Synthesis and biological activity of novel ester derivatives of N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid containing amide and keto function as inhibitors of glucosamine-6-phosphate synthase

Article abstract of DOI:10.1016/j.bmcl.2016.06.016

A short series of novel ester derivatives of N³-4-metoxyfumaroyl-(S)-2,3-diaminopropanoic acid (FMDP) containing amide or keto functions have been designed and synthesized. Their antifungal activity and inhibitory properties toward fungal glucosamine-6-phosphate synthase has also been evaluated. The obtained compounds 11–13 and 15–17 demonstrated good antifungal activity against Candida albicans. Compounds 11–13 displayed also potent inhibitory activity against fungal glucosamine-6-phosphate synthase, comparable to that of FMDP.

Full text of DOI:10.1016/j.bmcl.2016.06.016

Accepted Manuscript
Synthesis and biological activity of novel ester derivatives of N3-(4-metoxyfu-
maroyl)-(S)-2,3-diaminopropanoic acid containing amide and keto function as
inhibitors of glucosamine-6-phosphate synthase
Dorota Pawlak, Marta Schielmann, Marek Wojciechowski, Ryszard
Andruszkiewicz
PII:
S0960-894X(16)30627-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.016
BMCL 23969
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 April 2016
7 June 2016
8 June 2016
Please cite this article as: Pawlak, D., Schielmann, M., Wojciechowski, M., Andruszkiewicz, R., Synthesis and
biological activity of novel ester derivatives of N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid containing
amide and keto function as inhibitors of glucosamine-6-phosphate synthase, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and biological activity of novel ester derivatives of N3-(4-
metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid containing amide and keto
function as inhibitors of glucosamine-6-phosphate synthase.
Dorota Pawlak, Marta Schielmann, Marek Wojciechowski, Ryszard Andruszkiewicz*
Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology,
Gdańsk, Poland
Abstract
A short series of novel ester derivatives of N3-4-metoxyfumaroyl-(S)-2,3-diaminopropanoic acid
(FMDP) containing amide or keto functions have been designed and synthesized. Their antifungal
activity and inhibitory properties toward fungal glucosamine-6-phosphate synthase has also been
evaluated. The obtained compounds 11-13 and 15-17 demonstrated good antifungal activity against
Candida albicans. Compounds 11-13 displayed also potent inhibitory activity against fungal glucosamine-
6-phosphate synthase, comparable to that of FMDP.
In recent years, the number of invasive fungal infections have increased rapidly, especially in cases
of immunocompromised patients, such as patient with AIDS, after organ transplantation or cancer
chemotherapy.1 Systemic fungal infections are now significant causes of morbidity and mortality. None
of the available clinical chemotherapeutics fulfill all requirements for an ideal antifungal agent.2 There
are still need to search for new, potent and safe compounds, which may be used in the future as new
antifungal drugs.
Glucosamine-6-phosphate (GlcN-6-P) synthase has been proposed as a potential target for design of
antibacterial and antifungal agents. GlcN-6-P synthase catalyzes the first step in the biosynthesis of D-
glucosamine-6-phosphate, which is a precursor of a number of amino sugar-containing macromolecules,
including chitin and mannoproteins in fungi, peptidoglycan and lipopolysaccharides in bacteria and
glycoproteins in mammals.3
This enzyme was previously reported to be inhibited by a naturally occurring compound tetaine4 and
peptide A190095, and also by a number of chemically obtained compounds, especially electrophilic
ones6. In fact, not all of these inhibitors are selective; consequently they have ability to react with other
enzymes in the fungal cell. The most effective and selective compounds, which are the strong

inactivators of GlcN-6-P synthase, are the analogues of L-glutamine (the substrate of the enzyme) and
one of these compounds is N3-4-metoxyfumaroyl-(S)-2,3-diaminopropanoic acid (FMDP)7.
Despite the effectiveness of the enzyme inhibition, the FMDP inhibitor shows none or very poor
antimicrobial activity. This effect is caused by the high polarity of the inhibitor molecule. In order to
improve the antifungal activity of FMDP molecule several approaches have been proposed. Modification
of FMDP molecule by increasing its lipophilicity was taken into consideration. Latent esters derivatives
8,9 and analogues containing a keto function10 were described earlier. Recently, new amide derivatives of
FMDP as inhibitors of GlcN-6-P synthase with improved lipophilic properties and better antifungal
activities have been obtained and tested 11.
In continuation of our work on the synthesis of novel inhibitors of GlcN-6-P synthase we wish to
report the synthesis of novel ester derivatives of FMDP containing keto and amide functions (Fig.1 and
2) and evaluation of their anticandidal activities and inhibitory properties against fungal GlcN-6-P
synthase.
Figure 1 Structure of new keto-esters derivatives of FMDP
Figure 2 Structure of new amide-esters derivatives of FMDP

The target compounds were synthesized as shown in Scheme1 and 2. The syntheses started with the
preparation of N2-tert-butoxycarbonyl-N3-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (Boc-
FMDP) according to the method described earlier7. Boc-FMDP was used for the preparation of
compounds 1-10. The new esters derivatives 1-3 were obtained by reacting carboxylic acid salts (cesium
or NEt3 salts) with corresponding chloroketones (Scheme1). Ester conjugates 4-10 have been obtained
from the different hydroxyamides (which were previously synthesized from ethyl L-lactate and methyl
glycolate), using combination of DCC and DMAP as a coupling agent.12 The Boc- protected compounds
1-10 were treated with 4M HCl in dioxane to give hydrochloride salts of FMDP derivatives 11-19.
Scheme 1. Synthesis of FMDP ketoesters by using two different methods: method A: i (R-Cl,
NEt3,THF,NaIkat., r.t., 48h) and method B: ii (1. 20% aq Cs2CO3,H2O/MeOH, 2.DMF, R-Cl, r.t, 48h); iii
HCl/dioxane, r.t., 2h

Scheme 2. Synthesis of FMDP conjugates with hydroxyamides: i NHR1R2, r.t., 24h; ii DCC, DMAP, THF, r.t,
6h; iii HCl/dioxane, r.t., 2h
Compd
.
MIC (g/mL)
C. glabrata
DSM 11226
IC50 (M)
R1
R2
R3
C. albicans
S. aureus
E. coli
PCM
ATCC 10231
ATCC 29213
2560
11
H
CH3
-
0.25
0.5
0.5
128
1
512
>1024
>1024
>1024
>1024
1024
>1024
>1024
>1024
>1024
>1024
>1024
>1024
>1024
>1024
>1024
5.08± 0.15
4.91 ± 0.38
5.17 ± 0.13
201.94 ±21.67
26.98± 0.43
66.59± 4.11
82.1±3
12
CH3 CH3
512
-
13
H
C(CH3)3
512
-
14
H
H
H
>1024
>1024
>1024
>1024
>1024
>1024
>1024
15
H
H
CH3
CH3
H
16
H
CH3
H
2
512
17
CH3
CH3
8
>1024
>1024
>1024
>1024
18
H
CH3
CH3
-
16
199 ±3
19
CH3 CH3
256
>1024
>5000
FMDP
-
-
4.03± 0.26
Experiments were performed in triplicate; MIC’s values are the average of three independent
determinations. See Supplementary data for details
Table 1 Antimicrobial and inhibitory activity data for new compounds.

All of the obtained FMDP analogues were tested as inhibitors of purified GlcN-6-P synthase
(overproduced by Escherichia coli)13 and their IC50 values are presented in the Table 1.The ability of
compounds 11-19 to inhibit this enzyme was measured by determining the concentration of inhibitor
causing 50% inhibition of enzyme.
As can be seen from the Table 1 compounds 11, 12 and 13 showed almost the same inhibitory
activity as FMDP itself. Molecular modeling studies supported these observations.
All GlcN-6-P synthase glutamine domain inhibitors, as glutamine analogs known so far, share
similar structural features and in consequence similar pattern of interactions inside the binding pocket.
These include the interaction of 1-carboxyl group of the inhibitor molecule with Arg73 and His86, also
interaction of the 1-amino group of inhibitor with Gly99 and Thr76 and interaction of 2-amido group
with Gly99 and Trp74. Ligands 11,13 designed and presented in this paper have changed in this respect
(Fig 3).
Fig 3. Results of docking ligands 11 (right) and 13 (left) to the glutamine binding site of GlcN-6-P
synthase. Lower left hand corner miniature shows the pose of the ligand (HGA, N-hydroxyglutamine))
found in the crystal structure. Docked inhibitors are drawn with thick sticks while important residues of
the binding site are marked and drawn with thinner sticks.
Since the esterification of the carboxyl group significantly enlarged this part of the ligand, it is not
able to adopt anymore strictly the pose described for glutamine and its analogues described so far.

Instead, the introduced keto-ester substituent in ligand 11, occupies this spot in the binding site and in
consequence the ligand’s fumaroyl moiety is shifted out of the pocket, toward the hydrophobic channel
connecting the two binding sites of the enzyme. The introduced keto group mimics the interactions of
the 1-carboxyl group forming hydrogen bonds with Arg73, His86 and Thr124. The 1-amino group despite
the significant shift toward the binding site entrance is still able to form hydrogen bonds with Gly99 and
His97 backbone’s CO group. On the other hand, shifted esterified carboxyl group is also found in the
position normally occupied by glutamine amide moiety and thus can interact with Cys1 free amino
group. This effect is emphasized in case of the ligand 13. Due to the additional bulky t-butyl substituent,
which can hardly fit deep into the binding pocket, the pose of this ligand is moved even further toward
the binding site entrance with the keto carbonyl group interacting with the backbone NH of Gly99
residue. As the result of this ligand shift, not only the esterified carboxyl group is within the range of 2 Å
to Cys1 free amino group, but also 4-methoxy fumaroyl group reaches the Arg26 residue and forms
hydrogen bonds with its guanidinum moiety.
Surprisingly, this substantial change in the bound ligand pose forced by the keto esterification of the 1-
carboxyl group caused that the double bond of the fumaroyl moiety is facing directly the thiol group of
Cys1 facilitating the covalent bond formation between these two. This observation could explain the
noted high activity of the designed ligands in spite of the fact that they lack the free carboxylic group
serving as ligand’s anchor inside the binding site for the GlcN-6-P synthase glutamine domain inhibitors
known so far.
Previously we have also shown that simple amides of FMDP fit very well into the glutamine
binding site of glucosamine-6-phosphate synthase 11. Moreover, there is a correlation between the
enzyme inhibitory activity and anticandidal activity of the tested inhibitors. ( see Table1)
MIC’s were determined against yeast and bacteria species. Newly synthesized compound exerted
only poor antibacterial activity against S. aureus and E. coli and good antifungal activity when tested
against C. albicans ATCC 10231 strain. Out of the new analogues of FMDP, the esters derivatives with
keto function 11-13 exhibited the most potent anticandidal activity. In addition, FMDP analogues with
glycolamide containing alkyl substituent 15-16 have also interesting activity, much better than lactamide
analogues 17-19. Interestingly, compound 14 with primary amide showed weaker anticandidal activity
and simultaneously weak inhibitory activity in comparison with other glycol amides, i.e. compounds 15,
16, 17 and 18. The anticandidal activity of these compounds may be also attributed to their lipophilic
properties. More lipophilic compounds may be easily transported via lipidic cytoplasmic membrane by

simple diffusion to the cells. On the other hand the most lipophilic compound, i.e. 19 showed only a
slight anticandidal activity.
In summary, we have designed novel ester derivatives of FMDP containing keto or amide functions
as inhibitors of GlcN-6-P synthase. The 2-oxopropyl ester of FMDP 11 exhibited the highest anticandidal
activity out of the esters tested and the strongest inhibitory potency against fungal GlcN-6-p synthase.
On the other hand, ester with amide function, i.e. compound 15 showed the highest anticandidal
activity and moderate inhibitory activity out of the esters with amide function tested.
Acknowledgement
These studies were supported by The National Science Centre (NCN), Poland. Grant No. UMO-
2012/05/B/ST5/00291
References and notes
1
De Pauw,B.E.;Picazo, J.J. Int. J. of Antimicrob. Ag. 2008,32,167.
2 Borowski. E. Il Farm. 2000,55,206.
3 Milewski, S. Biochim Biophys Acta. 2002,1597, 173.
4
Milewski,S.; Chmara, H.;Borowski,E. Arch Microbiol. 1986, 145,234.
5 Molloy, B.B.; Lively, D,H.; Gale, R.M.; Gorman, M.; Boeck,L.D. J Antibiot. 1972 ,25,137.
6
Ghosh, S.;Blumenthal,H.J.; Davidson,E.; Roseman,S. J.Biol.Chem. 1960,235,1265.
7 Andruszkiewicz,R.;Chmara,H.;Milewski,S.; Borowski,E.Int. J. Peptide Protein Res. 1986,27,449.
8
Koszel,D.;Łącka,I.;Kozłowska-Tylingo, K.;Andruszkiewicz,R. J. Enzyme Inhib. Med. Chem. 2012,27,167.
9 Zgódka,D.; Jędrzejczak,R.; Milewski, S.; Borowski,E. Bioorg. Med. Chem. 2001, 99,931.

10
Walkowiak, A.; Wakieć, R.; Bontemps-Gracz, M.M.; Andruszkiewicz, R. J. Enzyme Inbib. Med. Chem. 2005,
20,439.
11 Pawlak, D.; Stolarska, M.; Wojciechowski, M.; Andruszkiewicz, R. Eur J Med Chem. 2015,90,577.
12 Fisher,C.H.; Ratchford,W.P. J Org Chem. 1950,15,317.
13Czarnecka, J.;Kwiatkowska ,K.;Gabriel,I.; Wojciechowski,M.;Milewski,S. J. Mol.Recognit. 2012,25, 564..
Graphical abstract