Partial synthesis of 14-deoxy-14-aminotriptolide

Article abstract of DOI:10.1016/j.tetlet.2016.03.060

Triptolide is a diterpene triepoxide isolated from Tripterygium wilfordii, a Chinese medicinal plant and possessing a wealth of biological activities. In order to produce more soluble derivatives of triptolide, its 14-amino analogue was prepared. Initial attempts at its preparation using classical displacements led to rearranged compounds and success was finally met using carefully controlled Borch conditions. 14-Deoxy-14-aminotriptolide was thus prepared for the first time and it was found 10 times less cytotoxic than the parent compound.

Full text of DOI:10.1016/j.tetlet.2016.03.060

Accepted Manuscript
Partial synthesis of 14-deoxy-14-aminotriptolide
El Bachir Kaloun, Christophe Long, Nicolas Molinier, Viviane Brel, Frédéric
Cantagrel, Georges Massiot
PII:
S0040-4039(16)30282-9
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.03.060
TETL 47448
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 January 2016
17 March 2016
18 March 2016
Please cite this article as: Kaloun, E.B., Long, C., Molinier, N., Brel, V., Cantagrel, F., Massiot, G., Partial synthesis
of 14-deoxy-14-aminotriptolide, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.03.060
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Partial synthesis of 14-deoxy-14-aminotriptolide
El Bachir Kalouna, Christophe Longb, Nicolas Molinierb, Viviane Brela, Frédéric
Cantagrelb and Georges Massiot*1b
a
Centre de Recherche en Oncologie Expérimentale, CRDPF, 3 avenue Hubert Curien, 31035 Toulouse Cedex 01,
France
b USR CNRS-Pierre Fabre No. 3388 ETaC CRDPF, 3 avenue Hubert Curien, 31035 Toulouse Cedex 01, France
Abstract
Triptolide is a diterpene triepoxide isolated from Tripterygium wilfordii, a chinese
medicinal plant and possessing a wealth of biological activities. In order to produce
more soluble derivatives of triptolide, its 14-amino analogue was prepared. Initial
attempts at its preparation using classical displacements led to rearranged compounds
and success was finally met using carefully controlled Borch conditions. 14-Deoxy-
14-aminotriptolide was thus prepared for the first time and it was found 10 times less
cytotoxic than the parent compound.
Keywords
Triptolide, Reductive amination, Aminotriptolide, Cytotoxicity
Triptolide (1) is a fascinating molecule embedding a highly reactive array of
functionalities, i.e. three contiguous oxiran rings and one  unsaturated lactone. It
was isolated in the seventies by Kupchan et al. from the root of Tripterygium wilfordii
Hook, a plant used as a Chinese traditional medicine.1 The compound displays a
broad spectrum of biological activities and shows promises in the treatment of various
disorders such as rheumatoid arthritis, chronic hepatitis and chronic nephritis,
ankylosing spondylitis, polycystic kidney disease and cancer.2 Triptolide is not a very
soluble molecule, it is highly toxic and therefore, derivatives have been prepared to
select candidates for clinical investigations. An excellent review has been published in
2012 on the subject and as far as SAR is concerned, it is concluded that the triepoxide
and the lactone ring are necessary for the activity.3 Worthy of note is the fact that
because of the extreme sensitivity of triptolide, most derivatives have to be prepared
by de novo total synthesis, with installation of the triepoxide in the last stages. In the
natural product, the most accessible function left for chemical modification, is the
alcohol at position C-14 and it has been the object of numerous derivatisations.
Among rare compounds which went into clinical trials, one can mention
Omtriptolide4,5 formed by succinylation and the prodrug Minnelide6,7,8
Numerous efforts have been deployed to transform the alcohol function of
triptolide into an amino group in order to increase water solubility. The most obvious
route implying amine or azide displacement of a sulfonate led to rearranged products,
while reductive amination of triptonide (the ketone derived from triptolide) gave
triptolide back, due to sluggish imine formation. Alternatively, Ritter reaction was
1 Corresponding author. Tel.: +33326913139; fax: +33326913166. E-mail address:
georges.massiot@univ-reims.fr.

found unsuitable due to the acid sensitive character of the triepoxide. This Letter
describes particular conditions, which allowed the preparation of the title compound
(2) through reductive amination, along with preliminary chemical derivatisations and
biological properties of this new molecule.
In a first series of reactions, triptolide mesylate (3) was treated with a variety
of primary amines and in all cases, secondary amines were obtained. As an example,
reaction with N-propylamine gave compound (4), mass and NMR spectra of which
13
showed disappearance of the mesyl group and presence of a N-propyl chain. The C
NMR spectrum presented signals for the three epoxides (C at  65.1, 64.3, 56.3 and
CH at 61.0, 58.5, 58.4) and a CH at  53.6 corresponding to the attachment of the
amino group. The structure was established by identification of the signals for the
amino side chain and by observation of a HMBC correlation between the NCH2 and
the CH at  53.6. This CH corresponded to a proton resonance at  3.20 appearing as
a double doublet with J= 3.6 and 0.9 Hz, the larger coupling of which was shared with
a doublet at  3.67. Clearly, this spectrum did not fit expected structure (4a) and
subsequently isomeric structure (4) appeared as the most reasonable alternative.
Configuration was deduced from coupling constants analysis and further
demonstrated by NOE correlations between H-14 and the isopropyl group and
between H-11 and the NCH2. It is presumed that, due to steric hindrance around the
mesylate, the opening of the epoxide by the amine is faster than the expected SN2 on
C-14. This is in accordance with results published by Liu et al., for the reaction
between benzylamine and triptolide,9 where the addition brings about C-12-C-13
epoxide opening, leading to diol (5) in the absence of a nucleofuge.
An alternative pathway for the formation of the title compound, was the
reductive amination of triptonide (6), easily prepared by Dess-Martin oxidation of
(1).10 Under normal conditions (NaBH4, NH3, MeOH, 60°C), it essentially led to
reduction back to triptolide, due to difficulties in forming the intermediate imine.
However, when the reaction was performed with ammonium acetate as the nitrogen
source and in the presence of molecular sieves, (2) was obtained in a moderate 16%
yield but accompanied with a fair amount of triptolide (35%), which was recycled.11
Compound 2 was identified by its spectral properties and in particular by 2D NMR
experiments, which showed the expected C-14 - H-14 correlations and indicated
that no rearrangement had occurred.12 The lactone ring proved to be untouched

and H-11 and H-12, detected by their coupling, were found attached to oxygen
bearing carbon. H-7 and H-14 had too similar chemical shifts to yield useful NOE
correlations. The configuration of C-14 was based on the fact that, the hydride could
only approach from the back side (exclusive reduction of triptonide into triptolide).
With 14-deoxy-14-aminotriptolide (2) in hand, a series of novel triptolide
analogues were prepared, by using the nucleophilicity of this nitrogen atom. N-formyl
derivative (7) was prepared using ethyl formate in 40% yield and classical conditions
were used to prepare N-mesyl (8), amide derivative (9), urea (10) and thioureas (11
and 12).
To examine whether the substitution affected their biological activities,
we evaluated the anticancer effects of these target compounds against five human
tumour cell lines. The results suggested that the position 14 had to be free from large
substituent. Compounds 8-12 with bulky group lost all the activity. Only N-formyl-
14-aminotriptolide (7) retained significant cytotoxicity, and was found as equipotent
as unsubstituted aminotriptolide (2). However, both were found to be less potent than
the original triptolide (1), which might be useful for certain applications.
A375
A549
HCT116
MCF7
Namalwa
Compound
yield
IC50 µM IC50 µM IC50 µM IC50 µM IC50 µM
0.0050
1.32
0.0052
NT
0.0047
0.772
0.356
>10
0.0017
1.34
NT
0.0100
2.05
16%
40%
24%
0.420
>10
0.354
>10
1.34
NT
>10
37%
62%
>10
>10
>10
>10
>10
>10
NT
NT
>10
>10

68%
6%
>10
>10
>10
>10
>10
>10
NT
NT
>10
>10
In summary, this work presents the preparation of C-14 aza triptolide and
derivatives using a reductive-amination procedure, and also the preparation of some
substituted derivatives. This work also demonstrates that high substitution on this
position C14 yields to the loss of biological activity. Interestingly, in two recent
publications Xu et al. described the preparation of novel derivatives of triptolide,
including a series of compounds in which an heterocycle was placed onto C-14 via an
additional methylene and which retained the full cytotoxicity of the natural
product.13,14
Acknowledgements:
This work was supported by Institut de Recherche Pierre Fabre, whose support is
gratefully acknowledged. The authors thank Drs Christian Bailly, Philippe Schmitt,
Frédéric Marion and Nguyen Thien for encouragements and fruitful discussions.

1 Kupchan, S. M. ; Court, W. A.; Dailey, Jr., R. G.; Gilmore, C. J.; Bryan, R. F. J. Am. Chem. Soc.
1972, 94, 7194-7195.
2 Qiu D.; Kao P. N.. Drugs R&D 2003; 4 (1): 1-18
3 Zhou, Z. L.; Yang, Y. X.; Ding, J.; Li, Y. C.; Miao, Z. H. Nat Prod. Rep. 2012, 29, 457-475.
4 Kitzen, J. J. E. M.; de Jonge, M. J. A.; Lamers, C. H. J.; Eskens, F. A. L. M.; van der Biessen, D.; van
Doorn, L.; ter Steeg, J.; Brandely, M.; Puozzo, C.; Vermeij, J. Eur. J. Cancer 2009, 45, 1764-1772.
5 Hurteloup, P. P.; Brandely-Talbot, M., PCT Int. Appl. (2008) WO 2008087202.
6 Rousalova, I.; Banerjee, S.; Sangwan, V.; 1, Evenson, K.; McCauley, J.A.; Kratzke, R.; Vickers,
S.M.; Saluja, A.K.; D’Cunha, J.; PLoS ONE, 2013, 8(10): e77411.
7 Banerjee, S.; Thayanithy, V.; Sangwan, V.; Mackenzie, T.N.; Saluja, A.K.; Subramanian, S. Cancer
Lett. 2013, 335, 412–420.
8 Chugh, R.; Sangwan, V.; Patil, S.P.; Dudeja, V.; Dawra, R.K.; Banerjee, S.; Schumacher, R.J.;
Blazar, B.R.; Georg, G.I.; Vickers, S.M.; Saluja, A.K. . Sci. Transl. Med.; 2012, 4, 156ra139.
9 Liu, J.; Chen, X.; Zhang, Y.; Miao, H.; Liu, K.; Li, L.; Zhong, D. Anal. Chim. Acta 2011 (689), 69–76.
10 Aoyagi, Y.; Hitotsuyanagi, Y.; Hasuda, T.; Fukaya, H.; Takeya, K.; Aiyama, R.; Matsuzaki, T.;
Hashimoto, S. Bioorg. Med. Chem. Lett. 2006, 16, 1947-1949.
11 Procedure for reductive amination of triptonide: ammonium acetate (4.3 g, 55.8 mmol) and sodium
cyanoborohydride (0.658 g, 10.46 mmol) were dissolved in 6 mL methanol in a sealed tube, at room
temperature and 3.53 g 4Å molecular sieves were added. Triptonide (500 mg, 1.395 mmol) dissolved in
120 mL THF was added under stirring and the reaction mixture was degassed by bubbling with argon
for 15 minutes and sealed. After 3 days of sonication, the suspension was filtered over Celite®, washed
with DCM and concentrated under reduced pressure. The residue was purified by HPLC to give 89 mg
(0.223 mmol, 16 % yield) of (2) as a white amorphous solid.
12 White powder, []20 -118.4 (c 0.17, MeOH); IR (film) max 3406, 3339, 2941, 2901, 1758, 1676,
D
1029, 887, 701cm-1; 1H NMR (CDCl3, 500 MHz)  4.82 (2H, m, H-19), 3.87 (1H, d, J = 3.2 Hz, H-11),
3.65 (1H, s, H-14), 3.62 (1H, d, J = 5.5 Hz, H-7), 3.44 (1H, d, J = 3.1 Hz, H-12), 2.79 (1H, m, H-5),
2.45 (1H, qt, J = 7 Hz, H-15), 2.29-2.22 (2H, m, H-2, H-6), 2.09 (1H, m, H-2), 1.97 (1H, dd, J = 15.0,
13.2 Hz, H-6), 1.50 (1H,m , H-1), 1.35 (1H, m, H-1), 1.09 (3H, s, H-20), 1.06 (3H, d, J = 6.6 Hz, H-
16), 0.76 (3H, d, J = 7.1 Hz, H-17); 13C NMR (MeOH, 125 MHz)  176.3 (C-18), 164.4 (C-4), 125.6
(C-3), 72.1 (C-19), 67.4 (C-13), C-9 (66.8), 63.3 (C-8), 57.3 (C-11), 56.0 (C-7), 53.0 (C-12), 50.9 (C-
14), 41.7 (C-5), 37.3 (C-10), 31.2 (C-1), 28.2 (C-15), 24.6 (C-6), 19.6 (C-16), 18.2 (C-2), 15.9 (C-17),
14.1 (C-20); HRESIMS m/z 360.1793 (calcd for C20H26NO5: 360.1805).
13 Xu, H. T.; Tang, H. Y.; Feng, H. J.; Li, Y. C. Eur. J. Med. Chem. 2014, 73, 46-55.
14 Xu, H. T.; Tang, H. Y.; Feng, H. J.; Li, Y. C. J. Org. Chem. 2014, 79, 10110-10122.

Graphical Abstract
Leave this area blank for abstract info.
Partial synthesis of
14-deoxy-14-aminotriptolide
El Bachir Kaloun, Christophe Long, Nicolas Molinier, Viviane Brel, Frédéric Cantagrel and Georges
Massiot

Highlights
Nucleophilic attack on triptolide mesylate yields epoxide opening and rearrangement
Borch reduction of triptonide yields aminotriptolide
Replacement of OH in triptolide by a primary amine strongly decreases cytotoxicity