Nitrosopurines en route to potently cytotoxic asmarines

Article abstract of DOI:10.1002/anie.201411493

A nitrosopurine ene reaction easily assembles the asmarine pharmacophore and transmits remote stereochemistry to the diazepine-purine hetereocycle. This reaction generates potent cytotoxins which exceed the potency of asmarine A (1.2 μM IC₅₀) and supersede the metabolites as useful leads for biological discovery.

Full text of DOI:10.1002/anie.201411493

Angewandte
Chemie
DOI: 10.1002/anie.201411493
Alkaloids
Nitrosopurines En Route to Potently Cytotoxic Asmarines**
Kanny K. Wan, Kotaro Iwasaki, Jeffrey C. Umotoy, Dennis W. Wolan,* and Ryan A. Shenvi*
Abstract: A nitrosopurine ene reaction easily assembles the
asmarine pharmacophore and transmits remote stereochemis-
try to the diazepine-purine hetereocycle. This reaction gener-
ates potent cytotoxins which exceed the potency of asmarine A
3. Thus, the driving force for investigation of the asmarines is
to determine their mechanism of action and chemical
reactivity within the cell. However, no material remains
[5]
from the original isolation work and the exact identity of the
[1b]
(
1.2 mm IC ) and supersede the metabolites as useful leads for
sponge is unknown.
Chemical synthesis is therefore the
5
0
biological discovery.
most feasible way to access material for biological study.
Procurement of these cytotoxic molecules represents
a challenge for synthesis despite efforts by Kashman, Schauss,
Tashiro, and Gundersen. Their difficulty arises partly from
the remoteness of the stereogenic tert-alkyl N-hydroxyamine
moiety (Figure 1), which frustrates stereocontrol relative to
the clerodane subunit. Furthermore, synthesis of the tert-alkyl
A
smarines A and B (1 and 2; Figure 1) were identified in
998 by Kashman and co-workers as the bioactive constitu-
[6]
1
ents of a Red Sea sponge (Raspailia sp.) extract, and exhibited
cytotoxicity against several cancer cell lines with a minimum
EC50 value of 1.2 mm and 120 nm, respectively. The asmar-
ines are unique among alkaloids by virtue of the embedded N-
[
1]
N-hydroxy-diazepinepurine pharmacophore is a challenge in
[2]
[6a,b,d]
hydroxypurine diazepine (primary pharmacophore) con-
nected by an ethyl bridge to a clerodane decalin (putative
secondary pharmacophore). Biosynthetically, the asmarines
derive from agelasines (e.g. 3), which are thought to exert
itself
and only one route exists. This successful strategy
[2]
by Kashman utilizes a carbocationic mode of ring closure
(30% HBr/AcOH at 1008C), which limits functional-group
compatibility and stereocontrol, and delivers analogues (e.g.
4–7) which, while bioactive, reach a maximum potency of
+
+
[3]
cytotoxicity by Na /K ATPase inhibition or by membrane
[4]
[7]
disruption. However, the uncharged purines 1 and 2 are
4 mm (GI , HT-29; Figure 2a). We aimed to secure efficient
5
0
likely to exert cytotoxicity through different mechanisms than
access to the asmarines to understand their structure–activity
relationship (SAR) and to provide simplified, high potency
analogues for further biological study.
A biomimetic strategy to close the seven-membered ring
from an agelasine-type intermediate 8 (Figure 2b) appeared
to offer high efficiency. However, control of the diazepine
stereochemistry during CꢀN bond formation presented
Figure 1. Asmarines may be derived from agelasines. [a] Against HT-29
[
3b]
cells. [b] Against MCF-7 cells.
[*] K. K. Wan, Dr. K. Iwasaki, Prof. Dr. R. A. Shenvi
Department of Chemistry, The Scripps Research Institute
10550 N. Torrey Pines Rd., La Jolla, CA 92037 (USA)
E-mail: rshenvi@scripps.edu
J. C. Umotoy, Prof. Dr. D. W. Wolan
Department of Molecular and Experimental Medicine and
Chemical Physiology, The Scripps Research Institute
1
0550 N. Torrey Pines Rd., La Jolla, CA 92037 (USA)
E-mail: wolani@scripps.edu
**] This work was supported by the NIH (GM104180 and the NSF GRFP
K.K.W.; DGE-1346837). We thank Dr. C. E. Moore and Prof. A. L.
[
(
Rheingold (UCSD) for X-ray crystallographic analysis. We thank
TSRI, Eli Lilly, Boehringer Ingelheim, Amgen, the Baxter Foundation,
and the Sloan Foundation for additional financial support.
Figure 2. a) Prior analogue work. b) Stereochemical relay and high
potency analogues through nitrosopurine ene reaction. [a] Cytotoxicity
against HT-29 cells (EC50 more appropriate).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201411493.
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a serious obstacle to this strategy. We wondered if a nitroso
ine was explored in a model system (Figure 3). The parent
compound 6-nitrosopurine 20 proved unstable and in our
[
8]
[9]
[17]
ene reaction of 8 might relay stereochemistry of the
decalin core to the tert-alkylamine stereocenter in 9, and thus
hands could not be isolated. However, we found that treat-
ment of the 6-N-hydroxyaminopurine 21 with MnO2 in
DMSO and in the presence of excess tetramethylethylene
gave the expected tert-alkyl methallyl hydroxylamine 22. A
variety of oxidants [I , Mn(OAc) , PhI(OAc) ] gave similar
[
10]
achieve the necessary Markovnikov selectivity. Herein we
report 1) the successful implementation of this strategy to
relay clerodane stereochemistry to the remote stereocenter,
2
) the ability of this strategy to probe the role of stereochem-
2
3
2
istry in SARs, and 3) the use of this nitrosopurine ene reaction
to synthesize simplified high potency asmarine analogues
which exhibit cytotoxicity at nanomolar levels against multi-
ple cancer cell lines.
results and on large scale PhI(OAc) proved more amenable
2
to purification. When these oxidation conditions were applied
to the N-hydroxyaminopurine 23 in a mixture of methanol
and dichloromethane, the targeted seven-membered diaze-
pine purine 24 was obtained as the sole product with no trace
of the eight-membered diazocine. Notably, this reaction could
be performed on gram-scale, thus demonstrating that the
A short route to the targeted 8 began with 6-chloro-4,5-
pyrimidinediamine (11) and 4-iodo-1-butyne (12; Scheme 1).
The diamine 11 was monoformylated to amide 13, and 12 was
[
11]
subjected to the Wipf-modified
tion,
Negishi carboalumina-
[
12]
with subsequent alkylation of the intermediate
organoaluminum with gaseous formaldehyde to yield the
[
13]
iodoalcohol 14. The formamide 13 was alkylated with 14 and
[
14]
heating effected ring closure to the chloropurine 15. This
alcohol was converted into the allylbromide 16 (see the
Supporting Information), which was then trapped with the
enolate generated from dissolving metal reduction of the
[15]
(
methyl)-Wieland–Miescher ketone ketal 17 (99% ee) in
[
16]
the presence of bis(2-methoxyethyl)amine to yield 18 as
a single stereoisomer. Selective displacement of the aryl-
chloride of 18 with hydroxylamine proved challenging since
condensation with the decalin ketone to form an oxime
occurred competitively. We reasoned that the oxime might be
cleaved later, and thus decided to push the reaction towards
the bis(hydroxyamin)ated product 19, an unforeseen but
providential choice (see Table 1).
Having obtained the agelasine scaffold 19, oxidation of
the 6-N-hydroxyaminopurine (HAP) moiety to a nitrosopur-
Figure 3. Proof-of-principle for nitrosopurine ene reactions. DMSO=
dimethylsulfoxide.
chemistry of nitrosopurines
(e.g. 25) is a practical means of
procuring material.
When applied to the agela-
sine-type compound 19, we also
obtained the diazepine 26
exclusively, but initial experi-
ments generated this as a 1:1
mixture
of
diastereomers
(entry 1, Table 1). Fortunately,
we discovered two influences
on diastereoselectivity in the
ene reaction. There was
a marked solvent effect associ-
ated with diastereoselectivity,
whereby more polar protic sol-
vents increased selectivity, and
water proved to be the most
selective (ethanol was added
for solubility). This effect was
not due to protonation or
hydrogen bonding, since nei-
ther acetic acid had a pro-
nounced effect (entry 6), nor
Scheme 1. Union of clerodane and purine cores, and a nitrosopurine ene reaction with remote
stereocontrol. Cp=cyclopentadiene, Ms=methanesulfonyl, THF=tetrahydrofuran.
2
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did the strongly hydrogen-bond-donating solvent trifluoroe-
Table 1: Solvent and substituent control stereoinduction.
[
18]
thanol (entry 7) when compared to ethanol (entry 3). There
was also a stark and serendipitous substituent effect: although
the incidental oxime caused high stereoselectivity, the ketone
2
7 and exocyclic methylene 28 showed almost no selectivity in
formation of 29 and 30, respectively (entries 10 and 11).
Application of the polarized diradical (PD) 31 model for the
[
19]
nitroso ene reaction of 19 suggests that developing positive
charge at the carbon atom in the transition state might be
stabilized by the proximal oxime (Figure 4). The conformer
3
2 should be lower in energy than 33, which suffers a steric
clash between the methyl and the aromatic ring. These
polarized transition states would be more populated in
solvents of high polarity (e.g., water), as predicted by Leach
[
a]
Entry
Y
Solvent
T [8C] Additive
(S)-13/(R)-13
[19]
and Houk.
1
2
3
4
5
6
7
8
9
NOH DMF
NOH CH Cl
2
22
22
22
–
–
–
–
1.5:1.0
1.5:1.0
3.8:1.0
3.0:1.0
2.1:1.0
2.7:1.0
2.3:1.0
4.7:1.0
7.9:1.0
1.2:1.0
1.1:1.0
This strategy allowed us to directly probe the contribution
of diazepine stereochemistry to cytotoxicity. The major
diastereomer 26 (Figure 5) exhibited higher potency against
2
NOH EtOH
NOH EtOH
NOH MeOH/CH
NOH EtOH
NOH TFE
NOH EtOH/H O (1:1)
NOH EtOH/H O (1:3)
O
CH2
ꢀ20
[
b]
2
Cl
2
(1:9)
4
22 NH OH
[
c]
22 AcOH
22
22
22
22
22
–
–
–
–
–
2
2
1
1
0
1
EtOH/H O (1:4)
EtOH/H O (1:4)
2
2
[
a] Determined by NMR spectroscopy and LCMS. [b] 1% NH OH.
4
[c] 1 equiv AcOH. DMF=N,N-dimethylformamide.
HT-29 cells (EC = 8 mm) than the minor isomer 34 (17 mm),
5
0
even though 34 corresponds to the stereochemistry of the
natural asmarines (determined by X-ray analysis; see the
Supporting Information). Since the difference in potency is
not profound, the stereochemistry in the diazepine seems to
Figure 4. Water might increase the polar character in a nitrosoene
polarized diradical (PD) and the transition state leading to it. Con-
formers of different energy are stabilized by the proximal oxime.
X=(-OC H O-).
[20]
2
4
Figure 5. Activity (EC , 48 h) against HT-29 cells. Although there is clear SAR, stereochemistry does not play a major role. X=(-OC H O-).
5
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2
4
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play only a minor role in the mechanism of action. Synthesis
also allowed us to probe the role of absolute stereochemistry.
We were surprised to find that ent-26 and ent-34 possessed
similar biological activity as their enantiomers (EC = 10 mm
for both). Similarly, the diketones 35 and ent-35 possess
activity which differs only twofold. Whereas this similarity
might suggest a nonspecific mechanism of action, it seems
more likely that the differences in potencies reflect cell
permeability, thus affecting accessibility to the target. As
such, replacement of the oxime with a more lipophilic
methylene delivers compounds with greater potency (36 and
chemistry had little influence on the activity of the asmarines,
we adapted the route shown in Scheme 1 to the late-stage,
divergent appendage of unnatural hydrophobic cores. As
shown in Figure 6, a short convergent sequence from 13 and
39 was devised to arrive at the iodide 40 on gram scale. From
39, a variety of substituents were added by a very effective
Negishi coupling, a noteworthy step given the acidic proton
on the hydroxyamino group and our observation that the N-
hydroxyamino purine will chelate metals. Each adduct (41–
48) was then cyclized by the nitrosopurine ene reaction to its
N-hydroxydiazepine purine (50–57) to generate a small
library of cytotoxic compounds.
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0
[
22]
3
7). In this case, the ꢀnaturalꢁ diazepine 37 is fivefold more
potent than the ꢀunnaturalꢁ diastereomer 36, and in fact
matches the potency against HT-29 cells reported for
asmarine A (EC = 1 mm). As control compounds, we also
Each molecule in Figure 6 was screened for cytotoxicity
against HT-29, Jurkat, and HeLa cells. While compounds with
truncated side chains (24, 49–53) show cytotoxicity, the
potency is weak, with or without an unsaturated linker (24
versus 49). Small rings like a methylenecyclobutane can be
generated in the ene reaction, thus highlighting the mildness
of the reaction conditions, which are in stark contrast to the
previously reported high temperature/strong acid method of
ring closure. Therefore, esters (52) and amino-acid (53) motifs
can be incorporated; the b,g-unsaturated amino ester carba-
mate does not migrate into conjugation under the reaction
conditions. This latter example shows some small stereose-
5
0
tested the NꢀH purine 38 and the uncyclized N-hydroxyami-
nopurine 19. Both compounds were unable to effectively
induce cell death after 72 hours. The inactivity of 19 and 37
suggests a different mechanism than that of the inhibition of
ATPases ascribed to the agelasines and their simplified
[
21]
analogues,
although further study will be necessary to
rule out this target.
Since the ultimate goal of our work was procurement of
material for biological study and it was clear that stereo-
Figure 6. A late-stage divergent route to unnatural asmarines and their activities (EC , 48 h) against HT-29 and HeLa cells. Potencies are on
5
0
a logarithmic scale.
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lectivity (2:1) associated with the presence of the carbamate
compare to methyl; 52), thus supporting the idea that
(
a Lewis-basic group is necessary for stereoinduction. How-
ever, none of these short, polar side chains show significant
potency. In contrast, large hydrophobic groups similar to the
clerodane cores of asmarines A and B induce high potency.
For example, compounds with 1- and 2-naphthyl substituents
(54 and 55) possess single-digit micromolar activity; 54 is
equipotent to asmarine A (1.1 mm against HT-29). 1-Ada-
mantyl (56) and 4-biphenyl (57) asmarines are more potent
with sub-micromolar activity against all three cell lines (471–
7
14 nm; 56 also inhibits HL60 cells at 199 nm, see Figure 8),
thus approaching the 120 nm IC50 value reported for asmar-
ine B (2) against HT29 cells. In fact, this study represents
a rare example of the deprioritization of isolated metabolites
in light of near-equipotent but simpler analogues. Readily
accessible asmarines 56 and 57 supersede the scarce metab-
olites 1 and 2 as useful tools for biochemical inquiry.
Figure 8. 1-Adamantyl-asmarine (56) and 4-biphenyl-asmarine (57)
possess nanomolar activity against seven cell lines.
Whereas there is some latitude in the choice of the
hydrophobic lobe, the N-hydroxy diazepine purine is more
conservative. The acyclic tert-alkyl N-hydroxyaminopurine 19
was completely inactive (Figure 7), thus indicating that a ring
Figure 7. Structural specificity for activity.
Figure 9. Reactivity of potential relevance to bioactivity.
is necessary. However, the ring-expanded N-hydroxy diazo-
cine purine 51 was similarly inactive. This eight-membered
ring was the unexpected anti-Markovnikov product of nitro-
sopurine ene reaction of cyclopropane 42, which reacts with
phosphorylation. Alternatively, we observed that the tert-
alkyl N-hydroxypurine is readily oxidized with mild reagents.
The acyclic purine 22 generates the stable nitroxide radical 59,
a vibrant red-orange crystalline solid whose structure was
“
twix” selectivity and avoids the alternative, highly strained
methylenecyclopropane (Figure 7).
[
23]
The potency of 56 and 57 is general, thus showing sub-
micromolar cytotoxicity against HL60 (leukemia), HEK 293,
MCF7 (breast cancer), and MDA-MB 231 (breast cancer) cell
lines, in addition to the HT29, Jurkat, and HeLa cell lines
confirmed by X-ray analysis. Interestingly, the nitroxides of
the diazepines (e.g. 24) are very unstable, cannot be isolated,
and instead appear to disproportionate to the corresponding
NꢀH diazepines, thus suggesting that, if generated, they may
(
Figure 8). The activity of 56 is surprising, since the saturated
react with a target in vivo.
analogue was reported by Kashman to possess very weak
activity against two cancer cell lines (NSCL A549 and
PANC1, EC > 27 mm). The activity of 57 is noteworthy
since installation of functional groups on the aromatic rings
should allow a variety of pull-down experiments.
To summarize, we have established a chemical platform
for the study of asmarine cytotoxins enabled by an unusual
but highly practical nitrosopurine ene reaction. This reaction
exhibits both high regioselectivity for the Markovnikov
product and exhibits high chemoselectivity. Identification of
a Lewis base (oxime) as a stereoelectronic control element in
the nitroso ene reaction may be generally useful for linear
stereocontrol. Use of the nitrosopurine ene reaction as
a simplifying element in the synthesis of cytotoxic asmarine
analogues enables very short syntheses with few redox
manipulations and no protecting groups. Notably, this route
is diversifiable at a late stage, and has generated potent
analogues with cytotoxicity in the nanomolar range. We have
also found that 1) increased potency may result from cell
5
0
This work builds a platform for the discovery of the
mechanism of action of the asmarines. We are entertaining
three hypotheses: noncovalent, covalent, and radical. The
latter two possibilities are supported by some experimental
[
1b]
[6b]
data. Kashman and co-workers and Ohba and Tashiro
have reported the instability of the N-hydroxypurine upon
acylation, wherein methanol adds to the purine ring and
cleaves the NꢀO bond (2!58; Figure 9). This mode of
reactivity may also be effected in vivo by acetylation or
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Angewandte
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permeability, but the N-hydroxypurine diazepine is required;
) the similar potency of 54 and 55 suggests the hydrophobic
moiety does not play a major role in target binding;
) stereochemistry at the diazepine plays a minor, but not
[6] a) D. Pappo, A. Rudi, Y. Kashman, Tetrahedron Lett. 2001, 42,
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dersen, Tetrahedron Lett. 2007, 48, 1931.
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activity, since an NꢀH analogue is inactive; and 5) the seven-
membered ring is required for activity, since acyclic variants
and a diazocine analogue show no cytotoxicity. These find-
ings, coupled with the development of a short, scalable, and
divergent route to asmarine analogues, and especially the
identification of potent the biphenyl asmarine 57 lay the
groundwork for identification of the mechanism of action of
these enigmatic metabolites.
[
7] Although labeled as cytotoxicity in Ref. [2], the use of GI50
indicates cytostatic effect. Based on the activity of our analogues,
we assume the authors observed cytotoxicity and meant to use
EC50
.
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8] W. Adam, O. Krebs, Chem. Rev. 2003, 103, 4131.
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Received: November 27, 2014
Published online: && &&, &&&&
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4
Keywords: alkaloids · ene reaction · natural products ·
structure–activity relationships · terpenoids
.
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Alkaloids
K. K. Wan, K. Iwasaki, J. C. Umotoy,
D. W. Wolan,*
R. A. Shenvi*
&&& — &&&
Nitrosopurines En Route to Potently
Cytotoxic Asmarines
Unnatural product: A nitrosopurine ene
reaction easily assembles the asmarine
pharmacophore and transmits remote
stereochemistry to the diazepine-purine
heterocycle. This reaction generates
potent cytotoxins which exceed the
potency of asmarine A and supersede the
metabolites as useful leads for biological
discovery.
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!