Methods for the synthesis of carbon-13 labelled acids and esters

Article abstract of DOI:10.1002/jlcr.1198

Syntheses of isotopically labelled putative biosynthetic intermediates to the natural products monocerin 1, hectochlorin 2 and strobilurin A 3 are described. For the preparation of [9,10-¹³C₂] dihydroisocoumarin 10, a stereoselective aldol condensation of ¹³C₂-acetylated chiral auxiliary 5 was used to assemble the labelled C9-C14 fragment. The preferred approaches to the syntheses of [1,2-¹³C₂]5,5-dichlorohexanoic acid 15 and the N-acetylcysteamine derivative of [1,2-¹³C₂]cinnamic acid 19 involved a Horner-Wadsworth-Emmons chain extension and Knoevenagel reaction, respectively. Copyright

Full text of DOI:10.1002/jlcr.1198

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Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 338–341.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1198
Short Research Article
Methods for the synthesis of carbon-13 labelled acids and
estersy
ANGELA C. JORDAN1,*, LORRAINE C. AXFORD2, JOHN R. HARDING1, YVONNE O’CONNELL2, THOMAS J. SIMPSON2
and CHRISTINE L. WILLIS2
1 Isotope Chemistry, Drug Metabolism and Pharmacokinetics Department, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK
2 School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK
Received 30 August 2006; Revised 2 November 2006; Accepted 22 November 2006
Abstract: Syntheses of isotopically labelled putative biosynthetic intermediates to the natural products monocerin 1,
hectochlorin 2 and strobilurin A 3 are described. For the preparation of [9,10-13C2]dihydroisocoumarin 10, a
stereoselective aldol condensation of 13C2-acetylated chiral auxiliary 5 was used to assemble the labelled C9-C14
fragment. The preferred approaches to the syntheses of [1,2-13C2]5,5-dichlorohexanoic acid 15 and the
N-acetylcysteamine derivative of [1,2-13C2]cinnamic acid 19 involved a Horner-Wadsworth-Emmons chain extension
and Knoevenagel reaction, respectively. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: aldol reaction; carbon-13 labelling; Horner–Wadsworth–Emmons; Knoevenagel
Introduction
metabolite is to use precursors with two carbon-13 labels
located at vicinal sites and to detect the 13C–13C coupling
by 13C-NMR spectroscopy.1 Thus efficient and flexible
methods are required to introduce the vicinal carbon-13
labels. Herein methods are described for the synthesis of
carbon-13 labelled putative biosynthetic intermediates to
the natural products monocerin 1, hectochlorin 2 and
strobilurin A 3 using three different approaches: a
Substrates incorporating stable isotopic labels have
proved valuable for a range of studies in bio-organic
chemistry and in particular for the elucidation of bio-
synthetic pathways. However, feeding studies to intact
organisms with labelled putative biosynthetic intermedi-
ates often lead to a variable level of incorporation of
isotopic label into the final metabolite. An effective method
to detect a low level of incorporation of carbon-13 into a
stereoselective
aldol
reaction,
Horner-Wadsworth-
Emmons chain extension and the Knoevenagel reaction.
Cl
Cl
O
N
S
O
O
N
OAc
MeO
O
MeO
OMe
O
O
O
MeO
O
O
HO
OH
O
S
1
2
3
Results and discussion
*Correspondence to: Angela C. Jordan, Isotope Chemistry, Drug
Metabolism and Pharmacokinetics Department, AstraZeneca, Mere-
side, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK.
E-mail: angela.jordan@astrazeneca.com
yProceedings of the Ninth International Symposium on the Synthesis
and Applications of Isotopically Labelled Compounds, Edinburgh,
16–20 July 2006.
Synthesis and incubation studies with [9,10-13C2]
dihydroisocoumarin 10
Monocerin 1 is a polyketide derived natural product
which has been isolated from a number of fungi
Copyright # 2007 John Wiley & Sons, Ltd.

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SYNTHESIS OF CARBON-13 LABELLED ACIDS AND ESTERS 339
S
S
S
TBDMS
i) Sn(OTf)2,1-ethylpiperidine,
O
*
O
*
O
O
*
OH
DCM, -40 0C
BOPCl, DMAP,
*CH3*CO2H
i) Et3N, MeNHOMe, DCM
ii) Butanal,-780C
ii) TBDMSOTf, 2,6-lutidine, DCM
iPr
N
HN
S
N
N
S
iPr
S
*
*
*
OMe
100%
88%
90% over 2 steps
4
5
6
7
sec-BuLi,THF, -72 0C
45%
Me
BnO
* = 13
C
8
CONMe2
OBn
*
*
iPr
iPr
*
BnO
HO
iPr
BnO
*
*
10
*
i) HCl/EtOH (1%), RT, 87%
9
3 N HCl/dioxane (1:1),
Reflux
ii) AcOH, Me4N.B(OAc)3H, 92%
O
OTBDMS
OH
O
OH OH
CONMe2
CONMe2
83%
10
9 OBn
OH
O
OBn
Scheme 1 Synthesis of [9,10-13C2]dihydroisocoumarin 10.
Synthesis of [1,2-13C2]5,5-dichlorohexanoic acid 15
including Dreschlera monoceras and Dreschlera rave-
nelii.2 Results from feeding studies using isotopically
labelled sodium acetate with D. ravenelii3 led to the
proposal that dihydroisocoumarin 10 is the first
polyketide synthase (PKS) free intermediate in the
biosynthesis of monocerin. Hence 10 was required in
isotopically labelled form for incorporation studies. A
stereoselective aldol reaction was used to prepare the
labelled C9-C14 fragment using the sulfur containing
thiazolidinethione auxiliary 4 (Scheme 1).
Hectochlorin 2 was isolated from the marine cyano-
bacterium Lyngbya majuscula and possesses a struc-
ture in accord with a mixed PKS-NRPS biosynthetic
origin.6 Of particular interest is the unusual dichlor-
ohexanoic acid side chain and
a
sample of
[1,2-13C2]5,5-dichlorohexanoic acid 15 was required
to investigate the biosynthesis of hectochlorin.
Acetylated chiral thiazolidinethiones, as illustrated
above, and other acetylated auxiliaries including
oxazolidinones and sultams have been used widely
to introduce vicinal 13C2 labels. Whilst these
starting materials could be considered for the synthesis
of 15, with no asymmetric centre present in the target,
a more efficient strategy proved to be assembly of the
required C6-framework via a Horner–Wadsworth–Em-
mons chain extension of protected 4-oxobutanal 11
which in turn was prepared from ethyl acetoacetate
(Scheme 2).
Originally the mixed anhydride generated from
sodium 13C2-acetate and pivaloyl chloride was used
to acylate the sodium salt of thiazolidinethione 4 giving
5 in 35% yield.4 Recently we have found that a more
efficient approach to 5 involves treatment of auxiliary 4
with 13C2-acetic acid, BOPCl and catalytic DMAP giving
the product in quantitative yield. A stereoselective aldol
reaction of acetylated auxiliary 5 with butanal in the
presence of tin triflate and 1-ethylpiperidine5 gave the
required alcohol 6 as a single diastereomer.4 Following
cleavage of the auxiliary and formation of the silyl
ether, the pivotal step was coupling the resultant
Weinreb amide 7 with the anion of benzamide 8
(prepared using sec-butyl lithium) to give 9 with
the required carbon skeleton of our target 10. Following
further functional group modifications including
deprotection of the silyl ether under mild conditions,
The ketonic carbonyl of ethyl acetoacetate was
protected as a cyclic acetal prior to reduction of the
ester with diisobutylaluminium hydride (DIBAL) giving
aldehyde 11 in 65% yield after purification by column
chromatography. Treatment of 11 with commercially
available [1,2-13C2]triethylphosphonoacetate gave
unsaturated ester 12. Reduction of 12 with Pd/C
under a hydrogen atmosphere, followed by deprotec-
tion of the acetal using cerium(III) chloride gave ethyl
[1,2-13C2]5-oxohexanoate 13 in 73% yield over the two
steps. To complete the synthesis of the target com-
pound 15, it was necessary to convert ketone 13 to
dichloride 14. Initially geminal dichlorination proved
to be problematic e.g. using either PCl5/DCM/H2O
a
stereoselective reduction and finally treatment
of the resultant dihydroxyamide with acid, the
target [9,10-13C2]dihydroisocoumarin 10 was isolated
in good yield. Incubation studies of 10 with D. ravenelii
showed that an exceptionally high incorporation
(60%) of intact isotopic label into monocerin had
occurred.4
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 338–341
DOI: 10.1002.jlcr

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340 A. C. JORDAN ET AL.
i) TsOH, HOCH CH OH,
2
Benzene, Reflux
58%
2
O
O
O
*
(EtO) PO*CH *CO Et
2
2
2
O
O
O
O
O
NaH,THF, N
2
66%
OEt ii) DIBAL, DCM, -78 °C
OEt
H
*
H
65%
11
12
i)
Pd/C, MeOH
2,
13
88%
ii) CeCl .7H O,NaI,
* =
C
3
2
Acetonitrile
84%
i) HTBDMSN-NHTBDMS,
0.01% Sc(OTf)
ii) CuCl, Et N, MeOH
3
LiOH
3
THF
O
O
*
O
O
Cl
Cl
O
Cl
Cl
H
2
52% over 2 steps
76%
*
*
OH
OEt
OEt
*
*
*
15
14
13
Scheme 2 Synthesis of [1,2-13C2]5,5-dichlorohexanoic acid 15.
*
O
O
O
*
13
=
C
*
HO2C
CO2H
17
H
*
*
Pyridine
N
DCC, DMAP,
HSNAC
Piperidine
57%
S
OH
H
*
*
67%
O
16
18
19
Scheme 3 Synthesis of the SNAC derivative of [2,3-13C2]cinnamic acid 19.
followed by alumina/SOCl72 or H2NNH2 ꢀ H2O/molecu-
lar sieves/MeOH followed by CuCl/Et3N/MeOH,8 none
of the required product 14 was isolated. Recently Myers
and Furrow9 have reported a valuable modification of
the Takeda conditions8 for the synthesis of gem-
dihalides from ketones. Using their reagent, prepared
by treatment of anhydrous hydrazine (care, potentially
explosive) with TBDMSCl, ketone 13 was converted to a
hydrazone which on reaction with CuCl/Et3N/MeOH
gave dichloro ester 14 in 52% yield. Finally hydrolysis
of the ester using lithium hydroxide completed the
synthesis of the target [1,2-13C2]5,5-dichlorohexanoic
acid 15.
attributed in part to SNAC acting as a mimic of
coenzyme A.11
A very simple 2-step procedure was used for the
synthesis of the required [2,3-13C2]thiol ester 19
involving a Knoevenagel reaction followed by thioester-
ification. First treatment of commercially available
[1-13C]benzaldehyde 16 and [2-13C]malonic acid 17
with a mixture of piperidine and pyridine at reflux gave
[2,3-13C2]cinnamic acid 18. This was followed by a
DCC, DMAP mediated coupling of the acid 18 with
N-acetylcysteamine giving the target compound 19 in
67% yield (Scheme 3).
Conclusion
Synthesis of N-acetylcysteamine thiol ester of
[2,3-13C2]cinnamic acid 19
Three methods have been used for the introduction of
vicinal carbon-13 labels into substrates required for
biosynthetic studies: an aldol reaction, Horner-
Wadsworth-Emmons chain extension and a Knoevena-
gel reaction. Each is efficient and lends flexibility for
the incorporation of vicinal carbon-13 labels into a
substrate. The labelled dihydroisocoumarin 10 was
incorporated into the fungal metabolite monocerin 1 at
an exceptionally high level. Feeding studies with
the labelled 5,5-dichlorohexanoic acid 15 and SNAC
cinnamic ester 19 are in progress.
Strobilurin A 3 is produced by the fungi Strobililurus
tenacellus and Oudemansilla mucida and is of mixed
biosynthetic origin.10 Feeding studies with labelled
precursors have shown that the strobilurins are poly-
ketide in origin with a benzoate starter extended by
acetate derived malonate units. The benzoate starter
unit itself appears to originate from the shikimate
pathway by degradation of phenylalanine to benzoyl-
CoA via cinnamic acid prior to assembly. To confirm
this proposed pathway the S-N-acetyl cysteamine
(SNAC) derivative of [2,3-13C2]cinnamic acid 19 was
required. It has been shown that often SNAC deriva-
tives are more readily incorporated into a biosynthetic
pathway than the parent carboxylic acid–this has been
Acknowledgements
We are grateful to the EPSRC (LCA and YOC) for
funding and to Professor W. H. Gerwick for collaborative
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 338–341
DOI: 10.1002.jlcr

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SYNTHESIS OF CARBON-13 LABELLED ACIDS AND ESTERS 341
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Gerwick WH. J Nat Prod 2002; 65: 866; Suntornch-
ashwej S, Chaichit N, Isobe M, Suwanborirux K.
J Nat Prod 2005; 68: 951.
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 338–341
DOI: 10.1002.jlcr