T. Akama et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
cooled to 15 °C, filtered and concentrated to give the crude product. The residue
was purified by column chromatography (SiO2, petroleum ether/ethyl acetate =
40/1 to 4:1) to give crude 81 (942 g) as a yellow oil. To a solution of 81 (1.20 kg,
3.77 mol) in MeOH (300 mL) and THF (6 L) was added NaBH4 (80 g, 2.11 mol) in
portions at 0 °C. Then the reaction mixture was stirred at 15 °C for 1 h. HPLC
showed 81 was consumed completely. The reaction solution was adjusted to pH
= 4 with 2 M HCl. The organic layer was removed in vacuum and the mixture
was filtered. The cake was washed with petroleum ether (5 L) and dried in
vacuum to give 82 (665 g, 80%) as a white solid. 1H NMR (400 MHz, DMSO-d6): d
9.18 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 5.00 (s, 2H), 4.30 (q, J
= 7.0 Hz, 2H), 2.68 (s, 3H), 1.33 (t, J = 7.0 Hz, 3H). To a mixture of 82 (867 g, 3.94
mol) in H2O (5 L) was added NaOH (394 g, 9.85 mol) in one portion. The solution
was heated at 40 °C for 3 h. HPLC showed 82 was consumed completely. This
batch was worked-up together with the other batches and acidified with 2 N HCl
to pH = 2. The solid was filtered and washed with H2O (10 L). The cake was dried
to give 83 (2.00 kg, yield 87%) as a white solid. 1H NMR (400 MHz, DMSO-d6): d
12.75 (s, 1H), 9.13 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 4.98 (s,
2H), 2.68 (s, 3H); HPLC purity: 100% at both 220 nm and 254 nm; MS (ESI+): m/z
= 193 (M+1). To a solution of 4-fluorobenzylalcohol (72, R1 = 4-fluorobenzyl,
290 g, 2.30 mol, 248.10 mL) and N-Boc-(S)-valine (73, R2 = isopropyl, R3 = H, 500
g, 2.30 mol) in dry DCM (6.0 L) were added DCC (854 g, 4.14 mol, 838 mL) and
DMAP (39.36 g, 322.19 mmol). The reaction mixture was stirred at 25 °C for 15
h. The mixture was filtered and washed with DCM (2 L) and concentrated to give
the crude product. The residue was purified by column chromatography (SiO2,
petroleum ether/ethyl acetate = 50:1 to 10:1) to give 4-fluorobenzyl (tert-
plates at 34 °C and 5% CO2. Culture media consisted of complete HMI-9 (IMDM)
with 20% bovine serum. To ensure log growth phase, trypanosomes were sub-
cultured at appropriate dilutions every 2–3 days. Bloodstream form
Trypanosoma vivax (T. vivax) cannot be cultured axenically, therefore
trypanosomes (T. vivax STIB719/ILRAD 560 strain) were harvested from
a
highly parasitemia mouse via cardiac puncture and used directly in the ex vivo
drug sensitivity assay on the same day. IC50 determination was carried out using
the Alamar Blue assay for T. congolense (72 h in vitro assay) and modified slightly
for T. vivax (48 h ex vivo assay). Parasite starting concentrations of 2 Â 105 (T. c.)
and 4 Â 105 (T. v.) were calculated respectively, using a cell analyzer system or
haemo-cytometer, followed by quantification at 536 nm excitation and 588 nm
emission wavelengths using a flow cytometer reader. Test compounds were
prepared as 10 mg/mL DMSO stocks for each assay run. Compounds were
assayed in at least three separate, independent test runs and an 11-point
dilution curve was used to determine the IC50 values. Data points were averaged
to generate sigmoidal dose-response curves and IC50 values were determined
using Softmax Pro 5.2 software.
7. Methods for testing compound efficacy in mouse models: In vivo mouse efficacy
studies were performed at the Swiss TPH, using established mouse models of
infection for T. c. and T. v. NMRI female mice were independently infected either
with 1 Â 105 T. c. parasites/mouse (STIB736/IL1180 strain) or 1 Â 104 T. v.
parasites/mouse (STIB719/ILRAD560 strain) using an intraperitoneal route.
Parasitemia was allowed to develop over 7 days (T. c.) or 3 days (T. v.),
respectively, before treatment was administered. Compounds were
administered via intraperitoneal injection in 10% DMSO/water. Four mice were
used per treatment group. Mice were monitored for the presence of
trypanosomes, via tail blood examination microscopically with twice-per-
week parasitemia checks for 60 days post treatment. Parasitemia was graded
on a scale of 0–3, with 0 indicating no trypanosomes seen in 20 fields of view, 1
indicating the presence of 1–5 trypanosomes per field, 2 indicating the presence
of 6–20 trypanosomes per field and 3 indicating greater than 20 trypanosomes
per field. Mice scoring 2 or 3 were immediately euthanized. After 60 days,
aparasitemic mice were considered cured. Untreated control mice survive on
average for 10 and 6 days post infection for T. c. and T. v., respectively. All in vivo
mouse experiments were conducted in accordance with the strict guidelines set
out by the Swiss Federal Veterinary Office, under the ethical approval of license
number #2813.
butoxycarbonyl)-L
-valinate (74, R1 = 4-fluorobenzyl, R2 = isopropyl, R3 = H, 708
g, 95% yield) as a white solid. 1H NMR (400 MHz, CDCl3): d 7.35 (dd, J = 8.2 & 5.5
Hz, 2H), 7.05 (t, J = 8.6 Hz, 2H), 5.19–5.08 (m, 2H), 5.01 (d, J = 8.4 Hz, 1H), 4.25
(dd, J = 8.4 & 4.4 Hz, 1H), 2.13 (dd, J = 11.9 & 6.2 Hz, 1H), 1.44 (s, 9H), 0.93 (d, J =
7.1 Hz, 3H), 0.84 (d, J = 7.1 Hz, 3H). The mixture of 4-fluorobenzyl (tert-
butoxycarbonyl)-
1.06 kg, 3.26 mol) in HCl/EtOAc (6.0 L) was stirred at 25 °C for 14 h. The
solvent was removed under reduced pressure to give 4-fluorobenzyl -valinate
L
-valinate (74, R1 = 4-fluorobenzyl, R2 = isopropyl, R3 = H,
L
hydrochloride (75, R1 = 4-fluorobenzyl, R2 = isopropyl, R3 = H, 780 g, 91% yield)
was obtained as a white solid. 1H NMR (400 MHz, CDCl3): d 8.90 (br. s, 3H), 7.37
(dd, J = 8.2 & 5.5 Hz, 2H), 7.03 (t, J = 8.4 Hz, 2H), 5.29–5.10 (m, 2H), 3.95 (br, s,
1H), 2.44 (dd, J = 11.0 & 6.6 Hz, 1H), 1.08 (dd, J = 10.1 & 7.1 Hz, 6H). To the
mixture of 83 (150 mg, 0.77 mmol), 75 (203 mg, 0.77 mmol) and DIEA (0.4 mL,
2.33 mmol) in DMF was added HATU (325 mg, 0.86 mmol). The mixture was
stirred at rt for 3 h. The crude product was purified by preparative TLC and
preparative HPLC to get the final product 5 (AN11736, 125 mg, 40% yield). 1H
NMR (400 MHz, DMSO-d6): d 9.03 (s, 1H), 8.57 (d, J = 7.2 Hz, 1H), 7.47–7.43 (m,
2H), 7.33 (d, J = 7.6 Hz, 1H), 7.23–7.18 (m, 3H), 5.21 (d, J = 12.4 Hz, 1H), 5.11 (d, J
= 12.4 Hz, 1H), 4.96 (s, 2H), 4.33 (t, J = 7.2 Hz, 1H), 2.42 (s, 3H), 2.19-2.10 (m, 1H),
0.94 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H); HPLC purity: 100% at both 220 nm
and 254 nm; MS (ESI+): m/z = 400 (M+1).
8. Method for testing compound efficacy in cattle: The cattle studies were conducted
in accordance with the method described for cattle by Eisler et al. (2001) in
studies conducted in fly-proof facilities and using T. congolense and T. vivax
isolates that had previously been confirmed resistant in cattle to diminazine (7
mg/kg live weight) and/or isometamidium (1 mg/kg live weight). Cattle studies
included negative (saline) controls, all assessments were made for 100 days post
treatment (unless animals relapsed sooner) and were conducted by staff blinded
(masked) to allocation of animals to treatment groups and in accordance with
the principles of veterinary good clinical practice (VICH, 2000).
6. Assay methods for determination of T. c. and T. v. IC50 values: Bloodstream-form
Trypanosoma congolense (T. congolense IL-3000 strain) were cultured in 24-well