Supplementary Note Small-molecule reversible Factor D ... · 1 Supplementary Note Small-molecule...

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Supplementary Note

Small-molecule reversible Factor D inhibitors targeting the alternative complement

pathway

Jürgen Maibaum1*

, Sha-Mei Liao2, Anna Vulpetti

1, Nils Ostermann

1, Stefan Randl

3, Simon

Rüdisser1, Edwige Lorthiois

1, Paul Erbel

1, Bernd Kinzel

1, Fabrice A. Kolb

4, Samuel Barbieri

1,

Julia Wagner1, Corinne Durand

1, Kamal Fettis

1, Solene Dussauge

1, Nicola Hughes

1, Omar

Delgado2, Ulrich Hommel

1, Ty Gould

2, Aengus Mac Sweeney

5, Bernd Gerhartz

1, Frederic

Cumin1, Stefanie Flohr

1, Anna Schubart

1, Bruce Jaffee

2, Richard Harrison

6, Antonio Maria

Risitano7, Jörg Eder

1 & Karen Anderson

2*

1Novartis Institutes for BioMedical Research, Novartis Pharma AG, Novartis Campus, CH-4056

Basel, Switzerland. 2Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue,

Cambridge, Massachusetts 02139, USA. 3Evonik Japan Co., Shinjuku Monolith 12F, 2-3-1

Nishi-Shinjuku, Shinjuku-ku, 163-0938 Tokyo, Japan. 4Pharma Research and Early

Development, Roche Innovation Center Basel, CH-4070 Basel, Switzerland. 5Drug Discovery

Department, Actelion Pharmaceuticals Ltd., CH-4123 Allschwil, Switzerland. 6Institute of

Infection and Immunity, School of Medicine, Cardiff University, Henry Wellcome Building,

Heath Park, Cardiff CF14 4XN, UK. 7University of Naples, Department of Clinical Medicine and

Surgery, Division of Hematology, Via S. Pansini 5, Postal Code Naples, Italy.

[*]Corresponding Authors e-mail: [email protected] and

[email protected].

Nature Chemical Biology: doi:10.1038/nchembio.2208

mailto:[email protected]

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Chemical synthesis. General information. Unless otherwise noted, all reagents were purchased

from commercial sources and used without further purification. Microwave reactions were

performed on a Biotage® Initiator+ synthesizer. Drying of organic phases was performed, if not

otherwise indicated, by filtration through either a Biotage Universal Phase Separator or Isolute®

Phase Separator cartridge. Purification by preparative high-performance liquid chromatography

(HPLC) was performed using a Waters Sunfire C18-ODB column (30x100 mm, particle size 5

µm) and the following eluant at a flow rate of 40 mL/min: 20–100% acetonitile (CH3CN) in H2O

over 20 min and then 100% CH3CN over 3 min (both CH3CN and H2O contained 0.1%

trifluoroacetic acid), if not otherwise indicated. 1H,

13C and

19F NMR were measured on various

Bruker Avance spectrometers at room temperature and data are reported as follows: chemical

shift (p.p.m., units) from an internal standard, multiplicity (s = singlet, d = doublet, dd = double

doublet, t = triplet, q = quartet, m = multiplet, and br = broad), coupling constant J (Hz), and

integration. IR analyses were performed in transmission with a Bruker Hyperion FTIR

microscope (Hyperion 2000) coupled with a Bruker Vertex 70 FTIR spectrometer. High-

resolution mass analyses were performed on a Q Exactive Plus mass spectrometer (Thermo

Scientific), coupled to an Ultimate 3000 UHPLC, using electrospray ionization after separation

by liquid chromatography; the elemental composition was derived from the averaged mass

spectra acquired at the high resolution of about 30’000 and high mass accuracy <1 ppm was

obtained by using a lock mass. Optical rotations were measured on a Perkin-Elmer 241

polarimeter. Analytical HPLC was performed using a Waters X-Bridge C18 column (3 x 50 mm,

2. m) and the following conditions: 10–98% CH3CN in water over 8.6 min, then 100% CH3CN

over 1.4 min (CH3CN and water containing 0.1% trifluoroacetic acid) at a flow rate of 1.4


3

mL/min, UV detection at 215 and 254 nm. TLC analyses were performed on precoated plates

(silica gel 60 F254, Merck Darmstadt, Germany) and spots were visualized with UV light.

Methyl (S)-2-((2-((3-(trifluoromethoxy)phenyl)carbamoyl)pyrrolidine-1-carboxamido)-

methyl)benzoate (2)

A round-bottomed flask was charged with Boc-L-proline (4.00 g, 18.6 mmol) and 130 mL

dimethylformamide (DMF). To this solution was added 3-(trifluoromethoxy)aniline (3.62 g, 20.4

mmol), HBTU (8.46 g, 22.3 mmol) and Hünig’s base (11.4 mL, 65.0 mmol). After stirring at

room temperature for 18 h, the mixture was concentrated under reduced pressure. The residue

was diluted with ethylacetate (AcOEt), and the organic phase was subsequently washed with 1M

HCl, a saturated aqueous NaHCO3 solution and brine (100 mL each). The organic phase was

dried (Na2SO4), concentrated in vacuo, and the crude residue was purified by flash

chromatography on silica gel (0–50% AcOEt in cyclohexane (cHex)) to afford (S)-tert-butyl 2-

((3-(trifluoromethoxy)phenyl)carbamoyl)pyrrolidine-1-carboxylate (8) (4.7 g, 68% yield) as

colorless oil. TLC (cHex:AcOEt, 1:1 v/v): RF = 0.65; HPLC tR = 4.94 min (purity >99% at 215

nm); 1H NMR (600 MHz, DMSO-d6) δ 10.26 (d, J = 11.2 Hz, 1H), 7.79 (s, 1H), 7.50 (d, J = 8.1

Hz, 1H), 7.41 (m, 1H), 7.00 (d, J = 8.2 Hz, 1H), 4.24 (dd, J = 8.4, 3.3 Hz) and 4.17 (dd, J = 8.2,

4.6Hz, 1H; rotamers), 3.41 and 3.32 (m, 2H; rotamers), 2.23–2.12 (m, 1H), 1.90–1.84 (m, 2H),

1.78 (m, 1H), 1.38 (s, 3H), 1.24 (s, 6H); 13

C NMR (151 MHz, DMSO-d6) δ 172.0 and 171.6,

153.6 and 153.1, 148.5, 140.7 and 140.7, 130.4, 120.1 (q, J = 256 Hz), 117.8 and 117.7, 115.2


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and 115.1, 111.3 and 111.2, 78.7 and 78.5, 60.5 and 60.1, 46.7 and 46.5, 30.9 and 30.1, 28.1 and

27.8, 23.9 and 23.4 (two rotamers); 19

F NMR (376 MHz, DMSO-d6) δ –56.7, –56.8 (rotamers);

IR (neat): 3284, 3109, 3007, 1699, 1664, 1615, 1568, 1496, 1257 cm-1

; HRMS (m/z): [M+H]+

calcd for C17H21F3N2O4, 375.1526; found, 375.1529.

To a solution of (S)-tert-butyl 2-((3-(trifluoromethoxy)phenyl)carbamoyl)pyrrolidine-1-

carboxylate (8) (4.00 g, 10.7 mmol) in CH2Cl2 (30 mL) was added trifluoroacetic acid (8.23 ml,

107 mmol). After stirring at room temperature for 3 h, the reaction mixture was diluted with

MeOH and then volatiles were evaporated under reduced pressure. The reaction mixture was

taken up in toluene and concentrated again in vacuo. This step was repeated three times to

azeotropically remove excess of trifluoroacetic acid. The thus obtained crude (S)-N-(3-

(trifluoromethoxy)phenyl)pyrrolidine-2-carboxamide trifluoroacetate salt (9) (4.1 g, 99% yield;

yellowish oil) was used without further purification in the next reaction step. HPLC tR = 2.79 min

(purity = 99% at 215 nm); 1H NMR (600 MHz, DMSO-d6) δ 11.11 (s, 1H), 9.89 (br s, 1H), 8.83

(br s, 1H), 7.79 (s, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.47 (t, J = 8.2 Hz, 1H), 7.08 (m, 1H), 4.43 (t, J

= 7.6 Hz, 1H), 3.28 (m, 2H), 2.39 (m, 1H), 2.1–1.9 (m, 3H); 13

C NMR (151 MHz, DMSO-d6) δ

167.9, 159.0 (q, J = 32 Hz), 148.6, 139.9, 130.7, 120.1 (q, J = 257 Hz), 118.2, 116.2, 59.8, 45.8,

29.6, 23.6; HRMS (m/z): [M+H]+ calcd for C12H13F3N2O2, 275.1002; found, 275.1004.

To an ice-cooled solution of methyl 2-(aminomethyl)benzoate (2.00 g, 12.1 mmol) in CH2Cl2 (50

mL) was added a saturated aqueous NaHCO3 solution (50 mL) and triphosgene (1.26 g, 4.24

mmol). The reaction mixture was stirred at 5 °C for 1 h. The organic phase was then separated

and the water phase was extracted with CH2Cl2. The combined organics were dried (Na2SO4),

filtered and evaporated to dryness to afford the crude methyl 2-(isocyanatomethyl)benzoate (10)

(1.87 g, 81% yield) as brownish solid, which was used without further purification in the next

reaction step.


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A round-bottomed flask was charged with (S)-N-(3-(trifluoromethoxy)phenyl)pyrrolidine-2-

carboxamide trifluoro acetate salt (9) (2.50 g, 6.44 mmol) and 130 mL tetrahydrofurane (THF).

To this solution was added triethylamine (1.80 mL, 12.9 mmol) and methyl 2-(isocyanato-

methyl)benzoate (10) (1.23 g, 6.44 mmol). The reaction mixture was stirred at room temperature

for 1 h. After removal of volatiles under reduced pressure, the mixture was diluted with AcOEt

(150 mL) and the organic layer was washed with 1M HCl (70 mL), dried (Na2SO4) and

evaporated in vacuo. Purification by flash chromatography on silica gel (0–50% AcOEt in cHex)

gave methyl (S)-2-((2-((3-(trifluoromethoxy)phenyl)carbamoyl)pyrrolidine-1-carboxamido)-

methyl)benzoate (2) (2.37 g, 80% yield) as a brown solid. Further purification of this material

was performed by preparative HPLC. The combined fractions containing the desired product

were concentrated in the presence of solid K2CO3 and the water phase was extracted twice with

CH2Cl2. The combined organics were dried and evaporated in vacuo to yield a white solid. TLC

(cHex:AcOEt, 1:1 v/v): RF = 0.35; HPLC tR = 4.82 min (purity >99%, determined at 215 nm);

[ ]25

D (deg cm3 g

-1 dm

-1) = –71.0 (c =1 g cm

-3 in MeOH);

1H NMR (600 MHz, DMSO-d6) δ

10.22 (s, 1H), 7.85–7.80 (m, 2H), 7.55–7.46 (m, 3H), 7.41 (t, J = 8.2 Hz, 1H), 7.35 (m, 1H), 6.99

(d, J = 8.1 Hz, 1H), 6.81 (t, J = 5.8 Hz, 1H), 4.61 (dd, J = 16.9, 5.9 Hz, 1H), 4.54 (dd, J = 16.9,

5.9 Hz, 1H), 4.37 (dd, J = 8.4, 2.8 Hz, 1H), 3.82 (s, 3H), 3.54 (m, 1H), 3.38 (m, 1H), 2.13 (m,

1H), 2.01–1.90 (m, 3H); 13

C NMR (151 MHz, DMSO-d6) δ 172.3, 167.2, 156.5, 148.5, 142.1,

140.8, 132.1, 130.3, 129.9, 128.2, 127.7, 126.4, 120.1 (q, J = 256 Hz), 117.7, 115.1, 111.2, 60.4,

52.0, 46.1, 41.8, 29.8, 24.2; 19

F NMR (376 MHz, DMSO-d6) δ –56.62; IR (neat): 3275, 3148,

2954, 1714, 1632, 1611, 1523, 1492, 1440, 1256, 1218, 1163, 1085 cm-1

; HRMS (m/z): [M+H]+

calcd for C22H22F3N3O5, 466.1584; found, 466.1585; analysis (% calcd, % found for

C22H22F3N3O5): C (56.77, 56.52), H (4.76, 4.92), N (9.03, 8.84).


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Characterization of fragment hit (3).

5-(Benzyloxy)-1H-indole-2-carboxamide (beige solid). HPLC: tR = 3.74 min; 1H NMR (600

MHz, DMSO-d6): δ 11.39 (br s, 1 H), 7.91 (br s, 1 H), 7.48 (d, J = 7.3 Hz, 2 H), 7.39 (t, J = 7.5

Hz, 2 H), 7.36–7.29 (m, 3 H), 7.17 (d, J = 2.0 Hz, 1 H), 7.03 (d, J = 1.4 Hz, 1H), 6.92 (dd, J =

8.8, 2.4 Hz, 1 H), 5.10 (s, 2 H); 13

C NMR (151 MHz, DMSO-d6): δ 162.8, 152.7, 137.6, 132.2,

131.9, 128.3, 127.6, 127.4, 114.9, 113.1, 103.6, 102.8, 69.6; IR (neat): 3422, 1672, 1608, 1528,

1449, 1429, 1281, 1022, 737, 695 cm-1

; HRMS (m/z): [M+H]+ calcd for C17H21F3N2O4,

267.1128; found, 267.1125.

3-(((2-Carbamoyl-1H-indol-5-yl)oxy)methyl)benzoic acid (4)

A mixture of ethyl 5-hydroxy-1H-indole-2-carboxylate [CAS 24985-85-1] (500 mg, 2.44 mmol),

tert-butyl 3-(bromomethyl)benzoate [CAS 126062-63-3; Enamine LLC, USA] (727 mg, 2.68

mmol) and potassium carbonate (673 mg, 4.87 mmol) in DMF (5 mL) was stirred in a round-

bottomed flask at room temperature for 60 h. The reaction mixture was diluted with EtOAc and

the organic phase was washed with water and brine, dried and concentrated under reduced


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pressure. Purification was performed by preparative HPLC (column: Waters Sunfire, C18-ODB,

5 µm, 30x100 mm; flow: 40 ml/min; eluent: 20–100% acetonitile/H2O/20 min, 100% CH3CN/3

min; both CH3CN and H2O contained 0.1% trifluoroacetic acid). Fractions containing the desired

product were combined and freeze-dried in high vacuo to afford ethyl 5-((3-(tert-

butoxycarbonyl)benzyl)oxy)-1H-indole-2-carboxylate (11) (530 mg, 55% yield) as a white solid.

HPLC tR = 6.30 min (purity >99% at 215 nm); 1H NMR (400 MHz, DMSO-d6) δ 11.77 (br s,

1H), 8.01 (s, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.38

(d, J = 8.9 Hz, 1H), 7.23 (s, 1H), 7.08–6.99 (m, 2H), 5.18 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.56

(s, 9H), 1.34 (t, J = 7.1 Hz, 3H); 13

C NMR (101 MHz, DMSO-d6) δ 165.2, 161.6, 153.2, 138.5,

133.3, 132.4, 131.9, 129.2, 128.8, 128.4, 128.1, 127.4, 117.0, 114.0, 107.7, 104.1, 81.3, 69.5,

60.8, 28.2, 14.7; IR (neat): 3289, 2978, 1709, 1692, 1530, 1386, 1366, 1179, 1163 cm-1

; HRMS

(m/z): [M+NH4]+

calcd for C23H29O5N2, 413.2071; found, 413.2077.

To a stirred solution of ethyl 5-((3-(tert-butoxycarbonyl)benzyl)oxy)-1H-indole-2-carboxylate

(11) (460 mg, 1.16 mmol) in EtOH (10 mL) was added water (2 mL) and KOH (131 mg, 2.33

mmol). After stirring at room temperature for 18 h, another aliquot of KOH (131 mg, 2.36 mmol)

dissolved in water (2 mL) was added and stirring was continued for 18 h. The reaction mixture

was then neutralized by addition of 1N HCl and concentrated to remove EtOH. The residue was

taken up in 1N HCl and extracted twice with EtOAc. The combined organic layers were washed

with brine, dried and concentrated under reduced pressure. Purification was peformed by

preparative HPLC to afford 5-((3-(tert-butoxycarbonyl)benzyl)oxy)-1H-indole-2-carboxylic acid

(12) (332 mg, 77% yield) as a white solid. HPLC tR = 5.24 min (purity >99% at 215 nm); 1H

NMR (400 MHz, DMSO-d6) δ 11.64 (br s, 1H), 8.00 (s, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.72 (d, J

= 7.7 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.07–

6.92 (m, 2H), 5.18 (s, 2H), 1.56 (s, 9H). 13

C NMR (101 MHz, DMSO-d6) δ 165.2, 163.1, 153.1,


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138.5, 133.2, 132.4, 131.9, 129.2, 128.7, 128.4, 127.5, 116.6, 113.9, 107.4, 104.2, 81.3, 69.5,

28.2; IR (neat) 3280, 2975, 1692 (br), 1535, 1434, 1369, 1254, 1218, 1163, 846, 750 cm-1

;

HRMS (m/z): [M+NH4]+


To a solution of 5-((3-(tert-butoxycarbonyl)benzyl)oxy)-1H-indole-2-carboxylic acid (12) (280

mg, 0.762 mmol) and Hünig’s base (0.399 mL, 2.29 mmol) in DMF (3 mL) was added HATU

(348 mg, 0.915 mmol) and NH4Cl (82 mg, 1.52 mmol). The resulting mixture was then stirred at

rt for 2h. The mixture was directly purified by preparative HPLC. Fractions containing the

desired product were combined and freeze-dried in high vacuo to deliver tert-butyl 3-(((2-

carbamoyl-1H-indol-5-yl)oxy)methyl)benzoate (13) (226 mg, 80% yield) as an off-white solid.

HPLC tR = 4.87 min (purity >99% at 215 nm); 1H NMR (400 MHz, DMSO-d6) δ 11.39 (br s,

1H), 8.00 (s, 1H), 7.93–7.83 (m, 2H), 7.72 (d, J = 7.7 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.37–7.28

(m, 2H), 7.18 (d, J = 2.2 Hz, 1H), 7.03 (s, 1H), 6.94 (dd, J = 8.9, 2.4 Hz, 1H), 5.18 (s, 2H), 1.56

(s, 9H); 13

C NMR (101 MHz, DMSO-d6) δ 165.3, 163.2, 152.9, 138.7, 132.7, 132.4 (2 C), 131.9,

129.1, 128.7, 128.4, 127.8, 115.3, 113.6, 104.2, 103.3, 81.3, 69.5, 28.2; IR (neat): 3420, 3360,

3251, 3052, 2927, 1702, 1672, 1605, 1542, 1495, 1236, 1172, 841, 757 cm-1

; HRMS (m/z):

[2M+H]+


To a solution of tert-butyl 3-(((2-carbamoyl-1H-indol-5-yl)oxy)methyl)benzoate (13) (180 mg,

0.491 mmol) in CH2Cl2 (4 mL) was added trifluoroacetic acid (1 mL) and the resulting mixture

was stirred at room temperature for 60 min. After removal of volatiles under reduced pressure,

the residue was purified by preparative HPLC to afford 3-(((2-carbamoyl-1H-indol-5-

yl)oxy)methyl)benzoic acid (4) (114 mg, 74% yield) as a white solid. HPLC tR = 2.87 min (purity

>99% at 215 nm); 1H NMR (400 MHz, DMSO-d6) δ 11.38 (br s, 1H), 8.05 (s, 1H), 7.94–7.87 (m,

2H), 7.73 (d, J = 7.7 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.37–7.25 (m, 2H), 7.18 (d, J = 2.3 Hz,

1H), 7.03 (d, J = 1.5 Hz, 1H), 6.94 (dd, J = 8.9, 2.4 Hz, 1H), 5.19 (s, 2H); 13

C NMR (101 MHz,


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DMSO-d6) δ 167.3, 162.9, 152.6, 138.3, 132.3, 132.0, 131.0, 128.8, 128.7, 128.3, 127.5, 115.0,

113.8, 109.9, 103.0, 69.2; IR (neat): 3434, 3320, 3214, 2931, 2529, 1883, 1690, 1641, 1575,

1491, 1434, 1313, 1284, 1235, 1201, 1167, 1121, 840, 804, 762, 747, 692 cm-1

; HRMS (m/z):

[M–H]– calcd for C17H13O4N2, 309.0881; found, 309.0885; analysis (% calcd, % found for

C17H14N2O4.0.5 H2O): C (63.94, 63.63), H (4.73, 4.44), N (8.77, 8.62).

1-(2-((1R,3S,5R)-3-(((R)-1-(3-Chloro-2-fluorophenyl)ethyl)carbamoyl)-2-

azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (6)

To a solution of 1H-pyrazolo[3,4-c]pyridine [CAS 271-47-6 ] (4.00 g, 33.6 mmol) in DMF (50

mL) were added iodine (12.8 g, 50.4 mmol) and potassium hydroxide (4.70 g, 84.0 mmol). The

reaction mixture was stirred at room temperature for 16 h. The mixture was then diluted with

10% aqueous sodium thiosulfate solution (20 mL) and water (20 mL), and the organic layer was

extracted with EtOAc (3x). The combined organics were washed with brine, dried and

concentrated to give 3-iodo-1H-pyrazolo[3,4-c]pyridine (14) (6.24 g, 68% yield) as a beige solid.

tR HPLC = 0.46 min (>99% purity at 215 nm); 1H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H),

8.30 (d, J = 5.6 Hz, 1H), 7.45 (d, J = 5.6 Hz, 1H); 13

C NMR (101 MHz, DMSO-d6) δ 139.4,

137.8, 135.8, 131.0, 114.5, 93.2; IR (neat): 3066, 2730, 2700,1509, 1455, 1328, 1246, 1030, 913,

810, 651 cm-1

; HRMS (m/z): [M+H]+ calcd for C6H5N3I, 245.9523; found, 245.9524.


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To a suspension of 3-iodo-1H-pyrazolo[3,4-c]pyridine (14) (6.24 g, 22.9 mmol) and potassium

carbonate (7.29 g, 52.7 mmol) in CH3CN (50 mL) was added dropwise tert-butyl bromoacetate

(4.06 mL, 27.5 mmol) at room temperature. The reaction mixture was heated to reflux for 2 h

with stirring and then cooled to ambient temperature. The precipitate was filtered off, washed

with CH3CN and the combined filtrates were concentrated under reduced pressure. The oily

residue was purified by flash chromatography on silica gel (25–50% EtOAc in cHex) to afford

tert-butyl 2-(3-iodo-1H-pyrazolo[3,4-c]pyridin-1-yl)acetate (15) (3.77 g, 43.5% yield) as a brown

solid. 1H NMR (400 MHz, DMSO-d6) δ 9.17 (d, J = 1.1 Hz, 1H), 8.36 (d, J = 5.6 Hz, 1H), 7.48

(dd, J = 5.6, 1.2 Hz, 1H), 5.48 (s, 2H), 1.42 (s, 9H).

A mixture of tert-butyl 2-(3-iodo-1H-pyrazolo[3,4-c]pyridin-1-yl)acetate (15) (3.76 g, 10.5

mmol), Zn(CN)2 (1.35 g, 11.5 mmol), Pd(dppf)Cl2 (0.85 g, 1.05 mmol), Pd2(dba)3 (0.96 g, 1.05

mmol), water (4 mL) and DMF (30 mL) was purged with argon and stirred at 100 °C for 16 h.

After cooling to room temperature, the mixture was diluted with EtOAc. The organic phase was

washed with water (1x), a saturated aqueous NaHCO3 solution (2x) and brine, dried and

concentrated. The oily residue was purified by flash chromatography on silica gel (0–50% cHex

in EtOAc) to deliver tert-butyl 2-(3-cyano-1H-pyrazolo[3,4-c]pyridin-1-yl)acetate (16) (1.25 g,

46% yield) as a brown solid. HPLC tR = 3.04 min (purity >99% at 215 nm); 1H NMR (400 MHz,

DMSO-d6) δ 9.42 (s, 1H), 8.52 (d, J = 5.7 Hz, 1H), 7.97 (d, J = 5.7 Hz, 1H), 5.67 (s, 2H), 1.43 (s,

9H); 13

C NMR (101 MHz, DMSO-d6) δ 166.5, 141.8, 137.2, 137.1, 128.4, 117.5, 113.2, 83.3,

52.6, 28.0; IR (neat): 3052, 2987, 2236, 1740, 1462, 1369, 1245, 1154, 1124, 846, 747 cm-1

;

HRMS (m/z): [M+H]+ calcd for C13H15O2N4, 259.1190; found, 259.1192.

A solution of tert-butyl 2-(3-cyano-1H-pyrazolo[3,4-c]pyridin-1-yl)acetate (16) (663 mg, 2.57

mmol) in trifluoroacetate (6 mL) was heated at 140 °C under microwave irradiation for 90 min.

The reaction mixture was concentrated under reduced pressure. The residue was suspended in


11

MeOH and volatiles were removed in vacuo to afford 2-(3-carbamoyl-1H-pyrazolo[3,4-

c]pyridin-1-yl)acetic acid trifluoroacetate salt (17) (860 mg, 90% yield) as a brown solid, which

was used without further purification in the next reaction step. HPLC tR = 0.21 min (purity >99%

at 215 nm); 1H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 1H), 8.39 (d, J = 5.6 Hz, 1H), 8.08 (d, J =

5.6 Hz, 1H), 7.92 (s, 1H), 7.57 (s, 1H), 5.52 (s, 2H); 13

C NMR (101 MHz, DMSO-d6) δ 169.5,

163.4, 140.5, 138.5, 136.0, 126.3, 115.7, 51.4; 19

F NMR (376 MHz, DMSO-d6) δ –73.5; IR

(neat): 3527, 3256, 3118, 1702, 1636, 1503, 1432, 1382, 1317, 1268, 1187, 739 cm-1

; HRMS

(m/z): [M+H]+ calcd for C9H9N4O3, 221.0669; found, 221.0669.

A round-bottomed flask was charged with (1R,3S,5R)-2-(tert-butoxycarbonyl)-2-aza-

bicyclo[3.1.0]hexane-3-carboxylic acid [CAS 197142-34-0; Neptune Technotrade, USA] (12.0 g,

52.8 mmol) and 300 mL CH2Cl2. To this solution was added HBTU (24.0 g, 63.4 mmol), Hünig’s

base (32.3 ml, 185 mmol) and (R)-1-(3-chloro-2-fluorophenyl)ethanamine HCl salt [CAS

1253792-97-0; APAC Pharmaceutical, LLC, USA] (12.2 g, 58.1 mmol). After stirring at room

temperature for 18h, the reaction mixture was diluted with CH2Cl2 and the organic layer was

washed with saturated aqueous NaHCO3 solution (50 mL) and 1M HCl (50 mL). The organic

phase was dried and concentrated under reduced pressure. Purification by flash chromatography

on silica gel (25–33% AcOEt in cHex) afforded (1R,3S,5R)-tert-butyl 3-((R)-1-(3-chloro-2-

fluorophenyl)ethylcarbamoyl)-2-azabicyclo[3.1.0]hexane-2-carboxylate benzoate (18) (19.0 g,

92% yield) as a colorless oil. TLC (cHex:AcOEt, 1:1 v/v): RF = 0.57; HPLC tR = 4.71 min

(purity = 98% at 215 nm); 1H NMR (500 MHz, DMSO-d6) δ 8.34 (d, J = 7.2 Hz, 1H), 7.50–7.15

(m, 2H), 7.18 (m, 1H), 5.13 (br m, 1H), 3.88 (br m, 1H), 3.32 (br m, 1H), 2.24 (br m, 1H), 2.01

(m, 1H), 1.63–1.08 (m, 13H), 0.73 (m, 1H), 0.39 (m, 1H); 13

C NMR (126 MHz, DMSO-d6) δ

170.6, 158.3 and 158.0, 154.5 (d, J = 247 Hz), 133.4, 128.8, 126.6, 125.1, 119.6, 78.9, 61.1, 42.4,

37.7, 33.3, 27.9, 21.0, 15.0, 14.1; 19

F NMR (376 MHz, DMSO-d6) δ –122.11, –122.65 (two


12

rotamers); IR (neat): 3300, 3075, 2978, 1685, 1547, 1455, 1177, 1134 cm-1

; HRMS (m/z):

[M+H]+ calcd for C19H24ClFN2O3, 383.1532; found, 383.1537.

To (1R,3S,5R)-tert-butyl 3-((R)-1-(3-chloro-2-fluorophenyl)ethylcarbamoyl)-2-aza-

bicyclo[3.1.0]hexane-2-carboxylate (18) (19.0 g, 49.6 mmol) in CH2Cl2 (350 mL) was added

trifluoroacetic acid (38.2 mL) and the reaction mixture was stirred at room temperature for 60 h.

The mixture was then diluted with MeOH and volatiles were evaporated under reduced pressure.

Excess of trifluoroacetic acid was removed azeotropically by repeated evaporation of a toluene

solution under reduced pressure to afford the crude (1R,3S,5R)-N-((R)-1-(3-chloro-2-fluoro-

phenyl)ethyl)-2-azabicyclo[3.1.0]hexane-3-carboxamide trifluoroacetate salt (19) (21.7 g, 90%

yield) as yellowish oil. HPLC tR = 2.39 min (purity = 98% at 215 nm); 1H NMR (500 MHz,

DMSO-d6) δ 9.01 (d, J = 7.4 Hz, 3H), 7.51 (m, 1H), 7.38 (m, 1H), 7.24 (m, 1H), 5.16 (m, 1H),

3.98 (dd, J = 10.7, 7.8 Hz, 1H), 3.27 (m, 2H), 2.49 (m, 1H; overlap with solvent), 2.00 (m, 1H),

1.81 (m, 1H), 1.42 (d, J = 7.0 Hz, 3H), 0.86 (m, 1H), 0.78 (m, 1H); 13

C NMR (126 MHz, DMSO-

d6) δ 165.9, 154.7 (d, J = 248 Hz), 132.6 (d, J = 14 Hz), 129.3, 126.4, 125.4, 119.7 (d, J = 17 Hz),

55.8, 43.3, 34.2, 29.9, 20.6, 14.9, 5.5; IR (neat): 3259, 3076, 2984, 1668, 1561, 1458 cm-1

;

HRMS (m/z): [M+H]+ calcd for C14H16ClFN2O, 283.1008 ; found, 283.1010.

A round-bottomed flask was charged with (1R,3S,5R)-N-((R)-1-(3-chloro-2-fluorophenyl)ethyl)-

2-azabicyclo[3.1.0]hexane-3-carboxamide trifluoroacetate salt (19) (21.7 g, 49.2 mmol) and

DMF (300 mL). To this solution was added 2-(3-carbamoyl-1H-pyrazolo[3,4-c]pyridin-1-

yl)acetic acid (17) (16.5 g, 49.2 mmol), HBTU (22.4 g, 59.1 mmol) and Hünig’s base (40.0 mL,

229 mmol). After stirring at room temperature for 18 h, the solvent (DMF) was evaporated in

vacuo and the residual oil was diluted with CH2Cl2 to afford a white precipitate. The solid was

filtered off, washed with CH2Cl2 and dried at 50 °C in vacuo for 1h to give the crude product as a

beige solid (22.7g). This material was then dissolved in CH2Cl2:MeOH, 9:1 (v/v), the organics


13

were washed with a saturated aqueous NaHCO3 solution (250 mL), dried and evaporated in

vacuo to afford an off-white solid (19.0 g (80% yield); HPLC purity >99% at 215 nm). To this

material (10.0 g) was added AcOEt (125 mL) and the mixture was gradually heated up to 55 °C

at a rate of 1°C/min with stirring (PolyBLOCK®

reactor, HEL Inc., USA) to provide a solution.

After 30 min of stirring at 55 °C, the mixture was cooled at a rate of 0.5°C/min to 5°C where it

was held for 30 minutes. This procedure was repeated several times by gradually reducing the

maximum heat temperature by about 2 °C/cycle, and the resulting solid was collected by vacuum

filtration. Drying at 40 °C in vacuo for 72 h provided 1-(2-((1R,3S,5R)-3-(((R)-1-(3-chloro-2-

fluorophenyl)ethyl)carbamoyl)-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)-1H-pyrazolo[3,4-

c]pyridine-3-carboxamide (6) (9.2 g) as white powder. Melting point 205.9 °C; TLC

(CH2Cl2:MeOH, 9:1 v/v): RF = 0.60; HPLC tR = 2.56 min (purity >99% at 215 nm); [ ]25

D (deg

cm3g

-1 dm

-1) = –95.7 (c = 1 g cm

-3 in MeOH);

1H NMR (500 MHz, DMSO-d6) δ 9.14 (s, 1H),

8.39 (d, J = 7.7, 1H), 8.38 (d, J = 5.6 Hz, 1H), 8.07 (d, J = 5.6 Hz, 1H), 7.88 (s, 1H), 7.54 (s, 1H),

7.43 (t, J = 6.9 Hz, 1H), 7.28 (t, J = 6.9 Hz, 1H), 7.09 (t, J = 7.9 Hz, 1H), 5.90 (d, J = 17.4 Hz,

1H), 5.62 (d, J = 17.3 Hz, 1H), 5.05 (m, 1H), 4.30 (dd, J = 9.0, 4.7 Hz, 1H), 3.71 (m, 1H), 2.25

(m, 1H), 2.14 (m, 1H), 1.89 (m, 1H), 1.33 (d, J = 7.0 Hz, 3H), 1.03 (m, 1H), 0.79 (m, 1H); 13

C

NMR (126 MHz, DMSO-d6) δ 170.0, 166.0, 163.0, 154.4 (d, J = 246 Hz), 140.0, 138.3, 137.6,

135.8, 133.3 (d, J = 14 Hz), 128.8, 126.0 (d, J = 4 Hz), 125.9, 125.2 (d, J = 4 Hz), 119.4 (d, J =

18 Hz), 115.2, 63.2, 51.9, 42.7, 36.8, 31.6, 21.1, 18.1, 17.6; 19

F NMR (376 MHz, DMSO-d6) δ –

122.6; IR (neat): 3406, 3239, 3204, 1682, 1652, 1610, 1552, 1467, 1455 cm-1

; HRMS (m/z):

[M+H]+ calcd for C23H22ClFN6O3, 485.1498 ; found, 485.1498; analysis (% calcd, % found for

C23H22ClF3N6O3): C (56.97, 56.64), H (4.57, 4.42), N (17.33, 17.28).


14

1-(2-((1R,3S,5R)-3-((6-Bromopyridin-2-yl)carbamoyl)-2-azabicyclo[3.1.0]hexan-2-yl)-2-

oxoethyl)-1H-indazole-3-carboxamide (7)

To a suspension of 1H-indazole-3-carboxylic acid amide [CAS 90004-04-9; J&W Pharmalab]

(12.5 g, 78.0 mmol) and potassium carbonate (25.7 g, 186 mmol) in CH3CN (375 mL) was added

dropwise tert-butyl bromoacetate (13.8 mL, 93.0 mmol) at room temperature. The reaction

mixture was heated to reflux for 4 h with stirring and then cooled to ambient temperature. The

precipitate was filtered off, washed with CH3CN and the combined filtrates were concentrated

under reduced pressure. The crude solid was suspended in Et2O and filtered to afford tert-butyl 2-

(3-carbamoyl-1H-indazol-1-yl)acetate (20) (20.3 g, 95% yield) as a white solid, which was used

without further purification in the next reaction step. HPLC tR = 1.72 min (99% purity at 254 nm)

(Waters XBridge C18, 2.5 m, 3x30mm, 10-98% CH3CN in water water over 3 min, then 98%

CH3CN over 0.5 min (CH3CN and water containing 0.1% trifluoroacetic acid) at a flow rate of

1.4 mL/min and 40 °C); 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 8.2 Hz, 1H), 7.72 (br s,

1H), 7.71 (d, J = 8.5 Hz, 1H), 7.47 (m, 1H), 7.46 (br s, 1H), 7.29 (t, J = 7.7 Hz, 1H), 5.37 (s, 2H),

1.42 (s, 9H); MS (UPLC/MS): 276.3 [M+H]+, 293.3 [M+18]

+.

A solution of tert-butyl 2-(3-carbamoyl-1H-indazol-1-yl)acetate (20) (40.6 g, 147 mmol) and

trifluoroacetic acid (114 mL, 1.48 mol) in CH2Cl2 (400 mL) was stirred at room temperature for

24 h. The reaction mixture was concentrated under reduced pressure. The residue was suspended

in Et2O and the resulting white suspension was stirred for 1 h, the precipitate was filtered off and


15

washed with Et2O to afford (3-carbamoyl-indazol-1-yl)-acetic acid (21) (34.4 g, 100% yield) as a

white solid, which was used without further purification in the next reaction step. UPLC tR =

1.24 min (purity = 97% at 210–440 nm) (Waters UPLC Acquity HSS T3 column (1.8µm,

2.1x50mm); eluent A: water+0.05% HCOOH+3.75 mMol ammonium acetate, eluent B:

CH3CN+0.04% HCOOH; gradient: 5–98% B over 9.4 min at a flow rate of 1.0 mL/min at 60°C;

1H NMR (600 MHz, DMSO-d6) δ 13.3 (br s, 1H), 8.20 (d, J = 8.2 Hz, 1H), 7.74 (d, J = 8.3 Hz,

1H), 7.73 (br s, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.38 (t, J = 7.45 Hz, 1H), 5.37 (s, 2H);

13C NMR (150 MHz, DMSO-d6) δ 169.4, 163.7, 141.4, 137.9, 126.7, 122.3, 122.2, 121.9, 110.45,

50.3; IR (neat): 3434, 3310, 2467, 1892, 1711, 1573, 1492, 1446, 1420, 1332, 1308, 1256, 1186,

1019, 819, 791, 773, 750 cm-1

. HRMS (m/z): [M+H]+ calcd for C10H10O3N3, 220.0717; found,

220.0717.

A round-bottomed flask was charged with (1R,3S,5R)-2-(tert-butoxycarbonyl)-2-aza-

bicyclo[3.1.0]hexane-3-carboxylic acid [CAS 197142-34-0; Neptune Technotrade, USA] (17.5 g,

77.0 mmol) and CH2Cl2 (420 mL). 1-Chloro-N,N,2-trimethylpropenylamine (11.2 mL, 85.0

mmol) was added at 0 °C under a nitrogen atmosphere. Formation of the acid chloride

intermediate was monitored by TLC after quenching of an aliquot with MeOH. After completion

of the reaction (2 h), 2-amino-6-bromopyridine (14.7 g, 85.0 mmol) was added at 0 °C, followed

by DIPEA (26.9 mL, 154 mmol), and the reaction mixture was further stirred for 24 h at room

temperature. The reaction mixture was concentrated under reduced pressure, MeOH was added

and volatiles were evaporated. CH2Cl2 was added and the resulting suspension was stirred for 1

h, the precipitate was filtered off and washed with CH2Cl2 to afford a first crop of (1R,3S,5R)-3-

(6-bromo-pyridin-2-ylcarbamoyl)-2-aza-bicyclo[3.1.0]hexane-2-carboxylic acid tert-butyl ester

(22) (17 g). The filtrate was concentrated under reduced pressure, suspended in CH2Cl2 and

filtered off to afford a second crop (5.1 g). The combined products (22.1 g, 74% yield; white


16

solid) were used without further purification in the next step. TLC (EtOAc): RF = 0.85; HPLC tR

= 5.27 min (purity = 97.5% at 210–450 nm) (Waters UPLC Acquity HSS T3 column (1.8µm,



1H NMR (600 MHz, DMSO-d6) δ 10.8 (m, 1H), 8.11 (m, 1H), 7.76 (m, 1H), 7.35 (d, J = 7.7 Hz,

1H), 4.13 (m, 1H), 3.42 (m, 1H), 2.30 (m, 1H), 2.11 (m, 1H), 1.59 (m, 1H), 1.41 (s, 3H), 1.25 (s,

6H), 0.74 (m, 1H), 0.40 (m, 1H); 13

C NMR (150 MHz, DMSO-d6) (rotamers observed at room

temperature) δ 171.1, 170.7, 154.8, 153.9, 152.1, 141.5, 138.8, 122.95, 112.2, 79.1, 61.0, 60.3,

37.4, 32.6, 31.95, 28.1, 27.75, 15.3, 14.9, 14.1, 14.0; IR (neat): 3212, 3117, 3082, 2975, 2938,

2870, 1714, 1662, 1594, 1568, 1528, 1476, 1430, 1394, 1377, 1366, 1301, 1193, 1153, 1127, 803

cm-1

; HRMS (m/z): [M+H]+ calcd for C16H21N3O3

79Br, 382.0761; found, 382.0761.

To (1R,3S,5R)-3-(6-bromo-pyridin-2-ylcarbamoyl)-2-aza-bicyclo[3.1.0]hexane-2-carboxylic acid

tert-butyl ester (22) (34.0 g, 89.0 mmol) in CH2Cl2 (500 mL) was added trifluoroacetic acid (68.4

mL, 888 mmol) and the reaction mixture was stirred at room temperature for 24 h. Volatiles were

evaporated under reduced pressure. Excess of trifluoroacetic acid was removed by repeated

evaporation of an EtOAc solution under reduced pressure to afford (1R,3S,5R)-2-aza-

bicyclo[3.1.0]hexane-3-carboxylic acid (6-bromo-pyridin-2-yl)-amide trifluoroacetate salt (23)

(33.8 g, 96% yield) as white solid. HPLC tR = 0.87 min (purity = 100% at 220 and 254 nm)

(Waters XBridge C18, 2.5 m, 3x30mm, 10–98% CH3CN in water water over 3 min, then 98%

CH3CN over 0.5 min (CH3CN and water containing 0.1% trifluoroacetic acid) at a flow rate of

1.4 mL/min and 40 °C); 1H NMR (400 MHz, DMSO-d6) δ 11.3 (br s, 1H), 9.47 (br m, 2H), 8.05

(d, J = 7.9 Hz, 1H), 7.83 (t, J = 7.9 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 4.15 (m, 1H), 3.38 (m, 1H;


17

overlap with H2O), 2.62 (dd, J = 12.7, 7.7 Hz, 1H), 2.10 (m, 1H), 1.83 (m, 1H), 0.84 (m, 2H);

MS: 282.1/284.1 [M+H]+, 563.1/565.2 [2M+H]

+.

A round-bottomed flask was charged with (3-carbamoyl-indazol-1-yl)-acetic acid (21) (11.1 g,

50.5 mmol), CH2Cl2 (600 mL) and DIPEA (13.0 mL, 76.0 mmol). To this solution was added a

solution of propylphosphonic anhydride in EtOAc (50 wt. %, 32.7 mL, 55.5 mmol). After stirring

at room temperature for 20 min, a solution of (1R,3S,5R)-2-aza-bicyclo[3.1.0]hexane-3-

carboxylic acid (6-bromo-pyridin-2-yl)-amide trifluoroacetate salt (23) (20.0 g, 50.5 mmol) was

added, follwed by dropwise addition of DIPEA (13 mL, 76 mmol) in CH2Cl2 (400 mL). After

stirring at room temperature for 4 h, the reaction mixture was poured into a saturated aqueous

solution of NaHCO3 and extracted with CH2Cl2. The combined organics were dried (Na2SO4),

filtered and concentrated under reduced pressure. MeOH (80 mL) was added and the suspension

was vigorously stirred for 1 h, followed by filtration to afford a beige solid (first cropof 10.2 g).

The filtrate was concentrated under reduced pressure, CH2Cl2 and 1 N aqueous HCl were added

and the two layers were separated. The organic phase was washed with brine, dried (Na2SO4),

filtered and concentrated. The crude residue was purified by flash chromatography on silica gel

(0–4% of MeOH in CH2Cl2) to afford a second crop of product (4 g). A round-bottomed flask

was charged with the combined products and MeOH (100 mL), and the mixture was vigourously

stirred at room temperature-. A fine suspension was formed which was filteredand dried under

high vacuum at 40 °C for 24 h to afford 1-(2-((1R,3S,5R)-3-((6-bromopyridin-2-yl)carbamoyl)-2-

azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)-1H-indazole-3-carboxamide (7) as a white solid (12.4

g, 51% yield). Melting point 228.5 °C; TLC (CH2Cl2/MeOH 95:5 v/v): RF = 0.4; HPLC tR = 3.96

min (purity = 100% at 210-450 nm) (Waters UPLC Acquity HSS T3 column (1.8µm,




18

[ ]25

D (deg cm3g

-1 dm

-1) = –139.1 (c = 1 g cm

-3 in MeOH/CH2Cl2 1:1 (v/v));

1H NMR (600 MHz,

DMSO-d6) (95/5 ratio of rotamers; coalescence observed at 100 °C; signals of the major rotamer

are given) δ 10.8 (s, 1H), 8.18 (d, J = 8.1 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.72 (t, J = 8.1 Hz,

1H), 7.71 (br s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.40 (br s, 1H), 7.33 (d, J

= 7.7 Hz, 1H), 7.27 (t, J = 7.4 Hz, 1H), 5.82 (d, J = 17.4 Hz, 1H), 5.49 (d, J = 17.4 Hz, 1H), 4.44

(m, 1H), 3.82 (m, 1H), 2.32 (m, 1H), 2.21 (m, 1H), 1.89 (m, 1H), 1.02 (m, 1H), 0.74 (m, 1H); 13

C

NMR (150 MHz, DMSO-d6) δ 170.2, 166.3, 163.7, 152.0, 141.6, 141.4, 138.7, 137.6, 126.4,

123.0, 122.3, 122.2, 121.8, 112.3, 110.6, 62.9, 51.0, 36.8, 31.35, 17.4, 17.3; IR (neat): 3412,

3283, 3034, 2947, 1712, 1656, 1573, 1543, 1436, 1401, 1312, 1201, 1158, 1133, 786, 745 cm-1

;

HRMS (m/z): [M+H]+ calcd for C21H19

79BrN6O3, 483.0775; found, 483.0776; analysis (% calcd,

% found for C21H19BrN6O3): Br (16.53, 16.36), C (52.19, 51.41), H (3.96, 3.95), N (17.39,

17.22).


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