SUPPLEMENTARY NOTES
A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy
Baptiste Ronan1, Odile Flamand1, Lionel Vescovi1, Christine Dureuil1, Laurence Durand1, Florence Fassy1, MarieFrance Bachelot1, Annabelle Lamberton1, Magali Mathieu2, Thomas Bertrand2, Jean-Pierre Marquette2, Youssef ElAhmad1, Bruno Filoche-Romme1, Laurent Schio1, Carlos Garcia-Echeverria1, Hélène Goulaouic1 and Benoit
Pasquier1*
1
Oncology Drug Discovery, Sanofi, Vitry Sur Seine, France
2
Structure, Design and Informatics, Sanofi, Vitry Sur Seine, France
*Address correspondence to: Dr. B. Pasquier, Sanofi, 13 Quai Jules Guesde, 94403 Vitry Sur Seine, France;
benoit.pasquier@sanofi.com
1
Synthetic protocols
The nomenclature of the compounds was carried out with the ACDLABS software, version 11.01. All solvents and
reagents obtained from commercial sources were used without further purification. Thin layer chromatography was
carried out on Merck silica gel 60 F254 glass plates. Flash chromatography was performed using prepacked Merck
silica gel cartridges (15−40 μm). The microwave oven used was a Biotage, InitiatorTM 2.0, 400 W max, 2450 MHz
instrument. The 1H NMR spectra at 400 MHz was performed on a Bruker Avance DRX-400 spectrometer with the
chemical shifts (δ in ppm) in the solvent dimethyl sulfoxide-d6 (d6-DMSO) referenced at 2.5 ppm at a temperature
of 303 K, and coupling constants (J) are given in hertz.
The mass spectra (MS) were obtained by methods A and B.
Method A: Waters UPLC-SQD instrument; ionization, positive or negative mode electrospray (ES+ and ES−) or
both; chromatographic conditions, column Acquity BEH C18 1.7 μM, 2.1 mm × 50 mm; solvents (A) H 2O (0.1%
formic acid), (B) CH3CN (0.1% formic acid); column temperature 50 °C; flow rate 1 mL/min; gradient (2 min) from
5% to 50% of B in 0.8 min; 1.2 min 100% of B; 1.85 min 100% of B; 1.95 min 5% of B; retention time = Tr (min).
Method B: Waters ZQ instrument; ionization, positive or negative mode electrospray (ES+ or ES−) or both;
chromatographic conditions, column XBridge C18 2.5 μM, 3 mm × 50 mm; solvents (A) H 2O (0.1% formic acid),
(B) CH3CN (0.1% formic acid); column temperature 70 °C; flow rate 0.9 mL/min; gradient (7 min) from 5% to
100%.
Purities for final compounds were measured using UV detection at 220 nm and are ≥95.0%.
Compounds 1 (SAR405), 2 (SAR209) and 3 (SAR404) were synthesized as following:
2
H2 3 bars; Pd/C 10%
rt; 24h
MeOH; H2O; 12N HCl
N
CH2(CO2Et)2
NaOMe
100°C; 75 min
H
N
Cl
+
O
N
H3N
H2 N
N
CF3
99%
100%
N
HO
CF3
N
H
N
4
5
N
NH
N
N
O
N
N
CF3
Cl
O
O
Cs2CO3
DMF
rt; 17 h
O
N
100°C; 41 h
N
35%
N
1) POCl3
65°C; 3h
1,2-dichloroethane
2) Separation of 2
enantiomers by chiral
chromatography
22%
Cl
O
CF3
N
N
N
H
86%
CF3
Cl
O
N
H
N
7
1
CF3
6
Cl
Cl
O
O
N
NH 110°C
1.5 h
O
N
N
N
83%
N
CF3
Cl
O
Cs2CO3
DMF
rt; 17 h
N
N
49%
N
N
H
N
CF3
O
8
3
Cl
Cl
HO
63%
Cs2CO3
DMF
100°C; 7 h
O
N
N
O
N
N
CF3
HO
2
(R,S)-[4-(trifluoromethyl)-1,4,5,6-tetrahydropyrimidin-2-yl]ammoniun chloride (4). To a solution of 2-amino4-(trifluoromethyl)pyrimidine (22 g, 135 mmol) in water (200 mL) and methanol (50 mL) were added 12N HCl (aq)
(50 mL) and 10% Pd/C (1.1 g). The reaction mixture was hydrogenated under 3 bars of H2 at room temperature for
24 h. The reaction mixture was filtered and the filtrate concentrated in vacuo. The crude residue was dried in vacuo
3
in presence of P2O5 to give compound 4 (27 g, 99% yield) as a grey powder: 1H NMR (400MHz, DMSO-d6) δ
(ppm): 8.96 (s, 1H), 8.58 (s, 1H), 7.42 (s, 2H), 4.41 (m, 1H), 3.37 (m, 1H), 3.20 (m, 1H), 2.12 – 1.98 (m, 2H); ES± :
[M+H]+ : m/z 168 ; Tr (min) = 0,17.
(R,S)-8-hydroxy-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one (5). Sodium methylate
(10 g, 185 mmol) was added to a stirred mixture of compound 4 (10 g, 49 mmol) in diethyl malonate (50 mL). The
reaction mixture was heated to 100°C for 75 min and then cooled to room temperature and concentrated in vacuo.
The crude residue was triturated in diethyl ether (50 mL), filtered and stirred in cooled water (20 mL). The aqueous
layer was made at pH 5-6 by the addition of 12N HCl (aq). The suspension was filtered and the crude residue was
washed with diethyl ether to give compound 5 (11.5 g, 100% yield) as a white powder: 1H NMR (400MHz, DMSOd6) δ (ppm): 10.8 (m broad, 1H), 8.48 (m broad, 1H), 4.81 (s, 1H), 4.29 (m, 1H), 4.12 (m, 1H), 3.31 (m, 1H), 2.20
(m, 1H), 2.08 (m, 1H); ES± : [M+H]+ : m/z 236 ; [M-H]- : m/z 234; Tr (min) = 0,26.
(S)-8-chloro-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one (6). To a suspension of
compound 5 (34 g, 145 mmol) in 500 mL of 1,2-dichloroethane was added phosphorus oxychloride (60 mL) at room
temperature. The reaction mixture was heated to 65°C for 3 h and then cooled to room temperature and concentrated
in vacuo. The crude residue was diluted with cooled water (100 mL) and ethyl acetate (400 mL). The aqueous layer
was made at pH 6 by the addition of 32% NaOH (aq). The layers were separated and the organic layer was dried
over magnesium sulfate and concentrated in vacuo. The crude orange residue was purified by flash column
chromatography (97-3 CH2Cl2/MeOH) to give (R,S)-8-chloro-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2a]pyrimidin-6-one (20 g, 55% yield) as a white powder: ES± : [M+H]+ : m/z 254 ; [M-H]- : m/z 252; Tr (min) =
0,51. Separation of two enantiomers was made by chiral chromatography, with Chiralpak AD as stationary phase
and EtOH / heptane (20-80) as mobile phase. The levogyre (in MeOH) enantiomer was concentrated to give 8.52 g
(23% yield) of (R)-2-chloro-8-(trifluoromethyl)-6,7,8,9-tetrahydro-4H-pyrimido[1,2-a]pyrimidin-4-one as a white
powder. The dextrogyre (in MeOH) enantiomer was concentrated to give 8.21 g (22% yield) of compound 6, as a
white powder: 1H NMR (400MHz, DMSO-d6) δ (ppm): 9.15 (s, 1H), 5.80 (s, 1H), 4.36 (m, 1H), 4.11 (m, 1H), 3.46
(m, 1H), 2.27 - 2.08 (m, 2H); ES± : [M+H]+ : m/z 254 ; [M-H]- : m/z 252; Tr (min) = 0,51; [α]D 25 589 nm = + 21.3 ±
0.5 (MeOH) ; [α]D 25 589 nm = - 21.8 ± 0.7 (DMSO).
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(S)-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one
(7). A mixture of compound 6 (9 g, 35.5 mmol) and (R)-3-methylmorpholine (10,77g, 106.5 mmol) was stirred at
100°C for 41 h. The crude residue was dissolved in AcOEt (100 mL) and washed with water (3 x 100 mL). The
combined organic layers were dried over sodium sulfate and concentrated in vacuo. The crude residue was purified
by flash column chromatography (gradient of 100 to 95-5 CH2Cl2/MeOH) to give compound 7 (9.75 g, 86% yield)
as a pale yellow powder: 1H NMR (400MHz, DMSO-d6) δ (ppm): 8.10 (m, 1H), 4.86 (s, 1H), 4.29 – 4.15 (m, 2H),
4.10 (m, 1H), 3.85 (dd, J=11.2 -3.4 Hz, 1H), 3.76 (dl, J=13.0 Hz, 1H), 3.64 (d, J=11.4 Hz, 1H), 3.51 (dd, J=11.4 –
3.1 Hz, 1H), 3.41 – 3.28 (m, 2H), 3.00 (td, J=13.0 – 3.8 Hz, 1H), 2.21 – 2.00 (m, 2H), 1.12 (d, J=6.8 Hz, 3 H); ES ±
(m/z): calc 318 - found [M+H]+ (m/z) 319, [M-H]- (m/z) 317; [α]D 25 589 nm = -48.0 ± 0.5 (DMSO).
(S)-8-(morpholin-4-yl)-2-(trifluoromethyl)-1,2,3,4-tetrahydropyrimido[1,2-a]pyrimidin-6-one (8). A mixture of
compound 6 (10 g, 39.43 mmol) and morpholine (43.50 g, 499.31 mmol) was stirred at 110°C for 1.5 h. The crude
residue was dissolved in AcOEt (50 mL) and washed with water (50 mL). The solid was filtered and washed with
diethyl ether (2 x 30 mL) to give compound 8 (10 g, 83% yield) as a white powder: 1H NMR (400MHz, DMSO-d6)
δ (ppm): 2.01 – 2.23 (m, 2 H), 3.31 – 3.35 (m, 1 H), 3.36 – 3.40 (m, 4 H), 3.58 – 3.62 (m, 4 H), 4.07 – 4.14 (m, 1 H),
4.19 – 4.29 (m, 1 H), 4.92 (s, 1 H), 8.14 (m, 1 H); ES ± (m/z): calc 304 - found [M+H]+ (m/z) 305, [M-H]- (m/z) 303;
[α]D 25 589 nm = +14.2 ± 0.6 (MeOH).
(2S)-1-[(5-chloro-3-pyridyl)methyl]-8-[(3R)-3-methylmorpholin-4-yl]-2-(trifluoromethyl)-3,4-dihydro-2Hpyrimido[1,2-a]pyrimidin-6-one (1). To a solution of compound 7 (400 mg, 1.26 mmol) in DMF (2,5 ml) were
added cesium carbonate (1.64 g, 5.03 mmol) and 3-chloro-5-(chloromethyl)pyridine (244 mg, 1.51 mmol). The
reaction mixture was stirred at rt for 17 h and concentrated in vacuo. The crude residue was dissolved in AcOEt (30
mL) and washed with water (3 x 25 mL). The combined organic layers were dried over magnesium sulfate and
concentrated in vacuo. The crude residue was purified by flash column chromatography (gradient of 94-3-3 to 92-44 CH2Cl2/MeOH/MeCN) to give compound 1 (195 mg, 35% yield) as a white powder: mp: 202.4 °C ; [α]D 25 589 nm
= -38.9 ± 0.8 (DMSO); 1HNMR (400 MHz, DMSO-d6) δ (ppm): 0.71 (d, J=6.6 Hz, 3 H), 2.34-2.45 (m, 2 H), 2.89
(dt, J=4.2 et 13.2 Hz, 1 H), 3.18-3.31 (m, 2 H), 3.38-3.46 (m, 2 H), 3.52 (d, J=11.6 Hz, 1 H), 3.76 (dd, J=3.2 et 11.0
Hz, 1 H), 4.01 (m, 1 H), 4.21 (m, 1 H), 4.60 (d, J=16.4 Hz, 1 H), 4.86 (m, 1 H), 4.89 (s, 1 H), 5.11 (d, J=16.4 Hz, 1
H), 7.82 (s broad, 1 H), 8.45 (s broad, 1 H), 8.49 (d, J=2.0 Hz, 1 H); 13C NMR (125.77MHz, DMSO-d6) δ (ppm):
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62.0, 159.7, 49.8, 146.3, 146.0, 135.8, 133.6, 130.8, 125.8, 77.5, 70.1, 65.7, 55.3, 50.6, 46.0, 38.9, 36.0, 18.5, 12.9;
ES- (m/z): [M-H+HCO2H]- 488; IR (KBr): 1658; 1574; 1533; 1440; 1170; 1137 cm-1; analysis (calcd; found for
C19H21ClF3N5O2): C(51.41; 51.92), H(4.77; 5.26), N(15.78; 15.13).
(2S)-1-[(2S)-2-hydroxy-2-phenyl-ethyl]-8-morpholino-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2a]pyrimidin-6-one (2). To a solution of compound 8 (400 mg, 1,31 mmol) in DMF (3ml) were added cesium
carbonate (1.5 g, 4.6 mmol) and (S)-2-chloro-1-phenyl-ethanol (415 mg, 2.63 mmol). The reaction mixture was
irradiated at 100°C under microonde during 7 h, filtered and concentrated in vacuo. The crude residue was dissolved
in AcOEt (30 mL) and washed with water 10ml. The organic layer was dried over magnesium sulfate and
concentrated in vacuo. The crude residue was purified by flash column chromatography (gradient of 95-3-3 to 92-44 CH2Cl2/MeOH/MeCN) to give compound 2 (350 mg, 63% yield) as a white powder: mp: 218 °C; [α]D 25 589 nm =
+10.1 ± 0.5 (MeOH); 1HNMR (400 MHz, DMSO-d6) δ (ppm): 2.18 – 2.30 (m, 1 H), 2.36 – 2.47 (m, 1 H), 3.08 (dd,
J=10.0 and 13.9 Hz, 1 H), 3.22 (dt, J=4.8 and 14.2 Hz, 1 H), 3.42 – 3.49 (m, 4 H), 3.63 – 3.69 (m, 4 H), 4.19 – 4.32
(m, 2 H), 4.76 – 4.85 (m, 1 H), 4.93 – 5.03 (m, 2 H), 5.70 (m, 1 H), 7.22 – 7.40 (m, 5 H); 13C NMR (125 MHz,
DMSO-d6) δ (ppm): 162.2, 160.5, 149.9, 142.9, 128.4, 127.3, 125.8 (q), 125.6, 77.0, 70.5, 65.8, 57.1, 56.2 (q), 44.3,
36.0, 18.1; ES± (m/z): [M+H]+ m/z 425, [M+HCOOH-H]- m/z 469, Tr (min)= 1.06; IR(CH2Cl2): 1656 ; 1568 ;
1354 ; 1171 ; 1135 cm-1 ; analysis (calcd ; found for C20H23F3N4O3): C(56.6; 56.54), H(5.46; 5.72), N(13.2; 12.92).
(2S)-1-[(5-chloro-3-pyridyl)methyl]-8-morpholino-2-(trifluoromethyl)-3,4-dihydro-2H-pyrimido[1,2a]pyrimidin-6-one (3). To a solution of compound 8 (300 mg, 0.99 mmol) in DMF (2 ml) were added cesium
carbonate (1.29 g, 3.96 mmol) and 3-chloro-5-(chloromethyl)pyridine (192 mg, 1.19 mmol). The reaction mixture
was stirred at rt for 17 h and concentrated in vacuo. The crude residue was dissolved in AcOEt (25 mL) and washed
with water (3 x 20 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo.
The crude residue was purified by flash column chromatography (gradient of 94-3-3 to 92-4-4
CH2Cl2/MeOH/MeCN) to give compound 3 (210 mg, 49% yield) as a white powder: mp: 170 °C ; [α]D 25 89 nm = 15.4 ± 0.6 (DMSO); 1HNMR (400 MHz, DMSO-d6) δ (ppm): 2.31- 2.41 (m, 2 H), 3.17 – 3.30 (m, 5 H), 3.40 – 3.51
(m, 4 H), 4.18 – 4.25 (m, 1 H), 4.68 (d, J=16.2 Hz, 1 H), 4.82 – 4.92 (m, 1 H), 4.98 (s, 1 H), 5.06 (d, J=16.2 Hz, 1
H), 7.84 (t, J=2.0 Hz, 1 H), 8.48 (d, J=2.0 Hz, 1 H), 8.51 (d, J=2.0 Hz, 1 H); 13C NMR (125.77MHz, DMSO-d6) δ
(ppm): 162.0, 160.1, 149.9, 146.5, 146.3, 135.7, 134.0, 130.7, 125.7 (q), 77.5, 65.6, 55.5 (q), 50.8, 44.2, 36.0, 18.6;
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ES- (m/z): retention time Tr (min)= 0.96, [M+H] + m/z 430, [M+HCOOH-H]- m/z 474; IR(KBr): 1652; 1580; 1526;
1149; 1040; 786 cm-1.
Conbination studies
Statistical analysis
Absolute IC40 is defined as the concentration of compound where the percentage of inhibition (I%) is equal to 40%.
This measurement allows determining the potential synergistic combinations using the statistical method described
hereunder. The interaction between SAR405 and Everolimus on cell lines was characterized using ray design
approach39 which allowed us to investigate synergy for several effective fractions of the compounds in the mixture,
the effective fraction being constant for each ray. The relative potency  was first estimated as

IC 40 A
where
IC 40 B
IC40A is the IC40 of the compound A (SAR405) and IC40B is the IC40 of the compound B (Everolimus). The
effective fraction for the ray i is then calculated as f i 
1
ci    1
where c i 
[B]
is the constant ratio of the
[A]
concentrations of the two compounds. It corresponds to the proportion defined in terms of unit of effect of each
compound alone according to their respective IC40 values. Among the 19 combination rays, all the rays for which
the effective fraction is in the range 0.08-0.97 were analyzed. A global non-linear model using NLMIXED
procedure of the software SAS V9.2 was applied to fit simultaneously the concentration-responses curves for each
ray using a 4-parameter logistic model corresponding to the following equation:
Yijk  E min i 
( E max i  E min i )
  ijk
Concij 

1  exp  mi log(
)
IC 50 i 

Yijk is the percentage of inhibition for the kth replicate of the jth concentration in the ith ray
Concij is the jth mixture concentration (sum of the concentrations of compound A and compound B) in the i
th
ray
Emini is the minimum effect obtained from i th ray
Emaxi is the maximum effect obtained from i th ray
IC50i is the IC50 obtained from i th ray
mi is the slope of the curve adjusted with data from i th ray
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ijk is the residual for the kth replicate of the jth concentration in the ith ray, ijk ~ N(0, 2)
Emin, Emax and/or slope were shared whenever it was possible without degrading the quality of the fit.
The combination index Ki of each ray and its 95% confidence interval was then estimated using the following
equation based on the Loewe additivity model:
CA
CB

 Ki
IC 40 A IC 40 B
where IC40A and IC40B are the concentrations of compound A and compound B necessary to obtain 40% of
inhibition for each compound alone and C A and CB are the concentrations of compound A and compound B in the
mixture necessary to obtain 40% of inhibition.
Additivity was then concluded when the confidence interval of the combination index (Ki) includes 1, significant
synergy was concluded when the upper bound of the confidence interval of Ki is less than 1 and significant
antagonism was concluded when the lower bound of the confidence interval of Ki is higher than 1.
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