9-norlignans: Occurrence, properties and their semisynthetic preparation from hydroxymatairesinol

Article abstract of DOI:10.3390/molecules24020220

Lignans, neolignans, norlignans and norneolignans constitute a large class of phenolic natural compounds. 9-Norlignans, here defined to contain a β-β' bond between the two phenylpropanoid units and to lack carbon number 9 from the parent lignan structure, are the most rarely occurring compounds within this class of natural compounds. We present here an overview of the structure, occurrence and biological activity of thirty-five 9-norlignans reported in the literature to date. In addition, we report the semisynthetic preparation of sixteen 9-norlignans using the natural lignan hydroxymatairesinol obtained from spruce knots, as starting material. 9-Norlignans are shown to exist in different species and to have various biological activities, and they may therefore serve as lead compounds for example for the development of anticancer agents. Hydroxymatairesinol is shown to be a readily available starting material for the preparation of norlignans of the imperanene, vitrofolal and noralashinol family.

Full text of DOI:10.3390/molecules24020220

molecules
Article
9-Norlignans: Occurrence, Properties and Their
Semisynthetic Preparation from Hydroxymatairesinol
Patrik Eklund * and Jan-Erik Raitanen
Johan Gadolin Process Chemistry Centre, Laboratory of Organic Chemistry, Åbo Akademi University,
Piispankatu 8, FIN-20500 Turku, Finland; jraitane@abo.fi
* Correspondence: paeklund@abo.fi; Tel.: +358-2-215-4720
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 November 2018; Accepted: 28 December 2018; Published: 9 January 2019
Abstract: Lignans, neolignans, norlignans and norneolignans constitute a large class of phenolic
natural compounds. 9-Norlignans, here defined to contain a
β–β’ bond between the two
phenylpropanoid units and to lack carbon number 9 from the parent lignan structure, are the most
rarely occurring compounds within this class of natural compounds. We present here an overview of
the structure, occurrence and biological activity of thirty-five 9-norlignans reported in the literature
to date. In addition, we report the semisynthetic preparation of sixteen 9-norlignans using the natural
lignan hydroxymatairesinol obtained from spruce knots, as starting material. 9-Norlignans are shown
to exist in different species and to have various biological activities, and they may therefore serve
as lead compounds for example for the development of anticancer agents. Hydroxymatairesinol is
shown to be a readily available starting material for the preparation of norlignans of the imperanene,
vitrofolal and noralashinol family.
Keywords: lignans; norlignans; 9-norlignans; semisynthesis; hydroxymatairesinol; bioactivity
1. Introduction
Many secondary metabolites, including lignans, flavonoids, and coumarins are formed from
phenylpropanoids originating from the well-known shikimic acid pathway. The parent structures are
usually further oxidized and arranged into various structures. Norlignans, a subclass of lignans lacking
one or more carbon atoms, seem to be a rather unknown class of natural products. Compared to
other phenylpropanoids their occurrence, biosynthetic pathway, and their properties are much
less reported in the literature. Especially 9-norlignans (lacking carbon 9 from the parent lignan
skeleton) with guaiacyl (3-methoxy-4-hydroxyphenyl) moieties have rarely been reported in the
literature, although they would be expected to be common norlignans as guaiacyl lignans are
the most abundant class of lignans. To the best of our knowledge, (+)-imperanene, vitrofolal E
and F, noralashinol A and C are the only guaiacyl type 9-norlignans which have been reported as
plant constituents [
dihydrodehydroxy-imperanenes have also been synthesized [5,6
1
–
4
]. The unnatural enantiomer, (
−
)-R-imperanene, dehydroxyimperanenes and
]. Normally, common substitution
patterns are found in norlignans, but the more rare 2,4,5-trisubstituted phenyl moieties seem to be
present in several 9-norlignans. In this paper we review the occurrence and properties of natural
compounds belonging to the class of 9-norlignans, and in addition, we report the semisynthesis of
9-norlignans from the natural lignan hydroxymatairesinol.
1.1. Classification and Nomenclature of Norlignans
The term lignan is defined as two phenyl propane units coupled together by a β–β’ bond, and if
the same structural units are coupled in any other ways the product is called a neolignan. If the
coupling between the units contains an ether function (fundamental parent structures) the compound
Molecules 2019, 24, 220; doi:10.3390/molecules24020220
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is called an oxyneolignan. According to the present recommendations by the International Union
of Pure and Applied Chemistry (IUPAC), the prefix nor (modification of the fundamental parent
structure) is used when the lignan, neolignan or oxyneolignan lack one or more carbon atoms [
However, in the literature there are today over 60 compounds named as norlignans although the
major part of these are lacking the ’coupling, which is the definition for the fundamental parent
7].
β–β
structure named lignan. In our opinion most of these structures should be named as norneolignans
or noroxyneolignans (prefix nor + fundamental parent structure). The choice of the numbering of
the lost carbon is also somewhat confusing. Numbering of the modified carbon skeleton (in this
case nor) should be performed so that the modification is expressed by the lowest locant (number,
unprimed). However, the choice of locants for the removal of a carbon atom (nor) is preferred in
the order: unprimed, higher number. Consequently, there is a conflict when assigning structures
to 9 or 7-norlignans or norneolignans. Although the exact definition of different norlignans seems
a bit confusing and vague, we here define a norlignan as a plant-derived compound consisting of
two phenylpropane units coupled in the propane moiety with a
carbon atoms.
β–β’ bond and missing one or more
Moreover, we consider 9-norlignans to be derived from a parental lignan skeleton and thus to
have a detectable ’ coupling and to lack one (or more, in the case of di(bis)norlignans) of the
β–β
terminal carbon atoms (carbon 9). In Figure 1, the fundamental structures of different norlignan and
norneolignan structures coupled in the propane moiety are displayed (oxyneolignans and others are
excluded, dotted lines in red indicate the missing carbon-9, the bold lines indicate the coupling in the
propane moiety).
Figure 1. Fundamental structures for the lignan and different 9-norlignan and 9-norneolignan
structures. Dotted (red lines) lines indicate the missing carbon-9 atom.
1.2. 9-Norlignans
According to the definition above, approximately 25 naturally occurring 9-norlignans can be
found in the literature. These are yateresinol (
1
) [
11] descuraic acid (
)-justiflorinol (11) [14], (+)-virgatyne (12) [15], the arylnaphthalene derivative 13 [16]
18], S-(+)-imperanene (16) [ ], 3-methoxy-imperanene (18) [19],
], tectonoelin A and B (21 22) [20], peperotetraphin (23) [21] the related
cyclobutane carboxylates 24 and 25 [22] and hyperione A and B (26 27) [23] (Figure 2). In addition,
8
], pachypostaudin A and B (
2,3) [9], aglacin H
(4
) [10], vitrofolal A,B,E, and F (
5
,6,
7,
8) [
2
,
9) [12], cestrumoside (glycoside)
(
10) [13], (
−
and the lactone 14 [17], compound 15
noralashinol A and C (19 20) [
[
1
,
3
,
4
,
,
some related 9-norlignans have been synthesized and some 9-norlignan like compounds have also
been reported [6,24,25]. In Figure 3, the structures of compounds 28–35 are shown.

Molecules 2019, 24, 220
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OMe
OMe
OMe
OMe
OH
O
O
MeO
MeO
OH
HO
OH
OMe
MeO
MeO
OMe
HO
OMe
OMe
MeO
OMe
OMe
MeO
MeO
OMe
OMe
OMe
OH
OMe
Yateresinol (1)
Aglacin H (4)
Pachypostaudin A (2)
Vitrofolal A (5)
Pachypostaudin B (3)
O
MeO
O
MeO
O
MeO
HO
O
OH
HO
OH
HO
HO
OH
OMe
OMe
OMe
OMe
OMe
OMe
MeO
OMe
OH
OH
Vitrofolal F (8)
HO
OH
O
Vitrofolal B (6)
Vitrofolal E (7)
Descuraic acid (9)
O
OH
HO
OH
O
OH
OH
OH
O
HO
HO
O
O
OGlu
HO
O
OH
OH
O
O
O
O
(+)-Virgatyne (12)
OH
Cestrumoside (10)
Justiflorinol (11)
O
13
O
MeO
MeO
HO
O
MeO
OH
OH
MeO
OH
O
HO
HO
HO
OH
OMe
MeO
OMe
OMe
OMe
OH
-(+)-Imperanene (16)
-(-)-Imperanene (17)
OH
3-Methoxy-imperanene (18)
Lactone (14)
OH
15
OH
S
R
OMe
OMe
HO
R
MeO
HO
MeO
HO
OH
MeO
O
O
OMe
OH
OH
OMe
OH
OH
Tectonoelin A (R=H) (21)
Tectonoelin B (R=OMe) (22)
Noralashinol C (19)
Noralashinol A (20)
O
O
O
OMe
MeO
O
OMe
O
O
O
O
O
O
O
O
O
OMe
OMe MeO
OMe MeO
O
7´ -(-)-Hyperione A (26)
7´S-(-)-Hyperione B (27)
R
24,25
Peperotetraphin (23)
Figure 2. The structures of previously reported 9-norlignans with natural origin.

Molecules 2019, 24, 220
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HO
O
OH
OH
OH
O
O
HO
OMe
OMe
OMe
OMe
OH
OMe
HO
HO
HO
O
MeO
O
MeO
OMe
MeO
HO
HO
Pouzolignan D (28)
Pouzolignan K (29)
Dysosmanorlignan A (30)
O
O
O
OMe
OMe
HO
HO
OH
OMe
HO
OMe
OH
OMe
OH
OMe
MeO
(S)-9´-dehydroxyimperanene (33)
(R)-9´-dehydroxyimperanene (32)
Dysosmanorlignan B(31)
OMe
HO
OMe
HO
OMe
OH
(R)-7,8-dihydro-9´-dehydroxyimperanene (34)
OMe
OH
(S)-7,8-dihydro-9´-dehydroxyimperanene (35)
Figure 3. Structures of synthetic 9-norlignans and 9-norlignan related compounds.
1.3. Occurrence and Biological Activity
Yateresinol ( ) was first isolated from the heartwood of Libocedrus yateensis in 1979 by Ertman
et al. [ ]. It has also been found in Cryptomerica japonica D. Don and found to be part of the wood
1
8
discoloring substances in sapwood [26]. Studies of butt-rot sugi wood suggested that the norlignans
may be important for antifungal properties but no clear effect has been shown [27]. Yateresinol has
been tested for cytotoxicity towards the human HL60 and Hepa G2 cancer cell lines [28] but was
shown to be inactive with the half maximal inhibitory concentration (IC50) values >20
The two 9,90-bisnorlignans
and (pachypostaudin A and B) have been isolated from stem bark
of Pachypodanthium staudtii Engl. et. Diels [ ]. The same bisnorlignans have also been isolated
µg/mL.
2
3
9
from bark of the related species Pachypodanthium confine Engl. et Diels [29]. Both species are trees
growing in the tropical zone of west and central Africa, and have been used in traditional African
medicine. However, no biological activity of the bisnorlignans has been reported. Aglacin H (4) has
been isolated from the bark of Aglaia cordata collected in Indonesia [10]. No other sources, and no
chemical and biological properties for this norlignan have so far been reported. Norlignans from Vitex
species, namely vitrofolal A, B, E, and F (compounds
These compounds were first isolated from Vitex rotundifolia [
been isolated from the seeds [30] and from the roots of Vitex negundo [31
have also been isolated from the fruits of Vitex cannabifolia [33]. Vitrofolal E (
reported in the stem and bark of Syringa pinnatifolia [ 34]. Vitrofolals have been shown to have various
biological activities. Compound was shown to have antibacterial activity against methicillin-resistant
Staphylococcus aureus at concentrations above 64 g/mL [ ]. Compounds and have also been
reported to have antioxidant activity by inhibition of lipid peroxidation using the ferric thiocyanate
method, and by scavenging of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). In both assays
and showed comparable or slightly higher antioxidative activity than -tocopherol, L-cysteine and
butylated hydroxyanisole (BHA) [33 35]. Vitrofolal E ( ) has also been shown to have moderate activity
in the in vitro cholinesterase inhibition assay [31] and to have tyrosinase inhibitory activity in the same
5–
8
) are norlignans of the arylnaphthalene type.
11]. Compounds have later also
32]. Compounds and
) was also recently
2
,
5–8
7
,
7
8
7
2,
7
µ
2
7
8
7
8
α
,
7

Molecules 2019, 24, 220
5 of 12
µ
M-range compared to known inhibitors [32]. Vitrofolal F (
8
) has been shown to have
α
-chymotrypsin
(serine protease) inhibitory effect and has thus been suggested as a specific natural inhibitor of this
enzyme [36]. The aryldihydronaphthoic acid derivative, descuraic acid ( ), was isolated from seeds of
9
Descurainia Sophia (L.) Webb ex Prantl, an annual/biennial herb [12]. Despite the structural similarities
with vitrofolals, no biological properties of this compound have been published to date, however it is
used in traditional medicine to alleviate common cold symptoms and to prevent asthma and oedema.
The 9-norlignan glycoside cestrumoside (10), was recently identified from methanol extract of Cestrum
diurnum L. leaves [13]. No biological effects for this compound were reported, although several
species of this plant have been used in traditional Chinese medicine, especially for treatment of burns
and swellings. However, recently 10 was claimed to be a protein kinase C inhibitor in an animal
feed additive [37]. A study of the cytotoxic effects of the arylnaphthalene lignans from Vietnamese
Acanthaceae, Justicia patentiflora revealed the structure of justiflorinol (11). However, the cytotoxic
evaluation showed that 11 does not have the same cytotoxic effect as structurally related lignans found
in this plant species [14]. Justiflorinol has also been isolated from leaves of Piper sanguineispicum and
tested for its antileishmanial activity and for cytotoxicity on MCF7 and Vero cells. No significant
biological activity was found [38]. (+)-Virgatyne (12) has been found in whole plant extracts of the
Taiwanese annual plant Phyllantus virgatus Frost. F. (Euphorbiaceae) [15]. This plant has traditionally
been used to protect the liver and 12 was tested for anti-hepatitis B virus in a MS-G2 cell line, but was
shown to be completely inactive [39].
Different liverwort species have been shown to contain dihydronaphthalene and naphthalene
lignans and norlignans. The arylnaphthalene derivative 13 was first isolated from Pellia epiphylla
and the structure was confirmed by chemical synthesis [16]. The same compound (13) was later
found in Jamesoniella autumnalis [40], Bazzania trilobata [41], Lepidozia reptans [42], Lepidozia incurvata,
Chiloscyphus polyanthos and Jungermannia exsertifolia ssp. cordifolia. In the last three species conjugates
with malic acid, shikimic acid and
activity has been reported.
α-L-rhamnose were also isolated [43], but so far no biological
The butyrolactone 9-norlignan (14) has been isolated from a methanol-water extract of leaves of
Cestrum parqui, originally a South American shrub, but frequently occurring also in the Mediterranean
region. Green cestrum (Cestrum parqui) has been shown to have phytotoxic effects and has been
studied as a natural herbicide. Compound 14 showed phytotoxic effects on Lactuca sativa (lettuce) and
Lycopersicon esculentum (tomato) by reducing the development of the plant [17].
Compound 15 was first identified in alkaline extraction (pulping) of Norway spruce (Picea abies)
roots. Later it was also shown to be a degradation product of the abundant guaiacyl type
dibenzylbutyrolactone lignan hydroxymatairesinol [18]. The degradation process has been studied
in detail and 15 can be obtained by alkaline treatment of hydroxymatairesinol in nearly quantitative
yields [
corresponding alcohol ((
has been found in the rhizomes of Imperata cylindrica. (+)-Imperanene was shown to have platelet
aggregation inhibitory effects and gave a complete inhibition, at 6
10−4 M concentration, of rabbit
platelet aggregation induced by thrombin [ ]. (+)-Imperanene, 3-methoxyimperanene (18) and their
5
]. The carboxylic acid has not been reported as a constituent in other plants, but the
−
)-imperanene (17)), although the other enantiomer (+)-imperanene 16,
×
1
glucosides have also been detected in rum distillate wastewater. In the same report, these compounds
were shown to be inhibitors of human tyrosinase activity (IC50 = 1.85 mM). (+)-Imperanene showed
the highest activity, indicating that the guaiacyl (3-methoxy-4-hydroxyphenyl) substitution pattern
is important for the activity [19]. Contrary to many other norlignans, imperanene has been a target
for several synthetic works. Noralashinol C (19) and noralashinol A (20), structurally related to the
arylnaphthalene norlignans vitrofolals, have been isolated from stem barks of Syringa pinnatifolia.
Compound 20 seems to be the acetal of
was inactive in a cytotoxicity assay using HepG2 hepatic cancer cells and 20 was inactive in the NO
production inhibitory assay [ ]. Tectonoelin A and B (21 22), and compound 14 have been isolated
from the leaves of Tectona grandis and shown to have growth inhibition activity in the etiolated wheat
7 (possibly formed by reaction with methanol). Compound 19
3
,4
,

Molecules 2019, 24, 220
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coleoptiles bioassay test [20]. Interestingly, both green cestrum and teak leaves seem to contain these
rare norlignans and display herbicidal effects. Cyclobutane-type norlignans peperoteraphin (23) and
two related isomers (24
and 25 were tested for cytotoxicity in human liver cancer cell lines HepG2, human lung cancer cell lines
A549, and human cervical cancer cell lines Hela. Moderate activity with IC50 values around 50
for all cell lines, were detected [21 22]. Hyperione A and B (26 27) have been isolated from Hypericum
,25) have been isolated from Peperomia tetraphylla (whole plant). Compounds 24
µM
,
,
chinense (whole herb). Hypericum species are known for their antibacterial activity but compounds 26
and 27 showed no antibacterial activity [23]. The occurrence and biological activities of 9-norlignans
are summarized in Table 1.
Table 1. 9-Norlignans, their occurrence and biological activity.
Compound
Number
1
Occurrence
Bioactivity
References
Libocedrus yateensis, Cryptomerica
japonica
Yateresinol
Antifungal
[8,26]
Pachypodanthium staudtii,
Pachypodanthium confine
Aglaia cordata
Pachypostaudin A
Pachypostaudin B
Aglacin H
2
3
4
5
6
7
8
[9,29]
[10]
Antibacterial, antioxidant,
cholinesterase inhibition, tyrosinase
inhibitory effects, chymotrypsin
inhibitory effects
Vitrofolal A
Vitrofolal B
Vitrofolal E
Vitrofolal F
Vitex rotundifolia, Vitex negundo,
Vitex cannabifolia, Syringa pinnatifolia
[2,11,30–34,
36]
Descuraic Acid
Cestrumoside
9
10
Descurainia Sophia
Cestrum diurnum L.
[12]
[13]
Justicia patentiflora, Piper
sanguineispicum
Phyllantus virgatus
(−)-Justiflorinol
(+)-Virgatyne
11
12
[14,38]
[15]
Pellia epiphylla, Jamesoniella
autumnalis, Bazzania trilobata,
Lepidozia reptans, Lepidozia incurvata,
Chiloscyphus polyanthos,
Jungermannia exsertifolia ssp.
cordifolia
Arylnaphthalene
13
[16,40–43]
Lactone
Compound
14
15
Cestrum parqui
Picea abies
Phytotoxic effects
[17]
[18]
Imperata cylindrica, sugarcane rum
distillate
Platelet aggregation inhibitory
effect, tyrosinase inhibitory effect
Tyrosinase inhibitory effect
(+)-Imperanene
16
[1,19]
[19]
3-Methoxy-Imperanene
Noralashinol C
Noralashinol A
Tectonoelin A
Tectonoelin B
Peperoteraphin
Cyclobutane
18
19
20
21
22
23
Sugarcane rum distillate
Syringa pinnatifolia
[3,4]
Tectona grandis
Herbicidal activity
Cytotoxic effect
[20]
Peperomia tetraphylla
Hypericum chinense
[21,22]
24
carboxylate
Cyclobutane
carboxylate
Hyperione A
Hyperione B
25
26
27
[23]
In addition to these compounds, some 9-norlignan-like compounds (although containing
additional carbon atoms) have been isolated (Figure 3). Pouzolignan D (28) and K (29) were isolated
from the aerial parts of Pouzolzia zeylanica (L.) Benn. var. microphylla (wedd.) W.T. Wang [24] and
dysosmanorlignans A (30) and B (31) from the roots of Dysosma versipellis [25]. No biological activity
has been reported for these compounds.
Several of these naturally occurring lignans have been the topic for total synthesis, however,
the synthetic methods are excluded from this literature review. Some semisynthetic methods as
well as the synthesis of interesting derivatives related to the presented norlignans will be discussed
briefly. For example, the synthesized dehydroxy- and dihydrodehydroxyimperanene isomers have
been evaluated for their plant growth regulatory effects, for larvicidal, antifungal, antibacterial and
cytotoxic activity, and compared to dihydroguaiaretic acid isomers. In most studies compounds
32–35 were less or equally active as dihydroguaiaretic acids. In the cytotoxicity study, using HL-60

Molecules 2019, 24, 220
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and HeLa cells, compound 35 showed the highest activity with IC50 values at approximately 6 µM,
suggesting that norlignans may serve as lead compounds for anticancer agents [6].
As previously mentioned, imperanene derivatives can also be synthesized from the natural lignan
hydroxymatairesinol and these compounds can be further transformed to vitrofolal and noralashinol
type norlignans. To expand the substrate scope for investigation of 9-norlignans and to perform
structure–activity relationship studies, we have undertaken the semisynthetic preparation of 15
different 9-norlignans with different functionalities using hydroxymatairesinol as starting material.
In this strategy we chose to introduce flexibility or rigidity to the basic skeleton. We also varied the
functionality at carbon-9 by the preparation of the carboxylic acid, the ester and the alcohol. In addition,
the skeleton was also aromatized by oxidation and two different substitution patterns at the aromatic
moieties were prepared. The semisynthesis of these compounds from hydroxymatairesinol is presented
in detail below.
2. Results
Semisynthesis of Norlignan Derivatives from Hydroxymatairesinol
The knots of Norway spruce (Picea abies (L.) H. Karst) have been shown to contain about
10% (w/w) of the dibenzylbutyrolactone hydroxymatairesinol (36, Scheme 1). Methods for the
separation of knots and the subsequent isolation of hydroxymatairesinol were developed during
the last decade. Today hydroxymatairesinol is isolated on kg-scale, which certainly makes it one
of the most readily available lignans in pure form for further studies. In our previous studies,
we have shown that hydroxymatairesinol is degraded to norlignan derivative 15 by strong alkali.
This retro-aldol-type reaction probably involves a formation of a quinone methide intermediate and
the loss of formaldehyde [5].
During degradation, two of the stereocentra of hydroxymatairesinol are destroyed, but the third
retains the R-configuration without isomerization, yielding an optically pure product in excellent
yields, usually over 95%. Further esterification and reduction of the obtained product (37), by lithium
aluminium hydride (LAH) affords the 9-norlignan ( )-imperanene (17), as previously reported.
−
However, the structure of 15 offers many possibilities for modification of the structure, which enabled
the preparation of a set of derivatives for structure-activity relationship (SAR) studies of their biological
effects (Scheme 1). The double bond was shown to be susceptible for protonation and Friedel–Crafts
ring closing to yield two diastereomers of the 6’-7-cyclo-9-norlignan (aryltetralin-type) 38 in a 3:4
ratio. The two diastereomers were not separable by column chromatography. Further modification
of the carboxylic acid to the ester 39 and the alcohol 40 was performed equally as in the case of
15 [5]. Oxidation of the cyclonorlignan 39 by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was
utilized to aromatize the aliphatic ring to obtain the corresponding arylnaphthalene vitrofolal-type
norlignan 41. This structure was also transformed into the acid 42 and the alcohol 43 by hydrolysis
and reduction, respectively. Oxidation of 38 or 40 by DDQ was proven to be unsuccessful. In the
case of 38, the structure was easily decarboxylated during the oxidation. The double bond of 15 was
also reduced by catalytic hydrogenation in a batch reactor using ethanol, Pd/C and hydrogen at a
slightly elevated pressure (2 bar) to give 44. Again, the ester 45 and the alcohol 46 were prepared
equally as for the other structures (Scheme 1). The permethylated derivative 48 was obtained by
methylation with MeI in dry acetone and K2CO3. The acid 47 and the alcohol 49 were prepared
accordingly. With these simple chemical modifications, we obtained a set of compounds (derivatives of
imperanenes, vitrofolals and noralashinols) with some structural alterations. In this strategy we chose
to introduce both flexibility, by reducing the double bond, and rigidity by introducing the aliphatic
ring, at the basic skeleton of imperanene. The nonflexible cyclic structures with a half-boat structure
38
structure (compounds 41
family. Finally, we chose to prepare the carboxylic acid with an ionizable group, the ester containing
–
40 adopted a certain non-planar conformation, and were therefore aromatized to yield a planar
–
43) resembling bioactive 9-norlignans of the vitrofolal and noralashinol

Molecules 2019, 24, 220
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the carbonyl function and the alcohol, for comparison of the effects of the functionality at C-9’. Some of
the derivatives were also submitted to methylation of the free phenolic groups for comparison of
the 3-methoxy-4-hydroxyphenyl and 3,4-dimethoxyphenyl moieties. The liverwort norlignan 13 was
obtained from 42 by treatment with AlCl3 in pyridine.
Scheme 1. Preparation of various 9-norlignans from hydroxymatairesinol. This generalized
scheme shows the fundamental chemical transformations. For more detailed information on the
interconversions and transformations including reaction conditions, see the supplementary material.
Compounds 15, 44, 48, 37, 38, 40 and 41 have previously been evaluated as inhibitors of multidrug
resistance protein 1 (MRP1)-mediated transport using human erythrocytes as model. This was
part of a larger study on polyphenolic compounds comprising lignans, norlignans, stilbenes and
flavonoids. Compound 15 was shown to be the most active one, with moderate activity at
IC50 = 50
is crucial for the activity. Furthermore, none of the norlignans showed detrimental effects up to
200 M, indicating a large therapeutic width. These compounds could therefore be interesting as
possible agents reversing multidrug resistance and to potentially sensitize cancer cells to anticancer
drugs [44]. A similar set of compounds (15 38 40 41, 44 and 48) were tested for estrogen and
antiestrogen activity in the yeast estrogen assay. Compounds 40 41 and 48 showed antiestrogenic
effects, albeit at quite high concentrations (100–500 M) [45]. Some of these norlignans were
µM. The structure-activity relationship study showed that the carbonyl function at C-9’
µ
,
,
,
,
µ
also studied for antimicrobial effects in comparison with the effective stilbene trans-pinosylvin
(trans-3,5-dihydroxystilbene) and its monomethylated derivative (trans-3-hydroxy-5-methoxystilbene).

Molecules 2019, 24, 220
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Although some effects were observed, it was concluded that the norlignans were ineffective against
Salmonella infantis, Listeria monocytogenes and Candida tropicalis [46].
3. Discussion and Conclusions
Norlignans of the C-9 type are an uncommon group of polyphenols in nature. Many of these
compounds can be found in plants used in traditional Asian medicine, however published scientific
data of their bioactivities is in general rather scarce. In most cases, their biological effects have been
tested in a few assays mainly for antioxidant, cytotoxic and antimicrobial activities. Clearly, a more
broad and systematic screening of bioactivity should be conducted to evaluate these compounds as
potential bioactive agents. 9-Norlignans belonging to the vitrofolal-, noralashinol-, and imperanene
families have however been shown to be bioactive and some of them could find potential in
treatment of cancer. Furthermore, these 9-norlignans have been shown to inhibit specific enzymes,
which warrants further investigations of these compounds. In our semisynthetic approach using
hydroxymatairesinol as starting material for the preparation of 9-norlignans, we have developed simple
chemical transformations to unsaturated, saturated, cyclic and aromatic structures closely resembling
those of the vitrofolal, noralashinol and imperanene family. The unsaturated compounds 15, 17 and 37
could also be seen as polyphenolic compounds with both trans-stilbene-like, and lignan-like structures,
which could make them interesting as antimicrobial compounds. The cyclic structures were obtained
in high yields by treatment with formic acid or trifluoroacetic acid. However, this Friedel–Crafts-type
reaction can also be performed in aqueous conditions, using mineral acids. The aromatization was
introduced by reaction with DDQ with moderate yields. DDQ was superior for this reaction due to its
benzylic hydride abstraction properties, yielding dehydrogenation of the product, rather than oxidative
polymerization. Hydrogenation of the double bond was facile, proceeding in high yields. To broaden
the substrate scope of the semisynthetic 9-norlignans, the initial carboxylic acid (of compound 15) was
esterified and reduced by conventional methods, in relatively high yields. By these interconversions the
carboxylic acid, the methyl ester and the alcohol derivatives were obtained for all the basic structures.
Some structures were also methylated at the phenolic positions, which gave us 16 norlignans with
different structures available for screening of biological activity. This semisynthetic route is not
restricted to these compounds and it offers numerous additional chemical modifications and functional
group interconversions. The biological testing of some selected semisynthetic 9-norlignans showed
that none of these compounds were highly active. However, compound 15 showed moderate activity
as an inhibitor of MRP1-mediated transport. The activity was decreased for all other derivatives,
which indicated that the double bond and the carboxylic acid function were important for the activity.
Due to the limited scope of biotesting for these semisynthetic 9-norlignans, no generalized results
of their bioactivity or results on structure-activity relationships can be proposed, without further
screening of biological activity.
4. Materials and Methods
All commercially available chemicals were used as supplied by the manufacturers.
Hydroxymatairesinol was isolated from Norway spruce (Picea abies (L.) Karst) knots by previously
described methods [5]. Knots of Norway spruce were separated, ground and freeze-dried prior
to extraction in a Soxhlet apparatus. The raw extract obtained with acetone-water (9:1 v/v),
after the removal of lipophilic extractives with hexane was purified by flash chromatography (eluent
CH2Cl2:EtOH 98:2 v/v) to yield hydroxymatairesinol.
GC analyses were performed on a standard gas chromatograph (Agilent Technologies, model 6850,
Santa Clara, CA, USA) equipped with a HP-5 column and a flame ionization (FI) detector. The samples
were silylated using hexamethyldisilazane-chlorotrimethylsilane in pyridine, prior to analyses.
Gas chromatography-mass spectrometry (GCMS) analyses were performed essentially the same
way (Agilent Technologies, model 7890A+5975C). High-resolution mass spectrometry (HRMS) were
recorded using a Micro Q-TOF (Bruker, Billerica, MA, USA) with electrospray ionization (ESI)

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operated in positive mode or with a ZAB-Spec high-resolution MS-ESI instrument (Fisons Instruments,
Ipswich, UK).
1H- and 13C-NMR spectra were recorded on an Avance instrument (Bruker) at 600.13 and
150.90 MHz, respectively. 2D NMR experiments (1H-1H COSY, HSQC, HMBC) were recorded
using standard pulse sequences and chemical shifts are reported downfield from tetramethylsilane.
Optical rotations were measured with a digital polarimeter model 241 (Perkin Elmer, Waltham, MA,
USA) using a 1 dm, 1 mL cell.
The inhibition of multidrug resistance protein 1-mediated transport was studied by
measurement of 20,70-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) efflux in human
erythrocytes. Erythrocytes were loaded with 20,70-bis-(carboxypropyl)-5-(6)-carboxyfluorescein
acetoxymethyl ester (BCPCF-AM) and incubated with or without the norlignans (10–200 mM).
The extracellular BCPCF fluorescence intensity was then measured and the IC50 values for BCPCF
efflux were determined from dose-response curves. The detailed experimental procedure and
results have previously been published [44]. The estrogen and antiestrogen activity was determined
using the yeast estrogen assay consisting of a transformed yeast strain (Saccharomyces cerevisiae).
Cells were pre-cultured and diluted in a medium containing chlorophenol red-β-D-galactopyranoside.
The suspensions with compounds were incubated in 96-well plates for three days before the absorbance
at 540 nm was measured. Estrogenic or anti-estrogenic activity was calculated by subtraction of
absorbance at 620 nm from that of 540 nm. Dose curves were plotted and the IC50 values were
calculated. 17β-Estradiol was used as reference. A more detailed description has been published by
Aehle et al. [45].
The antimicrobial activity tests were performed with Listeria monocytogenes L211, Salmonella infantis
EELA72 and Candida tropicalis 4068 using an automated incubator and turbidity reader to monitor the
microbial growth. The compounds were studied at equimolar concentrations (0.1–1 mM). The growth
was calculated by subtraction of the turbidity of the test culture from that of the control and expressed
as growth inhibition percentages. The detailed experimental procedure and results have previously
been published [46].
Supplementary Materials: The following are available online. Full experimental procedures for compounds
36–50, including analytical data.
Author Contributions: Conceptualization: P.E., methodology: P.E. and J.-E.R., formal analysis: P.E. and J.-E.R.,
investigation: P.E. and J.-E.R., resources: P.E., writing—original draft preparation: P.E., writing—review and
editing: P.E. and J.-E.R., visualization: P.E., supervision: P.E., project administration: P.E., funding acquisition: P.E.
Funding: This research was part of the BIOSTIMUL (Bioactive and wood-associated stilbenes as multifunctional
antimicrobial and health promoting agents) project. Financial support from the Finnish Funding Agency
for Technology (Tekes), from the Magnus Ehrnrooth foundation and from the Liv och Hälsa foundation is
gratefully acknowledged.
Acknowledgments: Annika Smeds is acknowledged for the HRMS analyses and Jan-Erik Lönnqvist for help with
the syntheses.
Conflicts of Interest: The authors declare no conflicts of interest.
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