Total synthesis of inubosin B

Article abstract of DOI:10.1016/j.tetlet.2020.152641

Inubosin B is the most active member of the acridine alkaloids isolated from Streptomyces sp. IFM 11440 culture. The inubosins initiate neuroregeneration via a neurogenine 2 pathway. In this work, we have described the total synthesis of inubosin B via two synthetic routes. The effects of various coupling, cyclization and reduction reactions are discussed including common pitfalls and side reactions. Reverse phase chromatography with TFA was crucial for the isolation of the product from aluminum ions present in the reduction media.

Full text of DOI:10.1016/j.tetlet.2020.152641

Journal Pre-proofs
Total Synthesis of Inubosin B
Mohamed Farouk Hamissa, Petr Niederhafner, Martin Šafař ík, Marta Telus,
Lenka Kolá řová, Leah Koutná, Hana Šestáková, Radko Souček, Jaroslav
Šebestík
PII:
S0040-4039(20)31146-1
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.152641
TETL 152641
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
21 October 2020
5 November 2020
8 November 2020
Please cite this article as: Farouk Hamissa, M., Niederhafner, P., Šafař ík, M., Telus, M., Kolá řová, L., Koutná,
L., Šestáková, H., Souček, R., Šebestík, J., Total Synthesis of Inubosin B, Tetrahedron Letters (2020), doi:
https://doi.org/10.1016/j.tetlet.2020.152641
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Total Synthesis of Inubosin B
a,b,
a,b
a
a
Mohamed Farouk Hamissa *, Petr Niederhafner , Martin Šafařík , Marta Telus , Lenka
a
a
a
a
a,
Kolářová , Leah Koutná , Hana Šestáková , Radko Souček , Jaroslav Šebestík *
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo náměstí 2,
1
6610 Prague 6, Czech Republic.
b
Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical
Technology, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague 6,
Czech Republic.
KEYWORDS: Neuroregeneration, Neurodegeneration, Acridine, Alkaloid, Total synthesis
Corresponding Authors
*
mfahamissa@hotmail.com and sebestik@uochb.cas.cz
1

Abstract: Inubosin B is the most active member of the acridine alkaloids isolated from
Streptomyces sp. IFM 11440 culture. The inubosins initiate neuroregeneration via a neurogenine
2
pathway. In this work, we have described the total synthesis of inubosin B via two synthetic
routes. The effects of various coupling, cyclization and reduction reactions are discussed
including common pitfalls and side reactions. Reverse phase chromatography with TFA was
crucial for the isolation of the product from aluminum ions present in the reduction media.
2

Introduction
Neurodegenerative diseases are characterized by the progressive loss of neurons leading
to neuron death.1-3 Currently, no efficient strategies for curing neurodegenerative diseases are
available, and all marketed drugs and treatment protocols instead focus on relieving symptoms
and slowing the rate of decline in patients.4-6 On the other hand, adult neurogenesis is the
differentiation of neural stem cells to neurons in order to compensate for the neuronal loss.7-9
Accordingly, the induction of neurogenesis can be a therapeutic strategy for neurodegenerative
diseases.2, 10, 11
Natural products are important for drug discovery and activity optimization.12-17 The
inubosins are a group of naturally occurring acridine alkaloids recently isolated from the extract
of a culture of Streptomyces sp. IFM 11440.18 They induce neurogenesis by promoting the
activity of neurogenin2 (Ngn2).18 In comparison to the reference drug baicalin and other
members of the inubosin family, inubosin B had superior activity in increasing mRNA
1
8
expression of Ngn2. The superior activity of inubosin B compared to inubosins A and C is due
to the absence of the hydroxyl group at position 5 of the acridine ring, as well as the presence of
the hydroxyl group at position 4 (Fig. 1).18
OH
OH
OH
N
N
N
OH
Inubosin A
OH
Inubosin B
OH
OH
Inubosin C
Figure 1. Structures of inubosins A, B, and C.
However, to the best of our knowledge, there are no synthetic routes towards inubosin B;
thus, we carried out a total synthesis of inubosin B via two methods. Furthermore, our synthetic
route could potentially be used for the development of inubosin-like compounds, which can be
activities.19,
20
tested
for
various
biological
Results and Discussion
For the synthesis of inubosin B, we proposed a retrosynthetic scheme based on the typical
reactivities of acridines.21 The alcoholic and phenolic groups can be transformed into the
3

corresponding ester and ether groups (1), respectively. The acridine ring is then opened to N-
phenyl-benzaldehyde (2). After N-C bond disconnection, commercially available 2-
bromobenzaldehyde (3) and ester (4) are obtained as the precursors. Finally, the amino group of
the ester is transformed to a nitro group, and the phenol and ester are deprotected to give
commercially available 3-hydroxy-4-nitrobenzoic acid (6).
CH OH
COOCH3
CHO
COOCH3
2
N
N
N
H
OH
Inubosin B
OCH3
OCH3
(1)
(2)
COOCH3
CHO
Br
COOH
COOCH3
+
H N
2
O N
2
O N
2
OCH3
OH
6)
OCH3
5)
(
(
(4)
(3)
Scheme 1. Retrosynthesis of inubosin B.
The synthesis of inubosin B began with the bis-methylation of 3-hydroxy-4-nitrobenzoic
2
2
acid (6) using methyl iodide. Then, the nitro group was reduced to the amine group (4) with Zn
and AcOH.23 The coupling of amine (4) and 2-bromobenzaldehyde (3) was carried out via
Buchwald-Hartwig amination using a suitable palladium catalyst (Scheme 2). We have found
that the combination of Pd (dba) with DPEPhos provided the highest yield of the desired
2
3
aldehyde 2 (Table 1).24, 25
COOH
COOCH3
COOCH3
HN
H CO
3
CHO
a
b
c
OH
OCH3
OCH3
NO2
NO2
NH2
COOCH3
6
5
4
2
4

o
Scheme 2. Synthesis of precursor 2. Reagents and conditions: (a) MeI, K CO , DMF, 40-45 C, 86%; (b)
2
3
Zn, AcOH, MeOH, RT, 88%; (c) 2-bromobenzaldehyde, Pd (dba) , DPEPhos (see Table 1), K CO ,
2
3
2
3
o
toluene, 90 C, 55%.
Table 1. Optimization of the Buchwald-Hartwig amination reaction to give 2.
Pd-source
Pd
Ligand
Ligand
loading
[mol%]
Yield
loading
[
mol%]
0.047
0.025
0.025
[%]
25
Pd(OAc)2
Pd (dba)
DPEPhos
DPEPhos
dppf
0.1
0.05
0.05
55
2
3
Pd (dba)
30
2
3
Deviations from the retrosynthetic scheme began to appear during acridine ring closure
2
4
(
Scheme 3). Using the AlCl -promoted method for acridine closure, we were able to cyclize
3
the acridine ring and to cleave the 4-methoxy group in one step to obtain methyl 4-
hydroxyacridine-2-carboxylate (1c) containing 3.46% of aluminum according to elemental
analysis which corresponds to a 1:3 complex (3.44% is the theoretical calculation for
Al(C H NO ) ). This metallo-complex is similar to the aluminum 8-hydroxyquinoline complex
1
5
10
3 3
tris(8-hydroxyquinolinate)aluminum.26-28 Alternatively, the ring was cyclized using p-toluene
sulfonic acid (p-TSA), which partially cleaved the methyl ester group leading to a mixture of the
minor 1a and the major 1b products. Presumably due to its zwitterionic nature, carboxylic acid
1
b remains in the aqueous phase during washing and is sparingly soluble in MeOH and insoluble
in CHCl and THF. In order to increase the yield of ester 1a, acid 1b was converted to 1a using
3
SOCl /MeOH (Scheme 3).
2
5

CHO
NH
OCH3
Route I
Route II
2
COOCH3
c
a
COOCH3
COOCH3
COOH
+
d
N
N
N
1c
OH
OCH3
1
a
OCH3
1b
b
e
CH2OH
CH2OH
CH3
CH3
CH2OH
h
+
+
N
N
N
N
N
OH
7b
OCH3
f
7a OCH3
7c
OH
OH
Inubosin B
Inubosin B.TFA
g
+
CH3
CH2Br
N
N
7
c.TFA OH
8
OH
o
Scheme 3. Total synthesis of inubosin B. Reagents and conditions: (a) AlCl , toluene, 90 C, 60%; (b)
3
o
o
DIBAL-H, THF, 25 C, inubosin B.TFA 15% and 7c.TFA 4%; (c) p-TSA, toluene , 90 C, 1a 32%, 1b
o
o
3
9 %; (d) SOCl /DMF, 50 C, then MeOH , 0-25 C, 78%; (e) DIBAL-H or LiAlH , THF (see Table 2);
2 4
o
(
f) conc. HBr, reflux, 44%; (g) DMSO, H O, 50 C, inubosin B.TFA 18%; (h) pH 5.5 using aqueous
2
NaOH, 93%.
The reduction of methyl 4-methoxyacridine-2-carboxylate (1a) to 2-hydroxymethyl-4-
methoxy-acridine (7b) was very challenging (Table 2). The reducing agent and the solvent
played an important role in the reaction: e.g. the use of LiAlH or DIBAL-H led to side reactions,
4
such as reduction of the alcohol to a methyl group and deprotection of the phenolic methoxy
group.
Typically, unsubstituted acridines or acridine-9-carboxylates are reduced to 9,10-
dihydroacridne using LiAlH4.29, 30 However, this was not observed in a detectable amount during
our synthesis, even after careful LC-MS analysis (for more details the ESI: LC-MS reduction of
o
1
a at -15 and 25 C). This difference in reactivity can be explained by calculation of the natural
bond orbitals. The inubosin B precursor 1a has LUMO containing some electron density at the
carbonyl group, which is susceptible to reduction, whereas the 9-substituted acridine esters has
6

the LUMO localized only on the ring due to steric distortion and the breakage of conjugation.
Thus, the hydride may initially react with the acridine ring in the case of alkyl acridine-9-
carboxylates, whereas compound 1a can react with hydride at the carboxyl position (Fig. S3),
which also has the highest positive natural charge.
Table 2. Reduction of ester 1a to alcohol 7b accompanied by over-reduction
COOCH3
R
Reducing agent
/
solvent
N
N
OCH3
OR'
1a
7a: R: CH , R': CH .
3
3
7
b: R: CH OH, R': CH
2
3.
7
c: R: CH , R': H.
3
Reducing
Molar ratio
Temp.
Time Average yield* [%]
agent/solvent
[
h]
7a
7b
10
15
15
7c
o
LiAlH /THF
1
1
25 C
4
4
o
LiAlH /THF
66 C
4
4
o
LiAlH /THF
1.5
1.5
2
25 C
1
4
o
LiAlH /THF
25 C
4
65
55
55
4
o
LiAlH /THF
66 C
1
20
20
4
LiAlH /THF
1.5
3
Microwave 0.25
4
o
DIBAL-H/hexane
DIBAL-H/hexane
DIBAL-H/toluene
DIBAL-H/toluene
DIBAL-H/THF
25 C
4
1
4
1
4
20
20
70
o
4
25 C
60
60
o
3
25 C
o
4
25 C
o
3
25 C
*
the average yields were calculated from 4 runs.
3
1
Acidic deprotection of the methoxy group using HBr led to brominated analogue 8,
which was unstable during chromatographic analysis and slowly reacted with the solvent. This
7

brominated analogue (8) was hydrolyzed using a dimethyl sulfoxide (DMSO) and water
3
2
mixture, followed by HPLC purification to obtain inubosin B as the trifluoroacetate salt.
Due to the additional steps involved in route II, we returned to the cyclization using
AlCl (route I) leading to 1c, followed by reduction using DIBAL-H in THF, which can lead
3
directly to inubosin B. However, monitoring of the reduction of 1c using a TLC system was
misleading and the ester (1c) was easily over reduced to the corresponding methyl acridine (7c).
The use of HPLC was determined to be optimal for both monitoring of the reaction
progress and for compound separation. Moreover, the purification using HPLC with reverse
phase chromatography (MeCN/H O mobile phase containing 0.5% TFA) led to the removal of
2
aluminum from the sample (0.03% according to elemental analysis), and inubosin B was
obtained as the trifluoroacetate salt in 15% yield (see Scheme 3). The HPLC-purified inubosin
B.TFA had distinct Rf values (hexane-acetone-AcOH, 6:3:0.1): 0.4 and (toluene-EtOH-TFA,
7
:2:0.2): 0.35 using 2D-TLC (ESI, Fig. S2). The last step was conversion of the salt to the free
base by adjusting the pH to 5.5 with 0.02 N aqueous NaOH to obtain inubosin B in 93% yield.
There was good agreement between the NMR spectra of naturally occurring and synthesized
inubosin B (see ESI for details).
In conclusion, we obtained inubosin B via two synthetic routes and observed that the
usage of reverse phase chromatography with the MeCN/H O mobile phase containing TFA led
2
to removal of the aluminum ions from the acridine samples. In the future, this total synthesis may
open the way towards inubosin analogues which are unavailable from natural resources.
Acknowledgments
This work was supported by the Czech Science Foundation (17-00121S), Research Project RVO
6
1388963, by European Regional Development Fund; OP RDE; Project: "Chemical biology for
drugging undruggable targets (ChemBioDrug)" (No. CZ.02.1.01/0.0/0.0/16_019/0000 729), and
computational grants of CESNET (LM2015042) and the CERIT-SC (LM2015085). MFH thanks
the Science and Technology Development Fund in Egypt (STDF-STF) no. 6458. LKl, LKu, and
HŠ thank the Open Science project of the Academy of Sciences of the Czech Republic.
Molecular visualization was carried out with the UCSF Chimera package (Pettersen et al. 2004).
8

Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the
University of California, San Francisco (supported by NIGMS P41-GM103311). We thank Dr.
Martin Dračinský, Ph. D for NMR measurement of the inubosin B free base, and Dr. Jan Ježek,
Ph. D for corrections of the English language.
The authors declare no conflict of interests.
Supplementary Data
1
13
Experimental procedures, ESI-HRMS, H and C-NMR spectra of all synthesized compounds,
HPLC traces of all synthesized acridines, 2D TLC for purified inubosin B.TFA, NMR
comparison for inubosin B, comparison of the LUMO orbital, and an excel sheet for the
reduction of 1a.
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2
Total Synthesis of Inubosin B
Mohamed Farouk Hamissa a,b,*, Petr Niederhafner a,b, Martin Šafařík , Marta Telus , Lenka
a
a
a
a
a
a
a,
Kolářová , Leah Koutná , Hana Šestáková , Radko Souček , Jaroslav Šebestík *
aInstitute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo náměstí 2,
1
6610 Prague 6, Czech Republic.
bDepartment of Chemistry of Natural Compounds, Faculty of Food and Biochemical
Technology, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague 6,
Czech Republic.
Graphical Abstract
1

Total Synthesis of Inubosin B
Mohamed Farouk Hamissa a,b,*, Petr Niederhafner a,b, Martin Šafařík , Marta Telus , Lenka
a
a
a
a
a
a
a,
Kolářová , Leah Koutná , Hana Šestáková , Radko Souček , Jaroslav Šebestík *
aInstitute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo náměstí 2,
1
6610 Prague 6, Czech Republic.
bDepartment of Chemistry of Natural Compounds, Faculty of Food and Biochemical
Technology, University of Chemistry and Technology, Prague, Technická 5, 166 28 Prague 6,
Czech Republic.
Highlights



Total synthesis of Inubosin B.
Over reduction reactions.
Reverse phase removal of aluminum from oxine like chelators.
1