Synthesis and antibacterial activity of 9-substituted minocycline derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.09.078

A number of 9-acylamino and 9-sulfonylamino derivatives of minocycline have been synthesized for structure-activity relationship studies. These compounds showed activity against both tetracycline-susceptible and tetracycline-resistant strains. Many of the

Full text of DOI:10.1016/j.bmcl.2005.09.078

Bioorganic & Medicinal Chemistry Letters 16 (2006) 400–403
Synthesis and antibacterial activity of 9-substituted
minocycline derivatives
Phaik-Eng Sum,a,* Adma T. Ross,a Peter J. Petersenb and Raymond T. Testab
aDepartment of Chemical and Screening Sciences, Wyeth Research, Chemical Sciences Building 222/3147,
401 N. Middletown Road, Pearl River, NY 10965, USA
bDepartment of Infectious Diseases, Wyeth Research, Building 200, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 15 August 2005; revised 20 September 2005; accepted 26 September 2005
Available online 21 October 2005
Abstract—A number of 9-acylamino and 9-sulfonylamino derivatives of minocycline have been synthesized for structure–activity
relationship studies. These compounds showed activity against both tetracycline-susceptible and tetracycline-resistant strains. Many
of the 9-sulfonylamino derivatives exhibited improved antibacterial activity against a number of tetracycline- and minocycline-
resistant Gram-positive pathogens.
Ó 2005 Elsevier Ltd. All rights reserved.
The first tetracycline was introduced in the 1950s as a
safe and effective broad-spectrum antibiotic, and it has
been more than 30 years since the last tetracycline anti-
biotic, minocycline (1), was introduced into the market.1
These agents have been important medical products for
the last 50 years, however, the utility of these agents has
gradually diminished in recent years due to the emer-
gence of tetracycline-resistant bacteria. Similarly, the
alarming incidence of pathogenic bacteria resistant to
other currently available antibiotics has caused concern
among medical professionals.2 Tetracyclines inhibit pro-
tein synthesis by interfering with the binding of amino-
acylated tRNA to the A-site of the 30S subunit. There
are two major resistance mechanisms associated with
tetracyclines: (1) ribosome protection mechanism medi-
ated by cytoplasmic protein that interacts with the ribo-
some, and (2) efflux of the antibiotics mediated by
membrane-spanning proteins (an active transport mech-
anism).3–6
glycylcyclines are also effective against pathogens
that are resistant to other antibiotics, for example, meth-
icillin-resistant
Staphylococci,
penicillin-resistant
Streptococcus pneumoniae, and vancomycin-resistant
Enterococci.8 Three of the glycylcyclines, DMG-
DMDOT 2, DMG-MINO 3, and TBG-MINO 4 (also
known as tigecycline or GAR-936), havÒe been investi-
gated extensively.9,10 Tigecycline (Tygacil ) was selected
for further development (Fig. 1) and was recently
approved by the FDA.
During the course of our research to discover com-
pounds with improved activity and pharmacokinetic
properties, we have synthesized a number of compounds
with C-9 modifications. These compounds were de-
signed based on previous SAR that modifications at
ring-D might lead to compounds with enhanced activi-
ty.11 The crystal structure of tetracycline binding to
30S ribosomal subunit was only recently determined
by Ramakrishnan and co-workers.12 The data obtained
from the crystal structure shared numerous similarities
with our hypothesis derived from SAR and many bio-
chemical experiments.13,14 Crystal structure of the tet
repressor in complex with DMG-DMDOT indicated
that the bulky and positively charged glycylamido group
interferes with amino acid side chains which were iden-
tified by mutagenesis as sensitive to Tc (tetracycline) rec-
ognition and to the mechanism of induction.15 Both
findings suggested that the hydrophilic domain of the
tetracycline molecule is important for binding to ribo-
some (Fig. 2) and modification at that region would
Previously, we reported a novel class of semi-synthetic
tetracyclines that has been referred to as Ôglycylcy-
clinesÕ.7 The unique feature of these agents was the abil-
ity to overcome both ribosomal protection (tetM) and
efflux resistance (tetA, tetB, tetC, tetD, tetK, etc.) mech-
anisms associated with tetracycline. Most importantly,
Keywords: Novel tetracyclines; Antibiotic; Resistant bacteria.
*
Corresponding author. Tel.: +1 845 602 3431; fax: +1 845 602
5561; e-mail: sump@wyeth.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.078

P.-E. Sum et al. / Bioorg. Med. Chem. Lett. 16 (2006) 400–403
401
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
OH
OH
a, b
1 Minocycline
9
NH2
NH2
H2N
OH
OH
OH
OH
OH
O
O
O
OH
O
O
O
6 9-Amino-mino
1 Minocycline
R
N(CH3)2
OH
N(CH3)2
N(CH3)2
OH
O
O
N
NH2
N
H
NH2
OH
OH
R
N
OH
O
O
O
OH
OH
H
OH
O
7
O
O
2 R = H (DMG-DMDOT)
3 R = N(CH3)2 (DMG-MINO)
R =
N(CH3)2
N(CH3)2
OH
O
H
N
NH2
N
H
F
N
OH
OH
7a
7b
7c
7d
OH
O
O
O
4 Tigecycline (Tygacil, or GAR-936)
F3C
H
S
N
Figure 1. Structures of minocycline (1), DMG-DMDOT (2), DMG-
MINO (3), and tigecycline or tygacilÒ (4).
7e
7f
7g
7h
N
HO
Hydrophobic region
N(CH3)2
7j
7i
OH
8
C
D
B
A
Scheme 1. Reagents and conditions: (a) HNO3/H2SO4, 0 °C, 1 h; (b)
10% Pd/C, H2, 40 psi.
O
9
OH
OH
OH
O
O
NH2
Hydrophilic region (interacting with ribosome)
6 9-Amino-mino
Figure 2. Possible interactions of 6-demethyl-6-deoxytetracycline with
30S ribosomal subunit.
a
interfere with the binding, while modification of
the hydrophobic region might produce compounds with
enhanced antibacterial activity.
N(CH3)2
N(CH3)2
OH
O
Br
NH2
N
OH
OH
H
Herein, we report the synthesis and antibacterial activity
of 9-acylamino and 9-sulfonylamino derivatives of min-
ocycline. The key intermediate was prepared using the
same method as described previously.7 Nitration of min-
ocycline 1 gave the 9-nitro-mino and subsequent catalyt-
ic reduction gave the desired 9-amino-mino 6.
Treatment of 9-amino-mino hydrochloride 6 with excess
sodium carbonate and appropriate acylating agents gave
the desired final products 7a–g and j (Scheme 1).
OH
O
O
O
8
b
N(CH3)2
N(CH3)2
OH
O
N
NH2
N
OH
OH
H
OH
O
O
O
7i
Treatment of 9-amino-mino 6 with 4-bromobutyrylchlo-
ride gave the intermediate 8. Compound 8 was then treat-
ed with excess dimethylamine to give 7i (Scheme 2). This
compound was designed to probe the spacing require-
ment between the carbonyl and the nitrogen of the glycyl
group. Compound 7h, reported previously, was included
for comparison.9
Scheme 2. Reagents: (a) 4-bromobutyrylchloride/DMPU; (b)
dimethylamine.
the recommendations of the National Committee for
Clinical Laboratory Standards.16 The arylacylamino
derivatives in general show good activity against
Gram-positive bacteria, with the best compound in this
series 7e exhibited MICs of 0.5–1.0 lg/mL. However,
this series was less active against Gram-negative
bacteria; most of the MICs are greater than the highest
concentration tested (>32 lg/mL) (Table 1).
The 9-sulfonylamino derivatives 9a–e were synthesized
by treatment of 9-amino-mino 6 with appropriate sulfo-
nyl chloride (Scheme 3). Final products were purified by
HPLC.
Compound 7g, designed with a methylene in place of
NH, is less active than 7h, suggesting that the basicity
The activities of the above new tetracycline derivatives
were determined by the agar dilution method following

402
P.-E. Sum et al. / Bioorg. Med. Chem. Lett. 16 (2006) 400–403
The sulfonamide derivatives show the most interesting
6 9-Amino-mino
in vitro activity. Almost all the compounds listed in
Table 3 exhibited potent activity against Gram-positive
pathogens and were considerably less active against
Gram-negative bacteria. Compounds 9b and e show
very good activity against Streptococcus. aureus strain
carrying the tetM-resistant determinant (MIC 0.06–
0.25 lg/mL). In general, these compounds with MICs
of 1 and 8 lg/mL, respectively, were moderately active
against the S. aureus strain carrying the tetK-resistant
determinant (efflux). The best compound in sulfon-
amide series is 9b, demonstrating good activity with
MICs against Gram-positive bacteria ranging from
0.06 to 1.0 lg/mL. The fact that the sulfonamide and
the acylamido derivatives are much less active against
Gram-negative bacteria than the glycylcyclines9 sug-
gests that in addition to possibly structural require-
ments, the basicity of the cyclopentylamino group of
7h, dimethylamino side chain in 3 (DMG-MINO),
and tert-butylamino group in 4 (tigecycline) at the
9-glycylamido side chain is most likely responsible for
the good activity against Escherichia coli. The poor
activities against E. coli that are still sensitive to mino-
cycline shown by acylamido derivatives 7a–g,j and
sulfonamide derivatives 9a–d suggest that these com-
pounds most likely are not being permeated into the
Gram-negative cell wall. The moderate activity (MIC
8 lg/mL) of 9e against E. coli carrying the tetB
determinant suggest that it is possible for the
compound to be able to gain entry into the bacteria
cell and is most likely to be pumped out by the efflux
protein.
RSO2Cl,
Na2CO3/THF
N(CH3)2
N(CH3)2
OH
NH2
RO2SHN
OH
OH
OH
O
O
O
9
S
R =
Cl
9a
9b
9c
S
N
HN
O
H3C
9e
Scheme 3. Sulfonylamino derivatives of minocycline.
9d
of cyclopentylamine is crucial for the potent broad-spec-
trum antibacterial activity (Table 2). Extension of the
carbon linker between the glycyl dimethylamino nitro-
gen and the carbonyl, for example compound 7i, led
to reduction in overall activity when compared to that
of the dimethylglycylamido derivative (DMG-MINO)
3, indicating that the spacing between the carbonyl
and the nitrogen is crucial for binding to the protein,
and that gylcyl moiety seems to be the optimal group
for the antibacterial potency in both Gram-positive
and Gram-negative strains.
Table 1. Antibacterial activity of 9-(arylacylamino)-7-(substituted)-6-demethyl-6-deoxytetracycline 7a–f
Organism
Minimal inhibitory concentration (MIC) lg/mL compound
7b 7c 7d 7e
7a
16
7f
S. aureus UBMS 90-1 (tetM)
S. aureus UBMS 90-2 (tetM)
S. aureus UBMS 90-3 (sensitive)
S. aureus UBMS 88-7 (tetK)
S. aureus Smith (sensitive)
E. faecalis ATCC 29212
E. coli UBMS 88-1 (tetB)
E. coli 88-2 (sensitive)
4
4
1
8
1
4
4
2
1
8
1
4
8
2
1
4
1
2
2
1
4
4
2
8
8
1
0.5
1
16
4
32
2
1
8
1
8
>32
>32
>32
>32
>32
>32
NT
>32
>32
>32
NT
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
NT
>32
E. coli UBMS 89-1 (tetM)
E. coli 89-2 (sensitive)
Table 2. Antibacterial activity of 9-(alkylacylamino)-7-(substituted)-6-demethyl-6-deoxytetracycline 7g–j
Organism
Minimal inhibitory concentration (MIC) lg/mL compound
7g
7h
7i
7j
DMG-MINO
GAR-936
S. aureus UBMS 90-1 (tetM)
S. aureus UBMS 90-2 (tetM)
S. aureus UBMS 90-3 (sensitive)
S. aureus UBMS 88-7 (tetK)
S. aureus Smith (sensitive)
E. faecalis ATCC 29212
8
8
4
8
4
8
0.25
NT
0.12
2
8
4
2
4
2
1
0.12
NT
0.12
NT
0.25
0.5
0.06
0.5
16
2
>32
0.5
0.25
0.12
0.25
0.25
0.25
0.06
0.06
0.25
0.25
0.12
0.25
0.12
0.5
4
32
>32
>32
>32
E. coli UBMS 88-1 (tetB)
E. coli UBMS 90-4 (tetM)
E. coli 90-5 (sensitive)
>128
>128
>128
>32
16
16
0.12
0.25

P.-E. Sum et al. / Bioorg. Med. Chem. Lett. 16 (2006) 400–403
403
Table 3. Antibacterial activity of 9-(sulfonylamino)-7-(substituted)-6-demethyl-6-deoxytetracycline 9a–e
Organism
Minimal inhibitory concentration (MIC) lg/mL compound
9a
9b
9c
9d
9e
Mino
S. aureus UBMS 90-1 (tetM)
S. aureus UBMS 90-2 (tetM)
S. aureus UBMS 90-3 (sensitive)
S. aureus UBMS 88-7 (tetK)
S. aureus Smith (sensitive)
E. faecalis ATCC 29212
E. coli UBMS 88-1 (tetB)
E. coli 88-2 (sensitive)
4
0.25
0.12
0.12
1
1
32
4
0.25
0.06
0.12
8
8
0.06
4
0.25
0.25
2
2
2
0.03
0.25
0.06
2
4
32
2
4
0.12
0.06
>32
>32
ND
>32
0.03
0.25
32
0.12
0.06
8
0.06
16
8
1
>32
>32
32
>32
16
8
2
0.25
>32
2
E. coli UBMS 89-1 (tetM)
E. coli 89-2 (sensitive)
ND
16
ND
16
ND
2
5. Levy, S. B. J. Antimicrob. Chemother. 1989, 24, 1.
6. Speer, B. S.; Shoemaker, N. B.; Salyers, A. A. Clin.
Microbiol. Rev. 1992, 5, 387.
7. Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad,
G. A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med.
Chem. 1994, 37, 184.
8. Testa, R. T.; Petersen, P. J.; Jacobus, N. V.; Sum, P.-E.;
Lee, V. J.; Tally, F. P. Antimicrob. Agents Chemother.
1993, 37, 2270.
9. Sum, P.-E.; Petersen, P. J. Bioorg. Med. Chem. Lett. 1999,
9, 1459.
In summary, the sulfonamide series is much more active
against Gram-positive bacteria than the acylated series.
The structure–activity relationship studies of these
compounds provided useful information on the structur-
al requirements for activity against Gram-negative
bacteria. It also indicated that it is possible to design
compounds with activity selectively just against Gram-
positive bacteria. The potent in vitro activity of some
of the sulfonamide derivatives (e.g., 9b,e) against resis-
tant Gram-positive bacteria makes them potential
candidates for the development of new antibiotics tar-
geting selectively just the Gram-positive pathogens.
10. Petersen, P. J.; Jacobus, N. V.; Weiss, W. J.; Sum, P.-E.;
Testa, R. T. Antimicrob. Agents Chemother. 1999, 43, 738.
11. Sum, P.-E.; Sum, F.-W.; Projan, S. J. Curr. Pharm. Des.
1998, 4, 119.
12. Broadersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.;
Morgan-Warre, R. T.; Wimberly, B. T.; Ramakrishnan,
V. Cell 2000, 103, 1143.
13. Rasmussen, B. A.; Gluzman, Y.; Tally, F. P. Antimicrob.
Agents Chemother. 1994, 38, 1658.
14. Bergeron, J.; Ammirati, M.; Danley, D.; James, L.;
Norcia, M.; Retsema, J.; Strick, C. A.; Su, W.-G.;
Sutcliffe, J.; Wondrack, L. Antimicrob. Agents Chemother.
1996, 40, 2226.
Acknowledgments
We thank Ms. Nilda Jacobus and Dr. Frank Tally for
their support and the analytical chemistry group for
analytical HPLC and spectral data. We also thank Dr.
Jerauld Skotnicki and Dr. Tarek Mansour for helpful
discussion during the preparation of the manuscript.
15. Orth, P.; Schnappinger, D.; Sum, P.-E.; Ellestad, G. A.;
Hillen, W.; Saenger, W.; Hinrichs, W. J. Mol. Biol. 1999,
285, 455.
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