Barbituric acid derivatives comprising cationic and lipophilic groups
11072588 · 2021-07-27
Assignee
Inventors
- Morten Bøhmer Strøm (Tromsø, NO)
- Annette Bayer (Tromsø, NO)
- Stig Olov Magnus Engqvist (Tromso, NO)
- Marianne Hagensen Paulsen (Tromsø, NO)
- Dominik Ausbacher (Tromsø, NO)
Cpc classification
C07D233/96
CHEMISTRY; METALLURGY
A61P31/00
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present invention relates to bioactive cyclic compounds and their use as antimicrobial agents. In particular, the present invention relates to barbiturate mimics of Eusynstyelamides or small antimicrobial peptides. The compounds of the invention are represented by Formula (I). (I) ##STR00001##
Claims
1. A compound of Formula (I) ##STR00016## or a stereoisomer, a tautomer, or a solvate thereof, wherein: X is CH.sub.2 or C═W; Y is CH.sub.2 or C═W; Z is a bond, CH.sub.2 or C═W; W is N, O or S; each R.sup.1, which may be the same or different, comprises 2-15 non-hydrogen atoms and at least one cationic group which has a net charge of at least +1 at pH 7; each R.sup.2, which may be the same or different, is lipophilic and comprises at least 7 non-hydrogen and non-fluorine atoms; alternatively the R.sup.2 groups are linked or fused to form a lipophilic group having a total of at least 14 non-hydrogen and non-fluorine atoms, or at least 12 non-hydrogen and non-fluorine atoms when cyclic groups within each group are fused together; at least one R.sup.2 group contains a cyclic group; and the compound has a net positive charge of at least +2 at pH 7.
2. The compound of claim 1, wherein W is O.
3. The compound of claim 1, wherein X is C═W and Y is C═W and Z is a bond or C═W.
4. The compound of claim 3, wherein Z is a bond.
5. The compound of claim 3, wherein Z is C═W.
6. The compound of claim 1, wherein the compound is a compound of Formula (II) ##STR00017## wherein R.sup.1 and R.sup.2 are as defined in claim 1.
7. The compound of claim 1, wherein R.sup.1 comprises a cationic amine group or a cationic imine group.
8. The compound of claim 1, wherein R.sup.1 comprises at least one of —NR.sub.3+, ═NR.sub.2+, —NR.sub.2+—, and ═NR.sup.+—, wherein each R is the same or different at each occurrence and is H or alkyl.
9. The compound of claim 8, wherein R.sup.1 comprises —NR.sub.3+ or —NR—C(═NR.sub.2+—NR.sub.2 as the cationic group, wherein R is the same or different and is H or alkyl.
10. The compound of claim 8, wherein R is H, CH.sub.3 or CH.sub.2CH.sub.3.
11. The compound of claim 1, wherein the R.sup.2 groups are not linked or fused together.
12. The compound of claim 11, wherein each R.sup.2 group comprises at least 8 non-hydrogen and non-fluorine atoms.
13. The compound of claim 12, wherein each R.sup.2 group comprises at least 9 non-hydrogen and non-fluorine atoms.
14. The compound of claim 1, wherein both R.sup.2 groups contain a cyclic group.
15. A formulation comprising a compound of Formula (I) as defined in claim 1 in admixture with a suitable diluent, carrier or excipient.
16. A method of treating a microbial infection, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I) as defined in claim 1.
17. The method of claim 16, wherein the microbial infection is bacterial or fungal infection.
18. A method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I) as defined in claim 1.
19. The compound of claim 1, wherein R.sup.1 is ##STR00018## wherein each R independently is H or alkyl, and n is 1-10.
20. The compound of claim 19, wherein R.sup.1 is ##STR00019## wherein R is H, CH.sub.3 or CH.sub.2CH.sub.3and n is 2-8.
21. The compound of claim 19, wherein each R.sup.2 group is selected from alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, haloalkoxy, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, each of which may optionally be substituted with a substituent selected from the group consisting of halo, —CN, —R.sup.4NO.sub.2, —R.sup.4OR.sup.3, —R.sup.4(═O)R.sup.3, —R.sup.4OC(═O)R.sup.3, —R.sup.4O.sub.2R.sup.3, —R.sup.4N(R.sup.3).sub.2, —R.sup.4(═O)N(R.sup.3).sub.2, —R.sup.4OC(═O)N(R.sup.3).sub.2, —R.sup.4NR.sup.3(═O)R.sup.3, —R.sup.4NR.sup.3(═O)OR.sup.3, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, haloalkoxy, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl; wherein R.sup.4 is a bond or alkyl; R.sup.3 is selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, haloalkoxy, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl.
22. The compound of claim 19, wherein R.sup.2 is optionally substituted phenyl, naphthyl or pyridine.
23. The compound of claim 19, wherein each R.sup.2 group is -L-R.sub.x, wherein: L is a bond, alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy or haloalkoxy; and R.sub.x is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group, optionally substituted with a substituent selected from the group consisting of halo, —CN, —R.sup.4NO.sub.2, —R.sup.4OR.sup.3, —R.sup.4(═O)R.sup.3, —R.sup.4OC(═O)R.sup.3, —R.sup.4O.sub.2R.sup.3, —R.sup.4N(R.sup.3).sub.2, —R.sup.4(═O)N(R.sup.3).sub.2, —R.sup.4OC(═O)N(R.sup.3).sub.2, —R.sup.4NR.sup.3(═O)R.sup.3, —R.sup.4NR.sup.3(═O)OR.sup.3, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, haloalkoxy, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl; wherein R.sup.4 is a bond or alkyl; R.sup.3 is selected from the group consisting of H, alkyl, alkenyl, alkynyl, heteroalkyl, alkoxy, haloalkoxy, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl.
24. The compound of claim 23, wherein L is C.sub.1-3 alkyl, R.sub.x is an optionally substituted group selected from the group consisting of phenyl, naphthyl and pyridine.
25. The compound of claim 21, wherein the compound is ##STR00020## wherein n is 1-10.
26. The compound of claim 1, which is selected from the group consisting of ##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
27. The compound of claim 19, which is ##STR00027##
28. The compound of claim 1, wherein the cationic group is a cationic amine or a cationic imine.
Description
(1) The invention will now be described by way of the following non-limiting Examples with reference to the Figures in which:
(2)
(3)
(4)
(5)
EXAMPLES
Example 1
(6) Antimicrobial Activity Against Bacterial Reference Strains
(7) Two series of amphipathic barbiturates were prepared by the methods set out below.
(8) Series 7 consisted of barbiturates with two cationic amino groups and series 8 encompassed barbiturates with two cationic guanidine groups (
(9) TABLE-US-00001 TABLE 1 Antimicrobial activity (MIC in μg/mL) against antibiotic susceptible Gram- positive and Gram-negative reference strains. Antimicrobial activity.sup.a Compound Mw.sup.b S. a C. g E. c P. a 7(i) 790.83 4 1 4 8 7(ii) 994.20 4 1 4 8 7(iii) 903.04 1 0.25 2 4 7(iv) 778.74 8 1 16 16 7(v) 814.72 4 1 16 8 7(vi) 950.61 8 1 8 8 8(i) 874.91 1 <0.13 2 4 8(ii) 1078.28 1 0.25 2 4 8(iii) 987.12 1 0.25 4 4 8(iv) 862.82 1 0.25 1 8 8(v) 898.77 1 0.25 1 4 Oxytetracycline 460.434 0.65 0.65 2.5 20 .sup.aBacterial reference strains: S. a Staphylococcus aureus ATCC 9144, C. g Corynebacterium glutamicum ATCC 13032, E. c Escherichia coli ATCC 25922, and P. a Pseudomonas aeruginosa PA01, DSM 19880 (ATCC 15692). .sup.bMolecular weight including 2 equiv. of CF.sub.3OO.sup.−, i.e. + Mw 228.05.
(10) For the amine barbiturates of series 7 the minimum inhibitory concentration (MIC) values ranged from 0.25-8 μg/mL against the Gram-positive strains S. aureus and C. glutamicum, and MIC values from 2-16 μg/mL against the Gram-negative bacteria E. coli and P. aeruginosa.
(11) Higher antimicrobial activity was therefore in general observed against the Gram-positive bacteria than against the Gram-negative bacteria, although the differences were marginal for the most potent amine barbiturates of series 7.
(12) The most potent amine barbiturate was 7(iii), which had two super-bulky lipophilic 3,5-di-tBu-benzylic side-chains and displayed MIC values in the very lower range of 0.25-4 μg/mL against all the reference strains.
(13) The two barbiturates 7(i) and 7(ii) were the second most potent derivatives displaying MIC values of 1-8 μg/mL against the reference strains. These had smaller lipophilic side-chains, and revealed a correlation between side-chain size and antimicrobial activity.
(14) Guanylation of the amine barbiturates of series 7 resulted in a striking increase in antimicrobial activity for the resulting guanidine barbiturates of series 8. The highly potent guanylated barbiturates of series 8 showed a narrow range in MIC values of <0.13-1 μg/mL against the Gram-positive strains S. aureus and C. glutamicum, and MIC 1-8 μg/mL against the Gram-negative bacteria E. coli and P. aeruginosa.
(15) The guanylated barbiturates thereby showed high potency against the Gram-positive reference strains and were considered equipotent. Antimicrobial activity against the Gram-negative reference strains revealed 8(i), 8(ii), and 8(v) as the most potent derivatives with MIC values 1-4 μg/mL against E. coli and P. aeruginosa.
(16) The guanidine analogue 8(iii) (MIC: 4 μg/mL) followed thereafter and was an analogue of the highly potent and super bulky amine barbiturate 7(iii).
(17) Antimicrobial Activity Against 30 Multi-Resistant Clinical Isolates
(18) The barbiturates were also screened against a panel of 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum β-lactamase-carbapenemase (ESBL-CARBA) production and colistin resistance.
(19) The antimicrobial activity (MIC in μg/ml) for the compounds tested is shown in Table 2 below. Toxicity is displayed as haemolytic activity against human RBCs (EC.sub.50 in μg/ml) and a selectivity index in parenthesis (EC.sub.50/MIC values) against individual isolates.
(20) TABLE-US-00002 TABLE 2 Compound 7(i) 7(ii) 7(iii) 7(iv) 7(v) 7(vi) RBC 143 83 <4 243 160 183 Clinical isolates Antimicrobial activity S. aureus N315 8 (18) 4 (21) 2 (<2) 8 (30) 8 (20) 16 (11) S. aureus NCTC 10442 8 (18) 4 (21) 2 (<2) 8 (30) 8 (20) 16 (11) S. aureus strain 85/2082 8 (18) 4 (21) 2 (<2) 8 (30) 4 (40) 16 (11) S. aureus strain WIS 8 (18) 4 (21) 2 (<2) 8 (30) 8 (20) 16 (11) S. aureus IHT 99040 8 (18) 4 (21) 2 (<2) 8 (30) 8 (20) 16 (11) E. faecium 50673722 8 (18) 4 (21) 2 (<2) 16 (15) 8 (20) 8 (23) E. faecium 50901530 4 (36) 4 (21) 2 (<2) 8 (30) 4 (40) 8 (23) E. faecium K36-18 8 (18) 8 (10) 2 (<2) 16 (15) 8 (20) 16 (11) E. faecium 50758899 8 (18) 4 (21) 2 (<2) 16 (15) 8 (20) 16 (11) E. faecium TUH50-22 8 (18) 2 (<2) 8 (30) 4 (40) 8 (23) E. coli 50579417 8 (18) 8 (10) 4 (<1) 16 (15) 16 (10) 16 (11) E. coli 50639799 8 (18) 8 (10) 4 (<1) 16 (15) 16 (10) 16 (11) E. coli 50676002 4 (36) 8 (10) 8 (<0.5 16 (15) 16 (10) 16 (11) E. coli 50739822 8 (18) 8 (10) 4 (<1) 16 (15) 16 (10) 16 (11) E. coli 50857972 4 (36) 8 (10) 4 (<1) 16 (15) 16 (10) 8 (23) P. aeruginosa K34-7 >32 (—) 16 (5) 8 (<0.5) 32 (8) 32 (5) 32 (6) P. aeruginosa K34-73 >32 (—) 16 (5) 16 (<2.5) 32 (8) 32 (5) 32 (6) P. aeruginosa K44-24 >32 (—) 16 (5) 8 (<0.5) >32 (—) 32 (5) 32 (6) P. aeruginosa 50692172 >32 (—) 16 (5) 8 (<0.5) 32 (8) 16 (10) 32 (6) P. aeruginosa 50692520 >32 (—) 16 (5) 8 (<0.5) 32 (8) 16 (10) 32 (6) K. pneumoniae K47-25* >32 (—) 16 (5) 16 (<2.5) >32 (—) >32 (—) >32 (—) K. pneumoniae K66-45 32 (4) 16 (5) 8 (<0.5) >32 (—) 32 (5) 32 (6) K. pneumoniae 50531633* 16 (9) 8 (10) 8 (<0.5) 32 (8) 16 (10) 32 (6) K. pneumoniae 50625602 16 (9) 8 (<0.5) >32 (—) 32 (5) 32 (6) K. pneumoniae 50667959 32 (4) 16 (5) 8 (<0.5) >32 (—) 32 (5) 32 (6) A. baumanii K12-21 16 (9) 16 (5) 4 (<1) 32 (8) 32 (5) 16 (11) A. baumanii K44-35 32 (4) 16 (5) 4 (<1) 32 (8) 32 (5) 32 (6) A. baumanii K47-42 16 (9) 16 (5) 4 (<1) 32 (8) 32 (5) 32 (6) A. baumanii K55-13 16 (9) 16 (5) 4 (<1) 32 (8) 32 (5) 32 (6) A. baumanii K63-58* 16 (9) 16 (5) 4 (<1) 16 (15) 16 (10) 32 (6) Compound 8(i) 8(ii) 8(iii) 8(iv) 8(v) RBC 77 56 <4 133 88 Clinical isolates Antimicrobial activity S. aureus N315 2 (39) 2 (28) 4 (<1) 8 (17) 2 (44) S. aureus NCTC 10442 2 (39) 2 (28) 2 (<2) 8 (17) 2 (44) S. aureus strain 85/2082 2 (39) 2 (28) 2 (<2) 8 (17) 2 (44) S. aureus strain WIS 2 (39) 2 (28) 2 (<2) 8 (17) 2 (44) S. aureus IHT 99040 2 (39) 2 (28) 2 (<2) 8 (17) 2 (44) E. faecium 50673722 2 (39) 4 (14) 2 (<2) 16 (8) 4 (22) E. faecium 50901530 4 (19) 2 (28) 2 (<2) 8 (17) 4 (22) E. faecium K36-18 2 (39) 4 (14) 2 (<2) 16 (8) 4 (22) E. faecium 50758899 2 (39) 4 (14) 2 (<2) 16 (8) 4 (22) E. faecium TUH50-22 2 (39) 2 (28) 2 (<2) 8 (17) 4 (22) E. coli 50579417 4 (19) 4 (14) 16 (<0.25) 16 (8) 8 (11) E. coli 50639799 4 (19) 4 (14) 8 (<0.5) 8 (17) 4 (22) E. coli 50676002 4 (19) 4 (14) 16 (<0.25) 8 (17) 4 (22) E. coli 50739822 4 (19) 4 (14) 8 (<0.5) 8 (17) 8 (11) E. coli 50857972 4 (19) 4 (14) 8 (<0.5) 8 (17) 4 (22) P. aeruginosa K34-7 16 (5) 8 (7) 16 (<0.25) 32 (4) 16 (6) P. aeruginosa K34-73 8 (10) 8 (7) 8 (<0.5) 32 (4) 8 (11) P. aeruginosa K44-24 16 (5) 8 (7) 16 (<0.25) 32 (4) 16 (6) P. aeruginosa 50692172 16 (5) 8 (7) 16 (<0.25) 32 (4) 16 (6) P. aeruginosa 50692520 16 (5) 16 (4) 16 (<0.25) 32 (4) 16 (6) K. pneumoniae K47-25* 4 (19) 4 (14) 16 (<0.25) 16 (8) 8 (11) K. pneumoniae K66-45 4 (19) 4 (14) 8 (<0.5) 16 (8) 4 (22) K. pneumoniae 50531633* 4 (19) 4 (14) 16 (<0.25) 16 (8) 8 (11) K. pneumoniae 50625602 4 (19) 4 (14) 16 (<0.25) 16 (8) 16 (6) K. pneumoniae 50667959 16 (5) 4 (14) 8 (<0.5) 16 (8) 4 (22) A. baumanii K12-21 4 (19) 4 (14) 4 (<1) 32 (4) 8 (11) A. baumanii K44-35 8 (10) 4 (14) 4 (<1) 32 (4) 8 (11) A. baumanii K47-42 8 (10) 4 (14) 4 (<1) 32 (4) 8 (11) A. baumanii K55-13 8 (10) 8 (7) 4 (<1) 32 (4) 8 (11) A. baumanii K63-58* 4 (19) 4 (14) 4 (<1) 32 (4) 8 (11) *Clinical isolates resistant to the antibiotic colistin.
(21) Toxicity was determined against human RBCs, and a selectivity index (SI) was defined as the RBC EC.sub.50 value divided by the MIC value against individual isolates. The antimicrobial potencies against the multi-resistant clinical isolates were as low as MIC 2-4 μg/ml for the most potent barbiturates and followed the same trends as against the antibiotic susceptible strains.
(22) The most potent broad-spectrum barbiturates were the amine barbiturate 7(iii), and the guanidine barbiturates 8(i), 8(ii), 8(iii), and 8(v). It was evident that the guanidine group was efficient as the cationic group by the large number of highly potent guanidine barbiturates compared to amine barbiturates. However, several of the guanidine barbiturates displayed almost twofold higher RBC toxicity compared to analogous amine barbiturates. The interplay between the two different cationic groups and the seven different lipophilic side-chains thereby influenced both antimicrobial potency and RBC toxicity.
(23) 7(i) displayed low haemolytic activity (EC.sub.50 143 μg/ml) resulting in a good SI of 18-36 with respect to its activity against S. aurues, E. faecium and E. coli.
(24) Among the three amine barbiturates 7(ii), 7(vi), and 7(iii) with 3,5-disubstituted benzylic side-chains, the super-bulky 7(iii) was most potent with MIC values of 2-16 μg/ml against all the 30 multi-resistant clinical isolates.
(25) For the remaining two 3,5-disubstituted barbiturates, 7(ii) was more potent than 7(vi) showing that two bromine atoms as bulky benzylic substituents were more efficient than having two trifluoromethyl groups. However, the calculated C log P of 7(ii) was lower than the calculated C log P for the less potent 7(vi), showing that not only lipophilic effects of the side-chains affected antimicrobial potency, but possibly also electronic effects. The brominated barbiturate 7(ii) was very potent against S. aurues, E. faecium and E. coli with MIC values of 4-8 μg/ml, and by its high SI of 10-21 was one of the most promising amine barbiturates prepared.
(26) The amine barbiturates 7(iv) and 7(v) contained naphthyl based side-chains, and both showed highest potency against the Gram-positive isolates of S. aurues and E. faecium with MIC values 4-16 μg/ml. The fluorine substituted barbiturate 7(v) was more potent than 7(iv) against isolates of E. faecium. The difference was one titre step of the concentration gradient used, but reflected also the higher side-chain C log P of 7(v) compared to 7(iv) (Table 2). Of importance was the very low haemolytic activity of both 7(iv) with EC.sub.50 243 μg/ml and 7(v) with EC.sub.50 160 μg/ml, which gave very high SI's of 20-40 against the Gram-positive isolates.
(27) As discussed above, guanylation of the barbiturates resulted in both increased antimicrobial potency and haemolytic activity of series 8.
(28) The larger guanidine group can form more intricate electrostatic and hydrogen-bonding interactions than a primary amine group, and thereby interact with both anionic and zwitterionic phospholipids. The increased antimicrobial activity and RBC toxicity may be explained by the guanidine groups ability to bind to both anionic phospholipids being the main constituent of bacterial membrane, and zwitterionic phospholipids being the main constituent in mammalian cell structure.
(29) With respect to side-chain structures, the same order of potency was observed for the guanidine barbiturates of series 8 as for the amine barbiturates of series 7. However, the guanidine series 8 represented a major increase in antimicrobial activity against the Gram-negative multi-resistant clinical isolates compared to the amine series 7. The 4-substituted barbiturate 8(i) displayed MIC values of 4-8 μg/ml against the Gram-negative multi-resistant clinical isolates of K. pneumoniae and A. baumanii, as well as MIC values of 2-4 μg/ml against S. aurues, E. faecium and E. coli. This represented up to 4-fold improvement in antimicrobial activity compared to the analogous amine barbiturate 7(i). Also higher antimicrobial activity against P. aeruginosa (MIC: 8-16 μg/ml) for 8(i) was accomplished by changing the cationic group to guanidine.
(30) The level of toxicity against human RBCs for the guanidine barbiturates of series 8 depended also on the specific side-chain structure in question. For the guanidine 8(i) twofold increased RBC toxicity was observed compared to the amine 7(i). However, the haemolytic activity of 8(i) was still low with EC.sub.50 of 77 μg/ml resulting in high SI's of 19-39 with respect to its high potency against multi-resistant S. aurues, E. faecium and E. coli.
(31) Of the two 3,5-disubstituted guanidine barbiturates 8(ii) and 8(iii) tested, 8(ii) was the overall most broad-spectrum barbiturate prepared throughout the study, and displayed MIC values of 2-16 μg/ml against all 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria. The haemolytic activity of 8(ii) (EC.sub.50: 56 μg/ml) was lower than expected compared to the general twofold increase in toxicity observed for the guanidine barbiturates and the haemolytic toxicity of its analogous amine counterpart 7(ii) (EC.sub.50: 83 μg/ml). Thus, the efficiency of the two bromine atoms as bulky substituents on the benzylic side-chains was once more demonstrated, and pointed to 8(ii) as one of the most promising broad-spectrum barbiturates prepared.
(32) The super-bulky barbiturate 8(iii) was among the most potent derivatives prepared.
(33) The antimicrobial potencies of the two naphthyl based guanidine barbiturates 8(iv) and 8(v), showed that the fluorinated 8(v) had highest broad-spectrum activity with MIC values of 2-16 μg/ml against all the 30 multi-resistant clinical isolates. The toxicity against human RBCs was also low for 8(v) (EC.sub.50: 88 μg/ml) and comparable to the most promising brominated amine barbiturate 7(ii). Due to the high potency of 8(v) it also displayed a higher SI of 11-44 against S. aurues, E. faecium and E. coli, and was thereby the overall second most selective derivative against the multi-resistant clinical isolates of S. aureus (SI: 44) of all barbiturates prepared.
(34) Barbiturate 8(v) was also among the most selective barbiturates against individual isolates of the Gram-negative bacteria. The less potent analogue 8(iv) showed MIC values of 8-16 μg/ml against S. aureus and E. faecium, and was thereby equipotent to its amine analogue 7(iv). The potency of 8(iv) against E. coli was higher than for 7(iv), but due to higher haemolytic activity the SI of the guanylated barbiturate 8(iv) was lower than for the amine barbiturate 7(iv). A stronger effect on RBC toxicity than antimicrobial activity was therefore observed by guanylation of 7(iv) to 8(iv).
(35) Against the three clinical isolates of K. pneumoniae K47-25, K. pneumoniae 50531633, and A. baumanii K63-58 that display resistance against the last-resort cationic antibiotic colistin, all the investigated amphipathic barbiturates displayed antimicrobial activity in the same range as against the colistin susceptible clinical isolates. The mechanism of resistance is thought to involve altered LPS composition and charge that affects the binding and mechanism of colistin, but it seemed not to have any major impact on the binding and activity of the most potent present amphipathic barbiturates.
(36) Chemicals and Equipment
(37) All reagents and solvents were purchased from commercial sources and used as supplied with the exception of starting material 1-(Bromomethyl)-4-fluoronaphthalen e, which was synthesized from the 4-Fluoro-1-naphthoic acid according to literature procedures.
(38) Anhydrous DMF was prepared by storage over 4 Å (4×10.sup.−10 m) molecular sieves. Reactions were monitored by thin-layer chromatography (TLC) with Merck pre-coated silica gel plates (60 F254). Visualization was accomplished with either UV light or by immersion in potassium permanganate or phosphomolybdic acid (PMA) followed by light heating with a heating gun.
(39) Purifications using normal phase flash chromatography were either done by normal column chromatography using Normalsil 60, 40-63 mm silica gel or by automated normal phase flash chromatography (Heptane/EtOAc) with the sample preloaded on a Samplet® cartridge belonging to a Biotage SP-1.
(40) Purification of reactions by reversed-phase (RP) C18 column chromatography (water with 0.1% TFA/acetonitrile with 0.1% TFA) was also executed on an automated purification module with the sample preloaded on a Samplet® cartridge. Analytical RPHPLC was carried out on a Waters 2695 Separations Module equipped with an XBridge™ C18 5 μm, 4.6 mm×250 mm column and analysed at wavelengths 214 and 254 nm with a Waters 996 PDA detector spanning from wavelengths 210 to 310 nm. The derivatives were eluted with a mobile phase consisting of water and acetonitrile, both containing 0.1% TFA. The gradient started at 10% acetonitrile (3 min), followed by a linear gradient to 90% acetonitrile over 17 min. The flow rate was 1 mL min-1. NMR spectra were obtained on a 400 MHz Bruker Avance III HD equipped with a 5 mm SmartProbe BB/1H (BB=19F, 31P-15N). Data are represented as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, h=heptet, m=multiplet), coupling constant (J, Hz) and integration.
(41) Chemical shifts (δ) are reported in ppm relative to the residual solvent peak (CDCl.sub.3: δH 7.26, and δC 77.16; CD3OD: δH 3.31 and δC 49.00). Positive and negative ion electrospray ionization mass spectrometry (ESI-MS) was conducted on a Thermo electron LTQ Orbitrap XL spectrometer.
(42) Synthesis
(43) Established methods for the synthesis of substituted barbiturates include the condensation of alkylated malonate esters with urea, cyclization with N-alkylated urea and diethyl malonate, Knoevenagel condensation of barbituric acid and aldehydes or ketones, and alkylation of barbituric acid. However, the latter procedure is unselective with regards to alkylation of the 1, 3 and 5 positions. The present inventors have found that the condensation of dialkylated malonate esters with urea followed by N-alkylation was the most successful strategy.
(44) As shown in Scheme 1, Symmetrically disubstituted malonates 3(i)-(vi) were obtained from diethyl malonate 2 by dialkylation with the appropriate benzylic halides, and were subsequently cyclized with urea by treatment with NaH or K.sub.2CO.sub.3 in DMF to provide 4(i)-(vi) in yields of 70-92%. Dry conditions were imperative to the yield. Cyclisation of malonate 3(vi) gave low yields (27%) due to decarboxylation under the reaction conditions. The 5,5-disubstituted barbiturates 4(i)-(vi) were alkylated with an excess of 1,4-dibromobutane under basic conditions (K.sub.2CO.sub.3 in DMF) to give N,N″-dialkylated alkyl bromide barbiturates 5(i)-(vi) in 40-96% yield. These were converted to the corresponding azides 6(i)-(vi) with NaN.sub.3 (2-3 equiv.) in DMF (68-100% yield). Reduction of the azides to amines with NaBH.sub.4 and a catalytic amount of a dithiol, and subsequent Boc-protection provided Boc-protected diamines after purification by flash chromatography. Deprotection with TFA provided the target amine barbiturates 7(i)-(vi) (>95% purity as determined by analytical C18 reversed phase HPLC). The amine barbiturates 7(i)-(v) were guanylated with N-Boc-1H-pyrazole-1-carboxamidine in THF and purified before the Boc-protecting group was removed. Purification by C18 reversed phase flash chromatography gave the TFA salts of the target guanylated barbiturates 8(i)-(v) with >95% purity.
(45) ##STR00010## ##STR00011## ##STR00012##
Detailed Synthesis
Dialkylated Malonate Ester—3(i)-(vi)
General Procedure:
(46) To a stirred solution of diethyl malonate in DMF (≈100 mg/mL) over Cs.sub.2CO.sub.3 (2.1-2.2 equiv.) or K.sub.2CO.sub.3 (3 equiv.) was added the alkyl halide (2 equiv.). The reaction was kept stirring at r.t. over night. The reaction mixture was diluted with EtOAc (30 mL) and washed with water (25 mL), aqu. 5% LiCl sol. (3×25 mL) and brine (25 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CH.sub.2Cl.sub.2 (20 mL) and adsorbed on Celite. The product was purified on a silica column using 1-5% EtOAc in pentane as mobile phase.
Diethyl 2,2-bis(4-tert-butylbenzyl)malonate-3(i)
(47) According to the general procedure, a stirred solution of diethyl malonate (3.43 g, 21.4 mmol) in DMF (25 mL) over K.sub.2CO.sub.3 (8.8 g, 64.2 mmol) was added 1-(bromomethyl)-4-tert-butylbenzene (10 g, 44 mmol). The reaction was kept stirring at r.t. overnight. The reaction mixture was diluted with EtOAc (80 mL) and washed with water (3×50 mL), aqu. 5% LiCl sol. (50 mL) and brine (50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in dichloromethane (20 mL) and adsorbed on to Celite. The product was purified on a silica column using 1-5% EtOAc in pentane as mobile phase to yield 3(i) (8.80 g, 90%) as a white solid.
(48) 1H NMR (400 MHz, CDCl.sub.3): δ 7.28 (d, J=8.3 Hz, 4H), 7.11 (d, J=8.4 Hz, 4H), 4.10 (q, J=7.1 Hz, 4H), 3.19 (s, 4H), 1.30 (s, 18H), 1.14 (t, J=7.1 Hz, 6H).
(49) 13i) NMR (101 MHz, CDCl.sub.3): δ 171.2, 149.7, 133.4, 129.9, 125.2, 61.2, 60.4, 38.6, 34.5, 31.5, 14.0.
(50) HRMS-ESI: C29H40NaO4+[M+Na]+ calcd: 475.2818, found: 475.2795.
Diethyl 2,2-bis(3,5-dibromobenzyl)malonate-3(ii)
(51) According to the general procedure, a stirred solution of diethyl malonate (460 mg, 2.9 mmol) in DMF (5 mL) over Cs.sub.2CO.sub.3 (2.0 g, 6.37 mmol) was added 1,3-dibromo-5(bromomethyl)benzene (2 g, 6.0 mmol). The reaction was kept stirring at r.t. over night. The reaction mixture was diluted with EtOAc (30 mL) and washed with water (25 mL), aqu. 5% LiCl sol. (3×25 mL) and brine (25 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CH.sub.2Cl.sub.2 (20 mL) and adsorbed on Celite. The product was purified on a silica column using 1-5% EtOAc in pentane as mobile phase to yield 3(ii) (1.17 g, 61%) as a white solid.
(52) 1H NMR (400 MHz, CDCl.sub.3): δ 7.56 (t, J=1.8 Hz, 2H), 7.24 (d, J=1.8 Hz, 4H), 4.15 (q, J=7.1 Hz, 4H), 3.11 (s, 4H), 1.20 (t, J=7.2 Hz, 6H).
(53) 13i) NMR (101 MHz, CDCl.sub.3): δ 170.0, 139.9, 132.8, 132.0, 122.7, 61.9, 60.0, 39.3, 13.9.
(54) HRMS-ESI: C21H20Br4NaO4+[M+Na]+ calcd: 674.7987, found: 674.7961.
Diethyl 2,2-bis(naphtalen-2-yl-methyl)malonate-3(iv)
(55) To a stirred solution of diethyl malonate (3.44 g, 21.5 mmol) in 15 mL CH.sub.2Cl.sub.2 at 0° C. was added DBU (3.3 mL, 22.6 mmol). Reaction mixture was stirred for 5 min before 2-(bromomethyl)naphtalene (5 g, 22.6 mmol) was added. The reaction was allowed to reach r.t. and was stirred over night. The reaction was concentrated and the crude product isolated as a brown oil. The oil was dissolved in EtOAc (30 mL) and washed with water (2×30 mL), 10% citric acid (30 mL), 10% NaHCO.sub.3 (30 mL) and brine (30 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated affording 4.83 g of almost pure monoalkylated diethyl malonate. To a suspension of NaH (774 mg, 32.2 mmol) in dry THF (15 mL) at 0° C. was added diethyl 2-(naphthalen-2-ylmethyl)malonate (4.8 g) dropwise as a solution in THF (15 mL). The resulting mixture was stirred for 10 min before 2-naphtyl methyl bromide (5 g, 22.6 mmol) was added. The reaction was allowed to reach r.t. and was stirred over night. The reaction mixture was cooled in icebath, the excess of NaH was quenched with 10% citric acid solution and the reaction mixture concentrated. The crude product was then dissolved in EtOAc and washed with 10% citric acid sol. (3×30 mL), 10% NaHCO.sub.3 sol. (2×30 mL) and brine (30 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated to yield crude 3(iv) (7.35 g, 78%).
(56) 1HNMR (400 MHz, CDCl.sub.3): δ 7.85-7.80 (m, 2H), 7.77 (d, J=8.1 Hz, 4H), 7.65 (d, J=1.7 Hz, 2H), 7.49-7.43 (m, 4H), 7.32 (dd, J=8.5, 1.7 Hz, 2H), 4.14 (q, J=7.1 Hz, 4H), 3.45 (s, 4H), 1.14 (t, J=7.1 Hz, 6H). HRMS-ESI: C29H29O4+[M+H]+ calcd: 441.2060, found: 441.2059.
Diethyl 2,2-bis((4-fluoronaphtalen-1-yl-methyl)malonate-3(v)
(57) According to the general procedure, a stirred solution of diethyl malonate (1.3 g, 8.16 mmol) in DMF (10 mL) over K.sub.2CO.sub.3 (3.36 g, 24.3 mmol) was added 4-F-napht-1-yl Bromo methane (4 g, 16.7 mmol). The reaction was kept stirring at r.t. over night. The reaction mixture was diluted with EtOAc (30 mL) and washed with water (3×20 mL), aqu. 5% LiCl sol. (20 mL) and brine (20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. In a round bottomed flask the brown solid crude product was dissolved in warm EtOH, capped with alumina foil and left for 4 days at r.t. Upon standing for an hour the product crashed out of the brown solution as a white solid (1.6 g, 41%).
(58) 1H NMR (400 MHz, CDCl.sub.3): δ 8.18-8.08 (m, 2H), 8.05-7.95 (m, 2H), 7.57-7.46 (m, 4H), 7.36 (dd, J=8.0, 5.5 Hz, 2H), 7.04 (dd, J=10.2, 8.0 Hz, 2H), 3.81 (s, 4H), 3.75 (q, J=7.2 Hz, 4H), 0.85 (t, J=7.1 Hz, 6H). 13i) NMR (101 MHz, CDCl.sub.3): δ 171.3, 158.1 (d, J=251.4 Hz), 134.2 (d, J=4.2 Hz), 128.9 (d, J=4.6 Hz), 127.6 (d, J=8.2 Hz), 126.8, 125.9 (d, J=2.1 Hz), 124.1-123.9 (m), 121.2 (d, J=6.0 Hz), 108.9 (d, J=19.7 Hz), 61.5, 59.8, 35.5, 13.6. HRMS-ESI: C29H26(iv)2NaO4+[M+Na]+ calcd: 499.1691, found: 499.1689. 4.2.2.
Diethyl 2,2-bis(3,5-bis(trifluoromethyl)benzyl)malonate-3(vi)
(59) According to the general procedure, a stirred solution of DEM (490 mg, 3.1 mmol) in DMF (5 mL) over Cs.sub.2CO.sub.3 (2.2 g, 6.83 mmol) was added 1-(bromomethyl)-3,5-bis(trifluoromethyl) benzene (2 g, 6.51 mmol). The reaction was kept stirring at r.t. over night. The reaction mixture was diluted with EtOAc (30 mL) and washed with water (25 mL), aqu. 5% LiCl sol. (3×25 mL) and brine (25 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CH.sub.2Cl.sub.2 (20 mL) and adsorbed on Celite. The product was purified on a silica column using 1-5% EtOAc in pentane as mobile phase to yield 3(vi) (0.89 g, 63%) as a white solid. 1H NMR (400 MHz, CDCl.sub.3): δ 7.79 (s, 2H), 7.71-7.54 (m, 4H), 4.10 (q, J=7.1 Hz, 4H), 3.32 (s, 4H), 1.13 (t, J=7.1 Hz, 6H).
(60) 13i) NMR (101 MHz, CDCl.sub.3): δ 169.8, 138.5, 131.8 (q, J=33.3 Hz), 130.9-130.2 (m), 123.3 (q, J=272.7 Hz), 121.5 (p, J=3.9 Hz), 62.2, 60.3, 40.3, 13.8. HRMS-ESI: C25H19F12O4−[M−H]− calcd: 611.1098, found: 611.1097.
5,5-bis(4-tert-butylbenzyl)pyrimidin-2,4,6(1H,3H,5H)-trione-4(i)
(61) To a stirred solution of urea (6.63 g, 110 mmol) at r.t. in anhydrous DMF (20 mL) was added NaH (660 mg, 27.5 mmol) and the reaction was stirred for 5 min. A solution of 3(i) (5(v), 11 mmol) in anhydrous DMF (20 mL) was added dropwise to the reaction mixture and the reaction was stirred over night. The reaction mixture was diluted with EtOAc (20 mL) and washed with 10% citric acid (100 mL), 10% NaHCO.sub.3 (50 mL), brine (50 mL), water (20 mL), and brine (2×50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was purified with automated flash chromatography (heptane/EtOAc) affording 4.09 g (88%) of the product 4(i) as a white powder.
(62) 1H NMR (400 MHz, MeOD4): δ 7.26 (d, J=7.7 Hz, 2H), 7.05 (d, J=7.6 Hz, 2H), 3.31 (s, 6H, overlap MeOD), 1.24 (s, 18H). 13i) NMR (101 MHz, MeOD4): δ 174.2, 151.5*, 133.5, 130.4, 126.4, 61.4, 45.0, 35.3, 31.7. *assumed overlap of two signals HRMS-ESI: C26H31N2O3−[M−H]− calcd: 419.2340, found: 419.2335.
5,5-bis(3,5-dibromobenzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione-4(ii)
(63) To a stirred solution of urea (1.83 g, 2.79 mmol) in anhydrous DMF (15 mL) was added NaH (183 mg, 7.6 mmol) and the resulting solution was stirred for 10 min before 3(ii) (2.0 g, 3.05 mmol) was added. The resulting mixture was stirred over night. The reaction was diluted with EtOAc (50 mL), washed with 10% citric acid sol. (3×25 mL), 10% NaHCO.sub.3 (2×30 mL), and brine (30 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The white solid was dissolved in chloroform (25 mL) and concentrated again, purified by flash chromatography to yield 4(ii) (1.52 g, 88%).
(64) 1H NMR (400 MHz, CDCl.sub.3): δ 7.82 (NH, s, 2H), 7.58 (t, J=1.8 Hz, 2H), 7.21 (d, J=1.5 Hz, 4H), 3.32 (s, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 170.0, 146.4, 137.7, 134.2, 131.5, 123.6, 59.9, 43.4. HRMS-ESI: C18H11 79Br4N2O3−[M−H]− calcd: 618.7509, found: 618.7501.
5,5-bis((naphtalen-2-yl)methyl)pyrimidine-2,4,6(1H,3H,5H)-trione-4(iv)
(65) NaH (9 mg, 0.37 mmol) was added to a stirred solution of urea (91 mg, 1.49 mmol) in anhydrous DMF (3 mL) at r.t. The reaction mixture was left to stir for 10 min before 3(iv) (66 mg, 0.15 mmol) was added slowly and the reaction was left to stir over night. The reaction mixture was diluted with EtOAc (20 mL) and washed with water (4×20 mL) followed by brine (20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CHCl.sub.3 and adsorbed onto celite before purification on a silica column using 0-5% EtOAc in CHCl.sub.3 as mobile phase to yield 4(iv) (50 mg, 82%).
(66) 1H NMR (400 MHz, CD3OD): δ 7.76-7.70 (m, 4H), 7.69 (d, J=8.6 Hz, 2H), 7.62 (s, 2H), 7.44-7.36 (m, 4H), 7.26 (dd, J=8.4, 1.7 Hz, 2H), 3.60 (s, 4H). 13i) NMR (101 MHz, CD3OD): δ 173.2, 149.5, 133.8, 133.1, 132.8, 129.1, 128.7, 128.1, 127.9, 127.8, 126.6, 126.4, 60.8, 45.1. HRMS-ESI: C26H19N2O3−[M−H]− calcd: 407.1417, found: 407.1400.
5,5-bis((4-fluoronaphtalene-1-yl)methyl)pyrimidine-2,4,6(1H,3H,5H)-trione-4(v)
(67) To a stirred solution of urea (630 mg, 10.49 mmol) in anhydrous DMF (4 mL) was added NaH (76 mg, 3.16 mmol) and the resulting solution was stirred for 10 min before 3(v) (500 mg, 1.05 mmol) was added slowly. The resulting mixture was stirred over night. The reaction mixture was diluted with 25 mL EtOAc and washed with 4×50 mL water followed by 20 mL brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CHCl.sub.3 and adsorbed onto celite before purification on a silica column using 0-5% EtOAc in CHCl.sub.3 as mobile phase to yield 4(v) (430 mg, 92%).
(68) 1H NMR (400 MHz, CDCl.sub.3): δ 8.23 (d, J=8.5 Hz, 2H), 8.14-8.04 (m, 2H), 7.64-7.49 (m, 4H), 7.46 (s, 2H), 7.29-7.26 (m, 2H), 7.00 (dd, J=9.9, 8.1 Hz, 2H), 4.05 (s, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 171.4, 158.7 (d, J=253.3 Hz), 146.8, 133.3 (d, J=4.5 Hz), 128.0 (d, J=8.7 Hz), 127.4, 126.7 (d, J=4.7 Hz), 126.5 (d, J=1.9 Hz), 124.4-124.1 (m), 121.3 (d, J=6.2 Hz), 109.1 (d, J=20.1 Hz), 59.8, 40.0. HRMS-ESI: C26H19N2O3−[M−H]− calcd: 407.1401, found: 407.1414 4.2.3.
5,5-bis(3,5-bis(trifluoromethyl)benzyl)pyrimidine-2,4,6(1H,3H,5H)-trione-4(vi)
(69) To a solution of urea (1.3 g, 21.6 mmol) in 20 mL anhydrous DMF was added NaH (128 mg, 5.3 mmol) and the resulting solution was stirred for 10 min before 3(vi) (1.0 g, 1.7 mmol) was added. The resulting mixture was stirred over night. The reaction was diluted with EtOAc 9 (50 mL), washed with 10% citric acid sol. (3×30 mL), 10% NaHCO.sub.3 (2×20 mL), and brine (30 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by automated flash chromatography to yield the product 4(vi) (0.27 g, 27%) as white powder.
(70) 1H NMR (400 MHz, CDCl.sub.3): δ 7.82 (s, 2H), 7.73 (s, 2H), 7.62-7.57 (m, 4H), 3.57 (s, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 169.8, 146.1, 136.3, 132.6 (q, J=33.6 Hz), 130.4-129.7 (m), 122.0 (q, J=272.8 Hz), 122.9-122.2 (m), 59.9, 43.5. HRMS-ESI: C22H11F12N2O3−[M−H]− calcd: 579.0584, found: 579.0583.
1,3-bis(4-bromobutyl)-5,5-bis(4-tert-butylbenzyl)-pyrimidine-2,4,6(1H,3H,5H)-trione-5(i)
(71) To a stirred solution of 4(i) (3.88 g, 9.23 mmol) at r.t. in DMF (50 mL) was added K.sub.2CO.sub.3 (5.12 g, 37 mmol) and 1,4-dibromobutane (10.9 mL, 92.5 mmol). The reaction mixture was stirred over night. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (100 mL). The crude product was purified with automated flash chromatography affording the product 5(i) (2.60 g, 40%) as a white powder.
(72) 1H NMR (400 MHz, CDCl.sub.3): δ 7.22 (d, J=7.8 Hz, 4H), 6.98 (d, J=7.9 Hz, 4H), 3.60 (t, J=6.9 Hz, 4H), 3.41 (s, 4H), 3.33 (t, J=6.4 Hz, 4H), 1.56 (p, J=7.3 Hz, 4H), 1.42 (p, J=7.7 Hz, 4H), 1.25 (s, 18H). 13i) NMR (101 MHz, CDCl.sub.3): δ 170.9, 150.7, 149.9, 131.9, 129.2, 125.5, 60.7, 45.0, 40.7, 34.5, 32.9, 31.4, 29.5, 26.2. HRMS-ESI: C34H46 79Br2N2NaO3+[M+Na]+ calcd: 711.1774, found: 711.1773.
1,3-bis(4-bromobutyl)-5,5-bis(3,5-dibromobenzyl)-pyrimidine-2,4,6-(1H,3H,5H)-trione-5(ii)
(73) To a stirred solution of 4(ii) (300 mg, 0.48 mmol) in DMF (6 mL) was added K.sub.2CO.sub.3 (265 mg, 1.92 mmol) and 1,4-dibromobutane (0.57 mL, 4.81 mmol). The reaction was stirred until completion was indicated by TLC (5% EtOAc in CHCl.sub.3). The reaction mixture was diluted with EtOAc (25 mL) and the K.sub.2CO.sub.3 was filtered off. The organic phase was washed with 10% citric acid sol. (30 mL), NaHCO.sub.3 (30 mL), water (3×30 mL) and brine (30 mL), dried with Na.sub.2SO.sub.4, filtered and concentrated resulting in an oil that slowly turned into white crystals. The crude product was dissolved in CHCl.sub.3 (30 mL) and adsorbed onto celite before purification on silica column using pentane: CH.sub.2Cl.sub.2 (7:3 to 1:1) to yield 5(ii) (347 mg, 80%) as white powder.
(74) 1H NMR (400 MHz, CDCl.sub.3): δ 7.54 (d, J=1.8 Hz, 2H), 7.14 (d, J=1.7 Hz, 4H), 3.65 (t, J=7.4 Hz, 4H), 3.38 (t, J=6.7 Hz, 4H), 3.33 (s, 4H), 1.77-1.61 (m, 4H), 1.58-1.43 (m, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 169.9, 149.1, 138.4, 133.9, 131.3, 123.4, 59.9, 44.2, 41.3, 32.7, 30.0, 26.7. HRMSESI: C26H26 79Br3 81Br3ClN2O3−[M+Cl]− calcd: 928.6671, found: 928.6669.
1,3-bis(4-bromobutyl)-5,5-bis(3,5-di-tert-butylbenzyl)-pyrimidine-2,4,6(1H,3H,5H)-trione-5(iii)
(75) To a stirred solution 10 of 4(iii) (0.86 g, 1.62 mmol) in DMF was added K.sub.2CO.sub.3 (1.2 g, 8.9 mmol). The reaction mixture was stirred for 5 min before addition of 1,4-dibromobutane (1.76 mL, 14.8 mmol). The reaction was stirred until completion was indicated by TLC (5% EtOAc in CHCl.sub.3). Then the reaction mixture was diluted with EtOAc (15 mL) and the K.sub.2CO.sub.3 was filtered off. The organic phase was washed with 10% citric acid sol. (30 mL), NaHCO.sub.3 (30 mL), water (3×30 mL) and brine (30 mL), dried with Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by automated flash chromatography to yield the product 5(iii) (0.64 g, 74%).
(76) 1H NMR (400 MHz, CDCl.sub.3): δ 7.26 (t, J=1.9 Hz, 2H), 6.89 (d, J=1.8 Hz, 4H), 3.59* (t, J=7.5 Hz, 4H), 3.46 (s, 4H), 3.23 (t, J=6.7 Hz, 4H), 1.51 (p, J=6.8 Hz, 4H), 1.35-1.23 (m, 40H). 13i) NMR (101 MHz, CDCl.sub.3): δ 171.0, 151.1, 150.0, 134.4, 123.7, 121.5, 60.5, 46.5, 40.9, 34.8, 32.4, 31.6, 29.7, 26.5. *distorted triplet. HRMS-ESI: C42H62 79Br2KN2O3+[M+K]+ calcd: 839.2759, found: 839.2725.
1,3-bis(4-bromobutyl)-5,5-bis(naphtalen-2-yl-methyl)-pyrimidine-2,4,6(1H,3H,5H)-trione-5(iv)
(77) To a stirred suspension of 4(iv) (200 mg, 0.49 mmol) and K.sub.2CO.sub.3 (273 mg, 1.95 mmol) in DMF (4 mL) was added 1,4-dibromobutane (0.57 mL, 4.9 mmol). The reaction was stirred until completion was indicated by TLC (5% EtOAc in CHCl.sub.3). Then the reaction mixture was diluted with EtOAc (25 mL) and the K.sub.2CO.sub.3 was filtered off. The organic phase was washed with 10% citric acid sol. (30 mL), NaHCO.sub.3 (30 mL), water (3×30 mL), and brine (30 mL), dried with Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CHCl.sub.3 (30 mL) and adsorbed onto celite before purification on silica column using 0-5% EtOAc in CHCl.sub.3 to yield 5(iv) (347 mg, 80%) as white powder.
(78) 1H NMR (400 MHz, CDCl.sub.3): δ 7.75 (dd, J=9.4, 6.4 Hz, 4H), 7.70 (d, J=8.4 Hz, 2H), 7.57 (s, 2H), 7.48-7.42 (m, 4H), 7.18 (dd, J=8.5, 1.7 Hz, 2H), 3.68 (s, 4H), 3.53 (t, J=6.7 Hz, 4H), 2.99 (t, J=6.2 Hz, 4H), 1.35-1.19 (m, 8H). 13i) NMR (101 MHz, CDCl.sub.3): δ 170.9, 149.6, 133.3, 132.7, 132.5, 128.8, 128.5, 127.8, 127.7, 127.2, 126.6, 126.3, 60.8, 45.8, 40.9, 32.8, 29.5, 26.3. HRMSESI: C34H34 79Br2N2NaO3+[M+Na]+ calcd: 699.0827, found: 699.0839.
1,3-bis(4-bromobutyl)-5,5-bis(4-F-naphtalene-1-ylmethyl) pyrimidine-2,4,6(1H,3H,5H)-trione-5(v)
(79) To a stirred suspension of 4(v) (242 mg, 0.54 mmol) and K.sub.2CO.sub.3 (300 mg, 2.17 mmol) in DMF (5 mL) was added 1,4-dibromobutane (0.64 mL, 5.4 mmol). The reaction was checked with TLC (CHCl.sub.3 Rf product 0.74, Rf starting material 0.11) and when no traces of starting material was visible the reaction mixture was diluted with EtOAc (25 mL) and the K.sub.2CO.sub.3 filtered off. The organic phase was washed with 10% citric acid sol. (30 mL), NaHCO.sub.3 (30 mL), water (3×30 mL) and brine (30 mL) dried with Na.sub.2SO.sub.4, filtered and concentrated yielding the crude as an oil. The crude product was dissolved in CHCl.sub.3 (30 mL) and adsorbed onto celite before purification on silica column using CHCl.sub.3 as mobile phase to yield 5(v) (237 mg, 61%) as a white solid.
(80) 1H NMR (400 MHz, CDCl.sub.3): δ 8.23 (d, J=8.6 Hz, 2H), 8.08 (d, J=8.3 Hz, 2H), 7.63 (t, J=7.7 Hz, 2H), 7.54 (t, J=7.6 Hz, 2H), 7.23 (dd, J=8.0, 5.5 Hz, 2H), 7.00 (dd, J=9.8, 8.1 Hz, 2H), 4.06 (s, 4H), 3.33 (t, J=7.2 Hz, 4H), 3.05 (t, J=6.6 Hz, 4H), 1.34-1.12 (m, 4H), 1.08-0.90 (m, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 170.96, 158.5 (d, J=253.3 Hz), 149.4, 133.2 (d, J=4.4 Hz), 128.0 (d, J=8.4 Hz), 127.4 (d, J=4.7 Hz), 127.2, 126.4 (d, J=2.1 Hz), 124.8 (d, J=2.7 Hz), 124.1 (d, J=15.7 Hz), 121.1 (d, J=6.0 Hz), 108.9 (d, J=20.0 Hz), 60.0, 40.9, 40.7, 32.7, 29.3, 25.9. HRMSESI: C34H32 79Br2F2N2NaO3+[M+Na]+ calcd: 735.0639, found: 735.0622. 4.2.4.
1,3-bis(4-bromobutyl)-5,5-bis(3,5-bis(trifluoromethyl)-benzyl)pyrimidine-2,4,6(1H,3H,5H)-trione-5(vi)
(81) To a stirred solution of 4(vi) (0.864 g, 1.57 mmol) in DMF (20 mL) was added K.sub.2CO.sub.3 (1.233 g, 8.93 mmol) and 1,4-dibromobutane (1.76 mL, 14.9 mmol). The reaction mixture was stirred for 48 h, diluted with EtOAc (30 mL) and washed with water (3×20 mL), 5% LiCl sol. (3×20), and brine (20 mL). The crude product was purified with automated flash chromatography to afford 5(vi) (0.64 g, 50%) as a white powder.
(82) 1H NMR (400 MHz, CDCl.sub.3): δ 7.79 (s, 2H), 7.53 (s, 4H), 3.59 (s, 4H), 3.57-3.51 (m, 4H), 3.26 (t, J=6.8 Hz, 4H), 1.67-1.55 (m, 4H), 1.43-1.29 (m, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 169.4, 148.4, 136.9, 132.2 (q, J=33.6 Hz), 130.0-129.4 (m), 122.9 (q, J=272.9 Hz), 122.1 (p, J=3.8 Hz), 59.7, 44.3, 41.1, 31.7, 29.6, 26.1. HRMS-ESI: C30H26 79Br3F12N2O3−[M+Br]− calcd: 926.9308, found: 926.9308.
1,3-bis(4-azidobutyl)-5,5-bis(4-tert-butylbenzyl)-pyrimidine-2,4,6(1H,3H,5H)-trione-6(i)
(83) To a stirred solution of bromide 5(i) (2.40 g, 3.47 mmol) in DMF (15 mL) was added NaN.sub.3 (678 mg, 10.4 mmol) and stirred for 18 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with water (4×50 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product 6(i) was isolated as a clear oil (2.16 g, 100%).
(84) 1H NMR (400 MHz, CDCl.sub.3): δ 7.20 (d, J=7.7 Hz, 4H), 6.97 (d, J=7.8 Hz, 4H), 3.59 (s, 4H), 3.40 (s, 4H), 3.21 (s, 4H), 1.37-1.28 (m, 8H), 1.24 (s, 18H). 13i) NMR (101 MHz, CDCl.sub.3): δ 171.0, 150.8, 150.0, 132.0, 129.3, 125.5, 60.7, 50.9, 45.1, 41.1, 34.6, 31.4, 26.0, 24.8. HRMS-ESI: C34H46N8O3Na+[M+Na]+ calcd: 637.3577, found: 637.3583.
1,3-bis(4-azidobutyl)-5,5-bis(3,5-dibromobenzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione-6(ii)
(85) To a stirred solution of 5(ii) (239 mg, 0.26 mmol) in DMF (3 mL) was added NaN.sub.3 (52 mg, 0.8 mmol). The reaction was stirred until completion was indicated by TLC (5% EtOAc in CHCl.sub.3). Then the reaction mixture was diluted with EtOAc (15 mL) and washed with water (2×20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CHCl.sub.3 and adsorbed onto celite before purification on silica column using 0-5% EtOAc in CHCl.sub.3 to yield 6(ii) (194 mg, 91%).
(86) 1H NMR (400 MHz, CDCl.sub.3): δ 7.54 (s, 2H), 7.14 (s, 4H), 3.73-3.58 (m, 4H), 3.33 (s, 4H), 3.31-3.22 (m, 4H), 1.58-1.29 (m, 8H). 13i) NMR (101 MHz, CDCl.sub.3): δ 169.9, 149.1, 138.4, 133.8, 131.4, 123.3, 59.9, 50.9, 44.2, 41.6, 26.1, 25.3. HRMS-ESI: C26H26 79Br4(ii) IN8O3−[M+Cl]− calcd: 848.8555, found: 848.8564.
1,3-bis(4-azidobutyl)-5,5-bis(3,5-di-tert-butylbenzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione-6(iii)
(87) To a stirred solution of 5(iii) (630 mg, 11 0.78 mmol) in DMF (10 mL) was added NaN.sub.3 (140 mg, 2.15 mmol). The reaction was stirred over night. When full conversion was reached according to MS, the reaction mixture was diluted with EtOAc (50 mL) and washed with water 4×50 mL. The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated affording the crude product 6(iii) (463 mg, 80%).
(88) 1H NMR (400 MHz, CDCl.sub.3): δ 7.25 (t, J=1.9 Hz, 2H), 6.87 (d, J=1.7 Hz, 4H), 3.56 (t, J=7.2 Hz, 4H), 3.45 (s, 4H), 3.14 (t, J=6.5 Hz, 4H), 1.25 (s, 44H). 13i) NMR (101 MHz, CDCl.sub.3): δ 171.1, 151.1, 150.0, 134.4, 123.8, 121.6, 60.6, 50.8, 46.5, 41.3, 34.8, 31.6, 25.9, 25.1. HRMS-ESI: C42H62N8NaO3+[M+Na]+ calcd: 749.4838, found: 749.4838.
1,3-bis(4-azidobutyl)-5,5-bis(naphthalen-2-yl)pyrimidine-2,4,6-(1H,3H,5H)-trione-6(iv)
(89) To a stirred solution of 5(iv) (509 mg, 0.75 mmol) in DMF (3 mL) was added NaN.sub.3 (146 mg, 2.25 mmol). The reaction was stirred until completion was indicated by TLC (5% EtOAc in CHCl.sub.3). Then the reaction mixture was diluted with EtOAc (20 mL) and washed with water (3×20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was dissolved in CHCl.sub.3 and adsorbed onto celite before purification on silica column using 0-5% EtOAc in CHCl.sub.3 to yield 6(iv) (194 mg, 91%).
(90) 1H NMR (400 MHz, CDCl.sub.3): δ 7.80-7.71 (m, 4H), 7.68 (d, J=8.4 Hz, 2H), 7.57 (s, 2H), 7.51-7.41 (m, 4H), 7.17 (d, J=8.4 Hz, 2H), 3.68 (s, 4H), 3.52 (t, J=7.0 Hz, 4H), 2.85 (t, J=6.6 Hz, 4H), 1.14 (p, J=7.4 Hz, 4H), 1.02 (p, J=7.0 Hz, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 170.9, 149.6, 133.4, 132.7, 132.5, 128.8, 128.5, 127.8, 127.7, 127.2, 126.6, 126.3, 60.8, 50.7, 45.8, 41.2, 25.8, 24.9. HRMS-ESI: C34H34N8NaO3+[M+Na]+ calcd: 625.2646, found: 625.2647.
1,3-bis(4-azidobutyl)-5,5-bis((4-fluoronaphtalen-1-yl)methyl)pyrimidine-2,4,6-(1H,3H,5H)-trione-6(v)
(91) To a stirred solution of 5(v) (166 mg, 0.23 mmol) in DMF (3 mL) was added NaN.sub.3 (45 mg, 0.69 mmol). The reaction was stirred over night until completion was indicated by TLC (CHCl.sub.3) Then the reaction mixture was diluted with EtOAc (20 mL) and washed with water (3×30 mL) and brine (30 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated to yield 6(v) (142 mg, 95%).
(92) 1H NMR (400 MHz, CDCl.sub.3): δ 8.23 (d, J=8.6 Hz, 2H), 8.08 (d, J=8.3 Hz, 2H), 7.62 (t, J=7.6 Hz, 2H), 7.54 (t, J=7.5 Hz, 2H), 7.22 (t, J=6.6 Hz, 2H), 6.98 (t, J=9.0 Hz, 2H), 4.06 (s, 4H), 3.33 (t, J=6.8 Hz, 4H), 2.94 (t, J=6.4 Hz, 4H), 1.10-0.74 (m, 8H). 13i) NMR (101 MHz, CDCl.sub.3): δ 170.9, 158.5 (d, J=253.4 Hz), 149.4, 133.2 (d, J=4.4 Hz), 127.9 (d, J=8.5 Hz), 127.4 (d, J=4.6 Hz), 127.2, 126.4 (d, J=1.9 Hz), 124.8 (d, J=2.6 Hz), 124.1 (d, J=15.7 Hz), 121.1 (d, J=6.1 Hz), 108.8 (d, J=20.0 Hz), 60.0, 50.7, 41.1, 40.7, 25.6, 24.4. HRMS-ESI: C34H32ClF2N8O3−[M+Cl]− calcd: 673.2259, found: 673.2259 4.2.5.
1,3-bis(4-azidobutyl)-5,5-bis(3,5-bis(trifluoromethyl)-benzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione-6(vi)
(93) To a stirred solution of 5(vi) (101 mg, 0.12 mmol) in DMF (1 mL) was added NaN.sub.3 (23 mg, 0.35 mmol). The reaction was stirred over night. When full conversion was reached according to MS the reaction mixture was diluted with EtOAc (15 mL) and washed with water (3×20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated to yield crude 6(vi) (63 mg, 68%) as white powder.
(94) 1H NMR (400 MHz, CDCl.sub.3): δ 7.78 (s, 2H), 7.53 (s, 4H), 3.59 (s, 4H), 3.57-3.48 (m, 4H), 3.19 (t, J=6.7 Hz, 4H), 1.42-1.31 (m, 4H), 1.31-1.20 (m, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 169.6, 148.6, 137.0, 132.3 (q, J=33.6 Hz), 129.9, 123.0 (q, J=272.9 Hz), 122.8-121.9 (m), 59.8, 50.6, 44.5, 41.6, 26.0, 24.9. HRMS-ESI: C30H26(ii) IF12N8O3−[M+Cl]− calcd: 809.1630, found: 809.1622.
1,3-bis(4-aminobutyl)-5,5-bis(4-tertbutylbenzyl) pyrimidin-2,4,6(1H,3H,5H)-trione-7(i)
(95) To a stirred solution of 6(i) (2.16 g, 3.52 mmol) and Et3N (0.98 mL, 7.05 mmol) in i-PrOH:THF (1:1, 10 mL) was added 1,3-propanedithiol (0.1 mL, 0.99 mmol). The mixture was stirred for 5 min before addition of NaBH.sub.4 (270 mg, 7.14 mmol). After 72 h reaction time, Boc2O (1.69 g, 7.74 mmol) and K.sub.2CO.sub.3 (1.94 g, 14.0 mmol) were added and the reaction was stirred for 18 h and evaporated, before EtOAc (20 mL) and water (15 mL) were added and stirred for 30 min. The organic phase was washed with water (3×15 mL) and brine (15 mL) and concentrated. The resulting crude was purified by automated flash chromatography and evaporated. The Boc-protected intermediate was deprotected with TFA (2.2 mL, 28.7 mmol) in CH.sub.2Cl.sub.2 (10 mL) for 18 h. The reaction mixture was concentrated and the crude product purified by RP automated flash chromatography and lyophilized to yield 7(i) (367 mg, 85%) as the TFA-salt.
(96) 1H NMR (400 MHz, CD3OD): δ 7.25 (d, J=7.1 Hz, 4H), 6.98 (d, J=7.2 Hz, 4H), 3.62-3.53 (m, 4H), 3.39 (s, 4H), 2.87 (t, J=7.4 Hz, 4H), 1.55-1.36 (m, 4H), 1.36-1.15 (m, 22H). 13i) NMR (101 MHz, CD3OD): δ 172.3, 163.0 (q, J=34.4 Hz, TFA), 151.9, 151.0, 133.5, 130.3, 126.5, 118.2 (q, J=292.8 Hz, TFA), 61.9, 45.9, 41.7, 40.0, 35.3, 31.7, 25.6, 25.5. HRMS-ESI: C34H51N4O3+[M+H]+ calcd: 563.3956, found: 563.3934.
1,3-bis(4-aminobutyl)-5,5-bis(3,5-dibromobenzyl)methyl)-pyrimidin-2,4,6(1H,3H,5H)-trione-7(ii)
(97) To a stirred solution of 6(ii) (810 mg, 0.99 mmol) and Et3N (0.32 mL, 2.29 mmol) in i-PrOH:THF (1:1, 5 mL) was added 1,3-propanedithiol (0.20 mL, 1.99 mmol). The mixture was stirred for 5 min before addition of NaBH.sub.4 (90 mg, 2.37 mmol). After 48 h reaction time, Boc2O (650 mg, 2.97 mmol) were added and the reaction mixture was stirred for 18 h and evaporated. The crude mixture was added EtOAc (15 mL) and water (15 mL) and stirred for 30 min. The organic phase was washed with water (3×15 mL) and brine (15 mL) and concentrated. The resulting crude was purified by automated flash chromatography and evaporated. The Boc-protected intermediate was deprotected with TFA (2 mL, 26 mmol) in CH.sub.2Cl.sub.2 (5 mL) for 18 h. The reaction mixture was concentrated and the crude product purified by RP automated flash chromatography and lyophilized to yield 7(ii) (374 mg, 38%) as the TFA-salt.
(98) 1H NMR (400 MHz, CD3OD): δ 7.66 (s, 2H), 7.23 (s, 4H), 3.68 (t, J=7.7 Hz, 4H), 3.43 (s, 4H), 3.08-2.82 (m, 4H), 1.76-1.48 (m, 4H), 1.49-1.32 (m, 4H). 13i) NMR (101 MHz, CD3OD): δ 171.2, 163.01 (q, J=34.4 Hz, TFA), 150.4, 140.5, 134.5, 132.7, 124.1, 118.24 (q, J=293.3 Hz, TFA). 61.2, 44.8, 42.2, 40.3, 26.3, 25.8. HRMS-ESI: C26H31 79Br4N4O3+[M+H]+ calcd: 762.9124, found: 762.9124.
1,3-bis(4-aminobutyl)-5,5-bis(3,5-di-tert-butylbenzyl)-pyrimidin-2,4,6(1H,3H,5H)-trione-7(iii)
(99) To a stirred solution of 6(iii) (405 mg, 0.55 mol) and Et3N (0.16 mL, 1.15 mmol) in i-PrOH:THF (1:1, 6 mL) was added 1,3-propanedithiol (0.12 mL, 1.15 mmol). The mixture was stirred for 5 min before addition of NaBH.sub.4 (44 mg, 1.16 mmol). After 72 h reaction time, Boc2O (490 mg, 2.25 mmol) was added and the reaction was stirred for another night, before diluted with EtOAc (10 mL) and water (10 mL) and stirred for 1 h. The organic phase was washed with water (3×15 mL) and brine (15 mL) and concentrated. The resulting crude was purified by automated flash chromatography and evaporated. The Boc-protected intermediate was deprotected with TFA (1.7 mL, 22.2 mmol) in CH.sub.2Cl.sub.2 (5 mL) for 6 h. The reaction mixture was concentrated and the crude product purified by RP automated flash chromatography and lyophilized to yield 7(iii) (154 mg, 31%) as the TFA-salt.
(100) 1H NMR (400 MHz, CD3OD): δ 7.31 (t, J=1.5 Hz, 2H), 6.89 (d, J=1.6 Hz, 4H), 3.59 (t*, 4H), 3.44 (s, 4H), 2.78 (t*, 4H), 1.40 (p, J=7.7 Hz, 4H), 1.26 (s, 36H), 1.17 (p, J=7.6 Hz, 4H). 13i) NMR (101 MHz, CD3OD): δ 172.3, 162.8 (q, J=34.7 Hz, TFA), 152.3, 151.1, 135.8, 124.7, 122.6, 118.1 (q, J=292.5 Hz, TFA), 61.8, 47.3, 42.0, 39.9, 35.6, 31.9, 25.9, 25.5. *distorted triplets. HRMS-ESI: C42H67N4O3+[M+H]+ calcd: 675.5211, found: 675.5211.
1,3-bis(4-aminobutyl)-5,5-bis(naphtalen-2-yl-methyl)-pyrimidin-2,4,6(1H,3H,5H)-trione-7(iv)
(101) To a stirred solution of 6(iv) (438 mg, 0.73 mmol) and Et3N (0.22 mL, 1.59 mmol) in i-PrOH:THF (1:1, 4 mL) was added 1,3-propanedithiol (0.1 mL, 0.99 mmol). The mixture was stirred for 5 min before addition of NaBH.sub.4 (68 mg, 1.81 mmol). After 72 h reaction time, Boc2O (333 mg, 1.53 mmol) and NaHCO.sub.3 (244 mg, 2.90 mmol) were added and the reaction was stirred for 18 h, before filtered through a pad of celite and concentrated. The resulting crude was purified by automated flash chromatography and evaporated. The Boc-protected intermediate (305 mg) was deprotected with TFA (2 mL, 26.1 mmol) in CH.sub.2Cl.sub.2 (5 mL). When MS showed full deprotection, the reaction mixture was concentrated and the crude product purified by RP automated flash chromatography and lyophilized to yield 7(iv) (287 mg, 90%) as the TFA-salt.
(102) 1H NMR (400 MHz, CD3OD): δ 7.90-7.68 (m, 6H), 7.60 (s, 2H), 7.52-7.43 (m, 4H), 7.19 (d, J=8.3 Hz, 2H), 3.70 (s, 4H), 3.59-3.50 (m, 4H), 2.56-2.37 (m, 4H), 1.30-0.96 (m, 8H). 13i) NMR (101 MHz, CD3OD): δ 172.2, 162.8 (q, J=35.2 Hz, TFA), 151.0, 134.7, 134.1, 134.0, 129.9, 129.4, 128.8, 128.7, 128.2, 127.6, 127.3, 118.1 (d, J=292.3 Hz, TFA), 62.0, 46.6, 41.7, 39.8, 25.6, 25.5. HRMS-ESI: C34H39N4O3+[M+H]+ calcd: 551.3017, found: 551.3020.
1,3-bis(4-aminobutyl)-5,5-bis((4-fluoronaphtalen-1-yl)methyl)pyrimidin-2,4,6(1H,3H,5H)-trione-7(v)
(103) To a stirred solution of 6(v) (67 mg, 0.105 mmol) and Et3N (0.03 mL, 0.21 mmol) in i-PrOH:THF (1:1, 4 mL) was added 1,3-propanedithiol (0.1 mL, 0.99 mmol). The mixture was stirred for 5 min before addition of NaBH.sub.4 (8 mg, 0.21 mmol). After 72 h reaction time, Boc2O (48 mg, 0.22 mmol) and NaHCO.sub.3 (35 mg, 0.42 mmol) were added and the reaction was stirred for 18 h, before filtered through a pad of celite and concentrated. The resulting crude was purified by automated flash chromatography and evaporated. The Boc-protected intermediate (72 mg) was deprotected with TFA (0.2 mL, 2.61 mmol) in CH.sub.2Cl.sub.2 (5 mL). When MS showed full deprotection, the reaction mixture was concentrated and the crude product purified by RP automated flash chromatography and lyophilized to yield 7(v) (82 mg, 89%) as the TFA-salt.
(104) 1H NMR (400 MHz, CDCl.sub.3): δ 8.34 (d, J=7.9 Hz, 2H), 8.07 (d, J=7.7 Hz, 2H), 7.74-7.53 (m, 4H), 7.38-7.19 (m, 2H), 7.08 (t, J=8.9 Hz, 2H), 4.13 (s, 4H), 3.39-3.33 (m, 4H), 2.60 (t, J=6.8 Hz, 4H), 1.20-1.00 (m, 4H), 0.94-0.71 (m, 4H). 13i) NMR (101 MHz, CDCl.sub.3): δ 172.2, 163.11 (q, J=34.1 Hz, TFA), 159.6 (d, J=251.5 Hz), 150.8, 134.5 (d, J=4.4 Hz), 129.3 (d, J=4.5 Hz), 128.4 (d, J=8.5 Hz), 128.2, 127.6 (d, J=1.1 Hz), 126.3 (d, J=2.4 Hz), 125.2 (d, J=15.6 Hz), 121.5 (d, J=6.2 Hz), 118.23 (q, J=292.8 Hz, TFA), 109.76 (d, J=20.2 Hz), 61.0, 41.7, 41.3, 39.9, 25.3, 25.1. HRMS-ESI: C34H37(iv)2N4O3+[M+H]+ calcd: 587.2828, found: 587.2828. 4.2.6.
1,3-bis(4-aminobutyl)-5,5-bis(3,5-bis(trifluoromethyl)-pyrimidin-2,4,6(1H,3H,5H)-trione-7(vi)
(105) To a stirred solution of 6(vi) (63 mg, 0.81 μmol) and Et3N (0.034 mL, 0.24 mmol) in i-PrOH:THF (1:1, 2 mL) was added 1,3-propanedithiol (0.10 mL, 0.99 mmol). The mixture was stirred for 5 min before 12 addition of NaBH.sub.4 (92 mg, 0.24 mmol). After 48 h reaction time, Boc2O (70 mg, 0.32 mmol) and K.sub.2CO.sub.3 (45 mg, 0.33 mmol) were added and the reaction was stirred for another night, before diluted with EtOAc (10 mL) and water (10 mL) and stirred for 1 h. The organic phase was washed with water (3×15 mL) and brine (15 mL) and concentrated. The resulting crude was purified by automated flash chromatography and evaporated. The Boc-protected intermediate was deprotected with TFA (2 mL, 26 mmol) in CH.sub.2Cl.sub.2 (5 mL) for 18 h. The reaction mixture was concentrated and the crude product purified by RP automated flash chromatography and lyophilized to yield 7(vi) (12 mg, 16%) as the TFA-salt.
(106) 1H NMR (400 MHz, CD3OD): δ 7.93 (s, 2H), 7.68 (s, 4H), 3.71 (s, 4H), 3.61-3.54 (m, 4H), 2.87-2.80 (m, 4H), 1.57-1.46 (m, 4H), 1.33-1.22 (m, 4H). 13i) NMR (101 MHz, CD3OD) δ 170.9, 150.1, 139.4, 133.0 (q, J=33.4 Hz, TFA), 131.6-131.1 (m), 124.6 (q, J=272.1 Hz, TFA), 123.0, 61.1, 44.8, 42.3, 40.0, 25.9, 25.7. HRMS-ESI: C30H31F12N4O3+[M+H]+ calcd: 723.2197, found: 723.2161.
1,1′-(4,4′-(5,5-bis(4-tert-butylbenzyl)-2,4,6-trioxodihydropyrimidine-1,3(2H,4H)-diyl)bis(butane-4,1-diyl))diguanidine-8(i)
(107) To a stirred solution of the TFA salt of 7(i) (129 mg, 0.16 mmol) in THF (2 mL) was added NaHCO.sub.3 (68 mg, 0.81 mmol) and N,N′-bis-Boc-1-guanylpyrazole (200 mg, 0.64 mmol). The reaction was stirred at r.t. for 48 h. The reaction mixture was concentrated, the crude product was dissolved in EtOAc (20 mL) and washed with 10% citric acid sol. (2×20 mL), 10% NaHCO.sub.3 sol. (20 mL) and brine (20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was purified by automated flash chromatography and the resulting Boc-protected intermediate was deprotected with TFA (1 mL) in CH.sub.2Cl.sub.2 for 18 h. The reaction mixture was concentrated and the crude was purified by RP automated flash chromatography and lyophilized to yield 8(i) (16 mg, 11%) as a white powder. 1H NMR (400 MHz, CD3OD): δ 7.24 (d, J=8.3 Hz, 4H), 6.98 (d, J=8.3 Hz, 4H), 3.58 (t, J=6.7 Hz, 4H), 3.39 (s, 4H), 3.13 (t, J=6.6 Hz, 4H), 1.39-1.29 (m, 8H), 1.24 (s, 18H). 13i) NMR (101 MHz, CD3OD) δ 172.4, 162.4 (q, J=35.6 Hz, TFA), 158.7, 151.9, 151.2, 133.4, 130.3, 126.4, 117.9 (q, J=291.5 Hz, TFA), 61.9, 45.9, 42.0, 41.9, 35.3, 31.7, 26.8, 25.8. HRMS-ESI: C36H55N8O3+[M+H]+ calcd: 647.4393, found: 647.4378.
1,1′-(4,4′-(5,5-bis(3,5-dibromobenzyl)-2,4,6-trioxodihydropyrimidine-1,3(2H,4H)-diyl)bis(butane-4,1-diyl))diguanidine-8(ii)
(108) o a stirred solution of the TFA salt of 7(ii) (360 mg, 0.362 mmol) in THF (5 mL) was added NaHCO.sub.3 (240 mg, 2.86 mmol) and N,N′-bis-Boc-1-guanylpyrazole (564 mg, 1.82 mmol). When MS showed full guanylation, the reaction mixture was filtered and concentrated. The crude product was dissolved in EtOAc (20 mL) and washed with brine (2×20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was purified by automated flash chromatography and the resulting Boc-protected intermediate was deprotected with TFA (0.2 mL) in CH.sub.2Cl.sub.2 (2 mL) for 18 h. The reaction mixture was concentrated and the crude was purified by RP automated flash chromatography and lyophilized to yield 8(ii) (44 mg, 11%) as a white powder.
(109) 1H NMR (400 MHz, CD3OD): δ 7.65 (t, J=1.8 Hz, 2H), 7.22 (d, J=1.7 Hz, 4H), 3.67 (t, J=7.2 Hz, 4H), 3.42 (s, 4H), 3.20 (t, J=6.7 Hz, 4H), 1.52-1.36 (m, 8H). □13i) NMR (101 MHz, CD3OD): δ 171.3, 163.0 (q, J=34.6 Hz, TFA), 158.6, 150.5, 140.5, 134.5, 132.6, 124.1, 118.2 (q, J=292.7 Hz, TFA), 61.2, 44.9, 42.6, 42.1, 27.0, 26.4. HRMS-ESI: C28H35 79Br2 81Br2N8O3+[M+H]+ calcd: 850.9525, found: 850.9532.
1,1′-(4,4′-(5,5-bis(3,5-di-tert-butylbenzyl)-2,4,6-trioxodihydropyrimidine-1,3(2H,4H)-diyl)bis(butane-4,1-diyl))-diguanidine-8(iii)
(110) To a stirred solution of the TFA salt of 7(iii) (118 mg, 0.13 mmol) in THF (3 mL) were added N,N′-bis-Boc-1-guanylpyrazole (245 mg, 0.79 mmol) and NaHCO.sub.3 (49 mg, 0.59 mmol) and stirred at r.t. until TLC (CHCl.sub.3) showed full conversion. The reaction mixture was diluted with EtOAc (5 mL) and washed with 10% citric acid sol. and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude was purified by automated flash chromatography and the resulting Boc-protected intermediate was deprotected with TFA (1.5 mL) in CH.sub.2Cl.sub.2 (1.5 mL) for 4 h. The reaction mixture was concentrated and the crude was purified by RP automated flash chromatography and lyophilized to yield 8(iii) (44 mg, 34%) as a white powder.
(111) 1H NMR (400 MHz, CD3OD): δ 7.30 (t, J=1.8 Hz, 2H), 6.89 (d, J=1.8 Hz, 4H), 3.58* (t, J=7.5 Hz, 4H), 3.44 (s, 4H), 3.06 (t, J=7.0 Hz, 4H), 1.38-1.14 (m, 44H). 13i) NMR (101 MHz, CD3OD): δ 172.4, 162.8 (q, J=35.2 Hz, TFA), 158.6, 152.3, 151.3, 135.8, 124.6, 122.6, 118.0 (q, J=292.3 Hz, TFA), 61.7, 47.3, 42.4, 41.8, 35.6, 31.9, 26.7, 26.2. *distorted triplet. HRMS-ESI: C44H71N8O3+[M+H]+ calcd: 759.5644, found: 759.5637.
1,1′-(4,4′-(5,5-bis(naphthalen-2-ylmethyl)-2,4,6-trioxodihydropyrimidine-1,3(2H,4H)-diyl)bis(butane-4,1-diyl))-diguanidine-8(iv)
(112) To a stirred solution of the TFA salt of 7(iv) (54 mg, 0.069 mmol) in THF (4 mL) were added N,N′-bis-Boc-1-guanylpyrazole (63 mg, 0.20 mmol) and NaHCO.sub.3 (41 mg, 0.48 mmol) and stirred at r.t. until TLC (CHCl.sub.3) showed full conversion. The reaction mixture was diluted with EtOAc (5 mL) and washed with 10% citric acid sol. (2×10 mL), and brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated. The Boc-protected intermediate was dissolved in CHCl.sub.3 and adsorbed onto Celite before purification on a silica column using CHCl.sub.3 as mobile phase. The Boc-protected intermediate (64 mg of a total of 104 mg, 0.057 mmol) was deprotected with TFA (0.2 mL) in CH.sub.2Cl.sub.2 (4 mL) until TLC showed full conversion. The reaction mixture was concentrated and purified by RP automated flash chromatography and lyophilized to yield 8(iv) (60 mg, 99%) as a white powder.
(113) 1H NMR (400 MHz, CD3OD): δ 7.85-7.69 (m, 6H), 7.59 (s, 2H), 7.53-7.40 (m, 4H), 7.20 (dd, J=8.4, 1.8 Hz, 2H), 3.69 (s, 4H), 3.54 (t, J=7.1 Hz, 4H), 2.80 (t, J=7.1 Hz, 4H), 1.21-1.08 (m, 4H), 1.08-0.98 (m, 4H). □ 13i) NMR (101 MHz, CD3OD): δ 172.3, 163.1 (q, J=34.3 Hz, TFA), 158.5, 151.0, 134.7, 134.1, 134.0, 129.8, 129.4, 128.7, 128.6, 128.3, 127.6, 127.3, 118.2 (q, J=293.0 Hz, TFA), 62.0, 46.6, 42.1, 41.8, 26.6, 25.9. HRMS-ESI: C36H43N8O.sub.3+[M+H]+ calcd: 635.3450, found: 635.3448.
1,1′-(4,4′-(5,5-bis((4-fluoronaphthalen-1-yl)methyl)-2,4,6-trioxodihydropyrimidine-1,3(2H,4H)-diyl)bis(butane-4,1-diyl))diguanidine-8(v)
(114) To a stirred solution of the TFA salt of 7(v) (35 mg, 43 μmol) in THF (3 mL) were added N,N′-bis-Boc-1-guanylpyrazole (38 mg, 122 μmol) and NaHCO.sub.3 (25 mg, 0.29 mmol) and stirred at r.t. until TLC (CHCl.sub.3) showed full conversion. The reaction mixture was diluted with EtOAc (5 mL) and washed with 10% citric acid sol. and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The Boc-protected intermediate was dissolved in CHCl.sub.3 and adsorbed onto Celite before purification on a silica column using CHCl.sub.3 as mobile phase. The Boc-protected intermediate (41 mg of a total of 95 mg, 0.038 mmol) was deprotected with TFA (0.1 mL) in CH.sub.2Cl.sub.2 (4 mL) until TLC showed full conversion. The reaction mixture was concentrated and purified by RP automated flash chromatography and lyophilized to yield 8(v) (20 mg, 52%) as a white powder.
(115) 1H NMR (400 MHz, CD3OD): δ 8.32 (d, J=8.6 Hz, 2H), 8.06 (d, J=7.9 Hz, 2H), 7.71-7.55 (m, 4H), 7.25 (dd, J=8.0, 5.5 Hz, 2H), 7.06 (dd, J=10.2, 8.1 Hz, 2H), 4.12 (s, 4H), 3.35 (t, J=7.2 Hz, 4H), 2.87 (t, J=7.1 Hz, 4H), 1.00 (p, J=7.2 Hz, 4H), 0.86 (p, J=7.4, 6.8 Hz, 4H). 13i) NMR (101 MHz, CD3OD): δ 172.2, 163.1 (q, J=34.1 Hz, TFA), 159.6 (d, J=251.6 Hz), 158.5, 150.9, 134.5 (d, J=4.3 Hz), 129.2 (d, J=4.6 Hz), 128.9 (d, J=8.5 Hz), 128.2, 127.6 (d, J=1.6 Hz), 126.2 (d, J=2.5 Hz), 125.2 (d, J=15.8 Hz), 121.5 (d, J=6.2 Hz), 118.2 (q, J=292.8 Hz, TFA), 109.7 (d, J=20.2 Hz), 60.9, 42.1, 41.8, 41.4, 26.5, 25.4. HRMS-ESI: C36H41F2N8O3+[M+H]+ calcd: 671.3264, found: 671.3244.
(116) Biological Test Methods
(117) Minimum Inhibitory Concentration (MIC) Assay
(118) Working solutions of the test derivatives were prepared with up to 100% DMSO and stored at −20° C. If necessary, the solutions were heated to 40-80° C. before testing to facilitate complete dissolution. Double-distilled water was used in all dilutions prepared. The final concentration of DMSO in the test series was ≤1% and did not affect the assay results. A microdilution susceptibility test was used for MIC determination according to Clinical and Laboratory Standards Institute, “Methods for Dilution Antimicrobial Sisceptibility Tests for Bacteria That Graw Aerobically”, Approved Standard M07-A9, edition 2012, with modifications as described by Igumnova et al (Bioorg Med Chem, 2016, 24 (22), 5884-5894).
(119) Briefly, the bacterial inoculum was adjusted to approximately 2.5-3×104 cells/mL in Mueller-Hinton broth (MHB, Difco Laboratories, USA), and incubated in a ratio of 1:1 with test derivatives in polystyrene 96-well flat-bottom microplates (NUNC, Roskilde, Denmark). Positive growth control (without test derivatives) and negative control (without bacteria) were included. The reference antibiotic was oxytetracycline hydrochloride (Sigma Aldrich, Saint Louis, Mo., USA). The microplates were incubated in an EnVision microplate reader (Perkin-Elmer, Turku, Finland) placed in an incubator set to 35° C. for 48 h. The MIC value was defined as the lowest concentration of derivative resulting in no bacterial growth as determined by OD.sub.600 measurement. All derivatives were tested in three parallels.
(120) Antimicrobial Screening Against Clinical Isolates
(121) The MIC was determined as explained above with some exceptions; working solutions of test derivatives were prepared from concentrated DMSO stocks stored at room temperature, the density of the bacterial inoculum was increased 40× to 1-1.2×106 cells/mL, enterococci were incubated in Brain Heart Infusion broth (BHIB, Difco Laboratories, USA), the microplates were incubated for 24 h, and the derivatives were tested in four parallels.
(122) Determination of Haemolytic Activity
(123) Hemolysis was determined using a heparinized fraction (1000 IU/mL) of freshly drawn blood. Blood collected in EDTA containing test tubes (Vacutest®, KIMA, Arzergrande, Italy) was used for determination of the hematocrit (hct). The heparinized blood was washed 3× with pre-warmed PBS and adjusted to a final hct of 4%. Derivatives in DMSO (50 mM) were added to a 96-well polypropylene V-bottom plate (NUNC, Fisher Scientific, Oslo, Norway) and serially diluted. The test concentration range was 500-4 μM with DMSO contents ≤1%. A solution of 1% triton X-100 was used as positive control for 100% hemolysis. As negative control a solution of 1% DMSO in PBS buffer was included. No signs of DMSO toxicity were detected. RBCs (1% v/v final concentration) were added to the well plate and incubated at 37° C. and 800 rpm for 1 h. After centrifugation (5 min, 3000 g), 100 μL of each well were transferred to a 96-well flat bottom plate and absorbance was measured at 545 nm with a microplate reader (VersaMax™, Molecular Devices, Sunnyvale, Calif., USA). The percentage of hemolysis was calculated as the ratio of the absorbance in the derivative-treated and surfactant treated samples, corrected for the PBS background. Three independent experiments were performed and EC.sub.50 values are presented as averages.
Example 2
(124) Further compounds of interest according to the invention are set out in
Example 3-Anti-Biofilm Activity
(125) S. epidermidis was used to assess the effects of the test compounds on inhibiting biofilm formation. Tryptic soy broth (TS; Merck, Darmstadt, Germany) was used as growth media.
(126) A culture of S. epidermidis grown overnight in TS was diluted with fresh TS containing 1% glucose (1:100). 50 μL aliquots were transferred to a 96-well microtiter plate, and 50 μL of the test compounds dissolved in water at different concentrations was added. After incubation at 37° C. overnight, the bacterial suspension was discarded carefully before the wells were washed with water. The plate was dried and the biofilm fixed by incubation for 1 hour at 55° C. before the cells attached to the surface were stained with 0.1% crystal violet (100 μL) for 5 minutes. The crystal violet solution was removed and the plate once more washed with water and dried at 55° C. for 1 hour. After adding 70 μL of 70% ethanol, the plate was incubated at room temperature for 10 minutes.
(127) Biofilm formation was observed by visually inspecting the plates. The MIC was defined as the lowest concentration where no biofilm formation was visible.
(128) An S. epidermidis suspension, diluted with 50 μL of water, was used as a positive control, and 50 μL Staphylococcus haemolyticus suspension with 50 μL of water was used as a negative control. A mixture of 50 μL water and 50 μL TS was used as assay control.
(129) TABLE-US-00003 Compound Anti-biofilm IC.sub.50 (μg/ml) 7(i) 4 7(ii) 6 7(iii) 1 7(iv) 4 7(v) 4 8(i) 1 8(ii) 1 8(iii) 2 8(iv) 1 8(v) 1
Example 4—Anti-Cancer Activity Against A2058 Cells
(130) Cytotoxicity of the test compounds was evaluated after 72 hour exposure to human Caucasian metastatic melanoma (A2058, ATCC CRL-11147) cells.
(131) A2058 cells were grown overnight (2,000 cells/well), and then incubated with the test compounds (range of concentrations) diluted in MEM Earle's supplemented with gentamycin (10 μg/mL), non-essential amino acids (1%), sodium pyruvate (1 mM), L-alanyl-Lglutamine (2 mM), but without FBS (total volume was 100 μl) for 24 hours. Ten μL of CellTiter 96® AQueous One Solution Reagent (Promega, Madison, Wis., USA) was added, and the plates were then further incubated for 1 hour.
(132) Absorbance was measured at 485 nm in a DTX 880 Multimode Detector. Results were calculated as % survival compared to negative (assay media) and positive (Triton X-100; Sigma-Aldrich) controls.
(133) TABLE-US-00004 Compound Anti-cancer IC.sub.50 (μg/ml) 7(i) 1 7(ii) 1 7(iii) 1 7(iv) 1 7(v) 1 8(i) 2 8(ii) 2 8(iii) 33 8(iv) 8 8(v) 8
(134) Compound 9 (below) was also tested, and found to have an IC.sub.50 value of 2.9 μg/ml.
(135) ##STR00013##
Example 4
(136) Methods of the synthesis of hydantoins following the “Bucherer-Bergs reaction for hydantoin synthesis” are well known in the art (e.g. Bucherer, H. T.; Brandt, W. J. Prakt. Chem. 1934, 140, 129; Bucherer, H. T.; Steiner, W. J. Prakt. Chem. 1934, 140, 291; and Bucherer, H. T.; Lieb, V. A. J. Prakt. Chem. 1934, 141, 5).
(137) This method, which is shown below, could be used for the synthesis of compounds of Formula (III). Specifically, as shown in scheme 2 below, the precursor hydantoins could be prepared by reacting the appropriate substituted carbonyl compounds with potassium cyanide (KCN) and ammonium carbonate [(NH4)2CO3], or from cyanohydrin and ammonium carbonate in a multiple component reaction.
(138) ##STR00014##
(139) For introducing the cationic R.sub.1 groups, the same methods discussed above for the synthesis of compounds 7(i)-8(v) (i.e. compounds of Formula (II)) could be used. One possible synthesis is shown in scheme 3 below.
(140) ##STR00015##