Antimycobacterial activities of 2,4-diamino-5-deazapteridine derivatives and effects on mycobacterial dihydrofolate reductase

Article abstract of DOI:10.1128/AAC.44.10.2784-2793.2000

Development of new antimycobacterial agents for Mycobacterium aviurn complex (MAC) infections is important particularly for persons coinfected with human immunodeficiency virus. The objectives of this study were to evaluate the in vitro activity of 2,4-di

Full text of DOI:10.1128/AAC.44.10.2784-2793.2000

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2000, p. 2784–2793
Vol. 44, No. 10
0
066-4804/00/$04.00ϩ0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Antimycobacterial Activities of 2,4-Diamino-5-Deazapteridine
Derivatives and Effects on Mycobacterial Dihydrofolate Reductase
3
1
2
2
3
WILLIAM J. SULING, LAINNE E. SEITZ, VIBHA PATHAK, LOUISE WESTBROOK, ESTHER W. BARROW,
3
3
2
2
SABRINA ZYWNO-VAN-GINKEL, ROBERT C. REYNOLDS, J. ROBERT PIPER, AND WILLIAM W. BARROW *
1
2
3
Bacteriology and Mycology Research Unit, Organic Chemistry, and Mycobacteriology Research Unit,
Southern Research Institute, Birmingham, Alabama 35205
Received 22 May 2000/Returned for modification 19 June 2000/Accepted 12 July 2000
Development of new antimycobacterial agents for Mycobacterium avium complex (MAC) infections is impor-
tant particularly for persons coinfected with human immunodeficiency virus. The objectives of this study were
to evaluate the in vitro activity of 2,4-diamino-5-methyl-5-deazapteridines (DMDPs) against MAC and to
assess their activities against MAC dihydrofolate reductase recombinant enzyme (rDHFR). Seventy-seven
DMDP derivatives were evaluated initially for in vitro activity against one to three strains of MAC (NJ168,
NJ211, and/or NJ3404). MICs were determined with 10-fold dilutions of drug and a colorimetric (Alamar Blue)
microdilution broth assay. MAC rDHFR 50% inhibitory concentrations versus those of human rDHFR were
also determined. Substitutions at position 5 of the pteridine moiety included ™CH , ™CH CH , and ™CH OCH
3
2
3
2
3
groups. Additionally, different substituted and unsubstituted aryl groups were linked at position 6 through a
two-atom bridge of either ™CH NH, ™CH N(CH ), ™CH CH , or ™CH S. All but 4 of the 77 derivatives were
2
2
3
2
2
2
active against MAC NJ168 at concentrations of <13 ␮g/ml. Depending on the MAC strain used, 81 to 87% had
MICs of <1.3 ␮g/ml. Twenty-one derivatives were >100-fold more active against MAC rDHFR than against
human rDHFR. In general, selectivity was dependent on the composition of the two-atom bridge at position 6
and the attached aryl group with substitutions at the 2؅ and 5؅ positions on the phenyl ring. Using this
assessment, a rational synthetic approach was implemented that resulted in a DMDP derivative that had
significant intracellular activity against a MAC-infected Mono Mac 6 monocytic cell line. These results
demonstrate that it is possible to synthesize pteridine derivatives that have selective activity against MAC.
Dihydrofolate reductase (DHFR) is a key enzyme in the
folate metabolic pathway that is necessary for the biosynthesis
of RNA, DNA, and protein. The enzyme is an important target
for medicinal chemistry (5), and inhibitors for it have been
used in anticancer (methotrexate [4]), antibacterial (trimetho-
prim [9]), and antimalarial (pyrimethamine [24]) chemother-
apy. DHFR is present in all cells and is necessary for the
maintenance of intracellular folate pools in a biochemically
active reduced state (18). Enzyme inhibition is effective be-
cause binding affinities for substrate analogs are so great that
they are not readily displaced by the natural substrates (18).
Inhibition results in depletion of intracellular reduced folates,
which are necessary for one-carbon transfer reactions. One-
carbon transfer reactions are important for the biosynthesis of
thymidylate, purine nucleotides, methionine, serine, glycine,
and many other compounds necessary for RNA, DNA, and
protein synthesis (13). Some bacteria have an uptake system
for folates, but most require de novo folate synthesis. Reduc-
tion of dihydrofolate to tetrahydrofolate is, however, a univer-
sal requirement (13).
Although DHFR does not represent a new target, there is
still enthusiasm for the development of improved derivatives of
this class of inhibitor (1, 5, 18, 22, 25, 26), particularly with
regard to mycobacteria (7, 15, 16, 17, 19, 28, 29; W. J. Suling,
R. C. Reynolds, J. R. Piper, V. Pathak, E. W. Barrow, L. E.
Gundy, S. Z. V. Ginkel, L. Westbrook, and W. W. Barrow,
Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother.,
abstr. 339, 1999). A unique feature of DHFR is the selectivity
that is possible in the design of inhibitors. This makes it an
ideal “old” target for rational and effective drug design for
antimycobacterial agents. The antifolate drug trimethoprim is
perhaps the best example of selectivity. However, trimetho-
prim is a poor inhibitor of Mycobacterium avium complex
(
MAC; 28). Results reported here support the hypothesis that
new antifolates can be developed for treatment of mycobacte-
rial infections.
The antifolate compounds evaluated in this investigation are
derivatives of 2,4-diamino-5-methyl-5-deazapteridine (DMDP),
having structures similar to the trimetrexate/piritrexim class of
antifolates (Fig. 1). Due to an interest in new antimycobacte-
rial drugs, several DMDP inhibitors were chosen from the
Southern Research Institute chemical repository for screening
against MAC. We had previously reported on antimycobacte-
rial activity by 12 of these derivatives for Mycobacterium tuber-
culosis and MAC (28). At the same time, DMDP derivatives
were also reported active against MAC by Shoen et al. (27). As
an extension of that work, we report on the results of further
studies with other newly synthesized DMDP derivatives with
regard to activity against MAC. Most of these derivatives
showed good activity against three strains of MAC, with MICs
ranging from Ͻ0.13 to 1.3 ␮g/ml. As a result, the derivatives
were also evaluated for their ability to inhibit MAC recombi-
nant DHFR (rDHFR) activity. This was compared to their
inhibition of purified human rDHFR. Evaluation of selective
enzyme inhibition by the 77 DMDP derivatives led to the
synthesis of a 78th derivative that showed significant activity
against intracellularly replicating MAC.
*
Corresponding author. Mailing address: Mycobacteriology Re-
(Some of this research was presented at the 39th Inter-
search Unit, Southern Research Institute, 2000 Ninth Ave. South, P.O.
Box 35205, Birmingham, AL 35205. Phone: (205) 581-2139. Fax: (205)
science Conference on Antimicrobial Agents and Chemother-
apy, San Francisco, Calif., 26 to 29 September 1999.)
5
81-2877. E-mail: barrow@sri.org.
2
784

VOL. 44, 2000
ANTIMYCOBACTERIAL ACTIVITIES OF 5-DEAZAPTERIDINES
2785
FIG. 1. Structures of trimetrexate and piritrexim, with positions numbered.
MATERIALS AND METHODS
1H-NMR (DMSO-d
CH CH CH ), 1.63 (m, 2H, 5Ј-CH
.67 (s, 3H, 5-CH ), 3.77 (t, 2H, 5Ј-CH
6
)
␦
0.93 (t, 3H, 2Ј-CH
CH CH ), 1.69 (m, 2H, 2Ј-CH
CH CH ), 3.85 (t, 2H, 2Ј-CH
2
2 2 3
CH CH ), 0.94 (t, 3H, 5Ј-
2
2
3
2
2
3
2
CH
CH
2
CH
CH
3
),
),
Synthesis of DMDP derivatives. The structures of the compounds used in the
present study are presented below (see Tables 2 through 5) and are numbered
consecutively as 1 through 78. The compounds were synthesized according to the
method of Piper et al. (22). The synthesis of each particular class of analogs was
carried out by the exact procedures that are well described by Piper et al. (22),
and these descriptions need not be duplicated here. In general, the appropriate
2
3
2
2
3
2
3
4
6
8
7
.28 (d, 2H, CH NH), 5.06 (t, 1H, CH NH), 6.06 (dd, 1H, 4Ј-H), 6.13 (d, 1H,
2
2
Ј-H), 6.25 (bs, 2H, 2 or 4-NH
.42 (s, 1H, 7-H). Analytical calculated for C21H N O ⅐ 0.5H O: C, 62.06; H,
28 6 2 2
.21; N, 20.67. Found C, 62.05; H, 6.95; N, 20.50.
MIC. MICs were determined against three strains of MAC (NJ168, serovar 1;
2
2
), 6.67 (d, 1H, 3Ј-H), 7.01 (bs, 2H, 2 or 4-NH ),
5
-substituted 5-deaza-6-bromomethylpteridine was reacted with the desired,
commercially available thiophenol or aniline to obtain the S- or N-linked ana-
logs, respectively. The 10-CH -linked analogs were prepared by Wittig coupling
NJ211, serovar 4/6; and NJ3404, serovar 4), using a colorimetric microdilution
broth assay as reported previously (6, 28, 32; Suling et al., 39th ICAAC, abstr.
2
3
39). The MAC strains were kindly provided by L. Heifets, National Jewish
of the reaction product of triphenylphosphine and 5-methyl-5-deaza-6-bromo-
methylpteridine with the appropriately substituted and commercially available
benzaldehyde derivative followed by catalytic hydrogenation (22). All compound
Center for Immunology and Respiratory Diseases, Denver, Colo. A frozen cul-
ture in 7H9 broth (Difco Laboratories, Detroit, Mich.), supplemented with ADC
enrichment (Difco) and 0.2% glycerol, was thawed and diluted in broth to about
2
1
structures were verified by mass and H-labeled nuclear magnetic resonance
5
ϫ 10 CFU/ml and used as the inoculum. The assay used a 96-well (U-shaped)
(
(
NMR) spectroscopy. Sample purity was assessed by thin-layer chromatography
TLC) and elemental analysis; all compounds gave single spots by TLC and were
microtiter plate and a format designed to accommodate seven compounds in
four log10 dilutions. The assay plates also contained uninoculated drug and
medium controls and viability controls. Each test compound was dissolved in
dimethyl sulfoxide (DMSO), then diluted in broth at twice the desired concen-
tration, and 0.05 ml was added to duplicate assay wells. The highest concentra-
tion of DMSO in the assay medium was 1.3%. DMSO did not affect growth at
this concentration. Each plate was then inoculated with 0.05 ml of standardized
culture and the plates were incubated at 37°C for 6 or 13 days, depending on the
assay strain. The REDOX indicator Alamar Blue (Acumed International, Inc.,
Westlake, Ohio) was then added to each well as a mixture with Tween 80, and the
plates were incubated for an additional 18 to 22 h. The plates were read in an
optical microtiter plate reader programmed to subtract the absorbance at 600 nm
from that at 570 nm to blank out turbidity and absorbance due to oxidized dye.
The MIC was reported as the lowest concentration of drug yielding a differential
absorbance of zero or less. This approximated the color change of blue to red
that was observed visually after metabolic reduction of dyes and represented the
concentration at which no visible growth occurred. Trimethoprim or ethambutol
was used as a positive control.
within acceptable combustion parameters (0.4%). It is notable that these com-
pounds do not have defined melting points, but generally decompose above
00°C. The full experimental details of these compounds are available in the
reference of Piper et al. (22) or will be reported elsewhere (unpublished data).
As examples of these preparations, both compound 1 (SRI-8686) and compound
8 (SRI-20094) are presented.
Preparation of compound 1. To dissolve as much material as possible, 2,4-
diamino-5-methyl-5-deaza-6-cyanopteridine (3.0 g, 0.015 mmol) was stirred with
glacial acetic acid (342 ml) with warming (Ͻ50°C). After a brief period, the
suspension was cooled to ambient temperature and both 2,5-diethoxyaniline
3
7
(
2.98 g, 0.016 mmol) and Raney Nickel (4.995 g of a 50% slurry in water) were
added, and the reaction was placed in a hydrogenation apparatus under H
pressure (atmospheric) with vigorous stirring. After several hours, the reaction
had absorbed the required H , and stirring was halted. The reaction was left
standing under H pressure overnight. After removal of remaining hydrogen, the
2
2
2
reaction mixture was filtered through a celite pad, and the solid was rinsed with
glacial acetic acid until the washings were clear. The filtrate was concentrated
Human rDHFR. Purified human rDHFR, which was generated using a recom-
binant system in Escherichia coli (23), was provided by Anatrace (Maumee,
Ohio). According to the supplier, the enzyme has properties identical to those
isolated from human cells as reported by Delcamp et al. (8), and it contained an
equimolar concentration of enzyme and dihydrofolate.
under vacuum and diluted with 342 ml of H
0 with concentrated ammonia. The resulting suspension was placed in the
refrigerator overnight, and the suspension was filtered the following morning to
yield 3.312 g of crude material after drying under vacuum (P , 25°C). This
material was further purified by dissolving in 250 ml of hot dimethylformamide
DMF) drying onto 16.56 g of flash silica gel (220 to 400 mesh) under high
vacuum, and then loading onto a flash chromatography column packed with
52 g of flash silica gel and 4:1 chloroform/methanol with 1% glacial acetic acid.
2
O, and the pH was adjusted to 9 to
1
2
O
5
Recombinant MAC DHFR. The MAC DHFR gene was cloned into the vector
pET15b at the NdeI, BamHI restriction sites and expressed in E. coli strain
BL21(DE3)pLysS as a fusion protein with a His tag (34). The plasmid was grown
in Luria-Bertani broth containing 34 ␮g of chloramphenicol per ml and 50 ␮g of
ampicillin per ml at 28°C to an optical density of 0.6 at 600 nm, then was induced
with 0.1 mM isopropyl ␤-D-thiogalactopyranoside for 20 h. The cells were har-
vested and lysed on ice in a buffer containing 5 mM imidazole, 500 mM NaCl, 20
(
3
Elution with the same solvent gave three separate pools of fractions containing
the major product. After evaporation, the middle pool gave 0.1343 g of highly
pure material showing a single spot by TLC (4:1 chloroform/methanol with 1%
ϩ
1
glacial acetic acid): FAB-MS m/z 369.0 (MϩH) , H-NMR (DMSO-d
6
) ␦ 1.24 (t,
H), 2.67 (s, 3H,
), 4.28 (d, 2H,
NH™), 6.05 (dd, 1H, 4Ј-H), 6.10 (d, 1H, 6Ј-H), 6.33
), 6.68 (d, 1H, 3Ј-H), 7.06 (bs, 2H, 2 or 4-NH ), 8.42 (s, 1H
-H). Analytical calculated for C19 ⅐ 0.5H O: C, 60.46; H, 6.68; N, 22.27.
Found C, 60.48; H, 6.80; N, 22.05.
2
mM Tris (pH 7.9), 20 ␮g of DNase per ml, 10 mM MgCl , 22 ␮g of leupeptin per
3
5
™
H 5Ј-OCH
-CH ), 3.86 (q, 2H, 5Ј-OCH
CH NH™), 5.13 (t, 1H, ™CH
2
CH
3
), 1.30 (t, 3H, 2Ј-OCH
2
CH
3
), 1.91 (s, CH
3
CO
CH
2
ml, 20 ␮g of pepstatin A per ml, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1%
Triton X-100. The insoluble proteins were removed by centrifugation, and the
soluble proteins were purified by binding the His tag portion of the fusion protein
to a His bind (Novagen, Inc., Madison, Wis.) resin column and elution with a 5
to 500 mM imidazole salt gradient in a buffer containing 500 mM NaCl and 20
mM Tris-NCl (pH 7.9). All steps were carried out at 4°C. Fractions with DHFR
activity were pooled, and the buffer was exchanged to 5 mM imidazole-500 mM
NaCl–20 mM Tris-HCl (pH 7.9) with a PD 10 column (Pharmacia), at 4°C. The
His tag fusion protein was then removed by cleavage with 0.5 U of thrombin per
mg of recombinant protein for 1 h on ice. The recombinant DHFR was stabilized
throughout the purification by the addition of bovine serum albumin (1 mg/ml)
and was stored at Ϫ70°C.
3
2
CH ), 3.94 (q, 2H, 2Ј-OCH
3
2
3
2
2
(
bs, 2H, 2 or 4-NH
2
2
7
H
24
N
6
O
2
2
Preparation of compound 78. As an alternative to the catalytic hydrogenation
method described previously, the synthesis of SRI-20094 represents a direct
coupling of an aniline derivative with 2,4-diamino-5-methyl-5-deaza-6-bromo-
methylpteridine. 2,5-Dipropoxyaniline (500 mg, 1.24 mmol) was dissolved in
DMF, and the solution was flushed with dry nitrogen. 2,4-Diamino-5-methyl-5-
deaza-6-bromomethylpteridine (500 mg, 2.38 mmol) was added, and the reaction
was stirred for 24 h. Next, the reaction was diluted with water and neutralized
with saturated NaHCO
3
. The suspension was filtered, and the solid was washed
DHFR assay and enzyme inhibition studies. DHFR activity of both human
and MAC enzymes was measured at 30°C as the decrease in absorbance at 340
nm (34). The reaction mixture (1 ml) contained 10 mM 2-mercaptoethanol, 0.1
mM NADPH, 0.1 mM dihydrofolate, 1 mM EDTA, 55 mM potassium phosphate
(pH 7), and enzyme. The reaction was initiated by the addition of dihydrofolate
after preincubation of the other components for 3 min. For inhibition assays,
various amounts of inhibitor were added to the mixture before the 3-min prein-
cubation period. The 50% inhibitory concentration (IC50) was determined from
a plot of the log10 of the drug concentration versus percent inhibition as the
with water followed by diethylether. Drying under vacuum yielded 210 mg of the
crude material, and this material was further purified by dissolving in a minimum
of hot DMF and drying on 2.0 g of silica gel (60 to 120 mesh) followed by column
chromatography. The powder was placed on a small column packed with silica
gel (60 to 120 mesh) loaded with 7:1 chloroform/methanol and 1% concentrated
NH
combined and dried to yield 35 mg of pure SRI-20094. Purity was confirmed by
4
OH. Elution with the same solvent gave several pure fractions that were
ϩ
TLC with the aforementioned solvent system: FAB-MS m/z 397.0 (MϩH)
,

2
786
SULING ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
amount of inhibitor required to inhibit the reaction by 50%. Each reported IC50
is the average of two or more determinations. The amount of enzyme used for
the inhibition assays was about 0.0024 U/ml, which yielded a mean rate plus or
minus standard deviation of Ϫ0.036 Ϯ 0.0046 per min for MAC DHFR and
Ϫ0.029 Ϯ 0.0026 for human DHFR. Linearity was maintained for Ͼ7 min. One
unit of enzyme is defined as the amount which reduces 1 ␮mol of dihydrofolate
against each strain, 39% had MICs of Յ0.13 ␮g/ml for NJ168,
and 64% and 71% had MICs of Յ0.13 ␮g/ml for NJ211 and
NJ3404, respectively. The antibacterial DHFR inhibitor tri-
methoprim had an MIC of Ͼ13 ␮g/ml for all three strains (data
not shown).
Ϫ1
per min using a molar extinction coefficient of 12,300 M (14). The selectivity
Enzyme inhibition assays with DMDP derivatives. Using
MAC and human rDHFR, 77 DMDP derivatives were evalu-
ated initially for their ability to inhibit enzyme activity. In this
assay, the IC s of trimethoprim, trimetrexate, and piritrexim
ratio was determined as the ratio of the human DHFR IC50 to the MAC DHFR
IC50
.
Intracellular efficacy. Intracellular efficacy for compounds 1 (SRI-8686) and
7
8 (SRI-20094) was assessed by using a previously described method involving
5
0
the Mono Mac 6 monocytic cell line (MM6; 2, 31). MM6 cells were obtained
from the German Collection of Microorganisms and Cell Cultures, Braun-
schweig, Germany. The MM6 was originally established by H. W. L. Ziegler-
Heitbrock, Institute of Immunology, University of Munich, Germany (33), and it
is a human acute monocytic leukemia cell line. This cell line has been previously
used by us (2, 31) and others (30) to examine effectiveness of antimycobacterial
drugs against intracellularly replicating M. tuberculosis. MM6 cells were main-
tained in RPMI 1640 containing 10% (vol/vol) fetal calf serum, 2 mM L-glu-
tamine, nonessential amino acids, 1 mM sodium pyruvic acid, and 9 ␮g of bovine
insulin (Sigma Chemical Co., St. Louis, Mo.) per ml (MM6 medium). Cells were
routinely assayed to verify absence of mycoplasma contamination using the
Gen-Probe Mycoplasma Rapid Detection System (Gen-Probe, San Diego, Cal-
if.).
for the human rDHFR were Ͼ350,000, 4.7, and 4.4 nM, re-
spectively. The IC s of the same three drugs for the MAC
50
rDHFR were 4,100, 1.0, and 1.4 nM, respectively (data not
shown).
The DMDP analogs contained modifications in three areas,
the 5 position, the linking atom in the 10 position of the bridge,
and the aromatic nucleus attached to the 10 position. The
results are reported in Tables 2 through 4 and are based on
structural similarity of derivatives in each group. Table 2 con-
tains the data obtained from those derivatives having a ™CN™
bridge and substitutions on the attached phenyl ring, denoted
as R2, R3, R4, R5, and R6. The 5-substituent (R1) also had
various modifications. Table 3 contains similar derivatives but
having alternative aromatic-ring systems instead of a phenyl
group attached to the bridge nitrogen. Table 4 contains deriv-
atives similar to those in Table 2 except that the bridge nitro-
Preparation of mycobacteria for infection of monocytes. Before infection of
MM6, MAC strain NJ3404 was initially grown in Middlebrook 7H9 (Difco)
containing 0.2% glycerol and 10% oleic acid albumin-dextrose-catalase (Difco).
After the mycobacteria had reached exponential phase, they were dispersed by
vortexing with glass beads, and clumps were allowed to settle for 30 min (20). The
supernatant was removed, aliquoted, frozen at Ϫ70°C, and then thawed and used
for infection by resuspension in appropriate cell culture medium. Actual CFUs
per milliliter were determined by preparation of serial dilutions in Dulbecco’s
phosphate-buffered saline (DPBS; Mediatech, Inc., Herndon, Va.) and plating
on 7H10 agar.
gen was replaced with either a ™CH ™ or ™S™.
2
Derivatives with modifications at the 5-position and substi-
tutions on the phenyl group. In the case of the 5-substituent
(R1), we evaluated 5-H, 5-CH , 5-CH CH , and 5-CH OCH .
Infection of monocytes. Before infection, MM6 cells were adjusted to 8 ϫ 105
cells per ml, and 0.5 ml per well was dispensed in 12-well tissue culture dishes
3
2
3
2
3
(
Corning Costar Corp., Cambridge, Mass.). The MAC cells were then added to
Only two compounds were tested that had the 5-H substitution
(compounds 26 and 32 [Table 2]); therefore, it was not possible
to make any conclusive evaluation regarding selectivity. It ap-
peared that the 5-H substitution may have modestly improved
selectivity; the selectivity ratio was increased only by about
threefold (compounds 26 versus 43 [Table 2]). The 5-H sub-
stitution, however, lowered the activity for both human and
the MM6 cells to achieve a final ratio of 10 mycobacteria per macrophage, with
5
a density of 4 ϫ 10 MM6 cells per 1.0 ml per well. After infection for 24 h, the
infected MM6 cells were collected by centrifugation (200 ϫ g) and washed twice
with DPBS to remove any unphagocytosed mycobacteria. The cells were then
5
replated at a density of 4 ϫ 10 cells per 1.0 ml per well, with appropriate wells
containing drug preparations, and the plates were incubated at 37°C with 5%
carbon dioxide. One milliliter of fresh medium was added at day 4, and infection
continued until CFU assay at day 7.
Determination of CFU. The determination of CFU at zero hour was conducted
by first lysing the monocytes with 0.25% (wt/vol) sodium dodecyl sulfate in DPBS
and then plating serial dilutions onto 7H10 agar plates (31). As a means of
decreasing viscosity, 5 ␮l (5 U of activity) of RQ DNase (Promega Corp. Mad-
MAC rDHFR when compared to the 5-CH substitution (com-
3
pound 26 versus 43 and compound 32 versus 18 [Table 2]).
Several direct comparisons were available in the 5-CH CH
2
3
ison, Wis.), with MgSO
4
(5 mM), was added to each well following addition of
and 5-CH
resulted in decreased activity for both enzymes versus their
-CH counterparts, and in contrast to the 5-H substitution,
2 3
OCH substitutions. These substitutions in general
sodium dodecyl sulfate, and the plates were incubated at 37°C for 20 min (31).
The plating procedure was repeated at 7 days, and CFU were enumerated after
5
3
1
0 to 14 days of incubation of cultures.
Macrophage viability assays. Cell viability was determined by means of an
they did not improve the selectivity ratio. For example, a com-
parison of compound 2 with compounds 6 and 29 (Table 2)
showed that substitution on the 5-position of a ™CH CH or
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [thiazolyl
blue]) cytotoxicity assay (Sigma), modified in our laboratory as described previ-
ously (2, 31). Viability is reported as a percentage of the nontreated cells.
2
3
™
CH OCH yielded a reduction in the selectivity ratio from
2 3
9
09 to 357 and 12.9, respectively. Thus, the optimum substitu-
tion at the 5-position was ™CH3.
RESULTS
An overall evaluation of the data presented in Table 2 sug-
In vitro activity of DMDP derivatives against MAC strains.
In vitro inhibition was evaluated against three MAC strains
gested that the 5-CH substitution and substitutions at the 2Ј
3
(R2) and 5Ј (R5) positions on the phenyl ring resulted in an
optimal selectivity ratio. The best examples for this comparison
are compounds 15, 7, 4, 2, and 1 (Table 2), which demonstrated
an increase in the selectivity ratio from 62 to 2,738, respec-
tively. This 44-fold improvement was obtained by extended
modifications at the 2Ј and 5Ј positions.
(
(
NJ168, NJ211, and NJ3404) for 50 of the DMDP derivatives
Table 1). MAC strain NJ168 was consistently the most resis-
tant of the three strains tested. With this strain, four derivatives
had MICs of Ͼ13 ␮g/ml (Table 1 [indicated by ϩ, derivatives
2
9, 45, 50, and 51]), while MAC strains NJ211 and NJ3404
responded with MICs in this range for only one (derivative 29)
and two (derivatives 29 and 50 [Table 1]) of the derivatives,
respectively (ϩ [Table 1]). The MICs of most of the 50 com-
pounds for all three strains were Յ1.3 ␮g/ml, with NJ168 hav-
ing the least (38 derivatives [76%]). Both strains NJ211 and
NJ3404 had 43 derivatives (86%) with MICs of Յ1.3 ␮g/ml.
Strain NJ168 was used to screen an additional 27 derivatives.
Of those 27 derivatives, 24 had MICs of Յ1.3 ␮g/ml (ϩϩϩ and
ϩϩϩϩ [Table 1]), for a total of 62 out of 77 (81%). More
significant is the fact that, out of all the derivatives tested
Derivatives with complex ring substituents attached to the
™CN™ bridge. Sixteen derivatives were evaluated with modifi-
cations involving heterocyclic ring substitutions attached to the
™CN™ bridge. Although many of the compounds had good
activity versus MAC DHFR, selectivity for the MAC compared
to the human enzyme was poor except for tetrahydronaphthyl-
linked compounds 52 and 53 (Table 3). The selectivity ratio for
these compounds was 374 and 153, respectively.
Derivatives involving modification of the bridge molecules.
We also evaluated modifications of the 10-position bridging

TABLE 1. Numerical listing of DMDP derivatives, along with their
Southern Research Institute (SRI) numbers
atom from a nitrogen to a ™CH ™ or ™S™. Although only 10
2
derivatives in this classification were examined, results sug-
gested that modifications at this position might result in im-
proved selectivity. For example, a comparison of compound 68
MIC rangesa for MAC strain
DMDP
SRI no.
derivative no.
NJ168
NJ211
NJ3404
(
Table 4) with 20 (Table 2) revealed that substitution of the
™
CH NH™ bridge with ™CH CH ™ caused a 66-fold decrease in
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
8686
8117
8687
8202
9734
8922
8229
9786
9674
9717
9643
8911
9672
9826
9018
9647
9782
9645
8574
8709
9675
9644
9787
9981
9980
7746
9827
9596
8758
8710
9733
8577
9646
9595
9600
8692
9614
9613
9676
9599
9612
8227
7714
9632
8766
9684
8228
8923
9683
8765
8759
8708
8691
9735
9468
9771
9449
7747
9487
9450
9824
7711
9825
9692
9439
9823
9673
9388
8455
9437
8451
8450
9490
8573
7896
9165
8453
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩ
ϩϩϩ
ϩϩϩϩ
ϩ
ϩϩϩ
ϩϩϩ
ϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ND
ND
ND
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ND
2
2
2
activity for the human rDHFR, resulting in an increase in the
selectivity ratio from 23.4 to 660 (28-fold). Interestingly, the
alkyl substitutions on the phenyl ring of the above compounds
are at the R3 and R5 positions. In contrast, the same does not
apply for those derivatives with substitutions on the R2 and R5
positions. For example, a comparison of compounds 76 (Table
ϩϩϩ
ϩϩϩϩ
ND
ND
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
ND
4) and 2 (Table 2), in which the ™CH NH™ bridge is substituted
2
ND
with ™CH CH ™, showed that this modest alteration resulted in
2
2
ϩϩϩϩ
ND
such a decrease in activity against the MAC rDHFR that the
selectivity ratio was reduced from 909 to 57 (16-fold).
A small number of compounds were evaluated wherein the
ϩϩϩϩ
ϩϩϩϩ
ND
™
X- linkage was replaced by ™S- (eight compounds [Table 4]).
ϩϩϩϩ
ND
It is interesting to note that all of the ™S-substituted linkages
were selective for the MAC enzyme. It is also notable that in
every comparison of pairs except one (compounds 70, 72, 73,
and 75 in Table 4 versus 43, 18, and 2, in Table 2 and 62 in
Table 3, respectively) the ™S-linkage was significantly more
selective than the 10-aza counterpart. Unfortunately, the one
exception is a critical case; the 2Ј,5Ј-dialkoxy (R2, R5) substi-
tution yielded the greatest selectivity in the 10-aza linkage, and
the 2Ј,5Ј-dimethoxy analog (compound 2 [Table 2]) is about
ninefold more selective than its ™S-linked counterpart (com-
pound 73 [Table 4]).
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ND
ND
ND
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩ
ϩϩϩϩ
ND
ϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ND
ϩ
ϩϩϩϩ
ND
ϩϩϩ
ND
ND
ND
ϩϩϩ
ND
ND
ND
A small sample set (Table 2, compounds 3, 19, and 30 versus
1
™
, 47, and 20, respectively) was screened in which both the
NH™ and ™NCH ™ were available with the same substitution
3
ϩϩϩϩ
ND
ϩϩϩϩ
ND
pattern for comparison. In two instances, the substitution of a
™CH3 for a ™H at the 10-aza group reduced selectivity by
increasing inhibition of the human enzyme with little or no
change in activity against MAC rDHFR. However, in one
instance (compound 19 versus 47), the activity against the
human enzyme was decreased by over 10-fold with a small
effect on the MAC enzyme inhibition, giving an almost 8-fold
increase in selectivity.
Intracellular efficacy of compound 1 (SRI-8686). Based on
the data presented in Tables 2 through 4, we chose the deriv-
ative with the highest selectivity ratio for intracellular efficacy
evaluation. Compound 1 was evaluated in the MM6 cells by
using concentrations of 0.1 and 1.0 ␮g/ml. Unfortunately, ex-
cessive loss of MM6 viability at the end of the 7-day experi-
mental period for the highest concentration (i.e., 1.0 ␮g) pre-
vented proper assessment of data and indicated that use of
higher concentrations would not be successful for the same
reason. Because of these results, we evaluated the enzyme data
ND
ND
ND
ND
ND
ND
ND
ND
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩ
ND
ϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩ
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩ
ϩϩ
ϩ
ϩϩ
ϩ
ϩϩϩ
ϩϩϩ
ϩϩ
ϩϩϩϩ
ϩϩ
ϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩ
ϩϩ
ϩϩϩ
ϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩ
(
Table 2) in an attempt to rationalize improvements that could
be made to reduce toxicity to the human cell line.
Based on our results with regard to substitutions at the R2
and R5 positions (Table 2), we hypothesized that further alkyl
extension at these positions would improve the selectivity ratio
by decreasing the activity for the human DHFR. If such a
modification could be made while maintaining activity for the
MAC DHFR, then it should be possible to demonstrate sig-
nificant intracellular efficacy because toxicity to the cell lines
would be reduced. As a result, compound 78 (SRI-20094) was
synthesized with ™O(CH ) CH substitutions at the R2 and R5
ϩϩϩϩ
ϩϩϩϩ
ND
ϩϩϩϩ
ϩϩϩϩ
ND
ND
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩ
2
2
3
positions. To demonstrate the rationale for our experimental
approach, this derivative is given in Table 5, along with similar
data already reported for other DMDP derivatives in the same
series (Tables 1 and 2). As the data suggest, further extension
at the R2 and R5 positions resulted in decreased activity
(threefold) for the human rDHFR with continued mainte-
ϩϩ
ϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
ϩϩϩ
a
ϩ, Ͼ13 ␮g/ml; ϩϩ, Ͼ1.3 to Յ13 ␮g/ml; ϩϩϩ, Ͼ0.13 to Յ1.3 ␮g/ml;
ϩϩϩϩ, Յ0.13 ␮g/ml. ND, not done.
2
787

2
788
SULING ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. DMDP derivatives with modifications at the 5 position (R1) and substitutions on the phenyl group (R2 through R6)
Substitution at DMDP position
R3 R4
IC50 (nM) for:
Derivative
SRI
Selectivity
a
c
b
no.
no.
ratio
R1
R2
R5
R6
Human DHFR
MAC DHFR
1
2
3
4
5
6
7
8
9
8686
8117
8687
8202
9734
8922
8229
9786
9674
9717
9643
8911
9672
9826
9018
9647
9782
9645
8574
8709
9675
9644
9787
9981
9980
7746
9827
9596
8758
8710
9733
8577
9646
9595
9600
8692
9614
9613
9676
9599
9612
8227
7714
9632
8766
9684
8228
8923
9683
8765
8759
™CH3
™OCH CH
™H
™H
™H
™H
™H
™Br
™H
™H
™H
™H
™CH3
™H
™H
™H
™OCH CH
™H
2,300
1,000
1,000
150
1,900
1,000
300
277
250
850
120
370
69
0.84
1.1
2,738
909
714
375
358
357
349
318
227
224
132
119
77
2
3
2
3
3
™CH3
™CH3
™CH3
™CH3
™OCH3
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™OCH3
™H
™H
™H
™CH3
™H
™H
™H
™H
™CH3
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
™H
c
™OCH CH
™OCH CH
1.4
2
3
2
™CH3
™CH3
™OCH3
™OCH3
™OCHF2
™F
™OCH3
™H
0.40
5.3
2.8
0.86
0.87
1.1
3.8
0.91
3.1
0.90
0.91
0.92
0.95
0.78
0.84
1.2
™CH CH
™OCH3
™CH3
™H
™CH3
™H
™H
™CH3
™F
™H
™CH3
™CH3
™CH3
™H
2
3
™CH3
™CH3
™CH3
™CH3
™CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
™Cl
™Cl
™OCH3
™F
™OCH3
™CH3
™Cl
™Cl
™H
™CH CH
2
3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™H
™C H
68
75
6
5
™H
™H
™F
™Cl
™H
57
62
46
42.3
32.1
30
23.4
22
44
33
27
c
™OCH3
™H
™CF3
™OCH3
™F
36
™OCH3 ™H
15
0.64
0.85
0.82
1.1
1.1
0.93
240
0.91
0.19
2,400
0.73
0.94
73
™CH3
™H
™H
™Cl
™H
™H
™H
™OCHF2
19
™H
17
20.7
18
™H
™H
20
™H
™OCH3 ™OCH CH
™H
18
16.5
16.1
16
2
3
™H
™OCH3 ™O(CH ) CH
™H
15
2 2
3
™H
™OCH3 ™OCH3
™OCH3
™H
3,900
14
™CH3
™CH3
™H
™F
™H
™H
™OCH3
™Cl
™H
15
™CH3
™OCH3
™H
™H
2.8
31,000
14.7
12.9
11.9
10.6
9.2
™CH OCH
™OCH3
™OCH3
™CH3
™H
2
3
c
™CH3
™CH3
™H
™OCH3 ™H
8.7
™OCH3
™H
™Cl
™CH3
™H
H
™H
™CH3
™Br
™H
™H
™H
™H
™Cl
™Cl
™Cl
™H
™H
™CH3
10
670
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™CH3
™Cl
8.0
0.89
0.79
0.90
0.70
0.87
0.85
0.70
0.82
0.60
0.93
0.82
0.92
1,200
0.86
0.66
0.88
0.82
550
9.0
7.7
Cl
6.1
6.7
5.1
6.1
5.9
4.6
5.2
3.7
5.5
4.7
5.0
™H
7.44
7.29
7.01
6.94
6.6
6.34
6.17
5.91
5.7
™OCH3 ™OCH3
™H
™CH3
™H
™H
™Cl
™Br
™Br
™Br
™H
™H
™CH3
™H
™H
™Br
™CH3
™H
™H
™H
™H
™OCH3 ™H
™CF3
™OCH3
™H
™H
™OCH3 ™OCH3
™H
™Cl
™Cl
5.43
5.1
™CH OCH
™H
™OCH3 ™OCH3
™H
6,100
2
3
™CH3
™CH3
™F
™H
™H
™H
™H
™H
™H
™H
™H
™CF3
™CF3
™CF3
™CF3
™OCH3
™H
4.1
2.7
3.6
3.2
4.8
™OCH3
™OCH3
™Br
4.1
™CH CH
4.1
2
3
™CH3
3.9
™CH OCH
™H
™H
™OCH3 ™OCH3
™H ™Cl
1,300
2,600
2.4
2
3
3
™CH OCH
1,500
1.76
2
a
b
c
Derivatives are listed in descending order by selectivity ratio (see last column).
Selectivity ratio equals IC50 human DHFR/IC50 MAC DHFR.
10
™
3
H group on ™N link is replaced with ™CH . SRI, Southern Research Institute.

TABLE 3. DMDP derivatives with complex ring substitutions attached to the ™CN bridge
IC50 (nM) for:
Derivative
SRI no.b
Selectivity
a
X
c
no.
ratio
Human DHFR
710
MAC DHFR
1.9
d
5
2
3
8708
374
153
5
8691
9735
142
0.93
320
5
4
5,300
16.6
5
5
5
6
9468
9771
10
0.76
280
13.2
12.1
3,400
5
5
7
8
9449
12
1.2
86
10.0
9.5
e
7747
820
5
9
9487
6.7
0.86
7.79
6
6
0
1
9450
9824
7.1
6.2
0.95
1.0
7.47
6.2
6
6
2
3
7711
9825
4.3
2.0
0.81
0.43
5.31
4.65
6
4
9692
4.0
0.87
4.6
6
6
5
6
9439
9823
4.3
3.8
0.98
0.88
4.39
4.3
6
7
9673
Ͼ25,300
620
a
b
c
d
e
Derivatives are listed in descending order by selectivity ratio (see last column).
SRI, Southern Research Institute.
Selectivity ratio ϭ IC50 human DHFR/IC50 MAC DHFR.
™
H group on ™N link is replaced with a ™CH
3
group.
™
CH group in position 5 is replaced with an ™H.
3
2
789

2
790
SULING ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
nance of activity for the MAC DHFR (Table 5). This yielded
a selectivity ratio of 7,300.
Intracellular efficacy of compound 78 (SRI-20094). Intracel-
lular efficacy of compound 78 was evaluated in MM6 cells
using concentrations of 0.5 and 1.5 ␮g/ml. Results revealed
that compound 78 was able to reduce MAC CFUs at 0.5 and
has been reported in other systems that the 5-H versus 5-CH3
substitution confers a greater decrease in activity against hu-
man DHFR than other DHFRs, resulting in higher selectivity
(12). In this study, only a modest increase in selectivity was
observed but always at the expense of enzyme inhibition. It is
clear from X-ray data in other systems that the 5-CH substi-
3
1
.5 ␮g/ml in the MM6 cell model (Fig. 2). Significant CFU
tution optimally occupies a hydrophobic pocket in the DHFR
enzyme. This observation is true for the human enzyme as well
as homology models derived for the mycobacterial enzymes
(R. C. Reynolds, unpublished results). The larger 5-substituent
™CH CH generally had only a modest effect on inhibition of
reductions of 0.65 and 0.73 log, respectively, were observed for
the 0.5- and 1.5-␮g/ml concentrations (P Ͻ 0.001) (Fig. 2). At
the end of the 7-day experimental period, viability of the MM6
cells was determined by the MTT assay (see above). Viability
of the cells treated with 0.5 and 1.5 ␮g/ml was 79 and 76% of
the nontreated control, respectively.
2
3
the human enzyme while slightly increasing the IC50 against
the MAC enzyme. Thus, the selectivity ratios generally dropped
slightly. When a larger substituent such as a 5-CH OCH was
2
3
tested, it was clear that enzyme inhibition was significantly and
negatively impacted, confirming that the 5-CH3 substitution
optimally fills this hydrophobic cleft within the DHFR enzyme.
In the bridge linker region (™X™), a variety of linkers were
DISCUSSION
Previous reports suggest that DMDP derivatives might be
developed into clinically useful antimycobacterial drugs (27,
8). We therefore conducted this study to determine the struc-
evaluated (X ϭ ™NH™ or ™NCH [Table 2]; X ϭ ™CH ™ and
2
3
2
™
S™ [Table 4]). However, the majority of derivatives contained
a 10-aza linkage. Our results with a small sample set (com-
pounds 3/1, 19/47, 30/20 [Table 2]), in which ™CH replaced the
ture-activity relationships of several DMDP derivatives as an-
timycobacterial agents against MAC and as selective inhibitors
of MAC DHFR. All derivatives were modified to test the
impact of key substitutions on binding and selectivity of the
compounds for mycobacterial DHFR versus the human en-
zyme, while retaining the critical 2,4-diaminopyrimidine por-
tion of the deazapteridine nucleus that is so critical for pro-
tonation in the active site of DHFR enzymes (3). The resulting
salt bridge between the inhibitor and enzyme forms the basis
for the potent binding and inhibition of these antifolate drugs.
Selectivity for eukaryotic versus prokaryotic DHFRs is gener-
ally driven through alterations outside of this critical binding
function (3). Therefore, while most of the DMDP derivatives
had good antimycobacterial activity against MAC, we found
that enzyme inhibition and selectivity for the MAC rDHFR
could be dramatically improved by making certain chemical
substitutions at strategic positions on the molecule.
3
™H on the bridge nitrogen, indicated generally that this sub-
stitution enhanced the inhibition of human DHFR while ac-
tivity decreased for MAC DHFR. Others have reported a
mixed trend in activity for other DHFRs with this substitution
on DMDP derivatives (10, 11). It is difficult to argue a trend
with such a small data set. It will be necessary to investigate a
small number of ™NCH ™ derivatives of other highly active,
3
selective ™N-linked analogs to further investigate the effect of
this substitution.
When we assayed a limited number of derivatives in which
the bridge nitrogen was replaced with a sulfur atom, we found
that the ™S-linked derivatives were more active against the
MAC DHFR than the human DHFR. Also, except for the
2Ј,5Ј-dimethoxy-substituted derivative 73 (Table 4), all of the
™S-linked derivatives yielded a higher selectivity ratio than
their ™N-linked counterparts. However, as mentioned previ-
In the case of the 5-substituent (R1 in Table 2), four groups
were evaluated, 5-H, 5-CH , 5-CH CH , and 5-CH OCH . It
3
2
3
2
3
TABLE 4. DMDP derivatives involving modification of the bridge molecules (™X™)
Modification at positionc
IC50 (nM) for:
Derivative
SRI no.b
Selectivity
ratiod
a
no.
X
R2
R3
R4
R5
R6
Human DHFR
MAC DHFR
68
69
70
71
72
73
74
75
76
77
9388
8455
9437
8451
8450
9490
8573
7896
9165
8453
™CH2
™S
™H
™OCH3
™H
™H
™OCH3
™OCH3
™OCH3
™OCH3
™H
™OCH3
™Cl
™H
™H
™H
™H
™H
™H
™H
990
440
1,200
500
590
2,500
420
440
2,700
850
1.5
1.5
4.5
2.7
3.6
23
660
293
267
185
164
109
108
95.7
57
™H
™OCH3
™S
™H
™OCH3
™H
™OCH3
™H
™Cl
™H
™H
™S
™H
™S
™H
™H
™S
™OCH3
™H
™H
™S
™H
3.9
4.6
47
™S
™1-Naphthyl
™H
™CH2
™S
™OCH3
™H
™H
™H
™OCH3
™CH3
™H
™H
™H
16
53
a
b
c
Derivatives are listed in decreasing order of selectivity ratio (see last column).
SRI, Southern Research Institute.
Substitutions on the phenyl group are designated as R2 through R6.
Selectivity ratio equals IC50 human DHFR/IC50 MAC DHFR.
d

VOL. 44, 2000
ANTIMYCOBACTERIAL ACTIVITIES OF 5-DEAZAPTERIDINES
2791
TABLE 5. DMDP derivatives in the series involving substitutions at the R2 and R5 positions
MICc of
MAC
(␮g/ml)
Substitution at position
R2 R5
IC50 (nM) for:
Derivative
SRI no.b
Selectivity
ratiod
a
no.
Human DHFR
MAC DHFR
1
7
4
2
1
7
5
9018
8229
8202
8117
8686
20094
™CH3
™CH3
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
ϩϩϩϩ
57
300
0.92
0.86
0.40
1.1
0.84
1.0
62
349
™OCH3
™CH3
™CH3
™OCH3
™OCH3
™OCH CH
™O(CH ) CH
2 2 3
150
375
™OCH3
1,000
2,300
7,300
909
2,738
7,300
™OCH CH
2
3
2
3
8
™O(CH ) CH
2
2
3
a
b
c
Derivatives are listed in ascending order by selectivity ratio (see last column).
SRI, Southern Research Institute.
ϩϩϩϩ, Յ0.13. MAC strain NJ3440 was used for comparison of MIC.
Selectivity ratio equals IC50 human DHFR/IC50 MAC DHFR.
d
ously, the 2Ј,5Ј-dialkoxy, 10-aza-linked derivatives had the
highest selectivity ratios. Thus, it may not be true that the
selectivity of any highly active sample can be improved by
changing the 10-aza linkage to a 10-thia linkage. A different
mode of binding for the ™S-linked compounds versus the highly
selective ON-linked-2Ј,5Ј-dialkoxy compounds in the MAC
enzyme may explain why the selectivity of some samples is
improved while the highly potent and selective 2Ј,5Ј-dialkoxy
compounds actually decrease. It may be possible, however, to
enhance the selectivity of other less selective but potent inhib-
itors by altering the linkage from nitrogen to sulfur. For ex-
ample, some of the halogen- and trifluoromethyl-substituted
compounds were active against both the MAC and human
enzymes. It will be worthwhile to make the equivalent ™S-
linked analogs to test this hypothesis (compare compound 72
[Table 4] versus 18 [Table 2]).
There are only two examples of a ™CH -substituted bridge
2
linkage in our data set (Table 4). With such a limited sample,
the results are equivocal in terms of a clear trend in enzyme
inhibition with this linkage alteration. Again, it would appear
that for the potent and selective 2,5-dimethoxy analog, this
alteration (compound 2 [Table 2] versus 76 [Table 4]) reduced
selectivity although this result came by decreasing activity
against both enzymes; human inhibition was decreased 2.7-fold
whereas MAC inhibition was reduced about 40-fold. In the
case of the 3,5-dimethoxy substitution (compound 20 [Table 2]
versus 68 [Table 4]), selectivity was enhanced from 23.4 to 660
by altering the ™NH™ linkage to a ™CH ™ linkage. Enzyme
2
inhibition was again lessened, but only slightly for MAC (0.64
nM versus 1.5 nM) versus about 66-fold for human enzyme (15
nM versus 990 nM). Therefore, substitution at the 3,5-position
of the ™C-linked deazapteridine system may be fruitful for
developing other active, selective DHFR inhibitors.
The remaining portion of the DMDP analogs that requires
discussion is the substituted aromatic ring (R2 through R6)
attached through the linkage to the 6 position of the deaza-
pteridine ring system (Table 2). In certain cases, these substi-
tutions have been discussed above, relative to other changes in
the molecule. In general, we next discuss the 5-CH ™10-aza
3
system, since more predictive data are available. Several sub-
stitution patterns stand out. The 2Ј,4Ј,6Ј-trisubstituted analogs
appeared to show good selectivity and activity (compounds 5
and 10 [Table 2]). No 2Ј,6Ј- or 4Ј,6Ј-disubstituted analogs were
evaluated in this study. Based on this small data set of com-
pounds, it will be worthwhile to pursue a modest number of
analogs to probe the 2Ј,4Ј-, 2Ј,6Ј-, 4Ј,6Ј-, and 2Ј,4Ј,6Ј-substitu-
tion patterns in order to enhance selectivity and potency. It was
the 2Ј,5Ј-dialkoxy substitution that showed a clear trend to-
wards higher selectivity while retaining nanomolar potency
against the MAC enzyme. The 2Ј,5Ј-dimethoxy analog (com-
pound 2 [Table 2]) was quite active against MAC (MIC Յ 0.13
␮g/ml). This analog inhibited MAC rDHFR at 1.1 nM versus
FIG. 2. Treatment of MAC-infected MM6 cells with compound 78 (SRI-
2
0094). MM6 cells were infected with MAC strain NJ3404 as described in
Materials and Methods. CFUs were determined in triplicate at 0 and 7 days for
nontreated ( E ) cells and those treated with 0.5 ␮g ( F ) of compound 78 per
ml and 1.5 ␮g ( ■ ) of compound 78 per ml. Values represent the mean plus or
minus standard error of the mean for triplicate assays. The differences between
the mean values for the cells treated with 0.5 ␮g and 1.5 ␮g of compound 78 per
ml versus the nontreated control cells were both significant at a P value of
Ͻ0.001. Significance was determined by one-way analysis of variance, and a
correction for multiple comparisons (posttest) was performed by the Tukey-
Kramer multiple-comparisons test using InStat (GraphPad Software).

2
792
SULING ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
1
,000 nM for the human enzyme, giving a good selectivity ratio
REFERENCES
of 909. Extension of the methoxy moiety at the R2 and R5
positions (i.e., diethoxy analog 1 [Table 2]) was similarly more
active against the MAC rDHFR (0.84 nM), while the activity
against the human enzyme decreased to 2,300 nM for a selec-
tivity ratio of 2,378. Further extension at the R2 and R5 posi-
tions resulted in a derivative with decreased potency for the
human rDHFR but continued activity for the MAC DHFR
1
. Baccanari, D. P., and L. F. Kuyper. 1993. Basis of selectivity of antibacterial
diaminopyrimidines. J. Chemother. 5:393–399.
2. Barrow, E. L. W., G. A. Winchester, J. K. Staas, D. C. Quenelle, and W. W.
Barrow. 1998. Use of microsphere technology for sustained and targeted
delivery of rifampin to Mycobacterium tuberculosis-infected macrophages.
Antimicrob. Agents Chemother. 42:2682–2689.
3. Blaney, J. M., C. Hansch, C. Silipo, and A. Vittoria. 1984. Structure-activity
relationships of dihydrofolate inhibitors. Chem. Rev. 84:333–407.
4
. Bleyer, W. A. 1978. The clinical pharmacology of methotrexate. New appli-
cations of an old drug. Cancer Treat. Rev. 41:36–51.
. Bowden, K., N. V. Harris, and C. A. Watson. 1993. Structure-activity rela-
tionships of dihydrofolate reductase inhibitors. J. Chemother. 5:377–388.
(compound 78 [Table 5]). This improvement reduced the tox-
icity of the series further and allowed for the demonstration of
intracellular efficacy in the MM6 cells. Clearly, other substitu-
tions at the 2Ј,5Ј position of the phenyl ring would be fruitful
in order to develop more selective and potent inhibitors of
MAC DHFR. Studies are in progress to improve the potency
of the DMDP derivatives. It is interesting to note that Piper et
al. (22) previously investigated the activity of several of the
DMDP derivatives reported here, but against Pneumocystis
carinii and Toxoplasma gondii DHFR. These investigators con-
cluded that, in general, compounds with a 2Ј,5Ј-disubstituted
phenyl group are better in potency and selectivity than com-
pounds substituted on other positions of the phenyl group.
In summary, our results support the conclusion that DMDP
derivatives can be developed which are highly active against
MAC and which have minimum toxicity to the host. However,
a comparison of our results with those reported by others for
trimethoprim (21) suggests that further improvement of the
selectivity ratio by about 10-fold is needed to achieve this goal.
Trimethoprim, a clinically effective antibacterial agent and
DHFR inhibitor, has reported IC s for E. coli DHFR of 5 to
5
6. Collins, L. A., and S. G. Franzblau. 1997. Microplate Alamar Blue assay
versus BACTEC 460 system for high-throughput screening of compounds
against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob.
Agents Chemother. 41:1004–1009.
7
. Czaplinski, K.-H., W. H a¨ nsel, M. Wiese, and J. K. Seydel. 1995. New ben-
zylpyrimidines: inhibition of DHFR from various species. QSAR, CoMFA
and PC analysis. Eur. J. Med. Chem. 30:779–787.
. Delcamp, T. J., S. S. Susten, D. T. Blankenship, and J. H. Freisheim. 1983.
Purification and characterization of dihydrofolate reductase from methotrex-
ate-resistant human lymphoblastoid cells. Biochemistry 22:633–639.
8
9. Finland, M., and E. H. Kass. 1973. Symposium on trimethoprim-sulfameth-
oxazole. J. Infect. Dis. 128:S425–S816.
0. Gangjee, A., O. Adair, and S. F. Queener. 1999. Pneumocystis carinii and
Toxoplasma gondii dihydrofolate reductase inhibitors and antitumor agents:
synthesis and biological activities of 2,4-diamino-5-methyl-6-[(monosubsti-
tuted anilino) methyl]-pyrido [2,3-d] pyrimidines. J. Med. Chem. 42:2447–
2455.
1. Gangjee, A., J. Shi, S. F. Queener, L. R. Barrows, and R. L. Kisliuk. 1993.
Synthesis of 5-methyl-5-deaza nonclassical antifolates as inhibitors of dihy-
drofolate reductases and potential antipneumocystis, antitoxoplasma, and
antitumor agents. J. Med. Chem. 36:3437–3443.
12. Gangjee, A., A. Vasudevan, S. F. Queener, and R. L. Kisliuk. 1996. 2,4-
Diamino-5-deaza-6-substituted pyrido[2,3-d]pyrimidine antifolates as potent
and selective nonclassical inhibitors of dihydrofolate reductases. J. Med.
Chem. 39:1438–1446.
13. Hartman, P. G. 1993. Molecular aspects and mechanism of action of dihy-
drofolate reductase inhibitors. J. Chemother. 5:369–376.
4. Hillcoat, B. L., P. F. Dixon, and R. L. Blakley. 1967. Effect of substrate
decomposition on the spectrophotometric assay of dihydrofolate reductase.
Anal. Biochem. 21:178–189.
15. Kansy, M., J. K. Seydel, M. Wiese, and R. Haller. 1992. Synthesis of new
2,4-diamino-5-benzylpyrimidines active against various bacterial species.
Eur. J. Med. Chem. 27:237–244.
1
1
5
0
5
8
1
7
nM while the IC s for rat liver DHFR are 2.6 ϫ 10 to 3.7 ϫ
50
5
0 nM (21). These data give a selectivity ratio of 32,500 to
4,000. The selectivity ratio for compound 78 was 7,300. Fur-
ther studies are in progress to improve the activity of DMDP
derivatives against MAC and to assess their efficacy in an
animal model. Providing that structural modifications can be
1
made to increase the IC5 for the human DHFR, while main-
0
taining selective potency against the MAC DHFR, we feel that
it will be possible to increase the selectivity ratio to levels
comparable to those observed in other bacterial systems with
trimethoprim. This should result in a DHFR inhibitor that can
be used effectively to treat MAC infections, providing that
other factors such as cell uptake of drug and pharmacologic
parameters are favorable.
1
6. Li, R., R. Sirawaraporn, P. Chitnumsub, W. Sirawaraporn, J. Wooden, F.
Athappilly, S. Turley, and W. G. J. Hol. 2000. Three-dimensional structure of
M. tuberculosis dihydrofolate reductase reveals opportunities for the design
of novel tuberculosis drugs. J. Mol. Biol. 295:307–323.
1
7. Locher, H. H., H. Schlunegger, P. G. Hartman, P. Angehrn, and R. L. Then.
1
996. Antibacterial activities of epiroprim, a new dihydrofolate reductase
inhibitor, alone and in combination with dapsone. Antimicrob. Agents Che-
It is important to note that, although initial in vitro screening
of these DMDP derivatives with MAC demonstrated good
activity (27, 28), no clear rational synthetic approach was pos-
sible until enzyme inhibition data were obtained. Preliminary
studies with M. tuberculosis have revealed that most of the
DMDP derivatives presented here show less in vitro activity
against whole cells (Suling et al., 39th ICAAC, abstr. 339). The
results of subsequent preliminary studies with M. tuberculosis
rDHFR have suggested that the lower activity is due to differ-
ences in inhibitor binding with the enzyme (W. J. Suling, un-
published data). Thus, additional studies utilizing recombinant
M. tuberculosis DHFR will be useful in organizing a rational
synthetic approach for improved activity against M. tuberculo-
sis. Those studies are also in progress.
mother. 40:1376–1381.
18. McCourt, M., and V. Cody. 1991. Conformational analysis of lipophilic
antifolates: crystal structure of 2-amino-4-oxo-6-adamantylpteridine and a
comparison of its binding to bacterial and avian dihydrofolate reductase.
J. Am. Chem. Soc. 113:6634–6639.
1
9. Meyer, S. C. C., S. K. Majumder, and M. H. Cynamon. 1995. In vitro
activities of PS-15, a new dihydrofolate reductase inhibitor, and its cyclic
metabolite against Mycobacterium avium complex. Antimicrob. Agents Che-
mother. 39:1862–1863.
0. National Committee for Clinical Laboratory Standards. 1995. Tentative
standard M24-T. Antimycobacterial susceptibility testing for Mycobacterium
tuberculosis. National Committee for Clinical Laboratory Standards, Wayne, Pa.
21. Ohemeng, K. A., and B. Roth. 1991. Receptor-based design of novel dihy-
drofolate reductase inhibitors: benzimidazole and indole derivatives. J. Med.
Chem. 34:1383–1394.
2. Piper, J. R., C. A. Johnson, C. A. Krauth, R. L. Carter, C. A. Hosmer, S. F.
Queener, S. E. Borotz, and E. R. Pfefferkorn. 1996. Lipophilic antifolates as
agents against opportunistic infections. 1. Agents superior to trimetrexate
and piritrexim against Toxoplasma gondii and Pneumocystis carinii in in vitro
evaluations. J. Med. Chem. 39:1271–1280.
3. Prendergast, N. J., T. J. Delcamp, P. L. Smith, and J. H. Freisheim. 1988.
Expression and site-directed mutagenesis of human dihydrofolate reductase.
Biochemistry 27:3664–3671.
4. Saxena, A. K., and M. Saxena. 1986. Advances in chemotherapy of malaria.
Prog. Drug Res. 30:221–280.
5. Schweitzer, B. I., A. P. Dicker, and J. R. Bertino. 1990. Dihydrofolate re-
ductase as a therapeutic target. FASEB J. 4:2441–2452.
26. Seydel, J. K. 1993. In vitro and in vivo results of brodimoprim and analogues
2
2
ACKNOWLEDGMENTS
2
This research was primarily funded by National Institutes of Health
NIH) grant AI41348 (W.W.B., Principal Investigator). Earlier work
(
on some of the DMDP derivatives was funded by NIH grants AI38706
and AI30279 (J.R.P., Principal Investigator).
The M. avium strains were kindly provided by L. Heifets, National
Jewish Center for Immunology and Respiratory Diseases, Denver,
Colo.
2
2

VOL. 44, 2000
ANTIMYCOBACTERIAL ACTIVITIES OF 5-DEAZAPTERIDINES
2793
alone and in combination against E. coli and mycobacteria. J. Chemother.
:422–429.
7. Shoen, C., O. Choromanska, R. C. Reynolds, J. R. Piper, and M. Cynamon.
998. In vitro activities of several diaminomethylpyridopyrimidines against
Mycobacterium avium complex. Antimicrob. Agents Chemother. 42:3315–
316.
tuberculosis and Mycobacterium avium complex. Antimicrob. Agents Chemo-
ther. 44:283–286.
31. Wright, E. L., D. C. Quenelle, W. J. Suling, and W. W. Barrow. 1996. Use of
Mono Mac 6 human monocytic cell line and J774 murine macrophage cell
line in parallel antimycobacterial drug studies. Antimicrob. Agents Chemo-
ther. 40:2206–2208.
5
2
1
3
3
2. Yajko, D. M., J. J. Madej, M. V. Lancaster, C. A. Sanders, V. L. Cawthon, B.
Gee, A. Babst, and W. K. Hadley. 1995. Colorimetric method for determining
MICs of antimicrobial agents for Mycobacterium tuberculosis. J. Clin. Micro-
biol. 33:2324–2327.
2
8. Suling, W. J., R. C. Reynolds, E. W. Barrow, L. N. Wilson, J. R. Piper, and
W. W. Barrow. 1998. Susceptibilities of Mycobacterium tuberculosis and My-
cobacterium avium complex to lipophilic deazapteridine derivatives, inhibi-
tors of dihydrofolate reductase. J. Antimicrob. Chemother. 42:811–815.
9. Then, R. L. 1993. History and future of antimicrobial diaminopyrimidines.
J. Chemother. 5:361–368.
3
3. Ziegler-Heitbrock, H. W. L., E. Thiel, A. F u¨ tterer, V. Herzog, and A. Wirtz.
2
3
1
988. Establishment of a human cell line (Mono Mac 6) with characteristics
of mature monocytes. Int. J. Cancer 41:456–461.
0. Tomioka, H., K. Sato, H. Kajitani, T. Akaki, and S. Shishido. 2000. Com-
parative antimicrobial activities of the newly synthesized quinolone WQ-
34. Zywno-vanGinkel, S., T. P. Dooley, W. J. Suling, and W. W. Barrow. 1997.
Identification and cloning of the Mycobacterium avium fol A gene, required
for dihydrofolate reductase activity. FEMS Microbiol. Lett. 156:69–78.
3
034, levofloxacin, sparfloxacin, and ciprofloxacin, against Mycobacterium