Total synthesis of tetracyclic kynurenic acid analogues isolated from chestnut honey

Article abstract of DOI:10.1016/j.tetlet.2017.12.015

A short and efficient synthesis of novel tetracyclic Kynurenic acid analogues, isolated from chestnut honey, is described. The crucial step of the strategy was a MW-assisted cyclization of enamines of ethyl dioxohexahydropyrrolizine and 2,3-dioxooctahydroindolizine carboxylates to obtain 2,3,6,11b-tetrahydro-1H-pyrrolizino[2,1-b]quinoline-5,11-dione and 5,8,91,011,11a-hexahydroindolizino[2,1-b]quinoline-6,12-dione, respectively. Because of its modular nature, the synthetic strategy can have value as a general method for the preparation of compounds containing these new heterocyclic scaffolds.

Full text of DOI:10.1016/j.tetlet.2017.12.015

Accepted Manuscript
Total synthesis of tetracyclic kynurenic acid analogues isolated from chestnut
honey
Raffaella Cincinelli, Giangiacomo Beretta, Sabrina Dallavalle
PII:
S0040-4039(17)31507-1
https://doi.org/10.1016/j.tetlet.2017.12.015
TETL 49516
DOI:
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
3 November 2017
29 November 2017
3 December 2017
Please cite this article as: Cincinelli, R., Beretta, G., Dallavalle, S., Total synthesis of tetracyclic kynurenic acid
analogues isolated from chestnut honey, Tetrahedron Letters (2017), doi: https://doi.org/10.1016/j.tetlet.
2017.12.015
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Total synthesis of tetracyclic kynurenic acid analogues isolated from chestnut honey
Raffaella Cincinelli, Giangiacomo Beretta, Sabrina Dallavalle
1

Total synthesis of tetracyclic kynurenic acid analogues isolated from chestnut honey
Raffaella Cincinellia*, Giangiacomo Berettab, Sabrina Dallavallea
aDepartment of Food, Environmental and Nutritional Sciences, Division of Chemistry and
Molecular Biology, Università degli Studi di Milano, via Celoria 2, I-20133 Milano, Italy
bDepartment of Environmental Science and Policy ESP, Università degli Studi di Milano, Via
Mangiagalli 25, 20133 Milano, Italy
*Corresponding Author
E-mail address: raffaellacincinelli@gmail.com (R. Cincinelli)
ABSTRACT
A short and efficient synthesis of novel tetracyclic Kynurenic acid analogues, isolated from chestnut
honey, is described. The crucial step of the strategy was a MW-assisted cyclization of enamines of
ethyl dioxohexahydropyrrolizine and 2,3-dioxooctahydroindolizine carboxylates to obtain
2,3,6,11b-tetrahydro-1H-pyrrolizino[2,1-b]quinoline-5,11-dione
and
5,8,9,10,11,11a-
hexahydroindolizino[2,1-b]quinoline-6,12-dione, respectively. Because of its modular nature, the
synthetic strategy can have value as a general method for the preparation of compounds containing
these new heterocyclic scaffolds.
Keywords: Kynurenic acid analogues, chestnut honey, natural products, microwave assisted
synthesis
Kynurenic acid (KYNA), an endogenous, non-selective antagonist of ionotropic glutamate
receptors, is commonly considered as a final metabolite of tryptophan via the kynurenine pathway
(KP).
Recent studies provided evidence for an important role of kynurenine pathway metabolites in
several neurological, psychiatric and neurodegenerative diseases, with particular emphasis on the
2

putative neuroprotective activity exerted by KYNA.1 The biological activity of KYNA is due to its
antagonizing capacity toward the glycine site of the NMDA (N-methyl-D-aspartate) receptors
and/or the cholinergic α7 nicotine receptors. However, concomitant or alternative involvement of
other pharmacological targets responsible for its electrophysiological and behavioral actions cannot
be excluded.2
KYNA is present in several food products, with the highest known concentrations found in chestnut
honey.3 6-hydroxy-KYNA and KYNA are also present in the plant kingdom with a taxa-specific
distribution of the former.4 The observation of significant levels of KYNA in different medicinal
plants suggested that it may contribute to their therapeutic potential, especially in the digestive
system.5
Interestingly, recent studies consistently demonstrated that chestnut honey also contains tetracyclic
KYNA derivatives, with a tetrahydropyrrolizinoquinolinedione (compound 1, figure 1) or a
hexahydroindolizinoquinolinedione (compound 2, figure 1) core.6 These substances originate
mainly from the nectar of male flowers of chestnut.7 Their biosynthesis is also intriguing, as they
probably derive from KYNA,7 differently from the isomeric fungal metabolite
tetrahydroquinolactacide,8 whose biosynthesis most likely starts from anthranilic acid.9
Currently, the biological function(s) and therapeutic potentiality of derivatives 1 and 2 are
unknown.
Hence, the aim of the present study was to set up a synthetic procedure for these new quinolinone
alkaloids which may - in principle - have value in the preparation of the natural compounds
themselves as well as in synthesizing analogues for structure-activity relationship studies.
11
10
8
1
O
O
2
3
1
4
10
7
11 11b
12
9
2
3
9
8
N
N
4
7
5
6
N
H
6
N
H
O
O
5
2
1
3

Figure 1. Structures of compounds 1 and 2 isolated from chestnut honey
Initially, we focused on the synthesis of compound 1. We envisioned that the most straightforward
approach could be based on the construction of the tetracyclic system starting from kynurenic acid.
Thus, disconnection at the amide bond level of the compound 1 gave as precursor the key
intermediate A, which should be accessible by metal-catalyzed cross-coupling reaction between a
functionalized kynurenic acid B and a boronic acid C (Scheme 1, path I)
O
I
+
(HO)2B
N
R''
N
R
COOMe
B
C
O
N
R''
COOR'
N
R
A
path I
1
O
2
10
11
9
8
3
NH
4
path II
Winterfeldt oxidation
path III
N
6H
1
7
O
5
O
N
R'''O
+
N
O
N
R
NH2
O
O
D
E
Scheme 1. Retrosynthetic analyses of compound 1
The N-benzyl protected coupling partner B (compound 5) was easily obtained by treatment of N-
benzylaniline 3 with DMAD (dimethyl acetylenedicarboxylate) in methanol at reflux, followed by
cyclization with Eaton’s reagent10 to give N-benzyl kynurenic acid methyl ester (4). Iodination11
provided intermediate 5 in good yield.
4

The N-Boc protected partner C (compound 6) was obtained from commercially available N-Boc
pyrrole-2- boronic acid by hydrogenation with PtO2 in ethyl acetate at room temperature (Scheme
2). Once prepared the two fragments, we had to couple them to obtain compound 7.
OH
B
O
N
O
N
O
N
H
HO
N
Ph
I
6
BocN
N
Boc
COOMe
a, b
c
x
COOMe
COOMe
Ph
3
4
Bu3Sn
BocN
Ph
Ph
5
7
8
d
O
O
e
x
N
Boc
COOMe
N
N
H
N
O
Ph
1
9
Scheme 2. Synthesis of compound 9. Reagents and conditions: a) DMAD, MeOH, reflux, 85%; b) P2O5/MeSO3H, N2,
50 °C, 82%; c) I2, CAN, N2, 50-80 °C, 98%; d) Pd(PPh3)4, NaHCO3, DME, 2-pyrrol boronic acid, N2, reflux, 64%; e)
H2 40 atm, Pd/C 10%, MeOH, rt.
Initial efforts to effect the carbon-carbon coupling reaction, carried out using Pd(PPh3)4 and
aqueous Na2CO3 in toluene at 80 °C, were unsuccessful. Various attempts of coupling made with
different bases (K2CO3, K3PO4, CsCO3) different phosphine ligands (Pd(dppf), Pd(dba)2) and a
variety of solvents (dioxane, THF and DME) always gave complex reaction mixtures.
Stille cross coupling reaction was also unsuccessful. Stannane 8 was prepared from N-Boc
pyrrolidine and n-Bu3SnCl with secBuLi in THF. Reaction of this latter with 5 in the presence of
Pd(PPh3)4 in dioxane12 did not provide the expected product (Scheme 2).
A possible explanation of these failures in coupling reactions is the poor reactivity of alkyl boronic
acids. Aryl-alkyl Suzuki-Miyaura cross-couplings are often complicated by reduction and
isomerization side reactions as a result of the presence of β-hydrogen atoms in the alkyl group.13
5

The use of N-Boc-2-pyrroleboronic acid instead of the corresponding alkyl boronic acid 6 easily
afforded the coupling product 9,14 but the successive reduction of the ring together with the removal
of the protecting benzyl group failed, even by hydrogenation at high pressure15 (Scheme 2).
Thus, an alternative route was pursued. We envisioned that the functional quinolone skeleton of
compounds 1 and 2 might be assembled by the application of the Winterfeldt oxidation of indoles.16
This reaction has provided an efficient method for indole-quinolone transformations in the total
synthesis of the potent antitumor agent camptothecin 17 and of the antibiotics quinolactacine A18 and
B.19
As reported by Husson and coworkers,20 pyrrolo[3,4-b]-quinoline-3,9-diones can be obtained by
Winterfeldt oxidation (O2, t-BuO-K+, DMF) of N(b) substituted tetrahydropyrido[3,4-b]indol-1-
ones. Thus, we identified compound D as a new key intermediate (Scheme 1, path II).
As the removal of benzyl group had been troublesome (see scheme 2), we chose the
(phenylsulfonyl)ethyl group for N-protection. Indeed, the group can be easily removed by treatment
with strong bases such as t-BuOK,21 which is also used to perform Winterfeldt oxidation.
Thus, commercially available indole-2-carboxylic acid methyl ester 10 was treated with 2-
phenylsulfonylethyl chloride in presence of NaH and DMF to obtain compound 11. Hydrolysis of
the ester to the corresponding acid 12 was achieved under basic conditions using LiOH in THF/H2O
1:1 (Scheme 3).
OMe
O
OMe
O
a
b
N
R
N
H
10
11
HO
OH
O
N
O
c
N
R
N
R
13
12
N
d
O
N
R
R = CH2CH2SO2Ph
14
6

Scheme 3. Synthesis of compound 14. Reagents and conditions: a) ClCH2CH2SO2Ph, NaH, DMF, N2, rt, 61%; b)
LiOH.H2O, THF/H2O 1:1, 98%; c) EDCI (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), HOBt
(hydroxybenzotriazole), prolinol, THF, N2, rt, 86%; d) TFA, 1,2-dichloromethane, 50 °C, N2, 12%.
The condensation of 12 with prolinol gave compound 13, which was cyclized by TFA in 1,2-
2
dichloroethane (Scheme 3) to obtain compound 14. However, the very poor yield of this reaction
2
prevented us to pursue this strategy.
In our last approach (Scheme 1, path III), retrosynthetic cleavage at the C4/C5a and NH/C2 bonds
dissected the target molecule 1 into an aniline and the intermediate E.23
Ethyl-2-pyrrolidinylacetate 17 was prepared in two steps in 32% overall yield by radical alkylation
of pyrrole with ethyl iodoacetate followed by reduction with rhodium on alumina in glacial acetic
acid. Alternatively, the compound was obtained in three steps from commercially available 2-
pyrrolidinone in an improved 53% yield.24 The synthesis of 1-carbethoxy-2,3-dioxopyrrolizidine 19
was carried out using Adams’ method.25 Reaction with diethyl oxalate in presence of sodium
ethoxide gave compound 19 in 80% yield. Condensation with aniline in toluene produced
compound 21 in good yield. Intramolecular cyclization of the enamine performed under various
conditions (P2O5/Me3SO3H, H2SO4 and PPA) did not allow us to obtain the desired compound,
giving only decomposition products, most probably due to the instability of enamines in an acidic
environment. When the reaction was performed thermally in a neutral medium (boiling diphenyl
ether) the desired compound 1 was obtained in low yield (24%) after a long reaction time (Scheme
4). To overcome this limitation we shifted to microwave heating. After several attempts (solvent
free26 and in acetonitrile at various temperatures between 130 °C and 180 °C), we found that the
desired compound could be obtained in a satisfactory 45% yield heating at 240 °C in DMF for 2h.
The compound was fully characterized by MS and NMR spectroscopy. Disappointingly, when we
compared the 1H NMR spectrum of compound 1 dissolved in CDCl3 (20 mg/mL) with the spectrum
of the natural compound reported in the literature in the same solvent,6 we found some
7

discrepancies. Specifically, the reported chemical shift of the aromatic proton on carbon 7 was 7.57
ppm, whereas in our compound the chemical shift of the same proton was 7.79 ppm. Analogously,
the signal of the NH proton was 9.74 ppm in the literature paper and 11.15 ppm in our spectrum. In
13C NMR spectrum of the isolated compound the signals of C6a and C10a were overlapped at 139.8
ppm, whereas the same signals in our spectrum were at 141.1 and 140.2 ppm.
However, we noticed that increasing the dilution of the sample (5mg/mL), the above reported
signals shifted upfield and at a concentration of 1mg/mL, the spectra of compound 1 completely
matched those reported in the literature (Figure 2). Moreover, the signal of the NH proton, sharp at
high concentration (20 mg/mL), became progressively broader during the dilution process, a
behaviour indicative of the reduced ability of the NH proton to form hydrogen bonds.
This evidence suggested that compound 1 in concentrated CDCl3 solution gives strong
intermolecular hydrogen bond interactions, which progressively weaken with the dilution of the
sample. The formation of hydrogen bonds was confirmed by the shift of NH and aromatic hydrogen
signals in the NMR spectra of compound 1 (10 mg/mL) run at different temperatures (10 °C, 15 °C,
25 °C). (see SI, figures 1S and 2S).
8

0.5 mg/mL
1 mg/mL
5 mg/mL
20 mg/mL
9

Figure 2. Effect of the concentration of compound 1 on the chemical shift of aromatic and N-H protons (1H NMR, 600
MHz, CDCl3, expanded region 7-12 ppm)
The developed synthetic protocol was exploited for the synthesis of the six-membered ring
compound 2. Ethyl 2-piperidinylacetate 18 was prepared by hydrogenation of the commercially
available 2-pyridylacetate 16 with PtO2 in ethanol and 6M HCl. Reaction with diethyl oxalate gave
the cyclized compound 20, which was then converted into the corresponding enamine 22 in 54%
yield. (Scheme 4). Microwave cyclization afforded compound 2, whose spectroscopic properties
matched with those of the natural compound isolated from chestnut honey.
N
H
O
a
b
n
EtO
O
O
n
c
15
EtO
N
H
O
N
EtO
O
O
n= 1 17
n= 2 18
n= 1 19
20
N
OEt
n= 2
16
d
O
n
O
n
EtO
HN
e
N
N
N
H
O
O
n= 1 1
2
n= 1 21
22
n= 2
n= 2
Scheme 4. Synthesis of compounds 1 and 2. Reagents and conditions: a) Rh/Al2O3 5%, CH3COOH, H2, 1 atm, 79% b)
PtO2, EtOH, HCl 6M, H2 1 atm; 74% c) diethyl oxalate, EtONa, EtOH, reflux, N2, 80% for 19 and 70% for 20; d)
aniline, toluene, reflux, 70% for 21 and 53% for 22; e) DMF, 245 °C, MW, 45% for 1 and 60% for 2.
To summarise, the first total synthesis of two novel tetracyclic KYNA derivatives isolated from
chestnut honey was designed and carried out. The crucial step of our strategy was a MW-assisted
cyclization of enamines of ethyl 2,3-dioxooctahydroindolizine and dioxohexahydropyrrolizine
carboxylates. Further synthetic studies and evaluation of biological activity of the obtained
tetracyclic compounds are in progress.
10

A. Supplementary data Supplementary data associated with this article, including experimental
procedures and copies of NMR spectra of the new compounds, can be found, in the online version,
at
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Research highlights
A convenient synthesis of naturally occurring kynurenic acid analogues is described.
Crucial step of the strategy was a MW-assisted cyclization of enamines.
The synthesis is convergent and can have value for the preparation of analogues
13