2.1. N-Aryl-Oxalamic Acids as FAHD1 Inhibitors
First, we synthesized exemplaric known compounds of scaffold
B (
Figure 3) derived from anilines functionalized with polar carboxy or nitro groups, which present the charge density into the space of the oxyanion hole in the FAHD1 catalytic cavity. The acylation of amino-substituted benzoic acids yields oxalamic acid methyl esters (
1a–
3a) in high yields and purity. Analogously, the acylation of nitro-substituted anilines afforded after saponification oxalamic acids
4b–
6b. The compounds obtained from the carboxy series revealed instantly a good-to-moderate inhibition of FAHD1-ODx activity (
Figure 4 and
Table 1).
In the series of methyl esters 1a–3a, we observed a remarkable change in activities as a function of the position of the carboxy substituent. The o-substituted 1a shows only a moderate inhibition (IC50 = 47 µM), whereas the m-substituted congener 2a was already comparable with the only known FAHD1 inhibitor oxalate (IC50 13 µM vs. 7 µM). The p-carboxy-substituted derivative 3a revealed a weak activity.
1b belongs (accordingly the design steps shown in
Figure 3) to a fumarylpyruvate (FMP) mimetic. The
m-phenyl carboxy group is presented at a similar position as in the FAHD1 substrate FMP, creating an additional interaction not accessible by
o- and
p-substituted congeners
2a and
3a, respectively. The
o-carboxy derivative
1a can be termed as a close mimetic of oxaloacetate. However, a six-membered hydrogen bonding between amide NH and oxygen of the
o-carboxylate creates a resonace stabilized flat structure that impairs the presentation of negative charge density to the oxyanion hole of FAHD1. This conformational penalty is obviously romoved upon saponification to the acid
1b, most probably due to electronic repulsion of the corboxy functions. Saponification of the FMP analog
2a to the corresponding acid
2b dit not change the inhibitory activity, suggesting that most of the binding energy of this derivative is carried by the
m-substituent. The
p-carboxy derivatives
3a and
3b present the
N-aryl substituent in a position not covered by substrates OAA or FMP, which explains their reduced activity.
The 2-oxalylamino-benzoic acid
1b is a known competitive inhibitor of protein-tyrosine phosphatase
β [
15]. The molecule behaved as a phosphate mimetic and has been used as a “minimal unit” mimetic for advanced and selective inhibitors of this enzyme, but
1b apparently could also serve well for FAHD1 inhibitors. The low molecular weight of
1b enables a broad derivatization for the establishment of a structure–activity relationship (SAR) for FAHD1. Somewhat surprising was that oxalamic acid methyl esters
1a–
3a kept active. Especially
meta-substituted
2a was equally potent copmpared to the
ortho-carboxylate presenting
1b. This finding is important, because acid functions are known to impair the cellular uptake. Ester derivatives could act as prodrugs, facilitating membrane crossing (for additional ester derivatives, see
Section 2.3). We investigated further substituents on the
N-aryl scaffold, such as hydroxy-, methoxy or halogen groups in various positions, but none of the synthesized compounds improved the IC
50 below 30 µM (selected results are provided in the
Supporting Materials).
In the next step, we investigated
α- and
β-naphthylamines as examples for annulated ring systems. It was found that only
β-substitution is appropriate for good inhibitors, wheras the
α-naphthyl congeners sustain a dramatic loss of activity (
Figure 5 and
Table 2).
N-
β-naphthyl-oxalamic acid methyl ester (
7a) and -acid (
7b) share a similarity with the
m-carboxyl substituted derivatives shown in
Figure 4 but still miss the charge presentation. The ester-acid pair
7a and
7b is a rare example where the saponification of the ester to the acid led to a significant loss of activity. The results warrant further investigations of annulation with heterocycles and substitution of the naphthyl moiety by polar functional groups.
Discouraging results were obtained from investigation of compounds where the
N-aryl ring was spacered by an alkyl (e.g.,
N-benzyl or
N-cycloalkyl motifs). Such derivatives revealed IC
50 > 100 µM (for examples, see the
Supporting Materials).
2.2. N-Pyridyl Substituted Oxalamic Acid Derivatives and Their N-Oxides as FAHD1 Inhibitors
In the following, we gave focus to synthesis of pyridine scaffolds (
D) and their
N-oxides (
E), which were deduced from the OAA substrate (
Figure 3). Heterocyclic
N-oxides have been investigated in numerous medicinal chemistry projects and revealed anticancer, anti-inflammatory, antibacterial, anti-HIV and antiparasitic activities, among many others [
16] Additionally, the
N-oxide moiety serves as an important molecular recognition motif in biological interactions.
We first synthesized 3-aminopyridine and 2-aminopyridine oxalamic acid methyl esters
9a and
10a (
Figure 6). Clean oxidation of the heterocyclic ring nitrogen afforded the corresponding
N-oxides
11a and
12a, which were saponified to the acids
11b and
12b. The compounds showed FAHD1 inhibitory activity, as proposed by the design strategy (
Table 3). Derivatives
9a,
11a and
11b obtained from the 3-aminopyridine building block turned out to be much superior in FAHD1 inhibitory potency compared to the 2-aminopyridine congeners
10a,
12a and
12b. The 3-aminopyridine-derived compounds can deliver negative charge density towards the space of the oxyanion hole of FAHD1, stabilizing the binding of the inhibitor. In contrast, the 2-aminopyridine congeners present the charge density opposite to the oxyanion hole. Rotation around the C–N bond cannot correct for this obstacle. There is no evidence for a tautomerism from the NMR-spectra of the 2-aminopyridine-derived compounds.
To study the effect of annulation of the pyridine ring, 2-amino or 3-aminoquinoline building blocks were applied in the synthesis. The modification was especially beneficial for the 2-aminoquinolines (
14a,
16a and
16b;
Figure 7 and
Table 4) compared to the nonanullated 2-aminopyridines (
10a,
12a and
12b;
Figure 6). The 3-aminoquinolines kept good activity with the 3-aminoquinoline
N-oxide
15b as the most active representative.
2.3. Variations Affecting the 1,2-Dicarbonyl Motif in Oxalamic Acid Derivatives
As disclosed by the single molecule X-ray diffraction analysis of FAHD1 with bound oxalate inhibitor, the 1,2-dicarbonyl motif is an essential structural element for bidentate binding to the magnesium cofactor. We explored several modifications in this motif. The oxalamic acid derivative
1a was cyclized at an elevated temperature or upon treatment with acetic acid anhydride to a 4-oxo-4
H-benzo[
d]-[1,3]oxazine derivative
17a [
17]. Analogously, the
ortho-amide-bearing methyl ester
18a was cyclized to methyl 4-oxo-1,4-dihydroquinazoline-2-carboxylate
19a [
18]. The latter one was saponified to the acid congener
19b (
Figure 8). All these small molecules inhibited FAHD1 ODx activity in the low µM IC
50 range (
Table 5). It must be considered that the highly active
17a is a reactive molecule able to acylate nucleophiles (but, so far, we have no evidence that
17a is modifying residues in the catalytic center of FAHD1).
Formation of cyclic
17a is propagated by a nucleophilic attack of 2-amide carbonyl oxygen on the activated
ortho-carboxyl group of
1a [
17]
, disclosing a significant polarization of the 2-amide function towards the oxygen atom, which supports strong bidentate binding to the enzyme cofactor and gains stability towards hydrolysis of the amide bond in oxalamic acids. The nucleophilic property of the 2-oxygen in oxalamic acid derivatives inspired us to investigate the reaction with a Lawesson reagent (CAS RN 19172-47-5). We found a clean specific exchange of the 2-carbonyl in oxalamic acid esters to the 2-thio congeners
20a–
22a. Saponification of the methyl esters afforded the acid derivatives
20b–
22b (
Figure 9).
However, these transformations did not improve the inhibitory activity, as summarized in
Table 6.
Change of oxalamic esters to oxalamic acid amides was also not a favorable transformation, as revealed by derivatives
23–25.
C-terminal cycloalkyl amide
25 was an inferior compound (
Figure 10 and
Table 7).
A surprising result became visible when we tested a variation in the terminal ester functionality. Change of methyl esters to
tert. butyl esters in the series of
ortho-,
meta- and
para-aminobenzoic acids yielded two highly potent inhibitors
26 and
28, whereas the
meta-substitution (
27) was not well-tolerated (
Figure 11 and
Table 8). Comparison of the
tert. butyl esters
26–28 with the corresponding methyl esters
1a–
3a (see
Figure 5 and
Table 1) reveals that the modification is beneficial for the methyl esters with lower activity.
1a gained a four-fold and
3a even a six-fold increase in activity, whereas the highly active
m-carboxy-substituted
2a sustains a twenty-fold loss in activity upon the change from a methyl to
tert. butyl ester.
However, the catalytic center of FAHD1 (as derived from the oxalate ligated FAHD1 X-ray structure) does not feature enough space for the accommodation of a hydrophobic and bulky
tert. butyl residue. Especially the activity increase of the
p-carboxy-substituted
28, which carries the charge presenting substituent at an unfavorable position (s.
3a,
Figure 4), points towards a drastic reorganization within the catalytic center of FAHD1 upon the binding of these structures. These findings need further investigations. The derivatizations of
tert. butyl esters were extended from the aminobenzoic acids to other scaffolds and resulted in additional compounds with high inhibitory activity (see the
Supporting Materials).
Starting from these observations, and despite the initially negative experience made with variations in the terminal amide region, we explored symmetric oxalamic acid diamides for FAHD1 inhibition.
2.4. N,N′-Diaryl-Oxalamic Acid Diamides
Surprisingly, the synthesis starting with 2-aminopyridine delivered highly active C
2-symmetrical compounds (e.g.,
29 and
30;
Figure 12 and
Table 9). Symmetry was not a prerequisite for high activity, as demonstrated by compounds
32 and
33. The C
2-symmetrical combination of
N,
N′-bis-3-aminopyridyl led to a significant loss of activity (
31). The combination of
N-2-aminopyridyl with
N′-3-aminopyridyl (
37) even resulted in a total loss of activity. These results point towards a so-far-undisclosed specific interaction of these compounds within FAHD1, which deserves further investigations. The
N,
N′-diamide scaffold cannot be compared with the compounds described in
Section 2.2, which are capable of bidentate binding to the Mg cofactor of FAHD1. The
N,N′-diamide molecules adopt a planar structure with the 1,2-carbonyls in
s-trans conformation [
19] (rotational barrier ~ 12 kcal/mol), which does not support bidentate Mg complexation. The interpretation of the activities may become even more complex when taking into account, that the oxalic acid diamides present recognition motifs for
β-sheets or backbone amide bonds (present, e.g., in the exposed FAHD1 lid domain).
We also synthesized novel
N-oxides of
N,
N′-diamides (
35–37) and achieved good results with 2-aminopyridine-based compounds
35 and
36, whereas the 3-aminopyridyl containing derivative
37 was found to be inactive. Obviously, the
N′-
m-toluidine amide in
37 destroys the activity of acid congener
11b (
Figure 6 and
Table 3), which represents one of the most active compounds found in this study. In contrast, the 2-aminopyridyl-derived
N′-
m-toluidine amide
36 gained significant activity compared to its acid congener
12b. The so-far-collected results for the
N,
N′-diamide scaffold deserve a broad extension of SAR studies with this novel FAHD1 inhibitor scaffold.
N,N′-dipyridyl-containing compounds could also be oxidized to corresponding bis-N,N′-oxides, but the compounds obtained were unfortunately not soluble.
Taking together, the data for tert. butyl esters and the N,N′-diaryl-oxalamic acid diamides, an inhibition mode different to oxalate, can be assumed for these scaffolds. The discovery of the scaffold expands the initially applied design strategy significantly. To clarify the binding mode of these molecules, we initiated already co-crystallization experiments to create a solid basis for the future design of second-generation FAHD1 inhibitors to be active in the nanomolar regime.