Potentiation of the fosmidomycin analogue FR 900098 with substituted 2-oxazolines against Francisella novicida

Article abstract of DOI:10.1039/c6md00365f

A library of 33 compounds was screened for potentiation of the antibiotic FR 900098 against the Francisella tularensis surrogate Francisella novicida. From the screen a highly potent 2-oxazoline adjuvant was discovered capable of potentiating FR 900098 with a 1000-fold reduction in MIC against the Francisella sub-species F. novicida and F. philomiragia.

Full text of DOI:10.1039/c6md00365f

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Potentiation of the Fosmidomycin analogue FR 900098 with substituted 2-oxazolines
against Francisella novicida†
Matthew D. Stephensa , Nisakorn Yodsanitb and Christian Melander*a
aDepartment of Chemistry, North Carolina State University, 2620 Yarbrough Drive,
Raleigh, NC 27695, USA
bDepartment of Chemistry, Mahidol University, 999 Phuttamonthon 4 Road, Salaya
73170, Thailand
ABSTRACT: A library of 33 compounds was screened for potentiation of the antibiotic
FR 900098 against the Francisella tularensis surrogate Francisella novicida. From the
screen a highly potent 2ꢀoxazoline adjuvant was discovered capable of potentiating FR
900098 with a 1000ꢀfold reduction in MIC against the Francisella subꢀspecies F.
novicida and F. philomiragia.
Oxazolines represent an important class of heterocylic compounds that are present
in a wide array of natural products.1 Additionally, oxazolines have many significant uses
such as: protecting group for carboxylic acids,2 ligands for asymmetric synthesis,3,4 and
living cationic ringꢀopening polymerization reactions.5-7 Synthesis of substituted
oxazolines is most commonly performed by one of three methods involving acid
chlorides,8 aldehydes,9 or nitriles.10
The fosmidomycin analogue FR 900098 is known to inhibit DXR (1ꢀdeoxyꢀdꢀ
xyluloseꢀ5ꢀphosphate reductoisomerase), the first committed step for the biosynthesis of
isoprenoids through the nonꢀmevalonate pathway.11,12 The nonꢀmevalonate pathway is
present in several pathogenic bacterial species, but is not present in humans making it an
attractive drug target.13ꢀ16 The production of isoprenoids through the nonꢀmevalonate
†The authors declare no competing interests.

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pathway has been shown to be essential for the survival of Francisella species as
mutations to the pathway are lethal.17,18
Previously, our group discovered an oxazoline containing compound (1a) capable
of potentiating FR 900098 against the Gramꢀnegative bacteria Francisella philomiragia
with a 256ꢀfold reduction in MIC (Figure 1).19 Building on this discovery we opted to
explore antibiotic potentiation against the commonly used Francisella tularensis
surrogate Francisella novicida.20 F. novicida is often used in place of F. tularensis since
both are genetically similar but does not require special containment facilities.21
Antibiotic resistance and alternative treatment options for F. tularensis are of great
interest due to its history as a biological weapon,22 extreme virulence,23 and
environmental persistance.24 Herein, we report the construction of a small library of
heterocyclic analogues capable of potentiating the phosphonic acid antibiotic FR 900098
against the Francisella subꢀspecies F. novicida.
Figure 1. Structures of the lead compound and antibiotic. Values in red are MIC value of
compound or antibiotic. Value in blue is MIC of antibiotic in the presence of 1a against F.
philomiragia.
The lead compound (1a) was tested against F. novicida and reduced FR 900098’s
MIC 4ꢀfold to 4 ꢁg mLꢀ1. Oxazoline analogues were then prepared by reacting 2ꢀ

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bromoethylamine and various acid chlorides in the presence of TEA at reflux (Scheme 1).
The first series of compounds synthesized were designed in order to investigate the effect
that the alkyl tail length had on antibiotic potentiation. When the tail was removed
entirely (R = H, 1b), no reduction in antibiotic MIC was observed (Figure 2).
Modification of the tail to methyl (1c), ethyl (1d), and propyl (1e) substituents similarly
delivered compounds that were unable to potentiate FR 900098. Increasing the tail length
by a methylene unit to a butyl chain (1f) increased antibiotic activity 4ꢀfold and lowered
the MIC to 4 ꢁg mLꢀ1. Interestingly, further extension to hexyl (1g) resulted in a marked
decrease in MIC to 0.0156 ꢁg mLꢀ1 a 1000ꢀfold overall reduction. Extension of the tail to
heptyl (1h) potentiated FR 900098 with an 8ꢀfold reduction to an MIC of 2 ꢁg mLꢀ1.
Scheme 1. General synthetic scheme for cyclization of oxazolines.
In order to synthesize the octyl and nonyl tails, 4ꢀoctyl (2i) and 4ꢀnonylbenzoyl
chloride (2j) needed to be synthesized due to lack of commercial availability. The acid
chlorides were synthesized by esterification of 4ꢀiodobenzoic acid (2a) in refluxing
MeOH under acid catalysis (Scheme 2). The methyl ester (2b) was then subjected to
Sonogashira coupling with 1ꢀoctyne or 1ꢀnonyne, giving alkynes 2c and 2d, which were
both hydrogenated with H2 over Pd/C in THF. The esters were then hydrolyzed with
LiOH in refluxing THF/H2O and upon acidification gave pure carboxylic acids 2g and
2h. The acids were subsequently converted to the acid chlorides by treatment with oxalyl

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chloride in CH2Cl2 and a catalytic amount of DMF. Finally, the acid chlorides were
cyclized to 1i and 1j via the approach outlined in Scheme 1. Testing of octyl (1i), nonyl
(1j) and decyl (1k) tail lengths revealed an 8ꢀfold reduction to an MIC of 2 ꢁg mLꢀ1 for
1i, but no MIC reduction was observed for 1j or 1k.
Scheme 2. Synthetic route to oxazolines 1i and 1j.
Scheme 3. Synthetic route for aryl head analogues.
Aside from alkyl substituents other functionalities were explored to probe the
oxazolines ability to potentiate the antibiotic FR 900098. Switching the phenyl core for
the heterocyclic pyridinyl core (1l) resulted in no change in antibiotic MIC. Incorporation
of a highly electron donating para – dimethyl amino group (1m) also yielded no change
in MIC, likewise for a para – azo motif (1n). Replacing the phenyl ring with a polycyclic
naphthyl ring (1o) was nonꢀadvantageous. The results were similar for strongly electron
withdrawing fluorine atoms in 1p and 1q, also being disadvantageous to MIC reduction,

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as was the introduction of a meta – bromine (1r). A cinnamoyl derivative (1s) was
synthesized which actually increased the MIC to 32 ꢁg mLꢀ1. Interestingly, increasing the
ring size of the oxazoline to a 6ꢀmembered derivative (1t) displayed modest activity,
reducing the MIC 4ꢀfold to 4 ꢁg mLꢀ1.
Scheme 4. Synthetic route to thiazolines.
At this point our studies revealed that enhanced activity was observed with alkyl
chains of pentyl, hexyl, heptyl and octyl, while other substitutions of the phenyl ring were
unsuccessful at potentiating antibiotic activity. The next series of analogues were
designed in order to establish the oxazolines role in activity. In order to assess whether
the oxazoline was required analogues of the pentyl and hexyl tails were synthesized with
oxazole head groups. Arylation of oxazole was achieved by reacting 2 equivalents of
oxazole with aryl bromides (3a and 4a) using Pd(OAc)2, KOH, and RuPhos in refluxing
toluene (Scheme 3).25 Neither of the oxidized derivatives 3b or 4b, showed any activity
as compared to the more saturated oxazolines. Other heterocylic heads were heads
incorporated into the oxazolines position by standard Suzuki coupling conditions. The
pyrimidine (3c) analogue moderately potentiated FR 900098 with an MIC of 4 ꢁg mLꢀ1,
while the pyridine (4c) derivative was much more active lowering the MIC to 0.5 ꢁg mLꢀ

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1
a 32ꢀfold MIC reduction. We next evaluated whether substitution of the oxazoline with
a thiazoline or imidazoline was tolerated. Thiazolines were synthesized by treatment of
acid chlorides 5a and 6a with NH4OH in MeOH affording primary amides 5b and 6b,
which were dehydrated to nitriles by refluxing SOCl2 (Scheme 4). Cyclization of the
nitriles was performed with 2ꢀaminoethanethiol to give thiazolines 5d and 6d, which
reduced antibiotic MIC to 1 ꢁg mLꢀ1 and 2 ꢁg mLꢀ1 a 16ꢀfold and 8ꢀfold reduction
overall, respectively. The imidazoline (7b) was synthesized by cyclization from 7a with
ethylenediamine and oxidation with NBS (Scheme 5).26 Further oxidation of 7b to the
imidazole (7c) was carried out by treatment with PhI(OAc)2.27 The imidazoline, however,
abrogated activity as did the more oxidized imidazole analogue. This loss in activity for
the imidazoline led us to investigate the completely saturated heterocycles 1,3ꢀdioxolane
(7d) and 1,3ꢀthiolane (7e). Acetalization and thioacetalization was carried out under
standard conditions using catalytic acid and azeotropic distillation. The completely
saturated analogues were inactive. Next, we sought to test the unmasked version of 1g,
the carboxylic acid (8). The acid was produced by hydrolysis of 6a and was found to be
unable to potentiate FR 900098 (Scheme 6). Increasing 8’s lipophilicity by esterification
with MeOH and catalytic acid failed to produce enhanced activity as the methyl ester (9).
Surprisingly, the previously synthesized 2ꢀaminoimidazole (10) analogue actually
increased the MIC to 32 ꢁg mLꢀ1, which has previously been shown to lower the MIC of
βꢀlactam antibiotics and colistin against other gramꢀnegative bacteria.28

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Scheme 5. Synthetic route toward imidazoles and acetals.
Scheme 6. Synthesis of the carboxylic acid (8) and methyl ester (9).
A time kill curve was constructed for 1g to determine whether it was acting
through a toxic or nonꢀtoxic mechanism. Analysis of the graph shows a slight delay in
growth for compound containing samples as compared to strictly bacterial samples at the
4 h and 6 h time points (SI Figure 1). After 6 h samples containing 1g began to grow at a
similar rate as the control and growth became identical after 24 h. This result provides
strong evidence that the oxazoline 1g is acting through a nonꢀbactericidal mechanism.
After discovering a highly active adjuvant for potentiation of FR 900098 against
F. novicida we sought to establish the effectiveness of 1g across other subꢀspecies of the
Francisella genus. Having previously succeeded in potentiating F. philomiragia with 1a
we tested 1g against that bacterium as well. Oxazoline 1g was slightly less active than the
original lead, lowering the antibiotic MIC to 32 ꢁg mLꢀ1 a 32ꢀfold reduction.

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Figure 2. Analogue library. Compounds were tested at 25% of their MIC value. Values in
red are MIC value of compound or antibiotic. Values in blue are MIC of antibiotic in
presence of compound against F. novicida. aCompound was tested at 40 ꢁM.
In conclusion, a diverse library of heterocycles was constructed in order to
investigate potentiation of the antibiotic FR 900098 against the F. tularensis surrogate F.
novicida. The SAR study led to several highly active heterocycles based on the lead 2ꢀ
oxazoline 1a that were able to potentiate FR 900098. The most potent adjuvant 1g
reduced the MIC 1000ꢀfold from 16 ꢁg mLꢀ1 to 0.0156 ꢁg mLꢀ1. Given the lipophilic
nature of 1g, coupled with the observation that activity falls off precipitously below 30
µM (data not shown), the mechanism of action driving FR 900098 potentiation by

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compound 1g most likely involves membrane permealization; however further studies are
necessary to validate this. Colony count enumeration provides evidence that strongly
suggests that it is acting through a nonꢀtoxic mechanism. Testing of 1g against F.
philomiragia demonstrates 1g’s ability to potentiate FR 900098 against multiple subꢀ
species across the Francisella genus.
Acknowledgements
The authors would like to thank the NIH (GM055769 and DE022350) and the Faculty of
Science, Mahidol University for their generous support of this work.
References
(1) Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M.
H.; Patchett, A. A.; Galloway, S. M.; Hyland, S. A.; Anderson, M. S.; Raetz, C. R.
Science 1996, 274, 980.
(2) Meyers, A. I.; Temple, D. L.; Haidukewych, D.; Mihelich, E. D. J. Org. Chem.
1974, 39, 2787.
(3) Hargaden, G. C.; Guiry, P. J. Chem. Rev. 2009, 109, 2505.
(4) McManus, H. A.; Guiry, P. J. Chem. Rev. 2004, 104, 4151.
(5) Adams, N.; Schubert, U. S. Adv. Drug Deliv. Rev. 2007, 59, 1504.
(6) Hoogenboom, R. Angew. Chem. Int. Ed. 2009, 48, 7978.
(7) Kobayashi, S.; Uyama, H. J. Polym. Sci. A Polym. Chem. 2002, 40, 192.
(8) Holerca, Marian N.; Percec, V. European J. Org. Chem. 2000, 2000, 2257.
(9) Ishihara, M.; Togo, H. Tetrahedron 2006, 63, 1474.
(10) Witte, H.; Seeliger, W. Angew. Chem. Int. Ed. Engl. 1972, 11, 287.
(11) Chofor, R.; Risseeuw, M. D.; Pouyez, J.; Johny, C.; Wouters, J.; Dowd, C.S.;
Couch, R. D.; Van Calenbergh, S. Molecules (Basel, Switzerland) 2014, 19, 2571.
(12) Proteau, P. J. Bioorg. Chem. 2004, 32, 483.
(13) Wiemer, A. J.; Hsiao, C. H.; Wiemer, D. F. Curr. Top. Med. Chem. 2010, 10, 1858.
(14) Jawaid, S.; Seidle, H.; Zhou, W.; Abdirahman, H.; Abadeer, M.; Hix, J. H.; van
Hoek, M. L.; Couch, R. D. PLoS ONE 2009, 4, e8288.
(15) Singh, N.; Cheve, G.; Avery, M. A.; McCurdy, C. R. Curr. Pharm. Des. 2007, 13,
1161.
(16) Sangari, F. J.; PérezꢀGil, J.; CarreteroꢀPaulet, L.; GarcíaꢀLobo, J. M.; Rodríguezꢀ
Concepción, M. Proc. Natl. Acad. Sci. USA 2010, 107, 14081.
(17) Gallagher, L. A.; Ramage, E.; Jacobs, M. A.; Kaul, R.; Brittnacher, M.; Manoil, C.
Proc. Natl. Acad. Sci. USA 2007, 104, 1009.
(18) Brown, A. C.; Parish, T. BMC Microbiol. 2008, 8, 78.

MedChemComm
Page 10 of 11
View Article Online
DOI: 10.1039/C6MD00365F
(19) Stephens, M. D.; Hubble, V. B.; Ernst, R. K.; van Hoek, M. L.; Melander, R. J.;
Cavanagh, J.; Melander, C. Med. Chem. Commun. 2016, 7, 128.
(20) Santic, M.; Molmeret, M.; Abu Kwaik, Y. Cell. Microbiol. 2005, 7, 957.
(21) Federal register 2012, 77, 61083.
(22) Dennis, D. T.; Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.; Ascher, M. S.;
Eitzen, E.; Fine, A. D.; Friedlander, A. M.; Hauer, J.; Layton, M.; Lillibridge, S. R.;
McDade, J. E.; Osterholm, M. T.; O'Toole, T.; Parker, G.; Perl, T. M.; Russell, P.
K.; Tonat, K. JAMA 2001, 285, 2763.
(23) Dai, S.; Mohapatra, N. P.; Schlesinger, L. S.; Gunn, J. S. Front. Microbiol. 2010, 1,
144.
(24) Berrada, Z. L.; Telford Iii, S. R. Arch. Microbiol. 2011, 193, 223.
(25) Strotman, N. A.; Chobanian, H. R.; Guo, Y.; He, J.; Wilson, J. E. Org. Lett. 2010,
12, 3578.
(26) Fujioka, H.; Murai, K.; Ohba, Y.; Hiramatsu, A.; Kita, Y. Tetrahedron Lett. 2005,
46, 2197.
(27) Ishihara, M.; Togo, H. Synlett 2006, 2006, 227.
(28) Worthington, R. J.; Bunders, C. A.; Reed, C. S.; Melander, C. ACS Med. Chem.
Lett. 2012, 3, 357.

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