Organic Letters
Letter
Scheme 2. Synthesis of 7,8-Dihydropteroate 3 and 2′-
Table 1. Air Oxidation Half-Lives of 3, 6, and 7 in DMSO-d6
and 1.5 N NaOD at 23 °C
Hydroxy-7,8-dihydropteroate 6
reaction mixture was lowered to ∼3. The compounds are stable
for months when stored at 4 °C and pH 2 as a precipitated
suspension of the reaction mixture which contains about 5% (w/
v) antioxidant ascorbic acid. If stored in this manner, the
compounds can be rapidly recovered to prepare a DMSO stock
solution for immediate biological evaluation as follows: the
suspension is centrifuged, and the residue is washed successively
by H2O, EtOH, and Et2O, then dried with a stream of argon, and
dissolved in DMSO. Less than 5% degradation of compound 7
was observed in 6 months when stored as described above. The
compounds are also stable when stored as solids (after Et2O
wash) at −80 °C in an argon filled amber vial.
With an authentic sample of 7, we proceeded to evaluate it for
inhibition of recombinant DfrA,15 the DHFR ortholog in M.
tuberculosis using a coupled steady-state kinetic spectroscopic
assayunderinitial velocityconditions thatmeasuresconsumption
of NADPH. These experiments confirmed 2′-hydroxy-7,8-
dihydrofolate 7 is a potent inhibitor of DfrA with respect to 7,8-
dihydrofolate. The inhibition constant (Ki) derived from this
analysis using the Cheng−Prushoff equation is 174 102 nM
(Figure S1). These results provide the first biochemical validation
for the mechanism of action of PAS, which innocently enters the
folate pathway exploiting the relaxed substrate specificity of the
mycobacterial enzymes DHPS and DHFS that together convert
PAS into 7, that in turn inhibits the downstream enzyme DHFR.
This further clarifies our understanding of this old drug first
introduced into clinical use nearly 80 years ago.
The naturally occurring reduced folate species are well-known
to undergo spontaneous oxidative degradation; however,
dihydrofolate species are relatively more stable than tetrahy-
drofolate species.16 Reduced folates are also much more prone to
airoxidation anddegradation at lowerpH, butat pHhigher than7
they are quite stable.17 In order to determine the solution stability
of these compounds under air, we studied the kinetics of air
oxidation. A 20 mM solution of each compound in DMSO-d6 or
1.5 N NaOD were exposed to air and incubated at room
temperature without exclusion of light, and the rate of oxidation
wasstudiedby1HNMR. Theratiosofoxidizedandreducedforms
were readily calculated by integration of C-7 (oxidized form: ∼8.8
ppm, reduced form: ∼3.8 ppm) and C-9 protons (oxidized form:
∼4.6 ppm, reduced form: ∼3.9 ppm) that exhibited diagnostic
and well resolved chemicals shifts for each species. Based on
NMR, a third compound was also formed over time which was
likely the 6-formylpterin.16 The oxidation followed first-order
kinetics, and the half-lives were determined (Table 1). Among the
three compounds examined, 2′-hydroxy-7,8-dihydropteroate 6
was the least stable in DMSO possessing a half-life of less than 2 h.
The other two compounds, by comparison, exhibited half-lives of
more than 5 h under aerobic conditions at ambient temperature.
a
Percent remaining in 24 h.
All three compounds displayed significantly improved stabilities
in 1.5 N NaOD. Thus, only 17% of the compound 7 was degraded
in 24 h under these alkaline conditions. Interestingly, DMSO
solutions of all three compounds turned dark red within 2 h,
whereas NaOD solutions persisted unchanged as light orange
solutions.
We next sought to develop a sensitive analytical method
employing high-performance liquid chromatography tandem
mass spectrometry (LC-MS/MS) that could be used for
subsequent studies to monitor intracellular levels of the natural
folate metabolite 3, and PAS derived biotransformation products
6 and 7. As the compounds were more stable under alkaline
conditions, a buffer system at pH 9 was employed for
chromatography. Additionally, the compounds showed superior
ionization in ESI negative mode revealing the [M − H]− peaks for
the parent molecular ions. Representative MS/MS spectra
including parent ions and major fragment ions used for multiple
reactionmonitoring(MRM)areprovidedinFigure2A−C. These
MS/MS spectra were obtained by direct infusion of 10 μM
aqueous acetonitrile (1:1, 10 mM ammonium acetate, pH 9.0)
solutions of 3, 6, and 7 into the ESI source of the mass
spectrometer at a flow rate of 10 μL/min. The major
fragmentation pattern observed for pteroates 3 and 6 was due
to the cleavage of the C9−N10 bond, which produced PABA (m/z
135.9) and PAS (m/z 151.8) anions, respectively, as well as the
pterin anion (m/z 175.9). The majority of fragment ions for
hydroxyfolate 7 were obtained from expulsion of the glutamoyl
moiety and the cleavage of C9−N10 bond. Next, to determine the
extent of matrix associated ion suppression in the biological
samples, the standard solutions were prepared by spiking
synthetic standards into a mycobacterial extract.18 We generated
a calibration curve for each compound, and the Limits of
Quantitation (LOQ) for each compound was identified (<6 ng/
mL, Figure 2D). The peak area was linear in the concentration
range of LOQ to 0.4 μg/mL with R2 > 0.99 (Supporting
Information) providing a large dynamic range for quantitation. A
representative chromatogram is shown in Figure 2, and a detailed
In summary, we have accomplished the first synthesis of the
PAS derived antifolate species 6 and 7 using a convergent
approach and shown both species are quite unstable under
aerobic conditions, but can be safely stored for months as a
suspension with 5% w/v ascorbic acid. We envision that the
strategy utilized here of obtaining the formylpterin by the
C
Org. Lett. XXXX, XXX, XXX−XXX
Dawadi, Surendra
