Total synthesis and in vivo evaluation of 8-deoxypumiliotoxin 193H

Article abstract of DOI:10.1080/14786419.2019.1636244

The total synthesis of both the double bond isomers of indolizine alkaloid 8-deoxypumiliotoxin 193H has been accomplished. Both the double bond isomers Z-4 and E-4 induced convulsions and inhibited neuro-muscular activity at a dose of 25 mg/kg after intra

Full text of DOI:10.1080/14786419.2019.1636244

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Total synthesis and in vivo evaluation of 8-
deoxypumiliotoxin 193H
Liga Zvejniece, Maija Dambrova & Gints Smits
To cite this article: Liga Zvejniece, Maija Dambrova & Gints Smits (2019): Total synthesis
and inꢀvivo evaluation of 8-deoxypumiliotoxin 193H, Natural Product Research, DOI:
10.1080/14786419.2019.1636244
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1636244
Total synthesis and in vivo evaluation of
8-deoxypumiliotoxin 193H
Liga Zvejniece, Maija Dambrova
and Gints Smits
Latvian Institute of Organic Synthesis, Riga, Latvia
ABSTRACT
ARTICLE HISTORY
Received 6 May 2019
Accepted 22 June 2019
The total synthesis of both the double bond isomers of indolizine
alkaloid 8-deoxypumiliotoxin 193H has been accomplished. Both
the double bond isomers Z-4 and E-4 induced convulsions and
inhibited neuro-muscular activity at a dose of 25 mg/kg after
intraperitoneal injection in mice. The lethal dose of Z-4 and E-4
was 100 mg/kg, indicating that 8-deoxypumiliotoxin 193H is 10-
times less toxic than the known pumiliotoxin (þ)-251 D.
KEYWORDS
Total synthesis;
stereoselectivity; in vivo
studies; deoxypumilio-
toxin 193H
1. Introduction
Amphibians possess a broad range of structurally unique naturally occurring com-
pounds (Simmaco et al. 1998; Daly 1998; Daly et al. 2004; Daly et al. 2005; Pukala
ꢀ
et al. 2006; Xu and Lai 2015; Rodrıguez et al. 2017; Dennison et al. 2018) with intrigu-
ing biological properties, including anticancer (Fornari Baldo et al. 2012), antimicrobial
(Simmaco et al. 1998; Cunha Filho et al. 2005; Wu et al. 2011; Wang et al. 2013;
Dennison et al. 2018), antifungal (Artika et al. 2015) and cardiotonic (Daly et al. 1985)
activities. The most frequently used source of these natural products is the skin secre-
tion of frogs that populate the forests of South America, Africa and Australia. These
amphibians were historically used to prepare poisoned arrows for hunting and warfare
CONTACT Gints Smits
gintssmits@osi.lv
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1636244
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
L. ZVEJNIECE ET AL.
Figure 1. Representative members of the PTX family.
(Jones 2007). Most of these alkaloids are sequestered unchanged from dietary arthro-
pods such as mites, ants, beetles, and millipedes (Saporito et al. 2009). Today, more
than 800 alkaloids have been characterized from the skins of dendrobatid frogs (Daly
et al. 2005). A broad family of indolizine alkaloids found in dendrobatid frogs are the
pumiliotoxins (PTX) (Pelletier 1999; Daly et al. 2005; Michael 2016). The first members
of this family, PTX A (1) and B (2), were isolated by Daly in the 1960s (Daly and Myers
1967); however, their structure remained unclear for almost 2 decades. In the 1980s,
the isolation and X-ray characterization of a simpler indolizine alkaloid PTX 251 D (3)
(Daly et al. 1980) allowed the resolution of the also structures of 1 and 2. The total
syntheses of PTX were pioneered by Overman (Overman and Bell 1981; Overman and
Goldstein 1984; Overman and Lin 1985; Franklin and Overman 1996) in the 1980s and
are still ongoing (Suryavanshi et al. 2014; Chou et al. 2014; Correia et al. 2016; Qu and
Helmchen 2017). PTX is known to inhibit sodium and potassium channels (Gusovsky
et al. 1992; Vandendriessche et al. 2008; Daly et al. 1990). The estimated minimum
lethal dose of PTX A (1) and B (2) in mice is 2.5 and 1.5 mg/kg, respectively (Daly and
Myers 1967), and for PTX (þ)-251 D (3) the lethal dose is 10 mg/kg. Moreover, this
effect is strongly dependent on the stereochemistry and substitution pattern of the
molecule (Daly et al. 1990; Daly et al. 2003). Additionally, PTX (þ)-251 D (3) induces
convulsions, apparent pain at the site of injection and pronounced long-lasting hyper-
activity in mice (Daly et al. 2003), while PTX A (1) and B (2) inhibit nerve-muscle activ-
ity (Daly and Myers 1967) (Figure 1).
A common structural motif of PTXs is an octahydroindolizine core that possess an
alkylidene substituent at the C-6 position. One of the simplest members of the PTX
alkaloid family is 8-deoxyPTX 193H (4), which is found in Scheloribates azumaensis
mites that populate Japan (Takada et al. 2005). This compound, however, has never
been isolated, and its structure was proposed only on the basis of a GC-MS fragmenta-
tion pattern and analogy to other PTX family members (Takada et al. 2005). Despite its
structural simplicity, 8-deoxyPTX 193 H (4) contains most of the characteristic structural
elements of PTX: an exocyclic Z- configuration double bond and an indolizine core
with 2 chiral centres, making it an interesting target from a medicinal chemistry per-
spective. Over the past decades, a number of different approaches have been reported
for the assembly of the indolizine core of PTX (Fox et al. 1991; Franklin and Overman
1996; Lin et al. 1996; O’Mahony et al. 2004; Pinho et al. 2013); however, a stereoselect-
ive introduction of the alkylidene moiety at the C-6 position poses a considerable chal-
lenge. The putative 8-deoxyPTX 193H (4) was first synthesized by our group in 2015
(Smits and Zemribo 2015) followed by a recent synthesis by Okada (Okada et al.

NATURAL PRODUCT RESEARCH
3
Scheme 1. Reagents and conditions: (a) i) NMM, EtOCOCl, THF, then CH2N2, rt, 75%; ii) CF3COOAg,
Et3N, tBuOH, rt, THF, rt, 72%; (b) i) H2, 10% Pd/C, EtOH, rt; ii) 7, DIPEA, THF, rt, 80% in 2 steps; (c)
i) TFA, DCM, rt; ii) HBTU, DMAP, 0.01 M, DCM, rt, 83% in 2 steps; (d) Bu2BOTf, Me2NEt, 1 h at 0 ꢁC,
then 1 h at rt, then 1 h at 50 ꢁC (MW), then BnOH, HBTU, 39% for Z-10, 9% for E-10.
2018), but the in vivo activity has never been evaluated. Herein, we report the total
synthesis of both the double bond isomers of 8-deoxyPTX 193H (4). The rota-rod, trac-
tion and chimney tests were utilized and body temperature was measured to assess
toxicity and determine the effects of both isomers on muscle strength and coordin-
ation in mice.
2. Results and discussion
Our approach towards the total synthesis of 8-deoxyPTX 193H (4) (Smits and Zemribo
2015) was based on a stereoselective Ireland–Claisen rearrangement developed earlier
by our group (Smits et al. 2015; Smits and Zemribo 2013) (Scheme 1).
Our synthetic studies towards the synthesis of 8-deoxyPTX 193H (4) began with an
Arndt-Eistert homologation of the commercial proline 5 giving the desired homopro-
line derivative 6 in a 54% yield over 2 steps. The Cbz-protecting group in 6 was fur-
ther cleaved by palladium catalysed hydrogenation, and the intermediate was
alkylated with allyl bromide 7 (Honda et al. 1987; Shklyaruck and Matiushenkov 2011)
furnishing the homoproline ester 8 in an 80% yield over 2 steps. Next, the ester 8 was
converted to the eight-membered lactone 9 first by cleavage of the tert-butyl ester,
followed by a macrolactonization under high dilution conditions. With the lactone 9 in
hand, the stage was set for the crucial step of this total synthesis, a stereoselective
Ireland–Claisen rearrangement. Despite our attempts to optimize this step (Smits and
Zemribo 2015), the desired indolizines 10 were obtained in moderate yields and ꢀ1:4
E-/Z- selectivity; however, we were able to separate the double bond isomers Z-10
and E-10 by flash column chromatography and focus on the total synthesis
(Scheme 2).
The synthesis of 8-deoxyPTX 193H Z-4 and its double bond isomer E-4 was accom-
plished in a 2-step sequence. First, the esters Z-10 and E-10 were reduced to the cor-
responding alcohols Z-11 and E-11, followed by tosylation and reduction of the
R
intermediate tosylates with a large excess of SuperhydrideV. Using this methodology
>50 mg of both 8-deoxyPTX 193H Z-4 and E-4 were synthesized and further used the
in vivo studies in mice.
To measure the effects of compounds on muscle strength and coordination, we uti-
lized the rota-rod, traction and chimney tests and measured body temperature in

4
L. ZVEJNIECE ET AL.
Scheme 2. Reagents and conditions: (a) DIBAL-H, DCM, -78 ꢁC, 90% for Z-11, 81% for E-11; (b)
R
V
TsCl, cat. DMAP, DIPEA, then Superhydride , THF, 50% for Z-4, 54% for E-4.
Table 1. The toxicological profile of Z-4 and E-4 in mice.
Muscle strength and
coordination
Body temperature
changes
Compounds
Dose, mg/kg
Convulsive behaviour
Mortality
Z-4
10
25
50
100
10
25
0/1
1/1
2/2
1/1
0/2
2/2
2/2
1/1
0/1
1/1
2/2
1/1
0/2
1/2
2/2
1/1
0/1
0/1
1/2
1/1
0/2
0/2
1/2
1/1
0/1
0/1
0/2
1/1
0/2
0/2
0/2
1/1
E-4
50
100
The compounds were administered i.p. at doses of 10, 25, 50 and 100 mg/kg. The effects were observed 30, 60, 120,
180 and 240 min after administration. The data are expressed as a n animals with effects/total animal n.
mice (see at Supplementary data) (Dambrova et al. 2008). The effects of compounds
were evaluated at 30, 60, 120, 180 and 240 min after intraperitoneal (i.p.) administra-
tion at doses of 10, 25, 50 and 100 mg/kg.
Z-4 and E-4 induced convulsion at a dose of 25 mg/kg. The convulsive behaviour in
mice started approximately 5 min after the compound administration and lasted
15 min. The compounds also induced seizures at higher doses of 50 and 100 mg/kg.
The inhibitory activity of Z-4 and E-4 on muscle strength and coordination were
observed at a dose of 25 mg/kg in the first 30 min and at a dose of 50 mg/kg in the
first 60 min period. In addition, both isomers decreased the rectal temperature at a
dose of 50 mg/kg during the first 60 min. The administration of Z-4 and E-4 at a dose
of 10 mg/kg did not bring about any effects. After i.p. administration of Z-4 and E-4 at
a dose of 100 mg/kg the mice died during the first 10 min (Table 1).
3. Conclusions
In summary, Z-4 and E-4, the double bond isomers of the putative 8-deoxyPTX 193H,
were synthesized using an Ireland–Claisen rearrangement as the stereochemistry
determining step. Both isomers Z-4 and E-4 administered at equal doses induced con-
vulsions, inhibited neuro-muscular responses and reduced body temperature in mice.
The lethal dose of 8-deoxyPTX 193H was 10-fold higher compared to PTX (þ)-251 D.
The less toxic properties of 8-deoxyPTX could be attributed to a simplified alkylidene
substituent at the C-6 position compared to PTX 251 D as well as PTX A and B.

NATURAL PRODUCT RESEARCH
5
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by the European Regional Development Fund (ERDF) project
No.1.1.1.2/VIAA/1/16/243 «Total Synthesis of Eleganine A and 17-nor-Excelsinidine».
ORCID
Maija Dambrova
https://orcid.org/0000-0002-1739-0928
Gints Smits
http://orcid.org/0000-0001-5044-4169
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