Supplementary Content S1

Isoform-selective thiazolo[5,4-b]pyridine S1P1 agonists possessing acyclic amino carboxylate head-groups Anthony B. Reed*, Brian A. Lanman, Susana Neira, Paul E. Harrington, Kelvin K. C. Sham, Mike Frohn, Alexander J. Pickrell, Andrew S. Tasker, Anu Gore, Mike Fiorino, Andrea Itano, Michele McElvain, Scot Middleton, Henry Morrison, Han Xu, Yang Xu, Min Wong, and Victor J. Cee

Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States

Supplementary content

Table SI-1. Statistical analysis of hS1P1 and hS1P3 data...........................................S2 Synthetic routes for 1–8d ...........................................................................................S3 Procedure for the synthesis of 8c ...............................................................................S14 hS1PR cellular assays ................................................................................................S18 Rat lymphocyte depletion study .................................................................................S18

Supplementary Content S2

Table SI-1. Statistical analysis of hS1P1 and hS1P3 data

Cmpd hS1P1 RI

(n)

hS1P1 RI

EC50 (μM)

hS1P1 RI EC50 SD

(for n=2) or SE (for n>2)

hS1P1 RI %efficacy

hS1P1 RI %efficacy

SD (for n=2) or SE (for

n>2)

hS1P3 Ca2+ (n)

hS1P3 Ca2+ EC50

(μM)

hS1P3 Ca2+ EC50

SD (for n=2) or SE (for n>2)

hS1P3 Ca2+

%efficacy

hS1P3 Ca2+ %efficacy

SD (for n=2) or SE (for

n>2)

AMG 369 14 0.002 0.0004 98 5 6 0.888 0.085 26 2

1 2 0.045 0.006 98 22 2 >25

2a 6 2.82 0.355 105 10 2 >25

2b 2 5.88 0.955 81 30 2 >25

2c 2 0.062 0.022 100 6 2 >25

2d 2 2.41 1.62 103 24 2 >25

3a 2 0.123 0.053 92 14 2 >25

3b 4 0.015 0.005 113 2 2 >2.5

3c 2 0.478 0.375 155 85 2 >25

3d 4 0.008 0.010 121 2 2 >2.5

4a 2 0.042 0.023 94 4 2 >2.5

4b 2 0.036 0.024 113 18 2 7.12 0.838 18 2

5a 2 0.003 0.002 103 7 2 1.56 0.159 17 1

5b 18 0.003 0.0005 96 3 20 >25

5c 2 0.179 0.124 128 14 2 >2.5

5d 2 0.196 0.056 153 1 2 >25

6 2 0.002 0.0003 102 4 2 1.44 0.017 11 1

7a 2 0.019 0.008 80 27 2 5.56 1.74 39 1

7b 2 0.0006 0.0002 100 0 4 0.834 0.191 31 8

7c 2 0.0005 0.0001 134 3 4 1.13 0.143 35 6

7d 2 0.002 0.001 110 16 2 >2.5

7e 2 0.002 0.001 102 1 2 >2.5

8a 2 0.0003 0.0001 128 6 2 >2.5

8b 2 0.0005 0.0003 115 4 2 >2.5

8c 2 0.0002 0.00001 114 8 6 >25

8d 2 0.003 0.002 93 11 4 >25

Supplementary Content S3

Synthetic Routes for 1–8d:

1:

(a) toluene 100 °C, 10 min, then add CSA, 100 °C, 9 h, 79%

2a:

(b) NaClO2, NaH2PO4, 2-methyl-2-butene/t-BuOH/H2O, rt, 2 h, 78%

2b:

(c) DCM, rt, 3 d, 30%; (d) H2 (1 atm), 10% Pd/C, THF, rt, 3 h, 98%; (e) NaOH, THF/water, rt, 16 h, 81%

2c:

Supplementary Content S4 (f) Cs2CO3, diethylmalonate, DMF, rt–35 °C, 2.5 h, 90%; (g) 5 N HCl, 120 °C, 3 h, 19%

2d:

(h) KOtBu, THF, rt, 16 h, 50%; (i) H2 (1 atm), 10% Pd/C, THF, rt, 16 h, 88%; (j) NaOH, THF/water, rt, 16 h,

69%

3a:

(k) thionyl chloride, 65 °C, 30 min; (l) sodium azide (1.2 M in water), Bu4N+Br-, DCM, 0 °C, 2 h (m) TFA,

DCE, 80 °C, 16 h, 37% over three steps; (n) K2CO3, MeOH/water (1:1), rt, 16 h, 72%

3b:

Supplementary Content S5

(o) CuSO4, DCM, rt, 16 h; (p) DIBAL in toluene, THF, rt, 15 min; (q) then add 4 N HCl in dioxane, rt, 10

min, 10% over three steps

3c:

N S

N

F

NH2

3c

9N S

N

F

NO2r s

(r) MeNO2, NH4OAc, acetic acid, 100 °C, 4 h, 92%; (s) Zn, THF/MeOH/AcOH/H2O (1:1:1:1), 60 °C, 8 h, 27%

3d:

(t) KOtBu, THF, 40 min, 0 - 22 °C, 70%; (u) H2 (50 psi), 10% Pd/C, wet, EtOH, rt, 16 h, 84%; (v) LiAlH4, THF,

0 °C, 15 min, 8%

4a:

Supplementary Content S6

(w) t-butyl 3-aminopropanoate HCl, NaOtBu, PdCl2(dba)3, Xantphos, toluene, 80 °C, 18 h then μw (30

min @ 130 °C, 30 min @ 150 °C), 53%; (x) TFA, DCM, rt, 19 h, 29%

4b:

(y) pyrrolidin-2-one, NaH (60% in mineral oil), DMF, 18%

5a:

(z) t-butyl glycine•HCl, NaBH3CN, AcOH, DIPEA, MeOH/DCM, rt, 2.5 h, >99%; (aa) TFA, DCM, rt, 3 h, 25%

5b:

Supplementary Content S7

(bb) β-alanine ethyl ester•HCl, NaBH3CN, AcOH, DIPEA, MeOH/DCM, rt, 2.5 h, 72%; (cc) LiOH hydrate,

THF/H2O, rt, 25 min, 92%

5c:

(dd) γ-aminobutanoic acid, NaBH3CN, AcOH, MeOH/DCM, rt, 2.5 h, 60%

5d:

(ww) 3-(methylamino)propanoic acid, NaBH3CN, AcOH, MeOH/DCM, 70 °C, 1 h, 36%

6:

Supplementary Content S8

(ee) glyoxylic acid monohydrate, DCM, rt, 5 h; (ff) 2 N HCl, reflux, 16 h, 9% over two steps

7a:

(gg) DIPEA, THF, rt, 30 min, 67%; (hh) LiOH hydrate, THF/water, rt, 30 min, 98%

(ii) EtOAc, 60 °C, 2 h (both used crude)

Supplementary Content S9

7b, d:

7c, e:

Supplementary Content S10

(jj) 3a, AcOH (5 eq), benzene, 80 °C, 1 h; (kk) SFC separation of regioisomers [column: Princeton Pyridine

(250 x 21 mm, 5 μm), mobile phase: 60:40 (A:B), A: liquid CO2, B: methanol]; 20–43% over two steps for (R), 50–70% over two steps for (S); (ll) TFA, DCM, 0 °C–rt , 1 h; 85–98% over two steps for (R), 76–90% over two steps for (S). Regioisomers assigned on the basis of nOe.

8a, b:

Supplementary Content S11

(mm) Cs2CO3, DMF, rt, 1 h, then add 10, 1.5 h, 35 °C; (nn) 5 N HCl, 120 °C, 1 h, 67% over two steps; (oo)

Boc2O, TEA, THF/water, rt, 18 h; (pp) chiral separation [SFC: Chiralpak AD-H (21 x 250 mm, 5 μm), A:

liquid CO2, B: ethanol (0.2% IPA), isocratic: 60:40 (A:B)], 52–55% over two stseps; (qq) TFA, DCM, rt, 5

min, 41–73%. Stereochemistry arbitrarily assigned.

Supplementary Content S12 8c,d:

(rr) TMSCH2MgCl, KOtBu, THF, 0 °C, 48%; (ss) benzophenone imine, DCM, rt, 85%; (tt) Cs2CO3, DMF, 35

°C; then 10, 25%; (uu) chiral separation [SFC: Chiralpak AD-H (21 x 250 mm, 5 μm), A: liquid CO2, B:

ethanol (0.2% IPA), isocratic: 60:40 (A:B)]; (vv) 5 N HCl, THF, 80 °C, 68–72% over two steps.

Stereochemistry arbitrarily assigned.

Endnote 14:

Supplementary Content S13 (xx) NaBH4, THF/MeOH, rt, 2 h, 75%; (yy) methyl 2-bromoacetate, NaH (60% in mineral oil), DMF, rt, 20

min, 31%; (zz) LiOH hydrate, THF/H2O, rt, 10 min, 86%

Endnote 15:

(aaa) 3a, acetic acid, benzene, 80 °C, 1 h, then SFC separation of regioisomers [column: Chiralpak IC (21 x

250 mm, 5 μm) mobile phase: 55:45 (A:B), A: liquid CO2, B: methanol]; (bbb) 0.1 N sodium hydroxide,

THF, rt, 36 h

For (S) isomers (not shown) (aaa) 3a, acetic acid, benzene, 80 °C, 1 h; then SFC separation of

regioisomers [column: Chiralpak IC (21 x 250 mm, 5μm) mobile phase: 55:45 (A:B), A: liquid CO2, B:

methanol]: 65% for 3-acetoxysuccinamic acid, 7% for 2-acetoxysuccinamic acid; (bbb) 0.1 N sodium

hydroxide, THF, rt, 36 h: 94% for 3-hydroxysuccinamic acid, 16% for 2-acetoxysuccinamic acid

Supplementary Content S14 Synthesis of (–)-2-amino-4-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-

yl)phenyl)-2-methylbutanoic acid (8c)

Synthesis of 2-(2-fluoro-4-vinylphenyl)-5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridine (10)

Step a: Trimethylsilylmethylmagnesium chloride (1.1 M solution in THF; 15 mL, 16 mmol) was added

dropwise to a solution of 3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-yl)benzaldehyde

(4.00 g, 11 mmol) in THF (80 mL) at 0 °C, and the resulting mixture was stirred at 0 °C for 30 min. 1 N

aqueous HCl was then added at 0 °C, and the resulting mixture was extracted with EtOAc (2x). The

combined extracts were dried over MgSO4, filtered, and concentrated in vacuo.

The residue was taken up in THF (100 mL) and cooled to 0 °C. A slurry of potassium t-butoxide (2.4 g, 21

mmol) in THF (12 mL) was slowly added, and the resulting brown mixture was stirred at 0 °C for 30 min.

1 N aqueous HCl was added to neutralize excess KOtBu, and the resulting mixture was extracted with

EtOAc. The extract was dried over MgSO4, filtered, and concentrated in vacuo. Chromatographic

purification of the residue (silica gel, 5–30% EtOAc/hexanes followed by 40–50% (3%

Et3N/EtOAc)/hexanes) gave an impure solid which was rinsed with ethyl ether and re-purified (silica gel

0–50% EtOAc/hexanes) to provide 2-(2-fluoro-4-vinylphenyl)-5-(1-phenylcyclopropyl)thiazolo[5,4-

b]pyridine (1.90 g, 48% yield). MS (ESI) m/z: 372.9 (M+1)+. 1H NMR (300 MHz, CHLOROFORM-d) δ ppm

1.37 - 1.42 (m, 2 H) 1.76 - 1.83 (m, 2 H) 5.43 (d, J=10.82 Hz, 1 H) 5.88 (d, J=17.54 Hz, 1 H) 7.01 (d, J=8.62

Hz, 1 H) 7.26 - 7.35 (m, 3 H) 7.35 - 7.39 (m, 2 H) 7.39 - 7.47 (m, 3 H) 8.02 (d, J=8.62 Hz, 1 H) 8.30 (t,

J=7.89 Hz, 1 H).

Synthesis of ethyl 2-(diphenylmethyleneamino)-4-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-

b]pyridin-2-yl)phenyl)-2-methylbutanoate (12)

Supplementary Content S15 Step b: In a sealed pressure flask, a mixture of benzophenone imine (0.93 mL, 5.5 mmol) and finely

ground rac-alanine ethyl ester hydrochloride (0.85 g, 5.5 mmol) in DCM (20 mL) was stirred at ambient

temperature for 24 h. The reaction mixture was subsequently vacuum filtered, and the filtrate was

diluted with ethyl ether, washed with water, dried over MgSO4, filtered, and concentrated in vacuo.

Chromatographic purification of the residue (silica gel, 0–25% EtOAc/hexanes) furnished ethyl 2-

(diphenylmethyleneamino)propanoate (1.36 g, 85% yield). 1H NMR (300 MHz, CHLOROFORM-d) δ ppm

1.26 (t, J=7.09 Hz, 3 H) 1.43 (d, J=6.72 Hz, 3 H) 4.10 - 4.24 (m, 3 H) 7.15 - 7.22 (m, 2 H) 7.28 - 7.38 (m, 3

H) 7.42 - 7.50 (m, 3 H) 7.61 - 7.68 (m, 2 H).

Step c: In a sealed pressure flask, a slurry of cesium carbonate (0.630 g, 1.93 mmol) and ethyl 2-

(diphenylmethyleneamino)propanoate (1.36 g, 4.83 mmol) in DMF was stirred at ambient temperature

for 2 h. 10 (0.600 g, 1.61 mmol) was added, and the resulting dark-red mixture was heated at 35 °C for

18 h. The reaction mixture was subsequently adsorbed onto silica gel and chromatographically purified

(silica gel, 0–10% EtOAc/hexanes) to give ethyl 2-(diphenylmethyleneamino)-4-(3-fluoro-4-(5-(1-

phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-2-methylbutanoate (12; 270 mg, 25% yield). 1H

NMR (300 MHz, CHLOROFORM-d) δ ppm 1.13 (t, J=7.09 Hz, 3 H) 1.39 (d, J=2.34 Hz, 2 H) 1.49 (br. s., 3 H)

1.79 (d, J=2.34 Hz, 2 H) 2.16 - 2.42 (m, 2 H) 2.76 - 2.90 (m, 1 H) 2.91 - 3.07 (m, 1 H) 3.61 - 3.84 (m, 2 H)

7.01 (d, J=8.62 Hz, 1 H) 7.08 - 7.21 (m, 4 H) 7.29 - 7.36 (m, 4 H) 7.36 - 7.47 (m, 7 H) 7.58 (d, J=7.16 Hz, 2

H) 8.02 (d, J=8.48 Hz, 1 H) 8.24 (t, J=7.97 Hz, 1 H).

Synthesis of (-)-2-amino-4-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-2-

methylbutanoic acid (8c)

Step d: Preparative SFC method:

Column: Chiralpak AD-H (21 x 250 mm, 5 μm)

A: Liquid CO2

B: Ethanol (0.2% IPA)

Isocratic: 60:40 (A:B)

Flow rate: 70 mL/min

Outlet Pressure: 100 bar

Analytical SFC chromatogram showing the separation of enantiomers

Supplementary Content S16

Peak 1 Peak 2

Fractions from peak 1 were combined and concentrated to give ethyl-2-(diphenylmethyleneamino)-4-(3-

fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-2-methylbutanoate. MS (ESI) m/z:

654.1 (M+1)+. 1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.13 (t, J=7.16 Hz, 3 H) 1.36 - 1.43 (m, 2 H)

1.48 (s, 3 H) 1.75 - 1.83 (m, 2 H) 2.14 - 2.40 (m, 2 H) 2.75 - 2.89 (m, 1 H) 2.92 - 3.06 (m, 1 H) 3.62 - 3.86

(m, 2 H) 7.00 (d, J=8.62 Hz, 1 H) 7.08 - 7.15 (m, 1 H) 7.17 (dd, J=5.99, 2.19 Hz, 3 H) 7.27 - 7.35 (m, 3 H)

7.36 (d, J=1.46 Hz, 1 H) 7.37 - 7.39 (m, 2 H) 7.41 (d, J=1.32 Hz, 3 H) 7.43 (d, J=1.32 Hz, 1 H) 7.44 - 7.47 (m,

1 H) 7.58 (d, J=8.48 Hz, 2 H) 8.02 (d, J=8.48 Hz, 1 H) 8.24 (t, J=7.89 Hz, 1 H).

Step e: 5 N aqueous HCl (3.00 mL, 0.307 mmol) was added to a solution of ethyl-2-

(diphenylmethyleneamino)-4-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-2-

methylbutanoate (peak 1 from chiral separation; 200 mg, 0.307 mmol) in THF (2 mL), and the resulting

mixture was heated to 80 °C for 18 h. THF was subsequently removed in vacuo, and the resulting

concentrate was diluted with water and adjusted to pH ~1 with 1 N aqueous HCl. The resulting

precipitate was collected by vacuum filtration, rinsed with water and ether, and dried in vacuo (50 °C, 2

h). Chromatographic purification (silica gel, 0–10% (5% AcOH/CHCl3)/MeOH) subsequently provided (–)-

2-amino-4-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-2-methylbutanoic acid

(8c ; 97 mg, 68% yield) as a white amorphous solid: MS (ESI) m/z: 462.0 (M+1)+. 1H NMR (300 MHz,

DMSO-d6) δ ppm 8.55 (br. s., 3 H), 8.19–8.34 (m, 2 H), 7.23–7.51 (m, 7 H), 7.05 (d, J=8.6 Hz, 1 H), 2.82–

3.00 (m, 1 H), 2.58–2.71 (m, 1 H), 2.08–2.20 (m, 2 H), 1.62–1.73 (m, 2 H), 1.52 (s, 3 H), 1.32–1.44 (m, 2

H). [α] –12.5 (c. 1.03, DMSO).

ee determination:

8c was dissolved in methanol and analyzed by analytical HPLC (see below). No enantiomeric

contaminant (i.e., 8d) was detected.

17-Jul-2008

Instrument:ZQSample Name:98413-10-3Login:wesleyb Date:17-Jul-200813:38:50

Tray05:04ADH_40_EtOH(0.2%DEA)_6min_4mL.mth

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75

AU

0.0

2.0e-2

4.0e-2

6.0e-2

8.0e-2

1.0e-1

1.2e-1

1.4e-1

1.6e-1

1.8e-1

2.0e-1

92858-AD-40-EtOH(DEA)-002 2: Diode Array 212

Range: 2.171e-1

Supplementary Content S17

Column: Chirobiotic T (150 x 4.6 mm, 5 μm)

Mobile phase: D = Ethanol:methanol:triethylamine:acetic acid (50:50:0.1:0.1)

Isocratic: 100% D

Flow rate: 1 mL/min

ee determination by polar organic HPLC

The specific rotation of 8d (and 8c)was measured using a Perkin Elmer Model 341 polarimeter

conditions :

sodium lamp at 589 nm

T = 32.7 °C

path = 100 mm

c = 15.57 mg/1.50 mL = 1.04

dissolved in DMSO

[α] +10 (c. 1.04, DMSO)

min0.5 1 1.5 2 2.5

mAU

-25

0

25

50

75

100

125

150

MWD1 D, Sig=230,8 Ref=off (485-487\94485A12.D)

Are

a: 12

37.5

1.9

40

min0.5 1 1.5 2 2.5

mAU

-25

0

25

50

75

100

125

150

MWD1 D, Sig=230,8 Ref=off (485-487\569TI-32.D)

Are

a: 828

.28

1.1

82

Are

a: 86

5.572

1.9

48

98593-5-2 AEE ~ 100%

Supplementary Content S18

hS1PR cellular assays

The hS1P1 receptor internalization assay was performed using a U2OS cell line expressing hS1P1-

eGFP chimeric protein (Thermo Scientific (BioImage), Søborg, Denmark). Upon compound treatment,

the hS1P1 receptor was internalized into the cytoplasm, forming GFP-containing endosomes. This event

was detected using an ArrayScan VTI automated microscope (Thermo Scientific Cellomics, Pittsburg, PA),

and the degree of receptor internalization was quantitated by counting the number of GFP-containing

endosomes per cell. hS1P1-eGFP expressing U2OS cells were starved in serum free media for two hours

prior to compound treatment. Compounds were incubated with the starved cells at 37 °C for one hour.

Compound-treated cells were subsequently fixed using 4% formaldehyde, and nuclei were stained using

Hoechst dye (Invitrogen/Molecular Probes, Cat. #H3570). The cells were then imaged by ArrayScan VTI,

and the potency and efficacy of the compounds were determined by plotting the number of GFP-

containing endosomes per cell against corresponding compound concentration.

The Ca2+-mobilization assay was performed using CHO cell lines stably co-expressing hS1P3

receptor and a chimeric Gq/i5 G-protein. S1P (a known agonist) or compound treatment of these cells

activated the PLC-β / IP3 pathway, triggering release of Ca2+ from intracellular stores (e.g., the ER). Cells

were loaded with Ca2+ sensitive fluorescent dye (Calcium Indicator Dye, Cat. #51-9000177BK, BD

Biosciences) and a fluorescence quencher (PBX Signal Enhancer, Cat. #51-9006254, BD Biosciences) prior

to compound treatment. Intracellular Ca2+ release resulted in Ca2+ binding to the dye and dye

fluorescence (520 nm peak emission wavelength) upon excitation at 480 nm. The level of receptor

activation was quantitated by measuring fluorescence intensity following compound treatment. In this

assay, cells were starved in medium containing charcoal/dextran stripped serum for 16–20 hours.

Compounds were added to cells loaded with Ca2+ sensitive dye and fluorescence quencher inside a

FLIPRTETRA® plate reader (Molecular Devices, Sunnyvale, CA) and the fluorescence signal was measured.

CHO cells expressing only the chimeric Gq/i5 G-protein were employed as a negative control. The

potency and efficacy of the compounds were determined by plotting fluorescence intensity against

corresponding compound concentration.

Rat lymphocyte depletion study

Female Lewis rats (250 grams, 6–8 wks) were received from Charles River Laboratories

(Wilmington, MA) and allowed to acclimatize for at least one week before being placed on

study. Rats (N = 5/group) were administered vehicle (20% Captisol, 1% HPMC, 1% Pluronic

F68/water, pH = 2) or compound (0.1, 0.3, or 1.0 mg/kg in 20% Captisol, 1% HPMC, 1% Pluronic

F68/water, pH = 2) by oral gavage (10 mL/kg). 24 h post-dose, animals were sacrificed by CO2

inhalation, and blood was collected by cardiac puncture. Approximately 1 mL of blood was

Supplementary Content S19 transferred to a Microtainer® hematology tube containing EDTA (Becton Dickinson, #365973)

for CBC analysis and 500 μL of plasma was placed in a Microtainer® tube containing heparin

(Becton Dickinson, #365958) for subsequent pharmacokinetic analysis (plasma exposure).

Differential cell counts were obtained using an Advia® 120 hematology system (Bayer

Diagnostics).