ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1991, p. 2238-2245

Vol. 35, No. 11

0066-4804/91/112238-08$02.00/0 Copyright © 1991, American Society for Microbiology

Crystal Structure and Molecular Structure of Mefloquine Methylsulfonate Monohydrate: Implications for a Malaria Receptor JEAN M. KARLEl* AND ISABELLA L. KARLE2 Department of Pharmacology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC 20307-5100,1 and Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375-50002 Received 28 February 1991/Accepted 29 July 1991

The crystal structure of (+)-mefloquine methylsulfonate monohydrate was determined by X-ray diffraction and was compared with the crystal structures of mefloquine hydrochloride and mefloquine free base. The conformation of mefloquine was essentially the same in all three crystalline environments and was not dependent on whether mefloquine was a salt or a free base. In mefloquine methylsulfonate monohydrate, the angle between the average plane of the quinoline ring and the average plane of the piperidine ring was 76.90. 0-1 distance was 2.730 ± 0.008 A (1 A-0.1 nm), which is close to the The intramolecular aliphatic N-13 0 distance found in the antimalarial cinchona alkaloids. The hydroxyl group formed a aliphatic N hydrogen bond with the water molecule, and the amine group formed hydrogen bonds with two different methylsulfonate ions. The crystallographic parameters for (±t)-mefloquine methylsulfonate monohydrate were as follows: C17H17F6N20' * CH3S03- * H20; Mr = 492.4; symmetry of unit cell, monoclinic; space group, P21/a; parameters of unit cell, a was 8.678 + 0.001 A, b was 28.330 ± 0.003 A, c was 8.804 ± 0.001 A, was 97.50 0.010; the volume of the unit cell was 2145.9 A3; the number of molecules per unit cell was 4; the calculated density was 1.52 g cm 3; the source of radiation was Cu Ka (A = 1.54178 A); ,I (absorption coefficient) was 20.46 cm-'; F(OOO) (sum of atomic scattering factors at zero scattering angle) was 1,016; room temperature was used; and the final R (residual index) was 6.58% for 1,740 reflections with F. > 3a (F). Since the mechanism of antimalarial action and the mechanism of mefloquine resistance may involve hydrogen bond formation between mefloquine and a cellular effector or transport proteins, the common conformation of mefloquine found in each crystalline environment may define the orientation in which mefloquine forms these potentially critical hydrogen bonds with cellular constituents. .

The continuing spread of multi-drug-resistant Plasmodium falciparum malaria (22) necessitates the development of new antimalarial drugs that are active against these protozoa. Mefloquine hydrochloride was developed by the Walter Reed Army Institute of Research and Roche Laboratories (Nutley, N.J.) as an alternate choice for the treatment of chloroquine-resistant malaria and is marketed under the trade name Lariam. However, since pockets of clinical resistance to mefloquine have been reported in both Africa (8) and southeastern Asia (2, 11), new drug therapies which overcome the resistance problem need to be developed. Understanding the mode of action and mode of resistance of mefloquine on a molecular level should aid the development of new treatment strategies. Although mefloquine alters the pH of the parasite's acid vesicles at nanomolar concentrations (13), the precise mode of action of mefloquine is not well defined. Structure-activity studies demonstrate that mefloquine loses its antimalarial activity if the amine and hydroxyl groups of mefloquine are acetylated (26). Formation of an 0-methyl or O-ethyl derivative as well as conversion of the saturated 2-piperidyl group to an unsaturated 2-pyridyl group also abolishes activity (26). This apparent structural requirement to have underivatized amine and hydroxyl groups suggests that these groups need to be free to hydrogen bond to cellular constituents. Thus, antimalarial activity may depend upon the ability of mefloquine *

Corresponding author.

a cellular "effector." In addition, structure-activity studies (5, 6, 26) on the 3- and 4-piperidyl isomers of mefloquine and of the 3,6-bis(trifluoromethyl)9-phenanthryl analog of mefloquine show that the physical placement of the hydroxyl and the amine groups with respect to each other is critical to antimalarial activity. These data further support the hypothesis that these compounds must hydrogen bond to cellular constituents to effect antimalarial activity and must do so with a specific geometry. At present, the mechanism(s) of resistance to mefloquine has not been elucidated (21). The three-dimensional structure of mefloquine defines how the mefloquine molecule physically interacts with a cellular effector or transport proteins. In order to determine the preferred conformation(s) of mefloquine, we undertook the comparison of the structure of mefloquine as both a free base and a salt in three different crystalline environments. The crystal structure of mefloquine methylsulfonate monohydrate (Fig. 1) was determined by X-ray diffraction and was compared with the crystal structures of mefloquine hydrochloride previously determined by us (lOa) and of mefloquine free base previously determined by Oleksyn and colleagues (18, 19). Although mefloquine appears to contain rotatable bonds, a single conformation of mefloquine was observed. Mefloquine assumed this conformation both as a free base and as a salt. Using this conformation, we diagramed the geometry of the interaction of mefloquine via hydrogen bond formation with cellular recep-

to hydrogen bond to

tors.

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X-RAY CRYSTAL STRUCTURE OF MEFLOQUINE

1991

2239

B

3

CH30 "G6HOH

2

ROH

0R

a tS

C

W

3

0

"H

2

~~4

FIG. 1. (A) Chemical structure and numbering scheme (in italics) of mefloquine methylsulfonate. The stereochemistry about C-l1 and C-12 is either 11R,12S or 11S,12R. (B) Chemical structure of quinidine salt.

TABLE 1. Fractional coordinates and thermal parameters Ueqa Fractional coordinate

Atom

F-1 F-2 F-3 F-4 F-5 F-6

y

z

(0.0004) (0.0005) (0.0005) (0.0004) (0.0004)

0.2936 (0.0001) 0.3021 (0.0001) 0.2583 (0.0001) 0.2888 (0.0001) 0.2896 (0.0001)

(0.0005) 0.3076 (0.0006) 0.3592 (0.0007) 0.3902 (0.0007) 0.3682 (0.0007) 0.3099 (0.0007) 0.2811 (0.0007) 0.2271 (0.0008) 0.2036 (0.0007) 0.2293 (0.0007) 0.2833 (0.0007) 0.3850 (0.0008) 0.2079 (0.0008) 0.4126 (0.0007) 0.5719 (0.0007) 0.6129 (0.0006) 0.7637 (0.0008) 0.8931 (0.0008) 0.8571 (0.0008) 0.7042 (0.0007) 0.1799 (0.0009) 0.4242 (0.0006) 0.2086 (0.0006) 0.3736 (0.0005) 0.1023 (0.0005) 0.2199 (0.0002) 0.1399 (0.0008) 0.6092 (0.0056) 0.5346 (0.0049) 0.3402 (0.0051) 0.0632 (0.0074)

0.2693 (0.0001) 0.2397 (0.0002) 0.2289 (0.0002) 0.1835 (0.0002) 0.1464 (0.0002) 0.1551 (0.0002) 0.1202 (0.0002) 0.1325 (0.0002) 0.1796 (0.0002) 0.2149 (0.0002) 0.2035 (0.0002) 0.2702 (0.0002) 0.2654 (0.0003) 0.0964 (0.0002) 0.0822 (0.0002) 0.0348 (0.0002) 0.0162 (0.0002) 0.0494 (0.0002) 0.0988 (0.0002) 0.1162 (0.0002) 0.3901 (0.0002) 0.0930 (0.0002) 0.4707 (0.0002) 0.4535 (0.0002) 0.4682 (0.0002) 0.4497 (0.0001) 0.0558 (0.0002) 0.0350 (0.0018) 0.0130 (0.0015) 0.0850 (0.0016) 0.0545 (0.0025) 0.0272 (0.0031)

-0.4296 (0.0004) -0.3108 (0.0005) -0.5097 (0.0005) 0.1452 (0.0005) 0.0215 (0.0004) 0.2554 (0.0004)

x

0.2535 0.4813 0.4445 0.3428 0.1145 0.1470

N-i C-2 C-3 C-4 C-4a

C-s C-6 C-7 C-8 C-8a C-9 C-10

C-1i

C-12 N-13

C-14 C-iS C-16

C-17 C-18 0-1 0-2 0-3 0-4 S W-1c H-13Ad H-13Bd

H-Old

-0.1542 (0.0006) -0.2819 (0.0007) -0.3281 (0.0007) -0.2342 (0.0007) -0.0936 (0.0007) 0.0132 (0.0007) 0.1467 (0.0007) 0.1827 (0.0007) 0.0824 (0.0007) -0.0609 (0.0007) -0.3836 (0.0008) 0.1251 (0.0007) -0.2788 (0.0007) -0.1932 (0.0007) -0.2494 (0.0005) -0.1654 (0.0008) -0.1811 (0.0008) -0.1278 (0.0008) -0.2188 (0.0008) 0.3097 (0.0009) -0.4382 (0.0005) 0.4446 (0.0005) 0.2502 (0.0005) 0.1777 (0.0005) 0.2948 (0.0002) -0.5333 (0.0008) -0.3565 (0.0058) -0.2357 (0.0048) -0.4831 (0.0051) -0.5808 (0.0074) -0.4929 (0.0102)

u,,b (A2) 0.057 (0.002) 0.079 (0.002) 0.080 (0.002) 0.066 (0.002) 0.058 (0.002) 0.067 (0.002) 0.038 (0.002) 0.038 (0.002)

0.04i (0.003) 0.035 (0.002) 0.038 (0.002) 0.047 (0.003) 0.054 (0.003) 0.048 (0.003) 0.040 (0.002) 0.035 (0.002) 0.045 (0.003) 0.048 (0.003) 0.038 (0.002) 0.036 (0.002) 0.037 (0.002) 0.052 (0.003) 0.054 (0.003) 0.054 (0.003) 0.042 (0.002) 0.069 (0.003) 0.045 (0.002) 0 .064 (0.002) 0.070 (0.002) 0.059 (0.002) 0.043 (0.001) 0.069 (0.003) 0.041 (0.017) 0.022 (0.014) 0.022 (0.014) 0.037 (0.025) 0.171 (0.038)

H-WlAd,e H WlBd,e 0.1647 (0.0096) a Values in parentheses are estimated standard deviations. b Valuesin Ua.q = aajaj where i and i are the summation indices, a is the length of the basis vector of the reciprocal lattice, and a is the basis vector of the direct lattice. c d I

Oxygen atom of water molecule. These atoms were refined isotropically. Hydrogen atom of water molecule.

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KARLE AND KARLE

TABLE 2. Bond lengths Bond length

Bond

(A)a

1.335 (0.007) F-1--C-9 ..................................... 8 F-3--C-9 ..................................... 1 .32 (0.008) 1.329 (0.007) F-5--C-10..................................... 1.301 (0.009) N-1--C-2 ..................................... 1.383 (0.009) C-2--C-3 ..................................... C-3--C-4 ..................................... 1.367 (0.009) 1.533 (0.009) C-4--C-11 ...................... 1.426 (0.009) C-4a--C-8a ..................................... C-6--C-7 ..................................... 1.391 (0.010) C-8--C-8a ............................... 1.438 (0.009) 1.539 (0.008) C-11--C-12 ..................................... 1.492 (0.008) C-12-N-13 ..................................... 1.511 (0.008) N-13--C-14 ..................................... 1.521 (0.010) C-15---C-16 ..................................... 1.733 (0.007) C-18-S ..................................... 0-3-S .1.444(0.005) ............

a

.

Bond length

Bond

(A)'

1.336 (0.008) F-2--C-9 1.338 (0.008) F-4-C-10 1.329 (0.008) F-6--C-10 1.348 (0.008) N-1--C-8a 1.509 (0.009) C-2---C-9 1.418 (0.009) C-4--C-4a 1.409 (0.009) C-4a--C-5 ............... 1.366 (0.010) C-5--C-6 ..................................... C-7--C-8 ..................................... 1.373 (0.009) 1.496 (0.010) C-8--C-10............................. 1.423 (0.008) C-11--0-1 ..................................... 1.522 (0.009) C-12--C-17 ..................................... 1.486 (0.010) C-14--C-15 ..................................... 1.516 (0.009) C-16--C-17 ..................................... 1.461 (0.005) 0-2-S 0-4-S .1.450(0.005) .....................................

.....................................

......................................

.....................................

.....................................

.....................................

..................

......

.....................................

Values in parentheses are estimated standard deviations.

MATERIALS AND METHODS

(+)-Mefloquine methylsulfonate monohydrate [DL-erythro-a-2-piperidyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol methylsulfonate monohydrate] was synthesized under contract by Aerojet Chemical Company (Sacramento, Calif.) and was crystallized from ethyl acetate-hexane. Diffraction data were collected from a clear colorless plate, 1.2 by 1.8 mm and 0.5 mm thick, in the 0-20 mode (25) to a maximum 20 value of 1100 on the four-circle diffractometer (R3m/micro Nicolet; Siemens, Madison, Wis.) with a graphite monochromator. The X-ray source was Cu Ka radiation (50 kV, 40 mA). The range of indices were as follows: h, -9--9; k, -30--*0; and 1, 0-*9. The total number of independent reflections was 2,706. The standard reflections 0,0,3, 0,12,0, and -1,-4,2 were monitored after every 60 intensity measurements. The standards remained constant within 2.2%. The lattice parameters were based on 25 centered reflections, with 20 values between 250 and 43°. The data were

corrected for Lorentz and polarization effects, but no correction for absorption or extinction was used. The structure was solved routinely by direct-phase determination (10). All of the nonhydrogen atoms were found in the first E map. Coordinates for hydrogen atoms attached to the oxygen and nitrogen atoms were found in the difference maps. Least-squares refinement was performed by using 1,740 reflections with Fo > 3ou(F,) (Rmerge = 0.012), where F,, is the observed structure factor. Coordinates for all atoms except the hydrogen atoms on the carbon atoms were refined (on F, where F is the structure factor) with a blocked cascade program in the SHELXTL system (24). The hydrogen atoms that were attached to the carbon atoms were placed in idealized positions and were allowed to ride with the carbon atoms. Anisotropic thermal parameters for the C, N, 0, and F atoms and isotropic thermal parameters for hydrogen atoms attached to the nitrogen and oxygen atoms were refined for a total of 310 parameters. The final values for R (residual index) and wR (a weighted correla-

TABLE 3. Bond angles Bond angle

Bond angle

(o)a

(oa)

116.5 (0.5) C-2-N-1--C-8a ....................................... 115.0 (0.6) N-1--C-2--C-9 ....................................... C-2--C-3--C-4 .........................................119.3 (0.6) 119.8 (0.6) C-3---C-4--C-11 ....................................... 125.1 (0.6) C-4--C-4a--C-5 ....................................... C-5--C-4a--C-8a ....................................... 119.5 (0.6) 121.2 (0.6) C-5--C-6--C-7 ....................................... C-7--C-8--C-8a ....................... ................ 119.9 (0.6) 120.1 (0.6) C-8a-- C-8--C-10 ....................................... 117.2 (0.6) N-1--C-8a- C-8 ....................................... C-4--C-11---C-12 ........................................ 110.8 (0.5) C-12--C-11--0-1 ....................................... 106.9 (0.5) C-11---C-12--C-17 ....................................... 115.1 (0.5) C-12-N-13--C-14 ....................................... 112.4 (0.4) 111.5 (0.6) C-14--C-15--C-16 ....................................... 111.5 (0.5) C-12--C-17--C-16 ....................................... C-18-S--0-3 ....................................... 107.1 (0.4) 106.0 (0.3) C-18-S--0-4 ....................................... 111.7 (0.3) 0-3-S--0-4 ....................................... a

Values in parentheses are estimated standard deviations.

N-1---C-2---C-3.125.2 (0.6) C-3--C-2--C-9 .119.8 (0.6) .119.2 (0.6) C-3--C-4--C-4a C-4a--C-4--C-11 .121.0 (0.5) C-4--C-4a--C-8a .115.4 (0.6) C-4a--C-5--C-6 .120.4 (0.6) C-6---C-7--C-8 .120.8 (0.6) C-7--C-8--C-10 .120.0 (0.6) N-1--C-8a--C-4a.124.5 (0.6) 118.3 (0.6) C-4a- C-8a--C-8 ............ 111.6 (0.5) C-4--C-11--0-1 .............. 108.1 (0.5) C-11--C-12-N-13 .............. 108.5 (0.5) N-13--C-12--C-17 .............. 110.5 (0.5) N-13--C-14--C-15 .............. 110.5 (0.5) C-15--C-16--C-17 .............. 106.9 (0.3) C-18-S--0-2 .............. 112.9 (0.3) 0-2-S- 0-3 .............. 111.8 (0.3) 0-2-S--0-4 .............. .

.

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2241

TABLE 4. Selected torsion angles for the mefloquine molecule Bond ~~ Torsion ~ ~ ~angle (¶10Bon

Bond Bond

C-2--C-3--C-4--C-11 .......................................... -175.8 C-11--C-4--C-4a-C-8a .......................................... 175.8 C-3-C-4--C-11- -1 .......................................... -19.6 C-4a--C-4-C-11- -1 .......................................... 162.9 C-4--C-11--C-12-C-17 .......................................... -54.8 O-1-C-11--C-12--C-17 .......................................... 67.0 C-17-C-12-N-13-C-14 .......................................... 58.0 N-13--C-12-C-17--C-16 .......................................... -57.1 N-13-C-14-C-15--C-16 .......................................... 55.2

Torsion angle (0)0 -2.8 C-11--C4--C-4a-C-5 ........................................... C-3-C-4-C-11-C-12 ..................... 99.4 C-4a-C-4-C-11--C-12 ........................................... -78.1 C-4--C-11--C-12-N-13 ........................................... -176.2 O-1--C-11-C-12-N-13 ........................................... -54.5 C-11--C-12-N-13--C-14 .......................................... -176.6 C-11--C-12--C-17--C-16 ........................................... -178.3 C-12-N-13-C-14-C-15 .......................................... -58.1 C-14--C-15--C-16--C-17 ........................................... -54.9 ......................

a Estimated standard deviations for the torsion angles range from 0.5 to 0.90.

tion factor) were 6.58 and 5.96%, respectively; w was calculated from the following: w = 1/[r2( F ) + 0.0006(F0)2]. The final difference in electron density was P max = 0.34 and P | mi = -0.30 eA3 (1 A = 0.1 nm). S = 1.2. (A/cr)max - 0.11, where p is the amplitude of the electron density, S is a goodness of fit value, and (A/v) is the amount of change in a parameter divided by the estimated standard deviation of a parameter. Atomic scattering factors were those incorporated in SHELXTL (24). All of the 'stereographic images of superimposed structures were created on an Evans and Sutherland PS390 graphics system (Salt Lake City, Utah) by using SYBYL software (Tripos, St. Louis, Mo.) and a Hewlett-Packard 7550A graphics plotter (Rockville,' Md.). RESULTS

Coordinates and thermal parameters (Ueq values) (see Table 1, footnote b, for the definition of Ueq) for the non-hydrogen atoms and coordinates for the refined hydrogen atoms are listed in Table 1, bond lengths are listed in Table 2, and bond angles are listed in Table 3. The bond length of the hydrogen atoms attached to the carbon atoms was kept fixed at 0.96 A throughout the refinement procedure. Selected torsion angles are listed in Table 4, and the numbering scheme for mefloquine methylsulfonate is shown in Fig. 1. Mefloquine crystallized as a secondary amine salt, with one water molecule and one methylsulfonate ion per mefloquine molecule. The hydrogen atom from the methylsulfonate group resided on the piperidine nitrogen atom, making the nitrogen tetrahedral (Fig. 2). The piperidine ring assumed a chair conformation, with atoms C-12, N-13, C-15, and C-16 being coplanar. The root mean square distance of these atoms from the average plane through these four atoms was 0.012 A. The angle between the average plane of the quinoline ring and the average plane of the piperidine ring was 76.90. The aryl group was equatorial to the piperidine ring. The N-13 to 0-1 intramolecular distance was 2.730 ± 0.008 A. A -54.5 ± 0.60 torsion angle for atoms O-1-C-11--C-12-N-13 demonstrated that mefloquine is gauche about the C-11-C-12 bond. The C-4-C11-C-12-N-13 torsion angle of -176.2 ± 0.50 placed the amine group as nearly as far as possible from the quinoline

ring.

Each hydrogen atom of the amine and hydroxyl groups of mefloquine and of the water molecule formed hydrogen bonds (Table 5). Each oxygen atom of the methylsulfonate

group and the oxygen atom of the water molecule accepted hydrogen bonds. Atom 0-2 of the methylsulfonate group accepted two hydrogen bonds, one from N-13 of mefloquine and one from the water molecule. The molecules were oriented in the crystal (Fig. 3) such that the hydrophilic groups formed chains of hydrogen bonds parallel to the a axis. In between these chains were the hydrophobic trifluoromethyl groups, the quinoline rings, and the hydrophobic sides of the piperidine ring and the methylsulfonate groups.

The three-dimensional conformation of mefloquine as its methylsulfonate salt was compared with the conformation'of mefloquine hydrochloride and mefloquine free base to establish the orientation of the pyrimidine ring with respect to the quinoline ring and the hydroxyl group. Figure 4 illustrates the superposition of four mefloquine molecules: the methylsulfonate'salt,-the hydrochloride salt (10a), and two conformations of mefloquine free base (18, 19). It is evident from Fig. 4 that all four mefloquine molecules share nearly the same conformation. Table 6 li'sts details of the four meflo-

FIG. 2. Space-filling drawing of the crystal structure of mefloquine as its methylsulfonate salt. The methylsulfonate ion is not shown. The nitrogen atoms are colored solid black, the oxygen atom is striped, and the fluorine atoms are dotted. Only the carbon and hydrogen atoms are white. A hydrogen atom originally from the methylsulfonate group is attached to N-13, the aliphatic nitrogen atom of mefloquine, giving the nitrogen atom a positive charge and making mefloquine a secondary amine salt. The spheres represent 75% of the van der Waals radii. Only one enantiomer of ,mefloquine is illustrated.

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FIG. 3. Packing diagram of mefloquine methylsulfonate monohydrate viewed down the c axis. The a axis is vertical, and the b axis is horizontal. The box represents the smallest repeating unit in the crystal. The dashed lines represent hydrogen bonds. The methylsulfonate ions were drawn with thicker lines to distinguish them from the mefloquine molecules.

mefloquine defines the direction of hydrogen bonding, the conformation of mefloquine may enable the further characterization of a possible common receptor site for the amino alcohol antimalarial agents. The direction of hydrogen bonding, as defined by the conformation of mefloquine found in the crystal structures, is illustrated in Fig. 5. Dots have been drawn around the portions of mefloquine that participate in hydrogen bonding at the van der Waals radius of the hydrogen atoms. Generally, the angle of a hydrogen bond, the donor-hydrogenacceptor angle, is 150 to 1800, although smaller angles do occur. Mefloquine, being a secondary amine, can form up to three hydrogen bonds, one with the hydroxyl group and two with the amine group. Consequently, both salts of mefloquine formed three intermolecular hydrogen bonds with either counterions or water solvate molecules. The position and conformation of the pyrimidine ring define the position of the hydrogen atoms of the amine group, since the amine group of a secondary amine salt should always be tetrahedral. Even though the hydrogen atoms of the hydroxyl groups, i.e., H-01, of both molecules are 0.37 A apart in Fig. 5, these hydrogen atoms can readily rotate into the same position. The hydrogen atoms of the hydroxyl groups in the

0-1 distance ranged from quine structures. The N-13 2.73 to 2.85 A. This range is close to the aliphatic N to hydroxyl 0 distance of 2.84 to 3.22 A exhibited by the active cinchona alkaloids in their crystalline state (1, 4, 7, 9, 12, 14-17, 23). For all four mefloquine molecules, the C-4-C-11-C-12-N-13 torsion angle was close to 1800, placing N-13 about as far away from the quinoline ring as possible. All four molecules were gauche about the C-11-C-12 bond, with N-13 positioned between the oxygen and hydrogen atoms attached to C-11. The average plane of the piperidine ring twisted away from the average plane of the quinoline ring by 68.3 to 76.90.

DISCUSSION The crystalline conformation of mefloquine as its methylsulfonate salt was virtually the same as the crystalline conformation of mefloquine as either its hydrochloride salt or as a free base (Fig. 4 and Table 6). Solution nuclear magnetic resonance spectra of both the free base and the HCl salt are also consistent with this conformation (3, 20). Thus, a preferred conformation for mefloquine was established. Since the three-dimensional conformation of

TABLE 5. Hydrogen bond distances and angles Distance Donor atom

N-13 N-13 0-1 W-1C

W-1C

Hydrogen atom

H-13A H-13B H-01

H-W1A H-W1B

Acceptor atom

0-2 0-4 W-1C 0-3 0-2

(A)a

Symmetry equivalent operations to obtain donor

acceptor

Donorhydrogenacceptor angle (O)b

2.06 1.86 1.92 2.08 1.96

153.0 170.8 162.8 168.2 157.1

0.5 + x, 0.5 - y, -1 + z 0.5 - x, -0.5 + y, -z x, y, z -0.5 + x, 0.5 - y, -1 + z 0.5 - x, -0.5 + y, -z

Donoracceptor

Hydrogen-

Hydrogen-

donor

2.925 2.785 2.712 2.810 2.809

0.94 0.94 0.82 0.74 0.90

Estimated standard deviations for the donor-acceptor, the hydrogen-donor, and the hydrogen-acceptor distances are near 0.008, 0.07, and 0.07 respectively. b Estimated standard deviations for the donor-hydrogen-acceptor angles are near 0.50. c W-1 is the oxygen atom of the water molecule, and H-W1A and H-W1B are the hydrogen atoms of the water molecule.

A,

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TABLE 6. Comparison of different crystal forms of mefloquine Torsion angle (°);

Mefloquine form

Methylsulfonate monohydrate

Hydrochlorideb Free basec Free basec

Angle

(0) between avg

N-13-..-1plnsoquooe C-4--C-11--C-12 O-1--C-11--C-12 H-01--0-1-..N-13 distance (A) panespiofrquinooines -N-13 -N-13 and piperidine rings -H-13 2.730 ± 0.008 2.791 ± 0.006 2.848 ± 0.004

-176.2 174.7 169.7 175.6

2.784 ± 0.004

± ± ± ±

0.5 0.3 0.3 0.3

-54.5 ± 0.6 -62.8 ± 0.4 -66.6 ± 0.3 -61.4 ± 0.3

-18.1, 82.6 -14.6, 93.1 77.4"

86.8d

76.9 69.5 76.2 68.3

a All of the structures in this table were racemates. The torsion angles listed are for one enantiomer and will be of opposite sign for the other enantiomer. b Derived from coordinates to be published elsewhere (lOa). Derived from coordinates obtained from the authors of reference 19. The crystal of mefloquine free base contained mefloquine molecules in two different conformations. Values are ± estimated tstandard deviations for a reported R factor of 3.8%. dToe mefloquine free base contains only one H-13 atom and, therefore, only one H-01-O-1...N-13-H-13 torsion angle.

two free base conformations of mefloquine are within 0.26 A of H-O1 in mefloquine methylsulfonate. Unlike the two hydrogen bonds formed with mefloquine's amine group, the cinchona alkaloids as tertiary amines can form only one hydrogen bond involving the amine group. The superposition of quinidine (light lines) with mefloquine (heavy lines) in Fig. 6 shows that H-13B of mefloquine's amine group is equivalent to the hydrogen atom of quinidine's tertiary amine salt. Both the hydroxyl hydrogen atom and the hydrogen atom of the amine salt of quinidine point in the s-ame direction as H-O1 and H-13B of mefloquine and, therefore, -can hydrogen bond in the same direction. The similar direction of hydrogen bonding is further demonstrated by the torsion angle of H-O- - * * N-H-N for quinidine sulfate of 12.80. The two mefloquine salts possess an H-O1--1 * N-13-H-13 torsion angle of ± 14.,6° and ±18.1° (Table 6). Using the hypothesis that binding of the amino alcohol antimalarial agents to a receptor site at minimum involves hydrogen bonding with both the amine and hydroxyl groups and4that the conformations of mefloquine and quinidine do

N

N

not substantially change upon interaction with cellular constituents, we can describe the' interaction of these amino alcohol antimalarial agents with cellular receptors. The complementary' sur,face to the dQt surfaces in Fig. 5 and 6 outlines the approximate geometry of the proposed receptor. The hydroxyl group can hydrogen bond to proton-accepting

functional side chains'of amino acids. Although mefloquine can form two hydroge'n bonds With its secondary amine group, only one hydrogen bond may form between mefloquine's amine group and a receptor (as in Fig. 6), since the cinchona alkaloids are limited to only one hydrogen bond with their tertiary amine group. In summary, the similarities of the crystalline conformations of mefloquine establish a preferred conformation for mefloquine in which the quinoline (Fig. 4, ring A) and pyrimidine (Fig. 4, ring 'B) rings are angled from each other by 68° to 770, the aryl group is equatorial to the piperidine ring, the aliphatic nitrogen atom is gauche with respect 'to the hydroxyl group and is positioned nearly as far as possible from the quinoline ring, and the aliphatic nitrogen "to hydroxyl oxygen distance is 2.73 to 2.85 A. This conformation of mefloquine superimposes with the crystalline conformation of quinidine salt, such that the hydrogen atoms of the hydroxyl groups and the amine groups of both molecules can form hydrogen bonds in identical directions. These confor-

B

N

N

FIG. 4. Stereodiagram of the superposition of four mefloquine molecules from the crystal structures of mefloquine methylsulfonate, mefloquine hydrochloride (lOa), and mefloquine free base (two conformations) (18, 19). Only one enantiomer of mefloquine is illustrated. The hydrogen atoms of the free base conformations, but not the amine hydrogen atom, were placed in idealized positions. The quinoline rings are labeled A, and the piperidine rings are labeled B. The diagram can be viewed in three dimensions with the aid of a stereoviewer (Hubbard Scientific Co., Northbrook, Ill.) or by holding the drawing steady approximately 45 cm from your eyes and allowing your eye muscles to relax until the center image comes into focus.

---a~~~~~~~

FIG. 5. Stereodiagram depicting the regions of hydrogen bonding possible with mefloquine. Atoms 0-1, C-11, C-12, and N-13 of mefloquine methylsulfonate and mefloquine hydrochloride (lOa) were superimposed. For each molecule, dots were drawn at the van der Waals radius of H-O1, H-13A, and H-13B, which are the hydrogen atoms of the hydroxyl and 'amine groups. Only one enantiomer of mefloquine is illustrated. Instructions for stereoviewing are given in the legend to Fig. 4.

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ANTIMICROB. AGENTS CHEMOTHER.

KARLE AND KARLE

A

B

1

FIG. 6. (A) Stereodiagram of mefloquine (heavy lines) superimposed with the cinchona alkaloid quinidine (light lines). Atoms 0-1, C-11, C-12, and N-13 of mefloquine methylsulfonate are superimposed with the corresponding atoms of quinidine sulfate (9). The dots are drawn at the van der Waals radius for H-O1 and H-13B of mefloquine methylsulfonate and the hydroxyl and amine hydrogen atoms of quinidine sulfate. Instructions for stereoviewing are given in the legend to Fig. 4. (B) Individual drawings of the two drawings which are superimposed in panel A. Since both molecules are salts, the amine group of both molecules contains the hydrogen atom of the salt and is tetrahedral.

mations may define the geometry of hydrogen bond formation between a receptor and amino alcohol antimalarial compounds. REFERENCES 1. Allen, F. H., 0. Kennard, and R. Taylor. 1983. Systematic analysis of structural data as a research technique in organic chemistry. Accounts Chem. Res. 16:146-153. 2. Boudreau, E. L., H. K. Webster, K. Pavanand, and L. Thosingha. 1982. Type II mefloquine resistance in Thailand. Lancet

ii':1335. 3. Carroll, F. I., and J. T. Blackwell. 1974. Optical isomers of aryl-2-piperidylmethanol antimalarial agents. Preparation, optical purity, and absolute stereochemistry. J. Med. Chem. 17:

210-219.

4. Carter, 0. L., A. T. McPhail, and G. A. Sim. 1967. Optically active organometallic compounds. Part I. Absolute configuration of (-)-1,1'-dimethylferrocene-3-carboxylic acid by x-ray analysis of its quinidine salt. J. Chem. Soc. Sect. A 1967:365373. 5. Chien, P. L., and C. C. Cheng. 1973. Further side-chain modification of antimalarial phenanthrene amino alcohols. J. Med.

Chem. 16:1093-1096.

6. Chien, P. L., and C. C. Cheng. 1976. Difference in antimalarial activity between certain amino alcohol diastereomers. J. Med. Chem. 19:170-172. 7. Doherty, R., W. R. Benson, M. Maienthal, and J. M. Stewart.

1978. Crystal and molecular structure of quinidine. J. Pharm. Sci. 67:1698-1700. 8. Felix, R., F. Gay, A. Lyocoubi, M. D. G. Bustos, B. Diquet, M. Danis, and M. Gentilini. 1990. Rdsistance croisde a la mefloquine et a l'halofantrine lors d'un paludisme a P. falciparum contracte en Sierre Leone. Bull. Soc. Pathol. Exot. 83:43-45. 9. Karle, I. L., and J. Karle. 1981. Anomalous dispersion of sulfur in quinidine sulfate, (C20H25N202)2SO4. H20: implications for structure analysis. Proc. Natl. Acad. Sci. USA 78:5938-5941. 10. Karle, J., and 1. L. Karle. 1966. The symbolic addition procedure for phase determination for centrosymmetric and noncentrosymmetric crystals. Acta Crystallogr. 21:849-859. 10a.Karle, J. M., and I. L. Karle. Acta Crystallogr. Sect. C, in press. 11. Karwacki, J. J., H. K. Webster, N. Limsomwong, and G. D. Shanks. 1989. Two cases of mefloquine resistant malaria in Thailand. Trans. R. Soc. Trop. M'ed. 83:152-153. 12. Kashino, S., and M. Haiso. 1983. Structure of quinidine, C20H25N202. Acta Crystallogr. Sect. C 39:310-312. 13. Krogstadt, D. J., P. H. Schlesinger, and I. Y. Gluzman. 1985. Antimalarials increase vesicle pH on Plasmodium falciparum. J. Cell Biol. 101:2302-2309. 14. Oleksyn, B. 1978. The environmental effect on the geometry of cinchonine molecule in the crystalline state. Acta Crystallogr. Sect. A 34:S77. 15. Oleksyn, B. 1982. The alkaloid cinchonidine. Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 38:1832-1834.

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16. Oleksyn, B., L. Lebioda, and M. Ciechanowicz-Rutkowska. 1979. The molecular and crystal structure of the alkaloid cinchonine. Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 35:440-444. 17. Oleksyn, B., K. M. Stadnicka, and S. A. Hodorowicz. 1978. The crystal structure and absolute configuration of cinchoninium tetrachlorocadmate (II) dihydrate. Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 34:811-816. 18. Oleksyn, B. J. 1982. The role of molecular geometry in the biological activity of cinchona alkaloids and related compounds, p. 181-190. In J. F. Griffin and W. L. Duax (ed.), Molecular structure and biological activity. Elsevier Science Publishing Co., Amsterdam. 19. Oleksyn, B. J., L. Lebioda, and J. Sliwinski. 1980. Crystal structure investigation of mefloquine, a new potent antimalarial drug, p. 200-204. In Z. Kaluski (ed.), Proceedings of the IlIrd Symposium on Organic Crystal Chemistry. University Adam Mickiewicz, Poznan, Poland. 20. Olsen, R. E. 1972. Antimalarial activity and conformation of

X-RAY CRYSTAL STRUCTURE OF MEFLOQUINE

21. 22. 23. 24.

25. 26.

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erythro- and threo-ot-(2-piperdyl)-3,6-bis(trifluoromethyl)-9-phenanthrenemethanol. J. Med. Chem. 15:207-208. Panisko, D. M., and J. S. Keystone. 1990. Treatment of malaria-1990. Drugs 39:160-189. Payne, D. 1987. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol. Today 3:241-246. Pniewska, B., and A. Suszko-Purzycka. 1989. Structure of quinine monohydrate toluene solvate. Acta Crystallogr. Sect. C 45:638-642. Sheldrick, G. M. 1980. SHELXTL. An integrated system for solving, refining and displaying crystal structures from diffraction data. University of Gottingen, Gottingen, Federal Republic of Germany. Stout, G. H., and L. H. Jensen. 1989. X-ray structure determination. A practical guide, 2nd ed. John Wiley & Sons, New York. Sweeney, T. R. 1981. The present status of malaria chemotherapy: mefloquine, a novel antimalarial. Med. Res. Rev. 1:281301.

Crystal structure and molecular structure of mefloquine methylsulfonate monohydrate: implications for a malaria receptor.

The crystal structure of (+/-)-mefloquine methylsulfonate monohydrate was determined by X-ray diffraction and was compared with the crystal structures...
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