Inhibition of Bacterial Dihydrofolate Reductase by 6-Alkyl-2,4-diaminopyrimidines

Abstract

A series of (±)-6-alkyl-2,4-diaminopyrimidine-based inhibitors of bacterial dihydrofolate reductase (DHFR) have been prepared and evaluated for biological potency against Bacillus anthracis and Staphylococcus aureus. Biological studies reveal attenuated activity relative to earlier structures lacking substitution at C6 of the diaminopyrimidine moiety, though minimum inhibitory concentration (MIC) values are in the 0.125–8 μg/mL range for both organisms. This effect was rationalized from previous three-dimensional X-ray structure studies that indicate the presence of a side pocket containing two water molecules adjacent to the main binding pocket. Because of the hydrophobic nature of the substitutions at C6 the main interactions are with protein residues Leu20 and Leu28. These interactions lead to a minor conformational change in the protein, which opens the pocket containing these waters such that it is continuous with the main binding pocket. These water molecules are reported to play a critical role in the catalytic reaction. This highlights a new area for inhibitor expansion within the limited architectural variation at the catalytic site of bacterial DHFR.

Introduction

It is well known that the arsenal of available antibiotics is not sufficient to meet the growing burden of bacterial resistance, and that fewer efforts are being made at a pharmaceutical level to pursue new substances with antibiotic properties.^[1–3] We have combined this need with that of biodefense in re-purposing the anti-folate target dihydrofolate reductase (DHFR, E.C. 1.5.1.3). Our recently reported^[4–6] library of dihydrophthalazine-containing compounds has demonstrated potency against DHFR in Bacillus anthracis. We are also monitoring the broad-spectrum potency of this series, and in particular for anti-staphylococcal activity. Staphylococcus aureus is a prominent human pathogen with increasing antibiotic resistance, such as methillicin- and vancomycin-resistant forms (MRSA and VRSA, respectively).^[7,8] Previous work from our group demonstrated a conserved potency of our initial compound RAB1 (1a) for S. aureus 29213, four MRSA strains and three VRSA strains.^[9] Compound 1b was also found to be highly active. We have now attempted to enhance the broad-spectrum potency by incorporating molecular modeling techniques interfaced with chemistry, microbiology and biochemistry, and X-ray crystallography.

The previously determined B. anthracis DHFR co-crystal structure complexed with 1a revealed a preference for the S-enantiomer at the single chiral center bearing a propyl moiety.^[5] This is in contrast to the co-crystal structure of S. aureus DHFR with 1a, which revealed an alternate position for the dihydrophthalazine portion of the inhibitor containing the chiral center. Binding limited one position to the S-enantiomer, while the alternate position residing in a shallow surface pocket could accommodate either enantiomer.^[9] The binding site residues in contact with RAB1 show little variation in identity between the S. aureus and B. anthracis species.^[5,10] However, the shape of the binding site in S. aureus DHFR is generally wider than that of B. anthracis DHFR, allowing formation of the secondary binding surface, which we observed to be influenced by residue changes on the periphery of the site. These changes include a loss of an aromatic stacking interaction with Phe151 due to a His residue at position 30 in contrast to a Phe or Tyr, as well as loss of electrostatic stabilization arising from Tyr to Phe changes at residues 47 and 68.^[10] These observations highlighted how residues from outside of the binding pocket could modulate interactions and, in this case, allow either enantiomer to be bound in S. aureus DHFR versus just the S-enantiomer as complexed with B. anthracis DHFR.^[5]

Despite these differences between the bacterial species, the diaminopyrimidine (DAP) moiety, which is well conserved among anti-DHFR inhibitors, complexes the equivalent protein side chains within the binding site.^[5,10] The arrangement of hydrogen bond participants around the DAP ring allows for precise placement amid surrounding amino acid side chains and requires participation of both the side chain and the main chain from the protein, regardless of species. Examination of structures of B. anthracis and S. aureus DHFR highlighted a small unoccupied pocket of ~50 ų adjacent to the C6 position of the DAP ring (Figure 1). In efforts to explore the potential occupancy of this pocket, a series of DAP-based antifolates bearing methyl, ethyl, and propyl groups at C6 of the DAP moiety were designed, synthesized, and evaluated for potency as inhibitors of bacterial DHFR. A similar approach has been reported for the anti-malarial compound pyrimethamine, which has poor antibacterial activity due to differences in the plasmodial and bacterial DHFR protein sequences^[10,11] as well as a series of anti-bacterial propargyl linked DHFR inhibitors.^[12]

Figure 1.

A pocket adjacent to the DAP moiety is present in bacterial DHFR proteins and is occupied by two water molecules. Surface renderings of the DHFR protein from (a) B. anthracis and (b) S. aureus, both with compound 1a,^[5,10] and (c) S. aureus complexed with compound 12c. Surfaces are colored based on Kyte-Doolittle hydrophobicity calculations, where orange is hydrophobic and green is polar. The pocket of interest is continuous with the substrate site only at the DAP ring position; above this, it is gated by opposing Leu residues (20 and 28). In (c), the movement of Leu28 results in a complete opening of this pocket with the substrate-binding site. In (b) the alternate position for the dihydrophthalazine moiety of 1a is shown. In all panels the position of the co-factor NADPH can be seen at the bottom of the substrate site and, in this view, to the right of it; it is not involved in the pocket or interactions with the alkyl substituents.

Results and Discussion

Chemistry

The synthetic strategy started with 2, which led to compounds 3–8, and with 9, which gave (±)-10 in Scheme 1. Compounds 8 and (±)-10a–b were then coupled to generate racemic 11a–c and 12a–c as depicted in Scheme 2. Since both 1a and 1b demonstrated excellent inhibitory properties in our previous studies,^[5,6] the propyl- and isobutenyl-substituted compounds 11a–c and 12a–c were targeted. The synthesis of DAP derivatives 8a–c involved a seven-step sequence from commercially available 5-iodovanillin (2). Methylation of the phenolic hydroxyl in 2, using potassium carbonate and dimethyl sulfate in DMF, furnished iododimethoxybenzaldehyde 3. Further conversion of 3 by reduction with sodium borohydride gave alcohol 4, and treatment with phosphorus tribromide produced bromide 5. Reaction of 5 with a series of β-keto esters, in the presence of sodium methoxide in ethanol, led to the benzyl-substituted keto esters 6a–c. Cyclization of 6a–c was accomplished in good yields using guanidine carbonate in boiling ethanol to give hydroxypyrimidines 7a–c, which were used directly in the next sequence. These 4-hydroxypyrimidines were converted to diaminopyrimidines 8a–c in two steps by refluxing the former in phosphorus oxychloride, followed by treatment with ethanolic ammonia at 165 °C. A similar strategy has been reported previously^[13] but did not involve an iodine-substituted substrate. In the current work, this sequence proceeded cleanly to give 8a–c in 80–91% yields.

Scheme 1.

Synthesis of 8a–c and 10a–b. a) (CH₃O)₂SO₂, K₂CO₃, DMF, 100 °C, 96% yield; b) NaBH₄, THF, 23 °C, 99% yield; c) PBr₃, Et₂O, 23 °C, 96% yield; d) RCOCH₂CO₂Et, NaOMe, EtOH, 78 °C, [a: R′ = CH₃, b: R′ = CH₂CH₃, c: R′ = CH₂CH₂CH₃], 60–68% yield; e) [H2N=C(NH₂)₂]₂CO₃, EtOH, 78 °C, 75–80% yield; f) POCl₃, 106 °C; g) NH₃, EtOH, 165 °C, 72–75% yield (two steps); h) RMgBr, THF, 50 °C [a: R = CH₂CH₂CH₃, b: R = CH=C(CH₃)₂]; i) CH₂=CHCOCl, Et₃N, THF, 23 °C, 40–50% yield (two steps).

Scheme 2.

Synthesis of 11a–c and 12a–c. a) (±)-10a, Pd(OAc)₂, N-ethylpiperidine, DMF, 140 °C, 81–84% yield; b) (±)-10b, Pd(OAc)₂, N-ethylpiperidine, DMF, 140°C, 78–84% yield.

Finally, the dihydrophthalazine coupling partners (±)-10a–b were obtained by reacting phthalazine (9) with the propyl and isobutenyl Grignard reagents, followed by N-acylation with acryloyl chloride in the presence of triethylamine as described in earlier reports.^[5,6]

The final coupling reactions involving 8a–c and 10a–b were accomplished using a palladium acetate-promoted Heck coupling in the presence of N-ethylpiperidine in anhydrous DMF solvent at 140 °C (see Scheme 2). Purification of the products by flash chromatography, followed by recrystallization, afforded the final racemic compounds 11 and 12 in yields ranging from 78–85%. Spectral and elemental analyses of these materials revealed that each existed as a solvate of water and/or ethanol.

Biological Activity

Assessment of the parent compounds and inhibitors with alkyl substituents at C6 of the DAP moiety were carried out with purified DHFR in an enzyme assay to gauge the level of effectiveness at competing with the endogenous substrate, dihydrofolic acid. The resulting IC₅₀ values were incorporated with the enzyme K_m value for the substrate in order to calculate a K_i value,^[14] allowing for direct comparison despite the different enzymatic efficiencies of B. anthracis DHFR and S. aureus DHFR (Table 1). A general trend of more potent anti-staphylococcal activity was maintained with K_i values generally four times lower for S. aureus than for B. anthracis. Substitutions from the C6 position of DAP increased the K_i value for each species, marking them as less effective inhibitors than the parent compounds, and a decrease in potency (higher K_i) was progressive with substitution length. In some instances, the limits of solubility precluded determination of the IC₅₀ (such as with 11b, 11c and 12c), and values are listed as greater than the top concentration tested.

Table 1.

Biological activity of 6-alkyl-DAP inhibitors^[a]

Ki, nM (SEM)			MIC, μg/mL B. anthracis
Compound	B. anthracis Sterne	S. aureus 29213	Sterne	S. aureus 29213
1a	9.4 (0.2)	1.2 (0.1)	1	0.125–0.25
11a	72.4 (0.6)	4.5 (0.1)	8	0.5
11b	> 2400	3.6 (0.1)	4	0.5–2
11c	> 2400	4.0 (0.1)	1	1–2
1b	8.3 (0.2)	1.8 (0.1)	0.5	0.125 – 0.25
12a	14.3 (0.2)	3.4 (0.1)	4	0.25 – 0.5
12b	25.4 (0.8)	2.9 (0.1)	4	0.25 – 0.5
12c	> 2500	4.1 (0.1)	2	2

^[a]The Ki value is calculated from IC₅₀ measurements with compensatory weight based on the K_m for the dihydrofolate substrate. These values are listed with the standard error of the mean (SEM). The minimum inhibitory concentration (MIC) is the concentration of inhibitor needed to stop all bacterial growth under defined conditions.

These compounds were also incubated in cultures of the two bacterial organisms to determine the minimum inhibitory concentration (MIC) required to inhibit bacterial growth (Table 1), which is a useful parameter for consideration in clinical applications. The MIC values for S. aureus progressively increased 10-fold between parent (1a and 1b) versus the C6 propyl substitution (11c and 12c), indicating decreased potency within the cellular system. Interestingly, for B. anthracis the MIC values follow the same trend of progressively decreasing potency only for the compounds bearing an isobutenyl at the chiral center (1b, 12a–c), which is 9 Å from the C6 position. The other series, containing a propyl at the chiral center, displays the same MIC value and thus potent inhibition for the parent (1a) as for the C6 propyl substitution 11c. However, the two intermediate substitutions, 11a and 11b, show an increase in MIC in excess of any expected experimental variation. This is difficult to rationalize in terms of structural elements as the K_i values do not reflect the same trend. Since the MIC measurements are in the context of whole cells, they require the compounds to cross cell membranes, and there are additional factors in the MIC assay that are absent in the K_i measurements. These factors clearly influence the resulting activity, and consequently another explanation could involve cross-reactivity with other member enzymes of the folate pathway, although this has not been demonstrated.

X-ray Data

X-ray crystallography was used to visualize the DAP modifications within the binding site of S. aureus DHFR for complexes with 12b and 12c (Table 2). Similar experiments with B. anthracis DHFR were not successful, as this protein has been observed to crystallize poorly or not at all in the absence of a tightly bound compound in the substrate site (unpublished observation). Due to the high degree of similarity of residues for each DHFR involved in binding, it is feasible to analyze the S. aureus DHFR co-crystal structure to draw generalizations about the mode of binding. The resulting electron density maps, obtained prior to crystallographic refinement with the inhibitor, clearly shows the positions of the appended atoms on the DAP ring within the S. aureus DHFR binding site (Figure 2a,b). For either 12b or 12c, the placement within the pocket is as expected, and contacts are conserved from those previously visualized with 1a.^[9] However, the substitutions at C6 complex along the hydrophobic edge of the identified pocket (Figure 1c). These did not protrude within the adjacent 50 ų pocket, where two highly conserved water molecules are located.

Table 2.

Statistics for macromolecular X-ray data collection and refinement

S.aureus DHFR + NADPH	(±)-12b	(±)-12c
PDB code	4FGH	4FGG
Data Collection
Space Group	P6122	P6122
Cell dimensions
a, b, c (Å)	78.9, 78.9, 106.2	79.0, 79.0, 107.5
α, β, γ (°)	90, 90, 120	90, 90, 120
Resolution (Å)	Inf – 2.50 (2.55–2.50)	Inf – 2.30 (2.36–2.30)
Rint	16.0 (48.4)	14.4 (50.4)
I/σI	19.6 (5.5)	22.8 (4.8)
Completeness (%)	100 (100)	100 (100)
Redundancy	36.0 (26.0)	34.6 (23.6)
Mosaicity	0.74	0.66
Wilson B-factor	34.0	24.8
Refinement
Resolution (Å)	68.4 – 2.5	68.4 – 2.3
Rwork/Rfree	19.2/24.6	19.7/24.5
Protein atoms	1324	1330
Ligand/NADPH atoms	40/48	40/48
Water atoms	69	143
B factor, protein	23.5	17.4
B factor, Ligand/NADPH	32.7/24.2	26.6/14.4
B factor, water	30.1	24.1
R.M.S.D. bond lengths (Å)	0.008	0.008
R.M.S.D. bond angles (°)	1.184	1.225

Figure 2.

Position and interactions of 6-alkyl substitutions within the binding site of S. aureus DHFR. (a) and (b) depict the electron density for the inhibitors 12b and 12c, respectively, prior to crystallographic refinement with ligand in place. Protein and ligand are shown as ball-and-stick models. Electron density is rendered as mesh with a 2Fo − Fc (blue) map at 1.2 σ and an Fo − Fc (green) map at 3.0 σ. These maps clearly indicate the position of the alkyl extensions in the absence of any model bias. Both (c) and (d) display distance measurements between atoms of inhibitor 12c (yellow carbon atoms, blue nitrogen atoms and red oxygen atoms) and protein side chains (colored as 12c but with grey carbon atoms) Leu20, Trp22, Asp27, and Leu28. The two water molecules discussed in the text are also depicted. The NADPH co-factor is visible behind the inhibitor at the top of the Figure; it is not involved in binding at the site of the 6-alkyl extensions.

Other computational and experimental studies have demonstrated the important role of water at so-called “hydration sites” in protein-ligand binding, analogous to the currently identified water molecules.^[15] Calculated energetics indicate the liberation of these two water molecules would require ~4 kcal/mol; this inhibitor series generally displays binding energies of 42–44 kcal/mol and are derived largely from van der Waals interactions. These highly conserved water molecules have been seen in previous DHFR structures and are well-characterized within the E. coli system. Such water is required for positioning the folate substrate relative to the nicotinamide co-factor, in addition to coordinating the critical catalytic residue Asp27 to maintain optimum reaction geometry.^[16,17] Trp22 is a secondary residue that also coordinates the water molecules, and it is not clear if its role is strictly geometric or if it assists in perturbation of the local pK_a as needed to promote protonation of the substrate via Asp27. It is also suggested that these two conserved waters may provide the catalytic proton, supporting their invariant positions among all DHFR structures known to date and their importance to the protein structure.

The C6 alkyl group on each structure is sandwiched between the face of the central dimethoxybenzene ring of the inhibitor, residues Leu20, Asp27 and Leu28, and the two highly conserved water molecules (Figure 2c,d). In addition, for the 12c complex, a rotameric change in side chain position of Leu28 is necessary to accommodate the inhibitor (Figure 1c). As a consequence, this movement alters the position of the distal end of the inhibitor by ~0.7 Å. The energetic costs of this different arrangement likely play a role in the decreased potency. Differences in biological activity between the two species probably arise due to structural elements that influence positioning at the opposite end of the inhibitor (the dihydrophthalazine containing the chiral center, ~9 Å away). Therefore, when relating these observations to the B. anthracis DHFR protein, the same mode of binding is expected, including the maintenance of the two water molecules within the pocket. Overall, these observations highlight the relative ease in re-ordering hydrophobic interactions, which are not dependent on the direction of atomic interactions, rather than displacing the specific hydrogen bonds mediated by the water molecules. Furthermore, the rotameric movement of Leu28 in the complex with 12c revealed a continuous opening of the pocket adjacent to the inhibitor-binding pocket (Figure 1c). This may prove very advantageous for further inhibitor design, and defines a minimum alkyl chain length to achieve this result.

Conclusions

In summary, alkyl extensions have been appended to the DAP moiety of propyl- and isobutenyl-substituted, dihydrophthalazine-based DHFR inhibitors, and their activities have been evaluated. Testing the potency against B. anthracis, a known bioterrorism agent, and S. aureus, a prominent human pathogen, revealed modestly reduced potency versus systems lacking substitution at C6 on the pyrimidine. This effect is rationalized based on the three-dimensional structures, which highlight the conservation of two water molecules within a small protein cavity adjacent to the main binding site. These water molecules are believed to play a critical role in the catalytic reaction and are not displaced by these inhibitors. The longest extension tested, a propyl group, was the minimum length needed to completely open this side pocket to the main binding site, which may be advantageous in design of more efficacious agents targeting bacterial DHFR.

Experimental Section

Chemistry

Commercial anhydrous N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were stored under dry nitrogen and transferred by syringe into reactions when required. Tetrahydrofuran (THF) was dried over potassium hydroxide pellets and distilled from lithium aluminium hydride prior to use. Potassium carbonate (K₂CO₃) was heated at 120 °C under high vacuum for a period of 16 h and stored in an oven at 90 °C before use. All other commercial reagents were used as received.

Unless otherwise specified, all reactions were run under dry nitrogen in oven-dried glassware. Reactions were monitored by thin layer chromatography on silica gel GF plates (Analtech No. 21521). Preparative separations were performed by flash column chromatography^[18] on silica gel (Davisil^® grade 62, 60–200 mesh) mixed with UV-active phosphor (Sorbent Technologies, No. UV-5). Band elution for all chromatographic separations was monitored using a hand held UV lamp. The saturated NaCl, NH₄Cl and NaHCO₃ used in work-up procedures refer to aqueous solutions. Melting points were uncorrected. FT-IR spectra were run as thin films on NaCl disks. ¹H- and ¹³C-NMR spectra were measured on a Varian GEMINI 300 instrument at 300 MHz (¹H) and 75 MHz (¹³C), respectively, and referenced to internal tetramethylsilane. Elemental analyses were performed by Atlantic Microlab, Inc.

5-Iodo-3,4-dimethoxybenzaldehyde (3)

This compound was prepared from 2 on a 0.27-mol scale using the method of Nimgirawath.^[19] The crude product was recrystallized (4:1 ethanol:water) to give 3 as a white solid (25.2 g, 96%): mp: 71–72°C (lit^[18] mp: 71–72°C); ¹H-NMR (CDCl₃): δ =9.83 (s, 1H), 7.85 (d, 1H, J = 1.7 Hz), 7.41 (d, 1H, J = 1.7 Hz), 3.93 (s, 3H), 3.92 (s, 3H); ¹³C-NMR (CDCl₃):δ =189.7, 154.2, 153.0, 134.7, 133.9, 111.0, 92.1, 60.7, 56.1. IR: ν = 2832, 2730, 2693 cm⁻¹; No IR or ¹³C-NMR data for 3 have been reported previously.

(3-Iodo-4,5-dimethoxyphenyl)methanol (4)

The method of Chowdhury and co-workers was modified.^[20] A solution of 25.0 g (85.0 mmol) of 3 and 100 mL of THF was treated portion-wise with 1.91 g (50.0 mmol) of sodium borohydride (1.91 g, 50.0 mmol) over 5 min, and the reaction was stirred at room temperature for 45 min. The crude reaction mixture was quenched with 100 mL of saturated NH₄Cl and extracted with ethyl acetate (3 × 125 mL). The combined organic layers were washed with 100 mL of saturated NaCl, dried (MgSO₄), and concentrated under vacuum to yield 4 as a thick colorless liquid (24.8 g, 99%): ¹H-NMR (CDCl₃): δ=7.27 (d, J = 1.6 Hz, 1H), 6.86 (d, J = 1.6 Hz, 1H), 4.53 (s, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 2.83 (br s, 1H); ¹³C-NMR (CDCl₃): δ=152.4, 147.7, 139.0, 128.3, 111.2, 92.1, 63.9, 60.3, 55.8; IR: ν = 3392, 2824 cm⁻¹.

5-(Bromomethyl)-1-iodo-2,3-dimethoxybenzene (5)

A stirred solution of 20.0 g (68.0 mmol) of 4 and 100 mL of dry ether was cooled to 0°C, and 20.2 g (7.0 mL, 74.8 mmol, 1.1 equiv) of phosphorus tribromide was added dropwise over 20 min. After the addition, stirring was continued for an additional 30 min to ensure complete conversion. The reaction mixture was quenched by dropwise addition of 200 mL of saturated NaHCO₃ over a period of 45 min. [Note: Quenching was done slowly to minimize frothing.] The reaction mixture was transferred to a separatory funnel, the layers were separated, and the aqueous layer was further extracted with ethyl acetate (3 × 150 mL). The combined extracts were washed with saturated NaCl, dried (MgSO₄), and concentrated under vacuum to yield 5 as a pale yellow solid (23.2 g, 96%): mp: 64–65°C; ¹H-NMR (CDCl₃): δ=7.37 (d, J = 1.6 Hz, 1H), 6.91 (d, J = 1.6 Hz, 1H), 4.40 (s, 2H), 3.87 (s, 3H), 3.83 (s, 3H); ¹³C-NMR (CDCl₃): δ=152.5, 149.0, 135.4, 130.7, 113.4, 92.2, 60.4, 56.0, 32.3; IR: ν = 2827 cm⁻¹.

Ethyl 2-(3-Iodo-4,5-dimethoxybenzyl)-3-oxobutanoate (6a)

The method of Chowdhury and coworkers was modified.^[20] To a solution of 8.75 g (8.57 mL, 67.0 mmol) of ethyl acetoacetate dissolved in 70 mL of dry ethanol was added 3.63 g (67.0 mmol) of powdered sodium methoxide, and the reaction mixture was warmed to 50°C over 30 min. To the warm mixture was added dropwise 20.0 g (56.0 mmol, 0.84 equiv) of 5, and the reaction was refluxed for 18 h. After cooling, the crude product was concentrated under vacuum and purified on a 100-cm × 3-cm silica gel column using 25% ethyl acetate in hexanes to give 6a as a colorless liquid (15.4 g, 68%): ¹H-NMR (CDCl₃): δ=7.16 (d, J = 1.6 Hz, 1H), 6.71 (d, J = 1.6 Hz, 1H), 4.17 (q, J = 7.3 Hz, 2H), 3.83 (s, 3H), 3.79 (s, 3H), 3.74 (t, J = 7.3 Hz, 1H), 3.06 (m, 2H), 2.23 (s, 3H), 1.25 (t, J = 7.3 Hz, 3H); ¹³C-NMR (CDCl₃): δ=201.9, 168.8, 152.3, 147.5, 136.2, 130.2, 113.5, 92.3, 61.6, 61.1, 60.3, 55.8, 32.9, 29.5, 14.0; IR: ν = 2828, 1736, 1716 cm⁻¹.

Ethyl 2-(3-Iodo-4,5-dimethoxybenzyl)-3-oxopentanoate (6b)

This compound was prepared as above using 9.69 g (67.0 mmol) of ethyl 3-oxovalerate dissolved in 70 mL of dry ethanol, 3.63 g (67.0 mmol) of powdered sodium methoxide, and 20.0 g (56.0 mmol, 0.84 equiv) of 5 to give 6b as a colorless liquid (14.1 g, 60%): ¹H-NMR (CDCl₃): δ=7.15 (d, J = 2.0 Hz, 1H), 6.69 (d, J = 2.0 Hz, 1H), 4.16 (q, J = 7.3 Hz, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 3.74 (t, J = 7.3 Hz, 1H), 3.06 (m, 2H), 2.62 (dq, J = 18.1, 7.3 Hz, 1H), 2.39 (dq, J = 18.1, 7.3 Hz, 1H), 1.23 (t, J = 7.3 Hz, 3H), 1.03 (t, J = 7.3 Hz, 3H); ¹³C-NMR (CDCl₃): δ=204.9, 168.9, 152.3, 147.6, 136.4, 130.3, 113.6, 92.3, 61.5, 60.3, 60.1, 55.9, 36.1, 33.2, 14.0, 7.5; IR: ν = 2823, 1740, 1714 cm⁻¹.

Ethyl 2-(3-Iodo-4,5-dimethoxybenzyl)-3-oxohexanoate (6c)

This compound was prepared as above using 10.6 g (67.0 mmol) of ethyl butyrylacetate dissolved in 70 mL of dry ethanol, 3.63 g (67.0 mmol) of powdered sodium methoxide, and 20.0 g (56.0 mmol, 0.84 equiv) of 5 to give 6c as a colorless liquid (15.6 g, 64%): ¹H-NMR (CDCl₃): δ=7.15 (d, J = 1.6 Hz, 1H), 6.70 (d, J = 1.6 Hz, 1H), 4.16 (q, J = 7.3 Hz, 2H), 3.82 (s, 3H), 3.79 (s, 3H), 3.74 (t, J = 7.3 Hz, 1H), 3.06 (m, 2H), 2.55 (dt, J = 17.6, 7.3 Hz, 1H), 2.36 (dt, J = 17.6, 7.3 Hz, 1H), 1.56 (sextet, J = 7.3 Hz, 2H), 1.23 (t, J = 7.3 Hz, 3H), 0.86 (t, J = 7.3 Hz, 3H); ¹³C-NMR (CDCl₃): δ=204.2, 168.7, 152.2, 147.5, 136.3, 130.2, 113.5, 92.2, 61.4, 60.3 (2C), 55.8, 44.5, 33.0, 16.7, 14.0, 13.4; IR: ν = 2824, 1740, 1714 cm⁻¹.

2-Amino-5-(3-iodo-4,5-dimethoxybenzyl)-6-methylpyrimidin-4-ol (7a)

To a solution of 10.0 g (24.6 mmol) of 6a in 30 mL of dry ethanol was added 17.7 g (98.5 mmol, 4.00 equiv) of guanidine carbonate, and the reaction mixture was refluxed for 18 h. The ethanol was evaporated to a minimal volume, 50 mL of ice cold water was added, and the reaction mixture was kept at 0°C for 30 min to give a white precipitate. The solid was filtered and washed thoroughly with 100 mL of water and 50 mL of ether, and was then dried under high vacuum for 6 h to give 7a as a white solid 7.40 g, 75%): mp: 185–186°C; ¹H-NMR (DMSO-d₆): δ=7.54 (br s, 1H), 7.08 (s, 1H), 6.97 (s, 1H), 6.58 (br s, 2H), 3.76 (s, 3H), 3.65 (s, 3H), 3.59 (s, 2H), 1.98 (s, 3H); ¹³C-NMR (DMSO-d₆):δ=169.6, 161.0, 158.4, 151.9, 145.9, 141.0, 128.6, 113.3, 108.6, 92.2, 59.7, 55.7, 29.8, 21.3; IR: ν = 3516-2358, 1654 cm⁻¹.

2-Amino-5-(3-iodo-4,5-dimethoxybenzyl)-6-ethylpyrimidin-4-ol (7b)

This compound was prepared as above using 10.0 g (23.8 mmol) of 6b and 17.7 g (95.2 mmol, 4.00 equiv) of guanidine carbonate in 30 mL of dry ethanol to give 7b as a white solid (7.70 g, 78%): mp: 190–191°C; ¹H-NMR (DMSO-d₆): δ=11.0 (br s, 1H), 7.07 (s, 1H), 6.94 (s, 1H), 6.54 (br s, 2H), 3.76 (s, 3H), 3.65 (s, 3H), 3.61 (s, 2H), 2.35 (q, J = 7.1 Hz, 2H), 1.01 (t, J = 7.1 Hz, 3H); ¹³C-NMR (DMSO-d₆):δ=167.2 (br), 163.8 (br), 153.9, 152.0, 146.2, 140.1, 128.6, 113.3, 109.0, 92.3, 59.8, 55.8, 28.8, 27.2 (br), 12.6; IR: ν = 3405-2390, 1666 cm⁻¹.

2-Amino-5-(3-iodo-4,5-dimethoxybenzyl)-6-propylpyrimidin-4-ol (7c)

This compound was prepared as above using 10.0 g (23.0 mmol) of 6c and 16.6 g (92.2 mmol, 4.00 equiv) of guanidine carbonate in 30 mL of dry ethanol to give 7c as a white solid (7.90 g, 80%): mp: 194–195°C; ¹H-NMR (DMSO-d₆): δ=7.16 (br s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 6.71 (br s, 2H), 3.76 (s, 3H), 3.65 (s, 3H), 3.61 (s, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.45 (sextet, J = 7.3 Hz, 2H), 0.84 (t, J = 7.3 Hz, 3H); ¹³C-NMR (DMSO-d₆): δ=169.0, 164.2, 157.8, 151.9, 145.9, 141.3, 128.7, 113.3, 108.6, 92.2, 59.8, 55.7, 35.9, 29.4, 21.4, 14.0; IR: ν = 3520-2320, 1654 cm⁻¹.

5-(3-Iodo-4,5-dimethoxybenzyl)-6-methylpyrimidine-2,4-diamine (8a)

A mixture of 6.00 g (15.0 mmol) of 7a in 15 mL of phosphorus oxychloridewas refluxed for 2 h. During this time, the suspension gradually became a light brown solution. The solution was cooled in an ice bath for 20 min and was then slowly added dropwise to 150 g of crushed ice with vigorous stirring to give a white precipitate. The solid was filtered under vacuum and washed thoroughly with 100 mL of water, 50 mL of 20% ethanol-water, and finally with 50 mL of ether to give 5.70 g (91%) of the corresponding chloride as a white solid. This product was contaminated with several minor impurities and proved difficult to purify. Thus, it was used directly in the next step.

A stirred suspension of 5.50 g (13.1 mmol) of the chloride in 80 mL of dry ethanol was cooled to 0°C, and ammonia gas was bubbled through the solution for 15–20 min. The resulting solution was transferred to a glass-lined 450-mL stainless steel pressure reactor (Paar No. 4760) and heated to 165°C for 16 h. The reaction was cooled, and the solvent was evaporated under vacuum. The crude product was purified by flash chromatography on a 70-cm × 3-cm silica gel column eluted with dichloromethane:methanol:triethylamine (95:5:1) to give 8a as a white solid (4.21 g, 80%): mp: 236–237°C; ¹H-NMR (DMSO-d₆): δ=6.99 (s, 1H), 6.91 (s, 1H), 6.51 (br s, 2H), 6.14 (br s, 2H), 3.77 (s, 3H), 3.69 (s, 2H), 3.65 (s, 3H), 2.08 (s, 3H); ¹³C-NMR (DMSO-d₆): δ=163.3, 159.6, 159.4, 152.0, 146.4, 138.3, 128.3, 113.4, 102.4, 92.6, 59.8, 55.8, 29.2, 20.1; IR: ν = 3420-2200, 1637 cm⁻¹.

5-(3-Iodo-4,5-dimethoxybenzyl)-6-ethylpyrimidine-2,4-diamine (8b)

This compound was prepared as above using 6.00 (14.4 mmol) of 7b and 15 mL of phosphorus oxychloride to give 5.61 g (90%) of the expected chloride as a tan solid. A stirred suspension of 5.50 g (12.6 mmol) of the chloride in 80 mL of dry ethanol was cooled to 0°C, treated with ammonia, and then heated to 165° C for 16 h. Purification by flash chromatography using dichloromethane:methanol:triethylamine (95:5:1) gave 8b as an off-white solid (4.20 g, 80%): mp: 242–243°C; ¹H-NMR (DMSO-d₆): δ=6.98 (d, J = 1.6 Hz, 1H), 6.91 (d, J = 1.6 Hz, 1H), 6.57 (br s, 2H), 6.23 (br s, 2H), 3.77 (s, 3H), 3.72 (s, 2H), 3.65 (s, 3H), 2.41 (q, J = 7.7 Hz, 2H), 1.04 (t, J = 7.7 Hz, 3H); ¹³C-NMR (DMSO-d₆):δ=164.1, 163.6, 159.6, 152.0, 146.4, 138.5, 128.3, 113.4, 101.6, 92.5, 59.8, 55.8, 28.8, 25.8, 12.9; IR: ν = 3460-2200, 1633 cm⁻¹.

5-(3-Iodo-4,5-dimethoxybenzyl)-6-propylpyrimidine-2,4-diamine (8c)

This compound was prepared as above using 6.00 g (14.0 mmol) of 7c and 15 mL of phosphorus oxychloride to give 5.70 g (91%) of the expected chloride as a tan solid. A stirred suspension of 5.50 g (12.3 mmol) of the chloride in 80 mL of dry ethanol was cooled to 0°C, treated with ammonia, and then heated to 165° C for 16 h. Purification by flash chromatography using dichloromethane:methanol:triethylamine (95:5:1) gave 8c as an off-white solid (4.31 g, 82%): mp: 233–234°C; ¹H-NMR (DMSO-d₆): δ=6.98 (s, 1H), 6.91 (s, 1H), 6.39 (br s, 2H), 6.06 (br s, 2H), 3.77 (s, 3H), 3.72 (s, 2H), 3.66 (s, 3H), 2.36 (t, J = 7.1 Hz, 2H), 1.49 (sextet, J = 7.1 Hz, 2H), 0.84 (t, J = 7.1 Hz, 3H); ¹³C-NMR (DMSO-d₆): δ=164.2, 163.4, 160.2, 151.9, 146.3, 138.8, 128.4, 113.4, 101.8, 92.5, 59.8, 55.8, 34.9, 29.0, 21.5, 13.9; IR: ν = 3480-2340, 1639 cm⁻¹.

(±)-(E)-3-{5-[(2,4-Diamino-6-methyl-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (11a)

To a stirred solution of 1.00 g (2.50 mmol) of 8a in 8 mL of dry DMF under nitrogen was added 627 mg (2.75 mmol, 1.10 equiv) of (±)-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one [(±)-10a]^[6] dissolved in 1 mL of DMF, followed by 310 mg (0.38 mL, 2.75 mmol, 1.10 equiv) of N-ethylpiperidine. To this solution was added 20 mg (0.089 mmol) of palladium(II) acetate, and the reaction mixture was heated at 140°C for 20 h. Isolation of the expected product was achieved by pouring the cooled reaction mixture directly onto a 50-cm × 2.5-cm silica gel flash chromatography column packed in dichloromethane. Impurities were eluted using dichloromethane, and the final product was collected using dichloromethane:methanol:triethylamine (97:3:1) as the eluent. Evaporation of the solvent gave a pale yellow solid, which was further purified using a 15-cm × 2-cm silica gel column, packed with dichloromethane and eluted with dichloromethane:methanol:triethylamine (97:3:1). This second chromatography removed yellow-colored impurities as well as several minor contaminants. Evaporation of the solvent gave 11a as a light purple solid (1.05 g, 84%): mp: 137–138°C; ¹H-NMR (DMSO-d₆): δ=7.94 (s, 1H), 7.84 (d, J = 15.9 Hz, 1H), 7.60 (d, J = 15.9 Hz, 1H), 7.57-7.37 (complex m, 4H), 7.12 (s, 1H), 6.87 (s, 1H), 6.76 (br s, 2H), 6.28 (br s, 2H), 5.84 (t, J = 6.6 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 2H), 3.74 (s, 3H), 2.17 (s, 3H), 1.54 (m, 2H), 1.19 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H); ¹³C-NMR (DMSO-d₆): δ=165.5, 163.5, 158.5, 152.5, 146.0, 142.9, 136.7, 135.9, 133.6, 131.7, 128.3, 127.8, 126.5, 126.1, 123.6, 117.9 (2C), 114.0, 103.1, 60.7, 55.7, 50.3, 22.6, 19.7, 17.8, 15.2, 13.6 (one aromatic C was unresolved); IR: ν = 3329, 3185, 1651, 1616 cm⁻¹; Anal. calcd for C₂₈H₃₂N₆O₃·3.5 H₂O·0.5 CH₃CH₂OH: C 57.60, H 6.33, N 13.88, found: C 57.75, H 6.43, N 13.54.

(±)-(E)-3-5-[(2,4-Diamino-6-ethyl-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (11b)

This compound was prepared as above using 1.00 g (2.42 mmol) of 8b, 606 mg (2.66 mmol, 1.10 equiv) of (±)-10a,^[6] 300 mg (0.36 mL, 2.66 mmol, 1.10 equiv) of N-ethylpiperidine, and 20 mg (0.089 mmol) of palladium(II) acetate dissolved in 9 mL of dry DMF under nitrogen to give 11b as a pale yellow solid (1.06 g, 85%): mp: 192–193°C; ¹H-NMR (DMSO-d₆): δ=7.94 (s, 1H), 7.84 (d, J = 15.9 Hz, 1H), 7.61 (d, J = 15.9 Hz, 1H), 7.57-7.36 (complex m, 4H), 7.12 (overlapping s, 2H and 1H), 6.88 (s, 1H), 6.60 (br s, 2H), 5.84 (t, J = 6.6 Hz, 1H), 3.78 (overlapping s, 3H and 2H), 3.74 (s, 3H), 2.52 (obscured, 2H), 1.53 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.16 (obscured, 2H), 1.10 (t, J = 7.1 Hz, 3H); ¹³C-NMR (DMSO-d₆):δ=165.5, 164.1, 157.4, 152.5, 146.1, 142.9, 136.6, 135.6, 133.6, 131.7, 128.3, 127.9, 126.5, 126.1, 123.6, 118.0, 117.9, 114.0, 102.7, 60.8, 55.8, 50.3, 36.8, 29.0, 24.8, 17.8, 13.6, 12.8 (one aromatic C was unresolved); IR: ν = 3329, 3185, 1651, 1616 cm⁻¹; Anal. calcd for C₂₉H₃₄N₆O₃·5.0 H₂O: C 57.60, H 6.33, N 13.90, found: C 57.67, H 6.33, N 13.61.

(±)-(E)-3-5-[(2,4-Diamino-6-propyl-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (11c)

This compound was prepared as above using 1.00 g (2.34 mmol) of 8c, 587 g (2.57 mmol, 1.10 equiv) of (±)-10a,^[6] 290 mg (0.35 mL, 2.57 mmol, 1.10 equiv) of N-ethylpiperidine, and 20 mg (0.089 mmol) of palladium(II) acetate dissolved in 9 mL of dry DMF under nitrogen to give 11c as an off-white solid (1.00 g, 81%): mp: 140–141°C; ¹H-NMR (DMSO-d₆): δ=7.93 (s, 1H), 7.85 (d, J = 15.9 Hz, 1H), 7.58 (d, J = 15.9 Hz, 1H), 7.57-7.35 (complex m, 4H), 7.09 (s, 1H), 6.88 (s, 1H), 6.50 (br s, 2H), 6.09 (br s, 2H), 5.84 (t, J = 6.6 Hz, 1H), 3.77 (overlapping s, 3H and 2H), 3.74 (s, 3H), 2.44 (t, J = 7.1 Hz, 2H), 1.53 (m, 2H), 1.16 (m, 4H), 0.87 (t, J = 7.1 Hz, 3H), 0.82 (t, J = 7.1 Hz, 3H); ¹³C-NMR (DMSO-d₆):δ=165.5, 163.6 (2C), 159.6, 152.4, 145.9, 142.8, 136.6, 136.4, 133.6, 131.7, 128.2, 127.8, 126.5, 126.1, 123.6, 117.9, 117.7, 114.1, 102.4, 60.8, 55.7, 50.3, 36.8, 34.7, 29.5, 21.5, 17.8, 13.9, 13.6; IR: ν = 3335, 3190, 1650, 1615 cm⁻¹; Anal. calcd for C₃₀H₃₆N₆O₃·2.75 H₂O: C 62.32, H 6.92, N 14.32, found: C 62.26, H 6.69, N 14.06.

(±)-(E)-3-{5-[(2,4-Diamino-6-methyl-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-one (12a)

This compound was prepared as above using 1.00 g (2.50 mmol) of 8a, 660 mg (2.75 mmol, 1.10 equiv) of (±)-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-one [(±)-10b],^[6] 310 mg (0.38 mL, 2.75 mmol, 1.10 equiv) of N-ethylpiperidine, and 20 mg (0.089 mmol) of palladium(II) acetate dissolved in 9 mL of dry DMF under nitrogen to give 12a as an off-white solid (1.02 g, 80%): mp: 165–166°C; ¹H-NMR (DMSO-d₆): δ=7.93 (s, 1H), 7.83 (d, J = 15.9 Hz, 1H), 7.56 (d, J = 15.9 Hz, 1H), 7.52 (m, 2H), 7.43 (d, J = 7.1 Hz, 1H), 7.30 (d, J = 7.1 Hz, 1H), 7.11 (s, 1H), 6.96 (br s, 2H), 6.86 (s, 1H), 6.52 (br s, 2H), 6.49 (d, J = 9.9 Hz, 1H), 5.24 (d, J = 9.9 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 2H), 3.73 (s, 3H), 2.19 (s, 3H), 1.96 (s, 3H), 1.60 (s, 3H); ¹³C-NMR (DMSO-d₆): δ=165.2, 163.7, 157.8, 152.5, 146.1, 142.1, 136.8, 135.6, 133.8, 133.5, 132.2, 128.2, 127.9, 126.3, 126.2, 123.1, 122.1, 118.0, 117.9, 114.0, 103.3, 60.7, 55.7, 49.2, 29.5, 25.3, 19.1, 18.4 (one aromatic C was unresolved); IR: ν = 3333, 3186, 1652, 1618 cm⁻¹; Anal. calcd for C₂₈H₃₂N₆O₃·2.5 H₂O·0.5 CH₃CH₂OH: C 62.15, H 6.94, N 14.47, found: C 62.44, H 7.04, N 14.60.

(±)-(E)-3-{5-[(2,4-Diamino-6-ethyl-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-one (12b)

This compound was prepared as above using 1.00 g (2.42 mmol) of 8b, 639 mg (2.66 mmol, 1.10 equiv) of (±)-10b,^[6] 300 mg (0.36 mL, 2.66 mmol, 1.10 equiv) of N-ethylpiperidine, and 20 mg (0.089 mmol) of palladium(II) acetate dissolved in 9 mL of dry DMF under nitrogen to give 12b as a pale yellow solid (1.06 g, 84%): mp: 192–193°C; ¹H-NMR (DMSO-d₆): δ=7.91 (s, 1H), 7.84 (d, J = 15.9 Hz, 1H), 7.53 (d, J = 15.9 Hz, 1H), 7.50 (m, 2H), 7.43 (d, J = 7.1 Hz, 1H), 7.30 (d, J = 7.1 Hz, 1H), 7.07 (s, 1H), 6.88 (s, 1H), 6.49 (d, J = 9.9 Hz, 1H), 6.29 (br s, 2H), 5.91 (br s, 2 H), 5.24 (d, J = 9.9 Hz, 1H), 3.77 (s, 3H), 3.76 (s, 2H), 3.73 (s, 3H), 2.45 (q, J = 7.3 Hz, 2H), 1.96 (s, 3H), 1.60 (s, 3H), 1.07 (t, J = 7.3 Hz, 3H); ¹³C-NMR (DMSO-d₆): δ=166.0, 165.2, 163.4, 160.6, 152.4, 145.9, 142.1, 136.8, 136.7, 133.8, 133.5, 132.2, 128.2, 127.8, 126.3, 126.2, 123.1, 122.2, 117.8, 117.6, 114.1, 101.6, 60.8, 55.7, 49.2, 29.5, 26.4, 25.3, 18.4, 13.0; IR: ν = 3329, 3182, 1651, 1615 cm⁻¹; Anal. calcd for C₃₀H₃₄N₆O₃·2.0 H₂O: C 64.04, H 6.81, N 14.90, found: C 64.05, H 6.72, N 14.64.

(±)-(E)-3-{5-[(2,4-Diamino-6-propyl-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-isobutenyl-2(1H)-phthalazinyl)-2-propen-1-one (12c)

This compound was prepared as above using 1.00 g (2.34 mmol) of 8c, 617 mg, 2.57 mmol, 1.10 equiv) of (±)-10b,^[6] 290 mg (0.35 mL, 2.57 mmol, 1.10 equiv) of N-ethylpiperidine, and 20 mg (0.089 mmol) of palladium(II) acetate dissolved in 9 mL of dry DMF under nitrogen to give 12c as an off-white solid (980 mg, 78%): mp: 138–139°C; ¹H-NMR (DMSO-d₆): δ=7.90 (s, 1H), 7.84 (d, J = 15.9 Hz, 1H), 7.50 (m, 3H), 7.43 (d, J = 7.1 Hz, 1H), 7.30 (d, J = 7.1 Hz, 1H), 7.05 (s, 1H), 6.88 (s, 1H), 6.48 (d, J = 9.9 Hz, 1H), 6.03 (br s, 2H), 5.69 (br s, 2H), 5.24 (d, J = 9.9 Hz, 1H), 3.76 (s, 3H), 3.73 (overlapping s, 2H and 3H), 2.39 (t, J = 7.1 Hz, 2H), 1.95 (s, 3H), 1.59 (s, 3H), 1.52 (sextet, J = 7.1 Hz, 2H), 0.86 (t, J = 7.1 Hz, 3H); ¹³C-NMR (DMSO-d₆): δ=166.4, 165.2, 163.2, 161.4, 152.4, 145.9, 142.1, 137.0, 136.8, 133.8, 133.5, 132.2, 128.2, 127.8, 126.24, 126.17, 123.1, 122.2, 117.8, 117.6, 11.42, 101.8, 60.8, 55.7, 49.2, 35.7, 29.8, 25.3, 21.6, 18.4, 14.1; IR: ν = 3340, 3177, 1656, 1606 cm⁻¹; Anal. calcd for C₃₁H₃₆N₆O₃·0.75 H₂O: C 66.41, H 7.18, N 14.99, found: C 66.26, H 6.91, N 14.74.

Biology

Initial studies utilizing molecular modeling were carried out by Sapient Discovery of San Diego, CA. These included calculation of energy values upon complexation using in-house software based on Monte-Carlo formalism.

All biological studies utilized stocks of racemic inhibitor dissolved in dimethyl sulfoxide (DMSO); the concentration of DMSO was kept ≤1% during all assay procedures. Enzyme preparation, crystallization, activity (IC₅₀ and K_i), and structure solution and refinements were carried out as in previous studies.^[6,9]

Briefly, recombinant DHFR protein was expressed in BL21 (DE3) E. coli cultures and purified by IMAC using a C-terminal 6-His tag. For crystallization, the 6-His tag was cleaved using an introduced thrombin site, and protein was further purified by size exclusion chromatography. Crystallization conditions were as previously published: 15% polyethylene glycol 6000 in a 0.1 M MES pH 6.5 buffer with 0.15 M sodium acetate. The enzymatic activity was assayed using a 0.05 M phosphate buffered pH 7.0 system also containing 5% glycerol, 10 mM EDTA, 1 mM β-mercaptoethanol at 30 °C with saturating NADPH and dihydrofolate. Conversion of dihydrofolate to tetrahydrofolate was detected with the redox-sensitive tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), which was measured by absorbance of 450 nm light. Inhibitors were tested with at least 6 concentrations, and the percent inhibition relative to a control reaction was used to construct a four-parameter curve fit (JC Jr.),^[21] providing a calculation of the IC₅₀.

MIC determinations were also described earlier^[9] and followed the protocol given in the Clinical Laboratory and Standards Institute (CLSI) manual.^[22] Inhibitors were tested as a series of 10 concentrations made in 2-fold dilutions. The MIC was determined as the lowest concentration of inhibitor that prevented bacterial growth (visually and at 600 nm) after a 20-h incubation at 37°C in atmospheric CO₂.

X-ray data were collected by Sapient Discovery of San Diego, CA, using a Bruker Proteum/R 6000 instrument suite that included a SMART6000 CCD detector. Data integration and scaling were carried out with the Proteum software suite.^[23] Scaling statistics (Table 1) were obtained with XPREP. The value for R_int as reported from this procedure included all data, in contrast to limiting to the strongest reflections as used in comparable programs.^[24] Crystallographic data were isomorphous with the previously determined structure of S. aureus DHFR co-crystallized with (±)-1,^[9] allowing the protein portion of the model (PDB code 3M08) to be used directly in the Phenix^[25] refinement program with visualization and model building using Coot.^[26] Figures were generated using the Chimera^[27] program. Coordinates and structure factors have been deposited with the Protein Databank^[28] and were given the identifier codes 4FGH [(±)-12b] and 4FGG [(±)-12c].