Discovery and Characterization of a Water-Soluble Prodrug of a Dual Inhibitor of Bacterial DNA Gyrase and Topoisomerase IV

Article abstract of DOI:10.1021/acsmedchemlett.5b00196

Benzimidazole 1 is the lead compound resulting from an antibacterial program targeting dual inhibitors of bacterial DNA gyrase and topoisomerase IV. With the goal of improving key drug-like properties, namely, the solubility and the formulability of 1, an

Full text of DOI:10.1021/acsmedchemlett.5b00196

Letter
pubs.acs.org/acsmedchemlett
Discovery and Characterization of a Water-Soluble Prodrug of a Dual
Inhibitor of Bacterial DNA Gyrase and Topoisomerase IV
Hardwin O’Dowd,*,† Dean E. Shannon,†,⊥ Kishan R. Chandupatla,† Vaishali Dixit,† Juntyma J. Engtrakul,†
Zhengqi Ye,† Steven M. Jones,† Colleen F. O’Brien,† David P. Nicolau,‡ Pamela R. Tessier,‡
Jared L. Crandon,‡ Bin Song,† Dainius Macikenas,†,# Brian L. Hanzelka,§ Arnaud Le Tiran,†,∇
Youssef L. Bennani,∥ Paul S. Charifson,† and Anne-Laure Grillot†
†Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, Massachusetts 02210, United States
‡Center for Anti-Infective Research and Development, Hartford Hospital, 80 Seymour Street, Hartford, Connecticut 06102, United
States
§Vertex Pharmaceuticals Incorporated, 2500 Crosspark Road, Bioventures Center, Coralville, Iowa 52241, United States
∥Vertex Pharmaceuticals Incorporated, 275 Boulevard Armand Frappier, Laval, QC H7V 4A7, Canada
S
* Supporting Information
ABSTRACT: Benzimidazole 1 is the lead compound resulting from an antibacterial
program targeting dual inhibitors of bacterial DNA gyrase and topoisomerase IV. With
the goal of improving key drug-like properties, namely, the solubility and the
formulability of 1, an effort to identify prodrugs was undertaken. This has led to the
discovery of a phosphate ester prodrug 2. This prodrug is rapidly cleaved to the parent
drug molecule upon both oral and intravenous administration. The prodrug achieved
equivalent exposure of 1 compared to dosing the parent in multiple species. The
prodrug 2 has improved aqueous solubility, simplifying both intravenous and oral
formulation.
KEYWORDS: Prodrug, DNA gyrase, topoisomerase IV, water soluble
The clinical use of phosphate esters as prodrugs is
ecently, we reported the discovery and characterization of
Rcompound 1, a dual inhibitor of bacterial DNA gyrase precedented;12,13 noteworthy examples include HIV protease
1
inhibitor fosamprenivir,14 anticonvulsant fosphenytoin,15 antie-
metic fosaprepitant,16 and antifungal fosfluconazole17 (Figure
2).
and topoisomerase IV2 with no cross resistance with the
extensively used fluoroquinolone antibiotics.3−6 Compound 1
was shown to possess potent in vitro antibacterial activity versus
clinically important pathogens and in vivo efficacy was
demonstrated versus S. aureus in a neutropenic rat thigh
infection model.1 For nosocomial bacterial infections, where
drug-resistance is particularly pronounced, the option of
intravenous drug delivery is highly desirable.7−11 Due to the
extremely poor aqueous solubility of 1, the identification of a
water-soluble prodrug became a priority. In this letter we
describe the discovery and characterization of the phosphate
ester prodrug 2 of compound 1 (Figure 1).
Compound 1 offers multiple points of attachment for a
phosphate containing promoiety, and initially, attachment to
the benzimidazole NH was considered. Phosphonooxymethyl
derivatives of benzimidazole anthelmintic drugs have been
reported.18,19 Attempts to apply the same strategy to 1 were not
successful, as none of the desired phosphonooxymethyl adducts
were observed upon reaction of 1 with either dibenzyl
chloromethyl phosphate or di-tert-butyl chloromethyl phos-
phate under a variety of conditions. Furthermore, it was
recognized that even if successful this approach would likely
suffer from a lack of regioselectivity with respect to the
benzimidazole nitrogens and potentially the urea nitrogens.
Accessing the phosphate ester of the tertiary alcohol proved
more straightforward. Following the reported synthesis of
fosfluconazole,17,20 1 was reacted with dibenzyl diisopropyl
phosphoramidite, and the resulting phosphite ester intermedi-
ate was oxidized in situ to provide the dibenzylphosphate
Received: May 14, 2015
Accepted: June 22, 2015
Figure 1. Compound 1 and its phosphate ester prodrug 2.
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00196
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ACS Medicinal Chemistry Letters
Letter
Figure 3. Aqueous solubility of 1 (blue) and phosphate prodrug 2
(orange) as a function of pH.
Table 1. MICs versus Select Pathogens; Gyrase and
a
Topoisomerase Kis
MIC (μg/mL)
Ki (μM)
E.
S. aureus faecalis
E.
S.
S.
faecium
pneumoniae S. aureus aureus
Figure 2. Phosphate prodrugs fosamprenivir, fosphenytoin, fosapre-
pitant, and fosfluconazole.
compd (29213) (29212) (49624)
(10015)
gyrase
topo IV
1
2
0.016
8
0.016
1
0.063
4
≤0.008
0.25
<0.009
<0.009
0.012
0.030
derivative 3 (Scheme 1). Hydrogenolysis of the benzyl groups
in the presence of sodium hydroxide gave the disodium salt of
a
ATCC #s in parentheses.
Despite inhibiting both enzymatic targets, the less potent
Scheme 1. Synthesis of Phosphate Ester Prodrug 2 from
Compound 1
a
MICs of 2 indicated that bioconversion to 1 would be
necessary for in vivo antibacterial activity. The assumption at
the outset was that the phosphate promoiety would be cleaved
by alkaline phosphatase (AP), which is present in high
abundance in both the intestine and the liver.22 Intravenous
administration of the prodrug would rely on AP cleavage in the
liver for conversion to the parent. Initially, the phosphate
prodrug pharmacokinetics were evaluated in rat; when
administered intravenously (1 mg/kg) the prodrug form was
rapidly converted to 1; 2 was below the level of quantitation at
2 h, while the presence of 1 in plasma was detectable at 4 h
postdose (Figure 4). Further pharmacokinetic studies in rat,
a
Reaction conditions: (a) (BnO)2P−NiPr2 (1.5 equiv), tetrazole (2
equiv), DMF, MeCN, rt for 18 h, then m-CPBA (1.6 equiv), 0 °C to
rt, 40 min, 85%; (b) (i) H2, Pd/C (0.07 equiv), aq NaOH (2 equiv),
EtOH, rt; (ii) aq HCl (2.2 equiv), MeOH, rt, 92%.
the phosphate ester 2; a solvent mixture of ethanol and aqueous
sodium hydroxide was necessary to ensure that both the
starting dibenzyl phosphate ester and deprotected phosphate
ester would remain in solution and allow removal of the
palladium catalyst. Conversion to the free acid form was
accomplished via treatment of the disodium salt with aqueous
hydrochloric acid in methanol.
As anticipated, the phosphate prodrug was much more water-
soluble than the parent. At pH 7, the aqueous solubility of the
prodrug 2 was approximately 75 mg/mL, >30,000-fold higher
than that of 1 (Figure 3).
Figure 4. Conversion of prodrug 2 (orange) to 1 (blue) following
intravenous administration of 2 in rat (1 mg/kg).
Appending the phosphate moiety to compound 1 renders the
resulting prodrug much less potent in vitro versus all pathogens
tested (Table 1). Interestingly, at the target-level, the prodrug 2
showed similar activity to that of the parent 1; Ki values versus
both S. aureus gyrase and topoisomerase IV enzymes21 are
shown in Table 1. This finding was rationalized based on the
established binding mode of the benzimidazole urea class in
both DNA gyrase and topoisomerase IV, i.e., the phosphate
moiety extends toward solvent.1,3 The lack of whole cell activity
is likely a result of the altered physicochemical properties of the
prodrug prohibiting it from reaching the desired targets, which
reside in the bacterial cytoplasm.
dog, and monkey demonstrated that a similar level of plasma
exposure (area under the curve, AUC) to the parent was
achievable dosing the prodrug intravenously versus dosing the
parent (Figure 5).
While the phosphate prodrug was designed primarily to
overcome issues with developing an intravenous formulation of
1, improving solubility via phosphate prodrugging has been
shown to result in improved oral bioavailability.14,23,24 Addi-
tionally, development of a single agent suitable for both oral
and IV administration versus developing two drug substances is
B
DOI: 10.1021/acsmedchemlett.5b00196
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ACS Medicinal Chemistry Letters
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Liver and intestinal S9 fractions were used to evaluate the
conversion of phosphate ester prodrug to parent29 and the
likelihood of prodrug conversion in humans. Prodrug 2 was
incubated over a concentration range from 1 to 1000 μM in
liver and intestinal S9 fractions from rat, monkey, dog, and
human. The concentration−time profiles of the parent 1
formed at various concentrations of the prodrug 2 were plotted
to give velocity of formation of the parent. The velocity of
formation of parent at each prodrug concentration was fitted to
the Michaelis−Menten equation to yield kinetic constants
(Table 3). Rank order of conversion in liver S9 fractions was rat
≈ human > monkey > dog. The rate of conversion of the
prodrug to parent in intestinal S9 fractions was higher. The
Vmax and Km in human intestinal S9 could not be accurately
determined since the velocity of formation of 1 did not saturate
over the range of concentrations in the experiment. However,
the rate of formation was clearly higher in human and monkey
than in rat and dog.
Figure 5. Plasma exposure of the parent 1 administered IV as 1 (blue
bar) versus as the prodrug 2 (orange bar) in rat, dog, and monkey (1
mg/kg nominal dose).
preferred. Oral delivery would require AP cleavage prior to
intestinal epithelial absorption, the phosphate ester being too
polar to be orally bioavailable;25−27 evaluation of the prodrug in
MDCK cells28 confirmed this to be the case (Table 2). Plasma
Formulation for both IV and oral delivery was simplified for
the prodrug versus 1. Table 4 shows the formulations used in
Table 2. Permeability of 1 versus Phosphate Ester Prodrug 2
in MDCK Cells
Table 4. Comparison of formulations used in PK studies
IV
PO
Papp (1 × 10−6 cm/sec) in MDCK wild-type
1
2
1
2
apical-to-basolateral
basolateral-to-apical
rat
25% DMA/30% PG/
5% tween
D5W 10% vitamin E-
TPGS
0.5%
MC
1
2
9.5
16.3
not permeable
not permeable
dog
20% captisol
D5W 10% vitamin E-
TPGS
0.5%
MC
monkey 20% captisol
D5W 20% cavitron/1% 0.5%
HPMC-AS MC
exposure of 1 via oral administration of the phosphate prodrug
was determined in rat, dog, and monkey (Figure 6). As
the PK studies presented above (Figures 3 and 4). For IV
delivery of the prodrug, formulation with 5% dextrose in water
(D5W) was sufficient to attain a stable solution. Methyl-
cellulose (MC, 0.5%) was found to be suitable for oral delivery.
An unmet medical need that could potentially be addressed
by an agent with the antibacterial spectrum of 1 is nosocomial
bacterial pneumonia caused by methicillin-resistant S. aureus.30
The antibacterial efficacy of the prodrug was evaluated in a
neutropenic mouse model of lung infection31 versus two
methicillin-resistant S. aureus (MRSA) isolates (Figure 7).
Neutropenic female CD-1 mice (6 per group) were infected
with the S. aureus strain of interest; 3 h postchallenge the
treatment phase was initiated, dosing 2 at 7.5, 15, and 30 mg/
kg BID PO. After 24 h of drug treatment the lungs were
evaluated for bacterial burden, as measured by bacterial colony-
forming units (CFU). Levofloxacin, a fluoroquinolone used to
treat pneumonia in the clinic, served as the control antibacterial
and was dosed TID subcutaneously (SC) to approximate
clinical dosing. The prodrug showed a dose-dependent decrease
in bacterial burden; at 30 mg/kg BID, the prodrug achieved >3
log reduction in CFU. The prodrug 2 is efficacious versus both
Figure 6. Plasma exposure of the parent 1 administered PO as 1 (blue
bar) versus as the prodrug 2 (orange bar) in rat (3 mg/kg), dog (3
mg/kg), and monkey (30 mg/kg).
anticipated minimal exposure of the prodrug was observed
when delivered orally (data not shown), improved oral
exposure of 1 was observed when dosing the prodrug 2 versus
dosing as the parent in rat, dog, and monkey. In the higher
species, dog and monkey, greater than 3-fold improvement in
exposure was demonstrated dosing the same nominal dose of
prodrug versus the parent.
Table 3. Michaelis−Menten Parameters for the Formation of 1 from Phosphate Ester Prodrug 2 in Both Liver and Intestine S9
Fractions
liver
intestine
Vmax (pmol/min/mg)
Km (μM)
Vmax (pmol/min/mg)
Km (μM)
rat
45.5 23
19.3 0.66
25.2 4.1
45.8 16
49.8 38
256 16
122 45
137 95
958 111
1162 387
1974 362
>1000
736 158
998 556
5177 1098
ND
dog
monkey
human
C
DOI: 10.1021/acsmedchemlett.5b00196
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ACS Medicinal Chemistry Letters
Letter
∇Institut de Recherches Servier, 11 rue des Moulineaux, 92150
Suresnes, France.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the following individuals for bioanalytical
and in vivo support: Hong Tsao, Elaine Kolaczkowski,
Stephanie Donahue, Ria Seliniotakis, Naran Bao, and Rebecca
Shawgo. The authors also thank Ralph Stearns, Francoise
Berlioz-Seux, Alice Tsai, and Peter Jones for scientific input and
discussions.
Figure 7. Oral efficacy of prodrug 2 versus two MRSA strains in a
mouse pneumonia model. Gray dotted lines indicate mean burden
from early control and vehicle treated groups. aDosed 10.6 mg/kg TID
SC.
ABBREVIATIONS
■
ADME, absorption, distribution, metabolism, and excretion;
AP, alkaline phosphatase; ATCC, American Type Culture
Collection; AUC, area under curve; BID, “bis in die”, twice a
day; CFU, colony forming units; DMA, dimethylacetamide;
HPMC-AS, hydroxypropyl methylcellulose acetate succinate;
IV, intravenous; MDCK, Madin−Darby canine kidney
epithelial cell line; MIC, minimum inhibitory concentration;
MRSA, methicillin-resistant S. aureus; PD, pharmacodynamics;
PG, propylene glycol; PK, pharmacokinetics; PO, “per os”, by
mouth; SC, subcutaneous; TID, “ter in die”, three times a day;
vitamin E-TPGS, D-α-tocopherol polyethylene glycol 1000
succinate
MRSA isolates, including the levofloxacin-resistant strain, S.
aureus 156. The MICs for 1 and levofloxacin, the control, versus
the two methicillin-resistant strains used are shown in Table 5.
Table 5. MICs (μg/mL) of 1 and Levofloxacin versus MRSA
Isolates Used in Mouse Pneumonia Model
1
levofloxacin
S. aureus 509
S. aureus 156 (levoR)
0.06
0.06
0.25
8
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development path of this novel antibacterial. The prodrug 2 is
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for the synthesis of 2 from 1, in vitro
ADME, and in vivo PK/PD protocols. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acsmedchemlett.5b00196.
AUTHOR INFORMATION
Corresponding Author
*Phone: 617 341 6028. E-mail: hardwin_odowd@vrtx.com.
■
Present Addresses
⊥DE Synthetics, 30 Dineen Drive, Fredericton, NB E3B 5A3,
Canada.
#Retrophin Incorporated, 12255 El Camino Real, Suite 250,
San Diego, California 92130, United States.
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