31)
157.6(2), C(27)-Ir(1)-S(1) 161.7(2), C(26)-Ir(1)-S(1) 159.3(2), Ir(1)-S(1)-C(20) 110.7(2).
5

Crystals of (3a) and (3b) suitable for X-ray diffraction analyses were obtained by
slow diffusion of hexane into a saturated solution of the corresponding compound in
CH2Cl2. Complexes (3a) and (3b) are isostructural, with molecular structures consisting in
the theophylline-imidazolylidene ligand coordinated to iridium (C-Ir), and the 1,5-
cyclooctadiene and a thiolate ligands completing the coordination sphere around the metal
center (Table 1, Figure 1). The geometry of the iridium atom can be described as a pseudo-
square planar as seen by the C(2)-Ir(1)-S(1) angle, being 92.0(1)º for (3a) and 91.0(1)º for
(3b). While the Ir-Ccarbene distances are 2.024(4) and 2.009(4) Å, for (3a) and (3b),
respectively.
Table 1. Structural and refinement data for compounds (3a) and (3b).
Compound (3a)
C29H27F5IrN4O2S
782.80
298(2)
0.71073
Monoclinic
P21/n
7.1495(3)
41.6834(16)
9.7013(4)
90
102.4180(10)
90
2823.5(2)
4
Compound (3b)
C29H28F4IrN4O2S
764.81
298(2)
0.71073
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (º)
β (º)
Triclinic
P-1
7.3026(4)
9.4501(5)
20.9232(10)
78.7458(14)
86.3229(14)
77.6856(14)
1383.19(12)
2
γ (º)
Volume (Å3)
Z
ρ calc (g/cm3)
μ (mm-1)
1.842
4.871
1.836
4.964
F (0 0 0)
1532
750
Crystal size (mm3)
2Θ range for data collection (º)
Max. and min. transmission
Reflections collected
Independent reflections
GOF
0.389 x 0.209 x 0.138
2.204, 25.343
0.7452 and 0.4910
35638
5165
1.258
0.315 x 0.064 x 0.037
2.245, 25.364
0.7452 and 0.4839
15479
5059
1.172
Data/restraints/parameters
R1 I ≥ 2σ (I)
wR2 I ≥ 2σ (I)
5165 / 0 / 382
0.0357
0.0718
5059 / 0 / 373
0.0306
0.0619
6

With the compounds in hand, we carried out preliminary in vitro evaluation of their
cytotoxic activity. The experiments were carried out following the sulforhodamine B
protocol, using a 25 μM solution of the compound in DMSO, using six human cancer cell
lines, namely: glial cells of nervous central system (U-251), prostate (PC-3), leukemia (K-
562), colon (HCT-15), breast (MCF-7) and lung (SKLU-1) (Table 2). The theophylline
derivatives (1) and (2) were inactive in practically all cell lines, while the Ir(I) complexes
performed fine. Interestingly, the thiolate derivatives (3a) and (3b) were more active than
(3), reaching values of inhibition above 98 % in all cases. It is well known that fluorinated
aromatic rings favor non-covalent interactions (e.g. π-π stacking), thus, the thiolate ligand
may play an important role in the activity of these species, probably via molecular
recognition or even helping in the transport process.
Since compound (3) was the more selective for PC-3 and SKLU-1, we determined
their IC50 values on these two human cancer cell lines. Having the IC50 value of (3) for PC-
3 (7.8 ± 0.4 μM) being slightly better than that found for cisplatin (8.4 ± 04 μM), but less
efficient for SKLU-1 (compare: (3) 10.7 ± 0.7 μM vs cisplatin: 4.3 ± 0.5 μM).
Table 2. Growth inhibition (%) of human cancer cells by theophylline derivatives (25 μM)*
Compound
U-251
PC-3
K-562
HCT-5
MCF-7
SKLU-1
1.9
4
---
---
---
22.3
(1)
(2)
---
1.4
---
---
---
8.9
7

---
99.5
99.4
55.8
100
55.8
100
69.3
100
100
100
(3)
100
(3a)
(3a)
100
98.4
100
100
100
100
*The results are the average of three runs.
3. Conclusions
In summary, we have synthesized a series of theophylline-based Ir(I) NHC
complexes. The molecular structures of the thiolate derivatives (3a) and (3b) were
unambiguously determined by different spectroscopic techniques and single crystal X-ray
diffraction analyses. The structures showed the theophylline-imidazolylidene ligand
coordinates to the Ir(I), and a thiolate and a 1,5-COD ligand complete their coordination
spheres.
The complexes were active against six human cancer cell lines. The presence of the
fluorinated-thiolate ligand improved the cytotoxic activity of the compounds, but decrease
their selectivity. The IC50 values of (3) were very similar to those found for cisplatin, being
in the micromolar scale.
8

We believe that we have a nice modular structure that deserves further studies,
involving the further change of other benzyl substituents and some other thiolate ligands.
These compounds in turn may allow us the determination of SAR, that combined with
potential mechanistic studied might shed light to better design other similar complexes with
better performance and selectivities in the fight of this global illness. Efforts aimed to
achieve these goals are currently under development in our laboratories.
4. Experimental Part
All chemical compounds were commercially obtained from Aldrich Chemical Co.
1
13
and used as received without further purification. The H and C{1H} NMR spectra were
recorded on a Bruker Ascend 500 spectrometer. Chemical shift are reported in ppm down
field of TMS using the residual signals in the solvent (CDCl3, 7.27 ppm) as internal
standard. Elemental analyses were performed on a Perkin Elmer 240. CHNS analyses were
performed in Thermo Scientific Flash 2000 elemental analyzed, using a Mettler Toledo
XP6 Automated-S Microbalance and sulfanilamide as standard (Thermo Scientific BN
217826, attained values N = 16.40 %, C = 41.91 %, H = 4.65 % and S = 18.63 %; certified
values N = 16.26 %, C = 41.81 %, H = 4.71 % and S = 18.62 %). MS-Electrospray
determinations were recorded on a Bruker Daltonics-Esquire 3000 plus Electrospray Mass
Spectrometer.
Synthesis of 7-benzyl-1,3-dimethylxanthine (1).
A solution of theophylline (1.082 g, 6.01 mmol) and KOH (0.374 g, 6.67 mmol) in
DMF (3 mL) was stirred at 85 ºC for 15 min. Then, benzyl bromide (0.790 mL, 6.61 mmol)
was added and the reaction mixture was heated at 110 ºC overnight. The reaction was
9

cooled to room temperature, followed by addition of 15 mL of water. The solid was filtered
and washed further with water. Yield: 1.275 g (79 %). The spectroscopic data obtained was
in good agreement with that reported [10].
Synthesis of 7-benzyl-1,3,9-trimethylxanthinium tetrafluoroborate (2).
To a solution of compound (1) (0.477 g, 1.76 mmol) in ClCH2CH2Cl (20 mL) at 0
ºC, was added [O(CH3)3]BF4 (0.379 g, 2.56 mmol). The resulting reaction mixture was kept
at 0 ºC for 1 h. Then, was allowed to reach room temperature and let stirring for 72 h. The
resulting solid was filtered and washed with CH2Cl2. Yield: 0.525 g (80 %). The
spectroscopic data obtained were similar to those reported in the literature [10].
Synthesis of [(NHC)IrCl(COD)] (3).
A solution of compound (2) (0.108 g, 0.29 mmol), [IrCl(COD)]2 (0.100 g, 0.15
mmol) and K2CO3 (0.040 g, 0.29 mmol)in THF (5 mL) was heated at 50 ºC for 24 h. Then,
the solution was cooled to room temperature and all the volatiles removed under high
vacuum. The crude solid was dissolved in CH2Cl2 and purified by column chromatography
using silica gel. Elution with a CH2Cl2/acetone mixture (96/4) afforded the separation of a
1
yellow band that contained the desired compound. Yield: 0.073 g (41 %). H NMR (500
MHz, CDCl3): δ 7.48 (d, 3JH-H = 7.1 Hz, 2H, CHAr), 7.31 (t, 3JH-H = 7.4 Hz, 2H, CHAr), 7.26
3
2
2
(d, JH-H = 6.8 Hz, 1H, CHAr), 6.13 (d, JH-H = 14.6 Hz, 1H, -CH2-), 5.91 (d, JH-H = 14.6
Hz, 1H, -CH2-), 4.89 – 4.79 (m, 1H, CHCOD), 4.75 – 4.65 (m, 1H, CHCOD), 4.49 (s, 3H, -
CH3), 3.79 (s, 3H, -CH3), 3.31 (s, 3H, -CH3), 2.97 – 2.88 (m, 1H, CHCOD), 2.66 – 2.51 (m,
1H, CHCOD), 2.39 – 2.12 (m, 3H, CH2 COD), 1.98 – 1.81 (m, 2H, CH2 COD), 1.77 – 1.65 (m,
13
2H, CH2 COD), 1.48 – 1.37 (m, 1H, CH2 COD). C NMR (126 MHz, CDCl3) δ 188.4 (Ir-
10

Ccarbene), 152.4 (C=O), 150.6 (C=O), 140.7 (Cteophylline), 136.6 (CAr), 128.4 (CHAr), 127.8
(CHAr), 127.7 (CHAr), 109.6 (Cteophylline), 88.4 (CHCOD), 87.3 (CHCOD), 53.5 (CHCOD), 53.2
(-CH2-), 52.8 (CHCOD), 39.0 (-CH3), 34.4 (CH2 COD), 32.3 (CH2 COD), 31.9 (-CH3), 30.2
(CH2 COD), 28.6 (CH2 COD), 28.4 (-CH3). Electrospray MS (20 V, m/z): 619.926 [M]+. Anal.
Calcd for C23H28ClIrN4O2 (620.16): C, 44.54; H, 4.55; N, 9.03. Found: C, 44.60; H, 4.57;
N, 9.00.
Synthesis of [(NHC)Ir(SC6F5)(COD)] (3a).
To a solution of compound (3) (0.031 g, 0.05 mmol) in acetone (15 mL) was added
dropwise a solution of [Pb(SC6F5)2] (0.019 g, 0.03 mmol) in acetone (15 mL). The resulting
reaction mixture was stirred at room temperature for 24 h. After this time, the reaction was
filtered over celite, and all the volatiles were removed under high vacuum, affording the
desired product. Yield: 0.025 g (64 %). 1H NMR (500 MHz, CDCl3) δ 7.33 – 7.23 (m, 5H,
2
2
CHAr), 6.02 (d, JH-H = 14.9 Hz, 1H, -CH2-), 5.74 (d, JH-H = 14.9 Hz, 1H, -CH2-), 4.46 (s,
3H, -CH3), 4.45 – 4.37 (m, 1H, CHCOD), 4.32 – 4.24 (m, 1H, CHCOD), 3.80 (s, 3H, -CH3),
3.34 (s, 3H, -CH3), 2.93 – 2.81 (m, 1H, CHCOD), 2.49 – 2.39 (m, 1H, CHCOD), 2.35 – 2.18
(m, 2H, CH2 COD), 2.16 – 2.05 (m, 1H, CH2 COD), 1.91 – 1.70 (m, 3H, CH2 COD), 1.70 – 1.61
(m, 1H, CH2 COD), 1.40 – 1.35 (m, 1H, CH2 COD). 13C NMR (126 MHz, CDCl3): δ 188.3 (Ir-
Ccarbene), 152.1 (C=O), 150.4 (C=O), 148.4 – 147.6 (m, CArF), 146.1 – 145.5 (m, CArF),
140.5 (Cteophylline), 137.0 (CAr), 136.8 – 135.7 (m, CArF), 128.5 (CHAr), 127.6 (CHAr), 126.9
(CHAr), 121.6, 109.5 (Cteophylline), 84.0 (CHCOD), 82.9 (CHCOD), 57.6 (CHCOD), 57.6
(CHCOD), 53.1 (-CH2-), 39.3 (-CH3), 34.1 (CH2 COD), 31.9 (CH2 COD), 31.7 (-CH3), 30.5
(CH2 COD), 28.7 (CH2 COD), 28.6 (-CH3). Electrospray MS (20 V, m/z): 785.326 [M+H]+.
11

Anal. Calcd for C29H28F5IrN4O2S (783.83): C, 44.44; H, 3.60; N, 7.15. Found: C, 44.54; H,
3.59; N, 7.17.
Synthesis of [(NHC)Ir(SC6F4H-4)(COD)] (3b).
To a solution of compound (3) (0.032 g, 0.05 mmol) in acetone (15 mL) was added
dropwise a solution of [Pb(SC6F4H-4)2] (0.018 g, 0.03 mmol) in acetone (15 mL). The
reaction mixture was stirred at room temperature for 24 h. After this time, the reaction was
filtered over celite, and all the volatiles removed under high vacuum, affording the desired
product. Yield: 0.021 g (53 %). 1H NMR (500 MHz, CDCl3): δ 7.33 – 7.24 (m, 3H, CHAr),
7.23 – 7.17 (m, 2H, CHAr), 6.63 – 6.52 (m, 1H, CHArF), 6.07 (d, 2JH-H = 15.2 Hz, 1H, -CH2-
), 5.67 (d, 2JH-H = 15.2 Hz, 1H, -CH2-), 4.62 – 4.51 (m, 1H, CHCOD), 4.49 – 4.38 (m, 4H, -
CH3 and CHCOD), 3.77 (s, 3H, -CH3), 3.34 (s, 3H, -CH3), 2.91 – 2.79 (m, 1H, CHCOD), 2.45
– 2.36 (m, 1H, CHCOD), 2.34 – 2.19 (m, 2H, CH2 COD), 2.15 – 2.05 (m, 1H, CH2 COD), 1.95 –
13
1.79 (m, 2H, CH2 COD), 1.78 – 1.60 (m, 2H, CH2 COD), 1.44 – 1.30 (m, 1H, CH2 COD). C
NMR (126 MHz, CDCl3): δ 188.5 (Ir-Ccarbene), 152.1 (C=O), 150.4 (C=O), 144.7 – 144.0
(m, CArF), 141.3 – 140.5 (m, CArF), 140.4 (Cteophylline), 137.3 (CAr), 135.8 – 135.2 (m, CAr-F),
128.5 (CHAr), 127.5 (CHAr), 126.5 (CHAr), 109.5 (Cteophylline), 101.3 (CHArF), 84.1 (CHCOD),
83.1 (CHCOD), 57.3 (CHCOD), 57.2 (CHCOD), 53.2 (-CH2-), 39.2 (-CH3), 34.2 (CH2 COD),
31.9 (CH2 COD), 31.7 (-CH3), 30.4 (CH2 COD), 28.7 (CH2 COD), 28.6 (-CH3). Electrospray MS
(20 V, m/z): 767.252 [M+H]+. Anal. Calcd for C29H29F4IrN4O2S (765.84): C, 45.48; H,
3.82; N, 7.32. Found: C, 45.50; H, 3.81; N, 7.38.
4.1 Cytotoxic evaluation
4.1.1 Cell lines culture and culture medium
12

The compounds were screened in vitro against human cancer cell lines: U-251
(human glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (chronic
myelogenous leukemia), HCT-15 (human colorectal adenocarcinoma), MCF-7 (human
mammary adenocarcinoma), SKLU-1 (human lung adenocarcinoma). Cell lines were
supplied by National Cancer Institute (USA). The human tumor cytotoxicity was
determined using the protein-binding dye sulforhodamine B (SRB) in microculture assay to
measure cell growth, as described in the protocols established by the NCI1 [37]. The cell
lines were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum, 2
mM L-glutamine, 10.000 units/mL penicillin G sodium, 10.000l g/ml streptomycin sulfate
and 25 μg/mL amphotericin B (Gibco) and 1% non-essential amino acids (Gibco). They
were maintained at 37 ºC in humidified atmosphere with 5% CO2. The viability of the cells
used in the experiments exceeds 95% as determined with trypan blue.
4.1.2 Cytotoxic assay
Cytotoxicity after treatment of the tumors cells and normal cell with the test
compounds was determined using the protein-binding dye sulforhodamine B (SRB) in
microculture assay to measure cell growth [37]. The cells were removed from the tissue
culture flasks by treatment with trypsin, and diluted with fresh media. Of these cell
suspension, 100 μL containing 5000–10,000 cell per well, were pipetted into 96 well
microtiter plates (Costar) and the material was incubated at 37 ºC for 24 h in a 5% CO2
atmosphere. Subsequently, 100 μL of a solution of the compound obtained by diluting the
stocks were added to each well. The cultures were exposed for 48 h to the compound at
concentrations 25 μM. After the incubation period, cells were fixed to the plastic
substratum by addition of 50 μL of cold 50% aqueous trichloroacetic acid. The plates were
13

incubated at 4 ºC for 1 h, washed with tap H2O, and air-dried. The trichloroacetic-acid-
fixed cells were stained by the addition of 0.4% SRB. Free SRB solution was the removed
by washing with 1% aqueous acetic acid. The plates were then air-dried, and the bound dye
was solubilized by the addition of 10 mM unbuffered tris base (100 μL). The plates were
placed on and shaken for 10 min, and the absorption was determined at 515 nm using a
ELISA plates reader (Bio-Tex Instruments).
4.2 Data collection and refinement for compound (3a) and (3b)
Crystals of (3a, CCDC 1977715) and (3b, CCDC 1977716) were grown from
Hexane/iPrOH and mounted on glass fibers, then placed on a Bruker Smart Apex II
diffractometer with a Mo-target X-ray source (λ = 0.71073Å). The detector was placed at a
distance of 5.0 cm from the crystals and frames were collected with a scan width of 0.5 in ω
and an exposure time of 10 s/frame. Frames were integrated with the Bruker SAINT
software package[38] using a narrow-frame integration algorithm. Non-systematic absences
and intensity statistics were used in monoclinic P21/n and triclinic P1 space groups
respectively. The structures were solved using Patterson methods using SHELXS-2014/7
program [39]. The remaining atoms were located via a few cycles of least squares
refinements and difference Fourier maps. Hydrogen atoms were input at calculated
positions and allowed to ride on the atoms to which they are attached. Thermal parameters
were refined for hydrogen atoms on the phenyl groups using a Ueq = 1.2 Å to precedent
atom. The final cycles of refinement were carried out on all non-zero data using SHELXL-
2014/7.[39] Absorption corrections were applied using SADABS program.[40]
Supplementary information
14

Supplementary data for compound (3a) and (3b) have been deposited at the
Cambridge Crystallographic Data Centre. Copies of this information are available free of
charge on request from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
(Fax:
+44-
1223-336033;
e-mail
deposit@ccdc.cam.ac.uk
or
www:
http://www.ccdc.cam.ac.uk) quoting the deposition numbers CCDC 1977715-1977716.
Acknowledgments
We would like to thank Dr. Francisco Javier Pérez Flores, Q. Eréndira García Ríos,
M.Sc. Lucia del Carmen Márquez Alonso, M.Sc. Lucero Ríos Ruiz, Q. María de la Paz
Orta Pérez, Q. Roció Patiño-Maya, Dr. Beatriz Quiroz García and Dr. Nuria Esturau
Escofet for technical assistance. H. V. would like to thank Programa de Becas
Posdoctorales-DGAPA-UNAM
for
postdoctoral
scholarships
(Oficio:
CJIC/CTIC/4751/2018). The financial support of this research by PAPIIT-DGAPA-UNAM
(PAPIIT IN210520) and CONACYT A1-S-33933 is gratefully acknowledged.
15

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Author Statement
Itzel Eslava-Gonzalez: Investigation. Hugo Valdés: Investigation, Writing - original
draft, Supervision. María Teresa Ramírez-Apan: Investigation. Miriam Rosario
Zermeño-Ortega: Investigation. Simón Hernandez-Ortega: Investigation. Alcives
Avila-Sorrosa: Investigation. David Morales-Morales: Conceptualization, Validation,
Supervision, Writing - original draft.
Graphical Abstract-Pictogram
Synthesis of Theophylline-Based Iridium(I) N-Heterocyclic Carbene
Complexes Including Fluorinated-Thiophenolate ligands.
Preliminary Evaluation of Their In Vitro Anticancer Activity.
19

aItzel Eslava-Gonzalez, aHugo Valdés*, aMaría Teresa Ramírez-Apan, aSimón Hernandez-
Ortega, bMiriam Rosario Zermeño-Ortega, cAlcives Avila-Sorrosa and aDavid Morales-
Morales*
aInstituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad
Universitaria, Ciudad de México, C.P. 04510, México. bUniversidad Autónoma de
Chihuahua, Facultad de Ciencias Químicas. Circuito Universitario, Campus Universitario
#2, Chihuahua, Chihuahua, C.P. 31125 México. cInstituto Politécnico Nacional, Escuela
Nacional de Ciencias Biológicas, Departamento de Química Orgánica. Carpio y Plan de
Ayala S/N, Colonia Santo Tomás, 11340 Ciudad de México, México
*Corresponding author. Tel.: +52 55 56224514; fax: +52 55 56162217.
E-mail address: damor@unam.mx (D. Morales-Morales)
CYTOTOXIC ACTIVITY
O
N
N
Ir
N
O
N
Cl
O
N
O
N
N
N
N
N
N
N
Ir
Ir
O
O
S
S
F
F
F
F
F
F
F
F
F
Highlights
Three theophylline-based Ir(I) N-heterocyclic carbene complexes were synthesized.
The complexes have excellent cytotoxic activities against different cancer cell lines.
The cytotoxic activity of the parent complex was enhanced by substitution of the
chloride ligand by a fluorinated thiophenol.
20

Declaration of interests
☒ The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
N/A
21