Combined drug design of potential Mycobacterium tuberculosis and HIV-1 inhibitors: 3’,4 ’ -di-substituted -4' H -spiro [isothiochromene-3,5'-isoxazol]-4(1 H )-one

We report herein the design and synthesis of 17 new spiroheterocycles 10-26 , on the basis of two hypothetical pharmacophore structures designed to interact with both of Mycobacterium tuberculosis bacteria and HIV-1 virus. The in vitro biological evaluation of these compounds allowed us to point out seven new potential non-nucleoside hits, with MIC values in the range of 6.25 µ g/mL and two new potential anti-HIV-1 inhibitors


Introduction
6] Chromanone heterocycles have also attracted much attention owing to their important pharmacological properties. 5Their high synthetic utility and pharmacological importance have prompted us to synthesize and to study the antitubercular activity of some new spiro-isoxazolidine compounds 10-26 (Scheme 1).
] From general structure/activity relationship observations, it appears that functionalized side chain(s) such as [X-(C)n-Y], where X,Y = O, N and n = 2 or 3, are crucial for bioactivity.These atoms or centres that have critical interactions with the bacterial cell receptor constitute the pharmacophore and are vital for antimicrobial activities.These interactions must have typically precise geometric requirements that are readily described in terms of the distances between the terminal atoms and their orientation in the pharmacophore sites (O 1 -C 1 -C 2 -N 1 -H 1c ) for (S,S)-2,2'-(1,2-ethanediyldiimino)dibutan-1-ol (Ethambutol) 9a and (O 1 -N 1 -C 1 O 5 ) for 2-phenyl-3-nitrozoimidazo[1,2-a]pyrimidine (IMP) 9b .The diahedral angle is around 1-3°.The (X--Y) distance is in the range of 2.5-2.8Å and the charges are different as schown in Figures 1 and 2.  As an extensive continuation of our study on the structure-antibacterial activity relationships in 3-nitroso-imidazo[1,2-a]pyridine (-pyrimidine) derivatives, 8-9b we performed an investigation of spiroheterocycles 10-26 because they represent an attractive model for a theoretical and experimental study of the pharmacophore and their medical applications because of the large variability and combination in their substituents (R 1 and R 2 ).Moreover, given the current interest in selective drugs through the development of molecules that recognize simultaneously specific tuberculosis and HIV-1 sites, we became interested in the combined synthesis, antitubercular and anti-HIV screenings of spiroheterocycles having two rigid pharmacophore systems (O=C-C-O and O-C-O-N) as it was postulated in Figure 3.The (X---Y) distance length of most significant pharmacophore site to inhibit Mycobacterium tuberculosis is probably (O 1 -C 2 -S 1 ) which possesses the requested geometrical parameters and alternative charges (Table 3).The molecules with the activity contain two substituted phenyl groups on each of the C 3 ' and C 4' atoms of the semi-conjugated isoxazoline ring.The replacement of the two phenyl groups by para-R 1 -phenyl and para-R 2 -phenyl in compound 10 or para-R 1 -phenyl in 15 results in a drastic change of antitubercular activity (from 23 to 91 %inhib.).The increase in activity could be due to the increase of the hydrophobic character that the alkyl and alkyloxy groups confer on the molecule.Hydrophobic molecules with rigid, planar structures such as aromatic rings, have been shown to have the ability to insert into membranes and induce localized permeability changes leading to leakage out of the membrane. 15The combination of methyl and nitro groups (18 and 24) while also hydrophobic and very easily inserted into the membrane, are much less likely to cause disruption of the lipid packing order.That can be explained by possible cell membrane disturbances as the nitro group is too small to have this type of effect.The enhanced antitubercular inhibition observed in the presence of (15, 16, 17, 19, 21, 22 and 24) is then more likely due to its interaction with some intracellular target.The presence of a strong or poor electron-withdrawing group must alter the nature of the compound in such a way as to promote binding to the target(s).

Evaluation of anti-HIV-1 activity in vitro
As shown in Table 4, two of these spiranic compounds show anti-HIV-1 activity.In this series however, neither the degree of lipophilicity (cLogP) nor the molecular polar surface area (TSPA) correlates positively with the anti-HIV-1 activity.These parameters were computed using AM1 geometries optimised using the GAUSSIAN03 package, 22 for which more information can be found elsewhere. 23nterestingly, 25-26 that has NO 2 groups has a smaller Log P value and a very high hydration and TPSA value (of 95 compared to 47.7).These are also the most active species, and should be related to these properties.Although, no relationship was obtained we can suggest that high Log P and hydration energies will lead to active molecules.A comparison of compounds with regard to their in vitro activity against HIV-1 reveals specificity notably when R1 is nitro substituent.Compounds 25 and 26 have 11 micromolar activity against HIV-1 while others have no activity against HIV-1.It appears that in this limited series, the presence of a more polar substituent on the phenyl of oxazoline ring (compound 25 and 26) enhances the activity (Table 5).

OSIRIS Calculations
The OSIRIS Property Explorer used in this paper is an integral part of Actelion's inhouse substance registration system.It lets you draw chemical structures and calculates on-the-fly various drug-relevant properties whenever a structure is valid.Prediction results of compounds 10-26 molecular properties (solubility, druglikness and drug-score) are abstracted (Table 6).
Properties with high risks of undesired effects like mutagenicity or a poor intestinal absorption are shown in red.Whereas a green color indicates drug-conform behaviour. 17 18 as a sum of fragment contributions.O-and N-centered polar fragments are considered.PSA has been shown to be a very good descriptor characterizing drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and blood-brain barrier penetration. 18rediction results of compounds 10-26 molecular properties (TPSA, GPCR ligand and ICM) are valued (Table 7).The 3',4'-di-unsubstituted-aryl spiro-isoxazoline compound 10 is among the least active substances to have been evaluated as antituberculosis agents in this series.Accordingly, an effort was initiated to establish a pharmacophore hypothesis to delineate the requirements of the active site via a comprehensive program of analogue synthesis and evaluation of the effects of structural modification(s) on anti-tuberculosis activity of 10.
We then set out to determine the resultant in vitro and in vivo effects of chemical alterations in each region.The unsubstituted phenyl-rings of 10 endowed anti-tuberculosis activity.The modulating anti-tuberculosis effect(s) of substituents having different electronegative properties, located at the para-phenyls sites comprising the two phenyl-rings of region III, were ascertained next.A chloro substituent located at the 4-position on the phenyl ring of 24 generated higher anti-tuberculosis activity relative to 26 and 10 in the Alamar test.However, in general p-nitrophenyl or p-methoxy-phenyl proved to be unhelpful as substituents R 2 .We postulate that the strong tendency to form a (O=C-C-O) dipolar pharmacophore site in the predominant (ketooxnium) form is likely to be responsible for the lack of biological activity observed with these semi π-conjugated isoxazoline derivatives.If this hypothesis is correct, by modifications of 10 we may be able to modulate the degree of interaction of the compound with Mycobacterium tuberculosis bacteria.

Petra calculations
PETRA is a program package comprising various empirical methods for the calculation of physicochemical properties in organic molecules.All methods are empirical in nature and have been developed over the last 20 years in the research group of Prof. J. Gasteiger. 19The following chemical effects can be quantified: heats of formation, bond dissociation energies, sigma charge distribution, π-charge distribution, inductive effect, resonance effect and delocalization energies and polarizability effect. 19o to check the apparent antitubercular activity of compounds (24, 19, 21, 22, 15, 17 and 16) and to look for evidence of structure-activity via Mycobacterium tuberculosis bacteria we performed Petra Calculations 19 with 10-26 (Table 8).The Petra software calculations confirmed that all compounds 10-26 have a clear preference for forming two antibacterial dipolar pharmacophore sites (O 4 =C 4 -C 3 -O 1' ) and (S 2 -C 3 -O 1' ) though their estimated partial π charges respectively for O 4 , O 1' are of different charges (-0.0827 e and +0.0309 e) for compound 21 for example.The Petra calculations with the other spiroisoxazoline 10-26 are shown and summarised as follows in Table 7.
Compound 21 has the same partial π-charges [+0.0275 for O 4 and -0.0827 for O 1' almost as tightly as 10 but its activity, as evidenced by a lack of observable antitubercular results, is considerably better (91% inhib.for 21 instead of 23% inhib.for 10).Comparison of the couples of compounds (11,15), (17,21) and (18,25) with both the same substituents shows no detectable change in the antitubercular activity.In the light of these observations, the lack of antitubercular activity by the latter seven compounds (15,16,17,19,21,22 and 24) is explicable in terms of the existence of dipolar pharmacophore site (O 4 =C 4 -C 3 -O 1' ).
A number of important points emerge concerning their antitubercular properties.The positive results we have recorded, while encouraging for purposes of new drug design, confirm that very likely most of these compounds could be used without great risk of toxicity in diverse antibacterial activity.Based on their structural properties, these compounds may be useful as antitubercular agents with high activity or as potential antiviral agent. 20RKAT USA, Inc.
These results prompt several pertinent observations: (i) This type of spiro-isoxazoline can furnish an interesting model for studying the interaction of spiro-isoxazoline antibiotics with DNA because the possible positive centre-bonding of a O 1 to the negatively charged centers of DNA is generally favoured; (ii) The rigid spiro geometric configuration enables us to prepare specific molecules for multi-therapeutic materials.

Experimental Section
Antitubercular evaluation Anti-tuberculosis activity assays were provided by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) at the Southern Research Institute, Birmingham, AL, USA.Screening was conducted at 6.25 µg/mL against Mycobacterium tuberculosis H37Rv (ATCC 27294) using the Microplate Alamar Blue Assay. 16Physicochemical proprieties of compounds 10-26 such as (cLogP, solubility, Druglikness, Drug-Score) and (cLogP, TPSA, GPCR Ligand, ICM, Kinase Inhibitor) and Atomic π−charges of heteroatoms are calculated by using respectively the Osiris, Molinspiration and Petra Calculations Programs.
The procedure used in anti-HIV screening (a).Candidate agent is disssolved in dimethyl sulfoxide (unless otherwise instructed) then diluted 1:100 in cell culture medium before preparing serial half-LoglC dilutions.T4 lymphocytes (CEM cell line) are added and after a brief interval HIV-1 is added, resulting in a I :200 final dilution of the compound.Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls.(b).Cultures are incubated at 37° in a 5% carbon dioxide atmosphere for 6 days.(c).The tetrazolium salt, XTT, is added to all wells, and cultures are incubated to allow formazan color development by viable cells.(d).Individual wells are analyzed spectrophotometrically to quantitate formazan production, and in addition are viewed microscopically for detection of viable cells and confirmation of protective activity.(e).Drug-treated virus-infected cells are compared with drugtreated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drug-containing wells without cells, etc.) on the same plate.(f).Data are ARKAT USA, Inc. reviewed in comparison with other tests done at the same time and a determination about activity is made.
General procedure 1 H and 13 C NMR spectra were recorded on a Bruker Spectrospin spectrometer operating at 80 MHz for 1 H (Université Paul Sabatier, Toulouse, France), an AC 200 spectrometer (operating at 200.12 MHz for 1 H, 50.32 MHz for l3 C), (Université de Franche-Compté, Besançon, France).Chemical shifts are listed in ppm and are reported relative to tetramethylsilane ( 1 H, 13 C), residual solvent peaks being used as internal standard.Complete assignments of the 13 C spectra required non-decoupled 13 C-NMR spectra with selective 1 H decoupling. Spectrometers, mass spectra on a Platform II Micro Mass spectrometer, Infrared spectra were obtained on a BECKMAN 310 spectrometer.Mass spectra on a HEWLETTPACKARD 5989A Mass spectrometer (70 eV) and elemental analysis (Université Paul Sabatier, Toulouse, France).

Figure 3 .
Figure 3. Possible interactions between antitubercular or anti-HIV-1 drugs and their specific biological targets.

Figure 3 .
Figure 3.A view of compound 20 with the atom-numbering scheme. 10

Figure 3 .
Figure 3.In vitro activity against HIV-1 results of active compound 25.

Table 1 .
Selected IR and 1 H NMR data of compounds 10-26

Table 2 .
Selected13C NMR data of compounds

ARKAT USA, Inc. Evaluation of anti-tuberculosis activity in vitro Fifteen compounds 10-26 have
been evaluated as anti-tuberculosis agents through the TAACF tuberculosis screening program, but only half of them have been shown to inhibit significantly the growth of Mycobacterium tuberculosis H 37 Rv using the Alamar assay at the first level adopted for in vitro screening.Just one compound 10 displayed modest in vitro activity (less than 50%).Not surprisingly, some of these spiro-isoxazoline derivatives are currently being modified in the goal to be examined at the in vivo stage of the tuberculosis screening program.The antituberculosis data are summarised in Table4.The most effective inhibitors are

Table 4 .
Antitubercular activity of spiro-isoxazoline compounds 10-26 a a the antitubercular screening of compounds 20 and 23 has not been done.

Table 5 .
Anti-HIV activity of selected compounds against HIV-1 cell line in vitro a (W): Wiener Index.b E HYD is the hydration energy in kcal/mol, the TPSA 21 is 47.7 for 11

, 14, 15, 16, 19, and
Molinspiration calculations cLogP (octanol/water partition coefficient) is calculated by the methodology developed byMolinspiration as a sum of fragment-based contributions and correction factors.The method is very robust and is able to process practically all organic, and most organometallic molecules.Molecular Polar Surface Area TPSA is calculated based on the methodology published by Ertl et al.
95.0 for 25-26.The octanaol/water partition coefficient is shown, where the values in parenthesis are regular LogP.

Table 6 .
OSIRIS calculations of spiro-isoxazoline compounds 10-26 b b The Osiris calculation of compounds 20 and 23 has not been done.