I. Toth, R. A. Hughes, G. Dekany, A. M. Hillery, P. Ward 685

Synthesis and Oral Uptake Studies of Lipidic and Glyco-Lipidic Conjugates of P-Lactam Antibiotics Istvan Toth*", Richard A. Hughes", Gyula Dekany", Anya M. Hillery", and Peter Wardb

The School of Pharmacy, University of London", 29-39 Brunswick Square, London WClN lAX,

Glaxo Group Research Limitedb, Greenford Road, Greenford, Middx UB6 OHE (UK)

Received September 27, 1993

Key Words: Amino acids, lipidic / Antibiotics / Drug delivery

Lipidic and glyco-lipidic cephalosporin conjugates l c and l i were synthesised as diastereomeric mixtures to improve their oral absorption. 2-(tert-Butoxycarbonylamino)tetradecanoic acid was condensed with thiazole Id and by following base- catalysed hydrolysis of the fully protected rac-le, the free acid rac-lf was produced. This was then used to acylate the carboxy-protected cephalosporin analog l g by using a modi- fied Vilsmeier reaction. Removal of the protecting groups of l b resulted in conjugate lc . Cephalosporin conjugate l i , which contains a sugar moiety in addition to a lipidic amino

acid residue, was synthesised to produce a drug conjugate with both lipophilic and hydrophilic properties [radiolabelled (14C) analogs la , l c and l i were also synthesised]. The radio- labelled parent l a and conjugates l c and l i were administe- red orally to rats and uptake of the radiolabel in the blood, various organs, urine and faeces was determined. The results showed that an increase in lipophilicity caused an increase in the oral uptake, suggesting that conjugation to lipidic amino acids and peptides is a useful approach to the impro- vement of the absorption of poorly absorbed drugs.

a-Amino acids with long hydrocarbon side chains, the so-called lipidic amino acids and their homooligomers the lipidic peptides, represent a class of compounds which com- bine structural features of lipids with those of amino acids[']. The lipidic amino acids and peptides can be coval- ently conjugated to or incorporated into poorly absorbed peptides and drugs. The resulting conjugates could be suf- ficiently hydrophobic to facilitate their passage across mem- branes. The underivatised hydrophobic conjugates were often insufficiently soluble in therefore to main- tain adequate water solubility, the lipidic amino acids were conjugated with hydrophilic

For the synthesis of lipidic cephalosporin conjugates, ra- cemic 2-aminotetradecanoic acid"] was used, hence the re- sultant conjugates l e and I f were racemates and the conju- gates lb, l c and lh-11 were diastereomeric mixtures.

Synthesis of Cephalosporin Conjugates

Ampicillin and cephalexin are both absorbed following oral administration, making the interpretation of the bio- logical activity of lipidic amino acid and peptide conjugates, of these antibiotics In contrast, the cephalospo- rin l a was not expected to be absorbed after oral adminis- trationL71. ruc-2-(tert-Butoxycarbonylamino)tetradecanoic acid[']

was condensed with the thiazole Id with the assistance of l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and 1- hydroxybenzotriazole to yield the fully protected le. Base- catalysed hydrolysis of the dipeptide l e gave the free acid l f , which was used to acylate the carboxy-protected com-

pound l g . A modification of the Vilsmeier reaction[7], was successful in condensing compound 3b to 4, thus affording the fully protected lipidic amino cephalosporin 1 b. The final step in the synthesis of cephalosporin conjugate l c was the removal of the amino (Boc) and carboxyl (diphenylmethyl) protecting groups with anhydrous TFA, using anisole as an electrophile scavenger. The synthesis of glycopeptide ce- phalosporin conjugate l i was carried out by using the reac- tion sequence described above. Compound 1 j['] was coupled to the thiazole Id and the resultant fully protected com- pound l k was saponified to give the acid 11. Acid 11 was condensed under Vilsmeier conditions with the cephalospo- rin analog l g . The resultant fully protected glycopeptide conjugate 1 h was partially deprotected with TFNanisole to give the desired conjugate l i .

Radiolabelled cephalosporin la, conjugates l c and l i were synthesised, containing a I4C atom in position 2 of the aminothiazole side chain. The initial step in the synthesis of the radiolabelled compound l c was the preparation of the labelled 2 from the nearly quantitative reaction of I4C- thiourea with ethyl 4-chloroaceto-acetate[81. From the radiolabelled ester Id, labelled l a , l c and l i were then syn- thesised by using the procedure described above.

Absorption of 14C-Labelled Lipidic Conjugates Following Oral Administration

The radioactivity detectable in the blood and organs fol- lowing oral administration of the parent l a was no greater than background, but compound l c demonstrated signifi- cant oral uptake. Total uptake of the conjugate after 1 hour in the blood and organs analysed constituted 14.2% (0.71

Liebigs Ann. Chern. 1994,685-688 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1994 0170-2041/94/0707-0685 $ 10.00+.25/0

686

X-

I. Toth, R. A. Hughes, G. Dekany, A. M. Hillery, P. Ward

H0 "x"

H3C CH3 -co

1

k

I

-N"TL&+ H H - - -Z

0 C HZOCO NHZ

-f H-co

[ C H Z I l l I CH3

r

- NH -

m

0 0 1 0

0 1 1 0

0 1 1 0

0 0 0 1

I I 1 1

1 1 1 1

1 1 0 0

1 I I 0

I 1 1 0

- NH

mg, 1.113 pmol) (Table 1, Figure 1) of the administered dose. The level of radiolabel of l c in the blood and organs remained high after 3 and 6 hours (10.1% and 13.8%, re- spectively) and slightly decreased after 12 hours (9.1%). Gastrointestinal tract contents were also analysed for radio- activity in an attempt to recover the entire administered

dose; 91.8%, 86.5% and 85.9% of dose administered were recovered after the 1-, 3- and 6-hour experiment, but only 57.4% were recovered after the 12-hour experiment. It is possible that after 6 hours compound l c was distributed to other sites in the body which were not selected for analysis. Compound l i also demonstrated significant oral uptake. In

0

15.0 7

COY

12.5

10.0

7.5

5.0

2.5

0 .o 0 2 4 6 8 1 0 1 2

Time (hour)

Table 1. Percentage of administered dose and distribution of compound l c (administered 5 mg, 7.84 pmol)

Organ Percentage uptake I h 3 h 6 h 12h

Blood 2.2 2.4 3.4 2.0 Liver 2.8 2.0 3.0 2.1 Kidney 1.5 0.7 0.8 0.4 Spleen 0.2 0.6 0.8 0.1 Stomach 1.2 1.4 0.4 0.2 Small intestine 5.4 2.5 3.9 1.2 Largc intestine 0.9 0.5 1.5 1.0 Stomach contents 47.6 52.1 1.2 1.6 Small intestine contents 27.9 20.2 1.2 I .4 Large intestine contents 2. I 4. I 69.7 27.4

- 15.2 Faeces - .

- 2.0 Urine - - Organ uptake 14.2 10.1 13.8 9.1

Figure 1. Organ uptake of compounds l i (open bar) and l c (hatched Recovered dose 91.8 86.5 85.9 54.7 bar) derived radioactivity

Liebigs Ann. Chem. 1994, 685-688

Lipidic and Glyco-Lipidic Conjugates of P-Lactam Antibiotics 687

Table 2. Percentage of administered dose and distribution of compound l i (administered 7.0 mg, 7.84 pnol)

Organ Percentage uptake I h 3 h 6 h 12h

Blood 1.3 1.0 1.0 1.3 Liver 0.3 0.4 0.9 1.1 Kidney 0.1 0.1 0.2 0.2 Spleen 0.1 0.1 0.2 0.2 Stomach 2.1 1.4 1.1 0.2 Small intestine 4.6 4.2 3.9 0.S Large intestine 0.5 0.4 1.9 1.3 Stomach contents 80.3 22.6 2.1 1.4 Small intestine contents 1.2 11.6 22.7 0.9 Large intestine contents 1.2 10.8 53.1 29.3

1 1 . 1 Faeces 0.7 Urine

Organ uptake 9.0 7.6 9.2 5.5 Recovered dose 91.7 52.6 87.1 48.2

. -

_ - -

this compound, the parent l a was conjugated to both a lipidic amino acid and a protected gulonic acid. The pres- ence of the sugar residue enhanced water solubility and the conjugate was administered as a clear solution. The blood and organ uptake of the compound showed a trend similar to that of compound lc. However, the introduction of a hydrophilic moiety resulted in lower uptake (Table 2, Fig- ure 1).

Experimental 'H-NMR Spectra: Varian XL-3600 and Bruker AM500 instru-

ments, 300 and 500 MHz, respectively; chemical shifts are reported in ppm downfield from internal TMS. - Mass spectra: VG Ana- lytical ZAB-SE instrument, fast-atom bombardment (FAB) ioni- sation. - The reaction progress of the reactions was monitored by thin-layer chromatography (TLC) on Kieselgel PFz54 by using dichloromethanelmethanol (10: 1) as the mobile phase. - Purifi- cation was achieved by flash chromatography through Kieselgel G with dichloromethanelmethanol (1O:OS). - Solvents were evapo- rated under reduced pressure with a rotary evaporator. - The 'H- NMR and MS data of the radiolabelled and the corresponding cold compounds were identical.

Method A. - Ethyl 2- {[(2R,2S) -2-tert-Butoxycarbamido]tetra- decanamido}thiazol-4-acetate (le): (2R,25')-2-(tert-butoxycarbami- do)tetradecanoic acid["l(O.223 g, 0.649 mmol), ethyl 2-aminothiaz- ole-4-acetate (Id) (100 mg, 0.649 mmol), triethylamine (66 mg, 0.65 mmol), l-ethyl-3-[3-(dimethylamino)propyl]carbodii~de hydro- chloride (135 mg, 0.65 mmol), 1-hydroxybenzotriazole hydrate (88 mg, 0.65 mmol) and tributylicosanoyl ammonium sulphate (3 mg) were stirred in CH2CI2 (10 ml) at 0°C for 1 h and at 20°C for 12 h. The reaction mixture was washed with water (3 X 10 ml), and the organic layer was dried (MgS04) and then concentrated. The residue was purified by flash chromatography to yield 0.072 g (40%) le. - 'H NMR (CDCI,): 6 = 6.80 ( lH, s, 5-H), 4.85 ( lH, m, OCONH), 4.30 (1 H, m, COCHNH) 4.19 (2H, q, OCH2CH3), 3.70 (2H, s, CH,CO), 1.95 ( lH, m, CHzCH), 1.61 ( lH, m,

(20H, m, 8 CH2, CH2CH, CH,CH), 0.88 (3H, t, CH3). - MS: mlz ("4) = 512 [M + H]+ (loo), 484 (2), 456 (20), 412 (15), 213

calcd. C 61.04, H 8.87, N 8.22; found C 61.31, H 8.82, N 8.07.

Method B. - 2- {((2S,2R)-2-tert-Butoxycarbamido]tetra- decanamido)thiazol-4-acetic Acid (If): Ethyl ester l e (100 mg, 0.31

CHZCH), 1.46 [9H, S, C(CH,),], 1.27 (3H, t, OCHZCH3), 1.25

(50), 198 (14), 187 (31), 113 (15), 57 (23). - C26H45N305S (511.6):

mmol) was dissolved in CH2C12 (5 ml) and a solution of 1 M NaOH in MeOHlwater (4: 1, 10 ml) added at room temperature. The pro- gress of the reaction was followed by TLC. After termination of the reaction, the organic solvents were removed in vacuo, the residual mixture was acidified with saturated citric acid solution to pH = 6 and subsequently extracted with CH2CI2. The organic layer was dried (MgS04), the solvent was evaporated and the residue purified by flash chromatography to yield 0.81 g (88%) of If. - 'H NMR (CDCl,): 6 = 6.76 (1 H, s, 5-H), 5.29 (1 H, m, OCONH), 4.40 (1 H, m, COCHNH), 3.68 (2H, s, CH,CO), 1.86 (1 H, m, CH,CH), 1.63 ( lH, m, CHzCH), 1.46 [9H, s, C(CH,),], 1.25 (20H, m, 8 CH,, CH2CH, CHzCH), 0.86 (3H, t, CH3). - MS: mlz (%) = 506 [M + Na]+ (16), 484 [M + HI+ (93), 428 (59), 406 (6), 384 (50), 242 (12), 198 (58), 185 (40), 159 (loo), 158 (17), 154 (16), 141 (12), 140 (ll), 139 (lo), 136 (13), 115 (13), 114 (19), 113 (30), 57 (80), 55 (15). - C24H41N305S (483.6): calcd. C 59.61, H 8.55, N 8.69; found C 59.61, H 8.42, N 8.55.

Method C. - (6R, 7R)-Diphenylmethyl 7- {2-( (2R,2S)-2-(tert- Butoxycarbamido) tetradecanamido] (thiazol-4-ylacetamido) 1-3- carbamoyloxymethyl-2,3-didehydro-8-oxo-S-thia-I -azabicyclo [4.2.O]octane-2-carboxylate (1 b) [A]: Oxalyl chloride (0.096 g, 0.065 ml, 0.753 mmol) was added to a solution of dimethylformam- ide (0.066 ml, 0.856 mmol) in dichloromethane (5 ml) at -20°C under nitrogen. After 1 min, the reaction vessel was transferred to an ice/water bath and the mixture stirred for 15 min. The suspen- sion was recooled to -20°C and compound If (0.331 g, 0.685 mmol) dissolved in dichloromethane (1 ml) added. The resulting solution was stirred at -5°C for 10 min, then recooled to -20°C and a solution of the tosylate salt of compound l g (0.419 g, 0.685 mmol) and N,N-dimethylaniline (0.414 g, 0.433 ml, 3.42 mmol) in dichloromethane added. The solution was allowed to warm to room temperature over a period of 30 min, then washed with 3% HCI, brine and dried. Evaporation of the solvent gave an oily resi- due which was purified by TLC; yield 0.240 g (40%). - 'H NMR (CDCI,): 6 = 7.87 (1 H, m, CONH), 7.44 (IOH, m, 10 Ph H), 6.96 ( lH, s, OCHPh2), 6.75 ( lH, d, 5'-H), 5.93 ( lH, m, CONH), 5.81 ( lH, m, 7-H), 5.35 ( lH , m, 6-H), 5.00 (2H, s, OCH2), 4.95 ( lH , m, OCONH), 4.70 (2H, m, CONH2), 4.30 ( lH, m, COCHNH), 3.64 (2H, s, CHICO), 3.48 (2H, q, 2 4-H), 1.86 (2H, m, CH,), 1.67 (2H, m, CH,), 1.45 [9H, s, OC(CH3)3], 1.27 (18H, m, 9 CH,), 0.89 (3 H, t, CH3). - MS: mlz (%) = 927 [M + Na]+ (19, 832 (1 I), 827

C 61.04, H 6.68, N 9.28; found C 60.98, H 6.57, N 9.17. (56), 806 (32), 184 (loo), 57 (30). - C46H60N60& (905.1): calcd.

Method D. - (6R. 7R)-7- {2-((2R,2S)-2-Aminotetradecanoyl- amino] (thiazol-4-ylacetamido) )-3-carbamoyloxymethy1-2,3- didehydro-8-oxo-S-thia-l-azabicyclo[4.2.O]octane-2-carboxylic Acid (lc): Compound l b (0.240 g, 0.265 mmol) was mixed with anisole (0.48 ml) and TFA (2.5 ml) added. The solution was stirred for 5 min, then water (0.24 ml) was added. After 10 s, diisopropyl ether (24 ml) was added and the resultant fine precipitate collected by filtration. The precipitate was washed thoroughly with diisopropyl ether and dried in vacuo; yield: 0.165 g (95%). - 'H NMR (CDC13/ CD30D): 6 = 6.80 (1 H, d, 5'-H), 5.78 (1 H, m, 7-H), 5.30 (1 H, m, 6-H), 5.00 (2H, s, OCH,), 4.32 (1 H, m, COCHNH), 3.64 (CH2CO), 3.47 (2H, q, 4-H), 1.86 (2H, m, CH,), 1.66 (2H, m, CHz), 1.27 (18H, m, 9 CH2), 0.89 (3H, t, CH,). - MS: m/z (YO) = 660 [M + Na]+ (36), 638 [M + HI+ (ll), 217 (31), 157 (loo), 57 (41). - C28H42N607SZ (638.8): calcd. C 52.64, H 6.62, N 13.15; found C 52.65, H 6.42, N 13.10.

Ethyl 2-( (2S,2R) -2- (2,3 : 4,6- Di- O-isopropylidene-2-keto-~-gulon- amido)tetradecanamido]thiazol-4-acetate (1 k): Compound 1 jI5l (0.400 g, 0.802 mmol) was condensed with ethyl 2-aminothiazol-4-

Liebigs Ann. Chem. 1994, 685-688

688 I. Toth, R. A. Hughes, G. Dekany, A. M. Hillery, P. Ward

acetate (Id) (0.213 g, 0.802 mmol) by using the procedure described in Method A; yield 0.402 g (75%). - ‘H NMR (CDC13): 6 = 7.44 ( lH, m, CONH), 6.80 ( lH, s, 5-H), 4.59 ( lH, d, 3’-H), 4.57 ( lH, m, COCHNH), 4.30 ( lH, dd, 4’-H), 4.18 (3H, m, 5‘-H, OCH2), 4.13 (2H, d, CH20), 3.70 (2H, s, CHzCO), 1.86 (2H, m, CHI), 1.63 (2H, m, CH2), 1.56, 1.53, 1.44, 1.34 (12H, 4 s, 4 CH3), 1.27 (3H, t, OCH2CH3), 1.24 (18H, m, 9 CH2), 0.88 (3H, t, CH3). - MS: mlz (“h) = 712 [M + 2 Na - H]+ (15), 690 [M + Na]+ (loo),

C33H53N309S (667.8): calcd. C 59.34, H 8.00, N 6.29; found C 59.10, H 7.88, N 6.20.

668 [M + HI+, 537 (l l) , 411 (ll), 389 (l l) , 324 (10). -

2-[ (2 R ,2S) -2- (2,3:4,6-Di-O-isopropylidene-2-keto-~-gulon- amido) tetradecanamido]thiazol-4-acetic Acid (1 I): Compound 1 k (0.20 g, 0.325 mmol) was treated with NaOH by using the pro- cedure described in Method B; yield 0.184 g (88%). - ‘H NMR (CDCI3): 6 = 7.50 ( lH, m, CONH), 6.80 ( lH, s, 5-H), 4.60 ( lH, d, 3’-H), 4.56 (1 H, m, COCHNH), 4.32 (1 H, dd, 4’-H), 4.18 (1 H, dt, 5’-H), 4.12 (2H, d, CH20), 3.70 (2H, s, CHzCO), 1.87 (2H, m, CH2), 1.67 (2H, m, CH2), 1.55, 1.53, 1.44, 1.35 (12H, 4 s, 4 CH3), 1.24 (18H, m, 9 CH2), 0.88 (3H, t, CH3). - MS: rnlz (YO) = 706 [M + 3 Na - 2H]+ (23), 684 [M + 2 Na - HI+ (93), 662 [M + Na]+ (loo), 322 (lo), 57 (14). - C31H49N309S (639.8): calcd. C 58.19, H 7.72, N 6.57; found C 58.08, H 7.60, N 6.41.

(6R, 7R)-Diphenylmethyl 7- {2-[(2R,2S)-2-(2,3:4,6-Di-O-isopro- pyl idene-2-keto-~-gut lonamido) tetradecanamido]( thiazol-4- ylacetamido) )-3-carbamoyloxymethy1-2,3-didehydro-8-oxo-5-thia-l- azabicyclo[4.2.0]octane-2-carboxylate (1 h): Compound 1 j (0.184 g, 0.288 mmol) was allowed to react with oxalyl chloride (0.040 g, 0.028 ml, 0.300 mmol) and dimethylformamide (0.026 g, 0.028 ml, 0.350 mmol). Then compound l g (0.127 g, 0.288 mmol) and di- methylaniline (0.174 g, 0.182 ml, 1.438 mmol) were added to the mixture. The crude product obtained was purified by using the pro- cedure described in Method C; yield 0.220 g (72%). - ‘H NMR (CDC13): 6 = 7.44 (lOH, m, 10 Ph H), 6.95 ( lH, s, OCHPh2), 6.75 (lH,d,5’-H),5.94(1H,m,CONH),5.81 (lH,m,7-H),5.35(lH, m, 6-H), 4.57 (2 H, m, COCHNH, 3”-H), 4.3 1 (1 H, dd, 4”-H), 4.13

s, CH,CO), 3.48 (2H, q, 2 4-H), 1.83 (2H, m, CH2), 1.65 (2H, m, CHZ), 1.53, 1.50, 1.44, 1.32 (12H, 4 s, 4 CH3), 1.25 (18H, m, 9 CHJ, 0.88 (3H, t, CH3). - MS: rnlz (%) = 1084 [M + Na]+ (12),

( lH , d, 5”-H), 4.10 (2H, d, CHZO), 4.08 (2H, d, CHZO), 3.67 (2H,

396 (13), 198 (26), 167 (loo), 136 (27), 77 (12), 59 (10). - C53H~&013S~ (1061.3): calcd. C 59.98, H 6.46, N 7.92; found C 59.72, H 6.38, N 7.85. (6R, 7R)-7- {2-[ (2R,2S) -2- (2,3:4,6-Di-O-isopropylidene-2-keto-

L-gulonamido) tetradecanamido] (thiazol-4-ylacetamido) )-3-car- bamoylmethyl-2,3-didehydro-8-0~0-5-thia-l -azabicyclo- [4.2.O]octane-2-carboxylic Acid (li): Compound l h (0.200 g, 0.188 mmol) was deprotected with TFA (2 ml) in the presence of anisole (0.4 ml) by using the procedure described in Method D; yield 0.160

5.81 ( lH, d, 7-H), 5.35 ( lH , m, 6-H), 4.58 ( lH , m, 3”-H, COCHNH), 4.31 ( lH , dd, 4-H), 4.13 ( lH, d, 5”-H), 4.10 (2H, d,

4-H), 1.86 (2H, m, CHz), 1.67 (2H, m, CHI), 1.53, 1.50, 1.43, 1.33 (12H, 4 s, 4 CH,), 1.25 (18H, m, 9 CH2), 0.88 (3H, t, CH3). - MS: rnlz (YO) = 917 [M + Na]+ (17), 845 (30), 217 (25), 157 (loo), 57 (46). - C40H58N6013S2 (895.0): calcd. C 53.67, H 6.53, N 9.39; found C 53.72, H 6.51, N 9.27.

Oral Dosing: A single dose of 7.84 pmol of compounds la, l c and l i in 1 ml of water vehicle (lc as a fine suspension) was admin- istered to each rat, and the blood and organs were examined using literature methods[9].

g (95Yo). - ‘H NMR (CDCl3lCD30D): 6 = 6.80 ( lH, d, 5’-H),

CHZO), 4.07 (2H, d, CHZO), 3.68 (2H, S, CH,CO), 3.46 (2H, q, 2

W. A. Gibbons, R. A. Hughes, A. Szeto, M. Charalambous, A. Aulabaugh, P. Mascagni, I. Toth, Liebigs Ann. Chem. 1990,

I. Toth, R. A. Hughes, P. Ward, M. A. Baldwin, K. J. Welham, A. M. McColm. D. M. Cox. W. A. Gibbons. Int. .I Pharm.

1175-1183.

1991, 73, 259-266. R. A. Hughes. I. Toth. P. Ward. A. M. McColm. D. M. Cox. W. A. Gi&on$, J Pharm. Sci. 1992, 81, 845-848: I. Toth, R. A. Hughes, P. Ward, D. M. Cox, A. M. McColm, G. J. Anderson, W. A. Gibbons, Int. .I Pharm. 1991, 77, 13-20. I. Toth, G. J. Anderson, R. Hussain, I. P. Wood, E. del Olmo Fernandez, P. Ward, W. A. Gibbons, Tetrahedron, 1992, 48, 923-930. W. M. M. Kirby, A. C. Kind, Ann. N Y Acad. Sci. 1967, 145, 291-297. R. Bell, M. W. Foxton, B. E. Looker, Eu. Pat. Appl. 181172, 1985; Chem. Abstr. 1986, 104, P 152823 v. E. Campaigne, T. P. Selby, .I Heterocycl. Chem. 1980, 17, 1255-1257. I. Toth, A. M. Hillery, I. P. Wood, C. Magnusson, P. Artursson, Int. .I Pharm. 1994, 102, 223-230.

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