F Nicoleau

D Parra
email: parra@math.univ-lyon1.fr

S Richard
email: richard@math.nagoya-u.ac.jp

Does Levinson's theorem count complex eigenvalues ?

Yes it does ! Indeed an extended version of Levinson's theorem is proposed for a system involving complex eigenvalues. The perturbed system corresponds to a realization of the Schrödinger operator with inverse square potential on the half-line, while the Dirichlet Laplacian on the half-line is chosen for the reference system. The resulting relation is an equality between the number of eigenvalues of the perturbed system and the winding number of the scattering system together with additional operators living at 0-energy and at infinite energy.

Introduction

Since its discovery by N. Levinson in 1949, the so-called Levinson's theorem has attracted a lot of interest and many researchers have generalized this relation between spectral and scattering theory. In the simplest situations, it corresponds to an equality between the number of bound states of a quantum mechanical system and an expression based on the scattering part of the system. Very often, this latter expression involves a regularization procedure, and in many cases some corrections must also be taken into account. Since the literature on the subject is very vast, we simply refer to the review papers [START_REF] Ma | The Levinson theorem[END_REF][START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF] and to the references mentioned therein.

Up to our knowledge, all these investigations have taken place in the context of self-adjoint operators in a Hilbert space since this framework is the natural one for quantum mechanics. On the other hand, non selfadjoint operators have recently been deeply studied, and in this context an extension of Levinson's theorem to non real eigenvalues seems a natural question. The purpose of this note is precisely to exhibit such a relation for a system involving non self-adjoint operators with a finite family of complex eigenvalues.

The system we consider consists in the operator

H = -∂ 2 x + m 2 - 1 4 1 x 2
in L 2 (R + ) with a complex parameter m and with a possibly complex boundary condition at x = 0. A large family of such operators have been recently analyzed in [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF] where their spectral and scattering theory have been constructed. For the reference system H 0 we simply consider the Dirichlet Laplacian on R + , but other choice are possible by using the chain rule. By particularizing some expressions obtained in [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF], explicit formulas for the wave operators and for the scattering operators for the pair (H, H 0 ) are directly available. Note that if H is not self-adjoint, the wave operators are not partial isometries and the scattering operator is not a unitary operator. Nevertheless, these operators can be defined and their properties studied.

Our approach for proving a Levinson's type theorem is based on the topological approach firstly introduced [START_REF] Kellendonk | Levinson's theorem for Schrödinger operators with point interaction: a topological approach[END_REF][START_REF] Kellendonk | The topological meaning of Levinson's theorem, half-bound states included[END_REF] and extensively reviewed in [START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF]. In this framework, Levinson's theorem corresponds to an index theorem in scattering theory, and the corrections come from the contribution of newly introduced operators living at threshold energies. For the model presented here, these corrections appear both at 0-energy and at energy corresponding to +∞. Accordingly, the number of eigenvalues of H will be equal to the winding number of the scattering operator together with the contributions of an operator living at 0-energy and of an operator living at ∞-energy. This relation and its explanation correspond to our main result.

This note is organized as follows: In Section 2 we introduce the model and recall some relations obtained in [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF]. The main result is presented in Section 3 which also contains some additional information on the wave operators. Finally, the algebraic framework and the few analytical proofs are provided in Section 4.

The model

The material of this section is borrowed from the reference [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF]. For any m ∈ C we consider the differential expression

L m 2 := -∂ 2 x + m 2 - 1 4 1 x 2 .
The minimal and maximal operators associated with it in

L 2 (R + ) are given by D(L max m 2 ) = {f ∈ L 2 (R + ) | L m 2 f ∈ L 2 (R + )} and D(L min m 2 ) is the closure of the restriction of L m 2 to C ∞ c (R + )
. These realizations satisfy the relation L min

m 2 * = L max m 2 . In addition if |Re(m)| < 1 then L min m 2 L max m 2 and D(L min m 2 ) is a closed subspace of codimension 2 of D(L max m 2 ). More precisely, if |Re(m)| ∈ (0, 1) and if f ∈ D(L max m 2 )
, then there exist a, b ∈ C such that:

f (x) -ax 1/2-m -bx 1/2+m ∈ D(L min m 2 ) around 0.
Here the expression g(x) ∈ D(L min m 2 ) around 0 means that there exists

ζ ∈ C ∞ c [0, ∞) with ζ = 1 around 0 such that gζ ∈ D(L min m 2 )
. Thus, for any κ ∈ C we define the family of operators H m,κ :

D(H m,κ ) = f ∈ D(L max m 2 ) | for some c ∈ C, f (x) -c κx 1/2-m + x 1/2+m ∈ D(L min m 2 ) around 0 .
Note that for simplicity the special case Re(m) = 0 is not considered in the present manuscript. Many properties of these operators have been exhibited in [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF]. For the spectral theory let us simply mention that H m,κ is self-adjoint if and only if m and κ are real. The operators H m,κ have a finite number of eigenvalues which are located in C \[0, ∞), and in addition one has [0, ∞) ⊂ σ(H m,κ ). A limiting absorption principle has been shown for these operators on (0, ∞), with a slight restriction if (m, κ) is an exceptional pair. We say that (m, κ) is an exceptional pair if κ = 0 and ±π ∈ Im 1 m Ln(ς) with

ς := κ Γ(-m) Γ(m) ,
and where Γ is the usual Γ-function and Ln the multivalued logarithm.

Still in the non-exceptional case, an incoming and an outgoing Hankel transformations F ∓ m,κ can be defined on C ∞ c (R + ) by the kernels

F ∓ m,κ (x, y) := e ∓i π 2 m 2 π J m (xy) -ςJ -m (xy) y 2 4 m 1 -ςe ∓iπm y 2 4 m .
with J m (z) := πz 2 J m (z) and J m the usual Bessel function. These operators extend then continuously to elements of B L 2 (R + ) . Now, for any bounded operator B with integral kernel B(x, y) let us set B # for its transpose, i.e. for the operator satisfying B # (x, y) = B(y, x). With this notation the operators

F ± m,κ satisfy F ±# m,κ F ∓ m,κ = 1l. In addition if we set 1l R + (H m,κ ) := F + m,κ F -# m,κ = F - m,κ F +# m,κ
one observes that this operator is a projection. The following equalities have also been proved in [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF]:

For any k ∈ C with Re(k) > 0 and -k 2 ∈ σ p (H m,κ ) one has (H m,κ + k 2 ) -1 1l R + (H m,κ ) = F ± m,κ (L 2 + k 2 ) -1 F ∓# m,κ = 1l R + (H m,κ )(H m,κ + k 2 ) -1
where L is the usual operator of multiplication by the variable in L 2 (R + ). Finally, whenever (m, κ) and (m ′ , κ ′ ) are not exceptional pairs, the wave operators for the pair of operators (H m,κ , H m ′ ,κ ′ ) can be defined by

W ± m,κ;m ′ ,κ ′ := F ± m,κ F ∓# m ′ ,κ ′ .
These operators satisfy the relations

W ∓# m,κ;m ′ ,κ ′ W ± m,κ;m ′ ,κ ′ = 1l R + (H m ′ ,κ ′ ) (1)
and

W ± m,κ;m ′ ,κ ′ W ∓# m,κ;m ′ ,κ ′ = 1l R + (H m,κ ) (2)
as well as the intertwining relation

W ± m,κ;m ′ ,κ ′ H m ′ ,κ ′ = H m,κ W ± m,κ;m ′ ,κ ′ .
The scattering operator is finally defined by

S m,κ;m ′ ,κ ′ := W -# m,κ;m ′ ,κ ′ W - m,κ;m ′ ,κ ′ .

The main result

From now on, let us fix a pair (m, κ) with |Re(m)| ∈ (0, 1) and κ ∈ C which is not exceptional. For the reference system we consider one of the simplest one, namely the Dirichlet Laplacian H D on the half-line. This operator is obtained from the general family for the indices ( 1 2 , 0). Note that by the chain-rule any other pair (m ′ , κ ′ ) can easily be used for the reference system.

In the following statement, we provide an alternative representation of the wave operator which is at the root of the algebraic framework presented in [START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF]. For that purpose, note first that for the special choice ( 1 2 , 0) the transformation .

F +# 1 2 ,0 ≡ F +
We finally consider the unitary group {U t } t∈R acting on any f ∈ L2 (R + ) as

[U t f ](x) = e t/
e i π 4 Ξ 1 2 (A) Ξ m (-A) -ςΞ -m (-A) H D 4 m e -i π 2 m 1 -ςe -iπm H D 4 m . (3)
Motivated by the formula obtained in the previous statement, let us now define the function of two variables: Γ m,κ; 1 2 ,0 :

R + × R by Γ m,κ; 1 2 ,0 (x, t) := e i π 4 Ξ 1 2 (t) Ξ m (-t) -ςΞ -m (-t) x 2 4 m e -i π 2 m 1 -ςe -iπm x 2 4 m .
Note that the condition (m, κ) is not an exceptional pair precisely prevents the denominator in the last factor to vanish. In addition, it is easily observed that this function is continuous on the square := [0, +∞] × [-∞, +∞], and therefore its restriction on the boundary of the square is also well defined and continuous. Note that this boundary is made of four parts:

= B 1 ∪ B 2 ∪ B 3 ∪ B 4 with B 1 = {0} × [-∞, +∞], B 2 = [0, +∞] × {+∞}, B 3 = {+∞} × [-∞,
+∞], and B 4 = [0, +∞] × {-∞}. Thus, the algebra C( ) of continuous functions on can be viewed as a subalgebra of

C [-∞, +∞] ⊕ C [0, +∞] ⊕ C [-∞, +∞] ⊕ C [0, +∞] (4)
given by elements (Γ 1 , Γ 2 , Γ 3 , Γ 4 ) which coincide at the corresponding end points, that is, Γ 1 (+∞) = Γ 2 (0), Γ 2 (+∞) = Γ 3 (+∞), Γ 3 (-∞) = Γ 4 (+∞), and Γ 4 (0) = Γ 1 (-∞). As shown in the following section one gets for κ = 0

Γ 1 (t) := Γ m,κ; 1 2 ,0 (0, t) = e i π 2 ( 1 2 -m) Ξ 1 2 (t)Ξ m (-t) if Re(m) > 0, e i π 2 ( 1 2 +m) Ξ 1 2 (t)Ξ -m (-t) if Re(m) < 0, (5)
Γ 2 (x) := Γ m,κ; 1 2 ,0 (x, +∞) = e iπ( 1 2 -m) 1 -ςe +iπm x 2 4 m 1 -ςe -iπm x 2 4 m , (6)
Γ 3 (t) := Γ m,κ; 1 2 ,0 (+∞, t) = e i π 2 ( 1 2 +m) Ξ 1 2 (t)Ξ -m (-t) if Re(m) > 0, e i π
In the special case κ = 0 one has Γ

1 (t) = Γ 3 (t) = e i π 2 ( 1 2 -m) Ξ 1 2 (t)Ξ m (-t),
Γ 2 (x) = e iπ( 1 2 -m) and Γ 4 (x) = 1. Let us now observe that the boundary of is homeomorphic to the circle S. Observe in addition that the restriction Γ m,κ; 1 2 ,0 of the function Γ m,κ; 1 2 ,0 to takes its values in C \ {0}. Then, since Γ m,κ; 1 2 ,0 is a continuous function on the closed curve and takes non-zero values, its winding number Wind(Γ m,κ; 1 2 ,0 ) is well defined. By convention, we shall turn around clockwise and the increase in the winding number is also counted clockwise. Let us stress that the contribution on B 3 has to be computed from +∞ to -∞, and the contribution on B 4 from +∞ to 0. Our main result now reads:

Theorem 2. If m ∈ C with |Re(m)| ∈ (0, 1) and if (m, κ) is not an excep- tional pair then Wind(Γ m,κ; 1 2 ,0 ) = number of eigenvalues of H m,κ . (9)
The above statement is a topological version of Levinson's theorem, and corresponds to an index theorem. In order to make the link with the usual formulation, it is necessary to consider the 4 contributions Γ j separately. Since Γ 1 (t) = Γ m,κ; 1 2 ,0 (0, t), the corresponding operator Γ 1 (A) can be understood as an operator related to the 0-energy of the scattering process. Similarly, Γ 3 (A) is an operator associated with the energy ∞ of the scattering process. On the other hand, the expression in [START_REF] Kellendonk | Levinson's theorem for Schrödinger operators with point interaction: a topological approach[END_REF]

Note that the special case κ = 0 is less interesting since S m,0; 1 2 ,0 is a constant and both sides of (9) in this case are equal to 0. Remark 4. For general m and κ in the range mentioned above, the computation of the r.h.s. of [START_REF] Rørdam | An introduction to K-theory for C * -algebras[END_REF] is rather involved and can take arbitrary large but finite values. Some useful information are provided in [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF]Prop. 5.3 & Lem. 5,[START_REF] Kato | Perturbation theory for linear operators[END_REF]. Equivalently, the computation of the winding number of the map x → S m,κ; 1 2 ,0 (x) is fairly tricky for complex numbers m and κ. Anyway, we can directly see from Corollary 3 that the computation of this winding number does not provide enough information for deducing the number of bound states of H m,κ , and that the correction +|Re(m)| can take arbitrary values in (0, 1).

The proofs

Before introducing the necessary algebraic framework, let us check the analytical part of our investigations. More precisely let us check some properties of the wave operators and of the function Γ m,κ; 1 2 ,0 . Proof of Lemma 1. We first look at the wave operator in the spectral representation of H D , or more precisely let us consider the operator

F * D W - m,κ; 1 2 ,0 F D = F * D F - m,κ F +# 1 2 ,0 F D = e i π 4 F * D F - m,κ with F * D = F D = Ξ 1 2 (-A)J (11)
and

F - m,κ = J Ξ m (A) -ςΞ -m (A) L 2 4 m e -i π 2 m 1 -ςe -iπm L 2 4 m . (12)
The unitary and self-adjoint transformation J :

L 2 (R + ) → L 2 (R + ) is defined by the formula Jf (x) = 1 x f 1
x for any f ∈ L 2 (R + ) and x ∈ R + Note that equalities (11) and ( 12 For showing that the function Γ m,κ; 1 2 ,0 extends to the square [0, +∞] × [-∞, +∞] let us compute its asymptotics. For the limits we recall from [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF]Eq. 4.25

] that Ξ 1 2 (∓∞)Ξ m ′ (±∞) = e ∓i π 2 ( 1 2 -m ′ ) .
One thus infers that for κ = 0 Γ m,κ; 1 2 ,0 (x, -∞) = e i π 4 e -i π (t)Ξ m (-t). The above expressions are summarized in equations ( 5) to [START_REF] Ma | The Levinson theorem[END_REF]. The case κ = 0 can be obtained similarly, and is simpler.

Let us now discuss the properties of the kernel and of the range of the wave operator W - m,κ; 1 2 ,0 . By taking into account [4, Thm. III. [START_REF] Kellendonk | Levinson's theorem for Schrödinger operators with point interaction: a topological approach[END_REF].17] on the separation of the spectrum into two components, and by using an alternative definition of the projection 1l R + (H m,κ ) in terms of the spectral density integrated on R + , as provided in [START_REF] Derezinski | On Schrödinger operators with inverse square potentials on the half-line[END_REF]Sec. 6.4], one infers that the subspace defined by 1l R + (H m,κ ) is complementary to the subspace generated by the eigenfunctions of the operator H m,κ . Since these eigenvalues are always in finite number the codimension of 1l R + (H m,κ )L 2 (R + ) is always finite. Similarly, since H D has no eigenvalue one gets 1l R + (H D ) = 1l. Then, by the properties (1) and ( 2) one infers that W - m,κ; 1 2 ,0 is a Fredholm operator. Summing up these information one gets :

Lemma 5. Let m ∈ C with |Re(m)| ∈ (0, 1) and assume that (m, κ) is not an exceptional pair. Then W - m,κ; 1 2 ,0 is a Fredholm operator with dim ker W - m,κ; 1 2 ,0 -dim cocker W - m,κ; 1 2 ,0 = -#σ p (H m,κ ).
The content of Theorem 2 can now be deduced either from [START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF]Thm. 4.4] (with n = -1) or from [START_REF] Cordes | Singular integral operators on a half-line[END_REF]Thm. 3]. Let us however make some comments. In [START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF] statements similar to Theorem 2 are provided for various models related to quantum mechanics. The main difference with these models is that here the operator H m,κ is not always self-adjoint. It follows that the wave operator W - m,κ; 1 2 ,0 is not always an isometry but only a Fredholm operator, and accordingly the operator Γ m,κ; 1 2 ,0 is not always a unitary operator but is still an invertible operator. However, for the algebraic construction used for obtaining a topological version of Levinson's theorem, these differences are perfectly manageable and the construction works in this case as well, see for example [START_REF] Rørdam | An introduction to K-theory for C * -algebras[END_REF]Rem. 8.1.7].

The main ingredient for the algebraic construction consists first in exhibiting a natural subalgebra of B L 2 (R + ) which contains the wave operator W - m,κ; 1 2 ,0 . By looking at the special representation obtained for the wave operator in (3) one deduces that W - m,κ; 1 2 ,0 belongs to the C * -algebra E (H D ,A) introduced in [START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF]Sec. 4.4]. This C * -algebra is generated by product of the form ψ(H D )η(A) with ψ ∈ C [0, ∞] and η ∈ C [-∞, ∞] . Our interest in this algebra comes from its easily understandable quotient through the ideal K L 2 (R + ) of compact operators on L 2 (R + ). In fact, this quotient is isomorphic to the C * -algebra C( ) which can be viewed as a subalgebra of the one introduced in (4). Thus, if we set q : E (H D ,A) → E (H D ,A) /K L 2 (R + ) for the quotient map, one gets that q W - m,κ; 1 2 ,0 = Γ m,κ; 1 2 ,0 = (Γ 1 , Γ 2 , Γ 3 , Γ 4 ) with Γ j introduced in ( 5) to [START_REF] Ma | The Levinson theorem[END_REF]. The final argument consists in borrowing the information from [START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF]Thm. 4.4] that the winding number of Γ m,κ; 1 2 ,0 is equal to (minus) the Fredholm index of the operator W - m,κ; 1 2 ,0 . By using Lemma 5 this leads directly to Theorem 2.

Alternatively, one can directly use the content of Theorem 3 of [START_REF] Cordes | Singular integral operators on a half-line[END_REF] once it is observed that the algebra presented in that paper corresponds to the algebra E (L,A) introduced in [START_REF] Richard | Levinson's theorem: an index theorem in scattering theory[END_REF]Sec. 4.4]. This algebra is isomorphic to the C * -algebra E (H D ,A) by a conjugation with the unitary map F D . The difference of a minus sign between the content of [2, Thm. 3] and Theorem 2 comes from the equality F * D AF D = -A which reverses part of the construction. Finally, let us also mention Theorem 4 in [START_REF] Breuer | On Banach algebras with σ-symbol[END_REF] which provides a similar abstract result but in a larger setting. Remark 6. A more analytical approach involving Jost function could also be used for proving Corollary 3. However, the flavor of an index theorem would be lost, and the contributions of the operators at 0-energy and at energy +∞ would not appear so explicitly.

1 2 2 )Γ( m+1-it 2 )

 122 ,0 corresponds to e i π 4 F D with F D the usual Fourier 4 sine transformation on R + . Let us also define for any t ∈ R the function Ξ m given by Ξ m (t) := e i ln(2)t Γ( m+1+it

2 - 1 4 2 + 1 4Corollary 3 .

 21213 corresponds to the scattering operator, or more precisely one has Γ 2 ( √ H D ) = S m,κ; 1 2 ,0 ≡ S m,κ; 1 2 ,0 (H D ), where S m,κ; 1 2 ,0 is the scattering operator for the pair (H m,κ , H D ), see [3, Sec. 6.5]. Now, by a slight adaptation of the proof of [5, Lem. 4] the contribution to the winding number coming from Γ 1 and Γ 3 are respectively equal to Re(m) and Re(m) when Re(m) > 0. Similarly, for Re(m) < 0 these two contributions are respectively equal to -Re(m) If m ∈ C with |Re(m)| ∈ (0, 1), if κ = 0 and if (m, κ) is not an exceptional pair one has Wind S m,κ; 1 2 ,0 (•) + |Re(m)| = number of eigenvalues of H m,κ .

  ) have been obtained in [3, Prop. 4.5 & Lem. 6.3]. By taking into account the relations F * D AF D = -A and F * D L 2 F D = H D one directly deduces formula (3).

  2 f e t x , ∀x ∈ R + which is usually called the unitary group of dilations. Its self-adjoint generator is denoted by A and is called the generator of dilations.

	Lemma 1. If m ∈ C with |Re(m)| ∈ (0, 1) and if (m, κ) is not an exceptional pair then the operator W -m,κ; 1 2 ,0 is equal to

( 1 2 -m) Ξ 1 2 (t)Ξ m (-t) if Re(m) < 0,(7)Γ 4 (x) := Γ m,κ; 1 2 ,0 (x, -∞) = 1.(8)

† Supported by JSPS Grant-in-Aid for Young Scientists A no 26707005.


Florent Olivon

Héliciane Palenzuela

Emmanuelle Girard-Valenciennes

Johan Neyts

Christophe Pannecouque

Fanny Roussi

Isabelle Grondin

Pieter Leyssen

Marc Litaudon

Heĺiciane Palenzuela

Antiviral activity of flexibilane and tigliane diterpenoids from Stillingia lineata

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

C hikungunya virus (CHIKV) is an alphavirus of the family Togaviridae that causes an acute illness associated with severe morbidity (virus-induced arthralgia, fever, myalgia, and rash). It is transmitted by virus-carrying Aedes mosquitoes (in particular, A. aegypti and A. albopictus). A recent resurgence of this disease and large-scale epidemics in Africa, the Indian subcontinent, Southeast Asia, and lately also South and Central America are probably linked to climate change, allowing mosquito vectors to thrive at latitudes and locations that they previously could not reach. 1,[START_REF] Hunter | Encyclopedia of Environmental Health[END_REF] Currently, no specific antiviral therapy is available, and treatment is focused on relief of symptoms. [START_REF] Caglioti | [END_REF][START_REF] Rezza | In Emerging Infectious Diseases[END_REF] In an effort to identify novel inhibitors of CHIKV replication, several species were studied earlier of the family Euphorbiaceae, namely, Trigonostemon cherrieri, [START_REF] Allard | [END_REF][6][7] T. howii, 8 Croton mauritianus, 9 and Euphorbia amygdaloides. 10 This work has led to the isolation of several daphnane-, jatrophane-, and tigliane-type diterpenoids, and 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to be the most potent and selective inhibitor of CHIKV replication, with an EC 50 of ∼3 nM and an SI near 2000. 8 Owing to the wide range of their biological activities and their structural diversity, diterpenes of the Euphorbiaceae are key natural products for the discovery of promising molecules in antiviral or anticancer therapies. [11][12][13][14] As an example, phorbol esters, which are protein kinase C modulators, 15 are well known to exhibit interesting antiviral properties, inter alia, against human immunodeficiency virus. [16][17][18] Recently, evaluation of the anti-CHIKV potential of 171 species from Reúnion Island using a virus-cell-based assay led to the selection of, among others, Stillingia lineata (Lam.) Mull. Arg. for further chemical and biological studies. [START_REF] Leyssen | Novel Plant Bioresources[END_REF] The genus Stillingia (Euphorbiaceae) is represented by 30 species distributed throughout Latin and North America and various islands of the Pacific and Indian Oceans. The leaf and latex extracts of some species have been reported to possess cytotoxic, [START_REF] Chapuis | [END_REF] proteolytic, 21 and antifungal 22 properties. The roots of Stillingia sylvatica are used for preparing a therapeutic composition to treat various ailments such as syphilis, bronchitis, constipation, hemorroids, diseases of the skin, and cancer. 23 Only three Stillingia species have been investigated chemically so far. 22,24,25 From the roots of S. sylvatica, Adolf and Hecker reported the isolation of diterpene esters of the daphnane and tigliane types. 24 More recently, Drager and colleagues reported the isolation of two novel diterpenes, tonantzitlolones A and B, sharing the rare flexibilane backbone, from S. sanguinolenta. 25 These compounds showed interesting cytotoxic activities against human kidney and breast cancer cell lines. 26 Tonantzitlolone A was also isolated from the aerial parts of Sebastania macrocarpa, another Euphorbiaceae species collected in Brazil. 27 In this paper are reported the LC-MS-guided isolation, structure elucidation, and anti-CHIKV properties of three known (1-3) and six new tonantzitlolones (4-9), tonantzitloic acid (10), and three new (11, 13, and 15) and two known tigliane diterpenes (12 and 14) isolated from the ethyl acetate extract of S. lineata leaves. The panel of viruses against which the antiviral activity of this compound class is studied was expanded with two additional members of the genus alphavirus (Sindbis virus: SINV; Semliki Forest virus: SFV), and for compounds 11-15 two members of the genus lentivirus (HIV-1 and HIV-2 viruses).

RESULTS AND DISCUSSION

The leaves and bark of S. lineata were collected in November 2009 in the district of Langevin (Reúnion Island). The EtOAc extracts obtained from the leaves and the bark of this species exhibited potent anti-CHIKV activities, with EC 50 values of 0.25 and 0.80 μg/mL, respectively. The chemical study of the EtOAc bark extract afforded three tonantzitlolones (1-3), of which 4′-acetoxytonantzitlolone (2) exhibited moderate activity on CHIKV replication. [START_REF] Techer | Criblage d'activiteś biologiques de plantes endeḿiques ou indigeǹes de La Reúnion -Recherche de molećules antivirales ciblant le virus du chikungunya[END_REF] With the aim of discovering additional members of this chemical series in this leaf EtOAc extract, a preliminary LC-HRESIMS study in the positive-ion mode was performed on the previously isolated tonantzitlolones A and B (1 and 2). The pseudomolecular ion peaks [M + Na] + and [M + H -H 2 O] + together with a fragment ion at m/z 333.2066 (C 20 H 29 O 4 ) were observed for both compounds. The latter corresponds to the loss of the side chain according to the fragmentation hypothesis depicted in Figure 1. The detection of this typical fragment ion is particularly interesting since it can be related to compounds that share the same C 20 -flexibilane macrocycle and can represent a qualitative marker for the presence of such compounds in extracts of S. lineata leaves.

The EtOAc leaf extract of S. lineata was defatted by a liquid/ liquid partition using acetonitrile and n-heptane and then subjected to normal-phase flash chromatography. All fractions were submitted to the previously mentioned LC-HRESIMS analysis. These experiments were shown to be particularly efficient and gave reliable ionizations, and all compounds showing an ion at m/z 333.2066 in their mass spectrum were isolated systematically. However, compound 6, with a nicotinate moiety, and compound 9 showed no such fragmentation. In parallel, the anti-CHIKV activity of all fractions was assessed, leading to the isolation of compounds 10-15. The 1 H and 13 C NMR data of compounds 4 to 10 and of 11, 13, and 15 are reported in Tables 1 and2, respectively. Tonantzitlolones A-C (1-3) were identified through comparison with NMR data reported in the literature, 25,[START_REF] Techer | Criblage d'activiteś biologiques de plantes endeḿiques ou indigeǹes de La Reúnion -Recherche de molećules antivirales ciblant le virus du chikungunya[END_REF] and their absolute configurations were assigned by ROESY correlations and comparison of specific rotation values with those reported in the literature. The R configuration of the stereogenic center C-4′ of compound 2 was determined by comparison of the NMR data given by Busch et al., 25 but the configuration of C-4′ of compound 3, postulated as R, remains uncertain.

Compound 4 showed a pseudomolecular ion at m/z 473.2523 [M + Na] + (calcd 473.2515) in the HRESIMS, corresponding to the molecular formula C 25 H 38 O 7 . From this formula and its spectroscopic data, closely comparable to those of 1, it was apparent that 4 has a similar structure but an aliphatic side chain that possesses two methyl groups instead of one methyl group and one ethyl group as for compound 1. Besides the characteristic macrocyclic moiety, the analysis of the 1 H NMR spectrum indicated the loss of the proton signal at 2.15 ppm (CH 2 -4′, q in 1). Instead, a methyl group at δ H 1.86 (CH 3 -4′, s), which correlated with C-2′, C-3′, and C-5′ (δ C 115.6, 158.6, and 20.6, respectively) in the HMBC spectrum, suggested a senecioate moiety, linked to the macrocycle at C-8 by observation of a correlation from H-8 to the C-1′ carbonyl (Figure 2). The relative configuration of 4 was defined by the inspection of the ROESY spectrum (Figure 3). Correlations between H-5/H-6β/H-7/H-8/H-10/H-18/H-12β/H-13β/H-14 suggested the β-orientation of these protons. A specific rotation value (+120, c 0.1, CHCl 3 ) of the same sign and magnitude as was obtained for compound 1 suggested that the absolute configuration should be the same as for tonantzitlolone A. Compound 4 was given the trivial name tonantzitlolone D.

Compound 5 (tonantzitlolone E) was assigned a molecular formula of C 25 H 38 O 8 on the basis of its 13 C NMR and HRESIMS data, suggesting the presence of an additional hydroxy group when compared with compound 4. In the HMBC spectrum, correlations from H 2 -4′ (δ H 4.11) to C-2′, C-3′, and C-5′ suggested the presence of a hydroxymethylene group at C-3′ to afford the 4′-hydroxysenecioate unit, linked to the macrocycle at C-8 by observation of a correlation from H-8 to carbonyl C-1′. Analysis of the ROESY spectrum and comparison with data reported indicated the same relative and absolute configurations to compound 4.

Compound [START_REF] Xu | [END_REF] Due to a preferential ionization of nonbonding electrons of the nitrogen atom of the nicotinate moiety, the in-source fragmentation of 6 did not lead to the cleavage of the side chain, as for the other members of this chemical series. The relative and absolute configurations of 6, similar to compound 4, were determined by inspection of ROESY correlations and through analysis of specific rotation values.

The HRESIMS of compound 7 (tonantzitlolone G) showed a pseudomolecular ion [M + Na] + at m/z 487.2664 (calcd 487.2672), corresponding to the molecular formula C 26 H 40 O 7 , the same as for compound 1. However, different chemical shifts were observed in the 1 H and 13 C NMR spectra of compounds 1 and 7. Both compounds were found to possess the same methyl senecioate unit, but a different macrocycle could be deduced from the analysis of the 1D-and 2D-NMR spectra of 7. Indeed, the loss of the 13 C NMR acetal resonance (δ C 97.2 in 4-6), which was replaced by a carbonyl at C-9 (δ 208.6) that correlated in the HMBC spectrum with H-8 (δ H 5.38) and H-10 (δ H 4.35), and the presence of a hydroxy group at C-5 (δ H/C 4.37/75.9) were the most obvious modifications observed for compound 7. These data suggested the opening of the lactol ring, leading to a slightly different C-15 macrocyclic moiety. Moreover, analysis of the NMR spectroscopic data of 7 (the key COSY and HMBC correlations are presented in Figure 2) indicated that 7 possesses the same C 20 -flexibilane macrocycle as tonantzitlolones 1-6. However, despite the presence of a strong ROESY correlation between H-5 (δ H 4.37) and H-3 (δ H 3.32), the relative configuration of the C-5 stereocenter remained ambiguous due to the flexibility of the macrocycle. The relative configurations of the other stereogenic centers were deduced from analysis of the ROESY spectrum (Figure 3). Compound 7 was named tonantzitlolone G.

Compound 8 (tonantztlolone H) showed in the HRESIMS a pseudomolecular [M + Na] + ion at m/z 545.2724 (calcd for C 28 H 42 O 9 Na, 545.2727), corresponding to the molecular formula C 28 H 42 O 9 . The molecular formulas of 2 and 8 are identical, and their 1 H and 13 C NMR data indicated that these compounds share the same 4′-acetoxymethylsenecioate side chain, but a different macrocycle. The NMR data related to the From this formula and its NMR spectroscopic data, which were comparable to those of 7, it was apparent that 9 has a structure similar to 7, as characterized by the presence of a methyl senecioate moiety and a hydroxy ketone macrocycle, but with an additional hydroxy group at C-1 and the shift of the Δ 1,2 double bond to positions C-2 and C-3. The shift of the double bond was supported by HMBC correlations between the methyl protons H 3 -16 (δ H 1.87) and the olefinic carbons C-2 and C-3 (δ C 144.5 and 133.0, respectively) and carbonyl C-4 (δ C 203.3). The E configuration of the double bond was confirmed by the absence of any ROESY correlation between H-2 and H-16. The chemical shifts of H/C-1 (δ H 4.17 and δ C 76.4) together with a COSY correlation between H-1 and H-2 indicated the presence of a hydroxy group at C-1. The relative configuration of 9 was deduced by analysis of the ROESY spectrum, but, as for compounds 7 and 8, the orientation of H-5 remains uncertain due to the flexibility of the macrocycle. Key 2D-NMR correlations observed for compound 9 are shown in Figures 2 and3.

It should be noted that compounds 7-9 were isolated in trace quantities, whereas the corresponding cyclic hemiacetals, tonantzitlolones A and B (1 and 2), were isolated in large amounts. The six-membered cyclic hemiacetals are generally favored due to conformational effects and a fast hemiacetalforming reaction. However, LC/MS and 1 H NMR analysis of compounds 1 and 2 stored at 4 °C in DMSO for three months showed the presence of an equilibrium mixture of cyclic hemiacetal and hydroxy ketone forms, in approximately 75:25 and 45:55 ratio for 7/1 and 8/2 mixtures, respectively.

The HRESIMS of compound 10 showed a pseudomolecular ion peak [M + Na] + at m/z 519.2557 (calcd 519.2570), which, in conjunction with the 13 C NMR spectroscopic data, was consistent with a molecular formula, C 26 H 40 O 9 , indicating seven indices of hydrogen deficiency. An initial analysis of the 1D-and 2D-NMR spectroscopic data of compound 10 indicated its planar structure to be substantially different from previous tonantzitlolones. To facilitate the structural determination of compound 10, the carbon numbering was kept unchanged. Its 13 C NMR spectrum, when compared with compound 7, revealed the presence of resonances for two carboxylic acids at δ 180.9 and 179.4 (C-4 and C-5, respectively) instead of a ketone and an oxymethine in 7, suggesting the opening of the planar macrocycle ring at C-4/C-5. Four spin systems, as depicted in Figure 2, were highlighted through analysis of the COSY spectrum, with one of these suggesting the presence of a methyl senecioate unit, similar to 7. Most of the correlations observed in the HMBC spectrum were comparable to those of 7 (Figure 2), suggesting that 10 has the same carbon chain as compound 7, including a tetrahydrofuran ring attached at C-11 and C-14, but with the two carboxylic acids at both ends of the carbon chain. The locations of the C-4 and C-5 carbonyl groups were determined from the HMBC correlations between C-4 (δ 180.9) and H-16 (δ 1.24) and H-3 (δ H 3.13) and between C-5 (δ 179.4) and H-6a (δ 2.20) and H-6b (δ 2.36). The cross-peak between H-8 and carbonyl C-1′ confirmed the link of the methyl senecioate unit at C-8. The trans configuration of the Δ 1,2 double bond was confirmed by the large coupling constant value between H-1 and H-2 (15.9 Hz). The relative configuration of carbons C-10, C-11, and C-14 was deduced from analysis of the ROESY spectrum of compound 10, but the configuration of the other carbons remained unknown due to the high molecular flexibility. Compound 10 was named tonantzitloic acid.

Compounds 11-15 were isolated from fractions F11, F12, and F14. They did not share the same MS/MS fragmentation pattern shown for the tonantzitlolone series, but instead, various losses of neutral fragments could be observed in the LC/MS experiments. The analysis of their NMR spectroscopic data indicated that these compounds possess a phorbol, a 4deoxyphorbol, or a 12-deoxyphorbol moiety. By comparison with NMR data reported in the literature, compounds 12 and 14 were identified as 12-O-acetylphorbol-13(2″-methyl)butyrate (12) 30 and 12-deoxyphorbol-13(2″-methyl)butyrate ( 14), 31 The NMR spectra of 11 showed typical signals for a 4αdeoxyphorbol moiety, as apparent from the characteristic chemical shifts of protons H-1 (δ 7.01, s), H-4 (δ 2.77, m), H-5α (δ 2.46 dd, 15.5, 5.0 Hz), H-5β (δ 3.43 d, 15.5 Hz), and H-7 (δ 5.09, brs). 32 The 13 C NMR signals for two carbonyls at δ 170.7 and 178.8 (C-1′ and C-1″, respectively) indicated the 4α-deoxyphorbol moiety to be substituted by two ester side chains. In the HMBC spectrum, a correlation between H-12 (δ 5.41) and the ester carbonyl signal C-1′ confirmed the acylation at C-12 by an acetate group. The values of the chemical shifts of the allylic protons H 2 -20 (δ 3.88 and 4.00) indicated the presence of a hydroxy group at C-20, 31 and the HMBC correlations between the proton resonance at δ 5.27 (OH-9, exchangeable with D 2 O) and C-1, C-9, and C-10 (δ 156.3, 78.2, and 47.6, respectively) indicated that a second hydroxy group is attached at C-9. Therefore, a second carbon chain could be connected to C-13 (δ 65.0) through the ester carbonyl C-1″ (δ 178.8). Finally, the HMBC correlation between H-2″ (δ 2.32) and C-1″ together with 1 H-1 H COSY correlations that linked H-2″, H-3″ α and β, H-4″, and H-5″ suggested the presence of a 2″-methylbutyrate unit. Analysis of the ROESY spectrum of 11 confirmed the relative configuration of 11 as similar to a known 4α-deoxyphorbol derivative. 31 Thus, compound 11 was established as 12-O-acetyl-4α-deoxyphorbol-13(2″-methyl)butyrate. The HRESIMS of compound 13 showed a pseudomolecular ion peak [M + Na] + at m/z 589.3115 (calcd 589.3141), which, in conjunction with the 13 C NMR data, was consistent with the molecular formula C 34 H 46 O 7 . Compound 13 provided spectroscopic data quite similar to those of 11. However, substantial modifications of the C-12 and C-13 acyl chains were evident. The 2″-methylbutyrate group in 11 was found to be replaced by a butyrate unit, with the acetyl group replaced by a nine-membered aliphatic side chain including six conjugated olefinic protons at δ 5.60, 6.63, 7.41, 6.48, 6.21, and 5.94 (C-2′ to C-7′) linked to C-12 through the carbonyl C-1′ (δ 166.3). The cis/trans/trans configuration of the double bonds was determined by analysis of the coupling constants of the protons of the C-2′/C-7′ spin system (see values in Table 2) together with correlations between H-2″/H-3″, H-3″/H-5″, H-4″/H-6″, and H-5″/H-7″ in the ROESY spectrum. The structure of 13 was established as 12β-O-[nona-2Z,4E,6E-trienoyl]-4α-deoxyphorbol-13-butyrate.

Compound 15 showed a pseudomolecular ion at m/z 617.3472 [M + Na] + (calcd for 617.3454) in the HRESIMS, corresponding to the molecular formula C 36 H 50 O 7 . Most of the NMR spectroscopic data were similar to those of 14, indicating that compound 15 possesses the same 4β-phorbol moiety but with a different acyl chain attached at C-13. The 13 C NMR spectroscopic data showed carbon resonances corresponding to six conjugated olefinic carbons at δ 124.0 (C-2′), 144.4 (C-3′), 137.4 (C-4′), 138.8 (C-5′), 140.2 (C-6′), and 132.9 (C-7′), a carbonyl at δ 200.8 (C-8′), an ester carbonyl at δ 168.6, and eight aliphatic carbons (one CH 3 and seven CH 2 ), forming a 16-membered aliphatic side chain. Analysis of the 1 H-1 H COSY and HMBC spectra supported the location of the three double bonds at C-2′, C-4′, and C-6′ and the ketone at C-8′. The all-trans configuration of the double bonds was determined by analysis of the coupling constants of the protons of the C-2′/C-7′ spin system (values in Table 2) together with correlations between H-2″/H-4″, H-4″/H-6″, H-3″/H-5″, and H-5″/H-7″ in the ROESY spectrum. The structure of 15 was therefore established as 12-deoxyphorbol-13-[8′-oxohexadeca-2E,4E,6E-trienoate].

The antiviral evaluation of compounds 1-15 was performed in virus-cell-based assays for three members of the genus alphavirus (CHIKV, SINV, and SFV), and the results are indicated in Table 3.

Phorbol esters 12 and 14 were found to be the most potent and selective inhibitors of CHIKV replication, as apparent from their lower EC 50 values and higher selectivity indices (EC 50 = 3.3 ± 0.3 μM, SI = 41 and EC 50 = 1.2 ± 0.2 μM, SI > 240, respectively), whereas tonantzitlolones B, C, and F (2, 3, and

6)

showed only moderate inhibitory activity on CHIKV replication. No compound exhibited significant antiviral activity against the SINV and SFV viruses. From the results obtained, regarding the 4-deoxyphorbols 11 and 13 and the phorbol 12, it was observed that the presence of an OH-4β group appears to be important for potent anti-CHIKV activity (12 vs 11), whereas the presence of an ester group at C-12 with a C 9 -chain fatty acid increased the antimetabolic effect on Vero cells (13 vs 11). In addition, regarding the 12-deoxyphorbols 14 and 15, the presence of a long C 16 -chain fatty acid promoted a strong antimetabolic effect. These results confirmed previous observations made, showing that the 12-deoxyphorbol prostratin possesses a favorable anti-CHIKV profile. 8 Since several phorbol esters have been reported to exhibit strong anti-HIV-1 activities, 17,18,33 compounds 11-15 were investigated for selective antiviral activity against HIV type-1 (III B ) and type-2 (ROD) lentiviruses. The results of this study are reported in Table 4. Compounds 12-15 showed a strong and selective antiviral effect on HIV-1 and HIV-2 virus replication, with EC 50 values in the submicromolar range. These results are comparable to those obtained in the CHIKV assays and confirmed that a long-chain ester moiety located at the C-12 or C-13 position on the phorbol diterpene core increased the antimetabolic effect. Comparison of the results obtained for the 12-deoxyphorbols 14 and 15 (which were comparable to the data of Huang et al. 33 ) indicated that compound 14, having a shorter side chain at C-13, showed higher selectivity indices for HIV-1 as well as for the HIV-2 virus due to a weaker antimetabolic effect. To better understand the structure-activity relationships within the phorbol series, other compounds of phorbol-or 12-deoxyphorbol-type should be isolated and evaluated biologically in these assays.

In a previous study performed by Busch and colleagues, 26 tonantzitlolones A (1) and B (2) were reported to show activity and selectivity against human kidney and breast cancer cell lines, but no EC 50 values were provided. Tonantzitlolones 1-9 and tonantzitloic acid 10 were therefore subjected to cytotoxic assays against the HCT-116 (human colon carcinoma), MCF-7 (human breast adenocarcinoma), and PC3 (human prostate) cancer cell lines. At 1 μM, none of the compounds had an inhibitory activity over 40% on the three cancer cell lines, whereas at 10 μM compounds 2-6 showed inhibition activities of 49, 50, 47, 61, and 68% for HCT-116 cells and 35, 41, 20, 49, and 40% for PC3 cells, respectively.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured at 25 °C on an Anton Paar MCP 300 polarimeter. UV spectra were recorded on a Varian Cary 100 UV-vis spectrophotometer. NMR spectra were recorded in CDCl 3 on a Bruker 600 MHz instrument (Avance 600) using a 1.7 mm microprobe for compounds 4, 5, 7 to 10, and 12 to 15, on a Bruker 500 MHz instrument (Avance 500) for plant extracts, fractions, and compounds 6 and 11, and on a Bruker 300 MHz instrument (Avance 300) for compounds 1 to 3. Chemical shifts (relative to TMS) are in ppm, and coupling constants are in Hz. SunFire analytical and preparative C 18 columns (250 × 4.6 mm and 250 × 19 mm i.d.; 10 μm Waters) and a Nucleodur PFP analytical column (250 × 4,6 mm i.d.; 5 μm Macherey-Nagel) were used for HPLC separations using a Dionex system equipped with a sample manager (Gilson 215 liquid handler), a column fluidics organizer, a binary pump (Dionex HPG-3200BX), a UV-vis diode array detector (190-600 nm, Dionex UVD340U), and a PL-ELS 1000 ELSD Polymer Laboratory detector. All solvents were purchased from Carlo Erba (France), and analytical plates (Si gel 60 F254) were from Merck (France). Prepacked GraceResolv silica cartridges were used for flash chromatography using a Teledyne Isco Combiflash Rf 200i. HRESIMS data were acquired using an Acquity Waters UPLC coupled to a Waters LCT Premier XE mass spectrometer. The UPLC system was equipped with a Waters Acquity PDA dectector. Separation was achieved on a BEHC 18 column (1.7 μm, 2.1 mm × 50 mm) at a flow rate of 0.6 mL/min. Elution was conducted with a H 2 O-CH 3 CN + 0.1% formic acid gradient as follows: 95:5 to 0:100 in 5.5 min. The ionization was carried out using an electrospray ionization source in the positive mode (range 80-1500 m/z).

Plant Material. Leaves of Stillingia lineata were collected in November 2009 in Langevin (La Reúnion, France) and authenticated by Prof. Dominique Strasberg (University of La Reúnion). A voucher specimen (RUN-100) has been deposited at the LCSNSA of the University of La Reúnion.

Extraction and Isolation. The dried leaves (224.2 g) of S. lineata were extracted with EtOAc (3 × 500 mL) to yield a crude extract (5.7 g) after concentration in vacuo at 40 °C. This extract was dissolved in 250 mL of acetonitrile and subjected to a liquid/liquid partition with n-heptane (3 × 250 mL) to afford 1.9 g of a MeCN-soluble fraction. This residue was subjected to silica gel column chromatography using a gradient of n-heptane-EtOAc-MeOH of increasing polarities (1:0:0 to 0:1:0 to 0:8:2, 50 mL/min) to afford 20 fractions, F1-F20, according to their TLC profiles. F1 (60.1 mg) was purified by preparative HPLC (SunFire C 18 , MeCN-H 2 O 90:10 + 0.1% formic acid at 20 mL/min) to afford compounds 1 (26.3 mg) and 4 (1.3 mg) (t R 8.0 and 6.5 min, respectively). Fraction F3 (96.2 mg) was subjected to preparative HPLC (SunFire C 18 , MeCN-H 2 O (80:20) + 0.1% formic acid at 20 mL/min) to yield compound 2 (29.4 mg) (t R 14.0 min). Purification of fraction F10 (70 mg) by preparative HPLC (SunFire C 18 , MeCN-H 2 O + 0.1% formic acid, 65:35 to 100:0, in 20 min, 20 mL/min) afforded a mixture of F10-1 (2.7 mg) and compound 3 (6.6 mg) (t R 7.0 and 13.5 min, respectively). F10-1 (2.7 mg) was separated by analytical HPLC (Nucleodur PFP, MeCN-H 2 O (50:50) + 0.1% formic acid at 1 mL/min), leading to compound 8 (0.9 mg) (t R 13.0 min). F11 (79.8 mg) was purified by preparative HPLC (SunFire C 18 , MeCN-H 2 O + 0.1% formic acid, 45:55 to 100:0, at 20 mL/min) to afford a mixture of F11-1 to -3 and compounds 5 (1.0 mg), 3 (1.8 mg), and 6 (2.9 mg) (t R 14.5, 16.0, 27.0, 21.5, 22.5, and 23.5 min, respectively). F11-1 (0.8 mg) was separated by analytical HPLC (Nucleodur PFP, MeCN-H 2 O (45:55) + 0.1% formic acid at 1 mL/min), leading to compound 9 (0.2 mg) (t R 14.5 min). F11-2 (3 mg) was separated by analytical HPLC (Nucleodur PFP, MeCN-H 2 O (55:45) + 0.1% formic acid at 1 mL/min), affording compound 10 (0.7 mg) (t R 8.0 min). Purification of fraction F11-3 (2.3 mg) by analytical HPLC (Nucleodur PFP, MeCN-H 2 O (65:35) + 0.1% formic acid at 1 mL/min) furnished compound 13 (0.5 mg) (t R 12.0 min). Fraction F12 (114.3 mg) was subjected to preparative HPLC (SunFire C 18 , MeCN-H 2 O + 0.1% formic acid, 50:50 to 100:0, in 20 min at 20 mL/min) to yield compounds 11 (1.3 mg) and 6 (1.2 mg) (t R 12.5 and 21.0 min, respectively). Purification of fraction F6 (21.7 mg) by preparative HPLC (SunFire C 18 , MeCN-H 2 O (60:40) + 0.1% formic acid, 20 mL/min) afforded compound 7 (0.7 mg) (t R 16.0 min). F14 (80 mg) was purified by preparative HPLC (SunFire C 18 , MeCN-H 2 O + 0.1% formic acid, 50:50 to 100:0, in 25 min at 20 mL/ min) to afford a mixture of F14-1 (1.5 mg) and compound 15 (0.4 mg) (t R 10.0 and 22.5 min, respectively). F14-1 (1.5 mg) was further separated by analytical HPLC (Sunfire C 18 , MeCN-MeOH (1:1)-H 2 O (75:25) at 1 mL/min), leading to compounds 14 (0.4 mg) and 12 (0.3 mg) (t R 7.5 and 10.0 min, respectively).

Tonantzitlolone D (4): colorless oil; [α] 24 D +120 (c 0.1, CHCl 3 ); UV (CH 2 Cl 2 ) λ max (log ε) 230 (3.90) nm; 1 H and 13 Virus-Cell-Based Anti-alphavirus Assay. The antiviral experiments have been performed in a biosafety screening facility that has been validated for handling chikungunya virus as well as the manipulation of molecules of unknown chemical safety risk. All studies have been performed by trained staff. Throughout the experiments, Vero (African green monkey kidney) cells were used. The following viruses were used: Chikungunya virus strain 899, Sindbis virus strain HRsp, and Semliki Forest virus strain Vietnam. Serial dilutions of the initial plant extracts, fractions, or compounds, as well as the reference compound, chloroquine, were prepared in assay medium [MEM Reg3 (cat. no. 19993013; Invitrogen), 2% FCS (Integro), 5 mL of 200 mM L-glutamine, and 5 mL of 7.5% sodium bicarbonate] and were added to empty wells of a 96-well microtiter plate (Falcon, BD). Subsequently, 50 μL of a 4× virus dilution in assay medium was added, followed by 50 μL of a cell suspension. This suspension, with a cell density of 25 000 cells/50 μL, was prepared from a Vero cell line subcultured in cell growth medium (MEM Reg3 supplemented with 10% FCS, 5 mL of L-glutamine, and 5 mL of sodium bicarbonate) at a ratio of 1:4 and grown for 7 days in 150 cm 2 tissue culture flasks (Techno Plastic Products). The assay plates were returned to the incubator for 6 or 7 days (37 °C, 5% CO 2 , 95-99% relative humidity), a time at which maximal virus-induced cell death or cytopathic effect was observed in untreated, infected controls.

Subsequently, the assay medium was aspirated, replaced with 75 μL of a 5% MTS (Promega) solution in phenol red-free medium, and incubated for 1.5 h. Absorbance was measured at a wavelength of 498 nm (Safire2, Tecan); optical densities (OD values) reached 0.6-0.8 for the untreated, uninfected controls. Raw data were converted to percentages of controls, and the EC 50 (50% effective concentration, or concentration that is calculated to inhibit virus-induced cell death by 50%) and CC 50 (50% antimetabolic concentration, or concentration that is calculated to inhibit the overall cell metabolism by 50%) values were derived from the dose-response curves. All assay conditions producing an antiviral effect that exceeded 50% were checked microscopically for minor signs of a cytopathic effect or adverse effects on the host cell (i.e., altered cell morphology). A compound was considered to elicit a selective antiviral effect on virus replication only when, following microscopic quality control, at least at one concentration of compound no cytopathic or any adverse effect is observed (image resembling untreated, uninfected cells). Multiple, independent experiments were performed.

Virus Cell-Based Anti-HIV Assay. Evaluation of the antiviral activity of the test compounds against HIV-1 strain (III B ) and HIV-2 strain (ROD) in MT-4 cells was performed using the MTT assay, as previously described. 34 Stock solutions (109 final concentration) of the test compounds were added in 25 μL volumes to two series of triplicate wells to allow simultaneous evaluation of their effects on mock-and HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds were made directly in flatbottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman Instruments, Fullerton, CA, USA). Untreated control HIV-and mock-infected cell samples were included for each sample. HIV-1 (III B ) or HIV-2 (ROD) stock (50 μL) at 100-300 CCIC 50 (50% cell culture infectious dose) or culture medium was added to microtiter tray wells. Mock-infected cells were used to evaluate the effect of test compound on uninfected cells in order to assess the cytotoxicity of the test compound. Exponentially growing MT-4 cells were centrifuged for 5 min at 1000 rpm, and the supernatant was discarded. The MT-4 cells were resuspended at 66 × 10 5 cells/mL, and an amount of 50 μL volume was transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically by the MTT assay. The MTT assay is based on the reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Across Organics, Geel, Belgium) by mitochondrial dehydrogenase of metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an eightchannel computer-controlled photometer (Multiscan Ascent Reader, Labsystem, Helsinki, Finland) at two wavelengths (540 and 690 nm). All data have been calculated using the median OD value of three wells.

Cell Culture and Proliferation Assay. Cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and were cultured according to the supplier's instructions. Human HCT-116 colorectal carcinoma and PC3 prostate adenocarcinoma cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 1% glutamine. MCF-7 breast adenocarcinoma cells were grown in DMEM supplemented with 10% FCS and 1% glutamine. Cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . Cell growth inhibition was determined by an MTS assay according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, the cells were seeded in 96-well plates (2.5 × 10 3 cells/well) containing 200 μL of growth medium. After 24 h of culture, the cells were treated with the tested compounds at 1 and 10 μM final concentrations. After 72 h of incubation, 40 μL of resazurin was added for 2 h before recording absorbance at 490 nm with a spectrophotometric plate reader. The percent cytotoxicity index [(OD 490 treated/OD 490 control) × 100] was calculated from three experiments. Taxotere (IC 50 's: 5 × 10 -8 M and 2 × 10 -9 M for HCT-116 and PC3 cell lines, respectively) and vinblastine (IC 50 : 5 × 10 -9 M for MCF-7 cell line) were used as positive controls.

ASSOCIATED CONTENT Supporting Information

1D-and 2D-NMR spectra and HRESIMS data for compounds 4 -12 and 14. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00116.

Chart 1 Figure 1 .

 11 Figure 1. Proposed fragmentation mechanism for tonantzitlolones A and B (1 and 2).

Figure 2 .

 2 Figure 2. Key COSY (bold) and HMBC (blue arrows) correlations of compounds 4, 7, 9, and 10.

Figure 3 .

 3 Figure 3. Key ROESY correlations (red arrows) of compounds 4, 7, 9, and 10.

Table 1 .

 1 NMR Spectroscopic Data (CDCl 3 ) for Compounds 4-10

		tonantzitlolone D (4)	tonantzitlolone E (5)		tonantzitlolone F (6)		tonantzitlolone G (7)
	position	δ C	δ H (J in Hz)	δ C	δ H (J in Hz)		δ C	δ H (J in Hz)		δ C	δ H (J in Hz)
	1	140.3 5.82 d (15.2)	140.3 5.82 d (15.2)		140.2 5.82 d (15.2)		138.4 5.52 d (15.7)
	2	126.9 5.20 dd (15.2, 9.8)	126.9 5.20 dd (15.2, 9.7)		127.2 5.26 dd (15.2, 9.6)		128.1 5.70 dd (15.7, 8.7)
	3	49.7 3.31 dq (9.8, 6.7)	49.7 3.32 dq (9.7, 6.8)		49.8 3.34 dq (9.6, 6.8)		47.4 3.32 dq (8.7, 6.8)
	4	211.7		211.6			211.1			214.0
	5	74.3 4.59 dd (11.7, 2.7)	74.4 4.59 dd (11.8, 2.5)		74.4 4.69 dd (11.8, 2.7)		75.9 4.37 brdd (5.5, 4.4)
	6α	28.9 1.33 m a	28.9 1.33 m a		29.2 1.45 ddd (13.6, 12.8, 11.8)	34.7 1.91 ddd (15.2, 4.4,
										3.2)
	6β	1.82 ddd (13.5, 4.0, 2.7)	1.83 ddd (13.6, 4.0, 2.7)	1.93 m a		1.98 m
	7	29.2 2.29 m		29.1 2.30 m			29.5 2.45 m			29.8 2.06 m
	8	73.2 4.86 d (2.2)	73.5 4.87 d (2.2)		75.6 5.11 d (2.4)		80.2 5.38 d (3.3)
	9	97.2		97.2			97.2			208.6
	OH-9	5.62 s		5.61 s			5.73 s
	10	78.3 3.38 d (6.3)	78.4 3.38 s			78.6 3.45 d (7.3)		78.8 4.35 s
	OH-10	3.12 d (6.3)	3.23 brs		3.12 d (7.3)
	11	87.7		87.7			87.9			85.0
	12α	37.5 2.40 dd (12.4, 7.6)	37.5 2.36 dd (12.6, 7.6)		37.5 2.37 dd (12.6, 7.7)		32.9 1.75 m a
	12β	1.51 ddd (12.4, 12.4, 7.2)	1.51 ddd (12.6, 12.3, 7.4)	1.50 ddd (12.6, 12.2, 7.3)	1.65 dd (9.2, 8.1)
	13α	28.2 2.00 dddd (12.4, 12.0, 11.3,	28.2 1.99 dddd (12.3, 12.0, 11.3,	28.3 1.95 dddd (12.2, 11.8, 11.3,	25.5 1.75 m a
		7.6)		7.6)			7.7)
	13β	1.73 ddd (12.0, 7.2, 5.0)	1.73 ddd (12.0, 7.4, 5.0)	1.74 ddd (11.8, 7.3, 5.1)		1.87 m a
	14	89.0 3.74 dd (11.3, 5.0)	89.0 3.74 dd (11.3, 5.0)		89.1 3.75 dd (11.3, 5.1)		87.1 3.74 dd (7.3, 6.5)
	15	38.9		38.9			39.0			39.7
	16	16.2 1.08 d (6.7)	16.2 1.08 d (6.8)		16.3 1.14 d (6.8)		20.1 1.18 d (6.8)
	17	17.2 0.81 d (6.9)	17.2 0.82 d (6.8)		17.3 0.89 d (7.0)		17.3 1.02 d (6.9)
	18	28.3 1.34 s		28.3 1.34 s			28.4 1.37 s			23.9 1.23 s
	19	25.6 0.87 s		25.6 0.88 s			25.7 0.90 s			25.6 0.85 s
	20	25.3 1.10 s		25.3 1.10 s			25.3 1.14 s			24.6 1.06 s
	1′	166.4		166.5			165.4			165.3
	2′	115.6 5.69 s		112.9 6.01 s			126.1			113.5 5.67 s
	3′	158.6		159.4			137.5 8.27 brd (7.7)		163.8
	4′	27.9 1.86 s		67.2 4.11 s			123.6 7.38 dd (7.7, 4.6)		34.0 2.15 q (7.5)
	5′	20.6 2.14 s		16.0 2.06 s			153.8 8.76 d (4.6)		12.0 1.05 t (7.5)
	6′						151.4 9.22 s			19.4 2.13 s
			tonantzitlolone H (8)			tonantzitlolone I (9)			tonantzitloic acid (10)
	position	δ C	δ H (J in Hz)	δ C		δ H (J in Hz)		δ C	δ H (J in Hz)
	1	138.1	5.51 d (15.7)		76.4	4.17 d (9.5)	136.3	5.55 d (15.9)
	2	128.2	5.71 dd (15.7, 8.8)	144.5	6.32 d (9.5)	128.2	5.48 dd (15.9, 6.6)
	3	47.4	3.33 dq (8.8, 6.8)	133.0				42.7	3.13 qd (7.0, 6.6)
	4	213.9			203.3			180.9
	5	75.8	4.38 brs		71.8	5.03 m		179.4
	OH-5					3.86 brd (3.9)
	6α	34.6	1.91 m a		34.6	2.13 m a			35.8	2.20 m a
	6β		1.99 ddd (7.0, 6.6, 6.0)						2.36 dd (15.7, 5.9)
	7	29.7	2.01 m		29.5	2.02 m			31.0	3.11 m
	8	80.6	5.39 d (2.9)		79.9	4.82 d (1.2)		79.5	5.50 brs
	9	208.5			207.0			206.4
	10	78.5	4.35 d (4.9)		82.9	4.33 d (4.9)		81.2	3.88 s
	OH-10		3.10 d (4.9)			3.78 d (4.9)
	11	85.0			85.8				85.4
	12α	32.7	1.74 m a		37.8	2.47 dd (12.5, 8.0)		34.8	2.18 m a
	12β		1.64 m			1.46 ddd (12.5, 12.2, 7.3)		1.68 m a
	13α	25.4	1.72 m a		29.7	1.84 dddd (12.2, 11.8, 11.4, 8.0)	27.0	1.68 m a
	13β		1.87 m a			1.68 ddd (11.8, 7.3, 4.5)			1.85 m
	14	87.1	3.73 dd (7.3, 6.4)	84.9	3.58 dd (11.4, 4.5)		87.1	3.73 dd (7.6, 6.6)
	15	39.6			43.9				39.3
	16	20.2	1.18 d (6.8)		12.4	1.87 s			17.2	1.24 d (7.0)
	17	17.3	1.01 d (6.4)		17.0	0.99 d (6.9)		19.2	1.11 d (6.9)
	18	23.9	1.22 s		27.1	1.32 s			20.5	1.19 s
	19	25.8	0.84 s		12.2	0.81 s			24.6	0.89 s

  ion peak at m/z 474.2461 (calcd for C 26 H 36 NO 7 , 474.2492). The 1 H and 13 C NMR data suggested that compounds 4 and 6 share the same flexibilane-type macrocyclic ring, but have a different side chain. The characteristic 1 H NMR signals at δ H 9.22 (H-6′, s), 8.76 (H-5′, d, 4.6 Hz), 8.27 (H-3′, brd, 7.7 Hz), and 7.38 (H-4′, dd, 7.7, 4.6 Hz), together with HMBC cross-peaks from H-3′, H-6′, and H-8 to the carbonyl C-1′ at δ C 165.4, revealed the presence of a nicotinyl ester moiety attached at C-8 on the macrocycle.

6 (tonantzitlolone F) gave a molecular formula of C 26 H 35 NO 7 , as supported by HRESIMS, showing a [M + H] +

Table 1

 1 Na] + ion peak at m/z 503.2635 (calcd 503.2621) corresponding to the molecular formula C 26 H 40 O 8 .

	. continued
			tonantzitlolone H (8)		tonantzitlolone I (9)		tonantzitloic acid (10)
	position	δ C	δ H (J in Hz)	δ C	δ H (J in Hz)	δ C	δ H (J in Hz)
	20	24.6	1.06 s	23.7	0.96 s	26.2	1.08 s
	1′	164.7		167.0		166.0
	2′	114.2	5.87 brs	112.8	5.69 s	113.8	5.74 s
	3′	158.6		164.9		163.2
	4′	73.7	5.23 q (6.6)	34.1	2.15 q a (7.4)	34.0	2.16 q (7.5)
	5′	19.4	1.33 d (6.6)	12.0	1.04 t (7.4)	12.0	1.05 t (7.5)
	6′	15.8	2.11 brs	19.4	2.08 s	19.4	2.10 s
	1″	170.2
	2″	21.5	2.08 s
	a Overlapped.

Table 2 .

 2 NMR Spectroscopic Data (CDCl 3 ) forCompounds 11, 13, and 15

				11			13			15
	position	δ C		δ H (J in Hz)	δ C		δ H (J in Hz)	δ C		δ H (J in Hz)
	1	156.3	CH	7.01 s	156.6	CH	7.04 s	161.6	CH	7.58 brs
	2	143.6	C		143.5	C		133.0	C
	3	213.4	C		213.8	C		209.6	C
	4	49.9	CH	2.77 m	49.8	CH	2.78 m	73.9	C
	5α	25.4	CH 2	2.46 dd (15.5, 5.0)	25.2	CH 2	2.47 dd (15.5, 4.7)	38.7	CH 2	2.45 d (19.0)
	5β			3.43 br d (15.5)			3.43 br d (15.5)			2.51 d (19.0)
	6	137.3	C		137.0	C		140.1	C
	7	126.6	CH	5.09 brs	126.9	CH	5.10 brs	130.5	CH	5.66 d (4.6)
	8	40.9	CH	1.93 brs	40.8	CH	1.93 brs	39.2	CH	3.00 brs
	9	78.2	C		78.2	C		76.3	C
	OH-9			5.27 s			5.30 s
	10	47.6	CH	3.48 m	47.6	CH	3.49 m	55.8	CH	3.26 brs
	11	43.7	CH	1.65 dq (10.4, 6.4)	43.6	CH	1.67 dq (10.4, 6.2)	36.4	CH	1.96 dq (6.8, 6.0)
	12α	76.2	CH	5.41 d (10.4)	74.9	CH	5.48 d (10.4)	31.9	CH	1.57 m
	12β									2.07 dd (14.8, 6.8)
	13	65.0	C		65.1	C		63.9	C
	14	37.5	CH	0.71 d (5.1)	37.1	CH	0.74 d (5.1)	32.8	CH	0.86 m
	15	25.7	C		25.4	C		23.2	C
	16	16.7	CH 3	1.17 s	16.6	CH 3	1.18 s	15.6	CH 3	1.06 s
	17	24.4	CH 3	1.18 s	24.4	CH 3	1.14 s	23.5	CH 3	1.19 s
	18	12.2	CH 3	1.06 d (6.4)	12.2	CH 3	1.06 d (6.2)	18.8	CH 3	0.86 m
	19	10.5	CH 3	1.76 s	10.5	CH 3	1.76 s	10.4	CH 3	1.75 brs
	20a	69.6	CH 2	3.88 d (12.4)	69.5	CH 2	3.87 d (12.3)	68.4	CH 2	3.95 d (12.8)
	20b			4.00 d (12.4)			4.00 d (12.3)			4.02 d (12.8)
	1′	170.7	C		166.3	C		168.6	C
	2′	21.3	CH 3	2.10 s	115.6	CH	5.60 d (11.3)	124.0	CH	5.98 d (15.2)
	3′				146.1	CH	6.63 dd (11.5, 11.3)	144.4	CH	7.33 ddd (15.2, 7.4, 3.0)
	4′				126.6	CH	7.41 dd (14.9, 11.5)	137.4	CH	6.64 dd (7.4, 3.0)
	5′				142.9	CH	6.48 dd (14.9, 10.9)	138.8	CH	6.64 dd (7.4, 3.0)
	6′				130.5	CH	6.21 dd (15.2, 10.9)	140.2	CH	7.17 ddd (15.2, 7.4, 3.0)
	7′				141.3	CH	5.94 dt (15.2, 7.2)	132.9	CH	6.31 d (15.2)
	8′				35.3	CH 2	2.11 td (7.4, 7.2)	200.8	C
	9′				22.3	CH 2	1.42 tq (7.4, 7.4)	41.5	CH 2	2.55 t (7.5)
	10′				13.9	CH 3	0.89 t (7.4)	24.4	CH 2	1.59 m
	11′-13′							29.4	CH 2	1.20-1.27 m
								29.5	CH 2	1.20-1.27 m
								29.6	CH 2	1.20-1.27 m
	14′							32.0	CH 2	1.20-1.27 m
	15′							22.9	CH 2	1.20-1.27 m
	16′							14.4	CH 3	1.85 t (7.1)
	1″	178.8	C		176.3	C
	2″α	41.5	CH	2.32 qt (7.0, 6.8)	36.5	CH 2	2.27 m
	2″β						2.30 m
	3″α	26.3	CH 2	1.41 qdd (7.5, 6.8, 6.8)	18.3	CH 2	1.62 tq (13.7, 7.4)
	3″β			1.70 qdd (7.5, 6.8, 6.8)
	4″	11.9	CH 3	0.90 t (7.5)	13.9	CH 3	0.92 t (7.4)
	5″	16.4	CH 3	1.09 d (7.0)

  whereas compounds 11, 13, and 15 are new. Compound 11 showed a pseudomolecular ion at m/z 497.2534 [M + Na] + (calcd for C 27 H 38 O 7 Na, 497.2515) in the HRESIMS, corresponding to the molecular formula C 27 H 38 O 7 .

Table 3 .

 3 Antimetabolic and Antiviral Activities of Compounds 1-15 in Vero Cells against CHIKV, SINV, and SFV Values are the median ± median absolute deviation calculated from at least three independent assays. b SI or window for antiviral selectivity is calculated as CC 50 Vero/EC 50 CHIKV, SINV, or SFV, respectively.

				CHIKV			SINV		SFV
	compound	CC 50 Vero a	EC 50	a	SI b	EC 50	a	SI b	EC 50 a	SI b
	1	nd c	>215			nd c			nd c
	2	122	12 ± 3	10.2	70		1.6	49 ± 4	2.5
	3	>210	24 ± 1	>9	109		>1.9	>208
	4	nd c	>222			nd c			nd c
	5	nd c	>107			nd c			nd c
	6	56 ± 6	1 9 ± 2	3	4 1 ± 5	1.4	69	0.8
	7	>215	168		>1.3	nd c			nd c
	8	nd c	>191			nd c			nd c
	9	nd c	>208			nd c			nd c
	10	nd c	>201			nd c			nd c
	11	105	77		1.4	nd c			nd c
	12	134	3.3 ± 0.3	41	>255			>255
	13	7.1 ± 1.4	1.4 ± 0.2	5.1	5.0 ± 0.8	1.4	>177
	14	>289	1.2 ± 0.2	>240	>289			>289
	15	13 ± 1	2.2 ± 1.5	5.9	11		1.2	>168
	chloroquine	89 ± 28	11 ± 7	8.1	11 ± 2	8.1	14 ± 2	6

a Data in μM. c nd = not determined.

Table 4 .

 4 Antimetabolic and Antiviral Activities of Compounds 11-15 in MT4 Cells against HIV-I (III B ) and HIV-II (ROD)

			HIV-I (III B )				HIV-II (ROD)
	compound	CC 50 (MT4) a	EC 50 a	SI b	CC 50	a	EC 50	a	SI b
	11	85 ± 18	>85		57 ± 27	13 ± 2	4
	12	102	0.271 ± 0.127	376	102		0.107 ± 0.035	949
	13	8.4 ± 0.9	0.233 ± 0.030	36	5.7 ± 1.7	0.174 ± 0.126	33
	14	116	0.129 ± 0.027	899	116		0.056 ± 0.020	2056
	15	13 ± 2	0.043 ± 0.003	299	25 ± 6	0.018 ± 0.003	1431
	nevirapine	>15	0.010 ± 0.008	>148			>15
	lamivudine	>87	3.9 ± 2.9	>22			15.5 ± 11.2	>5.6
	azidothymidine	>25	0.0071 ± 0.0004	>3516			0.0060 ± 0.0007	>4176
	dideoxyinosine	>50	10.8 ± 3.3	>4.6			9.6 ± 2.1	>5.2

a Data in μM. Values are the median ± median absolute deviation calculated from at least three independent assays. b SI or window for antiviral selectivity is calculated as CC 50 MT-4/EC 50 HIV (III B or ROD).

Table 1 ;

 1 C NMR, see HRESIMS m/z 473.2523 [M + Na] + (calcd for C 25 H 38 O 7 Na, 473.2515). Cl 2 ) λ max (log ε) 229 (3.88) nm; 1 H and 13 C NMR, see

	Tonantzitlolone E (5): colorless oil; [α] 24	D +108 (c 0.1, CHCl 3 );
	UV (CH 2

Table 1 ;

 1 HRESIMS m/z 489.2482 [M + Na] + (calcd for C 25 H 38 O 8 Na, 489.2464). Cl 2 ) λ max (log ε) 264 (3.53), 307 (3.51) nm; 1 H and 13 C NMR, see Table 1; HRESIMS m/z 474.2461 [M + H] + (calcd for C 26 H 36 NO 7 , 474.2492). Cl 2 ) λ max (log ε) 229 (3.82) nm; 1 H and 13 C NMR, see

	Tonantzitlolone F (6): colorless oil; [α] 24 D +76 (c 0.1, CHCl 3 ); UV
	(CH 2

Table 1 ;

 1 HRESIMS m/z 487.2664 [M + Na] + (calcd for C 26 H 40 O 7 Na, 487.2672). Cl 2 ) λ max (log ε) 229 (4.01) nm; 1 H and 13 C NMR, see Table 1; HRESIMS m/z 519.2557 [M + Na] + (calcd for C 26 H 40 O 9 Na, 519.2570). Cl 2 ) λ max (log ε) 230 (4.22) nm; 1 H and 13 C NMR, see Table 2; HRESIMS m/z 497.2534 [M + Na] + (calcd for C 27 H 38 O 7 Na, 497.2515). CHCl 3 ); UV (CH 2 Cl 2 ) λ max (log ε) 233 (3.87), 308 (4.19) nm; 1 H and 13 C NMR, see Table 2; HRESIMS m/z 589.3115 [M + Na] + (calcd for C 34 H 46 O 7 Na, 589.3141). CHCl 3 ); UV (CH 2 Cl 2 ) λ max (log ε) 202 (4.45), 314 (4.98) nm; 1 H and 13 C NMR, see Table 2; HRESIMS m/z 617.3472 [M + Na] + (calcd for C 36 H 50 O 7 Na, 617.3454).

	Tonantzitlolone H (8): colorless oil; [α] 24 (CH 2 Cl 2 ) λ max (log ε) 228 (3.90) nm; 1 H and 13 C NMR, see Table 1; D +74 (c 0.1, CHCl 3 ); UV HRESIMS m/z 545.2724 [M + Na] + (calcd for C 28 H 42 O 9 Na,
	545.2727).
	Tonantzitlolone I (9): colorless oil; [α] 24 (CH 2 Cl 2 ) λ max (log ε) 229 (3.95) nm; 1 H and 13 C NMR, see Table 1; D +24 (c 0.05, CHCl 3 ); UV HRESIMS m/z 503.2635 [M + Na] + (calcd for C 26 H 40 O 8 Na,
	503.2621).
	Tonantzitloic acid (10): colorless oil; [α] 24	D -38 (c 0.05, CHCl 3 );
	UV (CH 2 12-O-Acetyl-4α-deoxyphorbol-13-(2″-methyl)butyrate (11): col-
	orless oil; [α] 24 D -20 (c 0.1, CHCl 3 ); UV (CH 2 12β-O-[Nona-2Z,4E,6E-trienoyl]-4α-deoxyphorbol-13-butyrate (13): colorless oil; [α] 24 D -30 (c 0.1, 12-Deoxyphorbol-13-[8′-oxohexadeca-2E,4E,6E-trienoate] (15):
	colorless oil; [α] 24	D +40 (c 0.1,

ACKNOWLEDGMENTS

This work was supported by the Centre de Recherche et de Veille des Maladies E ḿergentes dans l'Oceán Indien (CRVOI), by the Regional Council of La Reúnion, and by the European Regional Development Fund (ERDF). The authors are very grateful to E. Boyer and D. Strasberg (UMR C_53 PVBMT) for their contribution to plant material identification and species collection. This work has also benefited from an "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-25-01). We also would like to acknowledge C. Collard, N. Verstraeten, and C. Vanderheydt for their excellent technical assistance in the acquisition of the antiviral data and Dr. J. Bignon and Mrs. H. Levaique, who performed the cytotoxic assays.

AUTHOR INFORMATION

Corresponding Author *Tel: + 33 1 69 82 30 85. Fax: + 33 1 69 07 72 47. E-mail: marc.litaudon@cnrs.fr.

Notes

The authors declare no competing financial interest.