75
Supporting Information © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006
Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006
S1
1,3,5-Triazepan-2,6-diones as Structurally Diverse and Conformationally Constrained Dipeptide Mimetics: Identification of
Malaria Liver Stage Inhibitors from a Small Pilot Library
Gersande Léna,[a] Eliette Lallemand,[b] Anne Charlotte Gruner,[b] Joel Boeglin,[a] Solveig Roussel,[a] Arnaud-Pierre Schaffner,[a] André Aubry,[c] Jean-François Franetich,[d] Dominique Mazier,[d] Irène Landau,[e] Jean-Paul Briand,[a] Claude
Diderjean,[c] Laurent Rénia,*[b] Gilles Guichard*[a]
[a] Immunologie et Chimie Thérapeutiques (ICT), Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France; Fax: Fax: (+33)^388-610-680; E-mail:[email protected]
[b] Immunohepatology Group, Institut Cochin, Paris, France; E-mail: [email protected]
[c] LCM3B, UMR-CNRS 7036, Groupe Biocristallographie
Université Henri Poincaré, Vandoeuvre, France
[d] INSERM U511, Groupe Hospitalier Pitié-Salpêtrière,Paris, France
[e] Equipe Parasitologie Comparée et Modèles Expérimentaux USM0307, Muséum National d’Histoire Naturelle, Paris France
S2
Page S2, Figure S1. 1H NMR spectrum of crude TFA·H2N-Phe-gSar-COOSu (3a) in CD3CN. Page S3, Figure S2. Polymer-supported-DIPEA (PS-DIPEA) mediated cyclization of carbamates 3 Page S4, Figure S3. Illustration of polymer-supported quenching (PSQ) for removal of electrophile (BnBr) after alkylation of urea nitrogens Page S5, Figure S4. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 4a Page S6, Figure S5. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 4i Page S7, Figure S6. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 4p Page S8, Figure S7. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 10a Page S9, Figure S8. Stability of monoalkylated 1,3,5-triazepin-2,6-dione 10a in DMSO-d6. Page S10, Figure S9. Stability of monoalkylated 1,3,5-triazepin-2,6-dione 4a in H2O. Page S11 to S72 : RP-HPLC traces, FT-IR, 1H-NMR and 13C-NMR spectra for all compounds 4-13. Page S73, Figure S10. Toxicity of the different 1,3,5-triazepin-2,6-diones towards hepatocytes in vitro
S3
Figure S1. 1H NMR spectrum of crude TFA·H2N-Phe-gSar-COOSu (3a) in CD3CN. Evidence for the presence of two equal populations with cis and trans conformation about the Phe-gSar peptide bond.
αCHa (Phe)
αCHb (Phe)
α1CHa (gSar)
α1CHb (gSar)
α2CHa (gSar)
α2CHb (gSar)
+H3NN
HN O
NO O
O
O
+H3NN
O
NH
O ON OO
3a trans 3a cis
S4
Figure S2. Polymer-supported-DIPEA (PS-DIPEA) mediated cyclization of carbamates 3
3 Xaa Xbb Yield 4 Purity by
(%) HPLC (%)
a Phe Sar 82 > 95
f Phg Sar 67 > 95
l Phe ( N Me ) Phe 69 > 98
H N
N
N O
O R 3 C H 2 C l 2 , r . t . , 2 4 h
H 2 N +
R 2
O
N R 3
R 4
N H
R 1
O
O N
O
O
3
C F 3 C O O - R 2
R 1
R 4
4 c y c l o ( X a a g X b b C O )
N
( 3 e q . )
5.0 10.0 15.0Minutes
0
50
100
150
mAU
HN
N
NH
O
O
4a
5.0 10.0 15.0Minutes
0
50
100
150
mAU
HN
N
NH
O
O
4a
5.0 10.0 15.0Minutes
0
100
200
300
400
500
mAU
5.0 10.0 15.0Minutes
0
100
200
300
400
500
mAU
HN
N
NH
O
O
4f
5.0 10.0 15.0Minutes
0.00
0.25
0.50
0.75
1.00
AU
4l
HN
N
NH
O
O
S5
Figure S3. Illustration of polymer-supported sequestration (PSS) for removal of electrophile (BnBr) after alkylation of urea nitrogens in 4a to give 8a.
NH
HS
N-(2-mercaptoethyl)aminomethyl PS
1.
2. filter
N
N
N
O
O6a
Br
N
N
N
O
O6a
S6
Figure S4. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 4a (cyclo-L-Phe-gSar-CO) with representative ROE connectivities in DMSO-d6.together with minimized structure (MM2 force field)
TToottaall eenneerrggyy ((MMMM22)):: -- 00 ..5511 KKccaall//mmooll
N
N
N
O
O
H1
H4 H2
H3a
4a
H3b
H2 H1 H3a H3b
H2 / H3a
H2 / H1
S7
Figure S5. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 4i (cyclo-L-Phe-L-gPro-CO) with representative ROE connectivities in DMSO-d6 together with minimized structure (MM2 force field)
H2 / H3 Total energy (MM2): + 4.71 kcal/mol
N
N
N
O
O
H1
H4 H2
H3
4i
H3 H2
H2 / H1
H3 / H4
H4 H1
S8
Figure S6. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 4p [cyclo-(D-Phe-L-gPro-CO)] with representative ROE connectivities in DMSO-d6 together with minimized structure (MM2 force field)
H5 H3
Total energy (MM2): + 8.6 kcal/mol
N
N
N
O
O
H1
H4 H2
H3
4p
H5a
H5b
H5/H3
H2/H1
H3/H4
H2 H1 H4
S9
Figure S7. Partial 500MHz ROESY spectrum of 1,3,5-triazepin-2,6-dione 10a with representative ROE connectivities in DMSO-d6.
αCH(Phe)/NH(Phe)
αCH(Phe)/αCHa (Sar)
αCHb (Sar)/βH (Bn)
N
N
NHb
Ha
H
O
O
βHβH
αH
10a
S10
Figure S8. Stability of monoalkylated 1,3,5-triazepin-2,6-dione 10a in DMSO-d6. A) Part of the 1H-NMR spectrum of 10a freshly dissolved in DMSO-d6. B) Part of the 1H-NMR spectrum of 10a after 4 years at room temperature in DMSO-d6.
Hd
ppm (t1)4.505.005.506.00
Ha
500 MHz
300 MHz
Hb Hc Hf He
N
N
N
O
O
Ha
Hb
Hd
Hc
He
Hf
10a
A
B
S11
Figure S9. Stability study of 1,3,5-triazepin-2,6-dione 4a in H2O. C18 RP-HPLC traces at 1h, 7days and 30 days
2.5 5.0 7.5 10.0 12.5 15.0 17.5Minutes
0.00
0.25
0.50
0.75
1.00
1.25AU
t = 1h
t = 7 d
t = 30 d
S12
S11-S72. RP-HPLC traces, FT-IR, 1H-NMR and 13C-NMR spectra for compounds 4,5 8-13.
S13
Cyclo(Phe-gSar-CO) (4a) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
40.4
45
50
55
60
65
70
75
80
85
90
95
100.0
cm-1
%T
3182.953049.29
2928.78
1651.70
1484.051453.241438.21
1396.841357.27
1275.76
1248.78
1213.90
1165.78
1127.401085.01
1043.02
945.18
911.23
838.29
782.64
742.80
700.07
3182.953049.29
2928.78
1651.70
1484.051453.241438.21
1396.841357.27
1275.76
1248.78
1213.90
1165.78
1127.401085.01
1043.02
945.18
911.23
838.29
782.64
742.80
700.07
2.5 5.0 7.5 10.0 12.5 15.0 17.5 Minutes -117
0 250 500 750
mAU 10.384 II+ II-
S14
ppm (f1)1.02.03.04.05.06.07.08.09.0
0
5000
10000
15000
20000
7.33
67.
332
7.32
87.
321
7.31
17.
306
7.27
07.
260
7.25
57.
248
7.24
17.
239
6.97
25.
293
5.21
75.
207
5.18
65.
176
4.87
54.
653
4.64
84.
642
4.63
64.
630
4.62
54.
620
4.02
54.
011
3.99
33.
979
3.37
13.
361
3.34
23.
332
3.08
1
2.83
12.
814
2.80
32.
785
1.00
0.97
1.00
0.98
1.993.25
1.00
1.00
2.98
1.03
ppm (f1)50100150
0
5000
10000
169.
938
157.
808
136.
210
129.
263
128.
948
127.
229
56.0
29
54.5
07
37.3
32
33.8
22
S15
Cyclo((D)Phe-gSar-CO) (4b)
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
38.9
45
50
55
60
65
70
75
80
85
90
95
98.2
cm-1
%T
3182.673049.43
2926.84
1651.99
1483.911453.36
1438.10
1396.931357.48
1275.62
1248.92
1165.67
1127.241084.91
1042.96
945.34
911.33
838.33
782.55
742.99
700.04
S16
ppm (t1)1.02.03.04.05.06.07.08.0
0
500
1000
1500
2000
7.33
27.
330
7.31
77.
307
7.30
37.
285
7.27
17.
265
7.25
87.
249
7.24
47.
237
7.23
5
7.01
75.
289
5.21
75.
207
5.18
65.
176
5.04
1
4.65
64.
652
4.64
54.
640
4.63
44.
628
4.62
44.
032
4.01
94.
001
3.98
83.
651
3.63
93.
626
3.37
23.
362
3.34
43.
333
3.08
02.
832
2.81
52.
801
2.78
6
1.00
1.00
3.10
1.29
1.01
0.971.02
0.86
5.32
ppm (f1)050100150200250
0
5000
10000
15000
169.
969
158.
125
136.
301
129.
247
128.
873
127.
146
55.9
79
54.4
76
37.2
0433
.778
S17
Cyclo(Val-gSar-CO) (4c) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
61.7
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98
99.5
cm-1
%T
3238.60
3079.97
2961.79
1651.86
1495.96
1440.73
1397.53
1369.25
1317.961298.20
1239.02
1185.041163.56
1126.50
1078.39
996.47
928.29859.58
827.36
764.10
676.02
2926.58
2875.94
2.5 5.0 7.5 10.0 12.5 15.0 17.
5 Minutes -0.14 0.00 0.25 0.50 0.75 1.00 1.25 AU 8.50
3 II+
S18
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
1000
2000
3000
4000
5000
6000
7.46
47.
260
5.60
05.
593
4.94
74.
929
4.89
54.
878
4.28
44.
263
4.23
34.
211
3.90
03.
884
3.87
33.
857
3.01
0
2.14
52.
123
2.10
12.
075
2.05
32.
030
1.03
71.
015
0.99
10.
969
1.00
1.04
1.05
3.10
1.10
6.46
0.98
0.97
ppm (f1)50100150
0
5000
10000
15000
20000
25000170.
219
158.
932
77.4
2477
.000
76.5
76
60.1
9955
.969
33.6
9529
.877
19.7
53
18.4
24
S19
Cyclo(Leu-gSar-CO) (4d) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
26.3
30
35
40
45
50
55
60
65
70
75
80
85
90
95
99.6
cm-1
%T
3579.38
3502.26
3219.323082.56
2927.51
1646.33
1516.16
1493.00
1466.99
1445.77
1399.49
1367.46
1342.94
1301.69
1244.93
1182.64
1161.77
1134.89
1107.02
1052.24
1025.84968.36
954.04
923.03
878.93
843.73
792.56
767.91
703.47
3326.58
1676.40
2.5 5.
0 7.5 10.0 12.5 15.
0 17.5
Minutes -73 0
100 200 300 400
m AU 9.37
3 I I + I I-
S20
ppm (t1)5.0
0
5000
10000
7.26
0
6.85
66.
854
5.29
25.
276
5.25
95.
224
5.20
8
4.95
94.
384
4.36
7
4.09
64.
073
4.04
44.
021
3.07
51.
884
1.86
51.
858
1.83
91.
813
1.79
41.
768
1.74
81.
727
1.72
41.
701
1.48
11.
462
1.45
51.
436
1.41
71.
410
1.39
21.
344
0.96
00.
951
0.93
80.
929
1.00
0.98
0.94
0.98
0.95
2.97
2.39
1.05
6.26
ppm (f1)50100150
0
5000
10000
15000
20000
25000
30000
35000170.
675
158.
231
77.4
2477
.000
76.5
77
56.1
73
51.0
06
39.9
66
33.7
53
24.3
82
22.8
1522
.017
S21
Cyclo(Dap(Fmoc)-gSar-CO) (4e) :
HN
N
NH
O
O
NHFmoc
2.5 5.
0 7.5 10.
0 12.5 15.
0 17.5
Minutes -50
0 50
100 150 200 250 300
m AU 13.737 I I
+ I I
-
S22
ppm (f1)1.02.03.04.05.06.07.08.09.0
0
500
1000
1500
7.81
87.
799
7.74
87.
637
7.61
87.
568
7.39
77.
379
7.34
67.
344
7.32
47.
315
7.29
77.
264
6.08
66.
054
6.04
46.
014
5.97
95.
966
5.87
25.
871
5.86
85.
863
5.85
25.
850
5.84
85.
847
5.84
55.
842
5.84
05.
837
5.83
55.
832
5.83
15.
828
5.82
45.
822
5.82
05.
440
5.43
25.
428
5.13
55.
131
5.12
35.
109
5.09
75.
084
4.50
54.
504
4.50
14.
495
4.34
34.
223
4.20
64.
191
3.99
73.
991
3.98
03.
968
3.95
93.
941
3.50
93.
489
3.46
23.
438
3.32
93.
327
3.32
53.
313
3.28
63.
278
3.26
43.
262
2.95
52.
900
2.17
02.
157
2.15
32.
098
1.93
91.
933
1.92
81.
913
1.90
91.
907
1.00
3.26
0.92
2.20
2.16
4.27
1.93
1.00
0.89
1.13
1.97
1.10
1.18
S23
Cyclo(Phg-gSar-CO) (4f) :
HN
N
NH
O
O
2.5 5.0 7.5 10.0 12.5 15.
0 17.5
Minutes
-0.14 0.00 0.25 0.50 0.75 1.00
AU 9.263 I I + I I
-
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
46.4
50
55
60
65
70
75
80
85
90
95
98.1
cm-1
%T 3278.72
2934.92
2349.59
2334.07
1674.34
1639.75
1489.56
1443.15
1394.78
1364.93
1334.89
1290.591249.22
1196.30
1152.00
1086.56
1023.30
1003.24978.86
922.22
825.74
771.92
758.01
710.87
697.21
669.32
3108.86
2956.96
S24
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
1000
2000
3000
4000
5000
6000
7000
8000
7.43
87.
417
7.39
57.
369
7.35
37.
346
7.33
67.
325
7.26
0
6.99
9
6.30
46.
282
5.27
65.
253
4.57
04.
551
4.51
84.
500
3.90
53.
882
3.85
33.
830
3.02
4
1.00
0.98
2.97
1.03
0.86
0.92
4.131.07
ppm (f1)50100150
0
5000
10000
168.
891
158.
019
137.
190
128.
348
127.
444
124.
294
76.6
2876
.407
76.2
0575
.781
61.2
43
54.0
74
33.2
30
S25
Cyclo((2-)Nal-gSar-CO) (4g) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
62.8
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
97.2
cm-1
%T
3233.20
1658.27
1457.08
1396.24
1270.95
1239.97
1155.74
1068.42
957.88894.92
859.55
823.35
746.85
667.913058.222921.51
3402.53
1486.79
2.5 5.
0 7.5 10.0 12.
5 15.0 17.5 Minutes -0.13
0.00 0.25 0.50 0.75 1.00 A
U 12.699 I I
+ I I
-
S26
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
5000
7.82
67.
806
7.79
87.
778
7.72
37.
491
7.48
47.
472
7.45
97.
452
7.38
57.
380
7.35
77.
352
7.26
0
6.57
3
5.29
65.
251
5.23
45.
199
5.18
2
4.81
84.
761
4.75
34.
744
4.73
34.
724
4.71
54.
707
4.04
14.
018
3.98
93.
966
3.54
53.
528
3.49
73.
480
3.09
5
3.01
22.
983
2.96
42.
935
1.67
2
1.00
1.00
0.931.03
0.99
2.961.03
0.92
1.001.99
1.002.98
ppm (f1)50100150
0
5000
10000
169.
922
157.
650
133.
576
133.
457
132.
504
128.
779
128.
241
127.
630
127.
586
126.
890
126.
380
125.
938
77.4
2477
.195
77.0
0076
.577
56.0
55
54.3
73
37.5
7233
.860
S27
Cyclo(Tic-gSar-CO) (4h) :
HN
N
N
O
O 2.5 5.0 7.5 10.0 12.5 15.0 17.5
Minutes -0.16 0.00 0.25 0.50 0.75 1.00 1.25
AU 10.911 I I
+ I I
-
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
44.2
50
55
60
65
70
75
80
85
90
95
98.8
cm-1
%T
3337.043297.17
2922.13
2320.90
1651.57
1484.04
1462.66
1433.54
1370.08
1355.641344.31
1323.39
1301.99
1243.81
1157.20
1112.11
1078.701043.95
993.35972.78
896.09
864.70834.03
791.44
778.70
750.45
719.69
667.91
3078.483007.59
2956.96
1641.01
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
44.2
50
55
60
65
70
75
80
85
90
95
98.8
cm-1
%T
3337.043297.17
2922.13
2320.90
1651.57
1484.04
1462.66
1433.54
1370.08
1355.641344.31
1323.39
1301.99
1243.81
1157.20
1112.11
1078.701043.95
993.35972.78
896.09
864.70834.03
791.44
778.70
750.45
719.69
667.91
3078.483007.59
2956.96
1641.01
S28
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
5000
10000
15000
200007.26
07.
243
7.23
67.
224
7.21
47.
207
7.19
57.
177
7.17
07.
159
7.15
07.
145
7.09
97.
078
7.05
85
.28
3
4.86
34.
844
4.81
34.
794
4.74
4
4.69
24.
555
4.54
14.
518
4.50
34.
490
4.43
84.
400
4.37
64.
349
4.32
63.
377
3.34
03.
325
3.28
83.
087
3.03
83.
024
1.001.041.021.041.03
4.101.01
1.063.620.47
GL2
55F1
ppm (f1)050100150200250
0
5000
10000
169.
984
159.
143
134.
387
134.
275
127.
634
127.
253
126.
678
125.
984
77.4
2477
.000
76.5
76
57.7
4356
.571
46.5
20
34.0
67
32.3
87
S29
Cyclo(Phe-gPro-CO) (4i) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
20.2
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
99.4
cm-1
%T
3196.543076.01
2961.412890.33
1713.63
1651.21
1480.751467.741451.77
1435.76
1412.38
1354.28
1335.08
1249.87
1196.83
1162.56
1124.07
1066.751031.25
1012.06970.60
933.54
917.01
903.61
849.07
779.92
746.18738.96
701.31
690.17
2.5 5.0 7.5 10.0 12.
5 15.0 17.
5 Minutes -91
0 100 200 300 400 500 600
mAU 10.911 I I + I I-
S30
ppm (t1)1.02.03.04.05.06.07.08.0
0
100
200
300
400
5007.35
87.
355
7.34
17.
286
7.28
27.
271
7.25
67.
205
7.20
27.
200
7.19
27.
188
6.98
36.
145
6.14
16.
139
5.75
55.
574
5.56
85.
561
4.66
94.
661
5.57
95.
556
4.68
34.
675
4.65
54.
647
3.59
73.
590
3.58
13.
574
3.56
73.
558
3.55
2
3.32
73.
274
3.26
03.
257
3.25
23.
242
3.23
43.
220
3.06
63.
054
3.03
83.
025
2.73
42.
719
2.70
62.
691
2.59
12.
507
2.50
42.
500
2.49
62.
493
2.01
42.
001
1.99
21.
989
1.98
01.
973
1.96
71.
961
1.94
71.
942
1.93
51.
927
1.92
11.
890
1.80
61.
799
1.79
21.
789
1.78
21.
775
1.23
51.
041
1.02
80.
875
0.86
1
1.00
0.97
1.012.31
1.03
0.96
0.95
0.92
0.86
1.971.900.88
2.131.88
ppm (f1)50100150
0
5000
10000
167.
753
157.
472
136.
271
129.
290
128.
935
127.
193
64.1
92
55.0
05
46.1
86
36.5
1933
.464
22.5
56
S31
Cyclo((2-)Nal-gPro-CO) (4j) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
46.8
50
55
60
65
70
75
80
85
90
95
98.5
cm-1
%T
3269.58
3057.39
2926.62
1650.62
1441.48
1327.84
1275.70
1244.121195.27
1181.791165.24
1143.86
1101.34
1034.21995.98
949.05
907.00862.44
826.02
791.90
750.56
739.12704.72
668.01
1461.51
2886.072951.89
3200.00
2.5 5.
0 7.5 10.0 12.5 15.0 17.5
Minutes -85 0
100 200 300 400 500 600 mAU 13.293 I I
+ I I-
S32
ppm (t1)5.0
0
1000
2000
3000
4000
5000
6000
7.81
57.
740
7.48
77.
480
7.46
87.
455
7.44
87.
396
7.39
17.
368
7.36
37.
260
6.53
06.
526
5.45
75.
441
5.29
6
4.95
6
4.68
64.
681
4.66
94.
660
4.65
24.
640
4.63
53.
850
3.83
63.
822
3.81
13.
798
3.78
53.
773
3.56
63.
548
3.53
13.
518
3.50
23.
493
3.48
13.
465
3.44
2
2.99
22.
963
2.94
42.
915
2.25
22.
230
2.21
82.
210
2.19
92.
178
2.16
82.
144
2.12
62.
120
2.09
92.
084
2.07
82.
073
2.05
82.
043
2.03
12.
026
1.95
01.
939
1.90
81.
897
1.88
51.
874
1.00
0.95
1.07
1.05
2.21
1.06
2.43
0.811.43
0.93
1.012.25
1.033.34
ppm (f1)50100150
0
5000
10000
15000
167.
695
157.
602
133.
725
133.
500
132.
519
128.
729
128.
316
127.
658
127.
045
126.
368
125.
931
77.4
6477
.041
76.6
17
64.2
53
54.9
39
46.2
41
36.7
12
33.3
34
22.5
89
S33
Cyclo(Tic-gPro-CO) (4k) :
HN
N
N
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
47.9
50
55
60
65
70
75
80
85
90
95
97.0
cm-1
%T
3221.863067.68
2966.79
1658.831636.55
1428.81
1357.481334.18
1283.17
1253.96
1188.001172.21
1145.71
1117.171099.16
1076.86
1033.30997.69
977.25
920.04893.96
872.89856.01
842.75
804.66
767.63
746.55
721.33
662.58
2916.45
3265.82
1471.62
1443.82
2.5 5.
0 7.5 10.
0 12.5 15.
0 17.5
Minutes -
0.16 0.00 0.25 0.50 0.75 1.00 1.25
AU 11.23
2 I I
+ I I-
S34
ppm (t1)5.0
0
5000
10000
7.24
67.
237
7.23
17.
219
7.20
87.
200
7.18
45.
615
5.60
75.
459
5.44
65.
436
5.42
75.
414
4.88
64.
836
4.59
24.
575
4.55
74.
540
4.38
84.
337
3.80
93.
786
3.76
83.
747
3.72
73.
602
3.57
93.
557
3.54
03.
517
3.37
43.
339
3.32
23.
287
3.10
63.
090
3.05
43.
037
2.68
7
2.33
62.
314
2.29
22.
272
2.25
02.
112
2.08
92.
070
2.04
82.
032
2.02
52.
135
1.99
21.
969
1.94
81.
926
1.90
51.
885
1.87
71.
870
1.85
51.
835
1.82
81.
813
1.80
71.
792
1.50
81.
459
1.41
81.
396
1.00
0.990.98
0.950.99
1.021.05
1.00
1.10
0.09
0.981.032.072.73
3.73
S35
Cyclo(Phe-g(N-Me)Phe-CO) (4l) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
40.3
45
50
55
60
65
70
75
80
85
90
95
98.0
cm-1
%T
3224.09
3064.18
2965.47
1652.00
1453.60
1368.87
1201.49
1145.08
1073.861046.87
909.86
846.55
747.45
698.45
3392.40
3311.39
2.5 5.0 7.5 10.0 12.5 15.0 17.5 Minutes -100
0 100 200 300 400 500 600 700
m AU 14.059 I I
+ I I
-
S36
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
10000
20000
30000
400007.
350
7.34
67.
340
7.32
77.
322
7.31
87.
311
7.29
97.
295
7.28
27.
280
7.27
57.
269
7.25
87.
253
7.24
27.
228
7.22
07.
213
7.20
27.
196
7.13
67.
122
5.70
75.
687
5.68
25.
662
5.64
15.
257
5.05
6
4.79
34.
788
4.77
54.
768
4.76
04.
748
4.74
23.
415
3.39
83.
367
3.35
0
3.09
33.
073
3.04
53.
025
3.00
92.
982
2.96
52.
851
2.84
32.
815
2.79
42.
767
1.00
1.720.95
1.01
0.98
1.573.111.34
1.0810.66
ppm (t1)50100150
0
10000
20000
30000
40000
170.
513
156.
768
136.
209
134.
659
129.
336
129.
224
129.
049
128.
715
127.
738
127.
341
63.9
20
54.5
55
37.8
9137
.468
27.0
64
S37
Cyclo(Phe-gHyp(Bn)-CO) (4m) :
HN
N
NH
O
O
BnO
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
39.0
45
50
55
60
65
70
75
80
85
90
95
99.1
cm-1
%T
3207.41
3064.88
2930.09
1651.88
1452.54
1409.93
1343.00
1198.071169.93
1091.541061.77
1028.81
976.99
920.09848.29
785.72
737.49
699.601484.26
2.5 5.0 7.5 10.0 12.5 15.0 17.5
Minutes -
0.12 0.00 0.25 0.50 0.75 1.00 AU 14.04
7 I I
+ I I
-
S38
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
500
1000
1500
2000
2500
3000
35007.35
57.
351
7.34
67.
328
7.32
27.
317
7.30
77.
276
7.26
67.
260
7.25
26.
617
5.58
75.
577
5.56
75.
556
5.60
85.
598
4.66
64.
551
4.54
34.
534
4.52
74.
513
4.48
64.
447
4.25
24.
242
4.2
31
4.21
53.
915
3.91
23.
907
3.87
03.
743
3.72
83.
701
3.68
63.
408
3.39
33.
359
3.34
4
2.89
32.
869
2.83
82.
807
2.79
02
.75
82.
747
2.62
12.
574
2.55
62.
549
2.54
42.
539
2.53
42.
526
2.52
12.
516
2.51
12.
489
2.48
12.
477
2.16
92.
016
2.00
71.
988
2.05
21.
970
1.29
71.
275
1.20
81.
232
1.18
4
1.00
1.10
0.94
1.03
2.990.91
0.99
0.90
10.47
1.150.29
0.201.18
1.49
ppm (f1)50100150
0
5000
10000
168.
378
157.
465
137.
222
136.
131
129.
227
128.
987
128.
559
128.
043
127.
759
127.
231
77.4
2477
.000
76.5
77
74.9
26
71.3
8463
.448
54.5
2850
.516
39.6
03
36.6
44
S39
Cyclo(Phe-gHyp-CO) (4n) : C14H17N3O3, pale brown solid, C18-RP-HPLC (A: 0.1% TFA in H2O, B: 0.08% TFA in MeCN, 0-100% B, 1.2 ml/min, 20 min): tR = 9.392 min; IR(solid): νmax (cm-1) = 3432 (OH), 3239 (NH), 3063 (Csp2), 2926 (Csp3), 1647, 1608 (CO), 1538 (C-O) ; HRMS(ESI): calculated for C14H18N3O3: 276.1348, found: 276.1341;
HN
N
NH
O
O
HO
2.5 5.0 7.5 10.0 12.5 15.0 17.5 Minutes -50
0 50
100 150 200 250 300 mAU 9.39
2 II+
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
60.0
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98
99.9
cm-1
%T
3239.85
1647.16
1608.16
1538.88
1487.14
1465.10
1415.69
1346.22
1195.06
1169.261057.09
1029.73
990.28
921.32
850.22
787.74
749.18740.36
702.14
671.96
3432.913063.29
2926.58
S40
Cyclo(Ala-gTic-CO) (4o) :
HN
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
54.0
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
9899.3
cm-1
%T
3456.56
3394.78
3237.55
2926.28
1641.33
1480.13
1456.12
1379.861342.34
1315.091293.80
1237.341225.11
1207.95
1171.81
1124.10
1091.49
1059.82
1035.74
913.38
859.04
773.10757.30
722.06698.86
1726.96
2.5 5.0 7.5 10.0 12.
5 15.0 17.
5 Minutes -97
0 100 200 300 400 500 600 700
mAU 10.433 I I + I I-
S41
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
1000
2000
3000
4000
7.28
97.
279
7.27
57.
271
7.25
67.
245
7.23
57.
223
7.21
4
5.74
55.
732
5.04
24.
740
4.68
8
4.62
1
4.56
94.
558
4.54
94.
536
4.52
7
3.25
83.
250
3.24
33.
207
3.19
3
2.93
92.
931
2.92
32.
889
2.87
3
2.16
21.
390
1.36
8
1.00
1.04
0.97
2.98
1.91
4.55
3.36
ppm (f1)50100150
0
5000
10000
169.
726
156.
934
134.
614
131.
154
128.
044
127.
919
127.
786
126.
438
77.4
2377
.000
76.5
76
59.3
84
48.8
27
42.3
19
33.8
89
16.6
62
S42
Cyclo((D)Phe-gPro-CO) (4p) :
HN
N
NH
O
O
2.5 5.
0 7.5 10.
0 12.5 15.
0 17.5
Minutes -82
0 100 200 300 400 500 mAU 10.62
7 I I + I I-
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.020.2
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
99.3
cm-1
%T
3240.66
2925.07
2882.82
1729.98
1669.43
1638.95
1484.43
1455.44
1402.72
1346.66
1309.12
1289.73
1246.43
1196.12
1157.00
1132.64
1093.81
1060.811029.58
995.78
905.23
850.22824.70
756.70
696.52
667.83
3331.64
S43
ppm (t1)1.02.03.04.05.06.07.08.0
0
100
200
300
400
500
7.29
47.
292
7.28
97.
281
7.27
87.
275
7.26
57.
263
7.23
27.
230
7.22
77.
220
7.21
57.
210
7.20
07.
178
7.17
57.
171
7.16
16.
298
6.28
6
4.92
34.
916
4.91
04.
903
3.96
93.
956
3.94
43.
931
3.51
53.
504
3.50
03.
491
3.48
13.
477
3.46
73.
278
3.26
43.
255
3.24
93.
241
3.22
6
3.06
93.
055
3.04
13.
026
2.50
42.
500
2.49
62.
493
2.06
5
2.02
42.
010
2.00
81.
999
1.99
61.
903
1.88
41.
830
1.80
61.
792
1.79
01.
670
1.65
91.
634
1.00
1.00
1.01
2.02
1.011.010.950.90
1.00
0.97
2.771.082.07
ppm (f1)50100150
0
5000
10000
167.
411
160.
674
136.
052
129.
614
128.
798
127.
384
63.8
1261
.298
46.6
9642
.162
32.6
86
21.9
09
S44
Cyclo(ProgVal)CO (4r) :
HN
NH
N
O
O
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
500
1000
1500
8.34
98.
334
6.73
26.
721
4.82
54.
813
4.72
04.
705
4.69
14.
839
4.79
83.
338
3.33
53.
332
3.32
63.
323
3.32
23.
318
3.31
63.
310
3.30
72.
507
2.50
42.
500
2.49
62.
493
2.25
52.
249
2.23
01.
889
1.87
91.
865
1.85
41.
827
1.81
31.
800
1.78
6
1.71
31.
711
1.70
01.
685
1.67
11.
666
2.28
82.
274
2.27
02.
263
2.26
02.
244
2.24
22.
235
2.21
70.
965
0.95
20.
942
1.14
6.43
1.07
1.12
1.03
0.96
4.76
S45
Cyclo(ProgLeu)CO (4s) :
HN
NH
N
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
47.4
50
55
60
65
70
75
80
85
90
95
99.2
cm-1
%T
3207.113056.09
2957.802925.30
2871.51
1689.33
1661.60
1620.30
1453.52
1386.62
1359.40
1340.271318.43
1279.831245.80
1202.74
1172.13
1136.06
1073.33
989.20
912.52
832.86
753.70
724.82695.52
1726.96
3260.75
S46
ppm (f1)1.02.03.04.05.06.07.08.09.0
0
10000
20000
30000
40000
50000
6.51
96.
495
5.56
35.
548
5.13
35.
115
5.11
05.
092
4.61
44.
591
4.56
83.
559
3.53
53.
511
3.49
12.
560
2.53
82.
532
2.51
72.
510
2.49
52.
488
2.47
32.
467
2.44
52.
072
2.05
02.
029
2.00
71.
987
1.96
41.
905
1.88
61.
867
1.84
51.
831
1.82
61.
807
1.77
91.
766
1.75
41.
744
1.73
11.
708
1.68
51.
517
1.49
41.
471
0.94
70.
929
0.92
50.
908
1.00
1.03
0.97
0.98
2.09
1.15
1.523.22
2.03
6.00
S47
Cyclo(Pro-gVal-CO-Pro-gVal-CO) (5r)
NH
NH
NHN
HN
N
O
O
O
O
ppm (f1)1.02.03.04.05.06.07.08.09.0
0
500
1000
1500
2000
2500
3000
4.30
94.
303
4.29
24.
286
4.23
54.
223
4.22
04.
209
3.65
43.
641
3.51
93.
516
3.51
03.
500
3.37
43.
360
3.35
63.
343
3.31
33.
310
2.27
52.
260
2.25
02.
235
2.06
22.
056
2.04
22.
036
2.03
02.
026
2.02
02.
007
1.99
31.
991
1.98
01.
967
1.96
21.
956
1.95
51.
908
1.89
71.
890
1.88
51.
013
1.00
00.
944
0.93
1
3.003.10
1.17
1.11
1.270.341.00
0.261.04
3.89
2.5 5.0 7.5 10.0 12.5 15.0 17.5
Minutes
-81 0 100 200 300 400 500
mAU 10.741 II+
S48
Cyclo(Pro-gLeu-CO-Pro-gLeu-CO) (5s)
NH
NH
NHN
HN
N
O
O
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
75.4
78
80
82
84
86
88
90
92
94
96
98
100.2
cm-1
%T
3313.69
2957.85
1651.841511.30
1386.70 1201.54
1172.481135.00
1057.52
919.47802.12
768.78
720.46
2870.88
S49
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
5000
7.30
15.
754
5.72
35.
350
5.32
5
4.17
44.
155
4.14
74.
129
3.59
93.
591
3.56
93.
282
3.26
13.
239
3.23
22.
173
2.16
12.
135
2.00
91.
985
1.95
61.
948
1.94
01.
932
1.92
41.
892
1.87
11.
851
1.82
11.
801
1.78
11.
760
1.66
11.
639
1.61
71.
596
1.58
71.
542
1.52
01.
501
1.47
80.
954
0.94
20.
932
0.92
00.
905
0.88
3
1.00
1.02
0.97
1.19
0.95
1.36
0.98
5.76
2.75
4.78
S50
1,3,7-Tribenzyl-5-methyl-[1,3,5]triazepane-2,6-dione (8a) :
N
N
N
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.019.6
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.5
cm-1
%T
3062.41
3028.972926.82
1638.35
1494.751471.91
1452.541424.30
1356.19
1251.94
1208.591177.93
1151.55
1115.801077.17
1028.62
965.93
850.33792.23
748.01731.65
696.67
1666.29
2.5 5.0 7.5 10.0 12.5 15.0 17.5 Minutes -
70 0
100 200 300 400 m A
U 16.825 I I
+ I I
-
S51
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
1000
2000
3000
4000
5000
60007.
366
7.36
17.
355
7.34
17.
317
7.30
77.
301
7.29
17.
285
7.27
77.
268
7.25
97.
246
7.24
07.
233
7.22
2
7.13
87.
133
7.11
1
5.22
94.
926
4.87
94.
774
4.72
4
4.55
74.
507
4.31
74.
267
4.16
74.
152
4.14
04.
126
4.03
23.
985
3.54
0
3.49
03.
287
3.27
23.
236
3.21
0
2.68
0
3.33
33.
318
3.19
03.
163
1.44
3
1.00
1.011.001.00
0.98
2.01
2.98
1.010.98
13.271.98
ppm (f1)50100150
0
10000
20000
30000
40000
50000
60000
70000
80000168.
984
163.
189
137.
268
136.
683
129.
761
128.
579
128.
451
128.
291
127.
911
127.
656
127.
259
126.
772
77.4
2577
.000
76.5
7566
.704
62.3
76
52.5
21
51.4
38
39.7
10
34.5
11
S52
5,6,8-Tribenzyl-hexahydro-3a,6,8-triaza-azulene-4,7-dione (8i) :
N
N
N
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
24.5
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.7
cm-1
%T
3029.37
2926.23
1667.85
1634.30
1495.83
1451.48
1419.05
1351.49
1280.40
1241.90
1203.24
1156.75
1077.80
1029.26969.15
941.09902.86
846.76
821.04799.93
733.00
697.05
3488.603083.54
3058.22
2951.89
2881.01
2.5 5.0 7.5 10.0 12.
5 15.0 17.5
Minutes -0.14 0.00
0.25 0.50 0.75 1.00
AU 17.304 I I + I I
-
S53
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
1000
2000
3000
4000
5000
60007.31
77.
312
7.30
47
.29
67.
292
7.28
87.
278
7.27
17.
264
7.25
97.
227
7.22
37.
218
7.20
57.
125
7.11
97.
098
5.59
75.
576
5.56
75.
545
4.66
64.
617
4.52
14.
466
4.38
54.
330
4.14
24.
130
4.11
34.
101
3.72
83.
718
3.70
03.
692
3.50
73.
496
3.47
33.
433
3.38
4
3.33
33.
320
3.28
63.
274
3.10
33.
075
3.05
73.
028
2.01
31.
998
1.98
81.
967
1.86
41.
852
1.84
21.
831
1.82
11.
810
1.43
91.
432
1.30
21.
269
3.76
63.
758
3.52
9
3.45
62.
248
2.12
42.
115
2.09
32.
081
2.07
32.
066
2.05
12.
044
2.03
51.
977
1.95
61.
947
1.93
51.
916
1.90
71.
894
1.88
41.
874
1.78
81.
782
1.77
51.
772
1.76
21.
755
1.73
41.
729
1.71
91.
712
1.70
31.
696
1.69
11.
676
1.67
0
1.00
0.972.08
1.05
1.010.851.151.081.08
13.562.12
1.991.541.33
ppm (t1)50100150
0
5000
10000
15000
20000
168.
004
165.
005
139.
017
137.
807
137.
503
129.
994
128.
617
128.
576
128.
375
128.
319
127.
434
126.
975
126.
895
126.
774
77.4
2377
.000
76.5
76
71.2
05
66.1
53
53.2
93
47.6
03
46.1
0139
.733
30.7
74
21.6
71
S54
(7-Benzyl-3-tert-butoxycarbonylmethyl-5-methyl-2,6-dioxo-[1,3,5]triazepan-1-yl)-acetic acid tert-butyl ester (9a) :
N
N
N
O
O
O
O
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
20.0
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.2
cm-1
%T
2976.96
2921.43
1739.55
1682.76
1637.29
1497.05
1469.75
1455.97
1437.83
1409.23
1386.14
1365.79
1285.061258.39
1225.791145.49
1079.11
1045.52
1013.27
987.85972.49
957.10
933.09921.60
892.90
844.26
794.10
776.18
751.45
728.00
698.96
3032.91
2850.63
2.5 5.0 7.5 10.0 12.5 15.0 17.5 Minutes -
108 0
250 500 750
m AU 16.551 I I + I I
-
S55
ppm (f1)5.0
0
5000
10000
15000
7.33
07.
324
7.30
67.
301
7.29
57.
288
7.27
67.
260
7.25
57.
241
7.21
75.
229
5.18
2
4.26
34.
216
4.09
84.
082
4.07
04.
054
4.04
63.
987
3.95
73.
899
3.83
93.
781
3.23
63.
223
3.19
63.
062
2.94
42.
885
1.46
61.
390
1.003.002.04
2.002.081.05
1.00
5.46
8.648.39
ppm (f1)50100150
0
5000
10000
15000
169.
169
168.
749
168.
605
161.
969
137.
173
129.
702
128.
614
127.
019
82.1
7781
.759
77.4
2377
.000
76.5
76
69.7
3965
.171
52.0
4151
.240
40.3
82
35.1
85
28.0
2227
.963
S56
(5-Benzyl-8-tert-butoxycarbonylmethyl-4,7-dioxo-octahydro-3a,6,8-triaza-azulen-6-yl)-acetic acid tert-butyl ester (9i) :
N N
O
O
O
O
O
O
2.5 5.
0 7.5 10.0 12.
5 15.0 17.5
Minutes -
109 0
250 500 750
mAU 17.005 I I
+ I I
-
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.038.4
40
45
50
55
60
65
70
75
80
85
90
95.7
cm-1
%T
2922.23
2853.22
1739.64
1679.61
1638.23
1470.26
1442.15
1393.71
1373.811364.91
1287.40
1221.09
1146.84
1108.59
1081.57
1042.91
988.27
974.04
949.37
844.23
796.59
750.66
736.06
698.55
686.85
668.312956.96
2977.21
S57
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
10000
20000
30000
40000
7.31
97.
302
7.29
67.
288
7.27
87.
260
7.23
67.
228
7.21
67.
205
5.72
15.
701
5.69
35.
672
5.29
5
4.12
84.
105
4.04
64.
000
3.98
93.
977
3.95
73.
941
3.80
83.
801
3.76
53.
737
3.70
53.
599
3.57
63.
563
3.54
33.
526
3.50
33.
312
3.30
13.
266
3.25
43.
216
3.18
43.
169
3.13
72.
806
2.74
72.
318
2.29
92.
284
2.26
92.
249
2.04
01.
994
1.96
71.
947
1.93
71.
924
1.91
91.
460
1.37
61.
903
1.89
81.
891
1.87
81.
864
1.85
21.
846
1.83
31.
820
1.80
01.
662
1.00
0.93
1.831.951.02
0.91
1.95
0.96
2.91
8.448.27
5.33
ppm (t1)50100150
0
10000
20000
30000
40000
169.
216
168.
607
167.
675
163.
885
137.
677
129.
811
128.
543
126.
856
81.8
6581
.651
77.4
2477
.000
76.5
77
71.5
6769
.665
52.6
59
46.7
8046
.414
40.4
67
30.9
5527
.982
21.4
07
S58
3,7-Dibenzyl-5-methyl-[1,3,5]triazepane-2,6-dione (10a) :
N
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
44.0
50
55
60
65
70
75
80
85
90
95.4
cm-1
%T
3199.15
3063.09
3027.39
2923.04
2853.63
1725.49
1653.06
1477.581452.45
1430.36
1398.03
1372.481354.55
1327.181289.73
1257.10
1158.35
1114.71
1076.49
1028.63996.71
951.88
927.65
905.70816.79
783.72
751.21
738.00
717.77
695.79
2956.96
3296.20
2.5 5.
0 7.5 10.0 12.5 15.
0 17.5 Minutes -0.12 0.00
0.25 0.50 0.75 1.00 AU 14.06
8 I I
+ I I
-
S59
ppm (t1)1.02.03.04.05.06.07.08.0
0
500
1000
7.36
27.
355
7.35
27.
349
7.34
57.
343
7.33
67.
334
7.32
97.
323
7.30
87.
305
7.29
37.
288
7.28
17.
277
7.27
27.
268
7.26
47.
260
5.15
95.
128
4.80
14.
771
4.59
04.
578
4.56
94.
560
4.41
44.
384
4.14
74.
115
3.37
33.
364
3.34
43.
336
2.84
72.
829
2.81
82.
800
2.64
8
1.00
1.02
1.981.00
1.02
1.01
1.113.01
10.07
S60
3-Benzyl-7-isopropyl-5-methyl-[1,3,5]triazepane-2,6-dione (10c) :
N
N
NH
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
30.9
35
40
45
50
55
60
65
70
75
80
85
90
95
100101.8
cm-1
%T
3393.04
3199.433063.40
2959.96
2920.03
2850.07
1647.00
1486.39
1468.241454.94
1433.73
1397.32
1371.571352.81
1330.081287.07
1256.47
1193.60
1168.26
1155.84
1119.001102.07
1083.13
1015.31
970.89954.45
944.04935.27
856.60
842.94
819.89
775.57759.72
746.32
703.63
685.95
2.5 5.0 7.
5 10.0 12.5 15.
0 17.5
Minutes -48 0
50
100 150 200 mAU 12.453 I I +
I I-
S61
ppm (f1)1.02.03.04.05.06.07.08.09.0
0
5000
10000
15000
7.37
07.
357
7.34
27.
325
7.30
37.
296
7.28
47.
271
7.26
0
4.88
04.
828
4.69
14.
741
4.53
24.
482
4.45
34.
402
3.94
63.
932
3.92
43.
910
2.61
9
2.25
72.
234
2.21
22.
189
2.16
72.
144
1.07
71.
054
1.04
11.
018
1.00
1.000.991.011.99
5.02
2.95
1.06
5.90
ppm (f1)50100150
0
10000
20000
30000
40000170.
032
158.
610
137.
573
127.
919
128.
913
128.
299
77.4
9577
.072
76.6
48
61.9
3460
.659
51.5
17
34.1
9030
.989
19.7
9318
.070
S62
5,8-Dibenzyl-hexahydro-3a,6,8-triaza-azulene-4,7-dione (10i) :
N
N
NH
O
O
2.5 5.0 7.
5 10.0 12.5 15.
0 17.5
Minutes
-50 0
50
100 150 200 250 300 mAU 14.567 I I +
I I-
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.037.2
40
45
50
55
60
65
70
75
80
85
90
95
99.7
cm-1
%T
3211.75
3029.90
2925.66
1635.68
1495.31
1446.55
1359.331295.09
1243.79
1202.63
1164.541141.08
1074.54
1029.15
967.48
943.00
826.16784.07
731.83
697.69
1663.76
S63
ppm (f1)1.02.03.04.05.06.07.08.09.0
0
5000
10000
15000
20000
25000
7.34
87.
342
7.33
07.
291
7.26
07.
255
7.23
17.
215
7.21
07.
188
5.71
35.
698
5.69
15.
676
5.12
05.
112
4.82
94.
773
4.56
6
4.51
0
4.43
74.
426
4.42
24.
412
4.40
64.
396
4.39
24.
381
3.67
73.
653
3.63
83.
630
3.61
43.
591
3.57
73.
554
3.53
23.
515
3.50
73.
492
3.45
93.
445
2.88
32.
852
2.83
52.
805
2.28
22.
267
2.26
12.
246
2.23
82.
223
2.21
62.
180
2.07
92.
060
2.02
91.
983
1.96
11.
859
1.83
71.
791
1.77
01.
764
1.000.99
1.03
1.04
0.98
10.90
3.10
0.99
0.94
2.101.10
ppm (t1)50100150
0
10000
20000
30000
40000
50000
60000
168.
351
160.
409
138.
746
136.
827
129.
572
128.
822
128.
750
127.
094
127.
058
126.
308
69.1
95
58.0
19
47.4
7745
.658
38.6
30
30.5
77
22.6
06
S64
(6-Benzyl-1-methyl-4,7-dioxo-[1,3,5]triazepan-3-yl)-acetic acid tert-butyl ester (11a) :
N
N
NH
O
O
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
24.7
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100.2
cm-1
%T
3221.82
3063.142975.14
2925.38
2349.95
1741.55
1678.81
1653.36
1603.91
1494.511442.48
1389.69
1365.75
1318.20
1259.97
1220.521154.71
1074.321048.08
1003.26
971.10
947.94
911.67
868.80
845.69
812.18
795.92
758.61
742.93
697.48
2.5 5.0 7.5 10.0 12.
5 15.0 17.5
Minutes -0.14 0.00 0.25 0.50 0.75 1.00
AU 13.817 I I
+ I I
-
S65
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
5000
10000
15000
20000
25000
30000
7.33
07.
311
7.28
17.
277
7.26
0
5.46
45.
412
5.29
64.
628
4.61
84.
604
4.59
74.
588
4.58
24.
305
4.24
6
4.19
34.
141
3.88
63.
827
3.39
63.
381
3.34
83.
333
3.13
0
3.06
32.
848
2.81
82.
801
2.77
01.
453
1.00
1.94
2.10
1.12
1.043.00
1.08
9.45
5.81
ppm (f1)50100150
0
5000
10000
15000169.
818
168.
913
157.
268
136.
114
129.
358
129.
030
127.
327
82.3
46
77.4
5877
.034
76.6
11
62.8
35
55.9
9951
.485
38.2
44
34.6
87
28.0
71
S66
(6-Isopropyl-1-methyl-4,7-dioxo-[1,3,5]triazepan-3-yl)-acetic acid tert-butyl ester (11c) :
N
N
NH
O
O
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
38.5
45
50
55
60
65
70
75
80
85
90
95
101.1
cm-1
%T
3226.15
3095.89
2921.77
2850.69
1734.66
1651.98
1478.99
1437.49
1399.951391.16
1370.57
1359.37
1309.161286.46
1258.68
1233.661154.88
1089.101012.57
980.41953.77
886.96
856.58
819.13791.58
765.42744.84
680.27658.75
3296.20
3392.40
2.5 5.0 7.
5 10.0 12.5 15.
0 17.5
Minutes
-50 0
50
100 150 200 250 mAU 12.184 I I + I I-
S67
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
5000
10000
15000
20000
7.26
0
5.11
0
5.05
84.
847
4.83
4
4.49
24.
440
4.22
2
4.16
33.
973
3.95
43.
940
3.93
03.
914
3.07
42.
220
2.19
72.
174
2.15
21.
446
1.43
51.
412
1.25
81.
229
1.05
51.
031
1.00
80.
853
0.82
8
1.00
6.83
9.42
1.20
3.00
2.12
1.06
0.94
0.98
ppm (f1)50100150
0
5000
10000
15000
20000
25000
30000
35000
169.
965
168.
913
157.
873
82.1
3477
.424
77.0
0076
.576
62.8
20
61.4
16
51.3
34
34.6
5530
.604
29.6
21
28.0
0119
.731
18.0
37
S68
(5-Benzyl-4,7-dioxo-octahydro-3a,6,8-triaza-azulen-8-yl)-acetic acid tert-butyl ester (11i) :
N
N
NH
O
O
O
O
2.5 5.0 7.5 10.0 12.5 15.0 17.5 Minutes
-50
0 50
100 150 200 250 mAU 14.333 I I + I I
-
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.035.0
40
45
50
55
60
65
70
75
80
85
90
95
99.5
cm-1
%T
3272.68
2927.012874.37
1735.43
1705.26
1674.47
1612.74
1489.05
1451.451415.02
1391.25
1375.64
1364.30
1330.65
1282.14
1230.80
1200.22
1161.17
1147.65
1084.75
1065.34
1033.391005.10
966.90
952.51932.59
909.40
888.33
855.83
825.07795.36
758.04
739.95
722.17
710.84
693.26
669.20
3058.22
S69
ppm (f1)5.0
0
10000
20000
30000
40000
50000
7.36
17.
343
7.33
77.
333
7.32
57.
313
7.30
67.
295
7.28
17.
276
7.26
07.
253
5.78
65.
773
5.76
55.
752
5.29
6
4.57
94.
573
4.46
14.
452
4.44
74.
438
4.42
94.
420
4.41
54.
401
3.77
03.
710
3.67
33.
648
3.62
33.
599
3.46
83.
454
3.42
03.
407
2.85
32.
820
2.80
52.
773
2.20
92.
186
2.17
82.
164
2.15
52.
143
2.13
32.
112
2.09
62.
082
2.06
62.
055
2.0
51
2.04
22.
035
2.02
11.
993
1.98
61.
942
1.93
51.
931
1.91
71.
911
1.45
4
1.00
0.94
2.231.94
9.04
1.841.00
1.840.89
0.92
5.39
S70
(7-Benzyl-1-carboxymethyl-5-methyl-2,6-dioxo-[1,3,5]triazepan-3-yl)-acetic acid (12) :
N
N
N
O
O
HO
HO
O
O
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
34.9
40
45
50
55
60
65
70
75
80
85
90
95
100.6
cm-1
%T
2927.86
1713.74
1618.79
1485.50
1454.651406.54
1325.13
1180.29
1079.22
1043.731030.01
959.26
882.07
797.32
758.15
720.29
700.85
3448.102622.782531.64
2. 5. 7. 10 12 15 17 Minu - 0
1 2 3
mA
X Y 13.2115 19.5
II
S71
S72
(6-Benzyl-1-methyl-4,7-dioxo-[1,3,5]triazepan-3-yl)-acetic acid (13) :
N
N
NH
O
O
HO
O
2.5 5.
0 7.5 10.
0 12.5 15.0 17.5 Minutes
-73 0
100 200 300 400
mAU 10.351 II+
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650.0
52.0
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98.1
cm-1
%T
3277.35
2924.15
1951.81
1702.53
1673.73
1608.55
1496.09
1454.94
1428.05
1397.70
1347.58
1312.16
1270.981256.99
1188.54
1166.00
1081.801056.41
1036.88
1000.22
962.26
929.61
878.05
861.07
800.81
761.50
751.24
724.46
698.78
658.21
2455.69
S73
ppm (t1)1.02.03.04.05.06.07.08.09.0
0
5000
7.35
07.
327
7.29
37.
270
7.24
47.
209
7.18
5
6.27
7
5.58
3
5.53
1
4.80
54.
785
4.76
84.
377
4.32
44.
105
4.04
6
4.00
7
3.94
93.
086
3.06
43.
039
3.01
7
2.95
02.
813
2.79
12.
766
2.74
4
2.50
62.
500
2.49
4
1.39
3
1.00
1.04
1.06
2.12
0.983.141.11
0.39
0.98
5.32
ppm (t1)50100150
0
5000
10000
15000
20000
25000
30000
35000171.
625
170.
496
156.
362
138.
049
129.
405
128.
029
126.
170
61.6
49
54.0
4850
.294
36.1
5733
.440
S74
Figure S10. Toxicity of the different 1,3,5-triazepin-2,6-diones towards hepatocytes in vitro. compounds were added at various concentrations to primary hepatocyte cultures and were incubated for 48 hours. Control wells were incubated with medium alone and values are shown for the different concentrations for comparative purpose. Toxicity was evaluated with a MTT assay, which determines mitochondrial activity. Live cells which possess active mitochondrial metabolized MTT into a formazan dye. The amount of formazan dye is detected by spectroscopy. Data are mean of duplicates and are presented in arbitrary units of optical density.
