(
17a and 17b, respectively) were alkylated with propargyl
Biochemical assay
All the hits were further tested in our laboratory for human
IMPDH2 inhibition according to protocols reported
chloride in presence of anhydrous K CO and DMF to yield 16
2
3
and 18a-b (Schemes 2-3). Substituted benzyl azides (20a-e,
Scheme 3) prepared from corresponding benzyl bromides (19a-
e), were further reacted with 16 and 18a-b) in tert-butanol and
1
6,17
previously.
The results are shown in Figure 6 and Table 3S
(Supplementary Data). Inhibition of tested compounds on
recombinant Mtb-GuaB2 enzymatic activity was assayed as
water (3:1 mixture) along with Cu(OAc)
compounds 21a-d and 22a-b (Scheme 4) in 55-88% yield (Table
). The synthetic procedures can be found in the Supporting Data
section.
2
to yield title
6
previously reported protocol.
1
Results and Discussion
a
Scheme 1 . Synthesis of arylalkyl amides 12-13
From our in-house library generated for the human and
Staphylococcus aureus IMPDH (SaIMPDH) inhibitor programs,
we selected 60 molecules based on their pharmacophoric features
for further screening against Mtb. Mycophenolic acid (1), a
prototypical IMPDH inhibitor, was included in this set since
MtbIMPDH inhibitors based on MPA structure (2, Figure 1) are
13
known. We also included 12 molecules (alkanoic acids)
structurally similar to MPA in the screening set. The molecular
property ranges for these molecules are shown in Table 2S
(Supplementary Data). The average logP for the screening set
a
was 2.899 (Min. 0.2022, Max. 5.325). The MPA analogs
exhibited lower logP values due to alkanoic acid functionality.
None of these molecules exhibited appreciable anti-Mtb activity
against Mtb H37Rv strain (MIC90 > 100 µM) (Table 1S). This
was not surprising owing to the higher lipophilicity required for
Reagents and conditions. a. substituted acid chloride, pyridine,
THF, 0 °C to RT, overnight.
a
Scheme 2 . Synthesis of 1,2,3-triazole analogs
crossing Mtb cell wall. The screening results for the hits (MIC90
≤
100 µM) are summarized in Table 1.
Of the nine hits, out of 73 molecules screened (Table 1), 21a,
1c and 22b (logP between 3.5 and 4.5) were two-fold more
2
a
Reagents and conditions. a. NH
2
NH
2
.H
2
O, EtOH, Reflux; b.
CO , DMF, 12 hrs
potent than others (13, 21d: logP between 2.5 and 3.5; and 21b:
logP >4.5) except 1 (logP 2.679). Compound 12 (logP 3.669)
exhibited MIC90 of 100 µM. This could be due to its lower
predicted logS (-0.228). Presence of a polar substituent (-OMe, -
triethyl orthoacetate; c. propagyl chloride, K
2
3
a
Scheme 3 . Synthesis of 1- and 2-(prop-2-ynyloxy)naph-thalene
rd
Cl) at 3 position on the arylalkyl moiety attached to triazole N
(
1
21a, 21c and 22b) could contribute to higher potency in the
,2,3-triazole series. Moving this substituent to the 4 position
th
led to complete loss of activity (MIC90> 100 µM) (Table 1S). A
planar [mono- or bicyclic (hetero)aromatic] substituent on the left
side of the linker was tolerated for anti-Mtb activity in the hits
a
Reagents and conditions. a. Propargyl chloride, K
2 hrs
2 3
CO , DMF,
1
(
12, 21c and 22b, Table 1). Replacing this aromatic ring with
alicyclic/spirocyclic ring(s) abolished the anti-Mtb activity (Table
S).
The hits were further taken up to test the activity against
a
Scheme 4 . Synthesis of title compounds
1
GuaB2 in target-specific whole-cell Mtb assays. The data of the
checkerboard assay (Anhydrotetracycline, ATc, vs compounds)
against guaB2 Tet-OFF are presented in Figure 5 (1, 12, 13 and
2
(
1a) and Figures 1S (21b, 21c and 21d) and 2S (22a and 22b)
Supplementary Data). The guaB2 Tet-OFF strain was a guaB2
cKD mutant in which guaB3 expression was unaffected by ATc
i.e., upon ATc treatment only guaB2 expression was down
a
Reagents and conditions
a. NaN
BuOH:H
3 2 2
, i-PrOH:H O (4:1); b. 16/18a/18b, Cu(OAc) , t-
(
2
O (3:1), RT, 8 hrs
6
regulated). The data clearly demonstrated that the transcriptional
silencing of guaB2 (upon ATc addition) in Mtb confers
hypersensitivity (shift of MICs from left to right) to the
compounds (more profound with 13, MIC90 reduced from 100
µM in WT strain to 12.5 µM upon depletion of guaB2 – both at
0.15 and 0.31 ng/mL ATc). Compounds 1, 12 and 13 definitely
were active against guaB2, whereas the activity of other
compounds at guaB2 could not be ascertained from the data
obtained from checkerboard assays.
Drug Susceptibility Testing
Unless indicated otherwise, minimum inhibitory concentration
(MIC) testing was carried out by broth microdilution using the
6
AlamarBlue (AB, Invitrogen) assay. For pairwise combination
(checkerboard) assays, a two-dimensional array of serial dilutions
of test compound and anyhydrotetracycline (ATc) was prepared
in 96-well plates, as previously described.
shown in Table 1 and Figure 5.
6,18
The results are
The hits were evaluated for antibacterial activity against five
bacterial strains at 60 to 146 μM (32 µg/mL) concentration. The
bacterial strains included one Gram-positive (Staphylococcus
aureus, MRSA) and four Gram-negative (Escherichia coli,
Klebsiella pneumoniae, Acinetobacter baumannii and
The hits (Table 1) were tested against SRMV2.6 strain (nsSNP
18
in guaB2, Y487C) which showed resistance to earlier lead
compound VCC234718, an isoquinoline sulfonamide. Here, no
significant deviation in the MICs was observed (Table 1),
suggesting the unique binding of the hits to the GuaB2 compared
to VCC234718. Further these hits were screened for Mtb-GuaB2
inhibition assay, where eight molecules inhibited Mtb-GuaB2 at
19
Pseudomonas aeruginosa). The results are summarized in Table
S (Supplementary Data).
3
50 μM. Molecules exhibiting more than 50% inhibition were