Fructose-1,6-bisphosphatase inhibitors. 2. Design, synthesis, and structure-activity relationship of a series of phosphonic acid containing benzimidazoles that function as 5′-adenosinemonophosphate (AMP) mimics

Article abstract of DOI:10.1021/jm901420x

Efforts to enhance the inhibitory potency of the initial purine series of fructose-1,6-bisphosphatase (FBPase) inhibitors led to the discovery of a series of benzimidazole analogues with human FBPase IC₅₀^s <100nM. Inhibitor 4.4 emerged as a lead compound based on its potent inhibition of human liver FBPase (IC₅₀ = 55 nM) and significant glucose lowering in normal fasted rats. Intravenous administration of 4.4 to Zucker diabetic fatty rats led to rapid and robust glucose lowering, thereby providing the first evidence that FBPase inhibitors could improve glycemia in animal models of type 2 diabetes. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901420x

J. Med. Chem. 2010, 53, 441–451 441
DOI: 10.1021/jm901420x
Fructose-1,6-bisphosphatase Inhibitors. 2. Design, Synthesis, and Structure-Activity Relationship of a
Series of Phosphonic Acid Containing Benzimidazoles that Function as 50-Adenosinemonophosphate
(AMP) Mimics
Qun Dang,*,† Srinivas Rao Kasibhatla,‡ Wei Xiao, Yan Liu,§ Jay DaRe, Frank Taplin, K. Raja Reddy, Gerard R. Scarlato,^
Tony Gibson,§ Paul D. van Poelje, Scott C. Potter, and Mark D. Erion
Departments of Medicinal Chemistry and Biochemistry, Metabasis Therapeutics, Inc., 11119 North Torrey Pines Road, La Jolla, California
92037. †Current address: Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, NJ 07065. ‡ Current address: Jubilant Biosys,
Bangalore, India. § Current address: Takeda San Diego Inc., San Diego, CA 92121. Current address: Neurocrine Biosciences, Inc., San Diego,
CA 92130. ^ Current address: Cambrex Corporation, East Rutherford, NJ 07073.
Received September 23, 2009
Efforts to enhance the inhibitory potency of the initial purine series of fructose-1,6-bisphosphatase
(FBPase) inhibitors led to the discovery of a series of benzimidazole analogues with human FBPase
IC50s < 100 nM. Inhibitor 4.4 emerged as a lead compound based on its potent inhibition of human liver
FBPase (IC50 = 55 nM) and significant glucose lowering in normal fasted rats. Intravenous adminis-
tration of 4.4 to Zucker diabetic fatty rats led to rapid and robust glucose lowering, thereby providing
the first evidence that FBPase inhibitors could improve glycemia in animal models of type 2 diabetes.
Introduction
inhibitors, we focused our attention on the N1 and N3 atoms
of the purine ring. Crystallographic data suggests that
neither N1 nor N3 appeared to form a hydrogen bond with
the FBPase binding site (Figure 2). Consequently, removal of
one or both of the nitrogens was considered unlikely to result
in a loss of binding affinity and more likely to improve
inhibitor potency by minimizing any losses due to desolva-
tion. Moreover, because many purine-containing nucleo-
tides form hydrogen bonds to one or both nitrogens, their
removal could enhance specificity for the AMP binding site
of FBPase over other AMP binding sites. The resulting
benzimidazoles were further optimized by attaching lipophi-
lic groups to the ring in order to produce favorable van der
Waals interactions with residues such as 177Met and 160Val.
Synthesis of Benzimidazole Analogues. The N1-substituted
4-aminobenzimidazole phosphonate analogues were pre-
pared from 5-diethylphosphono-2-furaldehyde (3)3 via a
four-step procedure as shown in Scheme 1. The cyclization
of aldehyde 3 with 3-nitro-benzene-1,2-diamine using the
FeCl3-promoted cyclization reaction developed for purine
synthesis17 gave benzimidazole 4 in 81% yield. The R1 group
was introduced via either Mitsunobu reactions with alcohols
or alkylation reactions with alkyl bromides using sodium
hydride as the base. The nitro group was subsequently
reduced to its corresponding amino group under hydrogena-
tion conditions to give compounds 5, and the phosphonate
diethyl esters were cleaved using TMSBr to yield compounds
1.1-1.17.
Fructose-1,6-bisphosphatase (FBPasea), a rate-limiting en-
zyme of the gluconeogenesis (GNG) pathway, has long been
recognized as a potential therapeutic target for the treatment
of type 2 diabetes mellitus (T2DM).1-3 Efforts over the past
two decades have produced several inhibitor series,4-10 but no
reports prior to our work of glucose lowering activity in
animal models of diabetes. Using a structure-guided drug
design strategy,3 we discovered CS-917 (2a, Figure 1), a
diamide prodrug11 of the potent and specific FBPase inhibitor
MB05032 (2),12 which demonstrated robust glucose lowering
activity in animal models13,14 and in patients with T2DM.15
This inhibitor series arose from our initial discovery of a series
of 8,9-disubstituted purines that functioned as AMP mimetics
capable of retaining the key binding interactions of AMP in
the allosteric binding site of FBPase while also forming
additional interactions designed to enhance potency and
specificity16 (Figure 2). An important step in the transforma-
tion of this modestly potent purine series (1a, IC50 = 0.8 μM)
into the thiazole series of compounds (2, IC50 = 0.016 μM)
was our discovery of a series of benzimidazole-based inhibi-
tors. Herein we report the design, synthesis, and structure-
activity relationships (SAR) of this compound series and
results from a study in the Zucker diabetic fatty rat, demon-
strating that FBPase inhibitors produce rapid and significant
glucose lowering in an animal of severe type 2 diabetes.
Inhibitor Design. In an effort to enhance the potency and
possibly the specificity of the purine series of FBPase
The C5-halo benzimidazole analogues were readily pre-
pared from compound 5a (wherein R1 is isobutyl group)
through direct halogenation reactions (Scheme 2). Thus,
treatment of 5a with NBS gave both C5-bromo (6a) and
C7-bromo (7a) isomers in 21 and 25% yields, respectively.
Similarly, chlorination of compound 5a with NCS produced
compounds 6b and 7b in 49 and 31% yields, respectively.
*To whom correspondence should be addressed. Phone: 732-594-
2018. Fax: 732-594-7444. E-mail: qun_dang@merck.com.
a Abbreviations: FBPase, fructose 1,6-bisphosphatase; OBAV, oral
bioavailability; T2DM, type 2 diabetes mellitus; EGP, endogenous
glucose production; GNG, gluconeogenesis; AMP, 50-adenosinemono-
phosphate; ZDF rats, Zucker diabetic fatty rats.
r
2009 American Chemical Society
Published on Web 11/30/2009
pubs.acs.org/jmc

442 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dang et al.
Treatment of compounds 6a-b with TMSBr gave com-
pounds 2.7 and 2.5, respectively. Compound 6a was also
used to prepare other C5 analogues, as depicted in Scheme 3.
Stille coupling of 6a with either vinyltin or allyltin gave
compounds 8a and 8b, respectively. Hydrogenation of com-
pounds 8a-b followed by TMSBr-mediated ester deprotec-
tion gave compounds 2.1 and 2.2, respectively.
Other 1,2-phenylenediamines (13a-b) were also subjected
to the four-step sequence described in Scheme 1 to give fully
Scheme 1a
The TMSBr-mediated ester deprotection of com-
pounds 7a and 7b gave compounds 2.8 and 2.6, respectively
(Scheme 4).
The four-step sequence described in Scheme 1 was also
used to prepare other benzimidazole analogues, as exempli-
fied in Scheme 5. Cyclization of 9a-b with aldehyde 3 under
the FeCl3-SiO2 reaction conditions gave compounds 10a
and 10b, respectively. N1-alkylations of compounds 10a-b
via Mitsunobu reactions followed by hydrogenation reac-
tions gave compounds 11a and 11b, respectively, and TMSBr-
mediated removal of the phosphonate esters gave compounds
2.3 and 2.9, respectively.
a Conditions and reagents: (i) 3-nitro-benzene-1,2-diamine,
FeCl3-SiO2, DMSO, 80 ꢀC, 2 h; (ii) (a) R1-OH, PPh3, DEAD or (b)
R1Br, NaH, DMF; (iii) H2, Pd-C, EtOH; (iv) TMSBr, CH2Cl2.
Treatment of 11a with boron tribromide resulted in re-
movals of both phosphonate diethyl esters and the methyl
group from the C5-methoxy leading to compound 2.4
(Scheme 6). On the other hand, N1-alkylation of compound
10b with various alkyl bromides, followed by hydrogenation
and TMSBr-mediated deprotection of phosphonate esters
gave compounds 2.10, 2.11, and 2.12 (Scheme 7).
Scheme 2
Double halogenation of compound 5a with excess NBS or
NCS followed by TMSBr-mediated phosphonate ester de-
protection gave the 5,7-dihalo-4-aminobenzimidazole ana-
logues 3.1 and 3.2, respectively (Scheme 8).
Halogenation of 11b with either NBS or NCS followed by
TMSBr-mediated removal of phosphonate esters gave com-
pounds 3.3 and 3.4, respectively (Scheme 9).
Scheme 3a
a Conditions and reagents: (i) Pd(PPh3)4, DMF vinyl- or allyl-SnBu3;
Figure 1. AMP Mimics as FBPase inhibitors.
(ii) H2, Pd-C, EtOH; (iii) TMSBr, CH2Cl2.
Figure 2. Purine 1a (B) mimicking AMP (A) interactions with human FBPase.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 443
Scheme 9a
Scheme 4
Scheme 5a
a Conditions and reagents: (i) NBS or NCS, DMF; (ii) TMSBr,
CH2Cl2.
Scheme 10a
a Conditions and reagents: (i) FeCl3-SiO2, DMSO, 80 ꢀC; (ii)
iBuOH, PPh3, DEAD; (iii) H2, Pd-C, EtOH; (iv) TMSBr, CH2Cl2.
a Conditions and reagents: (i) 3, FeCl3-SiO2, DMSO, 80 ꢀC; (ii)
iBuOH, PPh3, DEAD; (iii) H2, Pd-C, EtOH; (iv) TMSBr, CH2Cl2.
Scheme 6
Scheme 11a
Scheme 7a
a Conditions and reagents: (i) Pd(PPh3)4, DMF, vinyl-SnBu3; (ii)
TMSBr, CH2Cl2; (iii) CH2I2, Zn.
a Conditions and reagents: (i) NaH, DMF, RBr; (ii) H2, Pd-C,
EtOH; (iii) TMSBr, CH2Cl2.
was subjected to Simmons-Smith reaction followed by
TMSBr-mediated phosphonate ester deprotection yielding
analogue 3.9.
Scheme 8a
Suzuki couplings of 12b with various arylboronic acids
followed by hydrogenation and TMSBr-mediated deprotec-
tion of phosphonate esters gave analogues 4.1, 4.2, and 4.3
(Scheme 12). Alternatively, Stille couplings of 12b with either
vinyltin or allyltin followed by hydrogenation and TMSBr-
mediated deprotection of phosphate esters gave analogues
4.4 and 4.5.
Sonogashira couplings of 12b with various alkynes fol-
lowed by hydrogenation and TMSBr-mediated deprotection
of phosphonate esters gave analogues 4.6-4.9, Scheme 13.
Two methods were used to prepare prodrugs of lead
FBPase inhibitor 4.4 as depicted in Schemes 14 and 15.
The bisPOM18 and bisPOC19 phosphonic esters of 4.4 were
obtained via direct alkylation with either POM-I or POC-I to
produce prodrugs 5.1 and 5.3, respectively. The SATE20
and other phosphonic esters21 were prepared using the
a Conditions and reagents: (i) NBS or NCS, DMF; (ii) TMSBr,
CH2Cl2.
substituted benzimidazole analogues 3.5 and 3.6 as shown in
Scheme 10.
To fine-tune the 4-amino-5-fluorobenzimidazole series,
the C7 SAR was rapidly explored using compound 12b as
depicted in Schemes 11, 12, and 13. Stille coupling of 12b
with vinyltin gave compound 14. TMSBr-mediated removal
of phosphonate ester gave analogue 3.8. Alternatively, 14

444 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dang et al.
Scheme 12a
shown in Scheme 15. Coupling of the phosphonic dichloride
of 4.4 with L-alanine ethyl ester in the presence of Hunig’s
base gave the diamide prodrug 5.7, while stepwise coupling
of the phosphonic dichloride of 4.4 first with 2-methylala-
nine ethyl ester and then with ethyl glycolate gave pro-
drug 5.8.
In Vitro Evaluations of Benzimidazole Phosphonate Analo-
gues. Replacing the N1 and N3 nitrogen atoms in compound
1b with CH generates the benzimidazole analogue 1.4, which
exhibited identical FBPase inhibitory potency as 1b. This
observation validated our hypothesis that neither N1 nor N3
of our initial purine series of FBPase inhibitors are required
for binding affinity. To explore the SAR of the benzimida-
zole scaffold, we next prepared analogues wherein we at-
tached various alkyl and aryl groups to the benzimidazole
base. The analogues were evaluated as FBPase inhibitors of
both human liver and rat liver FBPase. In addition, inhibi-
tors were tested in primary rat hepatocytes for their ability to
inhibit glucose production (Table 1). Numerous N1-benzi-
midazole analogues were tested, but only 1.5 and 1.6 showed
slight improvement in potency compared to 1.4. The activity
of analogues 1.1-1.17 indicates that medium size alkyl
groups such as cyclopropylmethyl and cyclobutylmethyl
are preferred, while small (e.g., methyl) or large lipophilic
groups (benzyl, 4-tert-butyl-benzyl) are not well tolerated.
Consistent with previous reports, rat FBPase inhibitory
potency was 2- to 25-fold less than human FBPase potency.
In general, the rat liver FBPase potency for these compounds
correlates well with the relative inhibitory potency measured
in the primary rat hepatocyte assay, e.g., compounds 1.4 and
1.10 are potent inhibitors of rat liver FBPase and both of
them showed good efficacy at inhibiting glucose production
in rat hepatocytes. On the other hand, compounds 1.3, 1.6,
and 1.7 are equipotent compared to compound 1.10 against
rat liver FBPase but are 2-fold less potent in the cellular
assay. It is likely that these phosphonic acids penetrate cells
via organic anion transporters (OATs).22 Therefore, both rat
liver FBPase IC50 and OAT substrate specificity may be
determinants of potency at the cellular level.
a Conditions and reagents: (i) Pd(PPh3)4, DMF, p-R-C6H4-B(OH)2;
(ii) H2, Pd-C, EtOH; (iii) TMSBr, CH2Cl2; (iv) Pd(PPh3)4, DMF vinyl-
or allyl-SnBu3.
Scheme 13a
a Conditions and reagents: (i) Pd(PPh3)4, DMF, R-CtCH; (ii) H2,
Pd-C, EtOH; (iii) TMSBr, CH2Cl2.
Scheme 14a
Compared to a purine, the benzimidazole scaffold pre-
sents two additional positions for optimization. To improve
potency of 1.4, C5 and C7 SAR was explored with various
substituents and results are summarized in Table 2.
a Conditions and reagents: (i) R-I, Hunig’s base, DMF; (ii) SO2Cl2;
(iii) ROH, Hunig’s base, DMF.
Scheme 15a
The C5 SAR was explored with both electron-donating
(e.g., alkyl, hydroxy and alkoxy) and electron-withdrawing
(e.g., Cl) groups, analogues 2.1-2.5, 2.7, and 2.9, Table 2.
Addition of alkyl groups at C5 led to slight decrease in
potency, while C5-OMe and C5-OH analogues gave modest
improvement in potency. Halogenation at C5-position pro-
duced the most significant increase in potency, with fluoro
being the best, followed by chloro and bromo groups. On the
other hand, halogenation at the C7-position (analogues 2.6
and 2.8) was less effective compared to C5-halogenation.
Having identified C5-F as a preferred group, several N1-
substituents were then explored in order to improve FBPase
inhibitory potency further. Increasing lipophilicity via intro-
duction of additional methyl groups to the N1-isobutyl group
in compound 2.9 did not enhance potency (compounds 2.10
and 2.11, Table 2). On the other hand, converting the two
methyl groups into a cyclopropyl group in compound 2.9
produced 2-fold improvement in potency (compound 2.12,
Table 2). To optimize the benzimidazole scaffold further,
combination of subsituents at C5-, C6-, and C7-positions
were explored and results are summarized in Table 3 and 4.
a Conditions and reagents: (i) SO2Cl2; (ii) L-alanine ethyl ester (3
equiv) or ethyl glycolate (0.9 equiv) followed by 2-methylalanine ethyl
ester (2 equiv).
phosphonic dichloride coupling method. Treatment of 4.4
with thionyl chloride generated its corresponding phospho-
nic dichloride, which was treated with AcSCH2CH2OH or
4-(t-BuCO2)Ph-CH2OH or 4-hydroxymethyl-5-methyl-[1,3]-
dioxole-2-thione to give prodrugs 5.4, 5.5 and 5.6, respectively.
The phosphonic dichloride method was also used to
prepare the amino acid containing prodrugs 5.7 -5.8 as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 445
Table 1. SAR of N1-Benzimidazole Analoguesa
C5-position have similar activity compared to 2.9. The 5-
fluoro-6-chloro analogue 3.5 is 2-fold weaker than 2.9, but
the 5-bromo-6,7-dichloro analogue 3.6 lost significant
FBPase inhibition. These analogues indicate that the C5-
fluoro substitution is preferred, thus two analogues were
prepared to explore the C7-position in addition to the
presence of a C5-fluoro group. The C7-vinyl analogue 3.7
is a 2-fold weaker FBPase inhibitor compared to compound
2.9, while the C7-cyclopropyl 3.8 is ca. 2-fold more potent
than compound 2.9. This observation suggests further SAR
exploration at C7-position is warranted.
R
HL IC50, μM RL IC50, μM EC50, μM
1b
1.5
6
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
Me
Et
20
40
2
100
110
37
2.25
1.1
1.5
0.8
0.8
1.5
2.5
3.25
1
nPr
iBu
4
20
cyclopropyl-CH2-
cyclobutyl-CH2-
cyclopentyl-CH2-
cyclohexyl-CH2-
cycloheptyl-CH2-
20
2
100
40
2
45
20
20
2
100
100
25
Various R7 analogues of compound 3.8 were prepared and
evaluated, Table 4. Introduction of aryl groups did not
improve potency against human liver FBPase (compounds
4.1-4.3), and more importantly all three compounds failed
to lower blood glucose levels after intravenous (iv) adminis-
tration. Thus, alkyl groups were explored (compounds
4.4-4.10). The C7-ethyl analogue 4.4 is equipotent as com-
pound 3.8, and it produced significant glucose-lowering
effect after iv administration. On the basis of the potent
glucose-lowering effects and ease of synthesis, compound 4.4
was selected for further evaluations.
Selectivity Evaluations. The selectivity of compound 4.4
against five common enzymes with an AMP-binding
site were measured and compared to purine analogue 1b,
Table 5.
Benzimidazole 4.4 inhibits human liver FBPase with a 10-
fold improvement compared to the most potent purine
FBPase inhibitor 1b. Moreover, no effects were observed
at 4.4 concentrations 1000-fold above its FBPase IC50 for all
five AMP-binding enzymes and no significant effects were
observed in a selectivity screen against >70 enzymes,
ion channels, and receptors (Panlabs, Bothel, WA) at
10 μM of 4.4.
1.10 norbonyl
1.11 benzyl
5
20
20
20
20
20
20
20
100
100
100
100
100
100
100
1.12 4-tBu-benzyl
1.13 4-CF3-benzyl
1.14 4-Ph-benzyl
1.15 3-furnyl-CH2-
1.16 3-HO-benzyl
1.17 3-thienyl-CH2-
9.5
7
2.5
4.2
1.85
4
a HL, human liver FBPase; RL, rat liver FBPase; EC50, inhibition of
glucose production in primary rat hepatocytes.
Table 2. SAR of R5- and R7-Benzimidazole Analoguesa
R1
R5 R7 HL IC50, μM RL IC50, μM EC50, μM
1.4 iBu
2.1 iBu
2.2 iBu
2.3 iBu
2.4 iBu
2.5 iBu
2.6 iBu
2.7 iBu
2.8 iBu
2.9 iBu
H
Et
nPr
MeO
HO
Cl
H
Br
H
H
H
H
H
H
H
Cl
H
Br
H
H
H
H
1.5
2.5
3
4
20
20
2
20
50
50
16
25
15
24
14
14
0.7
0.5
0.2
0.9
0.4
0.4
0.1
0.15
0.85
0.055
20
2
NT
2
2
2.28
F
2.4
5
2.10 (Et)2CHCH2- F
2.11 (Et)2CH-
2.12 cPr-CH2-
0.85
2.5
F
F
9.5
3.3
Inhibition was demonstrated to occur through binding to
the AMP binding site based results from a 3H-labeled AMP
displacement assay, which showed that compound 4.4 dose-
dependently displaced AMP.16,23 The cellular efficacy of
these benzimidazole AMP mimetics was assessed in a glucose
production assay using freshly isolated primary rat hepato-
cytes.1 Compound 4.4 almost completely blocked glucose
production in primary rat hepatocytes at doses of 10 and
100 μM, Figure 3.
2.18
a HL, human liver FBPase; RL, rat liver FBPase; EC50, inhibition of
glucose production in primary rat hepatocytes.
Table 3. SAR of R5-, R6-, and R7-Benzimidazole Analoguesa
In Vivo Glucose Lowering Effects. To test whether FBPase
inhibitors effectively lowered glucose in a rodent model of
type 2 diabetes, we evaluated compound 4.4 in the Zucker
diabetic fatty (ZDF) rat. Administering 4.4 for 6 h by tail
vein infusion to 5 h-fasted ZDF rats (12 weeks of age) at a
dose of 10 mg/kg/h led to significant glucose lowering
(shown in Figure 4), which provided a strong piece of
evidence that FBPase inhibitor would be effective in treating
diabetic hyperglycemia.
Even though compound 4.4, being a phosphonic acid, was
expected to have low oral bioavailability (OBAV), it was
tested for oral efficacy in the normal fasted rat assay at doses
up to 100 mg/kg; plasma glucose levels were unchanged in
this study, indicating that 4.4 indeed has limited oral bio-
availability. The dianionic character of 4.4, similar to other
phosphonic acids, likely limits the oral absorption of com-
pound 4.4. Consequently, we prepared various phosphonic
acid prodrugs21 and evaluated them for OBAV or oral
efficacy (Table 6).
R5 R6
R7
HL IC50, μM RL IC50, μM EC50, μM
2.9
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
F
H
H
H
H
H
Cl
Cl
H
H
H
Br
0.1
1
2.28
2
2.4
14
14
3.6
5
Br
Cl
F
Cl
Cl
0.45
0.1
2
2
F
Br
0.13
0.225
2.09
2
F
H
Cl
16
NT
25
1.5
Br
F
10
NT
0.9
0.35
vinyl
cPr
0.28
0.06
F
a HL, human liver FBPase; RL, rat liver FBPase; EC50, inhibition of
glucose production in primary rat hepatocytes.
The C5- and C7-dibromo and -dichloro analogues 3.1 and
3.2 are much weaker compounds compared to 2.9, while
compounds 3.3 and 3.4 with either a chloro or a bromo group
at the C7-position in addition to the fluoro group at
The classic acyloxyalkyl ester prodrug 5.1 did not exhibit
significant improvement in OBAV compared to 4.4, most

446 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Table 4. SAR of R7-Benzimidazole Analogues of Compound 3.8a
Dang et al.
R7
HL IC50, μM
RL IC50, μM
EC50, μM
G-LOW, %
3.8
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
cPr
Ph
0.06
0.09
0.18
0.09
0.055
0.1
0.35
0.9
1.3
2
1.5
18
64
0
4-F-Ph
4-Cl-Ph
Et
21
0
>25
3.5
5.5
18
NT
64
60
28
NT
63
44
47
0.55
0.65
1.6
2.1
0.45
0.4
0.5
nPr
tBu(CH2)2-
(Me)2CH(CH2)3-
HO(CH2)3-
(Me)2N(CH2)3-
Cl(CH2)4-
0.21
0.1
23
1
0.08
0.055
0.07
9
3.1
a HL, human liver FBPase; RL, rat liver FBPase; EC50, inhibition of glucose production in primary rat hepatocytes; G-LOW, glucose lowering in the
normal fasted rats after iv administration.
Table 5. Selectivity of Purine 1b and Benzimidazole 4.4a
enzymes
benzimidazole 4.4
FBPase
adenosine kinase
IC50 = 0.055 μM
IC50 > 100 μM
IC50 > 100 μM
IC50 > 100 μM
EC50 >100 μM
EC50 >100 μM
adenylate kinase
AMP deaminase
glycogen phosphorylase
pPhosphofructokinase
a Inhibition of enzymes is reported as IC50, while activation of
enzymes is reported as EC50
.
Table 6. Evaluations of Prodrugs of Benzimidazole Analogue 4.4a
Figure 3. Inhibition of glucose production in hepatocytes by 4.4.
MW
LogP
TPSA
RB HB-A HB-D OBAV, %
4.4 381.4
5.1 609.6
5.2 646
2
124.32
154.92
128.90
173.38
203.42
187.06
154.92
160.52
157.72
5
15
16
17
11
15
17
15
15
7
11
11
13
11
9
4
2
0
2
2
2
2
4
3
1
4.35
3.29
2.16
3.23
3.79
7.37
3.13
3.63
2
2b
5.3 585.5
5.4 637.7
5.5 585.7
5.6 761.8
5.7 579.6
5.8 580.6
2
2
2
11
11
11
NDc
5
NDd
a OBAV, oral bioavailability determined using an urinary excretion
assay; LogP, TPSA (total polar surface area), RB (rotatable bond), HB-
A (hydrogen bond acceptor), HB-D (hydrogen bond donor) were
calculated using ADME Boxes version 3.5 (Pharma Algorithm,
Toronto, Canada). b Compound 5.2 is the HCl salt form of 5.1.
c Compound 5.6 was much weaker (EC50 > 20 μM) in the cellular assay
compared to 4.4, therefore it was not tested for OBAV. d Compound 5.8
did not lower glucose significantly in the normal fasted rat model after
oral administration, thus OBAV was not determined.
Figure 4. Glucose lowering in ZDF rats after intravenous infusion
(4.4, 10 mg/kg/h).
likely because 5.1 is quite lipophilic and therefore likely has
poor solubility. Syntheses of several other prodrugs
(5.3-5.8) also did not achieve acceptable OBAV. To under-
stand the structural features responsible for the observed low

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 447
OBAV, we determined the LogP, TPSA, RB, HB-A, and
HB-D. After review of the results (Table 6), we concluded
that the most likely limiting factor for the observed low
OBAV was the high molecular weight (MW), given that
molecules with MW > 500 tend to have lower OBAV.24
similar manner as compound 4 from 4-methoxy-3-nitro-1,2-
phenylenediamine (500 mg, 2.35 mmol) to give 10a (502 mg,
54% yield).
Diethyl [5-(5-Fluoro-4-nitro-1H-benzoimidazol-2-yl)-furan-2-
yl]-phosphonate (10b). This compound was prepared in a similar
manner as compound 4 from 4-fluoro-3-nitro-1,2-phenylene-
diamine (1.0 g, 5.82 mmol) to give 10b (1.7 g, 76% yield).
General Procedures N1-Alkylation of Benzoimidazoles. Di-
ethyl 5-(1-Isobutyl-4-amino-1H-benzoimidazol-2-yl)-furan-2-yl]-
phosphonate (5a). A solution of benzimidazole 4 (365 mg, 1
mmol) in anhydrous THF (5 mL) was treated with isobutyl
alcohol (1 mL, excess), triphenylphosphine (314 mg, 1.2 mmol),
and DEAD (209 mg, 1.2 mmol) at room temperature for 12 h.
The reaction mixture was evaporated, and the residue
was purified by flash chromatography on silica gel (1 cm ꢀ
15 cm, 55% EtOAc-hexane) to give diethyl 5-(1-isobutyl-4-
Conclusions
A structure-guided drug design approach was used to
improve the potency and selectivity profile of an early purine
phosphonate series of FBPase inhibitors. Replacements of
both N1 and N3 atoms in the purine scaffold with carbon
atoms produced new benzimidazole analogues with similar
FBPase inhibitory activity relative to analogues of the purine
series. Further optimization of the new benzimidazole scaf-
fold led to the discovery of several analogues with IC50 values
< 100 nM, which represents a significant improvement over
the early purine series of FBPase inhibitors (e.g., purine 1a,
IC50 = 0.8 μM). The SAR established by these benzimidazole
FBPase inhibitors proved to be instrumental for the discovery
of our first clinical candidate compound 2a (a diamide
prodrug of compound 2), which will be the subject of a
subsequent full paper reporting the design, synthesis, and
evaluation of thiazole FBPase inhibitors. Compound 4.4
emerged as a lead inhibitor based on its potent inhibition of
human liver FBPase (IC50 = 55 nM), enzyme specificity,
potent cellular activity (EC50 = 3.5 μM), and significant
glucose-lowering (64%) in normal fasted rats. Evaluation of
4.4 in the ZDF rat demonstrated for the first time that FBPase
inhibitors could significantly reduce glucose levels in an
animal model of T2DM.
nitro-1H-benzoimidazol-2-yl)-furan-2-yl]-phosphonate
(388
mg, 92%).
A solution of diethyl 5-(1-isobutyl-4-nitro-1H-benzoimida-
zol-2-yl)-furan-2-yl]-phosphonate (388 mg, 0.921 mmol) in
ethanol (10 mL) was treated with palladium on carbon (10%,
40 mg) and stirred at room temperature under 1 atm of hydrogen
for 24 h. The reaction mixture was filtered through a celite pad,
and the filtrate was evaporated to dryness. The crude material
was purified by flash chromatography on silica gel (2 cm ꢀ
15 cm, 75% EtOAc-hexane) to give 5a as a yellow solid
(372 mg, 95%).
Diethyl 5-(1-Isobutyl-4-amino-5-methoxy-1H-benzoimidazol-
2-yl)-furan-2-yl]-phosphonate (11a). This compound was pre-
pared in a similar manner as compound 5a from 10a (1.0 g, 2.5
mmol) to give 11a (98 mg, 9.3% yield).
Diethyl 5-(1-Isobutyl-4-amino-5-fluoro-1H-benzoimidazol-2-
yl)-furan-2-yl]-phosphonate (11b). This compound was prepared
in a similar manner as compound 5a from 10b (1.1 g, 2.8 mmol)
to give 11b (640 mg, 56% yield). 1H NMR (CDCl3): δ 0.94 (d,
6H, J = 6.6 Hz), 1.37 (t, 6H, J = 8.5 Hz), 2.1-2.3 (m, 1H),
4.0-4.3 (m, 6H), 4.36 (br s, 2H), 6.65 (dd, 1H, J1 = 8.8, J2 = 3.7
Hz), 7.02 (dd, 1H, J1 = 8.8, J2 = 8.8 Hz), 7.22 (m, 1H), 7.3
(m, 1H).
General Procedures for Halogenation of Benzimidazoles. Di-
ethyl 5-(4-Amino-5-bromo-1-isobutyl-1H-benzoimidazol-2-yl)-
furan-2-yl]-phosphonate (6a) and Diethyl 5-(4-Amino-7-bromo-
1-isobutyl-1H-benzoimidazol-2-yl)-furan-2-yl]-phosphonate (7a).
A solution of 5a (873 mg, 2.23 mmol) in anhydrous carbon
tetrachloride (30 mL) was treated with NBS (397 mg, 2.23
mmol) and AIBN (20 mg). The resulting reaction mixture was
heated to reflux for 12 h. The cooled reaction mixture was
evaporated to dryness, and the residue was purified by flash
chromatography on silica gel (3 cm ꢀ 15 cm, 30, 50, 75, 100%
EtOAc-hexane, gradient elution) to give compounds 6a (219
mg, 21%), 7a (261 mg, 25%), and diethyl [5-(4-amino-5,7-
dibromo-1-isobutyl-1H-benzoimidazol-2-yl)-furan-2-yl]-phos-
phonate (445 mg, 36%).
Diethyl 5-(4-Amino-5-chloro-1-isobutyl-1H-benzoimidazol-2-
yl)-furan-2-yl]-phosphonate (6b) and Diethyl 5-(4-amino-7-
chloro-1-isobutyl-1H-benzoimidazol-2-yl)-furan-2-yl]-phospho-
nate (7b). These compounds were prepared in a similar manner
as compound 6a and 7a from 5a (192 mg, 0.49 mmol) to give
compounds 6b (103 mg, 49% yield) and 7b (65 mg, 31% yield).
Diethyl 5-(4-Amino-5-vinyl-1-isobutyl-1H-benzoimidazol-2-
yl)-furan-2-yl]-phosphonate (8a). A solution of 6a (188 mg, 0.4
mmol) in anhydrous toluene (5 mL) was treated with tributyl-
vinyl-stannane (162.2 mg, 0.51 mmol) and palladium tetrakis-
(triphenylphosphine) (49.6 mg, 0.04 mmol) under nitrogen.
The resulting reaction mixture was heated to 80 ꢀC for 16 h.
The cooled reaction mixture was diluted with EtOAc (50 mL),
washed with saturated NaF (15 mL), brine (15 mL), dried
(MgSO4), and evaporated. The residue was purified by flash
Experimental Section
General Methods. Glassware for moisture-sensitive reactions
was flame-dried and cooled to room temperature under va-
cuum, and all reactions were carried out under a nitrogen
atmosphere. Anhydrous solvents were purchased and used
directly. TLC was performed on Merck Kieselgel 60 F254 plates,
and flash chromatography was performed on 230-240 mesh
EM Science silica gel 60. Melting points were recorded on a
Thomas-Hoover capillary melting point apparatus and were
uncorrected. Proton NMR spectra were obtained on a Varian
Gemini-200 operating at 200 MHz and recorded in δ with
tetramethylsilane (δ 0.00) as reference line internal standard.
All final compounds have purities >95%, determined either by
microanalyses (performed by Robertson Microlit Laboratories,
Inc., Madison, NJ) or via HPLC (UV detection at 280 nm). Low
resolution mass spectra were obtained from Mass Consortium
Corp., San Diego, CA.
General Procedures for the Preparation of N1-H Benzimida-
zoles. Diethyl [5-(4-Nitro-1H-benzoimidazol-2-yl)-furan-2-yl]-
phosphonate (4). A solution of 3-nitro-benzene-1,2-diamine (2,
153 mg, 1 mmol) and diethyl (5-formyl-furan-2-yl)-phospho-
nate3 (3, 348 mg, 1.5 mmol) in anhydrous DMSO (5 mL) was
treated with FeCl3-SiO2 (20 wt %, 1.62 g, 2.0 mmol).17 The
resulting reaction mixture was heated at 80 ꢀC for 2 h. The
cooled reaction mixture was diluted with EtOAc (60 mL) and
filtered through a silica gel plug (SiO2, 5 cm ꢀ 5 cm). The silica
gel was washed with EtOAc (3 ꢀ 20 mL). The combined filtrates
were evaporated to dryness, and the residue was purified
by flash chromatography (SiO2, 2 cm ꢀ 10 cm, 60, 80%
EtOAc-hexane, gradient elution) to give compound 4 as a
1
yellow solid (297 mg, 81%). H NMR (CDCl3): δ 11.07 (bs,
1H), 8.24-8.14 (m, 2H), 7.47-7.26 (m, 3H) 4.33-4.22 (m, 4H),
1.43 (t, 6H, J = 7.0 Hz).
chromatography on silica gel (2 cm
EtOAc-hexane) to give compound 8a (90 mg, 54%).
ꢀ
15 cm, 75%
Diethyl [5-(5-Methoxy-4-nitro-1H-benzoimidazol-2-yl)-furan-
2-yl]-phosphonate (10a). This compound was prepared in a

448 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dang et al.
Diethyl 5-(4-Amino-5-allyl-1-isobutyl-1H-benzoimidazol-2-
yl)-furan-2-yl]-phosphonate (8b). This compound was prepared
in a similar manner as 8a from 6a (188 mg, 0.4 mmol) to give 8b
(112 mg, 65%).
Diethyl 5-(1-Isobutyl-4-amino-5-fluoro-7-chloro-1H-benzo-
imidazol-2-yl)-furan-2-yl]-phosphonate (12a). A solution of 11b
(300 mg, 0.73 mmol) in anhydrous chloroform (4 mL) was
treated with NCS (147 mg, 1.1 mmol) at room temperature.
After stirring for 16 h, the reaction solution was evaporated and
the residue was purified by flash chromatography on silica gel
(3 cm ꢀ 15 cm, 75, 85, 100% EtOAc-hexane, gradient elution)
to give 12a as a yellow solid (190 mg, 59%).
(DMSO-d6): δ 0.69 (d, 6H, J = 6.6 Hz), 1.21 (t, 3H, J = 7.2 Hz),
1.8 (m, 1H), 2.82 (q, 2H, J = 7.2 Hz), 4.38 (d, 2H, J = 7.8 Hz),
6.83 (d, 1H, J = 12.8 Hz), 7.0 (m, 1H), 7.1 (m, 1H).
The crude 4.4 (114.7 g of combined batches) was treated with
a sodium hydroxide solution (1 M, 570 mL, 570 mmol) and
stirred at room temperature for 2 h. The resulting cloudy, dark-
orange solution was washed with EtOAc (2 ꢀ 350 mL), and the
dark-orange aqueous phase was diluted with methanol (350 mL)
and treated with activated carbon (Norit SA-3, 4.0 g). The
mixture was filtered twice through celite and once through filter
paper. The filtrate (1.4 L total volume) was treated with con-
centrated hydrochloric acid (48 mL, 570 mmol) dropwise with
vigorous stirring. A persistent yellow precipitate formed during
the second half of addition. After the addition was complete (45
min), the resulting mixture was stirred at room temperature for
2 h and then at 0 ꢀC for 1 h. The resulting solid was collected by
filtration and washed with MeOH-water (60:40, 100 mL) and
water (3 ꢀ 200 mL). The solid was dried under vacuum to give
4.4 as a fine yellow-orange powder (93.4 g, 86%); mp >220 ꢀC.
1H NMR (DMSO-d6): δ 0.68 (d, 6H, J = 6.6 Hz), 1.21 (t, 3H,
J = 7.2 Hz), 1.7-2.0 (m, 1H), 2.83 (q, 2H, J = 7.2 Hz), 4.37 (d,
2H, J = 7.4 Hz), 6.83 (d, 1H, J = 12.8 Hz), 7.02 (m, 1H), 7.12
(m, 1H). 13C NMR (DMSO-d6): δ 15.79, 19.25, 23.59, 30.47,
52.08, 112.66, 112.89, 113.1, 114.86, 115.00, 119.64, 120.10,
124.43, 124.73, 129.77, 133.49, 133.67, 143.15, 143.32, 147.65,
147.81, 148.02, 149.16, 153.66. [MH]þ calcd for C17H21N3O4PF
382; found 382. Anal. (C17H21N3O4PF) C, H, N.
Diethyl 5-(1-Isobutyl-4-amino-5-fluoro-7-bromo-1H-benzo-
imidazol-2-yl)-furan-2-yl]-phosphonate (12b). This compound
was prepared in a similar manner as compound 12a from 11b
(150 mg, 0.37 mmol) using NBS to give 12b (90 mg, 50% yield).
Diethyl [5-(5-Fluoro-1-isobutyl-4-nitro-7-vinyl-1H-benzoim-
idazol-2-yl)-furan-2-yl]-phosphonate (14). A solution of 12b
(80.0 g, 154 mmol) in DMF (650 mL) was treated with dichloro
bis(triphenylphosphine)palladium(II) (4.5 g, 6.4 mmol). While
nitrogen was bubbled through the stirred mixture, tributylvinyl
tin (51.5 mL, 176 mmol) was slowly added. After the addition
was complete (5 min), the mixture was warmed to 70 ꢀC and
stirred for 1 h with continued nitrogen purge. The mixture was
cooled to room temperature and concentrated under reduced
pressure. The resulting dark oil was dissolved in EtOAc (2.1 L)
and was treated with a mixture of sodium fluoride (113 g, 2.69
mol) in water (475 mL). After stirring for 16 h, the mixture was
diluted with water (350 mL) and filtered through a pad of celite
in a Buchner funnel (11 cm). The layers were separated and the
aqueous phase was extracted with EtOAc (800 mL). The com-
bined organic extracts were washed with water (600 mL) and
brine (600 mL) and were dried (MgSO4, 125 g), filtered, and
concentrated under reduced pressure to give a dark oil (88.6 g).
The crude material was purified by chromatography on silica gel
(1.75 kg, 6 in. diameter column), using 3:1 EtOAc-hexanes (16
L) as the eluting solvents to give 14 as a pale-orange powder
(66.7 g, 92%); mp 77-78.5 ꢀC. 1H NMR (DMSO-d6): δ 0.73 (d,
6H, J = 6.6 Hz), 1.25 (t, 3H, J = 7.0 Hz), 1.9 (m, 1H), 4.1 (m,
4H), 4.58 (d, 2H, J = 7.8 Hz), 5.7 (d, 1H, J = 9.4 Hz), 6.0 (d, 1H,
J = 11.5 Hz), 7.3-7.6 (series of m, 4H).
General Procedures for the Removal of Phosphonate Diesters.
Method a, TMSBr-Mediated Conditions. [5-(4-Amino-7-ethyl-5-
fluoro-1-isobutyl-1H-benzoimidazol-2-yl)-furan-2-yl]-phospho-
nic Acid (4.4). A solution of 14 (57.1 g, 122 mmol) in ethanol (425
mL) was treated with palladium on carbon (4.5 g) under a
continuous flow of nitrogen. The resulting mixture was stirred
under one atmosphere of hydrogen for 20 h at room tempera-
ture. The reaction mixture was flushed with nitrogen, filtered
through a pad of celite, and the filtrate was concentrated under
reduced pressure to provide a thick orange syrup. The material
was dissolved in toluene (250 mL) and filtered through a pad of
celite. The filtrate was concentrated under reduced pressure to
give an orange syrup (56 g, 100%). 1H NMR (DMSO-d6): δ 0.73
(d, 6H, J = 6.6 Hz), 1.05-1.35 (m, 6H), 1.85 (m, 1H), 2.82 (q,
2H, J = 7.0 Hz), 4.1 (m, 4H), 4.38 (d, 2H, J = 7.8 Hz), 5.18 (br s,
2H, exchangeable with D2O), 6.8 (d, 1H, J = 11.5 Hz), 7.2 (m,
1H), 7.4 (m, 1H). The material was used in the next step without
further purification.
The following compounds were prepared in a similar manner
as for compound 4.4:
4-Amino-1-methyl-2-[2-(5-phosphono)furanyl]benzimidazole
(1.1). mp >230 ꢀC. 1H NMR (D2O): δ 3.91 (s, 3H), 6.61-7.19
(m, 5H). Anal. (C12H12N3O4P 1H2O) C, H, N.
3
4-Amino-1-ethyl-2-[2-(5-phosphono)furanyl]benzimidazole
(1.2).). 1H NMR (D2O): δ 0.98 (t, J = 7.4 Hz, 3H), 4.19 (q, J =
7.4 Hz, 2H), 6.71-7.22 (m, 5H); mp >250 ꢀC. Anal. (C13-
H14N3O4P 1H2O) C, H, N.
3
4-Amino-L-propyl-2-[2-(5-phosphono)furanyl]benzimidazole
(1.3). 1H NMR (D2O): δ 0.78 (t, J = 7.2 Hz, 3H), 1.78 (m, 2H),
4.39 (t, J = 7.2 Hz, 2H), 6.61-7.21 (m, 5H); mp >230 ꢀC.
Anal. (C14H16N3O4P þ 1.25H2O) C, H, N.
4-Amino-l-isobutyl-2-[2-(5-phosphono)furanyl]benzimidazole
(1.4). 1H NMR (CD3OD): δ 0.71 (d, J = 7.2 Hz, 6H), 2.01 (m,
1H), 4.07 (d, J = 7.2 Hz, 2H), 6.71-7.09 (m, 5H). Anal. (C15
-
H18N3O4P 1.9H2O) C, H, N.
3
4-Amino-1-cyclopropylmethyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.5). mp >230 ꢀC. 1HNMR(D2O): δ0.22 (m, 2H), 0.95 (m,
2H), 1.25 (m, 1H), 4.26 (d, J = 7.0 Hz, 2H), 6.76-7.31 (m, 5H).
Anal. (C15H16N3O4P 0.75CH2Cl2) C, H, N.
3
4-Amino-1-cyclobutylmethyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.6). mp >230 ꢀC. 1H NMR (D2O): δ 1.61-1.88 (m, 6H),
2.78 (m, 1H), 4.42 (d, J = 7.0 Hz, 2H), 6.62-7.19 (m, 5H). Anal.
(C16H18N3O4P 0.5H2O) C, H, N.
3
4-Amino-1-cyclopentylmethyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.7). mp >230 ꢀC. 1H NMR (D2O): δ 0.98-1.50 (m, 8H),
2.23 (m, 1H), 4.23 (d, J = 7.2 Hz, 2H), 6.55-7.09 (m, 5H). Anal.
(C17H20N3O4P 1.4H2O) C, H, N.
3
4-Amino-1-cyclohexanemethyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.8). mp 210 ꢀC. 1H NMR (D2O): δ 0.98-1.85 (m, 11H),
4.21 (d, J = 7.3 Hz, 2H), 6.60-7.15 (m, 5H). Anal. (C18H22-
A solution of the above material in dichloromethane (500
mL) was cooled to 0 ꢀC and treated slowly with bromotri-
methylsilane (112 mL, 848 mmol). The solution was allowed to
warm to room temperature and was stirred for 16 h. The solvent
was evaporated under reduced pressure, and the resulting
residue was coevaporated with acetone (200 mL) to afford a
thick orange tar. The material was suspended in water (800
mL)-acetone (150 mL), and was stirred vigorously for 2 h. The
resulting solid was collected via filtration. The collected solid
was washed with water (3 ꢀ 150 mL) and dried under vacuum to
give crude 4.4 as a fine yellow powder (48.1 g, 103%). 1H NMR
N3O4P 0.5AcOH) C, H, N.
3
4-Amino-1-cycloheptanemethyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.9). mp 198 ꢀC. 1HNMR(D2O): δ1.08-1.60 (m, 12H), 2.02
(m,1H),4.21(d,J= 7.2 Hz, 2H), 6.61-7.16 (m, 5H). Anal. (C19H24-
N3O4P 0.5H2O) C, H, N.
3
4-Amino-1-norbornylmethyl-2-[2-(5-phosphono)furanyl]benzimi-
1
dazole (1.10). mp >230 ꢀC. H NMR (D2O): δ 0.67-2.02 (m,
11H), 4.41 (d, J = 7.4 Hz, 2H), 6.64-7.20 (m, 5H). Anal. (C19H22-
N3O4P 0.75H2O) C, H. N.
4-Amino-1-benzyl-2-[2-(5-phosphono)furanyl]benzimidazole (1.11).
3
mp >250 ꢀC. 1H NMR (DMSO-d6): δ 5.04 (s, 2H), 5.96 (s, 2H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 449
6.48-7.19 (m, 10H). Anal. (C18H14N3O4PNa2 1.6H2O) C, H,
4-Amino-5-fluoro-1-cyclopropylmethyl-2-[5-(2-phosphono)-
furanyl]benzimidazole (2.12). mp = 258-260 ꢀC. 1H NMR
(DMSO-d6): δ 0.1-0.4 (m, 4H), 1.35 (m, 1H), 4.58 (d, 2H, J =
3
N. Mass calcd for C18H16N3O4P = 369. Found: MHþ = 370;
MH- = 368.
4-Amino-1-(4-t-butylbenzyl)-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.12). mp = 246-249 ꢀC. 1H NMR (DMSO-d6): δ 1.18 (s,
9H), 5.90 (s, 2H), 6.56-7.26 (m, 11H). Anal. (C22H24N3-
O4P 0.3HBr 0.2PhMe) C, H, N. Mass calcd for C22H24N3-
7.0 Hz), 6.85-7.11 (m, 4H). Anal. (C15H15N3O4PF 0.3H2O)
3
C, H, N.
4-Amino-5,7-dibromo-1-isobutyl-2-[2-(5-phosphono)furanyl]ben-
zimidazole (3.1). mp >215 ꢀC. 1H NMR (D2O): δ 0.51 (d, J = 7.0
Hz, 6H), 1.81 (m, 1H), 4.29 (d, J = 7.4 Hz, 2H), 6.71 (m, 1H), 6.83
(m, 1H), 7.29 (s, 1H). Anal. (C15H16Br2N3O4P) C, H, N.
4-Amino-5,7-dichloro-1-isobutyl-2-[5-(2-phosphono)furanyl]ben-
zimidazole (3.2). mp = 205-207 ꢀC. 1H NMR (D2O): δ 0.63 (d,
J = 7.0 Hz, 6H), 1.91 (m, 1H), 4.41 (d, J = 7.4 Hz, 2H), 6.72 (m,
3
3
O4P = 425. Found: MHþ = 426; MH0 = 424.
4-Amino-1-(3-trifluoromethylbenzyl)-2-[2-(5-phosphono)furanyl]-
benzimidazole (1.13). mp 235-239 ꢀC. 1H NMR (DMSO-d6): δ
5.83 (s, 2H), 6.45-7.69 (m, 11H). Anal. (C19H15N3O4-
PF3 0.7HBr 0.6PhMe) C, H, N.
3
3
1H), 7.01 (m, 2H). Anal. (C15H16N3O4Cl2P 0.5H2O) C, H, N.
4-Amino-1-(4-phenylbenzyl)-2-[2-(5-phosphono)furanyl]benzimi-
3
1
4-Amino-5-fluoro-7-chloro-1-isobutyl-2-[5-(2-phosphono)furanyl]-
benzimidazole (3.3). mp = 220-225 ꢀC. 1H NMR (DMSO-d6): δ
0.76 (d, 6H, J =6.7 Hz), 1.9-2.1 (m, 2H), 4.58 (d, 2H, J = 7.4 Hz),
7.06 (m, 1H), 7.16 (d, 1H, J = 11.6 Hz), 7.2 (m, 1H). Anal.
dazole (1.14). mp >220 ꢀC. H NMR (D2O): δ 5.73 (s, 2H),
6.25-7.59 (m, 14H). Anal. (C24H20N3O4P 0.66H2O) C, H, N.
4-Amino-1-(3-furanylmethyl)-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.15). mp >230 ꢀC. 1H NMR (D2O): δ 5.48 (s, 2H),
6.24-7.42 (m, 8H). Mass calc. for C16H14N3O5P 358; obsd 358.
4-Amino-1-(3-hydroxybenzyl)-2-[2-(5-phosphono)furanyl]benzimi-
3
(C15H16N3O4FClP 0.9HBr) C, H, N.
3
4-Amino-5-fluoro-7-bromo-1-isobutyl-2-[5-(2-phosphono)furanyl]-
benzimidazole (3.4). mp = 195-200 ꢀC. 1H NMR (DMSO-d6): δ
0.77 (d, 6H, J = 6.7 Hz), 1.95-2.15 (m, 2H), 4.62 (d, 2H, J = 7.5
Hz), 7.1 (m, 1H), 7.22 (m, 1H), 7.31 (d, 1H, J = 11.6 Hz). Anal.
(C15H16N3BrFPO4) C, H, N.
1
dazole (1.16). mp 232-234 ꢀC. H NMR (D2O): δ 5.87 (s, 2H),
6.43-7.10 (m, 9H). Anal. (C18H16N3O5P 2H2O) C, H, N.
3
4-Amino-1-(3-thienylmethyl)-2-[2-(5-phosphono)furanyl]benzimi-
dazole (1.17). mp = 200-205 ꢀC. 1H NMR (D2O): δ 5.86 (s, 2H),
4-Amino-5-fluoro-6-chloro-1-isobutyl-2-[5-(2-phosphono)furanyl]-
benzimidazole (3.5). mp = 175-180 ꢀC. 1H NMR (DMSO-d6): δ
0.81 (d, 6H, J = 6.7 Hz), 1.9-2.1 (m, 1H), 4.24 (d, 1H, J = 7.0 Hz),
6.02 (br s, 2H), 6.95 (d, 1H, J = 9.7 Hz), 7.05 (m, 1H), 7.12 (m, 1H).
6.67-7.42 (m, 8H). Anal. (C16H14N3O4PS 1.7H2O) C, H, N.
3
4-Amino-5-ethyl-1-isobutyl-2-[5-(2-phosphono)furanyl]benzimi-
dazole (2.1). mp = 220-225 ꢀC. 1H NMR (DMSO-d6): δ 0.87 (d,
6H, J = 6.7 Hz), 1.17 (t, 3H, J = 7.3 Hz), 1.9-2.25 (m, 1H), 2.62
(t, 2H, J = 7.3 Hz), 4.29 (d, 1H, J = 7.3 Hz), 6.87 (d, 1H, J = 8.3
Hz), 7.01 (d, 1H, J = 8.3 Hz), 7.1 (m, 1H), 7.2 (m, 1H). Anal.
Anal. (C15H16N3ClFPO4 2H2O) C, H, N.
3
4-Amino-5-bromo-6,7-dichloro-2-[5-(2-phosphono)furanyl]ben-
zimidazole (3.6). mp = 224-225 ꢀC. 1H NMR (D2O): δ 0.64 (d,
J = 7.1 Hz, 6H), 1.98 (m, 1H), 4.51 (d, J = 7.3 Hz, 2H), 6.72 (m,
1H), 6.96 (m, 1H). Anal. (C15H15N3O4Cl2BrP 1HBr 0.9PhMe)
(C17H22N3O4P 0.5HBr) C, H, N.
3
4-Amino-5-propyl-1-isobutyl-2-[5-(2-phosphono)furanyl]benzimi-
dazole (2.2). mp = 207-210 ꢀC. 1H NMR (DMSO-d6): δ 0.85 (d,
6H, J = 6.6 Hz), 0.92 (t, 3H, J = 7.5 Hz), 1.4-1.7 (m, 2H),
1.95-2.2 (m, 1H), 2.55 (t, 2H, J = 7.0 Hz), 4.27 (d, 1H, J = 7.0
Hz), 6.87 (d, 1H, J = 8.2 Hz), 7.04 (d, 1H, J = 8.3 Hz), 7.09 (m,
3
3
C, H, N.
4-Amino-7-vinyl-5-fluoro-1-isobutyl-2-[5-(2-phosphono)furanyl]-
benzimidazole (3.7). mp = 238-242 ꢀC. 1H NMR (DMSO-d6): δ
0.83 (d, 6H, J = 6.6 Hz), 1.9-2.1 (m, 1H), 4.6 (d, 2H, J = 6.8 Hz),
5.75 (d, 1H, J = 9.3 Hz), 6.05-7.51 (several m, 5H). Anal.
2H), 7.21 (m, 1H). Anal. (C18H24N3PO4 2H2O) C, H, N.
3
4-Amino-5-methoxy-1-isobutyl-2-[5-(2-phosphono)furanyl]benzimi-
dazole (2.3). mp = 212-213 ꢀC. 1H NMR (D2O): δ 0.75 (d, J = 7.0
Hz, 6H), 2.09 (m, 1H), 3.80 (s, 3H), 4.18 (d, J = 7.0 Hz, 2H),
(C17H19N3O4FP 1.2H2O) C, H, N.
3
4-Amino-7-cyclopropyl-5-fluoro-1-isobutyl-2-[5-(2-phosphono)-
1
furanyl]-benzimidazole (3.8). mp 250-255 ꢀC (dec). H NMR
6.70-7.08 (m, 4H). Anal. (C16H20N3O5P H2O) C, H, N.
3
(DMSO-d6): δ 0.6-0.8 (m, 2H, buried under the d), 0.7 (d, 6H,
J = 6.7 Hz), 0.85-1.0 (m, 2H), 1.8-2.0 (m, 1H), 2.05-2.3 (m,
1H), 4.67 (d, 2H, J = 7.5 Hz), 6.74 (d, 1H, J = 12.7 Hz), 7.02 (m,
4-Amino-5-chloro-1-isobutyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (2.5). mp >240 ꢀC. 1H NMR (D2O): δ 0.71 (d, J = 7.2 Hz,
6H), 2.01 (m, 1H), 4.08 (d, J = 7.2 Hz, 2H), 6.71-7.10 (m, 4H).
1H), 7.13 (m, 1H). Anal. (C18H21N3O4FP 0.25 H2O) C, H, N.
Anal. (C15Hl7ClN3O4P 0.8H2O) C, H, N.
3
3
4-Amino-7-phenyl-5-fluoro-1-isobutyl-2-[5-(2-phosphono)furanyl]-
benzimidazole (4.1). mp 240-241 ꢀC (dec). 1H NMR (CD3OD þ 1
drop of NaOD): δ 0.3 (d, 6H, J = 6.6 Hz), 1.45 (m, 1H), 4.1 (d, 1H,
J = 6.8 Hz), 6.9 (m, 2H), 7.08 (m, 1H), 7.5-7.6 (m, 5H). Anal.
4-Amino-7-chloro-1-isobutyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (2.6). mp >239 ꢀC. 1H NMR (D2O): δ 0.72 (d, J = 7.0 Hz,
6H), 2.03 (m, 1H), 4.55 (d, J = 7.0 Hz, 2H), 6.51-7.08 (m, 4H).
Anal. (C15H17N3O4ClP 1HBr 1CH2Cl2) C, H, N.
3
3
(C21H21N3O4FP 0.05H2O) C, H, N.
4-Amino-5-bromo-1-isobutyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (2.7). mp >239 ꢀC. 1H NMR (D2O): δ 0.74 (d, J = 7.1 Hz,
6H), 2.05 (m, 1H), 4.13 (d, J = 7.1 Hz, 2H), 6.71-7.29 (m, 4H).
3
4-Amino-7-(p-fluorophenyl)-5-fluoro-1-isobutyl-2-[5-(2-phos-
1
phono)furanyl]-benzimidazole (4.2). mp 239-240 ꢀC (dec). H
NMR (CD3OD þ1 drop of NaOD): δ 0.32 (d, 6H, J = 6.6 Hz),
1.47 (m, 1H), 4.13 (d, 2H, J = 6.7 Hz), 6.8-7.0 (m, 2H), 7.05 (m,
1H), 7.1-7.3 (m, 1H), 7.3-7.6 (m, 3H). Anal. (C21H20-
N3O4F2P) C, H, N.
Anal. (C15H17N3O4BrP 0.5H2O) C, H, N.
3
4-Amino-7-bromo-1-isobutyl-2-[2-(5-phosphono)furanyl]benzimi-
dazole (2.8). mp >230 ꢀC. 1H NMR (D2O): δ 0.75 (d, J = 7.3 Hz,
6H), 2.05 (m, 1H), 4.15 (d, J = 7.3 Hz, 2H), 6.71-7.31 (m, 4H).
4-Amino-7-(p-chlorophenyl)-5-fluoro-1-isobutyl-2-[5-(2-phos-
Anal. (C15H17N3O4BrP 0.25H2O) C, H, N.
3
1
phono)furanyl]-benzimidazole (4.3). mp 235-236 ꢀC (dec). H
4-Amino-5-fluoro-1-isobutyl-2-[5-(2-phosphono)furanyl]benzimi-
dazole (2.9). mp = 230-235 ꢀC. 1H NMR (D2O): δ 0.84 (d, J =
6.6 Hz, 6H), 2.23 (m, 1H), 4.28 (d, J = 7.4 Hz, 2H), 6.77-7.16 (m,
NMR (DMSO-d6): δ 0.28 (d, 6H, J = 6.6 Hz), 1.3 (m, 1H), 3.97
(d, 2H, J = 6.8 Hz), 6.9-7.6 (series of m, 7H). Anal.
(C21H20N3O4FClP) C, H, N.
4H). Anal. (C15H17N3O4PF 0.8H2O) C, H, N.
3
4-Amino-5-fluoro-7-(n-propyl)-1-isobutyl-2-[5-(2-phosphono)-
furanyl]benzimidazole (4.5). mp 220-230 ꢀC (dec). 1H NMR
(DMSO-d6): δ 0.68 (d, 6H, J = 6.6 Hz), 0.92 (t, 3H, J = 7.0 Hz),
1.4-1.7 (m, 2H), 1.8 (m, 1H), 2.7 (t, 2H, J = 6.6 Hz), 4.33 (d, 2H,
J = 7.8 Hz), 6.8 (d, 1H, J = 12.8 Hz), 7.0 (m, 1H), 7.11 (m, 1H).
4-Amino-5-fluoro-1-(2-ethylbutyl)-2-[5-(2-phosphono)furanyl]-
benzimisdazole (2.10). mp = 178-182 ꢀC (dec). 1H NMR (DMSO-
d6): δ 0.74 (t, 6H, J = 7.0 Hz), 0.8-1.4 (m, 4H), 1.4-1.8 (m, 1H),
4.33 (d, 2H, J = 7.4 Hz), 6.85 (dd, 1H, J1 = 8.8, J2 = 3.5 Hz),
6.9-7.3 (series of m, 3H). Anal. (C17H21N3O4FP 1.0H2O) C, H, N.
3
Anal. (C19H23N3O4FP 0.85H2O) C, H, N.
4-Amino-5-fluoro-1-(3-pentyl)-2-[5-(2-phosphono)furanyl]benz-
imidazole (2.11). mp = 180-185 ꢀC (dec). 1H NMR (DMSO-d6):
δ 0.6.3 (t, 6H, J = 7.0 Hz), 1.6-2.3 (series of m, 4H), 4.5 (m, 1H),
6.83 (dd, 1H, J1 = 8.7, J2 = 3.6 Hz), 6.9 (dd, 1H, J1 = 8.7, J2 =
3
4-Amino-7-(3,3-dimethyl-n-butyl)-5-fluoro-1-isobutyl-2-[5-(2-
phosphono)furanyl]-benzimidazole (4.6). mp 200-205 ꢀC (dec).
1H NMR (DMSO-d6): δ 0.69 (d, 6H, J = 6.6 Hz), 0.97 (s, 9H),
1.4 (m, 2H), 1.9 (m, 1H), 2.7 (m, 2H), 4.36 (d, 2H, J = 6.8 Hz),
8.7 Hz), 7.0 (m, 2H). Anal. (C16H19N3O4FP 1.5H2O) C, H, N.
3

450 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Dang et al.
6.8 (d, 1H, J = 13.4 Hz), 7.02 (m, 1H), 7.12 (m, 1H). Anal.
(C21H29FN3O4P 0.75H2O) C, H, N.
4-Amino-7-(4-methylpentyl)-5-fluoro-1-isobutyl-2-[5-(2-phos-
phono)furanyl]-benzimidazole (4.7). mp 185-195 ꢀC (dec). H
NMR (DMSO-d6): δ 0.68 (d, 6H, J = 6.6 Hz), 0.85 (d, 6H, J =
6.6 Hz), 1.0-2.0 (series of m, 6H), 2.72 (m, 2H), 4.34 (d, 2H, J =
7.2 Hz), 6.8 (d, 1H, J = 12.4 Hz), 7.02 (m, 1H), 7.12 (m, 1H).
4H, J = 7.0 Hz), 4.45 (d, 1H, J = 7.8 Hz), 5.55 (bs, 2H), 5.77 (s,
2H), 5.84 (s, 2H), 6.82-7.35 (m, 3H). Anal. (C25H33N3O10FP)
C, H, N.
3
1
General Procedure for the Synthesis of Phosphonate Prodrugs
via Phosphonic Dichloride Coupling Reactions. 4-Amino-5-
fluoro-7-ethyl-1-isobutyl-2-{5-[2-bis(5-methyl-2-thione-1,3-dioxo-
len-4-yl)methylphosphono]furanyl}benzimidazole (5.6). A suspen-
sion of 4.4 (200 mg, 0.52 mmol) in thionyl chloride (1 mL) was
heated to reflux for 1 h. The cooled reaction solution was
evaporated to dryness. The residue was dissolved in anhydrous
chloroform (1.5 mL), cooled to 0 ꢀC, and treated with a solution
of 4-hydroxymethyl-5-methyl-[1,3]dioxole-2-thione25 (306 mg,
2.08 mmol) and pyridine (0.17 mL, 2.08 mmol) in chloroform
(1.5 mL) under nitrogen. The resulting reaction solution was
stirred at room temperature for 16 h. The reaction solution
was evaporated to dryness, and the residue was purified by
flash chromatography (SiO2, 2 cm ꢀ 15 cm, 50, 70, 90%
EtOAc-hexane, gradient elution) to give 5.6 as a yellow solid
(30 mg, 9%). 1H NMR (DMSO-d6): δ 0.82 (d, 6H, J = 6.6 Hz),
1.31 (t, J = 7.0 Hz, 3H), 1.99 (m, 1H), 2.23 (s, 6H), 2.89 (q, 2H,
J = 7.0 Hz), 4.21 (bs, 2H), 4.41 (q, 2H, J = 7.0 Hz), 4.97 (m, 4H),
Anal. (C21H29N3O4FP 0.25H2O) C, H, N.
3
4-Amino-7-(3-hydroxypropyl)-5-fluoro-1-isobutyl-2-[5-(2-phos-
1
phono)furanyl]-benzimidazole (4.8). mp 170-173 ꢀC. H NMR
(DMSO-d6): δ 0.68 (d, 6H, J = 6.6 Hz), 1.7-2.0 (m, 3H), 2.83 (t,
2H, J = 7.4 Hz), 3.45 (t, 2H, J = 6.0 Hz), 4.39 (d, 2H, J = 7.8
Hz), 6.8 (d, 1H, J = 12.8 Hz), 7.0 (m, 1H), 7.12 (m, 1H). Anal.
(C19H23N3O5FP 1H2O) C, H, N.
4-Amino-5-fluoro-7-[3-(N,N-dimethylamino)propyl]-1-isobu-
tyl-2-[5-(2-phosphono)-furanyl]benzimidazole (4.9). mp 208-
3
1
212 ꢀC (dec). H NMR (DMSO-d6): δ 0.71 (d, 6H, J = 6.6
Hz), 1.6-2.0 (m, 3H), 2.7 (br s, 6H), 2.8 (m, 2H), 3.0-3.2 (m,
2H), 4.34 (d, 2H, J = 7.8 Hz), 6.85 (d, 1H, J = 12.8 Hz), 7.05 (m,
1H), 7.1 (m, 1H). Anal. (C20H28N4O4FP 1HBr 2H2O) C, H, N.
3
3
4-Amino-5-fluoro-7-(4-chlorobutyl)-1-isobutyl-2-[5-(2-phosphono)-
furanyl]-benzimidazole (4.10). mp 210-220 ꢀC (dec). 1H NMR
(DMSO-d6): δ 0.69 (d, 6H, J = 6.6 Hz), 1.6-2.0 (m, 5H), 2.8 (m,
2H), 3.67 (t, 2H, J = 5.6 Hz), 4.34 (d, 2H, J = 7.8 Hz), 6.83 (d, 1H,
J = 12.8 Hz), 7.0 (m, 1H), 7.12 (m, 1H). Anal. (C19H24N3-
6.81-7.38 (m, 3H). Anal. (C27H29N3O8PS2F 0.25H2O) C, H, N.
3
4-Amino-5-fluoro-7-ethyl-1-isobutyl-2-{5-[2-bis(acetylthio-
ethyl)phosphono]-furanyl}benzimidazole (5.4). This compound
was prepared in a similar manner as compound 5.6 from
1
O4FClP 0.25H2O) C, H, N.
compound 4.4 and 2-acetylthioethanol. H NMR (CDCl3): δ
3
Method b. Boron Tribromide Mediated Hydrolysis. 4-Amino-
5-hydroxy-1-isobutyl-2-[5-(2-phosphono)furanyl]benzimidazole
(2.4). A solution of 11a (200 mg, 0.48 mmol) in anhydrous
dichloromethane (5 mL) was treated with a solution of boron
tribromide (1 M, 4.8 mL) in dichloromethane at -78 ꢀC. The
resulting reaction mixture was allowed to warm to room tem-
perature. After 16 h, the reaction solution was evaporated to
dryness, and the residue was treated with water (3 mL). The
precipitate was collected via filtration (washed with water and
MeOH) and dried under vacuum at 50 ꢀC to give compound 2.4
(20 mg, 12%); mp = 206-209 ꢀC. 1H NMR (D2O): δ 0.83 (d,
J = 7.0 Hz, 6H), 2.08 (m, 1H), 4.26 (d, J = 7.0 Hz, 2H),
0.84 (d, 6H, J = 6.6 Hz), 1.32 (t, J = 7.2 Hz, 3H), 2.01 (m, 1H),
2.34 (s, 6H), 2.91 (q, 2H, J = 7.0 Hz), 3.21 (t, J = 6.6 Hz, 4H),
4.22 (m, 4H), 4.49 (d, J = 7.0 Hz, 2H), 5.31 (bs, 2H), 6.85 (m,
1H), 6.91 (m, 1H), 7.32 (m, 1H). Anal. (C25H33N3-
O6PFS2 0.2CH2Cl2 0.1PhMe) C, H, N.
3
3
4-Amino-7-ethyl-5-fluoro-1-isobutyl-2-{2-[5-bis(4-trimethyl-
acetoxybenzyl)-phosphono]furanyl}benzimidazole (5.5). This
compound was prepared in a similar manner as compound 5.6
from compound 4.4 and 4-trimethylacetoxybenzyl alcohol.26 1
H
NMR (CDCl3): δ 0.68 (d, 6H, J = 6.6 Hz), 1.31 (m, 21H), 1.91
(m, 1H), 2.81 (q, 2H, J = 7.0 Hz), 4.39 (d, J = 7.0 Hz, 2H), 5.11
(m, 4H), 6.80-7.38 (m, 13H). Anal. (C41H49FN3O8P 1.2H2O)
3
6.67-7.25 (m, 4H). Anal. (C15H18N3O5P 2.7H2O) C, H, N.
C, H, N.
3
General Procedure for the Synthesis of Phosphonate Prodrugs
via Alkylation Reactions. 4-Amino-5-fluoro-7-ethyl-1-isobutyl-
2-[5-(2-bispivaloyloxymethyl-phosphono)furanyl]benzimidazole
(5.1). A suspension of 4.4 (400 mg, 1.05 mmol) in anhydrous
acetonitrile (5 mL) was treated with Hunig’s base (1.1 mL, 6.3
mmol) and iodomethyl pivolate (2.54 g, 10.5 mmol) at 0 ꢀC
under nitrogen. The resulting reaction solution was stirred at
0 ꢀC for 48 h. The reaction solution was evaporated, and the
resulting syrup was purified by flash chromatography (SiO2,
3 cm ꢀ 15 cm, 50, 70, 90% EtOAc-hexane, gradient elution) to
give 5.1 as a yellow solid (240 mg, 37%). 1H NMR (CDCl3): δ
0.80 (d, 6H, J = 6.6 Hz), 0.93-1.26 (m, 21H), 1.9-2.1 (m, 1H),
2.84 (q, 2H, J = 7.0 Hz), 4.43 (d, 2H, J = 7.0 Hz), 5.51 (bs, 2H),
5.63-5.85 (m, 4H), 6.72-7.38 (m, 3H). Anal. (C29H41N3O8PF)
C, H, N.
4-Amino-7-ethyl-5-fluoro-1-isobutyl-2-[5-({N,N0-(1-(S)-ethoxy-
carbonyl)ethyl}-phosphonodiamido)furanyl]benzimidazole (5.7).
This compound was prepared in a similar manner as compound
5.6 from compound 4.4 (500 mg, 1.3 mmol) and L-alanine ethyl
ester to give 5.7 as an orange oil (410 mg, 54%).26 1H NMR
(DMSO-d6): δ 0.70 (d, 6H, J = 6.6 Hz), 1.10-1.32 (m, 15H), 1.81
(m, 1H), 2.81 (q, 2H, J = 7.0 Hz), 3.81-4.04 (m, 6H), 4.39 (d, J =
7.0 Hz, 2H), 5.04 (bs, 2H), 5.10-5.31 (m, 2H), 6.89-7.22 (m, 3H).
Anal. (C27H39FN5O6P) C, H, N.
4-Amino-7-ethyl-5-fluoro-1-isobutyl-2-{2-[5-(O-ethoxycarbo-
nylmethyl-N-(1-methyl-1-ethoxycarbonyl)ethyl)phosphonamido]-
furanyl}benzimidazole (5.8). This compound was prepared in a
similar manner as compound 5.6 from compound 4.4 (300 mg,
0.79 mmol) by first reacting the phosphonic dichloride with ethyl
glycolate (1.0 equiv) and thenwithL-alanineethylester (2.0 equiv)
to give 5.8 as a yellow oil (70 mg, 15%). 1H NMR (CDCl3): δ 0.80
(d, 6H, J = 6.6 Hz), 1.22-1.61 (m, 12H), 1.98 (m, 1H), 2.88 (q,
2H, J = 7.0 Hz), 4.12-4.91 (m, 12H), 6.83 (d, J = 12.8 Hz, 1H),
4-Amino-5-fluoro-7-ethyl-1-isobutyl-2-[5-(2-bispivaloyloxy-
methylphosphono)-furanyl]benzimidazole Hydrogenchloride Salt
(5.2). A solution of 5.1 (50 mg, 1.05 mmol) in anhydrous benzene
(5 mL) was treated with a solution of hydrogenchloride in
dioxane (1 N, 0.16 mL) at room temperature. After 10 min,
the reaction solution was evaporated and the resulting syrup was
crystallized from hexane to give 5.2 as a yellow solid (30 mg,
5.31 (bs, 2H), 7.26 (m, 2H). Anal. (C27H38FN4O7P 0.1CH2Cl2)
3
C, H, N.
Biology Methods. FBPase enzyme assay, cellular glucose
production in rat hepatocyte assay, and glucose lowering
in Sprague-Dawley rats assay were carried out as reported
before.3,11,12
Glucose Lowering in Zucker Diabetic Fatty Rats. ZDF rats
were purchased from the Genetic Models Division of Charles-
Rivers (Indianapolis, IN) at the age of 10 weeks and allowed to
acclimate for 2 weeks prior to evaluation. Animals (n = 6) were
housed two per cage under a 12 h lighting cycle (7 a.m.-7 p.m.
light) and controlled temperature (22 ꢀC). They were fed Purina
5008 rat chow and drinking water ad libitum unless otherwise
1
57%); mp 100-104 ꢀC. H NMR (CDCl3): δ 0.71-1.32 (m,
24H), 1.9-2.1 (m, 1H), 2.94 (m, 2H), 3.53 (bs, 2H), 4.47 (m, 2H),
5.72-5.82 (m, 4H), 6.86-7.3 (m, 3H). Anal. (C29H42N3O8PFCl)
C, H, N, F, Cl, P.
4-Amino-5-fluoro-7-ethyl-1-isobutyl-2-{5-[2-(bis(ethyloxy-
carbonyloxymethyl)-phosphono]furanyl}benzimidazole (5.3).
This compound was prepared in a similar manner as 5.1 from
1
compound 4.4. H NMR (DMSO-d6): δ 0.81 (d, 6H, J = 6.6
Hz), 1.29 (m, 9H), 1.99 (m, 1H), 2.89 (q, 2H, J = 7.0 Hz), 4.21 (q,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 451
noted. On the morning of the experiment (7 a.m.), catheters were
inserted into the tail artery and vein for blood sampling and drug
administration, respectively. Rats were housed individually in
modified cages, which restricted access to the tail catheter sites,
and food was withheld for the remainder of the experiment. At
11 a.m., baseline blood glucose was measured from blood
obtained from the tail artery and an infusion of 4.4 at 10 mg/
kg/h or saline was initiated. Blood glucose was measured hourly.
4.4 was dissolved at a final concentration of 10 mg/mL in water
pH adjusted to 7.4 with 5 N NaOH.
(11) Dang, Q.; Kasibhatla, S. R.; Jiang, T.; Fan, K.; Liu, Y.; Taplin, F.;
Schulz, W.; Cashion, D. K.; Reddy, K. R.; van Poelje, P. D.;
Fujitaki, J. M.; Potter, S. C.; Erion, M. D. Discovery of phosphonic
diamide prodrugs and their use for the oral delivery of a series of
fructose 1,6-bisphosphatase inhibitors. J. Med. Chem. 2008, 51,
4331–4339.
(12) Dang, Q.; Kasibhatla, S. R.; Reddy, K. R.; Jiang, T.; Reddy, M. R.;
Potter, S. C.; Fujitaki, J. M.; van Poelje, P. D.; Huang, J.;
Lipscomb, W. N.; Erion, M. D. Discovery of potent and
specific fructose-1,6-bisphosphatase inhibitors and a series of
orally bioavailable phosphoramidase-sensitive prodrugs for the
treatment of type 2 diabetes. J. Am. Chem. Soc. 2007, 129,
15491–15502.
Acknowledgment. We thank Tao Jiang, Kevin Fan, and
Tom Hill for their contributions.
(13) van Poelje, P. D.; Potter, S. C.; Chandramouli, V. C.; Landau, B.
R.; Dang, Q.; Erion, M. D. Inhibition of fructose 1,6-bisphos-
phatase reduces excessive endogenous glucose production and
attenuates hyperglycemia in zucker diabetic Fatty rats. Diabetes
2006, 55, 1747–1754.
(14) Yoshida, T.; Okuno, A.; Izumi, M.; Takahashi, K.; Hagisawa, Y.;
Ohsumi, J.; Fujiwara, T. CS-917, a fructose 1,6-bisphosphatase
inhibitor, improves postprandial hyperglycemia after meal loading
in non-obese type 2 diabetic Goto-Kakizaki rats. Eur. J. Pharma-
col. 2008, 601, 192–197.
Supporting Information Available: Elemental analysis data
for all final compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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