Synthesis, characterization, X-ray crystallography analysis and cell viability study of (η6-p-cymene)Ru(NH2R)X2 (X = Cl, Br) derivatives

Article abstract of DOI:10.1016/j.poly.2021.115130

Studies over the past few decades demonstrate the potential for metallodrugs as bioactive therapeutics. Here, we describe six new ruthenium(II) complexes with the general motif of (η⁶-p-cymene)Ru(NH₂R)X_2, where NH₂R is either the influenza A antiviral drugs rimantadine or amantadine or the N-methyl-D-aspartate [NMDA] receptor antagonist, memantine and X = Cl or Br. All complexes were synthesized in high yield and purity and characterized by NMR spectroscopy and X-ray crystallography. Both the chlorine and bromine ruthenium(II) p-cymene complexes demonstrated cellular toxicity profiles similar to their respective free ligand, indicating that complexation to ruthenium(II) centers does not significantly increase toxicity of the bioactive ligand.

Full text of DOI:10.1016/j.poly.2021.115130

Polyhedron 200 (2021) 115130
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, characterization, X-ray crystallography analysis and cell
6
viability study of (
g
-p-cymene)Ru(NH R)X (X = Cl, Br) derivatives
2
2
a
b
c
d
a
Sarah L. McDarmont , Meredith H. Jones , Colin D. McMillen , Everett Clinton Smith , Jared A. Pienkos ,
Evan E. Joslin
a
b,
⇑
The University of Tennessee at Chattanooga, Department of Chemistry & Physics, 615 McCallie Avenue, Chattanooga, TN 37403, USA
Department of Chemistry, The University of the South, Sewanee, TN 37383, USA
Clemson University, Department of Chemistry, Clemson, SC 29634, USA
b
c
d
Department of Biology, The University of the South, Sewanee, TN 37383, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Studies over the past few decades demonstrate the potential for metallodrugs as bioactive therapeutics.
Received 13 January 2021
Accepted 22 February 2021
Available online 6 March 2021
6
Here, we describe six new ruthenium(II) complexes with the general motif of (
g
-p-cymene)Ru(NH
2
R)X2,
where NH
2
R is either the influenza A antiviral drugs rimantadine or amantadine or the N-methyl-
D
-aspar-
tate [NMDA] receptor antagonist, memantine and X = Cl or Br. All complexes were synthesized in high
yield and purity and characterized by NMR spectroscopy and X-ray crystallography. Both the chlorine
and bromine ruthenium(II) p-cymene complexes demonstrated cellular toxicity profiles similar to their
respective free ligand, indicating that complexation to ruthenium(II) centers does not significantly
increase toxicity of the bioactive ligand.
Keywords:
Coordination chemistry
Ruthenium(II) complexes
X-ray crystallography
Ó 2021 Elsevier Ltd. All rights reserved.
1
. Introduction
Transition metal complexes have recently gained interest for
sponding free ligands (Fig. 1) [6]. Whereas, similarly structured
bis-chelate complexes with magnesium(II), manganese(II), cobalt
(II), and zinc(II) with similar ligands have shown similar or better
HIV-1 inhibition profiles when compared to the free ligands [7].
their therapeutic potential [1]. One of the best known examples
is the platinum(II) salt cisplatin, which is commonly used as a
treatment for cancer [2]. More recently, gold complexes have been
investigated as anti-cancer agents, and have shown promising
results in clinical trials [3]. In addition to these precious metal spe-
cies, piano stool ruthenium(II) arene complexes have shown pro-
+
More recently, an amantadine (ATN) Cu (II) complex, {[ATNH ]
3 n
[CuCl ]} , was evaluated against a series of ATN-resistant influenza
A virus strains. This copper(II) complex was able to inhibit influ-
enza A virus strains that are not inhibited by amantadine or other
adamantane derivatives [8].
mise as chemotherapeutics and antimalarial drugs. Specific
In a recent perspective article, amantadine and its derivatives
(e.g., memantine and rimantadine) were suggested as potential
treatments for COVID-19 [9]. Though amantadine targets the influ-
enza A virus M2 proton-specific ion channel (M2) [10], the coron-
avirus envelope (E) protein could also function as an ion channel
[11]. Despite variable antiviral activity of amantadine, memantine,
and rimantadine against human and animal coronaviruses [12–17],
derivatization of these biologically active ligands to transition met-
als could increase efficacy. Therefore, due to the biological activity
6
examples include RAPTA-C [4] and
(g
-p-cymene)Ru(CQ)Cl
2
(
CQ = chloroquine) (Fig. 1) [5]. In particular, ruthenium-complexed
CQ was considerably more potent than free CQ against CQ-resis-
tant Plasmodium falciparum, suggesting that derivatization of exist-
ing FDA-approved therapeutics is a promising approach for some
compounds [5].
This strategy of attaching a bioactive ligand directly to the
metal center has been employed for the generation of antivirals.
For instance, Neamati et al. explored the bioactivity of the HIV-1
of amantadine [18], memantine (N-methyl-D-aspartate [NMDA]
integrase inhibitor analogs of diketo acid (DKA) and Elvitegravier
receptor antagonist) [19], rimantadine (active against influenza A
6
(
EVG) bound to a (
g
-p-cymene)ruthenium(II) fragment and found
virus) [20], and the well documented synthesis of ruthenium(II)
6
that the ruthenium(II) complexes were less active than the corre-
arene complexes, we envisioned the synthesis of (
g
-p-cymene)
ruthenium(II) complexes that contain these ligands. Indeed, the
structural literature contains a number of complexes of ruthe-
nium(II) chloride with extended amines bound opposite a p-cym-
⇑
Corresponding author.
E-mail address: eejoslin@sewanee.edu (E.E. Joslin).
https://doi.org/10.1016/j.poly.2021.115130
277-5387/Ó 2021 Elsevier Ltd. All rights reserved.
0

Fig. 1. Some biologically active inorganic compounds (ATN = amantadine).
ene group, including aniline [21], 4-methylaniline [21,22], benzy-
with paratone oil. Data were collected under nitrogen at 100 K
using Bruker D8 Venture diffractometer with Mo Ka
lamine [23], 2,6-diisopropylaniline [24], p-toluidine [25], and 2,6-
bis(diphenylmethyl)-4-methylaniline [26], as examples somewhat
related to the target amantadine, memantine, and rimantadine
that do not have additional traditional hydrogen bonding donor
or acceptor groups on the amine ligand. The structures of related
dibromide complexes of ruthenium(II) with p-cymene and
extended amines are, to our knowledge, not reported in the litera-
ture. Herein, we report the synthesis and characterization of these
a
a
(k = 0.71073 Å) microfocus source and a Photon 2 detector. Diffrac-
tion images were collected in 0.5° increments using phi and omega
scans. Instrument control, data processing, and scaling were per-
formed through the Apex 3 software suite (SAINT and SADABS rou-
tines) [28]. Symmetry analysis, structure solution, and structure
refinement were performed through the SHELXTL suite (XPREP,
SHELXT, and SHELXL routines) [29]. Structure refinement was per-
2
derivatives and their effect on cell viability. Both the chlorine and
formed by full-matrix least squares techniques on F . All non-
6
bromine (
g
-p-cymene)ruthenium(II) complexes demonstrated
hydrogen atoms were refined anisotropically. Hydrogen atoms
attached to carbon atoms were modeled in idealized locations
using appropriate riding models. The amine hydrogen atoms were
identified from the difference electron density maps and fully
refined. Crystallographic data for complexes 1–6 is given in Table 1.
cellular toxicity profiles similar to their respective free ligand, indi-
cating that the presence of a ruthenium(II) center does not signif-
icantly impact cellular toxicity. Overall, all complexes had 50%
cytotoxic concentrations (CC50) above 100
lM, providing a suitable
concentration window for future antiviral testing in cell culture.
Additionally, a chloroform solvate of complex 2, 2Á2CHCl
3
, was
obtained by allowing the chloroform/pentane system to evaporate
to dryness. Crystallographic data and structural figures of this sol-
vated species are provided in the SI.
2
. Material and methods
2.1. General methods
2.3. Viability measurements
Unless otherwise noted, all synthetic procedures were per-
formed under anaerobic conditions in a nitrogen-filled glovebox
DBT-9 (delayed brain tumor, murine astrocytoma clone 9) cells
or by using standard Schlenk techniques. 1H NMR, and C{ H}
13
1
[30] were maintained at 37 °C and 5% CO in Dulbecco’s modified
2
NMR (operating frequency 125 MHz) spectra were recorded on a
Eagle’s medium (DMEM) supplemented with 10% horse serum,
JOEL ECS or JOEL ECX 400 MHz spectrometer. All 1H and C{ H}
13
1
100 U/mL penicillin and streptomycin, and 10 mM HEPES. For via-
bility studies, DBT-9 cells were plated into opaque tissue culture-
treated 96-well plates at 20,000 cells/well approximately 24 h
prior to complex addition. Complexes were diluted to either
25 mM (complexes 3,6) or 100 mM (complexes 1,2,4,5) in DMSO.
Serial two-fold dilutions were generated in DMSO ranging from
100 mM to 0.098 mM. Complexes were diluted into DMEM at
1:1000 (100 mM to 0.098 mM); the final concentration of DMSO
was 0.4% (v/v) across all dilutions. Concentrations higher than
100 mM were not attempted, as DMSO can begin to affect cell via-
bility at or above 0.5% (v/v). DMEM was removed from all wells,
and 100 mL of DMEM containing the indicated concentration of
complex or vehicle [0.4% (v/v) DMSO] was added. There were four
1
NMR spectra are referenced against residual proton signals ( H
NMR) or the C resonances of the deuterated solvent (13C NMR).
13
6
[
(
g
-p-cy)RuBr
2
]
2
was synthesized according to a previously
reported procedure [27]. All other reagents were obtained from a
commercial source and used as received unless otherwise indi-
cated. All solvents were purchased as anhydrous solvents and used
as received.
2.2. X-ray crystallography
Single crystal X-ray diffraction data was obtained on complexes
1–6 using crystals mounted on low background cryogenic loops

S.L. McDarmont, M.H. Jones, C.D. McMillen et al.
Polyhedron 200 (2021) 115130
Table 1
Crystallographic Data for Complexes 1–6.
1
2
3
4
5
6
Empirical formula
F. W. (g/mol)
Crystal system
Space group
a (Å)
C
20
H
31Cl
2
NRu
C
22
H
35Cl
2
NRu
C
22
H
35Cl
2
NRu
C
20
H
31Br
2
NRu
C
22
H
35Br
2
NRu
C
22
H
35Br
574.40
monoclinic
P2 /c
2
NRu
457.43
triclinic
P-1
485.48
monoclinic
P2 /c
11.4061(7)
15.9222(10)
12.7305(7)
90
110.927(2)
90
2159.5(2)
4
485.48
tetragonal
I-4
26.109(3)
26.109(3)
6.4437(11)
90
546.35
triclinic
P-1
574.40
triclinic
P-1
1
1
7.2756(15)
11.620(2)
12.714(3)
71.013(8)
77.478(7)
79.387(7)
984.7(4)
2
7.3713(5)
7.8760(6)
6.7600(4)
27.8682(17)
36.107(2)
90
90.824(2)
90
6801.4(7)
12
1.683
b (Å)
c (Å)
11.7060(8)
13.1021(9)
69.288(2)
76.888(2)
76.047(2)
1013.73(12)
2
11.8014(9)
13.4882(11)
65.487(3)
76.608(3)
85.685(3)
1109.37(15)
2
a
(°)
b (°)
(°)
90
90
c
3
Volume (Å )
4392.5(13)
8
1.468
0.964
Z
3
D (calcd)(g/cm )
l
1.543
1.069
1.493
0.980
1.790
4.717
1.720
4.315
À1
, mm
4.223
h range,°
2.13–28.38
46,548
4914
0.0445
228
2.56–28.49
51,758
5467
0.0279
248
2.21–25.24
21,878
3973
0.0860
240
2.10–28.50
47,954
5126
0.0290
228
3.02–30.57
60,475
6763
0.0346
248
2.26–26.00
92,444
13,385
0.0456
733
Reflections coll.
Indep. reflections
R(int)
No. of parameters
No. of restraints
R indices
0
0
72
0
0
0
R
1
= 0.0178
wR = 0.0404
= 0.0207
wR = 0.0424
R
1
= 0.0168
wR = 0.0399
= 0.0186
wR = 0.0418
R
1
= 0.0900
wR = 0.1860
= 0.0952
wR = 0.1885
R
1
= 0.0143
wR = 0.0320
= 0.0159
wR = 0.0327
R
1
= 0.0165
wR = 0.0391
= 0.0179
wR = 0.0408
R
1
= 0.0299
wR = 0.0656
= 0.0391
wR = 0.0700
(
I greater than 2
r
(I))
2
2
2
2
2
2
R indices
R
1
R
1
R
1
R
1
R
1
R
1
(
all data)
2
2
2
2
2
2
Goodness of fit
Largest diff. peak/hole (e/Å )
CCDC deposition no.
1.094
0.506/-0.465
2,055,507
1.068
0.444/-0.484
2,055,508
1.120
1.435/-1.782
2,055,509
1.077
0.445/-0.461
2,055,510
1.066
0.488/-0.703
2,055,511
1.027
1.812/-0.988
2,055,512
3
biological replicates for each concentration of complex. Cells were
cultured as described above for 24 h post-addition. Viability was
determined using CellTiter-Glo according to manufacturer’s
instructions, and luminescence was measured using a BioTek Syn-
ergy HTX plate reader using a two-second integration time. All
graphs were generated using GraphPad Prism 9. All data were nor-
malized from 0% to 100% using GraphPad Prism 9. There was no
statistical difference between vehicle [0.4% (v/v) DMSO] and the
lowest concentration of each complex (0.098 mM). The box and
whisker plots represent all four biological replicates for each con-
centration, and none were removed during data processing and
analysis.
(CH
3
)
2
, 2.56 (s, 2H, NH
2
), 2.26 (s, 3H, p-cy-CH
3
), 2.13 (m, 3H, aman-
3
tadine), 1.82 – 1.50 (m, 12H, amantadine), 1.28 (d, J HH = 7 Hz, 6H,
1
3
CH(CH
95.41 (ipso-C of p-cy), 81.32 (CH-p-cy), 79.54 (CH-p-cy), 53.51,
44.53, 36.01, 29.62 (amantadine), 30.76 (CH(CH of p-cy), 22.25
(CH(CH of p-cy), 18.90 (CH of p-cy). Anal. Calc’d. for C20
NRu: C, 52.51; H, 6.83; N, 3.06 Found: C, 52.21; H, 6.74; N, 3.14.
3 2 3
) ). C NMR (101 MHz, CDCl ) d 103.02 (ispo-C of p-cy),
3 2
)
3
)
2
3
2
H31Cl -
2
.4.2. Synthesis of (p-cy)Ru(memantine)Cl
MemantineꢀHCl (212 mg, 0.001 mol) was dissolved in RO water
5 mL). KOH was added until the pH was greater than 12. After
0 min, dichloromethane (10 mL) was added and the organic layer
2
(2)
(
4
was isolated. The aqueous layer was extracted with dichloro-
2
2
.4. Synthesis
methane (2 Â 20 mL). The organic portions were combined and
4
dried with MgSO and the solvent was removed in vacuo to yield
.4.1. Synthesis of (p-cy)Ru(amantadine)Cl
2
(1)
memantine as a yellow oil (109 mg, 62% yield). Under nitrogen,
6
6
[
(g
-p-cy)RuCl
2
]
2
(500 mg, 0.816 mmol) was dissolved in chlo-
[(
form (6 mL) and stirred. To this, a solution of memantine
(109 mg, 0.608 mmol) in chloroform (2 mL) was slowly added.
2 2
g -p-cy)RuCl ] (128 mg, 0.209 mmol) was dissolved in chloro-
roform (15 mL). To this, a solution of amantadine (284 mg,
1
.89 mmol) in chloroform (5 mL) was added dropwise. The solu-
.
tion turned bright orange-red, and after 45 min, the solution was
slowly added to stirring hexanes (40 mL). An orange precipitate
formed and was collected on a fine-porosity frit and washed with
hexanes. The solid was dried in vacuo to yield a bright orange solid
After 30 min, the reaction solution was added to pentane
(50 mL). The resulting orange precipitate was collected on a fine
porosity frit and dried in vacuo (168 mg, 83% yield). Orange
block-like crystals were obtained by layering a chloroform solution
1
(
510 mg, 68% yield). Orange-red block-like crystals were obtained
of the complex with hexanes. H NMR (400 MHz, CDCl
3
) d 5.49 (d,
HH = 5.8 Hz, 2H, p-cy: CH),
), 2.58 (s, 2H, NH ), 2.26 (s,
), 2.21 (m, 1H, memantine), 1.66–1.02 (m, 12H,
1
3
3
by layering a chloroform solution of the complex with pentane. H
NMR (400 MHz, CDCl
JHH = 5.8 Hz, 2H, p-cy: CH), 5.45 (d, J
3.05 (sept, JHH = 6.9 Hz, 1H, CH(CH
3H, p-cy: CH
3
3
3
) d 5.53 (d, JHH = 6.0 Hz, 2H, p-cy: CH), 5.46
3
)
2
2
3
3
(
d, J HH = 6 Hz, 2H, p-cy: CH), 3.04 (sept, JHH = 7 Hz, 1H, p-cy: CH
3
2 2
g R)X Complexes.
6-p-cy)Ru(NH
Scheme 1. General Synthesis of (
3

S.L. McDarmont, M.H. Jones, C.D. McMillen et al.
Polyhedron 200 (2021) 115130
Fig. 2. Ruthenium(II) complexes 1–6.
Fig. 3. 1H NMR spectrum of (p-cy)Ru(amantadine)Cl
2
2 2 2 3
(1), (p-cy)Ru(amantadine)Br (4), (p-cy)Ru(memantine)Cl (2), and (p-cy)Ru(memantine)Br (5) in CDCl .
memantine), 1.30 (d, 3JHH = 7.0 Hz, 6H, CH(CH
)
), 0.89 (s, 6H,
) d 103.38 (ispo-C of p-
cy), 94.94 (ipso-C of p-cy), 81.67 (CH-p-cy), 79.17 (CH-p-cy),
3
2
4
and dried with MgSO and the solvent was removed in vacuo to
yield the rimantadine as a colorless oil (117 mg, 0.653 mmol).
1
3
memantine). C NMR (101 MHz, CDCl
3
Under nitrogen, a solution of rimantadine in chloroform (6 mL)
6
5
3
5.07, 50.66, 50.32, 43.05, 42.36, 32.98, 30.77, 30.38 (memantine),
0.10 (CH(CH of p-cy), 22.17 (CH(CH of p-cy), 18.78 (CH of p-
NRu: C, 54.43; H, 7.27; N, 2.89 Found:
was slowly added to a stirring solution of [(
g
-p-cy)RuCl
2
]
2
3
)
2
3
)
2
3
(133 mg, 0.216 mmol) in chloroform (2 mL). After stirring for
45 min, the reaction was added to hexanes (20 mL) to induce a pre-
cipitate. The resulting precipitate was collected on a frit and
washed with hexanes to yield an orange solid (156 mg, 72% yield).
cy). Anal. Calc’d. for C22
H35Cl
2
C, 54.43; H, 7.12; N, 2.89.
Orange columnar crystals were obtained by layering a dichloro-
2
.4.3. Synthesis of (p-cy)Ru(rimantadine)Cl
RimantadineꢀHCl (202 mg, 0.936 mmol) was dissolved in water
5 mL), and KOH was added until the pH was greater than 12. After
2
(3)
1
methane solution of the complex with hexanes.
H NMR
3
3
(
J
400 MHz, CDCl
HH = 6.5 Hz, 1H, p-cy: CH), 5.28 (d, J HH = 6.6, 1H, p-cy: CH), 5.16
3
) d 5.43 (d, J HH = 5.7 Hz, 1H, p-cy: CH), 5.29 (d, -
(
3
stirring for 30 min, dichloromethane (10 mL) was added and the
organic layer was collected. The aqueous layer was extracted with
dichloromethane (2 Â 20 mL). The organic portions were combined
3
3
(
d, J HH = 6.1 Hz, 1H, p-cy: CH), 3.20 (d, J HH = 10.3 Hz, 1H, NH
2
),
3
3
.03 (sept,
J HH = 7.0 Hz, 1H, p-cy: CH(CH ) ), 2.70 (m, 1H,
3 2
4

S.L. McDarmont, M.H. Jones, C.D. McMillen et al.
Polyhedron 200 (2021) 115130
Fig. 4. 1H NMR of (p-cy)Ru(rimantadine)Cl
(3) in CDCl .
2 3
Fig. 5. Structures of complexes 1–6 shown as 50% probability ellipsoids (hydrogen atoms omitted for clarity). The structure of complex 6 contains three unique molecules in
the asymmetric unit; only one is shown here for simplicity.
5

S.L. McDarmont, M.H. Jones, C.D. McMillen et al.
Polyhedron 200 (2021) 115130
rimantadine), 2.28 (s, 3H, p-cy: CH
3
), 2.09 (m, 1H, NH
2
), 2.02 (s, 3H,
rimantadine as a colorless oil (76.0 mg, 92% yield). Under nitrogen,
rimantadine), 1.79 1.40 (m, 12H, rimantadine), 1.30 (dd,
–
a solution of rimantadine (76.0 mg, 0.424 mmol) in chloroform
3
3
6
J
HH = 6.9, 4.9 Hz, 6H, CH(CH
3
)
2
), 1.23 (d, J HH = 6.6 Hz, 3H, riman-
tadine). C NMR (101 MHz, CDCl ) d 102.6 (ipso-C of p-cy), 96.0
ipso-C of p-cy), 81.9 (CH-p-cy), 81.6 (CH-p-cy), 81.2 (CH-p-cy),
(3 mL) was slowly added to a stirring solution of [(
g
-p-cy)RuBr
2
]
2
1
3
3
(100 mg, 0.127 mmol) in chloroform (5 mL). After stirring for
45 min, the reaction was added to hexanes (20 mL) to induce a pre-
cipitate which was collected on a frit and washed with hexanes to
yield an orange solid (99.0 mg, 66% yield). Orange-red columnar
(
7
3
8.9 (CH-p-cy), 63.7, 38.1, 36.9, 36.7, 28.2, 13.7 (rimantadine),
0.9 (CH(CH ), 23.0 (CH(CH ), 21.7 (CH(CH ), 18.9 (CH of p-
NRuÁ0.5H O: C, 53.44; H, 7.34; N,
.83 Found: C, 53.39; H, 7.21; N, 2.90.
3
)
2
3
)
2
)
3 2
3
cy). Anal. Calc’d. for C22
2
H
35Cl
2
2
crystals were obtained by layering a chloroform solution of the
1
) d 5.43 (d, 3
complex with hexanes. H NMR (400 MHz, CDCl
3
-
3
J
HH = 6.0 Hz, 1H, p-cy: CH), 5.26 (d, JHH = 6.8 Hz, 2H, p-cy: CH),
3
3
2
.4.4. Synthesis of (p-cy)Ru(amantadine)Br
2
(4)
(500 mg, 0.633 mmol) was dissolved in
(15 mL) in a round bottom flask. A solution of amantadine
220 mg, 1.45 mmol) in CHCl (5 mL) and added dropwise to the
stirring Ru-solution. The solution turned dark orange-red, and after
5 min, the solution was slowly added to stirring hexanes (40 mL).
5.18 (d,
NH ), 3.14 (sept,
rimantadine), 2.38 (s, 3H, p-cy: CH
3H, rimantadine), 1.79 – 1.42 (m, 12H, rimantadine), 1.31 (dd,
J
HH = 5.8 Hz, 1H, p-cy: CH), 3.33 (d,
J
HH = 11.0 Hz, 1H,
HH = 6.9 Hz, 1H, p-cy: CH(CH ), 2.68 (m, 1H,
), 2.13 (m, 1H, NH ), 2.02 (s,
6
3
[
(g
-p-cy)RuBr
2
]
2
2
J
3 2
)
CHCl
(
3
3
2
3
3
3
J
HH = 6.9, 4.5 Hz, 6H, CH(CH
tadine). C NMR (101 MHz, CDCl ) d 103.6 (ipso-C of p-cy), 96.3
3
)
2
), 1.23 (d, JHH = 6.7 Hz, 3H, riman-
1
3
4
3
A bright orange precipitate formed and was collected on a fine-
porosity frit and washed with hexanes. The solid was in vacuo to
yield a bright orange solid (543 mg, 79% yield). Orange columnar
(ipso-C of p-cy), 81.4 (CH-p-cy), 81.1 (CH-p-cy), 81.0 (CH-p-cy),
78.6 (CH-p-cy), 64.3, 38.1, 36.9, 36.7, 28.2, 13.8 (rimantadine),
31.3 (CH(CH
cy). Anal. Calc’d. C22
Found: C, 45.24; H, 6.01; N, 2.42.
3
)
2
), 23.2 (CH(CH
3
)
2
), 21.7 (CH(CH
3 2 3
) ), 19.8 (CH of p-
crystals were obtained by layering a chloroform solution of the
H35Br
2
NRuÁ0.5H
2
O: C, 45.29; H, 6.22; N, 2.40
1
complex with hexanes and pentane. H NMR (400 MHz, CDCl
3
) d
3
3
5
.51 (d, JHH = 6.0 Hz, 2H, p-cy:CH), 5.45 (d, J HH = 6.0 Hz, 2H, p-
3
cy:CH), 3.16 (sept, JHH = 7.0 Hz, 1H, p-cy:CH(CH
NH ), 2.36 (s, 3H, p-cy-CH ), 2.14 (m, 3H, amantadine), 1.83 –
.53 (m, 12H, amantadine), 1.27 (d, J HH = 7.0 Hz, 6H, CH(CH
3 2
) ), 2.67 (s, 2H,
3
. Results & discussion
2
3
3
1
3
)
2
).
) d 103.91 (ispo-C of p-cy), 95.75 (ipso-
C of p-cy), 81.14 (CH-p-cy), 79.09 (CH-p-cy), 53.99, 44.64, 44.63,
5.96, 29.61 (amantadine) 31.19 (CH(CH of p-cy),), 22.32 (CH
CH of p-cy), 19.77 (CH of p-cy). Anal. Calc’d. for C20 NRu:
C, 43.97; H, 5.72; N, 2.56 Found: C, 44.03; H, 5.88; N, 2.57.
3.1. Synthesis and NMR spectroscopy
1
3
C NMR (101 MHz, CDCl
3
6-p-cy)Ru(NH
2 2 2
R)Cl (NH R = amantadine (1),
The complexes (
g
3
3
)
2
6
memantine (2) and rimantadine (3)) and (
NH
synthesized by stirring [(
g
2 2
-p-cy)Ru(NH R)Br
(
3
)
2
3
H31Br
2
(
2
R = amantadine (4), memantine (5) and rimantadine (6)) were
6
6
g
-p-cy)RuCl
2
]
2
2 2
or [(g -p-cy)RuBr ] ,
2
2.4.5. Synthesis of (p-cy)Ru(memantine)Br (5)
MemantineꢀHCl (217 mg, 1.00 mmol) was dissolved in RO
water (5 mL). KOH was added until the pH was greater than 12.
After 40 min, dichloromethane (10 mL) was added and the organic
layer removed. The aqueous layer was extracted with dichloro-
methane (2 Â 20 mL). The organic portions were combined, dried
4
with MgSO , and the solvent was removed in vacuo to yield the
memantine as a yellow oil (75 mg, 42% yield). Under nitrogen,
6
[
(
g
-p-cy)RuBr
2
]
2
(110 mg, 0.139 mmol) was dissolved in chloro-
solution of memantine (75.0 mg,
.418 mmol) in chloroform (2 mL) was slowly added to the stirring
form (6 mL). To this,
a
0
6
solution of [(
2
g -p-cy)RuBr ]2. After 30 min, the reaction mixture
was added to pentane (50 mL), which produced a precipitate.
The orange solid was collected on a fine porosity frit and dried in
vacuo (92.5 mg, 61% yield). Orange-red tabular crystals were
obtained by layering a dichloromethane solution of the complex
1
3
with hexanes and pentane. H NMR (400 MHz, CDCl
3
) d 5.50 (d, -
3
J
HH = 5.8 Hz, 2H, p-cy: CH), 5.46 (d, JHH = 5.8 Hz, 2H, p-cy: CH), 3.18
sept, JHH = 6.9 Hz, 1H, CH(CH
3
(
3 2 2
) ), 2.69 (s, 2H, NH ), 2.36 (s, 3H, p-
cy: CH
tine), 1.30 (d, JHH = 6.9 Hz 6H, CH(CH
3
), 2.21 (m, 1H, memantine), 1.63–1.02 (m, 12H, meman-
3
3
)
2
), 0.89 (s, 6H, memantine)
) d 104.32 (ispo-C of p-cy), 95.32 (ispo-C
of p-cy), 81.49 (CH-p-cy), 78.75 (CH-p-cy) , 55.56, 50.86, 50.28,
3.09, 42.35, 33.03, 31.25, 30.39, 30.10 (memantine), 22.27 (CH
CH of p-cy), 19.66 (CH(CH of p-cy), 14.17 (CH of p-cy). Anal.
Calc’d. for C22 NRu: C, 46.00; H, 6.14; N, 2.44 Found: C, 46.20;
H, 6.14; N, 2.45
.
1
3
C NMR (101 MHz, CDCl
3
4
(
3
)
2
3
)
2
3
H35Br
2
2
.4.6. Synthesis of (p-cy)Ru(rimantadine)Br
RimantadineꢀHCl (101 mg, 0.466 mmol) was dissolved in water
5 mL), and KOH was added until the pH was greater than 12. After
0 min, dichloromethane (10 mL) was added and the organic layer
2
(6)
(
3
was removed. The aqueous layer was extracted with dichloro-
methane (2 Â 20 mL). The organic portions were combined, dried
Fig. 6. Structures of amantadine and memantine complexes viewed along the NAC
bond and bisecting the NH bonds.
with MgSO
4
, and the solvent was removed in vacuo to yield
2
6

S.L. McDarmont, M.H. Jones, C.D. McMillen et al.
Polyhedron 200 (2021) 115130
respectively, with excess amine in chloroform at room tempera-
ture. The complexes were isolated as orange solids in high purity
and yield through precipitation. (Scheme 1, Fig. 2).
and 5. These resonances appear as four doublets (5.43, 5.29, 5.28,
and 5.16 ppm), with two doublets overlapping to resemble a tri-
plet. The HSQC data (SI, Fig. S11) shows that each of these hydro-
gens correlate to a distinct carbon resonance. The HSQC data also
confirms the presence of two distinct (diastereotopic) proton reso-
The identity of complexes 1–6 were confirmed by 1H and 13
C
{1
, 2, 4, and 5 include two doublets (aromatic methine, p-cymene
3
H} NMR spectroscopy in CDCl . The resonances for complexes
1
2
nances for the NH hydrogens on the rimantadine ligand, located at
ligand), a septet (aliphatic methine, p-cymene), a singlet (methyl,
p-cymene), and a doublet (methyl, p-cymene) (Fig. 3). Spectro-
scopic differences between complexes 1, 2, 4, and 5 and their syn-
3.20 and 2.09 ppm. Variable temperature NMR from 298 K to 318 K
was conducted to confirm that the additional splitting in the aro-
matic protons was not due to hindered rotation of the p-cymene
6
6
1
thetic precursors, [(
g
-p-cy)RuCl
2
]
2
and [(g
-p-cy)RuBr
2
]
2
, include
ring. No change in the H NMR was observed (SI, Fig. S9) which
deshielded p-cymene resonances and increased second order cou-
confirms the additional resonances are due to the chiral center
on the ligand.
pling effects (i.e., roofing) for the aromatic methines on the p-cym-
6
ene. For complexes of the form (
g
-p-cymene)Ru(NH
2
R)X
2
(X = Cl,
Br), the resonances associated with the p-cymene ligand are influ-
enced by the halide on the complex (Fig. 3). For example, the septet
for complex 1 is at 3.04 ppm while the septet for complex 4 is at
3
.2. X-ray crystallography analysis
To verify the identity of the synthesized complexes and identify
3
.16 ppm. Resonances associated with the amine ligands are not
their structural features, single crystal X-ray diffraction was per-
formed on complexes 1–6. The structures of the complexes are
shown in Fig. 5. Potential rotation of the complexes’ building
blocks about certain bonds results in a variety of conformations
of the complexes in this series. This is demonstrated in Fig. 6 for
the amantadine and memantine complexes, which show variations
in the rotation of the p-cymene ligand as it coordinates to Ru, the
rotation about the Ru-N bond, and the rotation of the amantadine
and memantine ligands about the NAC bond. This occurs in concert
with different packing arrangements as the variations in the com-
plexes (for example, the size of Br versus Cl, and the presence of
methyl groups on memantine that are not present on amantadine)
are introduced (Fig. 7). It is interesting to note that only in the
as drastically impacted by the nature of the halide on the metal
center. Similar resonances are observed when NMRs are obtained
in DMSO d (SI, Fig. S15 and S16), which indicates these species
6
are stable in DMSO, the solvent used in cell viability studies (vide
infra).
The 1H NMR spectra of complexes 3 and 6 display additional
resonances due to the chiral center on rimantadine. This chiral cen-
ter causes the four aromatic methine hydrogens and the two iso-
propyl-methyl groups on the p-cymene ligand to be chemically
inequivalent. (Fig. 4). For instance, in complex 3, four resonances
are observed for the methine hydrogens on the p-cymene ligand,
instead of the two signals that are observed in complexes 1, 2, 4,
Fig. 7. Packing arrangements in complexes 1–6.
7

S.L. McDarmont, M.H. Jones, C.D. McMillen et al.
Polyhedron 200 (2021) 115130
amantadine complexes 1 and 4 are the analogous chloride and bro-
mide complexes isostructural. In the bromide series, the meman-
plexes 4–6, perhaps indicating a slight steric influence of the
larger bromide anions.
tine complex
5
exhibits
a
similar packing motif to the
In the complexes, NAHÁÁÁX and CAHÁÁÁX (X = Cl, Br) hydrogen
bonding both contribute to the intermolecular interactions that
stabilize the long-range packing (SI, Table 2). In complex 1 (and
the isostructural 4), one amine hydrogen atom and one chlorine
atom form hydrogen bonded dimers with their counterparts from
a neighboring molecule (SI, Fig. S19). The second chlorine atom
also extends the structure through a CAHÁÁÁCl interaction, where
pairs of these interactions also form dimers between different
neighboring molecules. Interactions involving p-cymene groups
of neighboring molecules complete the three-dimensional frame-
work of intermolecular interactions (SI, Fig. S20). These involve
CAHÁÁÁpi interactions between methyl hydrogen atoms and the
aromatic core of a neighboring p-cymene group (HÁÁÁC = 2.882 Å)
as well as offset piÁÁÁpi interactions where the aromatic regions
overlap (shortest CÁÁÁC = 3.333 Å). In complex 2, the Cl1 atom acts
as a hydrogen bond acceptor for two different CAHÁÁÁCl interactions
amantadine complex 4 (and the complexes have similar lattice
parameters), but it should be noted that there is a relative rotation
about the Ru-N bond between the complexes (given a common
NH -C orientation) that occurs to accomplish this. This leads to dif-
2
ferent intermolecular interactions.
The Ru-Cl bond lengths in 1–3 range from 2.405(5) Å to 2.4280
(
5) Å, similar to those in related Ru-p-cymene complexes [21–26],
and the Ru-Br bond lengths in 4–6 are, as expected, slightly longer,
ranging from 2.5427(4) Å to 2.5653(2) Å. The distance from Ru to
the centroid of the p-cymene ligand is consistent throughout this
series (ranging from 1.669(2) Å to 1.683(2) Å in 1–6), as are the
Ru-N bond lengths (2.125(16) Å to 2.1761(11) Å), and these are
also similar to related Ru-p-cymene complexes. The centroid-Ru-
N angle ranges from 135.2(3)° to 136.1(2)° in the chloride com-
plexes 1–3, and from 132.5(2)° to 135.8(2)° in the bromide com-
Fig. 8. Cell viability 24 h post-treatment with each complex. The amantadine, memantine, or rimantadine complexes and free ligands are grouped horizontally in A, B, and C,
respectively. DBT-9 cells were treated with each complex over a series of two-fold dilutions beginning at 100 M, with four replicates per concentration. Cell viability was
l
determined using the CellTiter-Glo Assay. Luminescence values are shown normalized from 0 to 100%, and the box and whisker plots represent the 25th to 75th percentiles
and min/max values, respectively.
8

S.L. McDarmont, M.H. Jones, C.D. McMillen et al.
Polyhedron 200 (2021) 115130
to the memantine and p-cymene groups of two different neighbor-
ing molecules. Pairs of both of these interactions form separate
dimers from a central molecule that extend the structure as chains
of dimers (SI, Fig. S21). The p-cymene groups of neighboring mole-
cules are not aligned for pi stacking interactions as they are in 1,
but they do support CAHÁÁÁpi interactions involving the hydrogen
atoms of methyl groups of two different neighboring molecules
4. Conclusions
Six new ruthenium(II) complexes containing bioactive ligands
were synthesized and characterized by NMR spectroscopy and X-
ray crystallography. Derivatives of (p-cymene)RuCl2 containing
other extended amines have been previously characterized by X-
ray crystallography, with the amantadine, memantine, and riman-
tadine derivatives here serving as new structural additions to this
(
one from a neighboring p-cymene group (HÁÁÁC = 2.863 Å) and
the other from a neighboring memantine group (HÁÁÁC = 2.892 Å)
to extend the structure in three dimensions. Likewise, complex 3
does not feature amine hydrogen bonding, but does again rely on
dimers of CAHÁÁÁCl hydrogen bonds to extend the structure in
one dimension along the c-axis. This time the dimers involve both
chlorine atoms of one molecule and two hydrogen atoms from the
aromatic core of the p-cymene group of a neighboring molecule (SI,
Fig. S22). In 5, the shortest hydrogen bonding contact to the bro-
family. The characterization of (p-cy)RuBr analogs reported in this
study are novel, and, in some cases, the Br-analogs display packing
2
arrangements that deviate from that of their Cl-congeners. Initial
cell viability studies indicate that complexation of these three
bioactive ligands to ruthenium(II) centers does not significantly
enhance cellular toxicity compared to their respective free ligands.
Furthermore, these studies provide a suitable window of concen-
trations at which to test potential antiviral activity and to deter-
mine whether these complexes have a tolerable selectivity index.
mine atoms again occurs through a CH
group, and pairs of these CAHÁÁÁBr interactions form dimers
Fig. S23). Unlike 2, where additional dimers were formed by
2
group of the memantine
(
CRediT authorship contribution statement
CAHÁÁÁCl interactions to the same chlorine atom, the additional
CAHÁÁÁBr dimers in 5 are formed through the second bromine atom.
This is more similar to what occurred in 1 and 4, where this bro-
mine atom interacts with a hydrogen atom of a neighboring p-
cymene group. This creates a one-dimensional motif. The pi inter-
actions in 5, however, are much less pronounced than in 1 and 4.
While the p-cymene groups are still aligned for a potential offset
piÁÁÁpi interaction, the shortest CÁÁÁC contact occurs at a much
longer distance (3.755 Å). In this way the piÁÁÁpi and CAHÁÁÁX inter-
actions involving the p-cymene group appear to have a more coop-
erative effect in 1 and 4 than in 5. Finally, complex 6 features
similar intermolecular interactions to 3, where in 6 the two bro-
mine atoms of a given molecule interact with two hydrogen atoms
on the aromatic core of the p-cymene group of a neighboring mole-
cule to produce one-dimensional chains. There are three such
unique chains in the structure, corresponding to the three unique
molecules in the asymmetric unit. While the molecules all have
different orientations relative to one another, all of these chains
propagate along the a-axis via the CAHÁÁÁBr interactions (SI,
Fig. S24).
Sarah L. McDarmont: Investigation, Validation, Formal analy-
sis, Writing - original draft, Writing - review & editing. Meredith
H. Jones: Investigation, Formal analysis, Writing - review & editing.
Colin D. McMillen: Methodology, Investigation, Formal analysis,
Resources, Visualization, Writing - original draft, Writing - review
&
editing. Everett Clinton Smith: Investigation, Formal analysis,
Writing - original draft, Writing - review & editing, Visualization,
Funding acquisition. Jared A. Pienkos: Conceptualization, Method-
ology, Investigation, Formal analysis, Resources, Writing - original
draft, Writing - review & editing, Funding acquisition. Evan E.
Joslin: Project administration, Methodology, Investigation, Formal
analysis, Visualization, Resources, Writing - original draft, Writing
-
review & editing, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
3.3. Cell viability studies
Acknowledgements
To determine the effect of complexes 1–6 or their free ligands
on cell viability, DBT-9 cells were treated with two-fold dilutions
of each complex or free ligand, and cell viability was measured
4 h post-treatment. Complexes 1–6 and their free ligands all have
0% cytotoxic concentrations (CC50) above 100 mM (Fig. 8), though
E.E.J and J.A.P would like to thank all the undergraduate stu-
dents listed for their hard work. E.E.J and E.C.S would like to thank
The University of the South for financial support. J.A.P would like to
thank the University of Tennessee at Chattanooga’s faculty grants
committee for funding.
2
5
the CC50 for memantine is likely close to 100
lM1 in DBT-9 cells.
Additionally, neither the chlorine nor bromine p-cymene ruthe-
nium(II) complexes significantly changed the concentration-
dependent cytotoxicity of the bioactive free ligand, at least out to
the concentrations tested. Concentrations above 100 mM were
not tested, as DMSO can begin to impact cell viability at 0.5% (v/
v) or above. The CC50 values for amantadine, rimantadine, and
memantine are cell-type specific, but are generally reported to be
around or greater-than 100 mM [31–33]. The CC50 for amantadine
in several eukaryotic cell lines, including human-derived lines,
ranges from approximately 95 to 850 mM [31]. CC50 values
of greater than 100 mM have been reported for rimantadine [32]
and memantine [33] in canine or murine cells, respectively, though
exact CC50 values were not provided. Together, these data indicate
that complexation of these three bioactive ligands to ruthenium(II)
centers does not significantly enhance cellular toxicity. Future
studies assessing potential antiviral activity will be needed to
determine whether these CC50 values are sufficient to obtain an
acceptable selectivity index.
Appendix A. Supplementary data
CCDC deposition numbers 2055507-2055513 contains the sup-
plementary crystallographic data for complexes 1–6 and 2Á2CHCl .
3
These data can be obtained free of charge via http://www.ccdc.-
cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallo-
graphic Data Center, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: (+44) 1223-336-033; or email: deposit@ccdc.cam.ac.uk. Sup-
plementary data to this article can be found online at https://doi.
org/10.1016/j.poly.2021.115130.
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