SPERMINE DERIVATIVES AS HEPARIN ANTIDOTES

Abstract

The invention relates to new spermine derivatives of Formula (I), wherein the meaning of the groups R.sub.1, R.sub.2, n and m is defined in the description, to a pharmaceutical composition thereof and to their use as heparin antidotes in the control of blood coagulation. The invention was developed based on the realization that it would be desirable to have improved heparin antidotes with good anticoagulant rates, based on simple, easy to produce synthetic small molecules.

Claims

1. A compound of Formula I: ##STR00008## wherein: n is an integer between 0 and 3; each m is independently an integer between 0 and 3; R.sub.1 is phenyl, naphthyl or fluorenyl, wherein R.sub.1 is optionally substituted by one to three groups R.sub.3; R.sub.2 is naphthyl or fluorenyl, wherein R.sub.2 is optionally substituted by one to three groups R.sub.3; each R.sub.3 is independently OH, OR.sub.4, halogen, C.sub.1-4 alkyl, CHO or OPO(OH).sub.2; and R.sub.4 is C.sub.1-4 alkyl, with the proviso that: if R1 is 2-methoxyphenyl, 2-hydroxyphenyl or 2-methoxy-1-naphthalen-1-yl, n=2 and m=1, then R2 is not 1-naphthyl or 2-methoxy-1-naphthalen-1-yl; and the compound of Formula I is not N1,N1-(butane-1,4-diyl)bis(N3-(naphthalen-1-ylmethyl)propane-1,3-diamine, or N1,N1-(butane-1,4-diyl)bis(N3-(naphthalen-2-ylmethyl)propane-1,3-diamine.

2. The compound of Formula I according to claim 1, wherein n is 2.

3. The compound of Formula I according to claim 1, wherein m is 1.

4. The compound of Formula I according to claim 1, wherein R.sub.4 is CH.sub.3.

5. The compound of Formula I according to claim 1, selected from: ##STR00009## ##STR00010##

6. The compound of Formula I according to claim 1 selected from: ##STR00011##

7. A pharmaceutical composition which comprises a compound of Formula I according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

8. (canceled)

9. A method of treating or preventing blood coagulation in a subject in need thereof, which comprises administering to said subject an effective amount of a compound of Formula I according to claim 1 or a pharmaceutically acceptable salt thereof.

10. The compound of Formula I according to claim 1, wherein n is 2; and m is 1.

11. The compound of Formula I according to claim 1, wherein: n is 2; m is 1 and R.sub.4 is CH.sub.3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1. (A) Fluorescence emission of 3AC upon titration with heparin. The inset shows the variation of the emission fluorescence at 307 nm against the equivalents of repeating units of Hep (with the fitting to the proposed binding model). (B) Schematic representation of the binding model between 3AC and Hep, with the corresponding stepwise equilibrium constants shown for each step. (C) Stacked partial .sup.1H NMR spectra of 3AC upon increasing addition of dp14 heparin (concentration of dp14 related to the disaccharide repeating unit).

[0056] FIG. 2. (A) Schematic representation of the in vitro enzymatic assay used to test the reversal effect of the heparin ligands. (B) Plot of the time evolution of the hydrolysis of the peptide substrate (485 M) under different conditions: all traces (0.085 g/mL of FX.sub.a and 0.02 IU/mL of AT.sub.III), all traces except solid circles (0.1 IU/mL of Hep), traces with symbols except X and solid circles (1 M of each ligand, legend in the inset). (C) Plot of the percent hydrolysis at 30 min. for some selected ligands, including the FX.sub.a and Hep control tests, as well as the results obtained with ciraparantag (cirap.).

[0057] FIG. 3. (A) Ex vivo coagulation test with freshly extracted mouse blood using Hep in combination with 3FF and 3AC antidotes. Molar concentrations of Hep are related to the disaccharide repeating units. (B) In vivo tail transection assay performed in mice to test the reversal of Hep activity by 3AC or 3FF. Statistically different (ANOVA) results (p<0.0001) are labelled.

EXAMPLES

General Methods

[0058] General: Reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka or Merck) and were used without further purification. Flash chromatographic purifications and preparative reversed-phase purifications were performed on a BiotageIsolera Prime equipment. TLCs were performed using 63 cm SiO.sub.2 pre-coated aluminum plates (ALUGRAM@SIL G/UV254)

[0059] Nuclear Magnetic Resonance (NMR): Spectroscopic experiments for the characterization of compounds were carried out on a Varian Mercury 400 instrument (400 MHz for .sup.1H and 101 MHz for .sup.13C). Chemical shifts (H) are quoted in parts per million (ppm) and referenced to the appropriate NMR solvent peak(s). 2D-NMR experiments COSY, HSQC and HMBC were used where necessary in assigning NMR spectra. Spin-spin coupling constants (J) are reported in Hertz (Hz). For the experiments performed in aqueous buffer a low molecular weight heparin was used: dp14 (from Iduron, prepared by high resolution gel filtration of partial heparin lyase digestion of high quality heparin, MW average4100). One- and two-dimensional (1D and 2D) NMR experiments were performed at 298 K on a 500 MHz Bruker AVANCEIII-HD equipped with a z-gradient (65.7 G cm.sup.1) inverse TCI-cryoprobe. Samples were dissolved in 5 mM Tris-d11 buffer with 50 mM NaCl (in D.sub.2O, pH 7.5, uncorrected pH meter reading). Bruker TopSspin 3.5pl6 standard pulse sequences were used for 1D and 2D experiments.

[0060] Liquid Chromatography coupled to Mass Spectrometry: Analyses were carried out at the IQAC Mass Spectrometry Facility, using a UPLC-ESI-TOF equipment: [Acquity UPLCBEH C.sub.18 1.7 mm, 2.1100 mm, LCT Premier X.sub.e, Waters]. (CH.sub.3CN+20 mM HCOOH and H.sub.2O+20 mM HCOOH) mixtures at 0.3 mL/min were used as mobile phase.

[0061] Surface Plasmon Resonance (SPR): Affinity experiments between inhibitors and heparin were performed on an Open SPR(Nicoya). All measurements were performed at 25 C. using a working buffer of 25 mM Tris at pH 7.5. Biotin-loaded sensor chips (NICOYA) were further functionalized with streptavidine (50 g/mL) and later with biotin-heparin (50 g/mL). Binding experiments to heparin were performed by injecting inhibitors at desired concentrations and at a rate of 40 L/min. Between binding assays, the surface was regenerated by exposure to an injection of 10 mM HCl. Fitting has been performed by Trace Drawer software using a one-to-one two-state algorithm, which considers a 1:1 binding with a further equilibrium like a conformational change. Results obtained from three independent experiments at three different concentrations of the ligands were fit globally to render the corresponding on/off rate constants and the apparent dissociation constants (K.sub.D.sup.app Table 1, as shown below).

[0062] Fluorescence spectroscopy titration: Fluorescence emission and excitation spectra were collected on a Photon Technology International Instrument, the Fluorescence Master Systems, using the Software Felix32 and cuvettes with 10 mm path length. Stock solutions of the corresponding binder (20 M) and heparin (18 IU/mL) were prepared in 1 mM Bis-Tris buffer at pH 7.5. Then, 2 mL of the binder solution was placed on a quartz cell and the emission fluorescence spectrum was measured upon excitation at 280 nm. Then, small volumes of the heparin stock solution were added to the cell, and the fluorescence spectra was acquired after each addition. For 3AC, the titration experiments were fitted using HypeSpec2008 software, which allows a non-linear global fitting of the full emission spectra to a binding mode as defined by the user.

[0063] Blood coagulation factor in vitro enzymatic assays: Recombinant antithrombin Ill and coagulation Factor X.sub.a were obtained from the Berichrom Heparin test, supplied by SIEMENS. Following the indications of the test kit, stock solutions were prepared as follows: Human antithrombin III (1 IU/mL), Factor X.sub.a reagent (0.4 g/mL, human plasma fraction with the additives Tris, sodium chloride and EDTA) and a chromogenic substrate specific for factor X.sub.a (4 mM of Z-D-Leu-Gly-ArgANBA-methyl amide). On the other hand, 4-nitroaniline (Sigma Aldrich) was added to the substrate solution at a concentration of 1.6 mM and was used as internal standard to quantify the hydrolysis of the chromogenic substrate. Heparin (sodium salt from porcine intestinal mucosa, polydisperse, from 6000 to 30000 Daltons) was purchased from Sigma-Aldrich. All compounds were dissolved in mili-Q water at the desired stock concentrations prior to start the assays and kept at 4 C. Various concentrations of the ligand, heparin (0.1 IU/mL), human antithrombin Ill solution (3.5 L) and Factor X.sub.a reagent solution (35 L) were added in order to an Eppendorf and brought to a total volume of 145 L. Then, 20 L of Reagent Substrate solution was added to start the experiment and the mixture was vigorously shaken at 25 C. 20 L samples were taken at minutes 5/10/15/20/30/50/80/120, diluted with 40 L of acetic acid (20% v/v), and injected in the analytical HPLC. The gradient ranged from 5% ACN (0.1% formic acid) in water (0.1% formic acid) to 100% ACN in 24 minutes using a 154.6 mm KROMAPHASE C.sub.18 5.0 m column (retention time of cleaved chromophore 9.1 min, retention time of chromogenic substrate 13.9 min, retention time of 4-nitroaniline 15.4 min). Concentrations of reagents have been adjusted compared with previous studies with the intention to slow down the total exhaustion of peptide substrate. In this way the activity can be more carefully modulated and any change is easier to detect. FX.sub.a/AT.sub.III activity was represented as percent of hydrolysis, which was calculated from the normalized area of cleaved peptide at 405 nm at each corresponding time point. Experiment carried out in absence of heparin was considered as maximum of activity while experiment with heparin and no ligand was considered as negative control (maximum inhibition of FX.sub.a by heparin). Concentration of heparin was selected for the measurement to render a significant inhibition within experimental time, while allowing the reaction to proceed.

[0064] Ex vivo blood coagulation assays: Freshly collected mouse blood was collected and directly used without further treatment. 300 L aliquots were prepared. To them, Hep (130 M), and various concentration of ligand (3AC or 3FF) were added. The samples of the different conditions were added to an Eppendorf tube and a picture was taken after 15 minutes. The clot formation was confirmed by turning around the Eppendorf vials.

[0065] In vivo tail transection assays with mice: Eight-weeks old CD-1 male mice (Janvier-Labs) were used for the study. Animals were housed under a 12 h light: dark cycle in an environmentally controlled room and free access to water and food. Mice were randomly assigned into 6 groups (n=6 for each group). Animals were anesthetized by i.p. injections of 0.465 mg/kg xylazine (Rompun, Bayer DVM) and 1.395 mg/kg ketamine (Imalgene 100, Merial Laboratorios). After anesthetizing them, different solutions were injected intravenously according to the assigned group: Saline group (only saline was given), Hep group (Hep injection, 100 IU/kg), 3AC (two groups, Hep injection followed by injection of 3AC at 2.2 mg/kg or 4.4 mg/kg) and 3FF (two groups, Hep injection followed by injection of 3FF at 2.2 mg/kg or 4.4 mg/kg). Two additional control groups (n=3) were included in which only the ligand (3AC or 3FF) at the highest dose (4.4 mg/kg) was injected. At 5 minutes after injections, tails were transected at 5 mm from the tip and immediately inserted in a tube containing 1 ml of saline buffer immersed in a water bath at 37 C., where the blood draining out of the wound was collected for 10 minutes. The blood draining out from the wound was collected in each tube. Tails were let bleed during 10 minutes. The total blood volume in each tube was quantified by spectrophotometry (absorbance at 414 nm) from a standard curve that was constructed with known volumes of blood hemoglobin concentration and corresponding absorbance value. Statistical analysis of the data was done with Kaleida Graph 4.5.4 using ANOVA.

Synthetic Protocols and Characterization Data

General Synthetic Scheme for Symmetric and Asymmetric Molecules:

##STR00007##

[0066] Compounds of Formula I may be prepared following different methods known by a person skilled in the field of organic synthesis. In particular, they may be obtained following the routes depicted in Scheme 1.

Homomers:

[0067] First, spermine or any other suitable polyamine reacts with 2 eq. of an arylaldehyde (R1) dissolved, for example in THF, to give the corresponding imine derivative. The imine reacts with a reductive reactant, such as NaBH.sub.3CN, to obtain the desired 3R.sub.1R.sub.1 compound of Formula I.

Heteromers

[0068] First, spermine or any other suitable polyamine reacts with 1 eq. of an arylaldehyde (R1) to give the corresponding imine derivative. The imine reacts with a reductive reactant, such as NaBH.sub.3CN, to obtain the desired 3R.sub.1.

[0069] Then, intermediate 3R.sub.1 compound dissolved in MeOH, is reacted with a second aryl aldehyde of interest R.sub.2, affording another imine, which after reduction gives the desired 3R1R2 compound of Formula I.

[0070] Purification of intermediates and/or final compounds of Formula (I), if required, can be carried out by HPLC at semipreparative level.

Detailed Synthesis and Characterization:

[0071] Compounds 3AA (2,2-(2,6,11,15-tetraazahexadecane-1,16-diyl)diphenol), 3BB (N.sup.1,N.sup.1-(butane-1,4-diyl)bis(N.sup.3-(2-methoxybenzyl)propane-1,3-diamine)) and 3AL (2-(16-(naphthalen-1-yl)-2,6,11,15-tetraazahexadecyl)phenol), used for comparative purposes were previously characterized in Carbajo, D., Prez, Y., Bujons, J. & Alfonso, I. Live-Cell-Templated Dynamic Combinatorial Chemistry. Angew. Chem. Int.

Ed. 59, 17202-17206 (2020).

Intermediates 3R1:

2-(((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)methyl)phenol (3A)

[0072] Spermine (120 mg, 0.594 mmol) was dissolved in 75 mL of THF.sub.anh at 0 C. 2-hydroxybenzaldehyde (58 L, 0.534 mmol) dissolved in 20 mL of MeOH was dropwise added and the reaction was stirred overnight. The day after, NaBH.sub.3CN (67 mg, 1.07 mmol) was added and stirred 24 h. Reaction was stopped by addition of 3 mL of H.sub.2O and 3 mL of HCl 1M. THF was carefully rotavapored and reaction crude was purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 77 mg of 3A were obtained as white powder (Yield=47%, Purity=91.1%).

[0073] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.2 (2H, m), 6.85 (2H, m), 4.11 (2H, s), 2.97 (12H, m), 1.94 (4H, m), 1.61 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 154.9, 131.7, 131.5, 120.6, 115.5, 114.8, 46.9, 44.4 (, 43.6, 36.4, 23.6, 22.6, 22.4.

[0074] MS: Calculated for C.sub.17H.sub.32N.sub.4O: 308.4700; found: 309.2684 (M+H).sup.+

4-(((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)methyl)phenol (3J)

[0075] Following an analogous procedure to that described for 3A but using 4-hydroxybenzaldehyde, intermediate 3J was obtained as white powder (Yield=66%, Purity=98.1%).

[0076] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.18 (2H, d, J=8 Hz), 6.78 (2H, d, J=8 Hz), 3.95 (2H, s), 2.92 (12H, m), 1.89 (4H, m), 1.58 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 156.7, 131.4, 123.3, 115.9, 50.8, 47.1, 47.0, 44.8 (C10), 44.6, 43.9, 36.7, 24.2, 23.3, 23.2.

[0077] MS: Calculated for C.sub.17H.sub.32N.sub.4O: 308.4700; found 309.2698 (M+H).sup.+

2-(((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)methyl)-4-bromo-6-methoxyphenol (3O)

[0078] Spermine (54 mg, 0.267 mmol) was dissolved in 25 mL of THF.sub.anh at 0 C. 5-bromo-2-hydroxy-3-methoxybenzaldehyde (54 mg, 0.24 mmol) dissolved in 10 mL of MeOH was dropwise added and the reaction was stirred overnight. The day after, NaBH.sub.3CN (30 mg, 0.48 mmol) was added and stirred 24 h. Reaction was stopped by addition of 2 mL of H.sub.2O and 2 mL of HCl 1M. THF was carefully rotavapored and reaction crude was purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 30 mg of 30 were obtained as brown powder (Yield=31%, Purity=97.3%).

[0079] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.18 (1H, s), 7.01 (1H, s), 4.11 (2H, s), 3.77 (3H, s), 2.98 (12H, m), 1.96 m (4H, H9, m), 1.65 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 148.4, 144.0, 125.2, 118.5, 116.6, 111.1, 56.3, 46.9, 45.8, C14), 44.4, 44.3, 36.4, 23.6, 22.7, 22.4.

[0080] MS: Calculated C.sub.18H.sub.33BrN.sub.4O.sub.2: 416.1787; found: 419.1703 and 417.1704 (M+H).sup.+

Compounds of 3R1R1 type

1,1-(2,6,11,15-tetraazahexadecane-1,16-diyl)bis(naphthalen-2-ol) (3FF)

[0081] Spermine (60 mg, 0.297 mmol) was dissolved in 25 mL of MeOH. 2-hydroxy-1-naphthaldehyde (193 mg, 1.039 mmol) was then added dissolved in 12 mL of MeOH. The solution was stirred 6 h. Then, NaBH.sub.3CN (126 mg, 2 mmol) was added and the reaction was stirred 24 h. After addition of H.sub.2O (2 mL) and HCl 1M (2 mL), the reaction was stirred 1 h. Solvent was removed by rotavaporation. Reaction mixture was purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA) to yield 78 mg (51%) of pure product 3FF (99% by HPLC). .sup.1H RMN (H.sub.2O/D.sub.2O): 7.86 (m, 6H), 7.54 (t, 2H, J=8 Hz), 7.36 (t, 2H, J=8 Hz), 7.18 (d, 2H, J=12 Hz), 4.65 (s, 2H), 3.14 (m, 4H), 3.00 (m, 8H), 2.04 (m, 4H), 1.62 (m, 4H). .sup.13C RMN (H.sub.2O/D.sub.2O): 154.1, 132.4, 132.1, 128.9, 128.4, 127.8, 123.7, 121.6, 117.3, 46.8, 44.4, 43.8, 41.6, 22.6, 22.4.

[0082] MS: Calculated for C.sub.32H.sub.42N.sub.4O.sub.2: 514.7140; found: 515.3389 (M+H).sup.+

2,2-(2,6,11,15-tetraazahexadecane-1,16-diyl)bis(naphthalen-1-ol) (3GG)

[0083] Spermine (20 mg, 0.099 mmol) was dissolved in 5 mL of MeOH at 0 C. 1-hydroxy-2-naphthaldehyde (40 mg, 0.215 mmol) was carefully added dissolved in 2 mL of MeOH. After 6 h, solution was clear red. NaBH.sub.3CN (28 mg, 0.444 mmol) was then added. The day after, solution had become brown. Reaction was stopped by addition of 0.5 mL of H.sub.2O and 0.5 mL of HCl 1M. Solvent was removed and crude was purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 8.5 mg of brown solid was obtained as 3GG pure product (Purity=97.4%, Yield=16%).

[0084] .sup.1H RMN (H.sub.2O/D.sub.2O): 8.06 (2H, dd), 7.82 (2H, dd), 7.48 (6H, m), 7.3 (2H, d), 4.33 (4H, s), 3.05 (4H, tr), 2.93 (8H, m), 1.99 (4H, m), 1.57 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 150.9, 135.0, 128.1, 127.8, 127.4, 126.3, 124.9, 121.4, 121.2, 113.1, 46.8, 44.4, 43.6, 22.6, 22.4.

[0085] MS: Calculated for C.sub.32H.sub.40N.sub.4O.sub.2: 514.3308; found: 515.3438 (M+H).sup.+

Compounds of 3R1R2 Type

2-(16-(2-hydroxyphenyl)-2,6,11,15-tetraazahexadecyl)naphthalen-1-ol (3AG)

[0086] 3A (53 mg, 0.171 mmol) was dissolved in 15 mL of THF.sub.anh at 0 C. 1-hydroxy-2-naphthaldehyde (35 mg, 0.205) was added dissolved in 1 mL of THF.sub.anh. After 4 h, NaBH.sub.3CN (24 mg, 0.377 mmol) was added and the reaction stirred overnight. The day after, reaction was stopped by addition of 0.5 mL of H.sub.2O and 0.5 mL of HCl 1M. Solvent was removed and crude purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 32 mg of 3AG were obtained as a light brown powder (Purity=97.4%, Yield=40%)

[0087] .sup.1H RMN (H.sub.2O/D.sub.2O): 8.06 (1H, dd, J1=3.4 Hz, J=6.3 Hz); 7.82 (1H, dd, J=3.4 Hz, J2=6.3 Hz); 7.49 (3H, m), 7.31 (1H, d, J=8.4 Hz3), 7.22 (2H, m,), 6.85 (2H, m), 4.32 (2H, s), 4.11 (2H, s), 3.00 (12H, m), 1.99 (4H, m), 1.59 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 155.0, 150.9, 135.0, 131.7, 131.5, 128.1, 127.8, 127.4, 126.3, 125.1, 121.4, 121.2, 120.5, 117.0, 115.5, 113.1, 46.9, 44.4, 43.6, 22.6, 22.4.

[0088] MS: Calculated for C.sub.28H.sub.40N.sub.4O.sub.2: 464.3151; found MS 465.3228 (M+H).sup.+

1-(16-(2-hydroxyphenyl)-2,6,11,15-tetraazahexadecyl)naphthalen-2-ol (3AF)

[0089] 3A (20 mg, 0.065 mmol) was dissolved in 10 mL of THF.sub.anh at 0 C. 2-hydroxy-1-naphthaldehyde (13 mg, 0.078 mmol) was added dissolved in 1 mL of THF.sub.anh. After 4 h, NaBH.sub.3CN (9 mg, 0.143 mmol) was added and the reaction stirred overnight. The day after, reaction was stopped by addition of 0.5 mL of H.sub.2O and 0.5 mL of HCl 1M. Solvent was removed and crude purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 6 mg of 3AF were obtained as a light brown powder (Purity >99%, Yield=19%)

[0090] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.90 (3H, m), 7.57 (1H, dd, J=8 Hz), 7.4 (1H, dd, J=8 Hz), 7.29 (2H, m), 7.21 (1H, d, J=9 Hz), 6.91 (2H, m), 4.68 (2H, s), 4.19 (2H, s), 3.17 (2H, m), 3.02 (10H, m), 2.08 (4H, m), 1.66 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 155.1, 154.2, 132.5, 132.2, 131.7, 131.6, 129.0, 128.5, 127.9, 123.8, 121.3, 120.6, 117.8, 117.1, 115.6, 114.9, 108.3, 46.9, 44.5, 43.9, 43.7, 41.7, 22.7, 22.5, 22.4.

[0091] MS: Calculated for C.sub.28H.sub.40N.sub.4O.sub.2: 464.3151; found: 465.3169 (M+H).sup.+

2-(16-(9H-fluoren-2-yl)-2,6,11,15-tetraazahexadecyl)phenol (3AC)

[0092] 3A (30 mg, 0.097 mmol) was dissolved in 5 mL of methanol at 0 C. 9H-fluorene-2-carbaldehyde (110 mg, 0.580 mmol) was added dissolved in 15 mL of methanol. The day after, NaBH.sub.3CN (70 mg, 1.15 mmol) was added and the reaction stirred overnight. Reaction was stopped by addition of 2 mL of H.sub.2O. Solvent was removed and crude purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 5 mg of 3AC were obtained as a white powder (Purity >99%, Yield=12%)

[0093] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.82 (2H, m), 7.56 (2H, m), 7.25 (3H, m), 7.21 (2H, m), 6.88 (2H, m), 4.18 (2H, s), 4.14 (2H, s), 3.85 (2H, s), 3.00 (12H, m), 2.01 (4H, m), 1.62 (4H, m). .sup.13C NMR (H.sub.2O/D.sub.2O): 155.0, 144.4, 143.9, 142.5, 140.2, 131.7, 131.6, 128.7, 128.6, 127.7, 127.1, 126.6, 120.5, 120.4, 120.3, 117.7, 115.5, 114.5, 51.4, 46.9, 44.4, 43.6, 36.3, 22.6, 22.4.

[0094] MS: Calculated for C.sub.31H.sub.42N.sub.4O: 486.7040; found: 487.4005 (M+H).sup.+

2-(16-(naphthalen-2-yl)-2,6,11,15-tetraazahexadecyl) (3AN)

[0095] 3A (15 mg, 0.0485 mmol) was dissolved in 5 mL of THF.sub.anh at 0 C. 2-naphtaldehyde (9 mg, 0.0582 mmol) was added dissolved in 1 mL Of THF.sub.anh. After 4 h, NaBH.sub.3CN was added and the reaction stirred overnight. The day after, reaction was stopped by addition of 0.5 mL of H.sub.2O and 0.5 mL of HCl 1M. Solvent was removed and crude purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 15 mg of 3AN were obtained as a white powder (Purity >99%, Yield=69%)

[0096] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.87 (m, 4H), 7.50 (m, 2H7.43 (d, J=8.44 Hz, 1H), 7.22 (m, 2H), 6.85 (tr, J=6.97 Hz, 2H), 4.3 (s, 2H), 4.12 (s, 2H), 3.00 (m, 12H), 1.98 (m, 4H), 1.59 (m, 4H). .sup.13C RMN (H.sub.2O/D.sub.2O): 155.0, 133.1, 132.7, 131.7, 131.5, 129.6, 129.1, 128.0, 127.7, 127.4, 127.0, 126.4, 120.5, 117.0, 115.5, 51.3, 46.9, 46.8, 44.4, 44.3, 43.7, 43.6, 22.6, 22.5, 22.4.

[0097] MS: Calculated for C.sub.28H.sub.40N.sub.4O: 448.3202; found: 449.3520 (M+H).sup.+

4-bromo-2-methoxy-6-(16-(naphthalen-1-yl)-2,6,11,15-tetraazahexadecyl)phenol (3LO)

[0098] 3O (15 mg, 0.036 mmol) was dissolved in 5 mL of THF.sub.anh at 0 C. 1-naphthaldehyde (6 L, 0.043 mmol) was added dissolved in 1 mL of THF.sub.anh. The day after, NaBH.sub.3CN (4.5 mg, 0.072 mmol) was added and the reaction stirred overnight. Reaction was stopped by addition of 0.5 mL of H.sub.2O and 0.5 mL of HCl 1M. Solvent was removed and crude purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 7 mg of 3LO were obtained as a brownish powder (Purity=98.7%, Yield=35%)

[0099] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.90 (3H, m), 7.50 (4H, m), 7.14 (1H, d J=4 Hz), 6.97 (1H, d, J=4 Hz), 4.65 (2H, s), 4.08 (2H, s), 3.73 (3H, s), 3.12 (2H, m), 2.96 (1OH, m), 1.98 (4H, m), 1.59 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 148.4, 144.1, 133.6, 130.8, 130.6, 129.5, 129.1, 127.5, 126.7, 126.3, 125.6, 125.3, 122.4, 118.6, 116.7, 111.2, 56.4, 48.1, 46.9, 45.9, 44.3, 22.7, 22.6, 22.4.

[0100] MS: Calculated C.sub.29H.sub.41BrN.sub.4O.sub.2: 557.5770; found: 557.2440 and 559.2421 (M+H).sup.+

1-(16-(5-bromo-2-hydroxy-3-methoxyphenyl)-2,6,11,15-tetraazahexadecyl)naphthalen-2-ol (3FO)

[0101] 3O (15 mg, 0.036 mmol) was dissolved in 5 mL of THF.sub.anh at 0 C. 2-hydroxy-1-naphthaldehyde (7 mg, 0.043 mmol) was added dissolved in 1 mL Of THF.sub.anh. The day after, NaBH.sub.3CN (4.5 mg, 0.072 mmol) was added and the reaction stirred overnight. Reaction was stopped by addition of 0.5 mL of H.sub.2O and 0.5 mL of HCl 1M. Solvent was removed and crude purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water 0.1% TFA). 5 mg of 3FO were obtained as a white powder (Purity >99%, Yield=25%)

[0102] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.86 (3H, m), 7.54 (1H, dd, J=7.5 Hz), 7.36 (1H, dd, J=7.5 Hz), 7.18 (2H, m), 7.01 (1H, d, J=2 Hz), 4.64 (2H, s), 4.12 (2H, s), 3.88 (3H, s), 3.12 (2H, m), 3.00 (10H, m) 2.03 (4H, m), 1.63 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 154.2, 148.4, 144.4, 132.5, 132.1, 129.0, 128.4, 127.9, 125.2, 123.7, 121.2, 117.3, 116.6, 114.8, 111.2, 108.2, 56.3, 46.9, 44.4, 43.8, 41.6, 22.6, 22.5.

[0103] MS: Calculated C.sub.29H.sub.41BrN.sub.4O3: 573.5760; found: 575.2441 and 573.2471 (M+H).sup.+

1-(16-(4-hydroxyphenyl)-2,6,11,15-tetraazahexadecyl)naphthalen-2-ol (3FJ)

[0104] 3J (23 mg, 0.074 mmol) was dissolved in 10 mL of THF.sub.anh at 0 C. After that, 2-hydroxy-1-naphthaldehyde (15 mg, 0.089 mmol) was added dissolved in 5 mL of THF.sub.anh. The day after, NaBH.sub.3CN (9 mg, 0.148 mmol) was added and the reaction was stirred overnight. Reaction was stopped by the addition of 1 mL of H.sub.2O and 1 mL of HCl 1M. Solvent was removed and crude was purified by reverse phase chromatography with a gradient of ACN (0.1% TFA) and water (0.1% TFA). 28 mg of 3FJ were obtained as a white fluffy solid (Purity >98.5%, Yield=82%).

[0105] .sup.1H RMN (H.sub.2O/D.sub.2O): 7.81 (3H, m), 7.5 (1H, dd, J1=7.5 Hz, J2=1 Hz), 7.32 (1H, dd, J1=7.5 Hz, J2=1 Hz), 7.19 (2H, d, J3=8.5 Hz), 7.14 (1H, d, J5=8 Hz), 6.79 (2H, d, J3=8.5 Hz), 4.6 (inside water, s), 4.02 (3H, s), 3.09 (2H, m), 2.95 (10H, m), 1.95 (4H, m), 1.58 (4H, m). .sup.13C RMN (H.sub.2O/D.sub.2O): 156.7, 154.2, 132.4, 132.1, 132.0, 131.6, 129.0, 128.4, 127.8, 123.7, 121.2, 117.3, 115.9, 108.2, 50.6, 46.9, 44.4, 43.7, 43.4, 41.7, 40.0, 22.6, 22.5.

[0106] MS: Calculated C.sub.28H.sub.40N.sub.4O.sub.2: 464.3151; found: 465.3310 (M+H).sup.+

Biophysical and Biochemical Tests Related to the Inhibition of the Heparin Activity.

[0107] Binding of the spermine derivatives to the heparin target. Surface Plasmon Resonance (SPR) on heparin-functionalized chips were used to determine the apparent dissociation constant of selected molecules (K.sub.D.sup.app, Table 1). The SPR-determined K.sub.D.sup.app for molecules containing an ortho phenol and a large aromatic ring (3AF and 3AG) display a K.sub.D.sup.app in the low micromolar range (entries 1 and 2). Quite remarkably, 3FF and 3AC render sub-micromolar affinity to Hep by SPR (entries 3 and 4). Moreover, two of the identified hits (3AC and 3FF) show higher affinity to Hep than ciraparantag (entry 5).

TABLE-US-00001 TABLE 1 Apparent dissociation constants, K.sub.D.sup.app (M), for the interaction of selected molecules with Hep. Entry Binder K.sub.D.sup.app (M) by SPR 1 3AF 2.62 0.20 2 3AG 1.86 0.12 3 3FF 0.797 0.24 4 3AC 0.567 0.013 5 Ciraparantag 1.53 0.12

[0108] Many binders are fluorescent and their emission spectra are perturbed by the interaction with Hep, allowing to obtain additional information about the supramolecular complexes in solution. The emission spectrum of 3AC is strongly reduced upon addition of up to 2 equivalents of the Hep disaccharide repeating units, slightly recovering upon additional Hep (FIG. 1A). Despite the complex titration isotherm, this could be fitted to a reasonable binding mode considering the disaccharide repeating unit of Hep as the binding motif (inset in FIG. 1A). The proposed binding model is depicted in FIG. 1B: 3AC forms a strong complex with one ligand per disaccharide repeating unit ([3AC-Hep] complex), which evolves to the [3AC-Hep.sub.2] complex upon additional Hep, or to a less specific species [3AC.sub.2-Hep] in the presence of excess 3AC. This binding model explains the observed fluorescence emission results: the proximity of the chromophores in the [3AC-Hep] and [3AC.sub.2-Hep]complexes accounts for the fluorescence quenching that is partially recovered when the 3AC bound molecules are spread along the Hep chain in the [3AC-Hep.sub.2] species. A putative K.sub.D.sup.app0.5 M can be estimated from the fluorescence binding data, which is in reasonable agreement with the SPR results.

[0109] The 3AC-Hep binding was also studied using .sup.1H NMR, in this case with a shorter heparin oligomer, dp14, which on average contains seven disaccharide repeating units. The titration of 3AC with dp14 (FIG. 1C) shows two different phases. At the beginning of the titration (low dp14: 3AC ratio), it has been observed a large broadening of the 3AC spectrum leading to the disappearance of some proton signals. Under these conditions, the [3AC.sub.2-dp14] and [3AC-dp14] complexes prevail (see FIG. 1B), which lead to an electrostatic charge compensation of dp14 favoring the aggregation of the species in solution. Addition of more equivalents of Hep produces the complete formation of [3AC-dp14.sub.2] species, which is clearly visible in the .sup.1H NMR spectrum by the growing of broad signals at a different chemical shift from those of 3AC alone. This titration experiment strongly correlates with the binding mode inferred from the fluorescence titration, while the slow exchange in the chemical shift NMR timescale is consistent with the very strong binding quantified by SPR and fluorescence spectroscopy.

[0110] In vitro inhibition of heparin activity. The effect of the binders as Hep antidotes can be tested in vitro using an enzymatic reaction related to the blood coagulation process (FIG. 2A). This test employs human recombinant coagulation factor X.sub.a (FX.sub.a) and antithrombin III (AT.sub.III), with the synthetic peptide Z-D-LGR-N(Me)ANBA as the substrate of the FX.sub.a protease to mimic the prothrombin to thrombin transformation that triggers blood coagulation. Heparin forms a ternary complex with AT.sub.III and FX.sub.a with a low hydrolytic activity, as a model for the anticoagulation effect of Hep. The addition of an efficient Hep binder dissociates the [FX.sub.a-Hep-AT.sub.III] complex, thus releasing the active form of FX.sub.a that cleaves the substrate. The peptide hydrolysis can be readily monitored by analytical HPLC thanks to the ANBA chromophore, as an accurate measurement of the reaction kinetics.

[0111] FIG. 2B shows the kinetic of hydrolysis of the peptide in different reaction conditions. In the absence of Hep (solid circles) the FXa exhibits the highest activity, which is clearly inhibited when Hep is present (0.1 IU/mL, solid squares). In parallel experiments, the addition of equal concentration (1 M) of each of the binders produces a different level of recovery of the FXa activity. Thus, the in vitro potency as Hep antidote grows in the series 3AL (X symbols)3AG (rhombuses)<3AF (open circles)<3FF (inverted solid triangles)<3AC (solid triangles). This trend nicely correlates with the binding data shown in Table 1. Actually, several selected molecules derived from spermine were tested in the enzymatic assay (FIG. 2C) most of them showing activity in the reversal of the heparin action. Also in the in vitro assays, 3FF and 3AC perform as the most active Hep antidotes, with 3FF being comparable with the drug ciraparantag (cirap.) and 3AC showing to be even more potent (FIG. 2C).

[0112] Reversing the anticoagulant activity of heparin: In order to check the efficacy of the antidotes in a more representative environment, 3FF and 3AC were tested in real blood coagulation assays. Thus, it has been monitored the coagulation of freshly extracted mouse blood in the absence or presence of Hep, and upon addition of different concentrations of 3FF and 3AC. By simply turning around the vials after 15 minutes (FIG. 3A), the differences in their viscosity are evident: the control samples are completely coagulated, Hep-containing vials remain liquid (the blood flows down), while 3FF and 3AC are able to revert the anticoagulant effect of Hep in a dose-response manner. Actually, at least qualitatively, 3AC seems to be a better antidote than 3FF in this simple ex vivo experiment, since a more robust clot was observed at equal doses.

[0113] 3FF and 3AC were tested in an in vivo coagulation model. It was thus performed the tail transection assay with mice after consecutive injection of Hep, and the drugs at two different doses each (FIG. 3B). The control groups injecting saline or Hep, and those corresponding to just the drugs were also included. The results clearly show that both 3FF and 3AC are efficient antidotes of Hep also in vivo, strongly reducing the bleeding of heparinized mice. Remarkably, the amount of blood loss is dose responsive for both drugs, and 3AC is a more potent reversal agent than 3FF. At the highest doses of 3FF/3AC here used, the blood loss of heparinized mice was reversed to values very similar to those of the control group injected with saline, strongly supporting the potential use of these molecules as Hep antidotes.