10.1002/cmdc.202000498
ChemMedChem
COMMUNICATION
were employed. The final steps towards successfully obtaining
the inhibitors are demonstrated in scheme 4. Boc deprotection of
coupled compounds 8a–c and 9a–c with 80% TFA in methylene
chloride afforded 10a–c and 11a–c respectively. N-formylation
was achieved by treatment with a formic acid and acetic
anhydride mixture at 0 °C for an hour. It should be noted that
under these conditions 5’-OH of ribose also gets formylated
resulting in low yields of desired product. A final benzyl
deprotection with Pd-C under H2 at 1 atm provided desired
compounds 14a–c and 15a–c. A similar set of reactions provided
compounds 14d–f and 15d–f (scheme 4).
The inhibitory capacities of the newly synthesized compounds
against adenylosuccinate synthetase are shown in Table 1. As
expected, compounds 14f and 15f were not active. With the
introduction of the N-formyl-N-hydroxy group (compounds 14e
and 15e), no inhibition of AdSS was observed. However, similar
derivatives (14d and 15d) with additional amide linkage showed
some inhibition. The compounds with a linear linker showed better
inhibition than compounds with more conformationally restricted
piperazine ring. Compound 14a with a 3-carbon linker showed
25% inhibition.
Having established the advantage for linearly linked compounds
over the conformationally restricted linkers, attempts were made
to increase the potency of inhibitors 14a and 15a. A 40% inhibition
of AdSS activity was observed with addition of one methylene
group (compound 14b) to the linear chain. The result of
compound 14b prompted us to further increase the chain length.
Interestingly, on increasing the distance to 5-carbon (14c)
between two moieties resulted in drastic loss of activity. These
experiments suggest that a bisubstrate inhibitor with a four-
carbon linker, as in 14b and 15b, span both the IMP and aspartate
catalytic domains more efficiently. Replacing adenosine (14a–f)
with 2,6-diaminopurine (15a–f) had a subtle effect on the
capabilities of these compounds to inhibit AdSS.
Table 1. Evaluation of bisubstrate inhibitors for activity against
adenylosuccinate synthetase.
R'
N
N
N
N
R
O
HO
HO
OH
R’
Compound
R
% Inhibitiona
DMSO
-
-
0
In summary, we have synthesized a series of bisubstrate
inhibitors with a purine nucleoside and a hadacidin moiety
separated by linkers of varying complexity and sizes.
Investigations of the biological profile of our target compounds
have shown that a linear spacer is more suitable than a cyclic
spacer and that the carbon chain length has an influence on the
activity. While, none of the derivatives showed significant AdSS
inhibition the study nevertheless provided information about the
size of linker suitable to covalently connect IMP and aspartate
inhibitors that probe aspartate. The best activity was obtained with
compound 14b bearing a C-4 linker that connects the adenosine
moiety to a N-formyl-N-hydroxyl group. We believe that the
described bisubstrate strategy could be useful in designing novel,
highly specific inhibitors for AdSS.
14a
15a
H
NH2
25
23
N
N
O
H
OH
H
14b
15b
H
NH2
40
34
N
N
O
O
OH
N
14c
15c
H
NH2
0
0
N
H
OH
O
14d
15d
H
NH2
18
20
N
N
N
O
OH
O
14e
15e
H
NH2
0
0
N
N
N
OH
14f
15f
H
NH2
0
0
N
NH
Hadacidin
100
-
-
Acknowledgements
a.
Tested at 10 mM
This work was supported by the American Cancer Society (IRG
85-001-22) at the University of Kentucky. The authors are grateful
to Mike Rosenbach and Dennis Carson of the University of
California, San Diego Moores Cancer Center for providing the
enzyme. G.I.E. would like to thank San Diego State University for
funding during the writing of this manuscript.
The ability of purine nucleotide linked hadacidin derivatives to
inhibit AdSS was demonstrated by HPLC detection.
Adenylosuccinate synthetase catalyzes the condensation of IMP
with aspartate forming adenylosuccinate monophosphate. The
enzymatic reaction also requires the conversion of GTP to GDP.
The mixture (100µl) contained, 20mM Hepes (pH 7.5), 50µM IMP,
25µM GTP, 500µM MgCl2, 10mM test compound and 2.0µg of
purified AdSS enzyme. The assay was initiated at room
temperature with the addition of either 100 µM or 500 µM of
aspartate. After 5 minutes, the reaction was stopped by freezing
in a methanol/dry ice-bath. The solution was allowed to reach
room temperature before 20µl of reaction mixture was injected
onto the HPLC. The mobile phase for the assay contained 65mM
potassium phosphate, 1mM PIC A, and 10% methanol. The buffer
has a pH of 4.4. The HPLC assay procedure allows separation of
all UV active components (GDP, GTP, and ASMP) of the mixture
at 266 nm. The amount of adenylosuccinate monophosphate
formed was quantified by the integration of peak areas over 3 runs.
Keywords: Lung cancer • small molecule • adenylosuccinate
synthetase • MTAP
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Tibrewal, Nidhi
