Design, synthesis, and structure–activity relationship studies of the anaephene antibiotics

Article abstract of DOI:10.1111/cbdd.13903

The natural products, anaephenes A (1) and B (2), were found to have antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). In this report, we expanded on our previous synthetic efforts by preparing a library of eighteen analogues in order to understand the structure–activity relationships (SAR) of this interesting class of natural products. These analogues were selected to explore the biological impact of structural variations in the alkyl chain and on the phenol moiety. Last, we further assessed the biological activity of anaephene B (2) and two additional analogues against other clinically relevant bacterial strains and the hemolytic activity of each and determined that these compounds act via a bactericidal mechanism. These studies led to the identification of compound 7, which was 4-fold more potent than the natural product (2) against MRSA (2 vs. 8?μg/ml) and a 2-hydroxypyridine analogue (18) which demonstrated equal potency compared with the natural product (2), albeit with a significant reduction in hemolytic activity (<1% vs. 80% at 100?μM).

Full text of DOI:10.1111/cbdd.13903

Article type
: Research Article
Design, Synthesis, and Structure-Activity Relationship
Studies of the Anaephene Antibiotics
David L. Kukla,† Juan Canchola,† Joseph D. Rosenthal, † and Jonathan J. Mills*,†
†Department of Chemistry, Illinois State University, Campus Box 4160, Normal, Illinois 61790-4160,
United States
Correspondence
Jonathan J. Mills, Department of Chemistry, Illinois State University, Normal, Illinois. Tel: (309)
438-7700. Email: jjmill5@ilstu.edu
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13903
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ABSTRACT:
The natural products, anaephenes A (1) and B (2), were found to have antimicrobial activity
against methicillin resistant Staphylococcus aureus (MRSA). In this report, we expanded on our
previous synthetic efforts by preparing a library of eighteen analogues in order to understand the
structure-activity relationships (SAR) of this interesting class of natural products. These analogues
were selected to explore the biological impact of structural variations in the alkyl chain and on the
phenol moiety. Lastly, we further assessed the biological activity of anaephene B (2) and two
additional analogues against other clinically relevant bacterial strains, the hemolytic activity of each
and determined that these compounds act via a bactericidal mechanism. These studies led to the
identification of compound 7, which was 4 fold more potent than the natural product (2) against
MRSA (2 vs. 8 μg/mL) and a 2-hydroxypyridine analogue (18) which demonstrated equal potency
compared to the natural product (2), albeit with a significant reduction in hemolytic activity (< 1% vs.
80% at 100 μM).
KEYWORDS: antibiotics, synthesis, Methicillin-resistant Staphylococcus aureus, Staphylococcus
aureus, natural products
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1. Introduction
Multi-drug resistant bacteria present a critical threat to human health and require the development of
novel antibiotics for treatment (Marston et al., 2016). Staphylococcus aureus was responsible for
119,000 infections and 20,000 deaths in the United States in 2017 (CDC, 2019). Methicillin-resistant
Staphylococcus aureus (MRSA) first emerged within 2 years of the clinical use of methicillin and
more recently has become a community-acquired infectious disease (Kluytmans-Vandenbergh et al.,
2006). MRSA has caused epidemics, such as the one involving the USA300 strain, and MRSA
infections result in over 23,000 deaths and over 1 million infections per year worldwide (Planet,
2017). There is also an increasing number of MRSA strains resistant to other antibiotics including
lipoglycopeptides, lipopeptides, aminoglycosides, oxazolidinones, tetracyclines, quinolones,
pleuromutilins and mupirocin (Watkins et al., 2019). Resistance to additional antibiotics makes
treatment difficult and presents an urgent need for the development of novel antibiotics.
Natural Products have a vast range of structural diversity and have provided scientists with new
molecular scaffolds for the development of new therapeutics for every disease (Patridge et al, 2016).
Antibacterial natural products provide excellent starting points for the development of novel
therapeutics to treat drug-resistant bacteria, considering their purpose is to interact with biological
systems (Shen, 2015). This is highlighted by the fact that several current classes of antibiotics are
derived from natural products including, β-lactams, tetracyclines, macrolides, glycopeptides and
aminoglycosides. The ability to develop novel drug molecules from natural products is hindered by
issues with isolation, supply and characterization. Total Synthesis of naturally occurring molecules
provides an opportunity to overcome these issues (Rossiter et al., 2017).
Recently, we reported the first total syntheses of the antibacterial natural products, anaephenes A (1)
and B (2) (Figure 1; Kukla et al., 2020). These compounds are part of the alkylphenol family which
have demonstrated a wide variety of biological activities. For example, monochasiol A (3), has been
shown to selectively inhibit the concanavalin A-induced interleukin-2 production in Jurkat cells,
which could have implications in treating autoimmune diseases (Figure 1; Kikuchi et al., 2017). Also,
phenolic compounds from essential oils, like eugenol, have demonstrated antibacterial activity against
both MSSA and MRSA (Das et al., 2016). While there have been reports of structure-activity
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relationship studies against drug-resistant bacteria on similar types of compounds such as, chromones
and cannabinoids, there are few reports on alkylphenol natural products (Appendino et al., 2008; Feng
et al., 2014; Li et al., 2021).
Figure 1. Examples of biologically active alkylphenol natural products.
In this study, our goal was to use our previously reported modular synthetic route towards the
anaephene natural products to rapidly generate analogues in order to understand the structure-activity
relationships and to improve the overall hydrophilicity of the compounds. Our strategy included
examining the chain length, degrees of unsaturation in the chain, the location of the unsaturations in
the chain, and three phenolic bioisosteres (Figure 2).
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Figure 2. Strategy to understand the structure-activity relationships for the anaephene natural
products.
2. Methods
2.1 General Experimental Procedures. 1H and 13C NMR spectra were obtained on a Bruker 400
MHz Avance III spectrometer or Bruker 500 MHz Avance III spectrometer in CDCl3 unless otherwise
noted. Chemical shifts are reported with the residual solvent peak used as an internal standard (CDCl3
= 7.26 ppm for 1H and 77.16 ppm for 13C). 1H NMR spectra were tabulated as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, bs = broad
singlet, dt = doublet of triplet, dd = doublet of doublets, ddd = doublet of doublets of doublets),
number of protons, and coupling constant(s). High-resolution mass spectra were obtained using
positive mode electrospray ionization (ESI+) on a Thermo Scientific Q Exactive hybrid quadripole-
Orbitrap mass spectrometer. Analytical HPLC was performed with a Thermo Finnigan Surveyor
HPLC utilizing a Thermo Scientific Hypersil GOLD column (5 μm, 100 mm × 4.6 mm) run with 80%
methanol and 20% 1.0 M acetic acid as the mobile phase at 1.0 mL/min to show the purity of all
tested compounds was > 95%. Reactions were monitored by TLC analysis (silica gel 60 F254, 250
mm layer thickness) and visualized with a 254 nm UV light. Flash chromatography on SiO2 was used
to purify the crude reaction mixtures and performed on a flash system utilizing pre-packed cartridges
and linear gradients. All starting materials and solvents were purchased from a commercial chemical
company and used as received.
2.2 General experimental procedure and characterization of compounds 7, 12a-12i, 13, 15-18.
General Synthetic procedure: To a solution of tert-butyl(3-iodophenoxy)dimethylsilane (1 eq) in
MeCN (0.2 M) was added terminal alkyne (3 eq) followed by CuI (0.2 eq) and PdCl2(PPh3)2 (0.05
eq). Then triethylamine (3 eq) was added and the reaction was heated at 60 °C for 2 hours. Then the
reaction was cooled to room temperature and tetrabutylammonium fluoride (TBAF) (3 eq) was added.
The reaction was stirred at room temperature overnight and then sat. NH4Cl was added. The aqueous
layer was extracted with EtOAc (3x), the combined organic layers were then washed with brine, dried
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(MgSO4), filtered and concentrated under reduced pressure. The residue was purified by flash column
chromatography (10-30% EtOAc:Hex) to afford the desired compound.
3-(undec-1-yn-1-yl)phenol (7). Yield: 39%; 1H NMR (CDCl3, 400 MHz) δH 7.14 (t, J = 7.8 Hz, 1H),
6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (dq, J = 2.6, 1.0 Hz, 1H), 4.68 (bs,
1H), 2.39 (t, J = 7.0 Hz, 2H), 1.60 (p, J = 7.3 Hz, 2H), 1.48-1.40 (m, 2H), 1.34-1.26 (m, 10H), 0.89 (t,
J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4, 115.0, 90.9, 80.3,
32.0, 29.7, 29.4, 29.3, 29.1, 28.9, 22.8, 19.5, 14.2; HRESIMS m/z 245.1902 [M+H]+ (calcd for
C17H25O, 245.1905).
3-(pent-1-yn-1-yl)phenol (12a). Yield: 41%; 1H NMR (CDCl3, 500 MHz) δH 7.15 (t, J = 7.9 Hz, 1H),
6.98 (dt, J = 7.6, 1.1 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.65
(bs, 1H), 2.37 (t, J = 7.0 Hz, 1H), 1.62-1.58 (m, J = 7.3 Hz, 2H), 1.05 (t, J = 7.4 Hz, 3H); 13C NMR
(CDCl3, 125 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4, 115.1, 90.6, 80.5, 22.3, 21.5, 13.7;
HRESIMS m/z 161.0964 [M+H]+ (calcd for C11H13O, 161.0966).
3-(hex-1-yn-1-yl)phenol (12b). Yield: 54%; 1H NMR (CDCl3, 500 MHz) δH 7.14 (t, J = 7.9 Hz, 1H),
6.98 (dt, J = 7.1, 0.8 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.74 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.73
(bs, 1H), 2.39 (t, J = 7.1 Hz, 1H), 1.59 (p, J = 7.3 Hz, 2H), 1.48-1.44 (m, J = 7.3 Hz, 2H), 0.95 (t, J =
7.3 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4, 115.0, 90.8, 80.3,
30.9, 22.1, 19.2, 13.8; HRESIMS m/z 175.1118 [M+H]+ (calcd for C12H15O, 175.1123).
3-(hept-1-yn-1-yl)phenol (12c). Yield: 73%; 1H NMR (CDCl3, 500 MHz) δH 7.14 (t, J = 7.8 Hz, 1H),
6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.70
(bs, 1H), 2.39 (t, J = 7.0 Hz, 2H), 1.61 (p, J = 7.3 Hz, 2H), 1.46-1.42 (m, 2H), 1.40-1.32 (m, 2H), 0.92
(t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4, 115.0, 90.8,
80.3, 31.3, 28.6, 22.4, 19.5, 14.1; HRESIMS m/z 189.1275 [M+H]+ (calcd for C13H17O, 189.1279).
3-(oct-1-yn-1-yl)phenol (12d). Yield: 70%; NMR data is consistent with the literature (Buckle et al.,
1985) 1H NMR (CDCl3, 500 MHz) δH 7.14 (t, J = 7.8 Hz, 1H), 6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86
(dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.64 (bs, 1H), 2.39 (t, J = 7.0 Hz, 2H),
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1.60 (p, J = 7.3 Hz, 2H), 1.48-1.42 (m, 2H), 1.36-1.29 (m, 4H), 0.91 (t, J = 7.0 Hz, 3H); 13C NMR
(CDCl3, 125 MHz) δC ; HRESIMS m/z 203.1432 [M+H]+ (calcd for C14H19O, 203.1436).
3-(non-1-yn-1-yl)phenol (12e). Yield: 30%; 1H NMR (CDCl3, 400 MHz) δH 7.14 (t, J = 7.8 Hz, 1H),
6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.66
(bs, 1H), 2.39 (t, J = 7.0 Hz, 2H), 1.62 (p, J = 7.3 Hz, 2H), 1.48-1.41 (m, 2H), 1.37-1.28 (m, 6H), 0.90
(t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4, 115.0, 90.8,
80.3, 31.9, 29.1, 29.0, 28.9, 22.8, 19.5, 14.2; HRESIMS m/z 217.1592 [M+H]+ (calcd for C15H21O,
217.1592).
3-(dec-1-yn-1-yl)phenol (12f). Yield: 61%; NMR data is consistent with the literature (Pu et al., 2013)
1H NMR (CDCl3, 500 MHz) δH 7.14 (t, J = 7.8 Hz, 1H), 6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J =
2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.69 (bs, 1H), 2.38 (t, J = 7.1 Hz, 2H), 1.59 (p, J
= 7.2 Hz, 2H), 1.44 (p, J = 7.2 Hz, 2H), 1.35-1.26 (m, 8H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 125 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4, 115.0, 90.8, 80.3, 32.0, 29.4, 29.3, 29.1,
28.9, 22.8, 19.5, 14.2; HRESIMS m/z 231.1746 [M+H]+ (calcd for C16H23O, 231.1749).
3-(dodec-1-yn-1-yl)phenol (12g). Yield: 38%; 1H NMR (CDCl3, 400 MHz) δH 7.14 (t, J = 7.8 Hz,
1H), 6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H),
4.65 (bs, 1H), 2.39 (t, J = 7.0 Hz, 2H), 1.59 (p, J = 7.3 Hz, 2H), 1.48-1.40 (m, 2H), 1.36-1.25 (m,
12H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4,
115.0, 90.8, 80.3, 32.1, 29.7, 29.7, 29.5, 29.3, 29.1, 28.9, 22.8, 19.5, 14.2; HRESIMS m/z 259.2058
[M+H]+ (calcd for C18H27O, 259.2062).
3-(tridec-1-yn-1-yl)phenol (12h). Yield: 43%; 1H NMR (CDCl3, 400 MHz) δH 7.14 (t, J = 7.8 Hz,
1H), 6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H),
4.79 (bs, 1H), 2.39 (t, J = 7.0 Hz, 2H), 1.60 (p, J = 7.3 Hz, 2H), 1.46-1.40 (m, 2H), 1.36-1.25 (m,
14H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δC 155.3, 129.6, 125.5, 124.4, 118.4,
115.0, 90.8, 80.3, 32.1, 29.8, 29.7, 29.7, 29.5, 29.3, 29.1, 28.9, 22.8, 19.5, 14.2; HRESIMS m/z
273.2214 [M+H]+ (calcd for C19H29O, 273.2218).
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3-(tetradec-1-yn-1-yl)phenol (12i). Yield: 45%; 1H NMR (CDCl3, 500 MHz) δH 7.14 (t, J = 7.8 Hz,
1H), 6.98 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H),
4.72 (bs, 1H), 2.39 (t, J = 7.0 Hz, 2H), 1.59 (p, J = 7.4 Hz, 2H), 1.44 (p, J = 7.2 Hz, 2H), 1.34-1.27
(m, 16H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δC 155.3, 129.6, 125.6, 124.5, 118.4,
115.0, 90.8, 80.3, 32.1, 29.8, 29.8, 29.8, 29.7, 29.5, 29.3, 29.1, 28.9, 22.8, 19.5, 14.2; HRESIMS m/z
287.2371 [M+H]+ (calcd for C20H31O, 287.2375).
4-(undec-1-yn-1-yl)phenol (13). Yield: 51%; 1H NMR (CDCl3, 400 MHz) δH 7.28 (d, J = 8.7 Hz, 2H),
6.74 (d, J = 8.7 Hz, 2H), 4.76 (bs, 1H), 2.37 (t, J = 7.0 Hz, 2H), 1.59 (p, J = 7.0 Hz, 2H), 1.44 (p, J =
7.4 Hz, 2H), 1.34-1.26 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δC 155.1,
133.2, 116.7, 115.4, 89.0, 80.2, 32.0, 29.6, 29.4, 29.3, 29.1, 29.0, 22.8, 19.5, 14.2; HRESIMS m/z
245.1902 [M+H]+ (calcd for C17H25O, 245.1905).
1-methoxy-3-(undec-1-yn-1-yl)benzene (15). 3-iodoanisole was used as the starting material. Yield:
38%; 1H NMR (CDCl3, 400 MHz) δH 7.18 (t, J = 7.8 Hz, 1H), 6.99 (dt, J = 7.7, 1.2 Hz, 1H), 6.93 (dd,
J = 2.5, 1.4 Hz, 1H), 6.83 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 3.79 (s, 3H), 2.40 (t, J = 7.1 Hz, 2H), 1.61
(p, J = 7.0 Hz, 2H), 1.45 (p, J = 7.1 Hz, 2H), 1.35-1.27 (m, 10H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR
(CDCl3, 100 MHz) δC 159.4, 129.3, 125.3, 124.3, 116.6, 114.2, 90.5, 80.6, 55.4, 32.0, 29.6, 29.4,
29.3, 29.1, 28.9, 22.8, 19.6, 14.2; HRESIMS m/z 259.2062 [M+H]+ (calcd for C18H27O, 259.2062).
6-(undec-1-yn-1-yl)-1H-indole (16). 6-iodoindole was used as the starting material. Yield: 48%; 1H
NMR (CDCl3, 500 MHz) δH 8.10 (bs, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.46 (s, 1H), 7.21 (dd, J = 3.1,
2.4 Hz, 1H), 7.17 (dd, J = 8.2, 1.3 Hz, 1H), 6.53-6.52 (m, 1H), 2.44 (t, J = 7.2 Hz, 2H), 1.63, (p, J =
7.1 Hz, 2H), 1.48 (p, J = 7.2 Hz, 2H), 1.37-1.29 (m, 10H), 0.90 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3,
125 MHz) δC 135.6, 127.5, 125.2, 123.8, 120.6, 117.5, 114.4, 103.0, 88.8, 81.8, 32.0, 29.7, 29.4, 29.4,
29.1, 29.1, 22.8, 19.7, 14.2; HRESIMS m/z 268.2062 [M+H]+ (calcd for C19H26N, 268.2065).
N-(3-(undec-1-yn-1-yl)phenyl)methanesulfonamide (17). N-(3-iodophenyl)methanesulfonamide was
used as the starting material. Yield: 56%; 1H NMR (CDCl3, 400 MHz) δH 7.27-7.25 (m, 1H), 7.23-
7.21 (m, 2H), 7.14 (dt, J = 7.6, 1.6 Hz, 1H), 6.29 (bs, 1H), 3.01 (s, 3H), 2.39 (t, J = 7.1 Hz, 2H), 1.60
(p, J = 7.0 Hz, 2H), 1.44 (p, J = 7.6 Hz, 2H), 1.34-1.26 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR
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(CDCl3, 100 MHz) δC 137.0, 129.8, 128.8, 126.0, 123.6, 119.9, 92.1, 79.7, 39.7, 32.0, 29.6, 29.4,
29.3, 29.1, 28.8, 22.8, 19.5, 14.2; HRESIMS m/z 322.1838 [M+H]+ (calcd for C18H28NO2S,
322.1841).
4-(undec-1-yn-1-yl)pyridin-2-ol (18). 4-Iodo-1,2-dihydropyridin-2-one was used as the starting
material. Yield: 45%; 1H NMR (CDCl3, 500 MHz) δH 13.04 (bs, 1H), 7.29 (bs, 1H), 6.57 (bs, 1H),
6.22 (bs, 1H), 2.40 (t, J = 7.1 Hz, 2H), 1.59 (p, J = 7.1 Hz, 2H), 1.42 (p, J = 7.3 Hz, 2H), 1.33-1.25
(m, 10H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δC ; HRESIMS m/z 246.1856
[M+H]+ (calcd for C16H24NO, 246.1858).
2.3 Experimental procedures and characterization of compounds 8, 11, 14.
3-undecylphenol (8). Procedure: Compound 7 (54.5 mg, 0.163 mmol) was dissolved in anhydrous
EtOH (1.4 mL) and then 10% Pd/C (3.50 mg, 0.0326 mmol) was added. The reaction flask was
equipped with a hydrogen ballon and stirred at room temperature overnight. The reaction was then
filtered through a pad of Celite and washed with EtOAc (10 mL). The mixture was then concentrated
and purified by flash column chromatography (9:1 hexanes:EtOAc) to afford 8 (28.4 mg, 52 % yield);
NMR data is consistent with the literature (Takaishi et al., 2004) 1H NMR (CDCl3, 400 MHz) δH 7.14
(t, J = 7.9 Hz, 1H), 6.76 (dd, J = 7.5, 0.5 Hz, 1H), 6.67-6.63 (m, 2H), 4.61 (bs, 1H), 2.56 (t, J = 7.6
Hz, 2H), 1.60 (p, J = 7.4 Hz, 2H), 1.34-1.26 (m, 16H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3,
100 MHz) δC 155.5, 145.1, 129.5, 121.1, 115.4, 112.6, 36.0, 32.1, 31.4, 29.8, 29.8, 29.7, 29.6, 29.5,
29.4, 22.8, 14.2; HRESIMS m/z 249.2216 [M+H]+ (calcd for C17H29O, 249.2218).
(E)-3-(undeca-5-en-1,10-diyn-1-yl)phenol (11). Procedure: Compound 6 (617 mg, 2.12 mmol) was
taken up in MeCN (4.1 mL) and then 4 Å molecular sieves (200 mg), N-methyl-morpholine oxide
(373 mg, 3.18 mmol) and tetrapropylammonium perruthenate (7.46 mg, 0.0212 mmol) were added.
The reaction was stirred for 24 hours at room temperature and then filtered, concentrated and purified
by flash column chromatography (3:1 hexanes:EtOAc) to provide compound 9. To a solution of 5-
(hex-5-yn-1-ylsulfonyl)-1-phenyl-1H-tetrazole (10; Kukla et al. 2020) (161 mg, 0.551 mmol) in THF
(3.3 mL) at −78 °C was added dropwise KHMDS (0.66 mL, 1.0 M in THF, 0.66 mmol). The reaction
mixture was stirred at −78 °C for 30 min, and then compound 9 (132 mg, 0.459 mmol) was added
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dropwise as a 1.0 M solution in THF. The reaction was then stirred at −78 °C for 2 h followed by
stirring at 0 °C for 2 h. Saturated NH4Cl (10 mL) was added, and the aqueous layer was extracted
with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried
(MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by flash column
chromatography (95:5 hexanes/EtOAc) to provide TBS-protected compound 11 (0.551 mmol). This
compound (87.6 mg, 0.248 mmol) was then dissolved in THF (1.2 mL), and tetrabutylammonium
fluoride was added as a 1 M solution in THF (0.75 mL, 0.75 mmol). The solution was stirred for 45
min at rt. Water (5 mL) was added, and the aqueous layer was extracted with EtOAc (3 × 5 mL). The
combined organic layers were washed with brine (5 mL), dried (MgSO4), filtered, and concentrated
under reduced pressure. The residue was purified by flash column chromatography (4:1
hexanes/EtOAc) to afford 11 (59.0 mg, 13% yield over three steps); 1H NMR (CDCl3, 500 MHz) δH
7.14 (t, J = 7.8 Hz, 1H), 6.97 (dt, J = 7.7, 1.2 Hz, 1H), 6.86 (dd, J = 2.5, 1.4 Hz, 1H), 6.75 (ddd, J =
8.2, 2.6, 0.9 Hz, 1H), 5.58-5.42 (m, 2H), 4.83 (bs, 1H), 2.45 (t, J = 7.2 Hz, 2H), 2.28 (q, J = 7.1 Hz,
2H), 2.20 (td, J = 7.2, 2.6 Hz, 2H), 2.14 (q, J = 7.1 Hz, 2H), 1.96 (t, J = 2.7 Hz, 1H), 1.62 (p, J = 7.2
Hz, 2H); 13C NMR (CDCl3, 125 MHz) δC 155.3, 130.8, 129.6, 129.5, 125.4, 124.4, 118.4, 115.1, 90.1,
84.8, 80.7, 68.5, 31.9, 31.6, 28.3, 19.9, 17.9; HRESIMS m/z 239.1432 [M+H]+ (calcd for C17H19O,
239.1436).
2-(undec-1-yn-1-yl)phenol (14). Procedure: To 2-iodophenol (S1) (200.0 mg, 0.909 mmol) and
K2CO3 (503 mg, 3.64 mmol) was added dry DMF (1.4 mL). Then chloromethyl methyl ether (0.10
mL, 1.36 mmol) was added at 0 °C, and the reaction was stirred overnight at room temperature. The
reaction mixture was then diluted with water (5 mL) and extracted with EtOAc (3 x 5 mL). The
combined organic layers were washed with brine (5 mL), dried (MgSO4), filtered, and concentrated.
The residue was purified by flash column chromatography (9:1 hexanes:EtOAc) to give 1-iodo-2-
(methoxymethoxy)benzene (S2) (0.544 mmol). S2 was then taken up in MeCN (2.6 mL) and 1-
undecyne (0.12 mL, 0.598 mmol), CuI (20.7 mg, 0.109 mmol), PdCl2(PPh3)2 (19.1 mg, 0.0272 mmol)
and triethylamine (0.25 mL, 1.81 mmol) were added. The reaction was heated to 60 °C and stirred for
16 hours. The reaction was cooled to room temperature, filtered and purified by flash column
chromatography (100% hexanes) to provide the MOM-protected compound (S3) (0.371 mmol). This
was then taken up in acetone (1.7 mL) and conc. HCl (0. 20 mL) was added. The reaction was stirred
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for 1 hour and then diluted with water. The reaction mixture was extracted with EtOAc (3 x 10 mL)
and the combined organic layers were washed with brine (5 mL), dried (MgSO4), filtered and
concentrated. The residue was purified by flash column chromatography (9:1 hexanes/EtOAc) to
afford 14 (24.7 mg, 12% yield over three steps); 1H NMR (CDCl3, 500 MHz) δH 7.29 (dd, J = 7.7, 1.6
Hz, 1H), 7.19 (td, J = 8.3, 1.7 Hz, 1H), 6.92 (dd, J = 8.3, 0.9 Hz, 1H), 6.84 (td, J = 7.5, 1.0 Hz, 1H),
5.79 (bs, 1H), 2.48 (t, J = 7.2 Hz, 2H), 1.63 (p, J = 7.6 Hz, 2H), 1.45 (p, J = 7.1 Hz, 2H), 1.35-1.25
(m, 10H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δC 156.7, 131.6, 129.7, 120.3, 114.4,
110.4, 98.2, 74.7, 32.0, 29.6, 29.4, 29.3, 29.1, 28.9, 22.8, 19.7, 14.2; HRESIMS m/z 245.1904 [M+H]+
(calcd for C17H25O, 245.1905).
2.4 Antibacterial Assays. Minimum inhibitory concentrations (MIC) were determined by broth
micro-dilution according to CLSI guidelines (CLSI, 2014). The test medium was lysogeny broth (LB).
S. aureus (ATCC 25923), MRSA (ATCC 33591), MRSA (ATCC BAA-44), S. epidermidis (ATCC
12228), S. epidermidis (ATCC 51625), E. faecalis (ATCC 29212), E. faecalis (ATCC 51299), P.
aeruginosa (ATCC 27853), and A. baumannii (ATCC 19606) were grown in LB for 6-8 h; this
culture was used to inoculate fresh LB (5 × 105 CFU/mL). The resulting bacterial suspension was
aliquoted (1 mL) and compound was added from a 10 mM DMSO stock to achieve the desired initial
starting concentration (128 μg/mL). Linezolid (from a 10 mM DMSO stock) was used as a positive
control with final concentrations ranging from 0.063 to 128 μg/mL. Inoculated media not treated with
compound served as the negative control. The plate was incubated under stationary conditions at 37
°C. After 16 h, minimum inhibitory concentration (MIC) values were recorded as the lowest
concentration of compound at which no visible growth of bacteria was observed, based on duplicate
plates performed in three separate experiments. To determine the minimum bactericidal concentration
(MBC), 100 μL of culture from wells containing no growth were plated onto tryptic soy agar and
incubated at 37 °C overnight. The highest dilution that resulted in 99.9% reduction in the cell count
was recorded as the MBC. The MBC values were determined in triplicate.
2.5 Hemolytic Activity. Hemolysis assays were performed on mechanically defibrinated sheep blood
(Hemostat Labs: DSB50). Defibrinated blood (1.5 mL) was placed into a microcentrifuge tube and
centrifuged for 10 min at 10,000 rpm. The supernatant was then removed and then the cells were
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resuspended in 1 mL of phosphate-buffered saline (PBS). The suspension was centrifuged, the
supernatant was removed, and cells were re-suspended two additional times. The final cell suspension
was then diluted 10-fold. Test compound solutions were made in PBS and then added to aliquots of
the 10-fold suspension dilution of blood. PBS was used as a negative control and a zero-hemolysis
marker. Triton X (a 1% sample) was used as a positive control serving as the 100% lysis marker.
Samples were then placed in an incubator at 37 °C while being shaken at 200 rpm for one hour. After
one hour, the samples were transferred to microcentrifuge tubes and centrifuged for 10 min at 10,000
rpm. The resulting supernatant was diluted by a factor of 4 in distilled water. The absorbance of the
supernatant was then measured with a UV spectrometer at a 540 nm wavelength.
3. Results
3.1 Synthesis and Antimicrobial Activity of Initial Anaephene Analogues.
Based on the synthetic route previously reported by our group, we first synthesized three analogues
that examined the removal and/or addition of unsaturations in the alkyl chain. This was accomplished
by starting with commercially available tert-butyl(3-iodophenoxy)dimethylsilane (4) and subjecting it
to a Sonogashira cross-coupling reaction with either 1-undecyne or pent-4-yn-1-ol to provide
compounds 5 and 6, respectively (Scheme 1a). Compound 5 was then treated with
tetrabutylammonium fluoride (TBAF) to remove the TBS protecting group, providing compound 7.
Compound 8 was then synthesized by subjecting 7 to hydrogenation conditions to give the fully
saturated alkyl chain. In order to synthesize compound 11, alcohol 6 was oxidized to aldehyde 9,
followed by a Julia-Kocienski olefination with sulfone 10 and followed by TBAF deprotection. The
minimum inhibitory concentration (MIC) values for compounds 7, 8 and 11 were determined against
MSSA and MRSA (Table 1). The fully saturated analogue, 8, had a 2-fold loss in potency compared
to anaephene B (2). Compound 11, containing an additional internal alkyne, was equipotent to the
natural product 2. Interestingly, compound 7, containing only the internal alkyne, was more potent
than 2, with MIC values of 2 μg/mL against both bacterial strains.
Scheme 1. (a) Synthetic route used to construct different anaephene analogues, (b) Structures of
analogues that contain variations in length of the alkyl chain and location of the hydroxyl substituent.a
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aReagents and conditions: (i) CuI, Et3N, PdCl2(PPh3)2, MeCN, 24 h, rt; (ii) 5, tetrabutylammonium
fluoride, THF, 45 min, rt.; (iii) 10% Pd/C, H2, EtOH, 48 h, rt; (iv) 6, tetrapropylammonium
perruthenate, N-methylmorpholine N-oxide, MeCN, 6 h, rt; (v) 10, KHMDS, THF, 4 h, -78 °C to 0
°C; (vi) tetrabutylammonium fluoride, THF, 45 min, rt.
This initial structure-activity relationship data on the varying degrees of unsaturation in the alkyl
chain demonstrated that the internal alkyne with no additional unsaturations was the most potent. To
further our understanding of the anaephene SAR, we next examined chain length while maintaining
the internal alkyne found in compound 7. To synthesize this set of analogues, a one pot, two-step
procedure was developed. Tert-butyl(3-iodophenoxy)dimethylsilane (4) was subjected to a
Sonogashira cross-coupling reaction for 2 hours at 60 °C with the corresponding terminal alkyne.
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After cooling to room temperature, TBAF was added and the reaction was stirred overnight to provide
compounds 12a-12i (Scheme 1b). The MIC values for compounds 12a-12i were then determined
against MSSA and MRSA (Table 1). We found that the potency increased as the alkyl chain became
longer from C5 (12a) to C11 (7) against MSSA and MRSA. Interestingly, as the chain length
increased from C12 to C14 (12g-12i), the activity quickly decreased against MSSA, but these
compounds remained active against MRSA. From this data, it was determined that C10 (12f) and C11
(7) were the optimal chain lengths for maximum potency. Because anaephene B (2) has a C11 alkyl
chain, we chose to keep this length for the next round of analogues. We next examined the
relationship between the hydroxyl substituent and alkyl chain. To synthesize the para analogue 13,
tert-butyl(4-iodophenoxy)dimethylsilane was subjected to the same one-pot, two-step procedure (vide
supra). Unfortunately, the ortho analogue 14 was unable to be synthesized via this route due to the
steric bulk of the TBS protecting group during the Sonogashira cross-coupling reaction. To
circumvent this, we synthesized the MOM protected compound, 1-iodo-2-(methoxymethoxy)benzene.
This compound successfully participated in the Sonogashira cross-coupling reaction and after
deprotection of the MOM group with HCl, the desired product (14) was obtained (Scheme S1).
Lastly, we synthesized compound 15 by subjecting 3-iodoanisole to a Sonogashira cross-coupling
reaction with 1-undecyne under than same reaction conditions used to synthesize the other analogues.
This allowed us to examine the importance of the hydrogen-bond donating ability of the hydroxyl
group. The MIC values for these compounds were assessed against MSSA and MRSA (Table 1). The
para analogue 13 was equipotent to compound 7, demonstrating no preference between the meta and
para relationships. However, the ortho analogue 14 displayed a significant reduction in potency and
the methylated analogue 15 was not active at 128 μg/mL.
Table 1. MIC Values of analogues 7, 8, 11, 12a-12i, and 13-18.a
Compound
S. aureus (ATCC 25923)
MRSA (ATCC 33591)
Anaephene B (2)
8
4
8
2
7
8
16
16
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11
8
>128
64
8
>128
64
16
8
12a
12b
12c
16
12d
8
12e
4
4
12f
4
2
12g
8
2
12h
64
2
12i
128
4
8
13
2
14
128
>128
>128
>128
8
64
>128
>128
>128
8
15
16
17
18
linezolid
0.5
1
aAll MIC values in μg/mL.
3.2 Synthesis and Antimicrobial Activity of Phenolic Bioisosteres.
Our initial SAR studies identified the optimal degrees of unsaturation and length of the alkyl chain, as
well as, the optimal position of the hydroxyl substituent. It has been well documented in drug
discovery that the bioavailability of phenolic compounds is inhibited due to the rapid production of
glucuronide and sulfated metabolites (De Souza et al., 2015). One way to overcome this problem is to
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utilize phenolic bioisosteres. The term bioisosterism was initially defined by Friedman and more
recently defined by Burger as “Compounds or groups that possess near-equal molecular shapes and
volumes, approximately the same distribution of electrons, and which exhibit similar physical
properties...” (Friedman, 1951; Burger, 1991). Some examples of phenolic bioisosteres include,
indoles, alkyl sulfonamides, hydroxypyridines, and benzimidazolones (Kawai et al., 2007). We
continued our SAR studies by looking at substituting the phenol moiety for with a bioisostere to
improve the overall drug-like properties of this class of molecules. We selected three different
isosteres, each with different overall properties, to provide additional SAR information on the
hydroxyl group. First, an indole isostere was selected because it maintains the hydrogen-bond
donating ability of the hydroxyl group but is not acidic like phenols. Second, a methylsulfonamide
isostere was selected because it maintains the hydrogen-bond donating ability and the acidity of
phenols. Third, a 2-hydroxypyridine isostere was selected to maintain the oxygen atom and increase
the hydrophilicity compared to phenols. Compounds 16, 17, and 18 were synthesized in a single step
by utilizing a Sonogashira cross-coupling reaction between 1-undecyne and 6-iodoindole, N-(3-
iodophenyl)methanesulfonamide or 4-iodopyridin-2(1H)-one, respectively (Scheme 2).
Scheme 2. Syntheses of phenolic bioisosteres.a
aReagents and conditions: (i) CuI, Et3N, PdCl2(PPh3)2, MeCN, 24 h, rt;
With these three isosteres in hand, we determined the MIC values against both MSSA and MRSA
(Table 1). Compounds 16 and 17, which both contain a nitrogen atom in place of the oxygen atom,
did not show any activity at 128 μg/mL. However, the 2-hydroxypyridine analogue 18 was equipotent
to anaephene B (2) and 2-fold weaker than compound 7 against MRSA.
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3.3 Additional Biological Testing of Anaephene B (2), analogue 7 and analogue 18.
Following the syntheses and MIC evaluations for each of the 18 analogues, compound 7 was found to
be one of the most potent. Compound 7 was 2-fold more potent than anaephene B (2) against MSSA
and 4-fold more potent against MRSA. Additionally, analogue 18 was equipotent to anaephene B (2),
which is interesting because the pyridine motif will provide an opportunity to improve the overall
drug-like properties of this class of molecules such as, increasing the hydrophilicity. Analogues 7 and
18, along with anaephene B (2), were evaluated against additional clinically relevant strains of
bacteria to further define the activity of these compounds.
Compounds 2, 7, and 18 all exhibited moderate to good potency against Gram-positive strains,
including a multi-drug resistant MRSA strain (BAA-44) and a vancomycin-intermediate E. faecalis
strain (ATCC 51299). Additionally, we evaluated these compounds against the Gram-negative
pathogens, A. baumannii (ATCC 19606) and P. aeruginosa (ATCC 27853) and they displayed no
activity. This may indicate a more specific mechanism of action for this class of compounds rather
than non-specific cell lysis.
Table 2. MIC values against additional clinically relevant bacterial strains of 2, 7, and 18.a
Strain
Anaephene B Compound 7
(2)
Compound
Linezolid
Ciprofloxacin
18
MRSA
4
2
8
1
1
nt
nt
nt
nt
0.5
1
(ATCC BAA-44)
S. epidermidis
(ATCC 51625)
E. faecalis
8
2
8
16
8
4
1
(ATCC 29212)
E. faecalis
8
4
16
1
(ATCC 51299)
P. aeruginosa
(ATCC 27853)
A. baumannii
128
>128
128
>128
128
>128
nt
nt
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(ATCC 19606)
ant = not tested. All MIC values in μg/mL.
Given that these molecules are amphipathic, we next evaluated the hemolytic activity of these
compounds to examine the effects on eukaryotic membrane integrity. Compounds 2, 7, and 18 were
subjected to a red blood cell hemolysis assay using mechanically defibrinated sheep blood (Liu et al.,
2007). Interestingly, these studies revealed that the hydroxypyridine analogue 18, possesses
significantly less hemolytic activity (< 1% at 100 μM) than anaephene B (2) (80% at 100 μM) or
analogue 7 (89% at 100 μM). This is a significant result, given the reduction in toxicity and the
increase in the therapeutic window. This also demonstrates the usefulness of bioisosteres to improve
the overall drug-like properties of a compound. Lastly, we determined the minimum bactericidal
concentration (MBC) of compounds 2, 7, and 18 against MSSA. The MBC values for 2, 7, and 18
were 16 μg/mL, 8 μg/mL, and 16 μg/mL, respectively. These results suggest that these compounds
work via a bactericidal mechanism (MBC ≤ 4x MIC; French, 2006).
4. Conclusions
Eighteen analogues (7, 8, 11, 12a-12i, 13-18) of the anaephene natural products were synthesized and
characterized. These analogues were found to possess varying antimicrobial activities against MSSA
and MRSA. Specifically, in 7, we found that an internal alkyne with no additional unsaturations in the
alkyl chain enhances antimicrobial activity against MRSA compared to anaephene B (2) (2 vs. 8
μg/mL). We also found the optimal alkyl chain length for activity against both MSSA and MRSA to
be between 10 (12f) and 11 (7) carbons with a meta (7) or para (13) relationship to the hydroxyl
substituent. These compounds displayed potent activity with MIC values of 4 μg/mL against MSSA
and 2 μg/mL against MRSA, which is only two-fold weaker than the FDA approved antibiotic,
linezolid. Lastly, we found that the incorporation of a 2-hydroxypyridine moiety, as in 18, still
possesses equal antimicrobial activity compared to anaephene B (2) however, compound 18 displayed
minimal hemolytic activity at 100 μM (< 1%), while anaephene B (2) and compound 7 displayed
significant hemolysis at 100 μM (80% and 89%, respectively). We also determined the MBC of these
compounds and these results suggest that these compounds exert their antimicrobial activities via a
bactericidal mechanism. This initial SAR study of the anaephene antibiotics provided analogues with
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improved potency, less toxicity, and provided a basis for continued study and development of this
class of natural products.
DATA AVAILABILTY STATEMENTS
The 1H and 13C NMR spectra and HPLC traces for all final compounds supporting this work are
available in the supplementary material of this article.
ACKNOWLEDGMENTS
Funding from the College of Arts and Sciences and the Department of Chemistry (ISU) are gratefully
acknowledged. Molecular assignments were made with assistance from high resolution MS
instrumentation acquired through support by the National Science Foundation MRI Program under
Grant No. CHE 1337497.
CONFLICT OF INTEREST
The authors declare no competing financial interests.
ORCID
Jonathan J. Mills: 0000-0001-6721-3690
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