Synthesis of minocycline hybrids

With minocycline as prototype tetracycline, 10 hybrids were synthesized with available plant-based aldehydes and ketones with a covalent linkage, which is summarized in Scheme 1.

Scheme 1.

Synthetic scheme for tetracycline hybrids

Structure–activity relationship studies of minocycline have suggested modification of the 9th position for enhancement of therapeutic activity. Nitration of the 9th position in tetracycline moiety and its subsequent reduction has been reported. We have followed the protocol by Zang et.al [40] with slight modifications. Various aldehydes/ketones which exhibited antibacterial activity in-vitro/in-vivo were selected for the study based on the literature reports [41]. So, an effort was made to covalently link minocycline with these compounds thereby producing hybrids. Reductive amination of selected aldehydes and ketones as mentioned in Table 1 with 9-amino minocycline in optimised conditions produced a set of 10 novel hybrids. Reductive amination proceeded smoothly for all compounds (except when citronellal was used) producing compounds listed in Table 1. Citronellal produced mixture of two major compounds which on characterisation using LC/MS were found to be a secondary amine product i.e. compound 4 as well as a its schiff base with 9-amino minocycline i.e. compound 10 as shown in Fig. 1. Both compounds were isolated using preparative HPLC and their in vitro antibacterial activities were established. Trials were run in order to synthesize the schiff base with other aldehydes also due to their novelty, but the synthesized compounds showed poor stability even at low temperatures and degraded within 1–2 days and hence were not included in the study. Surprisingly, citronellal imine is a stable compound and can be isolated and stored. It was noted that aliphatic amines reacted quickly when compared to aromatic amines and also better yields were obtained, with lesser side reactions. Normal phase column purification was not possible for the synthesized compounds as they tend to form chelates with ions present in silica gel and were bound to silica with no elution even at higher polarities. Hence reverse phase preparative HPLC was used to purify the compounds. All the compounds were characterized using mass and NMR spectroscopy.

Fig. 1Spectral elucidation of novel synthesized compounds Compound-1

4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-9-(propylamino)-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide; Red Solid, 1H NMR (400 MHz, DMSO-d6) δ 0.88 (m, 2H, propyl), 1.02(m, 2H, propyl), 1.12(d, 3H, propyl), 1.24 (s, 1H), 1.48 (m, 2H), 1.68 (m, 4H, -CH2-), 1.85 (m, 6H, -N(CH3)2), 2.49 (s, 2H,-CH2-), 2.58 (m, 6H, -N(CH3)2), 3.10 (m, 4H), 7.25 (s, 2H, -CONH2), MS(ESI) m/z 515 (M + H).

Compound-2

4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-9-((4-methoxybenzyl)amino)-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide; Red Solid, 1H NMR (400 MHz, DMSO-d6) δ 1.12 (d, 1H, -CH-), 1.24 (s, 2H, -CH2-), 2.04 (m, 4H, -CH2-), 2.4 (s, 6H, -N(CH3)2), 2.50 (d, 3H, -N(CH3)2), 3.80 (s, 3H, -OCH3), 3.89 (s, 3H, -N(CH3)2), 4.28 (s, 2H), 6.88 (m, 3H, aromatic), 7.01 (d, 2H, aromatic), 7.29 (s, 2H, -CONH2), 7.85 (d, 2H), 9.88 (s, 1H, -OH), MS(ESI) m/z 594 (M + H).

Compound-3

9-(cinnamylamino)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide;Red Solid, 1H NMR (400 MHz, DMSO-d6) δ 2.28 (s, 4H, -CH2-), 2.41 (m, 8H), 2.54 (s, 6H, -N(CH3)2), 3.48 (s, 1H, -CH-), 3.98 (s, 6H, -N(CH3)2), 6.32 (m, 2H, vinyl), 6.66 (m, 3H), 7.23 (s, 2H, -CONH2), 7.36 (m, 6H, aromatic), MS(ESI) m/z 589 (M + H).

Compound-4

4,7-bis(dimethylamino)-9-((3,7-dimethylocta-2,6-dien-1-yl)amino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide;Brown Solid, 1H NMR (400 MHz, DMSO-d6) δ 1.66–1.75 (m, 18H, aliphatic chain), 2.04–2.22 (m, 10H), 2.51–2.71 (m, 10H), 2.75(s, 1H), 3.48 (s, 1H), 3.71 (s, 1H), 5.06(s, 2H, vinyl), 7.24 (s, 2H, -CONH2), MS(ESI) m/z 609 (M + H).

Compound-5

4,7-bis(dimethylamino)-9-((3,7-dimethyloct-6-en-1-yl)amino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide;Brown Solid, 1H NMR (400 MHz, DMSO-d6) δ 0.86 (m, 6H, aliphatic), 1.24 (m, 6H, aliphatic), 1.56–1.67 (m, 12H, -aliphatic), 1.68–1.90 (m, 10H, aliphatic and -N(CH3)2), 2.56 (s, 6H, -N(CH3)2), 3.16 (s, 3H), 3.48 (s, 1H, -CH-), 5.05 (s, 1H, -CH-), 7.32 (s, 2H, -CONH2), MS (ESI) m/z 611 (M + H).

Compound-6

4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-9-(isopentylamino)-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide;Red Solid, 1H NMR (400 MHz, DMSO-d6) δ 0.86 (m, 6H, isovaleryl), 0.96 (s, 5H, isovaleryl), 1.24 (s, 3H), 1.33 (m, 2H), 1.54 (m, 2H), 1.68–1.91 (m, 8H), 2.24 (m, 3H, -NH(CH3)2), 2.55 (t, 3H, -NH(CH3)2), 2.62 (s, 2H, -CH2-), 3.10 (m, 2H), 7.28 (s, 2H, -CONH2), MS (ESI) m/z 543 (M + H).

Compound-7

9-(benzylamino)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide;Red Solid, 1H NMR (400 MHz, DMSO-d6) δ 1.12–1.13 (s, 12H), 2.47 (m, 7H, -NH(CH3)2), 3.61–3.67 (m, 2H, -CH2-), 4.33 (m, 4H), 7.36 (s, 2H, -CONH2), 7.53 (t, 2H), 7.62 (m, 1H), 7.87 (d, 1H), MS (ESI) m/z 563 (M + H).

Compound-8

9-(deca-2,4-dien-1-ylamino)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide;Brown Solid, 1H NMR (400 MHz, DMSO-d6) δ0.87 (t, 6H, aliphatic), 1.24–1.37 (m, 12H, aliphatic), 2.05 (m, 4H, -CH2-), 2.54(d, 8H,-N(CH3)2), 2.76 (m, 2H, -CH2-), 3.48 (s, 2H), 3.81 (m, 2H), 5.77 (m, 3H), 6.04 (m, 2H, vinyl), 6.25 (m, 1H), 6.62 (s, 1H), 7.20 (s, 2H, -CONH2), MS (ESI) m/z 609 (M + H).

Compound-9

9-(cyclohexylamino)-4,7-bis(dimethylamino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide; Dark Orange Solid, 1H NMR (400 MHz, DMSO-d6) δ1.13 (d, 3H), 1.24(m, 6H), 1.71–1.86 (m, 14H, cyclohexane merged), 2.03 (m, 3H), 2.33 (t, 2H), 2.41 (m, 1H, -CH-), 2.55 (d, 6H, -N(CH3)2), 3.23 (m, 1H), 3.64 (m, 1H), 7.24 (s, 2H, -CONH2), MS (ESI) m/z 555 (M + H).

Compound-10

4,7-bis(dimethylamino)-9-((3,7-dimethylocta-2,6-dien-1-ylidene)amino)-1,10,11,12a-tetrahydroxy-3,12-dioxo-3,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide;Brown Solid, 1H NMR (400 MHz, DMSO-d6) δ 0.88 (m, 1H), 0.95 (d, 2H), 1.12–1.13 (m, 18H, aliphatic), 1.27 (m, 3H, -N(CH3)2), 1.49 (m, 3H), 1.59 (s, 1H), 1.68 (s, 1H), 1.85 (m, 1H), 2.04 (s, 1H), 2.17 (s, 1H), 2.38 (m, 2H), 2.59 (m, 2H, -CH2-), 2.76 (m, 1H), 3.55 (m, 1H), 3.64 (m, 3H, -N(CH3)2), 3.74 (m, 1H, -CH-), 7.24 (s, 2H, -CONH2), MS (ESI) m/z 609 (M + H).