Antimycobacterial 1,4-napthoquinone natural products from Moneses uniflora

Article abstract of DOI:10.1016/j.phytol.2018.07.032

A new 1,4-naphthoquinone derivative, 5,8-dihydro-3-hydroxychimaphilin (4) and five known compounds (1, 2 and 5–7) were isolated from an extract of the Canadian medicinal plant Moneses uniflora that significantly inhibited the growth of Mycobacterium tuberculosis H37Ra. The structure of 4 was established through analysis of NMR and MS data and the absolute configuration of the glycone of 5 was determined by chemical transformation and comparison with standards prepared from D- and L-glucose. All compounds isolated were screened against Mycobacterium tuberculosis (H37Ra) and the mammalian HEK 293 cell line and, with the exception of compounds 5 and 7, exhibited marked selectivity in their bioactivity: Compound 1 exhibited potent antimycobacterial activity (IC₅₀ of 5.4 μM) and moderate cytotoxicity (IC₅₀ of 30 μM); compounds 2, 4 and 6 showed moderate antimycobacterial activity (IC₅₀ values from 28 to 47 μM) without affecting the viability of mammalian cells; compound 5 displayed moderate activity in both assays (IC₅₀ values of 44 and 55 μM respectively); and compound 7 was not active in either assay. These data suggest that the Moneses napthaquinone derivatives elicit biological responses in mycobacterial and mammalian cells through disparate modes of action that warrant further investigation.

Full text of DOI:10.1016/j.phytol.2018.07.032

Phytochemistry Letters 27 (2018) 229–233
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Antimycobacterial 1,4-napthoquinone natural products from Moneses
uniflora
a
a
b,c
d
b,c
Haoxin Li , Allyson Bos , Stéphanie Jean , Duncan Webster , Gilles A. Robichaud ,
a
a,e,⁎
John A. Johnson , Christopher A. Gray
Department of Biological Sciences, University of New Brunswick, Saint John, New Brunswick, E2L 4L5, Canada
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, New Brunswick, E1A 3E9, Canada
Atlantic Cancer Research Institute, Moncton, New Brunswick, E1C 8X3, Canada
Department of Medicine, Division of Infectious Diseases, Saint John Regional Hospital, Saint John, New Brunswick, E2L 4L2, Canada
Department of Chemistry, University of New Brunswick, 30 Dineen Dr, Fredericton, NB, E3B 5A3, Canada
a
b
c
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antimycobacterial activity
Medicinal plant
Moneses uniflora
Mycobacterium tuberculosis
Naphthoquinone
A new 1,4-naphthoquinone derivative, 5,8-dihydro-3-hydroxychimaphilin (4) and five known compounds (1, 2
and 5–7) were isolated from an extract of the Canadian medicinal plant Moneses uniflora that significantly
inhibited the growth of Mycobacterium tuberculosis H37Ra. The structure of 4 was established through analysis of
NMR and MS data and the absolute configuration of the glycone of 5 was determined by chemical transformation
and comparison with standards prepared from D- and L-glucose. All compounds isolated were screened against
Mycobacterium tuberculosis (H37Ra) and the mammalian HEK 293 cell line and, with the exception of compounds
Naphthohydroquinone glycoside
5
and 7, exhibited marked selectivity in their bioactivity: Compound 1 exhibited potent antimycobacterial ac-
tivity (IC50 of 5.4 μM) and moderate cytotoxicity (IC50 of 30 μM); compounds 2, 4 and 6 showed moderate
antimycobacterial activity (IC50 values from 28 to 47 μM) without affecting the viability of mammalian cells;
compound 5 displayed moderate activity in both assays (IC50 values of 44 and 55 μM respectively); and com-
pound 7 was not active in either assay. These data suggest that the Moneses napthaquinone derivatives elicit
biological responses in mycobacterial and mammalian cells through disparate modes of action that warrant
further investigation.
1
. Introduction
8-chlorochimaphilin (1 – 3), did not display activity against the same
Mycobacterium spp. when tested at lower amounts (100 μg per disk;
Moneses uniflora (L.) Gray (Ericaceae), commonly known as the one-
flowered wintergreen, single delight or wood nymph, is a small
3–10 cm high) perennial herb with a circumboreal distribution across
Saxena et al., 1996). In our hands, however, a methanolic extract of M.
uniflora collected on the east coast of Canada in New Brunswick showed
significant activity against M. tuberculosis H37Ra in a microplate-based
screening assay (mean growth inhibition ± SD = 99.6 ± 0.8% at
(
the northern hemisphere (Freeman, 2009). It is common in cool, moist
coniferous woods across Canada (Freeman, 2009; Hinds, 2000) and has
been used by the First Nations peoples for a variety of medicinal pur-
poses that include the treatment of tuberculosis (MacKinnon et al.,
−1
100 μgmL ; O’Neill et al., 2014) and prompted us to further in-
vestigate this plant. Bioassay guided fractionation of the extract led to
the isolation of a new 1,4-naphthoquinone derivative, 5,8-dihydro-3-
hydroxychimaphilin (4), three known 1,4-naphthoquinone derivatives
(1, 2 and 5) and two coumarins (6 and 7).
2009) and symptoms associated with the disease (Moerman, 1998,
2009). Indeed, Towers and co-workers reported that an extract of M.
uniflora collected from the Haida Gwaii archipelago off the north coast
of British Columbia, Canada, showed antimycobacterial activity when
tested at high titre in a disk diffusion screening assay against Myco-
bacterium tuberculosis and Mycobacterium avium (20 mg per disk;
McCutcheon et al., 1997); although the extract and three antibiotic
naphthoquinone constituents, chimaphilin, 3-hydroxychimaphilin and
2. Results and discussion
Compound 4 was isolated as orange needles that gave a sodiated
molecular (M + Na+) ion at m/z 227.0679, (calcd for C12
H
O
3
Na
+
:
12
227.0684) in the positive ion HRESIMS, consistent with a molecular
⁎
Corresponding author at: Department of Biological Sciences, University of New Brunswick, Saint John, New Brunswick, E2L 4L5, Canada.
E-mail address: cgray@unb.ca (C.A. Gray).
https://doi.org/10.1016/j.phytol.2018.07.032
Received 5 June 2018; Received in revised form 17 July 2018; Accepted 27 July 2018
1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

H. Li et al.
Phytochemistry Letters 27 (2018) 229–233
Table 3
Biological activities (MIC and IC50 values in μM [μg/mL]) of 1, 2 and 4–7.
Table 1
NMR Spectroscopic data of 5,8-dihydro-3-hydroxychimaphilin (4).a
Position
δ
C
, mult.
δ
H
(int., mult, J in Hz)
HMBC
COSY
Compounds
Mycobacterium tuberculosis H37Ra
HEK 293 cells
IC50 (95% CI)a
IC50 (95% CI)
1
2
3
4
5
187.6, s
116.9, s
150.9, s
183.0, s
24.6, t
MIC
1
20
5.44 (5.08–5.800)
[1.01 (0.95–1.080]
30.2
(26.1–35.0)
[5.63
(4.85–6.52)]
> 100
[ > 50]
> 100
[ > 50]
54.7
(41.2–71.58)
[19.1
(14.4–25.1)]
> 100
[ > 50]
> 100
3.01 (1H, m)
.06 (1H, m)
5.48 (1H, m)
4a, 6, 7, 8a
6
[3.125]
3
6
7
8
116.6, d
130.5, s
29.2, t
5, 8, 7-Me
6, 7-Me
2
4
5
125
[25]
125
[25]
300
47.3 (42.1–53.0)
[9.56 (8.52–10.7)]
28.2 (27.1–29.3)
[5.75 (5.53–5.98)]
43.5 (40.4–46.8)
b
2.99 (1H, m)
4a, 5 , 6, 7, 8a
3
.04 (1H, m)
8
4
2
7
3
a
a
-Me
-Me
-OH
141.9, s
135.1, s
8.2, q
1.94 (3H, s)
1.77 (3H, m)
6.91 (1H, s)
1, 2, 3
6, 7, 8
[100]
[15.2 (14.2–16.4)]
23.1, q
6, 8
b
1 , 2, 3, 4
6
250
32.8 (29.6–36.3)
[6.83 (6.16–7.56)]
> 100
[
50]
a
b
at 400 MHz for 1H and 100 MHz for 13C.
Recorded in CDCl
Weak correlation.
3
7
> 1000
[ > 200]
6.25 × 10
[ > 50]
[ > 50]
Rifb
–2
5.24 × 10
–3
NDc
3
(
4.74 × 10– – 5.80 × 10–3
)
formula of C12
H
12
O
3
and implying seven degrees of unsaturation. The
5.00 × 10–2
–3
[
]
[4.31 × 10
1
3
(3.90 × 10– – 4.77 × 10–3)]
3
C NMR spectrum showed 12 resonances (Table 1) that were indicative
DPTb
ND
ND
38.4
of a dimethylated naphthoquinone scaffold (Lee et al., 2001; Saxena
et al., 1996). The presence of two methylenes [δ 24.6 (C-5, t) and 3.01
(H -5, m) and 29.2 (C-8, t) and 2.99 (H -8, m) respectively] and a
2 2
(
[
(
21.2–69.9)
15.3
8.45–27.6)]
phenol [δ 150.9 (C-3, s) and 6.91 (3-OH, bs)] suggested that 4 was a
hydroxylated analogue of 5,8-dihydrochimaphilin (8; Lee et al., 2001).
HMBC correlations observed for 3-OH (to C-2, C-3 and C-4), 2-Me (to C-
a
CI = confidence interval.
Positive control: Rif = rifampin; DPT = deoxypodophyllotoxin.
ND = Not determined.
b
c
1, C-2 and C-3), H
2
-5 (to C-4a, C-6, C-7, and C-8a) and H
2
-8 (to C-4a, C-
-8 spin
5, C-6, C-7, and C-8a) together with the H
2
-5–H-6–7-Me–H
2
system revealed by the COSY spectrum allowed 4 to be identified as
methoxy-2,3,4,6-O-acetyl derivatives prepared from D- and L-glucose
25
25
5
,8-dihydro-3-hydroxychimaphilin.
Compound 5 was isolated as an optically active ([α]
([α]
D
D
= +184 and [α] = –175 respectively). Although 5 was first
2
5= –9.2)
reported as a natural product of Chimaphila umbellata in the patent
literature in 2002 (Hwang et al., 2002), details of its isolation did not
appear until 2015 and, whilst it was assumed to be the D-glucoside,
both the NMR data and the configuration of the glycone remained to be
assigned (Shin et al., 2015). Compounds 1, 2, 6 and 7 were identified as
chimaphilin (Saxena et al., 1996), 3-hydroxychimaphilin (Saxena et al.,
1996), isofraxetin (Artem’eva et al., 1973a; Artem’eva et al., 1973b;
Zhou et al., 2017), and umbelliferone (Gottlieb et al., 1979; Timonen
et al., 2011; Zolek et al., 2003) by HRMS and comparison of their NMR
data with literature values.
D
amorphous solid that was determined to have the molecular formula
C
C
+
18
H
H
22
O
O
7
from HRESIMS data (M + Na ion at m/z 373.1258; calcd for
Na : 373.1263). The structure and absolute stereochemistry
+
18
22
7
of 5 was established to be 4-hydroxy-2,7-dimethylnaphthylene-1-O- β-
D-glucopyranoside with the planar structure being revealed through
analysis of 1D and 2D NMR data (Table 2) and the configuration of the
glucose moiety assigned by comparison of the specific rotation of the α-
1
-methoxy-2,3,4,6-O-acetylglucopyranoside obtained from methano-
2
5
lysis and acetylation of the natural product ([α] = +68) with α-1-
D
The antimycobacterial activity and cytotoxicity of the compounds
isolated from M. uniflora was evaluated in vitro against M. tuberculosis
(H37Ra) and immortalized human embryonic kidney (HEK 293) cells
Table 2
NMR spectroscopic data of 4-Hydroxy-2,7-dimethylnaphthylene-1-O-β-D-glu-
(
Table 3). With the exception of umbelliferone, all of the compounds
copyranoside (5).a
inhibited the growth of M. tuberculosis; 3-hydroxychimaphilin (2), 5,8-
dihydro-3-hydroxychimaphilin (4), 4-hydroxy-2,7-dimethylnaphthy-
lene-1-O-β-D-glucopyranoside (5) and isofraxetin (6) were moderately
antimycobacterial whilst chimaphilin (1) exhibited makedly increased
activity (M. tuberculosis: MIC 20 μM, IC50 5.4 μM) comparable to that
reported for the most active antimycobacterial naphthoquinone natural
products (Salomon and Schmidt, 2012).
Position
δ
C
, mult.
δ
H
(int., mult, J in Hz)
HMBC
COSY
2-Me
1
2
3
4
5
6
7
8
143.5, s
129.5, s
110.7, d
151.0, s
123.2, d
126.9, d
136.7, s
122.4, d
124.1, s
130.5, s
106.4 d
75.9, d
78.1, d
71.8, d
77.8, d
62.9, t
6.55 (1H, s)
1, 4, 8a, 2-Me
7.98 (1H, d, 8.5)
7.18 (1H, dd, 8.5, 1.6)
4, 4a, 7
5, 8, 8a, 7-Me
6
5, 8, 7-Me
1,4-Naphthoquinones are well known to manifest widespread cy-
tototoxic effects through the generation of reactive oxygen species,
intracellular redox cycling and alkylation of glutathione as well as
nucleophilic sites in nucleic acids and proteins (Klotz et al., 2014;
Kumagai et al., 2012), and these largely unspecific mechanisms have
been used to rationalize antimycobacterial activity of quinones and
napthoquinones in the past (Tran et al., 2004). However, if the anti-
mycobacterial activity of the Moneses napthoquinones was affected by
the action of one or more of these general mechanisms, we would ex-
pect them to display similar profiles of bioactivity in both of our assays
rather than the marked differences that we observed. Whilst all of the
napthaquinones exhibited significant antimycobacterial activity, only
the naphthoquinones lacking a hydroxyl substituent at C-3 (1 and 5)
8.20 (1H, bs)
1, 6, 7-Me, 8a
6, 7-Me
8
4
1
2
3
4
5
6
a
a
'
'
'
'
'
'
4.74 (1H, d, 7.8)
3.64 (1H, m)
3.46 (1H, m)
3.43 (1H, m)
3.09 (1H, ddd, 9.3, 5.2, 2.5)
3.65 (1H, d, 11.7, 5.2)
1, 3'
1', 3'
2', 4'
3', 4'
2'
1', 3', 5'
2'
5'
4', 6'
5'
4', 5'
3
.75 (1H, dd, 11.7, 2.5)
2
7
-Me
-Me
17.8, q
22.0, q
2.44 (3H, s)
2.48 (3H, bs)
1, 2, 3
6, 7, 8
6, 8
a
Recorded in CD
3
OD at 400 MHz for 1H and 100 MHz for 13C.
230

H. Li et al.
Phytochemistry Letters 27 (2018) 229–233
displayed any appreciable level of cytotoxicity when evaluated against
the HEK 293 cell line. This structure activity relationship is in ac-
cordance with previous findings (D’Arcy et al., 1987; Inbaraj and
Chignell, 2004; Klaus et al., 2010; Klotz et al., 2014), and, given that
both 1 and 5 are C-2 methyl substituted, it would follow that the me-
chanism of cytotoxicity exhibited in the HEK 293 cells is predominantly
redox-based rather than a consequence of Michael-type alkylation of
nucleophilic cellular targets (Inbaraj and Chignell, 2004; Kumagai
et al., 2012). The marked difference observed in our bioassay data
therefore suggests that the antimycobacterial activity of the naptho-
quinones isolated from M. uniflora is being elicited through a more
specific mechanism of action such as DNA gyrase inhibition (Karkare
et al., 2013) that may warrant further in-depth study.
(23 mg) and 2 (6 mg). The CH
Sephadex LH-20 (1:1 CH Cl
portion of fraction D (47 of 72 mg) was subjected to normal phase
HPLC (17:3 hexane/EtOAc) to obtain 4 (12 mg). A portion of fraction
(30 of 85 mg) was subjected to normal phase HPLC (33: 17 hexane/
EtOAc) to give 6 (5 mg) and 7 (9 mg). A portion of the EtOAc fraction
(176 of 712 mg) was subjected to Sephadex LH-20 (1:1 CH Cl /MeOH)
resulting ten fractions (E – E10). Fraction E (20 mg) was subjected to
O) to obtain 5 (11 mg).
2
Cl
2
fraction (654 mg) was subjected to
2
2 1
/MeOH) to give ten fractions (D – D10). A
7
D
8
2
2
1
4
reverse phase HPLC (17:33 MeOH/H
2
3.3.1. Chimaphilin (1)
Yellow needles; IR (thin film) νmax 2956, 2924, 1667, 1599,
−
1 1
1298 cm ; H NMR (400 MHz, CDCl
7
3
) δ 7.93 (d, J = 7.9 Hz, 1H, H-5),
.87 (dq, 1.8, 0.6 Hz, 1H, H-8), 7.50 (ddq, J = 7.9, 1.8, 0.7 Hz, 1H, H-
3
. Experimental
6), 6.79 (q, J = 1.5 Hz, 1H, H-3), 2.48 (bs, 3H, 7-Me), 2.17 (d,
J = 1.5 Hz, 3H, 2-Me), consistent with literature values (Kosuge et al.,
1985; Saxena et al., 1995, 1996); 13C NMR (100 MHz, CDCl
) δ 186.0
s, C-1), 185.1 (s, C-4), 148.0 (s, C-2), 144.8 (s,C-7), 135.8 (d, C-3),
3.1. General experimental procedures
3
(
All solvents for extraction and fractionation were purchased from
134.5 (d, C-6), 132.2 (s, C-8a), 130.2 (s, C-4a), 127.0 (d, C-5), 126.4 (d,
C-8), 22.0 (q, 7-Me), 16.6 (q, 2-Me), data were consistent with literature
Fisher Scientific (Ottawa, ON, Canada). NMR solvents were purchased
from Sigma-Aldrich (Oakville, ON, Canada). Flash chromatography was
performed using a Biotage Flash + chromatography system on KP-Sil
values (Kagawa et al., 1992; Kosuge et al., 1985; Saxena et al., 1995,
1996); ESIHRMS m/z 187.0755 (M + H+), calcd for (C12
H
11
O
2
+ H ),
+
25 + S silica cartridges (40 − 63 μm, 60 Å) and size exclusion chro-
187.0759.
matography was performed with Sephdex LH-20 (25–100 μm). Semi-
preparative normal phase HPLC was performed on a Waters 510 pump,
a Waters R401 refractive index detector and a Phenomenex Luna silica
column (250 × 10 mm, 10 u, 100 Å) at a flow rate of 4.0 mL/min.
Optical rotations were determined on a Rudolph autopol III polarimeter
at 589 nm using a 5 cm sample cell. IR spectra were recorded on a
Perkin Elmer Spectrum Two FT-IR spectrometer as thin films on a so-
dium chloride disk. NMR spectra were recorded on an Agilent 400-MR
3.3.2. 3-Hydroxychimaphilin (2)
Yellow needles; IR (thin film) ν
max
3364, 2919, 2850, 1698,
−
1 1
1646 cm ; H NMR (CDCl ) δ 7.94 (d, J = 7.8 Hz, 1H, H-5), 7.90 (dq,
3
1.7, 0.6 Hz, 1H, H-8), 7.45 ((ddq, J = 7.8, 1.7, 0.7 Hz, 1H, H-6), 7.35
(bs, 1H, 3-OH), 2.48 (bs, 3H, 7-Me), 2.08 (s, 3H, 2-Me), consistent with
literature values (Saxena et al., 1995, 1996);13C NMR (100 MHz,
CDCl ) δ 185.4 (s, C-1), 181.0 (s, C-4) 153.3 (s, C-3), 146.4 (s, C-7),
3
DD2 instrument in CDCl
protonated solvent resonances (δ
9.000, respectively). HRMS was recorded on a Thermo LTQ Exactive
3
or CD
3
OD and were calibrated to residual
133.6 (d, C-6), 133.0 (s, C-8a), 127.4 (d, C-8), 127.2 (s, C-4a), 126.5 (d,
H
7.260 and 3.310; δ 77.160 and
C
C-5), 120.2 (s, C-2), 22.2 (q, 7-Me) 8.8 (q, 2-Me), consistent with lit-
4
erature values (Saxena et al., 1995, 1996); ESIHRMS m/z 203.0704 (M
+
+
instrument with an ESI source. Antimycobacterial susceptibility tests
were performed using modified Middlebrook 7H9 broth base (BBL™
MGIT™, Becton Dickinson, Mississauga, Ontario) in non-tissue culture
treated, low-binding, black 96-well microtitre plates sealed with
polyester films (50 μm). Fluorometric readings (in relative fluorescence
units, RFU) were recorded using a Molecular Devices Gemini EM dual-
scanning microplate spectrofluorometer with a 530 nm excitation filter
and a 590 nm emission filter operating in top-scan mode.
+ H ), calcd for (C12H11O3 + H ), 203.0708.
3.3.3. 5,8-Dihydro-3-hydroxychimaphilin (4)
Orange needles; IR (thin film) ν
max
3392, 2898, 1652, 1383,
−
1
1
13
1355 cm
; H and C NMR see Table 1; ESIHRMS m/z 227.0679
(M + Na+), calcd for (C12H12O3 + Na ), 227.0684.
+
3.3.4. 4-Hydroxy-2,7-dimethylnaphthylene-1-O-β-D-glucopyranoside (5)
2
5
D
Purple amorphous solid; [α] = –9.2 (c 0.8, MeOH); IR (thin film)
−
1
1
13
3.2. Plant material
νmax 3353, 2923, 1660, 1294, 1075 cm ; H and C NMR see Table 2;
+
+
ESIHRMS m/z 373.1258 (M + Na ), calcd for (C18
H
22
O
7
+ Na ),
Whole plants of M. uniflora were collected by hand from Saint
373.1263.
Léonard, New Brunswick, Canada (47°16.024′ N, 67°43.874′ W) in
October 2013. The whole plants were washed with water to remove
debris, freeze dried and stored at –20℃. Species identification was
confirmed by Dr. Stephen Clayden (New Brunswick Museum
Herbarium; voucher number NBM VP-37097).
3.3.5. Isofraxetin (6)
White needles; IR (thin film) ν
max
3345, 2927, 1692, 1582,
−
1 1
1121 cm
; H NMR (400 MHz, CDCl ) δ 7.61 (d, J = 9.5, 1H, H-4),
3
6.53 (s, 1H, H-8), 6.28 (d, J = 9.5, 1H, H-3) 3.94 (s, 3H, 7-OMe),
consistent with literature values (Artem’eva et al., 1973a; Artem’eva
1
3
.3. Extraction and isolation
et al., 1973b); H NMR (400 MHz, CD
6
3
OD) δ 7.83 (d, J = 9.4, 1H, H-4),
.71 (s, 1H, H-8), 6.21 (d, J = 9.4, 1H, H-3) 3.89 (s, 3H, 7-OMe),
consistent with literature values (Zhou et al., 2017);
1
3
The freeze dried M. uniflora (16 g) was ground to a powder and
C NMR
exhaustively extracted in MeOH for 8 h using a Soxhlet apparatus to
obtain crude extract (5.5 g) that exhibited significant anti-
mycobacterial activity and was subjected to bioassay-guided fractio-
nation. A portion of the crude extract (4.4 g) was fractionated using a
modified Kupchan solvent-solvent partition to give five fractions (Li
(100 MHz, CD OD, assignments marked with asterisks may be inter-
3
a
changed) δ 163.7 (s, C-2), 147.1 (s, C-7), 146.7 (d, C-4), 140.7 (s, C-
* *
8a ), 140.6 (s, C-5 ), 134.1 (s, C-6), 112.7 (d, C-3), 112.2 (s, C-4a),
+
101.1 (d, C-8), 56.8 (q, 7-OMe); ESIHRMS m/z 209.0445 (M + H ),
+
calcd for (C10
H
9
O
5
+ H ), 209.0450.
2 2
et al., 2012). The antimycobacterial hexane, CH Cl and EtOAc frac-
tions were further separated to obtain pure compounds. The hexane
fraction (511 mg) was subjected to silica flash column chromatography
eluting from 100% hexanes to 100% EtOAc with 10% increment re-
3.3.6. Umbelliferone (7)
Yellow needles; IR (thin film) νmax 3171, 1687, 1610, 1131 cm ; H
NMR (400 MHz, CD
J = 8.5 Hz, 1H, H-5), 6.79 (dd, J = 8.5, 2.3 Hz, 1H, H-6), 6.70 (bd,
J = 2.3 Hz, 1H, H-8), 6.18 (d, J = 9.5 Hz, 1H, H-3), consistent with
−
1 1
3
OD) δ 7.84 (bd, J = 9.5 Hz, 1H, H-4), 7.44 (d,
sulting eight fractions (H
1
- H
8
). A portion of fraction H
3
(46 of 93 mg)
was subjected to normal phase HPLC (9:1 hexane/EtOAc) to obtain 1
231

H. Li et al.
Phytochemistry Letters 27 (2018) 229–233
literature values (Timonen et al., 2011); 13C NMR (100 MHz, CD
Acknowledgements
3
OD) δ
163.7 (s, C-2), 163.2, (s, C-7) 157.2 (s, C-8a), 146.0 (d, C-4), 130.7 (d,
C-5), 114.5 (d, C-6), 113.1 (s, C-4a), 112.3 (d, C-3), 103.4 (d, C-8),
The authors would like to thank: Sean Haughian (University of New
Brunswick) for providing the M. uniflora used in this study; Stephen
Clayden (New Brunswick Museum) for confirming the identity of M.
uniflora; Larry Calhoun (University of New Brunswick) for 2D NMR data
collection; and Russel Kerr, Fabrice Berrue and Patricia Boland
(University of Prince Edward Island) for HRMS data collection.
Financial support for this research was provided by the Natural
Sciences and Engineering Research Council of Canada (Discovery Grant
to CAG), the New Brunswick Innovation Foundation (Research
Assistantship Initiative grants to CAG), and Horizon Health Network
(Heath Promotion Research Fund Tier II grant to DW, CAG and JAJ)
and is gratefully acknowledged.
consistent with literature values (Gottlieb et al., 1979; Timonen et al.,
+
2
011; Zolek et al., 2003); ESIHRMS m/z 163.0390 (M + H ), calcd for
+
(
9 7 3
C H O + H ), 163.0395.
3.4. Determination of the absolute stereochemistry of 5
Compound 5 (7 mg) was dissolved in 10 mL of anhydrous MeOH,
acetyl chloride (0.2 mL) was added and the reaction mixture was stirred
under reflux for two hours. The reaction mixture was concentrated in
vacuo and the residue partitioned between water and EtOAc. The aqueous
fraction was lyophilized to give the methyl glucoside (4 mg) that was
stirred overnight with acetic anhydride (3 mL) and DMAP (1 mg) in an-
hydrous pyridine (9 mL) at room temperature. EtOAc (30 mL) was added
to the reaction mixture before it was washed with 1 M HCl (3 × 40 mL),
Appendix A. Supplementary data
saturated sodium bicarbonate solution (3 × 40 mL) and distilled H
3 × 40 mL), dried (MgSO ) and concentrated in vacuo to give an 8:3
mixture (by H NMR) of the peracetylated α- and β-methylglucosides
2
O
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2018.07.032.
(
4
1
[
anomeric proton resonances at δ 4.95 (d, 3.7 Hz) and 4.43 (d, 7.9 Hz)
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