Discovery of a clinical candidate from the structurally unique dioxa-bicyclo[3.2.1]octane Class of sodium-dependent glucose cotransporter 2 inhibitors

Article abstract of DOI:10.1021/jm200049r

Compound 4 (PF-04971729) belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 inhibitors incorporating a unique dioxa-bicyclo[3.2.1]octane (bridged ketal) ring system. In this paper we present the design, synthesis, preclinical evaluation, and human dose predictions related to 4. This compound demonstrated robust urinary glucose excretion in rats and an excellent preclinical safety profile. It is currently in phase 2 clinical trials and is being evaluated for the treatment of type 2 diabetes.

Full text of DOI:10.1021/jm200049r

ARTICLE
pubs.acs.org/jmc
Discovery of a Clinical Candidate from the Structurally Unique
Dioxa-bicyclo[3.2.1]octane Class of Sodium-Dependent Glucose
Cotransporter 2 Inhibitors
Vincent Mascitti,* Tristan S. Maurer, Ralph P. Robinson, Jianwei Bian, Carine M. Boustany-Kari,
Thomas Brandt, Benjamin M. Collman, Amit S. Kalgutkar, Michelle K. Klenotic, Michael T. Leininger,
Andrꢀe Lowe, Robert J. Maguire, Victoria M. Masterson, Zhuang Miao, Emi Mukaiyama, Jigna D. Patel,
John C. Pettersen, Cathy Prꢀeville, Brian Samas, Li She, Zhanna Sobol, Claire M. Steppan,
Benjamin D. Stevens, Benjamin A. Thuma, Meera Tugnait, Dongxiang Zeng, and Tong Zhu
Groton Laboratories, Pfizer Global Research & Development, Eastern Point Road, Groton, Connecticut 06340, United States
S Supporting Information
b
ABSTRACT: Compound 4 (PF-04971729) belongs to a new
class of potent and selective sodium-dependent glucose cotran-
sporter 2 inhibitors incorporating a unique dioxa-bicyclo-
[3.2.1]octane (bridged ketal) ring system. In this paper we
present the design, synthesis, preclinical evaluation, and human
dose predictions related to 4. This compound demonstrated
robust urinary glucose excretion in rats and an excellent
preclinical safety profile. It is currently in phase 2 clinical trials and is being evaluated for the treatment of type 2 diabetes.
’ INTRODUCTION
usually associated with degradation by glucosidase enzymes
found in the gut and required to be administered as prodrugs.13
Clinical trials involving compounds from this class were aban-
doned. The susceptibility to glucosidase degradation was ele-
gantly addressed via the discovery of potent and selective C-aryl
glucoside SGLT2 inhibitors such as dapagliflozin (3; Figure 1).14
A number of compounds derived from the C-aryl glucoside class
have been identified and are now at various stages of clinical
development.15ꢀ22
Thus, to date, the SGLT2 inhibitor structural landscape has
been dominated by molecules belonging to either one of two
classes: O-aryl and C-aryl glucosides.15 In both cases, the majority
of effort has been spent on modifying the aglycon side chain due
to the relative ease of synthesis. However, changes to the aglycon
motif are often associated with increased lipophilicity and
molecular weight (with resultant implications in terms of phar-
macokinetics and/or safety), as well as introduction of structural
motifs associated with potential reactive metabolite formation.23
Additionally, some compounds from the C-aryl glucoside class
have tested micronucleus positive in vitro.24
The alarming and increasing prevalence of type 2 diabetes
along with the side effects associated with many current anti-
diabetic drugs (most notably hypoglycemia and weight gain) has
stimulated an intense effort to evaluate new mechanisms to
achieve glycemic control.1,2 Sodium-dependent glucose cotran-
sporter 2 (SGLT2) is a high-capacity, low-affinity transporter
expressed selectively in the S1 domain of the proximal tubule in
the kidney and is responsible for the reuptake of ∼90% of filtered
glucose.3,4 Sodium-dependent glucose cotransporter 1 (SGLT1),
on the other hand, is a low-capacity, high-affinity transporter that is
found in the kidney, the gut, and other tissues. SGLT1 plays a large
role in carbohydrate uptake from the diet.4,5 Inhibition of SGLT2
induces urinary glucose excretion (UGE) and thereby reduces
plasma glucose concentrations.6,7 This mechanism is the first to
address the phenomenon of the increased glucose reabsorption
observed in type 2 diabetic patients, which is a key component of
the “ominous octet” that characterizes hyperglycemia.8 Further-
more, since selective SGLT2 inhibition is a glucose-dependent
(thereby minimizing the risk of hypoglycemia) and insulin-inde-
pendent (thus potentially favoring pancreatic β-cell preservation)
mechanism that is associated with weight loss, it has emerged as a
very promising approach to the pathophysiologic treatment of type
2 diabetes.6,7,9,10
Herein we report the discovery of 4 (PF-04971729), a
compound from a new class of potent and selective SGLT2
inhibitors incorporating a structurally novel dioxa-bicyclo-
[3.2.1]octane ring system (Figure 2).25,26
Optimization of the natural product phlorizin (1; Figure 1), a
nonselective SGLT2 and SGLT1 inhibitor, led to the identifica-
tion of several selective O-aryl glucoside SGLT2 inhibitors;11,12
however, such inhibitors (e.g., sergliflozin, 2; Figure 1) were
Received: January 17, 2011
Published: March 30, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Advanced Intermediate 5a
Figure 1. From natural product to potent SGLT2 inhibitors.
a Reagents and conditions: (a) Swern oxidation; (b) NaOH, formalde-
hyde, i-PrOH/water, 23 °C (53%, two steps); (c) PMBBr, NaH, DMF,
0ꢀ60 °C (52%); (d) PdCl2, MeOH/CH2Cl2, 23 °C (78%); (e) Swern
oxidation(72%); (f) AlMe3, MeN(OMe)H2Cl, CH2Cl2, 0ꢀ23 °C (58%).
Scheme 2. Three-Step Analoguing Sequencea
Figure 2. Dioxa-bicyclo[3.2.1]octane SGLT2 inhibitors.
’ CHEMISTRY
Compounds from this class were prepared in an analogue-
friendly fashion starting from advanced intermediate Weinreb
amide 5.25 Compound 5 was synthesized on multigram scale
starting from D-glucose as shown in Scheme 1. Intermediate 627
was oxidized under Swern conditions to the corresponding
aldehyde, which, in a one-pot aldolꢀCannizzaro28 sequence,
produced intermediate 7 (53% yield over two steps). This two-step
protocol allowed for the installation of the tetrasubstituted carbon
(C-1) present in the final analogues. Protection of the primary
hydroxyl groups in 7 as p-methoxybenzyl (PMB) ethers followed
by allyl group removal gave lactol intermediate 8 (40% yield over
two steps). Oxidation of the lactol to the corresponding lactone 9
and subsequent treatment with N,O-dimethylhydroxylamine hydro-
chloride in the presence of trimethylaluminum provided advanced
intermediate 5 (42% yield over two steps).29
Intermediate 5, which presents the required carbon frame-
work in the proper oxidation state, was primed for the three-step
analoguing sequence as shown in Scheme 2. Nucleophilic addi-
tion of the appropriate organolithium reagent to 5 in THF
produced the corresponding cyclic lactol 10. Acid-promoted
one-pot PMB removal followed by stereoselective intramolecular
trapping of the putative oxonium ion intermediate gave com-
pound 11 containing the desired dioxa-bicyclo[3.2.1]octane ring
system.30 Finally, hydrogenolysis of the benzyl protecting groups
under transfer hydrogenation conditions yielded analogues 12
a Reagents and conditions: (a) ArLi, THF, ꢀ78 to 0ꢀ23 °C; (b) TFA,
anisole, CH2Cl2, 23 °C; (c) Pd black, HCO2H, EtOH/THF, 23 °C, then
HPLC separation.
and 13 as a mixture of diastereomers at C-4, which were
separated by HPLC. The results of single-crystal X-ray diffraction
analysis of representative derivatives 14 and 15 are shown in
Figures 3 and 4, respectively. The epimerization at C-4 may be
rationalized via the formation of a putative enol or enol ether
intermediate in the analoguing sequence. Although nonstereoselec-
tive, the three-step sequence allowed the rapid exploration of
structureꢀactivity relationships. Recently, we reported a stereoselec-
tive synthesis providing efficient access to compounds in the class.31
’ RESULTS AND DISCUSSION
During the course of our program, we developed a pharma-
cokineticꢀpharmacodynamic (PKPD) relationship model to
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Figure 5. Some properties of SGLT2 inhibitor 16.
Figure 3. ORTEP representation of the X-ray crystal structure of 14.
Figure 6. Relationship between predicted dose and human half-life for
compound 16. The open symbol represents the predicted human dose
(to achieve 60 g of UGE/24 h in healthy volunteers) obtained using the
PKPD model assuming a human volume of 1.9 L/kg and a clearance of
4.6 mL/min/kg. The dashed line represents the predicted relationship
between dose and t1/2,human obtained via a range of hypothetical volumes
between 0.5 and 9.0 L/kg. The solid line represents the predicted
relationship between dose and t1/2,human obtained via a range of
hypothetical clearances between 1.0 and 10 mL/min/kg.
Figure 4. ORTEP representation of the X-ray crystal structure of 15.
help predict a human dosing regimen that would induce near-
maximal theoretical UGE at steady state.32 In this model, human
PK predictions were based on allometric scaling from
rats after normalization for protein binding differences across
species.33 The PD component was likewise scaled from rats using
a differential equation model describing the rate of UGE as a
function of inhibitor concentration, inhibitor potency, plasma
glucose concentration, glomerular filtration rate, and glucose
reuptake.34
Previously, we described the synthesis and preclinical evalua-
tion of a series of C-5-spirocyclic C-glycoside SGLT2 inhi-
bitors.35 Unfortunately, in spite of relatively good potency and
selectivity for human SGLT2 [IC50(h-SGLT2) = 6.98 (
2.50 nM; n = 8],36 the lead compound 16 suffered from
suboptimal PK (Figure 5).37 Using rat to human allometry, the
predicted total plasma clearance (CLplasma) and steady-state
volume of distribution (Vss) in humans were respectively
4.6 mL/min/kg and 1.9 L/kg. This translated into a short
predicted human half-life (t1/2,human < 5.0 h) and a predicted
daily efficacious dose of >100 mg to produce near-maximal UGE
(60 g/24 h) in healthy volunteers. Furthermore, as we already
reported, simulations with the PKPD model also indicated
Given the goal to produce near-maximal UGE in the clinic
over 24 h at a daily dose of less than 100 mg, analogues with a
longer predicted t1/2,human were desired. Additional PKPD
simulations, varying respectively the distribution volume and
clearance, were performed to determine the most promising
approach for t1/2 and dose optimization (Figure 6). These simula-
tions indicated that targeting improvements in predicted clearance
was the most viable approach.38
Thus, decreasing the projected efficacious dose required
increasing t1/2,human via lowering of the predicted clearance in
humans. Studies analyzing the elimination mechanism(s) of 16
in rats revealed that CLplasma was largely mediated via hepatic
metabolism (CLhepatic), the renal clearance (CLrenal) being
generally low and passive (Figure 5).39 In vitro stability studies
on 16 in human hepatic tissue revealed a higher apparent intrinsic
clearance (CLint app)
40,41 in HHEP relative to HLM, suggestive of
non-cytochrome P450-mediated metabolic elimination. Indeed,
qualitative in vitro metabolite identification in HHEP confirmed
the formation of a single glucuronide conjugate of 16 (the
regioselectivity was not investigated).
that the predicted dose increases exponentially in relation to
37
decreasing t1/2,human
.
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Interestingly, replacement of the oxetane ring in 16 by an
azetidine, as in 17, produced a compound of similar lipophilicity
but with markedly reduced CLint app in HHEP (Figure 7). Mind-
ful of this observation, we proposed that the increased human
phase 2 metabolism of 16 was due to a lack of an H-bond donor
or polar group at the C-5 position. This hypothesis appeared to
be corroborated by the fact that C-aryl glucosides of similar
lipophilicities, such as 18 and 19, have a reduced CLint app
(∼2.0ꢀ4.0 μL/min/million cells) in HHEP. Furthermore,
although relatively good potency was observed for 16, it
appeared suboptimal when compared to that of 18 (IC50-
(h-SGLT2) = 2.39 ( 1.19 nM; n = 6).
All these observations led us to deprioritize the C-5-spirocyclic
C-glycoside SGLT2 inhibitors to focus our efforts on the dioxa-
bicyclo[3.2.1]octane class. It was hypothesized that the bridged
ketal system (Figure 2) would confer rigidity that could poten-
tially positively impact potency and selectivity. Moreover, it
would also allow introduction of a hydroxymethylene H-bond
donor group (in place of the spirocycle), which was anticipated
to help reduce the rate of human phase 2 metabolism (relative to
that of 16) and further improve potency.
A representative set of analogues is listed in Table 1 accom-
panied by in vitro data. Compounds from the class proved to be
potent and very selective SGLT2 inhibitors.42 The configuration
of the stereocenter at C-4 turned out to have a profound effect on
potency (∼50ꢀ100-fold) with a marked preference for the R
configuration (compare compounds 22 and 23 and compounds
4 and 24). Interestingly, compound 20, which bears the same
diarylmethane group as 16 and has lipophilicity similar to that
of 16 (as measured by ELogD),43 demonstrated comparable
CLint app in HLM but exhibited a marked decrease (>10-fold) in
CLint app measured in HHEP. The reduction in HHEP CLint app
to levels similar to those found in compounds 17, 18, and 19 was
also observed for other compounds from the class (Table 1).
Upon further profiling and in vivo PK experiments in
SpragueꢀDawley rats, compound 4 emerged as being very
promising (Table 2). It is interesting to note that, despite a
low predicted clearance in HHEP, compounds 20, 21, and 22
displayed high clearance in the rat. It is possible that subtle
species differences may exist between rats and humans with
regard to oxidative and conjugative metabolism, resulting in a
higher CLplasma in the rat in these instances. However, whereas
compounds 20ꢀ22 showed CLplasma similar to that of 16,
compound 4 showed a major improvement. In particular, a
marked decrease in CLplasma (∼5-fold) and an increase in t1/2
(∼2-fold) were observed. Although low, CLrenal [1.11 mL/min/
kg > (fuplasma) (GFR)] was indicative of some active renal
3
clearance. This was corroborated in vitro by the very weak uptake
of 4 by rOct2 and rOat3 transporters.44 On the other hand, there
was no uptake by hOAT1, hOAT3, or hOCT2 transporters.
Figure 7. In vitro CLint app considerations. CLint app(HHEP) values are
expressed per million cells.
Table 1. Representative Set of in Vitro Data
IC50 ( SDc,d (nM)
CLint app
b
e
fuplasma
compd R1
R2
C-4a ELogD human
rat
h-SGLT2
h-SGLTl
HLM [μL/min/mg] HHEP [μL/min/million]
20
21
22
23
4
Me OMe
Me OEt
R
R
R
S
2.6
3.0
3.2
3.9
3.6
3.8
0.206 0.186 1.07 ( 0.423 (n = 6)
1.11 ( 0.359 (n = 6)
506 ( 298 (n = 6)
958 ( 395 (n = 5)
546 ( 136 (n = 7)
>10000(n = 3)
<8.0
8.9
9.2
15
<2.1
<2.0
<2.0
2.3
Cl
Cl
Cl
Cl
OMe
OMe
OEt
0.882 ( 0.366 (n = 7)
43.1 ( 14.2 (n = 4)
0.064 0.040 0.927 ( 0.369 (n = 10)f 2050 ( 642 (n = 8)g
R
S
12
4.5
24
OEt
89.9 ( 28.2 (n = 3)
>10000 (n = 2)
a Configuration of the stereocenter at C-4. b Fraction unbound in plasma at 1000 ng/mL as measured by the equilibrium dialysis method. c The potency
at human SGLT (h-SGLT), reported as the arithmetic mean, was evaluated using a functional assay designed to detect the inhibition of methyl R-D-
glucopyranoside (AMG) uptake via the SGLT transporter expressed in CHO cells. d Number of runs indicated in parentheses. Apparent intrinsic
e
clearance determined by scaling t1/2 obtained from in vitro microsome or hepatocyte incubations. f IC50(h-SGLT2) = 0.877 nM (95% CI = 0.704ꢀ
1.09 nM) as the geometric mean of 10 replicates. g IC50(h-SGLT1) = 1960 nM (95% CI = 1460ꢀ2620 nM) as the geometric mean of eight replicates.
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Table 2. Rat IV PK Parametersa
compd
CLplasma (mL/min/kg)
Vss (L/kg)
tl/2 (h)
4
20
21
22
16
4.04
37.1
27.7
23.8
22.3
1.13
1.71
1.03
1.82
2.27
4.1
0.96
0.94
1.6
1.9
a PK experiments were conducted in male SpragueꢀDawley rats (n = 2
per group) at doses of 2 mg/kg (iv). iv formulation for 4: DMSO/
PEG400/30% SBECD (10/30/60, v/v/v). iv formulation for 16, 20, 21,
and 22: DMA/PG/50 mM Tris base (5/10/85, v/v/v).
Figure 9. Predicted steady-state doseꢀresponse curve for 4 in healthy
volunteers.
Figure 8. Rat 24 h UGE following treatment with 4. Male Spragueꢀ
Dawley rats (n = 5 per group) were randomized to receive one of six
doses of compound 4 (0.1, 1, 3, 10, 30, and 60 mg/kg) by oral gavage.
The formulation used was 20% (v/v) PEG400/24% (v/v) HPBCD.
Following compound administration, urine was collected over 24 h for
measurement of UGE. Simultaneously, drug exposure was assessed in
satellite animals (n = 2 per group).
Following 5.0 mg/kg oral administration to rats of a crystalline
form of 4 (as an L-pyroglutamic acid cocrystal), the maximal
systemic exposure and the time to reach peak concentrations
were 1.94 ( 0.185 μg/mL and 1.0 h, respectively. The oral
bioavailability (F) of 4 was 69%. Finally, from a safety perspec-
tive, we observed that 4 tested negative in the in vitro micro-
nucleus test.45
Figure 10. ORTEP representation of the X-ray crystal structure of a
cocrystal of 4.
in vivo IC50 was predicted to be 1.4 nM. Using single species
allometry, human F was estimated to be 65% for the L-pyroglu-
tamic acid cocrystal form of 4.
The promising profile of 4 prompted evaluation of its poten-
tial to induce UGE in vivo. Compared to the vehicle, the
compound caused a dose-responsive increase in UGE in rats
for all the doses tested (Figure 8). The maximal UGE (2554 (
141.1 mg/200 g of body weight) over 24 h was reached at a single
oral dose of 30 mg/kg corresponding to a free Cav of 408 nM
On the basis of the excellent preclinical data package and the
successful completion of regulatory toxicology studies, the
compound was advanced to clinical trials as the L-pyroglutamic
acid cocrystal form (mp = 142.5 °C) (Figure 10).25 Importantly,
the PKPD model proved very reliable in predicting the pharma-
cokinetics and pharmacological effects observed in healthy
volunteers during phase 1 trials. These clinical data will be
reported in due course.
(free AUC0ꢀ24 = 4280 ng h/mL).46 The free in vivo IC50 was
3
1.8 nM (95% CI = 1.0ꢀ2.6 nM), in good agreement with the rat
SGLT2 in vitro IC50 of 1.15 nM (95% CI = 0.757ꢀ1.74 nM;
n = 4).32
Having established the robust PD effect in rats, we then used
our SGLT2 PKPD model to predict the effect of compound 4 on
UGE in humans. Using allometric scaling, the predicted human
CLplasma and Vss were 1.7 mL/min/kg and 1.8 L/kg, respectively.
In turn, clearance and volume translated into a predicted
t1/2,human of 12 h. As shown by the predicted steady-state
doseꢀresponse curve for 4 (Figure 9), the increase of the key
parameter t1/2,human translated into a low predicted ED50 of 4 mg
once a day at steady state in healthy volunteers to produce a 32.5
g UGE/24 h (Emax = 65 g UGE/day).47 The corresponding free
’ CONCLUSION
We have identified a new class of potent and selective SGLT2
inhibitors incorporating a unique dioxa-bicyclo[3.2.1]octane
motif. The medicinal chemistry strategy focused on lowering of
the human dose through increasing t1/2 (via reducing the
clearance) and improving the potency. This strategy was sup-
ported by (1) innovative chemistry that allowed difficult, yet very
desirable, targets to be synthesized in an analogue-friendly
fashion and (2) the development of a PKPD model that helped
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confidently predict the human dose and reduce the discovery
cycle time. Overall, these efforts supported the advancement of
compound 4 into clinical development for type 2 diabetes
treatment.
(placed in a predried Biotage microwave vial, 10ꢀ20 mL, sealed with its
cap and placed under a positive stream of nitrogen gas) of 4-bromo-1-
chloro-2-(4-ethoxybenzyl)benzene (815 mg, 3.25 equiv) in anhydrous
tetrahydrofuran (2.9 mL) at ꢀ78 °C, and the resulting solution was
stirred at this temperature for an additional hour. A solution of 5
(600 mg) in anhydrous tetrahydrofuran (1.45 mL) was then added
dropwise over 1.3 h using a syringe pump, and the resulting mixture was
stirred at ꢀ78 °C for 1 h before being allowed to warm to ꢀ25 °C over
14 h [put in a deep Dewar covered with aluminum foil to maintain the
cold temperature (size of the Dewar: external diameter, 10 cm; internal
diameter, 8 cm; height, 9 cm)]. Diethyl ether was added, and the reaction
was quenched by dropwise addition of aqueous 1 M hydrochloric acid
solution. The resulting biphasic mixture was stirred at room temperature
for 15 min. The organic phase was separated, washed with brine, dried
over magnesium sulfate, filtered, and concentrated. Chromatography
over silica gel using a gradient of 10ꢀ40% ethyl acetate in heptane gave
intermediate (4S,5S)-3,4,5-tris(benzyloxy)-2-[4-chloro-3-(4-ethoxybenzyl)-
phenyl]-6,6-bis[[(4-methoxybenzyl)oxy]methyl]tetrahydropyran-2-ol as a
mixture of isomers (280 mg, 38% yield). HRMS: m/z calcd for C59H61-
O10ClNa (M þ Naþ) 987.3845, found 987.3840.
’ EXPERIMENTAL SECTION
Unless specified otherwise, starting materials are generally available
from commercial sources. NMR spectra were recorded on a Varian
Unity 400 (available from Varian Inc., Palo Alto, CA) at room
temperature at 400 MHz for proton. Chemical shifts are expressed in
parts per million (δ) relative to residual solvent as an internal reference
(for methanol-d, δH = 3.29 ppm and δC = 47.8 ppm). The peak shapes
are denoted as follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; q, quartet; m, multiplet; br s, broad singlet; 2s, two singlets; br d,
broad doublet. Electrospray (ES) ionization mass spectra were obtained
on a Waters ZMD instrument (carrier gas, nitrogen; solvent A, water/
0.01% formic acid; solvent B, acetonitrile/0.005% formic acid; available
from Waters Corp., Milford, MA). High-resolution mass spectrometry
(HRMS) was performed with an Agilent model 6210 time-of-flight
instrument. Where the intensity of single chlorine ions is described, the
expected intensity ratio was observed (approximately 3:1 for 35Cl/37Cl-
containing ions) and the intensity of only the lower mass ion is given.
Column chromatography was performed with either Baker silica gel
(40 μm; J.T. Baker, Phillipsburg, NJ) or silica gel 50 (EM Sciences,
Gibbstown, NJ) in glass columns or in Flash 40 Biotage columns (ISC,
Inc., Shelton, CT). MPLC (medium-pressure liquid chromatography)
was performed using a Biotage SP purification system or a Combiflash
Companion from Teledyne Isco; Biotage SNAP cartridge KPsil or
Redisep Rf silica (from Teledyne Isco) under low nitrogen pressure
was used. HPLC was performed using a Shimadzu 10A LCꢀUV or an
Agilent 1100 preparatory HPLC instrument. The tested compounds
were determined to be >95% pure by HPLC (see experimental details
related to each tested compound for the specific conditions). Except
where otherwise noted, all reactions were run under an inert atmosphere
of nitrogen gas using anhydrous solvents. Also, except where otherwise
noted, all reactions were run at room temperature (∼23 °C).
To a solution of the above intermediate (1.46 g) in dichloromethane
(31 mL) was added anisole (900 μL, ∼5 equiv) followed by 31 mL of a
solution of 20% trifluoroacetic acid in dichloromethane, and the result-
ing mixture was stirred at room temperature for 1 h. The mixture was
concentrated, and the crude material was chromatographed over silica
gel using a gradient of 10ꢀ30% ethyl acetate in heptane to afford
intermediate {(2S,3S)-2,3,4-tris(benzyloxy)-5-[4-chloro-3-(4-ethoxybenzyl)-
phenyl]-6,8-dioxa-bicyclo[3.2.1]oct-1-yl}methanol as a mixture of isomers
(670 mg, 63% yield). HRMS: m/z calcd for C43H44O7Cl (M þ Hþ)
707.2770, found 707.2765.
To a solution of the above intermediate (335 mg) in ethanol/
tetrahydrofuran (10 mL; 4/1, v/v) were added successively formic acid
(420 μL, 22 equiv) and palladium black (208 mg, 4 equiv), and the
resulting mixture was stirred at room temperature. After 1 h, additional
formic acid (420 μL, 22 equiv) and palladium black (208 mg, 4 equiv)
were added, and the mixture was allowed to stir for an additional hour at
room temperature. The palladium was filtered, and the crude mixture
obtained after evaporation of the solvent was purified by preparative
HPLC. Preparative HPLC: reversed-phase C18 Gemini column, 5 μm,
30 ꢁ 100 mm, 40 mL/min; gradient of acetonitrile/0.1% formic acid:
water/0.1% formic acid, 25ꢀ50% acetonitrile/0.1% formic acid over 18
min. UV detection: 220 nm. HPLC indicated a ratio of diastereomers of
1.1:1 (4:24).
(2R,3S,4S)-2,3,4-Tris(benzyloxy)-5-hydroxy-6-[(4-meth-
oxybenzyl)oxy]-5-[[(4-methoxybenzyl)oxy]methyl]hexanoic
Acid Methoxymethylamide (5). To a solution of 925 (10.4 g,
14.5 mmol) and N,O-dimethylhydroxylamine hydrochloride (1.77 g,
29.0 mmol) in dichloromethane (100 mL) at 0 °C was added dropwise a
2.0 M solution of trimethylaluminum in hexanes (14.5 mL, 29.0 mmol),
and the resulting solution was stirred at room temperature for 16 h. The
reaction mixture was cooled to 0 °C and quenched by slow addition of
aqueous 1 N hydrochloric acid solution. The resulting mixture was
allowed to stir for 1 h. The organic phase was separated and washed with
aqueous 1 N hydrochloric acid solution, dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The crude material
was purified by medium-pressure chromatography (gradient of 5ꢀ40%
ethyl acetate in heptane), yielding 6.5 g (58%) of product 5. 1H NMR
(400 MHz, CDCl3): δ (ppm) 2.62 (br s, 1H), 2.94 (br s., 3H), 3.23 (br s,
3H), 3.42 (d, J = 9.4 Hz, 1H), 3.50ꢀ3.60 (m, 3H), 3.75 (s, 3H), 3.77
(s, 3H), 4.03 (d, J = 6.9 Hz, 1H), 4.20 (dd, J = 6.9, 3.3 Hz, 1H), 4.31ꢀ4.44
(m, 5H), 4.46ꢀ4.51 (m, 2H), 4.53 (d, J = 12 Hz, 1H), 4.66 (d, J = 12 Hz,
1H), 4.80 (br d, J = 11.5 Hz, 1H), 4.87 (d, J = 11.4 Hz, 1H), 6.77ꢀ6.83
(m, 4H), 7.15ꢀ7.35 (m, 19H). HRMS: m/z calcd for C46H54NO10
(M þ Hþ) 780.3742, found 780.3708.
The fractions containing the product 4 were concentrated under
reduced pressure. The crude material was precipitated from ethyl acetate
and heptane. The resulting white solid was washed with heptane two
times and dried under reduced pressure. Yield: 60 mg, 29%. tR = 12.4
min. 1H NMR (400 MHz, CD3OD): δ (ppm) 7.43 (d, 1H, J = 1.9 Hz),
7.36 (dd, 1H, J = 8.3 and 2 Hz), 7.32 (d, 1H, J = 8.3 Hz), 7.08ꢀ7.04
(m, 2H), 6.79ꢀ6.75 (m, 2H), 4.12 (d, 1H, J = 7.5 Hz), 4.00 (s, 2H), 3.96
(q, 2H, J = 7.0 Hz), 3.81 (d, 1H, J = 12.5 Hz), 3.75 (dd, 1H, J = 8.3 and
1.3 Hz), 3.65 (d, 1H, J = 12.5 Hz), 3.63 (t, 1H, J = 8.2 Hz), 3.57 (dd, 1H,
J = 7.5 and 1.3 Hz), 3.52 (d, 1H, J = 8.0 Hz), 1.33 (t, 3H, J = 6.9 Hz). 13C
NMR (100 MHz, CD3OD): δ (ppm) 14.0, 38.1, 60.7, 63.2, 66.7, 71.9,
76.6, 78.1, 85.0, 108.4, 114.3 (2C), 125.9, 128.6, 129.3, 129.6 (2C),
131.6, 133.8, 137.4, 138.5, 157.7. HRMS: m/z calcd for C22H26O7Cl
(M þ Hþ) 437.1361, found 437.1360.
The fractions containing the product 24 were concentrated under
reduced pressure. The crude material was precipitated from ethyl acetate
and heptane. The resulting white solid was washed with heptane two
times and dried under reduced pressure. Yield: 30 mg, 15%. tR = 13.2
min. 1H NMR (400 MHz, CD3OD): δ (ppm) 7.48 (d, 1H, J = 1.9 Hz),
7.40 (dd, 1H, J = 8.1 and 1.9 Hz), 7.32 (d, 1H, J = 8.3 Hz), 7.08ꢀ7.03 (m,
2H), 6.80ꢀ6.74 (m, 2H), 4.04ꢀ3.99 (m, 3H), 3.95 (q, 2H, J = 7 Hz),
(1S,2S,3S,4R,5S)-5-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-
1-(hydroxymethyl)-6,8-dioxa-bicyclo[3.2.1]octane-2,3,4-triol
(4) and (1S,2S,3S,4S,5S)-5-[4-Chloro-3-(4-ethoxybenzyl)-
phenyl]-1-(hydroxymethyl)-6,8-dioxa-bicyclo[3.2.1]octane-
2,3,4-triol (24). n-Butyllithium (1.0 mL, 2.5 M (hexanes), 3.25 equiv)
was added dropwise (1 drop every 5 s) to an oxygen-degassed solution
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Journal of Medicinal Chemistry
ARTICLE
3.89ꢀ3.81 (m, 4H), 3.73 (d, 1H, J = 12.5 Hz), 3.49 (d, 1H, J = 7.3 Hz),
1.32 (t, 3H, J = 7 Hz). HRMS: m/z calcd for C22H26O7Cl (M þ Hþ)
437.1361, found 437.1358.
times and concentrated under reduced pressure. Yield: 37 mg, 27%. tR =
12.8 min. 1H NMR (400 MHz, CD3OD): δ (ppm) 7.50 (d, 1H, J = 1.9
Hz), 7.42 (dd, 1H, J = 8.3 and 1.9 Hz), 7.35 (d, 1H, J = 8.3 Hz), 7.12ꢀ
7.07 (m, 2H), 6.83ꢀ6.78 (m, 2H), 4.06ꢀ4.01 (m, 3H), 3.91ꢀ3.83 (m,
4H), 3.78ꢀ3.72 (m, 4H), 3.51 (d, 1H, J = 7.5 Hz). MS (LCꢀMS): m/z
423.3 (M þ Hþ, positive mode) 467.3 (M þ HCO2ꢀ, negative mode).
4-Nitrobenzoylation of 4 To Give 14. To a solution of 4 (10.6
mg, 0.024 mmol) in anhydrous tetrahydrofuran (300 μL) cooled at
0 °C were added N,N-diisopropylethylamine (30 μL, 7 equiv) and
4-(dimethylamino)pyridine (3 mg, 1 equiv) followed by p-nitrobenzoyl
chloride (27 mg, 6 equiv), and the resulting mixture was stirred at 60 °C
for 6 h. The mixture was cooled to room temperature, ethyl acetate and
water were added, and the organic phase was successively washed with
0.5 M aqueous hydrochloric acid solution and brine. The organic phase
was dried over magnesium sulfate, filtered, and concentrated, and the
crude material was purified by flash chromatography over silica gel,
eluting with a gradient of 10ꢀ50% ethyl acetate in heptane, to afford
(1S,2S,3S,4R,5S)-1-(Hydroxymethyl)-5-[3-(4-methoxybenzyl)-
4-methylphenyl]-6,8-dioxa-bicyclo[3.2.1]octane-2,3,4-triol
(20)25. This compound was obtained using procedures analogous to
the preparation of compounds 4 and 24. Preparative HPLC method:
reversed-phase C18 Phenomenex column, Luna, 5 μm, 150 ꢁ
21.20 mm, 20 mL/min; gradient of acetonitrile/0.1% formic acid:
water/0.1% formic acid, 20ꢀ60% acetonitrile/0.1% formic acid over
20 min. UV detection: 254 nm. The fractions containing the product
20 were concentrated under reduced pressure. The crude material was
precipitated from ethyl acetate and heptane. The resulting white solid
was washed with heptane two times and dried under reduced pressure.
1
Yield: 55 mg, 39%. tR = 10.9 min. H NMR (400 MHz, CD3OD): δ
(ppm) 7.33 (d, 1H, J = 1.6 Hz), 7.30 (dd, 1H, J = 7.6 and 1.6 Hz), 7.10
(d, 1H, J = 7.6 Hz), 7.02ꢀ6.98 (m, 2H), 6.79ꢀ6.75 (m, 2H), 4.13 (d,
1H, J = 7.4 Hz), 3.90 (s, 2H), 3.82 (d, 1H, J = 12.5 Hz), 3.77 (dd, 1H,
J = 8.2 and 1.2 Hz), 3.72 (s, 3H), 3.66 (d, 1H, J = 12.5 Hz), 3.65 (t, 1H,
J = 8.0 Hz), 3.59 (d, 1H, J = 7.8 Hz), 3.58 (dd, 1H, J = 7.5 and 1.5 Hz),
2.16 (s, 3H). HRMS: m/z calcd for C22H27O7 (M þ Hþ) 403.1751,
found 403.1737.
1
18 mg of product 14 (73% yield). H NMR (400 MHz, CDCl3): δ
(ppm) 8.33 (m, 2H), 8.28ꢀ8.12 (m, 8H), 8.07 (m, 2H), 8.00 (m, 2H),
7.91 (m, 2H), 7.45ꢀ7.40 (m, 2H), 7.34 (d, 1H, J = 8.2 Hz), 6.87 (m,
2H), 6.64 (m, 2H), 6.13 (d, 1H, J = 8.6 Hz), 6.06 (t, 1H, J = 8.3 Hz), 5.86
(d, 1H, J = 8.1 Hz), 4.81 (d, 1H, J = 8.3 Hz), 4.75 (d, 1H, J = 12.7 Hz),
4.60 (d, 1H, J = 12.8 Hz), 4.06 (d, 1H, J = 8.5 Hz), 3.98ꢀ3.90 (m, 4H),
1.39 (t, 3H, J = 7 Hz). Single crystals were obtained by slow recrystalliza-
tion from acetonitrile/2-propanol as the solvent. Mp = 211 °C.
4-Bromobenzoylation of 23 To Give 15. To a solution of 23
(11 mg, 0.026 mmol) in anhydrous tetrahydrofuran (600 μL) were
added at room temperature N,N-diisopropylethylamine (32 μL, 7 equiv)
and 4-(dimethylamino)pyridine (3 mg, 0.9 equiv) followed by
p-bromobenzoyl chloride (35 mg, 6 equiv), and the resulting mixture
was stirred at room temperature for 62 h. Ethyl acetate and water were
added, and the organic phase was successively washed with 0.5 M
aqueous hydrochloric acid solution and brine. The organic phase was
dried over magnesium sulfate, filtered, and concentrated, and the crude
material was purified by flash chromatography over silica gel, eluting
with a gradient of 15ꢀ30% ethyl acetate in heptane, to afford 27 mg of
product 15 (90% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) 7.82
(m, 2H), 7.74ꢀ7.64 (m, 4H), 7.58ꢀ7.46 (m, 8H), 7.42ꢀ7.34 (m, 4H),
7.29 (d, 1H, J = 8.3 Hz), 6.89 (m, 2H), 6.63 (m, 2H), 6.04 (dd, 1H, J =
9.6 and 1 Hz), 5.98 (dd, 1H, J = 9.6 and 4.4 Hz), 5.89 (d, 1H, J = 4.4 Hz),
4.70 (d, 1H, J = 12.4 Hz), 4.65 (d, 1H, J = 12.4 Hz), 4.60 (d, 1H, J =
8 Hz), 3.98ꢀ3.88 (m, 3H), 3.73 (s, 3H). Single crystals were obtained by
vapor diffusion techniques using heptane and ethyl acetate as solvents.
Mp = 191 °C.
(1S,2S,3S,4R,5S)-5-[3-(4-Ethoxybenzyl)-4-methylphenyl]-
1-(hydroxymethyl)-6,8-dioxa-bicyclo[3.2.1]octane-2,3,4-triol
(21)25. This compound was obtained using procedures analogous to
the preparation of compounds 4 and 24. HPLC preparative method:
reversed-phase C18 Phenomenex column, Luna, 5 μm, 150 ꢁ 21.20
mm, 20 mL/min; gradient of acetonitrile/0.1% formic acid:water/0.1%
formic acid, 20ꢀ60% acetonitrile/0.1% formic acid over 20 min. UV
detection: 254 nm. The fractions containing the product 21 were
concentrated under reduced pressure. The crude material was precipi-
tated from ethyl acetate and heptane. The resulting white solid was
washed with heptane two times and dried under reduced pressure. Yield:
20 mg, 38%. tR = 12.7 min. 1H NMR (400 MHz, CD3OD): δ (ppm) 1.34
(t, J = 6.9 Hz, 3H), 2.18 (s, 3H), 3.60 (d, J = 8 Hz, 2H), 3.66 (t, J = 8 Hz,
1H), 3.68 (d, J = 12.5 Hz, 1H), 3.78 (d, 1H, J = 8.8 Hz), 3.84 (d, J = 12.4
Hz, 1H), 3.92 (s, 2H), 3.97 (q, J = 7 Hz, 2H), 4.15 (d, J = 7.5 Hz, 1H),
6.77 (m, 2H), 7.00 (m, 2H), 7.12 (d, J = 7.7 Hz, 1H), 7.31 (dd, J = 7.9 and
1.4 Hz, 1H), 7.34 (s, 1H). MS (LCꢀMS): m/z 417.3 (M þ Hþ, positive
mode), 461.4 (M þ HCO2ꢀ, negative mode).
(1S,2S,3S,4R,5S)-5-[4-Chloro-3-(4-methoxybenzyl)phenyl]-
1-(hydroxymethyl)-6,8-dioxa-bicyclo[3.2.1]octane-2,3,4-triol
(22) and (1S,2S,3S,4S,5S)-5-[4-Chloro-3-(4-methoxybenzyl)-
phenyl]-1-(hydroxymethyl)-6,8-dioxa-bicyclo[3.2.1]octane-
2,3,4-triol (23)25. These compounds were obtained using procedures
analogous to the preparation of compounds 4 and 24. HPLC preparative
method: reversed-phase C18 Phenomenex column, Luna, 5 μm, 150 ꢁ
21.20 mm, 20 mL/min; gradient of acetonitrile/0.1% formic acid:water/
0.1% formic acid, 20ꢀ60% acetonitrile/0.1% formic acid over 20 min.
UV detection: 254 nm. HPLC indicated a ratio of diastereoisomers of
1.4:1 (22:23).
’ ASSOCIATED CONTENT
S
Supporting Information. X-ray crystallographic data for
b
14, 15, and an L-pyroglutamic acid cocrystal form of 4 and
additional discussion around the PKPD model. This material is
available free of charge via the Internet at http://pubs.acs.org.
The fractions containing the product 22 were concentrated under
reduced pressure. The crude material was precipitated from ethyl acetate
and heptane. The resulting white solid was washed with heptane two
times and concentrated under reduced pressure. Yield: 50 mg, 36%. tR =
12.1 min. 1H NMR (400 MHz, CD3OD): δ (ppm) 7.43 (s, 1H),
7.38ꢀ7.30 (m, 2H), 7.08 (d, 2H), 6.79 (d, 2H), 4.12 (d, 1H, J = 7.5 Hz),
4.01 (s, 2H), 3.81 (d, 1H, J = 12.5 Hz), 3.75 (d, 1H, J = 8.4 Hz), 3.73 (s,
3H), 3.66 (d, 1H, J = 11.7 Hz), 3.63 (t, 1H, J = 8.2 Hz), 3.57 (d, 1H,
J = 7.4 Hz), 3.52 (d, 1H, J = 7.8 Hz). HRMS: m/z calcd for C21H24O7Cl
(M þ Hþ) 423.1205, found 423.1192.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (860) 686-3455. Fax: (860) 715-0310. E-mail: vincent.
mascitti@pfizer.com.
’ ACKNOWLEDGMENT
We thank Paul DaSilva-Jardine, David Price, David Hepworth,
Margaret Chu-Moyer, Spiros Liras, Roger Ruggeri, Neeta Amin,
and Gianluca Nucci from Pfizer Global Research & Development
for stimulating discussions.
The fractions containing the product 23 were concentrated under
reduced pressure. The crude material was precipitated from ethyl acetate
and heptane. The resulting white solid was washed with heptane two
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ARTICLE
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ElogDoct: a tool for lipophilicity determination in drug discovery. 2. Basic
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(44) The in vitro transport assay used human embryonic kidney 293
cells (HEK293) transfected with the appropriate organic anion or cation
transporter.
(45) Test No. 487: In Vitro Mammalian Cell Micronucleus Test.
OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects;
Organisation for Economic Co-operation and Development: Paris,
2010; http://www.oecd.org/env/testguidelines. Compound 4 acts as
neither an aneugen nor a clastogen in the micronucleus assay.
(46) The vehicle group resulted in a UGE over 24 h of 16.4 (
4.69 mg/200 g of body weight.
(27) See ref 25. See also: Gent, P. A.; Gigg, R. The allyl ether as a
protecting group in carbohydrate chemistry. Part VI. The allyl ether as a
“temporary” protecting group in oligosaccharide synthesis. J. Chem. Soc.,
Perkin Trans. 1 1974, 1835–1839.
(47) Assuming a theoretical body weight of 70 kg and [MPG] =
(28) Schaffer, R. Branched-chain higher sugars. III. A 4-C-
(hydroxymethy1)-pentose. J. Am. Chem. Soc. 1959, 81, 5452–5454.
(29) Upon storage at room temperature for an extended period of
time, we observed that 5 cyclized back to lactone 9.
80 mg/dL. See the Supporting Information for more details.
(30) The addition of anisole avoids the formation of side products
arising from FriedelꢀCrafts-type reactions between the PMB cation and
the aryl ring of the substrate.
(31) Mascitti, V.; Prꢀeville, C. Stereoselective synthesis of a dioxa-
bicyclo[3.2.1]octane SGLT2 inhibitor. Org. Lett. 2010, 12, 2940–2943.
(32) For a more detailed discussion around the SGLT2 PKPD
model, see the Supporting Information.
(33) Hosea, N. A.; Collard, W. T.; Cole, S.; Maurer, T. S.; Fang,
R. X.; Jones, H.; Kakar, S. M.; Nakai, Y.; Smith, B. J.; Webster, R.;
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(34) This model assumes rapid drug equilibrium (plasma/urine),
simple direct inhibition, and plasma glucose concentration similar to the
measured fasting plasma glucose concentration. See the Supporting
Information for more details.
(35) Mascitti, V.; Robinson, R. P.; Prꢀeville, C.; Thuma, B. A.; Carr,
C. L.; Reese, M. R.; Maguire, R. J.; Leininger, M. T.; Lowe, A.; Steppan,
C. M. Syntheses of C-5-spirocyclic C-glycoside SGLT2 inhibitors.
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(36) Potency at human SGLT (h-SGLT) was evaluated using a
functional assay designed to detect the inhibition of methyl R-D-
glucopyranoside (AMG) uptake via the SGLT transporter expressed
in Chinese hamster ovary cells. See the Supporting Information for more
details. For 16, IC50(h-SGLT2) = 6.55 nM (95% CI = 4.69ꢀ9.15 nM)
as the geometric mean of eight replicates and IC50(h-SGLT1) = 1540
nM (95% CI = 1390ꢀ1700 nM) as the geometric mean of seven
replicates.
(37) Robinson, R. P.; Mascitti, V.; Boustany-Kari, C. M.; Carr, C. L.;
Foley, P. M.; Kimoto, E.; Leininger, M. T.; Lowe, A.; Klenotic, M. K.;
MacDonald, J. I.; Maguire, R. J.; Masterson, V. M.; Maurer, T. S.; Miao,
Z.; Patel, J. D.; Prꢀeville, C.; Reese, M. R.; She, L.; Steppan, C. M.; Thuma,
B. A.; Zhu, T. C-Aryl glycoside inhibitors of SGLT2: exploration of sugar
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(38) In addition to the difficulty in rationally designing and building
a structureꢀactivity relationship around Vss, attempts to increase Vss
were not pursued due to the relative insensitivity of the dose to changes
in this parameter.
(39) Total clearance is the sum of the hepatic clearance (CLhepatic),
renal clearance (CLrenal), and biliary clearance (CLbiliary). In first
approximation the CLbiliary component was neglected.
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