Dominique Monnet

Jordan Ninin

Benoit Clement

H Dominique Monnet

Benoît Clément

A global optimization approach to structured regulation design under Hinfinity constraints

I. INTRODUCTION

The H ∞ synthesis [START_REF] Zhou | Essentials of Robust Control[END_REF] is a method that formulates performance and robustness objectives as an optimization problem. The first methods developed to synthesize a controller from the H ∞ formulation [START_REF] Gahinet | A linear matrix inequality approach to H∞ control[END_REF], [START_REF] Doyle | Statespace solutions to standard h2 and H∞ control problems[END_REF] met difficulties to reach a large audience since synthesized controllers cannot be implemented efficiently in practice. Indeed, these methods provide full order controllers which have to be truncated by order reduction or stucturation [START_REF] Voinot | Unstationnary control of a launcher using observer-based structures[END_REF].

The synthesis of fixed order controllers, thus workable in real cases, is a challenging problem when meet robustness and performance constraints. The emergence of efficient methods to solve Linear Matrix Inequality problems (LMI) has encouraged formulating these constraints as LMI [START_REF] Boyd | MIMO PID tuning via iterated LMI restriction[END_REF], [START_REF] Sadeghpour | A toolbox for robust PID controller tuning using convex optimization[END_REF], [START_REF] Garpinger | A software tool for robust PID design[END_REF]. However, some problems cannot be cast into LMI and require other optimization methods to be solved, such as convex-concave optimization [START_REF] Hast | PID design by convex-concave optimization[END_REF] or non-smooth optimization [START_REF] Burke | Hifoo-a matlab package for fixed-order controller design and H∞ optimization[END_REF], [START_REF] Apkarian | Nonsmooth H∞ synthesis. Automatic Control[END_REF].

Global optimization [START_REF] Kearfott | An interval branch and bound algorithm for bound constrained optimization problems[END_REF] approaches present a major interest for non-convex problems. Indeed, global optimization methods provide an enclosure of the global optimum of a problem. Furthermore, computation times of global optimization methods are no more prohibitive due to a lot of studies [START_REF] Ninin | A reliable affine relaxation method for global optimization[END_REF], [START_REF] Bixby | A brief history of linear and mixed-integer programming computation[END_REF] to improve the convergence of global optimization algorithms. We choose to approach the H ∞ sensitivity problem under a structural constraint on the controller with global optimization algorithm based on interval arithmetic to cope with the non-convexity of this problem.

This paper is organized as follows. Section II proposes a definition of the H ∞ problem and the H ∞ sensitivity problem. Section III introduces interval arithmetic and proposes an algorithm to solve the H ∞ sensitivity problem. Section IV compares our results to those obtained with two other H ∞ synthesis methods. Section V concludes this paper. In this section, we recall the H ∞ synthesis problem. Then, we show how performance and robustness constraints can be expressed as H ∞ constraints and we introduce the H ∞ sensitivity problem. Finally, we introduce the notion of structured controller and we formulate the problem we propose to solve.

A. H ∞ problem

The H ∞ synthesis problem can be defined as proposed in [START_REF] Zames | Feedback and optimal sensitivity: Model reference transformations, multiplicative seminorms, and approximate inverses[END_REF]. Given an Linear Time Invariant (LTI) system P and a set K of LTI systems, find a controller from K which is a solution to the problem:

minimize K∈K ||F (P, K)|| ∞ subject to K internally stabilizes P (1)
where F (P, K) is the Linear Fractional Transform (LFT) that describes the closed loop system represented in Figure 1. In

F (P, K) =    T w→z1 (s)
. . .

T w→zp (s)    (2)
where T w→zj (s) = (T w1→zj (s), ..., T wn→zj (s)). T wi→zj (s) represents the channel from the i th external input to the j th error output. s = iω denotes the Laplacian variable, where i is the imaginary unit and ω ≥ 0 the pulsation. Problem 1 was solved with the DGKF algorithm [START_REF] Doyle | Statespace solutions to standard h2 and H∞ control problems[END_REF] based on the resolution of Ricatti equations, and was later reformulated as a Linear Matrix Inequalities problem [START_REF] Gahinet | A linear matrix inequality approach to H∞ control[END_REF].

B. From H ∞ constraints to H ∞ problem
In practice, Problem 1 is formulated from H ∞ constraints. Consider a LTI system G(s) and a controller K(s) in a tracking error regulation loop as shown in Figure 2, where e represents the tracking error signal, u the control, y the measure, r the reference, d the disturbance and n the noise. A signal zi = W i (s)z i denotes the weighted counterpart of the output signal z i .

r K G y d n + - + + e u W 1 W 2 W 3 ẽ ũ ỹ P Fig. 2. H∞ sensitivity approach.
The H ∞ norm of a LTI system that maps w ∈ L 2 into z ∈ L 2 represents the maximal gain of this system.

||T || ∞ = max w =0 ||z|| 2 ||w|| 2 = sup ω λ(T (iω)T (iω) * ), ( 3
)
where λ is the greatest eigenvalue and T (iω) * is the hermitian transpose of T (iω). Thus, minimizing ||T || ∞ means minimizing the maximal response of the system to inputs w.

The principle of H ∞ synthesis is to minimize infinity norm of objective channels T w→zi (s). Error outputs e, u and y are weighted with rational transfer functions W 1 (s), W 2 (s) and W 3 (s), such as non-desired behaviors of the closed loop system are penalized. For example, a non-desired behavior may be an important tracking error signal e at low frequencies (steady state). In this case, W 1 (s) penalizes the sensitivity function T w→e (k, s) at low frequencies but not at high frequencies where noise n occurs. An example of robust constraint would be to minimize the action of actuators at high frequencies. Thus, W 2 (s) would penalize T w→u (s) at these frequencies.

Once weighting functions W i (s) are defined, the objective is to find K(s) that minimizes every objectives ||T w→ zi (s)|| ∞ all at once, so that the closed loop system offers the desired behavior. In addition, K(s) must internally stabilize the closed loop system. In practice, H ∞ performance and robustness constraints are expressed as bounds on the gain of objective channels. These bounds are frequency templates designed from user specifications, and represent the inverse of weighting functions

W i (s): ∀ω, λ(T w→zi (iω)T w→zi (iω) * ) ≤ |W -1 i (s)| ⇐⇒ ||T w→zi (s)W i (s)|| ∞ ≤ 1 ⇐⇒ ||T w→ zi (s)|| ∞ ≤ 1 (4)
H ∞ constraints can be interpreted as a frequency dependent upper bound on the ratio between an error output and inputs. Ensuring that every H ∞ constraints are respected can be formulated as a Constraints Satisfaction Problem (CSP), find K, subject to

         ||T w→ z1 (s)|| ∞ ≤ 1 . . . ||T w→ zp (s)|| ∞ ≤ 1 K(s) internally stabilizes P (5)
This CSP problem can be considered as an optimization problem, that we call the H ∞ sensitivity problem:

minimize K max(||T w→ z1 (s)|| ∞ , ..., ||T w→ zp (s)|| ∞ ) subject to K(s) internally stabilizes P (6)
Indeed, if the minimum of Problem 6 is lower than one, the solution of Problem 6 is a feasible solution to Problem 5.

The classical way to compute a solution to Problem 5 is to solve the H ∞ problem. The system P (s) is built from G(s) and the weighting functions W i (s) as shown in Figure 2.

Then, a solution to Problem 5 can be computed by solving Problem 9. If ||F (P, K)|| ∞ is lower than one, then K is a solution to Problem 5. Indeed,

||F (P, K)|| ∞ = ||   
T w→ z1 (s) . . .

T w→ zp (s)    || ∞ ≥ max(||T w→ z1 (s)|| ∞ , ..., ||T w→ zp (s)|| ∞ ). (7)
For example, we can use [START_REF] Doyle | Statespace solutions to standard h2 and H∞ control problems[END_REF] or [START_REF] Gahinet | A linear matrix inequality approach to H∞ control[END_REF] to solve the H ∞ problem. However, these methods give solutions in the space of full-order controllers, which means that the order of the controller is equal to the one of P (s). Moreover, the more H ∞ constraints must be respected, the higher is the order of P (s). Therefore, controllers computed by these two methods are usually high orders.

Remark 1: Due to Equation ( 7), if the minimum of Problem 9 is greater than one, it does not imply that Problem 5 has no solution.

C. Structured H ∞ problem

In practice low-order controllers, possibly mixed with filters, are preferred because their dynamics can be interpreted easily. A structured controller depends on free parameters k ∈ K, where K denotes the space of controller parameters. For example, a PID controller depends on k p , k i and k d .

K(k, s) = k p + k i s + k d s, with k = (k p , k i , k d ) (8)
As F (P, K) represents the system P (s) interconnected with K(k, s), its elements T wi→ zj (k, s) depend on k. The structured H ∞ problem can be formulated as follows:

minimize k∈K ||F (P, K)|| ∞ subject to K(k, s) internally stabilizes P (9)
One method to solve this problem is the non-smooth synthesis [START_REF] Apkarian | Nonsmooth H∞ synthesis. Automatic Control[END_REF]. However, to our knowledge , no methods have been proposed to solve directly Problem 6 under a structural constraint on the controller. This problem can be expressed as follows,

minimize k∈K max(||T w→ z1 (k, s)|| ∞ , ..., ||T w→ zp (k, s)|| ∞ )
subject to K(k, s) internally stabilizes P (10) We propose to solve Problem 10 using a global optimization approach based on interval arithmetic.

III. GLOBAL OPTIMIZATION APPROACH BASED ON INTERVAL ARITHMETIC

In this section, we show that Problem 10 can be solved in a guaranteed way using global optimization based on interval arithmetic. Moreover, a global optimization approach enables to compute an enclosure of the minimum of the objective function. Thus a certificate of infeasibility of Problem 5 can be obtained. We first introduce interval arithmetic and the notion of inclusion function [START_REF] Jaulin | Applied Interval Analysis[END_REF]. Then, we reformulate Problem 10 as a min/max problem. After that we show how to tackle the maximization part of this min/max problem with interval arithmetic. Finally, we propose a branch and bound algorithm to solve Problem 10.

A. Interval Arithmetic

An Interval is a closed connected subset of R [START_REF] Jaulin | Applied Interval Analysis[END_REF]. Intervals are denoted using boldface letters x. A non-empty interval x can be represented by its endpoints:

x = [x, x] = {x : x ≤ x ≤ x} with x ∈ R ∪ {-∞}, x ∈ R ∪ {+∞} and x ≤ x.
The set of intervals is denoted by IR and the set of n-dimensional interval vectors, also called boxes, is denoted by IR n . The main advantage of interval arithmetic is the reliability of computations, in the sense that a guaranteed enclosure of the result is computed. This inclusion property is the fundamental theorem of interval arithmetic. Let g : R n → R m be a function. An inclusion function g : IR n → IR m of g is defined as follows,

∀x ∈ IR n , g(x) = {g(x), x ∈ x} ⊆ g(x) (11)
An inclusion function provides an upper and a lower bound on a function over an interval. Several techniques can be used to construct an inclusion function of every factorable function involving +, -, * , sin, exp, max, min, ... [START_REF] Tóth | Interval Method for Competitive Location Problems[END_REF]. Figure 3 illustrates the concept of inclusion function. It is important to note that g(x) in light gray in Figure 3 is an over approximation of g(x) displayed in dark gray, and is not the smallest possible box which encloses g(x).

x g(x)

g(x) g g Fig. 3. Inclusion function

B. Interval approach of the problem

The computation of ||.|| ∞ involves dealing with singular values (see Equation ( 3)). Unfortunately, an interval arithmetic approach does not allow to handle efficiently the computation of singular values. However, it is possible to avoid this difficulty by reformulating Problem 10. Indeed, ||T w→zj (k, s)|| ∞ can be formulated as the maximization of a scalar-valued function:

||T w→zj (k, s)|| ∞ = sup ω λ(T w→ zj (k, iω)T w→ zj (k, iω) * ) = sup ω λ(T w1→ zj (k, s), ...T wn→ zj (k, s))    T w1→ zj (k, s)
. . .

T wn→ zj (k, s)    = sup ω n i=1 |T wi→ zj (k, s)| 2 (12)
Then, Problem 10 can be reformulated as follows:

minimize k sup ω f (k, ω)
subject to K internally stabilizes P

where

f (k, ω) = max( n i=1 |T wi→ z1 (iω, k)| 2 , ..., n i=1 |T wi→ zp (iω, k)| 2 ). (14)
The internal stability constraint is generally expressed as a LMI [START_REF] Boyd | Linear Matrix Inequalities in System and Control Theory[END_REF]. Such a constraint cannot be mixed properly with a global optimization approach based on interval arithmetic. However, the stability constraint can be dealt with interval arithmetic using the Routh criterion on the characteristic polynomial of the state matrix of F (P, K) [START_REF] Walter | Guaranteed characterization of stability domains via set inversion[END_REF]. Indeed, the constraint "K(k, iω) internally stabilizes P " can be reformulated as a set of non-convex constraints on k, denoted R(k) ≤ 0. The loop shaping problem is formulated as a constrained min/max problem that can be solved using a branch and bound algorithm based on interval arithmetic.

C. Enclosure of the objective function

The objective function of Problem 13 involves the computation of f sup (k) = sup ω f (k, ω). To minimize this objective function using a branch and bound algorithm, we need to compute a lower and an upper bound of f sup over a box k. This bound must be as close as possible to the exact range of f sup to ensure the convergence of the branch and bound algorithm.

Using interval arithmetic, an inclusion function f of f (k, ω) can be built. Indeed, the operators +, * , abs, √ , max are well-defined [START_REF] Kearfott | An interval branch and bound algorithm for bound constrained optimization problems[END_REF]. In order to use interval arithmetic, we limit the study of sup ω f (k, ω) to a bounded set Ω.

Thus, we obtain the following equation:

∀k ∈ IR n , f sup (k) = sup ω∈Ω f (k, ω) : k ∈ k ⊆ f (k, Ω).
Unfortunately, the bounds obtained using the inclusion function directly over Ω are not close enough to f sup (k). That is why we subdivide Ω into several boxes ω i such as Ω = i ω i . Thus, we have the following result:

∀k ∈ IR n , f sup (k) ⊆ max i f (k, ω i ), max i f (k, ω i ) . f (k, ω) ω f sup (k) f sup (k) ω i × f (k, ω i ) Fig. 4. Computation of f sup (k).
Figure 4 illustrates the previous equation. Ω is discretized with non-overlapping intervals and f is evaluated for each of them. The highest lower bound and upper bound of the evaluations represented by dotted lines in Figure 4 give a guaranteed enclosure of f sup (k). Indeed, the function f sup , defined as follows, is an inclusion function of f sup :

∀k ∈ IR n , f sup (k) = max i f (k, ω i ), max i f (k, ω i ) .

D. Global Optimization Algorithm

We propose to solve Problem 13 with a branch and bound algorithm [START_REF] Kearfott | An interval branch and bound algorithm for bound constrained optimization problems[END_REF], [START_REF] Ninin | Global Optimization based on Interval Analysis: Affine Relaxation and Limited Memory[END_REF]. We denote µ the global minimum of Problem 13, K ⊆ K the initial domain of controller variables, lb µ and ub µ a lower and an upper bound of µ.

The principle of a branch and bound algorithm is to split K into smaller subsets and eliminate them if they do not satisfy the stability constraint or if it is certified that they do not contain the global solution. When the algorithm ends, we obtain:

• k * the best feasible solution found, • [lb µ , ub µ ] a reliable enclosure of the minimum µ,

• A certificate of infeasibility to Problem 5 if no feasible solution has been found or lb µ > 1.

1: while |ub µ -lb µ | ≥ and L = ∅ do 2:
Extract a box k from L 3:

Contract k using [START_REF] Ninin | A reliable affine relaxation method for global optimization[END_REF].

4:

Bissect k into k 1 and k 2 .

5:

for i = 1, 2 do 6:
Compute f sup (k i ).

7:

if f sup (k i ) ≤ ub µ then 8:
Add k i in L.

9:

end if 10:

Choose k ∈ k i that respects R(k) ≤ 0.

11:

if f sup (k) < ub µ then 12:
Update ub µ and k * = k. Update lb µ = min k∈L f sup (k).

16: end while Algorithm 1: Branch and Bound algorithm.

Algorithm 1 works as follows. A list of boxes L is initialized with K. At Line 2, the box k with the lowest lower bound f sup (k) is chosen from L. At Line 3, k is contracted on its feasible region using [START_REF] Ninin | A reliable affine relaxation method for global optimization[END_REF]. If the result is not empty, k is bisected in two non-overlapping boxes k 1 and k 2 . For k 1 and k 2 , f sup (k i ) is computed at Line 6 using Section III-C. If f sup (k) > ub µ , it is proved that the global minimum cannot belong to k, else the box is added to L. At Line 10, if possible, a point k that stabilizes P (s) is chosen in k. If f sup (k) is lower than the current value ub µ , k is a better solution and ub µ is updated. When Algorithm 1 stops, L contains a set of boxes that contain the global minimizer; [lb µ , ub µ ] provides a guaranteed enclosure of the global minimum µ; k * is the best known solution.

If (lb µ > 1) or if (L = ∅) ∧ (k * = ∅), it is certified that Problem 5 has no solution.

IV. EXAMPLE

The methodology is applied on a simple example and compared with methods presented in [START_REF] Apkarian | Nonsmooth H∞ synthesis. Automatic Control[END_REF] and [START_REF] Gahinet | A linear matrix inequality approach to H∞ control[END_REF]. We use the Matlab Robust Control toolbox where both of them are implemented.

A. Problem and results

We consider the second order LTI system:

G(s) = 1 s 2 + 1.4s + 1 ,
and a PID with a derivative filtering:

K(k, s) = k p + k i s + k d s 1 + s .
The vector of controller parameters is k = (k p , k i , k d ). Each parameter belongs to [-10, 10], then K = [-10, 10] 3 and the frequency domain is Ω = [10 -3 , 10 3 ]. We consider the regulation scheme of Figure 2 without input signals d and n.

It means that we have one input signal r and three weighted outputs z1 , z2 and z3 . Weighting functions are the following ones

W 1 (s) = 10s+100 1000s+1 , W 2 (s) = 10s+1 s+10 , W 3 (s) = 100s+1 s+10 . (15)
The templates of their inverses appear as dotted lines in Figure 5. We compute a controller that is a solution of Problem 5.

To do so, we solve Problem 1 with [START_REF] Gahinet | A linear matrix inequality approach to H∞ control[END_REF], Problem 9 with [START_REF] Apkarian | Nonsmooth H∞ synthesis. Automatic Control[END_REF] and Problem 10 with a global optimization approach. These methods are denoted respectively by H ∞ full, H ∞ struct and Global Optimization (GO) struct in the first column of Table I second column indicates computation times in second. The H ∞ norm of P (s) interconnected with the controller appears in the third column (see Figure 2). The value of the objective function of the sensitivity problem is indicated in the last column. The H ∞ full method gives a 5 th order controller denoted K H∞f ull , H ∞ struct provides the following solution: In the sequel, S H∞f ull = (I + G(s)K H∞f ull (s)) -1 denotes the sensitivity function defined with K H∞f ull , S H∞struct and S go are defined the same way replacing K H∞f ull by K H∞struct and K GO in the expression. Similarly, we defined the complementary sensitivity functions T H∞f ull = G(s)K H∞f ull (s)(I + G(s)K H∞f ull (s)) -1 , T H∞struct (s) and T go (s).

K H∞struct (k, s) = 0.

B. Discussion

H ∞ full method gives a result rapidly due to the convexity of the problem. As H ∞ structured method is based on local optimization, we run it 350 times with random start points such that the computation time is close to the one of GO structured. Considering ||F (P, K)|| ∞ criterion, H ∞ full gives a better result than H ∞ struct because K H∞f ull has a higher order. GO struct gives the worst result, but this method minimizes max i (||T w→ zi || ∞ ) instead of ||F (P, K)|| ∞ contrary to the other two methods. However, if we look at max i (||T w→ zi || ∞ ) criterion, one can remark that only the GO method is able to compute a solution lower than 1, i.e. to provide a controller that respects all the three frequency constraints ||T w→zi (s, k)|| ∞ ≤ 1, i ∈ {1, 2, 3}. This can be explained by the fact that both K(k, s) and G(s) act like low-pass filters and their outputs u and y are both weighted. This leads to coupled terms in P (s), thus P (s)P (s) * has got non-negligible terms outside its diagonal. Therefore, minimizing ||P (s)|| ∞ is a different problem than minimizing max Although the proportional and derivative coefficients of controllers computed by H ∞ struct and GO struct methods present significant differences, their Bode diagrams, shown in Figure 6 controller computed by H ∞ full shows a more complex behavior due to the high order. Despite the fact that controllers obtained with the three different methods present almost similar performances, this example shed a light on the fact that GO approach is not sensitive to coupled constraints, contrary to classical approaches which aim to minimize ||P (s)|| ∞ .

In this example, we chose fractional weighting functions W i (s) to compare our method with H ∞ full and H ∞ struct. However GO method can be extended to the case where the weighting functions W i (s) are not rational, provided that we have an inclusion function of f . One can remark that if weighting functions are not rational, P (s) is not linear anymore and the internal stability cannot be ensure with the Routh criterion. Nonetheless, internal stability of P (s) is not needed in practice, it suffices that K(s) internally stabilizes the closed loop system (i.e. without weighting functions on objective channels). Furthermore, an interval arithmetic approach allows to deal with parametric uncertainties. Indeed, if G(p, s) depends on uncertain parameters p defined as intervals, it is still possible to compute f (k, p, s) and therefore to run Algorithm 1. Ensuring a performance constraint with parametric uncertainties has already been investigated with interval arithmetic for SISO system [START_REF] Malan | Robust analysis and design of control systems using interval arithmetic[END_REF], but our approach is more general and can be applied to MIMO systems.

V. CONCLUSION

In this paper, we have introduced a method based on global optimization to solve the sensitivity problem given a structured controller. We show how to compute a guaranteed enclosure of the minimum of the loop shaping problem. Thus, it is possible to provide a certificate of infeasibility of the sub-optimal problem. We illustrated the advantage of a global optimization method compared to two classical approaches with an academic example. Indeed, our approach considers each weighted outputs separately so that our synthesis method is not sensitive to coupled constraints. Moreover, the approach proposed in this paper offers new prospects. It is possible to take parameters uncertainties into account without modification of the method. In addition, our method does not restrict the expression of weighting functions as rational ones. Even if the computation time can be improved, the proposed approach opens up new ways to synthesize and analyze robust controllers.

Figure 1 ,

 1 Fig. 1. Interconnected systems

i( 3 Fig. 5 . 1 1

 351 Fig. 5. Respect of frequency constraints.

Fig. 6 .

 6 Fig. 6. Bode diagrams of controllers.

  This work is supported by the city of Brest and the DGA (French Defense Procurement Agency).

D. Monnet is with Lab-STICC, ENSTA Bretagne, 2 rue Frano ¸is Verny, France dominique.monnet@ensta-bretagne.org J. Ninin is with Lab-STICC, ENSTA Bretagne, 2 rue Frano ¸is Verny, France jordan.ninin@ensta-bretagne.fr B. Clément is with Lab-STICC, ENSTA Bretagne, 2 rue Frano ¸is Verny, France benoit.clement@ensta-bretagne.fr II. STRUCTURED SYNTHESIS UNDER H ∞ CONSTRAINTS

  that presents the results obtained. The stop criterion of GO struct method is = 0.1. The

	Method	Cpu (s) ||F (P, K)||∞	max
	H∞ full	2	1.0258	1.01611
	H∞ struct	80	1.0411	1.04109
	GO struct	83	1.0811	0.99782
			TABLE I
			RESULTS OBTAINED.

i (||T w→ zi ||∞)


Justine Desroches

Charline Kieffer

Nicolas Primas

Sébastien Hutter

Armand Gellis

Hussein El-Kashef

Pascal Rathelot

Pierre Verhaeghe
email: pierre.verhaeghe@univ-tlse3.fr

Nadine Azas

Patrice Vanelle
email: patrice.vanelle@univ-amu.fr

Discovery of new hit-molecules targeting Plasmodium falciparum through a global SAR study

Keywords: Quinazoline, Quinoline, Quinoxaline, Thienopyrimidine, Trichloromethyl goup, Plasmodium falciparum, In vitro antiplasmodial activity, In vitro HepG2 cytotoxicity, Structure-Activity Relationships

come

Introduction

Cerebral malaria, caused by Plasmodium falciparum, is the leading cause of death among parasitic infections worldwide. According to the 2015 World Malaria Report [1], 214 million people were infected by Plasmodium in 2014, leading to 438.000 deaths out of which about 88% occurred in Africa, mainly in children under five. It is to note that a significant improvement of the situation has been noted since the beginning of the 21st century, thanks to the action of the WHO and the financial involvement of several non-governmental organizations.

However, the control of the infection is facing worldwide the emergence of drug-resistant strains of the parasite which turns into a major concern for the medical and scientific community. Indeed, the WHO recommended the treatment of P. falciparum malaria is based on an artemisinin-combination therapy (ACT), but resistances to artemisinin derivatives are emerging in Asia [START_REF] Phyo | Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study[END_REF], and it was demonstrated that they were responsible for therapeutic failures in several infected patients [START_REF] Spring | Dihydroartemisinin-piperaquine failure associated with a triple mutant including kelch13 C580Y in Cambodia: an observational cohort study[END_REF][START_REF] Paloque | Plasmodium falciparum: multifaceted resistance to artemisinins[END_REF]. Moreover, it was also highlighted that the African Anopheles gambiae mosquito could transmit such Asian resistant parasites [START_REF] Laurent | MANUSCRIPT Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa[END_REF], indicating a major worldwide spreading risk. Thus, research efforts have to be developed, in order to discover new chemical entities presenting novel mechanisms of action, to complete and guaranty the durable efficiency of ACTs.

Several research teams working in the field of antimalarial agents previously reported novel quinazoline derivatives displaying significant antiplasmodial activities, in particular when bearing an amino- In the light of these hit-molecules, within the aim of defining the antiplasmodial Structure Activity Relationships (SARs) of the corresponding series, 5 key-regions (I-V) were chosen and modulated, as summarized in Figure 2. Thus, for region I, we first investigated the role of the -CCl 3 group by replacing it by various substituents including closely related halogenated groups. For region II, we studied the influence of the combination of the halogen atoms on the aniline moiety of hit A. The effect of the methylation of the secondary amine nitrogen of hit A was then investigated in region III. Further modulations in region IV were made by replacing the quinazoline ring of hits A-D by a bio-isosteric thienopyrimidine. Indeed, starting from the natural quinazoline-based antimalarial molecule Febrifugine, such ring replacement was successfully carried out [START_REF] Kikuchi | Exploration of a New Type of Antimalarial Compounds Based on Febrifugine[END_REF]. Moreover, some recent thienopyrimidine derivatives demonstrated promising antiplasmodial activities [START_REF] Cohen | Discovery of new thienopyrimidinone derivatives displaying antimalarial properties toward both erythrocytic and hepatic stages of Plasmodium[END_REF][START_REF] Cabrera | 2,4-Diaminothienopyrimidines as Orally Active Antimalarial Agents[END_REF]. Finally, in region V, we looked for novel analogs, centered either on the quinoline nucleus, as this heterocycle is encountered in numerous antimalarial drugs such as quinine, chloroquine, mefloquine, amodiaquine, piperaquine and primaquine, or on the quinoxaline nucleus, taking advantage of previously reported results obtained in our team in the pyrrolo[1,2-a]quinoxaline series [START_REF] Primas | Synthesis and in vitro evaluation of 4-trichloromethylpyrrolo[1,2-a]quinoxalines as new antiplasmodial agents[END_REF]. The synthesis of all new derivatives, their in vitro biological evaluation and the resulting SARs is presented and discussed herein. was already reported in the literature, but to the best of our knowledge, it was never studied for its antiplasmodial potential [START_REF] Wang | A novel strategy to the synthesis of 4-anilinoquinazoline derivatives[END_REF]. The biological evaluation of the above prepared quinazolines is summed up in Table 1. The results of their in vitro biological evaluation are presented in Table 1. The influence of the hydrogen atom on the secondary amine, in region III, was also studied. For this purpose, a S N Ar reaction was performed between E and N-methyl-2,4dichloroaniline, affording the expected methylated compound 14 in low yield (Scheme 3).

The biological evaluation of compound 14 is summarized in Table 1.

Biological evaluation of the series belonging to regions I, II and III

These new derivatives were evaluated in vitro toward both their antiplasmodial activity against the K1 multi-resistant P. falciparum strain (determination of the IC 50 = inhibitory concentration 50%) and their cytotoxicity (determination of the CC 50 = cytotoxic concentration 50%) on the HepG2 human cell line. The results were compared with three commercial antimalarial reference-drugs (atovaquone, chloroquine and doxycycline) and a cytotoxic reference drug (doxorubicine). For all tested compounds, the corresponding selectivity indecies (SI) were calculated (SI = CC 50 / IC 50 ). The results are presented in Table 1.

The aim of the modulation of region I was to confirm that the trichloromethyl group, at position 2 of the quinazoline ring, was the only substituent providing the antiplasmodial activity. Indeed, the antiplasmodial activity was totally lost when replacing it with other groups, including closely related ones such as CF 3 or CHCl

Modulation of region IV: Biological evaluation

In region IV, the replacement of the quinazoline ring by thienopyrimidine scaffolds led to molecules displaying similar antiplasmodial activity as hit-molecules A-D, with IC 50 values in the submicromolar range (0.4 to 0.9 µM) (Table 2). However, all the thienopyrimidine derivatives, belonging either to the [3,2-d] or [2,3-d] series, appeared more cytotoxic (CC 50 = 0.7 to 13.3 µM) than the corresponding hit-molecules, leading to lower selectivity indecies.

Globally, SARs indicate that there is no significant difference between the two thienopyrimidine series, regarding both activity and cytotoxicity. In order to evaluate the influence of the pyrimidine moiety of the quinazoline ring onto the biological profile, we chose to synthesize some analogs in 4-substituted-2trichloromethylquinoline series bearing, at position 4, the same substituents a-d as the hitmolecules. Thus, starting from 4-chloro-2-trichloromethyquinoline H obtained by chlorination of 2-methylquinolin-4(1H)-one with PCl 5 and POCl 3 [START_REF] Alonso-Alija | Preparation of amino(monocyclic aroyl)pyridinones that inhibit p38 map kinase for use as antiinflammatory agents[END_REF], we first tried solventfree S N Ar reaction conditions [START_REF] Verhaeghe | Synthesis and in vitro antiplasmodial evaluation of 4-anilino-2-trichloromethylquinazolines[END_REF] with anilines but without any conversion. Compounds 30a and 30b were finally obtained by using a catalytic amount of conc. HCl in refluxing EtOH to obtain the desired compounds in 45 and 55% yields respectively. For the phenol c and thiophenol d, we generated the corresponding anions using NaH before reacting H. Thus, phenol derivative 30c and thiophenol derivative 30d were obtained, respectively, in 72 and 32% yields (Scheme 8). The biological evaluation of these molecules is summarized in Table 3.

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Scheme 8. Synthesis of 4-substituted-2-trichloromethylquinoline series. leading to 31a, 31b, 31c and 31d in, respectively, 19%, 5%, 85% and 83% respectively.

Another strategy to improve these low reaction yields was successful only with 3trifluoromethylaniline b, by reacting L in aniline b (without any solvent) under microwave heating in a sealed vial (69%). Then, from intermediates 31a-d, a final chlorination step was needed to transform the methyl group into a -CCl 3 one. This step was achieved using the classical route elaborated by our team using a PCl 5 /POCl 3 mixture, under microwave heating.

Unfortunately, only the ether and thioether derivatives 32c and 32d were obtained in good yields (70-72%) (Scheme 9). It seemed that the presence of the amine function of 31a and 31b inhibited the chlorination reaction. The results of the biological assays of this series are presented in Table 3.

Modulation of region V: biological evaluation.

Thus, the ring variation of region V afforded two series of compounds: a quinoline and a quinoxaline series.

Among the four tested compounds in quinoline series, it appeared that all exerted an antiplasmodial activity globally similar to the ones of hits A-D. However, the cytotoxicity toward the HepG2 human cell line was increased for all of the quinoline series, in comparison to the Hits A-D and particularly for aniline b (6.5 µM vs 150 µM for hit B). It must be pointed out that for quinoline 3d, a lack of solubility in the biological media was observed, as previously reported for hit D. Thus, the substitution of the quinazoline ring by a quinoline ring was globally detrimental.

Contrary to the quinoline series, the quinoxaline series appeared very promising. Thus, derivative 32c showed both a better antiplasmodial activity than its analog hit C (0.4 µM vs 1.1 µM) and a great improvement in the cytotoxic profile, its SI reaching 100, compared to 45 for hit C. Similarly, the same profile was observed for compound 32d with a 2-fold activity increase compared to hit D (0.45 µM vs 0.9 µM) and a SI of 77 without any solubility impairment, as observed for hit D.

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To conclude, we noted that for the region V, the suppression of nitrogen N-3 of the quinazoline led to generally more cytotoxic compounds while the replacement of the quinazoline moiety by a quinoxaline one was a significant improvement of the biological profile, compound 32c reaching a SI of 100.

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Finally, when comparing the biological activities of the synthetic intermediates prepared for studying region V, it was interesting to note that the intermediate E was not active (IC 50 = 54.5 µM) while intermediates H and K displayed a significant IC 50 (0.8 and 0.4 µM respectively). Noticeably, the intermediate K possessed a very good SI (265) thanks to a moderate value of its CC 50 . Moreover, the presence of a substituent at position 3 appeared to be mandatory for displaying a good antiplasmodial profile by comparison of compound I which displayed a SI of only 2 (Figure 6). It is to note that both derivatives 32c (IC 50 = 0.15 µg/mL) and K (IC 50 = 0.11 µg/mL) meet the hit to lead in vitro TDR criteria [START_REF] Nwaka | Advancing Drug Innovation for Neglected Diseases-Criteria for Lead Progression[END_REF] for "selectively active antiplasmodial agents": IC 50 <0.2 µg/mL, SI>100.

Conclusion

From previously identified antiplasmodial hits A-D, in 2-trichloromethyl-4-substitutedquinazoline series, new derivatives were synthesized in order to study the SARs in 5 precisely defined regions (I-V). The modulation of region I showed that the -CCl 3 group was mandatory for providing antiplasmodial activity in quinazoline series. In the same series, the study of regions II and III demonstrated that the optimal substitution of the aniline moiety by halogens atoms was 2,4-dichloro and that the N-methylation was detrimental toward activity .

Then, by modulating regions IV and V, it respectively appeared that the replacement of the quinazoline ring by a quinoline ring led to activity levels similar to the ones of Hits A-D but also to increased cytotoxicities values, while the replacement of the quinazoline ring by a quinoxaline ring highlighted derivatives 32c-d, displaying both a preserved activity and a reduced cytotoxicity, in comparison with hits A-D, as summarized in Table 4. These molecules now appear as new reference antiplasmodial hits. Finally, the great antiplasmodial potential of intermediate K was revealed. Both derivatives 32c (IC 50 = 0.15 µg/mL) and K (IC 50 = 0.11 µg/mL) meet the hit to lead in vitro TDR criteria [START_REF] Nwaka | Advancing Drug Innovation for Neglected Diseases-Criteria for Lead Progression[END_REF] for "selectively active antiplasmodial agents": IC 50 <0.2 µg/mL, SI>100, opening the way to the synthesis of novel lead-compounds. HRMS spectra were recorded on QStar Elite (Applied Biosystems SCIEX) spectrometer. PEG was the matrix for HRMS. The experimental exact mass was given for the ion which has the maximum isotopic abundance. Purity of synthetized compounds was checked with LC-MS analyses which were realized at the Faculté de Pharmacie de Marseille with a Thermo Scientific Accela High Speed LC System ® coupled with a single quadrupole mass spectrometer Thermo MSQ Plus ® . The RP-HPLC column used is a Thermo Hypersil Gold ® 50 × 2.1 mm (C18 bounded), with particles of 1.9 µm diameter. The volume of sample injected on the column was 1 µL. The chromatographic analysis, total duration of 8 min, is made with the gradient of following solvents: t = 0 min, water/methanol 50/50; 0 < t < 4 min, linear increase in the proportion of water to a ratio water/methanol 95/5; 4 < t < 6 min, water/methanol 95/5; 6 < t < 7 min, linear decrease in the proportion of water to return to a ratio 50/50 water/methanol; 6 < t < 7 min, water/methanol 50/50. The water used was buffered with 5 mM ammonium acetate. The times (t R ) of the molecules analyzed

General procedure for the preparation of N-aryl-2-

trichloromethylquinazolin-4-amine (2, 3, 7, 8 and 14) To a mixture of appropriate 4-chloroquinazoline (1 equiv) in isopropanol (10 mL) and appropriate aniline (0.8 equiv) were added a few drops of concentrated HCl. The vial was then sealed and heated at 70 °C for 2 h. The mixture was allowed to cool to room temperature and was poured into 20 mL of iced water. The mixture was then extracted twice with CH 2 Cl 2 .

The combined organic phases were washed twice with water, then dried (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure. The residue was then purified by silica gel column chromatography (Petroleum Ether/CH 2 Cl 2 1/1).

2-Chloromethyl-N-(2,4-dichlorophenyl)quinazolin-4-amine (2)

Starting from 4-chloro-2-chloromethylquinazoline [START_REF] Shishoo | Reaction of nitriles under acidic conditions: a novel, direct formation of condensed 4-chloropyrimidines[END_REF] (500 mg, 2.35 mmol) and 2,4dichloroaniline (304 mg, 1.88 mmol). 13 C NMR (50 MHz, CDCl 3 ) δ =160.9, 157.1, 149.9, 133.6, 128.9, 128.7, 128.0, 127.6, 127.5, 123.9, 122.9, 120.1, 113.9, 47. 13 C NMR (50 MHz, CDCl 3 ) δ =157.3, 152.1 (q, J = 36 Hz), 149. Hz, 2H), 7.71 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.6 Hz, 1H). 13

2-Chlorodifluoromethylquinazolin-4(3H)-one (5)

In a 100 mL round-bottom flask was successively introduced 2-chloro-N-(2-cyanophenyl)-2,2-difluoroacetamide 4 (500 mg, 2.17 mmol), 30% H 2 O 2 (2 mL) and a mixture of EtOH (6 mL) and water (8 mL). Then, NaOH (140 mg, 3.47 mmol) was added at 0 °C. The mixture was stirred at rt for 90 min and the volatiles were removed under vacuum. Water (100 mL) was added to the residue and the resulting mixture was acidified to pH = 4 with conc. HCl.

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The precipitate obtained was filtered, thoroughly washed with water and dried in an oven gave the 2-chlorodifluoromethylquinazolin-4(3H)-one [START_REF] Laurent | MANUSCRIPT Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa[END_REF]. Yield 80%. Beige powder. mp 246 °C. 1 H NMR (400 MHz, CDCl 3 ) δ = 10.44 (bs, 1H), 8.37 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 3.7 Hz, 2H), 7.67-7.61 (m, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ =135.6, 129.2, 128.7, 126.9. Five quaternary C were not observed under these experimental conditions. LC-MS (ESI, 35 eV): t R = 0.66 min, m/z 229 [M-H] -.

4-Chloro-2-chlorodifluoromethylquinazoline (6)

In a 20 mL vial was successively introduced 2-chlorodifluoromethylquinazolin-4(3H)-one 5 (500 mg, 2.17 mmol) and POCl 3 (1 mL, 10.8 mmol). The mixture was stirred at 140 °C for 10 min under microwave irradiation (800 W). After cooling at rt, the crude was purified by silica gel column chromatography (CH 2 Cl 2 ) to afford 4-chloro-2-chlorodifluoromethylquinazoline.

Yield

Representative procedure for the preparation of N-(2,4-

dihalogenophenyl)-2-trichloromethylquinazolin-4-amine (10-13) A 100 mL round-bottomed single-neck flask equipped with a condenser and a magnetic stir bar was charged with 4-chloro-2-trichloromethylquinazoline E [START_REF] Verhaeghe | Highly efficient microwave assisted α-trichlorination reaction of α-methylated nitrogen containing heterocycles[END_REF] (500 mg, 1.77 mmol), 2 equiv. of the appropriated dihalogeno-aniline (3.54 mmol) and 30 mL of isopropanol. The mixture was refluxed for 48 h. After cooling, water was added (50 mL), and the resulting solution was extracted three times with CH 2 Cl 2 . Then, the combined organic layers were washed with water and dried (Na 2 SO 4 ). Finally, the solvent was evaporated under reduced pressure. The residue was purified by trituration with CH 2 Cl 2 and filtration yielding the N-(2,4-dihalogenophenyl)-2-trichloromethylquinazolin-4-amines (10-13).

N-(2,4-dibromophenyl)-2-trichloromethylquinazolin-4-amine

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N-(4-bromo-2-chlorophenyl)-2-trichloromethylquinazolin-4-amine

N-(2,4-difluorophenyl)-2-trichloromethylquinazolin-4-amine (13)

Yield 70%. White powder. mp 165 °C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 9.00-8.87 (m, 1H), 8.12-8.08 (m, 1H), 7.95-7.66 (m, 4H), 7.07-6.92 (m, 2H). 13

2-Methylthieno[3,2-d]pyrimidin-4(3H)-one (16)

A solution of methyl 3-acetamidothiophene-2-carboxylate 15 [START_REF] Englert | Fragment-Based Lead Discovery: Screening and Optimizing Fragments for Thermolysin Inhibition[END_REF] (760 mg, 3.81 mmol) in

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4-Chloro-2-methylthieno[3,2-d]pyrimidine (17) [30]

To a solution of compound 16 (200 mg, 1.20 mmol) in POCl 3 (5.0 mL) were added a few drops of pyridine. The mixture was then refluxed for 24 h. After cooling down to room temperature, the solution was poured on ice and a saturated Na 2 CO 3 solution was added until the excess of POCl 3 was neutralized. The aqueous phase was then extracted with EtOAc, the combined organic phases were washed with brine, dried (Na 2 SO 4 ) and the solvent was

Methyl 3-(2,2,2-trichloroacetamido)thiophene-2-carboxylate (18)

To a solution of methyl 3-aminothiophene-2-carboxylate (500 mg, 3.18 mmol) and triethylamine (440 µL, 3.18 mmol) in tetrahydrofuran (4.0 mL) at 5 °C was added trichloroacetyl chloride (350 µL, 3.18 mmol). The reaction was stirred at 5 °C for 30 min.

Water (5.0 mL) was then added and the aqueous phase was extracted with EtOAc. The organic phase was then washed, dried (Na 2 SO 4 ) and the solvent was evaporated to afford

2-(Trichloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one (19)

HCl (g) was bubbled into acetic acid (1.23 mL) for 30 minutes. Then, nitrogen was swept across the solution. This solution was added to a mixture of methyl 3-aminothiophene-2carboxylate (3.0 g, 19.08 mmol) and trichloroacetonitrile (0.85 mL, 8.48 mmol) in acetic acid (1.86 mL). The reaction was then heated at 100 °C for 18 h. After cooling down to rt, the acetic acid was evaporated under reduced pressure and isopropanol (4.5 mL) was added. This solution was refluxed for 5 min, then refrigerated. The precipitate thus formed was filtered, and then purified by silica gel column chromatography (eluent: Petroleum Ether 100% + 10 Hz, 1 H), 7.37 (dd, J = 8.9, 2.3 Hz, 1 H). 13

N-(2,4-Dichlorophenyl)-2-(trichloromethyl)thieno[3,2-d]pyrimidin-4amine (21a)

2-(Trichloromethyl)-N-[3-(trifluoromethyl)phenyl]thieno[3,2-d]pyrimidin-4-amine (21b)

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To a solution of 4-chloro-2-(trichloromethyl)thieno [3,2-d]

4-[(4-Chlorophenyl)thio]-2-(trichloromethyl)thieno[3,2-d]pyrimidine (21d)

To a solution of 4-chlorothiophenol (100 mg, 0.69 mmol) in DMSO (0.5mL), NaH (33 mg, 1.39 mmol) was added portionwise under inert atmosphere. The mixture was stirred at room temperature for 20 mi. 4-Chloro-2-(trichloromethyl)thieno [3,2-d]pyrimidine 20 (200 mg, 0.69 mmol) was then added portionwise and the reaction was stirred for 24 h at rt. Water (1.0 mL) was added and the aqueous phase was then extracted with CH 2 Cl 2 . The organic phase was washed with water, brine and then dried (Na 2 SO 4 ). The solvent was evaporated and the residue was purified by silica gel column chromatography (eluent: Petroleum Ether 100% +

2-Methylthieno[2,3-d]pyrimidin-4(3H)-one (23)

To a solution of N-(3-cyanothiophen-2-yl)acetamide 22 (1.232 g, 7.41 mmol) and H 2 O 2 30% (6.5mL) in ethanol (22 mL) and water (4 mL), at 0 °C, was added NaOH in pellets (0.495 g, 12.4 mmol). The reaction was stirred at room temperature for 15 min and then heated at 55 °C until a homogeneous solution was obtained. After cooling to rt, the solvent was evaporated under reduced pressure. Water (10 mL) was added and the mixture was heated to 50 °C. After cooling to rt, a HCl 1N solution was added until pH 4. The precipitate was filtered, washed with water and dried in a vacuum oven at 40°C to afford 2-methylthieno [2,3-d]

2,2,2-Trichloro-N-(3-cyanothiophen-2-yl)acetamide (26)

To a solution of 2-aminothiophene-3-carbonitrile (1.08 g, 8.0 mmol) and trichloroacetyl chloride (0.89 mL, 8.0 mmol) in dioxane (15 mL), triethylamine (1.67 mL, 12.0 mmol) was added dropwise. The reaction was stirred at room temperature for 36 h. The aqueous phase was extracted with diethyl ether and the combined organic phases were washed with brine and dried (Na 2 SO 4 ). The solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: Petroleum Ether/EtOAc 9/1) to afford

N-(2,4-Dichlorophenyl)-2-(trichloromethyl)thieno[2,3-d]pyrimidin-4amine (29a)

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To a solution of 2,2,2-trichloro-N-( 7.92 (d, J = 8.1 Hz, 1 H), 7.60 (d, J = 5.9 Hz, 1 H), 7.52 (t, J = 7.9 Hz, 1 H), 7.48-7.40 (m, 2 H), 7.35 (d, J = 5.9 Hz, 1 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 167. 4, 160.4, 154.3, 138.7, 132.0, 131.3, 129.7, 127.6, 123.4, 120.9, 117.7, 116.6, 116.4, 97.

N-(2,4-Dichlorophenyl)-2-trichloromethylquinoline-4-amine (30a)

To a solution of 4-chloro-2-(trichloromethyl)quinoline H [START_REF] Kato | Trichloromethylquinolines: Synthesis and Reaction with Trimethyl Phosphite[END_REF] (281 mg, 1.0 mmol) and 2,4dichloroaniline (160 mg, 1.0 mmol) in ethanol (5.0 mL) were added a few drops of concentrated HCl. The vial was then sealed and heated at 100 °C overnight. The mixture was allowed to cool to rt and the organic phase was then washed with a solution of NaOH 2N. The aqueous phase was extracted with CH 2 Cl 2 . The combined organic phases were dried (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure. The residue was then purified by silica gel column chromatography (gradient: Petroleum Ether 100% + 10 mL of EtOAc every 100 mL of eluent) to afford 158 mg of the expected N- (2,4-

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2-(Trichloromethyl)-N-[3-(trifluoromethyl)phenyl]quinoline-4-amine (30b)

To a solution of 4-chloro-2-(trichloromethyl)quinoline H [START_REF] Kato | Trichloromethylquinolines: Synthesis and Reaction with Trimethyl Phosphite[END_REF] (281 mg, 1.0 mmol) and 3-(trifluoromethyl)aniline (170 µL, 1.0 mmol) in ethanol (5.0 mL) were added a few drops of concentrated HCl. The vial was then sealed and heated at 100 °C overnight. The mixture was allowed to cool to rt and the organic phase was then washed with a solution of NaOH 2N. The aqueous phase was extracted with CH 2 Cl 2 . The combined organic phases were dried (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure.

4-(4-Chlorophenoxy)-2-(trichloromethyl)quinoline (30c)

To a solution of 4-chlorophenol (141 mg, 1.1 mmol) in DMSO (1.0 mL), NaH (48 mg, 2.0 mmol) was added portionwise under inert atmosphere. The mixture was stirred at rt for 20 min. 4-chloro-2-(trichloromethyl)quinoline H [START_REF] Kato | Trichloromethylquinolines: Synthesis and Reaction with Trimethyl Phosphite[END_REF] (281 mg, 1.0 mmol) was then added portionwise and the reaction was stirred for 24 h at 50 °C. The aqueous phase was then extracted with CH 2 Cl 2 and the organic phase was washed with water, dried (Na 2 SO 4 ) and the solvent was evaporated. [START_REF] Kato | Trichloromethylquinolines: Synthesis and Reaction with Trimethyl Phosphite[END_REF] (281 mg, 1.0 mmol) was then added portionwise and the reaction was stirred for 24 h at rt. The aqueous phase was then extracted with CH 2 Cl 2 and the organic phase was washed with water, dried (Na 2 SO 4 ) and the solvent was evaporated. The residue was then purified by silica gel column chromatography (eluent:

(31a)

To a solution of 2-chloro-3-methylquinoxaline L [45] (1.0 g, 5.6 mmol) and 2,4dichloroaniline (0.91 g, 5.6 mmol) in anhydrous DMF (10 mL), Cs 2 CO 3 (1.82 g, 5.6 mmol) was added under inert atmosphere. The mixture was stirred at 70 °C for 24 h. After cooling, water then CH 2 Cl 2 were added. The organic layer was then washed five times with water and dried with Na 2 SO 4 . After filtration and evaporation, the resulting solid was purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 7/3 then 1/1) to afford N-( 2

2-((4-Chlorophenyl)thio)-3-(trichloromethyl)quinoxaline (32d)

To a solution of 2-((4-chlorophenyl)thio)-3-methylquinoxaline 31d (623 mg, 2.2 mmol) and PCl 5 (2.7 g, 13.0 mmol), POCl 3 was added to make a slurry (ca 6 mL). The mixture was then heated in a multimode microwave oven at 100 °C, 800 W for 20 min. After completion of the reaction, the mixture was poured into ice and was then neutralized with Na 2 CO 3. The resulting solution was extracted with CH 2 Cl 2 and dried with Na 2 SO 4 . After filtration and evaporation, the resulting solid was purified by silica gel column chromatography (eluent: Petroleum

Biology 4.2.1. In vitro Antiplasmodial evaluation

In this study, a K1 culture-adapted P. falciparum strain resistant to chloroquine, pyrimethamine and proguanil was used in an in vitro culture. Maintenance in continuous culture was done as described previously by Trager and Jensen [START_REF] Trager | Human malaria parasites in continuous culture[END_REF]. Cultures were maintained in fresh A+ human erythrocytes at 2.5% hematocrit in complete medium (RPMI 1640 with 25 mM HEPES, 25 mM NaHCO 3 , 10% of A+ human serum) at 37 °C under reduced O 2 atmosphere (gas mixture 10% O 2 , 6% CO 2 , and 84% N 2 ). Parasitaemia was maintained daily between 1% and 6%. The P. falciparum drug susceptibility test was carried

In vitro Cytotoxicity evaluation

HepG2 cell line was maintained at 37 °C, 6% CO 2 with 90% humidity in RPMI supplemented with 10% foetal bovine serum, 1% L-glutamine (200 mM) and penicillin (100 U/mL) / streptomycin (100 µg/mL) (complete RPMI medium). The evaluation of the tested molecules cytotoxicity on the HepG2 (hepatocarcinoma cell line purchased from ATCC, ref HB-8065) cell line was performed according to the method of Mosmann [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF] with slight modifications. Briefly, 5.10 3 cells in 100 µL of complete medium were inoculated into each well of 96-well plates and incubated at 37 °C in a humidified 6% CO 2 . After 24 h incubation, 100 µL of medium with various product concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added and the plates were incubated for 72 h at 37 °C. Triplicate assays were performed for each sample. Each plate-well was then microscope-examined for detecting possible precipitate formation before the medium was aspirated from the wells. 100 µL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (0.5 mg/mL in medium without FCS) were then added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 µL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (700 rpm) for 10 min. The absorbance was measured at 570 nm with 630 nm as reference wavelength using a

[ 6 ]

 6 , alkylamino-[7,8] or an aniline-substituent [9] at position 4 of the quinazoline ring. The research activity of our group is focused on the synthesis and anti-infective evaluation of new nitrogen-containing heterocycles [10]. We have intensively studied a large series of antiplasmodial derivatives based on the original 2-trichloromethylquinazoline scaffold. Thus, the synthesis and in vitro biological evaluation of a large quinazoline chemical library bearing an arylamino-[11,12], aryl-[13], phenoxy-[14], phenylthio-[15], sulfonamido-[16], alkynyl-[17], heteroarylamino-[18], benzyloxy-or alkoxy-[19] substituent at position 4 of the quinazoline ring, revealed several hit-molecules (A-D) which are presented in Figure 1.

Figure 1 .

 1 Figure 1. Antiplasmodial hit-molecules identified in 2-trichloromethylquinazoline series.

Figure 2 .

 2 Figure 2. Antiplasmodial SAR study involving 5 key-regions (I-V) from the quinazoline hits

Scheme 1 . 5 .Scheme 2 .

 152 Scheme 1. SAR modulation of region I: modification of the substituent at position 2 of the quinazoline ring.Reagents and conditions : (i) 2,4-dichloroaniline 0.8 equiv, conc. HCl cat., iPrOH, 70 °C, 2 h.

Scheme 3 .

 3 Scheme 3. Chemical modulations at regions II and III of the pharmacophore Reagents and conditions: (i) 2,4-dihaloaniline (2 equiv) or 2,4-dichloro-N-methylaniline (1.1 equiv), iPrOH, reflux, 2 h.

2 .

 2 Looking at the combination of the halogen substituents on the aniline ring at position 4 (region II), the antiplasmodial activity was maintained for brominated compounds 10-12, in comparison with hit A (respectively 0.3, 0.5 and 0.6 vs 0.4 µM), but the cytotoxicity was significantly increased, leading to less selective molecules. Contrary to the results obtained with brominated analogues, fluorinated analogue 13 presented the same cytotoxicity profile as hit A but was less active (IC 50 = 3.1 µM). Finally in region III, the methylation of the aniline moiety (compound 14) significantly impaired both antiplasmodial activity and cytotoxicity. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

a 2 . 3 .

 23 Doxorubicine was used as a cytotoxic reference-drug; b Atovaquone, Chloroquinine and doxycycline were used as antimalarial reference-drugs, c Selectivity indecies were calculated according to the formula : SI = HepG2 CC 50 / K1 IC 50 . d Highest concentration tested due to a lack of solubility. Region IV: Synthesis of thieno[3,2-d]pyrimidine series. The replacement of the phenyl ring of the quinazoline nucleus, by a thiophene nucleus as a bio-isostere, was next studied. We first tried to synthesize 4-substituted-2-(trichloromethyl)thieno[3,2-d]pyrimidine derivatives by a S N Ar reaction between the key chlorinated intermediate 20 and various nucleophilic species, corresponding to the substituents borne by hits A-D, respectively 2,4-dichloroaniline a, 3-trifluoromethylaniline b, 4-chlorophenol c and 4-chlorothiophenol d (Figure 3).

Figure 3 .(

 3 Figure 3. Thieno[3,2-d]pyrimidines initial retrosynthesis pathway.

Scheme 5 . 2 . 4 .

 524 Scheme 5. Synthesis of 4-substituted-2-(trichloromethyl)thieno[3,2-d]pyrimidine. Reagents and conditions: (i) aniline derivative a or b 1 equiv, conc. HCl cat., EtOH, 100 °C, 18 h, sealed vial; (ii) NaH 2 equiv, 4-chlorophenol c or 4-chlorothiophenol d 1 equiv, DMSO, rt or 50 °C, 24 h.

Figure 4 .Scheme 6 .Scheme 7 .

 467 Figure 4. Thieno[2,3-d]pyrimidines initial retrosynthesis pathway.

a 2 . 6 .

 26 Doxorubicine was used as a cytotoxic reference-drug; b Atovaquone, Chloroquinine and doxycycline were used as antimalarial reference-drugs, c Selectivity indecies were calculated according to the formula : SI = HepG2 CC 50 / K1 IC 50 . d Highest concentration tested due to a lack of solubility. Region V: Synthesis of quinoline series.

2 . 7 .

 27 Reagents and conditions : (i) aniline derivative 1 equiv, conc. HCl cat., EtOH, 100 °C, 18 h, sealed vial; (ii) NaH 2 equiv, 4-chlorophenol or 4-chlorothiophenol reagent 1 equiv, DMSO, rt or 50 °C, 24 h. Region V: Synthesis of quinoxaline derivatives. Previously, we reported that 2-trichloromethylquinoxaline I displayed an in vitro antiplasmodial activity (IC 50 = 1.5 µM), hindered by a significant in vitro cytotoxicity (CC 50 = 3.1 µM) [23]. Moreover, the introduction of a phenyl substituent at position 3 (compound J) gave a more selective derivative J (SI = 17.5 versus 2.1, respectively), thanks to a better M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT antiplasmodial IC 50 value (0.2 µM) [23]. Such interesting results noted with compound J prompted us to carry on the investigation of the antiplasmodial potential of the 3-substituted-2-trichloromethylquinoxaline scaffold, in continuation of the pharmacomodulation of the quinazoline moiety in region V (Figure 5).

Figure 5 .

 5 Figure 5. Rational for the synthesis of 2-substituted-3-trichloromethylquinoxaline derivatives.

Scheme 9 .

 9 Scheme 9. Synthesis of 2-substituted-3-trichloromethylquinoxaline series.Reagents and conditions: (i) 2,4-dichloroaniline, 4-chlorophenol or 4-chlorothiophenol 1 equiv, Cs 2 CO 3 1 equiv, anh. DMF, 70 °C, 12 h, sealed vial, N 2 ; (ii) 3-trifluoromethylaniline 7 equiv, 140 °C, MW, 45 min, sealed vial; (iii) PCl 5 6 equiv, POCl 3 as solvent, 100 °C, MW, 20 min, 800 W.

Figure 6 .

 6 Figure 6. Biological assessment of synthetic intermediates.

  M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

  Commercial reagents were used as received without additional purification. Melting points were determined on a Kofler bench and are uncorrected. Elemental analysis and HRMS were carried out at the Spectropole, Faculté des Sciences et Techniques de Saint-Jérôme, Marseille, France. NMR spectra were recorded on a Bruker ARX 200 spectrometer or a Bruker AV 250 spectrometer at the Faculté de Pharmacie de Marseille or a BRUKER Avance III nanobay 400 at the the Spectropole, Faculté des Sciences et Techniques de Saint-Jérôme, Marseille ( 1 H-NMR: 200, 250 or 400 MHz, 13 C-NMR: 50, 63 or 100 MHz). NMR references were the following: 1 H: CHCl 3 δ = 7.26, DMSO-d 6 δ = 2.50 and 13 C: CHCl 3 δ = 76.9, DMSO-d 6 δ = 39.5. Solvents were dried by conventional methods. The following adsorbent was used for column chromatography: silica gel 60 (Merck, particle size 0.063-0.200 mm, 70-230 mesh ASTM). TLC was performed on 5 cm × 10 cm aluminium plates coated with silica gel 60F-M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 254 (Merck) in an appropriate eluent. Visualization was made with ultraviolet light (234 nm).

  are indicated in min. The preparation of 4-chloro-2-trichloromethylquinazoline E [24], 2methyl-N-(2,4-dichlorophenyl)quinazolin-4-amine 1 [10], N-(2,4-dichlorophenyl)quinazolin-4-amine 9 [27], 4-chloro-2-trichloromethyquinoline H [43], methyl 3-acetamidothiophene-2carboxylate 15 [28], 4-chloro-2-chloromethylquinazoline F [24], 4-chloro-2dichloromethylquinazoline G [26], 4-chloro-2-trifluoromethylquinazoline [47], 2-chloro-3trichloromethylquinoxaline K [44] and 2-chloro-3-methylquinoxaline L [45] was achieved as described in the literature and characterization were consistent as reported in literature.

  White powder. mp 171 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.97 (d, J = 8.9 Hz, 1H), 8.21 (bs, 1H), 7.99-7.82 (m, 3H), 7.68-7.60 (m, 1H), 7.47-7.35 (m, 2H), 4.75 (s, 2H).

  Starting from 4-chloro-2-chlorodifluoromethylquinazoline 6 (500 mg, 2.01 mmol) and 2,4dichloroaniline (293 mg, 1.8 mmol). Yield 30%. White powder. mp 195 °C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.93 (d, J = 9.0 Hz, 1H), 8.38 (bs, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.00-7.92 (m, 2H), 7.79-7.74 (m, 1H), 7.48 (d, J = 2.5 Hz, 1H), 7.42-7.35 (m, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ =157.3, 155.6 (t, J = 29 Hz), 149.4, 134.2, 133.2, 130.0, 129.2, 129.0, 128.8, 128.2, 123.9, 122.9 (t, J = 292 Hz), 122.8, 120.1, 114.9. HRMS (ESI): m/z calcd. for C 15 H 8 Cl 3 F 2 N 3 [M+H] + : 374.9908. Found: 374.9909. LC-MS (ESI, 35 eV): t R = 4.58 min, m/z 376 [M+H] + .4.1.1.4. N-(2,4-dichlorophenyl)-2-(trifluoromethyl)quinazolin-4-amine(8) Starting from 4-chloro-2-trifluoromethylquinazoline [45] (500 mg, 2.15 mmol) and 2,4dichloroaniline (279 mg, 1.72 mmol). Yield 37%. White powder. mp 198 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.88 (d, J = 9.0 Hz, 1H), 8.29 (bs, 1H), 8.11-8.07 (m, 1H), 7.97-7.89 (m, 2H), 7.78-7.70 (m, 1H), 7.47-7.35 (m, 2H).

  Starting from 4-chloro-2-(trichloromethyl)quinazoline E[START_REF] Verhaeghe | Highly efficient microwave assisted α-trichlorination reaction of α-methylated nitrogen containing heterocycles[END_REF] (500 mg, 1.77 mmol) and Nmethyl-2,4-dichloroaniline (188 µL, 1.95 mmol). Yield 15%. Colorless oil. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.12-8.08 (m, 1H), 7.61-7.60 (m, 2H), 7.35-7.17 (m, 3H), 6.93-6.89 (m, 1H), 3.63 (s, 3H).13 C NMR (50 MHz, CDCl 3 ) δ =161.8, 160.1, 150.9, 143.1, 134.3, 133.1, 133.0, 131.3, 129.9, 129.5, 129.1, 127.1, 124.4, 114.9, 97.4, 41.3. HRMS (ESI): m/z calcd. for C 16 H 10 Cl 5 N 3 [M+H] + : 418.9317. Found: 418.9319. LC-MS (ESI, 35 eV): t R = 5.18 min, m/z 419.7 [M+H] + .4.1.2. Synthesis of 4-chloro-2-chlorodifluoromethylquinazoline (6) 4.1.2.1. 2-Chloro-N-(2-cyanophenyl)-2,2-difluoroacetamide (4) In a 250 mL round-bottom flask was successively introduced 2-aminobenzonitrile (2.0 g, 16.9 mmol), 4-(N,N-dimethylamino)pyridine (DMAP) (1.7 g, 13.3 mmol) and N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI.HCl) (3.4 g, 17.6 mmol) in CH 2 Cl 2 (50 mL). Then, 2-chloro-2,2-difluoroacetic acid (1.4 mL, 16.9 mmol) was added into the mixture and it was stirred for 24 h at rt. The solvent was evaporated under reduced pressure and water (200 mL) was added to the residue. The mixture was gentle heated at 40 °C until a homogeneous solution was obtained. After cooloing down to rt, conc. HCl was added to adjust the pH = 1. The resulting precipitate was filtered, thoroughly washed with water and dried in an oven leading 2-chloro-N-(2-cyanophenyl)-2,2-difluoroacetamide. Yield 15%. White powder. mp 84 °C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.37 (d, J = 8.5

  C NMR (50 MHz, DMSO-d 6 ) δ =160.7, 157.4, 153.2 (d, J = 260 Hz), 152.9 (d, J = 260 Hz), 149.6, 133.9, 130.2, 128.5, 123.5 (d, J = 8.8 Hz), 123.1 (dd, J = 9.5, 4.1 Hz), 120.1, 113.8, 111.4 (dd, J = 21.6, 3.6 Hz), 104.3-103 (m, 1CH), 97.6. Anal. Calcd for C 15 H 8 Cl 3 F 2 N 3 : C, 48.09; H, 2.15; N, 11.22. Found: C, 48.45; H, 1.99; N, 11.25. LC-MS (ESI, 35 eV): t R = 4.58 min, m/z 374 [M+H] + .

NH 4 OH

 4 25% (9.5 mL) was heated, in a sealed vial, at 105 °C for 3 h. After cooling to room temperature, concentrated HCl was added until pH 8. The precipitate thus formed was filtered, washed with water and dried in a vacuum oven at 40 °C to afford 2-methylthieno[3,2d]pyrimidin-4(3H)-one. No further purification necessary. Yield 63%. White solid. mp 91°C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 12.31 (bs, 1 H, NH), 8.12 (d, J = 5.2 Hz, 1 H), 7.29 (d, J = 5.2 Hz, 1 H), 2.36 (s, 3 H). 13 C NMR (63 MHz, DMSO-d 6 ) δ = 181.0, 158.8, 158.0, 156.3, 133.9, 124.5, 21.1. LC-MS (ESI, 35eV): t R = 0.64 min, m/z 167 [M+H] + .

  evaporated under reduced pressure. The residue was then purified by silica gel column chromatography (eluent: Petroleum Ether/EtOAc 8/2) to afford 4-chloro-2-methylthieno[3,2d]pyrimidine. Yield 97%. White solid. mp 84 °C. 1 H NMR (250 MHz, CDCl 3 ) δ = 7.96 (d, J = 5.5 Hz, 1H), 7.45 (d, J = 5.5 Hz, 1H), 2.78 (s, 3H). 13 C NMR (63 MHz, CDCl 3 ) δ = 164.7, 162.6, 154.7, 136.7, 127.9, 124.8, 25.5. LC-MS (ESI, 35 eV): t R = 1.74 min, m/z 185 [M+H] + .

methyl 3 -

 3 (2,2,2-trichloroacetamido)thiophene-2-carboxylate. No further purification necessary. Yield 99%. White solid. mp 244 °C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 11.42 (bs, 1 H), 8.06 (d, J = 5.4 Hz, 1 H), 7.89 (d, J = 5.4 Hz, 1 H), 3.88 (s, 3 H). 13 C NMR (63 MHz, DMSO-d 6 ) δ = 163.4, 158.1, 140.9, 133.5, 121.0, 113.6, 91.8, 52.2. LC-MS (ESI, 35 eV): t R = 4.11 min, m/z 300 [M+H] + .

  every 100 mL of eluent) to afford 2-(trichloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one. Yield 63%. White solid. mp 237 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 10.50 (bs, 1 H), 7.93 (d, J = 5.3 Hz, 1 H), 7.51 (d, J = 5.3 Hz, 1 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 157.7, 155.7, 151.8, 135.7, 126.1, 123.7, 92.0. LC-MS (ESI, 35 eV): t R = 0.78 min, m/z 267 [M-H] -. 4.1.8. 4-Chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine (20) To a solution of 2-(trichloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one 19 (200 mg, 0.74 mmol) in POCl 3 (3.0 mL) were added a few drops of pyridine. The reaction was refluxed for 1 h. After cooling to room temperature, the solution was poured on ice and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure. The residue was then purified by silica gel column chromatography (eluent: Petroleum Ether/EtOAc 8/2) to yield 4chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine. Yield 99%. White solid. mp 99 °C. 1 H NMR (200 MHZ, CDCl 3 ) δ = 8.21 (d, J = 5.5 Hz, 1 H), 7.74 (d, J = 5.5 Hz, 1 H). 13 C NMR (50 MHZ, CDCl 3 ) δ = 161.9, 161.7, 155.6, 139.0, 130.7, 125.6, 96.0. LC-MS (ESI, 35 eV): t R = 4.05 min (no ionization).

  To a solution of 4-chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine 20 (190 mg, 0.66 mmol) and 2,4-dichloroaniline (106 mg, 0.66 mmol) in ethanol (3.0 mL) were added a few drops of concentrated HCl. The vial was sealed and the reaction was heated at 100 °C for 24 h. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 8/2 + 10 mL of CH 2 Cl 2 every 100 mL of eluent) to afford N-(2,4-dichlorophenyl)-2-(trichloromethyl)thieno[3,2-d]pyrimidin-4amine. Yield 45%. White solid. mp 197 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.72 (d, J = 8.9 Hz, 1 H), 7.96 (d, J = 5.4 Hz, 1 H), 7.66 (d, J = 5.4 Hz, 1 H), 7.61 (s, 1 H), 7.48 (d, J = 2.3

4.1. 11 . 4 -( 4 -

 1144 Chlorophenoxy)-2-(trichloromethyl)thieno[3,2-d]pyrimidine (21c) To a solution of 4-chloro-2-(trichloromethyl)thieno[3,2-d]pyrimidine 20 (210 mg, 0.73 mmol) and 4-chlorophenol (281 mg, 2.19 mmol) in acetonitrile (5.0 mL) was added K 2 CO 3 (302 mg, 2.19 mmol). The reaction was heated at 160 °C for 1h10 under microwave irradiation. The mixture was poured onto a NaOH 10% solution. The aqueous phase was extracted with EtOAc. The organic phases were combined, washed with brine, dried (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure. The residue was then purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 8/2 + 10 mL of CH 2 Cl 2 every 100 mL of eluent) to yield 4-(4-chlorophenoxy)-2-(trichloromethyl)thieno[3,2-d]pyrimidine. Yield 39%. White solid. mp 161 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.09 (d, J = 5.4 Hz, 1 H), 7.70 (d, J = 5.4 Hz, 1 H), 7.45-7.31 (m, 4 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 163.5, 163.1, 161.5, 150.3, 136.9, 131.4, 129.9, 129.5, 125.2, 122.9, 117.7, 116.4, 96.4. Anal. Calcd for C 13 H 6 Cl 4 N 2 OS: C, 41.08; H, 1.59; N, 7.37. Found: C, 41.28; H, 1.72; N, 7.45. LC-MS (ESI, 35 eV): t R = 5.08 min, m/z 379 [M+H] + .

  CH 2 Cl 2 every 100 mL of eluent) to afford 4-[(4-chlorophenyl)thio]-2-(trichloromethyl)thieno[3,2-d]pyrimidine. Yield 7%. White solid. mp not determined. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.02 (d, J = 5.5 Hz, 1 H), 7.67-6.64 (m, 3 H), 7.45 (d, J = 8.8 Hz, 2 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 164.5, 161.1, 159.9, 137.0, 136.6, 136.4, 129.5, 127.5, 125.0, 124.7, 96.8. Anal. Calcd for C 13 H 6 Cl 4 N 2 S 2 : C, 39.41; H, 1.53; N, 7.07. Found: C, 39.75; H, 1.72; N, 7.20. LC-MS (ESI, 35 eV): t R = 5.36 min, m/z 395 [M+H] + .4.1.13. N-(3-Cyanothiophen-2-yl)acetamide (22)To a solution of 2-aminothiophene-3-carbonitrile (1.020 g, 8.21 mmol) in acetic anhydride (3.0 mL) were added a few drops of acetic acid. The reaction was heated at 120 °C for 10 min under microwave irradiation. The brown solid thus obtained was filtered, washed with water and dried in a vacuum oven at 40 °C to afford N-(3-cyanothiophen-2-yl)acetamide. No further purification necessary. Yield 90%. Brown solid. mp 210 °C (litt. 210-211 °C). 1 H NMR (200 MHz, DMSOd 6 ) δ = 11.70 (bs, 1 H), 7.13-7.12 (m, 2 H), 2.20 (s, 3 H). 13 C NMR (50 MHz, DMSO-d 6 ) δ = 168.4, 149.4, 124.7, 118.7, 114.9, 91.8, 22.5. LC-MS (ESI, 35 eV): t R = 0.81 min, m/z 165 [M-H] -.

pyrimidin- 4 (

 4 3H)-one. No further purification necessary. Yield 72%. White solid. mp 210 °C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 7.90 (s, 1 H), 7.39 (d, J = 5.8 Hz, 1 H), 6.93 (d, J = 5.8 Hz, 1 H), 2.19 (s, 3 H). 13 C NMR (50 MHz, DMSOd 6 ) δ = 166.9, 166.8, 145.8, 123.0, 115.8, 114.8, 23.3. LC-MS (ESI, 35 eV): t R = 0.65 min, m/z 167 [M+H] + . 4.1.15. 4-Chloro-2-methylthieno[2,3-d]pyrimidine (24) To a solution of 2-methylthieno[2,3-d]pyrimidin-4(3H)-one 23 (300 mg, 1.80 mmol) in POCl 3 (5.0 mL) were added a few drops of pyridine. The mixture was refluxed for 24 h and, after M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT cooling to rt, was poured onto ice. A saturated Na 2 CO 3 solution was added until complete neutralization of the excess of POCl 3 . The aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: Petroleum Ether/EtOAc 8/2) to yield 4-chloro-2-methylthieno[2,3d]pyrimidine. Yield 61%. Yellow solid mp 93 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 7.48 (d, J = 6.0 Hz, 1 H), 7.35 (d, J = 6.0 Hz, 1 H), 2.79 (s, 3 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 169.2, 163.0, 154.4, 126.8, 126.6, 119.5, 25.5. LC-MS (ESI, 35 eV): t R = 1.84 min, m/z 185 [M+H] + .

2, 2 , 2 -

 22 trichloro-N-(3-cyanothiophen-2-yl)acetamide. Yield 76%. White solid. mp 127 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 9.48 (s, 1 H), 7.08 (s, 2 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 158.7, 147.0, 124.8, 120.5, 113.4, 96.8, 90.8. LC-MS (ESI, 35 eV): t R = 0.80 min, m/z 267 (M -). HRMS (ESI): m/z calcd. for C 7 H 3 Cl 3 N 2 OS [M+H] + : 268.9104. Found: 268.9110. 4.1.17. 2-(Trichloromethyl)thieno[2,3-d]pyrimidin-4(3H)-one (27) Compound 2,2,2-trichloro-N-(3-cyanothiophen-2-yl)acetamide 26 (809 mg, 3.0 mmol) was added to a mixture of phosphoric acid and polyphosphoric acid (60 mL, 1:1). The solution was stirred at rt for 1 h and then heated at 70 °C for 3 h. After cooling to room temperature, the mixture was poured onto ice. The precipitate was filtered, washed with ice-cold water and dried in a vacuum oven at 40 °C to yield 2-(trichloromethyl)thieno[2,3-d]pyrimidin-4(3H)one. No further purification necessary. Yield 90%. White solid. mp 195 °C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 8.23 (s, 1 H), 7.56 (d, J = 5.8 Hz, 1 H), 7.25 (d, J = 5.8 Hz, 1 H). 13 C NMR (50 MHz, DMSO-d 6 ) δ = 167.4, 158.8, 144.6, 124.1, 119.2, 118.5, 91.6. LC-MS (ESI, 35 eV): t R = 0.69 min, m/z 267 [M-H] -.

  To a solution of 2,2,2-trichloro-N-(3-cyanothiophen-2-yl)acetamide 26 (320 mg, 1.18 mmol) and 3-(trifluoromethyl)aniline (200 µL, 1.18 mmol) in CH 2 Cl 2 (0.3 mL), AlCl 3 (160 mg, 1.18 mmol) was added portionwise. The mixture was stirred at rt for 72 h. The solvent was evaporated under reduced pressure and the residue 28b (LC-MS (ESI, 35 eV): t R = 4.99 min, m/z 430 [M+H] + ) was dissolved in ethanol (2.0 mL) and the solution was refluxed for 18 h. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 8/2 to 5/5) to afford 2-(Trichloromethyl)-N-(3-(trifluoromethyl)phenyl)thieno[2,3-d]pyrimidin-4-amine. Yield 15%. Pale blue solid. mp 124 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.42 (s, 1 H),

  2. Anal. Calcd for C 14 H 7 Cl 3 F 3 N 3 S: C, 40.75; H, 1.71; N, 10.18. Found: C, 41.03; H, 1.86; N, 10.24. LC-MS (ESI, 35 eV): t R = 5.22 min, m/z 412 [M+H] + . 4.1.20. 4-((4-Chlorophenyl)thio)-2-(trichloromethyl)thieno[2,3-d]pyrimidine (29d) M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT HBr (g) was bubbled into a solution of 2,2,2-trichloro-N-(3-cyanothiophen-2yl)acetamide 26 (270 mg, 1.0 mmol) and 4-chlorothiophenol (145 mg, 1.0 mmol) in diethyl ether (2.5 mL), at -20 °C, for 30 min. The bubbling was stopped when the mixture became homogeneous (bright yellow solution). The reaction was then stirred at rt for 18 h. Water (2.0 mL) was then added and the aqueous phase was extracted with EtOAc. The organic phase was dried (Na 2 SO 4 ) and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 8/2 + 5 mL of CH 2 Cl 2 every 100 mL of eluent) to afford a white solid 28d (86 mg) (LC-MS (ESI, 35 eV): t R = 4.46 min, m/z 413 [M+H] + ) which was dissolved in ethanol (1.0 mL) and the resulting solution was refluxed for 5 minutes. The solvent was evaporated under reduced pressure to yield 4-((4-chlorophenyl)thio)-2-(trichloromethyl)thieno[2,3-d]pyrimidine. No further purification necessary. Yield 22%. White solid. mp 150 °C (dec.). 1 H NMR (200 MHz, CDCl 3 ) δ = 7.70 (d, J = 6.0 Hz, 1 H), 7.61 (d, J = 8.6 Hz, 2 H), 7.46-7.42 (m, 3 H).

  35 eV): t R = 5.52 min, m/z 372 [M+H] + . 4.1.24. 4-((4-Chlorophenyl)thio)-2-(trichloromethyl)quinoline (30d) To a solution of 4-chlorothiophenol (159 mg, 1.1 mmol) in DMSO (1.0 mL), NaH (48 mg, 2.0 mmol) was added portionwise under inert atmosphere. The mixture was stirred at rt for 20 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT min. 4-Chloro-2-(trichloromethyl)quinoline H

, 4 -

 4 dichlorophenyl)-3-methylquinoxalin-2-amine. Yield 19%. White powder. mp 152 °C. 1 H NMR (250 MHz, CDCl 3 ) δ = 9.06 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.67-7.57 (m, 1H), 7.57-7.30(m, 4H), 2.78 (s, 3H).13 C NMR (63 MHz, CDCl 3 ) δ = 147.5, 145.2, 140.4, 138.0, 135.3, 129.6, 128.8, 128.4, 128.0, 127.6, 127.0, 126.4, 123.2, 121.4, 21.1. LC-MS (ESI, 35 eV): t R = 5.49 min, m/z 304 [M+H] + . 4.1.26. 3-Methyl-N-(3-(trifluoromethyl)phenyl)quinoxalin-2-amine (31b) A solution of 2-chloro-3-methylquinoxaline L [45] (500 mg, 2.8 mmol) and 3trifluoromethylaniline (2,5 mL, 20 mmol) in anhydrous DMF (10 mL) was heated at 140 °C in a sealed vial under microwave irradiation. After completion of the reaction (45 min), CH 2 Cl 2 was added, and the organic phase was washed successively with 1M HCl and brine. The organic layer was then dried with Na 2 SO 4 , filtered and evaporated. The resulting solid was purified by silica gel column chromatography (eluent: CH 2 Cl 2 ) to afford 3-Methyl-N-(3-(trifluoromethyl)phenyl)quinoxalin-2-amine. Yield 69%. Amber powder. mp 98 °C. 1 H NMR (250 MHz, CDCl 3 ) δ = 8.24 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.87 (dd, J = 8.1, 1.1 Hz, 1H), 7.78 (dd, J = 8.2, 1.0 Hz, 1H), 7.64 -M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT7.53 (m, 1H), 7.52-7.39 (m, 2H), 7.37-7.27 (m, 1H), 6.77 (bs, 1H, NH), 2.65 (s, 3H).13 C NMR (63 MHz, CDCl 3 ) δ = 147.7, 144.6, 140.3 (d, J = 3.2 Hz), 137.9, 131.5 (q, J = 33.1 Hz), 129.4 (d, J = 6.0 Hz), 128.2, 126.9, 126.1, 124.3 (q, J = 272.9 Hz), 122.8, 119.6 (d, J = 3.8 Hz), 116.6 (d, J = 3.4 Hz), 21.03. LC-MS (ESI, 35 eV): t R = 4.75 min, m/z 304 [M+H] + . 4.1.27. 2-(4-Chlorophenoxy)-3-methylquinoxaline (31c) To a solution of 2-chloro-3-methylquinoxaline L [45] (650 mg, 3.6 mmol) and 4chlorophenol (0.37 mL, 3.6 mmol) in anhydrous DMF (15 mL), Cs 2 CO 3 (1.19 g, 3.6 mmol) was added under inert atmosphere. The mixture was stirred at 70 °C overnight. After completion of the reaction, water was added, leading to a precipitate which was separated by filtration. The resulting precipitate was then thoroughly washed with water. The precipitate was dissolved in CH 2 Cl 2 and dried with Na 2 SO 4 . After filtration and evaporation, the resulting solid was purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 1/1) to afford 2-(4-chlorophenoxy)-3-methylquinoxaline. Yield 85%. Off-white powder. mp 108 °C. 1 H NMR (250 MHz, CDCl 3 ) δ = 7.69 (dd, J = 6.1, 3.7 Hz, 1H), 7.57 (dd, J = 6.3, 3.5 Hz, 2H), 7.46-7.38 (m, 2H), 7.28 -7.19 (m, 2H), 2.81 (s, 3H). 13 C NMR (63 MHz, CDCl 3 ) δ = 155.8, 151.5, 147.9, 139.6, 139.4, 130.6, 129.7, 129.4, 128.1, 127.6, 127.4, 123.3, 20.7. LC-MS (ESI, 35 eV): t R = 4.35 min, m/z 271 [M+H] + . 4.1.28. 2-((4-Chlorophenyl)thio)-3-methylquinoxaline (31d) To a solution of 2-chloro-3-methylquinoxaline L [45] (500 mg, 2.8 mmol) and 4chlorothiophenol (405 mg, 2.8 mmol) in anhydrous DMF (10 mL), Cs 2 CO 3 (912 mg, 2.8 mmol) was added under inert atmosphere. The mixture was stirred at 70 °C overnight. After completion of the reaction, water was added, leading to a precipitate which was separated by filtration. The resulting precipitate was then thoroughly washed with water. The precipitate was dissolved in CH 2 Cl 2 and dried with Na 2 SO 4 . After filtration and evaporation, the resulting solid was purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 1/1) to afford 2-((4-chlorophenyl)thio)-3-methylquinoxaline. Yield 83%. Beige powder. mp 118 °C. 1 H NMR (250 MHz, CDCl 3 ) δ = 7.96-7.92 (m, 1H), 7.72-7.68 (m, 1H), 7.60-7.54 (m, 4H), 7.44 (d, J = 6.6 Hz, 2H), 2.77 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ = 155.3, 151.4, 141.4, 140.0, 136.7, 135.6, 129.5, 129.2, 128.6, 128.3, 128.1, 127.2, 22.4. LC-MS (ESI, 35 eV): t R = 5.51 min, m/z 287 [M+H] + . 4.1.29. 2-(4-Chlorophenoxy)-3-(trichloromethyl)quinoxaline (32c) To a solution of 2-(4-chlorophenoxy)-3-methylquinoxaline 31c (624 mg, 2.8 mmol) and PCl 5 (2.88 g, 16.8 mmol), POCl 3 was added to make a slurry (ca 5 mL). The mixture was then M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT heated in a multimode microwave oven at 100 °C, 800 W for 20 min. After completion of the reaction, the mixture was poured into ice and was then neutralized with Na 2 CO 3. The resulting solution was extracted with CH 2 Cl 2 and dried with Na 2 SO 4 . After filtration and evaporation, the resulting solid was purified by silica gel column chromatography (eluent: Petroleum Ether/CH 2 Cl 2 7/3) to afford 2-(4-chlorophenoxy)-3-(trichloromethyl)quinoxaline. Yield 72%. White powder. mp 108 °C. 1 H NMR (250 MHz, CDCl 3 ) δ = 8.11 (d, J = 8.0 Hz, 1H), 7.72-7.63 (m, 3H), 7.38 (dd, J = 6.6, 3.5 Hz, 2 H), 7.22 (dd, J = 6.6, 3.5 Hz, 2 H). 13 C NMR (101 MHz, CDCl 3 ) δ = 152.5, 151.

Ether/CH 2

 2 Cl 2 9/1) to afford 2-((4-chlorophenyl)thio)-3-(trichloromethyl)quinoxaline. Yield 72%. Yellow powder. mp 92 °C. 1 H NMR (250 MHz, CDCl 3 ) δ = 8.15-8.04 (m, 1H), 7.76-7.64 (m, 3H), 7.56 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H).

  quantities of DNA in treated and control cultures of parasite in human erythrocytes according to a SYBR Green I fluorescence-based method[START_REF] Guiguemde | Chemical genetics of Plasmodium falciparum[END_REF] using a 96-well fluorescence plate reader. Compounds, previously dissolved in DMSO (final concentration less than 0.5% v/v) were incubated in a total assay volume of 200 µL of synchronized culture suspension (2% hematocrit and 0.4% parasitaemia) for 72 h in a humidified atmosphere (84% N 2 , 10% O 2 and 6% CO 2 ) at 37 °C, in 96-well flat bottom plates. Duplicate assays were performed for each sample. After incubation, 125 µL supernatant was discarded and cells were washed twice with 125 µL 1X PBS. 15 µL re-suspended cells were transferred to 96well flat bottom nonsterile black plates (Greiner Bio-one) already containing 15 µL of the SYBR Green I lysis buffer (2X SYBR Green I, 20 mM Tris base pH 7.5, 20 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-100). Negative control, treated by solvents (DMSO or H 2 O) and positive controls (atovaquone, chloroquine and doxycycline) were added to each set of experiments. Plates were incubated for 15 min at 37 °C and then read on a TECAN Infinite F-200 spectrophotometer with excitation and emission wavelengths at 485 and 535 nm, respectively. The concentrations of compounds required to induce a 50% decrease of parasite growth (IC 50 K1) were calculated from three independent experiments.

  ELx808 Absorbance Microplate Reader. DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The 50% cytotoxic concentration (CC 50 ) was determined from the dose-response curve by using the TableCurve software 2D v.5.0. CC 50 values represent the mean value calculated from three independent experiments.

Table 1 .

 1 In

	R 4
	N
	N	R 2

vitro antiplasmodial and cytotoxicity evaluation of compounds 1-3 and 7-14: Modulations at regions I, II and III.

Table 2 .

 2

	OH
	78%
	63%	97%	99%
	99%	63%

OH Scheme 4. Synthesis of key intermediate 20. Reagents and conditions: (i) Ac 2 O, AcOH cat., 120 °C, MW, 10 min; (ii) NH 4 OH 25%, 105 °C, 3 h, sealed vial; (iii) PCl 5 6 equiv, POCl 3 as solvent, 100 °C, MW, 20 min, 800 W; (iv) POCl 3 , pyridine cat., reflux 24 h (molecule 17) or 1 h (molecule 20); (v) Et 3 N, trichloroacetyl chloride, 5 °C, 30 min; (vi) AcOH saturated with gaseous HCl, trichloroacetonitrile, 100 °C, 18 h, evaporated then refluxed in iPrOH, 5 min.

Table 2 .

 2 In

	Molecule	Structure	HepG2 CC 50 (µM)	K1 P.falciparum IC 50 (µM)	Selectivity Index c
	21a		3.2	0.6	5.3
	21b		0.7	0.9	0.8
	21c		4.3	0.6	7.2
	21d		6.9	0.4	17.2
	29a		6.2	0.5	12.4
	29b		4.0	0.6	6.7
	29d		13.3	0.8	16.6
		Hit A	16	0.4	40
		Hit B	150	1.8	83
		Hit C	50	1.1	45
		Hit D	>25 d	0.9	>28
	Doxorubicine a	0.2	-	-
	Atovaquone b	> 15.

vitro antiplasmodial and cytotoxicity evaluation of compounds 21a-d and 29a-d: modulations at region IV

Table 3 .

 3 In

	Molecule	Structure	HepG2 CC 50 (µM)	K1 P.falciparum IC 50 (µM)	Selectivity Index c
	30a		11.7	1.3	9.0
	30b		6.5	1.3	5.0
	30c		31	1.5	20.7
	30d		>15.6 d	1.1	>14.2
	32c		40.2	0.4	100.5
	32d		38.6	0.5	77.2
		Hit A	16	0.4	40
		Hit B	150	1.8	83
		Hit C	50	1.1	45
		Hit D	>25 d	0.9	>28
		Doxorubicine a	0.2	-	-
		Atovaquone b	> 15.6 d	0.001	15600
		Chloroquine b	30	0.6	50
		Doxycycline b	20	6.0	3.3

vitro antiplasmodial and cytotoxicity evaluation of compounds 30a-d, and 32c-d: modulations at region V.

a Doxorubicine was used as a cytotoxic reference-drug; b Atovaquone, Chloroquinine and doxycycline were used as antimalarial reference-drugs, c Selectivity indecies were calculated according to the formula : SI = HepG2 CC 50 / K1 IC 50 . d Highest concentration tested due to a lack of solubility.

Table 4 .

 4 Summary of SAR data at regions IV and V.

	R
	IC 50	0,4	0.6	0.5	1.3	-
	CC 50	16	3.2	6.2	11.7	-
	SI	40	5.3	12.4	9.0	-
	IC 50	1,8	0.9	0.6	1.3	-
	CC 50	150	0.7	4.0	6.5	-
	SI	83	0.8	6.7	5.0	-
	IC 50	1.1	0.6	-	1.5	0.4
	CC 50	50	4.3	-	31	40.2
	SI	45	7.2	-	20.7	100.5
	IC 50	0.9	0.4	0.8	1.1	0.5
	CC 50	>25	6.9	13.3	>15.6 *	38.6
	SI	>28	17.2	16.6	>14.2	77.2

*Highest concentration tested due to a lack of solubility.

In bold, newly identified hit-molecules

  6. A quaternary C was not observed under these experimental conditions. Anal. Calcd for C 15 H 10 Cl 3 N 3 : C, 53.20; H, 2.98; N, 12.41. Found: C, 53.60; H, 3.12; N, 11.61. LC-MS (ESI, 35 eV): t R = 4.21 min, m/z 338 [M+H] + . White powder. mp 203 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 9.11 (d, J = 8.9 Hz, 1H), 8.30 (bs, 1H), 8.02-7.85 (m, 3H), 7.73-7.66 (m, 1H), 7.49-7.39 (m, 2H), 6.78 (s, 1H). 13 C NMR (50 MHz, CDCl 3 ) δ =160.6, 157.6, 149.6, 133.9, 133.6, 129.5, 128.7, 128.3, 128.2, 123.7, 122.9, 120.1, 114.5, 71.8. A quaternary C was not observed under these experimental conditions. Anal. Calcd for C 15 H 9 Cl 4 N 3 : C, 48.29; H, 2.43; N, 11.26. Found: C, 48.41; H, 2.29; N, 11.03. LC-MS (ESI, 35 eV): t R = 4.71 min, m/z 372 [M+H] + .

	4.1.1.2. 2-Dichloromethyl-N-(2,4-dichlorophenyl)quinazolin-4-amine (3)
	Starting from 4-chloro-2-dichloromethylquinazoline G [26] (500 mg, 2.02 mmol) and 2,4-
	dichloroaniline (259 mg, 1.62 mmol).
	Yield 42%.

  5, 134.2, 133.2, 130.1, 129.2, 129.1, 128.8, 128.2, 123.9, 122.8, 120.1, 119.8 (q, J = 275 Hz), 115.2. Anal. Calcd for C 15 H 8 Cl 2 F 3 N 3 : C, 50.30; H, 2.25; N, 11.73. Found: C, 50.39; H, 2.21; N, 11.80.

  C NMR (100 MHz, CDCl 3 ) δ =157.1 (t, J = 30 Hz), 137.6, 134.5, 132.7, 126.3, 121.6, 121.5, (t, J = 300 Hz), 115.3, 103.7. Anal. Calcd for C 9 H 5 ClF 2 N 2 O: C, 46.88; H, 2.19; N, 12.15. Found: C, 46.98; H, 2.13; N, 12.35. LC-MS (ESI, 35 eV): t R = 0.85 min, m/z 229 [M-H] -.

  75%. Beige powder. mp 90 °C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.38 (d, J = 8.3 Hz, 1H), 8.23 (d, J = 8.3 Hz, 1H), 8.12-8.08 (dt, J = 8.2, 1.2 Hz, 1H), 7.89 (dt, J = 8.2, 1.2 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ =164.2, 155.0 (t, J = 30 Hz), 150.4, 136.2, 130.9, 129.6, 126.1, 123.8, 122.0 (t, J = 292 Hz). Anal. Calcd for C 9 H 4 Cl 2 F 2 N 2 : C, 43.40; H, 1.62; N, 11.25. Found: C, 43.29; H, 1.56; N, 11.42. LC-MS (ESI, 35 eV): t R = 3.42 min, m/z 249 [M+H] + .

  , 119.5, 113.5, 97.7. Anal. Calcd for C 15 H 8 Br 2 Cl 3 N 3 : C, 36.59; H, 1.62; N, 8.46. Found:

	(10)
	Yield 55%. White powder. mp 243 °C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 10.32 (bs,
	1H), 8.55 (d, J = 8.2 Hz, 1H), 8.02-7.91 (m, 3H), 7.82-7.59 (m, 3H). 13 C NMR (50 MHz,
	DMSO-d 6 ) δ =160.0, 159.5, 148.9, 136.3, 134.7, 134.4, 131.1, 130.8, 128.5, 128.3, 123.1,
	122.5

C, 36.16; H, 1.53; N, 8.50. LC-MS (ESI, 35 eV): t R = 5.41 min, m/z 494 [M+H] + .

( 11 )

 11 Yield 63%. White powder. mp 222 °C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 10.38 (bs, J = 2.3 Hz, 1H), 7.44 (dd, J = 9.0, 2.3 Hz, 1H). 13 C NMR (50 MHz, DMSO-d 6 ) δ =160.6, 157.2, 149.6, 134.5, 134.0, 131.7, 130.3, 129.2, 128.8, 128.7, 122.8, 120.1, 120.0, 114.1, 97.5. Anal. Calcd for C 15 H 8 BrCl 4 N 3 : C, 39.86; H, 1.78; N, 9.30. Found: C, 40.01; H, 1.69; N, 9.26. LC-MS (ESI, 35 eV): t R = 4.82 min, m/z 450 [M+H] + .

	1H), 8.62 (d, J = 8.3 Hz, 1H), 8.07-7.97 (m, 2H), 7.93 (s, 1H), 7.87-7.80 (m, 1H), 7.75 -7.65
	(m, 2H). 13 C NMR (50 MHz, DMSO-d 6 ) δ =160.2, 159.7, 149.1, 135.0, 134.6, 132.1, 131.8,
	130.8, 130.6, 128.7, 128.5, 123.4, 119.3, 113.7, 97.9. Anal. Calcd for C 15 H 8 BrCl 4 N 3 : C,
	39.86; H, 1.78; N, 9.30. Found: C, 39.83; H, 1.70; N, 9.45. LC-MS (ESI, 35 eV): t R = 5.41
	min, m/z 450 [M+H] + .
	4.1.3.3. N-(2-bromo-4-chlorophenyl)-2-trichloromethylquinazolin-4-amine
	(12)
	Yield 70%. White powder. mp 223 °C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 9.09 (d, J
	= 9.0 Hz, 1H), 8.38 (s, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.00-7.89 (m, 2H), 7.77-7.70 (m, 1H),
	7.63 (d,

  pyrimidine 20 (150 mg, 0.52 mmol) and 3-(trifluoromethyl)aniline (87 µL, 0.52 mmol) in ethanol (2.5 mL) were added a few drops of concentrated HCl. The vial was sealed and the reaction was heated at 100°C for 24 h.

	The solvent was evaporated under reduced pressure and the residue was purified by silica gel
	column chromatography (eluent: Petroleum Ether/EtOAc 8/2) to yield 2-(trichloromethyl)-N-
	[3-(trifluoromethyl)phenyl]thieno[3,2-d]pyrimidin-4-amine.
	Yield 55%. White solid. mp 141 °C. 1 H NMR (200MHz, CDCl 3 ) δ = 8.42 (s, 1 H),
	7.88-7.83 (m, 2 H), 7.58-7.39 (m, 4 H). 13 C NMR (50 MHZ, CDCl 3 ) δ = 161.9, 161.0, 155.0,
	138.3, 133.9, 131.9, 131.3, 129.7, 125.7, 124.3, 121.3, 118.6, 115.7, 97.4. Anal. Calcd for
	C 14 H 7 Cl 3 F 3 N 3 S: C, 40.75; H, 1.71; N, 10.18. Found: C, 40.99; H, 1.80; N, 10.39. LC-MS
	(ESI, 35 eV): t R = 4.87 min, m/z 412 [M+H] +

  3-cyanothiophen-2-yl)acetamide 27 (200 mg, 0.74 mmol) and 2,4-dichloroaniline (118 mg, 0.74 mmol) in CH 2 Cl 2 (0.3 mL), AlCl 3 (100 mg, 0.74 mmol) was added portionwise. The mixture was stirred at rt for 72 h. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column

	chromatography (eluent: Petroleum Ether/EtOAc 7/3) to afford a white solid 28a (LC-MS
	(ESI, 35 eV): t R = 4.00 min, m/z 430 (M+H) + ) which was dissolved in ethanol (2 mL) and the
	resulting solution was refluxed for 4 h. The solvent was evaporated under reduced pressure
	and the residue was purified by silica gel column chromatography (eluent: Petroleum
	Ether/CH 2 Cl 2 8/2 to 5/5) to afford N-(2,4-dichlorophenyl)-2-(trichloromethyl)thieno[2,3-
	d]pyrimidin-4-amine.
	Yield 36%. White solid. mp 181 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.94 (d, J = 9.0
	Hz, 1 H), 7.77 (bs, 1 H), 7.64 (d, J = 6.0 Hz, 1 H), 7.47 (d, J = 2.3 Hz, 1 H), 7.37 (dd, J = 5.4,
	2.3 Hz, 2 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 167.3, 160.2, 153.8, 133.5, 128.9, 128.8, 128.2,
	127.9, 123.4, 122.6, 116.8, 116.2, 96.3. Anal. Calcd for C 13 H 6 Cl 5 N 3 S: C, 37.76; H, 1.46; N,
	10.16. Found: C, 37.95; H, 1.52; N, 10.39. LC-MS (ESI, 35 eV): t R = 5.26 min, m/z 412
	[M+H] + .

  13 C NMR (50, MHz, CDCl 3 ) δ = 166.5, 164.6, 159.5, 136.8, 136.3, 129.8, 129.5, 126.7, 125.1, 118.8, 96.7. Anal. Calcd for C 13 H 6 Cl 4 N 2 S 2 : C, 39.41; H, 1.53; N, 7.07. Found: C, 39.61; H, 1.61; N, 7.16. LC-MS (ESI, 35 eV): t R = 5.45 min, m/z 395 [M+H] + .

  Calcd for C 16 H 9 Cl 5 N 2 : C, 47.27; H, 2.23; N, 6.89. Found: C, 47.01; H, 2.28; N, 6.99. LC-MS (ESI, 35 eV): t R = 5.10 min, m/z 403 [M-H] -.

	dichlorophenyl)-2-
	trichloromethylquinoline-4-amine.
	Yield 39%. Orange powder. mp 121 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.22 (d, J =
	8.3 Hz, 1 H), 8.02 (d, J = 8.3 Hz, 1 H), 7.84-7.76 (m, 1 H), 7.68-7.64 (m, 2 H), 7.54-7.51 (m,
	1 H), 7.47 (s, 1 H), 7.35-7.29 (m, 1 H), 7.11 (s, 1 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 158.6,
	147.5, 147.0, 135.4, 131.0, 130.3, 129.7, 128.3, 127.8, 126.9, 122.5, 119.8, 99.7, 98.1. Anal.

  The residue was then purified by silica gel column chromatography (gradient: Petroleum Ether/CH 2 Cl 2 8/2 + 5 mL of CH 2 Cl 2

	every	100	mL	of	eluent)	to	afford	2-(trichloromethyl)-N-[3-
	(trifluoromethyl)phenyl]quinoline-4-amine.
		Yield 73%. Yellow powder. mp 159 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.22 (d, J =
	8.3 Hz, 1 H), 8.12 (d, J = 8.3 Hz, 1 H), 7.75 (t, J = 7.7 Hz, 1 H), 7.69 (s, 1 H), 7.57-7.42 (m, 6
	H). 13 C NMR (50 MHz, CDCl 3 ) δ = 157.8, 149.0, 146.4, 140.0, 132.8, 131.3, 130.6, 130.1,
	127.6, 125.3, 121.7, 120.3, 119.3, 119.1, 119.0, 98.8, 97.5. Anal. Calcd for C 17 H 10 Cl 3 F 3 N 2 : C,
	50.34; H, 2.48; N, 6.91. Found: C, 50.73; H, 2.69; N, 6.66. LC-MS (ESI, 35 eV) t R = 5.09
	min, m/z 403 [M-H] -.

  The residue was then purified by silica gel column chromatography

	(eluent	Petroleum	Ether/CH 2 Cl 2	6/4)	to	afford	4-(4-chlorophenoxy)-2-
	(trichloromethyl)quinolone.
		Yield 72%. White solid. mp 166 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.34 (d, J =
	8.3Hz, 1 H), 8.20 (d, J = 8.6 Hz, 1 H), 7.89-7.81 (m, 1 H), 7.71-7.64 (m, 1 H), 7.48 (d, J = 8.8
	Hz, 2 H), 7.22 (s, 1 H), 7.18 (d, J = 8.8 Hz, 2 H). 13 C NMR (50 MHz, CDCl 3 ) δ = 162.6,
	158.4, 152.6, 147.4, 131.3, 130.6, 130.0, 128.0, 122.2, 121.6, 121.0, 100.6, 97.7. Anal. Calcd
	for C 16 H 9 Cl 4 NO: C, 51.51; H, 2.43; N, 3.75. Found: C, 51.69; H, 2.53; N, 3.81. LC-MS (ESI,

  Anal. Calcd for C 16 H 9 Cl 4 NS: C, 49.39; H, 2.33; N, 3.60. Found: C, 49.49; H, 2.23; N, 3.53. LC-MS (ESI, 35 eV): t R = 5.76 min, m/z 388 [M+H] + .

	Petroleum Ether/CH 2 Cl 2 8/2) to afford 4-((4-chlorophenyl)thio)-2-(trichloromethyl)quinolone.
	Yield 32%. White powder. mp 151 °C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.24-8.18 (m,
	2 H), 7.87-7.78 (m, 1 H), 7.72-7.64 (m, 1 H), 7.57-7.46 (m, 5 H). 13 C NMR (50 MHz, CDCl 3 )
	δ = 156.8, 150.2, 145.4, 136.4, 136.1, 131.0, 130.8, 130.5, 129.3, 128.4, 127.7, 125.6, 123.2,
	114.1, 97.7.

  2, 142.4, 141.2, 137.0, 132.3, 131.1, 129.9, 129.8, 128.8, 127.1, 123.2, 94.9. LC-MS (ESI, 35 eV): t R = 5.63 min, (no ionization). HRMS (ESI): m/z calcd. for C 15 H 8 Cl 4 N 2 O [M+H] + : 372.9464. Found: 372.9461. Anal. Calcd for C 15 H 8 Cl 4 N 2 O: C, 48.16; H, 2.16; N, 7.49. Found: C, 48.37; H, 2.32; N, 7.42.

  13 C NMR (63 MHz, CDCl 3 ) δ = 152.9, 147.7, 142.0, 137.2, 137.0, 136.0, 132.1, 129.9, 129.8, 129.5, 128.7, 127.8, 96.7. LC-MS (ESI, 35 eV): t R = 6.59 min, m/z 389 [M+H] + . Anal. Calcd for C 15 H 8 Cl 4 N 2 S: C, 46.18; H, 2.07; N, 7.18. Found: C, 46.33; H, 2.26; N, 6.97.

Acknowledgement

This work was supported by Aix-Marseille Université and the CNRS. The authors thank Dr Vincent Remusat for the NMR spectra.