Spirocyclic compounds, potent CCR1 antagonists

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1883–1886

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Spirocyclic compounds, potent CCR1 antagonists Nafizal Hossain a,⇑, Svetlana Ivanova a,b, Jonas Bergare a, Tomas Eriksson a,b a b

Department of Medicinal Chemistry, R&I Innovative Medicines, AstraZeneca R&D, Pepparedsleden 1, Mölndal 431 83, Sweden Department of Medicinal Chemistry, AstraZeneca R&D Lund, Scheelevägen 1, Lund 221 87, Sweden

a r t i c l e

i n f o

Article history: Received 30 November 2012 Revised 25 December 2012 Accepted 28 December 2012 Available online 23 January 2013

a b s t r a c t Conformationally constrained spirocycles (17–23) and (31–36) were synthesised. In vitro data revealed that these compounds are CCR1 antagonists with sub-nanomolar potency. In a functional assay 22, 23 and 36 inhibited CCR1 mediated chemotaxis with an IC50 value of 2, 2.6 and 68 nM, respectively. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Chemokines Spirocycles Antagonists Chemotaxis

Chemokines are a large superfamily of cytokines which play an important role in leukocyte recruitment and activation.1–4 Chemokines transmit intracellular signals to their target cells by binding to the cell surface chemokine receptor and activating G-proteins coupled to the receptor. Chemokine receptors are viewed as attractive therapeutic targets for drug developments due to their central role in regulating leukocyte trafficking. The C–C chemokine receptor-1 (CCR1) and its major endogenous ligands MIP-1a (CCL3) and RANTES (CCL5) are believed to play an important role in chronic inflamatory diseases such as rheumatoid arthritis5,6 and multiple sclerosis.7,8 Small molecule chemokines receptor antagonists are of interest as potential therapeutic agents and the discovery of selective and highly potent chemokine receptor antagonists has previously been reported in the literature.9–13 CCR1 antagonists in clinical development have also been reviewed.14 Here, we report the design and synthesis of a novel class of spirocyclic CCR1 antagonists with sub-nanomolar potency and excellent in vitro metabolic stability. Previously, CCR1 antagonists of such as compound A (Fig. 1) were investigated.14,15 Compounds of this class were obtained from a parallel synthesis approach by reaction of 6-chlorobenzyl aminopiperidine with a variety of epoxides of type B (Fig. 1). In vitro data indicated that the NHAc substituent ortho to the phenol was important for CCR1 potency. The introduction of conformational constraint16a into the drug molecule to minimize the conformational entropy loss16b upon binding to the receptor may lead to compounds with enhanced potency. The replacement of 6-chlorobenzyl aminopiperidine of compound A (Fig. 1) with spirocycle 8 ⇑ Corresponding author. Tel.: +46 317761851; fax: +46 317763818. E-mail address: Nafi[email protected] (N. Hossain). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.12.095

or 9 was thus proposed to investigate the effect of conformational restriction on CCR1 potency. The intermediate spirocycles 8, 9 were prepared from disubstituted phenylbromide according to Scheme 1. Compound 1 was treated with iso-propylmagnesium chloride in THF at 0 °C to give intermediate 3 which was treated with commercially available tert-butyl 1-oxa-6-azaspiro[2.5] octane-6-carboxylate in the presence of copper(I) bromide dimethyl sulphide complex at 40 °C to give intermediate 5. Treatment of 5 with potassium tert-butoxide in 1,2-dimethoxypropane at 50 °C gave 7 in 67% overall yield. The Boc protecting group in 7 was removed by HCl treatment in THF to give 8 in 45% isolated yield. Compound 6 was obtained using similar reaction conditions as described for 5 followed by treatment with 48% aqueous HBr in acetic acid to afford 9 in 31% overall yield (Scheme 1). As the ortho N-acetyl group of compound A (Fig. 1) was shown to be important for CCR1 potency, a first attempt was made to select compounds that would mimic the ortho NHAc meta hydroxy substituted phenyl ring moiety of compound A (Fig. 1) and replacing 6-chlorobenzyl aminopiperidine with spirocycles 8 and 9. Therefore, compounds 22 and 23 were designed and synthesised as outlined in Scheme 2. Thus, 2-nitro-5-methoxyphenol was OH

X

O O

N N H HO

Cl

O NHAc

A

Y

B Figure 1.

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O R X

R

(i) X

Br

1. X = Cl R = F 2. X = F R = OMe

R

(ii)

MgBr

N

O

X OH 5. X = Cl R = F 6. X = F R = OMe

3. X = Cl R = F 4. X = F R = OMe O

(iii)/(iv)

N R X 7. X = Cl R = Boc (67%) 8. X = Cl R = H (45%) 9. X = F R = H (31%) 9a. X = H R = H

Scheme 1. Reagents and conditions: (i) iso-propylmagnesium chloride in THF, 0 °C, 0.5 h for 3, 30 °C, 16 h for 4 (quant.); (ii) tert-butyl 1-oxa-6-azaspiro [2.5] octane-6carboxylate, copper (I) bromide dimethyl sulphide complex, 40 °C, 18 h for 5 30 °C , 3 h for 6 (iii) potassium tert-butoxide, 1,2-dimethoxypropane, 40–50 °C, 40 h (67%); (iv) 48% aqueous HBr in acetic acid, reflux, 24 h (31%).

R NHAc HO

O

(i)

O

(ii)

O

N R

1

R

10. R1 = OMe (80%) 11. R1 = F (87%) 12. R1 = H

2

NHAc

O

OH

X

1

13. R1 = OMe (63%) 14. R1 = F (60%) 15. R1 = H (64%) 16. R1 = OAc

(iii)

NHAc

17. X = Cl R2 = OMe (97%) 18. X = Cl R2 = F (35%) 19. X = Cl R2 = H (36%) 20. X = F R2 = F (30%) 21. X = F R2 = H (70%) 22. X = Cl R2 = OH (42%) 23. X = F R2 = OH (30%)

Scheme 2. Reagents and conditions: (i) (2S)-oxiran-2-yl-methyl-3-nitrobenzene sulfonate, CS2CO3, DMF, rt, 24 h (60–64%); (ii) Spirocycles 8, 9, EtOH, 80 °C, 24 h, (30–97%); (iii) BBr3, CH2Cl2, 0–22 °C, 2.5 h (42%).

hydrogenated in the presence of Pd/C in THF and acetic anhydride to give 10 in high yield. Compound 10 was treated with (2S)-oxiran-2-yl-methyl-3-nitrobenzene sulphonate and Cs2CO3 to give corresponding epoxide 13 in 63% isolated yield. Epoxides 13 was opened by spirocycle 8 in ethanol to give 17 (97%). The methyl group in 17 was removed by treatment with BBr3 in CH2Cl2 to afford 22 in 42% isolated yield. Compound 23 was prepared in one pot two steps reaction. Thus, 16 was opened by spirocycle 9 in

ethanol to give 23 (30%) and the acetyl protecting group was removed during the course of the reaction without addition of any external base. In vitro biological data (Table 1) revealed 22 and 23 to be very potent CCR1 antagonists, nearly 10-fold more potent than non-conformationally restricted compound A. Compounds 22 and 23 were stable both in human liver microsomes and human hepatocytes and exhibited good permeability. However 22 and 23 were unstable in rat hepatocytes (Table 1).

Table 1 In vitro data of CCR1 antagonists.19 Entry

hCCR1 IC50 (lM)

hheps (ll/min/106 cells)

hmics (ll/min/ mg)

Caco2 (cm/ s  106)

rheps (ll/min/106 cells)

rmics (ll/min/ mg)

17 18 19 20 21 22 23 31 32 33 34 35 36

0.0009 0.00064 0.00096 0.00036 0.00096 0.00036 0.00052 0.006 0.009 0.032 0.00079 0.0014 0.018

n.a 6.1 2.5 9.3 11 <3 <3.6 n.a 3.5 n.a <3 <3 <3

55 13.5 <17 17.8 25.5 <10 <10 12.6 17.4 18.8 <10 <10 <10

24.4 17 26 18 16.5 13.5 6.6 n.a n.a n.a 2.7 2.7 1.6

n.a 17.5 26 12

n.a n.a n.a 29 n.a n.a n.a n.a n.a n.a <10 <10 14

Data represent the average values of duplicates or triplicates or multiplets ± standard deviation. N.a: not available. hheps: human hepatocytes. hmics: human microsomes. rheps: rat hepatocytes. rmics: rat microsomes.

25.7 17.6 n.a n.a n.a 4.2 <3 6.6

Chemotaxis MIP-1a IC50 (lM)

0.002 0.0026

0.068

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OMe NO2

O O

O

O

(i)

N

(ii)

X

O

N

1

R

OH

X

OMe 25. X = Cl R1 = NO2 (88%) 26. X = F R1 = NO2 (99%) 27. X = H R1 = NO2 (73%) 28. X = Cl R1 = NHAc (86%) 29. X = F R1 = NHAc (90%) 30. X = H R1 NHAc ((85%)

24

8. X = Cl 9. X = F 9a. X = H

2

R

NH2 O

(iii) (iv)

O O

N X

NH2

N NHAc

NHAc

O

X OMe

31. X = Cl R2 = OMe (13%) 32. X = F R2 = OMe (13%) 33. X = H R2 = OMe (19%) 34. X = Cl R2 = OH (32%) 35. X = F R2 = OH (39%) 36. X = H R2 = OH (40%)

31a: X = Cl (14%) 32a: X = F (9%) 33a: X = H (12%)

Scheme 3. Reagents and conditions: (i) EtOH, 88 °C, 4–20 h (73–99%); (ii) Pt/C, EtOAc, Ac2O, rt, 4 h (85–90%); (iii) N(Boc)2, Ph3P, DEAD, 0–22 °C, 24 h (12–19%); (iv) TFA, CH2Cl2, rt 1 h (100%).

Subsequently, we wanted to investigate if the meta hydroxy phenyl substitution of 22 and 23 is essential for CCR1 potency. Thus, we designed 18–21, synthesised according to Scheme 2. The intermediate 11 was prepared by hydrogenation of 2-nitro5-fluorophenol in the presence of Pd/C in THF and acetic anhydride in high yield. Phenols 11 and 12 were treated with (2S)-oxiran-2yl-methyl-3-nitrobenzene sulphonate and Cs2CO3 to give the corresponding epoxides 14 (60%) and 15 (64%) which were opened by the spirocycles 8 and 9 to give 18–21 in moderate yields. Biological data of 18–21 revealed that the meta hydroxy phenyl substituent in 22 and 23 could be replaced either by fluorine or a hydrogen without loss of CCR1 potency (Table 1). This indicates that meta hydroxy substitution of the phenyl is not required for CCR1 potency and it can provide a tool to modulate pharmacokinetic properties if needed. Spirocycles 17–21 are metabolically unstable. Nonetheless all compounds 17–21 have good permeability (Table 1). Next, we wanted to investigate whether the replacement of the aliphatic hydroxy group in the linker by other polar group such as primary amine would be tolerated with regard to CCR1 potency and what effect such a change would have on pharmacokinetic properties. Thus, we designed 34–36 which were synthesised according to Scheme 3. The epoxide 24 was prepared in good yield in a similar manner as described for 13–16. The ring opening reaction of 24 with spirocyle 8 or 9 or 9a17 in ethanol gave 25–27 in high yields. The hydrogenation of 25–27 in the presence of Pt/C and subsequent acetylation in the presence of acetic anhydride in ethyl acetate afforded 28–30 in excellent yields. The reaction of 28, 29 or 30 with N(Boc)2 in the presence of Ph3P and DEAD gave 31–33 along with 31a–33a18 in low yield after removal of the Boc group under acidic condition. Finally, BBr3 mediated demethylation of 31–33 gave 34–36 in low yield. In vitro data revealed that 34 and 35 were very potent CCR1 antagonists with good in vitro pharmacokinetic properties. Compounds 22, 23 and 36 were tested in a functional assay in which they inhibited CCR1 mediated chemotaxis with an IC50 value of 2, 2.6 and 68 nM, respectively (Table 1). In conclusion, when the 6-chlorobenzyl aminopiperidine moiety of compound A (Fig. 1) was replaced with spirocycles 8 and

9, to give 22 and 23, respectively, the CCR1 binding affinity was increased nearly 10-fold. When the meta hydroxy group in a substituted phenol (22 or 23) was replaced by fluorine (18 or 20) or with a hydrogen (19, 21) CCR1 potency was retained although in vitro metabolic stability was decreased. The meta hydroxy phenyl substitution on compound 22 could be methylated (17) without any loss of CCR1 potency but when meta hydroxy group in 34 was methylated (31) it lost some potency. The substitution pattern in the spirocycle 8 or 9 seems important for CCR1 potency. When chlorine or fluorine atom in 34 or 35 was replaced by a hydrogen (36) the CCR1 potency decreased more than tenfold probably due to reduced lipophilicity. The replacement of the hydroxy group (22 and 23) in the linker with a polar group such as a primary amine (34 and 35) was possible without losing CCR1 potency. In addition, rat hapatocyte stability was improved compared to 22 and 23. Compounds 22, 23, 34 and 35 are very potent CCR1 antagonists which exhibit good in vitro metabolic stability (22 and 23 are less stable in rat hepatocytes) and have moderate to good permeability. In addition these compounds inhibited CCL3 induced chemotaxis of THP-1 cells in a functional assay.20 In vivo results will be reported elsewhere. This investigation demonstrates the possible effectiveness of conformational restriction as an approach to improve drug binding affinity and functional potency. Acknowledgments We thank to our colleagues at the Departments of Biosciences and DMPK for generating data, Drs. Soren Andersen, Magnus Nilsson for critical reading of this manuscript and Matin Cooper for linguistic revision of the manuscript. We also thank Dr. Peter Sjö, Director of Department of Medicinal Chemistry, for his suggestions in writing this manuscript. References and notes 1. Strieter, R. M.; Standiford, T. J.; Huffnagle, G. B.; Colletti, L. M.; Lukacs, N. W.; Kunkle, S. L. J. Immunol. 1996, 156, 3583. 2. Luster, A. D. N. Engl. J. Med. 1998, 338, 436. 3. Gerard, C.; Rollins, B. J. Nat. Immunol. 2001, 2, 108.

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membranes of each batch was adjusted to 10% specific binding of 33 pM. [125I]MIP-1a human CCR1 binding assay: 100 ll of HEK-CCR1 membranes diluted in assay buffer pH 7.4 (137 mM NaCl (Merck), 5.7 mM glucose (Sigma) 2.7 mM KCl (Sigma), 0.36 mM NaH2PO4  H2O (Merck), 10 mM HEPES (Sigma), 0.1% (w/v) Gelatine (Sigma) with the addition of 17,500 units/L Bacitracin (Sigma) were added to each well of the 96 well filter plate (0.45 lM opaque Millipore), 12 ll of compound in assay buffer containing 10% DMSO was added to give final compound concentration of 1  10 5.5 to 1  10 9.5 M. 12 ll old human recombinant MIP-1a (R&D systems), 10 nM final concentration in assay buffer supplemented with 10% DMSO, was included in certain wells (without compound) as non specific binding control (NSB). 12 ll assay buffer with 10% DMSO was added to certain wells (without compound) to detect maximal binding (B0). 12 ll [125I] MIP-1a diluted in assay buffer to a final concentration in the wells of 33 pM, was added to all wells. The plates with lid were then incubated for 1.5 h at room temperature. After incubation the wells were emptied by vaccum filtration (MultiScreen Resist Vacuum Manifold System, Millipore) and washed once with 200 ll assay buffer. After the wash, all wells received an addition of 50 ll of scintillation fluid (OptiPhase Supermix, Wallac Oy). Bound [125I] MIP-1a was measured using a wallac Trilux 1450 MicroBeta counter.Calculation of percent displacement and IC50. The following equation was used to calculate percent displacement. Percent displacement = 1 {(cpm test cpm NSB)/ (cpmB0 cpmNSB)} where cpm test = average cpm in wells with membranes and compound and [125I] MIP-1a, NSB = average cpm in the wells with membranes and MIP-1a and [125I] MIP-1a (non specific binding). B0 = average cpm in well with membranes and assay buffer and [125I] MIP-1a (maximum binding). The molar concentration of compound producing 50% displacement (IC50) was derived using the Excel XL fit (version 2.0.9) to fit data to a 4-parameter logistics function. 20. Culture of THP-1 cells: Cells were thawed rapidly at 37 °C from frozen aliquots and resuspended in a 25 cm flask containing 5 ml of RPMI-1640 medium supplemented with Glutamax and 10% heat inactivated fetal calf serum without antibiotics (RPMI+10%HIFCS). At day 3 the medium is discarded and replaced with fresh medium. THP-1 cells are routinely cultured in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum and glutamax but without antibiotics. Optimal growth of the cells requires that they are passaged every 3 days and the minimum subculture density is 4  105 cells/ml. Chemotaxis assay Cells were removed from the flask and washed by centrifugation in RPMI + 10%HIFCS + glutamax. The cells were then resuspended at 2  107 cells/ml in fresh medium (RPMI + 10%HIFCS + glutamax) to which was added calcein-AM (5 ll of stock solution to 1 ml to give a final concentration of 5  10 6 M). After gentle mixing the cells were incubated at 37 °C in a CO2 incubator for 30 min. The cells were then diluted to 50 ml with medium and washed twice by centrifugation at 400g. Labelled cells were then resuspended at a cell concentration of 1  107 cells/ml and incubated with an equal volume of MIP-1a antagonist (10 10 M to 10 6 M final concentration) for 30 min at 37 °C in a humidified CO2 incubator. Chemotaxis was performed using Neuroprobe 96-well chemotaxis plates employing 8 lm filters (cat no.101–8). Thirty ll of chemoattractant supplemented with various concentrations of antagonists or vehicle were added to the lower wells of the plate in triplicate. The filter was then carefully positioned on top and then 25 ll of cells preincubated with the corresponding concentration of antagonist or vehicle were added to the surface of the filter. The plate was then incubated for 2 h at 37 °C in a humidified CO2 incubator. The cells remaining on the surface were then removed by adsorption and whole plate was centrifused at 2000 rpm for 10 min. The filter was then removed and the cells that had migrated to the lower wells were quantified by the fluorescence of cell associated calcein-AM. Cell migration was then expressed in fluorescence units after subtraction of the reagent blank and values were standardized to %migration by comparing the fluorescence values with that of a known number of labelled cells. The effect of antagonists was calculated as %inhibition when the number of migrated cells was compared with vehicle