Simultaneous structure-activity studies and arming of natural products by C-H amination reveal cellular targets of eupalmerin acetate

Article abstract of DOI:10.1038/nchem.1653

Natural products have a venerable history of, and enduring potential for the discovery of useful biological activity. To fully exploit this, the development of chemical methodology that can functionalize unique sites within these complex structures is highly desirable. Here, we describe the use of rhodium(II)-catalysed C-H amination reactions developed by Du Bois to carry out simultaneous structure-activity relationship studies and arming (alkynylation) of natural products at 'unfunctionalized' positions. Allylic and benzylic C-H bonds in the natural products undergo amination while olefins undergo aziridination, and tertiary amine-containing natural products are converted to amidines by a C-H amination-oxidation sequence or to hydrazine sulfamate zwitterions by an unusual N-amination. The alkynylated derivatives are ready for conversion into cellular probes that can be used for mechanism-of-action studies. Chemo- and site-selectivity was studied with a diverse library of natural products. For one of these - the marine-derived anticancer diterpene, eupalmerin acetate - quantitative proteome profiling led to the identification of several protein targets in HL-60 cells, suggesting a polypharmacological mode of action.

Full text of DOI:10.1038/nchem.1653

ARTICLES
PUBLISHED ONLINE: 19 MAY 2013 | DOI: 10.1038/NCHEM.1653
Simultaneous structure–activity studies and
arming of natural products by C–H amination
reveal cellular targets of eupalmerin acetate
Jing Li , Justin S. Cisar , Cong-Ying Zhou , Brunilda Vera , Howard Williams , Abimael D. Rodrıguez ,
1,2
3
1
4
1
4
´
Benjamin F. Cravatt3 and Daniel Romo1,2
*
Natural products have a venerable history of, and enduring potential for the discovery of useful biological activity. To fully
exploit this, the development of chemical methodology that can functionalize unique sites within these complex structures
is highly desirable. Here, we describe the use of rhodium(II)-catalysed C–H amination reactions developed by Du Bois to
carry out simultaneous structure–activity relationship studies and arming (alkynylation) of natural products at
‘unfunctionalized’ positions. Allylic and benzylic C–H bonds in the natural products undergo amination while olefins
undergo aziridination, and tertiary amine-containing natural products are converted to amidines by a C–H amination–
oxidation sequence or to hydrazine sulfamate zwitterions by an unusual N-amination. The alkynylated derivatives are
ready for conversion into cellular probes that can be used for mechanism-of-action studies. Chemo- and site-selectivity
was studied with a diverse library of natural products. For one of these—the marine-derived anticancer diterpene,
eupalmerin acetate—quantitative proteome profiling led to the identification of several protein targets in HL-60 cells,
suggesting a polypharmacological mode of action.
renewed realization of the intrinsic value of bioactive small functionality and often limited availability. One approach to acces-
molecules isolated from natural sources (that is, natural pro- sing natural products with strategically placed linkers is through
A
ducts) for drug discovery1 has spurred a renaissance in their total synthesis, which enables access to novel and unique attachment
study, including research on novel concepts for their efficient syn- sites, but this approach may not be applied rapidly to access varied
thesis2, the synthesis of natural product-like libraries (for a recent positions in complex natural products. Highly random derivatiza-
special issue on this topic, see ref. 3), the manipulation of biosyn- tion techniques involving photo-crosslinking of natural products
thetic pathways for synthetic biology, and their incorporation into to affinity matrices have been reported. However, these methods
high-throughput screens4. Natural products commonly demonstrate provide limited structure–activity relationship (SAR) data for sub-
high structural diversity, cell permeability and high affinity for their sequent drug development and no information regarding the site
cellular targets, arguably explaining why half the drugs in current of attachment in the event that cellular targets are not captured9.
clinical use are natural products or owe their intellectual origin to To fully exploit the inherent information content of complex, bio-
these bioactive small molecules5. Furthermore, the enduring value active natural products10,11, mild and generally applicable microscale
of natural products for advancing the knowledge of basic cell strategies for site-selective derivatization of native natural products
biology and their utility in the discovery of novel ‘druggable’ are required to enable SAR studies and the synthesis of natural
targets for human disease intervention cannot be overstated. In product-based cellular probes. Such probes have proven useful for
particular, natural products continue to facilitate the identification identifying the cellular targets of natural products and for providing
of both activators and inhibitors of proteins encoded in the a molecular-level understanding of how these small molecules exert
human genome in addition to a number of natural product-inspired their observed ameliorative or curative effects7.
molecules that are providing advances in this post-genomic era6,7.
We have recently described several mild, microscale methods for
Given that current pharmaceuticals are thought to access fewer simultaneous arming and SAR studies of natural products to
than 500 of the estimated 3000–10,000 potential therapeutic address these issues, including a Rh(II)-catalysed OH and NH inser-
targets for human disease intervention8, natural products hold tion of natural products bearing alcohols or amines12,13, a mild
great potential for the discovery of novel cellular targets and drug In(III)-catalysed iodination of arene-containing natural products14,
discovery. One approach for fully exploiting this potential requires and cyclopropanations of natural products bearing both electron-
the synthesis of natural product conjugates that contain a covalently rich and electron-deficient alkenes15. These functionalization
attached reporter tag, for example biotin or a fluorophore, appended methods are dependent on the presence of native functional
by a flexible linker at a position in the natural product that does not groups and are thus limited in terms of positional diversity. In
abrogate binding to putative cellular receptor(s). However, natural addition, existing functional groups in natural products are often
products are often challenging to functionalize in a chemo- and essential for maintaining biological activity. Accordingly, methods
site-selective manner because of their structural complexity, dense that enable the functionalization of C–H bonds would dramatically
1Department of Chemistry, Texas A&M University, PO Box 30012, College Station, Texas 77842-3012, USA, 2Natural Products LINCHPIN Laboratory, Texas
A&M University, PO Box 30012, College Station, Texas 77842-3012, USA, 3Scripps Research Institute, The Skaggs Institute for Chemical Biology, 10550
North Torrey Pines Road, La Jolla, California 92037, USA, 4Department of Chemistry, University of Puerto Rico, R´ıo Piedras Campus, PO Box 23346,
San Juan, PR 00931-3346, Puerto Rico. e-mail: romo@tamu.edu
*
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1
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1653
ARTICLES
H
Allylic
C H
unfunctionalized positions by Rh(II)-catalysed amination or aziridi-
nation processes with an alkynyl sulfamate reagent, which greatly
expands the methods available for direct functionalization of
native natural products. The described strategy provides a systematic
approach for further exploiting natural products for chemical gen-
etics and addressing the ‘small molecule target identification
problem’. The utility of this strategy is demonstrated by identifying
the cellular targets of the anticancer marine natural product eupal-
merin acetate (EuPA), by quantitative proteome profiling with an
alkyne-substituted derivative obtained by C–H amination.
–
Alkene
H
NP
X
α-heteroatom
H
Benzylic
–
C H
–
C H
1
Bioactive
natural product (NP)
In considering natural product derivatization via C–H amin-
ation, several issues were considered: (i) intermolecular C–H amin-
ation is significantly more challenging than intramolecular
processes16; (ii) high chemo- and site (chemosite) selectivity12 is
required for natural products, which often bear multiple functional
groups; and (iii) microscale reaction conditions (≤1 mg) may be
required, because natural products are often available only in
limited quantities. Mechanistic studies suggest that Rh-catalysed
C–H amination processes proceed via a concerted asynchronous
transition state involving a rhodium nitrene reactant and follow
the reactivity trend 38 . a-amino . a-ethereal ≥ benzylic .
28 ≫ 18 C–H bonds26.
MLn
–
C H amination
or aziridination
N
Y
'Metal nitrenoid'
H
N
Y
Y
and/or
NP
NP
HN
H
X
X
H
Results and discussion
2
3
In our initial studies we compared Du Bois and Lebel C–H amin-
ation conditions and were drawn to the former because of the
higher conversions observed with only 1.0 equiv. of nitrene precur-
sor, which greatly simplifies purification despite the need for a stoi-
chiometric oxidant. Building on the sulfamate design of Du Bois17,
we targeted a trichloroethyl substituted sulfamate with a terminal
alkyne side chain to enable subsequent conjugation to reporter
tags. Du Bois previously described the use of trichloromethyl substi-
tution on sulfamate nitrene precursors to prevent intramolecular
C–H amination and facilitate mild, reductive deprotection to the
primary amine. We therefore targeted sulfamate 9, which was
readily prepared by a two-step sequence from commercially avail-
able trichloromethyl-b-lactone (Fig. 2). In control experiments,
we verified the stability of the nitrene reagent sulfamate 9, which
could be recovered with good mass recovery. As an added benefit,
trichloromethyl substitution provides a unique isotopic pattern to
facilitate mass spectrometric identification of derivatized
natural products.
Alkynyl, C- or N-aminated
natural product derivative
Alkynyl, aziridinated
natural product derivative
N3
Rearrangements,
cyclizations,
alkyne-linker removal
Tag
Conjugation
4
N
'Remodelled
natural products'
Tag
N
N
H
N
Aminated or
aziridinated
natural products
Y
NP
X
5
Natural product-based cellular probes
Figure 1 | A two-step C–H amination or aziridination–conjugation sequence
for simultaneous arming and SAR studies of natural products.
For optimization studies, we chose carvone as a substrate, given
increase the range of available sites on natural products for functio- its potential for both allylic C–H amination and alkene aziridina-
nalization and improve the possibility of maintaining the bioactivity tion, and briefly studied various Rh(II) catalysts (Table 1).
of derivatives.
Rh2(esp)2 gave both allylic C–H amination and aziridination in a
Several mild and chemoselective functionalizations of C–H combined yield of 26% with 1.0 equiv. of nitrene precursor 9,
bonds adjacent to aryl groups, alkenes and heteroatoms (such as whereas Rh2(OAc)4, Rh2(OCOC8H15)4 and Rh2(TPA)4 gave aziridi-
O and N) have recently been described that make use of carbenoid nation products exclusively, in modest yields (23–26%) but with
or nitrenoid reagents in both an intramolecular and intermolecular high mass recovery. Although conversions were generally modest,
fashion16. Of particular interest to our efforts were intermolecular, the mass recovery of these derivatizations was excellent. In
Rh-catalysed C–H amination processes via metallo nitrenoids16, general, we view lower conversion with good mass recovery as a tol-
as described by the groups of Du Bois17,18, Lebel19 and others, erable balance when applying this reaction to sample-limited
which can also deliver aziridines from electron-rich olefins with natural products. Furthermore, in comparison with the overall
certain catalysts20,21. We envisioned that the application of C–H
aminations using a metal nitrenoid precursor bearing an alkyne to
derivatize native, bioactive natural products would enable simul-
NH2
ClSO2NCO,
HCOOH
CH3CN,
4
O
O
taneous arming and SAR studies of natural products at ‘unfunctio-
nalized’ positions. The attached alkyne enables subsequent
conjugation to various tags (for example, biotin or fluorophores),
providing natural product-based cellular probes useful for mode-
of-action studies (Fig. 1). In addition, rearrangements or cycliza-
tions of C–H amination products could lead to ‘remodelled’
natural products22–25, while deprotection would lead to aminated
S
6
EtN(iPr)2
CH2Cl2
O
OH
CCl3
O
O
NH2
0
23 °C;
3
3
+
O
N
H
N
H
Then 8
(CH3)2NC(O)CH3
CCl3
O
0
23 °C
(93%)
9
8
0
23 °C
(85%)
Cl3C
7
or aziridinated natural products for SAR studies. Here, we describe Figure 2 | Synthesis of alkynyl sulfamate 9. Ring opening of b-lactone 7
a two-step strategy for the functionalization of natural products at with alkynyl amine 6 and subsequent sulfamation of alcohol 8 provides 9.
2
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1653
ARTICLES
not derived from simple allylic oxidation by PhI(O2CtBu)2 and sub-
sequent condensation of the resulting aldehyde with sulfamate 9, a
control experiment without sulfamate was performed. This gave pri-
marily epoxidation of geraniol rather than allylic oxidation. Thus,
imine 13a may be derived from allylic C–H amination and sub-
sequent dehydrative elimination. Aziridination of geraniol was
site-selective for the less electron-rich alkene adjacent to the induc-
tively electron-withdrawing alcohol, suggestive of a possible
directing effect by the allylic alcohol. Benzylic C–H amination of
indane (14) occurred to give sulfamate 14a, as previously observed
with a more simple sulfamate17. Cinnarizine (15) gave site-selective
C–H amination on the piperazine ring adjacent to the less hindered
tertiary nitrogen, providing amidine 15a, presumably derived from
subsequent oxidation of the initial C–H amination product (Table 2,
entry 3). a-Pinene (16) underwent highly facially selective allylic
C–H amination, leading to sulfamate 16a as a single diastereomer
in 21% yield (Table 2, entry 4). As with indane, benzylic C–H amin-
ation occurred with b-estrone 3-methyl ether (17), providing a 4:1
mixture of diastereomeric sulfamates 17a in 40% yield (Table 2,
entry 5). In the case of parthenin (18, Table 2, entry 6), aziridination
was observed exclusively at the electron-deficient alkene, providing
Table 1 | Exploratory C–H amination with L-(2)-carvone.
Me
Me
Me
5 mol% catalyst
9 (1.0 equiv.)
O
O
O
3
+
NH
PhI(O2CtBu)2
MgO, C6H6
O
O
H
N
H
R =
23 o
C
Me
R
Me
O
N
S
CCl3
O
R
L-(–)-carvone
10
11
12
Entry Catalyst
C–H
amination
product (11)*
Aziridination
product (12)* starting
material
Recovered
1
Rh2(esp)2
Rh2(OAc)4
Rh2(OCOCF3)4
10%
Trace†
0
16%
26%
0
25%
23%
56%
68%
85%
67%
75%
2
3
4
5
Rh2(OCOC8H15)4 Trace†
Rh2 (TPA)4
0
*Isolated yield. †Only trace amounts of product were detected by LC/MS.
yields likely to be obtained by de novo synthesis, the yield of these aziridines 18a as a 1:1 mixture of diastereomers with low conver-
derivatives is quite acceptable for the goal of initial SAR and cellular sion. Site-selective allylic C–H amination occurred with both eupal-
probe synthesis, as only milligram to submilligram quantities of merolide (19) and EuPA (20), leading to the allylic sulfamates 19a
material are required.
and 20a as mixtures of diastereomers (d.r. ≈ 1:1), respectively
Although higher turnover numbers were reported by Du Bois (Table 2, entries 7 and 8).
when PhI(O2CCMe2Ph)2 was used as the oxidant18, we found that
Application of the amination conditions to amine-containing
PhI(O2CtBu)2 gave less complex reactions and both higher natural products led to either C–H amination/oxidation or a rare
product yields and mass recovery. The addition of inorganic bases N-amination leading to a zwitterionic betaine (‘inner salt’) (for an
(for example, K2CO3), Brønsted acids (for example, HOAc) or intramolecular example of N-amination with a sulfamate nitrene
Lewis acids (for example, In(OTf)3) did not lead to significant see ref. 27, and for an example of N-amination via substitution
increases in conversion, but did have a major impact on the chemos- using hydroxylamine derivatives see ref. 28), structurally verified
1
electivity of the reactions. For example, Brønsted and Lewis acid following extensive H NMR (Supplementary Table S3) and X-ray
additives (for example, HOAc and In(OTf)3) favoured C–H amin- analysis of an N-aminated adduct (Supplementary Fig. S1). The
ation, whereas the use of K2CO3 favoured alkene aziridination, an derivatization of (S,R)-noscapine led to C–H amination of the
observation important for altering chemoselectivity with complex N-methyl group of the isoquinoline ring and subsequent oxidation
natural products. Slow dropwise addition of a dilute solution of to give amidine 21a (E/Z ≈ 1:1, Table 3, entry 1). In addition, amin-
the oxidant did not improve conversion, and the use of MgO had ation of the isoquinoline nitrogen occurred to give betaine 21b. In a
.
.
no impact on conversion or yields. Thus, the direct addition of similar manner, dextromethorphan HBr H2O salt, in the presence of
the solid oxidant in one portion enabled reaction at higher concen- K2CO3 to provide the free base in situ, gave amidine 22a (E/Z ≈ 2:1,
trations (ꢀ0.13 M), an important consideration for microscale deri- Table 3, entry 2) and betaine 22b. In the case of cinchonidine,
vatizations, and this procedure dramatically improved efficiency. N-amination was observed at the tertiary quinuclidine nitrogen,
Although benzene was the optimal solvent, dichloromethane and the quinoline nitrogen and both positions leading to betaines 23a
a,a,a-trifluorotoluene were acceptable alternatives. Ultimately, the (verified by X-ray analysis, Supplementary Fig. S1, CCDC
conditions shown in the scheme in Table 1 (entry 1) were found 930772), 23b and 23c, respectively (Table 3, entry 3). Brucine also
to be optimal, so these conditions were subsequently applied in led to amination of the tertiary amine to give betaine 24a, and an
more complex settings.
eliminative oxidation with ring scission gave lactam 24b (diagnostic
13C signal at d ¼ 175.0; for further characterization data see
Scope of the Rh2(esp)2-catalysed C–H amination/alkene Supplementary Table S2) in 21% yield (Table 3, entry 4).
aziridination with a diverse set of natural products and drugs.
There is growing interest in structurally modifying or ‘remodel-
The optimized C–H amination/aziridination conditions were ling’ natural products to identify new chemical entities for screen-
applied to several commercially available natural products and ing29,30 by controlling chemosite selectivity of derivatizations using
drugs. Although conversions were modest in most cases, as with minimal peptides31 or organometallic catalysts12. In the course of
carvone, mass recovery was again generally excellent. As expected, these studies, we observed instances of unexpected cyclizations,
chemosite selectivity is highly substrate-dependent, and the rearrangements and insertions that led to novel natural product
conformation of the natural products in benzene probably dictates derivatives. As described above, brucine provided lactam 24b
the accessible positions for C–H amination or aziridination in (Table 3, entry 4) derived from a ring scission event. Carvone led
some cases, overriding the site selectivities anticipated based on to both allylic C–H amination and aziridination (Table 1), as
electronic considerations17. The structure and stereochemistry of described above. However, after prolonged storage or in the pres-
derivatives was determined by extensive one- and two- ence of base, the allylic sulfamate 11 underwent an aza-Michael
dimensional NMR spectroscopy (Supplementary pages S32–64).
reaction to provide the interesting bicyclic carvone derivative 25
Application of the optimized conditions to several natural pro- as an equilibrium 1:1 mixture with the starting allylic sulfamate
ducts and drugs led to either C–H amination/oxidation, amina- (Fig. 3a). Under standard conditions, gibberellic acid methyl ester
tion/dehydration or aziridination (Table 2). Functionalization of (GAME, 28) did not give the expected aziridine (Fig. 3b, entry 1);
geraniol (13) gave an ꢀ3:2 mixture of imine 13a (55%) and aziri- instead, ketone 31 was isolated in 55% yield (44% recovered starting
dine 13b (37%). To ensure that imine 13a (Table 2, entry 1) was material). We propose that initial aziridination occurred to give the
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1653
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Table 2 | Derivatives of natural products and drugs produced by C–H amination and aziridination.
Entry
Natural product/drug
Derivaties*,†,⊥
Entry
Natural product/drug
Derivaties
Me
Me
Me
Me
O
O
Me
H
Me
H
1
5
R
Me
OH
Me
N
H
Geraniol (13)
13a (E/Z = 8:1, 55%)
H
H
H
MeO
MeO
R
Me
Me
N
NHR
17a (d.r. 4:1, 40%)
(+51% rec. SM)
β-estrone 3-methyl ether (17)
Me
OH
13b (d.r. 1.2:1, 37%)
(0% rec. SM)
Me
HO
Me
HO
6
7
8
2‡
R
N
Me
Me
O
O
O
O
NHR
O
O
Indane (14)
14a (d.r. 1.5:1, 48%)
(+41% rec. SM)
Parthenin (18)
18a (d.r. 1:1, 8%)
(+90% rec. SM)
R
N
O
O
3
O
O
Ph
N
Ph
N
Me
Me
N
Ph
N
Ph
Me
O
Me
O
Ph
Ph
Me
HO
Me
HO
NHR
Cinnarizine (15)
15a (E/Z = 2:1, 21%)
(+74% rec. SM)
Eupalmerolide (19)
19a (d.r. 1:1, 52%)
(+15% rec. SM)
Me
Me
Me
Me
Me
O
Me
O
RHN
4
Me
Me
OAc
OAc
Me
Me
H
NHR
O
O
O
O
α-(–)-pinene (16)
16a (d.r. >19:1, 21%)
Me
Me
(+70% rec. SM)
Eupalmerin acetate (20)
(EuPA)
20a (d.r. 1:1, 28%, EuPAyne)
(+62% rec. SM)
*Reaction conditions: 2.0 equiv. 9, 5 mol% Rh2(esp)2, 4 equiv. Phl(O2CtBu)2, benzene, 23 8C, 30 min. †R ¼ SO3CH(CCl3)CH2C(O)NH(CH2)4CCH. ‡Diastereomers can be generated with achiral substrates, as in
this case with indane, because the sulfamate reagent bears a stereogenic centre. ⊥Rec. SM, recovered starting material.
expected aziridine 29, which underwent ring cleavage to afford a ter- ketone 31 derived from GAME (28) gave the primary amine 32 in
tiary carbocation 30, then a subsequent 1,2-pinacol rearrangement 85% yield, and the deprotection of betaine 24a proceeded smoothly
delivered ketone 31 (Fig. 3b). (For a related rearrangement of an to give the fairly stable hydrazine inner salt 34 in 76% yield.
epoxide derived from GAME (28) see ref. 32.) In efforts to isolate
the intermediate aziridine, a brief solvent study was undertaken Quantitative proteomic profiling of EuPA targets in living HL-60
and it was found that use of CH2Cl2 did deliver unprotected aziri- cells. EuPA has previously been shown to inhibit the proliferation of
dine 33 in 40% yield, accompanied by the rearranged ketone 31 several cancer cell lines including leukaemia and non-small-cell
(29%, Fig. 3b, entry 2). Use of a,a,a-trifluorotoluene significantly lung, ovarian, breast and colon cancer cell lines at sub- to low
suppressed the rearrangement (6%) and gave lower conversion to micromolar concentrations (GI50 ¼ 0.34–1.7 mM, where GI50 is
the unprotected aziridine 33 (23%, Fig. 3b, entry 3). Further optim- the concentration for 50% of maximal inhibition of cell
ization and exploration of the CH amination/aziridination con- proliferation) in the NCI 60 cancer cell line panel34. EuPA also
ditions were performed with GAME (Supplementary Table S1).
showed significant growth suppression in a mouse model of
We studied removal of the alkynyl sulfamate by N–S cleavage, malignant glioma xenografts35. Despite the potential of EuPA as
leading to the net addition of ‘NH’ (aziridination) or ‘NH2’ (C–H an anticancer therapeutic, the exact cellular target(s) of this
amination) to natural products, which could serve to improve the natural product are unknown.
water solubility of these products. Although several reductive
To demonstrate the utility of the described natural product deri-
methods for cleavage of trichloroethyl sulfamates could be vatization strategy and illustrate the concept of simultaneous SAR
applied17, we chose the very mild conditions developed by studies and arming of natural products, we considered the appli-
Ciufolini, using a 10% Cd/Pb couple33. Deprotection of bicyclic cation of quantitative proteomic profiling with the EuPA–alkyne
ketone 25 under these conditions gave a 3:1 mixture of the expected derivative 20a (Table 2, entry 8) to identify cellular targets of
deprotected azabicycle 26 and the ring-opened allylic amine 27 in EuPA in the HL-60 human acute myeloid leukaemia cell line.
75% combined yield. Similarly, deprotection of the rearranged Importantly, the described C–H amination of EuPA modified the
4
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1653
Table 3 | Derivatives of amine-containing natural products and drugs produced by C–H amination and N-amination.
ARTICLES
Entry
Natural product/drug
Derivatives*,†
Entry
Natural product/drug
Derivatives*,†
O
O
O
1
3
N
R
N
N
O
Me
H
N
H
+
H
OMe
H
H
N
H
O
OMe
O
HO
H
–
N
HO
R
R
H
O
O
N
OMe
OMe
MeO
MeO
N
Cinchonidine (23)
21a (E/Z = 1.3:1, 21%)
(S,R)-noscapine (21)
23a (19%)
–
O
O
N
R
+
N
Me
H
OMe
+
N
H
HO
H
H
O
N
–
HO
N
R
R
H
H
O
+
N
OMe
MeO
+
N
–
N
–
R
N
21b (37%)
(0% rec. SM)
23b (37%)
23c (6%)
(+37% rec. SM)
–
N
•HBr•H2O
R
2‡
4
R
N
+
N
Me
N
H
N
H
N
MeO
MeO
MeO
H
H
H
OMe
OMe
MeO
N
N
H
H
H
H
O
O
O
O
H
Dextromethorphan•HBr•H2O
22a (E/Z = 2:1, 27%)
(22)
Brucine (24)
24a (28%)
–
R
N
+
N
δ
13C 175.0 ppm
Me
H
OMe
O
N
H
MeO
MeO
H
H
22b (42%)
(+10% rec. SM)
N
O
O
24b (21%)
(0% rec. SM)
*Reaction conditions: 2.0 equiv. 9, 5 mol% Rh2(esp)2, 4 equiv. Phl(O2CtBu)2, benzene, 23 8C, 30 min. † R ¼ SO3CH(CCl3)CH2C(O)NH(CH2)4CCH. ‡C-H amination of dextromethorphan†HBr monohydrate was
performed in the presence of K2CO3 (6.0 equiv). ORTEP representation of the X-ray crystallographic structure of 23a; thermal ellipsoids are shown at 50% probability.
allylic position of the macrocycle far removed from the electrophilic these proteins, we performed competitive ABPP-SILAC (activity-
exocyclic alkene, the presumed pharmacophore, which probably based protein profiling-stable isotope labelling by amino acids in
serves as a Michael acceptor for nucleophilic residues of target pro- cell culture), a quantitative, mass spectrometry (MS)-based chemo-
teins. We first compared the antiproliferative properties of EuPA proteomic method that has been used to identify enzyme targets of
and EuPAyne (20a) in HL-60 cells, and found a decrease by only activity-based probes37,38 and small molecules in cells39,40 and
a factor of approximately five compared to the natural product (con- in vivo37,39. Using competitive ABPP-SILAC, we observed several
centration required to inhibit 50% of cell population IC50 ¼ 3.0 mM EuPAyne targets that were competed by pretreatment with EuPA,
versus 15 mM), suggesting that EuPAyne could serve as a chemical suggesting that these proteins are selective targets of EuPA
probe to profile the cellular targets of EuPA using quantitative, (Fig. 4d, Supplementary Table S4). Interestingly, the high-affinity
activity-based proteomic methods (Fig. 4a).
target Derlin-1 (DERL1) is associated with cancer cell proliferation,
To profile specific EuPA targets, we treated HL-60 cells with and cytochrome b5 type B (CYB5B)41 and thromboxane A synthase
EuPAyne (5 mM) and EuPA (0–15 mM). Following this treatment, (TBXAS1)42 are overexpressed in cancer43–45. These three high-affi-
the cells were homogenized and the probe adducts were conjugated nity targets were confirmed by overexpression in 293T cells and
to rhodamine-azide under copper-catalysed azide–alkyne were shown to interact specifically with EuPAyne 20a as they
cycloaddition conditions36, separated by SDS–polyacrylamide gel were competed out with EuPA in a concentration-dependent
electrophoresis, and visualized by in-gel fluorescent scanning. manner (Fig. 4e, Supplementary Fig. S2A,B). Thus, EuPAyne may
As shown in Fig. 4b, several EuPAyne bands compete in a prove to be a useful cellular probe for gaining further understanding
concentration-dependent manner with EuPA, indicating that they of the significance of these proteins in cancer proliferation.
are specific cellular targets of this natural product. To identify Another minor number of EuPA targets are either completely
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1653
ARTICLES
a
3 equiv. iPr2NEt
CH2Cl2, 1.5 h
O
H
10%Cd/Pb
NH4OAc, THF
R
H
HN
N
+
O
O
NH2
23 oC, 24 h
(75%)
Me
O
(1:1)
Me
NHR
(36% 11, 33% 25)
11
25
26
27
(3:1 ratio)
R = SO3CH(CCl3)CH2C(O)NH(CH2)4CCH
b
O
H
O
H
O
O
H
5 mol% Rh2(esp)2
9 (2.0 equiv.)
O
O
HO
O
HO
HO
O
OH
+
OH
NH
Me
Me
Me
PhI(OCOtBu)2
(4.0 equiv.)
solvent
NHR
MeO
MeO
MeO
O
O
Gibberellic acid methyl ester
23 oC
10%Cd/Pb
NH4OAc, THF
23 oC, 24 h
33 (d.r. = 2:1)
(GAME, 28)
31 (R = R)
32 (R = H)
(85%)
+
–H
Entry
Solvent
31
33
Recovered SM
C6H6
1
2
3
55%
29%
6%
0
44%
29%
67%
O
H
H
O
O
O
HO
O
+
CH2Cl2
HO
40%
23%
+ H
OH
OH
Me
C6H5CF3
Me
+
MeO
N
MeO
R
O
NHR
29
30
c
–
N
–
NH
R
+
+
N
N
MeO
MeO
MeO
MeO
10%Cd/Pb
NH4OAc, THF
H
H
H
N
N
23 oC, 4 h
(76%)
H
H
H
H
O
O
O
O
H
24a
34
Figure 3 | Natural product remodelling. a, Cyclization of the carvone C–H amination product via aza-Michael addition, leading to the bridged bicyclic
derivative 26. b, Rearrangement of a GAME-derived aziridine leading to the remodelled cyclopentanone 31 via pinacol rearrangement, and solvent study
leading to variations in product distribution. c, Reductive cleavage of the N–S bond in betaine 24a with Cd/Pb couple.
uncharacterized (NUDT8, NT5DC1) or have unknown molecular to a variety of tags in a subsequent step. Mild conditions were ident-
functions (CDKAL1).
ified for cleavage of the sulfamate group leading to net amination or
In summary, a method for performing simultaneous SAR studies aziridination of the parent natural product, which can improve
and arming of bioactive natural products through Du Bois C–H water solubility when required. The utility of this method for
amination and alkene aziridination is described using an alkynyl natural product derivitization was demonstrated by application to
sulfamate nitrene precursor and Rh2(esp)2 as catalyst. These derivi- EuPA, a marine-derived anticancer diterpene with unknown cellu-
tizations are mild enough to tolerate a number of functional groups lar targets. A unique, allylic C–H aminated derivative of EuPA
found in natural products, including alcohols, ethers, lactones, (IC50 ¼ 3.0 mM), coined EuPAyne (IC50 ¼ 15 mM), enabled the
lactams, esters, quinolines and piperazines. In several cases, the use of gel proteomic profiling of HL-60 cells for the initial analysis
described derivatization is found to be both chemo- and site-selec- of cellular protein targets in this cancer cell line. Competitive ABPP-
tive, enabling functionalization of allylic and benzylic positions via SILAC enabled quantitative proteome analysis of HL-60 cells and
C–H amination or alkenes through aziridination. In addition, the identification of several cellular protein targets of EuPA, includ-
N-aminations of tertiary amines occur readily, giving inner salts, ing several associated with cancer proliferation. The described deri-
and this process provides another avenue for tagging amine-con- vatization strategy greatly expands the methods available for direct
taining natural products. Future studies will explore the potential functionalization of native natural products and provides a rapid
utility of these inner salts for mode-of-action studies including pro- and systematic approach for further exploiting natural products
teomic profiling. For the purpose of SAR studies, the ability to for chemical genetics and addressing the ‘small molecule target
initially obtain low chemosite selectivity is ideal for sampling identification problem’. Other emerging methods for direct C–H
various positions of a given natural product in a tractable manner. functionalization46–48 are also expected to be applicable and will
In addition, attachment of an alkynyl substituent (‘arming’) potentially enable a truly universal approach for exploiting natural
enables direct conjugation of the derived natural product congeners products for chemical genetics26.
6
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1653
ARTICLES
a
b
IC50 (95% CI)
3.0 µM (2.4–3.7)
14 µM (8–25)
5 µM EuPAyne
2.5
1.5
EuPA
EuPAyne
15
5
1
0.5
0
EuPA (µM)
1.0
0.5
0.0
kDa
75
50
37
–1
0
1
2
3
log(concentration/µM)
25
20
c
Light cells
1. Click chemistry
2. Avidin enrichment
3. Trypsin digestion
4. LC-MS/MS
EuPA
EuPAyne
EuPAyne
Mix
Heavy cells
Mock
d
e
DERL1 CYB5B TBXAS1 transfected
Competition experiment
Control experiment
kDa
75
–
+
–
+
–
+
–
+
EuPA
Gene symbol
MW (kDa)
Ave. ratio
s.e.m.
Ave. ratio
s.e.m.
DERL1
CYB5B
NT5DC1
CPT1A
TAPBP
TBXAS1
TAP1
28.8/26.4
16.3
51.8
88.4/86.2
43.9/53.9
60.5
20
20
0
0
0.93
0.94
0.91
0.99
0.91
0.91
0.94
0.03
0.02
0.03
0.05
0.09
0.06
0.01
50
37
17.16
15.80
15.20
14.43
14.08
2.84
4.21
4.81
5.57
5.92
25
20
87.2
Figure 4 | Cytoxicity and proteomic profiling of EuPA and EuPAyne. a, Inhibition of HL-60 cell proliferation by EuPA and EuPAyne as measured using a
WST-1 assay. Error bars indicate the standard error from three replicates. b, In situ treatment of HL-60 cells with EuPAyne alone or in competition with EuPA
followed by conjugation to an azide-rhodamine reporter tag, SDS-PAGE and fluorescent scanning. Red arrows highlight competed signals. c, Schematic of
competitive ABPP-SILAC. Cells are enriched with heavy or light isotopic tags and treated with DMSO or EuPA, respectively, followed by the addition of
EuPAyne. EuPA-labelled proteomes are mixed, conjugated to biotin using click chemistry, enriched, and analysed by Multi-dimensional Protein Identification
Technology (MudPIT). d, List of putative EuPA targets in HL-60 cells. Average ratios are from duplicate runs and standard error results are reported. In the
control experiment, heavy and light cells are treated with EuPAyne alone. e, Validation of three high-affinity targets DERL1, CYB5B and TBXAS1 by labelling of
transiently transfected 293T cells with EuPAyne (5
mM)+EuPA (15 mM). Red arrows indicate the target’s expected molecular weight. These data illustrate
how EuPAyne can be used to profile the covalent molecular targets of EuPA in living cells.
with avidin beads, digested on-bead with trypsin, and analysed by liquid
Methods
chromatography–tandem MS (LC-MS/MS) using an LTQ-Orbitrap instrument.
Light and heavy signals were quantified from parent ion peaks (MS1) (see
Supplementary Information) and the corresponding proteins identified from
product ion profiles (MS2) using the ProLuCID search algorithm49. Control
experiments were carried out in the same manner with the exception that both heavy
and light cells were treated with EuPAyne alone.
Representative procedure for C–H amination/aziridination of complex natural
products. The natural product or drug (0.05 mmol, 1.0 equiv.), sulfamate 9
(0.1 mmol, 2.0 equiv.) and Rh2(esp)2 (0.0025 mmol, 0.05 equiv.) were mixed in a
small vial and evacuated under high vacuum for 10 min before being purged with
N2. (Note that if the natural product/drug is volatile, it should be added after this
evacuation process.) Benzene (0.4 ml) was added to give a green suspension
(0.125 M) and this mixture was stirred at 23 8C under N2 for 10 min. PhI(O2CtBu)2
(0.2 mmol, 4.0 equiv.) was then added in one portion and the reaction mixture was
vigorously stirred at 23 8C for an additional 30 min. The crude reaction mixture,
following initial analysis by liquid chromatography-mass spectrometry (LC-MS),
was loaded on a silica gel column directly without workup and purified by flash
column chromatography (eluting with EtOAc/hexane) to isolate the desired
product(s). For natural products only available in minute quantities, the reaction
could be successfully performed on a 1 mg scale using 0.2 ml of solvent
(concentration is ꢀ20 mM in this case). The amount of all other reagents is reduced
accordingly. If separation of sulfamate from the product is problematic, it can be
removed by extraction with 0.01 N (aq.) NaOH assuming stability of the natural
product to these conditions. The reaction mixture is dissolved in CH2Cl2 and
washed twice with 0.01 N (aq.) NaOH solution (0.2 vol.).
Received 10 December 2012; accepted 11 April 2013;
published online 19 May 2013
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Acknowledgements
The authors acknowledge support from the National Institutes of Health (NIH,
GM806307, to D.R.; CA087660, to B.C.; GM086271, to A.D.R.), the Welch Foundation
(A-1280, to D.R.), a Ray Kathren American Cancer Society fellowship (to J.S.C.) and a Ruth
L. Kirschstein National Research Service Award (F32GM095245-01 to J.S.C.). The Office of
the Vice President for Research, the College of Science and the Department of Chemistry at
Texas A&M provided seed funding for the TAMU Natural Products LINCHPIN
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Author contributions
D.R. and B.F.C. jointly conceived the study and supervised the work. D.R., B.F.C., J.L., J.S.C.
and C.Z. were involved with the design of the studies, performance of the experiments, and
analysis of the results. J.L., J.S.C. and D.R. wrote the manuscript. H.W. assisted with
acquisition and analysis of 2D and cryoprobe NMR data. A.D.R. and B.V. isolated and
purified samples of eupalmerolide and EuPA and assisted with the NMR analysis of the
resultant alkynylated derivatives. All authors edited the manuscript.
Additional information
Supplementary information and chemical compound information are available in the
online version of the paper. Reprints and permissions information is available online at
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Competing financial interests
The authors declare no competing financial interests.
8
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