Rifamycin antibiotics - New compounds and synthetic methods. Part 1: Study of the reaction of 3-formylrifamycin SV with primary alkylamines or ammonia

Article abstract of DOI:10.1016/S0040-4020(03)00184-4

Within the study covering the search for new methods of synthesis of rifamycin antibiotics, the reactions of 3-formylrifamycin SV (2) with primary amines or ammonia were studied. In the reaction of 2 with methylamine, unstable 3-methyliminomethylrifamycin SV (8) was formed, which was further oxidised to stable 3-methyliminomethylrifamycin S (9). In the reaction of 2 with ammonia, N,15-didehydro-15-deoxo-pyrimido-(4,5-b)rifamycin SV (10), a new compound with a chromophore system enlarged by a pyrimidine ring, was obtained. The product of its reduction with sodium borohydride - (11) - was also isolated. The structures of the compounds and an explanation of the synthesis mechanism have been proposed on the basis of mass spectrometry results as well as (1D) and (2D) ¹H- and ¹³C NMR analysis. In vitro antituberculous activity of the new compounds have been investigated.

Full text of DOI:10.1016/S0040-4020(03)00184-4

Tetrahedron 59 (2003) 1885–1893
Rifamycin antibiotics—new compounds and synthetic methods.
Part 1: Study of the reaction of 3-formylrifamycin SV
with primary alkylamines or ammonia
a
b
b
Krzysztof Bujnowski,a Ludwik Synoradzki, Eckhard Dinjus, Thomas Zevaco,
,
*
c
Ewa Augustynowicz-Kopec and Zofia Zwolska
c
´
aLaboratory of Technological Processes, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland
b
¨
Forschungszentrum Karlsruhe, Institut fur Technische Chemie, Bereich Chemisch-Physikalische Verfahren,
Postfach 3640, D-76021 Karlsruhe, Germany
cDepartment of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Płocka 26, 01-138 Warsaw, Poland
Received 15 August 2002; revised 13 January 2003; accepted 30 January 2003
Abstract—Within the study covering the search for new methods of synthesis of rifamycin antibiotics, the reactions of 3-formylrifamycin
SV(2) withprimary aminesor ammonia werestudied. In the reactionof2 withmethylamine, unstable 3-methyliminomethylrifamycin SV(8) was
formed, which was further oxidised to stable 3-methyliminomethylrifamycin S (9). In the reaction of 2 with ammonia, N,15-didehydro-15-
deoxo-pyrimido-(4,5-b)rifamycin SV (10), a new compound with a chromophore system enlarged by a pyrimidine ring, was obtained. The
product of its reduction with sodium borohydride—(11)—was also isolated. The structures of the compounds and an explanation of the
synthesis mechanism have been proposed on the basis of mass spectrometry results as well as (1D) and (2D) 1H- and 13C NMR analysis. In
vitro antituberculous activity of the new compounds have been investigated. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
the shortening of therapy duration from 18–24 months to
6–9 months. The mutation in the rpoB gene, which is
responsible for the coding in the b-subunit of the RNA
polymerase, is the genetic basis for the occurrence of
resistance phenomena to 3.12 In addition to their high
activity towards a broad spectrum of Gram-positive bacteria
and mycobacteria, rifamycins have also demonstrated the
capability to subdue certain types of viruses.13–15 It also has
been determined that rifamycins exhibit a specific hypo-
lipidemic action by decreasing lipid levels, especially
cholesterol in blood plasma and lipoproteins.16,17
The discovery of rifamycin antibiotics in the late 1950s1
resulted, in 1965, in the synthesis of rifampicin (3),2
introduced into medical care in 1968, which is still the most
important agent in the treatment of tuberculosis, leprosy and
diseases caused by the nontuberculous mycobacteria, e.g.
mycobacteriosis (Scheme 1). Rifamycins are distinguished
by a unique chemical structure, in particular, an aromatic
chromophore with an ansa chain3 and by a specific
antibacterial action, which consists in selective blocking
of the b-subunit of the DDRP enzyme (DNA-dependent
RNA polymerase) responsible for controlling the rebuilding
process of bacterial peptides.4–10 3 shows good in vitro and
in vivo activities against Mycobacterium tuberculosis,
Mycobacterium kansasii and Mycobacterium marinum.
It has moderate activity against Mycobacterium avium–
intracellulare complex and poor activity against rapid
growers Mycobacterium fortuitum and Mycobacterium
chelonae.11 3 is active against both intracellular and
extracellular M. tuberculosis. Its use in combination with
isoniazid in treatment regimes against tuberculosis allowed
As a result of these unusual properties, the rifamycin
derivatives are of great pharmaceutical interest. Therefore,
new biologically active semisynthetic rifamycins are the
targets of many studies. Clinical testing and studies of
structure–biological activity relations have shown that the
best activities are achieved if structural modifications are
made to the rifamycin S (5) or to its hydroquinonic
analogue, rifamycin SV (1), at the positions C-3 and C-4
of the aromatic chromophore. Another related compound,
3-formylrifamycin SV (2),18,19 is of great importance as
a key intermediate in the syntheses of semi-synthetic
rifamycin derivatives, including 3.
Keywords: rifamycins; ansa compounds; antibacterials; antibiotics;
cyclisation; NMR; pyrimidines; tautomerism.
The purpose of the research carried out by our group is to
search for new C-3-substituted derivatives of 2 exhibiting a
*
Corresponding author. Tel.: þ48-22-6605583; fax: þ48-22-6255317;
e-mail: krzysztof.bujnowski@chemix.ch.pw.edu.pl
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00184-4

1886
K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
Scheme 1. Structures of some common rifamycin derivatives.
high antibacterial activity, especially towards M. tuberculosis.
Three groups of reactions were studied in which 2 was the
starting material:
In the first step of the reaction of 2 with gaseous ammonia in
chloroform in the presence of acetic acid, the formation of
an intermediate was observed (probably of structure 12)
(TLC, intense blue spot), which quickly further reacted to
a new substance (10) (TLC, violet-brown spot, Rf 10,Rf
12 (Scheme 2). Compound (10) was isolated from chloro-
form in the form of violet needles. (The formation of 10
was also observed when the reaction was carried out in
lower aliphatic alcohols or tetrahydrofuran.) 10 was reduced
with sodium borohydride in anhydrous ethanol and the
orange coloured product (11) (TLC, orange spot, Rf 11.Rf
10) was isolated in an amorphous form and purified
(Scheme 2).
1. reactions with primary alkylamines or ammonia,20
2. with gaseous ammonia and acetone,21,22
3. with ammonia or amines and ketones or aldehydes.23
The scope of this work covers the isolation and spectro-
scopic characterisation of the products formed, the study of
the mechanisms involved in these new reactions as well as
the evaluation of their activity through antibacterial activity
tests. The results of the first part of the above mentioned
studies are presented in this paper.
The structures of the new rifamycin compounds (9), (10),
and of the already reported 1124,25 have been proposed on
the basis of the mass spectrometry and 1H, 13C NMR-
analysis in (D6)DMSO solutions (Scheme 2, Tables 1–3).
8 and 9 can be included into the known group of
3-iminomethylrifamycin SV2 and S derivatives. Criccho
et al. obtained a broad range of substances of type 8 as
hydrazone derivatives by the condensation of 2 with
hydrazine substrates having a primary amine group.26
Rifamycin S (5) was the starting material in another method
of synthesis of rifamycin SV (1) 3-iminomethyl deriva-
tives.27 11 was first synthesised by McCarthy et al.24,25 in a
reductive amination of 2 with ammonia in methanol in the
presence of sodium cyanoborohydride and hydrogen
chloride.
2. Results and discussion
2.1. Synthesis and purification
After the reaction of 2 with methylamine in chloroform in
the presence of acetic acid as a catalyst, the formation of a
product of presumed structure 8 was observed (TLC, intense
blue spot). (A similar course was found if the process was
carried out in lower aliphatic alcohol or tetrahydrofuran.)
This unstable substance gradually decomposes during
isolation from the reaction system, the hydrolysis to the
starting aldehyde (2) being the main decomposition path-
way observed (TLC). Compound (8) was isolated in the
form of a crude precipitate, followed by oxidation with
manganese dioxide in chloroform to obtain, after purifi-
cation, an amorphous, olive coloured, substance (9)
(quinonic analogue of 8) (Scheme 2). Other monoalkyl-
amines such as isopropylamine or tert-butylamine reacted
analogously.
2.2. NMR studies
Generally, the assignments of proton and carbon signals of
1
9–11 were based on (1D) H NMR and 13C NMR spectra,
on (2D) NMR 1H,1H COSY and 1H,13C COSY experiments
(the latter were optimised for a single bond coupling

K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
1887
Scheme 2. Reaction of 2 with primary alkylamines and ammonia.
1
constant) and, in the case of 10 and 11, H,13C HMBC
with 1,3,5-tri-t-butylhexahydro-1,3,5-triazine) occurs at
154.3 ppm. After expansion of the described fragment of
the DEPT 135 spectrum, separation of all the signals into
two distinct peaks was observed, e.g. the signal of C-28
appears at 121.35 and 121.14 ppm, C-18 at 125.48 and
correlation spectra (optimised for more than a single C–H
bond interaction) (Tables 1–3). In addition, DEPT 135
1
spectra as well as H NMR (H!D exchange) experiments
recorded for all the samples were used. The rest of signal
assignments were based on the literature28–33 and our own
spectroscopic database.
1
125.26 ppm. In the H,13C, COSY spectrum a correlation
signal H-10/C-10 was identified.0In the 1H NMR spectrum the
signal corresponding to the H-1 proton occurs in the form of
two partially overlapping doublets at ca. 8.54 ppm. Two
singlets are observed in this field after H!D exchange: a
larger one (8.59 ppm) and a smaller one (8.49 ppm). The
signal of the analogous C-3–CHvNþ proton in Tagushi’s
rifamycin derivative (CDCl3) appears at 8.98 ppm,31 and
that of the C-3–CHvN proton in rifampicin (CDCl3) at
8.25 ppm.32
Compound 9. In the DEPT 135 spectrum within the 100–
160 ppm range, the five signals of all the carbon atoms of
CHv groups of the ansa chain were0found. The sixth signal
(159.5 ppm) was assigned to the C-1 atom of the substituent
at C-3. In comparison, the signal of the analogous carbon
atom C-3–CHvNþ in the rifamycin derivative identified
by Taguchi et al.31 (reaction product of rifamycin S (5)

1888
K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
Table 1. 13C and 1H NMR data of 9 in (D6)DMSO
Atom C
d
Atom H
d
Multipl.
br
JH,H
1H,1H COSY correlations
3H-30 lr
H–N(amide)
10.71
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
180.6
111.0
112.4
179.9
101.2
167.3
101.8
177.3
HO-8
11.25
br
H-10
C-9
112.4
136.4
190.8
107.4
21.8
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
3H-13
3H-14
1.63
2.17
s
s
7.3
169.5
128.9
136.9
125.2
142.2
36.7
H-17
H-18
H-19
H-20
H-21
HO-21
H-22
H-23
HO-23
H-24
H-25
H-26
H-27
H-28
H-29
3H-30
3H-31
3H-32
3H-33
3H-34
6.52
6.50
6.24
2.30
3.08
4.47
1.37
3.14
2.92
1.60
5.08
1.24
3.43
5.25
6.30
2.09
0.85
0.53
0.88
20.03
a)
a)
a)
a)
dd
s
a)
a)
m
a)
dd
m
m
dd
a)
s
H-18
H-17, H-19
H-20
H-19, H-21, H-31
H-20, HO-21
H-21
H-23, 3H-32
H-22, H-24, HO-23
H-23
H-23, 3H-33
H-26
77.7
C-22
C-23
37.6
75.5
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-31
C-32
C-33
C-34
C-35
C-36
C-37
C-10
32.9
72.5
40.4
75.7
121.1
145.5
21.1
14.4
9.5
(25,24),2.0
(25,26)¼10.1
(27,28)¼7.2
(28,29)¼12.6
H-25, H-27, 3H-34
H-26, H-28
H-27, H-29
H-28
a)
d
a)
d
H-20
(32,22)¼6.0
(33,24)¼6.6
(34,26)¼6.0
10.4
9.3
H-24
H-26
172.6
20.6
56.0
159.5
37.5
3H-36
3H-37
H-10
3H-30
2.00
2.97
8.56
3.28
s
s
m
m
HO-8, 3H-30 lr
H–N(amide) lr, H-10 lr
C-30
Abbreviations: s: singlet, d: doublet, m: multiplet, br: broad, lr: long range correlation, a) this signal overlapped with another signal.
In the 13C NMR spectrum the signal of the C-30 carbon occurs
with respect to the one found at 166.7 ppm in the spectrum
at 37.5 ppm. In the H,13C, COSY spectrum the correlation
of rifamycin S (CDCl3).29 A similar difference, of ca.
13 ppm, occurs for the chemical shifts of the C-1 signal of
the sodium phenolate at 168.1 ppm and that of phenol alone
at 155.4 ppm (CDCl3).33
1
signal 3H-30/C-30 was identified. In the 1H NMR spectrum the
signal of the 3H-30 protons occurs as a doublet at 3.41 ppm.
0
1
Two different size signals of proton H-1 in the H NMR
spectrum after H!D exchange, as well as the splitting of
signals in the carbon spectrum indicate that 9 occurs in the
form of a mixture of two isomers, probably differing in the
(E)- and (Z)-configuration of the CvN bond of the imino
group. It appears that one of the two isomers quantitatively
prevails.
Compound 10. On the basis of the correlation signals
1
1
recorded in H,13C COSY and H,13C HMBC spectra, the
signal (155.3 ppm) in the 13C NMR spectrum was attributed
to the C-60 (sp2) carbon atom. The chemical shift of this
signal is analogous to the one found for the C-3–CHvNþ
carbon atom at 154.3 ppm in the substance produced by
Taguchi et al.31 and the chemical shift of the C-10–C-4vN-
3 carbon signal at 155.9 ppm in the quinazoline (CDCl3).34
In the 1H NMR spectrum of 10 the signal of the H-60 proton
occurs as a singlet (9.57 ppm), which does not disappear
after H!D exchange. In the 1H,13C HMBC spectrum
correlation signals ar0e observed originating from long range
couplings of the H-6 protons with carbon atoms: C-1, C-2,
C-3, C-4 and C-15 (Table 2).
In a (D6)DMSO solution of 9 a polar tautomeric form (B) in
which a proton is present at the nitrogen atom N-20 probably
dominates (Scheme 3). The correlation signals HO-8/H-10,
0
0
0
1
HO-8/3H-3 and H-1 /3H-3 in the H,1H COSY spectrum
and the splitting of signals of H-10, 3H-30 and HO-8 protons
in the 1HNMR spectrum (Table 1) are a result of this
tautomeric equilibrium. Thus, the chemical shift of the
signal at 177.3 ppm in the 13C NMR spectrum of 9,
tentatively assigned to C-8, is ca. 11 ppm low-field shifted
Assignment 15 signals of quaternary carbon atoms in the

K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
Table 2. 13C and 1H NMR data of 10 in (D6)DMSO
1889
Atom C
C-1
d
Atom H
d
Multipl.
JH,H
1H,1H COSY correlations
1H,13C HMBC correlations
150.4
HO-1
16.38
s
C-2
C-3
C-4
134.8
113.1
141.1
HO-4
HO-8
13.16
7.09
s
C-2, 3, 4
C-5
C-6
C-7
C-8
98.1
173.0
100.1
185.5
br
C-9
115.2
113.6
183.4
108.8
22.2
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
3H-13
3H-14
1.67
1.95
s
s
C-11, 12
C-6, 7, 8
7.4
160.2
135.5
130.0
126.4
137.1
38.1
H-17
H-18
H-19
H-20
H-21
HO-21
H-22
H-23
HO-23
H-24
H-25
H-26
H-27
H-28
H-29
3H-30
3H-31
3H-32
3H-33
3H-34
6.28
6.35
5.83
2.12
3.50
4.47
1.13
2.79
4.32
1.51
5.04
1.13
3.23
4.92
6.20
2.28
0.85
0.30
0.81
-0.37
d
(17,18)¼10.9
(18,19)¼15.7
(19,20)¼6.8
H-18
C-16, 19
C-16, 20
dd
dd
m
ddd
d
a)
ddd
d
m
dd
a)
dd
dd
d
H-17, H-19
H-18
3H-31
(20,21)¼8.0
72.2
(21,22)¼2.7
(HO-21,21)¼3.5
(22,23)¼2.4
HO-23 lr
H-23, 3H-32
H-22, HO-23
HO-21 lr, H-23
3H-33
H-26
H-25, 3H-34
H-28
C-22
C-23
37.4
75.8
(23,24)¼10.0
(HO-23,23)¼7.4
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-31
C-32
C-33
C-34
C-35
C-36
C-37
C-60
32.9
73.0
40.2
76.4
116.8
142.4
20.6
18.2
7.7
11.6
8.4
169.7
22.1
55.8
155.3
(25,24),1.0
(25,26)¼11.0
(27,26),1.0
(27,28)¼7.8
(28,29)¼12.7
C-35
C-28
H-27, H-29
H-28
C-12, 27, 28
C-15, 16, 17
C-19, 20, 21
C-22
C-24
C-25, 26, 27
s
d
d
d
(31,20)¼7.0
(32,22)¼6.9
(33,24)¼7.0
(34,26)¼7.0
H-20
H-22
H-24
d
3H-36
3H-37
H-60
1.98
2.91
9.57
s
s
s
C-35
C-27
C-1, 2, 3, 4, 15
13C NMR spectrum was made on the basis of the correlation
An unusual process has been noticed in the aromatic
chromophore of 10 in (D6)DMSO solutions. The molecular,
and thus magnetic, environment of C-8 and C-11 becomes
similar (a small difference in the chemical shifts of atoms
C-8 and C-11, 185.5 and 183.4 ppm, respectively, can still
be detected in the 13C NMR spectrum) (Table 2). This can
be correlated to a quick migration of the HO-8 proton and
the coexistence in solution of equal quantities of isomeric
forms (C) and (D) (Scheme 3). Taguchi et al. drew attention
to a similar phenomenon when interpreting the results of
NMR studies of a rifamycin derivative obtained from the
reaction of rifamycin S with 1,3,5-tri-t-butyl hexahydro-
1,3,5-triazine (CDCl3).31 This process might result from the
electron withdrawing influence of the newly formed
pyrimidine ring, condensed at C-2 and C-3 of the
chromophore, which consequently increases the acidity of
the phenol hydroxyl group at C-8. In the 13C NMR spectrum
of rifampicin (3) (CDCl3) the C-8 and C-11 signals occur at
169.2 and 195.4 ppm, respectively,32 which indicates the
absence of such a tautomeric equilibrium.
1
signals found in the H,13C HMBC spectrum, utilising the
literature28–32 and our own spectroscopic data (Table 2).
The C-15 signal (160.2 ppm) is high-field shifted by ca.
8–10 ppm with respect to the C-15 signal in various
rifamycin SV derivatives, in which this carbon atom is a part
of a tertiary or secondary amide group.31,32 This has been
ascribed to the formation of a new aromatic pyrimidine ring
as a part of a substituted quinazoline system in which the
C-15 carbon atom is bonded to two nitrogen atoms (Scheme
2). A similar chemical shift (160.7 ppm) was assigned for
the signal of the N-1–C-2vN-3 carbon atom of quinazoline
(CDCl3).34
Despite careful drying of the sample under vacuum,
supplementary signals were observed at 79.13 ppm in the
13C NMR spectrum and at 8.31 ppm in the 1H NMR
spectrum, these signals can be attributed to the presence of
chloroform in the crystals. This latter signal was absent
when CDCl3 instead of CHCl3 was used for the synthesis
and crystallisation of 10. This proves the formation of a
very stable adduct of 10 with chloroform.
Compound 11. The negative-phased signal of the C-60 atom

1890
K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
Table 3. 13C and 1H NMR data of 11 in (D6)DMSO
Atom C
C-1
d
Atom H
d
Multipl.
JH,H
1H,1H COSY correlations
1H,13C HMBC correlations
142.6
HO-1
HO-4
16.50
12.40
s
s
C-2
C-3
C-4
C-5
C-6
C-7
C-8
117.9
104.8
141.5
98.4
171.7
100.8
183.8
C-2,3,4,10
HO-8
11.62
s
C-9
113.3
112.4
186.0
108.2
21.7
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
3H-13
3H-14
1.75
2.03
s a)
s
C-11,12
C-6,8
7.1
157.3
124.4
123.1
134.3
141.6
39.5
H-17
H-18
H-19
H-20
H-21
HO-21
H-22
H-23
HO-23
H-24
H-25
H-26
H-27
H-28
H-29
3H-30
3H-31
3H-32
3H-33
3H-34
6.17
6.53
6.08
2.28
3.90
4.83
1.61
3.14
4.66
1.75
5.30
1.32
3.44
5.06
6.28
2.19
0.99
0.73
1.02
20.02
a)
dd
a)
m
m
d
m
a)
a)
a)
dd
m
d
dd
d
s
d a)
d
d a)
d
H-18
H-17, H-19
H-18
H-21, 3H-31
H-20
HO-23 lr
H-23, 3H-32
H-22, HO-23, H-24
HO-21 lr, H-23
H-23, 3H-33
H-26
71.0
C-22
C-23
37.4
75.8
C-21
C-35
C-28
C-24
C-25
C-26
C-27
C-28
C-29
C-30
C-31
C-32
C-33
C-34
C-35
C-36
C-37
33.7
73.0
40.0
76.4
116.3
142.2
20.2
18.1
8.2
12.8
8.2
170.0
20.7
56.0
(25,24),2.0
(25,26)¼10.8
(27,28)¼7.0
(28,29)¼12.7
H-25, 3H-34
H-28
H-27, H-29
H-28
C-12,28
C-15,16
C-20,21
C-22
C-24
C-26,27
H-20
H-22
H-24
(32,22)¼6.8
(34,26)¼6.7
3H-36
3H-37
H-10
2H-60
2.14
3.08
10.15
4.66
s
s
s
a)
C-35
C-27
C-60
38.7
C-4,10,15
in the newly formed dihydropyrimidine ring was found in
the DEPT 135 spectrum of this substance at 38.7 ppm. In the
1H NMR spectrum the signal of two protons bonded to C-60
occurs as a doublet at 4.66 ppm—as a result of the coupling
with H-10 (Table 3).
leading to a similar chemical environment for carbons C-8
and C-11 which can again be seen in the 13C NMR spectrum
(183.8 and 186.0 ppm, respectively).
11, contrary to 10, does not show a tendency to form an
adduct with chloroform.
Assignment of the quaternary carbon atoms present in 11
was made on the basis of the correlation signals found in the
1H,13C HMBC spectrum, by comparison with the spectrum
of 10 and the literature data.28–32 Thus, the signal at
157.3 ppm was assigned to the C-15 carbon atom in 11
(Table 3).
2.3. Synthesis of 10, mechanistic proposal
In the first stage of the reaction of 2 with ammonia an
intermediate is formed, most probably an iminomethylrifa-
mycin SV (12), which then undergoes an intramolecular
cyclisation to form the stable new derivative (10), with the
rifamycin chromophore enlarged by the newly formed
pyrimidine ring (Scheme 2).
1
In the H NMR spectrum of 11 four signals are present at
low field assigned to mobile protons (these signals disappear
after H!D exchange). Three signals were assigned to
the three hydroxyl protons of the chromophore and one, at
10.15 ppm, to the H-10 in the newly formed dihydropyrimi-
dine ring (Table 3).
This intramolecular cyclisation does not occur when another
substituent instead of a proton (e.g. an alkyl group) is
present in the imino group of the intermediate (Scheme 2).
The possibility for the imino group of the intermediate’s
carbocation to eliminate a proton is most probably required
for the formation of a pyrimidine system in 10.
A tautomeric exchange probably occurs in a (D6)DMSO
solution of 11, as found in the case of 10 (Scheme 3),

K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
1891
Scheme 3. Tautomeric equilibria in (D6)DMSO solution of 9–11.
2.4. Initial studies on the in vitro antibacterial properties
of 10 and 11
3. Conclusion
Unstable 3-alkyliminomethyl derivatives of rifamycin SV,
of type 8, which are formed in the reaction of 2 with
monoalkylamines, give stable quinonic 3-alkyliminomethyl
compounds of type 9 after oxidation. Compound (9)
probably occurs in the form of a mixture of two isomers,
differing in the (E)-and (Z)-configuration of the substituents
at the C-10vN-20 bond.
The obtained novel compounds were tested for antimicro-
bial and tuberculostatic activity in vitro against different
strains of M. tuberculosis. The following strains have been
used: (i) standard strain—M. tbc H37Rv; (ii) some wild
strains isolated from patients and sensitive to rifampicin
(3)—M. tbc 234, M. tbc 243; (iii) some wild strains isolated
from patients and resistant to rifampicin (3)—M. tbc 125,
M. tbc 220, M. tbc bovis. They were also tested against
different types of MOTT (Mycobacteria other than
M. tuberculosis) sensitive or resistant to rifampicin (3):
M. kansasii, M. scrofulaceum, M. avium intracellulare,
M. fortuitum. 10 shows a marked antituberculous activity
(MIC 0.5–1.0 mg/ml) against strains sensitive t o rifampicin
although lower than 3 itself (Table 4). 11 proved to be much
less active than 10. All strains of M. tuberculosis resistant to
3 and all strains of MOTT tested were resistant to 3, 10 and
11 at concentrations below 1.0 mg/ml.
N,15-Didehydro-15-deoxo-pyrimido-(4,5-b)rifamycin SV
(10)—a new derivative with an enlarged chromophore
system is the reaction product of 2 with ammonia, in which
the C-3 and C-15 carbon atoms of the backbone are linked
by a newly formed –CHvN– group, building a pyrimidine
ring. This results in a shortening of the ansa chain and leads
to a molecular structure which differs from the structure of
natural rifamycins.
The presented synthesis of 10 is also an interesting variant
Table 4. Antimycobacterial activity (MIC in mg/ml) of compounds 10 and 11 with reference to rifampicin (3). Protocol of the in vitro experiments
Strain
Compound
Concentration of compound (mg/ml)
0
0.03
0.06
0.12
0.25
0.5
1.0
3.1
M.tbc H37Rv
M. tbc 234
M. tbc 234
3
10
11
þþþ
þþþ
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
2
2
nt
3
10
11
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþ
þ
2
2
2
2
þþþ
2
2
þþþ
nt
nt
nt
þþþ
þþþ
þþþ
þþþ
þþ
þ
þþþ
þþþ
3
10
11
þþþ
þþþ
þþþ
þþ
þ
2
2
þþþ
nt
nt
nt
þþ
þ
þþþ
þþþ
(þ)—growth, (2)—no growth, nt—not tested.

1892
K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
of the synthesis of substituted pyrimidine rings as parts of
quinazoline systems. 10 is easily reduced with sodium
borohydride in ethanol to yield 11—a compound with a
dihydropyrimidine ring as the main product (Scheme 2).
0.5 h. After this time an orange coloured oxidation product
(9) was observed (TLC, CHCl3/MeOH 9:1). Manganese
dioxide was then filtered off from the reaction mixture. The
chloroform filtrate was dried with anhydrous sodium
sulphate and concentrated to ca. 4 ml. This concentrate
was purified by CC (CHCl3/MeOH 9:1 mixture as the
eluent). The selected fractions containing 9 were washed
with water, concentrated under vacuum to ca. 5 ml and
diluted with n-hexane, to give 9 as an amorphous
In a (D6)DMSO solution of 9–11 specific tautomeric
phenomena were observed (Scheme 3). 9, as a result of the
migration of the HO-8 proton to the N-20 nitrogen atom,
occurs mainly in the dipolar form (B). However, the
migration of the HO-8 proton to the OvC-11 carbonyl
group in 10 or 11 results in an equilibrium of the isomeric
forms of (C) and (D). In introductory studies it was found
that 10 shows clear action against M. tuberculosis.
1
precipitate. Yield: 0.15 g of 9 (14.6%). 13C and H NMR
data: Table 1. MS (ESI) m/z: 737.3 (100.0, (MþH)þ), 759.3
(66.7, (MþNa)þ). HRMS calcd for (MþH)þ C39H48N2O12
737.3280, found 737.3310, error 4.1 ppm. IR: 3460 (br.),
2970, 1730, 1650, 1590, 1405 cm21
.
4.1.2. N,15-Didehydro-15-deoxo-pyrimido-(4,5-b)rifa-
mycin SV (10). A solution of 5.8 g (ca. 8 mmol) of 2 in a
mixture of 50 ml of chloroform and 2.0 ml (ca. 40 mmol) of
acetic acid was saturated under stirring for 2 min with
gaseous ammonia at 40–458C. After 1 h of reaction at this
temperature, the initial aldehyde (2) underwent complete
conversion (TLC). The reaction mixture was first washed
with 150 ml of water; the pH of the aqueous phase was
adjusted during washing with 5% H2SO4 to ca. 6.0–7.0. The
chloroform solution was then washed with 150 ml of
aqueous Na2HPO4 (5%), and next dried with anhydrous
sodium sulphate. The solution was evaporated to ca. 30 ml
and left for 16 h at ca. 58C. The crystalline violet product
was filtered off, washed with cold (108C) chloroform and
dried under vacuum. Yield: 3.7 g of 10 (65.5%). 13C and 1H
NMR data: Table 2, MS: (ESI) m/z: 727.3 (100.0,
(MoxidisedþNa)þ)p, 729.3 (65.5, (MþNa)þ), 751.3 (21.4,
4. Experimental
4.1. General
The reagents used: 3-formylrifamycin SV (2) (Polfa—
Tarchomin) and methylamine, isopropylamine, t-butyl-
amine, ammonia, acetic acid (all-synthesis grade purity).
TLC: Merck Al-precoated silica gel 60 F254 (0.2 mm)
plates, a CHCl3/MeOH mixture (9:1) was used as the eluent.
CC: glass columns, Merck silica gel 60 (0.040–0.063 mm),
CHCl3/MeOH (9:1) as the mobile phase. NMR: the 1H and
13C NMR spectra were recorded on a Bruker AC 200 (1H:
200.13 MHz, 13C: 50.32 MHz) and Varian InovaUnity 400
spectrometer (1H: 400.20 MHz, 13C: 100.64 MHz) in
(D6)DMSO; chemical shift values d in ppm relative to
SiMe4 as internal reference; J in Hz. In order to
unequivocally attribute some coupling constants and
(MþNaþMeOH)þ)p p
.
HRMS calcd for (MþNa)þ
1
chemical shifts (1D) H NMR spectra were recorded on a
Bruker AM 500 instrument (1H: 500. 13 MHz). MS: mass
spectra by the ESI technique, containing high resolution ESI
MS, were recorded by employing a Mariner (PerSeptive
Biosystems) mass spectrometer. For analysis, samples were
dissolved in CH3OH. IR: IR spectra (KBr pellets) were
recorded on a Perkin–Elmer PE-577 (9) and Perkin–Elmer
16 PC (10, 11) spectrometers. Mp determination: the new
rifamycin derivatives have no definite melting points—they
do not melt up to 3008C, at ca. 1608C decomposition starts.
Biological tests: bacteriostatic properties of the compounds
were studied on agar Petri plates, Youmans liquid medium
containing 10% bovine serum. Rifampicin (3) (Ciba-Geigy)
has been used as a reference drug.
C38H46N2O11Na 729.2994, found 729.3011, error
2.4 ppm). ( p )—During MS analysis oxidation of 10
(hydroquinone) to the quinone form takes place. ( p p )—
Ion of the adduct with methanol. IR: 3432 (sharp), 2968,
1712 (sharp), 1648, 1622 cm21
.
4.1.3. N,15-Didehydro-15-deoxo-10,60-dihydropyrimido-
(4,5-b)rifamycin SV (11). 1.0 g (ca. 1.4 mmol) of 10 in
25 ml of dry ethanol was stirred at room temperature with
0.1 g (ca. 2.6 mmol) of NaBH4. After 2 h, the conversion of
10 was completed and the formation of a mixture of
products with a dominance of 11 (TLC, CHCl3/MeOH 9:1,
orange spot, Rf 11.Rf 10) could be noticed. The reaction
mixture was diluted with 30 ml of chloroform, followed by
cautious addition of 50 ml aqueous Na2HPO4 (5%). The
organic phase was then once again washed with 30 ml of
Na2HPO4 (5%), dried with anhydrous sodium sulphate and
evaporated to dryness, obtaining 0.8 g of a crude precipitate
of 11. It was then purified by CC (CHCl3/MeOH 88:12). 11
was obtained as an amorphous yellowish powder after
precipitation with n-hexane from concentrated selected
fractions of the eluate. Yield: 0.25 g of 11 (24.9%). 13C and
1H NMR data Table 3. MS: (ESI) m/z: 709.3 (100.0,
(MþH)þ). HRMS calcd for (MþH)þ C38H49N2O11
709.3331, found 709.3365, error 4.8 ppm. IR: 3416 (br.),
4.1.1. 3-[(Methylimino)methyl]rifamycin S (9). 5.0 g (ca.
7 mmol) of 3-formylrifamycin SV (2) was dissolved in a
solution of 40 ml chloroform and 1.6 g (ca. 27 mmol) acetic
acid. The reaction mixture was stirred at 30–358C and
saturated with methylamine. After 2.5 h the starting
aldehyde 2 reacted (TLC, CHCl3/MeOH, 9:1) to yield the
blue-violet substance (8) as the main product. The reaction
mixture was washed twice with 150 ml of water. The
remaining organic phase was dried with anhydrous sodium
sulphate, and after separating the drying agent, evaporated
to dryness, to obtain ca. 5.0 g of the crude product
containing 8 as the main component. 3-Formylrifamycin
SV (2) appeared among the precipitate components as a
result of the partial hydrolysis of 8. 1.0 g of the compound
(8)/the crude reaction mixture was dissolved in 25.0 ml of
chloroform and stirred with 0.6 g of manganese dioxide for
2974, 1714, 1646, 1602 cm21
4.2. Biological tests
See Table 4.
.

K. Bujnowski et al. / Tetrahedron 59 (2003) 1885–1893
1893
16. Traxler, Q.; Kump, W.; Mueller, K.; Fosch, W. J. Med. Chem.
1990, 33, 552.
Acknowledgements
17. Menear, K. A.; Dunnet, K. E.; Hastewell, J. G.; Eichler, H. G.;
Gibson, J. C.; Kimble, E. F.; Kump, W.; Taylor, P. W.
Arzneim.-Forsch./Drug Res. 1992, 42(II), 1125.
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The authors wish to express their gratitude to the Max-
Planck-Gesellschaft, Bundes Ministerium fu¨r Bildung und
Forschung (BMBF), and Polish Committee of Scientific
Research (KBN) for the financial support of our research.
We also would like to thank Professor Dr H. Bojarska-
Dahlig and Dr T. Mizerski for their valuable advice and
discussion.
20. Bujnowski, K.; Synoradzki, L.; Bojarska-Dahlig, H.; Dinjus,
E.; Gassner, F. Warsaw University of Technology. PL Pat.
Appl. P. 325572, 1998.
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E.; Leitner, W.; Gassner, F. Warsaw University of Tech-
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