Optimization of triazoles as novel and potent nonphlorizin SGLT2 inhibitors

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3774–3779

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Optimization of triazoles as novel and potent nonphlorizin SGLT2 inhibitors Xiaohui Du ⇑, Mike Lizarzaburu, Simon Turcotte, Taeweon Lee, Joanne Greenberg, Bei Shan, Peter Fan, Yun Ling, Julio C. Medina, Jonathan Houze Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA

a r t i c l e

i n f o

Article history: Received 9 February 2011 Revised 6 April 2011 Accepted 12 April 2011 Available online 20 April 2011

a b s t r a c t Previous efforts have led to the identification of a potent, selective, and nonphlorizin based SGLT2 inhibitor 1. This Letter describes efforts to further optimize the potency, microsomal stability, solubility and pharmacokinetic properties of this series of SGLT2 inhibitors. From these efforts, compounds 28 and 32 have improved solubility and pharmacokinetic properties compared to compound 1 Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Noncompetitive SGLT2 inhibitors Triazoles SAR Solubility Benzooxazinone head groups Indane tail group

Type 2 diabetes mellitus (T2DM) is a prevalent disease. It is estimated that the patient population has reached 285 million and is expected to increase to 438 million by 2030.1 It is characterized by hyperglycemia, which is a major risk factor for microvascular and macrovascular complications including retinopathy, nephropathy, neuropathy, and accelerated cardiovascular disease.2 Despite various therapeutic options, many patients demonstrate inadequate glycemic control and remain at risk for chronic complications.3 A novel approach to reduce plasma glucose in T2DM patients is by inhibition of glucose reabsorption in the kidney.4 Plasma glucose is normally filtered in the kidney in the glomerulus and actively reabsorbed in the proximal tubules. Sodium glucose co-transporter 2 (SGLT2)5 is predominantly expressed in the early S1 segment of the renal proximal tubules and is responsible for an estimated 90% of the glucose reuptake in the kidney. SGLT1,6 a related sodium-dependent glucose transporter is expressed in small intestine and in the more distal S3 segment of the renal proximal tubule. The remaining 10% of the glucose reuptake is likely mediated by SGLT1 and other subtypes. However, inhibition of SGLT1 could have adverse consequences in the GI tract illustrated by the hereditary syndrome glucose/galactose malabsorption (GGM). Therefore, selectivity over SGLT1 is preferred. It has been demonstrated that selective inhibition of SGLT2 resulted in urinary glucose excretion and exhibited a glucose-lowering effect.7 The majority of current SGLT2 inhibitors were based on phlorizin, a naturally-occurring O-aryl glycoside that has long been ⇑ Corresponding author. E-mail address: [email protected] (X. Du). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.04.053

known to cause glucosuria in animals and humans.8 However, phlorizin was not selective against SGLT1 and had poor metabolic stability. Subsequent research efforts by numerous institutions identified many phlorizin-based SGLT2-selective inhibitors9 such as dapagliflozin10 (Fig. 1). Recent phase III clinical trial results showed that dapagliflozin significantly improved fasting plasma glucose levels, lowered HbA1c level, and the body weight of the patients, thus demonstrating the utility of SGLT2 inhibitors as anti-diabetic agents.11 Our previous efforts12 resulted in a series of potent nonphlorizin based SGLT2 inhibitors exemplified by compound 1. It had a 10 nM IC50 against the human SGLT2 transporter in our [14C] glucose transporter assay13 and was selective against hSGLT1 (IC50 = 9 lM). However, this compound showed poor microsomal stability and high in vivo clearance in rat. Administration of compound 1 in 100 mg/Kg sc in mouse showed inconsistant results. Attempted Cl Cl HO

O

O Cl

HO

N

O N

OH OH

hSGLT2 IC50

OEt

N

F3 C

Dapagliflozin

1

3 nM

10 nM

Cl N

Figure 1. Structure of dapagliflozin and our nonphlorizin based inhibitor triazole 1.

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R1

R1 OH

R2

R2

NO2

R1

O O

a

O

b

OEt

R2

NO2

N H

I

O

II

O

c

R3

S

d

R3

OH

R3

NH

NH R4

III

IV

f

R3

N

R4

R4

R4 e

O

R3

N HO

N N

g

N N

VI

V 1

R

O

R4

Cl

R2

h

R3

N

N

O N

N N

N

N VIII

VII

R3 R4

Scheme 1. General synthesis of the triazole SGLT2 inhibitors. Representative yields in parentheses are for the preparation of compound 32. Reagents and conditions: (a) ethyl 2-bromoacetate, K2CO3, CH3CN (R1 = R2 = Me, 97%); (b) Fe, acetic acid, 75 °C (R1 = R2 = Me, 82%); (c) SOCl2, DCM, DMF (catalytic), amine (for R3COOH = dihydro-indene carboxylic acid, R4 = propylamine, 80%); (d) Lawesson’s reagent, toluene, 80 °C, 2 h. (74%); (e) formohydrazide, Hg(OAc)2, reflux, overnight (22%); (f) paraformaldehyde, xylene, 125 °C (57%); (g) SOCl2, DCM; (h) II, K2CO3, NaI, DMF, 65 °C (53%).

Table 1 Modifications of the C8-substituents

X 8 7 Cl

O

2

N

O N N

F3 C

Compound #

X group

a b

–Cl –COOMe –COOH –NH2 –NHCOMe –N(SO2Me)2 –NO2 –CN –SO2Me

Microsomal stabilityb

hSGLT2 inhibition IC50a

1 2 3 4 5 6 7 8 9

Cl N

(nM)

10 57 10000 36 9800 21 22 59 660

% Inhibition

(r/h) % remaining

100 100 95 100 95 100 100 100 99

<5/4 <5/<5 68/95 14/10 0.1/0.4 1/0.1 10/1 1.2/0.8 0.4/0.2

Inhibition of [14C] AMG transportation into the CHO cells that express hSGLT2. See Ref. 13 for assay protocol. See Ref. 14 for assay protocol. r: rat; h: human.

formulation for high dose delivery into mouse also failed. Both results are likely due to the poor solubility of this compound (1.6 lg/ mL in PBS buffer). In this Letter, we would like to report our further efforts in optimizing the SAR of this series to improve the pharmocokinetic properties of the inhibitors.

Incubation of compound 1 with rat microsomes showed that oxidation occurred both on the benzooxazinone head group and the chloro-phenylethyl tail group. Both these areas were targeted for modification with our initial goal being improving the microsomal stability and consequently the in vivo clearance.

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Table 2 SAR of the tail aromatic groups

Cl O Cl

N N

F3 C

Compound #

O N N R

R group

Microsomal stabilityb

hSGLT2 inhibition a

IC50 (nM)

% Inhibition

(r/h) % remaining

10

100

<5/4

10c

89

100

<5/<5

11

480

100

<5/<5

73

98

<5/<5

8

100

0/<5

23

100

5/<5

13

100

<5/7

3

100

2/20

10

100

3/3

18c

12

100

<5/8

19c

14

100

<5/<5

20

13

100

<5/<5

1

Cl

O 12

Cl 13

O

14

Cl N 15

N 16

Cl N 17

S

a b c

Inhibition of [14C] AMG transportation into the CHO cells that express hSGLT2. See Ref. 13 for assay protocol. See Ref. 14 for assay protocol. r: rat; h: human. Compounds are racemic.

Scheme 1 shows the general synthetic sequences to obtain our SGLT2 inhibitors. Benzooxazinone head groups II were generally made by reacting di-substituted nitro phenol with ethyl 2-bromoacetate followed by reductive cyclization of the nitro ester I. The synthesis of the remainder of the inhibitor started from the tail carboxylic acid. Coupling with a suitable amine generated the amide III which was converted to thioamide IV with Lawesson’s reagent. Triazole formation of IV with formohydrazide was catalyzed by mercury acetate. Subsequent hydroxymethylation of the triazole V and conversion to the chloride with thionyl chloride led to compound VII. Compound VII was then coupled under

basic conditions with benzooxazinones II to make the final inhibitor VIII. Polar groups were introduced onto the benzooxazinone to see if potency could be maintained and to improve microsomal stability (Table 1). Since C7 substitution was not well-tolerated12, attention was focused on C8 position. As shown in Table 1, the C-8 position tolerated a variety of polar groups including methylester (2), aniline (4), bis-sulfonamide (6), nitro (7) and cyano (8) groups. Compounds with highly polar carboxylic acid (3), acetamide (5), and sulfone (9) substitutions showed much weaker inhibition. Most compounds were not stable in rat and human liver micorsomes

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X. Du et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3774–3779 Table 3 Combinations of head benzooxazinones groups and tail aromatic groups improved microsomal stability

R1 O

Cl

N

O N N N

F3 C

N

R2

a b

Compound #

R1 group

R2 group

IC50a (nM) (hSGLT2 inhibition)

Microsomal stabilityb % remaining (m/r/h)

21 22 23 24

–NO2 –NO2 –NO2 –CN

–H –Cl –CF3 –H

41 7 7 110

78/19/73 77/6/51 41/29/68 70/28/80

Inhibition of [14C] AMG transportation into the CHO cells that express hSGLT2. See Ref. 13 for assay protocol. See Ref. 14 for assay protocol. m: mouse; r: rat; h: human.

except carboxylic acid 3. Selective compounds were tested for its stability in mouse liver micorsome for further comparison. Compounds 7 and 8 with strongly electron-withdrawing nitro and cyano groups showed slightly better mouse liver microsome stability with 25% and 16% remaining compared to less than 5% remaining of compound 1. Simultaneously, methyl substitutions and oxygen were introduced into the tail aromatic group area in an attempt to slow down benzylic oxidation by microsomes (Table 2). Mono-substitution at the benzylic position (10) was tolerated but di-substitution significantly decreased the potency as shown with compound 11. Oxygen replacement of the benzylic carbon in compound 12 also resulted in a significant loss of potency. Linker length variation between triazole and tail aromatic group was investigated. Compound 13 with a three atom linker maintained potency, but was not stable in microsomes. Therefore, the linker was truncated to see whether potency could be maintained and stability improved. Compound 14 with the aryl group directly attached to the triazole maintained potency. The chloro-phenyl group in compound 14 could be replaced by a more polar pyridine group (15, 16) and other heterocycles such as thiazole (17) with improvement in potency. Furthermore we sought to conformationally constrain the tail linker in an attempt to reduce the flexibility of the structures. A number of constrained analogs such as cyclopropyl linker 18, tetrahydronaphthalene 19 and indane 20 all showed potent SGLT2 inhibition. Compound 16 showed a slight improvement in human liver microsome stability. Compound 15 showed a slight improvement in mouse liver microsome stability with 15% remaining compared to less than 5% remaining of compound 1. The lack of general stability of these structures regardless of the tail aromatic groups reflected the ease of oxidation of thc 6,8-dichlorobenzoxazinone head group. Having examined each area of the molecule separately, we combined the most stable benzooxazinone head groups with our most stable tail aromatic groups (Table 3). Compound 21 with chloro-nitro substituted head group was more potent than compound 24 with chloro-cyano substituted head group. Compounds 22 and 23 with a substituted pyridine tail were consistently more potent

than compound 21 with a simple pyridine tail. All four combinations brought significant improvement in mouse and human liver microsomal stability. In addition to its potency and improved in vitro stability, compound 22 also showed a low clearance of 0.79 L/h/Kg in rat dosed 0.5 mg/Kg. However, when compound 22 was delivered into mouse or rat through high dose ip administration, it showed low exposure in both animal species. The poor solubility of this compound (2.2 lg/mL in PBS buffer) could be preventing its absorption in vivo. This result led us to focus on improving the solubility of our inhibitors to improve the potential exposure of our compounds. During our SAR optimization, N-propyl substitution on the triazole was found to be equally potent to the N-trifluoropropyl substitution. The substitution was therefore changed to N-propyl to reduce the hydrophobicity of the overall structures. In addition, another strategy to improve the solubility was to reduce the number of chlorine atoms in the inhibitors (Table 4). Compound 26 with a methyl group replacing the 6-chloro in the 6-chloro, 8-nitro bezooxazinone head group in compound 25 and compound 27 with a 6,8-dimethyl bezooxazinone head group both resulted in equally-potent inhibitors. Removing the chlorine in the tail pyridine moiety significantly improved solubility. Compound 28 had a marked improvement in solubility (64 lg/mL) while maintaining a potent inhibition of 12 nM. The loss of potency due to the removal of the chlorine in the tail moiety was compensated partially by the potent di-methyl bezooxazinone head group. One helpful discovery during the process was shown in compound 29. Though 3-methyl substitution decreased the inhibition of hSGLT2, the compound’s solubility was markedly increased presumably due to the greater dihedral angle between the triazole and the pyridine promoted by the methyl group. This led us to incorporate the linkers as shown in compounds 30–32 to disrupt the planar alignment of the triazole and pyridine to improve solubility. Indeed, compounds 30–32 all showed satisfactory solubility. Apart from high potency, compounds 28 and 32 were synthetically more accessible by having no chiral center in their structures compared to compounds 30 and 31. Both compounds 28 and 32 also had great selectivity against hSGLT1 with both IC50 being 30 lM on hSGLT1. These two compounds were evaluated for their rodent PK.

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Table 4 More SAR optimizations to improve the solubility of the inhibitors

head N N N

tail

Compound #

Head group

IC50a (nM)

Tail group

NO2

Solubility (lg/mL)

N

O 25

Cl

N

Cl

5

1

Cl

4

2

3

1.1

O

NO2

N

O 26

N

O N

O 27

N

Cl

O N

O 28

N

12

64

5100

80

8

19

10

23

5

35

O

NO2

N

O 29

Cl

N

O

O 30

N

O

O 31

N

O

O 32

N a

O

Inhibition of [14C] AMG transportation into the CHO cells that express hSGLT2. See Ref. 13 for assay protocol.

Table 5 High dose pharmacokinetic parameters of 28 and 32 in rat

SCa (rat)

a b

Compound #

28

32

Cmax (lM) AUC0–24 h (lM/h)b Tmax (h) Fraction unbound Dose (mg/Kg) Rat SGLT2 IC50 (nM) Microsomal stability % remaining (rat/human)

1.05 16.2 8 3% 100 3.5 <5/20

3.5 61.6 24 0.5% 70 3 <5/<5

For 28 and 32: n = 3. Due to the depot effect of both 28 and 32, AUC was calculated up to 24 h.

Despite relatively high microsomal turnover, it was felt that the improved solubility of compounds 28 and 32 would allow sufficient

intestinal absorption to saturate first-pass metabolism resulting in good plasma concentrations. Both compounds were formulated in high dose and dosed in mouse with sc administration. The PK parameters were shown in Table 5. At 100 mg/Kg, compound 28 reached nine-fold of IC50 at Cmax calculated by free unbound fraction. At 70 mg/Kg, compound 32 had about six-fold of IC50 at Cmax calculated by free unbound fraction. In conclusion, we have further optimized our triazole series of hSGLT2 inhibitors with a variety of head benzooxazinone and novel tail aromatic groups. Both areas tolerated polar groups such as a cyano group and a pyidine group. Many inhibitors had better potency and solubility compared to our previous lead inhibitor compound 1. More importantly, apart from the structural differences, compounds 28 and 32 both delivered micromolar plasma concentrations in rats. Future efforts in this series will focus on

X. Du et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3774–3779

introducing polar groups to the tail groups for better solubility, free fraction and potentially lower in vivo clearance. References and notes 1. Norman, P. Diabetes Pipeline: Intense Activity to Meet Unmet Need Insight Pharma Reports, Cambridge Health Institute, September 2010. 2. (a) Nazimek-Siewniak, B.; Moczulski, D.; Grzeszczak, W. J. Diabetes Complications 2002, 16, 271; (b) Kles, K. A.; Vinik, A. I. Curr. Diab. Rev. 2006, 2, 131; (c) Rahman, S.; Rahman, T.; Ismail, A. A.; Rashid, A. R. Diabetes Obes. Metab. 2007, 9, 767. 3. Blonde, L. Am. J. Manag. Care 2007, 13, S36. 4. Handlon, A. L. Expert Opin. Ther. Pat. 2005, 15, 1431. 5. (a) Wright, E. M.; Hirayama, B. A.; Loo, D. F. J. Intern. Med. 2007, 261, 32; (b) Santer, R.; Kinner, M.; Lassen, C. L.; Schneppenheim, R.; Eggert, P.; Bald, M.; Brodehl, J.; Daschner, M.; Ehrich, J. H. H.; Kemper, M.; Volti, S. L.; Neuhaus, T.; Skovby, F.; Swift, P. G. F.; Schaub, J.; Klaerke, D. J. Am. Soc. Nephrol. 2003, 14, 2873. 6. (a) Wright, E. M.; Hirayama, B.; Hazama, A.; Loo, D. D.; Supplisson, S.; Turk, E.; Hager, K. M. Soc. Gen. Physiol. Ser. 1993, 48, 229; (b) Wright, E. M.; Turk, E.; Hager, K.; Lescale-Matys, L.; Hirayama, B.; Supplisson, S.; Loo, D. D. F. Acta Physiol. Scand. Suppl. 1992, 146, 201. 7. (a) Oku, A.; Ueta, K.; Arakawa, K.; Ishihara, T.; Nawano, M.; Kuronuma, Y.; Matsumoto, M.; Saito, A.; Tsujihara, K.; Anai, M.; Asano, T.; Kanai, Y.; Endou, H. Diabetes 1999, 48, 1794; (b) Katsuno, K.; Fujimori, Y.; Takemura, Y.; Hiratochi, M.; Itoh, F.; Komatsu, Y.; Fujikura, H.; Isaji, M. J. Pharmacol. Exp. Ther. 2007, 320, 323; (c) Ueta, K.; Yoneda, H.; Oku, A.; Nishiyama, S.; Saito, A.; Arakawa, K. Biol. Pharm. Bull. 2006, 29, 114. 8. Ehrenkranz, J. R. L.; Lewis, N. G.; Kahn, C. R.; Roth, J. Diabetes Metab. Res. Rev. 2005, 21, 31. 9. Some of the most recent review and papers are: (a) Washburn, W. N. J. Med. Chem. 2009, 52, 1785; (b) Kakinuma, H.; Oi, T.; Hashimoto-Tsuchiya, Y.; Arai,

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