Synthesis and neurite growth evaluation of new analogues of honokiol, a neolignan with potent neurotrophic activity

Article abstract of DOI:10.1016/j.bmcl.2011.12.015

A versatile synthetic route is reported towards the preparation of new analogues for potent neurotrophic agent biaryl-type lignan honokiol. A focused 24-membered library of derivatives containing five different groups at 5′-position of honokiol has been prepared in fair to good overall yields. Compared to the natural product, or to analogues with a short alkyl chain in this position, these new derivatives have lost most of the neurotrophic activity.

Full text of DOI:10.1016/j.bmcl.2011.12.015

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1439–1444
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and neurite growth evaluation of new analogues of honokiol,
a neolignan with potent neurotrophic activity
Vemula Praveen Kumar a,b, R. Gajendra Reddy c, Duc Duy Vo b, Sumana Chakravarty c,
b,
Srivari Chandrasekhar a, , René Grée
⇑
⇑
a Division of Natural Product Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Université de Rennes 1, Laboratoire SCR, CNRS UMR 6226, Avenue du Général Leclerc, 35042 Rennes Cedex, France
c Chemical Biology/Division of Natural Product Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A versatile synthetic route is reported towards the preparation of new analogues for potent neurotrophic
agent biaryl-type lignan honokiol. A focused 24-membered library of derivatives containing five different
groups at 50-position of honokiol has been prepared in fair to good overall yields. Compared to the natural
product, or to analogues with a short alkyl chain in this position, these new derivatives have lost most of
the neurotrophic activity.
Received 7 November 2011
Revised 1 December 2011
Accepted 3 December 2011
Available online 9 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Honokiol
Suzuki–Miyaura cross coupling
Neurotrophic activity
Lignans
The root and stem bark of the oriental herb Magnolia officinalis1
(also known as Houpo) or Magnoliae obovata2 have been used in
traditional Chinese and Japanese medicines for treatment of vari-
ous diseases like flu, anxiety and allergy. Early research on these
traditional medicines has identified honokiol 1 and its structural
isomer magnolol 2 as the two main biphenyl-type neolignans ac-
tive compounds (Fig. 1).
The structure of honokiol 1 consists of a biphenyl skeleton with
ortho, para-C,C-coupling of para-allyl and ortho-allyl phenols.
Honokiol 1 exhibits anticancer,3 anti-inflammatory,4 anti-viral,5
anti-fungal,6 anti-oxidant7 and anxiolytic8,9 properties. Further, it
has a neuroprotective effect and promotes neurite outgrowth.2,8
Due to these biological properties, honokiol attracted a great inter-
est among biologists and chemists. Four total syntheses have been
reported till date for honokiol,9,10 and two for 40-O-methylhonoki-
ol.11 On the other hand, a few analogues have been already synthe-
sized for biological studies.5,12,13
a few compounds where double bonds were reduced and phenols
selectively protected.12 The results reveals that the 40-phenol and
5-allyl group are essential for neurotrophic activity. In agreement
with this proposal, it was found that 4-O-methylhonokiol pro-
motes neurite outgrowth, and this occurs through ERK activation.16
As part of our CNS program on the development of novel neuro-
trophic agents based on natural products, we selected new ana-
logues of honokiol as our target molecules. Based on literature
data indicated above, we designed derivatives with various R sub-
stituents at 50-allyl position of 1. Further, we have studied the ef-
fect of the substitution of two phenol groups by preparing
corresponding methyl ethers.
In this Letter, we describe the synthesis and biological evalua-
tion of a focused library of new analogues of honokiol, with hetero-
aromatic (pyrimidine, pyridine, pyrazole) R groups in position 50,
Small molecules, active as neurotrophic agents, appear to be of
much interest in the context of fast growing neurodegenerative
diseases.14 Honokiol has demonstrated neurotropic activity at
OH
0.1–1 l
M concentrations on the cultures of rat cortical neurons.9
OH
From a mechanistic point of view, this action seems to be related
to activation of extracellular signal-related kinases.15 A struc-
ture–activity relationship (Fig. 2) has been already performed on
HO
HO
Honokiol 1
Figure 1. Structures of honokiol 1 and magnolol 2.
Magnolol 2
⇑
Corresponding authors.
E-mail address: rene.gree@univ-rennes1.fr (R. Grée).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.015

1440
V. Praveen Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1439–1444
OH
OH (Me)
R
R1
4'
5'
5'
important for
bioactivity
less important
variability
tolerated
(R2 ) HO
(Me) HO
5
R = aromatic, heteroaromatic systems, COAr
Figure 2. Reported SAR studies and designed analogues of honokiol.
both as phenols and corresponding methylethers (Fig. 2). Further,
to explore the effect of a polar carbonyl group in this position,
we prepared also derivatives with a benzoyl system (compounds
g) as well as similar compounds with an acyl triazole group (deriv-
atives h) Scheme 3.
As shown in Scheme 1, we developed a convergent and versatile
synthetic route to produce designed analogues of 1, that is, com-
pounds from Series I, II and III depending upon the number of free
phenol groups. A Pd-catalyzed Suzuki–Miyaura coupling could be
employed to construct biaryls (3a–h, 4a–h, 5a–h) from bromides
(7a–h, 6a–h) and boronate 10. The aromatic bromides with various
obtained by treating propargyl ketone 8 with 2,3-dimethyl-1,
4-butadiene in toluene. Finally, a click reaction,19 performed be-
tween compound 8 and benzylchloride and sodium azide in water
at reflux for 24 h, gave the triazole 6h in 94% yield (Scheme 3).
The second series of intermediates, the demethylated com-
pounds 7a–h, were prepared in good yields by heating compounds
6a–h with borontrichloride–dimethylsulfide complex in dichloro-
ethane at reflux for 12–24 h (Scheme 3).
Next, the cross coupling partner boronic acid 10 was prepared
from 4-allyl anisole 11 in a one step process of lithiation followed
by reaction with trialkylborate (Scheme 4).17
heterocyclic and carbonyl groups at
a
-position to O-methyl/hydro-
The series I of biaryl compounds 5a–h have both phenol func-
tions protected as methyl ethers. They were synthesized using
Suzuki–Miyaura coupling reactions.20 The Pd(PPh3)4 catalyzed
cross coupling reaction between aryl bromides 6a–h and aryl boro-
nic acid 10 in 1,2-DME-H2O (8:2) in the presence of Na2CO3 at
reflux temperature for 16 h gave 5a–h in 76–92% yield (Scheme
5, Table 1). In the case of 6f (Table 1, entry 6) these reaction condi-
tions gave a lower yield, possibly due to base hydrolysis of ester
group. Finally, by changing solvent to benzene and base to potas-
sium carbonate, the biaryl derivative 5f was isolated in 82% yield.
xyl group could be easily obtained from propargyl ketone 8, which
should itself be synthesized from commercially available 5-bromo-
salicylaldehyde 9. The boronate 10 could be prepared from 4-allyl-
anisole 11 by a known procedure.17
The synthetic route started with the preparation of key inter-
mediate propargyl ketone 8. The O-methylation of 5-bromo salicyl-
aldehyde with dimethylsulfate in acetone in the presence of
Na2CO3, followed by the reaction with ethynyl magnesium bro-
mide gave propargyl alcohol 12 in good yield over two steps. Oxi-
dation of compound 12 with Jones’ reagent gave the desired ketone
8 in 78% yield (Scheme 2).
The stage was set for the preparation of the required intermedi-
ates. The propargyl ketone 8 was refluxed in acetonitrile with
several amidine hydrochlorides in the presence of Na2CO3 to give
corresponding pyrimidines 6a–c in fair to good yields. Then pyra-
zoles 6d,e were prepared in excellent yields by refluxing 8 with
hydrazines in ethanol for 12 h. The pyridine 6f was obtained in
78% yield by a Bohlmann–Rahtz reaction,18 by reacting 8 with ethy-
lacetoacetate, ammonium acetate and zinc bromide in toluene at
reflux for 24 h. On the other hand, the Diels–Alder product 6g was
OH
Br
OH OMe
O
OMe
Br
OHC
a, b
c
Br
9
12
8
Scheme 2. Synthesis of propargylic ketone 8. Reagents and conditions: (a) Me2SO4,
Na2CO3, acetone, reflux, 3 h, 92%; (b) ethynylMgBr, THF, rt, 16 h, 72%; (c) Jones
reagent, acetone, 0 °C, 78%.
OMe
OH
OMe
O
OMe
Br
OHC
R
Pd(0)
R
10
MeO
Br
Br
6 a-h
9
8
Series I (5 a-h)
OH
OH
B(OH)2
R
R
OH
MeO
MeO
Pd(0)
R
10
HO
MeO
11
10
Br
7 a-h
Series III (3 a-h)
Series II (4 a-h)
Scheme 1. Retrosynthesis of honokiol analogues.

V. Praveen Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1439–1444
1441
6a: R= Me, R1= Me, 85%
O
OR
Br
R1
f
f
f
1
7a
6b
: R= H, R = Me, 82%
N
N
N
N
OR
Br
N
1
: R= Me, R = Ph, 91.5%
7b: R= H, R1= Ph, 86%
Bn
a
e
1
6c
: R= Me, R = NH2, 52%
6h
: R= Me, 94%
f
7c: R= H, R1= NH2, 74%
O
OMe
Br
7h: R= H, 92%
d
R2
b
O
OR
8
N
H3C
H3C
N
OR
Br
6d: R= Me, R2= Me, 64%
7d: R= H, R2= Me, 91%
f
f
c
6e: R= Me, R2= Ph, 59%
Br
6g
: R= Me, 87%
EtOOC
H3C
7e: R= H, R2= Ph, 92%
f
OR
Br
7g: R= H, 95%
N
6f: R= Me, 78%
7f: R= H, 35%
f
Scheme 3. Reagents and conditions: (a) R1C = NH(NH2)ꢀHCl, sodium carbonate, acetonitrile, reflux, 8 h; (b) R2NHNH2, 4 Å MS, EtOH, reflux, 12 h; (c) ethylacetoacetate,
ammonium acetate, ZnBr2, toluene, reflux, 24 h; (d) 2,3-dimethyl-1,3-butadiene, toluene, 80 °C, 24 h; (e) benzylchloride, sodium azide, water, reflux, 24 h; (f) 2 equiv
BCl3ꢀDMS (2 M solution in dichloromethane), dichloroethane, 24 h.
The second series of molecules 3a–h were obtained in 48–95%
yields from adducts 4a–h through demethylation with borontri-
chloride–dimethyl sulfide complex in dichloroethane at reflux
(Scheme 6, Table 2).
B(OH)2
tBuLi, -78 °C- 0 °C, 2 h
MeO
MeO
B(iOPr)3, -78 °C- rt,
24 h, 45%
For biological evaluation,21,22,23 neurotrophic and cell viability
assays have been performed for all new compounds, by compar-
ison with honokiol, and the results were analyzed series wise. In
view of cell viability tests (data not shown) three compounds
were selected to report (see Fig. 3 and Supplementary data for
details).
11
10
Scheme 4. Synthesis of boronic acid 10.
The next compounds have either one phenol and one methoxy
group (series II) or two phenol groups (series III). The first deriva-
tives 4a–h were obtained in 62–89% yield by Suzuki–Miyaura cross
coupling reactions between aryl bromides 7a–h and aryl boronic
acid 10 in the presence of Pd(PPh3)4 (Scheme 6, Table 2).
For immunocytochemistry, after 48 h in culture with
treatment, cells were fixed with 4% paraformaldehyde at room
temperature for 15 min, and incubated in blocking buffer (2%
bovine serum albumin + 0.3% Triton X-100 in PBS) for 2 h at room
temperature. Primary antibody against beta III tubulin (1:300,
OMe
OMe
B(OH)2
R
R
Pd(PPh3)4, DME/H2O (8:2)
MeO
Na2CO3, 16 h
MeO
Br
6a-h
10
5a-h
Scheme 5. Synthesis of bisethers analogues 5a–h. For the nature of R groups and yields see Table 1.
OH
OH
OH
R
R
R
a
b
MeO
HO
Br
7a-h
4a-h
3a-h
Scheme 6. Reagents and conditions: (a) 10, Pd(PPh3)4, 1,2-DME-H2O (8:2), Na2CO3, reflux, 16 h; (b) BCl3ꢀDMS (2 M in dicloromethane), DCE, reflux.

1442
V. Praveen Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1439–1444
Table 1
Table 2
S. No.
1
R
Yielda (%)
89
S. No.
R
Product Yielda
(%)
Product Time Yielda
(%)
CH3
CH3
N
N
N
1
2
3
4a
4b
4c
82
79
72
3a
3b
3c
24
28
36
82
87
91c
N
N
N
5a
Ph
N
Ph
N
N
N
2
3
84
76
NH2
N
5b
NH2
N
CH3
N
N
4
5
6
4d
4e
4f
89
3d
3e
3f
18
18
36
95
94
48
5c
CH3
Ph
N
N
N
N
85
4
5
6
92
EtOOC
CH3
5d
62b
Ph
N
N
N
O
90
CH3
CH3
5e
7
8
4g
4h
86
81
3g
3h
12
18
93
69
EtOOC
O
82b
CH3
N
N
N
5f
N
O
Bn
CH3
CH3
a
b
c
7
85
84
Yields calculated after column chromatography.
Reaction carried out in benzene with K2CO3 as a base.
Reaction quenched with ammonium hydroxide solution.
5g
O
N
N
8
N
Bn
5h
a
Isolated yields after column chromatography.
Reaction carried out in benzene with K2CO3 as a base.
b
Millipore) was used to visualize neurons. Samples were incubated
with primary antibody in blocking buffer overnight at 4 °C.
Samples were washed with PBST (PBS with 0.1% Tween20) and
incubated with goat anti-mouse IgG conjugated to AlexaFlour
488 (1:400, Molecular Probs). Images were captured using an
Olympus IX70 (Olympus America, Melville, NY) inverted micro-
scope and with an Optronics MagnaFire (Goleta, CA) digital color
camera. Appropriate composite figures were produced using
Adobe Photoshop (Fig. 4).
Average neurite
Percent change
from control
Treatment
length per
neuron(µm)
-
60.27 5.5
62.97 7.02
119.06 20.84
101.30 10.08
66.05 5.43
67.14 6.87
66.16 5.02
Control
DMSO
NGF
honokiol
3f
We found only three compounds (dihydroxypyridine 3f,
dimethoxytriazole 5h and monomethoxymonohydroxymethyl
pyrazole 4d) having some neurotrophic effects at 0.1–1.0 lM
2.7
58.79
41.03
5.78
6.87
5.89
concentrations as shown below.
In summary, a versatile strategy was developed for the synthe-
sis of honokiol analogues through Suzuki–Miyaura cross coupling
reactions as key steps. In this paper we synthesized, in fair to good
overall yields, a focused library of 24 honokiol analogues with
different types of substituents at 50 position. Compared to the
natural product, or to analogues with a short alkyl chain in this
position, these new derivatives have lost most of the neurotrophic
activity. Synthesis and biological evaluation of new analogues of
honokiol and other polyphenol-type derivatives are under active
study in our groups and corresponding results will be reported in
due course.
5h
4d
Figure 3. Morphometric analysis of neurite outgrowth of differentiated Neuro2a
cells cultured for 48 h after treatment with NGF [200 ng/mL], DMSO [1%], EtOH
[0.5%], honokiol, 3f & 5h [1 lm] and 4d [0.1 lm]. The tabulated and bargraph data
were expressed as mean SEM where n = 60 and compared with control, however,
none of them are found to be having a significant neurotrophic effect.

V. Praveen Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1439–1444
1443
Figure 4. Immunocytochemical images, stained with beta III tubulin, showing optimum changes in neurite outgrowth of differentiated Neuro2a cells after treatment with
NGF [200 ng/mL], DMSO [1%], ethanol [0.5%], honokiol, 3f & 5h [1 lm] and 4d [0.1 lm] after 48 hours.
2. Fukuyama, Y.; Otoshi, Y.; Miyoshi, K.; Nakamura, K.; Kodama, M.; Nagasawa,
Acknowledgments
M.; Hasegawa, T.; Okazaki, H.; Sugawara, M. Tetrahedron 1992, 48, 377.
3. Kong, Z. L.; Tzeng, S. C.; Liu, Y. C. Bioorg. Med. Chem. Lett. 2005, 15, 163.
4. (a) Matsuda, H.; Kageura, T.; Oda, M.; Morikawa, T.; Sakamoto, Y.; Yoshikawa,
M. Chem. Pharm. Bull. 2001, 49, 716; (b) Liou, K.-T.; Shen, Y.-C.; Chen, C.-F.; Tsao,
C.-M.; Tsai, S.-K. Eur. J. Pharmacol. 2003, 475, 19; (c) Tse, K.-W.; Wan, C.-K.;
Shen, X.-L.; Yang, M.; Fong, W.-F. Biochem. Pharmacol. 2005, 70, 1443.
5. Amblard, F.; Govindarajan, B.; Lefkove, B.; Rapp, K. L.; Detorio, M.; Arbiser, J. L.;
Schinazi, R. F. Bioorg. Med. Chem. Lett. 2007, 17, 4428.
This research has been performed as part of the Indo-French
‘Joint Laboratory for Sustainable Chemistry at Interfaces’. We thank
CNRS, MESR, CSIR for support of this research. V.P.K. thanks for
financial assistance the CSIR, New Delhi in India and French Em-
bassy (Delhi) for support through the Sandwich thesis Program.
V.D.D. thanks Laboratoires Servier and Société de Chimie Théra-
peutique for a fellowship. R.G.R. thanks CSIR for Junior Research
Fellowship and S.C. likes to acknowledge the financial assistance
of Department of Biotechnology (DBT), India, in part, under
Ramalingaswami re-entry Fellowship.
6. Bang, K. H.; Kim, Y. K.; Min, B. S.; Na, M. K.; Rhee, Y. H.; Lee, J. P.; Bae, K. H. Arch.
Pharm. Res. 2000, 23, 46.
7. (a) Ogata, M.; Hoshi, M.; Shimotohno, K.; Urano, S.; Endo, T. J. Am. Oil Chem. Soc.
1997, 74, 557; (b) Shen, J.-L.; Man, K.-M.; Huang, P.-H.; Chen, W.-C.; Chen, D.-
C.; Cheng, Y.-W.; Liu, P.-L.; Chou, M.-C.; Chen, Y.-H. Molecules 2010, 15, 6452.
8. Fukuyama, Y.; Nakade, K.; Minoshima, Y.; Yokoyama, R.; Zhai, H.; Mitsumoto, Y.
Bioorg. Med. Chem. Lett. 2002, 12, 1163.
9. Esumi, T.; Makado, G.; Zhai, H.; Shimizu, Y.; Mitsumoto, Y.; Fukuyama, Y.
Bioorg. Med. Chem. Lett. 2004, 14, 2621. and references cited therein.
10. (a) Takeya, T.; Okubo, T.; Tobinaga, S. Chem. Pharm. Bull. 1986, 34, 2066; (b)
Chen, C.-M.; Liu, Y.-C. Tetrahedron Lett. 2009, 50, 1151; (c) Denton, R. S.; Scragg,
J. T.; Galofré, A. M.; Gui, X.; Lewis, W. Tetrahedron Lett. 2010, 66, 8029.
11. (a) Denton, R. M.; Scragg, J. T.; Saska, J. Tetrahedron Lett. 2011, 52, 2554; (b)
Kwak, J.-H.; Cho, Y. A.; Jang, J.-Y.; Seo, S.-Y.; Lee, H.; Hong, J. T.; Han, S.-B.; Lee,
K.; Kwak, Y.-S.; Jung, J.-K. Tetrahedron 2011, 67, 9401.
12. Esumi, T.; Makado, G.; Zhai, H.; Shimizu, Y.; Mitsumoto, Y.; Fukuyama, Y.
Bioorg. Med. Chem. Lett. 2004, 14, 2621.
13. (a) Kong, Z.-L.; Tzeng, C.-C.; Liu, Y.-C. Bioorg. Med. Chem. Lett. 2005, 15, 163; (b)
Luo, Y.; Xu, Y.; Chen, L.; Hu, J.; Peng, C.; Xie, D.; Shi, J.; Huang, W.; Xu, G.; Peng,
M.; Han, J.; Li, R.; Yang, S.; Wei, Y. Bioorg. Med. Chem. Lett. 2009, 19, 4702; (c)
Taferner, B.; Schuehly, W.; Huefner, A.; Baburin, I.; Wiesner, K.; Ecker, G. F.;
Supplementary data
Supplementary data (synthetic procedures and analytical data for
new compounds, biological tests) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.12.015.
References and notes
1. (a) Fujita, M.; Itokawa, H.; Sashida, Y. Chem. Pharm. Bull. 1972, 20, 212; (b) Song,
Q.; Fischer, N. H. J. Mex. Chem. Soc. 1999, 6, 211.

1444
V. Praveen Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1439–1444
Hering, S. J. Med. Chem. 2011, 54, 5349; (d) Ma, L.; Chen, J.; Wang, X.; Liang, X.;
Luo, Y.; Zhu, W.; Wang, T.; Peng, M.; Li, S.; Jie, S.; Peng, A.; Wie, Y.; Chen, L. J.
Med. Chem. 2011, 54, 6469.
19. (a) Li, P.; Wang, L. Lett. Org. Chem. 2007, 4, 23; (b) Rostovstev, V. V.; Green, L. G.;
Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596; (c) Tomoe, C. W.;
Christensen, C.; Medal, M. J. Org. Chem. 2002, 67, 3057; (d) Johnson, J. B.; Bercot,
E. A.; Williams, C. M.; Rovis, T. Angew. Chem., Int. Ed. 2007, 46, 4514; for a recent
review see: (e) Lutz, J.-F. Angew. Chem., Int. Ed. 2008, 47, 2182. and references
cited therein.
14. See for instance: Wilson, R. A.; Danishefsky, S. J. Acc. Chem. Res. 2006, 39, 539.
15. Zhai, H.; Nakade, K.; Oda, M.; Mitsumoto, Y.; Akagi, M.; Sakurai, J.; Fukuyama,
Y. Eur. J. Pharmacol. 2005, 516, 112.
16. Lee, Y. K.; Choi, I. S.; Kim, Y. H.; Kim, K. H.; Nam, S. Y.; Yun, Y. W.; Lee, M. S.; Oh,
K. W.; Hong, J. T. Neurochem. Res. 2009, 34, 2251.
17. Denton, R. M.; Scragg, J. T. Synlett 2010, 633.
18. (a) Bagley, M. C.; Dale, J. W.; Bower, J. Chem. Commun. 2002, 1682; (b)
Bohlmann, F.; Rahtz, D. Chem. Ber. 1957, 90, 2265; (c) Bagley, M. C.; Glover, C.;
Chevis, E. D. Synlett 2005, 649; (d) Bagley, M. C.; Glover, C. E.; Merritt, A. Synlett
2007, 2459. and references cited therein.
20. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
21. Evangelopoulos, M. E.; Weis, J.; Krüttgen, A. Oncogene 2005, 24, 3309.
22. Khaing, Z.; Kang, D.; Camelio, A. M.; Schmidt, C. E.; Siegel, D. Bioorg. Med. Chem.
Lett. 2011, 21, 4808.
23. Yamazaki, M.; Chiba, K. J. Health Sci. 2005, 51, 687.