COMPOUNDS FOR PROTECTION OF CELLS

Abstract

This invention is related to a compound with the structural formula (I)

##STR00001## wherein, R1, and R2 are independently selected from the group consisting of C.sub.1-C.sub.6 alkyl and is preferably methyl, ethyl, propyl or isopropyl; R3 is selected from the group consisting of CH.sub.2NHR.sub.9, C(═O)YR.sub.10, —CH2OH

##STR00002##

Claims

1. Compound with the structural formula (I) ##STR00018## wherein, R1, and R2 are independently selected from the group consisting of C.sub.1-C.sub.6 alkyl and is preferably methyl, ethyl, propyl or isopropyl; R3 is selected from the group consisting of CH.sub.2NHR.sub.9, C(═O)YR.sub.10, —CH2OH, ##STR00019## where * indicates the point of attachment of R.sub.3 to the remainder of the molecule; R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 are independently selected from the group consisting of H, —OH, alkyl, substituted alkyl, preferably hydroxyalkyl, aryl, substituted aryl, halogen, oxygen, heteroaryl, substituted heteroaryl; X is selected from the group consisting of H, O, S; Y is selected from the group consisting of O, NH, S; R.sub.9 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, preferably hydroxyalkyl, alkenyl, aryl, heteroaryl; R.sub.10 is selected from the group consisting of alkyl, substituted alkyl, preferably hydroxyalkyl or cyanoalkyl, aryl, OH; R.sub.11 and R.sub.12 together with the atom N to which they are attached form a saturated or unsaturated 3-8 membered ring, incorporating one or more additional, such as one, two, or three N, O, or S atoms; R.sub.13 and R.sub.14 together with the atom N to which they are attached form a saturated or unsaturated 3-8 membered ring, optionally substituted with alkylalcohol, and in case that R.sub.3 is CH.sub.2NHR.sub.9, C(═O)YR.sub.10, —CH2OH, or ##STR00020## than is R.sub.1 and R.sub.2 isopropyl.

2. Compounds according to claim 1, for use as a medicament.

3. Compounds for use in treatment or prophylaxis of ischemic stroke, cerebral seizure, thrombosis, embolism, hemorrhage, cardiovascular disease, arthritis, diabetes, cancer, in particular cancer related to aging, atherosclerosis, heart failure, myocardial infarctions, schizophrenia, bipolar disorder, fragile X syndrome, sickle cell disease, and chronic fatigue syndrome, chronic obstructive pulmonary disease (COPD), a neurodegenerative disease such as Alzheimer disease, Parkinson disease, Lou Gehrig's disease, Huntington's disease, hypothermia/reperfusion, hemorrhagic shock, infection diseases involved in the attack or increased breakdown of thrombocytes, such as haemorhorragic fever, in particular ebola, Marburg disease and chagas wherein the compound has the structural formula of (I) ##STR00021## wherein, R1, and R2 are independently selected from the group consisting of C.sub.1-C.sub.6 alkyl and is preferably methyl, ethyl, propyl or isopropyl; R3 is selected from the group consisting of —CH2OH, CH.sub.2NHR.sub.9, C(═O)YR.sub.10, ##STR00022## where * indicates the point of attachment of R.sub.3 to the remainder of the molecule; R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 are independently selected from the group consisting of H, —OH, alkyl, substituted alkyl, preferably hydroxyalkyl, aryl, substituted aryl, halogen, oxygen, heteroaryl, substituted heteroaryl; X is selected from the group consisting of H, ═O, ═S; Y is selected from the group consisting of O, NH, S; R.sub.9 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, preferably hydroxyalkyl or substituted hydroxyalkyl, alkylbenzylfluoride, alkenyl, aryl, substituted aryl, preferably haloaryl, heteroaryl; R.sub.10 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, preferably hydroxyalkyl or cyanoalkyl, haloalkyl, alkylamide, substituted alkylamide, aryl, substituted aryl, preferably nitrobenzyl, halobenzyl, alkylbenzoyl, OH, alkenyl, alkadienyl, alkylhalide, arylhalide, —CH2(C═O)O-alkyl, heteroaryl, substituted heteroaryl, —NH—CH.sub.2CH.sub.2CN; R.sub.11 or R.sub.12, is alkyl, substituted alkyl, preferably an alkylamine, or form together with the atom N to which they are attached a saturated or unsaturated 3-8 membered ring, optionally incorporating one or more additional, such as one, two, or three N, O, or S atoms, optionally substituted with an alkyl, alkylalcohol; R.sub.13 and R.sub.14 together with the atom N to which they are attached form a saturated or unsaturated 3-8 membered ring, optionally incorporating one or more additional, such as one, two, or three N, O, or S atoms, optionally substituted, preferably with an alkyl, alkylalcohol.

4. Compounds according to any one of the claims 1-3, for use in treatment or prophylaxis of ischemia/reperfusion injury.

5. Compounds according to any one of the claims 1-4, for use in treatment or prophylaxis of indications involved with oxidative stress induced cell damage.

6. Compounds according to any one of the claims 1-4, for preventing aggregation of platelets in the treatment or prophylaxis of arterial thrombosis, arterial fibrillation, pulmonary embolism (PE), deep vein thrombosis (DVT), or venous thromboembolism (VTE), congestive heart failure, stroke, myocardial infarction, genetic or acquired hypercoagulability, atherosclerosis, coronary artery disease, cerebrovascular disease cerebrovascular accident, peripheral artery occlusive disease (PROD).

7. Medium comprising compounds according to claim 1 and cells.

8. Medium according to claim 7, wherein the cells are blood platelets, mammalian cultured cells or mammalian primary cells.

9. Medium comprising compounds according to claim 1 and a tissue.

10. Method for protection of cells comprising adding a compound to a cell, wherein the compound has the structural formula of (I) ##STR00023## wherein, R1, and R2 are independently selected from the group consisting of C.sub.1-C.sub.6 alkyl and is preferably methyl, ethyl, propyl or isopropyl; R3 is selected from the group consisting of —CH2OH, CH.sub.2NHR.sub.9, C(═O)YR.sub.10, ##STR00024## where * indicates the point of attachment of R.sub.3 to the remainder of the molecule; R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 are independently selected from the group consisting of H, —OH, alkyl, substituted alkyl, preferably hydroxyalkyl, aryl, substituted aryl, halogen, oxygen, heteroaryl, substituted heteroaryl; X is selected from the group consisting of H, O, S; Y is selected from the group consisting of O, NH, S; R.sub.9 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, preferably hydroxyalkyl or substituted hydroxyalkyl, alkylbenzylfluoride, alkenyl, aryl, substituted aryl, preferably haloaryl, heteroaryl; R.sub.10 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, preferably hydroxyalkyl or cyanoalkyl, haloalkyl, alkylamide, substituted alkylamide, aryl, substituted aryl, preferably nitrobenzyl, halobenzyl, alkylbenzoyl, OH, alkenyl, alkadienyl, alkylhalide, arylhalide, —CH2(C═O)O-alkyl, heteroaryl, substituted heteroaryl, —NH—CH.sub.2CH.sub.2CN; R.sub.11 or R.sub.12, alkyl, substituted alkyl, preferably an alkylamine, or form together with the atom N to which they are attached a saturated or unsaturated 3-8 membered ring, optionally incorporating one or more additional, such as one, two, or three N, O, or S atoms, optionally substituted with an alkyl, alkylalcohol; R.sub.13 and R.sub.14 together with the atom N to which they are attached form a saturated or unsaturated 3-8 membered ring, optionally incorporating one or more additional, such as one, two, or three N, O, or S atoms, optionally substituted, preferably with an alkyl, alkylalcohol.

11. Method according to claim 10, wherein the protection of cells occurs during storage.

12. Method according to claim 11, wherein the storage occurs at a temperature below 37° C., in particular at room temperature, at about 4° C., at about −20° C. or at about −80° C.

13. Method according to any one of the claims 10 to 12, where the addition of the compound occurs prior to cooling the cells.

14. Method according to claim 8-11, wherein the cells are mammalian cultured cells, mammalian primary cells or blood platelets.

15. Method according to claim 8-11, wherein the cells are blood platelets and the compounds are Sul 100, 117, 118, 125, 126, 132, 136, 138, 139, 141, 142, 143, 144, 145.

16. Use of compounds according to claim 1 or 10, for storing cells at a temperature below 37° C., in particular at about 4° C., room temperature, about −20° C. or about −80° C.

Description

FIGURE DESCRIPTION

[0065] FIG. 1: Schematic overview of hypothermia/reperfusion injury inducing test where the compound of the invention is first added to the cells at 37° C., incubated for 1h, cooled at 4° C. during 24h, rewarmed and tested at 37° C.

[0066] FIG. 2: Trypan Blue absorption assay. NOD is used as a positive control. Sul 112 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone), Sul 121 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone) Sul 127 (methyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate), Sul 136 (2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid), Sul 89 (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), Sul 85 (((R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Sul 141 ((S)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid), Sul 142 ((R)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid). Sul 136 is a racemic mixture. Sul 141 and Sul 142 are respectively the S and R isomer. The compounds were added to SMAC cells at different concentrations. The absorption assay was performed after rewarming until 37° C. 4° C. means that the cells were first cooled until 4° C. C37° C. and C4° C. are tests of SMAC cells wherein the cells are maintained at 37° C. and first cooled at 4° C. and than rewarmed until 37° C., respectively without the presence of compounds.

EXAMPLE 1: PRESERVATION OF HEK CELLS

[0067] Material and Method:

[0068] Human Embryo Kidney (HEK) 293 cells were cultured in DMEM cell culture medium (Life Technologies, 41965-052) supplemented with fetal calf serum, penicillin and streptomycin. Cells were seeded at a density of 0.8-1.2E6 mL-1 in 25 cm2 polystyrene flasks, placed in a 37° C. CO2-regulated humidified incubator and were allowed to proliferate for 24 hours before commencing experiments.

[0069] The compounds SUL-090 (N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-091 (N-butyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-092 (6-hydroxy-N-isopropyl-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-093 ((E)-N-(3,7-dimethylocta-2,6-dien-1-yl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-095 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(morpholino)methanone), SUL-097 (N-(4-fluorobenzyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-098 (6-hydroxy-N—((S)-2-hydroxy-1-phenylethyl)-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-100 (6-hydroxy-2,5,7,8-tetramethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide), SUL-101 (6-hydroxy-N,2,5,7,8-pentamethyl-N-(2-(methylamino)ethyl)chroman-2-carboxamide), SUL-102 (6-hydroxy-2,5,7,8-tetramethyl-N-(3-(piperidin-1-yl)propyl)chroman-2-carboxamide), SUL-104 (6-hydroxy-2,5,7,8-tetramethyl-N-(3-nitrophenyl)chroman-2-carboxamide), SUL-106 (N-(4-fluorophenyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-107 (methyl 4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)benzoate), SUL-108 ((4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone), SUL-109 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone), SUL-111 (N—((R)-2-amino-2-oxo-1-phenylethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-112 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone), SUL-114 (N-(2-bromoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-117 (2-((butylamino)methyl)-2,5,7,8-tetramethylchroman-6-ol), SUL-118 (6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid), SUL-120 (6-hydroxy-N—((R)-1-hydroxypropan-2-yl)-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-121 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methanone), SUL-122 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-(2-hydroxyethoxy)ethyl)piperazin-1-yl)methanone), SUL-123 (N-(2-cyanoethyl)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-124 (6-hydroxy-N-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-125 ((R)—N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide), SUL-126 ((S)—N,6-dihydroxy-2,5,7,8-tetramethylchroman-2-carboxamide, SUL-128 (2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol), SUL-129 (2-((((S)-2-hydroxy-1-phenylethyl)amino)methyl)-2,5,7,8-tetramethylchroman-6-ol), SUL-130 (2,5,7,8-tetramethyl-2-(piperidin-1-ylmethyl)chroman-6-ol), SUL-131 (N,6-dihydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxamide), SUL-132 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone), SUL-134 (2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol), SUL-135 (2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-ol), SUL-136 (2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid), SUL-137 ((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone), SUL 138 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone), SUL-139 (2-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)acetic acid), SUL-140 (ethyl 2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetate), SUL-141 ((S)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid), SUL-142 ((R)-2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid), SUL-143 ((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid), SUL-144 ((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid), SUL-145 ((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid were tested.

[0070] The compounds were dissolved in dimethyl sulfoxide (DMSO) to several concentrations. Before commencing experiments, this stock solution was dissolved in pre-warmed DMEM and further diluted to obtain a concentration range from 10 nM-1 mM. The hypothermia rewarming injury protocol was used as follows and is summarized in FIG. 1. Cell culture medium was then replaced by these dilutions and cells were incubated in the presence of the compound for 1 hour. After incubation, lids were closed tightly and flasks were placed in a 4° C. room and cooled for 24 hours. After this cooling period, cells were placed back in the 37° C. incubator with lids closed and were allowed to rewarm for another 24 hours.

[0071] Assessment of Viability

[0072] After this rewarming period, microscopic images were taken to assess cell morphology (Nikon D5100, Nikon Diaphot-TMD). Cell culture medium was collected and centrifuged (4 minutes, 2000 rpm). The supernatant was collected and pH was immediately measured to prevent equilibration of the pH by the carbonate buffer in the medium, while the pelleted cells were resuspended in 5 mL of PBS. Cells remaining in the flask were washed twice with phosphate buffered saline (PBS) while the washing product was discarded. Subsequently, the cells were trypsinized by addition of 0.5 mL of trypsine and pooled with the resuspended pellet. This cell suspension was then stained at a final concentration of 0.2% trypan blue (Sigma, T8154), followed by manual assessment of viability and cell number on a Bürker-Türk hemocytometer. To conclude the assay, cell culture medium glucose levels were measured (Roche Accutrend Plus) as an indication of metabolic activity. All of these steps were performed with care to prevent mechanical stress to influence or alter the viability of the cells after cooling.

[0073] Results

[0074] Table 2 shows the results of the amount of cells that survived the process of cooling and rewarming with the addition of the compound according to the invention in different concentration. The amount represents the % cells that survived. The number of the compound corresponds with the compound as described in table 1. In the control, the same procedure was applied to two badges of HEK cells. The medium did not comprise a compound according to the invention. The amount of cells that survived depends on the used concentration and on the type of compound.

TABLE-US-00002 TABLE 2 10 nM 100 nM 1 μM 10 μM 100 μM 1 mM Mean SUL - 17 91.95 93.10 91.77 95.58 95.34 94 SUL - 83 0.00 79.38 70.12 55.74 1.11 41 SUL - 84 6.22 19.79 70.08 95.74 94.50 57 SUL - 85 1.23 0.00 1.43 84.88 78.26 33 SUL - 89 34.90 88.63 91.61 87.57 76 SUL - 90 8.67 91.34 90.25 77.92 20.41 58 SUL - 91 2.28 82.44 89.53 87.54 0.00 52 SUL - 92 3.13 64.58 87.14 86.47 1.55 49 SUL - 93 97.05 96.08 84.04 21.69 8.00 61 SUL - 94 98.63 97.31 96.35 89.02 0.00 76 SUL - 95 2.76 86.75 90.38 92.09 85.86 72 SUL - 96 5.62 96.34 93.53 96.88 0.00 58 SUL - 97 0.00 95.11 96.37 91.62 5.17 58 SUL - 98 90.99 97.81 97.54 88.49 1.02 75 SUL - 99 54.44 87.98 93.75 69.18 100.00 81 SUL - 100 77.78 76.87 63.25 68.77 55.61 0.00 57 SUL - 102 73.24 95.77 93.72 81.97 73.41 93.33 85 SUL - 103 99.74 98.10 96.85 98.31 10.27 0.00 67 SUL - 104 99.26 99.49 97.52 98.91 40.91 52.17 81 SUL - 105 94.72 95.01 80.50 96.11 32.86 50.64 75 SUL - 106 77.78 76.87 63.25 68.77 55.61 0.00 57 SUL - 107 4.42 95.74 92.72 94.77 37.62 28.92 59 SUL - 108 0.00 40.89 94.15 96.81 96.57 0.00 55 SUL - 109 92.64 98.05 97.01 94.93 93.73 87.37 94 SUL - 111 90.86 81.62 97.33 93.69 94.34 79.21 90 SUL - 112 96.26 96.52 95.38 92.97 93.06 0.00 79 SUL - 114 81.14 87.16 89.94 87.70 77.93 0.00 71 SUL - 117 0.72 89.29 93.56 84.79 90.09 0.00 60 SUL - 118 13.67 45.70 36.22 52.88 88.50 92.45 55 SUL - 120 17.65 65.27 95.54 91.63 88.73 81.46 73 SUL - 121 87.31 88.97 94.75 88.94 92.74 0.00 75 SUL - 122 5.15 22.54 85.60 85.17 88.50 55.56 57 SUL - 123 68.42 90.50 83.16 88.41 88.16 0.75 70 SUL - 125 8.09 0.00 97.27 96.65 88.44 22.50 52 SUL - 126 0.00 0.00 68.12 92.68 98.62 80.21 57 SUL - 127 0.00 0.00 97.56 96.67 89.19 92.05 63 SUL - 128 28.21 100.00 100.00 100.00 99.41 0.00 71 SUL - 129 0.00 5.65 88.80 89.92 91.52 0.00 46 SUL - 130 0.00 12.73 85.65 89.61 86.55 1.49 46 SUL - 131 2.99 1.59 22.52 91.10 75.38 0.00 32 SUL - 132 93.96 91.41 88.30 91.11 89.73 80.51 89 SUL - 134 87.43 86.34 87.74 86.58 88.82 36.51 79 SUL - 135 71.94 83.90 93.82 93.85 83.46 33.06 77 SUL - 136 99.60 100.00 98.37 99.52 99.35 100.00 99 SUL - 137 78.17 86.12 93.88 90.88 78.71 0.53 71 SUL - 138 92.84 91.74 77.01 91.71 93.30 86.94 89 SUL - 139 13.33 4.57 4.26 90.05 89.67 80.33 47 SUL - 140 8.70 17.95 95.65 97.00 94.63 0.00 52 SUL - 141 58.51 96.25 0.75 99.44 96.77 99.12 75 SUL - 142 0.00 73.02 98.51 99.16 99.42 91.89 77 SUL - 143 30.59 35.00 14.94 49.47 97.83 98.43 54 SUL - 144 4.08 18.27 21.57 25.93 91.01 92.59 42 SUL - 145 12.99 28.68 21.43 7.84 78.61 88.08 40 SUL - 146 0.96 17.00 90.63 80.65 85.49 0.00 46 DMSO 0.00 0.54 0.62 0.00 0.00 0.00  0 Control 4.71 0.00  2

EXAMPLE 2: PRESERVATION OF SMAC CELLS

[0075] Rat smooth muscle aortic cells (SMAC cells) were cultured in DMEM cell culture medium (Life Technologies, 41965-052) supplemented with fetal calf serum, penicillin and streptomycin. Cells were placed in a 37° C. CO2-regulated humidified incubator and were allowed to proliferate for 24 hours before commencing experiments. The compounds Sul 84, Sul 85, Sul 89, Sul 112, Sul 121, Sul 127, and Sul 136 were dissolved in DMSO to a final concentration of 100 mM. This solution was then dissolved in DMEM cell culture media and added to the cells at different concentrations, preincubated during 1 hour at 37° C. cooled down to 4° C. and kept at 4° C. during 24h. The cells were rewarmed during 1h until 37° C. and were tested. A Trypan blue test was performed, as described above for the HEK cells. In addition to this Trypan blue exclusion assay, an absorption assay was performed. The extend of Trypan blue absorption was used as an indication of the number of non-viable cells. Trypan blue was added to a 6-wells plate to a final concentration of 0.05% and cells were incubated at 37° C. for 5 minutes. Subsequently, excess dye was removed by carefully washing wells three times with cold PBS. After washing, 150 μL of 1% SDS was added to the wells to lyse the cells and free the trypan blue from any non-viable cells. Cell lysates were centrifuged and supernatant was subsequently transferred to a 96-wells plate. 1% SDS was used as a blank and absorption was measured at 595 nm. Cell death was expressed as a percentage of untreated 4° C. controls (100%).

[0076] FIG. 2 shows the results of a Trypan blue absorption assay to evaluate the amount of cells that survived the hypothermia and rewarming process. The viability of the SMAC cells was up to 90%.

[0077] Sul 136 is a racemic mixture. Sul 141 and Sul 142 are the R and S isomer, respectively. The S enantiomer has a better effect than the R enantiomer or the racemic mixture, even at lower concentration.

EXAMPLE 3: PRESERVATION OF BLOOD PLATELETS

[0078] Collection Via PRP

[0079] PRP platelets were harvested and 2.1 ml was suspended in Poly Propylene tubes of 5 ml, closed with a stop.

[0080] Several compounds according to the invention were added to the blood platelets directly after preparing the PRP plasma and incubated during 10 minutes at 37° C. or 30 minutes at room temperature. The blood platelets with the compound were then stored at 4° C.

[0081] A control was stored under shaking conditions at room temperature. Another control was stored at 4° C. without the addition of a compound. The compounds that were added are those listed in table 3.

[0082] A sample was taken out of each tube after 264 hours after addition of the compound and tested as follows:

[0083] The color and coagulation properties were visually analysed.

[0084] The thrombocyte cells that survived were counted and it was evaluated whether the thrombocytes were aggregated.

[0085] It was evaluated if the thrombocytes still were able to function after stimulation by the addition of ADP or under influence of collagen in a 96 well plate.

[0086] The adhesion was evaluated, meaning the ability of the platelets to bind collagen in a 6 well plate preincubated with collagen.

[0087] Via an ELISA assay, it was evaluated whether thromboxane was secreted from the platelets. Thromboxane facilitates aggregation and is produced by activated blood platelets.

[0088] Table 3 provides an overview of the results of the addition of the compounds to the blood platelets and evaluates the aggregation of the thrombocytes after addition of the compound according to the invention and cooling until 4° C.

TABLE-US-00003 TABLE 3 Name of compound (see also Final Table 1 for Concentration of Aggregation of blood the iupac Purity of compounds added to platelets after name) compound bloodplatelets storage SUL 100 >95%   30 mM | SUL-117 92% 30 mM + SUL 118 100%  30 mM | SUL-120 90% 30 mM + SUL-121 90% 30 mM + SUL-125 90% 30 mM + SUL-126 90% 30 mM + SUL-132 99.6%   30 mM + SUL-136 94% 30 mM + SUL-138 98.5%   30 mM + SUL-139 90-99% 30 mM + SUL-141 97% 30 mM + SUL 142 91% 30 mM || SUL-143 98% 30 mM + SUL-144 >90%   30 mM + SUL-145 95% 30 mM + −means that most of the thrombocytes were aggregated a + indicates that a few cells were still viable but some cells aggregated a ++ indicates that almost all cells were viable

[0089] Collection Via the Apheresis Method and PRP

[0090] Platelets were obtained via standard plateletpheresis procedures either on Haemonetics (donor 2611811 and donor 2611855) or on Cobe instruments (donor 2611770). According the protocol all units would be obtained with plateletpheresis procedure with the Cobe instrument, but from the three donations two were not passing the blood bank quality control criteria. Nevertheless, all platelet donations have a similar concentration and a similar platelet quality. In none of the platelet concentrates clots were found.

[0091] The freshly harvested platelets are weighed on a balance and for sampling of platelet concentrate a small PVC bag was seal-docked to the main platelet concentrate bag. Standard homogenization of platelet concentrate has to be done with a roller before sampling.

[0092] 300 μM suspension of Sul 136 is added with a procedure, which keeps the bag content sterile and the Sul 136 is kept in suspension by frequent whirling and stored at 37° C. before it is added to the freshly harvested platelets. The Sul 136 suspension is added to three platelet bags directly from the bag. After addition, the content of the platelet bag is gently mixed. After 2 hours the first sample is drawn with a seal-docked sample bag. The sample bag is disconnected by welding and from this sample bag the samples are taken for hematology analyzing, aggregometer tests and the flow-cytometry tests. Swirling in the bag is reported by experience of the laboratory worker in the standard format. Platelets without Sul 136 put on a flatbed shaker and stored at room temperature were used as control. Another control is stored at 4° C. Platelet samples in which Sul 136 is added, are stored without shaking in a refrigerator at 4° C.

[0093] Samples are taken after 2, 24, 48, 96, 168, 216, 264 hours and are stored for 7 weeks.

[0094] The samples are measured as follows:

[0095] Annexin V Test:

[0096] On a Beckman Coulter FC 500 flow cytometer using the Roche Annexin-V FLUOS Staining Kit

[0097] Procedure:

[0098] A mix of 20 μl Annexin-V fluorescein from Roche kit into 1 ml incubation buffer was made and 20 μl propidium iodide solution was added. 2 ml of platelet concentrate was centrifuged on standard Eppendorf centrifuge and the supernatant was discarded.

[0099] 1 ml PBS buffer was added and cell concentration was checked whether it was around 1.000.000. This was diluted 100 times with the prepared Annexin-V fluorescein and propidium iodide mix and incubated for 15 minutes. 500 μl incubation buffer from kit was added and cell analysis was made in a flowcytometer.

[0100] Platelet Activation with ADP and TRAP

[0101] Platelet activation was performed on a Beckman Coulter FC 500 flowcytometer using CD 41 PE, CD 62p FITC, IgG1 FITC/IgG1 PE antibodies of a Beckman Coulter kit.

[0102] Test Tube 1:

[0103] 40 μl Dilution buffer was brought in about 15 minutes to 37° C.; 10 μl IgG FITC/IgG PE+10 μl CD41 PE was added and mixed with Vortex, incubated 5 min at room temperature; 1000 μl cold HBSS buffer was added; measurement in flowcytometer was performed.

[0104] Test Tube 2:

[0105] 40 μl Dilution buffer was brought in about 15 minutes to 37° C.; 10 μl CD41 PE+5 μl CD62 FITC was added and mixed with Vortex, incubated during 5 min. at room temperature; 1000 μl cold HBSS buffer was added; measurement in flowcytometer was performed.

[0106] Test Tube 3:

[0107] 36 μl Dilution buffer+4 μl ADP was brought in about 15 minutes to 37° C.; 10 μl CD41 PE+5 μl CD62 FITC was added and mixed with Vortex, incubated during 5 min. at room temperature; 1000 μl cold HBSS buffer was added; measurement in flowcytometer was performed.

[0108] Test Tube 4:

[0109] 36 μl Dilution buffer+4 μl TRAP was brought in about 15 minutes to 37° C.; 10 μl CD41 PE+5 μl CD62 FITC was added and mixed with Vortex; incubated during 5 min. at room temperature; 1000 μl cold HBSS buffer was added; measurement in flowcytometer was performed.

[0110] Platelet aggregation was measured according to Platelet Aggregation Profiler PAP 8 (Mölab) using all reagents from Mölab.

[0111] Sample Preparation:

[0112] 3 ml platelet concentrate was centrifuged at 3000 RPM to get PPP. 1800 μl PPP and 600 μl PRP were mixed.

[0113] Test:

[0114] 225 μl PRP was brought in test tube with magnetic stirrer. 225 μl PPP+25 μl aquadest was brought in test tube without magnetic stirrer. PPP was used for basis. PRP was put during 2 min at 37° C. on a roller. Measurement with PRP in aggregometer was started after 30 seconds with 25 μl inductor. Measurement was performed during 6 min.

[0115] Visual determination of swirling was performed according to Bertolini, F. and Murphy, S. (1994) (A multicenter evaluation of reproducibility of swirling in platelet concentrates., Transfusion 34, 796-801.)

[0116] Results of Tests with PRP Collected Platelets

[0117] PRP platelets were tested with the addition of Sul 136. Table 4 shows the results of the number cells that survived 24, 48, 72, and 216 hours after storage at 4° C. The number of cells are given in % relative to the number of cells at time 0. 66% of the platelets stored at room temperature survived after 216 hours. Only 42.3% of the cells stored at 4° C. survived after 216 hours. The addition of Sul 136 resulted that substantial more platelets survived after storage of 72 and 216 hours at 4° C.

TABLE-US-00004 TABLE 4 Time in hours Sample 0 72 216 PRP Room Temp 100 80.7 66.1 PRP 4° C. 100 43.3 42.3 60 min 0.001 mM 100 83.8 86.1 60 min 0.01 mM 100 82.9 94.8 60 min 0.1 mM 100 76.3 77.6 60 min 1 mM 100 29.9 62.9 60 min 10 mM 100 51.8 43.4 10 min 0.001 mM 100 58.9 55.1 10 min 0.01 mM 100 66.0 52.1 10 min 0.1 mM 100 55.9 50.8 10 min 1 mM 100 59.1 59.3 10 min 10 mM 100 64.8 47.9

[0118] Table 5 shows the possibility of aggregation of the platelets collected with PRP and where Sul 136 was added, after stimulation with ADP. When 10 mM Sul 136 was added, the platelets still showed activity after stimulation with ADP. (+ means that the cells show aggregation and can thus be activated upon stimulation, − means no aggregation upon stimulation)

TABLE-US-00005 TABLE 5 Sample 0 h 24 h 48 h 72 h 216 h PRP KT + + + − − PRP 4° C. + − − − − 60 min 0.001 + − − − − mM 60 min 0.01 + − − − − mM 60 min 0.1 mM + − − − − 60 min 1 mM + − − − − 60 min 10 mM + + + + − 10 min 0.001 + − − − − mM 10 min 0.01 + − − − − mM 10 min 0.1 mM + − − − − 10 min 1 mM + − − − − 10 min 10 mM + + + + +

[0119] Table 6 shows the possibility of aggregation of the PRP platelets with Sul 136 after stimulation with collagen. When 10 mM Sul 136 was added, the platelets showed still activity after stimulation with collagen. (+ means that the cells show aggregation, ++ means strong aggregation, these platelets can thus be activated upon stimulation, − means no aggregation upon stimulation)

TABLE-US-00006 TABLE 6 Sample 0 h 24 h 48 h 72 h 216 h PRP KT + + + − − PRP 40° C. + − − − − 60 min 0.001 + − − − − mM 60 min 0.01 + − − − − mM 60 min 0.1 mM + − − − − 60 min 1 mM + − − − − 60 min 10 mM ++ ++ ++ ++ − 10 min 0.001 + − − − − mM 10 mM 0.01 + − − − − mM 10 min 0.1 mM + − − − − 10 min 1 mM + − − − − 10 min 10 mM ++ ++ ++ ++ ++

[0120] Results with Platelet Collected Via Apheresis

[0121] Table 7 shows the results of the test of the stored apherese blood platelets of donor 1(2611770) stored at room temperature on a flatbed shaker without Sul 136. The pH at day 12 was 6.3. The pH at day 19 was 5.7

[0122] Table 8 shows the results of the tests of the stored apherese blood platelets of donor 1, stored at 4° C. with the addition of Sul 136. The pH on day 12 was 6.2, the pH on day 19 was 5.9.

TABLE-US-00007 TABLE 7 Platelet aggregation Flow cytometry Platelets/ MPV Collagen ADP P-selectin Annexin Date μl fL (%) (%) MFI ADP TRAP (% positive) Day 1  1,316,000 8.2 35 10 1.99 6.54 12.8  3.13 Day 2  1,248,000 9.7 72  0 2.09 3.97 12.3  5.15 Day 3  1,400,000 9.0 27  0 2.17 4.00 11.7  5.72 Day 5  1,212,000 9.6  0  0 3.21 4.09 12.6  6.41 Day 8  1,216,000 9.6  0  0 3.47 4.53 10.2 16.70 Day 10 1,324,000 8.8  0  0 3.00 3.82 7.85 14.22 Day 12 1,528,000 9.2  0  0 4.34 4.78 6.51 21.45 Day 19 1,328,000 9.0  0  0 3.89 3.74 3.67 84.53

TABLE-US-00008 TABLE 8 Platelet aggregation Flow cytometry Platelets/ MPV Collagen ADP P-selectin Annexin Date μl fL (%) (%) MFI ADP TRAP (% positive) Day 1  1,000,000 10.6  0  0 2.09 3.07 3.57  4.51 Day 2  1,192,000  9.5  0  0 3.04 3.28 3.78 14.48 Day 3  1,240,000  9.2  0  0 3.73 4.12 4.95 18.24 Day 5  1,232,000  9.1  0  0 4.12 4.20 5.55 20.38 Day 8  1,352,000  9.3  0  0 4.82 4.83 5.52 34.56 Day 10 1,228,000  9.6  0  0 4.48 4.46 5.52 43.52 Day 12 1,204,000  9.8 17 12 4.57 4.43 4.42 56.91 Day 19 1,068,000  8.8 32 31 2.92 2.74 2.76 88.34

[0123] The flow cytometry tests were similar for the platelets stored at room temperature and the platelets stored at 4° C. with compound Sul 136. Moreover, the platelet aggregation upon stimulation with collagen and ADP was restored at day 12 and day 19 for platelets stored with compound Sul 136.

[0124] This result is comparable with the platelets stored at room temperature at day 1-3.

[0125] Table 9 and 10 show the results of the test performed with platelets form Donor 2. The platelets from table 9 were stored at 4° C. without Sul 136. The platelets from table 10 were stored at 4° C. with Sul 136.

TABLE-US-00009 TABLE 9 Platelet aggregation Flow cytometry Platelets/ MPV Collagen ADP P-selectin Annexin Date μl fL (%) (%) MFI ADP TRAP (% positive) Day 1  1,008,000 7.8 89 15 1.82 4.67 7.83  3.89 Day 2  792,000 6.5 83 36 2.50 5.62 7.18 10.83 Day 3  632,000 6.3 86 22 2.76 5.51 5.87 17.92 Day 5  824,000 6.5 82 27 3.43 6.44 7.30 24.04 Day 8  816,000 6.5 77 19 3.35 5.28 7.12 22.52 Day 10 684,000 6.5 83 13 3.00 4.12 5.13 22.78 Day 12 732,000 6.5 75 12 2.93 3.92 5.15 28.12 Day 19 868,000 6.5 24  9 2.81 2.90 3.54 34.18

TABLE-US-00010 TABLE 10 Platelet aggregation Flow cytometry Platelets/ MPV Collagen ADP P-selectin Annexin Date μl fL (%) (%) MFI ADP TRAP (% positive) Day 1  1,004,000 10.1 0 0 1.67 2.12 1.88  5.98 Day 2  1,272,000  7.6 0 0 2.13 2.53 2.35 10.96 Day 3  1,220,000  7.8 4 0 2.59 3.21 2.76 19.56 Day 5  1,252,000  8.0 0 0 2.95 3.19 3.16 32.11 Day 8  1,168,000  8.0 0 0 2.99 3.08 3.22 45.71 Day 10 1,104,000  8.1 0 0 2.76 2.83 2.96 53.96 Day 12 1,032,000  8.6 5 0 2.63 2.62 2.73 62.97 Day 19 860,000  8.8 0 0 2.01 1.99 2.14 72.67

[0126] Table 11 and 12 show the results of the test performed with platelets from Donor 3. The platelets from table 9 were stored at room temperature without Sul 136. The platelets from table 10 were stored at 4° C. with Sul 136.

TABLE-US-00011 TABLE 11 Platelet aggregation Flow cytometry Platelets/ MPV Collagen ADP P-selectin Annexin Date μl fL (%) (%) MFI ADP TRAP (% positive) Day 1  1,060,000  7.7 80 9 2.10 6.09 10.80  3.69 Day 2  1,004,000  9.6 81 0 2.18 3.81  9.20  3.87 Day 3  1,060,000  7.6 75 0 2.34 3.74  9.48  5.21 Day 5  964,000 10.1 34 0 2.80 4.29 10.80  6.12 Day 8  956,000  9.6 72 0 3.55 4.70 11.30  5.38 Day 10 976,000  9.3 23 0 3.30 4.36  8.44  8.09 Day 12 1,068,000  9.2 41 0 3.67 4.85  7.39 14.08 Day 19 1,028,000  9.7  0 0 4.76 4.56  4.59 90.52

TABLE-US-00012 TABLE 12 Platelet aggregation Flow cytometry Platelets/ MPV Collagen ADP P-selectin Annexin Date μl fL (%) (%) MFI ADP TRAP (% positive) Day 1  980,000 10.3 0 0 1.90 2.71 2.08  5.14 Day 2  1,188,000  7.7 0 0 2.82 3.49 3.62  8.68 Day 3  1,072,000  7.7 0 0 3.84 4.96 4.42 14.34 Day 5  1,068,000  8.0 0 6 4.50 4.84 5.04 27.77 Day 8  992,000  8.3 0 0 4.82 4.91 5.24 41.97 Day 10 892,000  8.3 0 0 4.37 4.25 5.04 62.65 Day 12 912,000  9.0 8 0 3.89 3.84 4.20 70.33 Day 19 692,000  9.5 0 0 2.22 2.37 2.01 80.99

[0127] Swirling Test

[0128] Still after 7 weeks Swirling is observed in the platelets stored with the compounds at 4 C

[0129] Swirling is not observed at platelets without added compounds stored at 4 C, measured after 24 hours.

EXAMPLE 4: SYNTHESIS OF THE COMPOUNDS

[0130] The compounds according to the invention are synthesized according to standard synthesis methods which are well known by a person skilled in the art.

[0131] SUL-0083, SUL-0084 and SUL-0085 are commercially available.

[0132] Synthesis of SUL 089-112, 114-117, 120-126, 128-130, 132, 134-135, 138, and 140.

[0133] Amidation of trolox was achieved by reaction with the appropriate amine in the presence of standard coupling reagents for amide formation, e.g., HATU and CDI. The corresponding amines were prepared by reduction of the amides formed with BH.sub.3

[0134] Hydroxamic acid derivatives were prepared by reaction with hydroxylamine/CDI. The synthesis of carbohydrazide analogues of trolox was achieved by reaction with (substituted) hydrazines. Enantiomeric/diastereomeric compounds were prepared starting from enantiomerically pure (R)- or (S)-Trolox or by means of chiral chromatography.

##STR00008##

[0135] Synthesis of SUL-118, SUL-119 en SUL-146

[0136] Oxidation of commercially available propofol with salcomine, a coordination complex of the salen ligand with cobalt, followed by reduction with NaBH.sub.4 afforded 2,6-diisopropylbenzene-1,4-diol Subsequent methylation with HCO/SnCl.sub.2/HCl and reaction with methyl methacrylate furnished SUL-146 (methyl 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate). Hydrolysis with LiOH yielded the carboxylic acid SUL-118 (6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid). The alcohol SUL-119 (2-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol) was obtained by reduction of SUL-146 with LiAlH.sub.4.

##STR00009##

[0137] Synthesis of SUL-131, SUL-133, SUL 137 en SUL-146

[0138] Starting from the carboxylic acid SUL-118 (6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid), the hydroxylamine was obtained by reaction with hydroxylamine using CDT as coupling reagent. Compounds SUL 133 ((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl)methanone) and SUL 137 ((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone) were prepared by reaction of SUL-118 with the appropriate piperazine derivative. Both coupling reagents HATU and CDI resulted in satisfactorily yields. SUL 139 (2-(4-(6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carbonyl)piperazin-1-yl)acetic acid) was prepared by a reductive amination of SUL 137 ((6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-yl)(piperazin-1-yl)methanone) with glyoxalic acid.

##STR00010##

[0139] Synthesis of SUL-136, SUL-141 and SUL-142.

[0140] Hydrolysis of SUL-140 (ethyl 2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetate) under N.sub.2 atmosphere furnished SUL-136 (2-(4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)piperazin-1-yl)acetic acid) in high yield. The enantiomers SUL-141 and SUL-142 were prepared according to the above-described conditions.

##STR00011##

[0141] Synthesis of SUL 143, 144 en 145

[0142] Amidation of trolox with (S)-methyl pyrrolidine-2-carboxylate (L-proline methyl ester) afforded, after column chromatography, two diastereoisomers. Subsequent hydrolysis of the individual diastereoisomers afforded SUL-144 ((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid, diastereomer 1) and SUL-145 ((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid, diastereomer 2). The racemic analogue SUL-143 ((2S)-1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylic acid) was obtained by mixing the esters of the individual diastereoisomers followed by hydrolysis of the ester moiety using LiOH.

##STR00012##

[0143] Amidation of Trolox (General Example)

[0144] SUL-108 ((4-butylpiperazin-1-yl)(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methanone). HCl. Trolox (11 g, 0.044 mol, 1 eq.) was suspended in acetonitrile (100-150 ml). CDI (8.6 g, 0.053 mol, 1.2 eq.) was added in portions. The reaction mixture was stirred for 0.5-1 hour at room temperature. After addition of 1-butylpiperazine (6.9 g, 0.048 mol, 1.1 eq.) the reaction mixture was stirred at 25-30° C. over the weekend. The reaction mixture was concentrated, H.sub.2O (200 ml) was added and the aqueous layer was extracted with EtOAc (4×). The combined organic layers were dried, filtered and concentrated. The crude product obtained was purified by column chromatography (DCM/10% MeOH) affording the compound aimed for (9 g product, 82% pure). Crystallization from EtOAc/heptanes afforded SUL-108 (6 g, 0.016 mol, 36% yield, 90% pure) as a white solid. The material obtained was dissolved in DCM (50-100 ml). HCl (4 M in dioxane, 8.8 ml, 0.0035 mol, 2.2 eq.) was added and the reaction mixture was stirred at room temperature over the weekend. The mixture was filtered, rinsed with DCM, and dried to afford the HCl salt of SUL-108 (6.3 g, 97-98% pure) as a white solid.

[0145] .sup.1H-NMR (CDCl.sub.3, in ppm): 0.93 (t, 3H), 1.38 (m, 2H), 1.58 (s, 3H), 1.67 (m, 2H), 2.09 (s, 3H), 2.12 (s, 3H), 2.15 (s, 3H), 2.50-3.20 (m, 14H). M.sup.+=375.3

[0146] Reduction of Trolox amides (general example)

[0147] SUL-128. (2-(((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methyl)-2,5,7,8-tetramethylchroman-6-431).HCl. BH.sub.3.THF in THF (16 ml, 0.0156 mol, 2 eq.) was cooled to T=0° C. A solution of SUL-112 ((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)((S)-2-(hydroxymethyl)pyrrolidin-1-yl)methanone; 2.6 g, 0.0078 mol, 1 eq.) in THF (50 ml) was added drop-wise and the reaction mixture was refluxed for 1 hour and cooled to room temperature overnight. The reaction mixture was cooled on an ice bath and HCl (6 M, 25 ml) was added drop-wise. DCM (100 ml) was added and the layers were separated. The aqueous layer was extracted with DCM (3×). The combined org. layers were dried over K.sub.2CO.sub.3 until no gas formation was noticed anymore. The organic phase was filtered and concentrated. The crude product was cooled on an ice bath, and NaOH (6M, 50 ml) was added drop-wise. After addition the reaction mixture was stirred for 1 hour and extracted with DCM (4×). The combined DCM layers were dried, filtered and concentrated to give 1.6 g crude product (20-40% pure). The material was purified by column chromatography affording SUL-128 (300 mg, 0.94 mmol, 12% yield, 90% pure). This was dissolved in DCM (10 ml) and cooled to T=0° C. (ice bath). HCl (4M in dioxane, 0.3 ml, 0.94 mmol, 1.2 eq.) was added and the reaction mixture was stirred at room temperature overnight. The solid formed was filtered, washed with Et.sub.2O and dried to afford the HCl salt of SUL-128 (300 mg, 90% pure) as a white solid (mixture of diastereomers).

[0148] .sup.1H-NMR (CDCl.sub.3, in ppm): 1.20-1.90 (m, 7H), 2.12 (s, 6H), 2.17 (s, 3H), 2.20-2.90 (m, 9H), 3.4-3.65 (m, 2H). M.sup.+=320.1

[0149] Synthesis of SUL-118 (6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic Acid).

[0150] Synthesis of 2,6-Diisopropylcyclohexa-2,5-diene-1,4-dione.

##STR00013##

[0151] Propofol 100 g, 561 mmol) was dissolved in DMF (250 mL). The solution was cooled to 0° C. while stirring. Salcomine (16.6 g, 51 mmol; 9 mol %) was added and the resulting reaction mixture was stirred 112 h overnight while warming to room temperature. The reaction mixture was poured in water (7 L). The resulting slurry was extracted with heptanes (5×1 L). The combined organic extracts were dried with Na.sub.2SO.sub.4. Concentration of the solution under vacuum afforded the crude 2,6-diisopropylcyclohexa-2,5-diene-1,4-dione (62.5 g; 325 mmol; 58% yield) as an oil. The product was used in the next step without further purification.

[0152] Synthesis of 2,6-Diisopropylbenzene-1,4-diol.

##STR00014##

[0153] Crude 2,6-diisopropylcyclohexa-2,5-diene-1,4-dione (62.5 g, 325 mmol) was dissolved in dichloromethane (300 mL) and methanol (100 mL). The solution was cooled to 0° C. with an ice bath. Sodium borohydride (4.5 g, 182 mmol) was added in portions. After the addition was complete the reaction mixture was stirred at room temperature overnight. Acetone (150 mL) was added to quench the excess of sodium borohydride. After 30 minutes stirring 2N aq. HCl (200 mL) was added. After stirring for 45 minutes the mixture was extracted with ethyl acetate (4×400 mL). The combined organic layers were dried with Na.sub.2SO.sub.4. Concentration of the solution under vacuum afforded crude 2,6-diisopropylbenzene-1,4-diol (64 g, 330 mmol) as a red oil in quantitative yield. The product was used in the next step without further purification.

[0154] Synthesis of 3,5-Diisopropyl-2-methylbenzene-1,4-diol.

##STR00015##

[0155] A mixture of 2,6-diisopropylbenzene-1,4-diol (64 g, 0.33 mol), paraformaldehyde (9.8 g, 0.327 mol), SnCl.sub.2 (217.9 g, 1.15 mol), concentrated aq. 37% HCl (0.6 L) and diisopropyl ether (2.5 L) was heated to reflux for 4 hours. After cooling to room temperature overnight the biphasic mixture was separated. The aqueous layer was extracted with TBME (2000 mL). The combined organic fractions were washed with 1N aq. HCl (1000 mL), water (1000 mL) and brine (1000 mL). The organic fractions were dried with Na.sub.2SO.sub.4 and concentrated under vacuum to give a 50:35 mixture of 3,5-diisopropyl-2-methylbenzene-1,4-diol and 2,6-diisopropyl-3,5-dimethylbenzene-1,4-diol (61 g oil) according to GCMS analysis. Purification by chromatography on silica gel (1200 mL) eluting with ethyl acetate/heptanes=97.5:2.5 (4000 mL), 95:5 (4000 mL) gave 3,5-diisopropyl-2-methylbenzene-1,4-diol 6 (16.6 g, 79.8 mmol; 24%: 83% pure) as an oil.

[0156] Synthesis of Methyl 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate.

##STR00016##

[0157] 3,5-diisopropyl-2-methylbenzene-1,4-diol (10.6 g, 50.9 mmol; 83% pure) was dissolved in methyl methacrylate (20 mL, 186 mmol). The solution was transferred to a Teflon tube in a Berghof reactor. Aqueous formaldehyde (10 mL; 37% wt. solution, stabilized with 10-15% MeOH) was added and the reaction mixture was heated to 180° C. (internal temperature) in the closed reactor for 5 hours while stirring. After cooling to ca. 40° C. The reaction mixture was poured in MeOH (200 mL) and the mixture was concentrated under vacuum. Purification by chromatography on silica gel (600 mL) eluting with ethyl acetate/heptanes=95:5 (5000 mL; TLC: Rf ˜0.2; spot stained with iodine vapor) gave the desired pure product methyl 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (10.0 g, 31.3 mmol, 61%).

[0158] Synthesis of 6-Hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic Acid (SUL-118).

##STR00017##

[0159] A mixture of purified methyl 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (8.3 g, 25.9 mmol) and lithium hydroxide monohydrate (4.3 g, 102.5 mmol; 4 eq.) in MeOH (100 mL), THF (100 mL) and water (25 mL) was heated for 30 minutes at ambient pressure while rotating with a rotary evaporator in a warm water bath at 60° C. The organic solvents were evaporated under vacuum. Water (150 mL) was added to the residue, followed by acetic acid (10 mL). A light orange mixture was obtained. Extraction with ethyl acetate (3×100 mL), drying of the combined organic fractions with Na.sub.2SO.sub.4 and concentration under vacuum gave the crude product as an orange solid. The solids were stirred with tBME (150 mL). A beige solid precipitated and an orange solution was obtained. Heptane (250 mL) was added and the mixture was stirred for 15 minutes. The mixture was filtered over a glass filter. The residual solids were washed with heptanes (2×50 mL) on the filter under suction. Drying of the solids under vacuum at 60° C. gave pure 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylic acid (SUL-118) as an off-white solid (3.1 g, 10.13 mmol; 39%, 100% pure). .sup.1H-NMR (CDCl.sub.3, in ppm): 1.38 (t, 12H), 1.52 (s, 3H), 1.87 (m, 1H), 2.20 (s, 3H), 2.30 (m, 1H), 3.20 (m, 1H), 3.38 (m, 1H). M+=307.10

EXAMPLE 5. SYNTHESIS OF SUL 119 (2-(HYDROXYMETHYL)-5,7-DIISOPROPYL-2,8-DIMETHYLCHROMAN-6-OL)

[0160] A solution of methyl 6-hydroxy-5,7-diisopropyl-2,8-dimethylchroman-2-carboxylate (500 mg, 1.56 mmol) in THF (12 mL) was added over 5 minutes with a syringe via a rubber septum to LiAlH.sub.4 (238 mg, 6.26 mmol; 4 eq.), pre-weighed in a dry 3-necked 100 mL roundbottomed flask under inert nitrogen atmosphere while stirring at room temperature. The exothermic addition of the ester was accompanied with gas evolution. After the addition was complete the resulting grey suspension was heated to reflux. After 3 hours the heating was stopped and the reaction was quenched by dropwise addition of EtOAc (6 mL; exothermic). Water (5 mL) was added in small portions, followed by 2N HCl (2 mL) followed by EtOAc (25 mL). The mixture was poured on Na.sub.2SO.sub.4 (ca. 50 g) and the slightly yellow organic layer was separated from the two-phase mixture. The aqueous phase was washed with EtOAc (50 mL) and the combined organic fractions were concentrated under vacuum to give the crude alcohol (530 mg) as a clear oil. Heptane (100 mL) was added and after concentration under vacuum the 2-(hydroxymethyl)-5,7-diisopropyl-2,8-dimethylchroman-6-ol (248 mg, 0.85 mmol, 54%, LCMS: 95.5% pure).

[0161] M+=293.2

EXAMPLE 6. SYNTHESIS OF SUL 139 (2-(4-(6-HYDROXY-5,7-DIISOPROPYL-2,8-DIMETHYLCHROMAN-2-CARBONYL)PIPERAZIN-1-YL)ACETIC ACID)

[0162] SUL-137 (440 mg, 1.17 mmol, 1 eq.,) was dissolved in MeOH (50 ml) and glyoxalic acid (216 mg, 2.35 mmol, 2 eq.) was added. The resulting mixture was stirred for 1 hour at room temperature and, subsequently, NaBH.sub.3CN (183 mg, 2.94 mmol, 2.5 eq.) was added. The reaction mixture was stirred at room temperature overnight. Acetic acid (few ml) was added and after stirring at room temperature for 0.5-1 hour, the reaction mixture was concentrated. The residue obtained was dissolved in EtOAc, washed with H.sub.2O (2×), dried, filtered and concentrated to afford SUL-139 (500 mg, 1.16 mmol, 98%, 91-92% pure) as a light yellow solid.

[0163] .sup.1H-NMR (CD.sub.3OD, in ppm): 1.33 (dd, 12H), 1.59 (s, 3H), 1.62 (m, 1H), 2.09 (s, 3H), 2-5-3.0 (m, 7H), 3.1-3.6 (m, 4H), 3.81 (bs, 2H), 4.28 (bs, 2H). M.sup.+=433.2.

EXAMPLE 7. SYNTHESIS OF SUL 136 (2-(4-(6-HYDROXY-2,5,7,8-TETRAMETHYLCHROMAN-2-CARBONYL)PIPERAZIN-1-YL)ACETIC ACID)

[0164] A 250 ml three-necked flask equipped with two septa (left and right) and a stopcock was charged with SUL-136 (15.5 g, 38.4 mmol) and THF/water (240 ml THF+80 ml water). The clear solution was stirred and degassed for at least 30 minutes by argon-bubbling, using an inlet tube equipped with a long syringe needle through the left septum; the right septum was equipped with a short needle and functioned as outlet. The degassed solution (which was maintained under argon) was cooled to 0° C. in an ice-bath and solid anhydrous LiOH (2.3 g, 96 mmol, 2.5 eq.) was added in one portion. The resulting reaction mixture was stirred for 2 hours at 0° C. after which is was neutralized by addition of a MeOH/water (3/1, v/v) slurry of Dowex-50WX8-200 ion-exchange resin; the final pH was approx 6. The Dowex resin was filtered off with suction and rinsed with 3 portions of MeOH/water (3/1, v/v). The filtrate was reduced in vacuo and to the wet product was added approx. 100 ml water. The resulting white aqueous suspension was freeze-dried overnight to afford SUL-136 (13.48 g, 93%. LCMS: 99.6%) as a white solid.

[0165] 1H-NMR (CD3OD, in ppm)): 1.60 (s, 3H), 1.65 (m, 1H), 2.05 (s, 3H), 2.10 (s, 6H), 2.55 (m, 2H), 2.62 (m, 1H), 3.0, (bs, 4H), 3.40 (bs, 2H), 3.65 (bs, 2H), 4.25 (bs, 2H). M+=377.1

EXAMPLE 8. SYNTHESIS OF SUL 144 ((2S)-1-(6-HYDROXY-2,5,7,8-TETRAMETHYLCHROMAN-2-CARBONYL)PYRROLIDINE-2-CARBOXYLIC ACID)

[0166] (2S)-methyl 1-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl)pyrrolidine-2-carboxylate (diastereomer 1, 3.5 g, 9.7 mmol) was dissolved in THF/H.sub.2O (60/20 mL). N.sub.2 was bubbled through the solution for 1 h. The mixture was cooled in an ice-bath and LiOH.H.sub.2O (1.01 g, 24.2 mmol, 2.5 eq.) was added. The reaction mixture was stirred under N.sub.2 at RT overnight. Dowex-50WX8-200 (washed 4× with MeOH/H.sub.2O 3:1) was added as a slurry in MeOH/H.sub.2O (3:1) until the pH-6. The mixture was filtered, washed with MeOH/H.sub.2O (3:1) and concentrated in vacuo. Demi H.sub.2O (50 mL) was added to the concentrate and the solution was freeze dried affording SUL-144 (3.4 g, 9.7 mmol, quant, 99.7% pure) as a off-white foam.

[0167] 1H-NMR (CDCl3): 1.60 (s, 3H), 1.65-2.30 (m, 14H), 2.60 (m, 2H), 2.81 (m, 1H), 3.49 (m, 1H), 4.01 (t, 1H), 4.50 (d, 1H). M+=348.1

EXAMPLE 9: SUL 109 FOR COLD ISCHEMIC TOLERANCE OF PORCINE HEARTS

[0168] This example studies if SUL 109 as additive to standard heart transplantation protocols will help to prolong the maximal cold ischemic protection of porcine hearts. For long term arrest, for example in case of transplantation, a commonly used solution is CUSTODIOL®. With CUSTODIOL® solution it is possible to keep a heart in a cold ischemic state for up to six hours before reperfusion with warm oxygenated blood is necessary.

[0169] Material and Methods

[0170] Two hearts were harvested from slaughterhouse pigs and treated as follows. Pigs were stunned by electrical shock to the head and exsanguinated by severing the superior caval vein. After exsanguinations the sternum was quickly opened and the heart and lungs were removed as a whole. The heart was immediately submersed in an ice cold bath and the aorta was cut proximal to the brachocephalic side branches. A 19 mm cannula was inserted in the aorta and tied off. The carefully de-aired cannulla was used to retrogradely administer cold cardioplegic solution. In the first heart, 2 liters of standard CUSTODIOL® with 5000 IU/l of heparin added was administered. The second heart was administered 2 liters of CUSTODIOL® with 5000 IU/l of heparin and 10 ml/l of SUL 109 in 0.9% NaCl at 75 μM. Both hearts were stored in plastic bags filled with the same solutions and transported to the lab on ice at approximately 4° C. Before preparation the hearts were stored at 4° C. for 24 hours. The next day both hearts were prepared to be mounted in the PhysioHeart platform as described in DeHart et al 2011. After de-airing the hearts, retrograde reperfusion of the aorta was started with warm oxygenated blood at 38° C. Both flow of blood and pressure at the aortic root were registered during the experiment.

[0171] Results and Findings

[0172] First the non SUL109 treated heart was reperfused with blood. This heart showed high vascular resistance at reperfusion resulting in a total coronary blood flow of 0.5 liters per minute at a set aortic pressure of 80 mmHg. Hardly any contractile activity of the heart muscle was visible after 5 minutes, only a slight motion of tissue resembling fibrillation. After some defibrillation by electric shock and assisting the heart with a pacemaker, a vague contraction of the lateral and anterior left ventricle was visible.

[0173] The second heart, which was treated with SUL109, was also reperfused with blood. Immediately after the start of reperfusion, the first remarkable difference with the non treated heart occurred. The muscle tissue of the treated heart initially was hard and stiff as the non treated heart but at reperfusion and warming up the heart gradually became less hard and stiff and almost feeling like a normal heart at reperfusion within 6 hours. This resulted in a lower vascular resistance, as observed by a coronary flow of 1 lpm at a perfusion pressure of 80 mmHg. The treated heart immediately displayed more contractile activity compared to the non treated one and after some defibrillation shocks a weak contractive unpaced pattern was observed.

[0174] Discussion

[0175] In the normal harvesting procedure of the PhysioHeart experiments, the hearts are arrested on a cold cardioplegic solution and transported to the LifeTec Group laboratories for further preparation and surgical interventions. Preparations can take up to 4 hours after which the heart is reactivated by reperfusion with warm oxygenated blood. In order to increase the preparational time, or to allow longer travel time before reperfusion, it would be very useful to be able to protect myocardial tissues during the cold ischemic time. This example shows a clear difference observed between the SUL109 treated heart and the non-treated heart, as the former showed an improved regular contractive activity of the myocardial tissue. Accordingly, using SUL 109 as an additive to standard heart transplantation protocols prolongs the cold ischemic protection.