Synthesis and antimicrobial activity of chiral quaternary N-spiro ammonium bromides with 3′,4′-dihydro-1′H-spiro[isoindoline-2,2′-isoquinoline] skeleton

Article abstract of DOI:10.2147/DDDT.S133250

A new class of highly functionalized tetrahydroisoquinolines with a quaternary carbon stereocenter was synthesized starting from an easily accessible l-tartaric acid. Nine strains of bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus m

Full text of DOI:10.2147/DDDT.S133250

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O r i g i n a l r e s e a r c h
synthesis and antimicrobial activity of chiral
quaternary N-spiro ammonium bromides
with 3′,4′-dihydro-1′h-spiro[isoindoline-2,2′-
isoquinoline] skeleton
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
5 July 2017
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Krzysztof Bielawski1
Katarzyna Leszczyńska2
Zbigniew Kałuża3
anna Bielawska4
Abstract: A new class of highly functionalized tetrahydroisoquinolines with a quaternary carbon
stereocenter was synthesized starting from an easily accessible l-tartaric acid. Nine strains of
bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans, Streptococcus
salivarius, Bacillus subtilis, Enterococcus faecalis, Moraxella catarrhalis, Escherichia coli,
Campylobacter jejuni) were used for the determination of minimal inhibitory concentration
(MIC) and minimal bactericidal concentration (MBC) of synthesized compounds. The influ-
ence of analyzed compounds on viability and induction of apoptosis in human skin fibroblasts
was determined. A majority of the synthesized compounds showed the strongest antibacterial
properties toward some gram-negative bacteria (M. catarrhalis and C. jejuni) with a high level
of selectivity. High antibacterial compounds have bactericidal activity ratio MBC/MIC #4.
Our studies also proved that the novel compounds do not possess cytotoxic and proapoptotic
potential in normal cells.
Olga Michalak3
Tamara Daniluk2
Olga staszewska-
Krajewska3
anna czajkowska4
Natalia Pawłowska1
agnieszka gornowicz4
1Department of synthesis and
Keywords: quaternary ammonium compounds, tetrahydroisoquinolines, antimicrobial activity,
antibiotic resistance
Technology of Drugs, 2Department
of Microbiology, Medical University
of Bialystok, Bialystok, 3institute of
Organic chemistry, Polish academy
of sciences,Warsaw, 4Department of
Biotechnology, Medical University of
Bialystok, Bialystok, Poland
Introduction
The growing numbers of bacterial strains resistant to currently used drugs constitute a
real threat to the health of global community. Multidrug-resistant (MDR), extensively
drug-resistant (XDR) and even pandrug-resistant (PDR) bacteria that are resistant to
all available antibiotics are considered to be responsible for serious hospital infec-
tions, particularly in immunocompromised individuals. The use of antibiotics is the
single most important factor leading to antibiotic resistance.1,2 Antibiotics are among
the most commonly prescribed drugs used in human medicine. However, up to 50%
of all antibiotics prescribed for people are not needed or are ineffective. Antibiotics
are also commonly used in feeding animals to prevent, control and treat disease and to
promote the growth of food-producing animals.3 Unfortunately, this dramatic increase
of antibiotic-resistant bacteria in recent years has no reflection in the number of new
antibacterial agents introduced into the market, thus limiting therapeutic options for
patients infected by such bacteria. Therefore, the development of new methods of
treatment for infections is one of the most urgent necessities in medicine today.4
Quaternary ammonium compounds (QACs) that belong to organic ionic chemical
agents are one model for the design of novel antibacterial compounds. In particular,
correspondence: agnieszka gornowicz
Department of Biotechnology, Medical
University of Bialystok, Kilinskiego 1,
15-089 Bialystok, Poland
Tel +48 85 748 5742
Fax +48 85 748 5416
email agnieszka.gornowicz@umb.edu.pl
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http://dx.doi.org/10.2147/DDDT.S133250

Bielawski et al
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they are an essential component of infection control practices Materials and methods
and are useful in the prevention of nosocomial infections.5 Nuclear magnetic resonance (NMR) spectra were recorded
Despite these applications, our knowledge of the molecular in CDCl3 at room temperature (except where indicated oth-
mechanism of action of QACs is very limited. In general, erwise) using a Varian VNMR500 spectrometer or Bruker
QACs have a more comprehensive spectrum of activity than Avance 500 (Bruker Corporation, Billerica, MA, USA).
antibiotics, and while antibiotics tend to have specific intra- Chemical shifts are quoted in parts per million relative
1
cellular targets these compounds may have multiple targets.6 to tetramethylsilane (TMS) for H NMR and CDCl3 for
A further exploration of QACs, which are more selective for 13C NMR. Coupling constants J are reported in Hertz. Infrared
bacterial cells than eukaryotic cells, is needed. Several natural (IR) spectra were obtained using a Fourier transform infrared
compounds having QAC moiety have shown antimicrobial (FTIR) Jasco 6200 or FTIR Spectrum 2000 (PerkinElmer
activity, including the ability to inhibit the biofilm formation.7 Inc., Waltham, MA, USA) and are reported in reciprocal
These include chelerythrine, sanguinarine and berberine from centimeters (cm−1). Mass spectra were recorded using an
the group of alkaloids which contain isoquinoline scaffold AMD-604 Intectra GmbH or a Mariner Perseptive Biosystem
and quaternary ammonium moiety (Scheme 1). Sanguinarine mass spectrometer. X-ray analysis was performed on Bruker
interacts with DNA, RNA and caps telomerases that leads AXS, APEX diffractometer (Bruker Corporation). Optical
to the rapid induction of apoptosis.8,9 Its antiplaque efficacy rotations were measured at 23°C with a Jasco P2000 digital
in humans was also proved, and sanguinarine has been used polarimeter. Thin-layer chromatography was performed using
as a component of mouthwashes and toothpastes in the UK precoated silica gel plates (Merck Kieselgel 60 F254 [Merck
and USA.10 Other studies based on sodium dodecyl sulfate Millipore, Billerica, MA, USA], 0.2 mm layer thickness).
polyacrylamide gel electrophoresis (SDS-PAGE) demon- Visualization of the developed chromatogram was performed
strated that berberine could destroy cellular proteins and lead by ultraviolet (UV) absorbance and cerium molybdate water
to damage in bacterial cells.11
solution. Flash chromatography was carried out using Merck
Collier et al12 found that decamethylene bisisoquinolinium Kieselgel (230–400 mesh). All air and moisture sensitive
bromide had a significant antimicrobial activity. Until now, a reactions were performed under an argon atmosphere in
number of isoquinolinium derivatives have been synthesized flame-dried glassware using anhydrous solvents. Most
and found to have a superior activity against gram-positive reagents were obtained from commercial suppliers and were
and gram-negative bacteria.13 These compounds have been used without further purification, unless noted otherwise. Tet-
shown to be antibacterial through membrane disruption14 as rahydrofuran (THF) was distilled from Na and benzophenone,
well as through binding to the cytosolic components, mainly and dichloromethane (DCM) and toluene were distilled from
nucleic acids.15 We have elaborated the procedure for the CaH2. The compounds 1, 4 and 5 were prepared according to
preparation of enantiopure hexahydropyrroloisoquinolines the literature methods.17,18 The same procedure was applied
from l-tartaric acid,16–18 and their further transformation for the synthesis of compounds 2 and 3.
into highly functionalized tetrahydroisoquinolines with a
general procedure for the preparation of
quaternary carbon stereocenter. The main task of this study
was to carry out the synthesis and assess the bacteriostatic and amino alcohols 6–9
bactericidal activity of chiral quaternary N-spiro ammonium Step I: Into a solution of diacetate 1–5 (5 mmol) in anhydrous
bromides with 3′,4′-dihydro-1′H-spiro[isoindoline-2,2′- methanol (50 mL), MeONa (5 mmol) was added at room tem-
isoquinoline] skeleton. The effect of tested compounds on the perature. Stirring was continued until disappearance of the
viability of human skin fibroblasts was also determined.
substrate (~0.5 h), and solid CO2 was added and concentrated
2
2
2
2
2
2
1
1
1
0H2
2
0H2
0H2
&KHOHU\WKULQH
2
0H2
%HUEHULQH
6DQJXLQDULQH
Scheme 1 natural bioactive Qacs that contain isoquinoline scaffold.
Abbreviation: Qacs, quaternary ammonium compounds.
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synthesis and antimicrobial activity
in vacuum. Crude product was dissolved in CH2Cl2, the pre- (s)-(1-(4-bromophenyl)-6,7-dimethoxy-1,2,3,4-
cipitate was filtered off and the filtrate was concentrated. tetrahydroisoquinolin-1-yl)metanol (8)
Step II: Crude diol was dissolved in CH3CN (25 mL), Yield 45%, amorphous solid. Purified on silica gel using
and a solution of NaIO4 (4.28 g, 20 mmol) in 38 mL H2O DCM:MeOH:NH3aq (95:5:0.5 → 90:10:0.5) as an eluent.
21
was added. Stirring was continued until disappearance of the [α]D =+33.6 (c =1, MeOH). IR (film): 3,322, 3,057, 2,999,
1
substrate (~4–6 days; thin layer chromatography control). 2,934, 2,833, 1,609, 1,515, 1,464 cm−1. H NMR (CDCl3):
Precipitate was filtered off and washed with ethyl acetate. The 2.55 (bm, 2H), 2.72 (dt, 1H, J =16.6, 5.1 Hz), 2.85 (m, 1H),
filtrate was concentrated in vacuo, approximately to 1/2 initial 2.94 (m, 1H), 3.04 (m, 1H), 3.74 (s, 3H), 3.88 (s, 3H), 3.95 (d,
volume and extracted with ethyl acetate (4×50 mL). Collected 1H, J =11.2 Hz), 4.11 (d, 1H, J =11.2 Hz), 6.41 (s, 1H), 6.65
extracts were dried with MgSO4, filtrated and concentrated (s, 1H), 7.23 (d, 2H, 8.6 Hz), 7.41 (d, 2H, 8.6 Hz). 13C NMR
in vacuo. Crude product was used in the next step.
(CDCl3): 29.19, 38.71, 55.81, 63.16, 67.25, 110.16, 111.97,
Step III: The obtained product in step II was dissolved in 121.44, 128.69, 128.97, 129.79, 131.40, 144.21, 147.54,
THF (30 mL), and during intensive stirring NaOH (1.20 g, 148.18. MS (EI, HR) m/z: (M+) Calcd. for C18H21NO3Br:
30 mmol) dissolved in water (30 mL) was added. Stirring 378.0705. Found: 378.0696.
was continued until it was judged that the reaction was over
(s)-(1-cyclohexyl-6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinolin-1-yl)methanol (9)
Yield 46%, amorphous solid. Purified on silica gel using
(thin layer chromatography control, 0.5–2 h). The solution
was diluted with water (50 mL) and extracted with DCM
(3×50 mL). Collected extracts were dried (MgSO4) and
concentrated in vacuo. Crude product was purified on silica
gel or via crystallization. Overall, the yield of amino alcohols
was in the range of ~40%–60%.
23
DCM:MeOH:NH3aq (95:5:0.5 → 90:10:0.5). [α]D =−62.9
(c =1, MeOH). 1H NMR (CDCl3): 0.9–1.94 (m, 11H), 2.01
(s, 3H), 2.71 (m, 1H), 2.84 (m, 1H), 3.23 (m, 1H), 3.32
(m, 1H), 3.80 (d, 1H, J =11.8 Hz), 3.85 (s, 3H), 3.86 (s, 3H),
4.08 (d, 1H, J =11.8 Hz), 6.58 (s, 1H), 6.63 (s, 1H), 6.82
(bs, 2H). 13C NMR (CDCl3): 25.51, 28.86, 29.28, 29.50,
29.91, 30.23, 30.78, 42.40, 49.31, 58.46, 58.83, 66.66, 68.76,
111.15, 114.13, 129.98, 150.47, 150.68, 180.24. MS (EI, HR)
m/z: (M+H+) Calcd. for C18H28NO3: 306.2065. Found:
306.2069.
(s)-(6,7-dimethoxy-1-methyl-1,2,3,4-
tetrahydroisoquinolin-1-yl)methanol (6)
Yield 42%, amorphous solid. Purified on silica gel using
DCM:MeOH:NH3aq = (95:5:0.5 → 90:10:0.5) as an eluent.
22
[α]D =−42.4 (c =1, MeOH). 1H NMR (CDCl3): 1.28 (s, 3H),
2.04 (s, 3H), 2.78 (m, 1H), 2.80 (m, 1H), 3.25 (m, 1H),
3.30 (m, 1H), 3.80 (d, 1H, J =11.8 Hz), 3.85 (s, 3H), 3.86
(s, 3H), 4.09 (d, 1H, J =11.8 Hz), 6.58 (s, 1H), 6.63 (s, 1H),
6.88 (bs, 2H). 13C NMR (CDCl3): 26.2, 29.20, 38.80, 58.36,
58.88, 66.64, 68.78, 111.12, 114.14, 129.99, 150.43, 150.66,
180.20. MS (EI, HR) m/z: (M+H+) Calcd. for C13H20NO3:
238.2055. Found: 238.2061.
general procedure for the preparation of
quaternary ammonium salts 11a–14c
Into a solution of amino alcohol (0.4 mmol) in MeCN or
DCM (2 mL), a proper dibromide (0.5 mmol) was added
followed by the addition of N,N-diisopropylethylamine
(DIPEA; 0.5 mmol). The reaction was run for 24 h at room
temperature, except for low-reactive cyclohexyl amino
alcohol 9, which required elevated temperature (60°C).
The reaction mixture was concentrated in vacuo, and crude
product was purified via crystallization or chromatography
on silica gel. Due to considerable broadening of some
NMR signals of ammonium salts with a bulky substituent at
C-1′ (aryl, cyclohexyl), only selected data are given.
(s)-(6,7-dimethoxy-1-(4-methoxyphenyl)-1,2,3,4-
tetrahydroisoquinolin-1-yl)methanol (7)
Yield 57%, amorphous solid. Purified on silica gel using
DCM:MeOH:NH3aq = (95:5:0.5 → 90:10:0.5) as an eluent.
22
[α]D =−26.7 (c =1, MeOH). 1H NMR (CDCl3): 2.72 (m, 2H),
2.88 (m, 1H), 2.96 (m, 1H), 3.05 (m, 1H), 3.73 (s, 3H),
3.78 (s, 3H), 3.89 (s, 3H), 3.96 (d, 1H, J =11.2 Hz), 4.17
(d, 1H, J =11.2 Hz), 6.46 (s, 1H), 6.64 (s, 1H), 6.82 (d, 1H, (s)-1′-(hydroxymethyl)-6′,7′-dimethoxy-1′-methyl-
J =8.8 Hz), 7.25 (d, 1H, J =8.8 Hz). 13C NMR (CDCl3): 3′,4′-dihydro-1′h-spiro[isoindoline-2,2′-isoquinolin]-
29.13, 38.61, 55.22, 55.80, 56.04, 63.24, 67.47, 110.31, 2-ium bromide (11a)
20
111.80, 113.62, 128.54, 129.10, 129.52, 136.92, 147.46, [α]D =+15.8 (c =1, MeOH). IR (film): 3,230, 3,010, 2,936,
148.01, 158.74. MS (EI, HR) m/z: (M+) Calcd. for C19H24NO4: 1,611, 1,523, 1,493, 1,408, 1,362 cm−1. 1H NMR (CD3OD):
329.1695. Found: 329.1693.
1.85 (s, 3H), 3.25 (m, 1H), 3.41 (m, 1H), 3.83 (s, 3H), 3.84
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(s, 3H), 3.89 (m, 1H), 3.98 (d, 1H, J =13.7 Hz), 4.21 (d, (m, 2H), 3.67 (s, 3H), 3.71–3.75 (m, 1H), 3.80 (s, 3H), 3.90
1H, J =13.7 Hz), 4.39 (m, 1H), 4.82 (d, 1H, J =15.5 Hz), (s, 3H), 4.04–4.09 (m, 1H), 4.38 (d, 14.4 Hz, 1H), 4.58
5.00 (d, 1H, J =15.5 Hz), 5.87 (d, 1H, J =15.6 Hz), 5.87 (s, (d, 13.6 Hz, 1H), 4.90 (d, 15.0 Hz, 1H), 5.14 (d, 13.6 Hz, 1H),
1H), 5.92 (s, 1H), 7.34 (d, 1H, J =7.1 Hz), 7.41 (m, 3H). 13C 5.52 (d, 14.4 Hz, 1H), 5.70 (d, 15.0 Hz, 1H), 6.58 (s, 1H),
NMR (CD3OD): 18.13, 23.48, 55.07, 55.43, 55.78, 63.76, 6.94–6.97 (m, 3H), 7.25–7.28 (m, 1H), 7.32–7.36 (m, 3H),
65.98, 67.09, 74.91, 109.82, 111.48, 122.48, 122.62, 123.61, 7.40–7.57 (m, 2H). 13C NMR (CD3OD) 23.9, 54.5, 54.7,
125.41, 128.75, 128.85, 132.11, 133.33, 148.51, 149.84. MS 55.1, 55.2, 63.0, 65.3, 65.6, 78.9, 111.0, 111.4, 113.6, 148.4,
(EI, HR) m/z: (M+) Calcd. for C21H26NO3: 340.1913. Found: 149.8, 161.0. MS (EI, HR) m/z: (M+) Calcd. for C27H30NO4:
340.1910.
432.2175. Found: 432.2173.
(s)-1′-(hydroxymethyl)-5,6,6′,7′-tetramethoxy-1′-
methyl-3′,4′-dihydro-1′h-spiro[isoindoline-2,2′-
(s)-1′-(hydroxymethyl)-5,6,6′,7′-tetramethoxy-1′-(4-
methoxyphenyl)-3′,4′-dihydro-1′h-spiro[isoindoline-
isoquinolin]-2-ium bromide (11b)
2,2′-isoquinolin]-2-ium bromide (12b)
22
21
[α]D =+14.5 (c =1, MeOH). IR (film): 2,989, 2,978, 2,039, [α]D =−2.33 (c =1, MeOH/DCM 8/2). IR (film): 3,402,
2,898, 2,771, 2,664, 1,608, 1,512, 1,467, 1,445 cm−1. H 3,244, 2,979, 2,942, 2,689, 1,725, 1,607, 1,515, 1,466 cm−1.
1
NMR (CD3OD): 1.84 (s, 3H), 3.41 (m, 1H), 3.80 (s, 3H), 3.84 1H NMR (CDCl3): 3.11–3.18 (m, 1H), 3.29–3.35 (br, 2H),
(s, 9H), 3.91 (m, 1H), 3.95 (d, 1H, J =13.7 Hz), 4.19 (d, 1H, 3.69 (s, 3H), 3.70–3.78 (m, 1H), 3.82 (s, 3H), 3.83 (s, 3H),
J =13.7 Hz), 4.39 (m, 1H), 4.72 (dd, 2H, J =7.9, 15.2 Hz), 3.84 (s, 3H), 4.18–4.27 (br, 1H), 4.65 (d, 13.6 Hz, 1H),
4.89 (d, 1H, J =14.4 Hz), 5.78 (d, 1H, J =15.4), 6.86 (s, 1H), 4.72–4.88 (br, 1H), 4.94 (d, 13.9 Hz, 1H), 5.19–5.34 (br,
6.90 (s, 1H), 6.91 (s, 1H), 7.0 (s, 1H). 13C NMR (CD3OD): 1H), 5.42–5.51 (br, 1H), 6.22 (br, 1H), 6.37 (br, 1H), 6.70
13
17.90, 23.51, 42.42, 54.46, 55.29, 55.31, 55.98, 64.07, (s, 1H), 6.73 (s, 1H), 6.84 (s, 1H), 6.86–6.70 (br, 1H). C
66.33, 67.12, 74.83, 105.62, 105.74, 109.81, 111.48, 123.68, NMR (CDCl3): 24.5, 53.7, 54.1, 55.4, 56.1, 56.2, 56.3, 56.3,
123.70, 124.96, 125.49, 148.49, 149.84, 150.39, 150.49. MS 64.9, 67.9, 72.8, 106.0, 106.7, 110.9, 111.6, 114.1, 148.1,
(EI, HR) m/z: (M+) Calcd. for C23H30NO5: 400.2124. Found: 149.7, 150.0, 150.3, 160.8. MS (EI, HR) m/z: (M+) Calcd.
400.2123.
for C29H34NO6: 492.2386. Found: 492.2386.
(s)-1′-(hydroxymethyl)-6′,7′-dimethoxy-1′-methyl-
3′,4′,5,7-tetrahydro-1′h-spiro[[1,3]dioxolo[4,5-f]
(s)-1′-(hydroxymethyl)-6′,7′-dimethoxy-1′-(4-
methoxyphenyl)-3′,4′,5,7-tetrahydro-1′h-spiro[[1,3]
dioxolo[4,5-f]isoindole-6,2′-isoquinolin]-2′-ium
isoindole-6,2′-isoquinolin]-2′-ium bromide (11c)
21
[α]D =+14.2 (c =1, MeOH). IR (film): 3,227, 2,963, 2,939, bromide (12c)
1
22
2,834, 1,611, 1,520, 1,481 cm−1. H NMR (CD3OD): 1.85 [α]D =−0.31 (c =1, MeOH/DCM 9/1). IR (film): 3,402,
(s, 3H), 3.28 (m, 1H), 3.41 (m, 1H), 3.85 (s, 6H), 3.92 3,244, 2,979, 2,942, 2,689, 1,725, 1,607, 1,515, 1,482 cm−1.
(m, 2H), 3.96 (d, 1H, J =13.6 Hz), 4.19 (d, 1H, J =13.6 Hz), 1H NMR (CDCl3): 3.24–3.41 (m, 1H), 3.69 (s, 3H), 3.83
4.38 (m, 1H), 4.68 (d, 2H, J =15.1 Hz), 5.73 (d, 1H, (s, 3H), 3.91 (s, 3H), 4.14 (br, 1H), 4.67 (br, 1H), 4.92
J =13.3 Hz), 6.01 (dd, 2H, J =0.9, 3.5 Hz), 6.77 (s, 1H), (br, 1H), 5.17 (br, 1H), 5.47 (br, 1H), 5.93–6.00 (m, 2H), 6.38
6.86 (s, 1H), 6.87 (s, 1H), 6.92 (s, 1H). 13C NMR (CD3OD): (s, 1H), 6.61 (s, 1H), 6.71–6.76 (m, 2H), 6.84–7.01 (br, 2H).
17.96, 23.83, 55.07, 55.42, 55.93, 63.32, 63.90, 67.07, 13C NMR (CDCl3): 24.4, 53.8, 55.4, 56.1, 56.2, 67.8, 101.9,
74.97, 101.97, 103.74, 102.83, 109.83, 111.48, 123.65, 103.5, 104.4, 110.9, 160.8. MS (EI, HR) m/z: (M+) Calcd.
124.71, 125.38, 125.99, 148.51, 148.98, 149.07, 149.86. for C29H30NO6: 476.2073. Found: 476.2078.
MS (EI, HR) m/z: (M+) Calcd. for C22H26NO5: 384.1811.
Found: 384.1811.
(s)-1′-(4-bromophenyl)-1′-(hydroxymethyl)-6′,7′-
dimethoxy-3′,4′-dihydro-1′h-spiro[isoindoline-2,2′-
isoquinolin]-2-ium bromide (13a)
(s)-1′-(hydroxymethyl)-6′,7′-dimethoxy-1′-(4-
methoxyphenyl)-3′,4′-dihydro-1′h-spiro[isoindoline-
22
[α]D =−1.44 (c =1, MeOH/DCM 9:1). IR (film): 3,213,
2,2′-isoquinolin]-2-ium bromide (12a)
2,925, 2,854, 2,685, 2,517, 2,490, 1,736, 1,612, 1,586,
[α]D =−0.85 (c =0.7, MeOH). IR (film): 3,227, 2,963, 2,939, 1,520, 1,489, 1,465 cm−1. 1H NMR (CD3OD): 3.33 (br, 1H),
21
2,834, 1,611, 1,520, 1,481 cm−1. 1H NMR (CD3OD):3.33–3.38 3.38–3.43 (m, 1H), 3.67 (s, 3H), 3.89 (s, 3H), 4.02 (br, 1H),
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synthesis and antimicrobial activity
4.40 (br, 1H), 4.60 (d, 13.5 Hz, 1H), 4.93 (d, 15.1 Hz, 1H), MS (EI, HR) m/z: (M+) Calcd. for C26H34NO3 408.2539.
5.14 (d, 13.5 Hz, 1H), 5.63 (br, 1H), 5.73 (d, 15.1 Hz, 1H), Found: 408.2529.
13
6.56 (s, 1H), 6.96 (s, 1H). C NMR (CD3OD): 23.8, 55.1,
63.1, 65.4, 66.1, 78.1, 111.0, 111.1, 148.6, 150.0. MS (EI,
HR) m/z: (M+) Calcd. for C26H27NO3Br: 480.1174. Found:
(s)-1′-cyclohexyl-1′-(hydroxymethyl)-5,6,6′,7′-
tetramethoxy-3′,4′-dihydro-1′h-spiro[isoindoline-
480.1174.
2,2′-isoquinolin]-2-ium bromide (14b)
24
[α]D =−13.9 (c =0.8, MeOH). IR (film): 3,230, 2,928, 2,854,
1,737, 1,612, 1,514, 1,465, 1,454, 1,350, 1,363, 1,226 cm−1.
1H NMR (CDCl3): 0.85–0.92 (m, 1H), 1.02–1.15 (m, 3H),
1.56–1.71 (m, 4H), 1.84–1.96 (m, 3H), 3.18–3.43 (m, 3H),
3.80 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H), 3.89 (s, 3H), 4.15
(d, 14.3 Hz, 1H), 4.67–4.84 (m, 3H), 5.83 (d, 14.3 Hz, 1H),
5.91 (d, 13.6 Hz, 1H), 6.63 (s, 1H), 6.70 (s, 1H), 6.96 (s, 1H),
7.04 (s, 1H). 13C NMR (CDCl3): 24.4, 25.3, 25.7, 27.5, 29.7,
46.0, 55.2, 55.9, 56.2, 56.3, 56.9, 65.6, 67.7, 69.1, 80.6,
105.5, 106.3, 111.4, 111.6, 148.0, 149.5, 150.2, 150.3. MS
(EI, HR) m/z: (M+) Calcd. for C28H38NO5: 468.2750. Found:
468.2740.
(s)-1′-(4-bromophenyl)-1′-(hydroxymethyl)-5,6,6′,7′-
tetramethoxy-3′,4′-dihydro-1′h-spiro[isoindoline-
2,2′-isoquinolin]-2-ium bromide (13b)
[α]D =+1.41 (c =1, MeOH/DCM 9:1). IR (film): 3,202,
3,004, 2,962, 2,939, 2,836, 1,722, 1,612, 1,586, 1,512,
22
1
1,466 cm−1. H NMR (CD3OD): 3.34–3.39 (m, 1H), 3.67
(s, 3H), 3.71–3.75 (m, 1H), 3.78 (s, 3H), 3.80 (s, 3H), 3.89
(s, 3H), 4.12 (br, 1H), 4.34 (br, 1H), 4.81 (d, 14.8 Hz, 1H),
5.07 (d, 13.4 Hz, 1H), 5.48 (br, 1H), 5.64 (d, 14.8 Hz, 1H),
6.56 (s, 1H), 6.80 (s, 1H), 6.56 (s, 1H), 6.88 6.56 (s, 1H), 6.95
6.56 (s, 1H). 13C NMR (CD3OD): 24.0, 55.7, 63.8, 65.7, 66.3,
78.9, 106.7, 106.7, 111.5, 111.8, 148.7, 150.3, 150.5, 150.5.
MS (EI, HR) m/z: (M+) Calcd for C28H31NO5Br: 540.1386.
Found: 540.1385.
(s)-1′-cyclohexyl-1′-(hydroxymethyl)-6′,7′-
dimethoxy-3′,4′,5,7-tetrahydro-1′h-spiro[[1,3]
dioxolo[4,5-f]isoindole-6,2′-isoquinolin]-2′-ium
bromide (14c)
(s)-1′-(4-bromophenyl)-1′-(hydroxymethyl)-6′,7′-
dimethoxy-3′,4′,5,7-tetrahydro-1′h-spiro[[1,3]
dioxolo[4,5-f]isoindole-6,2′-isoquinolin]-2′-ium
bromide (13c)
23
[α]D =−1.90 (c =1, MeOH). IR (film): 3,243, 2,935, 2,855,
1
2,691, 1,715, 1,611, 1,521, 1,483, 1,365 cm−1. H NMR
(CD3OD): 1.03 (br, 1H), 1.11–1.23 (br, 3H), 1.41–1.69
(br, 4H), 1.88 (br, 1H), 2.15–2.26 (m, 2H), 3.32–3.44
(m, 2H), 3.85 (s, 3H), 3.86 (s, 3H), 4.03–4.11 (br, 1H), 4.34
(d, 14.6 Hz, 1H), 4.50–4.70 (m, 3H), 5.51 (d, 14.5 Hz, 1H),
5.66 (d, 14.5 Hz, 1H), 5.95–6.02 (m, 2H), 6.67 (s, 1H), 6.87
(s, 1H), 6.91 (s, 1H), 6.94 (s, 1H). 13C NMR (CD3OD): 23.5,
25.6, 26.4, 27.0, 28.9, 31.3, 42.5, 45.3, 54.5, 56.5, 66.8, 67.8,
80.3, 111.6, 112.1, 147.4, 148.7. MS (EI, HR) m/z: (M+)
Calcd. for C27H34NO5: 452.2437. Found: 452.2429.
22
[α]D =+1.57 (c =1, MeOH). IR (film): 3,218, 2,972, 2,942,
2,684, 2,518, 2,492, 1,612, 1,520, 1,482, 1,396, 1,353,
1,298 cm−1. 1H NMR (CDCl3): 3.36 (br, 1H), 3.70 (s, 3H),
3.77 (br, 1H), 3.91 (s, 3H), 4.23 (br, 1H), 4.71 (br, 1H), 4.88
13
(br, 1H), 5.07 (br, 1H), 5.57 (br, 1H). C NMR (CDCl3):
24.5, 49.4, 54.1, 54.4, 56.1, 56.3, 64.6, 67.5, 72.8, 101.9,
103.6, 104.3. MS (EI, HR) m/z: (M+) Calcd. for C27H27NO5Br:
524.1073. Found: 524.1074.
(s)-1′-cyclohexyl-1′-(hydroxymethyl)-6′,7′-
dimethoxy-3′,4′-dihydro-1′h-spiro[isoindoline-2,2′-
Biological assays
antibiotics and bacterial strains
isoquinolin]-2-ium bromide (14a)
Norfloxacin (Nor) was obtained from Merck & Co., Inc.
24
[α]D =−13.9 (c =0.8, MeOH). IR (film): 3,547, 3,464, (Whitehouse Station, NJ, USA). Bacterial reference strains
3,408, 3,186, 3,009, 2,934, 2,855, 1,609, 1,520, 1,453 cm−1. with well-characterized properties from American Type
1H NMR (CDCl3): 0.99–1.23 (m, 4H), 1.29–1.41 (m, 1H), Culture Collection (ATCC, Manassas, VA, USA) were used
1.85–1.94 (m, 1H), 2.05–2.14 (m, 1H), 2.20–2.27 (m, 1H), in the study, as well as strains isolated from invasive infections
3.21–3.40 (m, 2H), 3.73–3.82 (m, 1H), 3.89 (s, 3H), 3.97 among in- and outpatients, from the collection of the Depart-
(s, 3H), 4.20 (d, 14.7 Hz, 1H), 4.75 (m, 1H), 4.83 (d, 14.1 Hz, ment of Microbiology, Medical University of Bialystok. All
1H), 5.86–5.93 (m, 2H), 6.70 (s, 1H), 7.03 (s, 1H). 13C NMR strains were stored according to the international standards
(CDCl3): 24.3, 25.7, 27.0, 27.4, 29.5, 31.2, 45.9, 55.0, 56.0, at −80°C in cryobank tubes (Mast Diagnostica, Reinfeld,
57.0, 58.9, 67.3, 68.9, 80.6, 111.3, 111.6, 147.9, 149.4. Germany), containing microporous beads suspended in a
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special hypertonic preserving solution, ensuring long-term on microtiter plates according to the European Committee on
storage and preservation of microorganisms, including fas- Antimicrobial Susceptibility Testing (EUCAST) or Clinical
tidious ones. Briefly, all bacterial strains were retrieved from and Laboratory Standard Institute (CLSI) guidelines. Briefly,
fresh bacterial agar cultures, suspended in the preserving Mueller-Hinton broth (MHB; Emapol, Gdansk, Poland)
solution, mixed with the beads and, after removing excess of and MHB supplemented with 5% horse blood and 20 mg/L
the solution, stored in a low-temperature freezer at −80°C. beta-nicotinamide adenine dinucleotide were used to culture
Prior to experiments, the bacterial strains under the study non-fastidious bacteria and fastidious bacteria, respectively.
were revitalized in Trypticase Soy Broth medium (BD, The chemical agents were diluted with ethanol to obtain the
Franklin Lakes, NJ, USA), a universal medium to culture stock solutions of concentration 400 mg/L. Subsequently,
various microorganisms, including aerobic and facultative serial dilutions from 200 to 0.195 mg/L were prepared in the
anaerobic bacteria as well as fungi. Subsequently, bacterial appropriate MHB and inoculated with bacterial suspensions
suspensions were prepared from fresh, 24-hour cultures, to obtain the final inoculum 105 CFU/mL. The MIC values
to obtain inoculum 108 colony-forming unit (CFU)/mL. were recorded after 24-hour incubation at 35°C. The highest
Antibacterial activity of the chemical agents was tested on dilution that inhibits bacterial growth was assumed as MIC.
the following bacterial species: bacterial ATCC reference All experiments were performed in triplicate.20–22
strains, gram-positive coccus Staphylococcus aureus ATCC
25923; Streptococcus pyogenes ATCC 19615; Streptococcus assessment of bactericidal activity
mutans ATCC 35668; Streptococcus salivarius ATCC 13419; MBCs that kill 99.9% of bacteria in the initial inoculum under
Enterococcus faecalis ATCC 29212; gram-negative coccus standardized in vitro conditions were used to determine the
Moraxella catarrhalis ATCC 25238 and gram-negative bactericidal activity of the chemical agents. To that end, after
rod Escherichia coli ATCC 25922 (facultative anaerobe); the estimation of MICs, bacteria from the dilutions were
Campylobacter jejuni ATCC33560 (microaerophile). Tests subcultured on agar media (Columbia Agar and sheep blood;
were performed using clinical bacterial isolates, including Oxoid Limited, Basingstoke, UK), and MBC values were
MDR strains: S. aureus (isolates from skin and soft tissue recorded after 24-hour incubation at 35°C. The lowest con-
infections); S. pyogenes (isolates from pharyngitis cases and centration that killed at least 99.9% of bacteria was assumed
skin infections); S. mutans and S. salivarius (isolates from as MBC. All experiments were performed in triplicate.23
periodontal pockets and oral mucous membranes); E. faecalis
(isolates from urinary tract infections); M. catarrhalis (isolates controls
from respiratory tract infections); E. coli (isolates from urinary The following internal quality controls were included in
tract infections and skin and wound infections); C. jejuni (iso- the experiments: control A – to determine the quality of
lates from feces of patients with diarrhea). Bacillus subtilis bacterial growth in broth media without the chemical agents
strains were isolated from food.
under study; control B – as the verification of the sterility
of microbiological media; control C – to determine in vitro
bacteriostatic activity (MIC) of Nor antibiotic for the bacte-
rial strains under study as the reference control, which were
assessment of bacterial susceptibility to
the chemical agents
A quantitative method, microdilution in liquid (broth) medium performed simultaneously with the planned experiments with
performed on microtiter plates, was used to determine the anti- the chemical agents.
bacterial activity of the chemical agents under study. Briefly,
the method allowed estimating in vitro minimal inhibitory red blood cell lysis
concentrations (MICs, mg/L) and minimal bactericidal con- The hemolytic activity of compounds 11a–c, 12a–c, 13a–c
centrations (MBCs, mg/L) of the tested agents. To that end, and 14a–c (0–200 mg/L), against human red blood cells
broth media containing serial dilutions of the chemical agents (Rockland Immunochemicals Inc., Limerick, PA, USA), was
were inoculated with tested bacterial strains, and after 24-hour tested. Cells were suspended in phosphate-buffered saline
incubation the turbidity of the medium was recorded.19
(PBS) and incubated for 1 h at 37°C after the addition of test
molecules. The hemoglobin concentration in supernatants
after centrifugation at 2,000× g was monitored by measuring
assessment of bacteriostatic activity
Susceptibility of non-fastidious bacteria and fastidious the absorbance at 540 nm. Then, 100% hemolysis (positive
bacteria to the chemical agents under study was estimated control) was taken from samples in which 2% Triton X-100
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synthesis and antimicrobial activity
was added.24 The research protocol was approved by the was conducted using the Origin 7.5 software (OriginLab
Committee for Ethics and Supervision on Human and Ani- Corporation, Northampton, MA, USA). Statistical dif-
mal Research, Medical University of Bialystok (Nr N/ST/ ferences in multiple groups were determined by one-way
ZB/15/001/2209 [153-09-535F]).
ANOVA followed by Tukey’s test. P,0.05 was considered
statistically significant.
Cell culture fibroblasts
Human skin fibroblasts (CCD 1112Sk) were obtained from
Results
ATCC. Cells were maintained in Dulbecco’s Minimal chemistry
Essential Medium (DMEM) supplemented with 10% fetal We envisioned that a new type of spiro-QACs can be obtained
bovine serum (FBS), 2 mM glutamine, 50 U/mL penicil- via double alkylation of easily available, by our methodol-
lin and 50 mg/mL streptomycin at 37°C in a humidified ogy, enantiopure amino alcohols with 1,2-bis(bromomethyl)
atmosphere containing 5% CO2. Cells were cultured in benzene. To verify this idea, several 10b(S) configurated
Costar flasks and grown in 5% CO2 at 37°C in high humid hexahydropyrroloisoquinolines (1–4) were obtained from
atmosphere to subconfluence (90%–95%). Subconfluent l-tartaric acid using our procedure16,17 (Scheme 2). Cyclo-
cells were treated with 0.05% trypsin and 0.02% EDTA in hexyl derivative 5 was prepared via Pd-catalyzed hydrogen
calcium-free PBS, counted in a hemocytometer and seeded at reduction of a 10β-phenyl substituted compound 4. There-
5×105 cells/well in six-well plates (Nunc) in 2 mL of growth fore, obtained pyrroloisoquinolines were submitted to base
medium. The cells were grown in a monolayer (80% conflu- hydrolysis of acetate esters. Isolated corresponding diols
ence) before being used for further analysis.
were oxidized with sodium periodate to give an epimeric
mixture of cyclic hemiaminals type A.18
cell viability assay
The masked dialdehydes A treated with a strong base
The growth inhibitory effects of the test compounds against (NaOH) underwent an intramolecular, highly chemoselec-
human skin fibroblast cells were measured by using 3-(4,5- tive Cannizzaro reaction, to give corresponding β-amino
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide alcohols 6–9. Finally, the title quaternary N-spiro ammonium
(MTT) assay. Compounds were dissolved in dimethyl sulfox- bromides 11a–14c were obtained by double alkylation
ide (DMSO) and screened at a range of concentrations against reaction of enantiopure amino alcohols 6–9 with 1,2-
normal cells. The cytotoxic activity was determined after bis(bromomethyl)benzene derivatives 10a–c (Scheme 3) in
24 h. All experiments were repeated at least three times.
the presence of DIPEA.
Fluorescent microscopy assay
antimicrobial activity
The cell viability was estimated 24 h after the addition of the The antimicrobial activity of compounds 11a–14c was tested
study compounds to assess apoptosis. The cell suspension against gram-positive cocci of the genus Staphylococcus,
(250 μL) was stained with 10 μL of the dye mixture (10 μM Enterococcus and Streptococcus and gram-positive bacilli of
acridine orange and 10 μM ethidium bromide), which was the genus Bacillus. We also tested the antimicrobial activity
prepared in PBS. Acridine orange (fluorescent DNA-binding of these compounds against gram-negative bacilli E. coli of
dye) intercalated into DNA, making it appear green, and the family Enterobacteriaceae and C. jejuni, as well as against
bound to RNA, staining it red/orange. Ethidium bromide gram-negative M. catarrhalis. Nor was used for the control
was only taken up by nonviable cells; its fluorescence pre- to analyse tested strains for their susceptibility/resistance.
dominated that of the acridine orange, making the chromatin The new quaternary ammonium salts inhibited the growth
of necrotic cells appear orange. Cells cultured in a drug-free of more than 95% of gram-positive and gram-negative bac-
medium were used as controls. Analysis was performed using teria tested in the concentration range from 12 to 200 mg/L
Nikon Eclipse Ti inverted microscope, and the results were (Tables 1 and 2). The results of antibacterial activity showed
analyzed with NIS-Elements software (both from Nikon that gram-negative bacteria were more susceptible toward
Instruments, Melville, NY, USA).
the tested compounds than gram-positive bacteria. The
compounds 11a–c showed MIC above 200 mg/L against
S. aureus and B. subtilis. In contrast, the compound 12a at
statistical analysis
All numerical data are presented as mean ± SD from at a concentration of 25 mg/L inhibited the growth of cocci
least three independent experiments. Statistical analysis S. aureus and S. mutans. Five of the compounds 12c, 13a,
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20H
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Scheme 2 synthesis of amino alcohols 6–9 from l-tartaric acid.
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Scheme 3 synthesis of compounds 11a–14c.
Abbreviation: DiPea, N,N-diisopropylethylamine.
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synthesis and antimicrobial activity
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13b, 14a, 14c showed inhibition of the growth of bacilli in
the range of 50–200 mg/L.
We observed more differences in the MIC and MBC
values of the tested compounds obtained from gram-negative
bacteria. Most of the tested compounds inhibited the growth
of E. coli at a concentration range of 100–200 mg/L. It was
found that only 13c inhibited the growth of a strain of E. coli by
the MIC =50 mg/L. The compounds 12a, 13a, 13b, 14a, 14b
inhibited the growth of M. catarrhalis at the lowest concentra-
tion (MIC value between 15 and 50 mg/L). The compounds
11c, 12b, 12c, 13c, 14c inhibited the growth of C. jejuni at the
lowest concentration (MIC in the range of 12–25 mg/L).
The structure–antimicrobial activity relationship of the
synthesized compounds revealed that the presence of phe-
nyl substituent at the quaternary carbon stereocenter in the
compounds 12a–c and 13a–c leads to higher activity than
those having cyclohexyl and methyl substituents. Among
compounds 12 and 13, those having methoxyphenyl moieties
showed greater activity than the compounds with bromophe-
nyl units. Substitution of the phenyl ring in positions 3 and 4
at isoindoline moiety does not influence the antimicrobial
activity significantly.
The selective antibacterial activity of the compounds is
often a desirable feature. The on-target therapies are recom-
mended antibiotic/chemotherapeutic agents with a narrow
spectrum of activity directed against specific pathogens,
without adversely affecting the physiologic flora saline envi-
ronment. Because the MBC is usually a multiple of the MIC
values, the ratio MBC/MIC is an additional indicator of anti-
microbial activity. High antibacterial compounds have bacteri-
cidal activity ratio MBC/MIC #4. Compounds 11a–14c have
MBC/MIC ratio of 1–4 for 100% of the strains (Table 3).
The very promising compound from the group of the
tested compounds was 12a. It possessed stronger antimicro-
bial potential than Nor against S. mutans and B. subtilis thus
might be an alternative to use in lower doses to receive a bac-
tericidal effect. The compounds 11c, 12b, 12c, 14c were more
active against C. jejuni in comparison with the control.
evaluation of cell toxicity
Nonspecific insertion of antibacterial QACs into host cell
membranes can cause toxicity. Host cell membrane permea-
bilization can be measured by the release of proteins such
as hemoglobin from the cytosol to the extracellular space.
We showed no significant membrane permeabilization in
the range at which the compounds 11a–14c have bacteri-
cidal activity by evaluating hemoglobin release (Figure 1).
However, an increase in hemoglobin release was observed
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synthesis and antimicrobial activity
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Figure 1 Toxicity of compounds 11a–c, 12a–c, 13a–c and 14a–c measured by rBc hemoglobin release from human red blood cells after the addition of 11a–c, 12a–c,
13a–c and 14a–c (0–200 μM), followed by incubation for 1 h at 37°c.
Note: Mean values ± sD from three independent experiments (n=3) performed in duplicate are presented.
Abbreviation: rBc, red blood cell.
with increasing concentration. The compound 11c was the an assessment of the dual acridine orange/ethidium bromide
strongest hemolytic agent, but even for 11c, bactericidal fluorescent staining, and visualized it under a fluorescent
concentrations against C. jejuni were below its minimal microscope (Figure 3). We can observe that live cells with
hemolytic concentration.
normal nuclei appeared uniformly green in the control as
well as after treatment with compounds 11a–14c using the
high concentration (200 μM). Dual acridine orange/ethidium
Viability of human skin fibroblasts
The viability of human skin fibroblasts was determined bromide fluorescent staining confirmed the results obtained
after 24 hours of incubation with tested compounds. Our by cell viability assay. We proved that all compounds had no
studies revealed that all compounds were not cytotoxic for effect on induction of apoptosis or necrosis in normal cells.
normal cells. The viability in all cases was similar to control
(untreated cells). We detected more than 95% of viable cells Discussion
after 24 hours of incubation with compounds 11a–14c used in Our novel chiral quaternary N-spiro ammonium bromides
three concentrations: 50, 100 and 200 μM (Figure 2). To check with 3′,4′-dihydro-1′H-spiroisoindoline-2,2′-isoquinoline
the influence of tested compounds on apoptosis, we carried out skeleton possess antimicrobial activity against gram-positive
100
80
60
40
20
0
Control 11a 11b 11c 12a 12b 12c 13a 13b 13c 14a 14b 14c
50 µM
100 µM
200 µM
Figure 2 Viability of human cultured fibroblasts exposed to various concentrations (50, 100 and 200 μM) of tested compounds for 24 hours.
Note: Mean values ± sD from three independent experiments (n=3) performed in duplicate are presented.
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ꢀꢃDꢁꢂꢃꢄꢄꢁ—0ꢅ
ꢀꢇDꢁꢂꢃꢄꢄꢁ—0ꢅ
ꢀꢃEꢁꢂꢃꢄꢄꢁ—0ꢅ
ꢀꢇEꢁꢂꢃꢄꢄꢁ—0ꢅ
ꢀꢃFꢁꢂꢃꢄꢄꢁ—0ꢅ
ꢀꢇFꢁꢂꢃꢄꢄꢁ—0ꢅ
Figure 3 The influence of tested compounds (200 μM) on the induction of apoptosis in human skin fibroblasts after 24 hours of incubation, obtained using dual AO/EB
fluorescent staining.
Note: images were taken using a microscope with an inverted camera at 100× magnification.
Abbreviation: aO/eB, acridine orange/ethidium bromide.
and gram-negative bacteria. We observed that the inhibition mutants of E. coli and S. typhimurium proved that the outer
of the growth of selected bacteria depends on the concentra- membrane of gram-negative bacteria acts as a barrier that
tion of tested compounds and there were differences between limits the entry of different types of antibacterial agents.40–43
sensitivity of selected bacteria species.
The cell membrane, lipopolysaccharides, in wild-type gram-
A majority of the synthesized compounds show the negative bacteria act as a barrier which limits the access of
strongest antibacterial properties toward some gram-negative hydrophobic molecules to phospholipid and thence to the cell
bacteria (M. catarrhalis and C. jejuni) with a high level of interior. Apart from the hydrophilic and hydrophobic input
selectivity. It has been shown for many years that QACs paths, the third way was proposed to be by cationic agents
are either membrane-active agents or the plasma mem- such as QAC and biguanides. It is argued that they damage
brane agents in yeasts.25 On the model proposed by Salton, the outer membrane, thus fostering their own uptake.6,44
microorganisms exposed to these cationic agents follow the Our in vitro data confirmed that all tested compounds do
specific sequence of events. The agent is adsorbed and pen- not possess cytotoxic and proapoptotic potency in normal
etrated into the cell wall, then the reaction with cytoplasmic cells such as human skin fibroblasts. Our study needs further
membrane occurs followed by membrane disorganization. examination to explain in detail the molecular mechanisms
The consequence of these molecular events is the degrada- of action of chiral quaternary N-spiro ammonium bromides
tion of proteins and nucleic acids and wall lysis caused by with 3′,4′-dihydro-1′H-spiro[isoindoline-2,2′-isoquinoline]
autolytic enzymes.26,27
skeleton. In further studies, we will try to check whether their
The composition of cell membrane layers depends on the mechanism of action appears to be associated with damage
organism type and may act as a permeability barrier, in which to the cell membrane or whether also to inhibition of DNA
there may be a reduced uptake.28–32 Teichoic acid and peptido- replication or binding to other components of the cell.
glycan are major components of staphylococci cell wall.6,33–38
None of these compounds are an effective barrier to the entry Conclusion
of antiseptics and disinfectants. The high molecular weight Our findings proved that all novel tested chiral quaternary
substances can easily pass through the cell wall of staphylo- N-spiro ammonium bromides with 3′,4′-dihydro-1′H-
cocci and vegetative Bacillus spp., and it could explain the spiro[isoindoline-2,2′-isoquinoline] skeleton inhibited the
high sensitivity of these strains to QAC agents.30,39 Studies growth of more than 95% of gram-positive and gram-negative
based on staphylococci and gram-negative bacteria as well as bacteria tested in the concentration range from 12 to
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synthesis and antimicrobial activity
16. Mostowicz D, Wójcik R, Dołęga G, et al. Diastereoselective synthesis
of 10b-substituted hexahydropyrroloisoquinolines from L-tartaric acid.
Creation of a quaternary carbon stereocentre via N-acyliminium ion
cyclization. Tetrahedron Lett. 2004;45:6011–6015.
17. Kałuża Z, Mostowicz D, Dołęga G, et al. 10b-Substituted hexahy-
dropyrroloisoquinolines: studies on diastereoselective formation of
a quaternary carbon stereocenter via N-Acyliminium ion cyclization.
Tetrahedron. 2006;62:943–953.
200 mg/L. A majority of the synthesized compounds show the
strongest antibacterial properties toward some gram-negative
bacteria (M. catarrhalis and C. jejuni) with a high level of
selectivity. High antibacterial compounds have bactericidal
activity ratio MBC/MIC #2. Our studies revealed that all
tested compounds are active only toward bacteria, but do not
possess cytotoxic and proapoptotic activity in normal cells
such as human skin fibroblasts.
18. Kałuża Z, Mostowicz D, Dołęga G, et al. A new route to optically pure
highly functionalized tetrahydroisoquinolines with a quaternary carbon
stereocenter. Tetrahedron. 2008;64:2321–2328.
19. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating
antimicrobial activity: a review. J Pharm Anal. 2016;6:71–79.
20. European Committee on Antimicrobial Susceptibility Testing [homep-
age on the Internet]. Breakpoints Tables for Interpretation of MICs and
Zones Diameters. Version 5.0. 2015. Available from: http://www.eucast.
org. Accessed June 14, 2017.
21. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bac-
teria that Grow Aerobically, Approved Standard. 9th ed. Wayne, PA:
Clinical and Laboratory Standards Institute; 2012. CLSI document
M07-A9.
Acknowledgments
We thank Dr Paweł Czerwiński for the help in the synthe-
sis of selected compounds. The authors are thankful to the
National Science Centre (Grant DEC-2012/07/B/NZ7/04382)
for financial support.
22. Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to
determine the minimal inhibitory concentration (MIC) of antimicrobial
substances. Nat Protoc. 2008;3(2):163–175.
Disclosure
The authors report no conflicts of interest in this work.
23. Jorgensen JH, Turnidge JD. Susceptibility test methods: dilution and
disk diffusion methods. In: Jorgensen J, Pfaller M, Carroll K, Funke G,
Landry M, Richter S, Warnock D, editors. Manual of Clinical
Microbiology. Eleventh ed. Washington, DC: ASM Press; 2015:
1253–1273.
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